Tyrosine kinase effects on adrenoceptor-stimulated cyclic AMP accumulation in preoptic area and hypothalamus of female rats: modulation by estradiol

These studies examined the functional interactions between adrenergic G-protein coupled receptors and protein tyrosine kinases in the preoptic area and hypothalamus, brain regions that regulate reproductive function in female rats, and evaluated whether in vivo treatment with estradiol for 2 days modulates the cross-talk between these two signaling pathways. In hypothalamic slices genistein, a general tyrosine kinase inhibitor, enhances norepinephrine-stimulated cAMP synthesis independent of estradiol treatment. Genistein appears to act by increasing beta-adrenoceptor signaling. At high norepinephrine concentrations, estradiol potentiates genistein enhancement of the cAMP response in hypothalamic slices. This interaction between estradiol and genistein appears to involve modification of alpha(2)-adrenoceptor signaling mechanisms. In preoptic area slices, genistein enhancement of norepinephrine-stimulated cAMP synthesis is only observed in estradiol-treated rats. In this brain region, genistein enhances cAMP accumulation by modifying alpha(1)- and/or alpha(2)-adrenoceptor rather than beta-adrenoceptor signaling. Genistein amplification of norepinephrine-stimulated cAMP synthesis is not mediated by interactions with estrogen receptors, or by regulation of adenylyl cyclase or phosphodiesterase activities. At the concentration used, genistein inhibits tyrosine phosphorylation in slices from both brain regions. Daidzein, an inactive analogue of genistein, fails to enhance the norepinephrine-stimulated cAMP response in either brain region independent of hormone treatment. These results suggest that protein tyrosine kinases regulate adrenergic responses in the hypothalamus and preoptic area. Moreover, the functional interaction between adrenergic G-protein coupled receptor signaling and protein tyrosine kinases is modified in a brain region and receptor subtype specific manner by estradiol.     

Efferents of the medial preoptic area in the guinea pig: An autoradiographic study

Medial preoptic axons were traced into the diagonal band of Broca and septum, particularly lateral septum. Other labeled fibers could be followed dorsally from medial preoptic area injections adjacent to the stria medullaris, and in the periventricular fiber system and the stria terminalis and its bed nucleus. The anterior and medial amygdaloid nuclei were labeled by fibers via the stria terminalis and others arching over the optic tract and through the substantia innominata. The lateral habenula was labeled. Labeled periventricular fibers reached the periventricular nucleus of the thalamus. Descending efferents were traced principally below the fornix and in the adjacent lateral hypothalamus to label the anterior hypothalamus, the tuberal nuclei, and median eminence. Axons of the medial preoptic area joined the medial part of the medial forebrain bundle and distributed to the reticular formation and the central gray of the midbrain and pons. A small amount of contralateral connections were described.     

Location of putative glutamatergic neurons projecting to the medial preoptic area of the rat hypothalamus

The medial preoptic area is a key structure in the neural control of reproduction. Considerable evidence has accumulated indicating that glutamatergic innervation of the area plays an important role in this control. Sources of the glutamatergic input are unknown. Present investigations were aimed at studying this question. [3H]D-aspartate, which is selectively taken up by high-affinity uptake sites at presynaptic endings that use glutamate or aspartate as a transmitter, and is transported back to the cell body, was injected into the medial preoptic area. The neurons retrogradely labelled with [3H]D-aspartate were detected autoradiographically. Labelled cells were found in several telencephalic and diencephalic structures, but not in the brainstem. Within the telencephalon, labelled neurons were detected in the lateral septum, bed nucleus of the stria terminalis and amygdala. Diencephalic structures included the medial preoptic area itself, hypothalamic paraventricular, suprachiasmatic, ventromedial, arcuate, ventral premammillary, supramammillary and thalamic paraventricular nuclei. All of them are known to project to this area. The findings provide the first neuromorphological data on the location of putative glutamatergic neurons projecting to the medial preoptic area. Furthermore, they indicate that local putative glutamatergic neurons as well as several telencephalic and diencephalic structures contribute to the glutamatergic innervation of the area.     

A lumpers versus splitters approach to sexual differentiation of the brain

Over 50 years of rigorous empirical attention to the study of sexual differentiation of the brain has produced sufficient data to reveal fundamental guiding principles, but has also required the generation of new hypotheses to explain non-conforming observations. An early emphasis on the powerful impact and essential role of gonadal steroids is now complemented by an appreciation for genetic contributions to sex differences in the brain. The organizing effects of early steroid hormones on reproductively relevant brain regions and endpoints are largely dependent upon neuronal aromatization of androgens to estrogens. The effect of estradiol is mediated via estrogen receptors (ER). The presence or absence of ER can restrict hormone action to select cells and either prevent or invoke cell death. Alternatively, ER activation can initiate signaling cascades that induce cell-to-cell communication and thereby transduce organizational steroid effects to large numbers of cells. However, the specific details by which cell death and cell-to-cell communication are achieved appear to be locally, even cellularly, unique and specific to that particular subpopulation. As the field moves forward the increasingly specific and detailed elucidation of mechanism challenges us to generate new guiding principles in order to gain a holistic understanding of how the brain develops in males and females.     

Estrogen receptor-immunostaining of neuronal cytoplasmic processes as well as cell nuclei in guinea pig brain

We have recently developed an immunocytochemical technique for staining estrogen receptors in cell nuclei of some cells in the guinea pig brain. With optimization of this technique, we have now been successful in staining estrogen receptor-immunoreactivity in processes of many neurons in the guinea pig brain that also contain estrogen receptor-immunoreactive cell nuclei. While reaction product is visible in cell nuclei under a wide variety of conditions, neuronal processes are darkly immunostained only with modifications of the procedure optimized for maximum sensitivity, for example, the multiple-bridge immunocytochemical procedure. Omission of Triton X-100 and/or dimethylsulfoxide, usually used to increase penetration of the antibodies, had no effect on immunostaining in processes or cell nuclei, and immunostaining was apparent in both vibratome-cut and freezing microtome-cut sections. Injection of estradiol, but not progesterone, caused the total loss of cytoplasmic estrogen receptor-immunostaining, consistent with the idea that the cytoplasmic immunostaining, like the cell nuclear immunostaining is due to the presence of estrogen receptors. In all neuroanatomical regions containing estrogen receptor-immunoreactive cell nuclei, associated processes of some, but not all cells were also immunostained. However, in certain areas, such as the midbrain central gray and the preoptic area, cytoplasmic staining was particularly dark. The cellular characteristics that result in immunostaining in some estrogen receptor-immunoreactive cells, and not in others is under investigation.     

Effects of tamoxifen, fluphenazine and estradiol on ATP levels in preoptic area and hypothalamic slices from ovariectomized rats

Tamoxifen, the major adjuvant drug treatment for estrogen-dependent breast cancer, has been shown previously to affect both estrogen-dependent and calcium/calmodulin-dependent pathways. In the current study, we developed an in vitro slice system to study the effects of tamoxifen on ATP levels in hypothalamic (HTH) and preoptic areas (POA) of the rat brain. Baseline data showed that, following a 2-h incubation, HTH and POA slices had comparable ATP levels to hippocampal slices, a system used extensively by researchers examining the metabolic responsiveness of the hippocampal region (HPC) of the brain. HTH–POA slice ATP levels remained steady for 2, 4 and 6 h, but fell to 11% of initial levels by 12 h. Neurons from HTH–POA slices incubated for 4 h appeared healthy and demonstrated robust protein synthesis as measured autoradiographically by incorporation of [³H]leucine. We explored the effects of tamoxifen (TAM), fluphenazine (FLU) and estradiol (E₂) on ATP levels in HTH and POA slices. The effects of TAM were complex: a 4-h incubation with 10⁻⁶ M TAM led to decreased ATP levels in HTH (but not POA), and a 4-h incubation with 10⁻⁸ M led to increased ATP levels in POA (but not HTH); a 15-min exposure to 10⁻⁶ M TAM decreased ATP levels in POA (but not HTH) slices, while the exposure of slices to the lower concentration of TAM was without effect in either area. As with higher concentrations of TAM, 4-h incubation with 10⁻⁶ M FLU decreased ATP levels in HTH (but not POA), while incubation with E₂ did not affect slice ATP levels. These data are consistent with the hypothesis that both TAM and FLU alter ATP levels in HTH slices via calmodulin- or calcium-mediated processes.     

α-Adrenergic regulation of androgen receptor concentration in the preoptic area of the rat

We examined the effect of the pharmacological disruption of the catecholaminergic system on the concentration of nuclear androgen receptor, as measured by the in vitro binding of methyltrienolone ([³H]R1881) to salt extracts of anterior pituitary (AP), preoptic are (POA) and medial basal hypothalamus (MBH). Treatment of gonadectomized male and female rats with the dopamine-ß-hydroxylase inhibitor, diethyldithiocarbamate (400 mg/kg b. wt.), 30 min before treatment with dihydrotestosterone (1 mg/animal) produced a decrease in the number of nuclear androgen receptor compared with saline-treated controls (P<0.05). This effect was specific for the POA and was not present 15 h after DHT treatment. There was no effect on cytosolic androgen receptor nor was there a drug effect on the apparent dissociation constant (K_d) of [³H]R1881 binding to hypothalamus-preoptic area cytosols. Treatment of intact males and castrated, testosterone-treated males with thea₁- anda₂-adrenergic antagonists, prazosin (5 mg/kg b. wt.) and yohimbine (2 mg/kg b. wt.), respectively, resulted in a significant decrease in the number of nuclear AR 2 h following drug treatment (P<0.05). There was no effect of the ß-adrenergic receptor antagonist propranolol (10 mg/kg b. wt.) when given to intact animals, nor was there an effect of idazoxan (5 mg/kg) when given to testosterone-treated animals. The effects of yohimbine and prazosin were restricted to the POA. None of the drugs competed with the binding of [³H]R1881 for the androgen receptor nor did they alter theK_d of cytosol or nuclear androgen receptor. These data provide evidence for an adrenergic interaction with the POA androgen receptor and suggest a role for catecholamines in modulating androgen sensitivity in the rat brain.     

Estrogen binding in nuclear and cytosolic extracts from brain and pituitary of middle-aged female rats

Estrogen binding was compared in brain and pituitary of long-term ovariectomized young and middle-aged (MA) female rats. Binding was quantified in both cytosolic and nuclear extracts to ascertain whether fractions of estrogen binding are altered in MA females. Estrogen binding detected in nuclear extracts from hypothalamus/preoptic area and anterior pituitary of MA females was significantly lower than levels detected in young females. In each case where an age-related decrease in nuclear estrogen binding was observed, an increased number of putative estrogen receptors was detected in the cytosolic extract. Therefore, the age-related decrease in nuclear estrogen binding did not appear to result from a simple decrease in total available cellular estrogen receptors. Rather these results suggest a decrease in the ability of putative estrogen receptors in aging females to remain tightly bound to nuclei after their isolation. The ability of estrogen receptor complexes from aging animals to bind to DNA was evaluated by DNA-cellulose chromatography in order to examine possible quantitative or qualitative differences in estrogen binding proteins with age. The data did not indicate that the properties of estrogen receptors themselves changed with age. It is possible, therefore, that age-related alterations may interfere with the interaction between the estrogen receptor complex and the nucleus.     

Fos expression within regions of the preoptic area, hypothalamus and brainstem during pregnancy and parturition

Vaginocervical stimulation, that occurs during mating or with the birth of pups, is believed to induce specific sexual and maternal behaviours in the rat as well as stimulating a number of neuroendocrine responses including the secretion of oxytocin, prolactin and luteinizing hormone. Since the medial preoptic area has been implicated in the induction of maternal behaviour, the expression of the immediate-early gene product Fos was compared between non-pregnant, late pregnant and parturient rats. Although no difference was detected in the number of Fos-positive neuronal profiles in the preoptic area of non-pregnant and late-pregnant rats, a large increase was observed in the medial preoptic nucleus and the anteroventral periventricular region, as well as in the hypothalamic supraoptic nucleus, of parturient rats. Double labelling for Fos and tyrosine hydroxylase immunoreactivity in the brainstem of parturient rats showed the activation of catecholaminergic neurons in both the nucleus of the tractus solitarius and in the ventrolateral medulla that may form part of the afferent pathway from the uterus and cervix to the preoptic area and hypothalamus.     

Presence of sex difference of cytochrome P-450 in the rat preoptic area and hypothalamus with reference to coexistence with oxytocin

Localization of female type cytochrome P-450 (F1) in the preoptic area and hypothalamus of the rat was examined immunocytochemically using antiserum against purified hepatic P-450 (F1). This antiserum recognizes both P-450 (F1) and P-450 (M3). Western immunoblotting using the antiserum demonstrated that female rat brain contains P-450 (F1) but not P-450 (M3), since microsomes from the brain and liver displayed only one immunoreactive band at 50 kD, coinciding with that of P-450 (F1) purified from female rat liver. On the other hand, the male brain has P-450 (M3) but not P-450 (F1), as liver- and brain-derived microsomes produced single band at 49 kD, which represents a mol. wt. identical to that of P-450 (M3) extracted from male rat liver. These results indicate that P-450 (F1)-like immunoreactivity (LI) occurs in the female rat brain, while P-450 (M3)-LI takes place in the male rat brain. Immunocytochemical analysis further demonstrated the detailed cellular localization of these two P-450-LIs in the preoptic area and hypothalamus of female and male rats. Localization of P-450 (F1)-LI in the female rat hypothalamus resembled that of P-450 (M3)-LI in the male rat hypothalamus. Magnocellular neurosecretory neurons in the paraventricular nucleus and supraoptic nucleus were labeled and were found to contain oxytocin but lack vasopressin when serial sections of these areas were analyzed. In addition, groups of immunoreactive cells were seen in the median preoptic nucleus, medial and lateral preoptic area, caudal portion of the bed nucleus of the stria terminalis, lateral hypothalamus at the level of the paraventricular nucleus, periventricular zone from the preoptic area to the paraventricular nucleus, and parvocellular portion of the paraventricular nucleus.     

Prostanoids in the preoptic hypothalamus mediate systemic lipopolysaccharide-induced hyperalgesia in rats

The systemic administration of lipopolysaccharide (LPS), an experimental model of systemic bacterial infection is known to modulate nociception. It increases the prostaglandin E₂ (PGE₂) levels in the preoptic area of the hypothalamus (POA) and the microinjection of PGE₂ into the POA and the neighboring basal forebrain induces hyperalgesia. We, therefore, hypothesized that the PGE₂ synthesized in these regions mediates intravenous (i.v.) LPS-induced hyperalgesia. To test this hypothesis, we microinjected cyclooxygenase (COX) inhibitors into several sites in the rat hypothalamus and observed their effects on the LPS (0.1–100 μg/kg, i.v.)-induced changes in nociceptive behavior as assessed by a plantar test. LPS (10 and 100 μg/kg, i.v.) reduced the paw-withdrawal latency at 90 min and 45–60 min after injection, respectively, both thus indicating a hyperalgesic effect. This hyperalgesia was observed only in the period before the development of fever which started 120–135 min after the LPS injection. The LPS (100 μg/kg, i.v.)-induced hyperalgesia was completely abolished by pretreatment with the microinjection of diclofenac (an inhibitor of COX-1 and 2) at 1.0 ng into the bilateral POA. Furthermore, it was also blocked by the microinjection of NS-398 (a selective COX-2 inhibitor) at 1.0 ng into the bilateral POA and the horizontal limb of the diagonal band of Broca (DBB), but not the lateral hypothalamic area, the paraventricular hypothalamic nucleus, and the ventromedial hypothalamic nucleus. These findings suggest that LPS (i.v.)-induced hyperalgesia is mediated predominantly through a COX-2 induced prostanoids in the POA and the DBB in rats.     

Reduction of neurotensin immunoreactivity in the amygdala in Alzheimer''s disease

The density of neurotensin immunoreactivity (NT-IR) was dramatically decreased in 6 of 12 amygdaloid nuclear subregions in patients with Alzheimer's disease (AD) compared to age-matched normals. Diminution of NT-IR was most pronounced in amygdaloid regions containing the greatest number of senile plaques. This contrasts to our previous findings of little, if any, loss of substance P or somatostatin immunoreactivity within these same regions. The present findings corroborate biochemical reports of a decrease in NT-IR in the AD amygdala and suggest that this peptide may be selectively affected relative to other neuropeptides.     

Distribution of androgen receptor-like immunoreactivity in the brains of intact and castrated male hamsters

The distribution of androgen receptor-like (AR) immunoreactivity was mapped in brains of (a) intact, sham-castrated and (b) castrated male hamsters. The pattern of AR-immunoreactive (AR-ir) staining was, in general, similar to that reported for gonadal steroid autoradiography of the male hamster brain. Moreover, with one exception, AR-like staining was similar in intact and castrated males, and occurred in the medial preoptic area, bed nucleus of stria terminalis, amygdala, hippocampus, thalamus, and several hypothalamic nuclei including the periventricular, supraoptic, and ventromedial nuclei, and median eminence. However, while AR-ir labeling was virtually absent in the lateral septum of intact males, it was clearly present in the lateral septum of castrated males. The view that androgen receptors in brain generally decline after castration received no support from this study.     

Glucose transporter immunoreactivity in the hypothalamus and area postrema

To study the glucose transporter (GT) protein in two glucose-sensitive areas of the rat brain, frozen coronal sections at the level of the median eminence (ME) and area postrema (AP) were stained immunocytochemically with an antibody raised against human erythrocyte glucose transporter. Immunoreactivity was mainly confined to blood vessels in most brain areas but was lacking in those of the ME and AP, which also lack a normal blood-brain barrier. This suggests that glucose entry into these brain areas, unlike others, is not limited or regulated by capillary glucose transport systems. Tancyte processes stained strongly for GT and for glycogen and thus may have an unusual glucose metabolism.     

Neural distribution of estrogen receptor immunoreactive cells in the female musk shrew

In this report we describe the neural distribution of estrogen receptor immunoreactivity (ER-IR) in the female musk shrew (Suncus murinus). The highest concentrations of neurons containing ER-IR were found in the preoptic areas, the lateral septum, the anterior arcuate and ventromedial nuclei of the hypothalamus, the medial nuclei of amygdala, and the midbrain central grey. Additional preoptic, hypothalamic, limbic and midbrain nuclei also contained ER-IR cells. The distribution of ER-IR was similar to that described in other mammals and birds, with several important differences. In the female musk shrew, there was little difference in ER-IR intensity or distribution when brains from gonadally intact and ovariectomized musk shrews were compared. In addition, long-term treatment with a supraphysiological dose of estradiol was required to detect a decrease in ER-IR intensity. Finally ER-IR was noted in both nuclear and cytoplasmic regions of cells in ovariectomized and gonadally intact musk shrews. The dense ER-IR noted in intact females as well as the presence of cytoplasmic stain may be due to the unusual relationship between estradiol, ovulation and sexual receptivity in this species.     

Immunocytochemical localization of nicotinic acetylcholine receptor in rat hypothalamus

Immunocytochemical localization of neuronal nicotinic acetylcholine receptor (nAChR) was examined in rat hypothalamus. Monoclonal antibody against α4 ACh-binding subunits of nAChR was used in the avidin-biotin-peroxidase complex (ABC) immunocytochemical method at both the light and electron microscopic levels. By light microscopy nAChR-like immunoreactivity was found in many neuronal cell bodies and their fibers in the paraventricular nucleus (PVN) and in many axons and axon terminals in the median eminence (ME). The immunoreactivity of nAChR was the most intense in the ME. By electron microscopy immunoreaction products occurred on the rough endoplasmic reticulum, nuclear envelope, cytoplasmic matrices and postsynaptic densities of synaptic junctions in some neurons in the parvocellular part of the PVN. In the external layer of the ME, nAChR-like immunoreactivity was found over the entire plasma membranes of many axon terminals. Involvement of nAChRs in the release of neurotransmitters and neuropeptides both in the PVN and the ME is discussed.     

Leptin receptors in estrogen receptor-containing neurons of the female rat hypothalamus

This study was undertaken to reveal whether integration of the peripheral signals, leptin and estradiol, that convey information on the metabolic state and gonadal function, respectively, might occur in the same hypothalamic neuronal perikarya. Light and electron microscopic immunolabeling for leptin receptors (LRs) and estrogen receptors (ERs) was carried out on hypothalamic sections of female rats. In the medial preoptic area, periventricular regions, including the parvicellular paraventricular nucleus, the arcuate nucleus and the ventromedial hypothalamic nucleus, all of the cells that expressed immunoreactivity for ERs were also immunopositive for LR. On the other hand, only a subpopulation of LR-containing cells was found to express ERs. The extensive colocalization of receptors for leptin and estrogen in neuronal perikarya of all parts of the hypothalamus suggests a closely coupled interaction between these peripheral signals in the regulation of a variety of behavioral and neuroendocrine mechanisms.     

Sex differences in the regulation of progesterone receptor isoforms expression in the rat brain

We studied the effects of estradiol (E2) and progesterone (P) on progesterone receptor (PR) isoforms gene expression in the brain of ovariectomised female and gonadectomised male rats by RT-PCR analysis. In female rats, the expression of both PR isoforms was induced by E2 and down-regulated by P in the hypothalamus, whereas in the preoptic area these changes were only observed in PR-B isoform. On the contrary, in the hippocampus and the olfactory bulb, PR-A was the isoform induced by E2. In these regions, P did not modify the expression of any PR isoform. In the cerebellum and the frontal cortex of female rats, no treatment modified PR isoforms expression. In contrast with female rats, in the male rat brain, PR isoforms expression was only modified in the cerebellum, where PR-A was induced by E2. These results demonstrate a clear sexual dimorphism in the regulation of PR isoforms expression by sex steroid hormones in the rat brain, suggesting that this sex difference contributes to the sexually dimorphic effects of P in the rat brain.     

Damage of the medial preoptic area impairs peripheral pilocarpine-induced salivary secretion

The existence of neural connections between the medial preoptic area (MPOA) and the salivary glands and the increase in salivation by thermal or electrical stimulation of the MPOA have suggested an important role of MPOA in the control of salivary gland function. Although direct cholinergic activation of the salivary glands induces salivation, recent studies have suggested that salivation produced by i.p. pilocarpine may also depend on the activation of central mechanisms. Therefore, in the present study, we investigated the effects of bilateral electrolytic lesions of the MPOA on the salivation induced by i.p. pilocarpine. Adult male Holtzman rats (n = 11-12/group) with bilateral sham or electrolytic lesions of the MPOA were used. One, five, and fifteen days after the brain surgery, under ketamine anesthesia, the salivation was induced by i.p. pilocarpine (1 mg/kg of body weight), and saliva was collected using pre-weighed small cotton balls inserted into the animal's mouth. Pilocarpine-induced salivation was reduced 1 and 5 days after MPOA lesion (341 +/- 41 and 310 +/- 35 mg/7 min, respectively, vs. sham lesions: 428 +/- 32 and 495 +/- 36 mg/7 min, respectively), but it was fully recovered at the 15th day post-lesion (561 +/- 49 vs. sham lesion: 618 +/- 27 mg/7 min). Lesions of the MPOA did not affect baseline non-stimulated salivary secretion. The results confirm the importance of MPOA in the control of salivation and suggest that its integrity is necessary for the full sialogogue effect of pilocarpine. However, alternative mechanisms probably involving other central nuclei can replace MPOA function in chronically lesioned rats allowing the complete recovery of the effects of pilocarpine.