Light-dark Test Research Articles

Hypothalamic-pituitary-adrenal (HPA) axis activity and anxiety-like behavior during aging: A test of the glucocorticoid cascade hypothesis in amyloidogenic APPswe/PS1dE9 mice

Alzheimer’s disease (AD) is a progressive, dementing, whole-body disorder that presents with decline in cognitive, behavioral, and emotional functions, as well as endocrine dysregulation. The etiology of AD is not fully understood but stress- and anxiety-related hormones may play a role in its development and trajectory. The glucocorticoid cascade hypothesis posits that levels of glucocorticoids increase with age, leading to dysregulated negative feedback, further elevated glucocorticoids, and resulting neuropathology. We examined the impact of age (from 2 to 10 months) and stressor exposure (predator odor) on hormone levels (corticosterone and ghrelin), anxiety-like behavior (open field and light dark tests), and memory-related behavior (novel object recognition; NOR), and whether these various measures correlated with neuropathology (hippocampus and cortex amyloid beta, Aβ) in male and female APPswe/PS1dE9 transgenic and non-transgenic mice. Additionally, we performed exploratory analyses to probe if the open field and light dark test as commonly used tasks to assess anxiety levels were correlated. Consistent with the glucocorticoid cascade hypothesis, baseline corticosterone increased with age. Predator odor exposure elevated corticosterone at each age, but in contrast to the glucocorticoid cascade hypothesis, the magnitude of stressor-induced elevations in corticosterone levels did not increase with age. Overall, transgenic mice had higher post-stressor, but not baseline, corticosterone than non-transgenic mice, and across both genotypes, females consistently had higher (baseline and post-stressor) corticosterone than males. Behavior in the open field test primarily showed decreased locomotion with age, and this was pronounced in transgenic females. Anxiety-like behaviors in the light dark test were exacerbated following predator odor, and female transgenic mice were the most impacted. Compared to transgenic males, transgenic females had higher Aβ concentrations and showed more anxiety-like behavior. Performance on the NOR did not differ significantly between genotypes. Lastly, we did not find robust, statistically significant correlations among corticosterone, ghrelin, recognition memory, anxiety-like behaviors, or Aβ, suggesting outcomes are not strongly related on the individual level. Our data suggest that despite Aβ accumulation in the hippocampus and cortex, male and female APPswePS1dE9 transgenic mice do not differ robustly from their non-transgenic littermates in physiological, endocrine, and behavioral measures at the range of ages studied here.

Synergistic effect of mild traumatic brain injury and alcohol aggravates neuroinflammation, amyloidogenesis, tau pathology, neurodegeneration, and blood-brain barrier alterations: Impact on psychological stress

After a mild traumatic brain injury (mTBI), victims often experience emotional/psychological stress such as heightened irritability, anxiety, apathy, and depression. Severe mental health complications are common in military populations following a combat-acquired TBI and intensified unhealthy alcohol use. The high prevalence of alcohol abuse among TBI victims underscores how alcohol abuse exacerbates emotional/psychological symptoms such as depression and anxiety. The experimental mTBI was induced in vivo by fluid percussion injury (15 psi) in mice and ethanol diet feeding continued for 28 days. We analyzed different biomarkers of the biochemical mechanisms and pathophysiology of neurological damage, and functional outcome of psychological stress by sucrose preference, and light-dark tests. We demonstrated that the synergistic effect of TBI and alcohol leads to psychological stress such as depression and anxiety. The studies showed that oxidative stress, amyloidogenesis, tau pathology, neuroinflammation, and neurodegeneration markers were elevated, and glial activation and blood-brain barrier (BBB) damage were exacerbated during the synergistic effect of TBI and alcohol. Further, we studied the biochemical mechanisms of psychological stress that showed the significant reduction of 5-HT1AR, neuropeptide-Y, and norepinephrine, and an increase in monoamine oxidase-a in the combined effect of TBI and alcohol. This work suggested that the combined TBI and alcohol-induced effect leads to depression and anxiety, via sequential biochemical changes that cause neuroinflammation, amyloidogenesis, tau pathology, neurodegeneration, and BBB alterations. This clinically relevant study will contribute to developing a comprehensive therapeutic approach for patients suffering from TBI and alcohol-mediated neurological damage and psychological stress.

Innate immune stimulation prevents chronic stress-induced depressive and anxiogenic-like behaviors in female mice

It has been reported that pre-stimulation of the innate immune system can prevent depressive and anxiogenic-like behaviors in chronically stressed male mice. However, it is unclear whether similar effects can be observed in female animals. In the present study, we investigated this question in female mice. Our results showed that a single injection of lipopolysaccharide (LPS; 100 μg/kg) one day before stress exposure prevented increased immobility time in the tail suspension test and forced swimming test and decreased sucrose intake in the sucrose preference test in chronic unpredictable stress (CUS)-treated female mice. The single LPS pre-injection (100 μg/kg) prevented the CUS-induced decrease in (i) time spent in open arms and number of entries into open arms in the elevated plus maze test, (ii) time spent in lit side in the light-dark test, and (iii) time spent in the central region of the open field in the open field test, along with no changes in locomotor activity. It was also found that the single LPS pre-injection in female mice prevented the CUS-induced increase in the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 mRNA in the hippocampus and medial prefrontal cortex. Inhibition of innate immune system stimulation by minocycline pretreatment abrogated the preventive effect of LPS on CUS-induced depressive and anxiogenic-like behaviors and neuroinflammatory responses in the hippocampus and medial prefrontal cortex in female mice. These results suggest that pre-stimulation of the innate immune system by LPS injection may prevent the development of behavioral abnormalities in female mice.

Anxiolytic-like effects of hochuekkito in lipopolysaccharide-treated mice involve interleukin-6 inhibition.

Hochuekkito (HET) is a Kampo medicine used to treat postoperative and post-illness general malaise and decreased motivation. HET is known to regulate immunity and modulate inflammation. However, the precise mechanism and effects of HET on inflammation-induced central nervous system disorders remain unclear. This study aimed to assess the effect of HET on inflammation-induced anxiety-like behavior and the mechanism underlying anxiety-like behavior induced by lipopolysaccharide (LPS). Institute of Cancer Research mice were treated with LPS (300 μg/kg, intraperitoneally), a bacterial endotoxin, to induce systemic inflammation. The mice were administered HET (1.0 g/kg, orally) once a day for 2 weeks before LPS treatment. The light-dark box test and the hole-board test were performed 24 h after the LPS injection to evaluate the effects of HET on anxiety-like behaviors. Serum samples were obtained at 2, 5, and 24 h after LPS injection, and interleukin-6 (IL-6) levels in serum were measured. Human and mouse macrophage cells (THP-1 and RAW264.7 cells, respectively) were used to investigate the effect of HET on LPS-induced IL-6 secretion. The repeated administration of HET prevented anxiety-like behavior and decreased serum IL-6 levels in LPS-treated mice. HET significantly suppressed LPS-induced IL-6 secretion in RAW264.7 and THP-1 cells. Similarly, glycyrrhizin, one of the chemical constituents of HET, suppressed LPS-induced anxiety-like behaviors. Our study revealed that HET ameliorated LPS-induced anxiety-like behavior and inhibited IL-6 release in vivo and in vitro. Therefore, we postulate that HET may be useful against inflammation-induced anxiety-like behavior.

A comparative analysis of Danionella cerebrum and zebrafish (Danio rerio) larval locomotor activity in a light-dark test.

The genus Danionella comprises some of the smallest known vertebrate species and is evolutionary closely related to the zebrafish, Danio rerio. With its optical translucency, rich behavioral repertoire, and a brain volume of just 0.6 mm3, Danionella cerebrum (Dc) holds great promise for whole-brain in vivo imaging analyses with single cell resolution of higher cognitive functions in an adult vertebrate. Little is currently known, however, about the basic locomotor activity of adult and larval Danionella cerebrum and how it compares to the well-established zebrafish model system. Here, we provide a comparative developmental analysis of the larval locomotor activity of Dc and AB wildtype as well as crystal zebrafish in a light-dark test. We find similarities but also differences in both species, most notably a striking startle response of Dc following a sudden dark to light switch, whereas zebrafish respond most strongly to a sudden light to dark switch. We hypothesize that the different startle responses in both species may stem from their different natural habitats and could represent an opportunity to investigate how neural circuits evolve to evoke different behaviors in response to environmental stimuli.

Anxiolytic effect of Anthemis nobilis L. (roman chamomile) and Citrus reticulata Blanco (tangerine) essential oils using the light-dark test in zebrafish (Danio rerio)

Ethnopharmacological relevanceThe anxiety disorders are the most prevalent mental health condition, and anxiety is considered the sixth cause of disability surpassing diabetes mellitus, chronic obstructive pulmonary disease, and osteoarthritis. Besides, the COVID-19 pandemic provided an increase in the number of psychiatric diseases diagnosis in all social layers around the world. About 55%–94% of patients diagnosed with anxiety disorders are treated with benzodiazepines, meanwhile benzodiazepines can promote several adverse effects. In this way, alternative therapies, such as essential oils may offer significant benefits in the treatment of patients with anxiety disorders. However, the anxiolytic effect of these essential oils must be proper evaluated appropriate as well as the suitable dosage and side effect need further research. Aim of the studyThe aim was to evaluate the anxiolytic effect of Roman chamomile (Anthemis nobilis L.) and tangerine (Citrus reticulata Blanco) essential oils using the light-dark test in adult zebrafish (Danio rerio). Material and methodsBoth essential oils were analyzed by GC-MS and the major compounds were identified. The anxiolytic effect was evaluated by light-dark test in adult zebrafish. ResultsThe results showed that roman chamomile essential oil has anxiolytic effect in adult zebrafish, whereas tangerine essential oil tends to reduce anxiety The major compounds of tangerine essential oil were limonene and γ-terpinene, and the major compounds of roman chamomile were pentadecyl-3-methyl-2-butenoate, hexadecyl-3-methyl-2-butenoate, 1-piperidinol and trans-1-ethyl-3-methyl-cyclopentane. ConclusionsThe present study demonstrated that this anxiolytic effect may be attributed to the synergistic effect of the compounds present in roman chamomile essential oil, particularly the major compounds. The roman chamomile essential oil at the highest concentration showed anxiolytic effect. The tangerine essential oil showed a tendency to reduce anxiety, but it was not statistically significative. In addition, roman chamomile and tangerine essential oils did not cause cause alteration in locomotion activity and exploratory ability of the fish.

Sex-dependent Differences of Emotional Status in a Rat Model of Prenatal Stress

Untoward events during pregnancy negatively affect offspring mental development. We investigated sex-dependent differences of emotional problems in offspring with a history of prenatal stress (PNS). Female rats demonstrated decreased anxiety-like behaviour in comparison to male controls, shown in the light-dark test. Male and female offspring of prenatally stressed mothers were characterized by higher anxiety levels, compared to unstressed controls. PNS induced depression-like behaviour in both sexes without differences among them, indicated with decreased intake of sweet solution in the sucrose consumption tests and increased immobility time spent in the forced swim test. Control females showed higher plasma corticosteroid (CORT) concentrantions after acute stress and decreased recovery (120 min after the stressor) than control males. Both male and female PNS-offspring were with elevated levels of CORT in the plasma, which remained high 120 min after application of the stressogenic procedure. This study evidenced that the unfavourable effect of PNS on emotional state correlated with impaired feedback mechanism of the hypothalamic-pituitary-adrenal (HPA) axis function both in male and female offspring.

Assessment of Binge-Like Eating of Unsweetened vs. Sweetened Chow Pellets in BALB/c Substrains.

Binge eating disorder (BED) is defined as chronic episodes of consuming large amounts of food in less than 2 h. Binge eating disorder poses a serious public health problem, as it increases the risk of obesity, type II diabetes, and heart disease. Binge eating is a highly heritable trait; however, its genetic basis remains largely unexplored. We employed a mouse model for binge eating that focused on identifying heritable differences between inbred substrains in acute and escalated intake of sucrose-sweetened palatable food vs. unsweetened chow pellets in a limited, intermittent access paradigm. In the present study, we examined two genetically similar substrains of BALB/c mice for escalation in food consumption, incubation of craving after a no-food training period, and compulsive-like food consumption in an aversive context. BALB/cJ and BALB/cByJ mice showed comparable levels of acute and escalated consumption of palatable food across training trials. Surprisingly, BALB/cByJ mice also showed binge-like eating of the unsweetened chow pellets similar to the escalation in palatable food intake of both substrains. Finally, we replicated the well-documented decrease in anxiety-like behavior in BALB/cByJ mice in the light-dark conflict test that likely contributed to greater palatable food intake than BALB/cJ in the light arena. To summarize, BALB/cByJ mice show binge-like eating in the presence and absence of sucrose. Possible explanations for the lack of selectivity in binge-like eating across diets (e.g., novelty preference, taste) are discussed.

NEUROPHARMACOLOGICAL ACTIVITY OF DOLICHANDRONE ATROVIRENS (ROTH) LEAF EXTRACTS IN SWISS ALBINO MICE

The aim of the present study was to evaluate the sedative and anxiolytic property of the leaves of the Dolichandrone atrovirens (D. atrovirens) The chloroform and ethanolic extracts of leaves were analysed for their phytochemical constituents by phytochemical screening and thin layer chromatography studies. The acute toxicity in albino mice showed no clinical signs of toxicity in the 2000 mg kg-1 dose administration orally. The extracts were orally administered to the group of animals to study the neuropharmacology activity, such as sleep induced by drugs methods, spontaneous motor co-ordination, locomotor activity, hole board test and light dark test. The extracts significantly (p<0.001) prolonged the sleeping time of mice, reduced motor co-ordination and anxiety. The results obtained from these experimental models confirmed that chloroform and ethanolic extracts of leaves of D. atrovirens possess central nervous depressant activity and anxiolytic activity. The ethanolic extract shows better activity compared to chloroform extract.

Granulocyte-macrophage colony-stimulating factor-triggered innate immune tolerance against chronic stress-induced behavioral abnormalities in mice

Pre-stimulation of the innate immune is considered a potential strategy to prevent chronic stress-induced behavioral abnormalities in animals. In this study, we investigated whether granulocyte–macrophage colony-stimulating factor (GM-CSF), an immunostimulant used in the clinic to treat diseases of the hematopoietic system, can prevent chronic stress-induced behavioral abnormalities in mice. Our results showed that a single intraperitoneal injection of GM-CSF (100 μg/kg) one day before stress exposure prevented the depression- and anxiety-like behaviors induced by chronic social defeat stress (CSDS) in mice, including preventing the CSDS-induced increase in the immobility time in the tail suspension test and forced swimming test and decrease in the time spent in the interaction zone in the social interaction test, as well as preventing the CSDS-induced decrease in the time spent (i) in open arms in the elevated plus maze test, (ii) on the illuminated side in the light–dark test, and (iii) in the central region of the open field test. The single GM-CSF preinjection (100 μg/kg) also prevented the CSDS-induced increase in the expression levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) mRNA in the hippocampus and medial prefrontal cortex of the mice. Further analysis showed that the preventive effect of GM-CSF on CSDS-induced depression- and anxiety-like behaviors and neuroinflammatory responses was abolished by pretreatment with minocycline (an innate immune inhibitor). These results indicate that a single low dose of GM-CSF before injection could be a potential way to prevent behavioral abnormalities induced by chronic stress in mice.

Male and Female C57BL/6 Mice Respond Differently to Awake Magnetic Resonance Imaging Habituation.

Traditionally, preclinical magnetic resonance imaging (MRI) has been performed in anesthetized animals. However, anesthesia has been shown to perturb normal brain function and physiology. Such effects limit our ability to detect subtle physiological alterations in disease models and treatment studies, thus hampering discovery and compromising generality of findings. Therefore, methods for awake animal MRI are needed to study the rodent brain in its natural physiological state, free of anesthetics. Current setups for awake animal MRI rely on restraining systems to avoid animal movement during scanning. To reduce restraint stress, animals are habituated to the scanner environment prior to MRI data collection. To date, however, most awake MRI studies employ male rodents only. This is a fundamental limitation as results obtained may be pertinent only to half of the population. We characterized training and habituation responses of male and female mice to provide improved, sex-dependent training procedures for awake mouse MRI. We recorded heart rate, monitored behavioral responses (body weight and fecal boli weight) and fecal corticosterone levels (FCM) as indicators of wellbeing and stress during a 14-day progressive habituation protocol. In addition, we also assessed discomfort levels and anxiety using the mouse grimace scale (MGS) and light/dark test (LDT), respectively. All scores were compared between both groups. We found that heart rate was significantly decreased after 10 and 11 days of training for both males and females, respectively. However, the specific time course for this decrease was significantly different between males and females, and females exhibited higher anxiety levels during habituation and 14 days after habituation than males. Lastly, we also found that mean FCM levels for both groups were decreased after 11 days of MRI habituation. The present work shows that mice can be successfully trained for extended MRI sessions which is necessary for many (particularly non-fMRI) studies. Importantly, we find that males and females differ in their response to awake MRI habituation, which should be considered in future awake MRI studies that aim to include male and female mice.

Phytochemical Evaluation and Pharmacological Activities of Antidesma Montanum Blume Leaf Extract

Abstract The demand for medicinal plants and their derived substances is increasing day by day due to their relevance in the context of drug discovery and development. The goal of this investigation is to assess the pharmacological and phytochemical potentials of the grossly underexplored Antidesma montanum Blume (Family: Phyllanthaceae). The methanolic extract of the leave of this plant was fractionated and then followed by initial screening of phytochemical. The investigation of the pharmacological potential, which includes antioxidant, antidiarrheal, anti-inflammatory, analgesic, anti-pyretic, and anxiolytic evaluations, was accomplished using an in vitro free radical scavenging assay with 2,2-diphenyl-1-picrylhydrazyl (DPPH), castor oil-induced diarrheal test, egg albumin test, acetic acid-induced writhing model, brewer’s yeast induced fever test, swing test, open field, and light-dark test, respectively. The investigation o phytochemicals proposes that the methanol extract of A. montanum possesses flavonoids, tannins, terpenoids, saponins, amino acids, fixed oils, and sterols. Pharmacological evaluation suggests that A. montanum possesses significant antioxidant, anti-diarrheal, anti-inflammatory, and analgesic effects. The methanol and chloroform fractions exhibited better DPPH radical scavenging activities with an IC50: 103 ± 0.05 and 108.7 ± 0.05 µg/ml, respectively. The methanol and chloroform fractions also showed anti-inflammatory capacities in the egg albumin (IC50 values: 89.10 ± 0.07 and 92.85 ± 0.07 µg/ml, respectively) model. The plant also showed anti-pyretic and anxiolytic activities in a dose-dependent manner. One of the possible sources of phytotherapeutic lead compounds is A. montanum. To extract and analyze the key bioactive components of this essential therapeutic plant, more research is required.

A novel behavioral paradigm to measure anxiety-like behaviors in zebrafish by the concomitant assessment of geotaxis and scototaxis

Pathological anxiety is a set of diseases characterized by specific clinical manifestations and the use of alternative models may provide novel insights in translational neurobehavioral research. In zebrafish, the separate performance of novel tank and light dark tests in different order to assess anxiety using a same animal may provide conflicting data due to the battery effect and/or time-drug-response and variability across tests. To improve data reliability, we aimed to characterize a novel behavioral paradigm to measure geotaxis and scototaxis as anxiety-like responses in the same trial. The novel apparatus consisted of four colored-compartments, with specific white- and black sections delimited in both bottom and upper areas of the tank. The main baseline responses of zebrafish in the novel apparatus were measured and animals were further exposed to modulators of anxiety. Zebrafish showed robust habituation to novelty stress during the 6-min trial with preference for the black section while exploring the top area. Fluoxetine (100 μg/L, 15 min) reduced geotaxis and scototaxis and ketamine (20 mg/L, 20 min) decreased geotaxis and increased the distance traveled in the black section while exploring the top, possibly due to the increased circling behavior. As anxiogenic modulators, conspecific alarm substance (3.5 mL/L, 5 min) exacerbated risk assessment, geotaxis, and scototaxis, whereas caffeine (10 mg/L, 15 min) increased geotaxis and exploration in the black section of the top area. Since important correlations were also found for relevant anxiety-like behaviors, our findings support the predictive validity of this novel paradigm to simultaneously assess geotaxis and scototaxis in zebrafish. Moreover, it fully adheres to the 3Rs principle of animal experimentation of reducing the number of subjects tested, execution time, also minimizing a potential battery effect.

When left is right: The effects of paw preference training on behaviour in mice

Spontaneous limb preferences exist in numerous species. To investigate the underlying mechanisms of these preferences, different methods, such as training, have been developed to shift preferences artificially. However, studies that systematically examine the effects of shifting preferences on behaviour and physiology are largely missing. Therefore, the aim of this study was to assess the impact of shifting paw preferences via training on spontaneous home cage behaviour, as well as anxiety-like behaviour and exploratory locomotion (Elevated plus maze test, Dark light test, Open field test, Free exploration test), learning performance (Labyrinth-maze) and stress hormones (fecal corticosterone metabolites) in laboratory mice (Mus musculus f. domestica). For this, we assessed spontaneous paw preferences of C57BL/6J females (Nambilateral = 23, Nleft = 23, Nright = 25). Subsequently, half of the individuals from each category were trained once a week for four weeks in a food-reaching task to use either their left or right paw, respectively, resulting in six groups: AL, AR, LL, LR, RL, RR. After training, a battery of behavioural tests was performed and spontaneous preferences were assessed again. Our results indicate that most mice were successfully trained and the effect of training was present days after training. However, a significant difference of preferences between RL and LL mice during training suggests a rather low training success of RL mice. Additionally, preferences of L mice differed from those of A and R mice after training, indicating differential long-term effects of training in these groups. Furthermore, left paw training led to higher levels of self-grooming, possibly as a displacement behaviour, and more time spent in the light compartment of the Dark light test. However, overall, there was no systematic influence of training on behavioural measures and stress hormones. Different explanations for this lack of influence, such as the link between training and hemispheric functioning or the intensity and ecological relevance of the training, are discussed.

Embryonic exposure to butylparaben and propylparaben induced developmental toxicity and triggered anxiety‐like neurobehavioral response associated with oxidative stress and apoptosis in the head of zebrafish larvae

Parabens are synthetic antimicrobial compounds used as a preservative for extending the shelf life of food, pharmaceutical and cosmetic products. The alkyl chain length of the paraben esters positively correlates with their antimicrobial property. Hence, long-chain paraben esters, namely butylparaben and propylparaben, are used in combination as they have better solubility and antimicrobial efficacy. Extensive use of parabens has now resulted in the ubiquitous presence of these compounds in various human and environmental matrices. During early life, exposure to environmental contaminants is known to cause oxidative-stress mediated apoptosis in developing organs. The brain being one of the high oxygen-consuming, metabolically active and lipid-rich organ, it is primarily susceptible to reactive oxygen species (ROS) and lipid peroxidation (LP) induced neuronal cell death. The primary cause for the impairment in cognitive and emotional neurobehvioural outcomes in neurodegenerative disease was found to be associated with neuronal apoptosis. The present study aimed to study butylparaben and propylparaben's effect on zebrafish during early embryonic stages. Besides this, the association between alteration in anxiety-like neurobehavioral response with oxidative stress and antioxidant status in head region was also studied. The study results showed variation in the toxic signature left by butylparaben and propylparaben on developmental parameters such as hatching rate, survival and non-lethal malformations in a time-dependent manner. Data from the light-dark preference test showed embryonic exposure to butylparaben and propylparaben to trigger anxiety-like behavior in zebrafish larvae. In addition, a significant increase in intracellular ROS and LP levels correlated with suppressed antioxidant enzymes: superoxide dismutases (SOD), catalases (CAT), Glutathione peroxidase (GPx), glutathione S-transferase (GST), and Glutathione (GSH) activity in the head region of the zebrafish larvae. Acetylcholinesterase (AChE) activity was also suppressed in the exposed groups, along with increased nitric oxide production. The overall observations show increased oxidative stress indices correlating with upregulated expression of apoptotic cells in a dose-dependent manner. Collectively, our findings reveal butylparaben and propylparaben as an anxiogenic neuroactive compound capable of inducing anxiety-like behavior through a mechanism involving oxidative-stress-induced apoptosis in the head of zebrafish larvae, which suggests a potential hazard to the early life of zebrafish and this can be extrapolated to human health as well.

Calorie restriction changes the anxiety-like behaviour of ageing male Wistar rats in an onset- and duration-dependent manner

Although initially recognized as a universally beneficial approach for the prevention of age-related impairments, the outcome of calorie restriction (CR) is now known to depend on several factors, most notably the age of the subject at the CR commencement, and CR duration. We aimed to examine if and how CR affects anxiety-like behaviour when it is introduced at middle age and late middle age. In addition, as the dopaminergic system is one of the main neurotransmitter systems involved in controlling anxiety, we examined the expression of dopamine receptors (D1R, D2R) in the cortex, striatum, and mesencephalon of male Wistar rats of varying ages. The study was performed on rats fed ad libitum (AL) or exposed to calorie restriction (60% of AL intake). Open field and light-dark tests were used to study anxiety-like behaviour, while PCR and Western blot were used to examine the expression of dopamine receptors. Calorie restriction implemented at middle-age led to variable outcomes on anxiety-like behaviour, while CR implemented at late middle age increased anxiety and decreased the availability of D2R levels in the cortex and mesencephalon. Taken together, these results advise caution when implementing calorie restriction late in life.

Brain systems in cocaine abstinence-induced anxiety-like behavior in rodents: A review

Cocaine use disorder (CUD) is a significant public health issue that generates substantial personal, familial, and economic burdens. Still, there are no FDA-approved pharmacotherapies for CUD. Cocaine-dependent individuals report anxiety during withdrawal, and alleviation of anxiety and other negative affective states may be critical for maintaining drug abstinence. However, the neurobiological mechanisms underlying abstinence-related anxiety in humans or anxiety-like behavior in rodents are not fully understood. This review summarizes investigations regarding anxiety-like behavior in mice and rats undergoing cocaine abstinence, as assessed using four of the most common anxiety-related assays: the elevated plus (or its derivative, the elevated zero) maze, open field test, light-dark transition test, and defensive burying task. We first summarize available evidence that cocaine abstinence generates anxiety-like behavior that persists throughout protracted abstinence. Then, we examine investigations concerning neuropeptide, neurotransmitter, and neuromodulator systems in cocaine abstinence-induced anxiety-like behavior. Throughout, we discuss how differences in sex, rodent strain, cocaine dose and dosing strategy, and abstinence duration interact to generate anxiety-like behavior.

A14 THE INTESTINAL MICROBIOTA CONTRIBUTES TO AGE-RELATED MEMORY DECLINE

BackgroundAge-related deterioration of cognitive function and memory capacity occur in both humans and rodents. For example, significant memory deficits have been reported in conventionally raised (SPF) old mice compared to conventionally raised young mice submitted to a spatial memory task (Prevot et al., 2019, Mol Neuropsychiatry 5, 84–97). Microbiota-to-brain signaling is now well established in mice and humans, but the extent to which it influences age-associated memory decline is unknown.AimsOur study examines whether the intestinal microbiota contributes to age-associated changes in brain function. We address the specific hypothesis that age-associated cognitive decline is attenuated in the absence of the intestinal microbiota.MethodsWe assessed anxiety-like and depressive-like behavior, locomotor activity and spatial memory performance in young germ-free (GF) mice (2–3 months of age, n=24) and senescent GF mice (13–27 months old, n=22) maintained in axenic conditions, and compared them to conventionally raised (SPF) mice of the same age. Anxiety-like behavior, locomotor activity and depressive-like behavior were measured using the light-dark preference, open-field, and tail suspension tests. We also used the Y-maze test based on a spontaneous alternation task to assess cognition, with the alternation rate as a proxy of spatial working memory. The age-associated inflammation was assessed with IL-6 cytokine plasma concentrations measured by ELISA.ResultsAnxiety-like behavior and depressive-like behavior did not change with the age regardless of the microbial status. However, old SPF mice traveled less distance (866.8 cm) than young SPF mice (1375 cm, p < 0.01) in the open-field. Similarly, old GF mice also traveled less distance (458.9 cm) than young GF mice (875.7 cm, p < 0.0001). In contrast to old SPF mice, old GF mice did not show memory impairment in the spatial memory task. Indeed, old SPF mice displayed lower alternation rate of 58.3%, compared to that found in young SPF mice (76.9%, p < 0.05) while both old and young GF mice had an identical alternation rate of 73.3% ( p > 0.05). In addition, IL-6 plasma levels revealed that old GF mice did not show signs of age-associated inflammation that was evident in old SPF mice (3.68 vs. 13.93 pg/ml, p < 0.05).ConclusionsWe conclude that the absence of age-related memory deficit in old germ-free mice is consistent with a role for the microbiota in age-related cognitive decline, likely mediated via the immune system, as suggested by the absence of age-associated inflammation in germ-free mice. We propose that novel microbiota-targeted therapeutic strategies may prevent or delay the cognitive decline of aging.Funding AgenciesCIHRBalsam Family Foundation

Indole-3-Carbinol Selectively Prevents Chronic Stress-Induced Depression-but not Anxiety-Like Behaviors via Suppressing Pro-Inflammatory Cytokine Production and Oxido-Nitrosative Stress in the Brain.

Indole-3-carbinol (I3C), a phytochemical enriched in most cruciferous vegetables, has been shown to display various biological activities such as anti-oxidative stress, anti-inflammation, and anti-carcinogenesis. In this study, we investigated the regulatory effect of I3C on chronic stress-induced behavioral abnormalities in mice. Results showed that repeated I3C treatment at the dose of 10, 30, and 60 mg/kg prevented chronic social defeat stress (CSDS)-induced behavioral abnormalities in the tail suspension test, forced swimming test, sucrose preference test, and social interaction test in mice, and did not affect CSDS-induced behavioral abnormalities in the elevated plus maze, light-dark test, and open-field test, suggesting that the I3C treatment selectively prevents the onset of depression- but not anxiety-like behaviors in chronically stressed mice. Further analysis demonstrated that repeated I3C treatment (60 mg/kg, 10 days) prevented CSDS-induced increases in levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) mRNA and protein, but did not affect CSDS-induced decreases in levels of IL-4, IL-10, and Ym-1 mRNA and/or protein in the hippocampus and prefrontal cortex, suggesting that I3C can selectively prevent chronic stress-induced pro-inflammatory but not anti-inflammatory responses in the brain. Further analysis showed that repeated I3C treatment (60 mg/kg, 10 days) prevented CSDS-induced increases in levels of nitrite and malondialdehyde (MDA), decreases in contents of glutathione (GSH), and decreases in levels of brain derived neurotrophic factor (BDNF) protein in the hippocampus and prefrontal cortex. These results demonstrated that I3C selectively prevents chronic stress-induced depression-like behaviors in mice likely through suppressing neuroinflammation and oxido-nitrosative stress in the brain.

The 5-HT6R agonist E-6837 and the antagonist SB-271046 reverse the psychotic-like behaviors induced by ketamine.

Schizophrenia is a serious mental disorder that affects 1% of the world's population. Although various therapeutic tools have been developed since the appearance of the first generation of antipsychotics, the effect of these agents does not manage to attenuate a significant part of psychotic symptoms. Ketamine is an anesthetic agent able to produce psychotic-like symptoms through the antagonism of the glutamatergic N-methyl-d-aspartic acid (NMDA) receptors (NMDARs). This drug has been widely used to study new pharmacological tools with potential antipsychotic properties. On the contrary, it is known that the 5-HT6 receptor agonist and antagonist drugs induce procognitive, anxiolytic and antidepressant effects in different preclinical models. Therefore, the aim of this study was to evaluate the behavioral actions of the 5-HT6 receptors' agonist E-6837 and the antagonist SB-271046, in ICR-CD1 mice previously treated with a subchronic ketamine scheme (10 mg/kg i.p. daily for 5 days). Results showed that repeated administration of ketamine induced recognition memory deficit, anxiogenic effects, obsessive-compulsive behaviors and stereotyped movements. The acute administration of both 5-HT6 agents reversed the memory deficit and induced a decrease in anxiety, whereas SB-271046 administration produced a decrease in climbing behavior. The injection of either of these 5-HT6 drugs had no effect in the light-dark test. Surprisingly, when these drugs were injected together with ketamine, anxiogenic actions were produced. Current findings suggest that both agonist and antagonist 5-HT6 drugs play an important role in modulating psychotic-like symptoms induced by the subchronic blockade of NMDAR.