Isabel C. Céspedes

Effects of chronic treatment with corticosterone and imipramine on fos immunoreactivity and adult hippocampal neurogenesis.

In a previous study we showed that rats chronically treated with corticosterone (CORT) display anxiogenic behavior, evidenced by facilitation of avoidance responses in the elevated T-maze (ETM) model of anxiety. Treatment with the tricyclic antidepressant imipramine significantly reversed the anxiogenic effects of CORT, while inhibiting ETM escape, a response related to panic disorder. To better understand the neurobiological mechanisms underlying these behavioral effects, analysis of c-fos protein immunoreactivity (fos-ir) was used here to map areas activated by chronic CORT (200 mg pellets, 21-day release) and imipramine (15 mg/kg, IP) administration. We also evaluated the number of cells expressing the neurogenesis marker doublecortin (DCX) in the hippocampus and measured plasma CORT levels on the 21st day of treatment. Results showed that CORT increased fos-ir in the ventrolateral septum, medial amygdala and paraventricular hypothalamic nucleus and decreased fos-ir in the lateral periaqueductal gray. Imipramine, on the other hand, increased fos-ir in the medial amygdala and decreased fos-ir in the anterior hypothalamus. CORT also decreased the number of DCX-positive cells in the ventral and dorsal hippocampus, an effect antagonized by imipramine. CORT levels were significantly higher after treatment. These data suggest that the behavioral effects of CORT and imipramine are mediated through specific, at times overlapping, neuronal circuits, which might be of relevance to a better understanding of the physiopathology of generalized anxiety and panic disorder.

Chronic unpredictable mild stress alters an anxiety-related defensive response, Fos immunoreactivity and hippocampal adult neurogenesis.

Previous results show that elevated T-maze (ETM) avoidance responses are facilitated by acute restraint. Escape, on the other hand, was unaltered. To examine if the magnitude of the stressor is an important factor influencing these results, we investigated the effects of unpredictable chronic mild stress (UCMS) on ETM avoidance and escape measurements. Analysis of Fos protein immunoreactivity (Fos-ir) was used to map areas activated by stress exposure in response to ETM avoidance and escape performance. Additionally, the effects of the UCMS protocol on the number of cells expressing the marker of migrating neuroblasts doublecortin (DCX) in the hippocampus were investigated. Corticosterone serum levels were also measured. Results showed that UCMS facilitates ETM avoidance, not altering escape. In unstressed animals, avoidance performance increases Fos-ir in the cingulate cortex, hippocampus (dentate gyrus) and basomedial amygdala, and escape increases Fos-ir in the dorsolateral periaqueductal gray and locus ceruleus. In stressed animals submitted to ETM avoidance, increases in Fos-ir were observed in the cingulate cortex, ventrolateral septum, hippocampus, hypothalamus, amygdala, dorsal and median raphe nuclei. In stressed animals submitted to ETM escape, increases in Fos-ir were observed in the cingulate cortex, periaqueductal gray and locus ceruleus. Also, UCMS exposure decreased the number of DCX-positive cells in the dorsal and ventral hippocampus and increased corticosterone serum levels. These data suggest that the anxiogenic effects of UCMS are related to the activation of specific neurobiological circuits that modulate anxiety and confirm that this stress protocol activates the hypothalamus-pituitary-adrenal axis and decreases hippocampal adult neurogenesis.

Physical therapy and psychological intervention normalize cortisol levels and improve vitality in women with endometriosis.

There is as yet no effective treatment for endometriosis. The objective of this study was to evaluate the effectiveness of submitting women with endometriosis and chronic pelvic pain to a therapeutic protocol involving physical and psychological therapy. Twenty-six female volunteers were submitted to a treatment protocol consisting of 2.5-h sessions, once a week for 10 weeks. We applied a Visual Analogue Scale, the Perceived Stress Questionnaire (PSQ), and the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). Morning, afternoon, and evening levels of cortisol were determined in saliva samples. The PSQ scores were significantly lower after treatment, whereas the scores for the SF-36 vitality and physical functioning domains were significantly higher. Salivary cortisol levels were higher after treatment in the samples collected in the morning, but not in those collected in the afternoon or evening. The post-treatment cortisol levels were similar to those reported for healthy women. There were positive correlations between vitality, role emotional, social functioning, and mental health, and negative correlations to perceived stress. In conclusion, the physical and psychological intervention protocol applied in this study to women suffering of endometriosis was effective in reducing perceived stress, normalizing cortisol levels, increasing vitality and improving physical functioning.

CRF type 1 receptors of the medial amygdala modulate inhibitory avoidance responses in the elevated T-maze

Corticotropin-releasing factor (CRF) plays a critical role in the mediation of physiological and behavioral responses to stressors. In the present study, we investigated the role played by the CRF system within the medial amygdala (MeA) in the modulation of anxiety and fear-related responses. Male Wistar rats were bilaterally administered into the MeA with CRF (125 and 250 ng/0.2μl, experiment 1) or with the CRFR1 antagonist antalarmin (25 ng/0.2 μl, experiment 2) and 10 min later tested in the elevated T-maze (ETM) for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. To further verify if the anxiogenic effects of CRF were mediated by CRFR1 activation, we also investigated the effects of the combined treatment with CRF (250 ng/0.2 μl) and antalarmin (25 ng/0.2 μl) (experiment 3). All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Results showed that CRF, in the two doses administered, facilitated ETM avoidance, an anxiogenic response. Antalarmin significantly decreased avoidance latencies, an anxiolytic effect, and was able to counteract the anxiogenic effects of CRF. None of the compounds administered altered escape responses or locomotor activity measurements. These results suggest that CRF in the MeA exerts anxiogenic effects by activating type 1 receptors, which might be of relevance to the physiopathology of generalized anxiety disorder.

Acute restraint differently alters defensive responses and fos immunoreactivity in the rat brain

Results from a previous study show that rats exposed to acute restraint display anxiogenic-like behavior, evidenced by facilitation of avoidance responses in the elevated T-maze (ETM) model of anxiety. In contrast, escape responses were unaltered by stress exposure. Since ETM avoidance and escape tasks seem to activate distinct sets of brain structures, it is possible that the differences observed with acute restraint are due to particularities in the neurobiological mechanisms which modulate these responses. In the present study, analysis of fos protein immunoreactivity (fos-ir) was used to map areas activated by exposure of male Wistar rats to restraint stress (30 min) previously (30 min) to the ETM. Corticosterone levels were also measured in stressed and non-stressed animals. Confirming previous observations restraint facilitated avoidance performance, an anxiogenic result, while leaving escape unaltered. Performance of the avoidance task increased fos-ir in the frontal cortex, intermediate lateral septum, basolateral amygdala, basomedial amygdala, lateral amygdala, anterior hypothalamus and dorsal raphe nucleus. In contrast, performance of escape increased fos-ir in the ventromedial hypothalamus, dorsolateral periaqueductal gray and locus ceruleus. Both behavioral tasks also increased fos-ir in the dorsomedial hypothalamus. Restraint significantly raised corticosterone levels. Additionally after restraint, fos-ir was predominantly seen in the basolateral amygdala and dorsal raphe of animals submitted to the avoidance task. This data confirms that different sets of brain structures are activated by ETM avoidance and escape tasks and suggests that acute restraint differently alters ETM behavior and the pattern of fos activation in the brain.

Chronic corticosterone administration facilitates aversive memory retrieval and increases GR/NOS immunoreactivity

Glucocorticoids are stress hormones that mediate the organisms reaction to stress. It has been previously proposed that the facilitation of emotional aversive conditioning induced by these hormones may involve nitric oxide-pathways. The purpose of the present study was to address this question. For that, male Wistar rats were surgically implanted with slow-release corticosterone (CORT) pellets (21 days) and tested in a step-down inhibitory avoidance task. Additional groups of animals were also submitted to the same treatment conditions and on the 21st day of treatment assayed for GR (glucocorticoid receptors)-nNOS (neuronal nitric oxide synthase) immunoreactivity (GRi-nNOSi) or measurements of plasma CORT. Results showed that CORT treatment induced facilitation of step-down inhibitory avoidance. This same treatment also significantly increased CORT plasma levels and GRi in the medial, basolateral and basomedial amygdala, in the paraventricular hypothalamic nucleus (PVN), in the ventral and dorsal dentate gyrus, in the ventral CA1 region and in the dorsal CA1 and CA3 regions. Furthermore, nNOSi and GRi-nNOSi were significantly increased by CORT treatment in the medial amygdala and basolateral amygdaloid complex, in the PVN, subiculum, in the dorsal CA3 region and in the ventral CA1 and CA3 regions. These results indicate that the facilitation of aversive conditioning induced by CORT involves GR-nNOS pathways activation, what may be of relevance for a better understanding of stress-related psychiatric conditions.

The activation and blockage of CRF type 2 receptors of the medial amygdala alter elevated T-maze inhibitory avoidance, an anxiety-related response.

Previous results show that the activation of CRF type 1 (CRFR1) receptors of the medial amygdala (MeA) induces anxiogenic-like effects. The present study investigates the role played by medial amygdala CRF type 2 receptors (CRFR2) in the modulation of anxiety and panic-related responses. Male Wistar rats were administered into the MeA with the CRFR2 agonist urocortin 2 (0.5 e 1.0μg/0.2μl, experiment 1) or with the CRFR2 antagonist astressin 2-B (60ng/0.2μl, experiment 2) and 10min later tested in the elevated T-maze (ETM) for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. In a third experiment, the effects of the combined treatment with urocortin 2 (1.0μg/0.2μl) and a sub-effective dose of astressin 2-B (30ng/0.2μl) were also investigated. All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Results showed that urocortin 2, in the highest dose administered (1.0μg/0.2μl), facilitated ETM avoidance, an anxiogenic-like effect. Astressin 2-B, also in the highest dose (60ng/0.2μl), significantly decreased avoidance latencies, an anxiolytic-like effect. The lower dose of astressin 2-B (30ng/0.2μl) did not induce anxiolytic-like effects but was able to counteract the anxiogenic-like effects of urocortin 2. None of the compounds administered altered escape responses or locomotor activity measurements. These results suggest that CRFR2 in the medial amygdala, as CRFR1, selectively modulate an anxiety-related response.

Dorsomedial hypothalamus CRF type 1 receptors selectively modulate inhibitory avoidance responses in the elevated T-maze

Corticotropin-releasing factor (CRF) plays a critical role in the mediation of physiological and behavioral responses to stressors. In the present study, we investigated the role played by the CRF system within the dorsomedial hypothalamus (DMH) in the modulation of anxiety- and panic-related responses. Male Wistar rats were administered into the DMH with CRF (125 and 250 ng/0.2 μl, experiment 1) or with the CRFR1 antagonist antalarmin (25 ng/0.2 μl, experiment 2) and 10 min later tested in the elevated T-maze (ETM) for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. To further verify if the anxiogenic effects of CRF were mediated by CRFR1 activation, we also investigated the effects of the combined treatment with CRF (250 ng/0.2 μl) and antalarmin (25 ng/0.2 μl) (experiment 3). All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Results showed that 250 ng/0.2μl of CRF facilitated ETM avoidance, an anxiogenic response. Antalarmin significantly decreased avoidance latencies, an anxiolytic effect, and was able to counteract the anxiogenic effects of CRF. None of the compounds administered altered escape responses or locomotor activity measurements. These results suggest that CRF in the DMH exerts anxiogenic effects by activating type 1 receptors, which might be of relevance to the physiopathology of generalized anxiety disorder.

CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress.

Corticotropin-releasing factor (CRF) acts to promote stress-like physiological and behavioral responses and is mainly expressed in the paraventricular hypothalamic nucleus (PVN). Urocortin 1 (Ucn1) is also a ligand to CRF type 1 and 2 receptors that has been associated with the stress response. Ucn1 neurons are primarily found in the Edinger-Westphal (EW) nucleus. It has been previously proposed that CRF and Ucn1 differently modulate stress responses to distinct types of stressors. The present study used male Wistar rats to compare the effects of acute restraint stress and unpredictable chronic stress (UCS) through Fos-immunoreactivity (Fos-ir) on CRF-containing neurons of PVN and Ucn1-containing EW centrally projecting neurons. Results showed that PVN neurons responded to both acute restraint and UCS. Also for the PVN, unspecific variables, dependent on the time animals remained in the laboratory, do not seem to alter Fos-ir, since no significant differences between acute and chronic control groups were found. On the other hand, EW neurons were only activated in response to acute restraint stress. Also, for this nucleus a significant difference was found between acute and chronic control groups, suggesting that unspecific variables, dependent on the time animals remain in the laboratory, interfere with the nucleus activation. These results suggest that CRF/Ucn1 neuronal circuits encompass two interconnected systems, which are coordinated to respond to acute stressors, but are differentially activated during chronic unpredictable stress.

Unpredictable chronic mild stress exerts anxiogenic-like effects and activates neurons in the dorsal and caudal region and in the lateral wings of the dorsal raphe nucleus

In previous studies, we verified that exposure to unpredictable chronic mild stress (UCMS) facilitates avoidance responses in the elevated T-maze (ETM) and increased Fos-immunoreactivity in different brain structures involved in the regulation of anxiety, including the dorsal raphe (DR). Since, it has been shown that the DR is composed of distinct subpopulations of serotonergic and non-serotonergic neurons, the present study investigated the pattern of activation of these different subnuclei of the region in response to this stress protocol. Male Wistar rats were either unstressed or exposed to the UCMS procedure for two weeks and, subsequently, analyzed for Fos-immunoreactivity (Fos-ir) in serotonergic cells of the DR. To verify if the anxiogenic effects observed in the ETM could be generalized to other anxiety models, a group of animals was also tested in the light/dark transition test after UCMS exposure. Results showed that the UCMS procedure decreased the number of transitions and increased the number of stretched attend postures in the model, an anxiogenic effect. UCMS exposure also increased Fos-ir and the number of double-labeled neurons in the mid-rostral subdivision of the dorsal part of the DR and in the mid-caudal region of the lateral wings. In the caudal region of the DR there was a significant increase in the number of Fos-ir. No significant effects were found in the other DR subnuclei. These results corroborate the idea that neurons of specific subnuclei of the DR regulate anxiety responses and are differently activated by chronic stress exposure.