Influence of gender and estrous cycle in the forced swim test in rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of gender and estrous cycle in the forced swim test in rats

The present work aimed at studying the influence of the estrous cycle in the forced swim test, an animal model of depression. For this, 44 male and female Wistar rats were divided into five groups according to the hormonal state in the first day of the study: metaestrus (N = 12), diestrus (N = 8), proestrus (N = 7), estrous (N = 6) and males (N = 11). They were housed in groups of five, with water and food ad libitum under a 12/12 h light/dark cycle. Females were screened daily for the estrous cycle. The animals were subjected to two swimming sessions in a glass cylinder with water up to 15 cm at 28±2º C. The data of the first five minutes of a 15-min first session were compared to those of a 5-min second session 24 h later. The results indicate that the latency to the first immobility was substantially reduced in the second session and was longer for females in diestrus and proestrus in the first session. The results also indicate that females in diestrus and proestrus exhibited less immobility than males in the first session; females in diestrus also exhibited less immobility than females in metaestrus. Females in metaestrus and diestrus, as well as males, did not present the decrease in total immobility times in the second session. The present results are analyzed in terms of differential effects of progesterone and estrogen on a learning component and an affective component.

Estrogen alters behavior and forebrain c-fos expression in ovariectomized rats subjected to the forced swim test

Estrogen has been implicated in brain functions related to affective state, including hormone-related affective disorders in women. Although some reports suggest that estrogen appears to decrease vulnerability to affective disorders in certain cases, the mechanisms involved are unknown. We used the forced swim test (FST), a paradigm used to test the efficacy of antidepressants, and addressed the hypotheses that estrogen alters behavior of ovariectomized rats in the FST and the FST-induced expression of c-fos, a marker for neuronal activity, in the rat forebrain. The behaviors displayed included struggling, swimming, and immobility. One hour after the beginning of the test on day 2, the animals were perfused, and the brains were processed for c-fos immunocytochemistry. On day 1, the estradiol benzoate-treated animals spent significantly less time struggling and virtually no time in immobility and spent most of the time swimming. Control rats spent significantly more time struggling or being immobile during a comparable period. On day 2, similar behavioral patterns with still more pronounced differences were observed between estradiol benzoate and ovariectomized control groups in struggling, immobility, and swimming. Analysis of the mean number of c-fos immunoreactive cell nuclei showed a significant reduction in the estradiol benzoate versus control groups in areas of the forebrain relating to sensory, contextual, and integrative processing. Our results suggest that estrogen-induced neurochemical changes in forebrain neurons may translate into an altered behavioral output in the affective domain.

Noradrenergic neurotransmission within the bed nucleus of the stria terminalis modulates the retention of immobility in the rat forced swimming test

The bed nucleus of the stria terminalis (BNST) is a limbic structure that has a direct influence on the autonomic, neuroendocrine, and behavioral responses to stress. It was recently reported that reversible inactivation of synaptic transmission within this structure causes antidepressant-like effects, indicating that activation of the BNST during stressful situations would facilitate the development of behavioral changes related to the neurobiology of depression. Moreover, noradrenergic neurotransmission is abundant in the BNST and has an important role in the regulation of emotional processes related to the stress response. Thus, this study aimed to test the hypothesis that activation of adrenoceptors within the BNST facilitates the development of behavioral consequences of stress. To investigate this hypothesis, male Wistar rats were stressed (forced swimming, 15 min) and 24 h later received intra-BNST injections of vehicle, WB4101, RX821002, CGP20712, or ICI118,551, which are selective α1, α2, β1, and β2 adrenoceptor antagonists, respectively, 10 min before a 5-min forced swimming test. It was observed that administration of WB4101 (10 and 15 nmol), CGP20712 (5 and 10 nmol), or ICI118,551 (5 nmol) into the BNST reduced the immobility time of rats subjected to forced swimming test, indicating an antidepressant-like effect. These findings suggest that activation of α1, β1, and β2 adrenoceptors in the BNST could be involved in the development of the behavioral consequences of stress. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Galanin N-Terminal fragment (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT in the Forced Swimming Test.

Galanin and Galanin (1-15) [GAL(1-15)] are implicated in anxiety- and depression related behaviors. Moreover, Galanin modulates 5-HT1A receptor (5-HT1AR) function at autorreceptor and postsynaptic level in the brain. In this study, we have analysed the ability of GAL(1-15) to modulate the effects of the 8-OH-DPAT agonist in the Forced Swimming Test (FST). Groups of rats were assessed in the FST. In the first set of experiments, to evaluate the interactions of 8-OH-DPAT and GAL(1-15), rats received subcutaneously (s.c) the effective doses of 8-OH-DPAT (0.25mg/Kg) 60min before the test and intracerebroventricularly (icv) GAL(1-15)1nmol 15min before the tests alone or in combination. In the second set of experiments, groups of rats received s.c. 8-OH-DPAT (0.125mg/Kg), icv GAL(1-15) 1nmol and icv the GALR2 antagonist M871 3 nmol alone or in combination. The locomotor activity was analysed in the open field test. GAL(1-15) 1nmol enhanced the antidepressant-like effects mediated by the effective dose of the 8-OH-DPAT. GAL(1-15) significantly decreased the immobility (p<0.05) and climbing (p<0.05) and increased the swimming (p<0.01) behaviour induced by an effective dose of 8-OH-DPAT (0.25mg/Kg) in FST. Moreover, after coadministration of GAL(1-15) and threshold dose of 8-OH-DPAT (0.125mg/Kg) a significant decreased appeared in immobility (p<0.01) and climbing (p<0.01) and increased the swimming behavior (p<0.001) vs 8-OH-DPAT group. Moreover, M871 blocked completely this interaction. These results indicate that GAL(1-15) enhances the antidepressant effects induced by 8-OH-DPAT in the FST. These findings may give the basis for the development of novel therapeutic drugs. This study was supported by Junta de Andalucía CVI6476.

The Effects of Insulin-Induced Moderate Hypoglycemia on Hippocampal Plasticity

Self-regulation of blood glucose in diabetics via insulin administration introduces the risk of hypoglycemia. Previous studies have shown hypoglycemia damages the dentate gyrus, an area of the hippocampus associated with anxiety- and depressive-like behavior. To date, only depressive-like behaviors have been observed following moderate hypoglycemia. This study sought to examine whether acute moderate hypoglycemia induces both behaviors due to high clinical comorbidity. One episode of moderate hypoglycemia was induced in a male Sprague-Dawley rat. Twenty-four hours later, hippocampal function was evaluated via the elevated plus maze and the forced swim test to assess anxiety-like and depressive-like behavior. Results, though not statistically significant, suggested that acute moderate hypoglycemia may increase anxiety- and depressive-like behavior. These findings may elucidate hypoglycemia-related behavioral changes.

Immediate and enduring effects of corticosterone administration during adolescence and adulthood on anxiety and depressive behavior in male rats

Previous research has shown that the stress hormone corticosterone can increase depressive and anxiety-like behavior in rats as well as dampen the HPA response to a novel stressor (Kalynchuk et aI., 2004; Johnson et aI., 2006). Several studies have also shown that adolescence is a period of increased sensitivity to the negative effects of stressors (reviewed in McCormick et aI., 2010), which are often the result of exposure to corticosterone, and yet there is no research to date examining the effects of corticosterone administration during adolescence. The purpose of these experiments is to determine both the immediate and enduring effects of prolonged exposure to corticosterone in adolescence and adulthood on anxiety-like behavior, depressive behavior, and the HPA response. In Experiment 1 adolescent and adult rats were administered an injection of 40 mg/kg of corticosterone or vehicle daily for 16 days. Ha l f of the rats were then tested on the elevated plus maze (EPM) one day after their last injection, and the following day were tested on the forced swim test (FST). After the FST, which is a stressor, blood samples were collected at three time points, and the plasma concentrations of corticosterone were determined using a radioimmunoassay. The remaining rats were left undisturbed for three weeks, and then underwent the same testing as the first group. Corticosterone treatment had little effect on anxiety-like and depressive behavior, but it did alter the HPA response to the FST. In those rats tested soon after the period of injections, corticosterone dampened the HPA response as compared to vehicle treated rats in both adolescent and adult treated rats. For the adolescent treated rats that were tested several weeks later, corticosterone treatment increased HPA response as compared to the vehicle treated rats, but the same was not true for the adult treated rats. I t was hypothesized that the lack of behavioral effects of the corticosterone treatment may be the result of the vehicle injections inducing a stress response and thereby both groups would have similarly altered behavior. In Experiment 2 rats were administered corticosterone dissolved in their drinking water with 2.5% ethanol, or jus t the 2.5% ethanol or plain water, to determine the effects of corticosterone treatment without a stressor present. The regular drinking water was replaced with treated water for 16 days either during adulthood or adolescence, and as before, rats were either tested in the FST one day after the water was removed or three weeks later. Again there was no effect of treatment on depressive behavior. Similar to what was observed in Experiment 1, corticosterone treatment dampened the HPA response to a stressor for the rats tested soon after the treatment period. However, in Experiment 2 there was no effect of treatment on HPA response in those rats tested several weeks after they were treated. These results indicate that corticosterone can have a lasting effect on the HPA when administered in adolescence by injections but not in drinking water, which is likely because of the different schedules of exposure and rates of absorption between the two administration methods.

Atténuation de la dépression post-infarctus par une diète riche en oméga-3 ou par la prise de probiotiques

L'Infarctus du myocarde (IM) provoque, chez le rat, une augmentation de l'apoptose dans le système limbique en plus d'induire des symptômes qui s'apparentent à la dépression chez l'humain. Nous avons démontré qu'une diète élevée en oméga-3 ou la prise de probiotiques pouvaient être efficaces pour réduire ces effets si ces interventions débutaient avant l'induction de l'ischémie myocardique. Cette étude a pour objectif de déterminer l'efficacité de ces interventions si elles débutent après l'ischémie myocardique. L’IM a été induit chez le rat mâle Sprague-Dawley par l’occlusion de l’artère coronaire descendante antérieure gauche pendant 40 minutes. À la suite de l’ischémie, les rats ont reçu des probiotiques (1 billion de bactéries vivantes de L. helveticus R0052 et de B. longum R0175) ou un véhicule dans leur eau de boisson en présence d'une diète élevée ou faible en oméga-3. À 3 jours post-IM, l’activité enzymatique de la caspase-3 et le nombre de cellules dUTP nick-end labelling (TUNEL) positives sont diminués dans les régions CA1 et le corps godronné de l’hippocampe ainsi que dans l’amygdale en présence de la diète élevée en oméga-3. La prise de probiotiques atténue également l’activité de la caspase-3 et le nombre de cellules TUNEL positives dans le corps godronné et l’amygdale médiane. À 2 semaines post-IM, le comportement dépressif évalué par 3 tests comportementaux (test d’interaction sociale, test de nage forcée et test d’évitement passif) a été observé chez le groupe recevant la diète faible en oméga-3 sans probiotiques et le comportement dépressif a été atténué avec la diète élevée en oméga-3 et/ou la prise de probiotiques. Les probiotiques ont augmenté les niveaux plasmatiques d’interleukine-4 (IL- 4) tandis que la diète élevée en oméga-3 a montré une diminution de la protéine chimiotactique monocytaire 1 (MCP-1). Ces résultats indiquent qu’une diète élevée en oméga-3 ou la prise de probiotiques, débutant à la suite de l’IM, s’avèrent bénéfiques pour atténuer la dépression et l’apoptose dans le système limbique.

Maternal weaning modulates emotional behavior and regulates the gut-brain axis

Evidence shows that nutritional and environmental stress stimuli during postnatal period influence brain development and interactions between gut and brain. In this study we show that in rats, prevention of weaning from maternal milk results in depressive-like behavior, which is accompanied by changes in the gut bacteria and host metabolism. Depressive-like behavior was studied using the forced-swim test on postnatal day (PND) 25 in rats either weaned on PND 21, or left with their mother until PND 25 (non-weaned). Non-weaned rats showed an increased immobility time consistent with a depressive phenotype. Fluorescence in situ hybridization showed non-weaned rats to harbor significantly lowered Clostridium histolyticum bacterial groups but exhibit marked stress-induced increases. Metabonomic analysis of urine from these animals revealed significant differences in the metabolic profiles, with biochemical phenotypes indicative of depression in the non-weaned animals. In addition, non-weaned rats showed resistance to stress-induced modulation of oxytocin receptors in amygdala nuclei, which is indicative of passive stress-coping mechanism. We conclude that delaying weaning results in alterations to the gut microbiota and global metabolic profiles which may contribute to a depressive phenotype and raise the issue that mood disorders at early developmental ages may reflect interplay between mammalian host and resident bacteria.

Estrogens, brain and behavior: studies in fundamental neurobiology and observations related to women's health

Mechanisms and consequences of the effects of estrogen on the brain have been studied both at the fundamental level and with therapeutic applications in mind. Estrogenic hormones binding in particular neurons in a limbic-hypothalamic system and their effects on the electrophysiology and molecular biology of medial hypothalamic neurons were central in establishing the first circuit for a mammalian behavior, the female-typical mating behavior, lordosis. Notably, the ability of estradiol to facilitate transcription from six genes whose products are important for lordosis behavior proved that hormones can turn on genes in specific neurons at specific times, with sensible behavioral consequences. The use of a gene knockout for estrogen receptor alpha (ERalpha) revealed that homozygous mutant females simply would not do lordosis behavior and instead were extremely aggressive, thus identifying a specific gene as essential for a mammalian social behavior. In dramatic contrast, ERbeta knockout females can exhibit normal lordosis behavior. With the understanding, in considerable mechanistic detail, of how the behavior is produced, now we are also studying brain mechanisms for the biologically adaptive influences which constrain reproductive behavior. With respect to cold temperatures and other environmental or metabolic circumstances which are not consistent with successful reproduction, we are interested in thyroid hormone effects in the brain. Competitive relations between two types of transcription factors - thyroid hormone receptors and estrogen receptors have the potential of subserving the blocking effects of inappropriate environmental circumstances on female reproductive behaviors. TRs can compete with ERalpha both for DNA binding to consensus and physiological EREs and for nuclear coactivators. In the presence of both TRs and ERs, in transfection studies, thyroid hormone coadministration can reduce estrogen-stimulated transcription. These competitive relations apparently have behavioral consequences, as thyroid hormones will reduce lordosis, and a TRbeta gene knockout will increase it. In sum, we not only know several genes that participate in the selective control of this sex behavior, but also, for two genes, we know the causal routes. Estrogenic hormones are also the foci of widespread attention for their potential therapeutic effects improving, for example, certain aspects of mood and cognition. The former has an efficient animal analog, demonstrated by the positive effects of estrogen in the Porsolt forced swim test. The latter almost certainly depends upon trophic actions of estrogen on several fundamental features of nerve cell survival and growth. The hypothesis is raised that the synaptic effects of estrogens are secondary to the trophic actions of this type of hormone in the nucleus and nerve cell body.

Anabolic-androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice

Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly, the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion, high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

Investigating the Effects of Mifepristone (RU486) in the Behavioral, Hormonal and Central Responses to Acute Stress

Depression is associated with glucocorticoid hypersecretion, due to dysfunction of the hypothalamo-pituitary-adrenocorticol axis (HPA-axis). Because excess glucocorticoids are associated with depressive-like features in humans, glucocorticoid receptor antagonists are currently being tested for antidepressant efficacy in clinical trials. In the current study the hypothesis that mifepristone (RU486), a glucocorticoid receptor antagonist, would decrease the neuroendocrine and central HPA-axis responses to an acute stressor and attentuate depressive like behavior in an animal model of behavioral helplessness (forced swim test) was tested. Adult male rats were treated with 10 mglkg RU486 (subcutaneous) for five days and then exposed to a IO-minute forced swim test (FST), conducted in Plexiglas cylinders. FST sessions were videotaped for later analysis of behavioral immobility. Plasma ACTH and corticosterone CORT were measured at 15min and 90min after FST cessation. Animals were perfused and brains were collected for immunocytochemical assessment of c-Fos expression in the medial prefrontal cortex (mPFC), a brain region implicated in both depression and central control of the HPA axis. RU486 significantly decreased peak ACTH and CORT concentrations following FST exposure. In addition, glucocorticoid negative feedback was at1enuated in RU486-treated animals exposed to the FST. Exposure to FST alone induced c-FOS expression in the mPFC, as measured by the number of c-Fos positive neurons. Treatment with RU486 significantly increased the number of rnPFC c-Fos positive cell following FST exposure. The behavioral data obtained from FST paradigm, demonstrated that RU486 decreased immobility in the FST illustrating the potential efficacy of this drug as an antidepressant. Collectively these data suggest that RU486 dampens HPA-axis responses to stress, possibly by enhancing the excitability of stress-inhibitory neurons in the mPFC. This is particularly exciting, given the fact that this neural region is associated with decreased neural activity during depression in humans.

Peripheral proinflammatory markers associated with ketamine response in a preclinical model of antidepressant-resistance

© 2015 Elsevier B.V. Ketamine, N-methyl- d-aspartate (NMDA) receptor antagonist and anti-inflammatory agent, has rapid therapeutic effects in a subset of patients with more intractable forms of depression. Irregular proinflammatory cytokine and acute-reactive protein levels have been reported in clinical and preclinical depression research. We explored the association between the rapid antidepressant-like effects of ketamine and peripheral proinflammatory profile in a model of antidepressant-resistance. Male Wistar rats were pre-treated with ACTH-(1-24) 100. μg/d or saline (0.9%) for 14. d. Antidepressant-like effects were assessed with the forced swim test (FST). Ketamine (10. mg/kg) significantly reduced immobility duration in saline-pretreated control animals. In contrast, a divergent response was observed in ACTH-pretreated antidepressant resistant animals, with 50% responders and 50% non-responders. Plasma samples were analyzed via enzyme-linked immunosorbent assay (ELISA) for interleukin 6 (IL-6), tumour necrosis factor alpha (TNFα) and C-reactive protein (CRP). Levels of CRP and TNFα differentiated ketamine responders and non-responders.

Avaliação comportamental e neuroquímica de ratos em dessincronização forçada: possíveis implicações para um modelo animal de oscilações no humor

Bipolar disorder has been growing in several countries. It is a disease with high mortality and has been responsible by the social isolation of the patients. Bipolar patients have alterations in circadian timing system, showing a phase shift in various physiological variables. There are several arguments demonstrating alterations in circadian rhythms may be part of the bipolar disorder pathophysiology. Given the necessity for further elucidation, the goal of this study was to validate the forced desynchronization protocol as an animal model for bipolar disorder. To do this, Wistar rats were submitted to a forced desynchronization protocol which consists in a symmetrical light dark cycle with 22h. Under this protocol, rats dissociate the locomotor activity rhythm into two components: one synchronized to the light / dark cycle with 22h, and another component with period longer than 24 hours following the animal endogenous period. These rhythms with different periods sometimes there is coincidence, which we named CAP (Coincidence Active Phase) and the opposite phase, non-coincidence, called NCAP (Non-Concidence Active Phase). The hypothesis is that in CAP animals present a mania-like behavior and animals in NCAP depressive-like behavior. We found some evidence described in detail throughout this thesis. In sum, the animals under forced desynchronization protocol were more stressed, showed an increase in stereotypic behaviors such as grooming and reduction in other behaviors such as risk assessment and vertical exploration when compared to the control group. The CAP animals showed increased locomotor activity, especially during the dark phase when compared to controls (rats under T24) and less depressive behavior in the forced swim test. The animals in NCAP showed a higher anxiety in elevated plus maze, but they don t have ahnedonia. The animals under dissociation have more labeled 5HT1A cells at the amygdala area, which appoint that they have more amygdala inhibition. Taking these data together, we could partially validated the forced desynchronization protocol as an animal model for mood oscillations