Sex differences in depolarizing actions of GABAA receptor activation in rat embryonic hypothalamic neurons.

GABAA receptor activation exerts trophic actions in immature neurons through depolarization of resting membrane potential. The switch to its classical hyperpolarizing role is developmentally regulated. Previous results suggest that a hormonally biased sex difference exists at the onset of the switch in hypothalamic neurons. The aim of this work was to evaluate sex differences in GABAA receptor function of hypothalamic neurons before brain masculinization by gonadal hormones. Hypothalamic cells were obtained from embryonic day 16 male and female rat foetuses, 2 days before the peak of testosterone production by the foetal testis, and grown in vitro for 9 days. Whole-cell and perforated patch-clamp recordings were carried out in order to measure several electrophysiological parameters. Our results show that there are more male than female neurons responding with depolarization to muscimol. Additionally, among cells with depolarizing responses, males have higher and longer lasting responses than females. These results highlight the relevance of differences in neural cell sex irrespective of exposure to sex hormones.

Body sodium overload modulates the firing rate and fos immunoreactivity of serotonergic cells of dorsal raphe nucleus.

In order to determine whether serotonergic (5HT) dorsal raphe nucleus (DRN) cells are involved in body sodium status regulation, the effect of a s.c. infusion of either 2 M or 0.15 M NaCl on 5HT DRN neuron firing was studied using single unit extracellular recordings. In separate groups of 2 M and 0.15 M NaCl-infused rats, water intake, oxytocin (OT) plasma concentration, urine and plasma sodium and protein concentrations were also measured. Also, to determine the involvement of particular brain nuclei and neurochemical systems in body sodium overload (SO), animals from both groups were perfused for brain immunohistochemical detection of Fos, Fos-OT and Fos-5HT expression. SO produced a significant increase in serotonergic DRN neuron firing rate compared to baseline and 0.15 M NaCl-infused rats. As expected, 2 M NaCl s.c. infusion also induced a significant increase of water intake, diuresis and natriuresis, plasma sodium concentration and osmolality, even though plasma volume did not increase as indicated by changes in plasma protein concentration. The distribution of neurons along the forebrain and brainstem expressing Fos after SO showed the participation of the lamina terminalis, extended amygdala, supraoptic and paraventricular hypothalamic nuclei in the neural network that controls osmoregulatory responses. Both Fos-OT immunoreactive and plasma OT concentration increased after s.c. hypertonic sodium infusion. Finally, matching the "in vivo" electrophysiological study, SO doubled the number of Fos-5HT immunolabeled cells within the DRN. In summary, the results characterize the behavioral, renal and endocrine responses after body sodium overload without volume expansion and specify the cerebral nuclei that participate at different CNS levels in the control of these responses. The electrophysiological approach also allows us to determine in an "in vivo" model that DRN 5HT neurons increase their firing frequency during an increase in systemic sodium concentration and osmolality, possibly to modulate sodium and water intake/excretion and avoid extracellular volume expansion.

Sex chromosome complement contributes to sex differences in bradycardic baroreflex response.

To investigate whether sex chromosome complement modulates bradycardic baroreflex response and contributes to the angiotensin II-bradycardic baroreflex sex differences, we used the four core genotype mouse model in which the effect of gonadal sex and sex chromosome complement is dissociated, allowing comparisons of sexually dimorphic traits among XX and XY females, as well as in XX and XY males. In conscious gonadectomized (GDX) MF1 transgenic mice we evaluated baroreflex regulation of heart rate in response to changes in blood pressure evoked by phenylephrine (1 mg/mL), angiotensin II (100 µg/mL), and sodium nitroprusside (1 mg/mL). The administration of phenylephrine in GDX-XY females resulted in a significantly lower baroreflex response when compared with the other genotypes (in beats · min(-1) · mm Hg(-1) [slopes of regression lines for GDX-XY females -3.56±0.37 versus -6.06±0.38, -6.37±0.54 and -6.70±0.34 for GDX-XY male, GDX-XX female, and GDX-XX male mice, respectively]) {F(1,19)=9.63; P<0.01}. In addition, in both GDX-XY males and females, the angiotensin II-bradycardic baroreflex response was attenuated when compared with heart rate changes in GDX-XX male and female mice (in beats · min(-1) · mm Hg(-1) [slopes of regression lines: -2.83±0.28 versus -5.76±0.26 in GDX-XY and GDX-XX mice, respectively]) {F(1,19)=13.91; P<0.005}. In contrast, reflex tachycardic responses to sodium nitroprusside were comparable in all of the genotypes. These data support the hypothesis that sex chromosome complement modulates reflex inhibition of heart rate to phenylephrine and angiotensin II. Elucidating the foundational sources of sexually dimorphic traits in the regulation of baroreceptor reflex may enable the design of more appropriate sex-tailored therapeutic treatments in the future.

Lack of feedback inhibition on rat basolateral amygdala following stress or withdrawal from sedative-hypnotic drugs.

Previous research has demonstrated that suppression of inhibition in projection neurons of the basolateral complex of the amygdala (BLA) represents an essential mechanism underlying the emergence of negative emotional responses, including exaggerated fear and anxiety. The present work evaluates inhibitory postsynaptic potentials (IPSPs) in pyramidal projection neurons of the BLA in rats subjected to either diazepam or ethanol withdrawal or uncontrollable stress. These are experimental paradigms conducive to a negative emotional state. In slices containing the BLA, IPSPs were studied using whole-cell patch clamp. In control animals, a small IPSP was evoked by sub-threshold stimulation of the external capsule. When an action potential (AP) was evoked by supra-threshold stimuli, IPSPs were considerably larger; these IPSPs were sensitive to blockade of GABA(A) receptors by picrotoxin. However, IPSPs were clearly reduced in diazepam- or ethanol-withdrawn and in stressed rats. Firing of an AP by a depolarizing pulse applied through the patch pipette consistently evoked an inhibitory postsynaptic current (IPSC) in the pyramidal neurons of control animals from all three experimental models; these IPSCs were mediated by GABA(A) receptor activation and were blocked after suppression of glutamatergic transmission. In contrast, no IPSCs were observed in slices from diazepam- or ethanol-withdrawn or stressed animals, although the depolarizing pulse regularly evoked an AP in pyramidal neurons. It is concluded that, in withdrawn or stressed rats, GABAergic disinhibition occurs due to attenuation or suppression of feedback inhibition.

Previous stress facilitates fear memory, attenuates GABAergic inhibition, and increases synaptic plasticity in the rat basolateral amygdala.

In experiments designed to investigate the relationship between stress and the acquisition of new fear memories, it was found that previous exposure to a restraint session increased fear conditioning in a contextual fear paradigm. Moreover, the infusion of bicuculline, a competitive antagonist of GABAA receptors, into the basolateral amygdala complex (BLA), but not into the central amygdaloid nucleus, induced the same behavioral effect. Pretreatment with midazolam (MDZ), a positive modulator of GABAA sites, prevented the facilitating influence on fear memory of both stress and GABAA receptor blockade in the BLA. These data suggest that facilitation of fear conditioning could be causally related to increased neuronal excitability attributable to depressed GABAergic inhibition in the BLA. To test this hypothesis, evoked potentials were studied in brain slices from stressed animals. Potentials evoked in the BLA by single stimuli applied to the external capsule showed multispike responses, suggestive of GABAergic disinhibition. These multiple responses were no longer evident after the slices were perfused with diazepam or if the stressed animals were pretreated with MDZ. In slices from stressed rats, paired-pulse inhibition (GABA dependent) was suppressed. Also, in stressed animals, long-term potentiation (LTP) was induced with a single train of high-frequency stimulation, which did not induce LTP in control rats. Moreover, MDZ pretreatment prevented the facilitating influence of stress on LTP induction. All of these findings support the hypothesis that previous stress attenuates inhibitory GABAergic control in the BLA, leading to neuronal hyperexcitability and increased plasticity that facilitates fear learning.

Increased fear learning coincides with neuronal dysinhibition and facilitated LTP in the basolateral amygdala following benzodiazepine withdrawal in rats.

Animals chronically administered with diazepam (DZM -- 2 mg/kg/day i.p.) or vehicle (VEH) for 21 days were tested in a fear-conditioning paradigm 4 days after the last administration. Increased freezing was observed in DZM withdrawn rats as compared to VEH injected control rats in both associative and nonassociative context and this increase was greatest for the DZM withdrawal group in the paired context. In animals anesthetized with urethane, single pulses in the medial prefrontal cortex evoked a field potential including a population spike (PS) in the basolateral complex of the amygdala (BLA) of control animals, whereas in DZM withdrawn animals multiple PSs were evoked. In brain slices from control rats, stimulation of the external capsule evoked a field potential including a PS in the BLA, whereas in DZM withdrawn rats multiple PSs were evoked. The amplitude of the PS was smaller in slices obtained from DZM withdrawn rats than from control rats, and paired pulse inhibition was significantly less in the former. Perfusion with DZM 2 microM of slices obtained from DZM withdrawn rats eliminated repetitive spiking. GABAergic blockade with 50 microM picrotoxin in control rats resulted in the appearance of multiple secondary PSs. In slices from DZM withdrawn rats high-frequency stimulation induced a highly significant potentiation that lasted at least 2 h (LTP), whereas in control rats the same stimulation did not induce LTP. Neuronal hyperexcitability leading to facilitated LTP observed in BLA of DZM withdrawn rats could be due to depressed GABAergic activity (dysinhibition). The increased synaptic plasticity may be at the root of the increased fear learning observed in withdrawn animals.

Estradiol regulates the slow Ca2+-activated K+ current in hippocampal pyramidal neurons.

The slow Ca2+-activated K+ current (sIAHP) was recorded in CA1 pyramidal neurons in hippocampal slices obtained from ovariectomized (OVX) or sham OVX (control) female rats. The sIAHP was significantly larger in cells from OVX rats than in cells from control rats. Superfusion with 5-100 nm 17beta-estradiol (E2) caused a progressive decrease in the sIAHP in cells from OVX rats but not in cells from control rats. In slices from OVX rats injected with 10 microg of E2 24 and 48 hr before they were killed, superfusion with E2 did not modify the sIAHP. In neurons from OVX rats, but not in neurons from control rats, E2 significantly increased both the number of action potentials and the burst duration generated by depolarizing pulses. The inactive isomer 17alpha-estradiol had no effect. The impermeant protein conjugate E2--BSA was as effective as free E2 at decreasing the sIAHP. Ca2+ spikes were also depressed by E2 in neurons from OVX rats, but not in control rats. A decrease in the intracellular Ca2+ signal, correlating with the inhibition of the Ca2+ spike and sIAHP produced by E2, was observed only in neurons from OVX rats. Our results indicate that ovariectomy increases the sIAHP and depresses excitability, whereas bath application or priming with E2 decreases the sIAHP, thus promoting excitability. These effects of E2 on the sIAHP and excitability, which are stereospecific and presumably mediated by membrane-bound receptors, could contribute to the hormonal regulation of synaptic plasticity and epileptiform activity as well as to learning and cognitive abilities dependent on the function of hippocampal neural circuits.

Sexual differentiation of the brain: genes, estrogen, and neurotrophic factors.

Based on evidence obtained during the past 50 years, the current hypothesis to explain the sexual dimorphism of structure and function in the brain of vertebrates maintains that these differences are produced by the epigenetic action of gonadal hormones. However, evidence has progressively accumulated suggesting that genetic mechanisms controlling sexual-specific neuronal characteristics precede, or occur in parallel with, hormonal effects. 1. In cultures of hypothalamic neurons taken from gestation day 16 (GD16) embryos, treatment of sexually segregated cultures with estradiol (E2) induces axon growth in neurons from male neurons, but not from female neurons. In these cultures treatment with E2 increased the levels of tyrosine kinase type B (TrkB) and insulin-like growth factor I (IGF-I) receptors in male but not in female neurons. This and other sex differences cannot be explained by differences in hormonal environment, because the donor embryos were obtained when gonadal secretion of steroids is just beginning, before the perinatal surge of testosterone that determines development of the male brain beginning at GD17/18. 2. The response to estrogen is contingent upon coculture with heterotopic glia (mostly astrocytes) from a target region (amygdala) harvested from same-sex fetuses at GD16, whereas in the presence of homotopic glia or in cultures without glia, E2 had no effect. It was concluded that the axogenic effect of E2 depends on interaction between neurons and glia from a target region and that neurons from fetal male donors appear to mature earlier than neurons from females, a differentiated response that takes place prior to divergent exposure to gonadal secretions. 3. The effects of target and nontarget glia-conditioned media (CM) on the E2-induced growth of neuronal processes of hypothalamic neurons obtained from sexually segregated fetal donors were also studied. Estrogen added to media conditioned by target glia modified the number of primary neurites and the growth of axons of hypothalamic neurons of males but not of females. 4. Neither the Type III steroidal receptor blocker tamoxifen nor Type I antiestrogen ICI 182,780 prevented the axogenic effects of the hormone. Estradiol made membrane-impermeable by conjugation to a protein of high molecular weight (E2-BSA) preserved its axogenic capacity, suggesting the possibility of a membrane effect responsible for the action of E2. 5. Western blot analysis of the tyrosine kinase type A (TrkA), type B (TrkB), type C (TrkC), and insulin-like growth factor (IGF-I R) receptors in extracts from homogenates of cultured hypothalamic neurons showed that in cultures of male-derived neurons grown with E2 and CM from target glia, the amounts of TrkB and IGF-I R increased notably. Densitometric quantification showed that these cultures had more TrkB than cultures with CM alone or E2 alone. On the contrary, in cultures of female-derived neurons, the presence of CM alone induced maximal levels of TrkB, which were not further increased by E2; female-derived neurons in all conditions did not contain IGF-I R. Levels of TrkC were not modified by any experimental condition in male- or female-derived cultures and Trk A was not found in the homogenates. These results are compared with similar data from other laboratories and integrated in a model for the confluent interaction of estrogen and neurotrophic factors released by glia that may contribute to the sexual differentiation of the brain.

Sexual receptivity after destruction of serotonergic terminals in hypothalamus or amygdala

Con el fin de localizar los sitios del cerebro donde las aferencias serotoninérgicas podrian regular las respuestas lordóticas, se inyectó la neurotoxina 5-7-dihidroxitriptamina en el núcleo ventromediano hipotalámo (VMN) o en la amígdala medial (AM) inducida por administración de estrógeno fue medida mediante el cociente lordótico, siendo significantemente mayor en animales que recibieron la neurotoxina en AM. Por el contrario, el cociente loraótico fue menor en animales inyectados en el VMN. No se encontraron diferencias significantes con los controles cuando los mismos animales fueron tratados con progesterona. Se propone que las aferencias serotoninérgicas actuan en el VMN facilitando las respuestas lordóticas, mientras que dichas aferencias tienen un efecto opuesto en AM. No es necesario que los terminales serotoninérgicos en VMN y AM estén intactos para que la progesterona ejerza su efecto potenciador de la receptividad sexual

Sexual receptivity after destruction of serotonergic terminals in hypothalamus or amygdala

Con el fin de localizar los sitios del cerebro donde las aferencias serotoninérgicas podrian regular las respuestas lordóticas, se inyectó la neurotoxina 5-7-dihidroxitriptamina en el núcleo ventromediano hipotalámo (VMN) o en la amígdala medial (AM) inducida por administración de estrógeno fue medida mediante el cociente lordótico, siendo significantemente mayor en animales que recibieron la neurotoxina en AM. Por el contrario, el cociente loraótico fue menor en animales inyectados en el VMN. No se encontraron diferencias significantes con los controles cuando los mismos animales fueron tratados con progesterona. Se propone que las aferencias serotoninérgicas actuan en el VMN facilitando las respuestas lordóticas, mientras que dichas aferencias tienen un efecto opuesto en AM. No es necesario que los terminales serotoninérgicos en VMN y AM estén intactos para que la progesterona ejerza su efecto potenciador de la receptividad sexual (AU)