Exploring Refinement Strategies for Single Housing of Male C57BL/6JRj Mice: Effect of Cage Divider on Stress-Related Behavior and Hypothalamic-Pituitary-Adrenal-Axis Activity

Introduction: Single housing of laboratory mice is a common practice to meet experimental needs, or to avoid intermale aggression. However, single housing is considered to negatively affect animal welfare and may compromise the scientific validity of experiments. The aim of this study was to investigate whether the use of a cage with a cage divider, which avoids physical contact between mice while maintaining sensory contact, may be a potential refinement strategy for experiments in which group housing of mice is not possible.Methods: Eight-week-old male C57BL/6JRj mice were single housed, pair housed or pair housed with a cage divider for four (experiment 1) or ten (experiment 2) weeks, after which we performed an open field test, Y-maze spontaneous alternation test, elevated plus maze test, an auditory fear conditioning task, and assessed responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis.Results: Housing conditions did not affect body weight, exploratory activity, anxiety, working memory, fear memory processing or markers for HPA-axis functioning in either experiment 1 or experiment 2. There was an increased distance traveled in mice housed with a cage divider compared to pair housed mice after 4 weeks, and after 10 weeks mice housed with a cage divider made significantly more arm entries in the Y-maze spontaneous alternation test.Conclusion: Taken together, our study did not provide evidence for robust differences in exploratory activity, anxiety, working memory and fear memory processing in male C57BL/6JRj mice that were single housed, pair housed or pair housed with a cage divider.

Apparatus design and behavioural testing protocol for the evaluation of spatial working memory in mice through the spontaneous alternation T-maze

Spatial working memory can be assessed in mice through the spontaneous alternation T-maze test. The T-maze is a T-shaped apparatus featuring a stem (start arm) and two lateral goal arms (left and right arms). The procedure is based on the natural tendency of rodents to prefer exploring a novel arm over a familiar one, which induces them to alternate the choice of the goal arm across repeated trials. During the task, in order to successfully alternate choices across trials, an animal has to remember which arm had been visited in the previous trial, which makes spontaneous alternation T-maze an optimal test for spatial working memory. As this test relies on a spontaneous behaviour and does not require rewards, punishments or pre-training, it represents a particularly useful tool for cognitive evaluation, both time-saving and animal-friendly. We describe here in detail the apparatus and the protocol, providing representative results on wild-type healthy mice.

Adolescent Binge-Type Ethanol Exposure in Rats Mirrors Age-Related Cognitive Decline by Suppressing Cholinergic Tone and Hippocampal Neurogenesis

Heavy alcohol consumption followed by periods of abstinence (i.e., binge drinking) during adolescence is a concern for both acute and chronic health issues. Persistent brain damage after adolescent intermittent ethanol exposure in rodents, a model of binge drinking, includes reduced hippocampal neurogenesis and a loss of neurons in the basal forebrain that express the cholinergic phenotype. The circuit formed between those regions, the septohippocampal pathway, is critical for learning and memory. Furthermore, this circuit is also altered during the aging process. Thus, we examined whether pathology in septohippocampal circuit and impairments in spatial behaviors are amplified during aging following adolescent intermittent ethanol exposure. Female and male rats were exposed to intermittent intragastric gavage of water (control) or 20% ethanol (dose of 5 g/kg) for a 2 days on/off cycle from postnatal days 25–55. Either 2 (young adult) or 12–14 (middle-age) months post exposure, rats were tested on two spatial tasks: spontaneous alternation and novel object in place. Acetylcholine efflux was assessed in the hippocampus during both tasks. There was no adolescent ethanol-induced deficit on spontaneous alternation, but middle-aged male rats displayed lower alternation rates. Male rats exposed to ethanol during adolescence had blunted behavioral evoked acetylcholine during spontaneous alternation testing. All ethanol-exposed rats displayed suppression of the cholinergic neuronal phenotype. On the novel object in place task, regardless of sex, ethanol-exposed rats performed significantly worse than control-treated rats, and middle aged-rats, regardless of sex or ethanol exposure, were significantly impaired relative to young adult rats. These results indicate that male rats display earlier age-related cognitive impairment on a working memory task. Furthermore, male rats exposed to ethanol during adolescence have blunted behavior-evoked hippocampal acetylcholine efflux. In addition, middle-aged and ethanol-exposed rats, regardless of sex, are impaired at determining discrete spatial relationship between objects. This type of pattern separation impairment was associated with a loss of neurogenesis. Thus, binge-type adolescent ethanol exposure does affect the septohippocampal circuit, and can accelerate age-related cognitive impairment on select spatial tasks.

Association between Novel Object Recognition/Spontaneous Alternation Behavior and Emission of Ultrasonic Vocalizations in Rats: Possible Relevance to the Study of Memory

Rats emit ultrasonic vocalizations (USVs) in situations with emotional valence, and USVs have also been proposed as a marker for memories conditioned to those situations. This study investigated whether USV emissions can predict and/or be associated with the behavior of rats in tests that evaluate unconditioned memory. To this end, rats were subjected to “tickling”, a procedure of heterospecific play that has emotional valence and elicits the emission of USVs, and afterwards evaluated in the novel object recognition test (NOR) and in the single trial continuous spontaneous alternation behavior (SAB) test in a Y-maze. The number of 22-kHz USVs (aversive) and 50-kHz USVs (appetitive) emitted in response to tickling and during NOR and SAB tests were scored, and the correlations among them and with rats’ behavior evaluated. Rats emitted 50-kHz USVs, but not 22-kHz USVs, during the NOR and SAB tests, and such calling behavior was not linked with the behavioral readouts indicative of memory function in either test. However, rats that prevalently emitted 22-kHz USVs in response to tickling displayed an impaired NOR performance. These findings suggest that measuring the emission of USVs could be of interest in studies of unconditioned memory, at least with regard to 22-kHz USVs.

Ziziphus jujuba (Rhamnaceae) Alleviates Working Memory Impairment and Restores Neurochemical Alterations in the Prefrontal Cortex of D-Galactose-Treated Rats

Alzheimer’s disease is a progressive cognitive dysfunction. However, pharmacological treatments are symptomatic and have many side effects, opening the opportunity to alternative medicine. This study investigated the antiamnesic effect of the aqueous extract of Ziziphus jujuba on D-galactose-induced working memory impairment in rats. Impairment of working memory was induced by subcutaneous (s.c.) injection of D-galactose (350 mg/kg/day) to rats for 21 days. These animals were then subjected to object recognition and Y-maze tests. Rats with confirmed memory impairment were treated per os (p.o.) with tacrine (10 mg/kg), aspirin (20 mg/kg, p.o.), extract (41.5, 83, and 166 mg/kg, p.o.), and distilled water (10 mL/kg, p.o.) daily for 14 days. At the end of the treatments, alteration in working memory was assessed using the above paradigms. Afterward, these animals were euthanized, and cholinergic, proinflammatory, and neuronal damage markers were analyzed in the prefrontal cortex. Rats administered D-galactose and treated with distilled water had impaired working memory (evidenced by decreased time spent on the novel object and discrimination index) and decreased spontaneous alternation in the Y-maze. D-galactose also decreased the levels of acetylcholinesterase and acetylcholine and increased the level of glial fibrillary acidic protein, ionized calcium-binding adapter molecule 1, tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interferon-gamma (IFN-γ). Treatment with the extract (166 mg/kg) reversed the time spent on the novel object and the discrimination index. It equally increased the percentage of spontaneous alternation. Neurochemical analysis revealed that the extract markedly alleviated acetylcholinesterase activity and neuroinflammation. These observations were corroborated by the reduction in neuronal loss. Taken together, these results suggest that Ziziphus jujuba aqueous extract possesses an antiamnesic effect. This effect seems to involve cholinergic and anti-inflammatory modulations. This, therefore, claims using this plant in the treatment of dementia in Cameroon subject to further studies and trials.

Comparison between the timing of the occurrence of taste sensitivity changes and short-term memory decline due to aging in SAMP1 mice

Several studies have suggested that cognitive impairment affects taste sensitivity. However, the mechanism behind this is still unclear. In this study, we focused on short-term memory. Using senescence-accelerated mouse prone 1 (SAMP1) mice, we compared whether the effects of aging are observed earlier in taste sensitivity or short-term memory. We used 8-week-old mice as the young group, and 70- and 80-week-old mice as aged groups. Taste sensitivity was evaluated using a 48-hour two-bottle preference test, and short-term memory was evaluated using the Y-maze test. SAMP1 mice showed apparently changes in taste sensitivity at 70-weeks-old. However, the influence of aging on spontaneous alternation behavior, which is indicative of short-term memory alterations, was not observed in 70-week-old mice. At 80-weeks-old, the influence of aging was observed, and spontaneous alternation behavior was significantly decreased. This suggests that age-dependent changes in taste sensitivity occur prior to short-term memory function decline. In addition, there was no significant influence of aging on the mRNA expression of long-term potentiation-related genes in the hippocampus of 80-week-old mice. Therefore, the age-related decline of short-term memory may not affect taste sensitivity.

Abstract P805: Inhibition of α5β1 Integrin as a Novel Therapeutic Approach in Experimental Vascular Dementia in Middle-Aged and Diabetic Mice

Bilateral carotid artery stenosis (BCAS), a model of vascular dementia (VaD), causes cognitive impairment due to white matter injury and blood-brain barrier (BBB) disruption. Although risk factors for VaD such as Type-2 diabetes and aging are clinically relevant for therapeutic discovery, they are rarely studied. Recently, we determined that inhibition of brain α5β1 integrin with the peptide ATN-161 improves BBB stability and functional outcome after cerebral ischemia in mice. Also, after 14 days BCAS in mice, we showed increased brain α5β1 integrin expression which correlates with BBB disruption. We hypothesize that middle age and / or diabetes will intensify post-BCAS brain α5β1 integrin expression, contributing to worsened cerebrovascular pathology and cognitive decline, and that inhibition of α5β1 integrin with ATN-161 would reduce brain α5β1 integrin level, stabilize the BBB, attenuate brain-vascular pathology, and protect against VaD. Methods: BCAS was induced with titanium micro-coils (ID: 0.18 mm) in young and middle-aged C57B6/J (13-15 weeks old; n=57 and 57-63 weeks old; n=57, respectively) as well as in young 13-weeks old diabetic (B6.BKS(D)-Leprdb /- J, n=27; 35-56 g) and genotype control (B6.BKS(D)-Leprdb /+ J, n=27; 28-36 g) mice for 14, 32 and 60 days. After BCAS or sham surgery, mice were randomly assigned to ATN-161 (1 mg/kg, i.p., 3x /week) or saline groups. Functional outcome measures: Y-maze spontaneous alternation, novel object recognition and nesting tests were performed at baseline, post-BCAS days 14, 30, 48 & 60. Brain samples were assessed by immunofluorescence and western blot, for the expression of α5β1 integrin, tight junction proteins, as well as markers for white matter integrity, astrocyte and microglia activation. While the analysis of functional tests, brain histology and western blots are underway, we have thus-far determined that ATN-161 treatment reduces (p<0.01) BCAS-elevated brain α5β1 integrin to sham control levels and improves (p<0.001, compared to saline controls) Y-maze spontaneous alternation in young B6 mice after 14 days. We expect to demonstrate that inhibition of α5β1 with ATN-161 will improve cerebrovascular pathology and cognitive outcome following BCAS in middle-aged B6 and type-2 diabetic mice.

THE OFFSPRING OF MOTHERS HARBORING APP/PS1 MUTATIONS PRESENTS EARLY CHANGES IN BRAIN METABOLISM AND MEMORY

Background: Previous studies suggested that individuals with a maternal history of Alzheimer’s Disease(AD) are at higher risk of developing AD than individuals with a paternal history of AD. One could suggest that intra-uterine interactions might be responsible for elevating this risk. In this context, AD rodent models are highly suited for improving our understanding of this matter since animals reach adulthood in a few months. Objective: Here, we aimed at investigating changes in memory-related processes and brain metabolism on the offspring born to transgenic mothers harboring human APP/PS1 mutations. Methods: Rats born to F344-AD(Tg-AD) and WT mothers were evaluated in two different time-points: ~5.5, and ~9.5 months. Y-maze test was used to evaluate spatial working memory and micro-PET [18F]FDG was used to assess brain glucose metabolism. Results: Y-maze test demonstrated that rats born to transgenic mothers presented memory disturbances, indexed by spontaneous alternation, at ~5.5 months and ~9.5 months (figure 1A), while rats born to WT mothers and Tg-AD fathers exhibited a decline in spontaneous alternation only at ~9.5 months. rats born to AD-Tg mothers present brain glucose hypermetabolism at ~9,5 months. Conclusion: Our findings demonstrate that rats born to AD-Tg mothers harboring APP/PS1 mutations present cognitive decline and brain glucose metabolism abnormalities earlier than those born to WT mothers. Further studies are needed to understand the biological basis behind this phenomenon.