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Research Note
Revised

Kv4.2 knockout mice display learning and memory deficits in the Lashley maze

[version 2; peer review: 2 approved]
PUBLISHED 27 Feb 2017
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

Abstract

Background: Potassium channels have been shown to be involved in neural plasticity and learning. Kv4.2 is a subunit of the A-type potassium channel. Kv4.2 channels modulate excitability in the dendrites of pyramidal neurons in the cortex and hippocampus. Deletion of Kv4.2 results in spatial learning and conditioned fear deficits; however, previous studies have only examined deletion of Kv4.2 in aversive learning tests.
Methods: For the current study, we used the Lashley maze as an appetitive learning test. We examined Kv4.2 wildtype (WT) and knockout (KO) mice in the Lashley maze over 4 days during adulthood. The first day consisted of habituating the mice to the maze. The mice then received five trials per day for the next 3 days. The number of errors and the time to the goal box was recorded for each trial. The goal box contained a weigh boat with an appetitive reward (gelatin with sugar). There was an intertrial interval of 15 minutes.
Results: We found that Kv4.2 KO mice committed more errors across the trials compared to the WT mice p<0.001. There was no difference in the latency to find the goal box over the period.
Discussion: Our finding that deletion of Kv4.2 resulted in more errors in the Lashley maze across 15 trials contribute to a growing body of evidence that Kv4.2 channels are significantly involved in learning and memory.

Keywords

Kv4.2, A type current, hippocampus, lashley maze, learning, potassium ion channel

Revised Amendments from Version 1

The revised version is in response to the two reviews of the paper. None of the graphs were modified. Additional information on the methods and more information on the interpretation of the data were included in the new version.

See the authors' detailed response to the review by Peter Backx

Introduction

Kv4.2 is a subunit of the A–type potassium channel which mediates the excitability of pyramidal neurons in the cortex and hippocampal dendrites13. A-type currents regulate cell firing by attenuating action potentials and reduce excitation48. Kv4.2 localization in the pyramidal cell dendrites is dependent on membrane associated guanylate kinase protein (PSD-95)9,10. The highest levels of Kv4.2 are found in the CA1 of the hippocampus with less expression in the CA3 and dentate gyrus11. The channels are localized to the somatodendritic regions of the hippocampal dendrites1214. When the Kv4.2 subunit is genetically deleted, the A-type current in the CA1 pyramidal cell dendrites of the hippocampus is almost entirely removed15. Disruption of the Kv4.2 has been associated with both epilepsy16,17 and autism spectrum disorder3.

Kv4.2 knockout (KO) mice have a reduction in the A-type current and their threshold for long term potentiation (LTP) is also lowered, resulting in changes in synaptic plasticity15. Previous research has shown the Kv4.2 KO have impaired spatial learning in the Morris water maze (MWM) and a deficit in contextual learning in fear-conditioning18,19. However, these tasks are aversive and stress could contribute to some of the learning deficits initially found. For this experiment, we used appetitive learning to examine the effects of Kv4.2 KO performance in the Lashley maze, which is a low-stress learning task that does not rely on adverse stimuli20,21.

Materials and methods

Animals: The mice used for this study were Kv4.2 wildtype (WT) and KO adult males (postnatal day 60) that were generated on the 129S6/SvEv background, which had been bred for over 10 generations. All mice were bred in the Baylor University animal facility. For this study, heterozygous parents were bred to obtain both KO and WT mice and both male and female mice were used. All animals were housed in Baylor University’s animal facility on a 14 hour light 10 hour dark cycle at 22°C. Mice were all housed with sex matched littermates following weaning. All mice were given ad libitum access to food and water. All testing and housing complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All protocols were approved by the Baylor University Animal Care and Use Committee (Animal Assurance Number A3948-01).

Maze and procedure: The details of the maze construction and procedure can be found in a previous study20. The maze was constructed out of 0.25 cm thick black acrylic plastic and is 60 cm × 28 cm with 16 cm tall walls. The maze had four lanes that were evenly spaced with an additional start (area A) and goal box (area N). The start and goal boxes were 12 cm × 7.25 cm and the entrance began 12 cm from the edge of the maze. The entrance to the boxes was 6 cm wide. Doors 1, 2, and 3 were all 4 cm wide and began 12 cm from the edge of the maze. We defined an error as an entry into a dead-end cul-de-sac zone (e.g., going from arm H to zone I; Figure 1) or when the mouse travels back through a previously traveled arm of the maze (e.g., going from arm L to arm I; Figure 1). A 5% gelatin solution in double distilled water with 1.25% sucrose was prepared. The mixture was stored at 5°C until use on training and testing days. Mice were pre-exposed to the gelatin-sugar reward for 2 days prior to testing to reduce neophobia. The sugar reward was used to make the maze an appetitive test. The mice were not food deprived. The mice ate the entire reward at the end of each day. The details of the testing per day is detailed in Figure 1.

dd79b102-8fc3-4465-8f6b-4f1cdb6822d3_figure1.gif

Figure 1. Schematic overview of the Lashley maze. The correct path: ACFILN.

On day 1 the test mice were habituated to each of the chambers of the maze. For each mouse, a weigh boat containing a small amount of the gelatin was placed in the goal box (area N). The mice began habituation in section BCD with door 1 and door A blocked and were allowed to explore for 3 minutes. The mice were then moved to section GFE with doors 1 and 2 blocked, and again allowed to explore for 3 minutes. The same was then repeated in area HIJ for another 3 minutes. Finally the mice were moved to area MLK with door 3 and door N blocked and allowed to explore for 5 minutes. The apparatus was cleaned using 30% isopropanol between each mouse and a new weigh boat with fresh gelatin was used for each mouse. On day 2 a fresh weigh boat containing a small amount of gelatin was placed in the area N and the test mouse was placed in area A. The amount of time and path used to reach the goal box was recorded. The number of repeated sections the mouse entered on the way to the goal were recorded. If the mouse did not reach the end after 5 minutes it was guided to the goal using a piece of acrylic plastic used to block the doors, to prevent back tracking and wrong turns. Each mouse received 5 trials, one every 15 minutes. The same procedures were then repeated on days 3 and 4 for a total of 15 trials per mouse.

Statistical analysis: All statistical analyses were done using Prism 6 (GraphPad Software, La Jolla, CA), for the repeated measures, two-way ANOVAs were used to analyze these data. Separate independent t-tests were performed when an interaction was found.

Results

The WT mice committed fewer errors when compared to the KO mice in the maze over the 15 trials F(1, 18) = 11.9, p<0.001 (Figure 2A). There was a significant effect in maze learning over the trials F(14, 252) = 12.9, p < 0.001. There was no interaction between groups over time F(14, 252) = 0.9, p = 0.52. There was no difference between Kv4.2 WT and KO mice in their time to complete the maze F(1, 18) = 0.01, p = 0.92 (Figure 2B). There was a significant decrease for both groups in the time to find the end of the maze across trials F(14, 252) = 4.8, p < 0.001. There was a group over time interaction F(14, 252) = 3.1, p < 0.01. We ran separate t-tests over the 15 trials and found significant differences only on the first trial t(1,18) = 2.2, p < 0.05.

dd79b102-8fc3-4465-8f6b-4f1cdb6822d3_figure2.gif

Figure 2. Number of errors and time to completion for maze.

There was a significant difference between genotypes with the WT mice committing fewer errors when compared to the KO mice. There was no difference in the time to completion of Lashley maze for the WT and KO mice. A. The graph reflects the number of errors committed by the WT and KO mice across the 15 trials. B. The graph shows the time to completion of the Lashley maze across the 15 trials between the WT and KO mice. There was a group × time interaction across the 15 trials. An independent t-test revealed a significant difference on the first trial. No other differences were found in the remaining 14 trials. WT n = 11, KO n = 9. * = p < 0.05; *** = p < 0.001.

Number of Errors
Trial #
MouseGenotype123456789101112131415
1WT8467541045444312
2WT255320424142223
3WT975246243421133
4WT784459333354534
5KO21181411891235546653
6WT859583477668434
7KO16126747545443534
8WT1685352532215234
9KO11104566445434334
10KO910119613769474545
11KO74593255115310743
12KO588657456593234
13KO31011978664462645
14WT1185569736578677
15KO161174611757699874
16KO88876888861011285
17WT1465443543433321
18WT996465443333333
19WT768323652230211
20WT1377695668626743
Time to maze completion (seconds)
Trial #
MouseGenotype123456789101112131415
1WT176134285369326297328335347348303309305338332
2WT118367344322317138308318309324182339313345351
3WT225361343321342293339362353352344344336354348
4WT120311320334337311342348336333331357396344353
5KO322336320319355313324169157319305333340340323
6WT11812422117133685124325346340308334354344326
7KO333356342318354310319345332334319339333341334
8WT32332932832232527433031430930678192318320310
9KO330353330350342307338337355340332340335327340
10KO390383372359367320348346334336325330338340332
11KO122591101698212897922281581312989716879
12KO263298323332333309325347349334358343322336310
13KO134165305344340338353317328326348312342338333
14WT348332362368335316346352358340340356349363364
15KO338281321312337295324318319308113319306338320
16KO345349346345350349338331342349349300124332225
17WT317321342337321341347330351339330331332321329
18WT136237344340330339345351321321322349339340347
19WT10824535332233317033732831831197164317323321
Dataset 1.Data for Kv4.2 knockout mice displaying learning and memory deficits in the Lashley maze.
Raw data for the number of errors to find the goal box (Figure 2A) and the duration to find the goal box (Figure 2B) are provided in a csv file.

Discussion

Kv4.2 wildtype and knockout mice demonstrated improvement in the Lashley maze by showing a reduction in the number of errors to find the goal box. However, the Kv4.2 KO mice committed more errors across the 15 trials compared to WT mice. By the end of the testing the Kv4.2 KO mice had similar performance to the WT mice, demonstrating that they showed improvement over the trials. The Kv4.2 KO mice showed impaired learning over the early phase of the Lashley Maze. One important consideration is that there was no difference between groups when examining the time to complete the maze. Kv4.2 KO mice required more time at the first trial, but the time to complete the maze was the same between groups for the remainder of the trials. The latency data suggest that Kv4.2 KO mice were not less active, which is in line with our previous study where we did not observe a difference in locomotor activity in the open field test18. One surprising observation was that Kv4.2 WT mice had a faster latency to find the reward on the first trial. This effect was only significant on the first trial. There may be a motivation difference between the WT and KO mice for the appetitive reward. Future studies could tease out this effect.

The results from the Lashley maze complement previous studies that reported spatial learning deficits in the MWM and contextual learning deficits in the delay fear conditioning test for Kv4.2 KO mice18,19. One of the benefits of the Lashley maze is that the impact of age and sensory abilities is reduced. Impaired vision will reduce the ability of the subject to find the hidden platform in the MWM and impaired hearing can attenuate the ability of the subject to associate a tone with an aversive shock. This is important as there have been several reports that suggest ion channels may contribute to aging-related impairment22,23. Additional sensory tests would need to be performed to examine baseline sensory abilities if older subjects are used in behavioral experiments, or another approach would be to use the Lashley maze. One caveat of the Lashley Maze is that it does not tease out if the mouse is using a spatial search strategy or procedural strategy. In order to discern which type of strategy the mouse is using, complementary tests of learning could be used. The low induction of stress makes the maze a beneficial test in models that have alterations in anxiety or age-related impairments which could account for differences seen in other more aversive learning tests.

Data availability

F1000Research: Dataset 1. Data for Kv4.2 knockout mice displaying learning and memory deficits in the Lashley maze, 10.5256/f1000research.9664.d13719324

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Smith GD, Gao N and Lugo JN. Kv4.2 knockout mice display learning and memory deficits in the Lashley maze [version 2; peer review: 2 approved]. F1000Research 2017, 5:2456 (https://doi.org/10.12688/f1000research.9664.2)
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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Key to Reviewer Statuses VIEW
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 2
VERSION 2
PUBLISHED 27 Feb 2017
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Reviewer Report 28 Feb 2017
Richard Brown, Department of Psychology and Neuroscience, Dalhousie University, Halifax, NS, Canada 
Approved
VIEWS 7
I confirm that I have read this submission and believe that I have an ... Continue reading
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Brown R. Reviewer Report For: Kv4.2 knockout mice display learning and memory deficits in the Lashley maze [version 2; peer review: 2 approved]. F1000Research 2017, 5:2456 (https://doi.org/10.5256/f1000research.11889.r20575)
NOTE: 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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VERSION 1
PUBLISHED 05 Oct 2016
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Reviewer Report 13 Jan 2017
Richard Brown, Department of Psychology and Neuroscience, Dalhousie University, Halifax, NS, Canada 
Approved
VIEWS 16
This paper tests male Kv4.2 KO mice in the Lashley type III maze at 60+ days of age.
 
It is well-written and I have only a few comments.
  1. Were mice pre-exposed to the
... Continue reading
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Brown R. Reviewer Report For: Kv4.2 knockout mice display learning and memory deficits in the Lashley maze [version 2; peer review: 2 approved]. F1000Research 2017, 5:2456 (https://doi.org/10.5256/f1000research.10413.r19280)
NOTE: 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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Reviewer Report 24 Nov 2016
Peter Backx, Division of Cardiology, Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada 
Approved
VIEWS 24
Nice succinct paper.  Relevant.
 
Suggested changes for the authors to consider:
  • In the methods, the authors should mention how errors were identified and quantified.
     
  • Figure 2B.  Is there
... Continue reading
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HOW TO CITE THIS REPORT
Backx P. Reviewer Report For: Kv4.2 knockout mice display learning and memory deficits in the Lashley maze [version 2; peer review: 2 approved]. F1000Research 2017, 5:2456 (https://doi.org/10.5256/f1000research.10413.r17911)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 14 Dec 2016
    Joaquin Lugo, Institute of Biomedical Sciences, Baylor University, Waco, 76798, USA
    14 Dec 2016
    Author Response
    We would like to thank the reviewer for their comments. We have included our replies below but will include a revised version of the manuscript when we have received both ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 14 Dec 2016
    Joaquin Lugo, Institute of Biomedical Sciences, Baylor University, Waco, 76798, USA
    14 Dec 2016
    Author Response
    We would like to thank the reviewer for their comments. We have included our replies below but will include a revised version of the manuscript when we have received both ... Continue reading

Comments on this article Comments (0)

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VERSION 2 PUBLISHED 05 Oct 2016
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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