Open Arm Exploration Research Articles

Cues to the usefulness of grooming behavior in the evaluation of anxiety in the elevated plus-maze.

behavior has been reported to be a response to novelty and other stressors. However, studies that sought to identify anxiety-related measures in the elevated plus-maze have not found grooming as a reliable index. Grooming has been implied with dearousal after a stressful experience.In the present work,a detailed investigation of this behavior was performed in a test with a longer duration (i.e., 10 min) than the usual duration in the elevated plus-maze. The level of anxiety was manipulated prior to the test by confining male rats to environments known to differ in the degree of aversive stimulation, including a familiar cage, a closed arm of the elevated plus-maze, and an open arm (i.e., the most aversive arm) of the elevated plus-maze. Grooming behavior was classified into three constituent elements: rostral grooming, head grooming, and body grooming. The groups were compared with regard to conventional measuresof the elevated plus-maze and grooming behavior. Prior confinement to an open arm led to a delayed effect of decreased open-arm exploration undetectable during the first half of the test compared with rats previously confined to a closed arm. Prior confinement to an open arm also increased the total duration of grooming during the second half of the test. These increases were found to be partially attributable to increases in the rostral element of grooming. The percentage of interrupted bouts was also found to discriminate the group previously confined to an open arm. These results indicate that prolongation of the session can reveal effects that are otherwise undetectable and that under such conditions some grooming measures can be useful in the evaluation of anxiety-like behavior. Keywords: elevated plus-maze, dearousal, anxiety, grooming behavior

Effect of berberine on depression- and anxiety-like behaviors and activation of the noradrenergic system induced by development of morphine dependence in rats.

The purpose of this study was to evaluate whether berberine (BER) administration could attenuate depression- and anxiety-like behaviors and increase corticotrophin-releasing factor (CRF) and tyrosine hydroxylase (TH) expression following chronic morphine withdrawal in rats. Male rats were exposed to chronic, intermittent, escalating morphine (10~50 mg/kg) for 10 days. After the last morphine injection, depression- and anxiety-like beahvior associated with morphine discontinuation persisted for at least three days during withdrawal without any change in ambulatory activity. Daily BER administration significantly decreased immobility in the forced swimming test and increased open-arm exploration in the elevated plus maze test. BER administration also significantly blocked the increase in hypothalamic CRF expression and TH expression in the locus coeruleus (LC) and the decrease in hippocampal brain-derived neurotrophic factor (BDNF) mRNA expression. Taken together, these findings demonstrated that BER administration significantly reduced morphine withdrawal-associated behaviors following discontinuation of repeated morphine administration in rats, possibly through modulation of hypothalamic CRF and the central noradrenergic system. BER may be a useful agent for treating or alleviating complex withdrawal symptoms and preventing morphine use relapses.

The anxiolytic effects of somatostatin following intra-septal and intra-amygdalar microinfusions are reversed by the selective sst2 antagonist PRL2903

Somatostatin (SST) is a polypeptide with two biological isoforms (SST14, and SST28), and five SST receptor subtypes (sst1-5). Together, they mediate a number of neural and hormonal functions. Recently, we found that intracerebroventricular (ICV), intra-amygdalar, and intra-septal microinfusions of SST14, SST28, and a selective sst2 receptor agonist L-779976 all produced anxiolytic-like effects in the elevated plus-maze, a widely used animal model of anxiety. The receptor specificity of these anxiolytic-like effects, however, has not been conclusively established.Accordingly, the anxiolytic effects of SST in the elevated plus-maze were assessed following intra-septal or intra-amygalar microinfusions of 1) SST (1.5μg per hemisphere), 2) the highly selective sst2 receptor antagonist PRL2903 (1.5μg per hemisphere), or 3) the combination of SST and PRL2903 (each 1.5μg per hemisphere). Antagonism of the anxiolytic effects of SST in the plus-maze by PRL2903 should result in open-arm exploration that is equivalent to that of 4) vehicle-injected control rats.Both intra-septal and intra-amygdalar microinfusions of SST produced anxiolytic effects in the elevated plus-maze, consistent with results found previously after ICV microinfusions (see Engin et al., 2008; Engin and Treit, 2009; Yeung et al., 2011). More importantly, infusion of PRL2903 completely reversed the anxiolytic effects of SST in both the amygdala and the septum. These results show that somatostatin's anxiolytic effects are mediated by sst2 receptors contained in the amygdala and septum of the rat brain.

Adolescent amphetamine exposure elicits dose-specific effects on monoaminergic neurotransmission and behaviour in adulthood

Despite the growing non-medical consumption of amphetamine (Amph) during adolescence, its long-term neurobiological and behavioural effects have remained largely unexplored. The present research sought to characterize the behavioural profile and electrophysiological properties of midbrain monoaminergic neurons in adult rodents after Amph exposure during adolescence. Adolescent rats were administered vehicle, 0.5, 1.5, or 5.0 mg/kg.d Amph from postnatal day (PND) 30-50. At adulthood (PND 70), rats were tested in an open-field test (OFT) and elevated plus maze (EPM), paralleled by in-vivo extracellular recordings of serotonin (5-HT), dopamine (DA) and norepinephrine (NE) neurons from the dorsal raphe nucleus, ventral tegmental area, and locus coeruleus, respectively. 5-HT firing in adulthood was increased in rats that had received Amph (1.5 mg/kg.d) during adolescence. At this regimen, DA firing activity was increased, but not NE firing. Conversely, the highest Amph dose regimen (5.0 mg/kg.d) enhanced NE firing, but not DA or 5-HT firing rates. In the OFT, Amph (1.5 mg/kg.d) significantly increased the total distance travelled, while the other doses were ineffective. In the EPM, all three Amph doses increased time spent in the open arms and central platform, as well as the number of stretch-attend postures made. Repeated adolescent exposure to Amph differentially augments monoaminergic neuronal firing in a dose-specific fashion in adulthood, with corresponding alterations in locomotion, risk assessment (stretch-attend postures and central platform occupancy) and risk-taking behaviours (open-arm exploration). Thus, adolescent Amph exposure induces long-lasting neurophysiological alterations that may have implications for drug-seeking behaviour in the future.

English

Withaferin-A dose-dependently (10 tob40 mg/kg) displayed anxiolytic activity, as measured by an increase in open arm exploration time in the elevated plus-maze (EPM), following intraperitoneal (i.p.) administration in rats. Acute administration of withaferin-A at 40.0 mg/kg significantly (P<0.05) increased open arm exploration time by 176% as compared to vehicle control, which is similar to the benzodiazepine diazepam at 1.0 and 3.0 mg/kg (191 and 200%, respectively). However, 24 h following sub chronic 5-day administration of diazepam twice daily (bid) at 3.0 mg/kg, diazepam was devoid of anxiolytic activity at 1.0 mg/kg, as measured by no difference in open arm exploration time when compared with vehicle control, while the 3.0 mg/kg dose still produced a significant (P<0.05) 175% increase in open arm exploration time. In contrast, following sub chronic administration of withaferin-A (40.0 mg/kg), a significant (P<0.01) enhancement in open arm exploration time was observed at 40.0 mg/kg (665% as compared to control). Therefore, withaferin-A resulted in anxiolysis which is similar to diazepam following acute administration in the EPM. However, following sub chronic administration unlike diazepam which showed an attenuation of anxiolytic activity, withaferin-A displayed enhanced anxiolytic efficacy and was devoid of tolerance.   Key words: Withaferin-A, anxiety, benzodiazepines, nitric oxide, elevated plus-maze.

Anti-nociceptive, anxiolytic and anticonvulsant effects of an aqueous leaf extract of Leea guineensis G. Don (Family: Leeaceae)

Leea guineensis G. Don (Family: Leeaceae) is an evergreen shrub or small tree used in Ghanaian traditional medicine to treat various ailments including epileptic fits and pain. However, little scientific evidence exists to support its use. The present study examined the anti-nociceptive, anti-anxiety and anticonvulsant effects of the aqueous leaf extract of L. guineensis (LGE) in murine models of pain (formalin test), anxiety (elevated plus-maze and light/dark box tests) and convulsion (pentylenetetrazole-, picrotoxin- and maximal electroshock-induced seizures tests). LGE (30 to 300 mg/kg,p.o.) and the positive control morphine (3 to 10 mg/kg, i.p.) exerted profound dose-dependent anti-nociceptive activity in both phases of the formalin test. LGE (30 to 300 mg/kg, p.o.) also showed anxiolytic effects similar to diazepam (0.1 to 1.0 mg/kg, i.p.) but contrary to pentylenetetrazole (3 to 30 mg/kg, i.p.). LGE increased the frequency and duration of open arm exploration while decreasing the protected forms of stretch-attend postures and head-dips in the elevated plus-maze. It also decreased the emergence latency of mice into the lit compartment of the light/dark box and increased the amount of time spent there. LGE (30 to 300 mg/kg, p.o) also exhibited significant anticonvulsant activity by protecting mice against pentylenetetrazole-, picrotoxin- and maximal electroshock-induced seizures. At all the doses used, LGE produced no motor deficits in mice. Together, the results suggest that the extract has anti-nociceptive, anxiolytic and anticonvulsant properties which support its traditional use. Key words: Leea guineensis, seizures, maximal electroshock, formalin test, elevated plus-maze, light/dark box.

The effects of phencyclidine (PCP) on anxiety-like behavior in the elevated plus maze and the light–dark exploration test are age dependent, sexually dimorphic, and task dependent

Previous research in our laboratory revealed sexually dimorphic effects of prior exposure to phencyclidine (PCP) on elevated plus maze behavior. In an attempt to examine the developmental time course of this effect and determine the extent to which it generalizes to other anxiety paradigms, young adult (61–64days old) and adult (96–107days old) male and female rats were treated with PCP (15mg/kg) or saline. Following a two week withdrawal period, animals were tested in either the elevated plus maze (EPM) or a light–dark exploration (LD) test. In adults, both tests revealed a sexually dimorphic effect driven by PCP-induced decreases in anxiety in females as indicated by increased time spent in the open arms of the EPM and in the lit compartment of the LD test and increased anxiety in males as indicated by decreased time spent in the lit compartment of the LD. In young animals, PCP pretreatment decreased open arm exploration in the elevated plus maze, indicating increased anxiety. However, PCP increased time spent in the light compartment in the light–dark exploration test, indicating decreased anxiety. Corticosterone levels measured 15min after the onset of the EPM failed to reveal an association between the behavioral effects of PCP and corticosterone levels. The results in adults substantiate the previously observed sexually dimorphic effect of PCP on elevated plus maze behavior in adults and indicate that the effect generalizes to another anxiety paradigm. The results in the younger animals suggest an age dependent effect of PCP on anxiety in general and indicate that behaviors in the elevated plus maze and the light–dark exploration test reflect dissociable psychobiological states.

Post-weaning environmental enrichment alters affective responses and interacts with behavioral testing to alter nNOS immunoreactivity

Challenging early life events can dramatically affect mental health and wellbeing. Childhood trauma and neglect can increase the risk for developing depressive, anxiety, and substance abuse disorders. Early maternal separation in rodents has been extensively studied and induces long-lasting alterations in affective and stress responses. However, other developmental periods (e.g., the pubertal period) comprise a critical window whereby social and environmental complexity can exert lasting changes on the brain and behavior. In this study, we tested whether early life environmental complexity impacts affective responses, aggressive behaviors, and expression of neuronal nitric oxide synthase (nNOS), the synthetic enzyme for nitric oxide, in adulthood. Mice were weaned into social+nonsocial enrichment, social only enrichment, or standard (isolated) laboratory environments and were tested in open field, elevated plus maze, forced swim, and resident–intruder aggression tests 60days later. Social+nonsocial enrichment reduced locomotor behavior and anxiety-like responses in the open field and reduced depressive-like responses in the forced swim test. Social housing increased open arm exploration in the elevated plus maze. Both social+nonsocial enrichment and social housing only reduced aggressive behaviors compared with isolation. Social+nonsocial enrichment also increased body mass gain throughout the study. Finally, socially-housed mice had reduced corticosterone concentrations compared with social+nonsocial-enriched mice. Behavioral testing reduced nNOS-positive neurons in the basolateral amygdala and the ventral lateral septum, but not in the social+nonsocial-enriched mice, suggesting that environmental complexity may buffer the brain against some environmental perturbations.

Infusions of neuropeptide Y into the lateral septum reduce anxiety-related behaviors in the rat

Neuropeptide Y (NPY) is one of the most abundant peptides in mammalian brain and NPY-like-immunoreactivity is highly expressed in the lateral septum, an area extensively involved in anxiety regulation. NPY counteracts the neurochemical and behavioral responses to acute threat in animal models, and intracerebroventricular (i.c.v.) administration of NPY at low doses is anxiolytic. Less is known about the specific contributions of the lateral septum to NPY-mediated anxiety regulation. In Experiment 1, the effects of infusions of NPY (1.5μg) into the lateral septum were investigated in three animal models of anxiety: the elevated plus-maze, novelty-induced suppression of feeding, and shock-probe burying tests. Experiment 2 examined the role of the NPY Y1 receptor in these models by co-infusing the Y1 antagonist BIBO 3304 (0.15μg, 0.30μg) with NPY into the lateral septum. In the elevated plus-maze, there were no changes in rats' open arm exploration, the index of anxiety reduction in this test. In the novelty-induced suppression of feeding test, rats infused with NPY showed decreases in the latency to consume a palatable snack in a novel (but not familiar) environment, suggesting a reduction in anxiety independent of increases in appetite. This anxiolysis was attenuated by co-infusion with BIBO 3304 (0.30μg) in Experiment 2. Lastly, rats infused with NPY showed decreases in the duration of burying behavior in the shock-probe burying test, also indicative of anxiety reduction. However, unlike in the feeding test, BIBO 3304 did not attenuate the NPY-induced anxiolysis in the shock-probe test. It is concluded that NPY produces anxiolytic-like actions in the lateral septum in two animal models of anxiety: the novelty-induced suppression of feeding, and shock-probe burying tests, and that this anxiolysis is dependent on Y1 receptor activation in the feeding test.

Microinjection of histamine into the cerebellar vermis impairs emotional memory consolidation in mice

The biogenic amine histamine is an important neurotransmitter in the central nervous system that has been implicated in learning and memory processes. Experimental evidence indicates that the role of the cerebellum may be more complex than the simple regulation of motor responses, and recent studies have demonstrated significant involvement of the cerebellum in emotional memory consolidation. This study investigated the effect of histamine microinjected into the cerebellar vermis on emotional memory consolidation in mice in the elevated plus-maze (EPM). The cerebellar vermis of male mice (Swiss Albino) were implanted with guide cannulae. The mice weighed between 25 and 30g. After three days of recovery, behavioral tests in the EPM were performed on two consecutive days; the testing periods were called, Trial 1 and Trial 2. Immediately after Trial 1, the animals received microinjections of histamine in the cerebellar vermis (0.54, 1.36, 2.72, and 4.07nmol/0.1μl). On both days, the test sessions were recorded to enable analysis of behavioral measures. The decrease in open arm exploration (% entries and % time spent in the open arms) in Trial 2 relative to Trial 1 was used as a measure of learning and memory. The data were analyzed using One-way Analysis of Variance (ANOVA) and Duncan's tests. The percentage of open arm entries (%OAE) and the percentage of time spent in the open arms (%OAT) were reduced in Trial 2 relative to Trial 1 for the control group; the same was true for the group that was microinjected with histamine at doses of 0.54 (%OAE and %OAT) and 1.36nmol (%OAT). However, when the animals received histamine at doses of 2.72 and 4.07nmol, their open arm exploration did not decrease. No significant changes were observed in the number of enclosed arm entries (EAE), an EPM index of general exploratory activity. These results suggest that there is a dose-dependent inhibitory effect of histamine microinjected into the cerebellar vermis on emotional memory consolidation.

Differential behavioral outcomes of 3,4-methylenedioxymethamphetamine (MDMA-ecstasy) in anxiety-like responses in mice

Anxiolytic and anxiogenic-like behavioral outcomes have been reported for methylenedioxymethamphetamine (MDMA or ecstasy) in rodents. In the present experiment, we attempted to identify behavioral, hormonal and neurochemical outcomes of MDMA treatment to clarify its effects on anxiety-related responses in 2-month-old Balb/c male mice (25-35 g; N = 7-10 mice/group). The behavioral tests used were open field, elevated plus maze, hole board, and defensive behavior against predator odor. Moreover, we also determined striatal dopamine and dopamine turnover, and serum corticosterone levels. MDMA was injected ip at 0.2, 1.0, 5.0, 8.0, 10, or 20 mg/kg. MDMA at 10 mg/kg induced the following significant (P < 0.05) effects: a) a dose-dependent increase in the distance traveled and in the time spent moving in the open field; b) decreased exploratory activity in the hole board as measured by number of head dips and time spent in head dipping; c) increased number of open arm entries and increased time spent in open arm exploration in the elevated plus maze; d) increased time spent away from an aversive stimulus and decreased number of risk assessments in an aversive odor chamber; e) increased serum corticosterone levels, and f) increased striatal dopamine level and turnover. Taken together, these data suggest an anxiogenic-like effect of acute MDMA treatment, despite the fact that behavioral anxiety expression was impaired in some of the behavioral tests used as a consequence of the motor stimulating effects of MDMA.

Identification of short-acting κ-opioid receptor antagonists with anxiolytic-like activity

The κ-opioid receptor plays a central role in mediating the response to stressful life events. Inhibiting κ-opioid receptor signaling is proposed as a mechanism for treating stress-related conditions such as depression and anxiety. Preclinical testing consistently confirms that disruption of κ-opioid signaling is efficacious in animal models of mood disorders. However, concerns about the feasibility of developing antagonists into drugs stem from an unusual pharmacodynamic property of prototypic κ-opioid receptor-selective antagonists; they inhibit receptor signaling for weeks to months after a single dose. Several fundamental questions include — is it possible to identify short-acting antagonists; is long-lasting inhibition necessary for efficacy; and is it safe to develop long-acting antagonists in the clinic. Here, we test representative compounds (AZ-ECPC, AZ-MTAB, and LY-DMPF) from three new chemical series of κ-opioid receptor ligands for long-lasting inhibition. Each compound dose-dependently reversed κ-opioid agonist-induced diuresis. However, unlike the prototypic antagonist, nBNI, which fully inhibited evoked diuresis for at least four weeks, the new compounds showed no inhibition after one week. The two compounds with greater potency and selectivity were tested in prenatally-stressed rats on the elevated plus maze, an exploration-based model of anxiety. Spontaneous exploration of open arms in the elevated plus maze was suppressed by prenatal stress and restored with both compounds. These findings indicate that persistent inhibition is not an inherent property of κ-opioid-selective antagonists and that post-stress dosing with transient inhibitors can be effective in a mood disorder model. This further supports κ-opioid receptor as a promising target for developing novel psychiatric medications.

Differential behavioral outcomes of 3,4-methylenedioxymethamphetamine (MDMA-ecstasy) in anxiety-like responses in mice

Anxiolytic and anxiogenic-like behavioral outcomes have been reported for methylenedioxymethamphetamine (MDMA or ecstasy) in rodents. In the present experiment, we attempted to identify behavioral, hormonal and neurochemical outcomes of MDMA treatment to clarify its effects on anxiety-related responses in 2-month-old Balb/c male mice (25-35 g; N = 7-10 mice/group). The behavioral tests used were open field, elevated plus maze, hole board, and defensive behavior against predator odor. Moreover, we also determined striatal dopamine and dopamine turnover, and serum corticosterone levels. MDMA was injected ip at 0.2, 1.0, 5.0, 8.0, 10, or 20 mg/kg. MDMA at 10 mg/kg induced the following significant (P < 0.05) effects: a) a dose-dependent increase in the distance traveled and in the time spent moving in the open field; b) decreased exploratory activity in the hole board as measured by number of head dips and time spent in head dipping; c) increased number of open arm entries and increased time spent in open arm exploration in the elevated plus maze; d) increased time spent away from an aversive stimulus and decreased number of risk assessments in an aversive odor chamber; e) increased serum corticosterone levels, and f) increased striatal dopamine level and turnover. Taken together, these data suggest an anxiogenic-like effect of acute MDMA treatment, despite the fact that behavioral anxiety expression was impaired in some of the behavioral tests used as a consequence of the motor stimulating effects of MDMA.

Pre-training anandamide infusion within the basolateral amygdala impairs plus-maze discriminative avoidance task in rats

Endocannabinoids (eCBs) modulate a variety of brain functions via activation of the widely expressed CB1 receptor. One site of high density of this receptor is the basolateral amygdala (BLA), a structure involved in the formation of aversive memories. The activation and blockade of CB1 receptors by systemic or hippocampal drug administrations have been shown to modify memory processing. However, little is known about the role of the BLA endocannabinoid system in aversive memories. Additionally, BLA endocannabinoid transmission seems to be related to emotional states, but the relevance of these effects to memory formation is still unknown. In this study we investigated the effects of the eCB anandamide (AEA) and the CB1 antagonist/inverse agonist AM251 infused into the BLA on the acquisition of an aversive memory task, concomitantly evaluating basal anxiety levels in rats. Male rats received pre-training micro-injection of AEA, AM251 or vehicle bilaterally into the BLA, and were studied with the plus-maze discriminative avoidance task (a paradigm that allows concomitant and independent evaluation of anxiety-like behavior and the memory of an aversive task). Our results showed that AEA into the BLA before training prevented memory retrieval 24h later, as evaluated by exploration of the aversive arm of the maze, while AM251 into the BLA did not interfere with animals’ performance. In addition, AEA had no effect on anxiety-like behavior (as evaluated by open arm exploration and risk assessment), while AM251 induced an anxiogenic effect. Our data indicate an important role of BLA CB1 receptors in aversive memory formation, and suggest that this involvement is not necessarily related to a possible modulation of anxiety states.

Basolateral amygdala inactivation impairs learned (but not innate) fear response in rats

Numerous studies have suggested that the amygdala is involved in the formation of aversive memories, but the possibility that this structure is merely related to any kind of fear sensation or response could not be ruled out in previous studies. The present study investigated the effects of bilateral inactivation of the amygdaloid complex in rats tested in the plus-maze discriminative avoidance task. This task concomitantly evaluates aversive memory (by discrimination of the two enclosed arms) and innate fear (by open-arm exploration). Wistar rats (3–5months-old) were implanted with bilateral guide cannulae into basolateral amygdala. After surgery, all subjects were given 1week to recover before behavioral experiments. Afterwards, in experiment 1, 15min prior to training, 0.5μl of saline or muscimol (1mg/ml) was infused in each side via microinjection needles. In experiment 2 the animals received injections immediately after the training session and in experiment 3 rats were injected prior to testing session (24h after training). The main results showed that (1) pre-training muscimol prevented memory retention (evaluated by aversive arm exploration in the test session), but did not alter innate fear (evaluated by percent time in open arms); (2) post-training muscimol impaired consolidation, inducing increased percent in aversive arm exploration in the test session and (3) pre-testing muscimol did not affect retrieval (evaluated by aversive enclosed arm exploration in the test session). The results suggest that amygdala inactivation specifically modulated the learning of the aversive task, excluding a possible secondary effect of amygdala inactivation on general fear responses. Additionally, our data corroborate the hypothesis that basolateral amygdala is not the specific site of storage of aversive memories, since retention of the previously learned task was not affected by pre-testing inactivation.

D-cycloserine enhances memory consolidation in the plus-maze retest paradigm.

Prior undrugged exposure to the elevated plus-maze (EPM) alters future behavioral strategy as well as responsivity to conventional anxiolytic agents. This EPM retest phenomenon appears to be dependent upon learning the spatial configuration of the maze on initial exposure and, in particular, the location of the relatively safe enclosed arms. As posttraining administration of the glycineB receptor partial agonist, D-cycloserine (DCS), has been shown to enhance the consolidation of many forms of memory, we have examined the effects of this compound on the EPM retest effect in male mice. The results of Experiment 1 confirmed that 5 min undrugged exposure to the EPM completed abolishes the anxiolytic efficacy of chlordiazepoxide (CDP; 15 mg/kg) on 24 hr retest. In Experiment 2, posttraining administration of DCS (7.5 and 15 mg/kg), but not CDP (15 mg/kg) or DCS (30 mg/kg), significantly and selectively increased time spent in the enclosed arms (and reciprocally decreased open arm exploration) on 24 hr retest, a finding consistent with an enhancement of consolidation. Experiment 3 used a modified EPM retest protocol to assess the effects of posttraining DCS (15 mg/kg) on behavioral responses to CDP (15 mg/kg) challenge on 24 hr retest. Using a 1-min prior exposure regimen that did not compromise the anxiolytic efficacy of CDP in control mice, the results showed that posttraining administration of DCS abolished the anxiolytic response to CDP challenge. These data strongly suggest that the EPM retest effect involves glycineB/NMDA receptor-dependent neuroplasticity. Further studies will be required to identify the neural circuitry involved.

Executive Functions of Postweaning Protein Malnutrition in Mice

It is well known that nutritional status during the fetal and/or lactation period is important for the development of the central nervous system (CNS). In contrast, the effect of malnutrition on postweaning development has not yet been thoroughly investigated. In the present study, we analyzed the behavioral and neuroanatomical effects of protein malnutrition (PM) postweaning in mice. Starting at 20-21 d of age, male ddY mice were maintained on a 5% casein diet (PM group) or 20% casein diet (control group) for 20 d. On the 20th d, body and brain weights of PM mice were lower than those of the control group. PM mice exhibited excessive alertness and spontaneous activity under novel conditions in the Irwin test. In addition, PM mice showed increased open arm exploration in the elevated plus maze compared to control mice. These results suggest that hyperactivity and reduced anxiety behavior or higher impulsiveness in PM mice could be due to an immature brain.

A Novel Elevated Plus-Maze Procedure to Avoid the One-Trial Tolerance Problem

The elevated plus-maze (EPM) test is one of the most commonly used behavioral assays to evaluate anxiety-related behavior in rodents. It is an economic test (5 min duration) without prior conditioning of the animals. The critical measure for anxiety is the time spent in the open arms of the maze. A confounding problem of the EPM is the so called one-trial tolerance (OTT), characterized by a marked decrease of open arm exploration in spite of treatment with anxiolytic acting benzodiazepines upon re-exposure to the EPM. This consistent finding is often raised as an evidence for the inappropriateness to re-test rodents in the EPM. However, a reliable re-test paradigm would broaden the usability and effectiveness of this test. Therefore, we tested how an extension of the inter-trial interval to 28 days (instead of the usual 24 h), and an additional change of the testing room would affect the open arm time and other behaviors on the EPM. In two experiments, drug-naive Wistar rats were exposed to the EPM on trial 1, and treated intraperitoneally with either vehicle or midazolam (0.25 mg/kg) 30 min before trial 2. Then, trial 2 (28 days after trial 1) was carried out in either the same testing room (Experiment 1) or in another unfamiliar room (Experiment 2). Twenty-eight days after trial 1 the open arm time of the rats in the vehicle treated control rats of both experimental groups was comparable to that of the first trial, independent of the testing room. Most importantly, we found that the treatment with the benzodiazepine midazolam had a significantly anxiolytic-like (i.e., increase of open arm time) effect in trial 2 only when conducted in the previously unfamiliar testing room (Experiment 2). We suggest that in order to reliably re-test the EPM and to prevent confounding effects due to the OTT, an inter-trial interval of 28 days and a change in testing rooms reinstates anxiolytic-like actions of benzodiazepines.

The anxiolytic-like effects of cannabidiol injected into the bed nucleus of the stria terminalis are mediated by 5-HT1A receptors

Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic-like effects in rodents and humans after systemic administration. Previous results from our group showed that CBD injection into the bed nucleus of the stria terminalis (BNST) attenuates conditioned aversive responses. The aim of this study was to further investigate the role of this region on the anxiolytic effects of the CBD. Moreover, considering that CBD can activate 5-HT1A receptors, we also verified a possible involvement of these receptors in those effects. Male Wistar rats received injections of CBD (15, 30, or 60 nmol) into the BNST and were exposed to the elevated plus-maze (EPM) or to the Vogel conflict test (VCT), two widely used animal models of anxiety. CBD increased open arms exploration in the EPM as well as the number of punished licks in the VCT, suggesting an anxiolytic-like effect. The drug did not change the number of entries into the enclosed arms of the EPM nor interfered with water consumption or nociceptive threshold, discarding potential confounding factors in the two tests. Moreover, pretreatment with the 5-HT1A receptor antagonist WAY100635 (0.37 nmol) blocked the effects of CBD in both models. These results give further support to the proposal that BNST is involved in the anxiolytic-like effects of CBD observed after systemic administration, probably by facilitating local 5-HT1A receptor-mediated neurotransmission.

The effects of pentylenetetrazol, chlordiazepoxide and caffeine in rats tested in the elevated plus-maze depend on the experimental illumination

The so-called anxiolytic and anxiogenic drugs are considered to cause, respectively, increases and decreases in plus-maze open arm exploration, without modifying locomotor activity occurring in the closed arms in an elevated plus-maze when the animals are tested in an illuminated environment. Simply testing animals in the dark also increases open arm exploration, which may be interpreted as an anxiolytic effect. We investigated the effects of two GABAergic drugs, pentylenetetrazol (10 and 20 mg/kg) and chlordiazepoxide (1.5 and 3 mg/kg), and one non-GABAergic drug, caffeine (10 and 30 mg/kg) on anxiety levels of rats tested in the elevated plus-maze under two illumination conditions, light or dark. All animals explored more the open arms in the dark. In the light, pentylenetetrazol decreased open arm exploration while chlordiazepoxide had the opposite effect. Neither pentylenetetrazol nor chlordiazepoxide had any effect in the dark. Caffeine, increased open arms exploration in both illumination conditions. These results indicate that light triggers aversion, a response mediated by GABA since the GABAergic drugs, but not caffeine, were ineffective when the rats were tested in the dark.