Co-cultures of cerebellar slices from mice with different <i>reelin</i> genetic backgrounds as a model to study cortical lamination

Adalberto Merighi; Laura Lossi

doi:10.12688/f1000research.126787.1

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Method Article

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

[version 1; peer review: 2 approved with reservations]

Adalberto Merighi ¹, Laura Lossi¹

PUBLISHED 17 Oct 2022

Author details Author details

¹ Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

Adalberto Merighi
Roles: Data Curation, Methodology, Validation, Writing – Original Draft Preparation

Laura Lossi
Roles: Conceptualization, Investigation, Methodology, Resources, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the NC3Rs gateway.

Abstract

Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler phenotype in mice, the first described cerebellar mutation. In homozygous mutants missing the Reelin gene (reln^-/-), neurons are incapable of correctly positioning themselves in layered brain areas such as the cerebral and cerebellar cortices. We here demonstrate that by employing ex vivo cultured cerebellar slices one can reduce the number of animals and use a non-recovery procedure to analyze the effects of Reelin on the migration of Purkinje neurons (PNs).
Methods: We generated mouse hybrids (L7-GFPrelnF1/) with green fluorescent protein (GFP)-tagged PNs, directly visible under fluorescence microscopy. We then cultured the slices obtained from mice with different reln genotypes and demonstrated that when the slices from reln^-/- mutants were co-cultured with those from reln^+/- mice, the Reelin produced by the latter induced migration of the PNs to partially rescue the normal layered cortical histology. We have confirmed this observation with Voronoi tessellation to analyze PN dispersion.
Results: In images of the co-cultured slices from reln^-/- mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than those generated from slices of reln^+/- animals. The mean roundness factor, area disorder, and roundness factor homogeneity were different when slices from reln^-/- mice were cultivated singularly or co-cultivated, supporting mathematically the transition from the clustered organization of the PNs in the absence of Reelin to a layered structure when the protein is supplied ex vivo.
Conclusions: Neurobiologists are the primary target users of this 3Rs approach. They should adopt it for the possibility to study and manipulate ex vivo the activity of a brain-secreted or genetically engineered protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the total avoidance of severe surgery (3Rs perspective).

Keywords

Reelin, Neuronal migration, Cerebellum, Purkinje neurons, Secreted proteins, Ex vivo methods, Cellular sociology, Voronoi tessellation

Corresponding authors: Adalberto Merighi, Laura Lossi

Competing interests: No competing interests were disclosed.

Grant information: Much of this work was supported by a grant (CRACK IT Solution Neuroinflammation and nociception in a dish) from the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs), London (UK).

Copyright: © 2022 Merighi A and Lossi L. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Merighi A and Lossi L. Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination [version 1; peer review: 2 approved with reservations]. F1000Research 2022, 11:1183 (https://doi.org/10.12688/f1000research.126787.1) First published: 17 Oct 2022, 11:1183 (https://doi.org/10.12688/f1000research.126787.1) Latest published: 02 Oct 2023, 11:1183 (https://doi.org/10.12688/f1000research.126787.2)

Research highlights

Scientific benefit(s):

• Co-culturing slices from animals with different reln genetic backgrounds allows studying ex vivo the effects of Reelin in cortical lamination
• Co-cultures can be pharmacologically manipulated and transfected with different types of fluorescent reporter proteins (FRP)
• They are amenable to electrophysiological recordings and immunocytochemical labeling

3Rs benefit(s):

• As several viable slices can be obtained from every single animal, these cultures substantially reduce the necessary number of mice in different experiments
• When a secreted molecule is to be studied (such as in the case of Reelin), this approach can be used to replace in vivo experiments where the substance has to be administered through more or less invasive routes, involving heavy surgery for molecules that are unable to pass the blood-brain barrier

Practical benefit(s):

• As experiments in vivo are more expensive than those in ex vivo/in vitro conditions, slice co-cultures are highly valuable in terms of cost vs. effectiveness
• They allow mid-throughput screening of different culture conditions, e.g., days in vitro, the chemical composition of the medium, etc., offering the possibility to save time and plan fewer in vivo confirmatory experiments, if necessary
• They are less technically demanding than in vivo experiments

Current applications:

• Study of the effect of Reln dosage on the differentiation of the laminated structures of the brain, primarily the cerebral and cerebellar cortex

Potential applications:

• Co-cultures can be used for the study of cerebellar neuronal wiring ex vivo, e.g., to reconstruct in a dish the olivo-cerebellar tract (climbing fibers) by cultivating together slices from cerebellum and medulla oblongata
• Co-cultures can be used to study the development and/or neurodegeneration in other areas of the brain and spinal cord

Introduction

Like any other animal tissue, the nervous tissue is made up of cells and the surrounding extracellular matrix. Proteins that are released from the neural cells consist of those in the extracellular matrix itself, as well as extracellular signaling and adhesion molecules.¹ Compared to neurons and neural precursor cells, glial cells release a comparatively modest amount of proteins with a narrower range of functions, and according to a two-dimensional (2D) gels and liquid chromatography/mass spectrometry study, about 22% of the proteins secreted by neural cells intervene in cell-to-cell interactions.¹

Reeler was the first discovered mouse cerebellar mutation,² it was distinguished by typical gait changes ("reeling"), and thus thereafter named. In recessive homozygous mutants (reln^-/-), Reelin, a large secreted extracellular matrix glycoprotein, was completely absent and proved to be required for the normal development of layered brain structures (i.e., the cerebral and cerebellar cortices) being directly involved in neuronal migration.³ Reelin absence causes severe cerebellar hypoplasia in reln^-/- mice. During the development of the cerebellar cortex, the granule cells synthesize and release the molecule into the neuropil, and Reelin acts as an attractant for correct migration and placement of the Purkinje neurons (PNs).³ Remarkably, only 5% of these neurons align into their typical location between the mature molecular and granular layers, 10% remain in the internal granular layer, and those left behind are distributed throughout the white matter of the medullary body in a rather compact central mass.⁴^–⁶ Differently from reln^-/- mice, heterozygous reln^+/-, and homozygous reln^+/+ animals, do not display obvious disturbances in cortical histology, although the size and number of the PNs, as well as their topology, may be somewhat altered also in the former.⁷

More recently, it was demonstrated that not only Reelin is implicated in neuronal migration but, after development, it intervenes in synaptogenesis, neuronal plasticity,⁸^–¹⁰ and several neuropsychiatric disorders.¹¹^–¹³ In addition, as Reelin is somehow the prototype of the brain extracellular matrix proteins because of its widely demonstrated intervention in the process of neural migration, there is a wide interest in gaining more information about its role in the normal and pathological brain. Finally, it seems reasonable to hold that the development of a reliable method to study the effects of Reelin on neuronal migration on live cells would be of benefit to the study of many other secreted brain proteins that regulate cell-to-cell interactions.

Several approaches are available for the study of these proteins. Among those in vitro, one can, for example, mention the above proteomic study, which was carried out on cortical neurons and astrocytes, as well as cell lines that were derived from dividing neural precursor cells of E16 rats.¹ Other approaches have used in vivo microdialysis combined with proteomics to discover new bioactive neuropeptides in the striatum¹⁴ or biopanning, an affinity selection technique that selects for peptides binding to a given target to identify proteins of the extracellular matrix.¹⁵ These and other more sophisticated secretome studies, for example,¹⁶ are very important in the initial identification of individual proteins in specific neural cell populations but do not offer any cues about their function and are not suitable to be used in longitudinal studies aiming to understand the effects of a given protein over time.

Longitudinal studies in vivo that are necessary to follow Reelin (and other brain-secreted proteins) intervention at different time points of development or in adulthood require a high number of animals at different ages to lead to conclusive and biologically relevant results. We here report on an ex vivo procedure to study the effect of Reelin on neuronal migration. Our procedure is based on the use of organotypic co-cultures of the mouse postnatal cerebellum¹⁷ but can be broadly employed in the study of the biological role of this (and other) secreted molecule in the brain. Alternatively, one could use three-dimensional (3D) cultures, but a reliable reconstruction of neural circuits is still very difficult to achieve and one should very well know these circuits in vivo such is e.g., the case of the retina.¹⁸ Moreover, the approach is usually expensive, time-consuming, and cellular and biomolecular analysis difficult to perform.¹⁹

The 3Rs relevance of our approach is primarily related to 1. The reduction of the number of experimental animals; 2. The refinement of the procedures eluding the administration in vivo of molecules with (potential) toxic effects and the use of heavy brain surgery (e.g., the intraventricular administration of substances that are unable to cross the blood-brain barrier and/or the need to implant osmotic pumps for sustained administration over time).

Potential end-users are neurobiologists chiefly interested in brain development and neurodegeneration from a structural, functional, and pharmacological point of view. Neuromodulation, i.e., the continuous change of synaptic network parameters, is required for adaptive neural circuit performance. This process is primarily based on the binding of a variety of secreted “modulatory” ligands to G protein-coupled receptors, which govern the operation of the ion channels affecting synaptic weights and membrane excitability.²⁰ The possibility to also apply our approach to studies on neuromodulation substantially widens the number of potentially interested researchers and opens yet unexplored avenues to implement the 3Rs principles.

The need for 3Rs research in these fields is supported by quantitative data. It is difficult to give an accurate estimate of the number of animals used for the purpose locally and worldwide. Yet one can reasonably hold that at least a 20% reduction (a figure based on the number of slices that can usually be generated per mouse) in their total number could be achieved by the adoption of this (and other) procedures ex vivo, as discussed in Ref. 17.

With specific regard to Reelin activity in normal and pathological conditions, a PubMed search (June 2022) with the string “reelin brain” gives back 1,499 results with a peak of 98 papers published in 2010 and a mean of 57 papers/year starting from 1993 (year of the first publication). A similar number of papers is retrieved from the Web of Science™ (1,349) and groups that have published at least two papers belong to 46 different countries. One has to consider that these figures increase substantially if the search is widened to secreted proteins more generally (i.e., 3,154 papers in PubMed for the string: secreted proteins AND “cell migration” AND brain). Although not often easy to glean from the Material and Methods section, in a typical publication in vivo, animal number ranges from 50 to 80 depending on the types of experiments, the number of experimental groups, and the approaches used.

The severity classification of our procedure as defined under the Directive 2010/63/EU is non-recovery.

Methods

Materials and methods

Mouse model

Ethical statement

All experimental procedures described here have been approved by the Italian Ministry of Health (n. 65/2016-PR del 21/01/2016 and n.1361.EXT.1 del 27/12/2016) and the Bioethics Committees of the University of Turin and the Department of Veterinary Sciences (DSV). The number of animals (8 reln^-/- and 8 reln^+/-) was kept to a minimum and all efforts were made to minimize their suffering.

Mouse housing and husbandry

Animals were housed in the facility of DSV under the following conditions: temperature 19–21 °C, humidity 55% ± 10%, light-dark cycle 12-12 h. Food (normal maintenance diet – meat-free rat and mouse diet SF00-100, Specialty Feeds, Glen Forrest Western Australia) and water (normal tap water) were given ad libitum. The bedding was non-sterile wood-chip. Environmental enrichment consisted of mini tubes, sizzle nests, and burrowing treats (Volkman Seed Small Animal Rodent Gourmet). Animals were bred in couples in a standard 484 cm² mouse cage. The mice themselves were not health-screened, the animal enclosure was free of the major rodent pathogens but some sentinels were positive for adventitious agents, i.e., mouse hepatitis virus after indirect fluorescent antibody (IFA) test and Multiplexed Fluorometric ImmunoAssay (MFIA) and Entamoeba sp. after annual mouse health monitoring (HM) Federation of European Laboratory Animal Science Associations (FELASA) screen.

Generation of L7-GFPrelnF1/mouse hybrids

Hybrids (L7-GFPrelnF1/) were generated by crossing L7-green fluorescent protein (GFP) (RRID:IMSR_JAX:004690) female mice (L7GFP^+/+) with Reeler heterozygous (reln^+/-) male mice (RRID:IMSR_JAX:000235).⁷ L7GFP^+/+ mice express GFP under the control of the L7 promoter.²¹^,²² As the L7 gene is specifically expressed by the PNs, these neurons are tagged by GFP, allowing their visualization without the need for immunocytochemical labeling. Before use, all animals were sexed and genotyped by routine methods to ascertain GFP expression (Note 1) and their appropriate reln genetic background.³

Methods for the model development

Preparation of organotypic co-cultures from L7-GFPrelnF1/of different genetic backgrounds

Experiments are reported in compliance with the ARRIVE guidelines,⁴² including randomization of samples in culture inserts (slices in a single insert came from different mice), blinding of the experimenter who performed image analysis with unblinding at end of image processing, and/or automation of quantification, as indicated in the following sections.

A step-to-step protocol for the preparation of cerebellar organotypic cultures (Figure 1A) has been deposited on protocols.io (https://dx.doi.org/10.17504/protocols.io.6qpvr67bbvmk/v1). This protocol is a refinement of previously published procedures from our laboratory.²³^,²⁴

Figure 1. General features of cerebellar cultures.

A: Flowchart showing the main steps for co-culture preparation (see protocols.io for details). B-D: Histological aspects of four DIV single- and co-cultured slices after immunostaining with different markers of cerebellar neurons and glia. B-C: Immunostaining of the cerebellar granule cells with PAX6 permits clear identification of these neurons (red) and the GFP-tagged PNs (green). D: Two co-cultured slices with different reln genetic backgrounds. The slices have originally plated at a distance from each other but tend to expand with time in vitro and thus are in contact in this image. The slice from an L7-GFPreln^-/-F1/mouse is indicated by the dotted white line. Note that in D PNs have been stained for calb 28k and thus appear yellowish-orange for the superimposition of the green GFP signal and the red calb 28k fluorescence. Abbreviations: calb 28k = 28kD calbindin; ChP = choroid plexus; CM = central mass; DIV = days in vitro; GFAP = Glial fibrillary acidic protein; GFP = green fluorescent protein; PAX-6 = paired-box protein PAX6; PCL = Purkinje cell layer; PNs = Purkinje neurons.

In the co-culture protocol, slices from reln heterozygous (reln^+/-) and homozygous (reln^-/-) hybrid mice were plated together. The positions of each genotypically identified slice in the insert were recorded so that they could be identified and monitored for the entire duration of the experiments (Note 2). Slices in each insert were numbered in a clockwise direction starting from a point indicated by a permanent mark on the bottom of the plastic dish and in the following analysis, the experimenter remained unaware of the matching between the slice number and the genotype of the donor mouse.

Qualitative analysis of PN migration

To analyze PN migration, organotypic cultures were photographed under a transmitted fluorescence light microscope. A series of six concentric circles spaced by 100 μm was superimposed on each photograph and roughly centered to the geometric center of the slice (Figure 2).

Figure 2. Temporal modifications of a single-cultured slice from an L7-GFPreln^-/-F1/mouse.

A: Low magnification view of the slice with superimposed concentric circles surrounding the center of the slice. B-F: Higher magnification of the same slice and its evolution over time. The apparent dispersion of the PNs is mainly due to the death of individual cells. The inserts in B-D show the histological features of two PNs at the periphery of the central mass. Note the reduction of fluorescence in D and the disappearance of the two cells in E-F. Arrows in the main panels point to the PNs shown in the inserts at higher magnification. Arrows in the inserts indicate the PN axon, arrowheads the main dendrites. Abbreviations: DIV = days in vitro; GFP = green fluorescent protein; PNs = Purkinje neurons.

Immunocytochemistry

A step-to-step protocol for the immunofluorescence staining of organotypic cultures can be found in Ref. 23. In this report, we simply show exemplificative staining with a rabbit anti-glial fibrillary acidic protein (GFAP – astrocytic marker) polyclonal antibody (Abcam Cat# ab7260, RRID: AB_305808), a mouse anti-28kD calbindin (a marker of the PNs) monoclonal antibody (Abcam Cat# ab9481, RRID: AB_2811302), and with a mouse anti-paired-box protein PAX6 (PAX6 – a marker of the differentiating granule cells) monoclonal antibody (Santa Cruz Biotechnology Cat# sc-81649, RRID: AB_1127044). All antibodies were used at dilutions ranging from 1:100 to 1:200.

Microscopy and photography

Cultures were photographed directly under a 10× or 20× objective of a Leika DM 6000 transmitted light microscope taking care not to expose them to environmental contaminants. Alternatively, they have been maintained in a microscope stage incubator fitted to a Leika SP5 Laser confocal microscope and photographed with a 20× lens (see Note 2).

Notes

1. Genotyping was done by sampling a small piece of the pinna so that at the same time it was possible to identify the subject and extract the genomic DNA.
2. Although not strictly necessary, one can use an incubator that is fitted to the microscope stage (see e.g., Figures 2 and 3 in Reference 25) to longitudinally monitor cultures and easily take photographs of the same slice so that individual microscopic fields can be easily recognized. Although this is ideal when it is necessary to pharmacologically challenge the cultures over time, it may be unpractical when several cultures must be processed together such is the case of the co-culture protocol here described.

Methods for the characterization and validation of the model

Cell dispersion was analyzed using Voronoi tessellation²⁶ aiming at demonstrating the precise relationship with cultural conditions, as this cellular sociology approach has proved useful in other biological contexts.²⁷

The numbers of technical repeats (individual slices from a single cerebellum) and independent biological repeats (organotypic cultures/co-cultures made by adding 3–6 individual slices to a single culture dish) are indicated in figure legends.

Cultures were obtained from two groups of mice: L7-GFPreln^(+/-)F1/ (n = 5) and L7-GFPreln^(-/-)F1 (n = 5). Cerebellar slices from these animals were subdivided into three groups:

1) single cultured L7-GFPreln^(+/-)F1/ slices, 2) single cultured L7-GFPreln^(-/-)F1/ slices, and 3) co-cultured L7-GFPreln^(+/-)F1/ slices + L7-GFPreln^(-/-)F1/ slices. In co-cultures, the ratio of L7-GFPreln^(+/-)F1/to L7-GFPreln^(-/-)F1/slices was 2:1/3:1. At least eight slices from each group were used for the analysis of cellular sociology (see below). The sample size (number of slices) was calculated using the G*Power calculator 3.1.9.4.²⁸ Input parameters were (unpaired t-test power calculator): tails, Two; parent distribution, Normal; α error probability, 0.05; power (1-β error probability), 0.95; effect size d, 2. The effect size was considered high based on qualitative observations and considering the mean area of the Voronoi polygons as the primary outcome. Output parameters were: non centrality r δ, 3.9088201; critical t, 2.1557656; Df, 13.2788745; sample size group 1, 8; sample size group 2, 8; Total sample size, 16; actual power, 0.9508778.

The eight slices/group of mice were randomly selected after sectioning the cerebella of at least five different animals.

The detailed procedure for calculation of Voronoi diagrams has been deposited on protocols.io (https://dx.doi.org/10.17504/protocols.io.yxmvmnrx6g3p/v1).

The parameters extracted from the analysis were: the mean area of Voronoi polygons (forms), the average roundness factor of those forms (RFav), the measure of the roundness factor homogeneity (RFH), and the measure of their area heterogeneity (area disorder (AD)). The last three parameters are characteristic of the population topography.²⁶

Statistics

We have used GraphPad Prism (RRID:SCR_002798) version 9.0.2 for Windows, GraphPad Software, San Diego, California USA, to assess the variations in the mean areas of Voronoi polygons, and RFav using a 95% confidence interval. Data were checked for outliers with the ROUT method (Q = 1) and normality with the Kolmogorov-Smirnov test. Further details of the tests used are given in figure legends. Inferential statistics were performed using ordinary one-way ANOVA followed by Tukey’s multiple comparison tests when data had a Gaussian distribution. The Brown-Forsythe test for equality of means and the Welch test was used for data sampled from populations with different variances.

Protocols

Protocol for establishing the co-culture model

The protocol below describes the step-by-step procedure required to establish and validate the long-term co-cultures of the postnatal murine cerebellum. With minimal modifications, it could be adapted to co-cultures of other areas of the brain, e.g., the hippocampus and entorhinal cortex or the cerebellum and the medulla oblongata containing the caudal (inferior) olivary nucleus. It stemmed from the single culture protocol previously developed in our laboratory.²³

Equipment

• Surgical instruments for brain dissection: universal scissors (length 13 cm), fine scissors straight and curved, Adson forceps, student anatomical standard pattern forceps, Dumont #7 forceps, gross anatomy blade (#20) and handle (#4), straight and curved spatulas, razor blades
• Dissecting microscope, e.g., Stereo microscope EZ4, Leica 10447197
• CO₂ incubator, e.g., Certomat CS-18 Sartorius BBI-8863385
• McIlwain tissue chopper with Petri dish modification Campden Instruments Model TC752-PD – see Note 1.
• Millicell-CM^® Cell Culture Inserts, 30 mm, hydrophilic PTFE, 0.4 μm, Merck, PICM0RG50
• Sterile 35-mm Petri dishes
• Nalgene^® vacuum filtration system, filter capacity 1000 mL, pore size 0.2 μm, Sigma-Aldrich, Z358207
• 500 μL disposable insulin syringes
• Sterile glass/disposable Pasteur pipettes
• Sterile filter paper dishes

Chemicals

• Pentobarbital sodium, Sigma-Aldrich, Y00021941
• D-(+)-Glucose, Sigma-Aldrich, G8270
• L-Ascorbic acid, Sigma-Aldrich, A92902
• Pyruvic acid, Sigma-Aldrich, 107360
• N-Methyl-D-glucamine, Sigma-Aldrich, M2004
• Sodium bicarbonate, Sigma-Aldrich, S5761
• Potassium chloride, Sigma-Aldrich, P3911
• Sodium phosphate monobasic, Sigma-Aldrich, S0751
• Calcium chloride, Sigma-Aldrich, C1016
• Magnesium chloride, Sigma-Aldrich, M8266
• Basal Medium Eagle, Sigma-Aldrich, B9638
• Horse serum, Sigma-Aldrich, H1138
• Hanks′ Balanced Salt solution, Sigma-Aldrich Catalog, H6648
• L-Glutamine solution, Sigma-Aldrich Catalog, G7513
• Antibiotic Antimycotic Solution (100×) Stabilized, Sigma-Aldrich, A5955
• Paraformaldehyde, powder, 95%, Sigma-Aldrich, 158127

Step 1: Preparation of solutions and culture medium (see Note 2)

1a. Stock solutions: 1 M CaCl₂; 1 M MgCl₂; 5% volume pentobarbital sodium in ddH₂O.

1b. Cutting solution: 130 mM n-methyl-D-glucamine Cl (NMDG); 24 mM NaHCO₃; 3.5 mM KCl; 1.25 mM NaH₂PO₄; 0.5 mM CaCl₂; 5 mM MgCl₂; 10 mM D-(+)-glucose; 1 mg/mL ascorbic acid; 2 mg/mL pyruvic acid.

To make 1 L, pour 850 mL of double-distilled water into a volumetric flask. Add 25.38 g NMDG, 2.017 g NaHCO₃, 261 mg KCl, 172 mg NaH₂PO₄, 1.80 g D-(+)-glucose, 1 g ascorbic acid, 2 g pyruvic acid. After complete dissolution SLOWLY add 5 mL MgCl₂ stock solution and 500 μL CaCl₂ stock solution. Bring to pH7.2-H7.4 with HCl. Sterile filter and store at 4 °C. The solution is stable for several months. Discharge if it becomes turbid. The addition of MgCl₂ and CaCl₂ is a critical step. If added too quickly, they precipitate making the solution cloudy. In this case, it must be discharged.

1c. Culture medium: 50% Basal medium Eagle (BME), 25% horse serum; 25% Hank's balanced salt solution (HBSS); 0.5% D-(+)-glucose; 0.5% L-glutamine (200 mM solution); 1% antibiotic antimycotic solution (100×).

To prepare 50 mL work under a laminar flow hood and use sterile glassware/plasticware. In a 100 mL cylinder add the components in the following order: 25 mL BME, 12.5 mL horse serum; 12.5 mL HBSS; 250 μL D-(+)-glucose; 250 μL L-glutamine; 500 μL antibiotic antimycotic solution. Transfer in a glass bottle and protect from light with aluminum foil. Store at 4 °C. Medium is stable for at least six months. Discharge if color changes and/or it becomes turbid.

1d. Fixative: Paraformaldehyde (PFA) 4% in 0.1 M phosphate buffer (PB), pH 7.4.

1e. Buffer solution: Phosphate-buffered saline (PBS) pH 7.4.

Step 2: Tissue sampling

Have ready the following: ice-cooled cutting solution; 50 mL sterile glass or plastic becker; 150 mm diameter sterile glass or plastic Petri dishes; sterile dissection/slice handling tools; sodium pentobarbital stock solution (room temperature); 500 μL disposable insulin syringes; sterile razor blades; sterile glass/disposable Pasteur pipettes; sterile filter paper dishes.

• Dissection of the brain and separation of individual slices after cutting (see Slice seeding below) should be carried out under sterile conditions as far as possible. If it is not possible to place the stereomicroscope under the laminar flow hood, dissection should be carried out under a simple plastic box opened in the front. The entire dissecting area should be cleaned and wiped off with 70% volume ethanol. During the production of slices, all procedures must be carried out in an ice-cold cutting solution. To keep the temperature a few degrees above 0 °C during the dissection, prepare some blocks of the frozen cutting solution to be added to the 4 °C chilled cutting solution contained in the Petri dish used to dissect the brain.
• Euthanize mice at the required post-natal age with an overdose of intraperitoneal sodium pentobarbital (60 mg/100 g body weight). Check for the absence of specific signs of life, i.e., absence of withdrawal reflexes that normally disappear within 5 min of the pentobarbital injection. When the animal is dead cut the head with scissors and drop it into a small plastic box or a 50 mL beaker filled with ice-cooled cutting solution (about 2–4 °C). Wait a couple of minutes for the head to be cooled and at the same time washed from the blood.
• Transfer the head to a glass Petri dish (10 cm diameter or more) filled with the clean cutting solution at 2–4 °C. Quickly remove the brain from the skull while the head is kept submerged in the ice-cooled cutting solution. To do so use straight fine scissors: insert scissors laterally in the foramen magnum and cut the bone at the basis of the skull on both sides of the brain, use a scalpel to make a transversal cut at the level of the olfactory bulbs, and lift the calvarium. Scoop out the brain with a curved spatula to prevent damage.
• Before separating the cerebellum from the other parts of the brain, completely remove the meninges with a pair of N.7 Dupont forceps.
• Isolate the cerebellum under the stereomicroscope: use a razor blade to make a transversal cut at the level of the mesencephalon and to separate the cerebellum from cerebellar peduncles connecting it to the cerebral trunk.
• Place the cerebellum on the stage of the tissue chopper within a drop of the ice-cooled cutting solution. Operate the chopper and cut 350 μm-thick parasagittal slices. Once terminated slicing, collect slices with a curved spatula (they are usually stuck together) and place them in a sterile 50-mm Petri dish filled with the ice-cooled cutting solution. Store at 4 °C until ready to separate slices. Slices should be separated and plated as soon as possible. We have stored slices for at least 30 min before slicing with no obvious detrimental effects on survival. However, it could be possible to culture slices that have been stored for longer.
• Separate individual slices under the stereomicroscope with a spatula and a needle, trying not to damage the tissue. During the entire procedure, slices must be submerged in the ice-cooled cutting solution. Discharge the damaged slices and/or very small (lateral) slices. If cutting was done smoothly, at least 10-12 slices should be obtained from a P5-P7 cerebellum. If the cerebellum is not submerged by an excess of the cutting solution, cutting with the chopper is easier. Set section thickness to any value between 200 μm - 400 μm after wiping out the solution with a piece of filter paper. Other cutting parameters, such as blade force, must be adjusted based on the type of chopper in use. With the McIlwain tissue chopper in use, we set the blade force knob at ¾ of its rotation clockwise, and the speed control knob at ½ of its rotation clockwise.
• Use a spatula with curved edges to collect slices and transfer them from the cutting stage of the chopper to the Petri dish.

Step 3 Slice seeding

• Before starting to seed slices onto the Millicell inserts bring the culture medium to room temperature and fill the required number of sterile 35-mm plastic Petri dishes with a 1.1 mL medium. Work under sterile conditions. The number of dishes required depends on the number of recovered slices, their size, and the experimental setup. In general, slices of the mouse post-natal cerebellum at day 5 have a maximum size of about 5 mm². Therefore, one can easily plate 5-6 slices (technical replicate when not co-culturing)/insert (experimental unit). Working with older animals or larger areas of the brain, i.e., the cerebral cortex allows plating a maximum of (roughly) three slices/insert.
• If planning co-culture experiments, like those described here, remember to have all slices ready, i.e., the L7-GFPreln^(+/-)F1/ slices and the L7-GFPreln^(-/-)F1/slices, before plating.
• Collect slices one by one and carefully lift them onto the dry Millicell membrane using a curved spatula. In co-culture experiments (Figure 1) carefully mark the positions of individual slices so that it will be possible to easily recognize them during subsequent manipulations. See Note 3.
• Once the required number of slices has been plated in the insert, place it inside a 35-mm Petri dish filled with the medium as indicated at the beginning of this section. Be careful to avoid air bubbles forming between the insert membrane and the medium, i.e., check that the membrane's lower surface is completely wet. Slices should be also wet but not submerged by the medium.
• Incubate at 34 °C in 5% volume CO₂ for up to 30 days in vitro (DIV) – see Note 4. Cultures can be maintained in vitro even longer, if necessary. Medium has to be changed twice a week. Allow slices to equilibrate to the in vitro conditions for at least 4 DIV before follow-up or starting a pharmacological treatment (if applicable), because during this initial interval there is a massive phase of cell death, as a consequence of the cutting procedure, see Ref. 29.

Notes

1. Slices can also be prepared with an oscillating vibratome. This is often required for subsequent electrophysiological studies as the cutting procedure is less destructive than chopping. However, cutting with the chopper is easier and less time-consuming, which is advantageous if one has to plate many slices in the course of a single experiment.
2. Several media are available and the best medium must be chosen according to the experimenter’s needs. Table 1 below compares the solutions/media in our protocol with two protocols used by other authors that have been employed to also cultivate adult brain slices.
3. To recollect the slice positions in the insert it is advisable to mark a reference point in the insert border with a waterproof pen and to make a drawing of the insert and the slices seeded inside.
4. Slices obtained from the cerebellum (and other central nervous system (CNS) areas) survive better at temperatures below 37 °C, hence the temperature settings of the incubator are important for survival. However, it should be noted that the neuroprotective effect of mild hypothermia on cultured neurons may obscure the action of certain apoptotic inductors if one is interested in the study of cell death.

Table 1. Protocols for the preparation of brain slices.

Procedure/Solutions	This protocol	Ullrich et al. (2011)³⁰	Schommer et al. (2017)³¹
Cutting solution	See text	No indication of a cutting solution	To prepare 50 mL: 40 mL Hibernate A 10 mL Horse Serum 0.5 mM L-Glutamine
Cutting	Chopper	Vibratome	Chopper
Growth medium	See text	50% MEM/HEPES 25% Horse Serum (inactivated) 25% Hank’s solution (HBSS) 2 mM NaHCO₃ 2 mM L-glutamine pH 7.2	To prepare 50 mL: Horse Serum 8 mL 400 μL antibiotic/antimycotic solution 40 mL Neurobasal A
Treatment medium (Day 1)	N/A	N/A	To prepare 50 mL: Horse Serum 8 mL 400 μL antibiotic/antimycotic solution 40 mL Neurobasal A
Treatment medium (Following days)	N/A	N/A	To prepare 50 mL: B27 suppl. 800 μL 400 μL antibiotic/antimycotic solution 40 mL Neurobasal A

Protocol for the characterization and validation of the model

This protocol is advantageous to analyze cellular migration and dispersion in longitudinal studies.

Starting from biological images, it can be used to study cellular sociology, i.e., to study the interactions of cells based on mathematical algorithms that rely on the analogies between cells and human societies.³² It relies on a model of parametrization and quantitation of cellular population topographies developed by Marcelpoil and Usson (1992).²⁶

Software

- Voronoi Diagram Generator by Frederik Brasz
- ImageJ (RRID:SCR_003070) by NIH
- FIJI (RRID:SCR_002285) (Image J) by NIH
- Microsoft Windows 10 by Microsoft

Step 1: Generation of Voronoi diagrams (see Note 1)

• Open the interactive Voronoi diagram (Thiessen polygon) generator. Figure 3 (left) shows the aspect of the generator mask.
• Upload the image to be analyzed (size must be 900×900 pixels and preferably saved as a PNG file). To do so your image has to be uploaded to the internet first (e.g., using Figshare or a personal website) so that it is possible to copy and paste its URL into the Voronoi generator. After uploading, the generator displays the image in its working space as shown in Figure 3 (right).
• Using the mouse, click above the center of each cell to generate the Voronoi polygons. Due to the thickness of the slice, it may be possible that two very close cells in the Z axis are not easily distinguished. This introduces an error that can be neglected considering that all slices are cut at the same initial thickness. In the end, you will obtain the image shown in Figure 4A. Save the image on your computer (right-click on the image and choose “save” from the drop-down menu).
• Choose “Visualization Normal” from the Visualization mode drop-down menu of the generator. The tessellation appears as shown in Figure 4B. Again, save the image on your computer (right-click on the image and choose “save” from the drop-down menu).
• Select “Hide sites” from the Options menu of the generator. The tessellation appears as shown in Figure 4C as the black dots corresponding to cell centers have disappeared. Again, save the image on your computer (right-click on the image and choose “save” from the drop-down menu).

Figure 3. Use of the Voronoi diagram generator.

Left – The mask of the Voronoi diagram generator with indications of its main commands and some hints for image elaboration. Right – The diagram generator with an uploaded example image of a single-cultured cerebellar slice from an L7-GFPreln^-/-F1/mouse. GFP = green fluorescent protein.

Figure 4. Elaboration of images for Voronoi analysis.

The image of Figure 1 is taken as an example to show the individual steps of the technique. A: Generation of Voronoi polygons over the microscope image. Note that the center points (black dots) correspond to the cell centers; B: Color visualization of Voronoi polygons with center points; C: Color visualization of Voronoi polygons without center points; D: Elimination of the open polygons, i.e., the polygons with one or more summits/sides outside the picture frame; E: Construction of the convex hull; F: Elimination of the polygons intersected by the convex hull. Note that the image in C (without center points) is used for this elaboration. This is because center points will be otherwise counted as particles by the ImageJ program in the subsequent elaboration; G: Elimination of the sides of the marginal polygons; H: Generation of the thresholded image to be elaborated by ImageJ with the Analyze Particles command; I: Generation of the overlay image with the indication of the number of each polygon analyzed by ImageJ. Note the number 1 circled in red at the center of the image. This number identifies the area in red in the following image; J: Image showing in red the area that ImageJ processes as a single particle. This area is discarded in the following elaborations; K: The values of Area, Perimeter, and Circularity (in red with gray background) of the first 25 particles (polygons) analyzed by ImageJ. In the example image processed here, ImageJ has analyzed a total of 318 particles of which particle #1 (highlighted in yellow) has to be discarded.

Step 2: Elimination of the marginal polygons

Due to the properties of the Voronoi partition, some polygons of the paving are not statistically representative of the set of polygons.²⁶ Those polygons, referred to as the marginal polygons are associated with points located on the border of the cell population and have one or more summits that do not contain total information on their “surround”. Such summits are created by points that belong to a half-plane outside the image area. Therefore, every point of the cell population whose associated polygon satisfies one of the two following conditions must not be taken into account in the subsequent computations:

- The polygon is open (the central point belongs to the convex hull) - see Figure 4D.
- At least one of the summits of the polygon is outside the convex hull - see Figure 4E.

The convex hull of a set of N points, i.e., the centers of the cells, is defined as the smallest convex set that contains all of the points. In the plane, this is a convex polygon.

• Elimination of the open polygons is carried out with Adobe Photoshop (RRID:SCR_014199) using the Magic wand tool to select and erase them from the image shown in Figure 4B. The result is shown in Figure 4D.
• Construct the convex hull from the image in Figure 4D. The convex hull is constructed with the Line tool by drawing segments that join the site points (cell centers) of the eliminated open polygons so that there are no concavities, as shown in Figure 4E.
• Using Photoshop, eliminate the polygons intersected by the convex hull and the polygons with open sides using the image of Figure 4C (without cell sites). The result is shown in Figure 4F.
• Cancel the sides of the marginal polygons. Use the Magic wand tool of Photoshop followed by the commands: Selection → Expand 2px; Selection → Contract 1px; Cancel; Modify →Stroke (color black) 2px. You should obtain an image in which the area of the marginal polygons is empty as in Figure 4G. This is the last elaboration that will be used for the subsequent steps of analysis.

Step 3: Analysis of Voronoi polygons

• Open the image to be analyzed with ImageJ. Set the appropriate scale with Analyze → Set scale.
• Run the following Macro by selecting Plugins → Macros → Run → Voronoi Macro (Box 1).
• The macro enhances image contrast (optional – line 1), converts the image into a black and white (B&W) 8-bit image (line 2), finds the edges of the Voronoi polygons (line 3), and optimizes their contrast (lines 4-6) as shown in Figure 4H. It then sets up the measurements necessary for the following analysis of polygons: Area, Shape descriptors, and Perimeter (line 7). It also permits the creation of an image (Figure 4I) with the overlay numerical indication of the individual polygons that the program has measured (Add to overlay and Display label). It also sets the number of Decimal places to 6 (line 7). Finally, the Macro performs the command Analyze Particles (line 8). Note the number 1 at the center of Figure 4I (encircled in red). This corresponds to the first counted particle that the program considers being the ensemble of the marginal polygons (highlighted in red in Figure 4J). Note that the red circle is only added here for clarity but not displayed at the end of the elaboration by ImageJ.
• At the end of the Macro, save all computed values in a .csv or a .xls file (according to the version of ImageJ used). This file must then be converted into a.xlsx Microsoft Excel file.

Box 1. Voronoi Macro.

run("Enhance Contrast…", "saturated=2");

run("8-bit");

run("Find Edges");

//run("Brightness/Contrast…");

setMinAndMax(0, 0);

run("Apply LUT");

run("Set Measurements…", "area perimeter shape limit display redirect=None decimal=6");

run("Analyze Particles…", "display summarize add in_situ");

Step 4: Analysis of data

• Open the .csv or .xls file generated by ImageJ with Microsoft Excel (RRID:SCR_016137). A table extracted from the file is shown in Figure 4K. It contains the following information: Column A: progressive numbering of the particles (polygons) counted by ImageJ; Column B: Identification of the image analyzed; Column C: Area (in μm² if the Set scale command has been set properly); Column D: Perimeter (in μm if the Set scale command has been set properly); Column E: Circularity (or Roundness factor); Columns F-H: Other shape descriptors computed by ImageJ that are not used in the analysis. Note that line 2 (highlighted in yellow) corresponding to Particle 1 must be deleted (as indicated above).
• Save the file as a.xlsx file.
• Open the.xlsx file in Microsoft Excel and calculate the following:
- ‐ Mean of area, perimeter, and circularity (roundness)
- ‐ Standard deviation of area, perimeter, and circularity (roundness)
- ‐ Area Disorder (AD)
- ‐ Roundness Factor Homogeneity (RFH)
The mean circularity (roundness) (RF_av) is computed directly by the ImageJ program using the following formula:
${RF}_{av} = \frac{1}{N} \sum_{i = 1}^{N} \frac{4 πA (X_{i})}{L {(X_{i})}^{2}}$

where A(X) is the area and L(X) is the perimeter of the N polygons generated by the Voronoi generator. RFav is a pure number (0 < RF_av ≤ 1).

The AD is calculated as follows:

AD = 1 - {(1 + \frac{σ_{A}}{A_{av}})}^{- 1}

where σ_A is the area standard deviation, and A_av is the mean area.

The RFH is calculated as follows:

RFH = {(1 + \frac{σ_{RF}}{{RF}_{av}})}^{- 1}

where σ_RF is the roundness factor standard deviation, and RF_av is the mean roundness factor.

Both are pure numbers with values >0 and ≤1.

• Transfer the values of RF_av, AD, and RFH to a new Microsoft Excel spreadsheet for subsequent statistical analysis.

Notes

1. It is possible to use several other Voronoi generators that can be found online as freeware or in dedicated programs. We found it particularly advantageous to use this generator because it is possible to directly upload the image to be analyzed and draw the sites, i.e., the cell centers, straight on it. As an alternative, it is possible to upload the X-Y coordinates of the sites in this and other generators. To do so one can use the ImageJ program and the Multipoint tool to obtain the spatial coordinates to be then uploaded to the Voronoi generator.

Results

Histology

Organotypic cultures from L7-GFPrelnF1/ (Figure 1A)³⁹^–⁴¹ permit a dynamic study of the effects of Reelin on neuronal migration and lamination of the cerebellar cortex. Thanks to GFP fluorescence, PNs can be visualized without the need for immunocytochemical labeling. In addition, our approach makes it unnecessary to use several groups of mice to be sacrificed at given postnatal ages to properly follow the cerebellar maturation. Figure 1B-D shows that, at the end of the experiments, cultures can be easily stained with two common markers of the cerebellar neurons. Figure 1D shows two co-cultured slices from a reln^+/- (top right) and a reln^-/- (bottom left). A comparison of the histology of the two slices permits clear identification of the phenotypical differences deriving from the different genetic backgrounds of the donor mice. Figure 2 shows the modifications over time in a single-cultured slice from an L7-GFPreln^-/-F1/mouse. After 29 days in vitro, the mass of the GFP fluorescent PNs tends to spread from the center of the slice but the neurons do not migrate to form a layered structure. Figure 5 shows that in co-cultures the histology of the slice derived from a homozygous (reln^-/-) mouse (Figure 5B-D) progressively changes to eventually become related to that from a heterozygous (reln^+/-) mouse (Figure 5A).

Figure 5. Histological aspects of single- and co-cultured slices after immunostaining with markers of PNs and glia.

A: The cerebellar histology of a slice from an L7-GFPreln^+/-F1/ mouse shows a cortical stratification that is similar to that of an early postnatal wild-type mouse in vivo. The PNs are stratified to form a PCL composed of several layers of these neurons. B: A single-cultured slice from an L7-GFPreln^-/-F1/ mouse shows a large central mass of PNs. C-D: Exemplificative temporal evolution of a slice from an L7-GFPreln^-/-F1/mouse co-cultured with slices from L7-GFPreln^+/-F1/mice. The PNs are spread from the central mass (C) and try to form a multilayer PCL similar to that in A. Concentric circles in A and D are 100 μm spaced. Note that in B-D PNs have been stained for calb 28k and thus appear yellowish-orange for the superimposition of the green GFP signal and the red calb 28k fluorescence. Abbreviations: calb 28k = 28kD calbindin; CM = central mass; DIV = days in vitro; GFAP, Glial fibrillary acidic protein (red in A and green in B-D); GFP, green fluorescent protein; PCL = Purkinje cell layer; PNs = Purkinje neurons.

Analysis of cellular sociology

Voronoi’s partition allowed for quantifying the dispersion of PNs in the presence or absence of Reelin. Starting from a set of points locating the position of the cell nuclei it was possible to obtain information on the order/disorder of the PN population (Figure 6). Polygon areas in single-cultured slices from reln^+/- animals (Figure 6A-C and Figure 7A-C) are larger than those from reln^-/- mice (Figure 6D-F and Figure 7A-C). Conversely, in co-cultures polygon areas in reln^-/- slices (Figure 6G-I and Figure 7A-C) become larger than in single-cultured reln^-/- slices, confirming a better dispersion of PNs in the presence of Reelin. RFav was not different in reln^-/- slices under different culture conditions indicating that the geometry of the polygons was unchanged (Figure 7D). We have also plotted AD and RFH in X/Y diagrams to show the spatial behavior of the PNs in the three groups of cultures (Figure 7E) and established that in the co-cultures the Reelin provided by the reln^+/- slices was sufficient to produce a measurable shift in the distribution of the PNs in reln^-/- slices from the pattern observed when slices are cultivated singularly.

Figure 6. Voronoi tessellation of slices.

Confocal images of the GFP-tagged PNs with superimposed Voronoi polygons (A, D, and G) that were generated and further elaborated as described in the Methods section and protocols.io. Images in B, E, and H show the initial elaboration of Voronoi polygons; those in C, F, and H show the last step of elaboration with the exclusion of the marginal polygons that are outside the convex hull. It can be seen that polygons are smaller and have a more homogeneous size in single cultured slices from L7-GFPreln^-/-F1/ mice (D-F), become larger and have less homogeneous sizes in co-cultured slices from L7-GFPreln^-/-F1/ mice (G-I), whereas slices from L7-GFPreln^+/-F1/mice display larger polygons of quite homogeneous sizes. Black dots in A-B, D-E, and G-H are the centers of the PNs. They have been cleared in the subsequent elaboration (C, F, and I) to avoid interference with automated counting. Abbreviations: GFP, green fluorescent protein; PCL = Purkinje cell layer; PNs = Purkinje neurons.

Figure 7. Quantitative analysis of Voronoi tessellation.

A-B: Descriptive statistics of the mean areas of Voronoi polygons. Note that there is little variability in the mean areas of polygons among singularly cultured slices obtained from reln^-/- homozygous mice as PNs remain aggregated into a central mass deep to the cerebellar cortex (see Figures 1, 2, 5); in the two other groups of cultures values are more dispersed and this indicates the dispersion of the PNs to form the PCL that will be typical of the mature cortex. A: Raw data plotted without any adjustment; B: Cleaned data after removing the two outliers identified in the co-cultured slices from reln^-/- homozygous mice with the ROUT method (Q = 1%). Error bars are 95% confidence interval. Data passed the Kolmogorov-Smirnov normality test. C: Ordinary one-way ANOVA [F(2, 20) = 8.966; P value = 0.0017] followed by Tukey’s multiple comparison test shows that in co-cultured slices from reln^-/- homozygous mice the mean polygon area is larger than in single-cultured slices from animals with the same genetic background (mean ± 95% CI: 1,361 ± 385 μm² versus 656 ± 195 μm², adjusted P value = 0.0287) and becomes closer to that of polygons in slices of reln^+/- heterozygous mice (mean ± 95% CI: 1,361 ± 385 μm² versus 1,663 ± 576 μm², adjusted P value = 0.4678). This observation confirms quantitatively the dispersion of the PNs in co-cultured slices from reln^-/- homozygous mice that lack Reelin but are exposed to the protein produced ex vivo by the slices from reln^+/- heterozygous mice. Also note the difference in mean areas of Voronoi polygons in single cultures of slices explanted from homozygous versus heterozygous mice (mean ± 95% CI: 56 ± 195 μm² versus 1,663 ± 576 μm², adjusted P value = 0.0014). n (number of slices from five different mice) = 8; * 0.05≤ adjusted P value >0.01; ** 0.001≤ adjusted P value >0.001. D: Brown-Forsythe [F* (2, 10.38) =11.41, P value = 0.0024] and Welch [W (2, 11.78) =11.94, P value = 0.0015) ANOVA tests of RFav. Since Voronoi polygons are convex, the average type of spatial occupation of the PNs is well-characterized by the RFav (mean circularity). In slices from single-cultured reln^-/- homozygous mice the RFav of the Voronoi polygons is higher than that of the polygons in slices from co-cultured slices of the same genetic background (mean ± 95% CI: 0.6733 ± 0.007 versus 0.6515 ± 0.0149, adjusted P value = 0.0314) and single-cultured reln^+/- heterozygous mice (mean ± 95% CI: 0.6733 ± 0.007 versus 0.6091 ± 0.0298, adjusted P value = 0.0082). On the other hand, the difference in RFav between single cultured slices from heterozygous mice and co-cultured slices from reln^-/- homozygous mice is not statistically significant (mean ± 95% CI: 0.6091 ± 0.0298 versus 0.6515 ± 0.0149, adjusted P value = 0.0718). The RF of a circle is 1 while that of a line is 0. Therefore, our analysis confirms mathematically that in single-cultured slices from reln^+/- heterozygous mice and in co-cultures of slices from reln^-/- homozygous mice there is a tendency to the alignment of the PNs, whereas in single-cultured slices from mice that lack Reelin the population of the PNs displays a spatial occupation consistent with the formation of a mass of cells in the cerebellar white matter. E: X-Y diagram showing topographical information of the PN population in the three experimental groups of cerebellar slices under the different culturing conditions reported in the Materials and Methods section. The X-axis displays the values of AD. AD varies when the value of the intrinsic disorder (i.e., the heterogeneity of the Voronoi polygon areas) increases, and a given value of AD corresponds to a given value of intrinsic disorder for any cell population. The Y-axis displays the values of RFH that varies in parallel to geometric disorder, i.e., the homogeneity/inhomogeneity of the circularity of the Voronoi polygons. Both AD and RFH vary from 0 to 1. A highly ordered population is characterized by values of AD and RFH, respectively, corresponding to 0 and 1²⁶, this means that all the polygons have the same area and circularity. The AD and RFH values are typical of a highly ordered population (high RFH, low AD) when one analyzes the clustered population of the PNs forming the central mass in the single-cultured reln^-/- slices. When the PNs align to eventually form a well-defined layer in the slices from reln^+/- heterozygous mice, the RFH diminishes and becomes closer to that of a line (=0), whereas the AD increases because the Voronoi polygons are small where the PNs tend to be aligned, but larger in the other parts of the slice (see Figure 4A-C). Note that in the co-cultured slices from reln^-/- mice, there is a shift towards the coordinates of the reln^+/- heterozygous mice. Abbreviations: PNs = Purkinje neurons; PCL = Purkinje cell layer; RF = roundness factor; RFav = mean roundness factor; AD = area disorder; RFH = roundness factor homogeneity.

Discussion

Here, not only we have been able to reduce the number of animals necessary to study ex vivo the effects of Reelin on cerebellar lamination, but also to avoid the use of the severe procedures that are necessary for in vivo longitudinal studies. Theoretically, our approach can be used for the study of the biological activities of any other brain-secreted protein, particularly if mutant and/or transgenic animals are available in which the protein under investigation is absent and thus it is possible to prepare co-cultures of the donor (normally expressing) and recipient (protein-lacking) slices. Several examples can be given to show the potential of our approach, a few of which are listed below. Remarkably, some of them refer to studies in Drosophila, a species that is easier to manipulate experimentally than mice and other mammals. For instance, the secreted neurotrophin Spätzle 3 was demonstrated to promote glial morphogenesis and neuronal survival and function in the fruitfly,³³ and, in rescue experiments, it had these effects only over very short distances. On the other hand, it was recently demonstrated that Slit, an evolutionarily conserved protein essential for brain development, acts at a long range and does not require processing by extracellular proteases in Drosophila.³⁴ Thus, our approach would be valuable to investigate the function of these two proteins in the mammalian brain, and, by plating slices at different distances, it could be useful to better establish their spatial range of action. Similarly, there is a growing interest in a better understanding of the function(s) of the Cyr61/CTGF/NOV (CCN) protein family in the nervous system.³⁵ CNN proteins bind directly to integrins and heparan sulfate proteoglycans and trigger multiple intracellular signaling pathways. At the cellular level, these proteins regulate gene expression and cell survival, proliferation, differentiation, senescence, adhesion, and migration, but little is known about their function in neural development. Likewise, co-cultures would be useful in studying the secretion of mutated huntingtin, which leads to neuronal degeneration in Huntington disease,³⁶ one of the most devastating neurodegenerative diseases. Our 3Rs approach could be primarily applied to neuroscience, although it is possible to envisage the preparation of slice cultures from organs such as the muscle, heart, liver, and solid tumors.

We have discussed in a previous publication the barriers for other potential end-users in the adoption of rodent ex vivo platforms in the field of neuroscience.¹⁷ In short, the main disadvantage of organotypic cultures lies in the disconnection of the explants from other areas of the brain with interruption of afferent and/or efferent pathways. Potential solutions to address/overcome these problems should be mainly sought in the reconstruction ex vivo of these connections by co-cultivating areas that are physiologically connected in vivo.³⁷^,³⁸

We believe it is important that this or similar approaches are adopted by others, as they can be used in medium throughput screening experiments preliminary to (if necessary) true experimentation in vivo. They have several scientific benefits,²⁹ such as the possibility to precisely control the experimental environment, pharmacologically manipulating the system with ease, the relative facility to perform longitudinal studies, the possibility to use several complementary techniques (genetic engineering, electrophysiology, immunocytochemistry) for biological characterization.

As partly discussed elsewhere previously,¹⁷ the potential of our approach in terms of animal reduction is remarkable as one can theoretically envisage reducing the number of experimental animals to at least one-fifth when aiming to characterize a single bioactive molecule.

Conclusions

This 3Rs approach is useful to study the effect of secreted biomolecules in a system modeling the in vivo condition but with remarkable benefits for animal reduction and refinement, avoiding the use of heavy surgery that is often necessary for the molecule(s) to reach the brain.

Data availability

Underlying data

Figshare: Voronoi analysis - Cultured Reln haplodeficient heterozygous mouse cerebellar slices, https://doi.org/10.6084/m9.figshare.21063616.³⁹

This project contains the following underlying data:

- Original images and images elaborated for Voronoi analysis of cerebellar slices from postnatal day 5-7 Reln haplodeficient heterozygous hybrid mice (L7-GFPreln^+/-F1/) cultured for 21 days in vitro.
- Area, Perimeter, and Circularity of individual Voronoi polygons. Area Disorder (AD), Roundness Factor Homogeneity (RFH), and Mean Roundness Factor (RFav) of the Voronoi polygon population.

Figshare: Voronoi analysis - Cultured Reln deficient homozygous mouse cerebellar slices, https://doi.org/10.6084/m9.figshare.21063517.⁴⁰

This project contains the following underlying data:

- Original images and images elaborated for Voronoi analysis of cerebellar slices from postnatal day 5-7 Reln deficient homozygous hybrid mice (L7-GFPreln^-/-F1/) cultured for 21 days in vitro.
- Area, Perimeter, and Circularity of individual Voronoi polygons. Area Disorder (AD), Roundness Factor Homogeneity (RFH), and Mean Roundness Factor (RFav) of the Voronoi polygon population.

Figshare: Voronoi analysis - Reln deficient homozygous mouse cerebellar slices co-cultured with Reln haplodeficient heterozygous mouse cerebellar slices, https://doi.org/10.6084/m9.figshare.21063280.⁴¹

This project contains the following underlying data:

- Original images and images elaborated for Voronoi analysis of cerebellar slices from postnatal day 5-7 Reln deficient homozygous hybrid mice (L7-GFPreln^-/-F1/) co-cultured for 21 days in vitro in the presence of slices from Reln haplodeficient homozygous hybrid mice (L7-GFPreln^+/-F1/).
- Area, Perimeter, and Circularity of individual Voronoi polygons. Area Disorder (AD), Roundness Factor Homogeneity (RFH), and Mean Roundness Factor (RFav) of the Voronoi polygon population.

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Reporting guidelines

Figshare: ARRIVE checklist for ‘Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination’, https://doi.org/10.6084/m9.figshare.21299211.⁴²

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

References

1. Schubert D, Herrera F, Cumming R, et al.: Neural cells secrete a unique repertoire of proteins. J. Neurochem. 2009 Apr; 109(2): 427–435. Epub 2009/02/10. eng. PubMed Abstract | Publisher Full Text | Free Full Text
2. Falconer DS: Two new mutants, ‘trembler’ and ‘reeler’, with neurological actions in the house mouse (Mus musculus L.). J. Genet. 1951; 1951(50): 192–201.
3. D'Arcangelo G, Miao GG, Chen SC, et al.: A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature. 1995 4/20/1995; 374(6524): 719–723. PubMed Abstract | Publisher Full Text
4. Mariani J, Crepel F, Mikoshiba K, et al.: Anatomical, physiological and biochemical sudies of the cerebellum from Reeler mutant mouse. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 1977 11/2/1977; 281(978): 1–28.
5. Heckroth JA, Goldowitz D, Eisenman LM: Purkinje cell reduction in the reeler mutant mouse: A quantitative immunohistochemical study. J. Comp. Neurol. 1989 1/22/1989; 279(4): 546–555. PubMed Abstract | Publisher Full Text
6. Yuasa S, Kitoh J, Oda S, et al.: Obstructed migration of Purkinje cells in the developing cerebellum of the reeler mutant mouse. Anat. Embryol. (Berl). 1993 10/1993; 188(4): 317–329. Publisher Full Text
7. Magliaro C, Cocito C, Bagatella S, et al.: The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse. Ann. Anat. 2016 9/2016; 207: 68–75. PubMed Abstract | Publisher Full Text
8. Hattori M, Kohno T: Regulation of Reelin functions by specific proteolytic processing in the brain. J. Biochem. 2021; 169(5): 511–516. PubMed Abstract | Publisher Full Text
9. Faini G, Del Bene F, Albadri S: Reelin functions beyond neuronal migration: from synaptogenesis to network activity modulation. Curr. Opin. Neurobiol. 2021 Feb; 66: 135–143. Epub 2020/11/17. eng. PubMed Abstract | Publisher Full Text
10. D'Arcangelo G: Reelin in the years: controlling neuronal migration and maturation in the mammalian brain. Adv. Neurosci. 2014; 2014: 1–19. Article ID 597395. Publisher Full Text
11. Lossi L, Castagna C, Granato A, et al.: The Reeler mouse: a translational model of human neurological conditions, or simply a good tool for better understanding neurodevelopment? J. Clin. Med. 2019 Dec 1; 8(12). PubMed Abstract | Publisher Full Text | Free Full Text
12. Ishii K, Kubo KI, Nakajima K: Reelin, and neuropsychiatric disorders. Front. Cell. Neurosci. 2016 2016; 10: 229. PubMed Abstract | Publisher Full Text | Free Full Text
13. D'Arcangelo G: Reelin mouse mutants as models of cortical development disorders. Epilepsy Behav. 2006 2/2006; 8(1): 81–90. PubMed Abstract | Publisher Full Text
14. Bernay B, Gaillard MC, Guryca V, et al.: Discovering new bioactive neuropeptides in the striatum secretome using in vivo microdialysis and versatile proteomics. Mol. Cell. Proteomics. 2009 May; 8(5): 946–958. PubMed Abstract | Publisher Full Text | Free Full Text
15. Umlauf BJ, Kuo JS, Shusta EV: Identification of brain ECM binding variable lymphocyte receptors using yeast surface display. Methods Mol. Biol. 2022; 2491: 235–248. PubMed Abstract | Publisher Full Text
16. Tüshaus J, Müller SA, Kataka ES, et al.: An optimized quantitative proteomics method establishes the cell type-resolved mouse brain secretome. EMBO J. 2020 Oct 15; 39(20): e105693. PubMed Abstract
17. Lossi L, Merighi A: The use of ex vivo rodent platforms in neuroscience translational research with attention to the 3Rs philosophy. Front. Vet. Sci. 2018; 2018(5): 164.
18. Hau KL, Lane A, Guarascio R, et al.: Eye on a dish models to evaluate splicing modulation. Methods Mol. Biol. 2022; 2434: 245–255. PubMed Abstract
19. Namchaiw P, Bunreangsri P, Eiamcharoen P: An in vitro workflow of neuron-laden agarose-laminin hydrogel for studying small molecule-induced amyloidogenic conditions.PLoS One.2022; 17(8): e0273458. PubMed Abstract
20. Smith SJ, von Zastrow M : A molecular landscape of mouse hippocampal neuromodulation. Front. Neural Circuits. 2022; 16: 836930. Epub 2022/05/24. eng. PubMed Abstract | Publisher Full Text | Free Full Text
21. Oberdick J, Levinthal F, Levinthal C: A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene. Neuron. 1988 7/1988; 1(5): 367–376. PubMed Abstract | Publisher Full Text
22. Zhang X, Baader SL, Bian F, et al.: High-level Purkinje cell-specific expression of green fluorescent protein in transgenic mice. Histochem.Cell Biol. 2001 6/2001; 115(6): 455–464. PubMed Abstract | Publisher Full Text
23. Alasia S, Merighi A, Lossi L:Transfection techniques and combined immunocytochemistry in cell cultures and organotypic slices.Merighi A, Lossi L, editors. Immunocytochemistry and Related Techniques. New York:Springer; 2015; pp. 329–355. Publisher Full Text
24. Alasia S, Cocito C, Merighi A, et al.: Real-time visualization of caspase-3 activation by fluorescence resonance energy transfer (FRET). Lossi L, Merighi A, editors. Neuronal Cell Death. New York:Humana Press/Springer Science+Business Media; 2015; pp. 99–114. Publisher Full Text
25. Lossi L, Castagna C, Merighi A:Neuronal cell death: an overview of its different forms in central and peripheral neurons.Lossi L, Merighi A, editors. Neuronal Cell Death. 1254 ed.New York:Springer;2015; pp. 1–18.
26. Marcelpoil R, Usson Y: Methods for the study of cellular sociology: Voronoi diagrams and parametrization of the spatial relationships. J. Theor. Biol. 1992/02/07/; 154(3): 359–369. Publisher Full Text
27. Nawrocki AR, Scherer PE: Keynote review: the adipocyte as a drug discovery target. Drug Discov. Today. 9/15/2005; 10(18): 1219–1230.
28. Faul F, Erdfelder E, Lang AG, Buchner A, et al.: G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods. 2007 May; 39(2): 175–191. PubMed Abstract | Publisher Full Text
29. Lossi L, Alasia S, Salio C, et al.: Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Prog. Neurobiol. 8/2009; 88(4): 221–245.
30. Ullrich C, Daschil N, Humpel C: Organotypic vibrosections: novel whole sagittal brain cultures. J. Neurosci. Meth. 9/30/2011; 201(1): 131–141. Publisher Full Text
31. Schommer J, Schrag M, Nackenoff A, et al.: Method for organotypic tissue culture in the aged animal. MethodsX. 2017; 2017(4): 166–171.
32. Ganesh S, Utebay B, Heit J, et al.: Cellular sociology regulates the hierarchical spatial patterning and organization of cells in organisms. Open Biol. 2020; 10(12): 200300.
33. Coutinho-Budd JC, Sheehan AE, Freeman MR: The secreted neurotrophin Spätzle 3 promotes glial morphogenesis and supports neuronal survival and function. Genes Dev. 2017 Oct 15; 31(20): 2023–2038. PubMed Abstract | Publisher Full Text | Free Full Text
34. Caipo L, González-Ramírez MC, Guzmán-Palma P, et al.: Slit neuronal secretion coordinates optic lobe morphogenesis in Drosophila. Dev. Biol. 2020 Feb 1; 458(1): 32–42. PubMed Abstract
35. Malik AR, Liszewska E, Jaworski J: Matricellular proteins of the Cyr61/CTGF/NOV (CCN) family and the nervous system. Front. Cell. Neurosci. 2015; 9: 237. Epub 2015/07/15. eng. PubMed Abstract | Publisher Full Text | Free Full Text
36. Trajkovic K, Jeong H, Krainc D: Mutant Huntingtin Is Secreted via a Late Endosomal/Lysosomal Unconventional Secretory Pathway. J. Neurosci. 2017 Sep 13; 37(37): 9000–9012. PubMed Abstract | Publisher Full Text | Free Full Text
37. Yamamoto N, Maruyama T, Uesaka N, et al.: Molecular mechanisms of thalamocortical axon targeting. Novartis Found. Symp. 2007; 288: 199–208. discussion -11, 76-81. PubMed Abstract
38. Corner MA, Baker RE, van Pelt J , et al.: Compensatory physiological responses to chronic blockade of amino acid receptors during early development in spontaneously active organotypic cerebral cortex explants cultured in vitro. Prog. Brain Res. 2005; 147: 231–248. PubMed Abstract | Publisher Full Text
39. Merighi A, Lossi L:Voronoi analysis - Cultured Reln haplodeficient heterozygous mouse cerebellar slices. figshare. [Dataset]. 2022. Publisher Full Text
40. Merighi A, Lossi L:Voronoi analysis - Cultured Reln deficient homozygous mouse cerebellar slices. figshare. [Dataset]. 2022. Publisher Full Text
41. Merighi A, Lossi L:Voronoi analysis - Reln deficient homozygous mouse cerebellar slices co-cultured with Reln haplodeficient heterozygous mouse cerebellar slices. figshare. [Dataset]. 2022. Publisher Full Text
42. Merighi A: ARRIVE guideline checklist for Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination by Adalberto Merighi and Laura Lossi. figshare. Journal Contribution. 2022. Publisher Full Text

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Author details Author details

¹ Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

Adalberto Merighi
Roles: Data Curation, Methodology, Validation, Writing – Original Draft Preparation

Laura Lossi
Roles: Conceptualization, Investigation, Methodology, Resources, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

Much of this work was supported by a grant (CRACK IT Solution Neuroinflammation and nociception in a dish) from the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs), London (UK).

Article Versions (2)

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Published: 02 Oct 2023, 11:1183

https://doi.org/10.12688/f1000research.126787.2

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https://doi.org/10.12688/f1000research.126787.1

© 2022 Merighi A and Lossi L. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Merighi A and Lossi L. Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination [version 1; peer review: 2 approved with reservations]. F1000Research 2022, 11:1183 (https://doi.org/10.12688/f1000research.126787.1)

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Open Peer Review

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Reviewer Report 18 Nov 2022

Pascale Chavis, INMED, INSERM, Aix-Marseille University, Marseille, France

Thomas Boudier, CENTURI, Aix-Marseille University, Marseille, France

Approved with Reservations

https://doi.org/10.5256/f1000research.139230.r153857

Summary:
The study reports the implementation of a co-culture method to analyze the effects of reelin and reduce animal use within the 3R framework. Hybrid mice were generated by crossing L7-GFP with heterozygous reeler to obtain GFP-tagged Purkinje neurons in different reelin backgrounds. Co-cultures of cerebellar slices from homozygous and heterozygous hybrids resulted in dispersion and morphology changes of GFP-tagged Purkinje neurons derived from slices bearing a homozygous background.

General:
The main concern is about the general scope of the paper and whether it is about ethical use of animals in biological experiments and animal welfare or about reelin neurobiology in the 3R framework. Although the two former issues are not to be overlooked, they should be developed in the discussion rather than in the introduction, highlights and results.

The authors also state that this approach will allow the study of reelin effects on cortical lamination and that the method will also be applicable to investigate the function of other brain secreted proteins. They should show that this is indeed the case either in co-cultures of other cortices from reelin hybrids or in cerebellar co-cultures from mutants of different proteins.

Strengths:
The authors developed an elegant technique highly suited for longitudinal and dynamic studies, implemented in the 3Rs framework. They provide a very detailed and exhaustive description of the method to allow replication in other labs.

Major points:
Methods:
Voronoi analysis: The authors perform Voronoi analysis to analyze spatial organization of cells. Although they give references to support their work, the theoretical paper is dated from 1992. Since then, framework and tools to study spatial organization, based on robust spatial statistics such as F, G, H and K functions, have been developed (see Lagache et al., 2013¹ for example).

The overall method is also extremely manual, involving at least 4 different softwares, and the initial cell detection is purely manual, along with the Voronoi adjustment. The removal of Voronoi zones of cells touching edges is justified, but no explanation is given on why the authors chose to favor convex area hence removing some additional cells in their study.

Quantitative analysis of PN migration: since slices expand with time (Figure 1), how is the center point precisely localized through different time points?

Data interpretation:
In co-cultures, changes in the histology of reln-/- slices are interpreted as resulting from the effect of Reelin produced by reln+/- slices. However, there is no clear demonstration of this, as this could be due to another secreted factor. To answer this point, reelin blockade (function or binding, etc...) should be achieved. This experiment would also have the advantage to validate the statement made in the highlight that co-cultures are amenable to pharmacological intervention or other type of manipulation/treatment.

In the same line of thought, one can wonder whether reln-/- slices phenotypical improvement would be better achieved if the donor mice were reln+/+ instead of reln+/-? This would validate the method further and also give an indication about time and/or dose effect.

Statistics:
In Figure 7, what is the variable: does each point represent one culture? How many polygons are included in each point, this could actually be a bias in condition where GFP-PN survival decrease with time such as in the reln-/- group.

Please, indicate in legend samples size i.e. number of cultures/dishes used.

In 7B, the distribution in the single reln+/-, clearly shows 2 groups in Voronoi polygons areas. Could you comment? Moreover, this kind of distribution undermines the use of means as presented in 7C.

Figures:
Figures 1 and 5 are somehow overlapping. The paper would gain clarity by showing in different figures temporal modifications and histological characterization of single cultures and co-cultures (similar to Figure 2).

Minor points:
What is the age of the mice used for slice preparation? And why are mice euthanized using sodium pentobarbital prior brain slicing (protocols.io.6qpvr67bbvmk/v1)?

What is the survival time of the single and co-cultures?

Is the rationale for developing the new method (or application) clearly explained?

Yes
Is the description of the method technically sound?

Yes
Are sufficient details provided to allow replication of the method development and its use by others?

Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

Yes

References

1. Lagache T, Lang G, Sauvonnet N, Olivo-Marin JC: Analysis of the spatial organization of molecules with robust statistics.PLoS One. 2013; 8 (12): e80914 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Pascale Chavis : Brain plasticity, Reelin neurobiology, Neuropsychiatric diseases. Thomas Boudier : Image processing, Image analysis.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

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Report a concern

Author Response 06 Oct 2023

Adalberto Merighi, Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

06 Oct 2023

Author Response

Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history ... Continue reading Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Summary:
The study reports the implementation of a co-culture method to analyze the effects of reelin and reduce animal use within the 3R framework. Hybrid mice were generated by crossing L7-GFP with heterozygous reeler to obtain GFP-tagged Purkinje neurons in different reelin backgrounds. Co-cultures of cerebellar slices from homozygous and heterozygous hybrids resulted in dispersion and morphology changes of GFP-tagged Purkinje neurons derived from slices bearing a homozygous background.
General:
The main concern is about the general scope of the paper and whether it is about ethical use of animals in biological experiments and animal welfare or about reelin neurobiology in the 3R framework. Although the two former issues are not to be overlooked, they should be developed in the discussion rather than in the introduction, highlights and results.
We fully understand this concern. A similar concern was also raised by Drs. Caruncho and Reive. The reason why the issues of the ethical use of animals in biological experiments and animal welfare have been developed in different parts of the manuscript other than the Discussion is to cope with the guidelines issued by the NC3Rs for Method papers. In response, we have fully revised the paper according to these observations as explained above in the response to the comments of the other two reviewers and in the preamble to our responses.
The authors also state that this approach will allow the study of reelin effects on cortical lamination and that the method will also be applicable to investigate the function of other brain secreted proteins. They should show that this is indeed the case either in co-cultures of other cortices from reelin hybrids or in cerebellar co-cultures from mutants of different proteins.
We agree with the reviewers that the experiments suggested would be indeed useful to confirm that our approach will permit studying 1. The effects of reelin in the other laminated areas of the brain or 2. The function of other secreted proteins. Yet, as regards the first issue, we have now thoroughly discussed previous experiments with approaches different from the one used here demonstrating the effects of Reelin on the lamination of the cerebral cortex. As regards the second issue, we have now softened our claims on the possible applications of our method to the study of other secreted brain proteins in another specific section of the discussion (Advantages and limitations of the organotypic culture approach to the study of neurodevelopment). We have also considered the issues raised here in a more thorough discussion of the strengths and weaknesses of our approach, as such a discussion was also suggested by Drs. Drs. Caruncho and Reive.
We are confident that the reviewers would agree with us that the experiments suggested would require a large amount of work that could the the subject of another paper and well beyond the purpose of this Method Paper (that as mentioned in our prologue was sponsored by the NC3Rs and was prepared according to the guidelines for the NC3Rs gateway). Apart from this, there are several theoretical and practical limitations to the possibility of performing the experiments above (we have addressed this point in response to the first comment under “Data Interpretation”).
That reelin produced by the cerebellar granule cells in slices from heterozygous or homozygous Reeler mice can diffuse in the culture medium was previously demonstrated after studies on chimeric mice and in Reeler cortical cultures. We have addressed this issue in the Discussion of the revised paper as follows:
Although the structural effects of the Reeler mutation are well known, little work has been done on the possibility of modifying Reelin-mediated deficits in live cells. A study in the Reeler-normal chimeric mice has previously demonstrated that the morphology and position of the cerebellar neurons and glial cells are controlled by extracellular environments but not by their genotype [11]. Subsequent work has shown that the protein produced by cortical explants of reln^+/+ or reln^+/- mice co-cultured with a Reeler-like ferret dysplastic cortex was capable of at least partly rescuing neuronal migration in the ferret explant [12]. Other studies, were carried out in the cerebral cortex and hippocampus and led to similar conclusions. It was thus shown that cortical layer development and orientation are modulated by relative contributions of Reelin-negative and -positive neurons in mouse chimeras [13] and that the possibility to restore a normal phenotype in reln^-/-slices co-cultured with wild-type slices was mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR) [14]. Our present observations are in full accord with these studies. A point left open for discussion regards the Reelin dosage necessary for the rescue of a normal phenotype in culture studies. In their chimeric mouse study, Yoshiki and Kusakabe qualitatively observed that only a few granule cells derived from a normal mouse were capable of promoting the alignment and the proper dendritic tree development of the PNs derived from the mutant. From their observations, these authors suggested that Reelin secreted from a single granule cell can affect the morphology of PNs in a wide area [11].
Our results quantitatively demonstrate that the Reelin produced by the slices obtained from haplodeficient mice is sufficient to restore the normal cerebellar phenotype. Therefore, our findings reinforce the idea that it may be possible to modify Reelin-mediated deficits through the experimental administration of the protein. Experiments have demonstrated that the medium obtained from cerebellar dissociated cultures from P5-8 normal mice as well as from cell extracts of the cultured cerebellum from these mice contained full-length Reelin [15]. These observations are fully in line with the demonstration that the protein is produced by the postmitotic granule cells of the deep external granular layer before they migrate to their final destination, the internal granular layer, where they lose Reelin immunoreactivity [16].
Strengths:
The authors developed an elegant technique highly suited for longitudinal and dynamic studies, implemented in the 3Rs framework. They provide a very detailed and exhaustive description of the method to allow replication in other labs.
Major points:
Methods:
Voronoi analysis: The authors perform Voronoi analysis to analyze spatial organization of cells. Although they give references to support their work, the theoretical paper is dated from 1992. Since then, framework and tools to study spatial organization, based on robust spatial statistics such as F, G, H and K functions, have been developed (see Lagache et al., 20131 for example).
Frankly, we do not see why Voronoi's analysis should be hampered by its relative age. Voronoi tesselation has been and still is widely employed in the study of the spatial organization of many diverse types of neuronal (and other) cell populations [17].
The paper quoted by the reviewers uses a modified Ripley’s K function to define the clustered, dispersed, or uniform distribution of points (i.e. clathrin endocytotic sites) in the 2D space [18]. Ripley's K-, H-, and L-functions are used primarily to identify the clustering of proteins in membrane microdomains (as is indeed the case for Lagache’s paper). Using this approach, aggregation (or clustering) is identified if the average number of proteins within a distance r of another protein is statistically greater than that expected for a random distribution [19]. Kiskowski et al. emphasized that “it is not entirely clear how the function may be used to quantitatively determine the size of domains in which clustering occurs” and have widely discussed the limitations and potential of Ripley's K in real-life scenarios”[19]. In the case of this study, where indeed the main issue under investigation is the clustering of the PNs, which is very high in the reln^-/-mice, one can raise reasonable doubts about the usefulness of applying Ripley's K in our conditions. Despite the above observations on the limitations of Ripley’s functions to modeling spatial clustering we have added a large bunch of new analyses based on the novel use of spatial statistics with ArcMap [geographical distribution tools (Central Feature, Mean Center, Median Center, Directional Distribution, and Standard Distance), pattern distribution tools (Average Nearest Neighbor, Getis-Ord General G, Ripley’s K function, and Global Moran’s I), and mapping clusters tools (Anselin Local Moran’s I, and Getis-Ord G*)] to compare their results with the conclusions that could be drawn solely from Voronoi analysis. As thoroughly discussed in the revised paper the two approaches yielded comparable results in support of our initial claims on the soundness of the method.
The overall method is also extremely manual, involving at least 4 different softwares, and the initial cell detection is purely manual, along with the Voronoi adjustment.
We have already at least partly addressed this comment in response to Drs. Caruncho and Reive observations (see Supplementary Material 4). In addition, we would like to stress here that the possibility of using an automated procedure is severely hampered by the extremely high compactness of the central mass of the PNs lying inside the cerebellar white matter. In other words, it seems to be very difficult to practically separate the individual PNs with a thresholding procedure based on color or gray histograms (see Fig. S4-2). In addition, the aforementioned paper by Lagache and collaborators uses a similar manual procedure for the initial detection of the regions of interest (ROIs) as reported in their Materials and Methods: “We first delimited cells’ contours by drawing polygonal Region of Interest (ROIs) with the Icy software [13] (http://icy.bioimageanalysis.org). We then used a wavelet-based detection method [28], implemented as a plugin Spot detector in Icy to extract the two-dimensional positions of putative endocytic spots at the cellular membrane…”.
The removal of Voronoi zones of cells touching edges is justified, but no explanation is given on why the authors chose to favor convex area hence removing some additional cells in their study.
We have addressed this comment also in response to similar observations made by Drs. Caruncho and Reive (see main text and Supplementary Material 3 and 4).
Quantitative analysis of PN migration: since slices expand with time (Figure 1), how is the center point precisely localized through different time points?
We believe that this comment applies to Figure 2 and thus refers to qualitative analysis. The center point was recorded using the memory system of the microscope stage at 4 DIV. Subsequent images were recorded using the memorized X-Y coordinates.
Data interpretation:
In co-cultures, changes in the histology of reln-/- slices are interpreted as resulting from the effect of Reelin produced by reln+/- slices. However, there is no clear demonstration of this, as this could be due to another secreted factor. To answer this point, reelin blockade (function or binding, etc...) should be achieved. This experiment would also have the advantage to validate the statement made in the highlight that co-cultures are amenable to pharmacological intervention or other type of manipulation/treatment.
We agree that, theoretically, the changes in the histology of co-cultured reln^-/-slice could be dependent on a different secreted factor. However, several published papers support our line of thought as we have discussed in the revised version of the paper (reported above). To our knowledge, no other molecules except Reelin and its downstream receptors and adaptor molecules have been demonstrated to be primarily responsible for the correct layered organization of the cerebellar cortex during postnatal development of altricial mammals, although the true mechanisms of PNs layering in the mature cerebellar cortex remain a matter of debate [20]. As authoritatively reviewed by Gabriella D’Arcangelo [21], the discoverer of Reelin and certainly one of the leading scientists in the field “In the cerebellum, Reelin is produced by granule cells in the external granule layer and is essential for the radial migration of the Purkinje neurons, which are born in the cerebellar ventricular zone and form at first a plate and then a single-cell layer. This assessment derives from a widespread analysis of cerebellar development in Reeler mice [2, 22-24], from Reelin gene and protein expression data [16, 25], and functional studies in organotypic cultures [26]”.
We likewise agree that the blockade of Reelin would have the advantage of validating our statement that co-cultures are amenable to pharmacological or other types of manipulation. However, this would require a huge amount of additional work that is prone to several worries about its practicability (e.g. the effectiveness of Reelin-blocking antibodies -when available -to penetrate the entire thickness of a slice). In addition, much of the results that may be stemming from these efforts would be mainly confirmatory of previous observations. We have now discussed in our revision the previous literature on the block of the Reelin function in cultured cerebellar slices that demonstrates a long-distance effect of the molecule in vitro.
In the same line of thought, one can wonder whether reln-/- slices phenotypical improvement would be better achieved if the donor mice were reln+/+ instead of reln+/-? This would validate the method further and also give an indication about time and/or dose effect.
We agree with this observation in theory, but previously published observations on mouse chimeras [11] led to the conclusion that very little Reelin production could be sufficient to rescue the Reeler phenotype. In addition, our main goal was to validate the co-culture method that will be surely of good use to answer these questions in future work.
Statistics:
In Figure 7, what is the variable: does each point represent one culture? How many polygons are included in each point, this could actually be a bias in condition where GFP-PN survival decrease with time such as in the reln-/- group.
The variable in the figure (now Figure 9) is a randomly chosen culture slice. The number of polygons included in each point is indicated in FigShare uploaded Excel files (.xlsx or .csv). Inspection of these files shows that the number of polygons remains very high in single cultured reln^-/- slices despite survival decrease thus excluding a bias.
Please, indicate in legend samples size i.e. number of cultures/dishes used.
Done
In 7B, the distribution in the single reln+/-, clearly shows 2 groups in Voronoi polygons areas. Could you comment? Moreover, this kind of distribution undermines the use of means as presented in 7C.
The existence of two groups of Voronoi polygon areas is explained by the layering of the cortex in the slices from heterozygous mice. We respectfully disagree with the observation that this kind of distribution undermines the use of the means in the now Figure 7C as 1. Analysis was supported by inferential statistics and 2. Results from ArcMap GIS analysis where the tessellations of images only included the hexagons with PNs inside (see Figures 6 and 10 in main text and Figs. S64-15 in Supplementary Material 6) led to fully comparable results as shown and discussed in the revised version of the paper.
Figures:
Figures 1 and 5 are somehow overlapping. The paper would gain clarity by showing in different figures temporal modifications and histological characterization of single cultures and co-cultures (similar to Figure 2).
In response, we have deleted panels A and B in the original Figure 1. In addition, we have shown other images of the cultures to better demonstrate their histological appearance (Fig. 10A-C) and Figure S2.
Minor points:
What is the age of the mice used for slice preparation? And why are mice euthanized using sodium pentobarbital prior brain slicing (protocols.io.6qpvr67bbvmk/v1)?
The mice were 5 days old. We have added this information to the revised paper. They were euthanized with sodium pentobarbital (SP) as routinely done in our laboratory. Injectable barbiturates are acceptable for use in mouse fetuses and neonates according to the AVMA GUIDELINES FOR THE EUTHANASIA OF ANIMALS: 2020 EDITION, whereas decapitation and cervical dislocation are acceptable with conditions according to the guidelines. In addition, a recent publication [27] has reviewed the effects of SP (and other euthanasia methods) on cells and tissues as well as on different analytes without finding adverse effects of SP versus decapitation and/or cervical dislocation in the brain. Therefore we prefer to use SP also following the recommendations of our Department’s Ethical Committee.
What is the survival time of the single and co-cultures?
The survival time was 30 days at maximum. We have added this information to the revised paper.

References (for both responses to reviewers)
1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Summary:
The study reports the implementation of a co-culture method to analyze the effects of reelin and reduce animal use within the 3R framework. Hybrid mice were generated by crossing L7-GFP with heterozygous reeler to obtain GFP-tagged Purkinje neurons in different reelin backgrounds. Co-cultures of cerebellar slices from homozygous and heterozygous hybrids resulted in dispersion and morphology changes of GFP-tagged Purkinje neurons derived from slices bearing a homozygous background.
General:
The main concern is about the general scope of the paper and whether it is about ethical use of animals in biological experiments and animal welfare or about reelin neurobiology in the 3R framework. Although the two former issues are not to be overlooked, they should be developed in the discussion rather than in the introduction, highlights and results.
We fully understand this concern. A similar concern was also raised by Drs. Caruncho and Reive. The reason why the issues of the ethical use of animals in biological experiments and animal welfare have been developed in different parts of the manuscript other than the Discussion is to cope with the guidelines issued by the NC3Rs for Method papers. In response, we have fully revised the paper according to these observations as explained above in the response to the comments of the other two reviewers and in the preamble to our responses.
The authors also state that this approach will allow the study of reelin effects on cortical lamination and that the method will also be applicable to investigate the function of other brain secreted proteins. They should show that this is indeed the case either in co-cultures of other cortices from reelin hybrids or in cerebellar co-cultures from mutants of different proteins.
We agree with the reviewers that the experiments suggested would be indeed useful to confirm that our approach will permit studying 1. The effects of reelin in the other laminated areas of the brain or 2. The function of other secreted proteins. Yet, as regards the first issue, we have now thoroughly discussed previous experiments with approaches different from the one used here demonstrating the effects of Reelin on the lamination of the cerebral cortex. As regards the second issue, we have now softened our claims on the possible applications of our method to the study of other secreted brain proteins in another specific section of the discussion (Advantages and limitations of the organotypic culture approach to the study of neurodevelopment). We have also considered the issues raised here in a more thorough discussion of the strengths and weaknesses of our approach, as such a discussion was also suggested by Drs. Drs. Caruncho and Reive.
We are confident that the reviewers would agree with us that the experiments suggested would require a large amount of work that could the the subject of another paper and well beyond the purpose of this Method Paper (that as mentioned in our prologue was sponsored by the NC3Rs and was prepared according to the guidelines for the NC3Rs gateway). Apart from this, there are several theoretical and practical limitations to the possibility of performing the experiments above (we have addressed this point in response to the first comment under “Data Interpretation”).
That reelin produced by the cerebellar granule cells in slices from heterozygous or homozygous Reeler mice can diffuse in the culture medium was previously demonstrated after studies on chimeric mice and in Reeler cortical cultures. We have addressed this issue in the Discussion of the revised paper as follows:
Although the structural effects of the Reeler mutation are well known, little work has been done on the possibility of modifying Reelin-mediated deficits in live cells. A study in the Reeler-normal chimeric mice has previously demonstrated that the morphology and position of the cerebellar neurons and glial cells are controlled by extracellular environments but not by their genotype [11]. Subsequent work has shown that the protein produced by cortical explants of reln^+/+ or reln^+/- mice co-cultured with a Reeler-like ferret dysplastic cortex was capable of at least partly rescuing neuronal migration in the ferret explant [12]. Other studies, were carried out in the cerebral cortex and hippocampus and led to similar conclusions. It was thus shown that cortical layer development and orientation are modulated by relative contributions of Reelin-negative and -positive neurons in mouse chimeras [13] and that the possibility to restore a normal phenotype in reln^-/-slices co-cultured with wild-type slices was mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR) [14]. Our present observations are in full accord with these studies. A point left open for discussion regards the Reelin dosage necessary for the rescue of a normal phenotype in culture studies. In their chimeric mouse study, Yoshiki and Kusakabe qualitatively observed that only a few granule cells derived from a normal mouse were capable of promoting the alignment and the proper dendritic tree development of the PNs derived from the mutant. From their observations, these authors suggested that Reelin secreted from a single granule cell can affect the morphology of PNs in a wide area [11].
Our results quantitatively demonstrate that the Reelin produced by the slices obtained from haplodeficient mice is sufficient to restore the normal cerebellar phenotype. Therefore, our findings reinforce the idea that it may be possible to modify Reelin-mediated deficits through the experimental administration of the protein. Experiments have demonstrated that the medium obtained from cerebellar dissociated cultures from P5-8 normal mice as well as from cell extracts of the cultured cerebellum from these mice contained full-length Reelin [15]. These observations are fully in line with the demonstration that the protein is produced by the postmitotic granule cells of the deep external granular layer before they migrate to their final destination, the internal granular layer, where they lose Reelin immunoreactivity [16].
Strengths:
The authors developed an elegant technique highly suited for longitudinal and dynamic studies, implemented in the 3Rs framework. They provide a very detailed and exhaustive description of the method to allow replication in other labs.
Major points:
Methods:
Voronoi analysis: The authors perform Voronoi analysis to analyze spatial organization of cells. Although they give references to support their work, the theoretical paper is dated from 1992. Since then, framework and tools to study spatial organization, based on robust spatial statistics such as F, G, H and K functions, have been developed (see Lagache et al., 20131 for example).
Frankly, we do not see why Voronoi's analysis should be hampered by its relative age. Voronoi tesselation has been and still is widely employed in the study of the spatial organization of many diverse types of neuronal (and other) cell populations [17].
The paper quoted by the reviewers uses a modified Ripley’s K function to define the clustered, dispersed, or uniform distribution of points (i.e. clathrin endocytotic sites) in the 2D space [18]. Ripley's K-, H-, and L-functions are used primarily to identify the clustering of proteins in membrane microdomains (as is indeed the case for Lagache’s paper). Using this approach, aggregation (or clustering) is identified if the average number of proteins within a distance r of another protein is statistically greater than that expected for a random distribution [19]. Kiskowski et al. emphasized that “it is not entirely clear how the function may be used to quantitatively determine the size of domains in which clustering occurs” and have widely discussed the limitations and potential of Ripley's K in real-life scenarios”[19]. In the case of this study, where indeed the main issue under investigation is the clustering of the PNs, which is very high in the reln^-/-mice, one can raise reasonable doubts about the usefulness of applying Ripley's K in our conditions. Despite the above observations on the limitations of Ripley’s functions to modeling spatial clustering we have added a large bunch of new analyses based on the novel use of spatial statistics with ArcMap [geographical distribution tools (Central Feature, Mean Center, Median Center, Directional Distribution, and Standard Distance), pattern distribution tools (Average Nearest Neighbor, Getis-Ord General G, Ripley’s K function, and Global Moran’s I), and mapping clusters tools (Anselin Local Moran’s I, and Getis-Ord G*)] to compare their results with the conclusions that could be drawn solely from Voronoi analysis. As thoroughly discussed in the revised paper the two approaches yielded comparable results in support of our initial claims on the soundness of the method.
The overall method is also extremely manual, involving at least 4 different softwares, and the initial cell detection is purely manual, along with the Voronoi adjustment.
We have already at least partly addressed this comment in response to Drs. Caruncho and Reive observations (see Supplementary Material 4). In addition, we would like to stress here that the possibility of using an automated procedure is severely hampered by the extremely high compactness of the central mass of the PNs lying inside the cerebellar white matter. In other words, it seems to be very difficult to practically separate the individual PNs with a thresholding procedure based on color or gray histograms (see Fig. S4-2). In addition, the aforementioned paper by Lagache and collaborators uses a similar manual procedure for the initial detection of the regions of interest (ROIs) as reported in their Materials and Methods: “We first delimited cells’ contours by drawing polygonal Region of Interest (ROIs) with the Icy software [13] (http://icy.bioimageanalysis.org). We then used a wavelet-based detection method [28], implemented as a plugin Spot detector in Icy to extract the two-dimensional positions of putative endocytic spots at the cellular membrane…”.
The removal of Voronoi zones of cells touching edges is justified, but no explanation is given on why the authors chose to favor convex area hence removing some additional cells in their study.
We have addressed this comment also in response to similar observations made by Drs. Caruncho and Reive (see main text and Supplementary Material 3 and 4).
Quantitative analysis of PN migration: since slices expand with time (Figure 1), how is the center point precisely localized through different time points?
We believe that this comment applies to Figure 2 and thus refers to qualitative analysis. The center point was recorded using the memory system of the microscope stage at 4 DIV. Subsequent images were recorded using the memorized X-Y coordinates.
Data interpretation:
In co-cultures, changes in the histology of reln-/- slices are interpreted as resulting from the effect of Reelin produced by reln+/- slices. However, there is no clear demonstration of this, as this could be due to another secreted factor. To answer this point, reelin blockade (function or binding, etc...) should be achieved. This experiment would also have the advantage to validate the statement made in the highlight that co-cultures are amenable to pharmacological intervention or other type of manipulation/treatment.
We agree that, theoretically, the changes in the histology of co-cultured reln^-/-slice could be dependent on a different secreted factor. However, several published papers support our line of thought as we have discussed in the revised version of the paper (reported above). To our knowledge, no other molecules except Reelin and its downstream receptors and adaptor molecules have been demonstrated to be primarily responsible for the correct layered organization of the cerebellar cortex during postnatal development of altricial mammals, although the true mechanisms of PNs layering in the mature cerebellar cortex remain a matter of debate [20]. As authoritatively reviewed by Gabriella D’Arcangelo [21], the discoverer of Reelin and certainly one of the leading scientists in the field “In the cerebellum, Reelin is produced by granule cells in the external granule layer and is essential for the radial migration of the Purkinje neurons, which are born in the cerebellar ventricular zone and form at first a plate and then a single-cell layer. This assessment derives from a widespread analysis of cerebellar development in Reeler mice [2, 22-24], from Reelin gene and protein expression data [16, 25], and functional studies in organotypic cultures [26]”.
We likewise agree that the blockade of Reelin would have the advantage of validating our statement that co-cultures are amenable to pharmacological or other types of manipulation. However, this would require a huge amount of additional work that is prone to several worries about its practicability (e.g. the effectiveness of Reelin-blocking antibodies -when available -to penetrate the entire thickness of a slice). In addition, much of the results that may be stemming from these efforts would be mainly confirmatory of previous observations. We have now discussed in our revision the previous literature on the block of the Reelin function in cultured cerebellar slices that demonstrates a long-distance effect of the molecule in vitro.
In the same line of thought, one can wonder whether reln-/- slices phenotypical improvement would be better achieved if the donor mice were reln+/+ instead of reln+/-? This would validate the method further and also give an indication about time and/or dose effect.
We agree with this observation in theory, but previously published observations on mouse chimeras [11] led to the conclusion that very little Reelin production could be sufficient to rescue the Reeler phenotype. In addition, our main goal was to validate the co-culture method that will be surely of good use to answer these questions in future work.
Statistics:
In Figure 7, what is the variable: does each point represent one culture? How many polygons are included in each point, this could actually be a bias in condition where GFP-PN survival decrease with time such as in the reln-/- group.
The variable in the figure (now Figure 9) is a randomly chosen culture slice. The number of polygons included in each point is indicated in FigShare uploaded Excel files (.xlsx or .csv). Inspection of these files shows that the number of polygons remains very high in single cultured reln^-/- slices despite survival decrease thus excluding a bias.
Please, indicate in legend samples size i.e. number of cultures/dishes used.
Done
In 7B, the distribution in the single reln+/-, clearly shows 2 groups in Voronoi polygons areas. Could you comment? Moreover, this kind of distribution undermines the use of means as presented in 7C.
The existence of two groups of Voronoi polygon areas is explained by the layering of the cortex in the slices from heterozygous mice. We respectfully disagree with the observation that this kind of distribution undermines the use of the means in the now Figure 7C as 1. Analysis was supported by inferential statistics and 2. Results from ArcMap GIS analysis where the tessellations of images only included the hexagons with PNs inside (see Figures 6 and 10 in main text and Figs. S64-15 in Supplementary Material 6) led to fully comparable results as shown and discussed in the revised version of the paper.
Figures:
Figures 1 and 5 are somehow overlapping. The paper would gain clarity by showing in different figures temporal modifications and histological characterization of single cultures and co-cultures (similar to Figure 2).
In response, we have deleted panels A and B in the original Figure 1. In addition, we have shown other images of the cultures to better demonstrate their histological appearance (Fig. 10A-C) and Figure S2.
Minor points:
What is the age of the mice used for slice preparation? And why are mice euthanized using sodium pentobarbital prior brain slicing (protocols.io.6qpvr67bbvmk/v1)?
The mice were 5 days old. We have added this information to the revised paper. They were euthanized with sodium pentobarbital (SP) as routinely done in our laboratory. Injectable barbiturates are acceptable for use in mouse fetuses and neonates according to the AVMA GUIDELINES FOR THE EUTHANASIA OF ANIMALS: 2020 EDITION, whereas decapitation and cervical dislocation are acceptable with conditions according to the guidelines. In addition, a recent publication [27] has reviewed the effects of SP (and other euthanasia methods) on cells and tissues as well as on different analytes without finding adverse effects of SP versus decapitation and/or cervical dislocation in the brain. Therefore we prefer to use SP also following the recommendations of our Department’s Ethical Committee.
What is the survival time of the single and co-cultures?
The survival time was 30 days at maximum. We have added this information to the revised paper.

References (for both responses to reviewers)
1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 06 Oct 2023

Adalberto Merighi, Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

06 Oct 2023

Author Response

Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history ... Continue reading Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Summary:
The study reports the implementation of a co-culture method to analyze the effects of reelin and reduce animal use within the 3R framework. Hybrid mice were generated by crossing L7-GFP with heterozygous reeler to obtain GFP-tagged Purkinje neurons in different reelin backgrounds. Co-cultures of cerebellar slices from homozygous and heterozygous hybrids resulted in dispersion and morphology changes of GFP-tagged Purkinje neurons derived from slices bearing a homozygous background.
General:
The main concern is about the general scope of the paper and whether it is about ethical use of animals in biological experiments and animal welfare or about reelin neurobiology in the 3R framework. Although the two former issues are not to be overlooked, they should be developed in the discussion rather than in the introduction, highlights and results.
We fully understand this concern. A similar concern was also raised by Drs. Caruncho and Reive. The reason why the issues of the ethical use of animals in biological experiments and animal welfare have been developed in different parts of the manuscript other than the Discussion is to cope with the guidelines issued by the NC3Rs for Method papers. In response, we have fully revised the paper according to these observations as explained above in the response to the comments of the other two reviewers and in the preamble to our responses.
The authors also state that this approach will allow the study of reelin effects on cortical lamination and that the method will also be applicable to investigate the function of other brain secreted proteins. They should show that this is indeed the case either in co-cultures of other cortices from reelin hybrids or in cerebellar co-cultures from mutants of different proteins.
We agree with the reviewers that the experiments suggested would be indeed useful to confirm that our approach will permit studying 1. The effects of reelin in the other laminated areas of the brain or 2. The function of other secreted proteins. Yet, as regards the first issue, we have now thoroughly discussed previous experiments with approaches different from the one used here demonstrating the effects of Reelin on the lamination of the cerebral cortex. As regards the second issue, we have now softened our claims on the possible applications of our method to the study of other secreted brain proteins in another specific section of the discussion (Advantages and limitations of the organotypic culture approach to the study of neurodevelopment). We have also considered the issues raised here in a more thorough discussion of the strengths and weaknesses of our approach, as such a discussion was also suggested by Drs. Drs. Caruncho and Reive.
We are confident that the reviewers would agree with us that the experiments suggested would require a large amount of work that could the the subject of another paper and well beyond the purpose of this Method Paper (that as mentioned in our prologue was sponsored by the NC3Rs and was prepared according to the guidelines for the NC3Rs gateway). Apart from this, there are several theoretical and practical limitations to the possibility of performing the experiments above (we have addressed this point in response to the first comment under “Data Interpretation”).
That reelin produced by the cerebellar granule cells in slices from heterozygous or homozygous Reeler mice can diffuse in the culture medium was previously demonstrated after studies on chimeric mice and in Reeler cortical cultures. We have addressed this issue in the Discussion of the revised paper as follows:
Although the structural effects of the Reeler mutation are well known, little work has been done on the possibility of modifying Reelin-mediated deficits in live cells. A study in the Reeler-normal chimeric mice has previously demonstrated that the morphology and position of the cerebellar neurons and glial cells are controlled by extracellular environments but not by their genotype [11]. Subsequent work has shown that the protein produced by cortical explants of reln^+/+ or reln^+/- mice co-cultured with a Reeler-like ferret dysplastic cortex was capable of at least partly rescuing neuronal migration in the ferret explant [12]. Other studies, were carried out in the cerebral cortex and hippocampus and led to similar conclusions. It was thus shown that cortical layer development and orientation are modulated by relative contributions of Reelin-negative and -positive neurons in mouse chimeras [13] and that the possibility to restore a normal phenotype in reln^-/-slices co-cultured with wild-type slices was mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR) [14]. Our present observations are in full accord with these studies. A point left open for discussion regards the Reelin dosage necessary for the rescue of a normal phenotype in culture studies. In their chimeric mouse study, Yoshiki and Kusakabe qualitatively observed that only a few granule cells derived from a normal mouse were capable of promoting the alignment and the proper dendritic tree development of the PNs derived from the mutant. From their observations, these authors suggested that Reelin secreted from a single granule cell can affect the morphology of PNs in a wide area [11].
Our results quantitatively demonstrate that the Reelin produced by the slices obtained from haplodeficient mice is sufficient to restore the normal cerebellar phenotype. Therefore, our findings reinforce the idea that it may be possible to modify Reelin-mediated deficits through the experimental administration of the protein. Experiments have demonstrated that the medium obtained from cerebellar dissociated cultures from P5-8 normal mice as well as from cell extracts of the cultured cerebellum from these mice contained full-length Reelin [15]. These observations are fully in line with the demonstration that the protein is produced by the postmitotic granule cells of the deep external granular layer before they migrate to their final destination, the internal granular layer, where they lose Reelin immunoreactivity [16].
Strengths:
The authors developed an elegant technique highly suited for longitudinal and dynamic studies, implemented in the 3Rs framework. They provide a very detailed and exhaustive description of the method to allow replication in other labs.
Major points:
Methods:
Voronoi analysis: The authors perform Voronoi analysis to analyze spatial organization of cells. Although they give references to support their work, the theoretical paper is dated from 1992. Since then, framework and tools to study spatial organization, based on robust spatial statistics such as F, G, H and K functions, have been developed (see Lagache et al., 20131 for example).
Frankly, we do not see why Voronoi's analysis should be hampered by its relative age. Voronoi tesselation has been and still is widely employed in the study of the spatial organization of many diverse types of neuronal (and other) cell populations [17].
The paper quoted by the reviewers uses a modified Ripley’s K function to define the clustered, dispersed, or uniform distribution of points (i.e. clathrin endocytotic sites) in the 2D space [18]. Ripley's K-, H-, and L-functions are used primarily to identify the clustering of proteins in membrane microdomains (as is indeed the case for Lagache’s paper). Using this approach, aggregation (or clustering) is identified if the average number of proteins within a distance r of another protein is statistically greater than that expected for a random distribution [19]. Kiskowski et al. emphasized that “it is not entirely clear how the function may be used to quantitatively determine the size of domains in which clustering occurs” and have widely discussed the limitations and potential of Ripley's K in real-life scenarios”[19]. In the case of this study, where indeed the main issue under investigation is the clustering of the PNs, which is very high in the reln^-/-mice, one can raise reasonable doubts about the usefulness of applying Ripley's K in our conditions. Despite the above observations on the limitations of Ripley’s functions to modeling spatial clustering we have added a large bunch of new analyses based on the novel use of spatial statistics with ArcMap [geographical distribution tools (Central Feature, Mean Center, Median Center, Directional Distribution, and Standard Distance), pattern distribution tools (Average Nearest Neighbor, Getis-Ord General G, Ripley’s K function, and Global Moran’s I), and mapping clusters tools (Anselin Local Moran’s I, and Getis-Ord G*)] to compare their results with the conclusions that could be drawn solely from Voronoi analysis. As thoroughly discussed in the revised paper the two approaches yielded comparable results in support of our initial claims on the soundness of the method.
The overall method is also extremely manual, involving at least 4 different softwares, and the initial cell detection is purely manual, along with the Voronoi adjustment.
We have already at least partly addressed this comment in response to Drs. Caruncho and Reive observations (see Supplementary Material 4). In addition, we would like to stress here that the possibility of using an automated procedure is severely hampered by the extremely high compactness of the central mass of the PNs lying inside the cerebellar white matter. In other words, it seems to be very difficult to practically separate the individual PNs with a thresholding procedure based on color or gray histograms (see Fig. S4-2). In addition, the aforementioned paper by Lagache and collaborators uses a similar manual procedure for the initial detection of the regions of interest (ROIs) as reported in their Materials and Methods: “We first delimited cells’ contours by drawing polygonal Region of Interest (ROIs) with the Icy software [13] (http://icy.bioimageanalysis.org). We then used a wavelet-based detection method [28], implemented as a plugin Spot detector in Icy to extract the two-dimensional positions of putative endocytic spots at the cellular membrane…”.
The removal of Voronoi zones of cells touching edges is justified, but no explanation is given on why the authors chose to favor convex area hence removing some additional cells in their study.
We have addressed this comment also in response to similar observations made by Drs. Caruncho and Reive (see main text and Supplementary Material 3 and 4).
Quantitative analysis of PN migration: since slices expand with time (Figure 1), how is the center point precisely localized through different time points?
We believe that this comment applies to Figure 2 and thus refers to qualitative analysis. The center point was recorded using the memory system of the microscope stage at 4 DIV. Subsequent images were recorded using the memorized X-Y coordinates.
Data interpretation:
In co-cultures, changes in the histology of reln-/- slices are interpreted as resulting from the effect of Reelin produced by reln+/- slices. However, there is no clear demonstration of this, as this could be due to another secreted factor. To answer this point, reelin blockade (function or binding, etc...) should be achieved. This experiment would also have the advantage to validate the statement made in the highlight that co-cultures are amenable to pharmacological intervention or other type of manipulation/treatment.
We agree that, theoretically, the changes in the histology of co-cultured reln^-/-slice could be dependent on a different secreted factor. However, several published papers support our line of thought as we have discussed in the revised version of the paper (reported above). To our knowledge, no other molecules except Reelin and its downstream receptors and adaptor molecules have been demonstrated to be primarily responsible for the correct layered organization of the cerebellar cortex during postnatal development of altricial mammals, although the true mechanisms of PNs layering in the mature cerebellar cortex remain a matter of debate [20]. As authoritatively reviewed by Gabriella D’Arcangelo [21], the discoverer of Reelin and certainly one of the leading scientists in the field “In the cerebellum, Reelin is produced by granule cells in the external granule layer and is essential for the radial migration of the Purkinje neurons, which are born in the cerebellar ventricular zone and form at first a plate and then a single-cell layer. This assessment derives from a widespread analysis of cerebellar development in Reeler mice [2, 22-24], from Reelin gene and protein expression data [16, 25], and functional studies in organotypic cultures [26]”.
We likewise agree that the blockade of Reelin would have the advantage of validating our statement that co-cultures are amenable to pharmacological or other types of manipulation. However, this would require a huge amount of additional work that is prone to several worries about its practicability (e.g. the effectiveness of Reelin-blocking antibodies -when available -to penetrate the entire thickness of a slice). In addition, much of the results that may be stemming from these efforts would be mainly confirmatory of previous observations. We have now discussed in our revision the previous literature on the block of the Reelin function in cultured cerebellar slices that demonstrates a long-distance effect of the molecule in vitro.
In the same line of thought, one can wonder whether reln-/- slices phenotypical improvement would be better achieved if the donor mice were reln+/+ instead of reln+/-? This would validate the method further and also give an indication about time and/or dose effect.
We agree with this observation in theory, but previously published observations on mouse chimeras [11] led to the conclusion that very little Reelin production could be sufficient to rescue the Reeler phenotype. In addition, our main goal was to validate the co-culture method that will be surely of good use to answer these questions in future work.
Statistics:
In Figure 7, what is the variable: does each point represent one culture? How many polygons are included in each point, this could actually be a bias in condition where GFP-PN survival decrease with time such as in the reln-/- group.
The variable in the figure (now Figure 9) is a randomly chosen culture slice. The number of polygons included in each point is indicated in FigShare uploaded Excel files (.xlsx or .csv). Inspection of these files shows that the number of polygons remains very high in single cultured reln^-/- slices despite survival decrease thus excluding a bias.
Please, indicate in legend samples size i.e. number of cultures/dishes used.
Done
In 7B, the distribution in the single reln+/-, clearly shows 2 groups in Voronoi polygons areas. Could you comment? Moreover, this kind of distribution undermines the use of means as presented in 7C.
The existence of two groups of Voronoi polygon areas is explained by the layering of the cortex in the slices from heterozygous mice. We respectfully disagree with the observation that this kind of distribution undermines the use of the means in the now Figure 7C as 1. Analysis was supported by inferential statistics and 2. Results from ArcMap GIS analysis where the tessellations of images only included the hexagons with PNs inside (see Figures 6 and 10 in main text and Figs. S64-15 in Supplementary Material 6) led to fully comparable results as shown and discussed in the revised version of the paper.
Figures:
Figures 1 and 5 are somehow overlapping. The paper would gain clarity by showing in different figures temporal modifications and histological characterization of single cultures and co-cultures (similar to Figure 2).
In response, we have deleted panels A and B in the original Figure 1. In addition, we have shown other images of the cultures to better demonstrate their histological appearance (Fig. 10A-C) and Figure S2.
Minor points:
What is the age of the mice used for slice preparation? And why are mice euthanized using sodium pentobarbital prior brain slicing (protocols.io.6qpvr67bbvmk/v1)?
The mice were 5 days old. We have added this information to the revised paper. They were euthanized with sodium pentobarbital (SP) as routinely done in our laboratory. Injectable barbiturates are acceptable for use in mouse fetuses and neonates according to the AVMA GUIDELINES FOR THE EUTHANASIA OF ANIMALS: 2020 EDITION, whereas decapitation and cervical dislocation are acceptable with conditions according to the guidelines. In addition, a recent publication [27] has reviewed the effects of SP (and other euthanasia methods) on cells and tissues as well as on different analytes without finding adverse effects of SP versus decapitation and/or cervical dislocation in the brain. Therefore we prefer to use SP also following the recommendations of our Department’s Ethical Committee.
What is the survival time of the single and co-cultures?
The survival time was 30 days at maximum. We have added this information to the revised paper.

References (for both responses to reviewers)
1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Summary:
The study reports the implementation of a co-culture method to analyze the effects of reelin and reduce animal use within the 3R framework. Hybrid mice were generated by crossing L7-GFP with heterozygous reeler to obtain GFP-tagged Purkinje neurons in different reelin backgrounds. Co-cultures of cerebellar slices from homozygous and heterozygous hybrids resulted in dispersion and morphology changes of GFP-tagged Purkinje neurons derived from slices bearing a homozygous background.
General:
The main concern is about the general scope of the paper and whether it is about ethical use of animals in biological experiments and animal welfare or about reelin neurobiology in the 3R framework. Although the two former issues are not to be overlooked, they should be developed in the discussion rather than in the introduction, highlights and results.
We fully understand this concern. A similar concern was also raised by Drs. Caruncho and Reive. The reason why the issues of the ethical use of animals in biological experiments and animal welfare have been developed in different parts of the manuscript other than the Discussion is to cope with the guidelines issued by the NC3Rs for Method papers. In response, we have fully revised the paper according to these observations as explained above in the response to the comments of the other two reviewers and in the preamble to our responses.
The authors also state that this approach will allow the study of reelin effects on cortical lamination and that the method will also be applicable to investigate the function of other brain secreted proteins. They should show that this is indeed the case either in co-cultures of other cortices from reelin hybrids or in cerebellar co-cultures from mutants of different proteins.
We agree with the reviewers that the experiments suggested would be indeed useful to confirm that our approach will permit studying 1. The effects of reelin in the other laminated areas of the brain or 2. The function of other secreted proteins. Yet, as regards the first issue, we have now thoroughly discussed previous experiments with approaches different from the one used here demonstrating the effects of Reelin on the lamination of the cerebral cortex. As regards the second issue, we have now softened our claims on the possible applications of our method to the study of other secreted brain proteins in another specific section of the discussion (Advantages and limitations of the organotypic culture approach to the study of neurodevelopment). We have also considered the issues raised here in a more thorough discussion of the strengths and weaknesses of our approach, as such a discussion was also suggested by Drs. Drs. Caruncho and Reive.
We are confident that the reviewers would agree with us that the experiments suggested would require a large amount of work that could the the subject of another paper and well beyond the purpose of this Method Paper (that as mentioned in our prologue was sponsored by the NC3Rs and was prepared according to the guidelines for the NC3Rs gateway). Apart from this, there are several theoretical and practical limitations to the possibility of performing the experiments above (we have addressed this point in response to the first comment under “Data Interpretation”).
That reelin produced by the cerebellar granule cells in slices from heterozygous or homozygous Reeler mice can diffuse in the culture medium was previously demonstrated after studies on chimeric mice and in Reeler cortical cultures. We have addressed this issue in the Discussion of the revised paper as follows:
Although the structural effects of the Reeler mutation are well known, little work has been done on the possibility of modifying Reelin-mediated deficits in live cells. A study in the Reeler-normal chimeric mice has previously demonstrated that the morphology and position of the cerebellar neurons and glial cells are controlled by extracellular environments but not by their genotype [11]. Subsequent work has shown that the protein produced by cortical explants of reln^+/+ or reln^+/- mice co-cultured with a Reeler-like ferret dysplastic cortex was capable of at least partly rescuing neuronal migration in the ferret explant [12]. Other studies, were carried out in the cerebral cortex and hippocampus and led to similar conclusions. It was thus shown that cortical layer development and orientation are modulated by relative contributions of Reelin-negative and -positive neurons in mouse chimeras [13] and that the possibility to restore a normal phenotype in reln^-/-slices co-cultured with wild-type slices was mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR) [14]. Our present observations are in full accord with these studies. A point left open for discussion regards the Reelin dosage necessary for the rescue of a normal phenotype in culture studies. In their chimeric mouse study, Yoshiki and Kusakabe qualitatively observed that only a few granule cells derived from a normal mouse were capable of promoting the alignment and the proper dendritic tree development of the PNs derived from the mutant. From their observations, these authors suggested that Reelin secreted from a single granule cell can affect the morphology of PNs in a wide area [11].
Our results quantitatively demonstrate that the Reelin produced by the slices obtained from haplodeficient mice is sufficient to restore the normal cerebellar phenotype. Therefore, our findings reinforce the idea that it may be possible to modify Reelin-mediated deficits through the experimental administration of the protein. Experiments have demonstrated that the medium obtained from cerebellar dissociated cultures from P5-8 normal mice as well as from cell extracts of the cultured cerebellum from these mice contained full-length Reelin [15]. These observations are fully in line with the demonstration that the protein is produced by the postmitotic granule cells of the deep external granular layer before they migrate to their final destination, the internal granular layer, where they lose Reelin immunoreactivity [16].
Strengths:
The authors developed an elegant technique highly suited for longitudinal and dynamic studies, implemented in the 3Rs framework. They provide a very detailed and exhaustive description of the method to allow replication in other labs.
Major points:
Methods:
Voronoi analysis: The authors perform Voronoi analysis to analyze spatial organization of cells. Although they give references to support their work, the theoretical paper is dated from 1992. Since then, framework and tools to study spatial organization, based on robust spatial statistics such as F, G, H and K functions, have been developed (see Lagache et al., 20131 for example).
Frankly, we do not see why Voronoi's analysis should be hampered by its relative age. Voronoi tesselation has been and still is widely employed in the study of the spatial organization of many diverse types of neuronal (and other) cell populations [17].
The paper quoted by the reviewers uses a modified Ripley’s K function to define the clustered, dispersed, or uniform distribution of points (i.e. clathrin endocytotic sites) in the 2D space [18]. Ripley's K-, H-, and L-functions are used primarily to identify the clustering of proteins in membrane microdomains (as is indeed the case for Lagache’s paper). Using this approach, aggregation (or clustering) is identified if the average number of proteins within a distance r of another protein is statistically greater than that expected for a random distribution [19]. Kiskowski et al. emphasized that “it is not entirely clear how the function may be used to quantitatively determine the size of domains in which clustering occurs” and have widely discussed the limitations and potential of Ripley's K in real-life scenarios”[19]. In the case of this study, where indeed the main issue under investigation is the clustering of the PNs, which is very high in the reln^-/-mice, one can raise reasonable doubts about the usefulness of applying Ripley's K in our conditions. Despite the above observations on the limitations of Ripley’s functions to modeling spatial clustering we have added a large bunch of new analyses based on the novel use of spatial statistics with ArcMap [geographical distribution tools (Central Feature, Mean Center, Median Center, Directional Distribution, and Standard Distance), pattern distribution tools (Average Nearest Neighbor, Getis-Ord General G, Ripley’s K function, and Global Moran’s I), and mapping clusters tools (Anselin Local Moran’s I, and Getis-Ord G*)] to compare their results with the conclusions that could be drawn solely from Voronoi analysis. As thoroughly discussed in the revised paper the two approaches yielded comparable results in support of our initial claims on the soundness of the method.
The overall method is also extremely manual, involving at least 4 different softwares, and the initial cell detection is purely manual, along with the Voronoi adjustment.
We have already at least partly addressed this comment in response to Drs. Caruncho and Reive observations (see Supplementary Material 4). In addition, we would like to stress here that the possibility of using an automated procedure is severely hampered by the extremely high compactness of the central mass of the PNs lying inside the cerebellar white matter. In other words, it seems to be very difficult to practically separate the individual PNs with a thresholding procedure based on color or gray histograms (see Fig. S4-2). In addition, the aforementioned paper by Lagache and collaborators uses a similar manual procedure for the initial detection of the regions of interest (ROIs) as reported in their Materials and Methods: “We first delimited cells’ contours by drawing polygonal Region of Interest (ROIs) with the Icy software [13] (http://icy.bioimageanalysis.org). We then used a wavelet-based detection method [28], implemented as a plugin Spot detector in Icy to extract the two-dimensional positions of putative endocytic spots at the cellular membrane…”.
The removal of Voronoi zones of cells touching edges is justified, but no explanation is given on why the authors chose to favor convex area hence removing some additional cells in their study.
We have addressed this comment also in response to similar observations made by Drs. Caruncho and Reive (see main text and Supplementary Material 3 and 4).
Quantitative analysis of PN migration: since slices expand with time (Figure 1), how is the center point precisely localized through different time points?
We believe that this comment applies to Figure 2 and thus refers to qualitative analysis. The center point was recorded using the memory system of the microscope stage at 4 DIV. Subsequent images were recorded using the memorized X-Y coordinates.
Data interpretation:
In co-cultures, changes in the histology of reln-/- slices are interpreted as resulting from the effect of Reelin produced by reln+/- slices. However, there is no clear demonstration of this, as this could be due to another secreted factor. To answer this point, reelin blockade (function or binding, etc...) should be achieved. This experiment would also have the advantage to validate the statement made in the highlight that co-cultures are amenable to pharmacological intervention or other type of manipulation/treatment.
We agree that, theoretically, the changes in the histology of co-cultured reln^-/-slice could be dependent on a different secreted factor. However, several published papers support our line of thought as we have discussed in the revised version of the paper (reported above). To our knowledge, no other molecules except Reelin and its downstream receptors and adaptor molecules have been demonstrated to be primarily responsible for the correct layered organization of the cerebellar cortex during postnatal development of altricial mammals, although the true mechanisms of PNs layering in the mature cerebellar cortex remain a matter of debate [20]. As authoritatively reviewed by Gabriella D’Arcangelo [21], the discoverer of Reelin and certainly one of the leading scientists in the field “In the cerebellum, Reelin is produced by granule cells in the external granule layer and is essential for the radial migration of the Purkinje neurons, which are born in the cerebellar ventricular zone and form at first a plate and then a single-cell layer. This assessment derives from a widespread analysis of cerebellar development in Reeler mice [2, 22-24], from Reelin gene and protein expression data [16, 25], and functional studies in organotypic cultures [26]”.
We likewise agree that the blockade of Reelin would have the advantage of validating our statement that co-cultures are amenable to pharmacological or other types of manipulation. However, this would require a huge amount of additional work that is prone to several worries about its practicability (e.g. the effectiveness of Reelin-blocking antibodies -when available -to penetrate the entire thickness of a slice). In addition, much of the results that may be stemming from these efforts would be mainly confirmatory of previous observations. We have now discussed in our revision the previous literature on the block of the Reelin function in cultured cerebellar slices that demonstrates a long-distance effect of the molecule in vitro.
In the same line of thought, one can wonder whether reln-/- slices phenotypical improvement would be better achieved if the donor mice were reln+/+ instead of reln+/-? This would validate the method further and also give an indication about time and/or dose effect.
We agree with this observation in theory, but previously published observations on mouse chimeras [11] led to the conclusion that very little Reelin production could be sufficient to rescue the Reeler phenotype. In addition, our main goal was to validate the co-culture method that will be surely of good use to answer these questions in future work.
Statistics:
In Figure 7, what is the variable: does each point represent one culture? How many polygons are included in each point, this could actually be a bias in condition where GFP-PN survival decrease with time such as in the reln-/- group.
The variable in the figure (now Figure 9) is a randomly chosen culture slice. The number of polygons included in each point is indicated in FigShare uploaded Excel files (.xlsx or .csv). Inspection of these files shows that the number of polygons remains very high in single cultured reln^-/- slices despite survival decrease thus excluding a bias.
Please, indicate in legend samples size i.e. number of cultures/dishes used.
Done
In 7B, the distribution in the single reln+/-, clearly shows 2 groups in Voronoi polygons areas. Could you comment? Moreover, this kind of distribution undermines the use of means as presented in 7C.
The existence of two groups of Voronoi polygon areas is explained by the layering of the cortex in the slices from heterozygous mice. We respectfully disagree with the observation that this kind of distribution undermines the use of the means in the now Figure 7C as 1. Analysis was supported by inferential statistics and 2. Results from ArcMap GIS analysis where the tessellations of images only included the hexagons with PNs inside (see Figures 6 and 10 in main text and Figs. S64-15 in Supplementary Material 6) led to fully comparable results as shown and discussed in the revised version of the paper.
Figures:
Figures 1 and 5 are somehow overlapping. The paper would gain clarity by showing in different figures temporal modifications and histological characterization of single cultures and co-cultures (similar to Figure 2).
In response, we have deleted panels A and B in the original Figure 1. In addition, we have shown other images of the cultures to better demonstrate their histological appearance (Fig. 10A-C) and Figure S2.
Minor points:
What is the age of the mice used for slice preparation? And why are mice euthanized using sodium pentobarbital prior brain slicing (protocols.io.6qpvr67bbvmk/v1)?
The mice were 5 days old. We have added this information to the revised paper. They were euthanized with sodium pentobarbital (SP) as routinely done in our laboratory. Injectable barbiturates are acceptable for use in mouse fetuses and neonates according to the AVMA GUIDELINES FOR THE EUTHANASIA OF ANIMALS: 2020 EDITION, whereas decapitation and cervical dislocation are acceptable with conditions according to the guidelines. In addition, a recent publication [27] has reviewed the effects of SP (and other euthanasia methods) on cells and tissues as well as on different analytes without finding adverse effects of SP versus decapitation and/or cervical dislocation in the brain. Therefore we prefer to use SP also following the recommendations of our Department’s Ethical Committee.
What is the survival time of the single and co-cultures?
The survival time was 30 days at maximum. We have added this information to the revised paper.

References (for both responses to reviewers)
1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 03 Nov 2022

Hector J. Caruncho, Division of Medical Sciences, University of Victoria, Victoria, BC, Canada

Brady Reive, University of Victoria, Victoria, BC, Canada

Approved with Reservations

https://doi.org/10.5256/f1000research.139230.r153860

Summary of study:
Hybrid mice were generated by crossing male heterozygous Reelin mutants with L7-GFP females, resulting in both homozygous and heterozygous Reeler hybrids with GFP-tagged Purkinje neurons. Cerebellum slices from both groups were cultured and co-cultured together. The morphology and clustering of cultured cells were compared and found differences to cell morphology and organization between homozygous Reeler mutant cultures versus co-cultures also including heterozygous Reeler tissue.

Strengths and weaknesses:

Strengths: Interesting original work. Low animal numbers needed. Relatively simple yet elegant combination of techniques. Thorough descriptions of methods to help readers apply to their own purposes/replication.

Weaknesses:
General: A bit strange that the conclusion is drawn pertaining to potential applications of cell cultures in general (I say that because although they’re not intended, it seems to speak to most forms of cell culture rather than specific to this technique). If this is a ground-breaking technique, it might be more valuable to show that it can be applied to more than one protein. Many proteins relate to cell survival, regulation of signaling, etc. as opposed to cell migration, so it’s a bit difficult to understand how this technique would benefit the study of other aspects of cell function. I suppose part of the issue for me is the absence of discussion of limitations to the model, so as I am imagining all sorts of limitations without the authors discussion or recognition of them, I’m left not understanding if/how the limitations would be explained. Some other limitations include: can you conclude that application of Reelin would resolve the layering deficits in homozygous Reelers through this model? I’m not understanding how the authors conclude cells originating from the homozygous slice show improved organization in co-cultures, as opposed to showing that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing altered organization because you a) can’t verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells); b) there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells. Maybe it wasn’t clear enough how co-cultures are grown together to allow for differentiation.

General comments on:

Methods: Good overall. My only concerns: Polygon selection and removal of the top polygon, which corresponds to the culmination of data (or all accounted for polygons). I’m not understanding why that information wouldn’t be valuable for comparing sets of polygons (I understand it’s included de facto in the sum of all selected polygons) but it would be good to show the amalgamations are similar across cultures (i.e. I would expect less stacking/layering of cells occurs in smaller montages of polygons compared to bigger ones corresponding to larger numbers of cells, which would impact nearest neighbour analyses etc.). Verification that the initial selection of cells is accurate is not done, and in fact is explained as being negligible. “Using the mouse, click above the center of each cell to generate the Voronoi polygons. Due to the thickness of the slice, it may be possible that two very close cells in the Z axis are not easily distinguished. This introduces an error that can be neglected considering that all slices are cut at the same initial thickness.”
It isn’t clear why initial thickness of section would dictate how one would treat stacking of grown cells. The stacking isn’t uniform just because the initial slice was uniform (at least it very much appears there exists more stacking in the central mass relative to the periphery. Also, it matters entirely because those clicks define the cell sizes, which is one of the outputs for statistical comparisons.

The inability to verify which slice cells originate should be described/explained. Is it possible to use a secondary reporter to compare and contrast within co-cultures? What would it mean that you can’t verify if so?

Is it true the imaging is done at the central mass? I had a difficult time understanding because there is also a section that states the coordinates images could be taken at. I’m very curious if there’s an impact of image location/bias involved. Is the origin selected by the researcher, are there methods for ensuring similarly sized clusters of cells are being imaged? Is it randomized? How many cells are removed in each?
I found this comment particularly interesting: “It is possible to use several other Voronoi generators that can be found online as freeware or in dedicated programs.” They acknowledge the existence of other modeling systems, would be valuable to know if the results are similar., i.e. the impact of the modeling choice. Even a visual demonstration to compare the resulting polygons would be a powerful addition (to answer the obvious question about validity of Voronoi method, how important is accurate cell selection, is there an impact of user experience, etc.).

Data interpretation: I think my biggest concern is lack of description of limitations/how perceivable limitations might be overcome, and secondary, that the discussion and conclusion only includes the word Reelin once, in the first sentence…otherwise the paper is about animal welfare? If the focus is going to be about how to reduce animal sizes, more information should be provided about litter sizes, culling, survival of mutants, etc. and how litter survival might be a limitation for studying other proteins using this method (or not). More information would be required about how many cultures can be derived from a single animal (i.e. size of slice versus size of structure at age of collection, etc.) Either it’s about Reelin, which I think it should be, and I think is interesting, or it’s a methods paper, in which case I think it would be much more valuable to show positive results in at least 2 proteins of interest in a way that allows for comparing the cultures to really show the applications of this technique. I think it might be limited to studying proteins with a very specific subset of functions, so maybe choosing another protein with a vastly different function of Reelin would be a good choice to really highlight the value of this technique. Instead, I think re-writing the discussion and conclusion to focus on results about Reelin would be more appropriate. What can the authors infer about the possibility of Reelin application to modify Reelin-mediated deficits? What can the authors infer about Reelin’s function in regulating cell morphology, beyond simple layering/migration results? Are these results consistent with the in vivo known functions of Reelin? What does this mean? Would you expect there would be differences in non-Purkinje cells? What other sets of cells you can study with this technique? Are there basket cells, granular cells etc. in the culture that we can’t visualize? Why the cerebellum? Is there interaction between the various cells in culture (both PN to PN and PN to any other non-GFP cells)? How might you evaluate cell interactions using this model in the future? What other directions would you go? Just so much to talk about and instead we were told we can save an undetermined, unspecified number of animals…but to learn what? Instead of providing 2 highly specific proteins that could be studied with this method, why not provide a sweeping generalized set of proteins you might have interest in studying with this technique? Usually, the issue is people over-exclaim their success, but in this case, I think not enough effort is given to really dive into what this study indicates about Reelin or what else can be done with the method. If this method is so great, then there should be no shortage of things to tell the reader that you learned about Reelin in applying the technique, instead of how many fewer animals might be needed in future hypothetical studies.

Language: Good language and writing. No considerable issues noted (grammar, spelling, awkward word choice, etc.).

Figures and Tables: Very thorough and good quality figures and figure descriptions. No issues.

Statistics: Only issue with stats described earlier: pertaining to non-randomized imaging location (coordinates in the dish essentially) leads to selection bias. Alternative suggestions would include: a) create a grid and take an average of all polygons in multiple grid boxes to get a variety of polygon origins within the analysis. Additionally, it would be valuable to demonstrate whether or not peripheral polygons are truly similar to the central mass related polygons, in other words, a within culture analysis would also be valuable (is there more or less homogeneity of cultures from Reelin homo- or heterozygous mice?). Combining a perceived selection bias with unconfirmed cell selection, the reader might reasonably be concerned the results aren’t the most meaningful or generalizable.

Ethics: No concerns.

Is the rationale for developing the new method (or application) clearly explained?

Yes
Is the description of the method technically sound?

Yes
Are sufficient details provided to allow replication of the method development and its use by others?

Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neuropharmacology; Reelin neurobiology; Animal models; Mood and Psychotic disorders

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Author Response 06 Oct 2023

Adalberto Merighi, Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

06 Oct 2023

Author Response
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history ... Continue reading
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Weaknesses:
General: A bit strange that the conclusion is drawn pertaining to potential applications of cell cultures in general (I say that because although they’re not intended, it seems to speak to most forms of cell culture rather than specific to this technique). If this is a ground-breaking technique, it might be more valuable to show that it can be applied to more than one protein. Many proteins relate to cell survival, regulation of signaling, etc. as opposed to cell migration, so it’s a bit difficult to understand how this technique would benefit the study of other aspects of cell function. I suppose part of the issue for me is the absence of discussion of limitations to the model, so as I am imagining all sorts of limitations without the authors discussion or recognition of them, I’m left not understanding if/how the limitations would be explained.
We thank the reviewers for these very useful observations. We have revised the paper by adding a thorough discussion of the limitations of the technique and trying to avoid a too strong generalization of its applicability.
Some other limitations include: Can you conclude that application of Reelin would resolve the layering deficits in homozygous Reelers through this model? I’m not understanding how the authors conclude cells originating from the homozygous slice show improved organization in co-cultures, as opposed to showing that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing altered organization because you a) can’t verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells); b) there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells. Maybe it wasn’t clear enough how co-cultures are grown together to allow for differentiation.
We are sorry that we have not been able to clarify these issues in the original version of the paper. We believe that, from our observations, we can indeed conclude that the reelin secreted from the cerebellar granule cells in the slices from heterozygous mice (reelin haplodeficient) has a positive effect on the organization of the cerebellar cortex in the slices obtained from homozygous mice (lacking reelin).
Our belief is based on the following:

Faulty Reelin signaling in the cerebellum causes Purkinje neurons (PN) migration to be disrupted and, as a result, granule cell precursor (GCPs) proliferation to be diminished – for a recent review see [1]. Morphological observations in vivo have long ago demonstrated that the Reeler mouse, which is devoid of Reelin, displays an altered stratification of its cerebellar and cerebral cortices, as well as of other layered areas of the brain. Specifically in the cerebellum, these alterations affect the migration and correct positioning of the PNs that, in the homozygous reln^-/- mouse, remain clustered together in a deep cellular mass embedded into the medullary body of the white matter [2]. Conversely, in reln^+/-and reln^+/+ mice the PNs migrate in an outward direction to eventually form a monolayer of cells between the molecular and granule cell layer of the mature cerebellar cortex, i.e. both genotypes display a normal cerebellar architecture, although we have recently demonstrated that there are very subtle differences in the position of the PNs between reln^+/-and reln^+/+mice [3]. The effects of the PNs on the GCP proliferation are mediated by Sonic Hedgehog (reviewed in [4]) and we have subsequently demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to a reduction of cortical size and cellularity, the latter being linked to quantitative differences in the extent of GCP proliferation and apoptosis between normal and Reeler mice [5]. The interactions between the PNs and the GCPs are thus quite complex: from one side the PNs stimulate the proliferation of GCPs and, from the other, the CGPs produce and release Reelin in the intercellular space, and the glycoprotein is essential for the correct positioning of the PNs.

If we understood well the reviewers’ thoughts, they suggest that we cannot claim that “the cells originating from the homozygous slice show improved organization in co-cultures” as it is equally possible “that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing an altered organization” but this in total conflict with the above-reported literature.

In addition, the reviewers believe that we are overinterpreting our findings because:

We could not “verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells”. However, we never suggested that there was a migration of PNs from one slice to the other, and there are no pieces of evidence from previous studies that GFP is exchanged between cells in the L7 mouse. The PN-specific gene coding L7 or pcp2 (Purkinje cells specific protein-2) was first described in 1988 by two independent groups [6, 7], and since then it has been of paramount importance for genetic targeting of these neurons [8]. Anyway, even if one assumes that the GFP can be transferred between cerebellar neurons, the protein should preferentially reach the neurons of the cerebellar nuclei which are in contact with the PN axons, as the retrograde transsynaptic transfer occurs only when a fusion protein with a non-toxic fragment of tetanus toxin is used for genetic engineering/transfection, see e.g. [9].

“there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells”. Although Occam’s razor is based on the idea that the simplest explanation is often the best one, we don’t see how the reviewers may conclude that we are simply comparing heterozygous (PN) cells between each other, since they derive from genotyped animals and no exchange of PNs occurs among slices. On the contrary, the simplest explanation of our findings is indeed the one put forward in the original paper.

Yet, we fully agree with the referees that “it wasn’t clear enough how co-cultures are grown together to allow for differentiation” and we have amended the manuscript by better explaining how co-cultures were established and how they could influence each other by adding some supplementary information to the paper (Supplementary Material 1).
General comments on:
Methods: Good overall. My only concerns: Polygon selection and removal of the top polygon, which corresponds to the culmination of data (or all accounted for polygons). I’m not understanding why that information wouldn’t be valuable for comparing sets of polygons (I understand it’s included de facto in the sum of all selected polygons) but it would be good to show the amalgamations are similar across cultures (i.e. I would expect less stacking/layering of cells occurs in smaller montages of polygons compared to bigger ones corresponding to larger numbers of cells, which would impact nearest neighbour analyses etc.).
The principle at the basis of Voronoi tessellation is that a plane can be divided into regions close to each of a given set of objects, in our case the centers of the GFP-tagged PNs. Cell centers are mathematically referred to as sites (or seeds or generators). For each site, there is a corresponding region, called a Voronoi cell (polygon), consisting of all points of the plane closer to that seed than to any other. In our case, PNs lay on a Euclidean plane (2D) and their centers form a discrete set of points. The marginal polygons must be excluded from analysis because one or two (when in the corners of the microscope image) of their sides are indeed extending to the infinite (i.e. they are OPEN polygons) as they are defined by the perimeter of the image and NOT by the existence of another site outside the microscopic field (See Fig. S3-1 in Supplementary Material 3).

In other words, the software designs these polygons only because the image is a finite portion of the space and the marginal polygons do not derive from the algorithm at the basis of the tessellation. Similarly, one can explain the need to eliminate the polygons that are intersected by the convex hull (asterisks in Fig. S3-1). The convex hull of a finite set PP of points in the plane can be defined as the unique convex polygon whose vertices are points from PP and which contains all points of PP. It can be demonstrated that a Voronoi polygon is unbounded if and only if one of its points is on the convex hull (indicated by asterisks in the figure). As a corollary, the convex hull can be computed from the Voronoi diagram in linear time [10]. Being unbounded, the polygons intersecting the convex hull do not have a finite area and thus cannot the used in the analysis. We have tried to better explain these concepts in the revised paper and Supplementary Material 3. Additionally, in Supplementary Material 3 we have used a different Voronoi generator that automatically computes the convex hull (Fig. S3-2) and demonstrated that there are no statistically significant differences with our original procedure to determine the hull (Fig. S3-3).
Verification that the initial selection of cells is accurate is not done, and in fact is explained as being negligible. “Using the mouse, click above the center of each cell to generate the Voronoi polygons. Due to the thickness of the slice, it may be possible that two very close cells in the Z axis are not easily distinguished. This introduces an error that can be neglected considering that all slices are cut at the same initial thickness.”
It isn’t clear why initial thickness of section would dictate how one would treat stacking of grown cells. The stacking isn’t uniform just because the initial slice was uniform (at least it very much appears there exists more stacking in the central mass relative to the periphery. Also, it matters entirely because those clicks define the cell sizes, which is one of the outputs for statistical comparisons.
Unfortunately, we have not been clear in explaining our point regarding the position of the GFP-tagged cells along the Z-axis of the section. We agree with the referees that stacking is not uniform, but what we signified in saying that selection errors were negligible was that if two or more cells are perfectly stacked one on top of the others we could visualize and take into consideration only the cells in the plane of focus as we used a 2D (and not a 3D) Voronoi analysis. To better clarify this point we have amended the text of the revised manuscript and added explanatory information in Supplementary Material 2.
The inability to verify which slice cells originate should be described/explained. Is it possible to use a secondary reporter to compare and contrast within co-cultures? What would it mean that you can’t verify if so?
If we understand well this comment signifies (also according to the comment above about the impossibility of verifying the origin of the cells as a consequence of a theoretical exchange of GFP from cell to cell) that we could not identify with certainty the phenotype of the GFP-tagged PNs in slices. We have substantially rebutted this possibility in responding to the previous comment. In addition, we don't see the need for a secondary reporter to compare and contrast within co-cultures. In the original paper, although not strictly necessary, we used calbindin 28k (a well-established marker of the PNs) to unambiguously prove the nature of the GFP-tagged neurons and we obtained a 100% coexistence of both tags (see e.g. Fig. 1 where the fluorescence of the positive neurons is yellow/orange for the sum of the green fluorescence of GFP and the red fluorescence of calbindin-immunostaining). This said, we believe is 100% positive that we could verify the origin of the cells in individual slices. To make this clearer, we have better explained how we have seeded the slices in the culture dish to allow for their unequivocal identification in the revised manuscript and Supplementary Material 1.
Is it true the imaging is done at the central mass? I had a difficult time understanding because there is also a section that states the coordinates images could be taken at. I’m very curious if there’s an impact of image location/bias involved. Is the origin selected by the researcher, are there methods for ensuring similarly sized clusters of cells are being imaged? Is it randomized? How many cells are removed in each?
Unfortunately, we have been unclear about these issues. We have added all the info about the above points in Supplementary Material 1 and Supplementary Material 4 (also in response to the comment below).
I found this comment particularly interesting: “It is possible to use several other Voronoi generators that can be found online as freeware or in dedicated programs.” They acknowledge the existence of other modeling systems, would be valuable to know if the results are similar., i.e. the impact of the modeling choice. Even a visual demonstration to compare the resulting polygons would be a powerful addition (to answer the obvious question about validity of Voronoi method, how important is accurate cell selection, is there an impact of user experience, etc.).
We found this comment particularly useful. We have briefly addressed this issue in the revised text and added a more detailed explanation in Supplementary Material 4.
Data interpretation: I think my biggest concern is lack of description of limitations/how perceivable limitations might be overcome
We have now described the limitations of the techniques in the main text and Supplementary Materials.
and secondary, that the discussion and conclusion only includes the word Reelin once, in the first sentence…otherwise the paper is about animal welfare? If the focus is going to be about how to reduce animal sizes, more information should be provided about litter sizes, culling, survival of mutants, etc. and how litter survival might be a limitation for studying other proteins using this method (or not). More information would be required about how many cultures can be derived from a single animal (i.e. size of slice versus size of structure at age of collection, etc.) Either it’s about Reelin, which I think it should be, and I think is interesting, or it’s a methods paper, in which case I think it would be much more valuable to show positive results in at least 2 proteins of interest in a way that allows for comparing the cultures to really show the applications of this technique. I think it might be limited to studying proteins with a very specific subset of functions, so maybe choosing another protein with a vastly different function of Reelin would be a good choice to really highlight the value of this technique. Instead, I think re-writing the discussion and conclusion to focus on results about Reelin would be more appropriate. What can the authors infer about the possibility of Reelin application to modify Reelin-mediated deficits? What can the authors infer about Reelin’s function in regulating cell morphology, beyond simple layering/migration results? Are these results consistent with the in vivo known functions of Reelin? What does this mean? Would you expect there would be differences in non-Purkinje cells? What other sets of cells you can study with this technique? Are there basket cells, granular cells etc. in the culture that we can’t visualize? Why the cerebellum? Is there interaction between the various cells in culture (both PN to PN and PN to any other non-GFP cells)? How might you evaluate cell interactions using this model in the future? What other directions would you go? Just so much to talk about and instead we were told we can save an undetermined, unspecified number of animals…but to learn what? Instead of providing 2 highly specific proteins that could be studied with this method, why not provide a sweeping generalized set of proteins you might have interest in studying with this technique? Usually, the issue is people over-exclaim their success, but in this case, I think not enough effort is given to really dive into what this study indicates about Reelin or what else can be done with the method. If this method is so great, then there should be no shortage of things to tell the reader that you learned about Reelin in applying the technique, instead of how many fewer animals might be needed in future hypothetical studies.
According to the comments above we have substantially rewritten the discussion that has now a section in which we take into consideration the significance of this study in terms of Reelin biology.
Statistics: Only issue with stats described earlier: pertaining to non-randomized imaging location (coordinates in the dish essentially) leads to selection bias. Alternative suggestions would include: a) create a grid and take an average of all polygons in multiple grid boxes to get a variety of polygon origins within the analysis. Additionally, it would be valuable to demonstrate whether or not peripheral polygons are truly similar to the central mass-related polygons, in other words, a within-culture analysis would also be valuable (is there more or less homogeneity of cultures from Reelin homo- or heterozygous mice?). Combining a perceived selection bias with unconfirmed cell selection, the reader might reasonably be concerned the results aren’t the most meaningful or generalizable.
We have clarified these points in response to some of the previous comments in particular in Supplementary Materials 1-4. We have also added a within-culture analysis by the use of several GIS tools.

References (for both responses to reviewers)

1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Weaknesses:
General: A bit strange that the conclusion is drawn pertaining to potential applications of cell cultures in general (I say that because although they’re not intended, it seems to speak to most forms of cell culture rather than specific to this technique). If this is a ground-breaking technique, it might be more valuable to show that it can be applied to more than one protein. Many proteins relate to cell survival, regulation of signaling, etc. as opposed to cell migration, so it’s a bit difficult to understand how this technique would benefit the study of other aspects of cell function. I suppose part of the issue for me is the absence of discussion of limitations to the model, so as I am imagining all sorts of limitations without the authors discussion or recognition of them, I’m left not understanding if/how the limitations would be explained.
We thank the reviewers for these very useful observations. We have revised the paper by adding a thorough discussion of the limitations of the technique and trying to avoid a too strong generalization of its applicability.
Some other limitations include: Can you conclude that application of Reelin would resolve the layering deficits in homozygous Reelers through this model? I’m not understanding how the authors conclude cells originating from the homozygous slice show improved organization in co-cultures, as opposed to showing that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing altered organization because you a) can’t verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells); b) there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells. Maybe it wasn’t clear enough how co-cultures are grown together to allow for differentiation.
We are sorry that we have not been able to clarify these issues in the original version of the paper. We believe that, from our observations, we can indeed conclude that the reelin secreted from the cerebellar granule cells in the slices from heterozygous mice (reelin haplodeficient) has a positive effect on the organization of the cerebellar cortex in the slices obtained from homozygous mice (lacking reelin).
Our belief is based on the following:

Faulty Reelin signaling in the cerebellum causes Purkinje neurons (PN) migration to be disrupted and, as a result, granule cell precursor (GCPs) proliferation to be diminished – for a recent review see [1]. Morphological observations in vivo have long ago demonstrated that the Reeler mouse, which is devoid of Reelin, displays an altered stratification of its cerebellar and cerebral cortices, as well as of other layered areas of the brain. Specifically in the cerebellum, these alterations affect the migration and correct positioning of the PNs that, in the homozygous reln^-/- mouse, remain clustered together in a deep cellular mass embedded into the medullary body of the white matter [2]. Conversely, in reln^+/-and reln^+/+ mice the PNs migrate in an outward direction to eventually form a monolayer of cells between the molecular and granule cell layer of the mature cerebellar cortex, i.e. both genotypes display a normal cerebellar architecture, although we have recently demonstrated that there are very subtle differences in the position of the PNs between reln^+/-and reln^+/+mice [3]. The effects of the PNs on the GCP proliferation are mediated by Sonic Hedgehog (reviewed in [4]) and we have subsequently demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to a reduction of cortical size and cellularity, the latter being linked to quantitative differences in the extent of GCP proliferation and apoptosis between normal and Reeler mice [5]. The interactions between the PNs and the GCPs are thus quite complex: from one side the PNs stimulate the proliferation of GCPs and, from the other, the CGPs produce and release Reelin in the intercellular space, and the glycoprotein is essential for the correct positioning of the PNs.

If we understood well the reviewers’ thoughts, they suggest that we cannot claim that “the cells originating from the homozygous slice show improved organization in co-cultures” as it is equally possible “that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing an altered organization” but this in total conflict with the above-reported literature.

In addition, the reviewers believe that we are overinterpreting our findings because:

We could not “verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells”. However, we never suggested that there was a migration of PNs from one slice to the other, and there are no pieces of evidence from previous studies that GFP is exchanged between cells in the L7 mouse. The PN-specific gene coding L7 or pcp2 (Purkinje cells specific protein-2) was first described in 1988 by two independent groups [6, 7], and since then it has been of paramount importance for genetic targeting of these neurons [8]. Anyway, even if one assumes that the GFP can be transferred between cerebellar neurons, the protein should preferentially reach the neurons of the cerebellar nuclei which are in contact with the PN axons, as the retrograde transsynaptic transfer occurs only when a fusion protein with a non-toxic fragment of tetanus toxin is used for genetic engineering/transfection, see e.g. [9].

“there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells”. Although Occam’s razor is based on the idea that the simplest explanation is often the best one, we don’t see how the reviewers may conclude that we are simply comparing heterozygous (PN) cells between each other, since they derive from genotyped animals and no exchange of PNs occurs among slices. On the contrary, the simplest explanation of our findings is indeed the one put forward in the original paper.

Yet, we fully agree with the referees that “it wasn’t clear enough how co-cultures are grown together to allow for differentiation” and we have amended the manuscript by better explaining how co-cultures were established and how they could influence each other by adding some supplementary information to the paper (Supplementary Material 1).
General comments on:
Methods: Good overall. My only concerns: Polygon selection and removal of the top polygon, which corresponds to the culmination of data (or all accounted for polygons). I’m not understanding why that information wouldn’t be valuable for comparing sets of polygons (I understand it’s included de facto in the sum of all selected polygons) but it would be good to show the amalgamations are similar across cultures (i.e. I would expect less stacking/layering of cells occurs in smaller montages of polygons compared to bigger ones corresponding to larger numbers of cells, which would impact nearest neighbour analyses etc.).
The principle at the basis of Voronoi tessellation is that a plane can be divided into regions close to each of a given set of objects, in our case the centers of the GFP-tagged PNs. Cell centers are mathematically referred to as sites (or seeds or generators). For each site, there is a corresponding region, called a Voronoi cell (polygon), consisting of all points of the plane closer to that seed than to any other. In our case, PNs lay on a Euclidean plane (2D) and their centers form a discrete set of points. The marginal polygons must be excluded from analysis because one or two (when in the corners of the microscope image) of their sides are indeed extending to the infinite (i.e. they are OPEN polygons) as they are defined by the perimeter of the image and NOT by the existence of another site outside the microscopic field (See Fig. S3-1 in Supplementary Material 3).

In other words, the software designs these polygons only because the image is a finite portion of the space and the marginal polygons do not derive from the algorithm at the basis of the tessellation. Similarly, one can explain the need to eliminate the polygons that are intersected by the convex hull (asterisks in Fig. S3-1). The convex hull of a finite set PP of points in the plane can be defined as the unique convex polygon whose vertices are points from PP and which contains all points of PP. It can be demonstrated that a Voronoi polygon is unbounded if and only if one of its points is on the convex hull (indicated by asterisks in the figure). As a corollary, the convex hull can be computed from the Voronoi diagram in linear time [10]. Being unbounded, the polygons intersecting the convex hull do not have a finite area and thus cannot the used in the analysis. We have tried to better explain these concepts in the revised paper and Supplementary Material 3. Additionally, in Supplementary Material 3 we have used a different Voronoi generator that automatically computes the convex hull (Fig. S3-2) and demonstrated that there are no statistically significant differences with our original procedure to determine the hull (Fig. S3-3).
Verification that the initial selection of cells is accurate is not done, and in fact is explained as being negligible. “Using the mouse, click above the center of each cell to generate the Voronoi polygons. Due to the thickness of the slice, it may be possible that two very close cells in the Z axis are not easily distinguished. This introduces an error that can be neglected considering that all slices are cut at the same initial thickness.”
It isn’t clear why initial thickness of section would dictate how one would treat stacking of grown cells. The stacking isn’t uniform just because the initial slice was uniform (at least it very much appears there exists more stacking in the central mass relative to the periphery. Also, it matters entirely because those clicks define the cell sizes, which is one of the outputs for statistical comparisons.
Unfortunately, we have not been clear in explaining our point regarding the position of the GFP-tagged cells along the Z-axis of the section. We agree with the referees that stacking is not uniform, but what we signified in saying that selection errors were negligible was that if two or more cells are perfectly stacked one on top of the others we could visualize and take into consideration only the cells in the plane of focus as we used a 2D (and not a 3D) Voronoi analysis. To better clarify this point we have amended the text of the revised manuscript and added explanatory information in Supplementary Material 2.
The inability to verify which slice cells originate should be described/explained. Is it possible to use a secondary reporter to compare and contrast within co-cultures? What would it mean that you can’t verify if so?
If we understand well this comment signifies (also according to the comment above about the impossibility of verifying the origin of the cells as a consequence of a theoretical exchange of GFP from cell to cell) that we could not identify with certainty the phenotype of the GFP-tagged PNs in slices. We have substantially rebutted this possibility in responding to the previous comment. In addition, we don't see the need for a secondary reporter to compare and contrast within co-cultures. In the original paper, although not strictly necessary, we used calbindin 28k (a well-established marker of the PNs) to unambiguously prove the nature of the GFP-tagged neurons and we obtained a 100% coexistence of both tags (see e.g. Fig. 1 where the fluorescence of the positive neurons is yellow/orange for the sum of the green fluorescence of GFP and the red fluorescence of calbindin-immunostaining). This said, we believe is 100% positive that we could verify the origin of the cells in individual slices. To make this clearer, we have better explained how we have seeded the slices in the culture dish to allow for their unequivocal identification in the revised manuscript and Supplementary Material 1.
Is it true the imaging is done at the central mass? I had a difficult time understanding because there is also a section that states the coordinates images could be taken at. I’m very curious if there’s an impact of image location/bias involved. Is the origin selected by the researcher, are there methods for ensuring similarly sized clusters of cells are being imaged? Is it randomized? How many cells are removed in each?
Unfortunately, we have been unclear about these issues. We have added all the info about the above points in Supplementary Material 1 and Supplementary Material 4 (also in response to the comment below).
I found this comment particularly interesting: “It is possible to use several other Voronoi generators that can be found online as freeware or in dedicated programs.” They acknowledge the existence of other modeling systems, would be valuable to know if the results are similar., i.e. the impact of the modeling choice. Even a visual demonstration to compare the resulting polygons would be a powerful addition (to answer the obvious question about validity of Voronoi method, how important is accurate cell selection, is there an impact of user experience, etc.).
We found this comment particularly useful. We have briefly addressed this issue in the revised text and added a more detailed explanation in Supplementary Material 4.
Data interpretation: I think my biggest concern is lack of description of limitations/how perceivable limitations might be overcome
We have now described the limitations of the techniques in the main text and Supplementary Materials.
and secondary, that the discussion and conclusion only includes the word Reelin once, in the first sentence…otherwise the paper is about animal welfare? If the focus is going to be about how to reduce animal sizes, more information should be provided about litter sizes, culling, survival of mutants, etc. and how litter survival might be a limitation for studying other proteins using this method (or not). More information would be required about how many cultures can be derived from a single animal (i.e. size of slice versus size of structure at age of collection, etc.) Either it’s about Reelin, which I think it should be, and I think is interesting, or it’s a methods paper, in which case I think it would be much more valuable to show positive results in at least 2 proteins of interest in a way that allows for comparing the cultures to really show the applications of this technique. I think it might be limited to studying proteins with a very specific subset of functions, so maybe choosing another protein with a vastly different function of Reelin would be a good choice to really highlight the value of this technique. Instead, I think re-writing the discussion and conclusion to focus on results about Reelin would be more appropriate. What can the authors infer about the possibility of Reelin application to modify Reelin-mediated deficits? What can the authors infer about Reelin’s function in regulating cell morphology, beyond simple layering/migration results? Are these results consistent with the in vivo known functions of Reelin? What does this mean? Would you expect there would be differences in non-Purkinje cells? What other sets of cells you can study with this technique? Are there basket cells, granular cells etc. in the culture that we can’t visualize? Why the cerebellum? Is there interaction between the various cells in culture (both PN to PN and PN to any other non-GFP cells)? How might you evaluate cell interactions using this model in the future? What other directions would you go? Just so much to talk about and instead we were told we can save an undetermined, unspecified number of animals…but to learn what? Instead of providing 2 highly specific proteins that could be studied with this method, why not provide a sweeping generalized set of proteins you might have interest in studying with this technique? Usually, the issue is people over-exclaim their success, but in this case, I think not enough effort is given to really dive into what this study indicates about Reelin or what else can be done with the method. If this method is so great, then there should be no shortage of things to tell the reader that you learned about Reelin in applying the technique, instead of how many fewer animals might be needed in future hypothetical studies.
According to the comments above we have substantially rewritten the discussion that has now a section in which we take into consideration the significance of this study in terms of Reelin biology.
Statistics: Only issue with stats described earlier: pertaining to non-randomized imaging location (coordinates in the dish essentially) leads to selection bias. Alternative suggestions would include: a) create a grid and take an average of all polygons in multiple grid boxes to get a variety of polygon origins within the analysis. Additionally, it would be valuable to demonstrate whether or not peripheral polygons are truly similar to the central mass-related polygons, in other words, a within-culture analysis would also be valuable (is there more or less homogeneity of cultures from Reelin homo- or heterozygous mice?). Combining a perceived selection bias with unconfirmed cell selection, the reader might reasonably be concerned the results aren’t the most meaningful or generalizable.
We have clarified these points in response to some of the previous comments in particular in Supplementary Materials 1-4. We have also added a within-culture analysis by the use of several GIS tools.

References (for both responses to reviewers)

1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 06 Oct 2023

Adalberto Merighi, Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

06 Oct 2023

Author Response
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history ... Continue reading
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Weaknesses:
General: A bit strange that the conclusion is drawn pertaining to potential applications of cell cultures in general (I say that because although they’re not intended, it seems to speak to most forms of cell culture rather than specific to this technique). If this is a ground-breaking technique, it might be more valuable to show that it can be applied to more than one protein. Many proteins relate to cell survival, regulation of signaling, etc. as opposed to cell migration, so it’s a bit difficult to understand how this technique would benefit the study of other aspects of cell function. I suppose part of the issue for me is the absence of discussion of limitations to the model, so as I am imagining all sorts of limitations without the authors discussion or recognition of them, I’m left not understanding if/how the limitations would be explained.
We thank the reviewers for these very useful observations. We have revised the paper by adding a thorough discussion of the limitations of the technique and trying to avoid a too strong generalization of its applicability.
Some other limitations include: Can you conclude that application of Reelin would resolve the layering deficits in homozygous Reelers through this model? I’m not understanding how the authors conclude cells originating from the homozygous slice show improved organization in co-cultures, as opposed to showing that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing altered organization because you a) can’t verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells); b) there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells. Maybe it wasn’t clear enough how co-cultures are grown together to allow for differentiation.
We are sorry that we have not been able to clarify these issues in the original version of the paper. We believe that, from our observations, we can indeed conclude that the reelin secreted from the cerebellar granule cells in the slices from heterozygous mice (reelin haplodeficient) has a positive effect on the organization of the cerebellar cortex in the slices obtained from homozygous mice (lacking reelin).
Our belief is based on the following:

Faulty Reelin signaling in the cerebellum causes Purkinje neurons (PN) migration to be disrupted and, as a result, granule cell precursor (GCPs) proliferation to be diminished – for a recent review see [1]. Morphological observations in vivo have long ago demonstrated that the Reeler mouse, which is devoid of Reelin, displays an altered stratification of its cerebellar and cerebral cortices, as well as of other layered areas of the brain. Specifically in the cerebellum, these alterations affect the migration and correct positioning of the PNs that, in the homozygous reln^-/- mouse, remain clustered together in a deep cellular mass embedded into the medullary body of the white matter [2]. Conversely, in reln^+/-and reln^+/+ mice the PNs migrate in an outward direction to eventually form a monolayer of cells between the molecular and granule cell layer of the mature cerebellar cortex, i.e. both genotypes display a normal cerebellar architecture, although we have recently demonstrated that there are very subtle differences in the position of the PNs between reln^+/-and reln^+/+mice [3]. The effects of the PNs on the GCP proliferation are mediated by Sonic Hedgehog (reviewed in [4]) and we have subsequently demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to a reduction of cortical size and cellularity, the latter being linked to quantitative differences in the extent of GCP proliferation and apoptosis between normal and Reeler mice [5]. The interactions between the PNs and the GCPs are thus quite complex: from one side the PNs stimulate the proliferation of GCPs and, from the other, the CGPs produce and release Reelin in the intercellular space, and the glycoprotein is essential for the correct positioning of the PNs.

If we understood well the reviewers’ thoughts, they suggest that we cannot claim that “the cells originating from the homozygous slice show improved organization in co-cultures” as it is equally possible “that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing an altered organization” but this in total conflict with the above-reported literature.

In addition, the reviewers believe that we are overinterpreting our findings because:

We could not “verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells”. However, we never suggested that there was a migration of PNs from one slice to the other, and there are no pieces of evidence from previous studies that GFP is exchanged between cells in the L7 mouse. The PN-specific gene coding L7 or pcp2 (Purkinje cells specific protein-2) was first described in 1988 by two independent groups [6, 7], and since then it has been of paramount importance for genetic targeting of these neurons [8]. Anyway, even if one assumes that the GFP can be transferred between cerebellar neurons, the protein should preferentially reach the neurons of the cerebellar nuclei which are in contact with the PN axons, as the retrograde transsynaptic transfer occurs only when a fusion protein with a non-toxic fragment of tetanus toxin is used for genetic engineering/transfection, see e.g. [9].

“there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells”. Although Occam’s razor is based on the idea that the simplest explanation is often the best one, we don’t see how the reviewers may conclude that we are simply comparing heterozygous (PN) cells between each other, since they derive from genotyped animals and no exchange of PNs occurs among slices. On the contrary, the simplest explanation of our findings is indeed the one put forward in the original paper.

Yet, we fully agree with the referees that “it wasn’t clear enough how co-cultures are grown together to allow for differentiation” and we have amended the manuscript by better explaining how co-cultures were established and how they could influence each other by adding some supplementary information to the paper (Supplementary Material 1).
General comments on:
Methods: Good overall. My only concerns: Polygon selection and removal of the top polygon, which corresponds to the culmination of data (or all accounted for polygons). I’m not understanding why that information wouldn’t be valuable for comparing sets of polygons (I understand it’s included de facto in the sum of all selected polygons) but it would be good to show the amalgamations are similar across cultures (i.e. I would expect less stacking/layering of cells occurs in smaller montages of polygons compared to bigger ones corresponding to larger numbers of cells, which would impact nearest neighbour analyses etc.).
The principle at the basis of Voronoi tessellation is that a plane can be divided into regions close to each of a given set of objects, in our case the centers of the GFP-tagged PNs. Cell centers are mathematically referred to as sites (or seeds or generators). For each site, there is a corresponding region, called a Voronoi cell (polygon), consisting of all points of the plane closer to that seed than to any other. In our case, PNs lay on a Euclidean plane (2D) and their centers form a discrete set of points. The marginal polygons must be excluded from analysis because one or two (when in the corners of the microscope image) of their sides are indeed extending to the infinite (i.e. they are OPEN polygons) as they are defined by the perimeter of the image and NOT by the existence of another site outside the microscopic field (See Fig. S3-1 in Supplementary Material 3).

In other words, the software designs these polygons only because the image is a finite portion of the space and the marginal polygons do not derive from the algorithm at the basis of the tessellation. Similarly, one can explain the need to eliminate the polygons that are intersected by the convex hull (asterisks in Fig. S3-1). The convex hull of a finite set PP of points in the plane can be defined as the unique convex polygon whose vertices are points from PP and which contains all points of PP. It can be demonstrated that a Voronoi polygon is unbounded if and only if one of its points is on the convex hull (indicated by asterisks in the figure). As a corollary, the convex hull can be computed from the Voronoi diagram in linear time [10]. Being unbounded, the polygons intersecting the convex hull do not have a finite area and thus cannot the used in the analysis. We have tried to better explain these concepts in the revised paper and Supplementary Material 3. Additionally, in Supplementary Material 3 we have used a different Voronoi generator that automatically computes the convex hull (Fig. S3-2) and demonstrated that there are no statistically significant differences with our original procedure to determine the hull (Fig. S3-3).
Verification that the initial selection of cells is accurate is not done, and in fact is explained as being negligible. “Using the mouse, click above the center of each cell to generate the Voronoi polygons. Due to the thickness of the slice, it may be possible that two very close cells in the Z axis are not easily distinguished. This introduces an error that can be neglected considering that all slices are cut at the same initial thickness.”
It isn’t clear why initial thickness of section would dictate how one would treat stacking of grown cells. The stacking isn’t uniform just because the initial slice was uniform (at least it very much appears there exists more stacking in the central mass relative to the periphery. Also, it matters entirely because those clicks define the cell sizes, which is one of the outputs for statistical comparisons.
Unfortunately, we have not been clear in explaining our point regarding the position of the GFP-tagged cells along the Z-axis of the section. We agree with the referees that stacking is not uniform, but what we signified in saying that selection errors were negligible was that if two or more cells are perfectly stacked one on top of the others we could visualize and take into consideration only the cells in the plane of focus as we used a 2D (and not a 3D) Voronoi analysis. To better clarify this point we have amended the text of the revised manuscript and added explanatory information in Supplementary Material 2.
The inability to verify which slice cells originate should be described/explained. Is it possible to use a secondary reporter to compare and contrast within co-cultures? What would it mean that you can’t verify if so?
If we understand well this comment signifies (also according to the comment above about the impossibility of verifying the origin of the cells as a consequence of a theoretical exchange of GFP from cell to cell) that we could not identify with certainty the phenotype of the GFP-tagged PNs in slices. We have substantially rebutted this possibility in responding to the previous comment. In addition, we don't see the need for a secondary reporter to compare and contrast within co-cultures. In the original paper, although not strictly necessary, we used calbindin 28k (a well-established marker of the PNs) to unambiguously prove the nature of the GFP-tagged neurons and we obtained a 100% coexistence of both tags (see e.g. Fig. 1 where the fluorescence of the positive neurons is yellow/orange for the sum of the green fluorescence of GFP and the red fluorescence of calbindin-immunostaining). This said, we believe is 100% positive that we could verify the origin of the cells in individual slices. To make this clearer, we have better explained how we have seeded the slices in the culture dish to allow for their unequivocal identification in the revised manuscript and Supplementary Material 1.
Is it true the imaging is done at the central mass? I had a difficult time understanding because there is also a section that states the coordinates images could be taken at. I’m very curious if there’s an impact of image location/bias involved. Is the origin selected by the researcher, are there methods for ensuring similarly sized clusters of cells are being imaged? Is it randomized? How many cells are removed in each?
Unfortunately, we have been unclear about these issues. We have added all the info about the above points in Supplementary Material 1 and Supplementary Material 4 (also in response to the comment below).
I found this comment particularly interesting: “It is possible to use several other Voronoi generators that can be found online as freeware or in dedicated programs.” They acknowledge the existence of other modeling systems, would be valuable to know if the results are similar., i.e. the impact of the modeling choice. Even a visual demonstration to compare the resulting polygons would be a powerful addition (to answer the obvious question about validity of Voronoi method, how important is accurate cell selection, is there an impact of user experience, etc.).
We found this comment particularly useful. We have briefly addressed this issue in the revised text and added a more detailed explanation in Supplementary Material 4.
Data interpretation: I think my biggest concern is lack of description of limitations/how perceivable limitations might be overcome
We have now described the limitations of the techniques in the main text and Supplementary Materials.
and secondary, that the discussion and conclusion only includes the word Reelin once, in the first sentence…otherwise the paper is about animal welfare? If the focus is going to be about how to reduce animal sizes, more information should be provided about litter sizes, culling, survival of mutants, etc. and how litter survival might be a limitation for studying other proteins using this method (or not). More information would be required about how many cultures can be derived from a single animal (i.e. size of slice versus size of structure at age of collection, etc.) Either it’s about Reelin, which I think it should be, and I think is interesting, or it’s a methods paper, in which case I think it would be much more valuable to show positive results in at least 2 proteins of interest in a way that allows for comparing the cultures to really show the applications of this technique. I think it might be limited to studying proteins with a very specific subset of functions, so maybe choosing another protein with a vastly different function of Reelin would be a good choice to really highlight the value of this technique. Instead, I think re-writing the discussion and conclusion to focus on results about Reelin would be more appropriate. What can the authors infer about the possibility of Reelin application to modify Reelin-mediated deficits? What can the authors infer about Reelin’s function in regulating cell morphology, beyond simple layering/migration results? Are these results consistent with the in vivo known functions of Reelin? What does this mean? Would you expect there would be differences in non-Purkinje cells? What other sets of cells you can study with this technique? Are there basket cells, granular cells etc. in the culture that we can’t visualize? Why the cerebellum? Is there interaction between the various cells in culture (both PN to PN and PN to any other non-GFP cells)? How might you evaluate cell interactions using this model in the future? What other directions would you go? Just so much to talk about and instead we were told we can save an undetermined, unspecified number of animals…but to learn what? Instead of providing 2 highly specific proteins that could be studied with this method, why not provide a sweeping generalized set of proteins you might have interest in studying with this technique? Usually, the issue is people over-exclaim their success, but in this case, I think not enough effort is given to really dive into what this study indicates about Reelin or what else can be done with the method. If this method is so great, then there should be no shortage of things to tell the reader that you learned about Reelin in applying the technique, instead of how many fewer animals might be needed in future hypothetical studies.
According to the comments above we have substantially rewritten the discussion that has now a section in which we take into consideration the significance of this study in terms of Reelin biology.
Statistics: Only issue with stats described earlier: pertaining to non-randomized imaging location (coordinates in the dish essentially) leads to selection bias. Alternative suggestions would include: a) create a grid and take an average of all polygons in multiple grid boxes to get a variety of polygon origins within the analysis. Additionally, it would be valuable to demonstrate whether or not peripheral polygons are truly similar to the central mass-related polygons, in other words, a within-culture analysis would also be valuable (is there more or less homogeneity of cultures from Reelin homo- or heterozygous mice?). Combining a perceived selection bias with unconfirmed cell selection, the reader might reasonably be concerned the results aren’t the most meaningful or generalizable.
We have clarified these points in response to some of the previous comments in particular in Supplementary Materials 1-4. We have also added a within-culture analysis by the use of several GIS tools.

References (for both responses to reviewers)

1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Weaknesses:
General: A bit strange that the conclusion is drawn pertaining to potential applications of cell cultures in general (I say that because although they’re not intended, it seems to speak to most forms of cell culture rather than specific to this technique). If this is a ground-breaking technique, it might be more valuable to show that it can be applied to more than one protein. Many proteins relate to cell survival, regulation of signaling, etc. as opposed to cell migration, so it’s a bit difficult to understand how this technique would benefit the study of other aspects of cell function. I suppose part of the issue for me is the absence of discussion of limitations to the model, so as I am imagining all sorts of limitations without the authors discussion or recognition of them, I’m left not understanding if/how the limitations would be explained.
We thank the reviewers for these very useful observations. We have revised the paper by adding a thorough discussion of the limitations of the technique and trying to avoid a too strong generalization of its applicability.
Some other limitations include: Can you conclude that application of Reelin would resolve the layering deficits in homozygous Reelers through this model? I’m not understanding how the authors conclude cells originating from the homozygous slice show improved organization in co-cultures, as opposed to showing that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing altered organization because you a) can’t verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells); b) there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells. Maybe it wasn’t clear enough how co-cultures are grown together to allow for differentiation.
We are sorry that we have not been able to clarify these issues in the original version of the paper. We believe that, from our observations, we can indeed conclude that the reelin secreted from the cerebellar granule cells in the slices from heterozygous mice (reelin haplodeficient) has a positive effect on the organization of the cerebellar cortex in the slices obtained from homozygous mice (lacking reelin).
Our belief is based on the following:

Faulty Reelin signaling in the cerebellum causes Purkinje neurons (PN) migration to be disrupted and, as a result, granule cell precursor (GCPs) proliferation to be diminished – for a recent review see [1]. Morphological observations in vivo have long ago demonstrated that the Reeler mouse, which is devoid of Reelin, displays an altered stratification of its cerebellar and cerebral cortices, as well as of other layered areas of the brain. Specifically in the cerebellum, these alterations affect the migration and correct positioning of the PNs that, in the homozygous reln^-/- mouse, remain clustered together in a deep cellular mass embedded into the medullary body of the white matter [2]. Conversely, in reln^+/-and reln^+/+ mice the PNs migrate in an outward direction to eventually form a monolayer of cells between the molecular and granule cell layer of the mature cerebellar cortex, i.e. both genotypes display a normal cerebellar architecture, although we have recently demonstrated that there are very subtle differences in the position of the PNs between reln^+/-and reln^+/+mice [3]. The effects of the PNs on the GCP proliferation are mediated by Sonic Hedgehog (reviewed in [4]) and we have subsequently demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to a reduction of cortical size and cellularity, the latter being linked to quantitative differences in the extent of GCP proliferation and apoptosis between normal and Reeler mice [5]. The interactions between the PNs and the GCPs are thus quite complex: from one side the PNs stimulate the proliferation of GCPs and, from the other, the CGPs produce and release Reelin in the intercellular space, and the glycoprotein is essential for the correct positioning of the PNs.

If we understood well the reviewers’ thoughts, they suggest that we cannot claim that “the cells originating from the homozygous slice show improved organization in co-cultures” as it is equally possible “that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing an altered organization” but this in total conflict with the above-reported literature.

In addition, the reviewers believe that we are overinterpreting our findings because:

We could not “verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells”. However, we never suggested that there was a migration of PNs from one slice to the other, and there are no pieces of evidence from previous studies that GFP is exchanged between cells in the L7 mouse. The PN-specific gene coding L7 or pcp2 (Purkinje cells specific protein-2) was first described in 1988 by two independent groups [6, 7], and since then it has been of paramount importance for genetic targeting of these neurons [8]. Anyway, even if one assumes that the GFP can be transferred between cerebellar neurons, the protein should preferentially reach the neurons of the cerebellar nuclei which are in contact with the PN axons, as the retrograde transsynaptic transfer occurs only when a fusion protein with a non-toxic fragment of tetanus toxin is used for genetic engineering/transfection, see e.g. [9].

“there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells”. Although Occam’s razor is based on the idea that the simplest explanation is often the best one, we don’t see how the reviewers may conclude that we are simply comparing heterozygous (PN) cells between each other, since they derive from genotyped animals and no exchange of PNs occurs among slices. On the contrary, the simplest explanation of our findings is indeed the one put forward in the original paper.

Yet, we fully agree with the referees that “it wasn’t clear enough how co-cultures are grown together to allow for differentiation” and we have amended the manuscript by better explaining how co-cultures were established and how they could influence each other by adding some supplementary information to the paper (Supplementary Material 1).
General comments on:
Methods: Good overall. My only concerns: Polygon selection and removal of the top polygon, which corresponds to the culmination of data (or all accounted for polygons). I’m not understanding why that information wouldn’t be valuable for comparing sets of polygons (I understand it’s included de facto in the sum of all selected polygons) but it would be good to show the amalgamations are similar across cultures (i.e. I would expect less stacking/layering of cells occurs in smaller montages of polygons compared to bigger ones corresponding to larger numbers of cells, which would impact nearest neighbour analyses etc.).
The principle at the basis of Voronoi tessellation is that a plane can be divided into regions close to each of a given set of objects, in our case the centers of the GFP-tagged PNs. Cell centers are mathematically referred to as sites (or seeds or generators). For each site, there is a corresponding region, called a Voronoi cell (polygon), consisting of all points of the plane closer to that seed than to any other. In our case, PNs lay on a Euclidean plane (2D) and their centers form a discrete set of points. The marginal polygons must be excluded from analysis because one or two (when in the corners of the microscope image) of their sides are indeed extending to the infinite (i.e. they are OPEN polygons) as they are defined by the perimeter of the image and NOT by the existence of another site outside the microscopic field (See Fig. S3-1 in Supplementary Material 3).

In other words, the software designs these polygons only because the image is a finite portion of the space and the marginal polygons do not derive from the algorithm at the basis of the tessellation. Similarly, one can explain the need to eliminate the polygons that are intersected by the convex hull (asterisks in Fig. S3-1). The convex hull of a finite set PP of points in the plane can be defined as the unique convex polygon whose vertices are points from PP and which contains all points of PP. It can be demonstrated that a Voronoi polygon is unbounded if and only if one of its points is on the convex hull (indicated by asterisks in the figure). As a corollary, the convex hull can be computed from the Voronoi diagram in linear time [10]. Being unbounded, the polygons intersecting the convex hull do not have a finite area and thus cannot the used in the analysis. We have tried to better explain these concepts in the revised paper and Supplementary Material 3. Additionally, in Supplementary Material 3 we have used a different Voronoi generator that automatically computes the convex hull (Fig. S3-2) and demonstrated that there are no statistically significant differences with our original procedure to determine the hull (Fig. S3-3).
Verification that the initial selection of cells is accurate is not done, and in fact is explained as being negligible. “Using the mouse, click above the center of each cell to generate the Voronoi polygons. Due to the thickness of the slice, it may be possible that two very close cells in the Z axis are not easily distinguished. This introduces an error that can be neglected considering that all slices are cut at the same initial thickness.”
It isn’t clear why initial thickness of section would dictate how one would treat stacking of grown cells. The stacking isn’t uniform just because the initial slice was uniform (at least it very much appears there exists more stacking in the central mass relative to the periphery. Also, it matters entirely because those clicks define the cell sizes, which is one of the outputs for statistical comparisons.
Unfortunately, we have not been clear in explaining our point regarding the position of the GFP-tagged cells along the Z-axis of the section. We agree with the referees that stacking is not uniform, but what we signified in saying that selection errors were negligible was that if two or more cells are perfectly stacked one on top of the others we could visualize and take into consideration only the cells in the plane of focus as we used a 2D (and not a 3D) Voronoi analysis. To better clarify this point we have amended the text of the revised manuscript and added explanatory information in Supplementary Material 2.
The inability to verify which slice cells originate should be described/explained. Is it possible to use a secondary reporter to compare and contrast within co-cultures? What would it mean that you can’t verify if so?
If we understand well this comment signifies (also according to the comment above about the impossibility of verifying the origin of the cells as a consequence of a theoretical exchange of GFP from cell to cell) that we could not identify with certainty the phenotype of the GFP-tagged PNs in slices. We have substantially rebutted this possibility in responding to the previous comment. In addition, we don't see the need for a secondary reporter to compare and contrast within co-cultures. In the original paper, although not strictly necessary, we used calbindin 28k (a well-established marker of the PNs) to unambiguously prove the nature of the GFP-tagged neurons and we obtained a 100% coexistence of both tags (see e.g. Fig. 1 where the fluorescence of the positive neurons is yellow/orange for the sum of the green fluorescence of GFP and the red fluorescence of calbindin-immunostaining). This said, we believe is 100% positive that we could verify the origin of the cells in individual slices. To make this clearer, we have better explained how we have seeded the slices in the culture dish to allow for their unequivocal identification in the revised manuscript and Supplementary Material 1.
Is it true the imaging is done at the central mass? I had a difficult time understanding because there is also a section that states the coordinates images could be taken at. I’m very curious if there’s an impact of image location/bias involved. Is the origin selected by the researcher, are there methods for ensuring similarly sized clusters of cells are being imaged? Is it randomized? How many cells are removed in each?
Unfortunately, we have been unclear about these issues. We have added all the info about the above points in Supplementary Material 1 and Supplementary Material 4 (also in response to the comment below).
I found this comment particularly interesting: “It is possible to use several other Voronoi generators that can be found online as freeware or in dedicated programs.” They acknowledge the existence of other modeling systems, would be valuable to know if the results are similar., i.e. the impact of the modeling choice. Even a visual demonstration to compare the resulting polygons would be a powerful addition (to answer the obvious question about validity of Voronoi method, how important is accurate cell selection, is there an impact of user experience, etc.).
We found this comment particularly useful. We have briefly addressed this issue in the revised text and added a more detailed explanation in Supplementary Material 4.
Data interpretation: I think my biggest concern is lack of description of limitations/how perceivable limitations might be overcome
We have now described the limitations of the techniques in the main text and Supplementary Materials.
and secondary, that the discussion and conclusion only includes the word Reelin once, in the first sentence…otherwise the paper is about animal welfare? If the focus is going to be about how to reduce animal sizes, more information should be provided about litter sizes, culling, survival of mutants, etc. and how litter survival might be a limitation for studying other proteins using this method (or not). More information would be required about how many cultures can be derived from a single animal (i.e. size of slice versus size of structure at age of collection, etc.) Either it’s about Reelin, which I think it should be, and I think is interesting, or it’s a methods paper, in which case I think it would be much more valuable to show positive results in at least 2 proteins of interest in a way that allows for comparing the cultures to really show the applications of this technique. I think it might be limited to studying proteins with a very specific subset of functions, so maybe choosing another protein with a vastly different function of Reelin would be a good choice to really highlight the value of this technique. Instead, I think re-writing the discussion and conclusion to focus on results about Reelin would be more appropriate. What can the authors infer about the possibility of Reelin application to modify Reelin-mediated deficits? What can the authors infer about Reelin’s function in regulating cell morphology, beyond simple layering/migration results? Are these results consistent with the in vivo known functions of Reelin? What does this mean? Would you expect there would be differences in non-Purkinje cells? What other sets of cells you can study with this technique? Are there basket cells, granular cells etc. in the culture that we can’t visualize? Why the cerebellum? Is there interaction between the various cells in culture (both PN to PN and PN to any other non-GFP cells)? How might you evaluate cell interactions using this model in the future? What other directions would you go? Just so much to talk about and instead we were told we can save an undetermined, unspecified number of animals…but to learn what? Instead of providing 2 highly specific proteins that could be studied with this method, why not provide a sweeping generalized set of proteins you might have interest in studying with this technique? Usually, the issue is people over-exclaim their success, but in this case, I think not enough effort is given to really dive into what this study indicates about Reelin or what else can be done with the method. If this method is so great, then there should be no shortage of things to tell the reader that you learned about Reelin in applying the technique, instead of how many fewer animals might be needed in future hypothetical studies.
According to the comments above we have substantially rewritten the discussion that has now a section in which we take into consideration the significance of this study in terms of Reelin biology.
Statistics: Only issue with stats described earlier: pertaining to non-randomized imaging location (coordinates in the dish essentially) leads to selection bias. Alternative suggestions would include: a) create a grid and take an average of all polygons in multiple grid boxes to get a variety of polygon origins within the analysis. Additionally, it would be valuable to demonstrate whether or not peripheral polygons are truly similar to the central mass-related polygons, in other words, a within-culture analysis would also be valuable (is there more or less homogeneity of cultures from Reelin homo- or heterozygous mice?). Combining a perceived selection bias with unconfirmed cell selection, the reader might reasonably be concerned the results aren’t the most meaningful or generalizable.
We have clarified these points in response to some of the previous comments in particular in Supplementary Materials 1-4. We have also added a within-culture analysis by the use of several GIS tools.

References (for both responses to reviewers)

1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253
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Hector J. Caruncho, University of Victoria, Victoria, Canada

Brady Reive, University of Victoria, Victoria, Canada
Pascale Chavis, Aix-Marseille University, Marseille, France

Thomas Boudier, Aix-Marseille University, Marseille, France

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The authors have made appropriate revisions and have provided comprehensive responses to all of our concerns.

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Pascale Chavis: Brain plasticity, Reelin neurobiology, Neuropsychiatric diseases. Thomas Boudier: Image processing, Image analysis.

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Hector J. Caruncho, Division of Medical Sciences, University of Victoria, Victoria, BC, Canada

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I consider the revisions made by the authors as appropriate and recommend the article be considered as 'Approved'.

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Pascale Chavis, INMED, INSERM, Aix-Marseille University, Marseille, France

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Is the rationale for developing the new method (or application) clearly explained?

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Are sufficient details provided to allow replication of the method development and its use by others?

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If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

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Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

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References

1. Lagache T, Lang G, Sauvonnet N, Olivo-Marin JC: Analysis of the spatial organization of molecules with robust statistics.PLoS One. 2013; 8 (12): e80914 PubMed Abstract | Publisher Full Text

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06 Oct 2023

Adalberto Merighi, Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

Before responding point-to-point to the reviewers’ observations that as a whole were very useful and supportive to ameliorate our work, we allow ourselves to put beforehand here a brief history of the paper submission and its main purposes. We initially wrote this paper as a Brief Report in response to the NC3Rs center's call for papers written by the NC3Rs grantees to be published in their Gateway of F1000 Research. According to the call rules, papers were subjected to the Center’s check for their suitability for submission according to the mission of the organization. After this check, not only the paper was approved to be eligible for APC funding, but we were asked to expand it in the form of a full Method Paper, as it was then submitted to the journal.
We believe this is the reason why it ended up displaying some mix between its focus on the 3Rs principles and the neurobiology issues that could be raised according to its results. We are grateful to the reviewers who spotted this primary fault of the original manuscript and we have done our best to correct it.
Having said so, we allow ourselves to stress that some of the requests made by the referees (use of cultures from wild-type mice, extension of the study to other areas of the brain, block of Reelin ex vivo) would have indeed required to produce a significant additional amount of work that could be hardly considered within the NC3Rs framework. This is because the Reeler mouse is a suffering phenotype (at least according to Italian animal welfare regulations) and we were not in the position to ask for additional simply confirmative experiments using these mice. Namely, as we have discussed in the section of the Discussion of the revised paper entitled “Insights on Reelin function in Neurodevelopment” several other papers have been published in which the block of the protein function in vitro resulted in at least a partial rescue of the normal cerebellar phenotype, as we have shown here. Moreover, the study of other areas of the brain affected by the Reeler mutation was beyond our original purpose, and the use of the approach here proposed (as discussed in response to this specific comment below) would have required the generation of a specific mouse strain with some sort of useful fluorescent tagging of the cell population of putative interest.
As all the above comments were surely aimed at strengthening the soundness of the method that we have proposed, we decided to pursue a different approach to validate the original Voronoi analysis and employed a series of spatial statistic tools that, up to now, are rarely used in neurobiology to analyze the dispersions of the Purkinje neurons in our cultures. Not only did these tools confirm the results of the Voronoi analysis, but gave interesting additional information on the mechanisms of migration of the Purkinje neurons during postnatal cerebellar development.
Thus, we are confident that the reviewers will share our point of view and support the publication of the paper in its thoroughly revised form.

Summary:
The study reports the implementation of a co-culture method to analyze the effects of reelin and reduce animal use within the 3R framework. Hybrid mice were generated by crossing L7-GFP with heterozygous reeler to obtain GFP-tagged Purkinje neurons in different reelin backgrounds. Co-cultures of cerebellar slices from homozygous and heterozygous hybrids resulted in dispersion and morphology changes of GFP-tagged Purkinje neurons derived from slices bearing a homozygous background.
General:
The main concern is about the general scope of the paper and whether it is about ethical use of animals in biological experiments and animal welfare or about reelin neurobiology in the 3R framework. Although the two former issues are not to be overlooked, they should be developed in the discussion rather than in the introduction, highlights and results.
We fully understand this concern. A similar concern was also raised by Drs. Caruncho and Reive. The reason why the issues of the ethical use of animals in biological experiments and animal welfare have been developed in different parts of the manuscript other than the Discussion is to cope with the guidelines issued by the NC3Rs for Method papers. In response, we have fully revised the paper according to these observations as explained above in the response to the comments of the other two reviewers and in the preamble to our responses.
The authors also state that this approach will allow the study of reelin effects on cortical lamination and that the method will also be applicable to investigate the function of other brain secreted proteins. They should show that this is indeed the case either in co-cultures of other cortices from reelin hybrids or in cerebellar co-cultures from mutants of different proteins.
We agree with the reviewers that the experiments suggested would be indeed useful to confirm that our approach will permit studying 1. The effects of reelin in the other laminated areas of the brain or 2. The function of other secreted proteins. Yet, as regards the first issue, we have now thoroughly discussed previous experiments with approaches different from the one used here demonstrating the effects of Reelin on the lamination of the cerebral cortex. As regards the second issue, we have now softened our claims on the possible applications of our method to the study of other secreted brain proteins in another specific section of the discussion (Advantages and limitations of the organotypic culture approach to the study of neurodevelopment). We have also considered the issues raised here in a more thorough discussion of the strengths and weaknesses of our approach, as such a discussion was also suggested by Drs. Drs. Caruncho and Reive.
We are confident that the reviewers would agree with us that the experiments suggested would require a large amount of work that could the the subject of another paper and well beyond the purpose of this Method Paper (that as mentioned in our prologue was sponsored by the NC3Rs and was prepared according to the guidelines for the NC3Rs gateway). Apart from this, there are several theoretical and practical limitations to the possibility of performing the experiments above (we have addressed this point in response to the first comment under “Data Interpretation”).
That reelin produced by the cerebellar granule cells in slices from heterozygous or homozygous Reeler mice can diffuse in the culture medium was previously demonstrated after studies on chimeric mice and in Reeler cortical cultures. We have addressed this issue in the Discussion of the revised paper as follows:
Although the structural effects of the Reeler mutation are well known, little work has been done on the possibility of modifying Reelin-mediated deficits in live cells. A study in the Reeler-normal chimeric mice has previously demonstrated that the morphology and position of the cerebellar neurons and glial cells are controlled by extracellular environments but not by their genotype [11]. Subsequent work has shown that the protein produced by cortical explants of reln^+/+ or reln^+/- mice co-cultured with a Reeler-like ferret dysplastic cortex was capable of at least partly rescuing neuronal migration in the ferret explant [12]. Other studies, were carried out in the cerebral cortex and hippocampus and led to similar conclusions. It was thus shown that cortical layer development and orientation are modulated by relative contributions of Reelin-negative and -positive neurons in mouse chimeras [13] and that the possibility to restore a normal phenotype in reln^-/-slices co-cultured with wild-type slices was mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR) [14]. Our present observations are in full accord with these studies. A point left open for discussion regards the Reelin dosage necessary for the rescue of a normal phenotype in culture studies. In their chimeric mouse study, Yoshiki and Kusakabe qualitatively observed that only a few granule cells derived from a normal mouse were capable of promoting the alignment and the proper dendritic tree development of the PNs derived from the mutant. From their observations, these authors suggested that Reelin secreted from a single granule cell can affect the morphology of PNs in a wide area [11].
Our results quantitatively demonstrate that the Reelin produced by the slices obtained from haplodeficient mice is sufficient to restore the normal cerebellar phenotype. Therefore, our findings reinforce the idea that it may be possible to modify Reelin-mediated deficits through the experimental administration of the protein. Experiments have demonstrated that the medium obtained from cerebellar dissociated cultures from P5-8 normal mice as well as from cell extracts of the cultured cerebellum from these mice contained full-length Reelin [15]. These observations are fully in line with the demonstration that the protein is produced by the postmitotic granule cells of the deep external granular layer before they migrate to their final destination, the internal granular layer, where they lose Reelin immunoreactivity [16].
Strengths:
The authors developed an elegant technique highly suited for longitudinal and dynamic studies, implemented in the 3Rs framework. They provide a very detailed and exhaustive description of the method to allow replication in other labs.
Major points:
Methods:
Voronoi analysis: The authors perform Voronoi analysis to analyze spatial organization of cells. Although they give references to support their work, the theoretical paper is dated from 1992. Since then, framework and tools to study spatial organization, based on robust spatial statistics such as F, G, H and K functions, have been developed (see Lagache et al., 20131 for example).
Frankly, we do not see why Voronoi's analysis should be hampered by its relative age. Voronoi tesselation has been and still is widely employed in the study of the spatial organization of many diverse types of neuronal (and other) cell populations [17].
The paper quoted by the reviewers uses a modified Ripley’s K function to define the clustered, dispersed, or uniform distribution of points (i.e. clathrin endocytotic sites) in the 2D space [18]. Ripley's K-, H-, and L-functions are used primarily to identify the clustering of proteins in membrane microdomains (as is indeed the case for Lagache’s paper). Using this approach, aggregation (or clustering) is identified if the average number of proteins within a distance r of another protein is statistically greater than that expected for a random distribution [19]. Kiskowski et al. emphasized that “it is not entirely clear how the function may be used to quantitatively determine the size of domains in which clustering occurs” and have widely discussed the limitations and potential of Ripley's K in real-life scenarios”[19]. In the case of this study, where indeed the main issue under investigation is the clustering of the PNs, which is very high in the reln^-/-mice, one can raise reasonable doubts about the usefulness of applying Ripley's K in our conditions. Despite the above observations on the limitations of Ripley’s functions to modeling spatial clustering we have added a large bunch of new analyses based on the novel use of spatial statistics with ArcMap [geographical distribution tools (Central Feature, Mean Center, Median Center, Directional Distribution, and Standard Distance), pattern distribution tools (Average Nearest Neighbor, Getis-Ord General G, Ripley’s K function, and Global Moran’s I), and mapping clusters tools (Anselin Local Moran’s I, and Getis-Ord G*)] to compare their results with the conclusions that could be drawn solely from Voronoi analysis. As thoroughly discussed in the revised paper the two approaches yielded comparable results in support of our initial claims on the soundness of the method.
The overall method is also extremely manual, involving at least 4 different softwares, and the initial cell detection is purely manual, along with the Voronoi adjustment.
We have already at least partly addressed this comment in response to Drs. Caruncho and Reive observations (see Supplementary Material 4). In addition, we would like to stress here that the possibility of using an automated procedure is severely hampered by the extremely high compactness of the central mass of the PNs lying inside the cerebellar white matter. In other words, it seems to be very difficult to practically separate the individual PNs with a thresholding procedure based on color or gray histograms (see Fig. S4-2). In addition, the aforementioned paper by Lagache and collaborators uses a similar manual procedure for the initial detection of the regions of interest (ROIs) as reported in their Materials and Methods: “We first delimited cells’ contours by drawing polygonal Region of Interest (ROIs) with the Icy software [13] (http://icy.bioimageanalysis.org). We then used a wavelet-based detection method [28], implemented as a plugin Spot detector in Icy to extract the two-dimensional positions of putative endocytic spots at the cellular membrane…”.
The removal of Voronoi zones of cells touching edges is justified, but no explanation is given on why the authors chose to favor convex area hence removing some additional cells in their study.
We have addressed this comment also in response to similar observations made by Drs. Caruncho and Reive (see main text and Supplementary Material 3 and 4).
Quantitative analysis of PN migration: since slices expand with time (Figure 1), how is the center point precisely localized through different time points?
We believe that this comment applies to Figure 2 and thus refers to qualitative analysis. The center point was recorded using the memory system of the microscope stage at 4 DIV. Subsequent images were recorded using the memorized X-Y coordinates.
Data interpretation:
In co-cultures, changes in the histology of reln-/- slices are interpreted as resulting from the effect of Reelin produced by reln+/- slices. However, there is no clear demonstration of this, as this could be due to another secreted factor. To answer this point, reelin blockade (function or binding, etc...) should be achieved. This experiment would also have the advantage to validate the statement made in the highlight that co-cultures are amenable to pharmacological intervention or other type of manipulation/treatment.
We agree that, theoretically, the changes in the histology of co-cultured reln^-/-slice could be dependent on a different secreted factor. However, several published papers support our line of thought as we have discussed in the revised version of the paper (reported above). To our knowledge, no other molecules except Reelin and its downstream receptors and adaptor molecules have been demonstrated to be primarily responsible for the correct layered organization of the cerebellar cortex during postnatal development of altricial mammals, although the true mechanisms of PNs layering in the mature cerebellar cortex remain a matter of debate [20]. As authoritatively reviewed by Gabriella D’Arcangelo [21], the discoverer of Reelin and certainly one of the leading scientists in the field “In the cerebellum, Reelin is produced by granule cells in the external granule layer and is essential for the radial migration of the Purkinje neurons, which are born in the cerebellar ventricular zone and form at first a plate and then a single-cell layer. This assessment derives from a widespread analysis of cerebellar development in Reeler mice [2, 22-24], from Reelin gene and protein expression data [16, 25], and functional studies in organotypic cultures [26]”.
We likewise agree that the blockade of Reelin would have the advantage of validating our statement that co-cultures are amenable to pharmacological or other types of manipulation. However, this would require a huge amount of additional work that is prone to several worries about its practicability (e.g. the effectiveness of Reelin-blocking antibodies -when available -to penetrate the entire thickness of a slice). In addition, much of the results that may be stemming from these efforts would be mainly confirmatory of previous observations. We have now discussed in our revision the previous literature on the block of the Reelin function in cultured cerebellar slices that demonstrates a long-distance effect of the molecule in vitro.
In the same line of thought, one can wonder whether reln-/- slices phenotypical improvement would be better achieved if the donor mice were reln+/+ instead of reln+/-? This would validate the method further and also give an indication about time and/or dose effect.
We agree with this observation in theory, but previously published observations on mouse chimeras [11] led to the conclusion that very little Reelin production could be sufficient to rescue the Reeler phenotype. In addition, our main goal was to validate the co-culture method that will be surely of good use to answer these questions in future work.
Statistics:
In Figure 7, what is the variable: does each point represent one culture? How many polygons are included in each point, this could actually be a bias in condition where GFP-PN survival decrease with time such as in the reln-/- group.
The variable in the figure (now Figure 9) is a randomly chosen culture slice. The number of polygons included in each point is indicated in FigShare uploaded Excel files (.xlsx or .csv). Inspection of these files shows that the number of polygons remains very high in single cultured reln^-/- slices despite survival decrease thus excluding a bias.
Please, indicate in legend samples size i.e. number of cultures/dishes used.
Done
In 7B, the distribution in the single reln+/-, clearly shows 2 groups in Voronoi polygons areas. Could you comment? Moreover, this kind of distribution undermines the use of means as presented in 7C.
The existence of two groups of Voronoi polygon areas is explained by the layering of the cortex in the slices from heterozygous mice. We respectfully disagree with the observation that this kind of distribution undermines the use of the means in the now Figure 7C as 1. Analysis was supported by inferential statistics and 2. Results from ArcMap GIS analysis where the tessellations of images only included the hexagons with PNs inside (see Figures 6 and 10 in main text and Figs. S64-15 in Supplementary Material 6) led to fully comparable results as shown and discussed in the revised version of the paper.
Figures:
Figures 1 and 5 are somehow overlapping. The paper would gain clarity by showing in different figures temporal modifications and histological characterization of single cultures and co-cultures (similar to Figure 2).
In response, we have deleted panels A and B in the original Figure 1. In addition, we have shown other images of the cultures to better demonstrate their histological appearance (Fig. 10A-C) and Figure S2.
Minor points:
What is the age of the mice used for slice preparation? And why are mice euthanized using sodium pentobarbital prior brain slicing (protocols.io.6qpvr67bbvmk/v1)?
The mice were 5 days old. We have added this information to the revised paper. They were euthanized with sodium pentobarbital (SP) as routinely done in our laboratory. Injectable barbiturates are acceptable for use in mouse fetuses and neonates according to the AVMA GUIDELINES FOR THE EUTHANASIA OF ANIMALS: 2020 EDITION, whereas decapitation and cervical dislocation are acceptable with conditions according to the guidelines. In addition, a recent publication [27] has reviewed the effects of SP (and other euthanasia methods) on cells and tissues as well as on different analytes without finding adverse effects of SP versus decapitation and/or cervical dislocation in the brain. Therefore we prefer to use SP also following the recommendations of our Department’s Ethical Committee.
What is the survival time of the single and co-cultures?
The survival time was 30 days at maximum. We have added this information to the revised paper.

References (for both responses to reviewers)
1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

38 Views

03 Nov 2022 | for Version 1

Hector J. Caruncho, Division of Medical Sciences, University of Victoria, Victoria, BC, Canada

Brady Reive, University of Victoria, Victoria, BC, Canada

38 Views Cite this report Responses(1)

Approved With Reservations

Is the rationale for developing the new method (or application) clearly explained?

Yes
Is the description of the method technically sound?

Yes
Are sufficient details provided to allow replication of the method development and its use by others?

Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neuropharmacology; Reelin neurobiology; Animal models; Mood and Psychotic disorders

Respond to this report

Responses (1)

Author Response

06 Oct 2023

Adalberto Merighi, Department of Veterinary Sciences, University of Turin, Grugliasco, 10095, Italy

Faulty Reelin signaling in the cerebellum causes Purkinje neurons (PN) migration to be disrupted and, as a result, granule cell precursor (GCPs) proliferation to be diminished – for a recent review see [1]. Morphological observations in vivo have long ago demonstrated that the Reeler mouse, which is devoid of Reelin, displays an altered stratification of its cerebellar and cerebral cortices, as well as of other layered areas of the brain. Specifically in the cerebellum, these alterations affect the migration and correct positioning of the PNs that, in the homozygous reln^-/- mouse, remain clustered together in a deep cellular mass embedded into the medullary body of the white matter [2]. Conversely, in reln^+/-and reln^+/+ mice the PNs migrate in an outward direction to eventually form a monolayer of cells between the molecular and granule cell layer of the mature cerebellar cortex, i.e. both genotypes display a normal cerebellar architecture, although we have recently demonstrated that there are very subtle differences in the position of the PNs between reln^+/-and reln^+/+mice [3]. The effects of the PNs on the GCP proliferation are mediated by Sonic Hedgehog (reviewed in [4]) and we have subsequently demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to a reduction of cortical size and cellularity, the latter being linked to quantitative differences in the extent of GCP proliferation and apoptosis between normal and Reeler mice [5]. The interactions between the PNs and the GCPs are thus quite complex: from one side the PNs stimulate the proliferation of GCPs and, from the other, the CGPs produce and release Reelin in the intercellular space, and the glycoprotein is essential for the correct positioning of the PNs.
If we understood well the reviewers’ thoughts, they suggest that we cannot claim that “the cells originating from the homozygous slice show improved organization in co-cultures” as it is equally possible “that amongst the co-culture, the cells originating from the heterozygous Reeler are the only cells showing an altered organization” but this in total conflict with the above-reported literature.
In addition, the reviewers believe that we are overinterpreting our findings because:
1. We could not “verify which slice they originate (both reported with the same protein, i.e. GFP, which can also be exchanged between cells”. However, we never suggested that there was a migration of PNs from one slice to the other, and there are no pieces of evidence from previous studies that GFP is exchanged between cells in the L7 mouse. The PN-specific gene coding L7 or pcp2 (Purkinje cells specific protein-2) was first described in 1988 by two independent groups [6, 7], and since then it has been of paramount importance for genetic targeting of these neurons [8]. Anyway, even if one assumes that the GFP can be transferred between cerebellar neurons, the protein should preferentially reach the neurons of the cerebellar nuclei which are in contact with the PN axons, as the retrograde transsynaptic transfer occurs only when a fusion protein with a non-toxic fragment of tetanus toxin is used for genetic engineering/transfection, see e.g. [9].
2. “there’s no change from heterozygous and co-cultures, supporting the “Occam’s razor” hypothesis that it’s really a comparison of heterozygous cells against other heterozygous cells”. Although Occam’s razor is based on the idea that the simplest explanation is often the best one, we don’t see how the reviewers may conclude that we are simply comparing heterozygous (PN) cells between each other, since they derive from genotyped animals and no exchange of PNs occurs among slices. On the contrary, the simplest explanation of our findings is indeed the one put forward in the original paper.

Yet, we fully agree with the referees that “it wasn’t clear enough how co-cultures are grown together to allow for differentiation” and we have amended the manuscript by better explaining how co-cultures were established and how they could influence each other by adding some supplementary information to the paper (Supplementary Material 1).
General comments on:
Methods: Good overall. My only concerns: Polygon selection and removal of the top polygon, which corresponds to the culmination of data (or all accounted for polygons). I’m not understanding why that information wouldn’t be valuable for comparing sets of polygons (I understand it’s included de facto in the sum of all selected polygons) but it would be good to show the amalgamations are similar across cultures (i.e. I would expect less stacking/layering of cells occurs in smaller montages of polygons compared to bigger ones corresponding to larger numbers of cells, which would impact nearest neighbour analyses etc.).
The principle at the basis of Voronoi tessellation is that a plane can be divided into regions close to each of a given set of objects, in our case the centers of the GFP-tagged PNs. Cell centers are mathematically referred to as sites (or seeds or generators). For each site, there is a corresponding region, called a Voronoi cell (polygon), consisting of all points of the plane closer to that seed than to any other. In our case, PNs lay on a Euclidean plane (2D) and their centers form a discrete set of points. The marginal polygons must be excluded from analysis because one or two (when in the corners of the microscope image) of their sides are indeed extending to the infinite (i.e. they are OPEN polygons) as they are defined by the perimeter of the image and NOT by the existence of another site outside the microscopic field (See Fig. S3-1 in Supplementary Material 3).

In other words, the software designs these polygons only because the image is a finite portion of the space and the marginal polygons do not derive from the algorithm at the basis of the tessellation. Similarly, one can explain the need to eliminate the polygons that are intersected by the convex hull (asterisks in Fig. S3-1). The convex hull of a finite set PP of points in the plane can be defined as the unique convex polygon whose vertices are points from PP and which contains all points of PP. It can be demonstrated that a Voronoi polygon is unbounded if and only if one of its points is on the convex hull (indicated by asterisks in the figure). As a corollary, the convex hull can be computed from the Voronoi diagram in linear time [10]. Being unbounded, the polygons intersecting the convex hull do not have a finite area and thus cannot the used in the analysis. We have tried to better explain these concepts in the revised paper and Supplementary Material 3. Additionally, in Supplementary Material 3 we have used a different Voronoi generator that automatically computes the convex hull (Fig. S3-2) and demonstrated that there are no statistically significant differences with our original procedure to determine the hull (Fig. S3-3).
Verification that the initial selection of cells is accurate is not done, and in fact is explained as being negligible. “Using the mouse, click above the center of each cell to generate the Voronoi polygons. Due to the thickness of the slice, it may be possible that two very close cells in the Z axis are not easily distinguished. This introduces an error that can be neglected considering that all slices are cut at the same initial thickness.”
It isn’t clear why initial thickness of section would dictate how one would treat stacking of grown cells. The stacking isn’t uniform just because the initial slice was uniform (at least it very much appears there exists more stacking in the central mass relative to the periphery. Also, it matters entirely because those clicks define the cell sizes, which is one of the outputs for statistical comparisons.
Unfortunately, we have not been clear in explaining our point regarding the position of the GFP-tagged cells along the Z-axis of the section. We agree with the referees that stacking is not uniform, but what we signified in saying that selection errors were negligible was that if two or more cells are perfectly stacked one on top of the others we could visualize and take into consideration only the cells in the plane of focus as we used a 2D (and not a 3D) Voronoi analysis. To better clarify this point we have amended the text of the revised manuscript and added explanatory information in Supplementary Material 2.
The inability to verify which slice cells originate should be described/explained. Is it possible to use a secondary reporter to compare and contrast within co-cultures? What would it mean that you can’t verify if so?
If we understand well this comment signifies (also according to the comment above about the impossibility of verifying the origin of the cells as a consequence of a theoretical exchange of GFP from cell to cell) that we could not identify with certainty the phenotype of the GFP-tagged PNs in slices. We have substantially rebutted this possibility in responding to the previous comment. In addition, we don't see the need for a secondary reporter to compare and contrast within co-cultures. In the original paper, although not strictly necessary, we used calbindin 28k (a well-established marker of the PNs) to unambiguously prove the nature of the GFP-tagged neurons and we obtained a 100% coexistence of both tags (see e.g. Fig. 1 where the fluorescence of the positive neurons is yellow/orange for the sum of the green fluorescence of GFP and the red fluorescence of calbindin-immunostaining). This said, we believe is 100% positive that we could verify the origin of the cells in individual slices. To make this clearer, we have better explained how we have seeded the slices in the culture dish to allow for their unequivocal identification in the revised manuscript and Supplementary Material 1.
Is it true the imaging is done at the central mass? I had a difficult time understanding because there is also a section that states the coordinates images could be taken at. I’m very curious if there’s an impact of image location/bias involved. Is the origin selected by the researcher, are there methods for ensuring similarly sized clusters of cells are being imaged? Is it randomized? How many cells are removed in each?
Unfortunately, we have been unclear about these issues. We have added all the info about the above points in Supplementary Material 1 and Supplementary Material 4 (also in response to the comment below).
I found this comment particularly interesting: “It is possible to use several other Voronoi generators that can be found online as freeware or in dedicated programs.” They acknowledge the existence of other modeling systems, would be valuable to know if the results are similar., i.e. the impact of the modeling choice. Even a visual demonstration to compare the resulting polygons would be a powerful addition (to answer the obvious question about validity of Voronoi method, how important is accurate cell selection, is there an impact of user experience, etc.).
We found this comment particularly useful. We have briefly addressed this issue in the revised text and added a more detailed explanation in Supplementary Material 4.
Data interpretation: I think my biggest concern is lack of description of limitations/how perceivable limitations might be overcome
We have now described the limitations of the techniques in the main text and Supplementary Materials.
and secondary, that the discussion and conclusion only includes the word Reelin once, in the first sentence…otherwise the paper is about animal welfare? If the focus is going to be about how to reduce animal sizes, more information should be provided about litter sizes, culling, survival of mutants, etc. and how litter survival might be a limitation for studying other proteins using this method (or not). More information would be required about how many cultures can be derived from a single animal (i.e. size of slice versus size of structure at age of collection, etc.) Either it’s about Reelin, which I think it should be, and I think is interesting, or it’s a methods paper, in which case I think it would be much more valuable to show positive results in at least 2 proteins of interest in a way that allows for comparing the cultures to really show the applications of this technique. I think it might be limited to studying proteins with a very specific subset of functions, so maybe choosing another protein with a vastly different function of Reelin would be a good choice to really highlight the value of this technique. Instead, I think re-writing the discussion and conclusion to focus on results about Reelin would be more appropriate. What can the authors infer about the possibility of Reelin application to modify Reelin-mediated deficits? What can the authors infer about Reelin’s function in regulating cell morphology, beyond simple layering/migration results? Are these results consistent with the in vivo known functions of Reelin? What does this mean? Would you expect there would be differences in non-Purkinje cells? What other sets of cells you can study with this technique? Are there basket cells, granular cells etc. in the culture that we can’t visualize? Why the cerebellum? Is there interaction between the various cells in culture (both PN to PN and PN to any other non-GFP cells)? How might you evaluate cell interactions using this model in the future? What other directions would you go? Just so much to talk about and instead we were told we can save an undetermined, unspecified number of animals…but to learn what? Instead of providing 2 highly specific proteins that could be studied with this method, why not provide a sweeping generalized set of proteins you might have interest in studying with this technique? Usually, the issue is people over-exclaim their success, but in this case, I think not enough effort is given to really dive into what this study indicates about Reelin or what else can be done with the method. If this method is so great, then there should be no shortage of things to tell the reader that you learned about Reelin in applying the technique, instead of how many fewer animals might be needed in future hypothetical studies.
According to the comments above we have substantially rewritten the discussion that has now a section in which we take into consideration the significance of this study in terms of Reelin biology.
Statistics: Only issue with stats described earlier: pertaining to non-randomized imaging location (coordinates in the dish essentially) leads to selection bias. Alternative suggestions would include: a) create a grid and take an average of all polygons in multiple grid boxes to get a variety of polygon origins within the analysis. Additionally, it would be valuable to demonstrate whether or not peripheral polygons are truly similar to the central mass-related polygons, in other words, a within-culture analysis would also be valuable (is there more or less homogeneity of cultures from Reelin homo- or heterozygous mice?). Combining a perceived selection bias with unconfirmed cell selection, the reader might reasonably be concerned the results aren’t the most meaningful or generalizable.
We have clarified these points in response to some of the previous comments in particular in Supplementary Materials 1-4. We have also added a within-culture analysis by the use of several GIS tools.

References (for both responses to reviewers)

1             Jossin, Y.: ‘Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation’, Biomolecules, 2020, 10, (6)
2             Mariani, J., Crepel, F., Mikoshiba, K., Changeux, J.P., and Sotelo, C.: ‘Anatomical, Physiological and Biochemical Studies of the Cerebellum from Reeler Mutant Mouse’, Philosophical Transactions of the Royal Society of London B: Biological Sciences, 1977, 281, (978), pp. 1-28
3             Magliaro, C., Cocito, C., Bagatella, S., Merighi, A., Ahluwalia, A., and Lossi, L.: ‘The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse’, Ann Anat, 2016, 207, pp. 68-75
4             Ruiz i Altaba, A., Palma, V., and Dahmane, N.: ‘Hedgehog–GLI signaling and the growth of the brain’, Nature Reviews Neuroscience, 2002, 3, (1), pp. 24-33
5             Cocito, C., Merighi, A., Giacobini, M., and Lossi, L.: ‘Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum’, Frontiers in Cellular Neuroscience, 2016, 10, pp. 141
6             Oberdick, J., Levinthal, F., and Levinthal, C.: ‘A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene’, Neuron., 1988, 1, (5), pp. 367-376
7             Nordquist, D., Kozak, C., and Orr, H.: ‘cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons’, The Journal of Neuroscience, 1988, 8, (12), pp. 4780-4789
8             Sługocka, A., Wiaderkiewicz, J., and Barski, J.J.: ‘Genetic Targeting in Cerebellar Purkinje Cells: an Update’, Cerebellum, 2017, 16, (1), pp. 191-202
9             Maskos, U., Kissa, K., St Cloment, C., and Brûlet, P.: ‘Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice’, Proc Natl Acad Sci U S A, 2002, 99, (15), pp. 10120-10125
10           Preparata, F.P., and Shamos, M.: ‘Computational Geometry: An Introduction’ (Springer New York, 1993. 1993)
11           Yoshiki, A., and Kusakabe, M.: ‘Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras’, Int.J Dev Biol., 1998, 42, (5), pp. 695-700
12           Schaefer, A., Poluch, S., and Juliano, S.: ‘Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex’, Dev Neurobiol, 2008, 68, (5), pp. 590-604
13           Hammond, V.E., So, E., Cate, H.S., Britto, J.M., Gunnersen, J.M., and Tan, S.S.: ‘Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras’, Cereb Cortex., 2010, 20, (9), pp. 2017-2026
14           Zhao, S., Chai, X., Bock, H.H., Brunne, B., Förster, E., and Frotscher, M.: ‘Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1’, J Comp Neurol, 2006, 495, (1), pp. 1-9
15           D’Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T.: ‘Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody’, The Journal of Neuroscience, 1997, 17, (1), pp. 23-31
16           Miyata, T., Nakajima, K., Aruga, J., Takahashi, S., Ikenaka, K., Mikoshiba, K., and Ogawa, M.: ‘Distribution of a reeler gene-related antigen in the developing cerebellum: An immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice’, Journal of Comparative Neurology, 1996, 372, (2), pp. 215-228
17           Galli-Resta, L., Novelli, E., Kryger, Z., Jacobs, G.H., and Reese, B.E.: ‘Modelling the mosaic organization of rod and cone photoreceptors with a minimal-spacing rule’, European Journal of Neuroscience, 1999, 11, (4), pp. 1461-1469
18           Lagache, T., Lang, G., Sauvonnet, N., and Olivo-Marin, J.C.: ‘Analysis of the spatial organization of molecules with robust statistics’, PLoS One, 2013, 8, (12), pp. e80914
19           Kiskowski, M.A., Hancock, J.F., and Kenworthy, A.K.: ‘On the use of Ripley's K-function and its derivatives to analyze domain size’, Biophys J, 2009, 97, (4), pp. 1095-1103
20           Rahimi-Balaei, M., Bergen, H., Kong, J., and Marzban, H.: ‘Neuronal Migration During Development of the Cerebellum’, Frontiers in Cellular Neuroscience, 2018, 12
21           D'Arcangelo, G.: ‘Reelin in the Years: Controlling Neuronal Migration and Maturation in the Mammalian Brain’, Advances in Neuroscience, 2014, vol. 2014, Article ID 597395, 19 pages. doi:10.1155/2014/597395
22           Goffinet, A.M.: ‘The embryonic development of the cerebellum in normal and reeler mutant mice’, Anat Embryol (Berl), 1983, 168, (1), pp. 73-86
23           Mikoshiba, K., Nagaike, K., Kohsaka, S., Takamatsu, K., Aoki, E., and Tsukada, Y.: ‘Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro’, Developmental Biology, 1980, 79, (1), pp. 64-80
24           Inoue, Y., Maeda, N., Kokubun, T., Takayama, C., Inoue, K., Terashima, T., and Mikoshiba, K.: ‘Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400’, Neuroscience Research, 1990, 8, (3), pp. 189-201
25           D'Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I., and Curran, T.: ‘A protein related to extracellular matrix proteins deleted in the mouse mutant reeler’, Nature, 1995, 374, (6524), pp. 719-723
26           Miyata, T., Nakajima, K., Mikoshiba, K., and Ogawa, M.: ‘Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody’, The Journal of Neuroscience, 1997, 17, (10), pp. 3599-3609
27           Shomer, N.H., Allen-Worthington, K.H., Hickman, D.L., Jonnalagadda, M., Newsome, J.T., Slate, A.R., Valentine, H., Williams, A.M., and Wilkinson, M.: ‘Review of Rodent Euthanasia Methods’, Journal of the American Association for Laboratory Animal Science, 2020, 59, (3), pp. 242-253

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Schubert D, Herrera F, Cumming R, et al.: Neural cells secrete a unique repertoire of proteins. J. Neurochem. 2009 Apr; 109(2): 427–435. Epub 2009/02/10. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[2] 2. Falconer DS: Two new mutants, ‘trembler’ and ‘reeler’, with neurological actions in the house mouse (Mus musculus L.). J. Genet. 1951; 1951(50): 192–201.

[3] 3. D'Arcangelo G, Miao GG, Chen SC, et al.: A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature. 1995 4/20/1995; 374(6524): 719–723. PubMed Abstract | Publisher Full Text

[4] 4. Mariani J, Crepel F, Mikoshiba K, et al.: Anatomical, physiological and biochemical sudies of the cerebellum from Reeler mutant mouse. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 1977 11/2/1977; 281(978): 1–28.

[5] 5. Heckroth JA, Goldowitz D, Eisenman LM: Purkinje cell reduction in the reeler mutant mouse: A quantitative immunohistochemical study. J. Comp. Neurol. 1989 1/22/1989; 279(4): 546–555. PubMed Abstract | Publisher Full Text

[6] 6. Yuasa S, Kitoh J, Oda S, et al.: Obstructed migration of Purkinje cells in the developing cerebellum of the reeler mutant mouse. Anat. Embryol. (Berl). 1993 10/1993; 188(4): 317–329. Publisher Full Text

[7] 7. Magliaro C, Cocito C, Bagatella S, et al.: The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse. Ann. Anat. 2016 9/2016; 207: 68–75. PubMed Abstract | Publisher Full Text

[8] 8. Hattori M, Kohno T: Regulation of Reelin functions by specific proteolytic processing in the brain. J. Biochem. 2021; 169(5): 511–516. PubMed Abstract | Publisher Full Text

[9] 9. Faini G, Del Bene F, Albadri S: Reelin functions beyond neuronal migration: from synaptogenesis to network activity modulation. Curr. Opin. Neurobiol. 2021 Feb; 66: 135–143. Epub 2020/11/17. eng. PubMed Abstract | Publisher Full Text

[10] 10. D'Arcangelo G: Reelin in the years: controlling neuronal migration and maturation in the mammalian brain. Adv. Neurosci. 2014; 2014: 1–19. Article ID 597395. Publisher Full Text

[11] 11. Lossi L, Castagna C, Granato A, et al.: The Reeler mouse: a translational model of human neurological conditions, or simply a good tool for better understanding neurodevelopment? J. Clin. Med. 2019 Dec 1; 8(12). PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. Ishii K, Kubo KI, Nakajima K: Reelin, and neuropsychiatric disorders. Front. Cell. Neurosci. 2016 2016; 10: 229. PubMed Abstract | Publisher Full Text | Free Full Text

[13] 13. D'Arcangelo G: Reelin mouse mutants as models of cortical development disorders. Epilepsy Behav. 2006 2/2006; 8(1): 81–90. PubMed Abstract | Publisher Full Text

[14] 14. Bernay B, Gaillard MC, Guryca V, et al.: Discovering new bioactive neuropeptides in the striatum secretome using in vivo microdialysis and versatile proteomics. Mol. Cell. Proteomics. 2009 May; 8(5): 946–958. PubMed Abstract | Publisher Full Text | Free Full Text

[15] 15. Umlauf BJ, Kuo JS, Shusta EV: Identification of brain ECM binding variable lymphocyte receptors using yeast surface display. Methods Mol. Biol. 2022; 2491: 235–248. PubMed Abstract | Publisher Full Text

[16] 16. Tüshaus J, Müller SA, Kataka ES, et al.: An optimized quantitative proteomics method establishes the cell type-resolved mouse brain secretome. EMBO J. 2020 Oct 15; 39(20): e105693. PubMed Abstract

[17] 17. Lossi L, Merighi A: The use of ex vivo rodent platforms in neuroscience translational research with attention to the 3Rs philosophy. Front. Vet. Sci. 2018; 2018(5): 164.

[18] 18. Hau KL, Lane A, Guarascio R, et al.: Eye on a dish models to evaluate splicing modulation. Methods Mol. Biol. 2022; 2434: 245–255. PubMed Abstract

[19] 19. Namchaiw P, Bunreangsri P, Eiamcharoen P: An in vitro workflow of neuron-laden agarose-laminin hydrogel for studying small molecule-induced amyloidogenic conditions.PLoS One.2022; 17(8): e0273458. PubMed Abstract

[20] 20. Smith SJ, von Zastrow M : A molecular landscape of mouse hippocampal neuromodulation. Front. Neural Circuits. 2022; 16: 836930. Epub 2022/05/24. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[21] 21. Oberdick J, Levinthal F, Levinthal C: A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene. Neuron. 1988 7/1988; 1(5): 367–376. PubMed Abstract | Publisher Full Text

[22] 22. Zhang X, Baader SL, Bian F, et al.: High-level Purkinje cell-specific expression of green fluorescent protein in transgenic mice. Histochem.Cell Biol. 2001 6/2001; 115(6): 455–464. PubMed Abstract | Publisher Full Text

[23] 23. Alasia S, Merighi A, Lossi L:Transfection techniques and combined immunocytochemistry in cell cultures and organotypic slices.Merighi A, Lossi L, editors. Immunocytochemistry and Related Techniques. New York:Springer; 2015; pp. 329–355. Publisher Full Text

[24] 24. Alasia S, Cocito C, Merighi A, et al.: Real-time visualization of caspase-3 activation by fluorescence resonance energy transfer (FRET). Lossi L, Merighi A, editors. Neuronal Cell Death. New York:Humana Press/Springer Science+Business Media; 2015; pp. 99–114. Publisher Full Text

[25] 25. Lossi L, Castagna C, Merighi A:Neuronal cell death: an overview of its different forms in central and peripheral neurons.Lossi L, Merighi A, editors. Neuronal Cell Death. 1254 ed.New York:Springer;2015; pp. 1–18.

[26] 26. Marcelpoil R, Usson Y: Methods for the study of cellular sociology: Voronoi diagrams and parametrization of the spatial relationships. J. Theor. Biol. 1992/02/07/; 154(3): 359–369. Publisher Full Text

[27] 27. Nawrocki AR, Scherer PE: Keynote review: the adipocyte as a drug discovery target. Drug Discov. Today. 9/15/2005; 10(18): 1219–1230.

[28] 28. Faul F, Erdfelder E, Lang AG, Buchner A, et al.: G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods. 2007 May; 39(2): 175–191. PubMed Abstract | Publisher Full Text

[29] 29. Lossi L, Alasia S, Salio C, et al.: Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Prog. Neurobiol. 8/2009; 88(4): 221–245.

[30] 30. Ullrich C, Daschil N, Humpel C: Organotypic vibrosections: novel whole sagittal brain cultures. J. Neurosci. Meth. 9/30/2011; 201(1): 131–141. Publisher Full Text

[31] 31. Schommer J, Schrag M, Nackenoff A, et al.: Method for organotypic tissue culture in the aged animal. MethodsX. 2017; 2017(4): 166–171.

[32] 32. Ganesh S, Utebay B, Heit J, et al.: Cellular sociology regulates the hierarchical spatial patterning and organization of cells in organisms. Open Biol. 2020; 10(12): 200300.

[33] 33. Coutinho-Budd JC, Sheehan AE, Freeman MR: The secreted neurotrophin Spätzle 3 promotes glial morphogenesis and supports neuronal survival and function. Genes Dev. 2017 Oct 15; 31(20): 2023–2038. PubMed Abstract | Publisher Full Text | Free Full Text

[34] 34. Caipo L, González-Ramírez MC, Guzmán-Palma P, et al.: Slit neuronal secretion coordinates optic lobe morphogenesis in Drosophila. Dev. Biol. 2020 Feb 1; 458(1): 32–42. PubMed Abstract

[35] 35. Malik AR, Liszewska E, Jaworski J: Matricellular proteins of the Cyr61/CTGF/NOV (CCN) family and the nervous system. Front. Cell. Neurosci. 2015; 9: 237. Epub 2015/07/15. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[36] 36. Trajkovic K, Jeong H, Krainc D: Mutant Huntingtin Is Secreted via a Late Endosomal/Lysosomal Unconventional Secretory Pathway. J. Neurosci. 2017 Sep 13; 37(37): 9000–9012. PubMed Abstract | Publisher Full Text | Free Full Text

[37] 37. Yamamoto N, Maruyama T, Uesaka N, et al.: Molecular mechanisms of thalamocortical axon targeting. Novartis Found. Symp. 2007; 288: 199–208. discussion -11, 76-81. PubMed Abstract

[38] 38. Corner MA, Baker RE, van Pelt J , et al.: Compensatory physiological responses to chronic blockade of amino acid receptors during early development in spontaneously active organotypic cerebral cortex explants cultured in vitro. Prog. Brain Res. 2005; 147: 231–248. PubMed Abstract | Publisher Full Text

[39] 39. Merighi A, Lossi L:Voronoi analysis - Cultured Reln haplodeficient heterozygous mouse cerebellar slices. figshare. [Dataset]. 2022. Publisher Full Text

[40] 40. Merighi A, Lossi L:Voronoi analysis - Cultured Reln deficient homozygous mouse cerebellar slices. figshare. [Dataset]. 2022. Publisher Full Text

[41] 41. Merighi A, Lossi L:Voronoi analysis - Reln deficient homozygous mouse cerebellar slices co-cultured with Reln haplodeficient heterozygous mouse cerebellar slices. figshare. [Dataset]. 2022. Publisher Full Text

[42] 42. Merighi A: ARRIVE guideline checklist for Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination by Adalberto Merighi and Laura Lossi. figshare. Journal Contribution. 2022. Publisher Full Text

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Abstract

Keywords

Research highlights

Introduction

Methods

Materials and methods

Figure 1. General features of cerebellar cultures.

Figure 2. Temporal modifications of a single-cultured slice from an L7-GFPreln^-/-F1/mouse.

Statistics

Protocols

Table 1. Protocols for the preparation of brain slices.

Figure 3. Use of the Voronoi diagram generator.

Figure 4. Elaboration of images for Voronoi analysis.

Box 1. Voronoi Macro.

Results

Histology

Figure 5. Histological aspects of single- and co-cultured slices after immunostaining with markers of PNs and glia.

Analysis of cellular sociology

Figure 6. Voronoi tessellation of slices.

Figure 7. Quantitative analysis of Voronoi tessellation.

Discussion

Conclusions

Data availability

Underlying data

Reporting guidelines

References

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Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Abstract

Keywords

Research highlights

Introduction

Methods

Materials and methods

Figure 1. General features of cerebellar cultures.

Figure 2. Temporal modifications of a single-cultured slice from an L7-GFPreln-/-F1/mouse.

Statistics

Protocols

Table 1. Protocols for the preparation of brain slices.

Figure 3. Use of the Voronoi diagram generator.

Figure 4. Elaboration of images for Voronoi analysis.

Box 1. Voronoi Macro.

Results

Histology

Figure 5. Histological aspects of single- and co-cultured slices after immunostaining with markers of PNs and glia.

Analysis of cellular sociology

Figure 6. Voronoi tessellation of slices.

Figure 7. Quantitative analysis of Voronoi tessellation.

Discussion

Conclusions

Data availability

Underlying data

Reporting guidelines

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated

Keep up to date with the NC3Rs

Thank you!

Figure 2. Temporal modifications of a single-cultured slice from an L7-GFPreln^-/-F1/mouse.