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A Comprehensive Behavioral Test Battery to Assess Learning and Memory in 129S6/Tg2576 Mice
Open accessAbstract
Transgenic Tg2576 mice overexpressing human amyloid precursor protein (hAPP) are a widely used Alzheimer's disease (AD) mouse model to evaluate treatment effects on amyloid beta (Aβ) pathology and cognition. Tg2576 mice on a B6;SJL background strain carry a recessive rd1 mutation that leads to early retinal degeneration and visual impairment in homozygous carriers. This can impair performance in behavioral tests that rely on visual cues, and thus, affect study results. Therefore, B6;SJL/Tg2576 mice were systematically backcrossed with 129S6/SvEvTac mice resulting in 129S6/Tg2576 mice that lack the rd1 mutation. 129S6/Tg2576 mice do not develop retinal degeneration but still show Aβ accumulation in the brain that is comparable to the original B6;SJL/Tg2576 mouse. However, comprehensive studies on cognitive decline in 129S6/Tg2576 mice are limited. In this study, we used two dementia mouse models on a 129S6 background--scopolamine-treated 129S6/SvEvTac mice (3-5 month-old) and transgenic 129S6/Tg2576 mice (11-13 month-old)-to establish a behavioral test battery for assessing learning and memory. The test battery consisted of five tests to evaluate different aspects of cognitive impairment: a Y-Maze forced alternation task, a novel object recognition test, the Morris water maze, the radial arm water maze, and a Y-maze spontaneous alternation task. We first established this behavioral test battery with the scopolamine-induced dementia model using 129S6/SvEvTac mice and then evaluated 129S6/Tg2576 mice using the same testing protocol. Both models showed distinctive patterns of cognitive impairment. Together, the non-invasive behavioral test battery presented here allows detecting cognitive impairment in scopolamine-treated 129S6/SvEvTac mice and in transgenic 129S6/Tg2576 mice. Due to the modular nature of this test battery, more behavioral tests, e.g. invasive assays to gain additional cognitive information, can easily be added.
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Antenatal Hypoxia Accelerates the Onset of Alzheimer’s Disease Pathology in 5xFAD Mouse Model
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“, 2016) and novel object recognition (NOR) test (Wolf et al., 2016) were conducted in a square test box (40 × 40 × 40 cm3).”
MethodInfluentialRaony, Í., Domith, I., Lourenco, M. V., Paes‐de‐Carvalho, R., & Pandolfo, P. (2022) · Progress in Neuro-Psychopharmacology and Biological Psychiatry · doi:10.1016/j.pnpbp.2022.110555 · Cited by 20
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Neuroinflammation Induced by Transgenic Expression of Lipocalin-2 in Astrocytes
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“The spontaneous alternation measurement using a Y-maze was used as a test for habituation and spatial working memory (Wolf et al., 2016).”
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The prevention of home-cage grid climbing affects muscle strength in mice
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“T maze spontaneous alternation The T maze spontaneous alternation test was run according to the procedure reported by Wolf and colleagues [40].”
Disease modification upon 2 weeks of tofacitinib treatment in a mouse model of chronic epilepsy
Hoffman, O. R., Koehler, J. L., Espina, J. E. C., Patterson, A. M., Gohar, E. S., Coleman, E. M., Schoenike, B. A., Espinosa‐García, C., Paredes, F., Varvel, N. H., et al. (2025) · Science Translational Medicine · doi:10.1126/scitranslmed.adt0527 · Cited by 20
Neuroepigenetic Changes in DNA Methylation Affecting Diabetes-Induced Cognitive Impairment
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“The spontaneous alternation Y-maze was used to evaluate the spatial working memory in 14-week-old mice as previously described with few modifications (38).”
MethodMahnashi, M. H., Ayaz, M., Alqahtani, Y. S., Alyami, B. A., Shahid, M. M., Alqahtani, O. S., Kabrah, S. M., Zeb, A., Ullah, F., & Sadiq, A. (2023) · Journal of Ethnopharmacology · doi:10.1016/j.jep.2023.116786 · Cited by 18
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“Mice were placed into the centre of the maze (3 arms at a 120 ̊ angle from each other, arms 30cm long and 10cm wide; walls and floor opaque and 10cm high) and their behaviour video-recorded for 5 min [39] or 8 minutes [26, 44, 45] for subsequent analysis.”
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Enhanced synaptic plasticity and spatial memory in female but not male FLRT2-haplodeficient mice
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MethodThe Nanotheranostic Researcher’s Guide for Use of Animal Models of Traumatic Brain Injury
McDonald, B. Z., Gee, C. C., & Kievit, F. M. (2021) · Journal of Nanotheranostics · doi:10.3390/jnt2040014 · Cited by 15
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Cognitive decline, Aβ pathology, and blood–brain barrier function in aged 5xFAD mice
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“1) as previously described [23].”
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Mechanosensitive ion channels and inflammation: key links in cellular signal transduction
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“A decrease in spontaneous alternation indicates a deficit in spatial working memory, which is a common cognitive impairment in AD [173].”
Zamarbide, M., Martínez‐Pinilla, E., Gil-Bea, F. J., Yanagisawa, M., Franco, R., & Pérez‐Mediavilla, A. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23073533 · Cited by 13
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“For this set of assessments (Figure 4), we used the Y maze, the novel object recognition (NOR), and the block test, designed to assess memory and learning [22].”
MethodAhmed, T., Ishaq, S., Siyar, S., Basri, R., Liaqat, A., & Hameed, A. (2022) · Current Molecular Pharmacology · doi:10.2174/1874467215666220928110557 · Cited by 12
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“less than three arm entries during the first minute of the second Y-maze trial were also excluded (see SI Methods and (Wolf et al., 2016)).”
BackgroundMice Recognise Mice in Neighbouring Rearing Cages and Change Their Social Behaviour
Ueno, H., Takahashi, Y., Mori, S., Murakami, S., Wani, K., Matsumoto, Y., Okamoto, M., & Ishihara, T. (2024) · Behavioural Neurology · doi:10.1155/2024/9215607 · Cited by 11
“Behavioural experiments in mice have long been an important test and are widely practised worldwide [46, 58].”
Youssef, Y. A., Tammam, S. N., El-Shenawy, B. M., Ilyas, S., Gad, A. A., Farag, K. S., Mathur, S., & Abdel-Kader, R. M. (2025) · European Journal of Pharmaceutics and Biopharmaceutics · doi:10.1016/j.ejpb.2025.114778 · Cited by 12
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Postnatal therapeutic approaches in genetic neurodevelopmental disorders
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“…1999, 2008; Moy et al., 2008), OCD (Burguière et al., 2015; Monteiro and Feng, 2016), cognition properties (Brooks et al., 2005; Crawley, 2008; Wolf et al., 2016), alterations in social behavior (Crawley, 1999, 2008; Moy et al., 2008; Netser et al., 2009), motor functions and anxiety…”
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“Both Y maze and MWM tests are commonly used to assess memory and cognition deficits (Wolf et al. 2016).”
Heat Shock Protein 70 as a Sex-Skewed Regulator of α-Synucleinopathy
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“Cognitive deficits were assessed by the Y-maze test [88] and anxiety-related behaviors were tested in an open field arena [87, 89, 90].”
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“In line with the mentioned functions, microtubules provide a platform for information storage and memory consolidation in neurons (Dent and Baas 2014; Dent 2017; Kaganovsky and Wang 2016; Woolf 2006).”
BackgroundRomero, A., Pérez, T., Santibáñez, N., Vega, M., & Miranda, P. (2020) · Aquaculture · doi:10.1016/j.aquaculture.2020.736170 · Cited by 10
Sáenz-Martínez, D. E., Santana, P., Aróstica, M., Forero, J. C., Guzmán, F., & Mercado, L. A. (2021) · Developmental & Comparative Immunology · doi:10.1016/j.dci.2021.104182 · Cited by 10
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A Concomitant Muscle Injury Does Not Worsen Traumatic Brain Injury Outcomes in Mice
Sun, M., Brady, R. D., van der Poel, C., Apted, D., Semple, B. D., Church, J. E., O'Brien, T. J., McDonald, S. J., & Shultz, S. R. (2018) · Frontiers in Neurology · doi:10.3389/fneur.2018.01089 · Cited by 9
“Spatial cognition was assessed in Y-maze as previously described (26, 40).”
MethodAlfheeaid, H., Alhowail, A., Ahmed, F., Zaki, A., & Alkhaldy, A. (2023) · Brain Science · doi:10.3390/brainsci13020165 · Cited by 11
“The Y-maze is a behavioral test used to assess the ability of an animal to recognize familiar places and its tendency to explore new pl ces [18].”
BackgroundMethodSketriené, D., Battista, D., Lalert, L., Kraiwattanapirom, N., Thai, H. N., Leeboonngam, T., Knackstedt, L. A., Nithianantharajah, J., Sumithran, P., Lawrence, A. J., et al. (2022) · Addiction Biology · doi:10.1111/adb.13206 · Cited by 10
“receptor mediated currents as compared to NMDA previously observed(5) but a body of literature exists demonstrating a role for NAc GluA2-lacking (presumably GluA1-containing) AMPAR in incubation of drug craving and enduring vulnerability to relapse.(9,16,32)”
ResultTekam, C. K. S., Majumdar, S., Kumari, P., Prajapati, S. K., Sahi, A. K., Shinde, S., Singh, R., Samaiya, P. K., Patnaik, R., Krishnamurthy, S., et al. (2022) · Toxicology · doi:10.1016/j.tox.2022.153409 · Cited by 9
Saadullah, M., Fakhar-e-alam, M., Muzammil, S., Asif, M., Siddique, M. H., Rashad, M., Atif, M. M., Sayyid, A. H., & Afzal, M. (2023) · Journal of King Saud University - Science · doi:10.1016/j.jksus.2023.102785 · Cited by 9
Graham, D. L., Meyer, J. S., & Stanwood, G. D. (2018) · Elsevier eBooks · doi:10.1016/b978-0-12-809405-1.00025-0 · Cited by 9
Comparative Behavioral Correlation of High and Low-Performing Mice in the Forced Swim Test
Valencia, S. T., Gonzales, E. L., Adil, K. J., Jeon, S. J., Kwon, K. J., Cho, K. S., & Shin, C. Y. (2019) · Biomolecules & Therapeutics · doi:10.4062/biomolther.2018.210 · Cited by 9
Acute Fasting Does Not Induce Cognitive Impairment in Mice
Zheng, H., Ton, H. T., Yang, L., Liufu, N., Dong, Y., Zhang, Y., & Xie, Z. (2019) · Frontiers in Neuroscience · doi:10.3389/fnins.2019.00896 · Cited by 9
“These results indicated that the acute fasting enhanced the spatial recognition memory of the mice as compared to the control condition, consistent with the findings from the other studies (Rayatnia et al., 2011; Wolf et al., 2016).”
ResultSorial, M. E. S., Abdelghany, R. M., & Sayed, N. S. E. (2025) · Inflammopharmacology · doi:10.1007/s10787-025-01695-0 · Cited by 8
“Percentage of time spent in and the percentage of visits to the novel arm were calculated (Martini et al. 2014; Sanderson et al. 2009; Wolf et al. 2016).”
de Tena, I. B., Moreno‐Rodríguez, M., Llorente‐Ovejero, A., Benito, S. M., Martínez‐Gardeazabal, J., Onandia‐Hinchado, I., Manuel, I., Giménez‐Llort, L., & Rodríguez‐Puertas, R. (2022) · European Journal of Neuroscience · doi:10.1111/ejn.15642 · Cited by 6
“…et al., 2010; McIlwain et al., 2001; von Kortzfleisch et al., 2019), the use of behavioral batteries that include several consecutive tests is a usual approach for the evaluation of fear, anxiety, cognition, and learning in rodents (Blanchard et al., 2003; Wolf et al., 2016; You et al., 2019).”
BackgroundCruz-Carrillo, G., Trujillo-Villarreal, L. A., Ángeles-Valdéz, D., Concha, L., Garza‐Villarreal, E. A., & Camacho‐Morales, A. (2022) · Neuroscience · doi:10.1016/j.neuroscience.2022.12.021 · Cited by 8
Micco, S. D., Ciaglia, T., Salviati, E., Michela, P., Kostrzewa, M., Musella, S., Moriello, A. S., Sarno, V. D., Smaldone, G., Matteo, F. D., et al. (2024) · European Journal of Medicinal Chemistry · doi:10.1016/j.ejmech.2024.116298 · Cited by 8
Subarachnoid hemorrhage in C57BL/6J mice increases motor stereotypies and compulsive-like behaviors
Nanegrungsunk, D., Ragozzino, M. E., Xu, H., Haselton, K. J., & Paisansathan, C. (2020) · Neurological Research · doi:10.1080/01616412.2020.1841481 · Cited by 8
Gnanaprakash, M., Staniszewski, A., Zhang, H., Pitstick, R., Kavanaugh, M. P., Arancio, O., & Nicholls, R. E. (2021) · Journal of Alzheimer s Disease · doi:10.3233/jad-200462 · Cited by 9
“impairments caused by the rd1 mutation and to exhibit more consistent behavioral performance than animals maintained in the original mixed B6;SJL background [52, 53].”
BackgroundQuerfurth, H. W., Slitt, A. L., DiCamillo, A., Surles, N. O., DeBoef, B., & Lee, H. (2022) · SSRN Electronic Journal · doi:10.2139/ssrn.4105279 · Cited by 8
Trivedi, M. K., Branton, A., Trivedi, D., Mondal, S. C., & Jana, S. (2023) · BMC Nutrition · doi:10.1186/s40795-023-00767-0 · Cited by 7
“The novelty test was used to determine the time spent and the number of entries in each arm [12].”
MethodKhan, N. S., Isaza, J. U., Rouhi, N., Jamani, N. F., Jabeen, S., Gill, A. K., Tsutsui, M., Visser, F., & Sargin, D. (2024) · ACS Chemical Neuroscience · doi:10.1021/acschemneuro.3c00626 · Cited by 8
Ontawong, A., Nehra, G., Maloney, B. J., Vaddhanaphuti, C. S., Bauer, B., & Hartz, A. M. S. (2025) · International Journal of Molecular Sciences · doi:10.3390/ijms26094352 · Cited by 7
“Specific luminal NBD-CSA fluorescence was determined as the difference between the total luminal fluorescence and fluorescence in the presence of the P-gp specific inhibitor PSC833 (5 µ M final concentration).”
Self-citationHu, S. X., Lopez-Robles, Y., Shen, G., Liu, E., Zhang, L., & Ma, Q. (2025) · Cells · doi:10.3390/cells14151202 · Cited by 7
“The data were analyzed via NIH ImageJ software 1.54e with the MouBeAT plugin [34] by an investigator blinded to experimental groups.”
Augmented impulsive behavior in febrile seizure-induced mice
Remonde, C. G., Gonzales, E. L., Adil, K. J., Jeon, S. J., & Shin, C. Y. (2022) · Toxicological Research · doi:10.1007/s43188-022-00145-1 · Cited by 7
Twitto‐Greenberg, R., Zaltsman, S. L., Michaelson, D. M., Liraz, O., Lubitz, I., Atrakchi, D., Shemesh, C., Ashery, U., Cooper, I., Harari, A., et al. (2023) · Neurobiology of Aging · doi:10.1016/j.neurobiolaging.2023.09.005 · Cited by 7
Suszyńska-Zajczyk, J., Witucki, Ł., Perła‐Kaján, J., & Jakubowski, H. (2024) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2024.1322844 · Cited by 7
Chartampila, E., Elayouby, K. S., Leary, P., LaFrancois, J. J., Alcantara‐Gonzalez, D., Jain, S. S., Gerencer, K., Botterill, J. J., Ginsberg, S. D., & Scharfman, H. E. (2024) · eLife · doi:10.7554/elife.89889 · Cited by 19
“…used to evaluate cognitive deficits in >9 months-408 old Tg2576 mouse model (i.e., Morris Water Maze, Radial Arm Water Maze, Y-409 Maze; (Yassine et al., 2013; A. Wolf et al., 2016)), provided an opportunity to capture cognitive deficits in animals as young as three months-old (Duffy et al., 2015).”
Owumi, S. E., Chimezie, J., Emmanuel, P. D., Okeibuno, A. C., & Owoeye, O. (2024) · BMC Neuroscience · doi:10.1186/s12868-024-00922-8 · Cited by 8
Cpne5 is Involved in Regulating Rodent Anxiety Level
Ding, X., Wang, H., Lü, Q., Hu, Z., Feng, S., Wu, Y., Yang, H., Wu, H., Fan, W., & Fan, M. (2017) · CNS Neuroscience & Therapeutics · doi:10.1111/cns.12674 · Cited by 7
“To investigate the role of Cpne5, 11 female homozygotes and seven female wild-type mice from littermate were used to perform behavioral test, including open field (OF), Y-maze, novelty recognition (NR), light–dark box (LDB), elevated plus maze (EPM), and elevated platform (EP) task [7].”
MethodA Novel Thermal-Visual Place Learning Paradigm for Honeybees (Apis mellifera)
Scheiner, R., Frantzmann, F., Jäger, M., Mitesser, O., Helfrich‐Förster, C., & Pauls, D. (2020) · Frontiers in Behavioral Neuroscience · doi:10.3389/fnbeh.2020.00056 · Cited by 7
“…a link to vertebrate studies because setups like the Morris Water Maze or the Radial Arm Maze have been used there for many years to study place learning and spatial tasks related to both basic and applied, clinical research (Savonenko et al., 2005; Vorhees and Williams, 2006; Wolf et al., 2016).”
BackgroundSalt as a non-caloric behavioral modifier: A review of evidence from pre-clinical studies
Beaver, J. N., & Gilman, T. L. (2021) · Neuroscience & Biobehavioral Reviews · doi:10.1016/j.neubiorev.2021.10.007 · Cited by 7
Danduga, R. C. S. R., Reddy, D. S., Seshadri, S. M., Has, K. S. S., & Kola, P. K. (2018) · Europe PMC (PubMed Central) · doi:10.22038/ijbms.2018.22647.5756 · Cited by 7
“Percentage of spontaneous alternations (%SA) was calculated using the formula (15)”
MethodUpadhya, M. A., Stumpf, A., O'Brien-Cairney, J., Gómez, C. C., Döring, J. H., Hoffmann, J., Mueller, S. G., Fukata, Y., van Hoof, S., Dhangar, D., et al. (2025) · Brain Behavior and Immunity · doi:10.1016/j.bbi.2025.02.019 · Cited by 7
“Behavioral tests were conducted between days 10 and 14 after surgery following established protocols (Jurek et al., 2019; Wolf et al., 2016) All tasks were performed and assessed by trained experimenters under blinded conditions and contained Open Field (locomotion and anxiety), Rotarod (motor…”
Mehanna, S., Hassan, N. H., Ibrahim, M. A., Mohammed, F. F., & Hassanen, E. I. (2025) · Biological Trace Element Research · doi:10.1007/s12011-025-04654-6 · Cited by 6
“Several brain regions influence this behavior, including the hippocampus, prefrontal cortex, and basal forebrain [43].”
An 8-cage imaging system for automated analyses of mouse behavior
Hernández, T. D. R., Joshi, N. R., Gore, S. V., Kreiling, J. A., & Créton, R. (2023) · Scientific Reports · doi:10.1038/s41598-023-35322-1 · Cited by 6
“A ‘Summary’ table shows the following 15 behaviors in table format: (1) M1 = movement during 1st hour, (2) MD = movement during daytime (average of hours 2–6), (3) M7 = movement in the 7th hour (1st hour of nighttime), (4) MN = movement during nighttime (average of hours 8–12), (5) N-D = The difference between nightti…”
BackgroundJino, K., Miyamoto, K., Kanbara, T., Unemura, C., Horiguchi, N., & Ago, Y. (2023) · Psychopharmacology · doi:10.1007/s00213-023-06510-8 · Cited by 6
“In a previous study by Burgin et al. (2010), the intravenous administration of D159687 led to pro-cognitive effects in a dose-dependent manner (0.03–0.1 µg/kg for the scopolamine-impaired Y-maze test and 0.3–10 µg/ kg for the novel object recognition test), rather than having a bell-shaped effect.”
InfluentialMatteucci, A., Ricceri, L., Fabbri, A., Fortuna, A., Travaglione, S., Guidotti, M., Martinelli, A., Villa, M., Pricci, F., Maroccia, Z., et al. (2019) · Neuroscience · doi:10.1016/j.neuroscience.2019.05.021 · Cited by 5
Ethanol Exacerbates the Alzheimer’s Disease Pathology in the 5xFAD Mouse Model
Mohammed, H. E., Nelson, J. C., & Marshall, S. A. (2024) · Neuroglia · doi:10.3390/neuroglia5030020 · Cited by 5
“…such as an NOR task with multiple familiar objects, should be performed in the future; additionally, a more comprehensive battery of behavioral assessments that measure cognitive flexibility should be performed to truly assess whether ethanol may accelerate AD-related cognitive decline [44,75,76].”
Russart, K. L. G., Huk, D. J., Nelson, R. J., & Kirschner, L. S. (2018) · Hormones and Behavior · doi:10.1016/j.yhbeh.2017.12.012 · Cited by 4
“Therefore, this task is a sensitive measure of spatial memory (Wolf et al., 2016).”
Assessment of Memory Function in Pilocarpine-induced Epileptic Mice
Park, K.-M., Kim, J., Choi, I., & Cho, K. (2020) · Journal of Visualized Experiments · doi:10.3791/60751 · Cited by 5
Metadata
- Authors
- Wolf, A., Bauer, B., Abner, E. L., Ashkenazy-Frolinger, T., & Hartz, A. M. S.
- Affiliations
- University of Minnesota, Duluth (US)
- University of Kentucky (US)
- Corresponding author
- Hartz, Anika M. S.
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2016-01-25
- Volume
- 11
- Issue
- 1
- Pages
- e0147733
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC4726499
- OpenAlex
- W2426168356
- Cited by
- 281
- References
- 89
- Citations by year
- 2026: 16 · 2025: 20 · 2024: 28 · 2023: 37 · 2022: 30 · 2021: 33 · 2020: 37 · 2019: 28 · 2018: 40 · 2017: 6 · 2016: 6
- FWCI
- 21.46
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UKnowledge (University of Kentucky) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- University of Kentucky (P30 AG028383, AG028383)
- National Institute on Aging (AG-028383, P30AG028383, R01AG039621)
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
- Life Sciences › Neuroscience › Cognitive Neuroscience › Memory and Neural Mechanisms
- Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
- Keywords
- mouse models of Alzheimer's disease, amyloid precursor protein overexpression, retinal degeneration, scopolamine-induced dementia, cognitive impairment, learning and memory, novel object recognition, Morris water maze, radial arm water maze, spontaneous alternation, amyloid-beta pathology
- MeSH terms
- Animals, Behavior, Animal, Male, Memory, Mice, Transgenic, Maze Learning, Mice
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