Reversal learning paradigm reveals deficits in cognitive flexibility in the <i>Fmr1 </i>knockout male mouse

Suzanne O. Nolan; Joaquin N. Lugo

doi:10.12688/f1000research.14969.1

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Research Article

Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse

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

Suzanne O. Nolan¹, Joaquin N. Lugo ^1-3

PUBLISHED 07 Jun 2018

Author details Author details

¹ Department of Psychology and Neuroscience, Baylor University, Waco, TX, 76798, USA
² Institute of Biomedical Studies, Baylor University, Waco, TX, 76798, USA
³ Department of Biology, Baylor University, Waco, TX, 76798, USA

Suzanne O. Nolan
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Joaquin N. Lugo
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Validation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Loss of FMR1 is associated with Fragile X syndrome, amongst the most prevalent inherited intellectual disability. Despite extensive research in this area, previous studies have failed to detect consistent evidence of cognitive impairments in the Morris water maze (MWM) task in the Fmr1 knockout (KO) mouse. However, few studies have examined cognitive flexibility in a reversal form of the MWM task, which may illuminate subtle learning deficits.
Methods: Adult male Fmr1 wildtype (WT) and KO mice were bred and tested in the MWM reversal paradigm. The testing paradigm consisted of two blocks per day, with 4 trials per block to locate a hidden platform. After the last trials on the fourth day of testing, the animals were given a probe trial with the platform removed. The following week, the location of the platform was switched to the opposite quadrant and the animals received 2 more days of testing, with 4 blocks in total.
Results: As expected, Fmr1 KO mice did not display a learning deficit during the acquisition phase, F_genotype(1, 24) = 0.034, p = 0.854, and performed similarly on the probe trial, F_genotype(1, 23) = 0.024, p = 0.877. However, during the reversal phase of learning, Fmr1 KO mice showed deficits in their ability to learn the new location of the platform, F_genotype(1, 23) = 3.93, p = 0.059. Further independent samples t-testing revealed that KO animals displayed significantly higher latency to reach the hidden platform during the third trial, t(23) = -2.96, p < 0.01.
Conclusions: While previous studies have not demonstrated deficits in spatial memory in the Fmr1 KO model, it is possible that the acquisition phase of the task is less sensitive to deficits in learning. Future studies using this model to evaluate therapeutic interventions should consider utilizing the MWM reversal paradigm.

Keywords

Cognitive flexibility, Autism, Morris water maze, Prefrontal cortex, FMRP

Corresponding author: Joaquin N. Lugo

Competing interests: No competing interests were disclosed.

Grant information: This research was supported by funding from the National Institutes of Health, National Institute of Neurological Disorders and Stroke [NS088776]
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2018 Nolan SO and Lugo JN. 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. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Nolan SO and Lugo JN. Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:711 (https://doi.org/10.12688/f1000research.14969.1) First published: 07 Jun 2018, 7:711 (https://doi.org/10.12688/f1000research.14969.1) Latest published: 07 Jun 2018, 7:711 (https://doi.org/10.12688/f1000research.14969.1)

Introduction

Fragile X syndrome (FXS) is a neurodevelopmental disorder, caused by a trinucleotide expansion mutation in the FMR1 gene, and is also one of the most prevalent inherited forms of intellectual disability¹. FXS is often modeled using the Fmr1 knockout (KO) mouse, which can be characterized by several behavioral phenotypes, including alterations in sociability and deficits in fear memory^2–4. Aside from deficits in spatial and non-spatial learning, one understudied facet of intellectual disability is the ability to incorporate new information into existing learning, termed cognitive flexibility. Cognitive flexibility can be studied in rodents using a variant of the Morris water maze (MWM) paradigm^5,6. In the MWM reversal paradigm, the location of the hidden platform is moved, and the latency to adjust to the new location is measured. As expected, several reports find evidence of impairments in reversal learning in the Fmr1 KO mouse across multiple strains, the C57BL/6J backcrossed strain^7,8 and the albino C57BL/6J background². However, other studies have been unable to replicate these findings, and further investigation points to the possibility of background strain differences⁹. Previous reports have not detected any impairments in reversal learning in the FVB.129 strain¹⁰. However, it may be that this paradigm is perhaps even more sensitive to methodological differences. The current study adds to this literature by using the FVB.129 strain in a previously utilized paradigm.

Methods

Animals

Male Fmr1^+/+ and female Fmr1^+/- FVB.129P2-Pde6b+Tyrc-ch Fmr1tm1Cgr/J (Stock No: 004624, The Jackson Laboratory, Bar Harbor, ME, USA) mice were used as breeders (9 total breeding pairs) to produce the following groups: male WT and male KO pups. Breeding pairs were of the following groupings: WT Female/WT Male (n = 2), KO Female/KO Male (n = 5), WT Female/KO Male (n = 2). Genotype was determined from toe clippings taken prior to age postnatal day (PD) 12 (Mouse Genotype, Escondido, CA, USA). The final sample sizes were as follows: n_{male WT} = 10, n_{male KO} = 16. Target samples sizes (n = 10) were calculated a priori using a power calculation in G*Power 3.1 with the following parameters: f = 0.50 (large effect), α = 0.05, power (1 – β) = 0.80, for the F family of tests with two groups and 8 repeated measures (trials). All pups were housed in individual cages (Allentown Caging PC7115HT, Allentown, PA, USA), filled with sani-chip bedding (7090 Teklad, Envigo, Somerset, NJ, USA). Prior to weaning on PD21, pups were housed with parents (1 male and 2 females) and littermates (up to 12 pups). Following weaning, subjects were housed with mixed genotype littermates, no more than 5 to a cage. The light cycle was kept at 12 hr. light, and the colony room was kept at an ambient temperature of 22° C. Animals had ad libitum access to food and water. All procedures performed were in accordance with Baylor University Institutional Animal Care and Use Committee (Animal Assurance Number A3948-01), as well as the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All efforts were made to ameliorate any stress and harm to the animals, specifically by habituating animals to the testing apparatus and room prior to trial recordings.

Morris water maze

All behavioral testing was conducted during the light cycle, specifically between 8 am and 5 pm. The methods for the current study were adapted as closely as possible from earlier studies of this behavior in the Fmr1 KO mouse (represented in Figure 1)⁹. Briefly, a 1.3 m diameter white pool was filled with water and made opaque through the addition of non-toxic white paint (Item LT3010, S&S Worldwide, Connecticut). The hidden platform measured 14.5 cm × 14.5 cm and was submerged approximately 2 cm below the water level. The testing paradigm consisted of two blocks per day for 4 days, with 4 trials in each block, for each mouse to test the ability to locate a hidden platform. The mice were habituated to the testing room in their holding cages for 30 minutes prior to the onset of testing. The amount of time spent in each quadrant for each trial was recorded with a ceiling-mounted video camera (Ganz YCH-02, Cary, NC, USA), and analyzed using automated tracking software (Ethovision XT 6, Noldus, Wageningen, Netherlands). After the last trial on the fourth day of testing was completed, the animals were given a probe trial. The probe trial involved removing the platform and allowing the subjects to explore the maze for 60 seconds. During the probe trial, the number of times the animal crossed the location of the hidden platform and the duration of time in each quadrant was calculated. Testing resumed on day 8 after a 3-day rest period. On day 8, the platform was placed in the opposite quadrant from the previous location that housed the hidden platform. Testing progressed as with the initial acquisition phase, with 2 blocks per day for 2 days. On the final day of testing, a visible platform was used to evaluate visual performance as well as swim speed. The visible platform was a two-tiered platform similar to the initial platform, with a second higher tier platform that extended 9.5 cm above the lower platform, allowing the animal to see the platform. The differences in methodology from the cited source were as follows: only four days of acquisition were conducted and the testing paradigm was lengthened to account for a consolidation period between the learning and reversal trials (See Figure 1 for a description of the testing paradigm). One KO animal was excluded from analysis due to a seizure during this task.

Figure 1. Overview of the testing paradigm.

A. An overview of the set-up of the testing arena. B. An overview of the progression of testing days.

Statistical analysis

Statistical analysis was performed in the form of a one-way analysis of variance (ANOVA) with one between-subjects factor (Genotype [wildtype, knockout]) and one within-subjects factor (Trial). All data were analyzed using GraphPad Prism Software 7.0 (San Diego, CA, USA) or IBM SPSS Statistics 23 (Aramonk, NY, USA).

Results

Fmr1 KO mice show no impairment in acquisition of spatial memory

To investigate the effect of genotype on hippocampal spatial memory, animals were tested in the MWM paradigm (Dataset 1). During the 8 blocks of learning trials (Figure 2A), there was a significant within-subjects effect of trial for latency to reach the platform, F(3.56, 85.47) = 30.15, p < .0005. Trial results did not interact significantly with genotype, F(3.56, 85.47) = 1.33, p = 0.24, suggesting both groups learned the location of the platform similarly. Between-subjects analyses indicated no effect of genotype, F(1, 24) = 0.03, p = 0.85. Further independent samples t-testing revealed no differences between WT and KO at any of the 8 different trials, p > 0.05.

For the probe trial (Dataset 2), as expected, male KO mice demonstrated similar time spent in the target quadrant, F(1,23) = 0.02, p = 0.88 (Figure 2B), compared to male wildtype (WT). Further independent samples t-testing revealed no differences in duration in any of the quadrants, p > 0.05.

Figure 2. Performance in the Morris water maze in Fmr1 knockout males.

A. Fmr1 knockout males show no deficits in acquisition in performance. B. Performance during the probe trial is not impaired in the Fmr1 knockout males. C. When subjected to a reversal learning paradigm, Fmr1 knockout mice display increased latency to the new platform location, demonstrating deficits in cognitive flexibility. Knockout – KO, Wild type - WT.

Loss of Fmr1 impairs ability to update existing learning with new platform location

The week following the initial learning trials, animals were tested in the reversal learning paradigm (Dataset 3). During the 4 blocks of learning trials (Figure 2C), a one-way ANOVA with repeated measures revealed a significant within-subjects effect of trial, F(3, 69) = 3.8, p < 0.05. Trial results did not interact significantly with genotype, F(3, 69) = 1.28, p = 0.29. However, between-subjects analyses indicated a marginal effect of genotype, F(1, 23) = 3.93, p = 0.059 (Figure 2C). Further independent samples t-testing revealed that KO animals displayed significantly higher latency to reach the hidden platform during the third trial, t(23) = -2.96, p < 0.01. Together, these results demonstrate that Fmr1 KO males demonstrate decreased learning and altogether a lack of cognitive flexibility across all trials of the MWM reversal task.

Impairments were not due to deficits in vision or motor capabilities

Visible platform information was also assessed to ensure differences were not due to deficits in vision (Dataset 4). Results were analyzed using a repeated-measures ANOVA across the four visible platform trials on latency to the platform across the two blocks of trials. Results indicated no effect of block, F(1, 24) = 1.341, p = 0.26, nor an interaction of block and genotype, F(1, 24) = 0.0005, p = 0.98. There was not a significant effect of genotype on latency to the platform during these trials either, F(1, 24) = 0.98, p = 0.33 (Figure 3A). Altogether, these data suggest that differences in latency to the platform could not be attributed to deficits in visual perception in the Fmr1 KO male mouse. Moreover, differences in latency to the platform on the previous trials could not be attributed to impairments in swimming abilities, as no differences in swim speed were detected during the visible trials, t(11.17) = 1.526, p = 0.16 (Figure 3B).

Figure 3. Performance in the Morris water maze visible trials.

A. Fmr1 knockout males showed no deficits in latency to the platform across the two blocks of the visible platform test. B. Fmr1 knockout males showed no deficits in swim speed across the visible platform test. Knockout – KO, Wild type - WT.

Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data			Average Duration	Average Duration	Average Duration	Average Duration	Average Duration	Average Duration	Average Duration	Average Duration	Average Velocity
Trial #	Duration	subject	block	direction	Total	Mean	Subject	Genotype	Block 1	Block 2	Block 3	Block 4	Block 5	Block 6	Block 7	Block 8	All 8 Blocks
					cm	cm/s	124	KO	48.22322275	34.43443375	19.06906875	12.0120115	10.9609605	16.691691	13.538538	12.537537	21.5976605
Trial 1	60.16016	124	1	E	633.2249946	20.87762096	124	KO	59.984985	34.209209	16.84184125	21.87187125	9.85985925	10.1351345	10.46046	13.38838775	22.77836917
Trial 2	60.16016	124	1	W	602.5090273	19.35391002	133	KO	46.39639625	23.0980975	12.98798725	14.664664	7.807807	8.2582575	11.71171125	9.85985925	27.26860168
Trial 3	23.023022	124	1	S	207.3483097	17.26174456	134	KO	26.02602575	10.53553475	27.82782725	16.541541	10.53553475	5.8558555	7.75775725	7.85785725	25.88363122
Trial 4	49.549549	124	1	N	455.5212091	16.13707957	134	WT	47.82282225	16.666666	22.22222175	10.91091025	11.611611	18.76876825	8.75875825	15.01501425	27.44120934
Trial 49	53.953953	124	2	S	622.3887475	20.31915438	150	WT	25.575575	17.99299225	9.409409	8.90890825	7.9579575	8.383383	7.382382	6.381381	23.59443469
Trial 50	16.616616	124	2	W	203.0753864	21.13252475	151	KO	45.295295	12.23723675	19.84484425	15.59059025	11.9119115	7.85785725	12.31231175	12.11211175	28.63869212
Trial 51	46.946946	124	2	N	498.4885107	18.24138763	157	KO	40.16516475	40.54054025	12.93793725	9.6346345	23.498498	25.40039975	17.89289225	22.57257225	27.30209852
Trial 52	20.22022	124	2	E	185.1071711	15.80529845	158	WT	27.27727675	18.643643	10.0600595	7.507507	5.505505	10.93593525	7.93293225	8.53353325	27.85379228
Trial 1	6.106106	124	3	E	77.2886173	27.57548115	160	WT	17.26726675	16.76676625	10.71071025	13.28828775	18.9689685	9.18418375	9.409409	14.2142135	27.38955179
Trial 2	3.203203	124	3	S	34.72472505	17.34501751	161	KO	54.479479	31.85685625	31.881881	42.292292	10.91091025	14.43943875	14.1641635	17.1921915	25.07076942
Trial 3	28.628628	124	3	N	332.0396484	19.86272623	167	KO	46.22122075	55.58058025	19.0940935	16.64164125	6.95695625	20.645645	11.88688625	21.9969965	26.04063588
Trial 4	38.338338	124	3	W	389.2762109	17.8388498	168	KO	33.383383	16.8168165	8.43343275	12.637637	8.658658	6.9569565	10.83583525	13.8638635	29.18735602
Trial 49	7.807807	124	4	W	110.5983547	26.30661792	177	KO	31.306306	32.9829825	23.1731725	44.49449425	17.592592	24.97497425	7.8828825	9.43443375	28.32697843
Trial 50	12.212212	124	4	N	204.3344168	30.01913465	178	KO	14.31431375	22.6726725	22.44744675	27.57757725	20.32032	17.2672665	17.6426425	21.371371	29.13973879
Trial 51	7.107107	124	4	E	56.96009507	15.80641403	184	WT	36.73673625	29.72972925	15.74074	26.45145075	6.0310305	17.06706675	11.0610605	13.938938	23.7113124
Trial 52	20.92092	124	4	S	214.5022149	19.84145424	186	WT	39.31431375	8.08308225	34.8098095	7.05705625	11.86186125	9.1591585	3.503503	18.2182175	28.39247078
Trial 1	22.722722	124	5	E	282.669289	21.23207551	188	WT	28.37837775	13.76376325	28.35335275	11.686686	30.0300295	23.27327275	12.562562	15.31531475	24.73035054
Trial 2	7.407407	124	5	N	79.43332537	20.34714192	189	WT	22.0470465	30.205205	11.68668625	14.2642635	20.72072025	9.684684	6.83183125	17.642642	30.16752414
Trial 3	4.404404	124	5	S	47.26954876	17.48972692	197	KO	34.8598595	18.74374325	30.755755	16.3163155	7.9829825	11.28628575	4.4294285	8.40840775	27.09124933
Trial 4	9.309309	124	5	W	107.8963321	18.58422677	198	KO	30.2552545	23.97397325	13.63863825	11.711711	5.98098025	12.1621615	7.1071065	6.25625575	27.44491403
Trial 49	36.236236	124	6	N	463.2618478	20.94110966	203	WT	60.135135	30.65565475	12.8378375	21.546546	9.70970925	11.9869865	12.1871865	16.0660655	22.80465199
Trial 50	13.513513	124	6	W	152.8808382	21.51098642	204	WT	49.09909875	18.39339275	43.7687685	41.9919915	34.8098095	25.32532475	15.21521475	37.06206175	24.39882581
Trial 51	5.005004	124	6	E	78.92979502	34.28301251	229	KO	25.8008005	17.9679675	16.46646575	13.538538	12.337337	11.411411	4.90490425	8.53353275	26.68884999
Trial 52	12.012011	124	6	S	125.6971795	19.31868176	230	KO	32.3823815	12.0870865	21.92192125	9.83483425	6.731731	7.407407	9.509509	4.654654	28.28967034
Trial 1	3.003002	124	7	S	48.1274651	25.3049171	231	KO	60.11011	25.9759755	12.81281225	9.15915875	10.18518475	10.83583525	12.21221175	6.506506	28.27398499
Trial 2	4.104104	124	7	W	54.77437209	22.79983284
Trial 3	20.42042	124	7	N	229.8141232	20.68327478
Trial 4	26.626626	124	7	E	299.0682218	19.40059148
Trial 51	2.502502	124	8	W	49.40604669	37.96668461
Trial 52	6.906906	124	8	E	93.23005694	23.88124195
Trial 53	20.32032	124	8	S	234.9142956	21.33447983
Trial 54	20.42042	124	8	N	232.3888858	22.32273594
Trial 5	59.95996	125	1	E	653.5735313	19.66626903
Trial 6	60.16016	125	1	W	626.2073511	19.01462082
Trial 7	59.85986	125	1	S	540.8274449	17.59890796
Trial 8	59.95996	125	1	N	545.7236131	17.41782499
Trial 53	38.138138	125	2	S	504.0163579	22.37832449
Trial 54	13.313313	125	2	W	130.0586046	16.65749324
Trial 55	59.95996	125	2	N	687.2841052	21.19125478
Trial 56	25.425425	125	2	E	328.8861944	21.06136675
Trial 5	47.347347	125	3	E	606.4564929	23.39188637
Trial 6	3.703703	125	3	S	34.75322312	15.09498521
Trial 7	7.907907	125	3	N	93.22083404	20.69502133
Trial 8	8.408408	125	3	W	100.4288725	22.29519799
Trial 53	10.61061	125	4	W	175.6750419	26.193937
Trial 54	40.54054	125	4	N	548.8694804	23.63449935
Trial 55	7.407407	125	4	E	119.1451281	23.33843568
Trial 56	28.928928	125	4	S	352.0193404	22.54277933
Trial 5	2.602602	125	5	E	76.47149161	47.74691898
Trial 6	4.804804	125	5	N	69.26596161	24.71310666
Trial 7	24.524524	125	5	S	289.6315623	20.37618419
Trial 8	7.507507	125	5	W	73.79589674	18.90309998
Trial 53	12.012011	125	6	N	114.0185826	19.63871982
Trial 54	5.905905	125	6	W	54.95854062	21.11676358
Trial 55	7.407407	125	6	E	89.30140414	23.47687675
Trial 56	15.215215	125	6	S	176.2005017	22.28155105
Trial 5	11.411411	125	7	S	192.4572051	27.46639573
Trial 6	13.513513	125	7	W	159.0286984	22.69568179
Trial 7	6.706706	125	7	N	54.00473122	21.58030444
Trial 8	10.21021	125	7	E	133.9274373	21.57959987
Trial 55	2.802802	125	8	W	25.13687727	20.92647126
Trial 56	8.708708	125	8	E	156.5404143	28.95997814
Trial 57	3.003002	125	8	S	60.13654303	33.37580768
Trial 58	39.039039	125	8	N	453.7330852	21.89754915
Trial 9	60.16016	133	1	E	696.7506028	23.51533156
Trial 10	29.129129	133	1	W	349.6950104	21.83407943
Trial 11	36.136136	133	1	S	415.1632627	20.43094249
Trial 12	60.16016	133	1	N	737.1701374	21.04094289
Trial 57	4.904904	133	2	S	51.12348547	23.21470822
Trial 58	14.014014	133	2	W	179.6799606	25.28173443
Trial 59	55.055054	133	2	N	755.8783891	24.758115
Trial 60	18.418418	133	2	E	232.5842808	24.20329687
Trial 9	17.217217	133	3	E	225.4413666	24.47998391
Trial 10	11.911911	133	3	S	210.3497459	26.94095021
Trial 11	6.906906	133	3	N	100.1680776	26.33364954
Trial 12	15.915915	133	3	W	236.1159572	24.31751277
Trial 97	19.919919	133	4	W	289.8066981	27.57303944
Trial 98	6.706706	133	4	n	87.04236338	20.22216653
Trial 99	26.026025	133	4	E	310.5817369	26.29416026
Trial 100	6.006006	133	4	s	56.17326816	18.70570658
Trial 9	6.906906	133	5	E	108.1981104	31.79114285
Trial 10	6.006005	133	5	N	110.2269335	35.52152322
Trial 11	13.613613	133	5	S	265.0437538	34.83929192
Trial 12	4.704704	133	5	W	70.96617921	27.26740479
Trial 57	8.408408	133	6	N	194.3711185	35.95866048
Trial 58	4.004003	133	6	W	61.51476138	32.34384635
Trial 59	13.613613	133	6	E	230.4128535	30.6910015
Trial 60	7.007006	133	6	S	86.15100787	26.08023511
Trial 9	11.211211	133	7	S	146.7300157	24.84462934
Trial 10	14.314314	133	7	W	259.487243	29.79629274
Trial 11	12.912912	133	7	N	205.2040548	27.33318799
Trial 12	8.408408	133	7	E	168.9739466	38.36477746
Trial 59	15.515515	133	8	W	258.5362692	29.34973382
Trial 60	6.406406	133	8	E	135.0999945	38.5614087
Trial 61	4.604604	133	8	S	53.13566164	23.07936397
Trial 62	12.912912	133	8	N	188.0478388	27.62643344
Trial 13	4.104104	134	1	E	149.9132073	65.11453821
Trial 14	60.16016	134	1	W	664.9208441	20.00770772
Trial 15	12.312312	134	1	S	176.3360866	26.29250385
Trial 16	27.527527	134	1	N	276.5077893	18.05432553
Trial 61	21.821821	134	2	S	301.3467933	25.08711985
Trial 62	5.905905	134	2	W	55.73526849	21.4152265
Trial 63	6.406406	134	2	N	85.33513526	23.68049907
Trial 64	8.008007	134	2	E	79.78296585	18.53561882
Trial 13	25.825825	134	3	E	364.0731185	25.6133212
Trial 14	60.16016	134	3	S	675.8848697	21.16642571
Trial 15	18.318318	134	3	N	197.6640034	20.14963355
Trial 16	7.007006	134	3	W	76.31428848	16.94178015
Trial 61	17.217217	134	4	W	249.4260139	23.95928518
Trial 62	24.824824	134	4	N	313.7391338	24.87502247
Trial 63	15.515515	134	4	E	155.5499966	20.44663566
Trial 64	8.608608	134	4	S	121.2465525	26.33158077
Trial 13	11.811811	134	5	E	228.0912827	29.59262779
Trial 14	14.514514	134	5	N	184.7709602	23.66491125
Trial 15	10.010009	134	5	S	164.2696939	28.79042753
Trial 16	5.805805	134	5	W	75.54157646	20.39622496
Trial 61	7.207207	134	6	N	144.1887078	29.39685442
Trial 62	4.304304	134	6	W	76.12990335	26.22544899
Trial 63	2.502502	134	6	E	52.46017044	34.93849012
Trial 64	9.409409	134	6	S	133.1273008	25.57581186
Trial 13	7.507507	134	7	S	113.8715929	25.85402504
Trial 14	6.506506	134	7	W	86.8072669	27.10012837
Trial 15	7.107107	134	7	N	87.24774523	24.21125533
Trial 16	9.909909	134	7	E	183.9280441	31.68003089
Trial 63	6.006005	134	8	W	53.12508051	16.08242037
Trial 64	12.412412	134	8	E	275.7961492	29.31068998
Trial 65	5.505505	134	8	S	137.3610784	30.49417946
Trial 66	7.507507	134	8	N	120.2024834	27.29144837
Trial 17	60.06006	150	1	E	813.4181524	22.14181685
Trial 18	54.554554	150	1	W	499.7910556	17.04065682
Trial 19	48.748748	150	1	S	508.3650165	19.83815099
Trial 20	27.927927	150	1	N	325.376256	19.58137283
Trial 65	43.743743	150	2	S	653.5064239	25.70287979
Trial 66	10.71071	150	2	W	95.07203578	17.26853383
Trial 67	6.506506	150	2	N	108.4742102	29.28805742
Trial 68	5.705705	150	2	E	87.95843896	27.45952961
Trial 17	22.222222	150	3	E	305.1638886	28.49147273
Trial 18	1.501501	150	3	S	22.70521463	25.20278232
Trial 19	29.229229	150	3	N	401.9505596	24.04482402
Trial 20	35.935935	150	3	W	472.8143164	22.81844287
Trial 65	5.805805	150	4	W	86.750677	30.95141429
Trial 66	10.91091	150	4	N	173.4170229	29.36331407
Trial 67	13.513513	150	4	E	246.9625404	44.85740021
Trial 68	13.413413	150	4	S	248.7193877	34.03708531
Trial 17	15.615615	150	5	E	234.4884954	28.22337142
Trial 18	9.509509	150	5	N	161.4125404	31.61788443
Trial 19	4.504504	150	5	S	46.82347094	21.26211488
Trial 20	16.816816	150	5	W	197.9790055	22.47510962
Trial 65	21.321321	150	6	N	324.2506839	24.53988693
Trial 66	14.014013	150	6	W	195.4212212	23.80802427
Trial 67	14.614614	150	6	E	205.1031142	26.96025533
Trial 68	25.125125	150	6	S	323.299425	22.27420833
Trial 17	14.614614	150	7	S	178.6344088	24.11566143
Trial 18	6.006006	150	7	W	77.10883838	26.56267366
Trial 19	5.605605	150	7	N	88.90846714	29.60651878
Trial 20	8.808808	150	7	E	119.2102821	28.35503221
Trial 67	18.818818	150	8	W	260.5552418	25.02832742
Trial 68	2.602602	150	8	E	96.81907379	74.40179603
Trial 69	14.614614	150	8	S	286.788627	27.02847546
Trial 70	24.024023	150	8	N	323.6176216	23.77162485
Trial 21	15.015014	151	1	E	255.2319396	34.45632769
Trial 22	60.16016	151	1	W	675.8365801	20.27509234
Trial 23	6.506506	151	1	S	68.26937901	22.00037395
Trial 24	20.62062	151	1	N	213.9418462	21.80896641
Trial 69	29.829829	151	2	S	326.2143208	21.72587975
Trial 70	17.617617	151	2	W	219.8200187	21.96002018
Trial 71	15.615615	151	2	N	182.0112848	21.9071352
Trial 72	8.908908	151	2	E	127.4414014	27.67695003
Trial 21	16.816816	151	3	E	218.5491021	22.2786267
Trial 22	11.511511	151	3	S	180.8711482	27.37731961
Trial 23	6.406406	151	3	N	105.5282704	28.49264235
Trial 24	2.902903	151	3	W	38.97525656	24.33516934
Trial 69	5.905905	151	4	W	75.28223518	26.8596298
Trial 70	10.31031	151	4	N	166.4050265	33.24761166
Trial 71	11.411411	151	4	E	110.7795858	18.14243849
Trial 72	8.008007	151	4	S	75.01549613	19.72117185
Trial 21	2.202202	151	5	E	26.41836049	21.99329109
Trial 22	15.615615	151	5	N	202.1066056	22.68589948
Trial 23	4.404404	151	5	S	54.26675712	18.69395997
Trial 24	9.609609	151	5	W	100.8919874	19.01718609
Trial 69	15.915915	151	6	N	263.9110969	27.75234064
Trial 70	6.106106	151	6	W	61.94359383	20.62721441
Trial 71	8.408408	151	6	E	130.7385616	29.68359248
Trial 72	3.103103	151	6	S	26.317439	13.83741753
Trial 21	2.502502	151	7	S	25.08066553	17.89685844
Trial 22	3.103103	151	7	W	38.49384129	27.46812736
Trial 23	18.218218	151	7	N	245.2384459	25.25703028
Trial 24	5.705705	151	7	E	76.44576406	24.63526743
Trial 71	4.104104	151	8	W	68.13492121	21.27086999
Trial 72	7.907907	151	8	E	122.6346734	25.52333853
Trial 73	5.205205	151	8	S	52.43267458	16.36881685
Trial 74	8.308308	151	8	N	129.3242759	30.04534426
Trial 1	31.831831	157	1	E	475.8334928	27.16329967
Trial 2	60.06006	157	1	W	673.8026654	20.52222175
Trial 3	60.16016	157	1	S	665.2435472	19.26313852
Trial 4	29.129129	157	1	N	285.1065937	17.36715769
Trial 55	6.406406	157	2	S	83.43029487	24.51378232
This is a portion of the data; to view all the data, please download the file.

Dataset 1.Learning Trial Data.

This datasheet contains the raw data exported from the Ethovision program (Columns A – G) as well as the transformed dataset that was used for analysis for the learning trials (Columns J – T)

Raw Data				Distance moved	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	In zone	Velocity	Organized Data
					Arena	Arena	Inner	Inner	Outer	Outer	OQuad	OQuad	LQuad2	LQuad2	OZone	OZone	RZone	RZone	Tzone	Tzone	LZone	LZone	TQuad1	TQuad1	RQuad	RQuad				Duration in Quad	Duration in Quad	Duration in Quad	Duration in Quad
	subject	block	direction	Total	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Duration	Frequency	Mean	Subject	Genotype	Opposite	Left	Target	Right
				cm	s		s		s		s		s		s		s		s		s		s		s		cm/s	150	WT	13.613613	15.115116	16.816817	14.614614
Trial 1	124	probe	N	827.2509154	60.16016	1	46.146147	8	14.014013	9	8.308308	4	8.208208	3	2.102102	3	1.801801	4	1.201202	3	0.600601	1	25.625625	8	18.018019	7	24.45039738	151	WT	5.805805	6.206205	29.32933	18.71872
Trial 2	125	probe	N	852.5328268	60.16016	1	44.644645	10	15.515515	10	8.608609	5	5.705705	3	0.2002	1	2.102103	5	3.203204	6	0.4004	1	26.626627	8	19.219219	9	23.72368797	160	WT	9.60961	17.217217	24.224225	8.908908
Trial 3	133	probe	N N	1009.724444	60.16016	1	49.44945	5	10.71071	5	8.108108	7	24.924924	11	1.201201	3	0.700702	2	1.601602	3	2.502501	5	22.422422	10	4.704706	5	29.23811234	161	WT	3.403403	4.704704	30.930931	20.920922
Trial 4	134	probe	N	814.213819	60.16016	1	54.254256	8	5.905904	8	5.205206	4	21.621621	11	-	0	0.5005	1	3.403404	9	4.804805	8	31.031031	9	2.302302	1	24.72338691	184	WT	5.305305	13.513513	34.534536	6.706706
Trial 5	150	probe	N	796.782791	60.16016	1	57.657659	4	2.502501	4	13.613613	12	15.115116	8	0.3003	1	0.1001	1	0.2002	1	1.601603	3	16.816817	8	14.614614	12	24.41674164	186	WT	11.711712	20.620619	19.819821	7.907908
Trial 6	151	probe	N	754.8791595	60.06006	1	56.456456	3	3.603604	3	5.805805	7	6.206205	6	-	0	1.8018	6	1.901902	4	-	0	29.32933	11	18.71872	11	22.24556175	188	WT	15.615615	6.606608	23.523523	14.414414
Trial 1	157	probe	N	925.2804685	60.16016	1	44.544545	10	15.615615	10	9.60961	6	19.619619	9	-	0	0.1001	1	2.202203	5	1.901901	4	25.825825	8	5.105106	5	26.9491395	189	WT	8.308309	19.619617	23.823826	8.408408
Trial 2	158	probe	N	908.6026902	60.06006	1	44.644645	9	15.415415	10	7.507508	4	6.706707	4	-	0	1.501501	3	2.702704	6	0.7007	2	21.021022	8	24.824823	8	24.93664948	203	WT	6.306305	10.410411	24.724724	18.71872
Trial 3	160	probe	N	959.4301602	59.95996	1	53.753752	6	6.206208	6	9.60961	6	17.217217	9	0.600601	2	0.600601	1	3.003003	9	1.401402	4	24.224225	10	8.908908	7	28.78290244	204	WT	17.717717	12.912912	12.312313	17.217218
Trial 4	161	probe	N	842.8445021	59.95996	1	51.551551	11	8.408409	11	3.403403	3	4.704704	3	-	0	3.203203	4	3.603603	8	0.3003	1	30.930931	10	20.920922	9	24.98521767	124	KO	8.308308	8.208208	25.625625	18.018019
Trial 5	167	probe	N	771.5915146	60.16016	1	36.036038	13	24.124122	13	11.811811	6	24.624624	7	1.801802	4	0.500501	1	1.301302	4	0.4004	1	15.715717	6	8.008008	4	24.47047003	125	KO	8.608609	5.705705	26.626627	19.219219
Trial 6	168	probe	N	984.9348379	60.16016	1	47.147148	8	13.013012	9	12.612613	8	12.612611	7	1.7017	5	0.3003	1	0.800801	3	0.800801	3	17.917918	7	17.017018	9	28.11285682	133	KO	8.108108	24.924924	22.422422	4.704706
Trial 7	184	probe	N	832.1007258	60.06006	1	49.849849	9	10.210211	9	5.305305	3	13.513513	8	0.8008	1	1.001001	1	3.403401	10	1.2012	3	34.534536	10	6.706706	4	24.23524297	134	KO	5.205206	21.621621	31.031031	2.302302
Trial 8	186	probe	N	935.3671959	60.06006	1	53.553555	5	6.506505	5	11.711712	9	20.620619	10	1.301302	3	0.4004	1	1.301302	4	1.801803	5	19.819821	8	7.907908	6	27.24290702	157	KO	9.60961	19.619619	25.825825	5.105106
Trial 9	188	probe	N	810.5966978	60.16016	1	50.350351	9	9.809809	9	15.615615	7	6.606608	7	0.8008	2	1.301301	4	2.402402	6	0.100101	1	23.523523	8	14.414414	8	23.81723042	158	KO	7.507508	6.706707	21.021022	24.824823
Trial 10	189	probe	N	985.2540259	60.16016	1	50.65065	8	9.50951	8	8.308309	6	19.619617	9	1.101101	3	0.8008	3	1.401402	4	0.8008	2	23.823826	11	8.408408	7	27.96218871	167	KO	11.811811	24.624624	15.715717	8.008008
Trial 11	176	probe	N	811.4817807	60.16016	1	49.249251	6	10.910909	6	7.607608	5	14.814816	8	1.401401	2	-	0	4.804804	8	0.7007	2	35.135134	10	2.602602	5	23.29512099	168	KO	12.612613	12.612611	17.917918	17.017018
Trial 12	177	probe	N	817.2970633	60.16016	1	53.953954	5	6.206206	5	11.611614	8	21.02102	9	0.4004	1	0.600601	1	4.504505	7	2.002001	5	20.720721	7	6.806805	6	24.59276168	176	KO	7.607608	14.814816	35.135134	2.602602
Trial 13	178	probe	N	908.0274112	60.06006	1	43.643644	10	16.416416	10	13.213213	6	23.923923	9	0.7007	2	0.400401	1	0.900902	2	2.602603	6	15.415416	7	7.507508	4	27.40542208	177	KO	11.611614	21.02102	20.720721	6.806805
Trial 1	197	probe	N	890.0505115	60.16016	1	49.14915	10	11.01101	9	9.709709	4	18.318319	9	1.201203	3	0.7007	2	4.604604	7	2.1021	5	26.626627	9	5.505505	5	25.2602424	178	KO	13.213213	23.923923	15.415416	7.507508
Trial 2	198	probe	N	834.1395355	60.06006	1	57.057058	4	3.003002	4	10.810811	8	11.711711	9	1.701703	4	0.4004	2	2.702703	5	0.900902	2	25.025024	11	12.512514	9	25.09957071	197	KO	9.709709	18.318319	26.626627	5.505505
Trial 3	203	probe	N	772.2139012	60.16016	1	49.949949	7	10.210211	7	6.306305	6	10.410411	5	0.700701	2	1.601601	2	3.803805	6	0.2002	1	24.724724	9	18.71872	9	22.29600535	198	KO	10.810811	11.711711	25.025024	12.512514
Trial 4	204	probe	N	936.3513733	60.16016	1	21.42142	9	38.73874	10	17.717717	7	12.912912	5	0.400401	1	-	0	0.900902	2	0.500501	1	12.312313	5	17.217218	7	27.92283915	229	KO	5.305304	26.126127	22.222223	6.306306
Trial 5	229	probe	N	953.3006843	59.95996	1	41.741744	12	18.218216	12	5.305304	4	26.126127	8	0.4004	1	0.8008	2	1.501501	4	2.302303	5	22.222223	9	6.306306	5	26.82668573	231	KO	12.012012	19.51952	23.123123	5.505505
Trial 6	230	probe	N	70.84334598	12.912912	1	-	0	12.912912	1	11.51151	3	-	0	-	0	-	0	-	0	-	0	-	0	1.401402	2	58.97714337

Dataset 2.Probe Trial Data.

This datasheet contains the raw data exported from the Ethovision program (Columns A - AB) as well as the transformed dataset that was used for analysis for the probe trial (Columns AC – AI)

Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data			Average Duration	Average Duration	Average Duration	Average Duration
	In zone				Distance moved	Velocity	Subject	Genotype	Block 1	Block 2	Block 3	Block 4
	Arena						150	WT	12.512512	20.14514475	5.505505	9.759759
	Duration	subject	block	direction	Total	Mean	151	WT	13.9389385	10.61061	11.661661	19.0190185
	s				cm	cm/s	160	WT	32.65765725	45.99599575	21.72172125	22.0470465
Trial 1	36.736736	124	R1	N	440.2199575	20.36017206	161	WT	25.40039975	11.48648575	4.45445375	10.28528475
Trial 2	6.506506	124	R1	E	143.1804723	34.0565138	184	WT	29.679679	7.332332	6.1561555	16.0910905
Trial 3	16.116116	124	R1	S	187.0261613	21.98107791	186	WT	19.344344	7.9829825	8.2832825	7.0070065
Trial 4	22.122122	124	R1	W	234.9306267	18.62664016	188	WT	12.9629625	5.83083025	5.605605	5.95595525
Trial 50	35.735735	124	R2	W	517.0300528	26.35270243	189	WT	5.73073025	15.16516475	7.1321315	3.95395325
Trial 51	5.105105	124	R2	N	79.87055588	23.46784708	203	WT	19.84484425	8.583583	6.606606	15.54054
Trial 52	5.305305	124	R2	S	86.02025478	29.63251709	204	WT	17.14214125	26.551551	13.7637635	12.98798725
Trial 53	17.517517	124	R2	E	230.4685297	24.75678443	124	KO	20.37037	15.9159155	9.709709	27.52752675
Trial 1	22.822822	124	R3	E	296.7881596	22.46147785	125	KO	20.8458455	14.18918875	31.88188125	30.83083
Trial 2	2.402402	124	R3	W	37.26548141	28.63711434	133	KO	12.9879875	18.2182175	21.346346	14.0140135
Trial 4	9.709709	124	R3	S	170.7594563	36.29547337	134	KO	39.18918875	21.82182125	24.299299	28.35335275
Trial 5	3.903903	124	R3	N	45.23416707	26.58172779	158	KO	33.43343275	8.0330325	13.73873825	12.28728675
Trial 50	7.307307	124	R4	S	116.6898603	30.67716617	167	KO	14.539539	13.26326275	16.29129075	10.485485
Trial 51	15.015014	124	R4	W	210.4368486	24.73250975	168	KO	5.80580525	21.496496	38.58858825	20.395395
Trial 52	40.84084	124	R4	N	496.1994817	21.36653384	176	KO	11.761761	10.71070975	18.64364275	8.433433
Trial 54	46.946946	124	R4	E	566.7601652	21.69322917	177	KO	23.198198	24.549549	23.423423	15.565565
Trial 5	31.131131	125	1	N	406.9260539	22.8381432	178	KO	15.71571525	24.349349	15.9409405	14.63963925
Trial 6	27.627627	125	1	E	302.458004	21.58252796	197	KO	27.0270265	28.628628	25.60060025	25.85085
Trial 7	21.621621	125	1	S S	256.2603254	20.15779826	198	KO	43.84384325	32.8078075	23.2732725	15.29028975
Trial 8	3.003003	125	1	W	38.13660117	21.16579382	229	KO	18.1681675	28.303303	5.40540475	11.636636
Trial 54	30.63063	125	2	W	428.4586969	24.04664489	230	KO	9.88488425	9.2092085	5.8308305	8.00800775
Trial 55	15.315315	125	2	N	186.6476196	21.6814981	231	KO	25.07507475	14.48948925	9.159159	12.66266225
Trial 56	4.504504	125	2	S	60.76363863	20.93202377
Trial 57	6.306306	125	2	E	78.86642456	21.29393263
Trial 6	54.454454	125	3	E	739.503786	24.79074669
Trial 7	18.818818	125	3	W	228.9905152	23.34301694
Trial 8	7.507507	125	3	S	90.0696174	21.94622933
Trial 9	46.746746	125	3	N	569.8264916	20.40346771
Trial 55	57.957957	125	4	S	799.3848704	25.19197489
Trial 56	6.806806	125	4	W	91.71051528	24.11021133
Trial 57	22.022021	125	4	N	342.2475444	28.02502155
Trial 58	36.536536	125	4	E	442.561754	22.78964646
Trial 9	17.817817	133	1	N	285.8453587	29.13871838
Trial 10	4.604604	133	1	E	54.81577386	26.07662824
Trial 11	26.126126	133	1	S	367.8461846	24.33631692
Trial 12	3.403403	133	1	W	31.92598362	16.78636291
Trial 58	5.905905	133	2	W	99.4279006	33.10947952
Trial 59	13.713713	133	2	N	223.6060264	29.78431697
Trial 60	28.528528	133	2	S	418.3748288	25.02731972
Trial 61	24.724724	133	2	E	350.7396092	24.33256632
Trial 10	44.344344	133	3	E	678.04395	27.76090386
Trial 11	12.612612	133	3	W	199.9535901	28.94981702
Trial 12	20.12012	133	3	S	239.3758587	25.44002649
Trial 13	8.308308	133	3	N	99.15742806	24.76457265
Trial 59	26.826826	133	4	S	468.8629266	30.81540148
Trial 60	16.016016	133	4	W	241.3625645	26.4968228
Trial 61	3.403403	133	4	N	76.55200297	42.4863584
Trial 62	9.809809	133	4	E	152.7937952	27.25732122
Trial 13	27.627627	134	1	N	389.0897389	23.41571051
Trial 14	54.354354	134	1	E	689.5646337	23.9192792
Trial 15	59.359359	134	1	S	709.115431	21.53211198
Trial 16	15.415415	134	1	W	209.4281472	21.79362228
Trial 62	7.507507	134	2	W	141.0277814	29.97591411
Trial 63	3.703703	134	2	N	49.21779473	20.48690553
Trial 64	28.228228	134	2	S	401.494127	24.1622052
Trial 65	47.847847	134	2	E	646.8651551	24.38560026
Trial 14	20.22022	134	3	E	334.2591366	32.4198885
Trial 15	25.225225	134	3	W	365.3213625	24.82694178
Trial 16	35.335335	134	3	S	432.2911634	22.97122115
Trial 17	16.416416	134	3	N	252.9583883	27.17263547
Trial 63	38.838838	134	4	S	618.5452198	28.34525491
Trial 64	54.854854	134	4	W	670.8188945	22.19033568
Trial 65	3.103103	134	4	N	49.19236986	24.57158957
Trial 66	16.616616	134	4	E	196.0135228	23.59246432
Trial 17	30.93093	150	1	N	459.5265572	24.94929567
Trial 18	4.104104	150	1	E	82.34371417	35.76582439
Trial 19	5.505505	150	1	S	67.31104017	21.01366078
Trial 20	9.509509	150	1	W	116.7957676	20.83553356
Trial 66	4.504504	150	2	W	35.91361241	17.08460936
Trial 67	56.356356	150	2	N	801.7273822	24.9509544
Trial 68	5.405405	150	2	S	63.03831245	22.49117064
Trial 69	14.314314	150	2	E	201.7562526	23.99458798
Trial 18	4.404404	150	3	E	63.32392538	27.50459207
Trial 19	3.303303	150	3	W	42.8093431	21.38326927
Trial 20	11.511511	150	3	S	140.1993551	23.73882809
Trial 21	2.802802	150	3	N	57.869987	36.13260927
Trial 67	13.713713	150	4	S	217.0955641	31.89390041
Trial 68	6.006005	150	4	W	75.53911946	23.5823606
Trial 69	14.814814	150	4	N	205.020361	25.28583122
Trial 70	4.504504	150	4	E	68.284986	28.42364244
Trial 21	12.312312	151	1	N	209.3240159	29.45277697
Trial 22	15.715715	151	1	E	226.187067	25.10676123
Trial 23	15.515515	151	1	S	154.7943016	19.57461337
Trial 24	12.212212	151	1	W	115.1449904	17.97340562
Trial 70	19.119119	151	2	W	289.7867311	29.24213502
Trial 71	3.703703	151	2	N	37.44158896	15.58507426
Trial 72	8.908908	151	2	S	111.5784128	22.29336191
Trial 73	10.71071	151	2	E	136.0148576	24.70525394
Trial 22	8.108108	151	3	E	79.96860242	17.75302122
Trial 23	18.818818	151	3	W	262.4365486	24.27537754
Trial 24	9.809809	151	3	S	161.5349087	26.45467235
Trial 25	9.909909	151	3	N	135.4323418	28.78658994
Trial 71	36.036035	151	4	S	455.9148672	23.35687036
Trial 72	25.425425	151	4	W	328.1348226	24.10343984
Trial 73	3.503503	151	4	N	26.6412324	14.785874
Trial 74	11.111111	151	4	E	152.0548733	25.31713827
Trial 5	44.844844	158	R1	N	643.3518249	24.16198201
Trial 6	1.701701	158	R1	E	13.53764617	15.02679135
Trial 7	59.059058	158	R1	S	759.7447696	23.35338124
Trial 8	28.128128	158	R1	W	345.6774567	22.13664733
Trial 57	16.316316	158	R2	W	227.2346084	25.22305477
Trial 58	3.403403	158	R2	N	80.76603694	40.34265095
Trial 59	7.907907	158	R2	S	124.9384768	26.55608774
Trial 60	4.504504	158	R2	E	97.79676893	44.40865416
Trial 5	26.126126	158	R3	E	487.0007126	31.59180014
Trial 6	7.907907	158	R3	W	113.0805978	26.89700869
Trial 7	17.417417	158	R3	S	254.7472003	24.95024285
Trial 8	3.503503	158	R3	N	122.6536856	64.49006245
Trial 57	2.902903	158	R4	S	48.25728344	26.78280156
Trial 58	7.807807	158	R4	W	116.3611503	27.67733293
Trial 59	3.903903	158	R4	N	71.10330321	35.51611742
Trial 60	34.534534	158	R4	E	556.9194837	26.62021492
Trial 9	4.904904	160	R1	N	77.11832143	33.49619321
Trial 10	60.16016	160	R1	E	977.9316451	28.0733845
Trial 11	5.505505	160	R1	S	77.5399399	24.98786413
Trial 12	60.06006	160	R1	W	700.8800962	20.35404812
Trial 61	60.16016	160	R2	W	981.7093261	27.86157206
Trial 62	3.503503	160	R2	N	62.93691995	29.94001728
Trial 63	60.16016	160	R2	S	814.0119627	24.2745714
Trial 64	60.16016	160	R2	E	803.4949565	23.06584392
Trial 9	41.741741	160	R3	E	735.5399173	30.11494721
Trial 10	8.808808	160	R3	W	136.5285872	26.22923277
Trial 11	25.325325	160	R3	S	362.9939411	26.86154588
Trial 12	11.011011	160	R3	N	210.3977275	30.90989851
Trial 61	23.323323	160	R4	S	396.913833	30.50129649
Trial 62	30.93093	160	R4	W	462.5701208	26.10778084
Trial 63	5.605605	160	R4	N	30.63395841	10.20111049
Trial 64	28.328328	160	R4	E	535.2714415	27.14397113
Trial 13	3.503503	161	R1	N	51.05243595	26.84286027
Trial 14	5.905905	161	R1	E	95.45072404	29.7985432
Trial 15	46.046045	161	R1	S	678.990065	24.75587148
Trial 16	46.146146	161	R1	W	619.6925433	22.26881412
Trial 65	3.803803	161	R2	W	61.31401791	26.63162196
Trial 66	13.613613	161	R2	N	242.3938632	29.89524426
Trial 67	24.624624	161	R2	S	430.9531706	28.32382122
Trial 68	3.903903	161	R2	E	83.12333173	41.52013253
Trial 13	3.803803	161	R3	E	78.03236107	43.3079823
Trial 14	6.406406	161	R3	W	103.4183429	26.49100645
Trial 15	5.605605	161	R3	S	83.55455304	25.294233
Trial 16	2.002001	161	R3	N	0.856036755	0.777437441
Trial 65	3.603603	161	R4	S	51.42192767	28.53917342
Trial 66	6.706706	161	R4	W	102.7686483	29.3331298
Trial 67	2.402402	161	R4	N	32.14937923	29.19749984
Trial 68	28.428428	161	R4	E	466.7645281	28.43278368
Trial 17	12.612612	167	R1	N	192.422511	27.85942314
Trial 18	9.609609	167	R1	E	216.5170079	40.05564679
Trial 19	6.506506	167	R1	S	3.735300275	0.867805532
Trial 20	29.429429	167	R1	W	464.5148244	25.4972768
Trial 69	10.91091	167	R2	W	152.881983	24.24271317
Trial 70	13.313313	167	R2	N	307.4666937	36.13639883
Trial 71	9.509509	167	R2	S	120.3975103	22.69377652
Trial 72	19.319319	167	R2	E	387.6227848	32.81654316
Trial 17	40.040039	167	R3	E	684.931032	29.24127899
Trial 18	6.706706	167	R3	W	101.4155656	25.97800363
Trial 19	5.305305	167	R3	S	107.9056205	29.13451772
Trial 20	13.113113	167	R3	N	191.1306301	26.89287144
Trial 69	3.003003	167	R4	S	154.9943653	67.32150742
Trial 70	21.421421	167	R4	W	296.4974133	26.92736573
Trial 71	1.601601	167	R4	N	14.58255966	18.20998959
Trial 72	15.915915	167	R4	E	257.9339498	27.70710273
Trial 21	2.202202	168	R1	N	31.91736828	19.92842475
Trial 22	4.704704	168	R1	E	117.1796929	53.21026205
Trial 23	6.406406	168	R1	S	96.16815639	25.96539838
Trial 24	9.909909	168	R1	W	168.9382742	26.78877501
Trial 73	15.115115	168	R2	W	259.2537177	30.11561837
Trial 74	22.822822	168	R2	N	427.9157865	29.68666722
Trial 75	31.731731	168	R2	S	510.5233029	26.28931663
Trial 76	16.316316	168	R2	E	254.3688063	25.66812981
Trial 21	60.16016	168	R3	E	1086.837426	29.58449175
Trial 22	60.16016	168	R3	W	879.0059328	24.12436742
Trial 23	23.723723	168	R3	S	385.3808036	27.30464516
Trial 24	10.31031	168	R3	N	181.3701876	28.31074707
Trial 73	42.542542	168	R4	S	789.1025032	29.08906491
Trial 74	5.305305	168	R4	W	80.03784957	24.98683126
Trial 75	28.528528	168	R4	N	490.6865418	29.0056682
Trial 76	5.205205	168	R4	E	70.73171992	26.17075206
Trial 41	28.928928	176	R1	N	499.6522056	29.36190911
Trial 42	4.004003	176	R1	E	62.01122576	22.94415963
Trial 43	11.111111	176	R1	S	211.9380174	32.57326376
Trial 44	3.003002	176	R1	W	36.9770324	19.44213996
Trial 93	15.015014	176	R2	W	189.0343232	24.21092882
Trial 94	14.014013	176	R2	N	227.2088096	28.7318466
Trial 95	8.808808	176	R2	S	185.9267643	32.58611275
Trial 96	5.005004	176	R2	E	141.662036	45.65175091
Trial 41	17.017016	176	R3	E	297.9194794	31.32858421
Trial 42	13.613613	176	R3	W	121.3781998	17.07841575
Trial 43	8.008007	176	R3	S	99.06651814	19.03220507
Trial 44	35.935935	176	R3	N	515.633242	24.76527618
Trial 93	5.605605	176	R4	S	88.16314647	28.41129322
Trial 94	7.907907	176	R4	W	113.2593265	24.59696226
Trial 95	12.012012	176	R4	N	162.5995074	24.24431837
Trial 96	8.208208	176	R4	E	203.9524044	39.9506938
Trial 45	60.06006	177	R1	N	933.5095192	26.19596511
Trial 46	7.207207	177	R1	E	182.7544081	41.49357316
Trial 47	14.114114	177	R1	S	174.9226962	23.61457103
This is a portion of the data; to view all the data, please download the file.

Dataset 3.Reversal Trial Data.

This datasheet contains the raw data exported from the Ethovision program (Columns A – G) as well as the transformed dataset that was used for analysis for the reversal trials (Columns J – O)

Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data	Raw Export Data					Average Duration	Average Duration	Average Duration	Average Duration	Average Duration	Average Duration
Trial	subject	Genotype	block	direction	Total	Duration	Mean	Subject	Genotype	Average Velocity Of 4 Visible Trials	Trial 1	Trial 2	Trial 3	Trial 4	Block 1	Block 2
					cm	s	cm/s	150	WT	30.18801296	6.106106	5.405405	2.302302	4.704704	5.7557555	3.503503
						Arena	Velocity	151	WT	35.81644256	8.508508	6.806806	5.005004	4.104104	7.657657	4.554554
Trial 29	124	2	V1	W	57.83514873	4.004003	23.11091988	160	WT	33.25915	18.018	6.30631	11.1111	3.9039	12.162155	7.5075
Trial 30	124	2	V1	S	489.1090766	29.629629	23.95196149	161	WT	25.808925	10.01	4.6046	5.3053	2.5025	7.3073	3.9039
Trial 53	124	2	V2	N	75.1612109	4.304304	31.28586242	184	WT	27.624875	19.7197	6.10611	25.8258	5.005	12.912905	15.4154
Trial 54	124	2	V2	E	722.948579	52.052051	23.44888019	186	WT	38.75585	19.8198	3.1031	6.10611	3.3033	11.46145	4.704705
Trial 31	125	2	V1	W	58.43447972	2.902902	29.18803542	188	WT	39.0888	2.002	14.1141	18.5185	19.1191	8.05805	18.8188
Trial 32	125	2	V1	S	94.14201599	3.903903	40.89039663	189	WT	29.188975	14.3143	9.60961	60.0601	25.1251	11.961955	42.5926
Trial 55	125	2	V2	N	517.7344716	39.439439	24.05658809	203	WT	22.436525	23.6236	6.30631	29.9299	13.013	14.964955	21.47145
Trial 56	125	2	V2	E	246.7315276	12.812812	33.30875925	204	WT	25.304725	30.3303	7.50751	15.6156	15.015	18.918905	15.3153
Trial 33	133	2	V1	W	95.98487295	5.705705	29.96526527	124	KO	25.449406	4.004003	29.629629	4.304304	52.052051	16.816816	28.1781775
Trial 34	133	2	V1	S	62.30435029	3.603603	28.29182299	125	KO	31.86094485	2.902902	3.903903	39.439439	12.812812	3.4034025	26.1261255
Trial 57	133	2	V2	N	25.18668715	3.103103	19.35501971	133	KO	25.87680524	5.705705	3.603603	3.103103	5.005004	4.654654	4.0540535
Trial 58	133	2	V2	E	77.76305737	5.005004	25.89511299	134	KO	27.72482685	8.008007	5.305305	13.513513	11.611611	6.656656	12.562562
Trial 35	134	2	V1	W	98.01542795	8.008007	21.75941938	157	KO	33.786425	3.6036	15.3153	45.8458	10.01	9.45945	27.9279
Trial 36	134	2	V1	S	76.77081996	5.305305	23.24062059	158	KO	26.695625	13.7137	27.1271	5.2052	57.0571	20.4204	31.13115
Trial 59	134	2	V2	N	232.324307	13.513513	30.94561738	167	KO	23.274075	60.0601	2.5025	60.1602	7.80781	31.2813	33.984005
Trial 60	134	2	V2	E	227.426069	11.611611	34.95365007	168	KO	26.852325	60.1602	60.0601	46.7467	32.4324	60.11015	39.58955
Trial 37	150	1	V1	W	86.2405737	6.106106	23.28494458	176	KO	30.205425	10.8108	6.00601	4.004	6.40641	8.408405	5.205205
Trial 38	150	1	V1	S	64.41918527	5.405405	22.98385286	177	KO	27.257075	5.005	4.8048	5.10511	4.6046	4.9049	4.854855
Trial 61	150	1	V2	N	65.86674908	2.302302	36.55605428	178	KO	28.533225	21.6216	40.3403	24.8248	12.4124	30.98095	18.6186
Trial 62	150	1	V2	E	102.505916	4.704704	37.92720012	197	KO	26.492025	6.10611	3.4034	21.9219	7.70771	4.754755	14.814805
Trial 39	151	1	V1	W	94.84216884	8.508508	20.59724519	198	KO	26.4284	6.50651	6.10611	20.6206	3.3033	6.30631	11.96195
Trial 40	151	1	V1	S	75.00355911	6.806806	20.81350508	229	KO	28.647775	4.4044	6.70671	12.7127	3.5035	5.555555	8.1081
Trial 63	151	1	V2	N	128.8300548	5.005004	45.96474419	230	KO	28.2025	44.5445	3.6036	19.2192	9.00901	24.07405	14.114105
Trial 64	151	1	V2	E	89.51386621	4.104104	55.89027578	231	KO	27.420175	12.9129	4.9049	5.40541	7.50751	8.9089	6.45646
Trial 1	157	2	V1	W	121.053	3.6036	57.5867
Trial 2	157	2	V1	S	319.705	15.3153	33.2693
Trial 27	157	2	V2	N	670.991	45.8458	24.6441
Trial 28	157	2	V2	E	121.925	10.01	19.6456
Trial 3	158	2	V1	W	218.151	13.7137	27.2416
Trial 4	158	2	V1	S	425.523	27.1271	27.0762
Trial 29	158	2	V2	N	61.7497	5.2052	26.8208
Trial 30	158	2	V2	E	821.428	57.0571	25.6439
Trial 5	160	1	V1	W	316.784	18.018	31.6467
Trial 6	160	1	V1	S	110.927	6.30631	34.6301
Trial 31	160	1	V2	N	200.02	11.1111	31.7175
Trial 32	160	1	V2	E	91.2011	3.9039	35.0423
Trial 7	161	1	V1	W	128.285	10.01	27.2673
Trial 8	161	1	V1	S	73.4725	4.6046	24.4664
Trial 33	161	1	V2	N	78.1998	5.3053	22.9769
Trial 34	161	1	V2	E	39.9752	2.5025	28.5251
Trial 9	167	2	V1	W	921.178	60.0601	25.2818
Trial 10	167	2	V1	S	15.3419	2.5025	11.7897
Trial 35	167	2	V2	N	954.819	60.1602	26.4962
Trial 36	167	2	V2	E	115.277	7.80781	29.5286
Trial 11	168	2	V1	W	854.178	60.1602	25.6253
Trial 12	168	2	V1	S	838.133	60.0601	23.9227
Trial 37	168	2	V2	N	763.724	46.7467	27.3462
Trial 38	168	2	V2	E	543.712	32.4324	30.5151
Trial 21	176	2	V1	W	227.346	10.8108	37.2325
Trial 22	176	2	V1	S	75.4442	6.00601	25.9892
Trial 47	176	2	V2	N	61.4159	4.004	29.2164
Trial 48	176	2	V2	E	105.125	6.40641	28.3836
Trial 23	177	2	V1	W	79.1084	5.005	29.2701
Trial 24	177	2	V1	S	82.1347	4.8048	31.5587
Trial 49	177	2	V2	N	54.7333	5.10511	19.5281
Trial 50	177	2	V2	E	60.2702	4.6046	28.6714
Trial 25	178	2	V1	W	363.854	21.6216	30.5454
Trial 26	178	2	V1	S	624.251	40.3403	27.2326
Trial 51	178	2	V2	N	437.764	24.8248	28.962
Trial 52	178	2	V2	E	191.942	12.4124	27.3929
Trial 13	184	1	V1	W	360.692	19.7197	32.1724
Trial 14	184	1	V1	S	102.227	6.10611	29.1784
Trial 39	184	1	V2	N	312.163	25.8258	22.1171
Trial 40	184	1	V2	E	64.9408	5.005	27.0316
Trial 15	186	1	V1	W	373.839	19.8198	31.3837
Trial 16	186	1	V1	S	85.912	3.1031	50.486
Trial 41	186	1	V2	N	48.1745	6.10611	16.0421
Trial 42	186	1	V2	E	108.621	3.3033	57.1116
Trial 17	188	1	V1	W	80.8337	2.002	80.753
Trial 18	188	1	V1	S	160.498	14.1141	22.9053
Trial 43	188	1	V2	N	303.1	18.5185	25.4451
Trial 44	188	1	V2	E	335.533	19.1191	27.2518
Trial 19	189	1	V1	W	215.802	14.3143	28.3666
Trial 20	189	1	V1	S	218.677	9.60961	38.3259
Trial 45	189	1	V2	N	859.433	60.0601	25.783
Trial 46	189	1	V2	E	381.584	25.1251	24.2804
Trial 53	197	2	V1	W	86.4356	6.10611	26.9841
Trial 54	197	2	V1	S	40.2516	3.4034	20.1057
Trial 67	197	2	V2	N	369.007	21.9219	29.9706
Trial 68	197	2	V2	E	124.427	7.70771	28.9077
Trial 55	198	2	V1	W	82.5033	6.50651	24.976
Trial 56	198	2	V1	S	84.2853	6.10611	24.0574
Trial 69	198	2	V2	N	273.073	20.6206	25.2593
Trial 70	198	2	V2	E	59.7594	3.3033	31.4209
Trial 57	203	1	V1	W	339.377	23.6236	23.8759
Trial 58	203	1	V1	S	85.0909	6.30631	22.37
Trial 71	203	1	V2	N	385.321	29.9299	23.4717
Trial 72	203	1	V2	E	156.378	13.013	20.0285
Trial 59	204	1	V1	W	442.786	30.3303	24.9911
Trial 60	204	1	V1	S	91.1555	7.50751	24.612
Trial 73	204	1	V2	N	239.827	15.6156	25.762
Trial 74	204	1	V2	E	204.449	15.015	25.8538
Trial 61	229	2	V1	W	70.3173	4.4044	28.0988
Trial 62	229	2	V1	S	113.975	6.70671	29.9635
Trial 75	229	2	V2	N	209.63	12.7127	31.2568
Trial 76	229	2	V2	E	40.4757	3.5035	25.272
Trial 63	230	2	V1	W	727.022	44.5445	27.7212
Trial 64	230	2	V1	S	55.3212	3.6036	27.633
Trial 77	230	2	V2	N	335.429	19.2192	29.6543
Trial 78	230	2	V2	E	139.147	9.00901	27.8015
Trial 65	231	2	V1	W	217.02	12.9129	28.1562
Trial 66	231	2	V1	S	63.0264	4.9049	26.2348
Trial 79	231	2	V2	N	62.0096	5.40541	25.8115

Dataset 4.Visible Platform Data.

This datasheet contains the raw data exported from the Ethovision program (Columns A – H) as well as the transformed dataset that was used for analysis of the visible platform trials (Columns K – S)

Discussion

As expected, deletion of Fmr1 did not impact initial spatial learning in the MWM. The current study did, however, demonstrate impairments in cognitive flexibility in the Fmr1 KO. As previously mentioned, other studies have not before detected such changes, and this discrepancy could be attributed to methodological differences¹⁰. In the aforementioned study, training occurred over 8 days, with only 3 training trials per day, and the reversal paradigm consisted of 4 days, with 3 trials per day. Furthermore, in support of our findings, deficits in long-term potentiation in the prefrontal cortex have been demonstrated in the Fmr1 KO mouse, the area on which the ability to adapt to a new location in this task is dependent on 11–13. Moreover, this ability to adapt to a new location is mediated through multi-synaptic connections between the hippocampus and the prefrontal cortex¹³. This proposed mechanism further supports our findings of no change to the initial spatial learning phase, as lesions to this area did not impact initial learning performance in spatial navigation¹¹.

The current study provides preliminary evidence that could be applied to tease apart subtle differences between the male and female Fmr1 KO phenotype, which has been difficult to conclusively evaluate. Future studies should expand upon these findings in females, as many studies have demonstrated a sex-specific effect of loss of Fmr1 on behavior (discussed in a recent review¹⁴)^2–4,15. Overall, this study corroborates and extends previous evidence of impaired cognitive flexibility in the male Fmr1 KO mouse.

Data availability

Dataset 1– “Learning Trial Data – CSV.csv”

This datasheet contains the raw data exported from the Ethovision program (Columns A – G) as well as the transformed dataset that was used for analysis for the learning trials (Columns J – T). http://dx.doi.org/10.5256/f1000research.14969.d206068¹⁶

Dataset 2 – “Probe Trial Data – CSV.csv”

This datasheet contains the raw data exported from the Ethovision program (Columns A - AB) as well as the transformed dataset that was used for analysis for the probe trial (Columns AC – AI). http://dx.doi.org/10.5256/f1000research.14969.d206069¹⁷

Dataset 3 – “Reversal Trial Data – CSV.csv”

This datasheet contains the raw data exported from the Ethovision program (Columns A – G) as well as the transformed dataset that was used for analysis for the reversal trials (Columns J – O). http://dx.doi.org/10.5256/f1000research.14969.d206076¹⁸

Dataset 4 – “Visible Platform Data – CSV.csv”

This datasheet contains the raw data exported from the Ethovision program (Columns A – H) as well as the transformed dataset that was used for analysis of the visible platform trials (Columns K – S). http://dx.doi.org/10.5256/f1000research.14969.d206077¹⁹

Competing interests

No competing interests were disclosed.

Grant information

This research was supported by funding from the National Institutes of Health, National Institute of Neurological Disorders and Stroke [NS088776].

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements

We would like to thank Gregory Smith for his initial training guidance. We would also like to thank Conner Reynolds, Samantha Hodges, Matthew Binder, and Andy Holley for their critical review of the paper.

Faculty Opinions recommended

References

1. Hagerman RJ, Hagerman PJ: Fragile X syndrome: diagnosis, treatment, and research. 3rd ed. Baltimore: Johns Hopkins University Press. xii. 2002; 540. Reference Source
2. Baker KB, Wray SP, Ritter R, et al.: Male and female Fmr1 knockout mice on C57 albino background exhibit spatial learning and memory impairments. Genes Brain Behav. 2010; 9(6): 562–74. PubMed Abstract | Publisher Full Text
3. Ding Q, Sethna F, Wang H: Behavioral analysis of male and female Fmr1 knockout mice on C57BL/6 background. Behav Brain Res. 2014; 271: 72–8. PubMed Abstract | Publisher Full Text | Free Full Text
4. Nolan SO, Reynolds CD, Smith GD, et al.: Deletion of Fmr1 results in sex-specific changes in behavior. Brain Behav. 2017; 7(10): e00800. PubMed Abstract | Publisher Full Text | Free Full Text
5. Morris R: Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984; 11(1): 47–60. PubMed Abstract | Publisher Full Text
6. Fmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium. Cell. 1994; 78(1): 23–33. PubMed Abstract | Publisher Full Text
7. Kooy RF, D'Hooge R, Reyniers E, et al.: Transgenic mouse model for the fragile X syndrome. Am J Med Genet. 1996; 64(2): 241–5. PubMed Abstract | Publisher Full Text
8. D'Hooge R, Nagels G, Franck F, et al.: Mildly impaired water maze performance in male Fmr1 knockout mice. Neuroscience. 1997; 76(2): 367–76. PubMed Abstract | Publisher Full Text
9. Paradee W, Melikian HE, Rasmussen DL, et al.: Fragile X mouse: strain effects of knockout phenotype and evidence suggesting deficient amygdala function. Neuroscience. 1999; 94(1): 185–92. PubMed Abstract | Publisher Full Text
10. Leach PT, Hayes J, Pride M, et al.: Normal Performance of Fmr1 Mice on a Touchscreen Delayed Nonmatching to Position Working Memory Task. eNeuro. 2016; 3(1): pii: ENEURO.0143-15.2016. PubMed Abstract | Publisher Full Text | Free Full Text
11. Martin HGS, Lassalle O, Brown JT, et al.: Age-dependent long-term potentiation deficits in the prefrontal cortex of the Fmr1 knockout mouse model of fragile X syndrome. Cereb Cortex. 2016; 26(5): 2084–2092. PubMed Abstract | Publisher Full Text
12. Ventura R, Pascucci T, Catania MV, et al.: Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex. Behav Pharmacol. 2004; 15(5–6): 433–442. PubMed Abstract | Publisher Full Text
13. de Bruin JP, Sànchez-Santed F, Heinsbroek RP, et al.: A behavioural analysis of rats with damage to the medial prefrontal cortex using the morris water maze: evidence for behavioural flexibility, but not for impaired spatial navigation. Brain Res. 1994; 652(2): 323–333. PubMed Abstract | Publisher Full Text
14. Romano E, Cosentino L, Laviola G, et al.: Genes and sex hormones interaction in neurodevelopmental disorders. Neurosci Biobehav Rev. 2016; 67: 9–24. PubMed Abstract | Publisher Full Text
15. Reynolds CD, Nolan SO, Jefferson T, et al.: Sex-specific and genotype-specific differences in vocalization development in Fmr1 knockout mice. Neuroreport. 2016; 27(18): 1331–1335. PubMed Abstract | Publisher Full Text | Free Full Text
16. Nolan SO, Lugo JN: Dataset 1 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source
17. Nolan SO, Lugo JN: Dataset 2 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source
18. Nolan SO, Lugo JN: Dataset 3 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source
19. Nolan SO, Lugo JN: Dataset 4 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source

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Author details Author details

¹ Department of Psychology and Neuroscience, Baylor University, Waco, TX, 76798, USA
² Institute of Biomedical Studies, Baylor University, Waco, TX, 76798, USA
³ Department of Biology, Baylor University, Waco, TX, 76798, USA

Suzanne O. Nolan
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Joaquin N. Lugo
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Validation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This research was supported by funding from the National Institutes of Health, National Institute of Neurological Disorders and Stroke [NS088776]
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (1)

version 1

Published: 07 Jun 2018, 7:711

https://doi.org/10.12688/f1000research.14969.1

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© 2018 Nolan SO and Lugo JN. 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. Data associated with the article 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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Nolan SO and Lugo JN. Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse [version 1; peer review: 1 approved, 2 approved with reservations] F1000Research 2018, 7:711 (https://doi.org/10.12688/f1000research.14969.1)

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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

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PUBLISHED 07 Jun 2018

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Reviewer Report 16 Jul 2018

Lisa Monteggia, Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

Approved

https://doi.org/10.5256/f1000research.16297.r34793

The current study examines male Fmr1 knockout mice in the Morris Water maze. This group focuses on the FVB strain as previous work has shown background strain differences can impact the phenotype of Fmr1 knockouts. The authors show that the ... Continue reading

The current study examines male Fmr1 knockout mice in the Morris Water maze. This group focuses on the FVB strain as previous work has shown background strain differences can impact the phenotype of Fmr1 knockouts. The authors show that the male Fmr1 knockout mice have no impairment in acquisition of spatial memory but do show differences in the reversal learning paradigm that are not attributed to vision or motor differences. In the reversal learning paradigm, the authors show that during the 4 blocks of reversal learning the Fmr1 KOs show a rather stable latency to find the platform in contrast to the WT mice that show the expected decrease to reach the platform with increasing trials. The authors highlight the finding that male Fmr1 knockout mice have deficits in reversal learning as assessed in the Morris water maze but not in acquisition, demonstrating a link to a specific form of learning in this paradigm. The study is straightforward and the data clearly presented.

A couple of comments the authors should further clarify:
The authors should elaborate on the breeding scheme as they are not directly comparing littermate controls.

Based on the authors discussion point regarding potential sex differences between male and female Fmr1 KO mice, is there data to suggest differences in cognitive flexibility between the males and female KOs?

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report 02 Jul 2018

Andre Fenton, Center for Neural Science, New York University, New York, NY, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.16297.r34795

I am afraid that I did not find this manuscript and the results to be compelling.
The experimental design is straightforward but the analysis is questionable and the report is not as scholarly as it should be.

... Continue reading

I am afraid that I did not find this manuscript and the results to be compelling.
The experimental design is straightforward but the analysis is questionable and the report is not as scholarly as it should be.

Statistics are used questionably. 1) t tests are used for post-hoc comparisons, when they are not justified, For example, post-hoc t tests are performed after ANOVA has failed to find relevant differences. 2) Furthermore, the alpha level for post-hoc t tests seem not to be corrected for multiple comparisons. The reported significant difference on trial 3 may not maintain after correcting alpha for multiple comparisons 0.05/4, although it might - a reader can’t know. Regardless, what is the justification for the t tests after the ANOVA results were not significant?

Dataset 1: Given no effects of genotype or interaction in the ANOVA, what justifies data snooping as follows: "Further independent samples t-testing revealed no differences between WT and KO at any of the 8 different trials, p > 0.05.” ?

Dataset 2: Given the ANOVA was not significant for a 1-way ANOVA, there is no justification for a t test and in fact it is numerically equivalent to the F-test, so even more so not warranted.
"Further independent samples t-testing revealed no differences in duration in any of the quadrants, p > 0.05.”

The manuscript states that "Fmr1 KO mice showed deficits in their ability to learn the new location of the platform, Fgenotype (1, 23) = 3.93, p = 0.059.” Why is p= 0.059 indicative of a deficit?

The Introduction is written as if the water maze is the only way to test spatial memory and cognitive flexibility, when of course it is not.

The Discussion states "The current study did, however, demonstrate impairments in cognitive flexibility in the Fmr1 KO. As previously mentioned, other studies have not before detected such changes“ Remarkably, the authors fail to consider the work from the Fenton lab which explicitly has investigated cognitive flexibility in Fmr1 KO mice (Radwan et al., 2015 Neurobiol. Dis.; Dvorak et al., 2018, PLoS Biol¹.) along with electrophysiological correlates of the ability.

Something is wrong with the initial statement: "Male Fmr1+/+ and female Fmr1+/- FVB.129P2-Pde6b+Tyrc-ch Fmr1tm1Cgr/J (Stock No: 004624, The Jackson Laboratory, Bar Harbor, ME, USA) mice were used as breeders (9 total breeding pairs) to produce the following groups:” because WT mice cannot contribute to breeding involving a KO male.

Fig 1B should have the days indicated, as well as the rest period.

Fig. 2C shows poor learning even in WT, despite the significant effect of trial. Could the paradigm simply not be robust in mice? There is a literature on this possibility, see Wolfer et al., 1998
News Physiol Sci².).

What justification is there for the assertion that "Moreover, this ability to adapt to a new location is mediated through multi-synaptic connections between the hippocampus and the prefrontal cortex13.”?
What is the mechanism that is referenced by the statement "This proposed mechanism further supports our findings of no change to the initial spatial learning phase, as lesions to this area did not impact initial learning performance in spatial navigation11.”?

The final statement seems untrue given that the authors claim to find a reversal deficit but say there had not been one detected previously "Overall, this study corroborates and extends previous evidence of impaired cognitive flexibility in the male Fmr1 KO mouse.”

What is "t-testing" should this not simply be stated as "t tests” ?

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

1. Dvorak D, Radwan B, Sparks FT, Talbot ZN, et al.: Control of recollection by slow gamma dominating mid-frequency gamma in hippocampus CA1.PLoS Biol. 2018; 16 (1): e2003354 PubMed Abstract | Publisher Full Text
2. Wolfer DP, Stagljar-Bozicevic M, Errington ML, Lipp HP: Spatial Memory and Learning in Transgenic Mice: Fact or Artifact?. News Physiol Sci. 1998; 13: 118-123 PubMed Abstract

Competing Interests: No competing interests were disclosed.

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

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Reviewer Report 20 Jun 2018

Laura Smith, Department of Neuroscience & Experimental Therapeutics, Texas A&M Health Science Center, Bryan, TX, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.16297.r34794

The study authored by Nolan and Lugo investigates the ability of Fmr1 KO mice on the FVB.129 background to demonstrate proper reversal learning in the Morris water maze (MWM). Background strain appears to make a large difference in observed cognitive ... Continue reading

The study authored by Nolan and Lugo investigates the ability of Fmr1 KO mice on the FVB.129 background to demonstrate proper reversal learning in the Morris water maze (MWM). Background strain appears to make a large difference in observed cognitive deficits in mice lacking expression of the Fmr1 gene, and this study makes a valuable contribution to the literature by assessing MWM reversal learning in Fmr1 KO mice on the pure FVB background.

Suggested revisions:

The authors refer to papers showing impaired reversal learning in Fmr1 KO mice on the C57BL/6J strain and cite D’Hooge et al., 1997¹ and Kooy et al., 1996² However, in each of these studies the authors specify that only pigmented offspring were selected for testing, suggesting that these mouse lines were not fully backcrossed to C57BL/6J. Of note, stem cells used in the creation of transgenic mice have often been derived from the 129 strain. In any case, the authors should likely revisit this statement, and instead may wish to report Paradee et al. (1999)³ which used fully backcrossed C57BL/6 KO mice.
The statement that “few studies have examined cognitive flexibility in a reversal form of the MWM task” (presumably in Fmr1 KO mice) seems inaccurate, as most studies using MWM to assess Fmr1 KO mice actually include this task (Paradee et al., 1999; Kooy et al., 1996; D’Hooge et al., 1997; The Dutch-Belgian Fragile X Consortium paper, 1994⁴; Baker et al., 2010)⁵. However, as the authors state, it had not been properly assessed on the FVB background.
The abstract conclusion says that “previous studies have not demonstrated deficits in spatial memory in the Fmr1 KO model.” However, multiple previous assessments of Fmr1 KO mice in the MWM have shown minor acquisition differences compared to WT mice (e.g., Paradee et al., 1999; Kooy et al., 1996, Am J Med Genet; The Dutch-Belgian Fragile X Consortium paper, 1994). Please consider these and also deficits observed in the plus-shaped water maze (e.g., Dobkin et al., 2000⁶; Van Dam et al., 2000⁷) and radial arm maze (Mineur et al., 2002⁸) to perhaps soften this statement appropriately.
Consider changing “disability” to “disabilities” in first sentence of the abstract.
Please add more detail about the Morris water maze task. For instance, did mice receive consecutive trials within each block? Was entry quadrant varied or static over each mouse’s trials within (and across) blocks? Was the temperature of the water controlled? What were the visual cues, where were they positioned with respect to each quadrant, and were they proximal or distal? If animals were artificially colored for detection in the maze, please specify. Were animals dried and/or warmed between or after trials?
It may be helpful to label days on Fig. 1B.
In the first Results paragraph-- in the absence of a significant main effect or interaction involving genotype, further independent samples t-testing between WT and KO should probably not be reported.
In the Discussion, the authors point to deficits in Fmr1 KO prefrontal cortex LTP with regard to the observed reversal learning deficit, which is fine. However, using this same logic to claim support for lack of observed differences in acquisition, based on findings that PFC is not relevant to acquisition, is somewhat flawed. It may be more appropriate (e.g., in the sentence regarding PFC and learning a new location) to simply acknowledge that "PFC is not required for acquisition" and allow the reader to intuit the point (that seems to be intended) more subtly. If this topic will be discussed, other brain regions likely involved in MWM acquisition (e.g., hippocampus), and that also show significant plasticity-related variations in Fmr1 KO compared to WT mice, should be included.
The authors’ point that this investigation should be made in females is well-received. However, it is not immediately clear how these data in male Fmr1 KO mice provide “preliminary evidence that could be applied to tease apart subtle differences between the male and female Fmr1 KO phenotype.” Perhaps this point can be further clarified or removed?
In the attached data sets, it is not clear why a few animal IDs are repeated. Specifically, please see the “Learning trial” set, and the “transformed” data on the right-hand side of the screen. Subjects 124 and 134 are listed twice, but all other IDs appear once. Also, a little more explanation, either directly on the data sheets or elsewhere in the manuscript, for the trial names, etc. seen here may be beneficial to users of these data.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. D'Hooge R, Nagels G, Franck F, Bakker CE, et al.: Mildly impaired water maze performance in male Fmr1 knockout mice.Neuroscience. 1997; 76 (2): 367-76 PubMed Abstract
2. Kooy R, D'Hooge R, Reyniers E, Bakker C, et al.: Transgenic mouse model for the fragile X syndrome. American Journal of Medical Genetics. 1996; 64 (2): 241-245 Publisher Full Text
3. Paradee W, Melikian HE, Rasmussen DL, Kenneson A, et al.: Fragile X mouse: strain effects of knockout phenotype and evidence suggesting deficient amygdala function.Neuroscience. 1999; 94 (1): 185-92 PubMed Abstract
4. Fmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium.Cell. 1994; 78 (1): 23-33 PubMed Abstract
5. Baker KB, Wray SP, Ritter R, Mason S, et al.: Male and female Fmr1 knockout mice on C57 albino background exhibit spatial learning and memory impairments.Genes Brain Behav. 2010; 9 (6): 562-74 PubMed Abstract | Publisher Full Text
6. Dobkin C, Rabe A, Dumas R, El Idrissi A, et al.: Fmr1 knockout mouse has a distinctive strain-specific learning impairment.Neuroscience. 2000; 100 (2): 423-9 PubMed Abstract
7. Van Dam D, D'Hooge R, Hauben E, Reyniers E, et al.: Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice.Behav Brain Res. 2000; 117 (1-2): 127-36 PubMed Abstract
8. Mineur YS, Sluyter F, de Wit S, Oostra BA, et al.: Behavioral and neuroanatomical characterization of the Fmr1 knockout mouse.Hippocampus. 2002; 12 (1): 39-46 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: fragile X; autism; addiction; dendritic structure and plasticity

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

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Laura Smith, Texas A&M Health Science Center, Bryan, USA
Andre Fenton, New York University, New York, USA
Lisa Monteggia, University of Texas Southwestern Medical Center, Dallas, USA

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16 Jul 2018 | for Version 1

Lisa Monteggia, Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

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Approved

The current study examines male Fmr1 knockout mice in the Morris Water maze. This group focuses on the FVB strain as previous work has shown background strain differences can impact the phenotype of Fmr1 knockouts. The authors show that the male Fmr1 knockout mice have no impairment in acquisition of spatial memory but do show differences in the reversal learning paradigm that are not attributed to vision or motor differences. In the reversal learning paradigm, the authors show that during the 4 blocks of reversal learning the Fmr1 KOs show a rather stable latency to find the platform in contrast to the WT mice that show the expected decrease to reach the platform with increasing trials. The authors highlight the finding that male Fmr1 knockout mice have deficits in reversal learning as assessed in the Morris water maze but not in acquisition, demonstrating a link to a specific form of learning in this paradigm. The study is straightforward and the data clearly presented.

A couple of comments the authors should further clarify:
The authors should elaborate on the breeding scheme as they are not directly comparing littermate controls.

Based on the authors discussion point regarding potential sex differences between male and female Fmr1 KO mice, is there data to suggest differences in cognitive flexibility between the males and female KOs?

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

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Reviewer Report

13 Views

02 Jul 2018 | for Version 1

Andre Fenton, Center for Neural Science, New York University, New York, NY, USA

13 Views Cite this report Responses(0)

Approved With Reservations

I am afraid that I did not find this manuscript and the results to be compelling.
The experimental design is straightforward but the analysis is questionable and the report is not as scholarly as it should be.

Statistics are used questionably. 1) t tests are used for post-hoc comparisons, when they are not justified, For example, post-hoc t tests are performed after ANOVA has failed to find relevant differences. 2) Furthermore, the alpha level for post-hoc t tests seem not to be corrected for multiple comparisons. The reported significant difference on trial 3 may not maintain after correcting alpha for multiple comparisons 0.05/4, although it might - a reader can’t know. Regardless, what is the justification for the t tests after the ANOVA results were not significant?

Dataset 1: Given no effects of genotype or interaction in the ANOVA, what justifies data snooping as follows: "Further independent samples t-testing revealed no differences between WT and KO at any of the 8 different trials, p > 0.05.” ?

Dataset 2: Given the ANOVA was not significant for a 1-way ANOVA, there is no justification for a t test and in fact it is numerically equivalent to the F-test, so even more so not warranted.
"Further independent samples t-testing revealed no differences in duration in any of the quadrants, p > 0.05.”

The manuscript states that "Fmr1 KO mice showed deficits in their ability to learn the new location of the platform, Fgenotype (1, 23) = 3.93, p = 0.059.” Why is p= 0.059 indicative of a deficit?

The Introduction is written as if the water maze is the only way to test spatial memory and cognitive flexibility, when of course it is not.

The Discussion states "The current study did, however, demonstrate impairments in cognitive flexibility in the Fmr1 KO. As previously mentioned, other studies have not before detected such changes“ Remarkably, the authors fail to consider the work from the Fenton lab which explicitly has investigated cognitive flexibility in Fmr1 KO mice (Radwan et al., 2015 Neurobiol. Dis.; Dvorak et al., 2018, PLoS Biol¹.) along with electrophysiological correlates of the ability.

Something is wrong with the initial statement: "Male Fmr1+/+ and female Fmr1+/- FVB.129P2-Pde6b+Tyrc-ch Fmr1tm1Cgr/J (Stock No: 004624, The Jackson Laboratory, Bar Harbor, ME, USA) mice were used as breeders (9 total breeding pairs) to produce the following groups:” because WT mice cannot contribute to breeding involving a KO male.

Fig 1B should have the days indicated, as well as the rest period.

Fig. 2C shows poor learning even in WT, despite the significant effect of trial. Could the paradigm simply not be robust in mice? There is a literature on this possibility, see Wolfer et al., 1998
News Physiol Sci².).

What justification is there for the assertion that "Moreover, this ability to adapt to a new location is mediated through multi-synaptic connections between the hippocampus and the prefrontal cortex13.”?
What is the mechanism that is referenced by the statement "This proposed mechanism further supports our findings of no change to the initial spatial learning phase, as lesions to this area did not impact initial learning performance in spatial navigation11.”?

The final statement seems untrue given that the authors claim to find a reversal deficit but say there had not been one detected previously "Overall, this study corroborates and extends previous evidence of impaired cognitive flexibility in the male Fmr1 KO mouse.”

What is "t-testing" should this not simply be stated as "t tests” ?

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

1. Dvorak D, Radwan B, Sparks FT, Talbot ZN, et al.: Control of recollection by slow gamma dominating mid-frequency gamma in hippocampus CA1.PLoS Biol. 2018; 16 (1): e2003354 PubMed Abstract | Publisher Full Text
2. Wolfer DP, Stagljar-Bozicevic M, Errington ML, Lipp HP: Spatial Memory and Learning in Transgenic Mice: Fact or Artifact?. News Physiol Sci. 1998; 13: 118-123 PubMed Abstract

Competing Interests

No competing interests were disclosed.

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

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Reviewer Report

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20 Jun 2018 | for Version 1

Laura Smith, Department of Neuroscience & Experimental Therapeutics, Texas A&M Health Science Center, Bryan, TX, USA

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Approved With Reservations

The study authored by Nolan and Lugo investigates the ability of Fmr1 KO mice on the FVB.129 background to demonstrate proper reversal learning in the Morris water maze (MWM). Background strain appears to make a large difference in observed cognitive deficits in mice lacking expression of the Fmr1 gene, and this study makes a valuable contribution to the literature by assessing MWM reversal learning in Fmr1 KO mice on the pure FVB background.

Suggested revisions:

The authors refer to papers showing impaired reversal learning in Fmr1 KO mice on the C57BL/6J strain and cite D’Hooge et al., 1997¹ and Kooy et al., 1996² However, in each of these studies the authors specify that only pigmented offspring were selected for testing, suggesting that these mouse lines were not fully backcrossed to C57BL/6J. Of note, stem cells used in the creation of transgenic mice have often been derived from the 129 strain. In any case, the authors should likely revisit this statement, and instead may wish to report Paradee et al. (1999)³ which used fully backcrossed C57BL/6 KO mice.
The statement that “few studies have examined cognitive flexibility in a reversal form of the MWM task” (presumably in Fmr1 KO mice) seems inaccurate, as most studies using MWM to assess Fmr1 KO mice actually include this task (Paradee et al., 1999; Kooy et al., 1996; D’Hooge et al., 1997; The Dutch-Belgian Fragile X Consortium paper, 1994⁴; Baker et al., 2010)⁵. However, as the authors state, it had not been properly assessed on the FVB background.
The abstract conclusion says that “previous studies have not demonstrated deficits in spatial memory in the Fmr1 KO model.” However, multiple previous assessments of Fmr1 KO mice in the MWM have shown minor acquisition differences compared to WT mice (e.g., Paradee et al., 1999; Kooy et al., 1996, Am J Med Genet; The Dutch-Belgian Fragile X Consortium paper, 1994). Please consider these and also deficits observed in the plus-shaped water maze (e.g., Dobkin et al., 2000⁶; Van Dam et al., 2000⁷) and radial arm maze (Mineur et al., 2002⁸) to perhaps soften this statement appropriately.
Consider changing “disability” to “disabilities” in first sentence of the abstract.
Please add more detail about the Morris water maze task. For instance, did mice receive consecutive trials within each block? Was entry quadrant varied or static over each mouse’s trials within (and across) blocks? Was the temperature of the water controlled? What were the visual cues, where were they positioned with respect to each quadrant, and were they proximal or distal? If animals were artificially colored for detection in the maze, please specify. Were animals dried and/or warmed between or after trials?
It may be helpful to label days on Fig. 1B.
In the first Results paragraph-- in the absence of a significant main effect or interaction involving genotype, further independent samples t-testing between WT and KO should probably not be reported.
In the Discussion, the authors point to deficits in Fmr1 KO prefrontal cortex LTP with regard to the observed reversal learning deficit, which is fine. However, using this same logic to claim support for lack of observed differences in acquisition, based on findings that PFC is not relevant to acquisition, is somewhat flawed. It may be more appropriate (e.g., in the sentence regarding PFC and learning a new location) to simply acknowledge that "PFC is not required for acquisition" and allow the reader to intuit the point (that seems to be intended) more subtly. If this topic will be discussed, other brain regions likely involved in MWM acquisition (e.g., hippocampus), and that also show significant plasticity-related variations in Fmr1 KO compared to WT mice, should be included.
The authors’ point that this investigation should be made in females is well-received. However, it is not immediately clear how these data in male Fmr1 KO mice provide “preliminary evidence that could be applied to tease apart subtle differences between the male and female Fmr1 KO phenotype.” Perhaps this point can be further clarified or removed?
In the attached data sets, it is not clear why a few animal IDs are repeated. Specifically, please see the “Learning trial” set, and the “transformed” data on the right-hand side of the screen. Subjects 124 and 134 are listed twice, but all other IDs appear once. Also, a little more explanation, either directly on the data sheets or elsewhere in the manuscript, for the trial names, etc. seen here may be beneficial to users of these data.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. D'Hooge R, Nagels G, Franck F, Bakker CE, et al.: Mildly impaired water maze performance in male Fmr1 knockout mice.Neuroscience. 1997; 76 (2): 367-76 PubMed Abstract
2. Kooy R, D'Hooge R, Reyniers E, Bakker C, et al.: Transgenic mouse model for the fragile X syndrome. American Journal of Medical Genetics. 1996; 64 (2): 241-245 Publisher Full Text
3. Paradee W, Melikian HE, Rasmussen DL, Kenneson A, et al.: Fragile X mouse: strain effects of knockout phenotype and evidence suggesting deficient amygdala function.Neuroscience. 1999; 94 (1): 185-92 PubMed Abstract
4. Fmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium.Cell. 1994; 78 (1): 23-33 PubMed Abstract
5. Baker KB, Wray SP, Ritter R, Mason S, et al.: Male and female Fmr1 knockout mice on C57 albino background exhibit spatial learning and memory impairments.Genes Brain Behav. 2010; 9 (6): 562-74 PubMed Abstract | Publisher Full Text
6. Dobkin C, Rabe A, Dumas R, El Idrissi A, et al.: Fmr1 knockout mouse has a distinctive strain-specific learning impairment.Neuroscience. 2000; 100 (2): 423-9 PubMed Abstract
7. Van Dam D, D'Hooge R, Hauben E, Reyniers E, et al.: Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice.Behav Brain Res. 2000; 117 (1-2): 127-36 PubMed Abstract
8. Mineur YS, Sluyter F, de Wit S, Oostra BA, et al.: Behavioral and neuroanatomical characterization of the Fmr1 knockout mouse.Hippocampus. 2002; 12 (1): 39-46 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

fragile X; autism; addiction; dendritic structure and plasticity

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

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[1] 1. Hagerman RJ, Hagerman PJ: Fragile X syndrome: diagnosis, treatment, and research. 3rd ed. Baltimore: Johns Hopkins University Press. xii. 2002; 540. Reference Source

[2] 2. Baker KB, Wray SP, Ritter R, et al.: Male and female Fmr1 knockout mice on C57 albino background exhibit spatial learning and memory impairments. Genes Brain Behav. 2010; 9(6): 562–74. PubMed Abstract | Publisher Full Text

[3] 3. Ding Q, Sethna F, Wang H: Behavioral analysis of male and female Fmr1 knockout mice on C57BL/6 background. Behav Brain Res. 2014; 271: 72–8. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Nolan SO, Reynolds CD, Smith GD, et al.: Deletion of Fmr1 results in sex-specific changes in behavior. Brain Behav. 2017; 7(10): e00800. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Morris R: Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984; 11(1): 47–60. PubMed Abstract | Publisher Full Text

[6] 6. Fmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium. Cell. 1994; 78(1): 23–33. PubMed Abstract | Publisher Full Text

[7] 7. Kooy RF, D'Hooge R, Reyniers E, et al.: Transgenic mouse model for the fragile X syndrome. Am J Med Genet. 1996; 64(2): 241–5. PubMed Abstract | Publisher Full Text

[8] 8. D'Hooge R, Nagels G, Franck F, et al.: Mildly impaired water maze performance in male Fmr1 knockout mice. Neuroscience. 1997; 76(2): 367–76. PubMed Abstract | Publisher Full Text

[9] 9. Paradee W, Melikian HE, Rasmussen DL, et al.: Fragile X mouse: strain effects of knockout phenotype and evidence suggesting deficient amygdala function. Neuroscience. 1999; 94(1): 185–92. PubMed Abstract | Publisher Full Text

[10] 10. Leach PT, Hayes J, Pride M, et al.: Normal Performance of Fmr1 Mice on a Touchscreen Delayed Nonmatching to Position Working Memory Task. eNeuro. 2016; 3(1): pii: ENEURO.0143-15.2016. PubMed Abstract | Publisher Full Text | Free Full Text

[11] 11. Martin HGS, Lassalle O, Brown JT, et al.: Age-dependent long-term potentiation deficits in the prefrontal cortex of the Fmr1 knockout mouse model of fragile X syndrome. Cereb Cortex. 2016; 26(5): 2084–2092. PubMed Abstract | Publisher Full Text

[12] 12. Ventura R, Pascucci T, Catania MV, et al.: Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex. Behav Pharmacol. 2004; 15(5–6): 433–442. PubMed Abstract | Publisher Full Text

[13] 13. de Bruin JP, Sànchez-Santed F, Heinsbroek RP, et al.: A behavioural analysis of rats with damage to the medial prefrontal cortex using the morris water maze: evidence for behavioural flexibility, but not for impaired spatial navigation. Brain Res. 1994; 652(2): 323–333. PubMed Abstract | Publisher Full Text

[14] 14. Romano E, Cosentino L, Laviola G, et al.: Genes and sex hormones interaction in neurodevelopmental disorders. Neurosci Biobehav Rev. 2016; 67: 9–24. PubMed Abstract | Publisher Full Text

[15] 15. Reynolds CD, Nolan SO, Jefferson T, et al.: Sex-specific and genotype-specific differences in vocalization development in Fmr1 knockout mice. Neuroreport. 2016; 27(18): 1331–1335. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. Nolan SO, Lugo JN: Dataset 1 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source

[17] 17. Nolan SO, Lugo JN: Dataset 2 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source

[18] 18. Nolan SO, Lugo JN: Dataset 3 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source

[19] 19. Nolan SO, Lugo JN: Dataset 4 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source

Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse

Abstract

Keywords

Introduction

Methods

Animals

Morris water maze

Figure 1. Overview of the testing paradigm.

Statistical analysis

Results

Fmr1 KO mice show no impairment in acquisition of spatial memory

Figure 2. Performance in the Morris water maze in Fmr1 knockout males.

Loss of Fmr1 impairs ability to update existing learning with new platform location

Impairments were not due to deficits in vision or motor capabilities

Figure 3. Performance in the Morris water maze visible trials.

Discussion

Data availability

Competing interests

Grant information

Acknowledgements

References

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