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Brief Report
Negative/null result

Neuronal subset-specific Pten-deficient mice do not exhibit deficits in sensorimotor gating processes

[version 1; peer review: 1 approved with reservations, 1 not approved]
PUBLISHED 08 Oct 2019
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Abstract

Background: Deficits in sensorimotor gating have been reported in individuals with autism spectrum disorder (ASD), as well as in ASD murine models. However, this behavior has been rarely examined in the neuronal subset-specific (NS)-Pten knockout (KO) model of ASD. NS-Pten KO mice exhibit hyperactivity of the PI3K/AKT/mTOR signaling pathway which is implicated in the onset of autistic deficits. This study investigates the potential relationship between PI3K/AKT/mTOR signaling and deficits in sensorimotor gating.   
Methods: To assess sensorimotor gating in NS-Pten KO mice we utilized a three-day paradigm. On day 1 (habituation) the mice were administered 80 repetitions of a 120-dB startle stimulus. On day 2, prepulse inhibition was measured with 90 trials of the startle stimulus that was paired with a smaller (70, 75, or 80 dB) prepulse stimulus. Day 3 was assessed one week later, consisting of randomized startle trials and trials with no stimulus and was used to determine the startle threshold.
Results: No significant difference between NS-Pten KO or wildtype (WT) mice was found for habituation (p > 0.05). No significant differences were found between groups when assessing the percentage of prepulse inhibition at 70, 75, and 80 dB (p > 0.05). There was also no difference in startle threshold between groups (p > 0.05).
Conclusion: Our study found that the NS-Pten KO model does not display significant deficits in sensorimotor gating processes. The present findings help to elucidate the relationship between PI3K/AKT/mTOR hyperactivation and sensory reactivity.

Keywords

autism, pten, macrocephaly, ASD, sensorimotor

Introduction

Sensorimotor gating is the ability of a sensory stimulus to suppress a motor response1. It can be measured by assessing prepulse inhibition (PPI), wherein a weak auditory stimulus inhibits a startle response that is induced by the following presentation of a loud sound2. Deficits in PPI have been widely reported in various neurological conditions, including autism spectrum disorder (ASD)35. Similar to humans, impairments in PPI have been reported in ASD models such as Fmr1 and Cntnap2-knockout (KO) mice; however, the underlying mechanism is unknown6,7. Pten mutant mice are another model of autism and can be used to investigate the connection between a cell signaling pathway commonly implicated in ASD, the PI3K/AKT/mTOR pathway, and specific autistic-like deficits8. In the present study, we use neuronal subset-specific (NS)-Pten KO mice that exhibit hyperactivation of the PI3K/AKT/mTOR pathway in the cortex, hippocampus, and cerebellum, and assess PPI in order to further elucidate the potential relationship between PI3K/AKT/mTOR signaling and deficits in sensorimotor gating9.

Methods

Subjects

Male and female mice on a FVB based mixed background were obtained from Baylor College of Medicine and have been bred for more than 10 generations at Baylor University. Heterozygous NS-Pten males (n=6) and females (n=12) were used to breed NS-Pten wildtype (WT) and KO pups (RRID: MGI:3714016). The housing for the breeders consisted of two females housed with one male. Genotype was determined from toe clippings taken on postnatal day (PD) 10 (performed by Mouse Genotype, Escondido, CA, USA). On PD 21, animals were weaned and housed with mixed genotype littermates in groups of n=3–5 in cages (Allentown Caging PC7115HT, Allentown, PA, USA) filled with sani-chip bedding (7090 Teklad, Envigo, Somerset, NJ, USA) kept in a room on a 12-hr light/dark diurnal cycle held at 22°C. Mice had ad libitum access to food and water. All animals were tested at 9–10 weeks of age between the hours of 10:00 and 11:30 a.m. A total of 29 male mice were assessed, 17 NS-Pten KO and 12 WT mice. The target sample size was determined by, and is in accordance with, the PPI literature1012. The final sample sizes were as follows: day 1: n=12 WT, n=17 KO, day 2: n=12 WT, n=13 KO, day 3: n=9 WT, n=9 KO. All test procedures were carried out in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by Baylor University’s Institutional Animal Care and Use Committee. Once the experiment concluded, mice were placed into a CO2 chamber and euthanized.

Sensorimotor gating assessment

Sensorimotor gating was assessed via the SR-LAB system, which consists of a 15 × 14 × 18 inch isolation cabinet, a plexiglass cylinder (3.2-cm diameter) mounted on a sensor platform, a standard speaker used to generate white noise, and a high-frequency speaker used to generate stimuli (San Diego Instruments, San Diego, CA, USA). The paradigm consisted of three separate testing days: habituation, prepulse inhibition, and startle response, and was conducted as previously described6.Each test day is further detailed in the Figure 1 legend. To eliminate potential confounds during testing, background sound levels were maintained at 68 dB and the experimenter was not present.

5d5700a2-a6f6-4da4-b432-c791d926769e_figure1.gif

Figure 1. Habituation, prepulse inhibition, and startle threshold in NS-Pten KO mice.

(a) On the first day of testing, the animal was acclimated to the room for 30 minutes then was placed inside the cylinder for a 5-minute habituation period, which was followed by 80 startle stimuli delivered at a fixed interval of 15 seconds. The startle stimulus was a 40-ms, 120-dB noise burst, with a rise/fall time of less than 1 ms. We found that there was no significant difference in habituation between KO and WT mice (p > 0.05). (b) Day 2 of testing occurred 24 hours after day 1 and tested prepulse inhibition. Once the mice were in the apparatus, there was a 5-minute habituation phase that was followed by 20 presentations of a 40-ms, 120-dB noise burst that had a fall time of less than 1 ms. In the prepulse phase, mice were presented with 90 trials consisting of three prepulse intensities, 70, 75, and 80 dB. Each prepulse was 20 ms in duration with a rise/fall time of less than 1 ms and were spaced 15 seconds apart. We found no difference in the percentage of prepulse inhibition between groups following prepulses of 70, 75, or 80 dB (p > 0.05). (c) One week after the prepulse session, the startle threshold was assessed. Following the 5-minute habituation period, the mice were presented with 99 trials of 11 trial types. These include a no stimulus trial and 10 startle stimuli trials ranging from 75–120 dB at 5 dB intervals. The startle stimuli are 40 ms noise bursts with a rise/fall of less than 1 ms. The 11 trial types were pseudorandomized, with each trial type being presented once in a block of the 11 trials. We observed no difference in startle threshold between NS-Pten KO and WT mice (p > 0.05). Data are presented as the mean ± standard error of the mean (SEM).

Statistical analysis

GraphPad Prism 7 software (La Jolla, CA) or SPSS 21.0 (IBM, USA) were used to analyze the data. Repeated-measure ANOVAs were run for habituation, prepulse inhibition, and startle threshold. No post-hoc tests were performed. A total of n=4 KO mice were excluded from the day 2 analysis and n=11 mice (3 WT and 8 KO) were excluded from the day 3 analysis due to protocol malfunction or death as a result of the severity of the knockout. A value of p < 0.05 was considered significant for each statistical test.

Results

When assessing the sensorimotor gating paradigm, no main effects were found for habituation (F[1,27] = 0.17, p >0.05), prepulse inhibition (F[1,23] = 2.65, p >0.05) or startle threshold (F[1,16] = 2.33, p >0.05). There were also no interactions for habituation (F[7,189] = 0.91, p >0.05), prepulse inhibition (F[2,46] = 0.71, p >0.05), or startle threshold (F[10,160] = 1.94, p >0.05) (Figure 1a–c). Raw results for each procedure on each day for every animal are available as Underlying data13.

Discussion

The NS-Pten KO mice did not exhibit significantly different sensorimotor gating from WT mice. A previous study by Kwon et al. (2006) assessed neuron-specific enolase (Nse)-Pten KO mice in a variation of the PPI protocol and reported a decrease in percent inhibition at 4 dB but no differences at 8 or 16 dB10. Our study assessed percent inhibition at 70, 75, and 80 dB, per established protocol, and found no differences at these intensity6. Therefore, this indicates that there may only be changes in percent inhibition in Pten mutant mice when the prepulse is comparatively quiet, as no impairments are reported for dB levels higher than 4 dB. Additionally, in accordance with our study, no differences in prepulse inhibition have been reported in the BTBR and Shank1 mouse models of autism14,15. This indicates that alterations in sensory reactivity may be a less sensitive measure of an autistic-like phenotype and may also only be present in particular ASD models.

Overall, the current study found that hyperactivity of the PI3K/AKT/mTOR pathway does not result in sensorimotor gating deficits in NS-Pten KO mice, suggesting that the pathway may not directly affect prepulse inhibition. This conclusion is supported by a prior study that assessed PPI in a transgenic mouse model of tuberous sclerosis complex, another model of ASD and mTOR hyperactivation, which similarly reported no deficits in prepulse inhibition between WT and KO mice16. Taken together, these studies indicate that despite mTOR’s contribution to an autistic-like phenotype, it does not significantly contribute to the onset of sensorimotor gating deficits in several different ASD models. Ultimately, our study is in support of the literature and helps to further elucidate the relationship between hyperactivation of the PI3K/AKT/mTOR pathway and deficits in sensory reactivity.

Data availability

Underlying data

Figshare: Neuronal subset-specific Pten-deficient mice do not exhibit deficits in sensorimotor gating processes. https://doi.org/10.6084/m9.figshare.9885401.v113.

This project contains the following underlying data:

  • PPI Day1 Pten Raw Data 9-6.xlsx (raw data from all experiments performed for all animals; day 1).

  • PPI Day 2 Pten Raw Data 9-6.xlsx (raw data from all experiments performed for all animals; day 2).

  • PPI day 3 Pten Raw Data 9-6.xlsx (raw data from all experiments performed for all animals; day 3).

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

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Binder MS, Nolan SO and Lugo JN. Neuronal subset-specific Pten-deficient mice do not exhibit deficits in sensorimotor gating processes [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2019, 8:1727 (https://doi.org/10.12688/f1000research.20604.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe 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 approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 08 Oct 2019
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Reviewer Report 08 Nov 2019
Maarten van den Buuse, School of Psychology and Public Health, La Trobe University, Melbourne, Vic, Australia 
Approved with Reservations
VIEWS 21
This Brief Report shows that a specific Pten-deficient mouse model shows no change in prepulse inhibition compared to wildtype controls. This is discussed in the context of literature about changes in PPI in humans with autism and in autism animal ... Continue reading
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HOW TO CITE THIS REPORT
van den Buuse M. Reviewer Report For: Neuronal subset-specific Pten-deficient mice do not exhibit deficits in sensorimotor gating processes [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2019, 8:1727 (https://doi.org/10.5256/f1000research.22655.r54867)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 11 Feb 2020
    Joaquin Lugo, Department of Psychology and Neuroscience, Baylor University, Waco, 76798, USA
    11 Feb 2020
    Author Response
    Reviewer 2. The original comments are first followed by a bullet point with our response.

    Abstract, background:
    "has been rarely examined” - does this mean it has been examined once before? What ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 11 Feb 2020
    Joaquin Lugo, Department of Psychology and Neuroscience, Baylor University, Waco, 76798, USA
    11 Feb 2020
    Author Response
    Reviewer 2. The original comments are first followed by a bullet point with our response.

    Abstract, background:
    "has been rarely examined” - does this mean it has been examined once before? What ... Continue reading
Views
29
Cite
Reviewer Report 16 Oct 2019
Benjamin K. Yee, Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hong Kong, China 
Not Approved
VIEWS 29
Whether sensorimotor gating, as evaluated by the prepulse inhibition (PPI) of the acoustic startle reflex paradigm, is attenuated or exaggerated in ASD is still controversial. The present study attempted to investigate this using a mutant mouse model. Specifically, neuronal deletion ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Yee BK. Reviewer Report For: Neuronal subset-specific Pten-deficient mice do not exhibit deficits in sensorimotor gating processes [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2019, 8:1727 (https://doi.org/10.5256/f1000research.22655.r55104)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 11 Feb 2020
    Joaquin Lugo, Department of Psychology and Neuroscience, Baylor University, Waco, 76798, USA
    11 Feb 2020
    Author Response
    Reviewer comments are first followed by a bullet point of our response.
     

     
    No attempt was made to examine sex difference, while it is highly relevant to ASD.
    • Thank you
    ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 11 Feb 2020
    Joaquin Lugo, Department of Psychology and Neuroscience, Baylor University, Waco, 76798, USA
    11 Feb 2020
    Author Response
    Reviewer comments are first followed by a bullet point of our response.
     

     
    No attempt was made to examine sex difference, while it is highly relevant to ASD.
    • Thank you
    ... Continue reading

Comments on this article Comments (0)

Version 4
VERSION 4 PUBLISHED 08 Oct 2019
Comment
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
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