Metestrus Females Research Articles

Cycle-dependent sex differences in expression of membrane proteins involved in cerebrospinal fluid secretion at rat choroid plexus

BackgroundFemale sex is a known risk factor of brain disorders with raised intracranial pressure (ICP) and sex hormones have been suggested to alter cerebrospinal fluid (CSF) dynamics, thus impairing ICP regulation in CSF disorders such as idiopathic intracranial hypertension (IIH). The choroid plexus (CP) is the tissue producing CSF and it has been hypothesized that altered hormonal composition could affect the activity of transporters involved in CSF secretion, thus affecting ICP. Therefore, we aimed to investigate if expression of various transporters involved in CSF secretion at CP were different between males and females and between females in different estrous cycle states. Steroid levels in serum was also investigated.MethodsFemale and male rats were used to determine sex-differences in the genes encoding for the transporters Aqp1 and 4, NKCC1, NBCe2, NCBE; carbonic anhydrase enzymes II and III (CA), subunits of the Na+/K+-ATPase including Atp1a1, Atp1b1 and Fxyd1 at CP. The estrous cycle stage metestrus (MET) and estrous (ES) were determined before euthanasia. Serum and CP were collected and subjected to RT-qPCR analysis and western blots. Serum was used to measure steroid levels using liquid chromatography tandem mass spectrometry (LC–MS/MS).ResultsSignificant differences in gene expression and steroid levels between males and ES females were found, while no differences were found between male and MET females. During ES, expression of Aqp1 was lower (p < 0.01) and NKCC1 was higher in females compared to males. CAII was lower while CAIII was higher in ES females (p < 0.0001). Gene expression of Atp1a1 was lower in ES compared to male (p = 0.0008). Several of these choroidal genes were also significantly different in MET compared to females in ES. Differences in gene expression during the estrus cycle were correlated to serum level of steroid hormones. Protein expression of AQP1 (p = 0.008) and CAII (p = 0.035) was reduced in ES females compared to males.ConclusionsThis study demonstrates for the first time that expression at CP is sex-dependent and markedly affected by the estrous cycle in female rats. Further, expression was related to hormone levels in serum. This opens a completely new avenue for steroid regulation of the expression of CSF transporters and the close link to the understanding of CSF disorders such as IIH.

Sexual dimorphic distribution of G protein-coupled receptor 30 in pain-related regions of the mouse brain.

Sex differences in pain sensitivity have contributed to the fact that medications for curing chronic pain are unsatisfactory. However, the underlying mechanism remains to be elucidated. Brain-derived estrogen participates in modulation of sex differences in pain and related emotion. G protein-coupled receptor 30 (GPR30), identified as a novel estrogen receptor with a different distribution than traditional receptors, has been proved to play a vital role in regulating pain affected by estrogen. However, the contribution of its distribution to sexually dimorphic pain-related behaviors has not been fully explored. In the current study, immunofluorescence assays were applied to mark the neurons expressing GPR30 in male and female mice (in metestrus and proestrus phase) in pain-related brain regions. The neurons that express CaMKIIα or VGAT were also labeled to observe overlap with GPR30. We found that females had more GPR30-positive (GPR30+) neurons in the primary somatosensory (S1) and insular cortex (IC) than males. In the lateral habenula (LHb) and the nucleus tractus solitarius (NTS), males had more GPR30+ neurons than females. Moreover, within the LHb, the expression of GPR30 varied with estrous cycle phase; females in metestrus had fewer GPR30+ neurons than those in proestrus. In addition, females had more GPR30+ neurons, which co-expressed CaMKIIα in the medial preoptic nucleus (mPOA) than males, while males had more than females in the basolateral complex of the amygdala (BLA). These findings may partly explain the different modulatory effects of GPR30 in pain and related emotional phenotypes between sexes and provide a basis for comprehension of sexual dimorphism in pain related to estrogen and GPR30, and finally provide new targets for exploiting new treatments of sex-specific pain.

Palatable high-fat diet intake influences mnemonic and emotional aspects in female rats in an estrous cycle-dependent manner.

Worldwide, the excessive consumption of fat and/or sugar has increased considerably. Palatablehigh-fat diets (HFDs) lead to metabolic disturbances and obesity, and impact emotional and cognitive processes. Previous studies in rodent models suggested that HFDs often cause multiple behavioral alterations, such as learning and memory deficits, and anxiety-like behaviors. Different sexes imply different behavioral and cognitive abilities; yet, most of these studies dealt with male or ovariectomized rats. We evaluated HFD effects in female rats submitted to different behavioral tasks, considering the effects of endogenous hormonal variations throughout estrous cycle. Female Wistar rats in each phase of the estrous cycle using commercial chow (CC) or HFD for 32days. During treatment, behavioral assessments using sucrose preference (SP), elevated plus-maze (EPM), open field (OF) and novel-object recognition (NOR). At the end of the behavioral tests, animals were euthanized, and performed an immunohistochemical analysis of the brains by brain-derived neurotrophic factor (BDNF) and tyrosine hydroxylase (TH). The main results demonstrated that (1) HFD-fed rats had higher body mass gain and food intake, without altering caloric intake, (2) rats in diestrus had lower sucrose intake, (3) females in metestrus and diestrus showed deficits in the novel-object recognition memory. Furthermore, TH-immunoreactivity decreased in the dorsal striatum and BDNF in the hippocampus in HFD-fed females. These results suggest that HFD alters neurochemical and metabolic aspects that may induce phase-dependent behavioral changes in female rats.

Cyclic changes and actions of progesterone and allopregnanolone on cognition and hippocampal basal (stratum oriens) dendritic spines of female rats

Ovarian steroids modulate the neuronal structure and function during the estrous cycle, contrasting peak effects during the proestrus cycle and low effects during the metestrus cycle. An ovariectomy (OVX) decreases gonadal hormones and tests the effects of substitutive therapies.We studied female rats with a normal estrous cycle and we also studied the effects of systemic progesterone (P4, 4.0 mg/kg) or its reduced metabolite allopregnanolone (ALLO, 4.0 mg/kg, both for 10 days) in females who had had an OVX 16.5 weeks prior to the study (long-term OVX) with the novel object recognition test (NORT) for associative memory. The dendritic shape and spine density in Golgi-impregnated basal dendrites (stratum oriens) of hippocampal pyramidal neurons was also studied.Proestrus females had a better performance than metestrus or OVX females in short-term memory (tested 1 h after the acquisition phase). Proestrus and metestrus females showed better results than OVX females for long-term memory (24 h after the initial phase). Both P4 and ALLO recovered the cognitive impairment induced by long-term OVX. Also, proestrus females had a higher density of dendritic spines than metestrus females, OVX reduced the density of spines when compared to intact females, whereas both P4 and ALLO treatments increased the dendritic spine density, number of dendritic branches along the dendritic length, and branching order compared to vehicle. These data add the dendrites of the stratum oriens as an additional site for naturally occurring changes in spine density during the estrous cycle and evidence the actions of progestins in both behavioral recovery and the structural dendritic rearrangement of hippocampal pyramidal neurons in long-term OVX female rats.

Endocannabinoids and Fear-Related Behavior in Mice Selectively Bred for High or Low Alcohol Preference.

Alcohol use disorders (AUDs) have a high incidence of co-morbidity with stress-related psychopathologies, such as post-traumatic stress disorder (PTSD). Genetic and pharmacological studies support a prominent role for the endocannabinoid system (ECS) in modulating stress-related behaviors relevant to AUDs and PTSD. Mouse lines selectively bred for high (HAP) and low (LAP) alcohol preference show reproducible differences in fear-potentiated startle (FPS), a model for PTSD-related behavior. The first experiment in this study assessed levels of the endocannabinoids, anandamide (AEA) and sn-2 arachidonylglycerol (2-AG), in the prefrontal cortex (PFC), amygdala (AMG), and hippocampus (HIP) of male and female HAP1 and LAP1 mice following the expression of FPS to determine whether ECS responses to conditioned-fear stress (FPS) were correlated with genetic propensity toward high or low alcohol preference. The second experiment examined effects of a cannabinoid receptor type 1 agonist (CP55940) and antagonist (rimonabant) on the expression of FPS in HAP1 and LAP1 male and female mice. The estrous cycle of females was monitored throughout the experiments to determine if the expression of FPS differed by stage of the cycle. FPS was greater in male and female HAP1 than LAP1 mice, as previously reported. In both experiments, LAP1 females in diestrus displayed greater FPS than LAP1 females in metestrus and estrus. In the AMG and HIP, AEA levels were greater in male fear-conditioned HAP1 mice than LAP1 mice. There were no line or sex differences in effects of CP55940 or rimonabant on the expression of FPS. However, surprisingly, evidence for anxiogenic effects of prior treatment with CP55940 were seen in all mice during the third drug-free FPS test. These findings suggest that genetic differences in ECS function in response to fear-conditioning stress may underlie differences in FPS expression in HAP1 and LAP1 selected lines.

Effect of Sex and Estrous Cycle on Nicotine withdrawal Syn- drome in the Rat

Introduction: Severity of withdrawal syndrome in women during smoking cessation has reportedly been influenced by menstrual phase. There are few studies of female rats with their four-day estrous cycle. Methods: Histological examination determined the precise estrous phase at time of testing. The subjects were 14 qualifying female rats and 8 male rats, all five to six months old. Slides of vaginal fluid were examined for estrous phaseidentifying cell types. Nicotine withdrawal was evaluated at either the proestrus phase (n=7), a follicular portion of the cycle, and the metestrus phase (n=7) a luteal portion. Rats were continuously infused with 9 mg/kg/day s.c. nicotine bitartrate. On the seventh day, each subject was challenged with 1 mg/kg of the nicotinic antagonist mecamylamine, a dose that precipitates a vigorous withdrawal syndrome only in nicotine-dependent rats. Subjects were observed over 30 min. on a standard checklist of somatically expressed withdrawal behaviors. Results: Male rats displayed 26.00 ± 3.64 withdrawal signs (M ± SEM), while female rats in proestrus exhibited a similar 28.86 ± 3.39 signs. Female rats in metestrus displayed 41.57 ± 5.38 signs. One-way ANOVA revealed a significant difference among groups, p=0.039. Post hoc comparisons revealed significant differences between metestrus and proestrus females, p=0.049 and between metestrus females and males, p=0.016, but not between proestrus females and males. Conclusions: The results are consistent with reports of menstrual phase influence on withdrawal severity in smoking cessation, providing a laboratory model for studying this issue and its potential treatment. Implications: In the large literature on nicotine withdrawal in the rat, there are relatively few studies on female rats and hardly any on the effects of the estrous cycle on physical dependence and withdrawal. The results are consistent with reports on the menstrual cycle affecting withdrawal severity in women undergoing smoking cessation, supporting the translational relevance of the rat nicotine dependence model. The methods utilized here expand the ability of rat physical dependence models to compare the sexes and the estrous phases in nicotine physical dependence and withdrawal.

Localization of CART-Positive Neurons in the Amygdaloid Body and the Relationship between Their Immunoreactivity and the Sex Steroid Level

This is the first study reporting the locations of neurons expressing CART peptide (cocaine-amphetamine-regulated transcript) throughout all the nuclear and paleocortical formations of the amygdaloid body (AB) and demonstrating the effects of sex steroids on immunoreactivity. The immunocytochemical reaction was performed on frontal brain sections from adult rats (seven females in estrus, seven females in metestrus, and seven males). The proportions of immunoreactive neurons to the total number of neurons in adjacent sections stained by the Nissl method were assessed in estrus and metestrus. In the dorsomedial and posterior cortical nuclei and the lateral capsular subnucleus of the central field, the relative numbers of immunoreactive neurons at estrus were significantly greater than at metestrus. These results provide evidence of the involvement of the olfactory and integrative centers of the AB in the pathogenesis of drug dependence and show that new and efficient methods of gene therapy might be developed using the intranasal route for drug administration.

Sex differences in inflammation evoked by noxious chemical, heat and electrical stimulation

Neurogenic inflammation (NI) is a feature of several inflammatory pain conditions in which females are overrepresented. Therefore, we asked if there are sex differences in the inflammatory response evoked by well known neurogenic stimuli. We compared the amount of plasma extravasation (PE), a measure of inflammation, in the hindpaw skin of male and female rats caused by subcutaneous injection of capsaicin, application of noxious heat (51 °C water bath) or electrical stimulation of the saphenous nerve. We also compared the amount of PE in males and females evoked by substance P (SP), the principal neurogenic mediator of PE. PE was quantified using a video camera and digital image analysis to measure changes in reflectance (pixel intensity, PI) of skin due to accumulation of extravasated Evans blue (EB) dye. The increase in PI induced by capsaicin was significantly greater in females compared to males ( p < 0.001) and in estrus, diestrus, and metestrus females compared to proestrus females. The time to reach maximal capsaicin-induced PE was two times longer in estrus, diestrus, and metestrus females compared to males ( p < 0.05). PE induced by heat was also significantly greater in females compared to males ( p < 0.001), however, there was no sex-related difference in PE induced by electrical stimulation or by injection of SP. These findings show that females have a greater inflammatory response when inflammation is induced by capsaicin and noxious heat suggesting possible sex-related changes in TRPV-1 receptor mediated mechanisms. These results add to the growing list of sex difference responses to noxious somatic stimulation.

Luteinizing Hormone Beta Promoter Stimulation by Adenylyl Cyclase and Cooperation with Gonadotropin-Releasing Hormone 1 in Transgenic Mice and LBetaT2 Cells1

Rat luteinizing hormone beta (Lhb) gene transcription is stimulated by hypothalamic gonadotropin-releasing hormone 1 (GnRH1), and this response may be modulated by other signaling pathways such as cAMP. Here we characterize the ability of cAMP, alone or with GnRH1, to stimulate Lhb gene transcription in mouse pituitary and clonal gonadotroph cells. Both cAMP and pituitary adenylyl cyclase-activating peptide increase GnRH1 stimulation of luciferase activity in pituitaries of mice expressing the rat Lhb-luciferase transgene, suggesting cAMP and GnRH1 pathways interact in vivo. cAMP stimulation of the Lhb-luciferase transgene was similar between females in metestrus and proestrus, but GnRH1 stimulation was greater at proestrus. Additive effects with combined treatments were observed at metestrus and proestrus. Elevated intracellular cAMP stimulated Lhb promoter activity in LbetaT2 clonal gonadotroph cells, alone and with GnRH1. In LbetaT2 cells, cAMP stimulation of the Lhb promoter was eliminated by inhibition of protein kinase A (PKA); GnRH1 stimulation was partially suppressed by either PKA or protein kinase C inhibitors. Only the proximal GnRH1-responsive region of the promoter was required for cAMP stimulation, and mutation of the 3' NR5A1 site diminished the response. Regulation of primary mRNA transcripts from the endogenous Lhb gene by cAMP and GnRH1 correlated with results from the Lhb-luciferase transgene or transfected promoter. Occupancy of the endogenous promoter by EGR1 was increased by GnRH1 with or without forskolin, but forskolin alone had little effect. Thus, cAMP stimulation of Lhb promoter activity, and enhancement of GnRH1 stimulation, occurs in multiple physiological states independent of steroid status, via a PKA-dependent mechanism.

Effects of 17β-estradiol and flutamide on splenic macrophages and splenocytes after trauma-hemorrhage

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8–12 weeks old), underwent laparotomy and hemorrhagic shock (35.0 ± 5.0 mm Hg for 90 min) and received 17β-estradiol (50 μg/25 g), flutamide (625 μg/25 g) or 17β-estradiol + flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-α, IL-6, IL-10, and IFN-γ) by splenic MΦ and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MΦ was observed following trauma-hemorrhage compared to shams. Administration of 17β-estradiol, flutamide and 17β-estradiol + flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MΦ. The TNF-α productive capacity, however, was only restored by 17β-estradiol and 17β-estradiol + flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MΦ IL-10 release. Anti-CD3 stimulation, administration of 17β-estradiol and 17β-estradiol + flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-α, IL-6 and IFN-γ compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17β-estradiol administration alone.

Effects of 17β-estradiol and flutamide on inflammatory response and distant organ damage following trauma-hemorrhage in metestrus females

We hypothesized that administration of androgen receptors antagonist flutamide following trauma-hemorrhage (T-H) in metestrus females will maintain immune function and reduce remote organ damage under those conditions. Female B57BL/J6 mice (metestrus state, 8-12 weeks old) underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mmHg for 90 min) and then received 17beta-estradiol (E2; 50 microg/25 g), flutamide (625 microg/25 g), or E2 + flutamide. Four hours after resuscitation, plasma cytokine and chemokine (TNF-alpha, IL-6, IL-10, IFN-gamma, and MCP-1) concentrations and their release in vitro by hepatic and pulmonary tissue macrophages (M Phi) were determined by flow cytometry. Organ damage was assessed by edema formation (wet-to-dry weight ratio) and neutrophil infiltration [myeloperoxidase (MPO) activity]. Administration of E2, flutamide, or E2 + flutamide following T-H resulted in a significant decrease in systemic TNF-alpha, IL-6, and MCP-1 concentrations under those conditions. This was accompanied by significantly decreased in vitro TNF-alpha release by Kupffer cells after administration of E2, flutamide, or E2 + flutamide. The in vitro release of proinflammatory cytokines by alveolar M Phi, however, was reduced significantly only by the addition of E2 or E2 + flutamide but not by the addition of flutamide. A significant decrease in pulmonary and hepatic edema formation as well as neutrophil infiltration in the lung was observed after E2, flutamide and E2 + flutamide administration. In contrast, hepatic neutrophil infiltration was only significantly reduced following E2 and E2 + flutamide administration. Thus, although flutamide does not produce synergistic, salutary effects with E2, its administration in females following T-H also produces salutary effects on the immune and organ function, similar to E2 administration under those conditions.

Lactated Ringer’s Solution and Hypertonic Saline Improve Survival in Uncontrolled Hemorrhagic Shock in Female Rats in Metestrus

In the present investigation the effect of fluid treatment in uncontrolled hemorrhagic shock after massive splenic injury (MSI) was comparatively studied in male and female rats. The anesthetized animals were randomly divided into three groups: in group 1 MSI was induced in males, in group 2 MSI was induced in females in proestrus, in group 3 MSI was induced in females in metestrus. Each group was divided into four subgroups: a) Sham-operated, b) MSI untreated (UT), c) MSI treated with 40 ml/kg lactated Ringer's solution (RL), and d) MSI treated with 5 ml/kg NaCl 7.5% (HTS). Total blood loss (TBL) in groups 1b, 2b, and 3b was 31.7 +/- 3.6%, 33.1 +/- 2.6%, and 36.7 +/- 2.6%, respectively, and mean survival time (MST) was 143.7 +/- 25.3 min, 174.8 +/- 10.4 min, and 67.8 +/- 11.4 min (P < 0.01 versus group 2b), respectively. TBL in groups 1c, 2c, and 3c increased to 52.4 +/- 5.5% (P < 0.02 versus UT), 48.6 +/- 1.6% (P < 0.02 versus UT), and 48.8 +/- 4.1% (P < 0.02 versus UT), respectively, and MST decreased to 126 +/- 19.4 min, (P < 0.05 versus UT), and 136.8 +/- 13.0 min (P < 0.05 versus UT) in groups 1c and 2c, respectively, and increased in group 3c to 120.4 +/- 23.3 min (P < 0.05 versus UT). TBL in groups 1d, 2d, and 3d was 31.3 +/- 4.8%, 38.0 +/- 4.2%, and 40.6 +/- 3.7%, respectively, and MST increased to 198.5 +/- 13.9 min (P < 0.05 versus UT) in group 1d, decreased to 128.4 +/- 17.2 min (P < 0.01) in group 2d, and increased to 102.6 +/- 19.0 min (P < 0.002 versus group 1d) in group 3d. RL infusion significantly increased blood loss in all three groups, reduced survival time in males and female rats in proestrus, but significantly improved survival in females in metestrus. HTS treatment did not alter blood loss in all three groups, but significantly improved survival in females in metestrus and males.

Spatial reference memory and neocortical neurochemistry vary with the estrous cycle in C57BL/6 mice.

Estrous cycle-related variations of spatial reference memory and neurochemistry in intact female mice were examined. Spatial reference memory was tested in cycling females, ovariectomized (OVX) females, and males by using a 1-day water maze protocol. Choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities were measured in the hippocampus and neocortex. Estrus females exhibited worse spatial acquisition and 30-min retention than did proestrus and metestrus females, higher neocortical ChAT activity than proestrus females, and higher neocortical GAD activity than OVX females and males. Neocortical, rather than hippocampal, neurochemistry was more sensitive to hormonal modulation, suggesting that hormonal mediation of neocortical function may play a critical role in regulating spatial reference memory in female mice.

Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat.

We have investigated genetic transmission of increased sensitivity to focal cerebral ischemia and the influence of gender in the stroke-prone spontaneously hypertensive rat (SHRSP). Halothane-anesthetized, 3- to 5-month-old male and female Wistar-Kyoto rats (WKY), SHRSP, and the first filial generation rats (F1 crosses 1 and 2) underwent distal (2 mm) permanent middle cerebral artery occlusion (MCAO) by electrocoagulation. Infarct volume was measured by using hematoxylin-eosin-stained sections and image analysis 24 hours after ischemia and expressed as a percentage of the volume of the ipsilateral hemisphere. Infarct volume in males and females grouped together were significantly larger in SHRSP, F1 cross 1 (SHRSP father), and F1 cross 2 (WKY father), at 36.6+/-2.3% (mean+/-SEM, P<0.001, n=15), 25.4+/-2.4% (P<0.01, n=14), and 33. 9+/-1.6% (P<0.001, n=18), respectively, compared with WKY (14+/-2%, n=17). Male F1 cross 1 (18.9+/-2.4%, n=6) developed significantly smaller infarcts than male F1 cross 2 (32.8+/-2%, n=8, P<0.005). Females, which underwent ischemia during metestrus, developed larger infarcts than respective males. A group of females in which the cycle was not controlled for developed significantly smaller infarcts than females in metestrus. Thus, the increased sensitivity to MCAO in SHRSP is retained in both F1 cross 1 and cross 2 hybrids, suggesting a dominant or codominant trait; response to cerebral ischemia appears to be affected by gender and stage in the estrous cycle. In addition, the male progenitor of the cross (ie, SHRSP versus WKY) influences stroke sensitivity in male F1 cohorts.

Female reproductive cycle influences plasma volume and protein restitution after hemorrhage in the conscious rat.

The present study was designed to test the hypothesis that gender and female reproductive cycle phase influence the restitution of blood volume following blood loss. The experiments were performed in conscious 9- to 11-wk-old Sprague-Dawley rats subjected to a slow hemorrhage of 19 ml/kg over 40 min. The effect of hemorrhage was compared in male rats and in female rats in either proestrus (Pro) or metestrus (Met). In comparison with either metestrus females or males, females hemorrhaged on the morning of proestrus showed a significantly larger overall decrease in percent hematocrit (Pro, -17 +/- 1; Met, -10 +/- 1; male, -13 +/- 1; mean +/- SE), increase in plasma volume (Pro, to 151 +/- 15% of initial volume; Met, 104 +/- 8%; male, 120 +/- 4%), and increase in total plasma protein content (Pro, to 164 +/- 14% of initial content; Met, 101 +/- 5%; male, 132 +/- 5%) over the 21-h posthemorrhage recovery period. Proestrus females also showed a larger increase in plasma osmolality during the first 2.5-h posthemorrhage than either metestrus females or males (P < 0.05). In addition, basal hematocrit and total blood volume (51Cr-tagged erythrocyte method) were higher in females studied on the morning of proestrus than in metestrus females. These results indicate that the female reproductive cycle is an important variable in basal blood volume regulation and in plasma protein and plasma volume restitution following blood loss.

The actions of diazepam and serotonergic anxiolytics vary according to the gender and the estrous cycle phase

The anxiolytic effect of diazepam (0.5, 1.0 and 2.0 mg/kg), buspirone (2.5 and 5.0 mg/kg) indorenate (2.5 and 5.0 mg/kg) and ipsapirone (5.0 and 10.0 mg/kg) was evaluated in male and female rats during the proestrus and metestrus phases. The burying behavior test was used to measure the anxiety levels. In this test, increases in the behavior latency are interpreted as prolonged reactivity, while reductions in the burying behavior are considered to reflect anxiolytic states. Diazepam increases in burying behavior latency were consistently higher than those observed after serotonergic anxiolytics. Buspirone, at no dose tested, affected the burying behavior latency, while indorenate and ipsapirone had only minor effects. Male individuals were more sensitive than females to the actions of diazepam on burying behavior. The serotonergic anxiolytics produce similar responses in both sexes. Metestrus females were much less sensitive to the action of all anxiolytics on burying behavior latency than proestrus females. Proestrus females were highly sensitive to the actions of diazepam on burying latency as compared both with males and metestrus females. Data show that a larger gender and within females variation occurs after treatment with diazepam as compared with the serotogergic anxiolytics. The results are discussed considering the relationships between ovarian hormones and the GABA-benzodiazepinic and serotonergic systems.