Progesterone receptor,estrogen receptor alpha,and the type II glucocorticoid receptor are coexpressed in the same neurons of the ovine preoptic area and arcuate nucleus: a triple immunolabeling study

The neuroendocrine reproductive and stress axes are known to be closely linked, but the mechanisms underlying these links remain poorly understood. In the ovine brain, GnRH neurons do not contain type II glucocorticoid (GR), progesterone (PR), or alpha estrogen (ERalpha) receptors. We sought to determine whether PR, ERalpha, and GR coexist within the same hypothalamic neurons. A triple immunocytochemical study, involving antisera raised in three different species, was performed on cryostat sections from ovariectomized ewes treated either with estradiol and progesterone or with progesterone alone. All PR-immunoreactive neurons contained ERalpha, and about 95% of ERalpha were PR immunoreactive in the preoptic area and arcuate nucleus. Although the PR with ERalpha colocalization ratio was not affected by the steroid treatments, immunolabeling for PR was weaker in animals that did not receive estradiol. Numerous PR- and ERalpha-immunoreactive cells contain GR. PR+ERalpha+GR-immunoreactive cells represent 70% of PR, 65% of ERalpha, and 72% of GR in the preoptic area and 70% of PR, 66% of ERalpha, and 63% of GR in the arcuate nucleus. These results suggest that estrogen, progesterone, and glucocorticoids may influence the activity of the same neurons to modulate both reproductive and stress axes.     

Cardiovascular regulation and expressions of NO synthase-tyrosine hydroxylase in nucleus tractus solitarius of ovine fetus

The purpose of this study was to examine cardiovascular responses to fourth cerebral ventricle (4V) administration of nitroglycerin (NTG) or an inhibitor of nitric oxide (NO) synthase (NOS) in the near-term ovine and to determine whether, during birth, neuronal NOS (nNOS) is induced in noradrenergic A1 neurons in the medial nucleus tractus solitarius (mNTS). In chronically instrumented fetal sheep, 4V injection of NTG (1.2 nmol), an NO donor, produced an arterial blood depressor and a moderate decrease in heart rate. Arterial blood pressure is increased by 4V administration of NG-nitro-L-arginine methyl ester (10 nmnol), an inhibitor of NOS, in fetuses. Sections of the medulla from fetuses and newborn lambs were examined by using immunolabeling with tyrosine hydroxylase (TH) antibody combined with NADPH diaphorase (NADPHd) histochemistry, a marker of nNOS activity. The NADPHd-positive cells and TH-positive cells containing NADPHd reactivity were significantly increased in the mNTS of newborns compared with the fetuses. The results suggest that during birth, there is upregulation of NADPHd/nNOS in the noradrenergic neurons of mNTS resulting in a centrally mediated reduction of fetal arterial blood pressure.     

Distribution of NADPH Diaphorase-Exhibiting Primary Afferent Neurons in the Trigeminal Ganglion and Mesencephalic Trigeminal Nucleus of the Rabbit

1. Nitric oxide (NO) is highly reactive gaseous molecule to which many physiological and pathological functions have been attributed in the central (CNS) and peripheral (PNS) nervous system. The present investigation was undertaken to map the distribution pattern of the enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), and especially its neuronal isoform (nNOS) in the population of primary afferent neurons of the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN) of the rabbit.2. In order to identify neuronal structures expressing nNOS we applied histochemistry to its specific histochemical marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd).3. We found noticeable amount of NADPHd-exhibiting primary afferent neurons in TG of the rabbit under physiological conditions. The intensity of the histochemical reaction was highly variable reaching the maximum in the subpopulation of small-to-medium-sized neurons. The large-sized neurons were only weakly stained or actually did not posses any NADPHd-activity. In addition, NADPHd-positive nerve fibers were detected between clusters of the ganglionic cells and in the peripheral branches of the trigeminal nerve (TN). NADPHd-exhibiting MTN neurons were noticed in the whole rostrocaudal extent of the nucleus even though some differences were found concerning the ratio of NADPHd-positive versus NADPHd-negative cell bodies. Similarly, we observed striking diversity in the intensity of NADPHd histochemical reaction in the subpopulations of small-, medium-, and large-sized MTN neurons.4. The predominant localization of NADPHd in the subpopulation of small-to-medium-sized TG neurons which are generally considered to be nociceptive suggests that NO probably takes part in the modulation of nociceptive inputs from the head and face. Furthermore, we tentatively assume that NADPHd-exhibiting MTN neurons probably participate in transmission and modulation of the proprioceptive impulses from muscle spindles of the masticatory muscles and mechanoreceptors of the periodontal ligaments and thus provide sensory feedback of the masticatory reflex arc.     

Distribution and morphological features of nitrergic neurons in the porcine large intestine

The distribution of nitric oxide synthase (NOS), an enzyme involved in the synthesis of the presumed non-adrenergic noncholinergic inhibitory neurotransmitter nitric oxide (NO), was demonstrated in the enteric nervous system of the porcine caecum, colon and rectum. Techniques used were NOS-immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-histochemistry. Throughout the entire large intestine, NOS-immunoreactive (IR) and NADPHd-positive neurons were abundant in the myenteric and outer submucous plexus. In the inner submucous plexus, only a small number of positive neurons were found in the caecum and colon, while a moderate number was observed in the rectum. The nitrergic neurons in the porcine enteric nerve plexuses were of a range of sizes and shapes, with a small proportion showing immunostaining for vasoactive intestinal polypeptide. Varicose and non-varicose NOS-IR and NADPHd-positive nerve fibres were present in the ganglia and connecting strands of all three plexuses. Nerve fibres were also numerous in the circular muscle layer, scarce in the longitudinal muscle coat and negligible in the mucosal region. The abundance of NOS/NADPHd in the intrinsic innervation of the caecum, colon and rectum of the pig implicates NO as an important neuronal messenger in these regions of the gastrointestinal tract.     

NADPH-diaphorase histochemistry in the terminal abdominal ganglion of the crayfish

Nitric oxide (NO) has an important modulatory role on the processing of sensory signals in vertebrates and invertebrates. In this investigation we studied the potential sources of NO in the terminal abdominal ganglion of the crayfish, Pacifastacus leniusculus, using NADPH-diaphorase (NADPHd) histochemistry, with NADPHd acting as a marker for NO synthase (NOS). In the terminal ganglion a mean of 27 strongly labelled NADPHd-positive cell bodies were found, and of these 80% [of stained cell bodies] [corrected] occurred in three regions located in antero-lateral, central and posterior parts of the ganglion. Ventral and antero-ventral commissures as well as specific dorsal and ventral areas of the dendritic neuropil showed positive staining. Intense labelling was seen in the ventro-medial tract, and in the connective between the terminal ganglion and the 5th abdominal ganglion. In addition, some motor neurones and neurones with branches in the sensory commissures were NADPHd positive. Our finding that NADPHd-positive cells occur in consistent patterns in the terminal abdominal ganglion implies that NO may have a role in mechanosensory processing in the crayfish.     

Estrogen receptor and progesterone receptor-immunoreactive cells are not co-localized with gonadotropin-releasing hormone in the brain of the female mink (Mustela vison)

The distribution of gonadal steroid (estrogen, progesterone) receptors in the brain of the adult female mink was mapped by immunocytochemistry. Using a monoclonal rat antibody raised against human estrogen receptor (ER), the most dense collections of ER-immunoreactive (IR) cells were found in the preoptic/anterior hypothalamic area, the mediobasal hypothalamus (arcuate and ventromedial nuclei), and the limbic nuclei (amygdala, bed nucleus of the stria terminalis, lateral septum). Immunoreactivity was mainly observed in the cell nucleus and a marked heterogeneity of staining appeared from one region to another. A monoclonal mouse antibody raised against rabbit uterine progesterone receptor (PR) was used to identify the PR-IR cells in the preoptic/anterior hypothalamic area and the mediobasal hypothalamus (arcuate and ventromedial nuclei). This study also focused on the relationship between cells containing sex-steroid receptors and gonadotropin-releasing hormone (GnRH) neurons on the same sections of the mink brain using a sequential double-staining immunocytochemistry procedure. Although preoptic and hypothalamic GnRH neurons were frequently in close proximity to perikarya containing ER or PR, they did not themselves possess receptor immunoreactivity. The present study provides neuroanatomical evidence that GnRH cells are not the major direct targets for gonadal steroids and confirms for the first time in mustelids the results previously obtained in other mammalian species.     

Regulation by estradiol of hypothalamic somatostatin gene expression: possible involvement of somatostatin in the control of luteinizing hormone secretion in the ewe

In the ewe, the mediobasal hypothalamus (MBH) is the primary central site for estradiol to generate the preovulatory GnRH/LH surges and sexual behavior. This area contains numerous neurons expressing the estradiol receptor alpha, distributed in the ventromedial nucleus (VMN) and the infundibular nucleus (IN). A large proportion of these neurons express somatostatin, making this neuropeptide a potential candidate for transmission of the estradiol signal to the GnRH neurons located in the preoptic area. We tested this hypothesis using ovariectomized ewes that had been subjected to an artificial estrous cycle. In the first experiment, 22 h after progesterone removal, ewes received estradiol (treated ewes) or empty implants (control ewes) for 4 h and then were killed. Using in situ hybridization, we showed that this short estradiol treatment increased the somatostatin mRNA amount by about 50% in the VMN and 42% in the IN. In the second experiment, preovulatory estradiol signal was replaced by somatostatin intracerebroventricular (ICV) administration. This treatment abolished LH pulsatility and dramatically decreased the mean basal level of LH secretion while it did not affect the mean plasma GH concentration. We demonstrated that an increase in somatostatin mRNA occurs at the time of the negative feedback effect of estradiol on LH secretion during the early stage of the GnRH surge induction. As ICV somatostatin administration inhibits the pulsatile LH secretion by acting on the central nervous system, we suggest that somatostatin synthesized in the MBH could be involved in the estradiol negative feedback before the onset of the preovulatory surge.     

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

Histological characterization of gonadotropin-releasing hormone (GnRH) in the hypothalamus of the South American plains vizcacha (Lagostomus maximus)

In contrast to most mammalian species, females of the South American plains vizcacha, Lagostomus maximus, show an extensive suppression of apoptosis-dependent follicular atresia, continuous folliculogenesis, and massive polyovulation. These unusual reproductive features pinpoint to an eventual peculiar modulation of the hypothalamo-hypophyseal-gonadal axis through its main regulator, the gonadotropin-releasing hormone (GnRH). We explored the hypothalamic histological landscape and cellular and subcellular localization of GnRH in adult non-pregnant L. maximus females. Comparison to brain atlases from mouse, rat, guinea pig and chinchilla enabled us to histologically define and locate the preoptic area (POA), the ventromedial nucleus, the median eminence (ME), and the arcuate nucleus (Arc) of the hypothalamus in vizcacha's brain. Specific immunolocalization of GnRH was detected in soma of neurons at medial POA (MPA), ventrolateral preoptic nucleus, septohypothalamic nucleus (SHy) and Arc, and in beaded fibers of MPA, SHy, ventromedial hypothalamic nucleus, anterior hypothalamic area and ME. Electron microscopy examination revealed GnRH associated to cytoplasmic vesicles of the ME and POA neurons, organized both in core and non-core vesicles within varicosities, and in neurosecretory vesicles within the myelinated axons of the MPA. Besides the peculiar and unusual features of folliculogenesis and ovulation in the vizcacha, these results show that hypothalamus histology and GnRH immune-detection and localization are comparable to those found in other mammals. This fact leads to the possibility that specific regulatory mechanisms should be in action to maintain continuous folliculogenesis and massive polyovulation.     

Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

Somatostatin-14 neurons in the ovine hypothalamus: colocalization with estrogen receptor alpha and somatostatin-28(1-12) immunoreactivity,and activation in response to estradiol

Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor alpha (ERalpha), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ERalpha, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% +/- 2%) and ventromedial nucleus (VMN; 29% +/- 1%) expressed ERalpha. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1-12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% +/- 3%; no estradiol, 19% +/- 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% +/- 3%; arcuate, 72% +/- 5%; VMN, 81% +/- 5%) than in the control animals (periventricular, 22% +/- 1%; arcuate, 29% +/- 3%; VMN, 31% +/- 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.     

Neuronal inputs from the hypothalamus and brain stem to the medial preoptic area of the ram: neurochemical correlates and comparison to the ewe

The retrograde tracer, FluoroGold, was used to trace the neuronal inputs from the septum, hypothalamus, and brain stem to the region of the GnRH neurons in the rostral preoptic area of the ram and to compare these imputs with those in the ewe. Sex differences were found in the number of retrogradely labeled cells in the dorsomedial and ventromedial nuclei. Retrogradely labeled cells were also observed in the lateral septum, preoptic area, organum vasculosum of the lamina terminalis, bed nucleus of the stria terminalis, stria terminalis, subfornical organ, periventricular nucleus, anterior hypothalamic area, lateral hypothalamus, arcuate nucleus, and posterior hypothalamus. These sex differences may partially explain sex differences in how GnRH secretion is regulated. Fluorescence immunohistochemistry was used to determine the neurochemical identity of some of these cells in the ram. Very few tyrosine hydroxylase-containing neurons in the A14 group (<1%), ACTH-containing neurons (<1%), and neuropeptide Y-containing neurons (1-5%) in the arcuate nucleus contained FluoroGold. The ventrolateral medulla and parabrachial nucleus contained the main populations of FluoroGold-containing neurons in the brain stem. Retrogradely labeled neurons were also observed in the nucleus of the solitary tract, dorsal raphe nucleus, and periaqueductal gray matter. Virtually all FluoroGold-containing cells in the ventrolateral medulla and about half of these cells in the nucleus of the solitary tract also stained for dopamine beta-hydroxylase. No other retrogradely labeled cells in the brain stem were noradrenergic. Although dopamine, beta-endorphin, and neuropeptide Y have been implicated in the regulation of GnRH secretion in males, it is unlikely that these neurotransmitters regulate GnRH secretion via direct inputs to GnRH neurons.     

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex were investigated in rat tongue by cytochemical, immunocytochemical, and ultrastructural methods to evaluate the possible presence of different neuronal subpopulations. Immunostaining for neurofilaments and protein gene product 9.5 revealed ganglionic cell bodies and nerve fibers. A large part of the neurons were positive at immunostaining for neuronal nitric oxide synthase (NOS), vesicular acetylcholine transporter (VAChT), or vasoactive intestinal peptide (VIP). A small subset of nerve fibers revealed immunoreactivity for cholecystokinin. Axons traveling under the lingual epithelium were evidenced by their content of calcitonin gene-related peptide (CGRP) or substance P (SP). Cell bodies positive for SP or CGRP were not detected. Using methods of co-localization, three different neuronal classes were detected. The main population was composed of AChE/NADPH-diaphorase (NADPHd)-positive cells. Small groups of acetylcholine esterase (AChE)-positive/NADPHd-negative cells were visible. Isolated neurons were AChE-negative/NADPHd-positive. The results of co-localization experiments for VAChT/NOS were consistent with those obtained by cytochemical co-localization of AChE and NADPHd. Experiments of co-localization for peptidergic and nitrergic structures revealed CGRP- and SP-immunoreactive fibers in the vallate papilla/von Ebner gland ganglion. In conclusion, the results demonstrated in the VP/VEG complex peptidergic, cholinergic, and nitrergic neurons. The presence of different neuronal subclasses suggests that a certain degree of functional specialization may exist.     

Interplay of KNDy and nNOS neurons: A new possible mechanism of GnRH secretion in the adult brain

Reproduction in mammals is favoured when there is sufficient energy available to permit the survival of offspring. Neuronal nitric oxide synthase expressing neurons produce nitric oxide in the proximity of the gonadotropin-releasing hormone neurons in the preoptic region. nNOS neurons are an integral part of the neuronal network controlling ovarian cyclicity and ovulation. Nitric oxide can directly regulate the activity of the GnRH neurons and play a vital role neuroendocrine axis. Kisspeptin neurons are essential for the GnRH pulse and surge generation. The anteroventral periventricular nucleus (AVPV), kisspeptin neurons are essential for GnRH surge generation. KNDy neurons are present in the hypothalamus's arcuate nucleus (ARC), co-express NKB and dynorphin, essential for GnRH pulse generation. Kisspeptin-neurokinin B-dynorphin (KNDy) neuroendocrine molecules of the hypothalamus are key components in the central control of GnRH secretion. The hypothalamic neurons kisspeptin, KNDy, nitric oxide synthase (NOS), and other mediators such as leptin, adiponectin, and ghrelin, play an active role in attaining puberty. Kisspeptin signalling is mediated by NOS, which further results in the secretion of GnRH. Neuronal nitric oxide is critical for attaining puberty, but its direct role in adult GnRH secretion is poorly understood. This review mainly focuses on the role of nNOS and its interplay with KNDy neurons in the hormonal regulation of reproduction.     

Estrogen modulation of K(+) channel activity in hypothalamic neurons involved in the control of the reproductive axis

Here we report on the progress we have made in elucidating the mechanisms through which estrogen alters synaptic responses in hypothalamic neurons. We examined the modulation by estrogen of the coupling of various receptor systems to inwardly rectifying and small conductance, Ca(2+)-activated K(+) (SK) channels. We used intracellular sharp-electrode and whole-cell recordings in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples mu-opioid receptors from G protein-gated inwardly rectifying K(+) (GIRK) channels in beta-endorphin neurons, manifest by a reduction in the potency of mu-opioid receptor agonists to hyperpolarize these cells. This effect is blocked by inhibitors of protein kinase A and protein kinase C. Estrogen also uncouples gamma-aminobutyric acid (GABA)(B) receptors from the same population of GIRK channels coupled to mu-opioid receptors. At 24 h after steroid administration, the GABA(B)/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area (POA) is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of alpha(1)-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these POA GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of both arcuate and POA neurons, among which gonadotropin-releasing hormone (GnRH) neurons are particularly sensitive. These findings indicate a richly complex yet coordinated steroid modulation of K(+) channel activity that serves to control the excitability of hypothalamic neurons involved in regulating the reproductive axis.     

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.