Role of state-dependency in memory impairment induced by acute administration of midazolam in mice

Although the memory deficits produced by pre-training benzodiazepines administration have been extensively demonstrated both in humans and in animal studies, there is considerable controversy about the involvement of the state-dependency phenomenon on benzodiazepines-induced anterograde amnesia. The present study aimed to characterize the role of state-dependency on memory deficits induced by the benzodiazepine midazolam (MID) in mice submitted to the plus-maze discriminative avoidance task (PM-DAT). This animal model concomitantly evaluates learning and retention of discriminative avoidance task, exploratory habituation as well as anxiety-like behavior and motor activity. Mice received 2mg/kg MID before training and/or before testing in the PM-DAT. Pre-training (but not pre-test) MID administration impaired the retention of the discriminative avoidance task, which was not counteracted by a subsequent pre-test administration of this drug, thus refuting the role of state-dependency. Conversely, the pre-training administration of MID also led to an impairment of the habituation of exploration in the PM-DAT (an animal model of non-associative memory). This habituation deficit was state-dependent since it was absent in pre-training plus pre-test MID treated mice. Concomitantly, MID pre-training administration induced anxiolytic effects and diminished the aversive effectiveness of the aversive stimuli of the task, leading to an impairment of the acquisition of the discriminative avoidance task. Our findings suggest that pre-training benzodiazepine administration can impair the retention of different types of memory by producing specific deleterious effects on learning or by inducing state-dependent memory deficits.     

Coping style and stress hormone responses in genetically heterogeneous rats: comparison with the Roman rat strains

The purpose of the present study was to evaluate for the first time the stress-induced hypothalamus-pituitary-adrenal (HPA), adrenocorticotropic hormone (ACTH), corticosterone and prolactin responses of the National Institutes of Health genetically heterogeneous rat stock (N/Nih-HS rats) in comparison with responses of the relatively high and low stress-prone Roman Low- (RLA-I) and High-Avoidance (RHA-I) rat strains. The same rats were also compared (experiment 1) with respect to their levels of unconditioned anxiety (elevated zero-maze test), novelty-induced exploratory behavior, conditioned fear and two-way active avoidance acquisition. In experiment 2, naive rats from these three strains/stocks were evaluated for “depressive-like” behavior in the forced swimming test. N/Nih-HS and RLA-I rats showed significantly higher post-stress ACTH, corticosterone and prolactin levels than RHA-I rats. N/Nih-HS rats also presented the highest context-conditioned freezing responses, extremely poor two-way avoidance acquisition and very low novelty-induced exploratory behavior. Experiment 2 showed that, compared to RHA-I rats, N/Nih-HS and RLA-I rats displayed significantly less struggling (escape-directed) and increased immobility responses in the forced swimming test. Factor analysis of data from experiment 1 showed associations among behavioral and hormonal responses, with a first factor comprising high loadings of elevated zero-maze variables and lower loadings of conditioned fear, two-way avoidance acquisition and hormonal measures, while a second factor mainly grouped conditioned fear and two-way avoidance acquisition with novelty-induced exploration and post-stress prolactin. Thus, regarding their anxiety/fearfulness, passive coping style, “depressive-like” and stress-induced hormonal responses the N/Nih-HS rats resemble the phenotype profiles of the relatively high-anxious and stress-prone RLA-I rat strain.     

In the rat forced swimming test,chronic but not subacute administration of dual 5-HT/NA antidepressant treatments may produce greater effects than selective drugs

The rat forced swimming test (FST) distinguishes selective serotonin (5-HT) and selective noradrenaline (NA) reuptake-inhibitors, which respectively increase swimming and climbing behaviours. However, NA-system-mediated inhibition of 5-HT-induced swimming prevents dual 5-HT/NA reuptake-inhibition to produce concurrently climbing with swimming. Since adaptative neurochemical processes occur in the treatment of depression, we examined the influence of long-term antidepressant treatment on these interactions. METHODS: (1) Selective [fluoxetine: 10 mg/kg; desipramine: 10 mg/kg] and non-selective [milnacipran: 40 mg/kg; mirtazapine: 20 mg/kg] antidepressants were administered subacutely (3inj) and chronically (17inj) over 16 days. (2) A subacute fluoxetine-desipramine combination (10-10 mg/kg) was administered in rats that were pre-treated with chronic-desipramine (10 mg/kg per day, 14 days). (3) NA-system-mediated interactions were further examined by combining the alpha(2)-receptor agonist clonidine (5, 10, 20, 200 microg/kg) with 10 mg/kg fluoxetine. RESULTS: (1) Long-term treatment with either fluoxetine or desipramine does not modify the behavioural response produced by their subacute administration. (2) In contrast, whereas subacute-milnacipran increases climbing solely, chronic-milnacipran produces greater anti-immobility effects and increases both climbing and swimming behaviours. Similarly, the fluoxetine-desipramine combination produces climbing solely, but increases both climbing and swimming behaviours in animals pre-treated with chronic-desipramine. Chronic but not subacute-mirtazapine increases swimming behaviour. (3) clonidine dose-dependently antagonizes fluoxetine-induced anti-immobility effects and swimming behaviour. CONCLUSIONS: Chronic enhancement of NA-transmission alters NA-system-mediated inhibition of 5-HT-induced behaviour in the FST, which may involve alpha(2)-receptors.     

Concomitant development of oral dyskinesia and memory deficits in reserpine-treated male and female mice

It has been suggested that reserpine-induced oral dyskinesia in rats may provide a new animal model of tardive dyskinesia. Both cognitive deficits and gender have been associated with the development of tardive dyskinesia. The aim of the present study was to investigate the effects of reserpine administration on the development of orofacial dyskinesia and on plus-maze discriminative avoidance task (DAT-an animal model of associative learning) in male and female mice. Male and female mice received 1.0 mg/kg reserpine or saline subcutaneously on day 1. On days 3, 6 and 8, the frequency of vacuous chewing movements (VCM) was quantified. On day 6, the DAT conditioning was performed, in a modified elevated plus-maze. In one of the enclosed arms, the animals received aversive stimulation (light and noise). On day 8, a test session was performed and the time spent by the animals in each of the enclosed arms was recorded. Our results showed that reserpine-treated male and female mice presented significantly higher VCM when compared with respective control groups in all observation days. On day 6, reserpine-treated female mice presented significantly higher VCM when compared with male mice injected with this drug. The DAT test performed on day 8 showed that the time spent in the aversive arm by saline-treated mice was significantly lower than the time spent in the non-aversive arm. This difference was not observed for reserpine-treated mice. Our results demonstrate the development of reserpine-induced oral dyskinesia in both male and female mice. While this oral dyskinesia is accompanied by a cognitive deficit in both genders, female mice tended to have more severe oral dyskinesia. It is suggested that reserpine-induced oral dyskinesia may provide a quick, simple and efficient mouse model of tardive dyskinesia.     

Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats

Despite being potent anxiolytic agents, benzodiazepines (BDZ) sometimes show reduced therapeutic efficacy in stressed rodents. However, the effectiveness of norepinephrine reuptake inhibitors (NRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) or other anxiolytic interventions, e.g., exercise, remained elusive. Here, we demonstrated that male rats subjected to restraint stress for 4 weeks showed decreases in percent open arm time and open arm entry, as determined by elevated plus-maze test (EPM). Increases in inhibitory avoidance trial 2 and outer zone time were also observed in elevated T-maze (ETM) and open field test (OFT), respectively. To evaluate the anxiolytic-like actions of exercise and anxiolytic drugs, stressed rats were subjected for 4 weeks to swimming or daily gavage with 2mg/kg diazepam (BDZ), or 10mg/kg fluoxetine (selective serotonin reuptake inhibitor), reboxetine (NRI), or venlafaxine (SNRI). In EPM, the open arm activity was higher in the swimming, reboxetine-treated and venlafaxine-treated groups as compared to age-matched controls, while diazepam and fluoxetine were without effect. In ETM, a reduction in avoidance latency was observed only in swimming and venlafaxine-treated groups. However, the combined swimming and pharmacological treatment showed no additive anxiolytic-like effect. It could be concluded that restraint stress induced anxiety-like behaviors, which were not responsive to diazepam or fluoxetine, whereas reboxetine, venlafaxine and swimming showed anxiolytic-like actions in stressed rats.     

Antidepressant effects of pramipexole, a novel dopamine receptor agonist

Summary Pramipexole (2-amino-4,5,6,7-tetrahydro-6-propyl-amino-benzthiazole-dihydrochloride), a new dopamine receptor agonist with preference for D₃ compared to D₂ and D₄ receptors, was tested in rats in respect of its potential antidepressant activity. In the forced swimming test the drug under study, given three times in rats, reduced the immobility time. In the forced swimming test, joint treatment with antidepressants (imipramine, amitriptyline) and pramipexole evoked a more potent effect than any of the drugs given alone; however, the locomotor hyperactivity was weaker after joint administration. Citalopram and fluoxetine, inactive per se in the forced swimming tests, visibly enhanced the antidepressant-like effect of pramipexole but, on the other hand, they attenuated the locomotor hyper-activity evoked by the drug. Repeated treatment with pramipexole (0.3 or 1 mg/kg, twice daily for 14 days) increased the locomotor activity measured at 1h after the last dose. Repeated administration of pramipexole (as above) potentiated the D-amphetamine- or quinpirole-induced locomotor hyperactivity.The obtained results indicate that, in the tests used, pramipexole evokes effects similar to those of typical antidepressants and, at the same time, enhances their activity (the forced swimming test in rats); therefore it may be regarded as a potential antidepressant drug.     

Effects of reserpine on the plus-maze discriminative avoidance task: dissociation between memory and motor impairments

We investigated the effects of reserpine (0.1-0.5 mg/kg) on the performance of mice in the plus-maze discriminative avoidance task (DAVT), which simultaneously evaluates memory and motor activity. All doses induced memory impairment (increased aversive arm time) but only 0.5 mg/kg reserpine decreased locomotion (entries in enclosed arms). The results suggest that the DAVT evaluation in reserpine-treated mice can be a useful model for studying cognitive deficits accompanied by motor impairments.     

Isolation rearing induced fear-like behavior without affecting learning abilities of Wistar rats.

1. Isolation-reared rats display fear-like behavior and depressive-like behavior in several behavioral tasks, suggesting that isolation rearing may model certain aspects of human psychopathologies. 2. After weaning (20 days old), male and female Wistar rats were isolation-reared during 20, 50 or 70 days. After that, they were tested in the elevated plus maze test, and in the open field test. Another group of isolation-reared rats (70 days of isolation) were tested in an auto-shaping task. 3. Isolation-reared rats displayed high levels of fear-like behavior in the elevated plus-maze test, and hyperlocomotion in the open field test. But, isolation-reared rats learned an auto-shaping task. 4. In conclusion, isolation rearing induced fear-like behavior, without affect learning abilities of rats.     

Acute and long-term consequences of single MDMA administration in relation to individual anxiety levels in the rat

Our previous work has shown that normal male Wistar rats can differ systematically in their behavioral response to the elevated plus-maze (EPM), where animals with high (HA) or low anxiety (LA) levels can be identified based on the percentage of time spent in the open arms. These animals also differ in other behavioral tests (e.g. active avoidance), and in their serotonin levels in the ventral striatum. Here, we tested whether such HA and LA rats might respond differently to the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). This drug can affect psychomotor activation and anxiety; effects which are probably due to its pronounced serotonergic and dopaminergic impacts in the rat brain. Based on a routine screening procedure in the plus-maze, male Wistar rats were divided into HA and LA sub-groups, in which rectal temperature was measured. Thirty minutes after the i.p. injection of MDMA (7.5 or 15 mg/kg) or vehicle, they were again tested in the plus-maze. During the next 3 weeks, the animals underwent further behavioral tests (plus-maze, open field, active avoidance, forced swimming) to test for possible long-term consequences of MDMA. Rectal temperature was found to be higher in LA than HA rats and was especially increased with the higher dose of MDMA (15 mg/kg). In the acute plus-maze test, the lower dose of MDMA led to an anxiogenic-like profile, whereas the higher dose led to an anxiolytic-like profile, both in HA and LA rats. Possible long-term consequences of MDMA were only tested with 7.5 mg/kg MDMA, since the 15 mg/kg dose led to a high level of lethality. The analysis of open field, plus-maze (performed after 9-12 days), and forced swimming behavior (performed after 20-21 days) did not provide indications for lasting effects of MDMA. In contrast, active avoidance learning was impaired in LA- but not HA-rats treated with MDMA. A single injection of MDMA does not only have acute effects on anxiety and psychomotor activation, but can also have some prolonged or delayed task-dependent behavioral consequences. The detection of such sequels can require that individual differences are taken into account and here, determining anxiety levels in the EPM seems to serve as a useful approach.     

Sleep deprivation impairs emotional memory retrieval in mice: influence of sex

The deleterious effects of paradoxical sleep deprivation on memory processes are well documented. However, non-selective sleep deprivation occurs more commonly in modern society and thus represents a better translational model. We have recently reported that acute total sleep deprivation (TSD) for 6 h immediately before testing impaired performance of male mice in the plus-maze discriminative avoidance task (PM-DAT) and in the passive avoidance task (PAT). In order to extend these findings to females, we examined the effect of (pre-test) TSD on the retrieval of different memory tasks in both male and female mice. Animals were tested using 3 distinct memory models: 1) conditioning fear context (CFC), 2) PAT and 3) PM-DAT. In all experiments, animals were totally sleep-deprived by the gentle interference method for 6h immediately before being tested. In the CFC task and the PAT, TSD induced memory impairment regardless of sex. In PM-DAT, the memory impairing effects of TSD were greater in females. Collectively, our results confirm the impairing effect of TSD on emotional memory retrieval and demonstrate that it can be higher in female mice depending on the memory task evaluated.     

The plus-maze discriminative avoidance task: a new model to study memory-anxiety interactions. Effects of chlordiazepoxide and caffeine

The plus-maze discriminative avoidance paradigm is a new animal model of learning/memory that provides simultaneous information about anxiety. Mice are conditioned to choose between the two enclosed arms (in one of which light and noise are presented as aversive stimuli) while avoiding the two open arms of the apparatus. The test has the advantage of measuring, at the same time and in the same animals, learning/memory (by the percent time spent in aversive enclosed arm - PTAV) and anxiety (by the percent time spent in the open arms - PTO). The effects of chlordiazepoxide and caffeine on learning/memory and anxiety of mice tested in this paradigm were investigated. Chlordiazepoxide (5 mg/kg) significantly increased and caffeine (20 mg/kg) significantly decreased PTO during the training session, suggesting an anxiolytic and an anxiogenic effect, respectively. In the test session, chlordiazepoxide- or caffeine-treated mice presented higher PTAV, suggesting amnestic effects. Given together, chlordiazepoxide plus caffeine did not alter PTO, and the amnesic effect produced by each drug was no longer observed. It is concluded that learning/memory depends on an optimum emotional level. The plus-maze discriminative avoidance model appears to be a useful test to investigate this critical relationship between learning/memory and anxiety.     

l-histidine induces state-dependent memory deficit in mice mediated by H1 receptor

This study investigated the role of H₁ receptor in the state-dependent memory deficit induced by l-histidine (LH) in mice using Trial 1/2 protocol in the elevated plus-maze (EPM). The test was performed for two consecutive days: Trial 1 (T1) and Trial 2 (T2). Before both trials, mice received a combined injection i.p. of saline + saline (SAL/SAL), 500 mg/kg l-histidine + saline (LH/SAL), 500 mg/kg l-histidine + 16 mg/kg chlorpheniramine (LH/CPA) or saline + 16 mg/kg chlorpheniramine (SAL/CPA). The trials were performed in the EPM 10 min after the last injection. Each animal was placed in the center of the maze facing the open arm and had five minutes to explore it. On both days, test sessions were videotaped. The behavioral measures were scored from videotape. Data were analyzed based on Analysis of Variance (ANOVA) and the Fisher’s LSD test. The data showed no effects on anxiety since there was no difference between the SAL/SAL and the other groups in Trial 1, respectively, open arm entries (OAE), open arm time (OAT) and their percentages (%OAE and %OAT). During Trial 2, OAE, OAT, %OAE and %OAT were reduced in mice treated with SAL/SAL, LH/CPA and SAL/CPA, while the group LH/SAL did not show any difference in these measures. No significant changes were observed in enclosed arm entries (EAE), an EPM index of general exploratory activity. Thus, it can be suggested that LH induces emotional memory deficit and the treatment with chlorpheniramine was able to revert this effect, suggesting this action of LH was mediated by the H₁ receptor.     

Spironolactone and low-dose dexamethasone enhance extinction of contextual fear conditioning

Glucocorticoids play a role in memory formation, and they may contribute to memory changes in stress-related mental disorders, such as posttraumatic stress disorder. Cortisol may act through mineralocorticoid (MR) or glucocorticoid (GR) receptors, and the objective of the present study was to evaluate the effects of the MR antagonist spironolactone, the GR antagonist mifepristone, the MR agonist fludrocortisone, and the GR agonist dexamethasone on the extinction of contextually conditioned fear in rats. Propranolol was used as a positive control. As expected, propranolol administered before the test session increased memory extinction. Pre-test administration of spironolactone and low-dose dexamethasone also increased the extinction of an aversive memory, whereas fludrocortisone impaired extinction. High-dose dexamethasone and mifepristone were found to have no effect in this model. Post-test spironolactone treatment impaired aversive memory extinction. These results indicate that MR and GR are related to extinction of aversive memories, and MR blockade may be a promising candidate for the treatment of stress-related memory disorders.     

LPS inhibits the effects of fluoxetine on depression-like behavior and hippocampal neurogenesis in rats

Depressed patients with increased inflammatory cytokines in peripheral blood have been reported to be more likely to exhibit treatment resistance. However, it is unknown whether the inflammation influences the action of antidepressant drugs. Here, we investigated the influence of lipopolysaccharide (LPS) on the antidepressant action of fluoxetine in depressive rats induced by chronic unpredictable mild stress (CUMS). In this study, we first modified the CUMS paradigm by administration of LPS daily before the stressor, and then investigated the influence of inflammation on the antidepressant action of fluoxetine. The effects of stress exposure and antidepressant treatment were assessed by behavioral testing (sucrose preference test, forced swimming test, novelty suppressed feeding test) and hippocampal BrdU labeling. The CUMS-induced behavioral changes can be reversed by 4-week fluoxetine treatment. Fluoxetine also increased the hippocampal neurogenesis in the depressive rats. Pretreatment with LPS, to mimic inflammation, had no significant effect on depressive behavior but attenuated the antidepressant action of fluoxetine significantly. Thus, our results suggest that the inflammation might play a certain role in the pathophysiology of antidepressant treatment resistance.     

Sexually dimorphic responses of rats to fluoxetine in the forced swimming test are unrelated to the function of the serotonin transporter in the brain

Due to the prevalence of depression in women, female rats may be a better models for antidepressant research than males. In male rats, fluoxetine inhibited the serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) which is reducing the immobility time in the repeated forced swimming test (rFST). The performance of female rats in this test is unknown. In this study, responses of male and female rats in the rFST under chronic treatment with fluoxetine and the function of SERT in their brains were examined. Wistar rats received oral fluoxetine (females: 0, 1, 2.5, or 5 mg kg^-1 day^-1; males: 0 or 2.5 mg kg^-1 day^-1; in sucrose 10%, 1.5 ml/rat) 1 hr before the test daily for 12 days over the course of the rFST. rFST consisted of a 15 min pretest followed by 5 min sessions of swimming at 1 (test), 7 (retest 1), and 14 (retest 2) days later. SERT functioning was assessed by ex vivo assays of the frontal cortex and hippocampus of rats. Fluoxetine reduced immobility time of males in the rFST while it failed to do so in females. In vitro treatment with fluoxetine inhibited the uptake of 5-HT of both sexes similarly, while in vivo chronic administration of fluoxetine failed to do so. In summary, rats responded to the chronic treatment with fluoxetine in a sexually dimorphic fashion during the rFST despite the functioning of SERT in their brains remaining equally unchanged. Hence, our data suggest that sexually dimorphic responses to fluoxetine in rFST may be unrelated to the function of SERT in rat brains.     

Chronic repetitive transcranial magnetic stimulation is antidepressant but not anxiolytic in rat models of anxiety and depression

Transcranial magnetic stimulation (TMS) has been proposed as a treatment for depression and anxiety disorders. While the antidepressant effect has been modelled in animals, there have been few attempts to examine a possible anxiolytic effect of repetitive TMS (rTMS) in animal models. We administered 18 days of rTMS to male Sprague-Dawley rats. On days 10 through 18, rats were tested in several anxiety models (social interaction, emergence, elevated plus-maze, and predator odor avoidance) and in the forced swim test. No group differences were apparent on any of the anxiety models, while TMS produced an antidepressant effect in the forced swim test. Interestingly, on day 1 of the forced swim test, the home cage control group displayed increased swimming behaviour compared with sham-treated animals, suggesting an observable level of stress may have accompanied sham treatment. The results from the forced swim test suggested that TMS had modest antidepressant properties, but it did not show anxiolytic properties in the models examined. The study also suggested that stress associated with handling should be taken into account in the interpretation of TMS studies in animals.     

Dissociations in dopamine release in medial prefrontal cortex and ventral striatum during the acquisition and extinction of classical aversive conditioning in the rat

Dual perfusion in vivo brain microdialysis was used to monitor extracellular levels of dopamine in the medial prefrontal cortex and ventral striatum during the acquisition and extinction of a classical aversive conditioning paradigm in rats. The main finding was a dissociation in the pattern of release in the two brain areas. The first stimulus–footshock pairing elicited large increases in cortical dopamine over baseline levels that were much greater than the increases elicited by different stimuli of equivalent salience that were unpaired with footshock. In contrast, dopamine levels in ventral striatum were unchanged under these conditions. Over the next two pairings, there was a decline in the cortical response and an increase in the response in ventral striatum. The first presentation of the aversive conditioned stimulus in a separate context elicited the largest response in ventral striatum. Post-conditioning, the cortical response to the conditioned stimulus was smaller than that elicited by the initial stimulus–footshock pairing and was equivalent in magnitude to that elicited by stimuli unpaired with footshock. Over the final two conditioned stimuli presentations, in the absence of the footshock reinforcer (extinction), responses declined in both brain areas. Simultaneous monitoring of behaviour indicated that the neurochemical events were accompanied by effective aversive learning, as indexed by conditioned freezing responses. The data are discussed in terms of the hypothesis that medial prefrontal cortex is especially engaged during novel circumstances which may, potentially, require new learning, whilst ventral striatal dopamine more closely follows the expression of conditioned responding during learning and extinction.     

Acute and chronic anxiogenic-like response to fluoxetine in rats in the elevated plus-maze: modulation by stressful handling

While antidepressants are widely prescribed to humans for the treatment of anxiety, the results achieved with animal anxiety models are conflicting. The experimental procedure and the prior test history of the animals are critical parameters that are largely susceptible to influence the results and their interpretation. We compared the effect of 5mg fluoxetine administered to six groups of rats subjected to the psychopharmacological test of the elevated plus-maze, under experimental conditions designed to demonstrate the effect of handling and one daily injection on the response to fluoxetine. The results show that for animals with the same recent experience, fluoxetine, when administered once or over a period of 15 days, induces anxiogenic-like behaviour. On the other hand, our results also show that stressful handling has an anxiolytic-like effect modulating the anxiogenic-like effect of fluoxetine, without eliminating it altogether.