Gabriela Díaz-Véliz

Effects of the estrous cycle and ovarian hormones on behavioral indices of anxiety in female rats

The influence of the estrous cycle and the effects of exogenous administration of estradiol and progesterone on level of anxiety were studied in intact and ovariectomized rats. Intact Sprague-Dawley female rats were classified according to the stages of estrous cycle. Another group of rats was ovariectomized bilaterally and, 14 days after surgery, they received estradiol benzoate (10 micrograms/kg, s.c.) and/or progesterone (25 mg/kg, s.c.) or corn oil (1 ml/kg). The behavioral tests began 3 h after estradiol or 6 h after progesterone and consisted of: (1) exploration of an elevated plus-maze; and (2) retention of a passive avoidance response. Open-arm exploration of the plus-maze varied according to light intensity and the stages of the estrous cycle. There was a slight increase in open-arm exploration by rats in metestrus, under high light intensity. Low light intensity increased the exploration of the open arms by rats in proestrus and estrus, compared to the other phases of the cycle. Retention of the passive avoidance response was inhibited during proestrus and estrus. Progesterone increased open-arm exploration of the plus-maze under high light conditions, whereas estradiol antagonized this effect. Retention of passive avoidance was inhibited after estradiol or progesterone injection. These results suggest that the behavioral indices of anxiety can vary across the estrous cycle, that low light intensities have anxiolytic-like effects, and that the sensitivity to this effect is higher during proestrus and estrus. This could be explained through modulatory effects of ovarian hormones upon behavioral indices of anxiety.

Influence of the estrous cycle, ovariectomy and estradiol replacement upon the acquisition of conditioned avoidance responses in rats☆

The changes in the acquisition of conditioned avoidance responses (CARs) and the performance of some spontaneous behaviors were examined across the estrous cycle of female rats. CARs were facilitated during diestrus, impaired at proestrus and practically abolished at estrus and metestrus. Motor activity and head shaking were minimally affected with the stages of the cycle. Motor activity was increased at metestrus and head shaking decreased at estrus. At 14 days following ovariectomy, there was a significant enhancement of CARs which was antagonized by the daily administration of estradiol benzoate (10 micrograms/kg) for three days. Ovariectomy also increased grooming behavior and estradiol replacement returned grooming to its basal level. The results suggest an inhibitory control of estradiol on CARs and grooming. The involvement of other hormones which also varied across the estrous cycle and its interaction with brain catecholamine systems, particularly dopamine, are discussed.

Ketanserin and anxiety levels : Influence of gender, estrous cycle, ovariectomy and ovarian hormones in female rats

The influence of gender, estrous cycle, ovariectomy and ovarian hormones on the behavioral effects of the 5-HT2 receptor antagonist, ketanserin (KET), was studied. Intact males, female rats in the four stages of the estrous cycle and ovariectomized (OVX) female rats 14 days after surgery were used. The OVX rats received progesterone [PROG, 25 mg/kg, subcutaneously (SC)] and/or estradiol benzoate (EB, 10 micrograms/kg, SC). KET (3 mg/kg, SC) was injected 30 min before testing. All the animals were subjected to the following behavioral tests: exploration of an elevated plus-maze and retention of a passive-avoidance response. KET enhanced the exploration of the open arms in diestrous female rats but inhibited this behavior during the other stages of the cycle and in OVX rats injected either with oil or EB. This dose of KET was ineffective in males and in OVX rats injected with PROG. Furthermore, KET inhibited the retention of the passive avoidance response in males, in diestrous and metestrous female rats and in OVX rats injected with oil. In estrous females and in OVX rats injected with EB, KET enhanced the passive-avoidance response. These results demonstrate that the sensitivity to KET differs with the gender, estrous cycle and hormonal treatment and suggest that central serotonergic activity is influenced by the hormonal status of the animal.

Chronic fluoxetine treatment induces structural plasticity and selective changes in glutamate receptor subunits in the rat cerebral cortex

It has been postulated that chronic administration of antidepressant drugs induces delayed structural and molecular adaptations at glutamatergic forebrain synapses that might underlie mood improvement. To gain further insight into these changes in the cerebral cortex, rats were treated with fluoxetine (flx) for 4 weeks. These animals showed decreased anxiety and learned helplessness. N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit levels (NR1, NR2A, NR2B, GluR1 and GluR2) were analysed in the forebrain by both western blot of homogenates and immunohistochemistry. Both methods demonstrated an upregulation of NR2A, GluR1 and GluR2 that was especially significant in the retrosplenial granular b cortex (RSGb). However, when analysing subunit content in postsynaptic densities and synaptic membranes, we found increases of NR2A and GluR2 but not GluR1. Instead, GluR1 was augmented in a microsomal fraction containing intracellular membranes. NR1 and GluR2 were co-immunoprecipitated from postsynaptic densities and synaptic membranes. In the immunoprecipitates, NR2A was increased while GluR1 was decreased supporting a change in receptor stoichiometry. The changes of subunit levels were associated with an upregulation of dendritic spine density and of large, mushroom-type spines. These molecular and structural adaptations might be involved in neuronal network stabilization following long-term flx treatment.

Effects of estradiol replacement in ovariectomized rats on conditioned avoidance responses and other behaviors

The acquisition of conditioned avoidance responses along with spontaneous behaviors were studied in ovariectomized rats. Fourteen days after ovariectomy, they were injected subcutaneously with one of the following doses of estradiol benzoate: 0.2, 2 or 20 microgram/rat. Behavioral tests were applied 3, 24, 48 or 72 hours after estradiol treatment. Although estradiol 2 microgram/rat induced a decrease in acquisition of conditioned avoidance responses at all times tested, this effect was maximum at 48 h. Estradiol 0.2, and 20 microgram/kg decreased and stimulated, respectively, the acquisition performance, as tested 3 h after injection. All doses increased global motility and rearing behavior. This hypermotility disappeared at 24 h, but it was observed again at 48 and 72 h after estradiol 0.2 and 20 microgram/rat. The hormone also induced an increase in head shaking and a decrease in grooming. Although the behavioral changes are more significant in presence of very low serum levels of estradiol, they seem to be triggered by the previous increase in the estradiol levels. The possible sites and mechanisms of action of estradiol on behavior are discussed.

Desipramine prevents stress-induced changes in depressive-like behavior and hippocampal markers of neuroprotection.

Extracellular signal-regulated kinases (ERKs) are widely implicated in multiple physiological processes. Although ERK1/2 has been proposed as a common mediator of antidepressant action in naive rodents, it remains to be determined whether the ERK1/2 pathway plays a role in depressive disorder. Here, we investigated whether chronic restraint stress (14 days) and antidepressant treatment [desipramine (DMI), 10 mg/kg intraperitoneally] induce changes in animal behavior and hippocampal levels of phospho-ERK1/2 and its substrate phospho-cAMP response element-binding protein (CREB). The results indicated that stress-induced depressive-like behaviors were correlated with an increase in P-ERK1/2 and P-CREB in the hippocampus evaluated by immunoblot analysis. As an indication of CREB activity, we evaluated changes in mRNA levels of its target genes. Brain-derived neurotrophic factor (BDNF) mRNA was reduced by stress, an effect prevented by DMI only in the CA3 area of hippocampus. Bcl-2 mRNA was reduced in all hippocampal regions by stress, an effect independent of DMI treatment. However, immunoblot from hippocampal extracts revealed that stress increased BCL-2 levels, an effect prevented by chronic DMI. These results suggest that ERKs and BDNF may be altered in depressive disorder, modifications that are sensitive to DMI action. In contrast, the stress-induced increase in BCL-2 may correspond to a neuroprotective response.

Anxiolytic and antidepressant-like effects of the hydroalcoholic extract from Aloysia polystachya in rats.

Behavioral effects of a hydroalcoholic extract from leaves of Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) were studied in female Sprague-Dawley rats. The extract was administered intraperitoneally and its effects on spontaneous motor activity (total motility, locomotion, rearing and grooming behavior) were monitored. Anxiolytic-like properties were studied in the elevated plus-maze (EPM) test and the possible antidepressant-like actions were evaluated in the forced swimming test (FST). The results revealed that high doses of the extract (25 and 50 mg/kg, i.p.) caused a significant decrease in total motility, locomotion, rearing and grooming behavior. All doses injected (from 1.56 to 50 mg/kg) increased the exploration of the EPM open arms in a similar way to that of diazepam (1 mg/kg, i.p.). In the FST, the extract (12.5, 25 and 50 mg/kg) was as effective as fluoxetine (10 mg/kg, i.p.) and imipramine (12.5 mg/kg, i.p.) in reducing immobility, along with a significant increase in swimming and climbing, respectively. These results suggest that some of the components of the hydroalcoholic extract of A. polystachya, such as thujone and carvone among others, may have sedative, anxiolytic and antidepressant-like properties which deserve further investigation.

Gender, Estrous Cycle, Ovariectomy, and Ovarian Hormones Influence the Effects of Diazepam on Avoidance Conditioning in Rats

This study examines whether the hormonal condition of the rat modifies the effects of diazepam (0.25 and 1.0 mg/kg) on avoidance conditioning and other behavioral responses. Acquisition of a conditioning avoidance response (CAR) and spontaneous motor behaviors were assessed in intact male, in intact diestrous and estrous females, and in ovariectomized (OVX) rats injected with estradiol (2 microg/rat, SC) or progesterone (5 mg/rat, SC). A higher dose (1.0 mg/kg) of diazepam significantly impaired the acquisition of CARs in diestrous, OVX, OVX + progesterone, and male rats. Conversely, both doses of diazepam significantly improved the acquisition of CAR in estrous rats and in OVX rats injected with estradiol. These effects on conditioning avoidance were not accompanied with equivalent changes in spontaneous motor behaviors. Motor activity and grooming behavior decreased in all experimental groups after administration of 1.0 mg/kg of diazepam. On the contrary, diazepam 0.25 mg/kg increased motor activity in estrous, OVX + estradiol, and OVX + progesterone rats after, whereas grooming behavior was not affected in any group. These findings suggest a physiological influence of ovarian steroid hormones in modifying the benzodiazepine effects on conditioning avoidance and motor activity. The results are discussed considering that ovarian steroids may interact with diazepam on the GABA(A)/benzodiazapine/chloride ionophore complex, modifying the coupling between benzodiazepine sites and GABA(A) receptors.

Behavioral effects of manganese injected in the rat substantia nigra are potentiated by dicumarol, a DT-diaphorase inhibitor

The purpose of this study was to evaluate the contribution of DT-diaphorase inhibition to in vivo neurodegenerative effects of dopamine (DA) oxidation to the corresponding o-quinones. The neurotoxicity to nigrostriatal DA neurons was induced by injection of manganese pyrophosphate (Mn(3+)) complex as a prooxidizing agent alone or together with the DT-diaphorase inhibitor dicumarol into the right rat substantia nigra. The behavioral effects were compared with those induced after selective lesions of dopaminergic neurons with 6-hydroxydopamine (6-OHDA). Intranigral injection of Mn(3+) and Mn(3+) plus dicumarol produced significant impairment in motor behavior compared with control animals. However, the effect seen in the Mn(3+) plus dicumarol injected group was significantly more severe than that observed in the Mn(3+) alone injected group. In motor activity and rearing behavior, the simultaneous injection of Mn(3+) plus dicumarol produced a 6-OHDA-like impairment. Similar effects were observed in the acquisition of a conditioned avoidance response (CAR). Dicumarol significantly impaired avoidance conditioning although without affecting the motor behavior. The behavioral effects were correlated to the extent of striatal tyrosine hydroxylase (TH)-positive fiber loss. Rats receiving unilateral intranigral Mn(3+) and Mn(3+) plus dicumarol injections exhibited a significant reduction in nigrostriatal TH-positive fiber density in medial forebrain bundle compared with the contralateral noninjected side. In conclusion, this study provides evidence that the neurotoxicity of Mn(3+) in vivo is potentiated by DT-diaphorase inhibition, suggesting that this enzyme could play a neuroprotective role in the nigrostriatal DA systems.

Progesterone effects on the acquisition of conditioned avoidance responses and other motoric behaviors in intact and ovariectomized rats

This study demonstrates a significant impairment in the acquisition of conditioned avoidance responses in female rats during their estrus phase. Progesterone (PROG 5 mg) injected 6 h prior to the test, significantly enhanced the performance exhibited by rats at estrus, but not at diestrus. In ovariectomized rats, the acquisition of conditioned avoidance responses was similar to the exhibited during diestrus and this behavior was depressed by a single dose of estradiol benzoate (EB 2 micrograms) injected 48 h prior to the test. PROG antagonized the avoidance depression induced by EB, but it was not able to induce changes in the acquisition of conditioned avoidance response in ovariectomized rats without EB pretreatment. Estradiol appears to be the principal ovarian steroid modulating the acquisition of an avoidance task, whereas PROG seems to have a secondary role in this behavior, regulating the actions of estradiol on the brain. PROG failed to induce consistent changes in some spontaneous motor behaviors in intact and ovariectomized rats.