This report evaluates the effects of chlordiazepoxide, a benzodiazepine commonly prescribed to manage anxiety-related disorders in adolescent/pediatric populations, on elevated plus maze (EPM) performance in juvenile mice. This approach was taken because chlordiazepoxide produces anxiolytic-like effects in multiple models in adult rodents, however, less is known about the behavioral effects of this benzodiazepine in juveniles. Thus, we administered a single intraperitoneal injection of chlordiazepoxide (0, 5, or 10 mg/kg) to postnatal day 35 male C57BL/6 mice. Thirty minutes later, mice were allowed to explore the EPM for 5-min. We found that chlordiazepoxide-treated mice (5 and 10 mg/kg) spent more time exploring the open arms of the EPM. No differences in velocity (cm/s) or distance traveled (cm) were observed between the groups. These results indicate that chlordiazepoxide induces anxiolytic-related behavior in adolescent male mice.

Cognitive impairment is a commonly observed complication following myocardial infarction; however, the underlying mechanisms are still not well understood. The most recent research suggests that extracellular signal-regulated kinase (ERK) plays a critical role in the development and occurrence of cognitive dysfunction-related diseases. This study aims to explore whether the ERK inhibitor U0126 targets the ERK/Signal Transducer and Activator of Transcription 1 (STAT1) pathway to ameliorate cognitive impairment after myocardial infarction. To establish a mouse model of myocardial infarction, we utilized various techniques including Echocardiography, Hematoxylin-eosin (HE) staining, Elisa, Open field test, Elevated plus maze test, and Western blot analysis to assess mouse cardiac function, cognitive function, and signal transduction pathways. For further investigation into the mechanisms of cognitive function and signal transduction, we administered the ERK inhibitor U0126 via intraperitoneal injection. Reduced total distance and activity range were observed in mice subjected to myocardial infarction during the open field test, along with decreased exploration of the open arms in the elevated plus maze test. However, U0126 treatment exhibited a significant improvement in cognitive decline, indicating a protective effect through the inhibition of the ERK/STAT1 signaling pathway. Hence, this study highlights the involvement of the ERK/STAT1 pathway in regulating cognitive dysfunction following myocardial infarction and establishes U0126 as a promising therapeutic target.

The elevated plus maze is the most widely used paradigm to evaluate anxiety-associated behavioral alterations in rodent models of central nervous system (CNS) disorders. Unconditioned aversive behavior for open and elevated areas is a measure of anxiety and can be assessed by the plus maze. Plus maze consists of perpendicularly arranged open arms and closed arms crossed in the middle with a central platform. Rodents are allowed to explore the maze between the open and closed arms. The number of entries and time spent in the open arms and the closed arms are used as indicators for the anxiety nature of the animals. Transfer latency is a memory indicator that measures the amount of time it takes to move an animal from an open arm to a closed arm. This chapter describes the pretest conditions, materials required, and protocol for the conductance and evaluating the results for the anxiety and cognition-related behavior in rodents.

BACKGROUND: Centella asiatica (L.) Urban, is a medicinal herb with rich history of traditional use in Indian subcontinent. This herb has been valued for its diverse range of medicinal properties including memory booster, and also as a folk treatment for skin diseases, wound healing and mild diuretic.
OBJECTIVE: Aging is a gradual and continuous process of natural decay in the biological systems, including the brain. This work aims to evaluate the effectiveness of ethanolic extract of Centella asiatica (CAE) on age-associated cognitive impairments in rats, as well as the underlying mechanism.
METHODS: Rats were allocated into five distinct groups of 5 animals each: Young rats (3 months old rats), middle-aged (m-aged) rats (13-14 months old), and the remaining three groups were comprised of m-aged rats treated with different concentrations of CAE, viz., 150, 300, and 450 mg/kg b. w., orally for 42 days. Y-maze, open field, novel object recognition, and elevated plus maze tests were used to assess animal behavior. The malondialdehyde (MDA), superoxide dismutase (SOD), and acetylcholinesterase (AChE) assays; and H&E staining were done in the rat brain to assess the biochemical and structural changes. CAE was also subjected to HPLC analysis, in vitro antioxidant and anti-cholinergic activity. The active compounds of CAE were docked with AChE and BuChE in molecular docking study.
RESULTS: The results showed that CAE treatment improves behavioral performance; attenuates the age-associated increase in MDA content, SOD, and AChE activity; and reduces neuronal loss. In vitro study showed that CAE has concentration-dependent antioxidant and anti-AChE activity. Furthermore, the presence of Asiatic acid and Madecassic acid in CAE and their good binding with cholinergic enzymes (in silico) also suggest the anticholinergic effect of CAE.
CONCLUSIONS: The findings of the current study show that the anticholinergic and antioxidant effects of CAE are attributable to the presence of Asiatic acid and Madecassic acid, which not only provide neuroprotection against age-associated cognitive decline but also reverse it.

Neuroimaging data in humans and neurobiological studies in rodents have suggested an involvement of the insular cortex (IC) in anxiety manifestations. However, the local neurochemical mechanisms involved are still poorly understood. Corticotropin-releasing factor (CRF) neurotransmission has been described as a prominent neurochemical mechanism involved in the expression of anxiety-like behaviors, but the brain sites related are poorly understood. Additionally, several findings indicate that control of physiological and behavioral responses by the IC occurs in a site-specific manner along its rostrocaudal axis. Thus, this study is aimed at evaluating the effect of CRF receptor agonism and antagonism within the anterior and posterior subregions of the IC in controlling anxiety-related behaviors in the elevated plus maze (EPM). For this, independent groups (six groups) of animals received bilateral microinjections of vehicle, the selective CRF1 receptor antagonist CP376395, or CRF into either the anterior or posterior subregions of the IC. Ten minutes later, the behavior in the EPM was evaluated for five minutes. Treatment of the anterior IC with CP376395, but not with CRF, increased the time and number of entries into the open arms of the EPM. CRF, but not the CRF1 receptor antagonist, microinjected into the posterior IC also increased exploration of the EPM open arms. Taken together, these data indicate that CRFergic neurotransmission in the anterior IC is involved in the expression of anxiety-related behaviors in the EPM. This neurochemical mechanism does not seem to be activated within the posterior IC during exposure to the EPM, but the effects caused by CRF microinjection indicate that activation of CRF receptors in this IC subregion might evoke anxiolytic-like effects.

Ultrasonic vocalizations (USV) are emitted by both young pups and adult rats to convey positive or negative emotional states. These USV manifestations are contingent on factors including developmental stage, situational requirements, and individual dispositions. Pups emit 40-kHz USV when separated from their mother and litter, which function to elicit maternal care. Conversely, adult rats can produce 50-kHz USV in response to stimuli that elicit reward-related states, including natural rewards, stimulant drugs, and reward-predictive stimuli. The present study aims to investigate whether pup 40-kHz USV can serve as predictors of behaviors related to positive or negative states in adult rats. Both male and female Wistar pups were initially tested on the 11th postnatal day and subsequently in adulthood. There was no significant difference in the number of 40-kHz ultrasonic vocalizations between male and female pups. However, cocaine elicited more 50-kHz USV and hyperactivity in adult females compared to males. Notably, cocaine increased the proportion of step and trill USV subtypes in both adult males and females. Interestingly, this effect of cocaine was stronger in females that were in the diestrus, compared to the estrus phase. In males, a significant positive correlation was found between pup 40-kHz USV and lower anxiety scores in adult male but not female rats tested on the elevated plus-maze test. Furthermore, no significant correlation was found between pup 40-kHz and adult 50-kHz USV in both males and females, whether in undrugged (saline) or in cocaine-treated rats. It is possible that the 40-kHz USV emitted by pups predicted reduced anxiety-like behavior only for male rats because they could elicit maternal care directed specifically to male pups. These findings suggest that 40-kHz USV can serve as an indicator of the emotional link between the rat mother and male pups. Indeed, this suggests that maternal care exerts a positive influence on the emotional state during adulthood.

The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbred polygenic model of childhood absence epilepsy (CAE), which, as their non-epileptic control (NEC) rats, are derived from Wistar rats. While the validity of GAERS in reproducing absence seizures is well established, its use as a model for CAE psychiatric comorbidities has been subject to conflicting findings. Differences in colonies, experimental procedures, and the use of diverse controls from different breeders may account for these disparities. Therefore, in this study, we compared GAERS, NEC, and Wistar bred in the same animal facility with commercially available Wistar (Cm Wistar) as a third control.
We performed hole board (HB) and elevated plus maze (EPM) tests that were analyzed with standard quantitative and T-pattern analysis in male, age-matched Cm Wistar and GAERS, NEC, and Wistar, bred under the same conditions, to rule out the influence of different housing factors and provide extra information on the structure of anxiety-like behavior of GAERS rats.
Quantitative analysis showed that GAERS and NEC had similar low anxiety-like behavior when compared to Cm Wistar but not to Wistar rats, although a higher hole-focused exploration was revealed in NEC. T-pattern analysis showed that GAERS, NEC, and Wistar had a similar anxiety status, whereas GAERS and NEC exhibited major differences with Cm Wistar but not Wistar rats. EPM results indicated that GAERS and NEC also have similar low anxiety compared to Cm Wistar and/or Wistar rats. Nevertheless, the analysis of the T-pattern containing open-arm entry showed GAERS and Wistar to be less anxious than NEC and Cm Wistar rats.
To summarize, comorbid anxiety may not be present in male GAERS rats. This study also highlighted the importance of including a control Wistar group bred under the same conditions when evaluating their behavior, as using Wistar rats from commercial breeders can lead to misleading results.

Relapsing-remitting multiple sclerosis (RRMS) is an autoimmune neurological disease and is the most common subtype of MS. In addition, it is associated with the development of depression and anxiety. To date, depressive- and anxiety-like behaviours were only studied using models of progressive MS, which causes severe motor alterations. Thus, we sought to standardise the depressive and anxiety-like behaviours in an RRMS model induced by experimental autoimmune encephalomyelitis (RR-EAE) in mice. The RR-EAE model was induced in C57BL/6 female mice using myelin oligodendrocyte glycoprotein (MOG35-55) antigen and Quillaja saponin (Quil A) as an adjuvant. The immunisation of RR-EAE did not induce locomotor alteration but caused relapsing-remitting induction of clinical scores in mice until 35 post-immunization (p.i.). Also, increased levels of tumour necrosis factor alpha (TNF-α), astrocyte marker (GFAP), and microglial markers (IBA-1) were detected in the prefrontal cortex at 35 p.i. of RR-EAE. In the open field test, RR-EAE mice showed decreased time spent at the centre and sniffing behaviour (at days 21 and 34 p.i.). Also, on day 35 p.i. the RR-EAE group spent less time in the open arms and had decreased open-arm entries compared to control mice in the elevated plus maze (EPM) test, confirming the anxiety-like behaviour. At day 36° p.i. in the tail suspension test, mice showed depression-like behaviour with decreased latency time and increased immobility time. Thus, the RR-EAE model mimics the neuroinflammatory and behavioural features of the RRMS, including depression- and anxiety-like symptoms.

Exposure to an enriched environment (EE) has been reported to generate multiple beneficial effects in rodents, including - among the many - amelioration of anxiety-related behaviors. The present study investigated whether living in an EE produced anxiolytic effects also in selectively bred Sardinian alcohol-preferring (sP) rats. The relevance of this research question relied on two factors: sP rats displayed an inherent, high anxiety-like state under different experimental conditions; exposure to EE reduced operant, oral alcohol self-administration in sP rats. Starting from weaning, male sP rats were kept under 3 different housing conditions: impoverished environment (IE; single housing with no environmental enrichment); standard environment (SE; 3 rats/cage with no environmental enrichment); EE (6 rats/cage with various elements of environmental enrichment). At the age of approximately 80 days, rats were exposed to an elevated plus maze test for assessment of anxiety-related behaviors. Compared to IE and SE rats, EE rats displayed higher basal levels of exploratory activity (i.e., increased number of entries into closed arms). Compared to IE and SE rats, EE rats also displayed a less \"anxious\" profile, as suggested by the increase in percent number of entries into open arms (OAs), percent time spent in OAs, number of head dips, and number of end-arm explorations in OAs. These data extend the protective (anxiolytic) effects of EE to a proposed animal model of comorbid alcohol use disorder and anxiety disorders.

Vasopressin (AVP) and oxytocin (OXT) are neuropeptides produced by magnocellular neurons (MCNs) of the hypothalamus and secreted through neurohypophysis to defend mammals against dehydration. It was recently demonstrated that MCNs also project to limbic structures, modulating several behavioral responses.
We found that 24 h of water deprivation (WD) or salt loading (SL) did not change exploration or anxiety-like behaviors in the elevated plus maze (EPM) test. However, rats deprived of water for 48 h showed reduced exploration of open field and the closed arms of EPM, indicating hypoactivity during night time. We evaluated mRNA expression of glutamate decarboxylase 1 (Gad1), vesicular glutamate transporter 2 (Slc17a6), AVP (Avpr1a) and OXT (Oxtr) receptors in the lateral habenula (LHb), basolateral (BLA) and central (CeA) amygdala after 48 h of WD or SL. WD, but not SL, increased Oxtr mRNA expression in the CeA. Bilateral pharmacological inhibition of OXTR function in the CeA with the OXTR antagonist L-371,257 was performed to evaluate its possible role in regulating the EPM exploration or water intake induced by WD. The blockade of OXTR in the CeA did not reverse the hypoactivity response in the EPM, nor did it change water intake induced in 48-h water-deprived rats.
We found that WD modulates exploratory activity in rats, but this response is not mediated by oxytocin receptor signaling to the CeA, despite the upregulated Oxtr mRNA expression in that structure after WD for 48 h.