Inducible nitric oxide synthase gene expression in the brain during systemic inflammation.

Inducible nitric oxide synthase (iNOS) is a transcriptionally regulated enzyme that synthesizes nitric oxide from L-arginine that has a key role in the pathophysiology of systemic inflammation and sepsis. Transgenic animals with a null mutation for the iNOS gene are resistant to hypotension and death caused by Escherichia coli lipopolysaccharide (LPS). The regulation of peripheral iNOS has been well studied in sepsis, but little is known about iNOS regulation in the brain during systemic inflammation or sepsis. We know that at baseline there is no detectable iNOS gene expression in the brain, but a detailed neuroanatomical study reveals that early in the course of systemic inflammation there is a profound induction of iNOS messenger RNA in vascular, glial and neuronal structures of the rat brain, accompanied by the production of nitric oxide (NO) metabolites in brain parenchyma and cerebrospinal fluid (CSF). We propose that the spillover of nitrite into the CSF has the potential to be a diagnostic marker for systemic inflammation and sepsis. Pharmacological interventions aimed at regulating iNOS function in the brain might represent a new treatment strategy in sepsis. Brain iNOS may be relevant to the pathophysiology, diagnosis and treatment of systemic inflammation and sepsis.

A receptor in pituitary and hypothalamus that functions in growth hormone release.

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

Pathophysiological significance of the obese gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats: evidence for loss of its satiety effect in VMH-lesioned rats.

To explore the pathophysiological significance of the obese (ob) gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats, we examined the synthesis and secretion of leptin and its satiety effect in VMH-lesioned rats compared with those in sham-operated rats. Northern blot analysis revealed that ob gene expression is markedly augmented in the mesenteric and sc white adipose tissue, but remained unchanged in the epididymal white adipose tissue during the development of obesity in VMH-lesioned rats. Plasma leptin levels were relatively constant in sham-operated rats, but were elevated during the development of obesity in VMH-lesioned rats. In sham-operated rats, a single i.v. (1.0 mg/rat) or intracerebroventricular (2.0 micrograms/rat) injection of recombinant human leptin reduced food intake and body weight gain in sham-operated rats. By contrast, no significant effect on food intake or body weight gain was observed in VMH-lesioned rats. The present study provides evidence that VMH-lesioned rats overproduce leptin and increase its release but cannot respond to it and suggests that the loss of its satiety effect contributes to the development of obesity and the obesity-related phenotypes in VMH-lesioned rats.

Neuropeptide-Y innervation of beta-endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis.

Central administration of neuropeptide-Y (NPY) inhibits pituitary LH release in ovariectomized rats and stimulates LH release in intact and ovariectomized rats pretreated with ovarian steroids. Although the precise neural mechanism of this dual effect of NPY is not known, experimental evidence suggests an underlying interaction between hypothalamic NPY and the inhibitory beta-endorphin (beta END) systems in the neuroendocrine regulation of pituitary LH release in the rat. The present study was undertaken to examine the morphological basis of the interaction between these two peptidergic systems in the hypothalamus. Sections of the mediobasal hypothalamus of colchicine-pretreated female rats were double immunostained for NPY and beta END and examined by light and electron microscopy. The light brown diaminobenzidine reaction was used to visualize beta END cells, while NPY neurons were labeled with a dark blue nickel ammonium sulfate-intensified diaminobenzidine reaction. Under the light microscope, a dense network of NPY-immunoreactive axons and axon terminals was observed in close apposition with beta END-immunoreactive neurons throughout the medial basal hypothalamus. Electron microscopic examination revealed that NPY-immunoreactive boutons formed axosomatic and axo-dendritic synaptic connections with beta END cells. A majority of these synaptic membrane specializations appeared asymmetrical [corrected]. In light of the previous evidence of excitatory and inhibitory effects on LH release and the existence of direct synaptic connections between NPY and LHRH neurons in the hypothalamus, the current results imply that the dual effects of NPY on LH secretion may involve modulation of LHRH secretion, both by the direct route and indirectly through the hypothalamic beta END system.

Immunohistochemical localization of insulin-like growth factor-II (IGF-II) and IGF-binding protein-2 during development in the rat brain.

The insulin-like growth factors (IGF-I and IGF-II) are peptide growth factors with both growth-promoting and insulin-like activities. In the nervous system, the expression of both IGF-I and IGF-II messenger RNAs (mRNAs) is developmentally regulated, with IGF-I expression highest during puberty, and IGF-II levels peaking during the perinatal period. The IGFs interact with and are modulated by a group of six binding proteins, the IGF-binding proteins (IGFBP-1 to IGFBP-6). IGFBP-2 mRNA is most prevalent in the nervous system, where, like IGF-II, its expression correlates with a period of brain growth. In the current study, cells containing IGF-II and IGFBP-2 were identified within the developing nervous system of the rat on embryonic day 12 (E12), E14, E18, and postnatal day 1 and in the adult. IGF-II immunoreactivity was detected within the mesenchymal core of the choroid plexus at all ages examined and was also observed in the developing leptomeninges. IGF-II appeared transiently in the central nervous system in presumptive glia of the hippocampus and medial basal hypothalamus and in a small population of neurons in the brain stem. IGFBP-2 was consistently observed in the epithelium of the choroid plexus as well as in the epithelia of the developing otic and olfactory placodes. While these results confirm the developmental expression of IGF-II and IGFBP-2 mRNA in the central nervous system, discrepancies exist between these data and those using molecular techniques. This may be due in part to differential sites of IGF-II and IGFBP-2 production compared to sites of action.

The acute suckling stimulus induces expression of neuropeptide Y (NPY) in cells in the dorsomedial hypothalamus and increases NPY expression in the arcuate nucleus.

Elevated neuropeptide Y (NPY) levels in the hypothalamus have been reported during lactation in the rat. The increase in NPY neuronal activity may be important in modulating a number of changes in hypothalamic neuronal function that are associated with lactation. The aims of the present study were to determine 1) if NPY neurons in the hypothalamus can be activated by the suckling stimulus; and 2) the time course of the activation in response to the suckling stimulus. In the first experiment, lactating rats were deprived of their 8-pup litters on day 9 post partum for 48 h. On day 11, the animals were divided into three groups and exposed to the suckling stimulus for varying periods of time up to 24 h. NPY neuronal activity was assessed by measuring changes in NPY messenger RNA (mRNA) levels, using in situ hybridization. NPY mRNA levels in the caudal portion of the hypothalamic arcuate nucleus (ARH) were approximately doubled by 24 h of suckling. NPY mRNA levels in the rostral portion of the ARH were not affected by suckling throughout the time examined. In addition to increased NPY mRNA in the ARH, resuckling for as little as 3 h induced NPY mRNA expression in cells located dorsal and lateral to the compact zone of the dorsomedial nucleus of the hypothalamus (DMH). NPY expression in these cells was not observed in the nonresuckled controls. These data demonstrate that the acute suckling stimulus activates two specific populations of NPY neurons in the hypothalamus: in the caudal portion of the ARH and in the DMH. The increased NPY neuronal activity may play an important role in modulating changes in hypothalamic regulation of hormone secretion and food intake.

Down-regulated STAT3 messenger ribonucleic acid and STAT3 protein in the hypothalamic arcuate nucleus of the obese leptin-deficient (ob/ob) mouse.

Leptin is a weight-reducing hormone produced by adipose tissue, which reduces food intake via hypothalamic leptin receptors and the JAK-STAT signaling pathway. In vivo studies have shown that leptin activates specifically STAT3 in the hypothalamus. We have studied the cellular localization of STAT3 messenger RNA (mRNA) and STAT3 protein in the mouse mediobasal hypothalamus using, respectively, in situ hybridization and immunohistochemistry. Strong STAT3 mRNA and STAT3 immunoreactivity was demonstrated in neurons located in the ventral part of the mouse arcuate nucleus. Comparison of STAT3 mRNA levels in the arcuate nucleus of lean control mice and obese leptin-deficient ob/ob mice showed that the levels of STAT3 mRNA in the arcuate nucleus were significantly lower (31% less in ob/ob mice), compared with control mice. Hybridization with a probe specific for STAT3alpha mRNA showed that the down-regulated STAT3 expression in the arcuate nucleus of ob/ob mice is represented by STAT3alpha. There was a marked difference in numbers and intensity of STAT3-immunoreactive cell bodies, with virtually no STAT3-immunoreactive cell bodies in the mediobasal hypothalamus of ob/ob mice, compared with control mice. Direct double-labeling immunofluorescence histochemistry of sections from control mice, combining a goat antiserum raised against a peptide sequence present in all leptin receptor isoforms (Ob-R) or a guinea pig anti-serum generated to a peptide sequence specific for Ob-Rb with rabbit STAT3 antiserum, demonstrated colocalization of STAT3 and Ob-R as well as colocalization of STAT3 and Ob-Rb, in many cell bodies of the arcuate nucleus. The results suggest that circulating leptin acts via leptin receptor-/STAT3-containing neurons in the ventral arcuate nucleus and that congenital leptin deficiency, as seen in obese ob/ob mice, results in a down-regulation of STAT3 mRNA and protein levels.

Neuroendocrine aging in the female rat: the changing relationship of hypothalamic gonadotropin-releasing hormone neurons and N-methyl-D-aspartate receptors.

The reproductive axis undergoes alterations during aging, resulting in acyclicity and the loss of reproductive function. In the hypothalamus, changes intrinsic to GnRH neurons may play a critical role in this process, as may changes in inputs to GnRH neurons from neurotransmitters such as glutamate. We investigated the effects of age and reproductive status on neuroendocrine glutamatergic NMDA receptors (NRs), their regulation of GnRH neurons, and their expression on GnRH neurons, in female rats. First, we quantified NR subunit messenger RNAs (mRNAs) in preoptic area-anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH), the sites of GnRH perikarya and neuroterminals, respectively. In POA-AH, NR1 mRNA levels varied little with age or reproductive status. NR2a and NR2b mRNA levels decreased significantly between cycling and acyclic rats. In MBH, NR mRNAs all increased with aging, particularly in acyclic animals. Second, we tested the effects of N-methyl-D,L-aspartate (NMA) on GnRH mRNA levels in POA-AH of aging rats. NMA elevated GnRH mRNA levels in young rats, but decreased them in middle-aged rats. Third, we quantified expression of the NR1 subunit on GnRH perikarya in aging rats using double label immunocytochemistry. NR1 expression on GnRH cell bodies varied with age and reproductive status, with 30%, 19%, and 46% of GnRH somata double labeled with NR1 in young proestrous, middle-aged proestrous, and middle-aged persistent estrous rats, respectively. Thus, 1) the expression of hypothalamic NR subunit mRNAs correlates with reproductive status; 2) changes in NR subunit mRNA levels, if reflected by changes in protein levels, may result in alterations in the stoichiometry of the NR during aging, with possible physiological consequences; 3) the effects of NR activation on GnRH mRNA switches from stimulatory to inhibitory during reproductive aging; and 4) expression of the NR1 subunit on GnRH perikarya changes with reproductive status. These molecular, physiological, and cellular neuroendocrine changes are proposed to be involved in the transition to acyclicity in aging female rats.

Sexually dimorphic expression of calcitonin gene-related peptide (CGRP) immunoreactivity by rat mediobasal hypothalamic neurons.

Although the hypothalamic arcuate nucleus is a sexually dimorphic region of the rat brain, there are no reports of sex differences in the number of neurons containing specific neuropeptides within this structure. As cells synthesizing calcitonin gene-related peptide (CGRP) have been shown to exhibit sex differences in other steroid-receptive regions of the rat brain, we examined whether the CGRP-immunoreactive cells located in the mediobasal hypothalamus may also be sexually dimorphic. Immunostaining of sections from male and female colchicine-treated rats revealed a small population of CGRP-immunoreactive cells distributed throughout the arcuate nucleus. Immunoreactive cells were also detected in the lateral hypothalamic perifornical region, dorsomedial, posterior periventricular and ventral tuberomammillary nuclei, and zona incerta. Cell count analysis revealed approximately twice as many CGRP-immunoreactive cell profiles in the rostral (P < 0.01), middle (P < 0.001), and caudal (P < 0.01) thirds of the arcuate nucleus of male rats compared with females. A significant sex difference in immunoreactive cell numbers (male > female) was also detected within the caudal dorsomedial nucleus (P < 0.05) but not in the posterior periventricular nucleus, perifornical region and zona incerta. Although fibers immunoreactive for CGRP were identified in low density throughout the mediobasal hypothalamus, only female rats displayed prominent fiber staining in the periventricular region. Double-labelling immunofluorescence experiments revealed that the CGRP-immunoreactive cells within the zona incerta, but not the hypothalamus, were also immunoreactive for tyrosine hydroxylase; at least 60% of the A13 dopaminergic neurons co-express CGRP. These results provide evidence that sex differences exist in the number of specific neuropeptide-synthesizing cells within the hypothalamic arcuate nucleus and provide further examples of cell populations expressing CGRP immunoreactivity in a sexually dimorphic manner.

Special relationship of gamma-aminobutyric acid to the ventromedial nucleus of the hypothalamus during embryonic development.

The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus for regulating homeostatic, neuroendocrine, and behavioral functions. We conducted immunocytochemical analyses by using antisera directed against gamma-aminobutyric acid (GABA), its synthetic enzyme glutamic acid decarboxylase (GAD67), GABA-A receptor subunits (alpha2, beta3, epsilon), estrogen receptor-alpha, and Neuropeptide Y (NPY) in the region of the VMH in embryonic mice to identify potential patterning elements for VMH formation. Cells and fibers containing GABA and GAD67 encircled the primordial VMH as early as embryonic day 13 (E13) when the cytoarchitecture of the VMH was not recognizable by Nissl stain. At E16-17 the cytoarchitecture of the VMH became recognizable by Nissl stain as GABAergic fibers invaded the nucleus, continued postnatally, and by adulthood the density of GABAergic fibers was greater inside than outside the VMH. GABA-A receptor subunit expression (beta3 by E13 and alpha2 by E15) within the primordial VMH suggested potential sensitivity to the surrounding GABA signal. Brain slices were used to test whether fibers from distal or proximal sites influenced VMH development. Coronal Vibratome slices were prepared and maintained in vitro for 0-3 days. Nissl stain analyses showed a uniform distribution of cells in the region of the VMH on the day of plating (E15). After 3 days in vitro, cellular aggregation suggesting VMH formation was seen. Nuclear formation in vitro suggests that key factors resided locally within the coronal plane of the slices. It is suggested that either GABA intrinsic to the region nearby the VMH directly influences the development and organization of the VMH, or along with other markers provides an early indicator of pattern determination that precedes the cellular organization of the VMH.

Galanin-R1 receptor in anterior and mid-hypothalamus: distribution and regulation.

The distribution and regulation of galanin-R1 receptor (GAL-R1-R) mRNA has been studied in the anterior and mid-diencephalon by using in situ hybridization. Moreover, possible colocalization of GAL-R1-R mRNA and prepro-galanin or vasopressin mRNAs has been analyzed at the cellular level using double in situ hybridization methodology. Many nuclei in the hypothalamus expressed GAL-R1-R mRNA, including the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Strong expression was also seen in the same sections in various areas outside of the diencephalon. The distribution patterns are similar to those described in earlier studies. Double labeling experiments showed GAL-R1-R mRNA in vasopressin neurons in the PVN and SON. Moreover, GAL-R1-R mRNA and prepro-galanin mRNA were colocalized in several hypothalamic nuclei. GAL-R1-R mRNA levels showed a high degree of plasticity. Thus, salt loading resulted in a marked increase in GAL-R1-R mRNA levels in the PVN and SON and a moderate decrease was seen during lactation. In contrast, hypophysectomy caused a decrease in GAL-R1-R mRNA levels. Differential effects of colchicine were recorded with a decrease of GAL-R1-R mRNA in the magnocellular hypothalamic neurons. After salt loading or during lactation, GAL-R1-R mRNA and prepro-galanin mRNA were regulated in parallel, whereas their levels changed in opposite directions after hypophysectomy and colchicine injection. In conclusion, GAL-R1-Rs are present in several hypothalamic nuclei, partly in neurons synthesizing galanin. The receptors are regulated in a specific fashion in the various nuclei, depending on the stimulus applied. The results suggest that the effect of galanin in the hypothalamus partly depends on the state of receptor expression.

Distribution of estrogen receptor-immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss).

Using antibodies against the hormone binding domain of the trout estrogen receptor (ER), the distribution of ER-immunoreactive (ER-IR) cells was studied in the brain of maturing diploid and triploid female rainbow trout using a streptavidin-biotin-peroxidase method followed by a nickel-intensified diaminobenzidine reaction. This technique resulted in an excellent signal/background ratio allowing unambiguous identification of positive cells. In all animals, ER-IR cells were consistently located in three brain regions, the ventral telencephalon, the anterior ventral preoptic region, and the mediobasal hypothalamus. About 250 ER-IR cells were observed in the ventral and dorsal parts of the ventral telencephalon. In the anterior nucleus preopticus periventricularis, about 2400 ER-IR cells were observed surrounding the preoptic recess. In the posterior hypothalamus, approximately 2700 ER-IR cells were located in the anterior, posterior and inferior divisions of the nucleus lateralis tuberis and in the nucleus saccus vasculosus. In these regions cell nuclei exhibiting different densities of staining were observed and absolutely no labeling of cytoplasmic processes was detected. These results are in partial agreement with those obtained either after injection of tritiated-estradiol in other teleots species or in situ hybridization of ER mRNAs in trout. In particular, no immunoreactivity was observed in the thalamic region nor in the nucleus posterioris periventricularis. These data indicate that target cells for estradiol are essentially located in brain regions involved in the neuroendocrine control of pituitary functions and having direct connections with the hypophysis.

Effect of interleukin-1beta on gonadotropin-releasing hormone (GnRH) and GnRH receptor gene expression in castrated male rats.

Increasing evidence suggests that interleukin-1beta (IL-1beta) regulates luteinizing hormone (LH) release primarily through modulation of the gonadotropin-releasing hormone (GnRH) neuronal activity. This study was undertaken to elucidate the effect of IL-1beta on GnRH as well as GnRH receptor (GnRHR) gene expression in the preoptic area. IL-1beta (100 ng/rat) or saline was administered into the lateral ventricle of castrated rats. RNA samples were isolated from micropunches of the preoptic area and mediobasal hypothalamus from individual brain slices and GnRH mRNA levels in the preoptic area and GnRHR mRNA levels in the mediobasal hypothalamus were determined by competitive reverse transcription-polymerase chain reaction (RT-PCR) protocols. Serum LH concentrations were decreased from 1 h to 3 h after IL-1beta treatment, but rebounded at 5 h, while serum concentrations of follicle-stimulating hormone (FSH) and prolactin were not altered. There were no significant changes in GnRH mRNA levels from the micropunched preoptic area, while GnRHR mRNA levels from the preoptic area and mediobasal hypothalamus micropunch samples, but not in the anterior pituitary, showed a pattern similar to the serum LH profile following i.c.v. administration of IL-1beta. We then examined the effect of IL-1beta on the translational efficiency of the GnRH mRNA. After the separation and fractionation of polyribosome-associated cytoplasmic RNA from the hypothalamic fragments containing the preoptic area-anterior hypothalamic area of control (saline-treated) and IL-1beta-treated group 3 h after administration, GnRH transcript levels were examined from the each fraction. IL-1beta decreased the translational efficiency of the transcribed GnRH mRNA. These results clearly demonstrate that central administration of IL-1beta suppresses the translational activity of GnRH mRNA. Moreover, GnRHR may play an important role in the modulation of GnRH neuronal activity through GnRHR-expressing neurones (or glia) in the hypothalamus.

Growth hormone-releasing hormone (GHRH)-GH-somatic growth and luteinizing hormone (LH)RH-LH-ovarian axes in adult female transgenic mice expressing human GH gene.

We have examined alterations in the hypothalamo-pituitary GH-somatic growth axis and the hypothalamo-pituitary LH-ovarian axis in a line of transgenic ICR mice expressing human GH (hGH) under the influence of the whey acid protein promoter. Transgenic female mice weighed twice as much as control females and were infertile. The size of the anterior pituitary (AP) was 1/3 that of the controls. In transgenic mice, acinar cells in the mammary and mandibular glands displayed hGH-immunoreactivity, and plasma hGH was detected by radioimmunoassay. In the medial basal hypothalamus (MBH) of transgenic females, the immunoreactive-GHRH level was decreased (P<0.01). There was a corresponding reduction in the number of GHRH-immunoreactive neurons in the arcuate nucleus (ARC) and in the immunostaining of GHRH nerve terminals in the median eminence. The level of somatostatin (SRIH) in the MBH was increased (P<0.05), and SRIH-immunoreactive neurons in the periventricular nucleus (PeV) were increased in size and number in transgenic mice. The MBH level of LHRH in transgenic animals was greater (P<0.01) than in controls, although there was no apparent difference in the number of LHRH-immunoreactive neurons or in LHRH level in the preoptic area. There are fewer SRIH- and LHRH-immunoreactive neurons in the ARC in transgenic mice. Cells in the AP for GH, PRL, and LH were fewer in transgenic mice. The ovary suffered disturbance of follicular development and of corpora lutea formation. These results demonstrate that chronic overproduction of hGH may profoundly affect the organization of the GHRH/SRIH-GH-somatic growth axis and the LHRH-LH-ovarian axis due to reduction of GHRH-, SRIH- and LHRH-neurons in the ARC and increase of SRIH-neurons in the PeV.

Effects of orchidectomy on levels of the mRNAs encoding gonadotropin-releasing hormone and other hypothalamic peptides in the adult male rhesus monkey (Macaca mulatta).

The testicular regulation of luteinizing hormone (LH) secretion in the adult rhesus monkey is mediated by an indirect action of testosterone to decelerate pulsatile gonadotrophin releasing hormone (GnRH) release. Whether this negative feedback action of testosterone involves regulation of GnRH gene expression is unknown. Therefore, the effect of bilateral orchidectomy on hypothalamic levels of the mRNA encoding this hypophysiotropic factor was examined. The feedback action of testosterone is generally considered to be mediated through non-GnRH cells, and the present experiment provided the opportunity to also examine testicular influences on mRNAs encoding putative hypothalamic factors implicated in the testicular regulation of LH secretion. Adult male rhesus monkeys were orchidectomized (n=5) or sham-orchidectomized (n=5) and killed 6 weeks later, after a castration-induced hypersecretion of LH was established. Separate preoptic and mediobasal hypothalamus containing areas were collected, and levels of GnRH mRNA, as well as those of mRNAs encoding pro-opiomelanocortin (POMC), the gamma-aminobutyric acid (GABA) synthesizing enzymes (glutamic acid decarboxylase 65 and 67; GAD65 and GAD67, respectively), neuropeptide Y, galanin and transforming growth factor (TGF)alpha, were quantified using RNase protection assay. Values were expressed in terms of optical density relative to that of cyclophilin mRNA levels. Bilateral orchidectomy produced a significant increase in GnRH mRNA levels that was restricted to the mediobasal hypothalamus and that was associated with a significant decrease in POMC, GAD65 and GAD67 mRNA levels in this region of the hypothalamus. In contrast, neuropeptide Y, galanin and TGFalpha mRNA levels were not affected by castration. These results indicate that, in the monkey, the deceleration of pulsatile GnRH release that is imposed by the testis, and presumably mediated by testosterone, is associated with a concomitant down regulation of GnRH gene expression in the mediobasal hypothalamus. They also support the notion that this hypothalamic feedback action may be mediated by POMC-and GABA-producing neurones in the mediobasal hypothalamus.

Orphanin FQ/nociceptin in the ventromedial nucleus facilitates lordosis in female rats.

The localization of opioid receptor-like (ORL-1) orphan receptor in the ventromedial nucleus of the hypothalamus (VMH) suggested a role for this opioid system in the regulation of lordosis behavior. Recently, the ligand for ORL-1, orphanin FQ/nociceptin (OFQ/N), has been characterized and also demonstrated to be present in the VMH. The present experiments examined whether OFQ/N in the VMH facilitates lordosis behavior in estrogen-primed, sexually unreceptive female rats, and whether estrogen regulates ORL-1 levels in the VMH. Estrogen was shown to increase ORL-1 immunoreactivity in the VMH, and microinfusions of OFQ/N into the VMH facilitated lordosis behavior in a dose-dependent manner.

Morphological evidence for a substance P projection from medial septum to hippocampus.

The medial septal nucleus provides one of the major afferents to the hippocampal formation. The two major types of neurons present in the medial septum are cholinergic and GABAergic, but other types of neurons are also present. A small population of substance P-containing neurons is present along the border between the medial and lateral septum, but it is unclear whether these project to the hippocampus. The present study, by employing both anterograde and retrograde tracing techniques, combined with immunocytochemistry for substance P, provides direct morphological evidence for a substance P projection from the lateral region of medial septum to a portion of CA2/3a, which is restricted to the mid-septotemporal portion of the hippocampus.

Androgen-dependent modulation of calbindin-D28K in hypothalamic tissue during prenatal development.

We examined the influence of androgens on fetal medial basal hypothalamic and preoptic area (MBH-POA) calbindin-D28K levels (via Western analysis) by treating pregnant rats with testosterone or flutamide, (an androgen receptor blocker). MBH-POA calbindin-D28K levels were significantly decreased in flutamide-treated male fetuses, whereas, MBH-POA calbindin-D28K levels were significantly increased in testosterone-treated female fetuses compared to controls. These results suggest that MBH-POA calbindin-D28K is modulated during prenatal development by androgens.

Immunocytochemical evidence for noradrenergic regulation of estrogen receptor concentrations in the guinea pig hypothalamus.

The noradrenergic system interacts with steroid hormones to influence female sexual behavior and gonadotropin release in rodents. Using a double label immunocytochemical procedure for estrogen receptors and dopamine-beta-hydroxylase, we investigated the anatomical relationships between noradrenergic neurons and estrogen receptor-immunoreactive (ER-IR) cells in the brain of ovariectomized female guinea pigs. Dopamine-beta-hydroxylase-immunoreactive (DBH-IR) varicosities were closely associated with ER-IR cells throughout the hypothalamus and preoptic area. This anatomical relationship was observed with almost 80% of the ER-IR cells in the ventrolateral hypothalamus (VLH), an area involved in the regulation of female sexual behavior in guinea pigs. Furthermore, the presence of closely associated DBH-IR varicosities was related to staining intensity of ER-IR neurons. In the rostral VLH, ER-IR neurons with closely associated DBH-IR varicosities stained more darkly than ER-IR neurons lacking this association, suggesting noradrenergic regulation of basal levels of cellular estrogen receptors. These findings provide neuroanatomical evidence suggestive of noradrenergic regulation of estrogen receptor levels in the hypothalamus of female guinea pigs.

Role of NMDA receptors in hypothalamic facilitation of feline defensive rage elicited from the midbrain periaqueductal gray.

The present study tested the hypothesis that the pathway from the medial hypothalamus to the midbrain periaqueductal gray (PAG) subserving defensive rage behavior in the cat facilitates the occurrence of this response when elicited from the PAG by utilizing excitatory amino acids as a neurotransmitter or neuromodulator. Cannula electrodes were implanted into the PAG for the elicitation of defensive rage behavior as well as for microinjections of excitatory amino acid antagonists and N-methyl-D-aspartic acid (NMDA). Monopolar stimulating electrodes were also implanted into the medial hypothalamus from which this response could also be elicited and, when stimulated at subthreshold levels for elicitation of behavior, could also facilitate the occurrence of PAG elicited defensive rage. Initially, dual stimulation of the PAG and medial hypothalamus facilitated the occurrence of defensive rage elicited from the PAG. Then, the identical dual stimulation paradigm was repeated with the same current parameters following the infusion of various antagonists for different receptors into the PAG defensive rage sites. The results indicate that infusion of either kynurenic acid [(0.1-2.0 nmol), a non-selective excitatory amino acid receptor antagonist] or D-2-amino-7-phosphonoheptanoic acid (AP7) [(0.1-2.0 nmol), a specific NMDA receptor antagonist], produced a dose and time dependent blockade of the facilitatory effects of medial hypothalamic stimulation. In contrast, microinjections of relatively larger doses of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [(4 nmol), a non-NMDA receptor (quisqualate and kainate) antagonist] or atropine [(4.4 nmol), a muscarinic receptor antagonist] had little effect upon medial hypothalamically elicited facilitation of the PAG response. In a second experiment, NMDA [0.1-1.0 nmol] was microinjected directly into PAG defensive rage sites in the absence of medial hypothalamic stimulation. In these animals, drug infusion mimicked the effects of dual stimulation by producing a dose and time dependent decrease in response latencies. A third experiment was designed to further test the hypothesis by neuroanatomical methods. Here, the retrograde label, Fluoro-Gold, was microinjected into defensive rage sites within the PAG and following a survival time of 5-6 days, the animals were sacrificed. The brains were then processed for immunocytochemical analysis of cells that immunoreact positively for aspartate and glutamate. The results indicated the presence of many retrogradely labelled and immunocytochemically positive cells within the rostro-caudal extent of the medial hypothalamus as well as others that were double labelled.(ABSTRACT TRUNCATED AT 400 WORDS)