Anxiety-like Behavioral Responses Research Articles

Diazepam effects on anxiety-related defensive behavior of male and female high and low open‐field activity inbred mouse strains

Open-field activity is a commonly used measure of anxiety-related behavior in rodents. The inbred High and Low Activity strains of mice, selected for extreme differences in open-field activity, have been used as a genetic model of anxiety-related behaviors. These selected strains have been thoroughly studied through extensive behavioral testing, quantitative trait locus (QTL) mapping, whole-genome sequencing, and RNA sequencing, to uncover phenotypic and genotypic differences related to anxiety-related behavior. However, the effects of anxiolytic drugs on anxiety-related behavior in these strains have not been studied previously. This study allowed us to expand on previous findings to further characterize the anxiety-related behavior of these unique strains, using an anxiolytic drug. The goal of this study was to determine whether the treatment of adult male and female High Activity (low anxiety) and Low Activity (high anxiety) mice with diazepam, an agonist at the benzodiazepine allosteric site on the GABAA receptor and a drug commonly prescribed to treat anxiety disorders in humans, led to decreases in anxiety-like defensive behavioral responses as assessed in the open-field test (OFT) and elevated plus-maze (EPM). We tested the effects of three doses of diazepam (0, 0.5, 1.0, 3.0 mg/kg, i.p.), given 30 min before behavioral testing to one High Activity strain (H2) and two Low Activity strains (L1 and L2). There was an anxiolytic effect of diazepam observed in the High Activity strain, with more entries into the open arms of the elevated plus-maze, an effect similar to that seen in common mouse strains. However, the only anxiolytic effect of diazepam seen in the Low Activity strains was a reduction in stretch attend posture (SAP). Low Activity strains also displayed freezing behavior in both the OFT and EPM. The combination of the observed freezing behavior, that was not reduced by diazepam, and the reduction in SAP seen with diazepam, suggests a more complex phenotype that includes a component of innate fear in addition to anxiety-related risk assessment behaviors. Since fear and anxiety are distinguishable traits, and both contribute to human anxiety disorders, these results provide novel insight about interpretation of previous genetic and phenotypic differences observed between the High and Low Activity strains.

Wistar-Kyoto rats and chronically stressed Wistar rats present similar depression- and anxiety-like behaviors but different corticosterone and endocannabinoid system modulation

The interplay of social, psychological, and biological stresses can trigger mental health conditions such as major depressive disorder (MDD), adjustment disorder, and posttraumatic stress disorder (PTSD). The endocannabinoid system (ECS), comprising endocannabinoids and cannabinoid receptors, is the critical pathway that mediates responses to stress stimuli. This study aimed to investigate the ECS's impact on responding to chronic social instability stress (SIS). Wistar (WIS) rats and an endogenously depressed rat model, Wistar-Kyoto (WKY), were used to evaluate depression- and anxiety-like behavioral responses, cognitive function, hormone levels, and ECS function. The animals in the stress group (WIS-STS and WKY-STS) were exposed to TMT (predator odor) for 10 mins (two exposures in total: one in light cycle and one in dark cycle) and daily roommate changes (30 days in total), while the control group (CTL) rats were exposed to a sham odor stimulus (distilled water) and did not undergo roommate changes.The results in the open field test suggest that WKY rats had significantly lower locomotor activity than WIS rats. In contrast, WKY rats and chronically stressed WIS rats presented similar depression- and anxiety-like behaviors and impaired cognitive function in the elevated plus maze, forced swimming test, and novel objective recognition test. However, chronic SIS did not exacerbate these behavioral changes in WKY rats. ELISA and Western blot analysis indicated that chronic SIS did not induce further upregulation of endocannabinoids and CB1R downregulation in WKY rats compared to WIS rats. In addition, the Luminex assay revealed that WKY rats showed a higher resilience on the HPA-axis modulation towards chronic SIS, distinguished by the hyperactivity of the HPA-axis modulation in WIS rats.Overall, the study revealed that the chronic SIS animal model (stressed WIS rats) and an animal model of endogenous depression (WKY rats) can generate similar behavioral changes in anxious behavior, behavioral despair, and cognitive impairment. Both animal models present hyperactivity of the ACTH modulation and ECS activity, while WKY rats are more resilient on CORT modulation towards chronic SIS.

Cannabidiol Treatment Shows Therapeutic Efficacy in a Rodent Model of Social Transfer of Pain in Pair-Housed Male Mice

Introduction: Prosocial behavior refers to sharing emotions and sensations such as pain. Accumulated data indicate that cannabidiol (CBD), a nonpsychotomimetic component of the Cannabis sativa plant, attenuates hyperalgesia, anxiety, and anhedonic-like behavior. Nevertheless, the role of CBD in the social transfer of pain has never been evaluated. In this study, we investigated the effects of acute systemic administration of CBD in mice that cohabited with a conspecific animal suffering from chronic constriction injury. Furthermore, we assessed whether repeated CBD treatment decreases hypernociception, anxiety-like behavior, and anhedonic-like responses in mice undergoing chronic constriction injury and whether this attenuation would be socially transferred to the partner. Materials and Methods: Male Swiss mice were Housed in pairs for 28 days. On the 14th day of living together, animals were then divided into two groups: cagemate nerve constriction (CNC), in which one animal of each partner was subjected to sciatic nerve constriction; and cagemate sham (CS), subjected to the same surgical procedure but without suffering nerve constriction. In Experiments 1, 2, and 3 on day 28 of living together, the cagemates (CNC and CS) animals received a single systemic injection (intraperitoneally) of vehicle or CBD (0.3, 1, 10, or 30 mg/kg). After 30 min, the cagemates were subjected to the elevated plusmaze followed by exposure to the writhing and sucrose splash tests. For chronic treatment (Exp. 4), sham and chronic constriction injury animals received a repeated systemic injection (subcutaneous) of vehicle or CBD (10 mg/kg) for 14 days after the sciatic nerve constriction procedure. On days 28 and 29 sham and chronic constriction injury animals and their cagemates were behaviorally tested. Results and Conclusion: Acute CBD administration attenuated anxiety-like behavior, pain hypersensitivity, and anhedonic-like behavior in cagemates that cohabited with a pair in chronic pain. In addition, repeated CBD treatment reversed the anxiety-like behavior induced by chronic pain and enhanced the mechanical withdrawal thresholds in Von Frey filaments and the grooming time in the sucrose splash test. Moreover, repeated CBD treatment effects were socially transferred to the chronic constriction injury cagemates.

Non-Dose-Dependent Relationship between Antipredator Behavior and Conspecific Alarm Substance in Zebrafish

A series of behavioral detection paradigms have been developed for zebrafish (Danio rerio) to examine anxiety-like behavioral responses. Among them, the novel tank diving test is rapidly gaining popularity in translational neuroscience and behavioral research for the investigation of psychopharmacological activity focusing on stress. Zebrafish respond to conspecific epidermal-released alarm substances with antipredator reactions. Although the alarm responses of zebrafish were well characterized in a novel tank diving experiment, the relationship between the intensity of the alarm behavior and the concentration of the alarm substance needed to be understood more adequately. In the current paper, we investigated the behavioral phenotypes and potencies of zebrafish elicited by the serial dilution of an alarm substance in the novel tank diving test. Using a video-tracking assisted behavioral quantification approach, we demonstrated no linear concentration-dependent relationship between antipredator behavior and skin extracts, suggesting that an optimal concentration induced each typical behavioral response. The results showed that the freezing duration (%) significantly increased when stimulated with 104-fold times dilutions of skin extract (equivalent 5 × 10−5 fish/L), while erratic movements (%), time in the bottom half (%), and latency to the upper half (s) significantly elevated when stimulated with 103-fold times dilutions (equivalent 5 × 10−4 fish/L). Therefore, the concentration threshold for an alarm substance that elicited innate fear behavior in zebrafish was presumed to be an equivalent concentration of approximately 5 × 10−4 fish/L. The conclusions may fill a knowledge gap between the innate fear response triggered by injured skin and a novel tank diving paradigm that provides insights into the characterization of alarm substance, behavioral responses, and physiological response mechanisms in zebrafish.

Enrofloxacin exposure induces anxiety-like behavioral responses in zebrafish by affecting the microbiota-gut-brain axis

The ubiquitous presence of antibiotic residues in aqueous environments poses a great potential threat to aquatic organisms. Nevertheless, the behavioral effects of environmentally realistic levels of antibiotics remain poorly understood in fish species. In this study, the behavioral impacts of enrofloxacin, one of typical fluoroquinolone antibiotics that is frequently detected in aquatic environments, were evaluated by the classic light-dark test (LDT) and novel tank task (NTT) in zebrafish. Furthermore, the effects of enrofloxacin exposure on the microbiota-gut-brain axis were also assessed to reveal potential affecting mechanisms underlying the behavioral abnormality observed. Our results demonstrated that zebrafish exposed to 60 μg/L enrofloxacin for 28 days took significantly longer to enter the stressful area of the testing tank and spent significantly less time there in both the LDT and NTT, indicating abnormal anxiety-like behaviors induced by the exposure. In addition, exposure to enrofloxacin at 6 and 60 μg/L resulted in a significant elevation in Bacteroidetes and a marked decline in the Firmicutes/Bacteroidetes ratio of the gut microbiota. Moreover, the intestinal contents of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), glucagon-like peptide 1 (GLP-1), and 5-hydroxytryptamine (5-HT) in zebrafish were significantly upregulated, whereas those of plasma adrenocorticotropic hormone (ACTH) and cortisol (COR) were markedly downregulated upon enrofloxacin exposure. Incubation of zebrafish with a high dose of enrofloxacin (60 μg/L) also resulted in evident increases in the contents of corticotropin-releasing hormone (CRH), brain-derived neurotrophic factor (BDNF), and neuropeptide Y (NPY) in the brain. Fortunately, no significant alteration in the expression of glial fibrillary acidic protein (GFAP) was detected in the brain after enrofloxacin exposure. Our findings suggest that the disruption of the microbiota-gut-brain axis may account for enrofloxacin-induced anxiety-like behaviors in zebrafish. Since the disruption of microbiota-gut-brain axis may give rise to various clinical symptoms, the health risk of antibiotic exposure deserves more attention.

MDMA treatment paired with a trauma-cue promotes adaptive stress responses in a translational model of PTSD in rats

MDMA (3,4-methylenedioxymethamphetamine), a synthetic ring-substituted amphetamine, combined with psychotherapy has demonstrated efficacy for the treatment of chronic posttraumatic stress disorder (PTSD) patients. This controlled prospective study aimed to assess the bio-behavioral underpinnings of MDMA in a translational model of PTSD. Rats exposed to predator-scent stress (PSS) were subjected to a trauma-cue at day 7 shortly after single-dose MDMA injection (5 mg/kg). The elevated plus maze and acoustic startle response tests were assessed on day 14 and served for classification into behavioral response groups. Freezing response to a further trauma-reminder was assessed on Day 15. The morphological characteristics of the dentate gyrus (DG) and basolateral amygdala (BLA) were subsequently examined. Hypothalamic–pituitary–adrenal axis and 5-hydroxytryptamine involvement were evaluated using: (1) corticosterone measurements at 2 h and 4 h after MDMA treatment, (2) Lewis strain rats with blunted HPA-response and (3) pharmacological receptor-blockade. MDMA treatment was effective in attenuating stress behavioral responses only when paired with memory reactivation by a trauma-cue. The effects of the treatment on behavior were associated with a commensurate normalization of the dendritic cytoarchitecture of DG and BLA neurons. Pretreatment with RU486, Ketanserin, or Pindolol prevented the above improvement in anxiety-like behavioral responses. MDMA treatment paired with memory reactivation reduced the prevalence rate of PTSD-phenotype 14 days later and normalized the cytoarchitecture changes induced by PSS (in dendritic complexities) compared to saline control. MDMA treatment paired with a trauma-cue may modify or update the original traumatic memory trace through reconsolidation processes. These anxiolytic-like effects seem to involve the HPA axis and 5-HT systems.

Cell-Type Specific Deletion of CB2 Cannabinoid Receptors in Dopamine Neurons Induced Hyperactivity Phenotype: Possible Relevance to Attention-Deficit Hyperactivity Disorder.

DAT-Cnr2 mice are conditional knockout (cKO) animals that do not express cannabinoid CB2 receptors (CB2R), in midbrain dopamine neurons. The hyperactivity phenotype of DAT-Cnr2 cKO mice were paradoxically reduced by low dose of amphetamine. Here, we report on the locomotor activity analysis in male and female adolescent (PND 30 ± 2) mice in basal conditions and in response to different doses of amphetamine, using the Open Field (OF), Elevated Plus-Maze (EPM) tests and the Novel Object Recognition (NOR) task as a putative model of attention deficit hyperactivity disorder (ADHD). Results showed that both male and female adolescent DAT-Cnr2 mice displayed significant increases in distance traveled in the OF test compared with WT mice. However, 2 mg/kg dose of amphetamine reduced the distance traveled by the DAT-Cnr2 but was increased in the WT mice. In the EPM test of anxiety-like behavioral responses, DAT-Cnr2 spent more time in the open arms of the maze than the WT mice, suggesting a reduction in anxiety-like response. DAT-Cnr2 mice showed significant increase in the number of unprotected head dips in the maze test and in the cliff avoidance reaction (CAR) test demonstrating impulsivity and risky behavior. DAT-Cnr2 mice also exhibited deficient response in the delay decision making (DDM), with impulsive choice. Both DAT-Cnr2 and WT were able to recognize the new object in the NOR task, but the exploration by the DAT-Cnr2 was less than that of the WT mice. Following the administration of 2 mg/kg of amphetamine, the similarities and differential performances of the DAT-Cnr2 and WT mice in the EPM test and NOR task was probably due to increase in attention. Microglia activation detected by Cd11b immunolabelling was enhanced in the hippocampus in DAT-Cnr2 cKO than in WT mice, implicating neuro-immune modulatory effects of CB2R. The results demonstrates that DAT-Cnr2 cKO mice with cell-type specific deletion of CB2R in midbrain dopaminergic neurons may represent a possible model for studying the neurobiological basis of ADHD.

Anxiety, fear, panic: An approach to assessing the defensive behavior system across the predatory imminence continuum

In order to effectively thwart predation, antipredator defensive behaviors must be matched to the current spatio-temporal relationship to the predator. We have proposed a model where different defensive responses are organized along a predatory imminence continuum (PIC). The PIC is a behavior system organized as a sequence of innately programmed behavioral modes, each representing a different interaction with the predator or threat. Ranging from low threat to predator contact, the PIC categorizes defense modes as pre-encounter, post-encounter, and circa-strike, corresponding to states of anxiety, fear, and panic, respectively. This experiment examined if the same significant stressor caused overexpression of all defensive responses along the PIC, including anxiety-like behavior, freezing, and panic-like responses. Female and male mice were exposed to acute stress that consisted of a series of ten pseudorandomly presented unsignaled footshocks (or no shocks). Mice were subsequently tested on a battery of tasks to assess stress effects on pre-encounter (anxiety-like), post-encounter (fear), and circa-strike (panic-like) behaviors. Results revealed that following stress, mice exhibited increased anxiety-like behavior shown through reduced average velocity within a modified open field. Furthermore, stressed mice showed increased fear following a single footshock in a new context as well as an increase in reactivity to white noise in the original stress context, with stressed mice exhibiting a more robust circa-strike-like response than controls. Therefore, significant stress exposure influenced the defensive states of anxiety, fear, and panic across the predatory imminence continuum. This research could therefore reveal how such responses become maladaptive following traumatic stress in humans.

Dioclea Altissima Seed Lectin (DAL) Prevents Anxiety-like Behavioral Responses in Adult Zebrafish (Danio Rerio): Involvement of GABAergic and 5-HT Systems.

Plant lectins have shown promising neuropharmacological activities in animal models. This study evaluated the effect of Dioclea altissima seed lectin (DAL) on adult zebrafish behavior. Zebrafish (n=6/group) were treated (i.p.; 20 μL) with DAL (0.025; 0.05 or 0.1 mg/mL), vehicle or diazepam (DZP) and submitted to several tests (open field, light/dark preference or novel tank). Flumazenil, pizotifen or granisetron were administered 15 min before DAL (0.05 mg/mL), and the animals were evaluated on light/dark preference test. It was also verified whether the DAL effect depended on its structural integrity and ability to interact with carbohydrates. DAL decreased the locomotor activity of adult zebrafish (0.025; 0.05 or 0.1 mg/mL), increased the time spent in the upper region of the aquarium (0.025 mg/mL), and decreased the latency time of adult zebrafish to enter the upper region on the novel tank test. DAL (0.05 mg/mL) also increased their permanence in the light zone of the light/dark preference test. The effect of DAL was dependent on carbohydrate interaction and protein structure integrity and was prevented by pizotifen, granizetron and flumazenil. DAL was found to have an anxiolytic-like effect mediated by the 5-HT and GABAergic receptors.

Enrofloxacin Exposure Induces Anxiety-Like Behavioral Responses in Zebrafish by Affecting the Microbiota-Gut-Brain Axis

Enrofloxacin Exposure Induces Anxiety-Like Behavioral Responses in Zebrafish by Affecting the Microbiota-Gut-Brain Axis

Sex Differences in BNST and Amygdala Activation by Contextual, Cued, and Unpredictable Threats.

Fear and anxiety can be described as emotional and physical responses to predictable and unpredictable threats. While the amygdala is necessary for context and cued fear conditioning, the bed nucleus of the stria terminalis (BNST) is important for anxiety-like behavior and conditioned responses to diffuse and/or unpredictable threats. However, we still lack knowledge about how the BNST and amygdala nuclei act in coordination. Moreover, the incidence of anxiety disorders and posttraumatic stress disorder (PTSD) is substantially higher in women than in men, but most studies of fear conditioning are conducted in male rodents. Here, we asked whether the BNST and the lateral, basal, and central nuclei of the amygdala are active during the expression of fear conditioning in male and female rats using FOS immunohistochemistry. We first show that the BNST is indeed involved in context fear expression in males, but not in females. The lateral amygdala was active in both sexes during context fear expression. We next trained animals using tone cues paired with an unconditioned stimulus (US), or tone cues which were unpaired with the US, and thus nonpredictive. Females displayed greater fear expression to these unpaired tones than males. FOS was upregulated in both the BNST and the basal amygdala during fear expression to unpaired tones in both sexes. The differential processing of fear responses by males and females highlights the need to acknowledge sex differences in conditioned fear memory.

Oxytocin in the anterior cingulate cortex attenuates neuropathic pain and emotional anxiety by inhibiting presynaptic long-term potentiation.

Oxytocin is a well-known neurohypophysial hormone that plays an important role in behavioral anxiety and nociception. Two major forms of long-term potentiation, presynaptic LTP (pre-LTP) and postsynaptic LTP (post-LTP), have been characterized in the anterior cingulate cortex (ACC). Both pre-LTP and post-LTP contribute to chronic-pain-related anxiety and behavioral sensitization. The roles of oxytocin in the ACC have not been studied. Here, we find that microinjections of oxytocin into the ACC attenuate nociceptive responses and anxiety-like behavioral responses in animals with neuropathic pain. Application of oxytocin selectively blocks the maintenance of pre-LTP but not post-LTP. In addition, oxytocin enhances inhibitory transmission and excites ACC interneurons. Similar results are obtained by using selective optical stimulation of oxytocin-containing projecting terminals in the ACC in animals with neuropathic pain. Our results demonstrate that oxytocin acts on central synapses and reduces chronic-pain-induced anxiety by reducing pre-LTP.

Evaluation of the effects of altitude on biological signatures of inflammation and anxiety- and depressive-like behavioral responses

Over sixteen million people suffer from a depressive episode annually in the United States, with females affected at twice the rate of males. Little is known about the effects of exposure to high altitude on the risk of development of major depressive disorder, despite reports of higher suicide rates at higher altitudes. We hypothesize that exposure to hypobaric hypoxia at high altitude increases endophenotypes of self-directed suicidal violence, including biological signatures of chronic inflammation and vulnerability to anxiety-like and depressive-like behavioral responses in a sex-specific manner. Biological signatures of inflammation, including granulocyte:lymphocyte ratios, monocyte cell counts, and monocyte:lymphocyte ratios were assessed using complete blood count data, anhedonia, and anxiety- and depressive-like behavioral responses were evaluated. We assessed biological signatures of inflammation and behavioral responses in the open-field test, sucrose preference test, and modified Porsolt forced swim test in young adult male and female Long-Evans and Sprague Dawley rats. All tests were conducted near sea level (374 ft [114 m] elevation) and at moderate-high altitude (5430 ft [1655 m] elevation) during acclimation periods of one, two, three, four, and five weeks following shipment from a sea level animal breeding facility (N = 320, n = 8 per group). Exposure to moderate-high altitude induced a biological signature of increased inflammation, as evidenced by main effects of altitude for: 1) increased granulocyte:lymphocyte ratio; 2) increased count and relative abundance of circulating monocytes; and 3) increased monocyte:lymphocyte ratios. Exposure to moderate-high altitude also increased anhedonia as assessed in the sucrose preference test in both male and female rats, when data were collapsed across strain and time. Among male and female Long Evans rats, exposure to moderate-high altitude increased immobility in the forced swim test, without changing anxiety-like behaviors in the open-field test. Finally, granulocyte:lymphocyte ratios were correlated with anhedonia in the sucrose preference test. These data are consistent with the hypothesis that hypobaric hypoxia at moderate-high altitude induces persistent endophenotypes of self-directed suicidal violence including biological signatures of inflammation, anhedonia, and depressive-like behavioral responses.

Glucocorticoid Receptor Antagonist Mifepristone Does Not Alter Innate Anxiety-Like Behavior in Genetically-Selected Marchigian Sardinian (msP) Rats.

Marchigian Sardinian alcohol-preferring (msP) rats serve as a unique model of heightened alcohol preference and anxiety disorders. Their innate enhanced stress and poor stress-coping strategies are driven by a genetic polymorphism of the corticotropin-releasing factor receptor 1 (CRF1) in brain areas involved in glucocorticoid signaling. The activation of glucocorticoid receptors (GRs) regulates the stress response, making GRs a candidate target to treat stress and anxiety. Here, we examined whether mifepristone, a GR antagonist known to reduce alcohol drinking in dependent rats, decreases innate symptoms of anxiety in msPs. Male and female msPs were compared to non-selected Wistar counterparts across three separate behavioral tests. We assessed anxiety-like behavior via the novelty-induced hypophagia (NIH) assay. Since sleep disturbances and hyperarousal are common features of stress-related disorders, we measured sleeping patterns using the comprehensive lab monitoring system (CLAMS) and stress sensitivity using acoustic startle measures. Rats received an acute administration of vehicle or mifepristone (60 mg/kg) 90 min prior to testing on NIH, acoustic startle response, and CLAMS. Our results revealed that both male and female msPs display greater anxiety-like behaviors as well as enhanced acoustic startle responses compared to Wistar counterparts. Male msPs also displayed reduced sleeping bout duration versus Wistars, and female msPs displayed greater acoustic startle responses versus male msPs. Importantly, the enhanced anxiety-like behavior and startle responses were not reduced by mifepristone. Together, these findings suggest that increased expression of stress-related behaviors in msPs are not solely mediated by acute activation of GRs.

Long-term Effects of Maternal Separation on Anxiety-Like Behavior and Neuroendocrine Parameters in Adult Balb/c Mice

Introduction: Disruption of maternal care using maternal separation (MS) models has provided significant evidence of the deleterious long-term effects of early life stress. Several preclinical studies investigating MS showed multiple behavioral and biomolecular alterations. However, there is still conflicting results from MS studies, which represents a challenge for reliability and replicability of those findings. Objective: To address that, this study was conducted to investigate whether MS would affect anxiety-like behaviors using a battery of classical tasks, as well as central and peripheral stress-related biomarkers. Methods: Male Balb/c mice were exposed to MS from postnatal day (PND) 2 to 14 for 180-min per day. Two independent cohorts were performed to evaluate both baseline and anxiety-like behavior responses to MS at PND60. We performed composite scores to evaluate MS effects on anxiety and risk assessment phenotypes. Also, we assessed mRNA gene expression in the medial pre-frontal cortex (mPFC) of glucocorticoid and mineralocorticoid receptors (GR and MR) using real-time PCR and peripheral corticosterone levels (CORT) to investigate possible neurobiological correlates to anxiety behaviors. Results: We found increased anxiety-like behavior and decreased risk assessment and exploratory behaviors in MS mice. The animals exposed to MS also presented a decrease in MR mRNA expression and higher levels of CORT compared to controls. Conclusions: Our findings reinforce the body of evidence suggesting that long-term MS induces effects on anxiety and risk assessment phenotypes following the exposure to a standardized MS protocol. Moreover, MS affected the expression of MR mRNA and induced significant changes on CORT response. This data highlights that the reprograming MS effects on HPA axis could be mediate by MR gene expression in mPFC and chronic overactivity of peripheral CORT levels.

Comparing the effects of two different strains of mycobacteria, Mycobacterium vaccae NCTC 11659 and M. vaccae ATCC 15483, on stress-resilient behaviors and lipid-immune signaling in rats

Stress-related disorders, such as posttraumatic stress disorder (PTSD), are highly prevalent and often difficult to treat. In rodents, stress-related, anxiety-like defensive behavioral responses may be characterized by social avoidance, exacerbated inflammation, and altered metabolic states. We have previously shown that, in rodents, subcutaneous injections of a heat-killed preparation of the soil-derived bacterium Mycobacterium vaccae NCTC 11659 promotes stress resilience effects that are associated with immunoregulatory signaling in the periphery and the brain. In the current study, we sought to determine whether treatment with a heat-killed preparation of the closely related M. vaccae type strain, M. vaccae ATCC 15483, would also promote stress-resilience in adult male rats, likely due to biologically similar characteristics of the two strains. Here we show that immunization with either M. vaccae NCTC 11659 or M. vaccae ATCC 15483 prevents stress-induced increases in hippocampal interleukin 6 mRNA expression, consistent with previous studies showing that M. vaccae NCTC 11659 prevents stress-induced increases in peripheral IL-6 secretion, and prevents exaggeration of anxiety-like defensive behavioral responses assessed 24 h after exposure to inescapable tail shock stress (IS) in adult male rats. Analysis of mRNA expression, protein abundance, and flow cytometry data demonstrate overlapping but also unique effects of treatment with the two M. vaccae strains on immunological and metabolic signaling in the host. These data support the hypothesis that treatment with different M. vaccae strains may immunize the host against stress-induced dysregulation of physiology and behavior.

Embryonic Exposure to Ethanol Increases Anxiety-Like Behavior in Fry Zebrafish.

Fetal alcohol spectrum disorder (FASD) is an umbrella term to describe the effects of ethanol (Eth) exposure during embryonic development, including several conditions from malformation to cognitive deficits. Zebrafish (Danio rerio) are a translational model popularly applied in brain disorders and drug screening studies due to its genetic and physiology homology to humans added to its transparent eggs and fast development. In this study, we investigated how early ethanol exposure affects zebrafish behavior during the initial growth phase. Fish eggs were exposed to 0.0 (control), 0.25 and 0.5% ethanol at 24h post-fertilization. Later, fry zebrafish (10days old) were tested in a novel tank task and an inhibitory avoidance protocol to inquire about morphology and behavioral alterations. Analysis of variance showed that ethanol doses of 0.25 and 0.5% do not cause morphological malformations and did not impair associative learning but increased anxiety-like behavior responses and lower exploratory behavior when compared to the control. Our results demonstrate that one can detect behavioral abnormalities in the zebrafish induced by embryonic ethanol as early as 10days post-fertilization and that alcohol increases anxious behavior during young development in zebrafish.

An acetylcholinesterase inhibitor, donepezil, increases anxiety and cortisol levels in adult zebrafish.

A potent acetylcholinesterase inhibitor, donepezil is a cognitive enhancer clinically used to treat neurodegenerative diseases. However, its complete pharmacological profile beyond cognition remains unclear. The zebrafish (Danio rerio) is rapidly becoming a powerful novel model organism in neuroscience and central nervous system drug screening. Here, we characterize the effects of 24-h donepezil administration on anxiety-like behavioral and endocrine responses in adult zebrafish. We evaluated zebrafish anxiety-like behaviors in the novel tank, the light-dark and the shoaling tests, paralleled by assessing brain acetylcholinesterase activity and whole-body cortisol levels. Overall, donepezil dose-dependently decreased zebrafish locomotor activity in the novel tank test and reduced time in light in the light-dark test, likely representing hypolocomotion and anxiety-like behaviors. Donepezil predictably decreased brain acetylcholinesterase activity, also increasing whole-body cortisol levels, thus further linking acetylcholinesterase inhibition to anxiety-like behavioral and endocrine responses. Collectively, these findings suggest negative modulation of zebrafish affective behavior by donepezil, support the key role of cholinergic mechanisms in behavioral regulation in zebrafish, and reinforce the growing utility of zebrafish models for studying complex behavioral processess and their neuroendocrine and neurochemical regulation.

Environmental enrichment during adolescence heightens ethanol intake in female, but not male, adolescent rats that are selectively bred for high and low ethanol intake during adolescence

ABSTRACT Background: Discriminating between adolescents who will eventually have ethanol use problems from those who do not is important. Environmental enrichment is a promising approach to reduce drug-related problems, but its impact on ethanol’s effects and intake is being scrutinized. Objective: We tested the effects of environmental enrichment on ethanol intake, preference, and anxiety-like response as well as shelter seeking and risk-taking behaviors. Methods: Experiment 1 examined ethanol intake, preference, and anxiety-like responses in 46 male and 54 female Wistar rats that were derived from a short-term breeding program that selected for high and low ethanol drinking during adolescence (ADHI2 and ADLO2 lines, respectively). Shelter-seeking and risk-taking behaviors were assessed (Experiment 2) in ADHI2 and ADLO2 rats (73 males, 76 females) reared under environmental enrichment or standard housing conditions and given doses of ethanol (2.5 g/kg, intraperitoneal) for 3 weeks. Environmental enrichment was applied on postnatal days 21–42. Ethanol intake was measured on postnatal days 42–68. Anxiety-like behavior and exploratory responses were assessed using the light-dark box and multivariate concentric square field test. Results: In Experiment 1, environmental enrichment increased ethanol intake in female, but not male, ADHI2 and ADLO2 rats (p < 0.05). In the baseline measurement of Experiment 2, ADHI2 rats exhibited reduced risk-taking and increased anxiety-like behavior (p < .05). After exposure to environmental enrichment the ADHI and ADLO rats, both males and females, exhibited increased risk-taking and exploratory behavior (p < 0.05). Conclusions: Environmental enrichment appears to increase ethanol intake in female rats by promoting the exploration of new environments or stimuli. The findings indicate that environmental enrichment increased ethanol intake in female, but not male, rats. Clinical programs that treat alcohol use disorder by emphasizing environmental stimulation should be designed with caution.

Chronic exposure to bisphenol S induces oxidative stress, abnormal anxiety, and fear responses in adult zebrafish (Danio rerio)

Bisphenol S (BPS) is increasingly used in a wide range of industrial and consumer products, resulting in its ubiquitous distribution across the environment, including aquatic ecosystems. Although it is commonly known as a weak/moderate estrogenic compound, there has been a growing acknowledgment of the potential of BPS to cause toxicity by inducing oxidative stress. Oxidative stress is a major participant in the development of anxiety-like behaviors in humans and animals. Therefore, the present study was designed to examine the impact of BPS on anxiety-like behavior and fear responses in adult zebrafish and also to elucidate the possible linkage between the BPS neurotoxicity and oxidative status of the brain. To this end, adult male and female zebrafish were exposed to 0 (control), 1, 10, and 30 μg/L of BPS and 1 μg/L of 17-β-estradiol (E2) for 75 days. Following exposure, changes in anxiety and fear-related responses were evaluated by applying a novel tank test and by exposing focal fish to chemical alarm cues. Additionally, we evaluated the expression of multiple antioxidant genes in the zebrafish brain. Our results indicate that BPS, irrespective of exposure concentration, and E2 significantly decreased bottom-dwelling behavior and the latency to enter the upper water column. Furthermore, exposure to the highest concentration of BPS and E2 induced a significant decrease in fear-related responses. The impaired anxiety and reduced fear-related responses were associated with a down-regulation in the transcription of genes involved in enzymatic antioxidant defense. Taken together, our results suggest that chronic exposure to BPS impairs anxiety and fear responses in adult zebrafish, possibly by inducing oxidative stress in the brain.