The probe trial in a water maze test should not exceed two times

The probe trial in a water maze test should not exceed two times | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The probe trial in a water maze test should not exceed two times Xiaodong Han, Xiaobin Liu, Yanna Jiang, Shuai Wu, Zhe Zhang, Cheng Gao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4134660/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract We performed two types of water maze tests (a visual-cued and an acoustic-cued) to determine the appropriate number of probe trials for a water maze test. Three probe trials were applied in the probe trial phase of each experiment. The observational indexes included the time of the first crossing and the number of crossing . In both types of the tests, the results showed that the time of the first crossing increased significantly from the second to the third probe trials (P < 0.05), while the number of crossing decreased significantly from the second to the third probe trials (P 0.05). Therefore, we believe that the number of probe trials in a water maze test should be one or two but no more than two times. Biological sciences/Neuroscience Biological sciences/Physiology Health sciences/Neurology Morris water maze probe trial transfer test neurodegenerative disorder Alzheimer’s disease Figures Figure 1 Introduction The Morris water maze (MWM) test is a classic test to measure spatial learning and memory in rodents. The procedures of the MWM test or its derivatives typically include two phases 1 , 2 : a latency acquisition phase (hidden platform test) and a probe trial phase (transfer test). In the latency acquisition phase, animals are subjected to three or four trials per training session or day. In each trial, a different starting quadrant is used to release the animal into the water. In the probe trial phase, the platform is removed from the pool and the animal is placed in the water from any of the three quadrants that did not contain the platform and allowed to search for the now absent platform within a specified time; the time elapsed when the animal crosses the former platform location for the first time, the number of crossing, the time the animal spends and the path length the animal travels in the former platform quadrant are usually measured 3 , 4 . A question arising here is, do we still need to place the animals in water at different starting locations during the probe trial phase? Or how many probe trials should we do in each probe trial session? At present, different researchers take different approaches. For instance, Dr. Terry 5 recommended two successive trials for each probe trial session and a 90 s duration per trial in experiments using rats. On the other hand, Dr. Blokland 6 suggested using a shorter duration (30 s) for a trial, but many laboratories employ a 60 s duration for a single probe trial, such as Dr. Song’s laboratory 7 . A major concern about this issue is that an increased number of probe trials might result in memory extinction. Richard Morris, the pioneer of MWM, has discussed this topic in his foundational review paper “Developments of a water-maze procedure for studying spatial learning in the rat” 1 . Morris first used one animal from his experiment as an example. This rat underwent two transfer tests: 60 s (transfer test 1) and 24 h (transfer test 2) after the last trial of the hidden platform test. The rat spent more than 50% of its time in the former platform quadrant during transfer test 1, but only 36% of that during transfer test 2. In addition, the animal crossed the former platform location relatively more often during transfer test 1 than during transfer test 2. It seemed that repeated tests led to memory extinction in the rat. Nevertheless, in a subsequent test in which 21 rats were used (7 per group) and three transfer tests were conducted, he did not find evidence of memory extinction 1 . In the present study, we carried out a visual-cued water maze experiment and an acoustic-cued water maze experiment, with 20 mice in each, to address the issue of the appropriate number of trials in a probe trial session. Results Research design Forty mice were randomly divided into two groups: G1, visual-cued water maze; G2, acoustic-cued water maze. Each experiment lasted for 7 days. Days 1 to 6 were for latency acquisition, during which a hidden platform was used and each mouse underwent three trials a day with each trial using a different starting quadrant to place the animal in the water to find the hidden platform (using the visual or the acoustic cue). The time (in seconds) an animal spent finding the platform was recorded as escape latency; if an animal failed to find the platform within 60 s, the time was recorded as 60 s. The seventh day was the phase of probe trial. On this day, the hidden platform was removed and the animal was released into water in any quadrant other than the one where the hidden platform had been located, and the animal’s searching behavior within 60 s was observed. When the mouse reached the former platform location for the first time, the time it used was recorded as the first crossing . The number of times the animal crossed the former platform location was recorded as the crossing number . The above procedures were the same for the two types of water maze tests except for the cues. The relative position of the cues and the platform was kept constant throughout an experiment. The first crossing time increased with the number of probe trials in both types of water maze tests In the probe trial phase of both the visual-cued and acoustic-cued water maze tests, three trials were conducted for each mouse. The first crossing time in the three trials was collected. In both the visual-cued and acoustic-cued tests, the mean first crossing time increased from the first trial to the second trial and to the third trial (in general, the shorter the time, the better the memory); ANOVA and post hoc analysis for both tests revealed that the difference between the first and second trials was not statistically significant (P > 0.05), but the difference between the second and third trials was statistically significant (P < 0.05). See Tables 1 and 2 and Fig. 1 A. Table 1 Comparison of the first crossing and crossing number in the probe trials of the visual-cued water maze experiment (mean ± SD) Trials n First crossing (s) Crossing number First trial 20 21.82±18.97 1.70±1.17 Second trial 20 28.93±19.47* 1.60±1.05* Third trial 20 45.58±17.52** 0.80±1.28** F value - 8.531 3.547 P value - 0.001 0.035 Note: *The mean difference is not statistically significant at the 0.05 level compared with the first trial; **The mean difference is statistically significant at the 0.05 level compared with both the first trial and the second trial. Table 2 Comparison of the first crossing and crossing number in the probe trials of the acoustic-cued water maze experiment (mean ± SD) Trials n First crossing (s) Crossing number First trial 20 20.95 ± 21.42 2.35 ± 1.84 Second trial 20 29.05 ± 25.39* 1.85 ± 2.13* Third trial 20 44.95 ± 21.44** 0.80 ± 1.11** F value - 5.722 4.093 P value - 0.005 0.022 Note: *The mean difference is not statistically significant at the 0.05 level compared with the first trial; **The mean difference is statistically significant at the 0.05 level compared with the first and second trials. The crossing numbers decreased with the number of probe trials in both types of water maze tests In the probe trial phase of both the visual-cued and acoustic-cued water maze tests, each mouse was subjected to three trials. The number of crossing in the three trials was recorded. In both tests, the mean number of crossing decreased from trial to trial (a larger number of crossing generally indicates a better memory). ANOVA and post hoc analysis for both tests revealed that the difference between the first and second trials was not statistically significant (P > 0.05), but the difference between the second and third trials was statistically significant (P < 0.05). See Tables 1 and 2 and Fig. 1 B. Discussion The MWM test has traditionally been used to test spatial learning and memory in rodents, and is now also frequently used in the study of neurodegenerative disorders such as Alzheimer’s disease 8 – 10 . Accurate parameters, such as the time of the first crossing and the number of crossing, obtained in the probe trial phase are essential for a better evaluation of animal’s retrieval memory, and these parameters may be significantly affected by the frequency of probe trials. Here we performed two types of water maze experiments, a visual-cued and an acoustic-cued, to test the effects of the probe trial numbers on animal’s behavior in searching for former platform location in the probe trial phase. For both water maze tests, we carried out three probe trials for each mouse with each trial lasted for 60 s. We measured the time of the first crossing (of the former platform location) and the number of crossing, and used these parameters to analyze the effects of the probe trial frequency on animal’s behavior in water maze searching. In regarding to these two parameters, a smaller first crossing time and a larger number of crossing typically indicate a better memory of the former platform location. The results of our experiments showed that the time of the first crossing increased and the number of crossing decreased with consecutive probe trials, and the increase of the first crossing time and the decrease of the number of crossing were not statistically significant between the first and second trials but statistically significant between the second and third trials. The increased first crossing time and the decreased crossing numbers may be the results of memory extinction (i.e., failing to find the platform again and again in the former platform location may result in the deletion of the previously formed memory of the platform location). In summary, we conducted two types of water maze tests (a visual-cued and an acoustic-cued) to determine the appropriate number of probe trials for a water maze test. We found that an animal’s memory of the former platform location decreased significantly from the second to third probe trials but not from the first to second trials; thus, we suggest that the number of trials per probe trial session should be one or two but no more than two times. Materials and Methods Animals and grouping Twelve-week-old female KM mice were used (the KM mice are white). The animals were maintained on a 12 h light/12 h dark cycle with free access to food and water. Forty mice were randomly divided into two groups: G1 and G2, 20 in each group; G1 was used for the visual-cued water maze, and G2 was used for the acoustic-cued water maze. The experimental protocol and the use of the animals were in accordance with the guidelines and regulations of Yan’an University and were approved by the institutional academic committee. Apparatus The water maze tank and the video tracking system (software Xeye V3.2) are products of Beijing Zhongshi Dichuang Technology Development Co., Ltd. The tank is 120 cm in diameter and 40 cm in height. The interior wall of the tank is white and the bottom is black. The platform is 19 cm high, and its top surface area is 9 cm in diameter. The platform is painted black. To make the platform invisible, water was filled to a depth of ~ 19.7 cm and 20 ml of a kind of black food coloring agent (Shandong Kaibei Food Co., Ltd.) was added to the water. The temperature of the water was maintained at 25 ± 1°C during the experiments. Visual and sound cues The visual cue was black paper cut in a square with a side length of 12 cm, which was affixed on the interior wall of the tank, and its bottom side was 3 cm above the water surface. The sound cue was created by a Bluetooth speaker that was fixed to the outside of the tank brim. A buzzer sound with a frequency of about 1000 Hz was used. The intensity of the buzzer sound was adjusted to approximately 40 dB. Procedures Day 0 (preparation). The mouse was placed on the invisible platform in the pool (with neither visual nor sound cues) and allowed to remain there for 15 s. The animal was placed in the water and allowed to search for and climb onto the platform. After the animal climbed onto the platform it was removed from the pool. If the mouse failed to find the platform within 60 s, it was guided onto the platform and allowed to stay there for 10 s, and then was removed from the pool (avoiding removing the animal directly from the water). The animal was wiped with a dry towel and placed in a home cage. The purpose of these operations is to eliminate animals that have swimming, visual, or hearing deficits. It also allows the animals to know that there is a platform in the pool and that it can be rescued from the platform. Day 1 (latency acquisition phase). The animal was placed on the invisible platform in the pool (with a visual cue for the visual-cued experiment or a sound cue for the acoustic-cued experiment) and was allowed to stay there for 15 s, the purpose of which was to allow the animal to learn the position of the platform relative to the cues; this step was omitted for all the remaining trials. The animal was lowered into the water in any of the other three quadrants without the platform, with the animal facing the tank wall, and allowed to search for the platform. After the animal climbed onto the platform, it was allowed to stay there for 10 s before being removed. The animal was dried with a towel and placed in a cage with clean, dry and warm bedding (the cage was placed on a heating blanket). If the animal did not find the platform within 60 s, it was gently directed onto the platform and then removed after 10 s. These steps were repeated in the other two quadrants. A video tracking system was used to follow the animal in the pool. When the animal reached the platform, the time spent on the platform was recorded as the escape latency. If an animal failed to find the platform within 60 s, the escape latency was recorded as 60 s. Day 2 (latency acquisition phase). The platform was moved to another quadrant in the pool, and the visual and sound cues were also moved so that their position relative to the platform remained the same, which was to ensure that the animals would rely only on the defined visual or sound cue to locate the hidden platform instead of any other possible cues such as smell. Then the same steps as on day 1 were followed, but the animals were placed in the water in a quadrant order different from that on day 1. Day 3 ~ Day 6 (latency acquisition phase). The same procedures as on Day 1 and Day 2 were repeated. Day 7 (probe trial or transfer test phase). The platform was removed from the pool. The animal was placed in the water in any of the other three quadrants facing the tank wall. The following parameters were collected both manually and by the video tracking system: the time the animal spent reaching the former platform location for the first time ( first crossing ); the number of times the animal crossed the former platform location ( crossing number ). The above steps were repeated in the other two quadrants for the same animal. Statistical analysis SPSS 26.0 was used for the statistical analysis. One-way ANOVA (analysis of variance) combined with post hoc tests were used to analyze the data. P < 0.05 was considered to indicate statistical significance. Declarations Competing interests: The authors declare that they have no competing interests. Author Contribution Z.Y.F. designed the research, analyzed the data, and wrote the paper; X.H., X.L., Y.J., S.W., Z.Z., and C.G. performed the research or contributed materials. All authors reviewed the final manuscript. Acknowledgements: Supported by the NSFC grant 81760732 and Shaanxi Provincial Department of Science and Technology Project 2022SF-393. Data Availability The datasets used and/or analysed during the current study are included in the supplementary information file [a compressed file]. References Morris, R. Developments of a water-maze procedure for studying spatial learning in the rat. Journal of neuroscience methods 11, 47–60, doi: 10.1016/0165-0270(84)90007-4 (1984). Fu, Z. Y. Development of a acoustic-cued water maze experiment. PNAS USA Under review (2024). Vorhees, C. V. & Williams, M. T. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nature protocols 1, 848–858, doi: 10.1038/nprot.2006.116 (2006). Othman, M. Z., Hassan, Z. & Che Has, A. T. Morris water maze: a versatile and pertinent tool for assessing spatial learning and memory. Experimental animals 71, 264–280, doi: 10.1538/expanim.21-0120 (2022). Terry, A. V., Jr. in Methods of Behavior Analysis in Neuroscience (ed J. J. Buccafusco) (CRC Press/Taylor & Francis. Copyright © 2009, Taylor & Francis Group, LLC., 2009). Blokland, A., Geraerts, E. & Been, M. A detailed analysis of rats' spatial memory in a probe trial of a Morris task. Behav Brain Res 154, 71–75, doi: 10.1016/j.bbr.2004.01.022 (2004). Bromley-Brits, K., Deng, Y. & Song, W. Morris water maze test for learning and memory deficits in Alzheimer's disease model mice. Journal of visualized experiments: JoVE, doi: 10.3791/2920 (2011). Shi, Y. et al. Bis(9)-(-)-Meptazinol, a novel dual-binding AChE inhibitor, rescues cognitive deficits and pathological changes in APP/PS1 transgenic mice. Translational neurodegeneration 7, 21, doi: 10.1186/s40035-018-0126-8 (2018). Liu, E. et al. Enriched gestation activates the IGF pathway to evoke embryo-adult benefits to prevent Alzheimer's disease. Translational neurodegeneration 8, 8, doi: 10.1186/s40035-019-0149-9 (2019). Wang, J. et al. Pharmacological inhibition of asparaginyl endopeptidase by δ-secretase inhibitor 11 mitigates Alzheimer's disease-related pathologies in a senescence-accelerated mouse model. Translational neurodegeneration 10, 12, doi: 10.1186/s40035-021-00235-4 (2021). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4134660","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":288167525,"identity":"d45adbbb-cf55-4a0f-82bd-149e5277f3ec","order_by":0,"name":"Xiaodong Han","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Han","suffix":""},{"id":288167528,"identity":"4e2d1bef-dcfc-4b8e-9f0a-39a01b8f9508","order_by":1,"name":"Xiaobin Liu","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Xiaobin","middleName":"","lastName":"Liu","suffix":""},{"id":288167530,"identity":"916a8459-443e-43a5-bf2d-8b946705aafc","order_by":2,"name":"Yanna Jiang","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Yanna","middleName":"","lastName":"Jiang","suffix":""},{"id":288167532,"identity":"37f2cdec-3783-4209-87c7-ece2edc3501a","order_by":3,"name":"Shuai Wu","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Wu","suffix":""},{"id":288167534,"identity":"d6bac0af-cd29-4961-9e7b-c8b7a3f541f4","order_by":4,"name":"Zhe Zhang","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Zhang","suffix":""},{"id":288167535,"identity":"2c37147c-092d-4206-8bb5-3da17a3e61b6","order_by":5,"name":"Cheng Gao","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Gao","suffix":""},{"id":288167536,"identity":"4ec2fa83-8f5e-4d0c-b65e-34a56dbc9690","order_by":6,"name":"Zhaoying Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYDACCRBhAMTMBxgfQIQSiNXClsBsQIIWBrAWNgmitMjPbn74mKfgjl0DG49ZNU/NYQZ+9hwDhp87cGthnHPM2JjH4FkySMttnmOHGSR73hgw9p7BrYVZIsFMmsfgcDKDfA9QC9thBoMbOQbMjG24tbBJpH+DaAHaUszz7zCDPSEtPBI5YFvsQFqYeduAtkgQ0CIhkVNsOMfgcAIDG1ux5Ny+dB6JM88KDvbi0SI/I33jgzd/DtszsDFv/PDmm7Ucf3vyxgc/8WgBASYeBobE/QcgDB6QyAH8GoAB/YOBwR7GGAWjYBSMglGAAQD3mUd9rwZ89AAAAABJRU5ErkJggg==","orcid":"","institution":"Yan'an University","correspondingAuthor":true,"prefix":"","firstName":"Zhaoying","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2024-03-20 06:41:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4134660/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4134660/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54283709,"identity":"09b4f050-2394-4c9f-bc70-a9f5d1515953","added_by":"auto","created_at":"2024-04-08 09:46:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the first crossing time (A) and the crossing number (B) in three probe trials of the visual-cued and acoustic-cued water maze experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e 1) Error bars represent the standard error of the mean. 2) *P\u0026gt;0.05 vs. the first trial; **P\u0026lt;0.05 vs. both the first trial and the second trial. 3) Also see Tables 1 and 2 for details.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4134660/v1/e6086717160692a1bd6a79a3.png"},{"id":57072339,"identity":"4907deb9-2684-4340-8be8-b70854581e7d","added_by":"auto","created_at":"2024-05-24 08:32:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":458382,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4134660/v1/bed8e2a4-0c8b-4d4e-beac-b28dc36dc02e.pdf"},{"id":54283711,"identity":"d537c2ee-3b31-413c-b272-75d47a33b524","added_by":"auto","created_at":"2024-04-08 09:46:25","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35497,"visible":true,"origin":"","legend":"","description":"","filename":"Rawdata.rar","url":"https://assets-eu.researchsquare.com/files/rs-4134660/v1/272792d37dd80f50640a038c.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"The probe trial in a water maze test should not exceed two times","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Morris water maze (MWM) test is a classic test to measure spatial learning and memory in rodents. The procedures of the MWM test or its derivatives typically include two phases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e: a latency acquisition phase (hidden platform test) and a probe trial phase (transfer test). In the latency acquisition phase, animals are subjected to three or four trials per training session or day. In each trial, a different starting quadrant is used to release the animal into the water.\u003c/p\u003e \u003cp\u003eIn the probe trial phase, the platform is removed from the pool and the animal is placed in the water from any of the three quadrants that did not contain the platform and allowed to search for the now absent platform within a specified time; the time elapsed when the animal crosses the former platform location for the first time, the number of crossing, the time the animal spends and the path length the animal travels in the former platform quadrant are usually measured\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. A question arising here is, do we still need to place the animals in water at different starting locations during the probe trial phase? Or how many probe trials should we do in each probe trial session? At present, different researchers take different approaches. For instance, Dr. Terry\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e recommended two successive trials for each probe trial session and a 90 s duration per trial in experiments using rats. On the other hand, Dr. Blokland\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e suggested using a shorter duration (30 s) for a trial, but many laboratories employ a 60 s duration for a single probe trial, such as Dr. Song\u0026rsquo;s laboratory\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA major concern about this issue is that an increased number of probe trials might result in memory extinction. Richard Morris, the pioneer of MWM, has discussed this topic in his foundational review paper \u0026ldquo;Developments of a water-maze procedure for studying spatial learning in the rat\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMorris first used one animal from his experiment as an example. This rat underwent two transfer tests: 60 s (transfer test 1) and 24 h (transfer test 2) after the last trial of the hidden platform test. The rat spent more than 50% of its time in the former platform quadrant during transfer test 1, but only 36% of that during transfer test 2. In addition, the animal crossed the former platform location relatively more often during transfer test 1 than during transfer test 2. It seemed that repeated tests led to memory extinction in the rat. Nevertheless, in a subsequent test in which 21 rats were used (7 per group) and three transfer tests were conducted, he did not find evidence of memory extinction\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, we carried out a visual-cued water maze experiment and an acoustic-cued water maze experiment, with 20 mice in each, to address the issue of the appropriate number of trials in a probe trial session.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eResearch design\u003c/h2\u003e\n\u003cp\u003eForty mice were randomly divided into two groups: G1, visual-cued water maze; G2, acoustic-cued water maze. Each experiment lasted for 7 days. \u003cstrong\u003eDays 1 to 6\u003c/strong\u003e were for latency acquisition, during which a hidden platform was used and each mouse underwent three trials a day with each trial using a different starting quadrant to place the animal in the water to find the hidden platform (using the visual or the acoustic cue). The time (in seconds) an animal spent finding the platform was recorded as escape latency; if an animal failed to find the platform within 60 s, the time was recorded as 60 s. \u003cstrong\u003eThe seventh day\u003c/strong\u003e was the phase of probe trial. On this day, the hidden platform was removed and the animal was released into water in any quadrant other than the one where the hidden platform had been located, and the animal\u0026rsquo;s searching behavior within 60 s was observed. When the mouse reached the former platform location for the first time, the time it used was recorded as the \u003cspan class=\"Underline\"\u003efirst crossing\u003c/span\u003e. The number of times the animal crossed the former platform location was recorded as the \u003cspan class=\"Underline\"\u003ecrossing number\u003c/span\u003e. The above procedures were the same for the two types of water maze tests except for the cues. The relative position of the cues and the platform was kept constant throughout an experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe first crossing time increased with the number of probe trials in both types of water maze tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the probe trial phase of both the visual-cued and acoustic-cued water maze tests, three trials were conducted for each mouse. The first crossing time in the three trials was collected. In both the visual-cued and acoustic-cued tests, the mean first crossing time increased from the first trial to the second trial and to the third trial (in general, the shorter the time, the better the memory); ANOVA and post hoc analysis for both tests revealed that the difference between the first and second trials was not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but the difference between the second and third trials was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). See Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of the first crossing and crossing number in the probe trials of the visual-cued water maze experiment (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eTrials\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eFirst crossing (s)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eCrossing number\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eFirst trial\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e21.82\u0026plusmn;18.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e1.70\u0026plusmn;1.17\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eSecond trial\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e28.93\u0026plusmn;19.47*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e1.60\u0026plusmn;1.05*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eThird trial\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e45.58\u0026plusmn;17.52**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e0.80\u0026plusmn;1.28**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eF value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e8.531\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e3.547\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eP value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e0.035\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote:\u0026nbsp; *The mean difference is not statistically significant at the 0.05 level compared with the first trial; **The mean difference is statistically significant at the 0.05 level compared with both the first trial and the second trial.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of the first crossing and crossing number in the probe trials of the acoustic-cued water maze experiment (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTrials\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFirst crossing (s)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCrossing number\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFirst trial\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.95\u0026thinsp;\u0026plusmn;\u0026thinsp;21.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSecond trial\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29.05\u0026thinsp;\u0026plusmn;\u0026thinsp;25.39*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThird trial\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e44.95\u0026thinsp;\u0026plusmn;\u0026thinsp;21.44**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.722\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.093\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eP value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.022\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003eNote: *The mean difference is not statistically significant at the 0.05 level compared with the first trial; **The mean difference is statistically significant at the 0.05 level compared with the first and second trials.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe crossing numbers decreased with the number of probe trials in both types of water maze tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the probe trial phase of both the visual-cued and acoustic-cued water maze tests, each mouse was subjected to three trials. The number of crossing in the three trials was recorded. In both tests, the mean number of crossing decreased from trial to trial (a larger number of crossing generally indicates a better memory). ANOVA and post hoc analysis for both tests revealed that the difference between the first and second trials was not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but the difference between the second and third trials was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). See Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe MWM test has traditionally been used to test spatial learning and memory in rodents, and is now also frequently used in the study of neurodegenerative disorders such as Alzheimer\u0026rsquo;s disease\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Accurate parameters, such as the time of the first crossing and the number of crossing, obtained in the probe trial phase are essential for a better evaluation of animal\u0026rsquo;s retrieval memory, and these parameters may be significantly affected by the frequency of probe trials.\u003c/p\u003e \u003cp\u003eHere we performed two types of water maze experiments, a visual-cued and an acoustic-cued, to test the effects of the probe trial numbers on animal\u0026rsquo;s behavior in searching for former platform location in the probe trial phase. For both water maze tests, we carried out three probe trials for each mouse with each trial lasted for 60 s. We measured the time of the first crossing (of the former platform location) and the number of crossing, and used these parameters to analyze the effects of the probe trial frequency on animal\u0026rsquo;s behavior in water maze searching.\u003c/p\u003e \u003cp\u003eIn regarding to these two parameters, a smaller first crossing time and a larger number of crossing typically indicate a better memory of the former platform location. The results of our experiments showed that the time of the first crossing increased and the number of crossing decreased with consecutive probe trials, and the increase of the first crossing time and the decrease of the number of crossing were not statistically significant between the first and second trials but statistically significant between the second and third trials. The increased first crossing time and the decreased crossing numbers may be the results of memory extinction (i.e., failing to find the platform again and again in the former platform location may result in the deletion of the previously formed memory of the platform location).\u003c/p\u003e \u003cp\u003eIn summary, we conducted two types of water maze tests (a visual-cued and an acoustic-cued) to determine the appropriate number of probe trials for a water maze test. We found that an animal\u0026rsquo;s memory of the former platform location decreased significantly from the second to third probe trials but not from the first to second trials; thus, we suggest that the number of trials per probe trial session should be one or two but no more than two times.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and grouping\u003c/h2\u003e \u003cp\u003eTwelve-week-old female KM mice were used (the KM mice are white). The animals were maintained on a 12 h light/12 h dark cycle with free access to food and water. Forty mice were randomly divided into two groups: G1 and G2, 20 in each group; G1 was used for the visual-cued water maze, and G2 was used for the acoustic-cued water maze. The experimental protocol and the use of the animals were in accordance with the guidelines and regulations of Yan\u0026rsquo;an University and were approved by the institutional academic committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eApparatus\u003c/h2\u003e \u003cp\u003eThe water maze tank and the video tracking system (software Xeye V3.2) are products of Beijing Zhongshi Dichuang Technology Development Co., Ltd. The tank is 120 cm in diameter and 40 cm in height. The interior wall of the tank is white and the bottom is black. The platform is 19 cm high, and its top surface area is 9 cm in diameter. The platform is painted black. To make the platform invisible, water was filled to a depth of ~\u0026thinsp;19.7 cm and 20 ml of a kind of black food coloring agent (Shandong Kaibei Food Co., Ltd.) was added to the water. The temperature of the water was maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C during the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVisual and sound cues\u003c/h2\u003e \u003cp\u003eThe visual cue was black paper cut in a square with a side length of 12 cm, which was affixed on the interior wall of the tank, and its bottom side was 3 cm above the water surface. The sound cue was created by a Bluetooth speaker that was fixed to the outside of the tank brim. A buzzer sound with a frequency of about 1000 Hz was used. The intensity of the buzzer sound was adjusted to approximately 40 dB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProcedures\u003c/h2\u003e \u003cp\u003e \u003cb\u003eDay 0 (preparation).\u003c/b\u003e The mouse was placed on the invisible platform in the pool (with neither visual nor sound cues) and allowed to remain there for 15 s. The animal was placed in the water and allowed to search for and climb onto the platform. After the animal climbed onto the platform it was removed from the pool. If the mouse failed to find the platform within 60 s, it was guided onto the platform and allowed to stay there for 10 s, and then was removed from the pool (avoiding removing the animal directly from the water). The animal was wiped with a dry towel and placed in a home cage. The purpose of these operations is to eliminate animals that have swimming, visual, or hearing deficits. It also allows the animals to know that there is a platform in the pool and that it can be rescued from the platform.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDay 1 (latency acquisition phase).\u003c/b\u003e The animal was placed on the invisible platform in the pool (with a visual cue for the visual-cued experiment or a sound cue for the acoustic-cued experiment) and was allowed to stay there for 15 s, the purpose of which was to allow the animal to learn the position of the platform relative to the cues; this step was omitted for all the remaining trials. The animal was lowered into the water in any of the other three quadrants without the platform, with the animal facing the tank wall, and allowed to search for the platform. After the animal climbed onto the platform, it was allowed to stay there for 10 s before being removed. The animal was dried with a towel and placed in a cage with clean, dry and warm bedding (the cage was placed on a heating blanket). If the animal did not find the platform within 60 s, it was gently directed onto the platform and then removed after 10 s. These steps were repeated in the other two quadrants.\u003c/p\u003e \u003cp\u003eA video tracking system was used to follow the animal in the pool. When the animal reached the platform, the time spent on the platform was recorded as the escape latency. If an animal failed to find the platform within 60 s, the escape latency was recorded as 60 s.\u003c/p\u003e \u003cp\u003e\u003cb\u003eDay 2 (latency acquisition phase).\u003c/b\u003e The platform was moved to another quadrant in the pool, and the visual and sound cues were also moved so that their position relative to the platform remained the same, which was to ensure that the animals would rely only on the defined visual or sound cue to locate the hidden platform instead of any other possible cues such as smell. Then the same steps as on day 1 were followed, but the animals were placed in the water in a quadrant order different from that on day 1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDay 3\u0026thinsp;~\u0026thinsp;Day 6 (latency acquisition phase).\u003c/b\u003e The same procedures as on Day 1 and Day 2 were repeated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDay 7 (probe trial or transfer test phase).\u003c/b\u003e The platform was removed from the pool. The animal was placed in the water in any of the other three quadrants facing the tank wall. The following parameters were collected both manually and by the video tracking system: the time the animal spent reaching the former platform location for the first time (\u003cb\u003efirst crossing\u003c/b\u003e); the number of times the animal crossed the former platform location (\u003cb\u003ecrossing number\u003c/b\u003e). The above steps were repeated in the other two quadrants for the same animal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS 26.0 was used for the statistical analysis. One-way ANOVA (analysis of variance) combined with post hoc tests were used to analyze the data. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.Y.F. designed the research, analyzed the data, and wrote the paper; X.H., X.L., Y.J., S.W., Z.Z., and C.G. performed the research or contributed materials. All authors reviewed the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eSupported by the NSFC grant 81760732 and Shaanxi Provincial Department of Science and Technology Project 2022SF-393.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are included in the supplementary information file [a compressed file].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMorris, R. Developments of a water-maze procedure for studying spatial learning in the rat. Journal of neuroscience methods 11, 47\u0026ndash;60, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0165-0270(84)90007-4\u003c/span\u003e\u003cspan address=\"10.1016/0165-0270(84)90007-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu, Z. Y. Development of a acoustic-cued water maze experiment. PNAS USA Under review (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVorhees, C. V. \u0026amp; Williams, M. T. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nature protocols 1, 848\u0026ndash;858, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nprot.2006.116\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2006.116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOthman, M. Z., Hassan, Z. \u0026amp; Che Has, A. T. Morris water maze: a versatile and pertinent tool for assessing spatial learning and memory. Experimental animals 71, 264\u0026ndash;280, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1538/expanim.21-0120\u003c/span\u003e\u003cspan address=\"10.1538/expanim.21-0120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerry, A. V., Jr. in \u003cem\u003eMethods of Behavior Analysis in Neuroscience\u003c/em\u003e (ed J. J. Buccafusco) (CRC Press/Taylor \u0026amp; Francis. Copyright \u0026copy; 2009, Taylor \u0026amp; Francis Group, LLC., 2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlokland, A., Geraerts, E. \u0026amp; Been, M. A detailed analysis of rats' spatial memory in a probe trial of a Morris task. Behav Brain Res 154, 71\u0026ndash;75, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbr.2004.01.022\u003c/span\u003e\u003cspan address=\"10.1016/j.bbr.2004.01.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBromley-Brits, K., Deng, Y. \u0026amp; Song, W. Morris water maze test for learning and memory deficits in Alzheimer's disease model mice. Journal of visualized experiments: JoVE, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/2920\u003c/span\u003e\u003cspan address=\"10.3791/2920\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, Y. \u003cem\u003eet al.\u003c/em\u003e Bis(9)-(-)-Meptazinol, a novel dual-binding AChE inhibitor, rescues cognitive deficits and pathological changes in APP/PS1 transgenic mice. Translational neurodegeneration 7, 21, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40035-018-0126-8\u003c/span\u003e\u003cspan address=\"10.1186/s40035-018-0126-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, E. \u003cem\u003eet al.\u003c/em\u003e Enriched gestation activates the IGF pathway to evoke embryo-adult benefits to prevent Alzheimer's disease. Translational neurodegeneration 8, 8, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40035-019-0149-9\u003c/span\u003e\u003cspan address=\"10.1186/s40035-019-0149-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, J. \u003cem\u003eet al.\u003c/em\u003e Pharmacological inhibition of asparaginyl endopeptidase by δ-secretase inhibitor 11 mitigates Alzheimer's disease-related pathologies in a senescence-accelerated mouse model. Translational neurodegeneration 10, 12, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40035-021-00235-4\u003c/span\u003e\u003cspan address=\"10.1186/s40035-021-00235-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Morris water maze, probe trial, transfer test, neurodegenerative disorder, Alzheimer’s disease","lastPublishedDoi":"10.21203/rs.3.rs-4134660/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4134660/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe performed two types of water maze tests (a visual-cued and an acoustic-cued) to determine the appropriate number of probe trials for a water maze test. Three probe trials were applied in the probe trial phase of each experiment. The observational indexes included the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003etime of the first crossing\u003c/span\u003e and the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003enumber of crossing\u003c/span\u003e. In both types of the tests, the results showed that the time of the first crossing increased significantly from the second to the third probe trials (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the number of crossing decreased significantly from the second to the third probe trials (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Meanwhile, the differences between the first and the second probe trials were not statistically significant regarding both the time of the first crossing and the number of crossing (both P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Therefore, we believe that the number of probe trials in a water maze test should be one or two but no more than two times.\u003c/p\u003e","manuscriptTitle":"The probe trial in a water maze test should not exceed two times","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 09:46:19","doi":"10.21203/rs.3.rs-4134660/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0f61d980-d213-41df-a437-14ba8f7b7929","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30341871,"name":"Biological sciences/Neuroscience"},{"id":30341872,"name":"Biological sciences/Physiology"},{"id":30341873,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2024-05-24T08:24:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-08 09:46:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4134660","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4134660","identity":"rs-4134660","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}