Projections of RFamide-related Peptide-3 Neurones in the Ovine Hypothalamus, with Special Reference to Regions Regulating Energy Balance and Reproduction

RFamide-related peptide-3 (RFRP-3) is a neuropeptide produced in cells of the paraventricular nucleus and dorsomedial nucleus of the ovine hypothalamus. In the present study, we show that these cells project to cells in regions of the hypothalamus involved in energy balance and reproduction. A retrograde tracer (FluoroGold) was injected into either the arcuate nucleus, the lateral hypothalamic area or the ventromedial nucleus. The distribution and number of retrogradely-labelled RFRP-3 neurones was determined. RFRP-3 neurones projected to the lateral hypothalamic area and, to a lesser degree, to the ventromedial nucleus and the arcuate nucleus. Double-label immunohistochemistry was employed to identify cells receiving putative RFRP-3 input to cells in these target regions. RFRP-3 cells were seen to project to neuropeptide Y and pro-opiomelanocortin neurones in the arcuate nucleus, orexin and melanin-concentrating hormone neurones in the lateral hypothalamic area, as well as orexin cells in the dorsomedial nucleus and corticotrophin-releasing hormone and oxytocin cells in the paraventricular nucleus. Neurones expressing gonadotrophin-releasing hormone in the preoptic area were also seen to receive input from RFRP-3 projections. We conclude that RFRP-3 neurones project to hypothalamic regions and cells involved in regulation of energy balance and reproduction in the ovine brain.     

Location of the Neuroendocrine Gonadotropin-Releasing Hormone Neurons in the Monkey Hypothalamus by Retrograde Tracing and Immunostaining*,**

In order to localize neuroendocrine gonadotropin-releasing hormone (GnRH) neurons in the monkey hypothalamus, four juvenile cynomolgus macaques (one female, three males) were each given two or three microinjections (0.2 to 0.3 μl per site) of the retrograde tracer wheat germ agglutinin-apoHorseradish peroxidase-10 nm colloidal gold into the superficial, median eminence region of the infundibular stalk. Five to 15 days following surgery, the brains were fixed by perfusion and vibratomed at 40 μm in the frontal plane. Every 12th section was immunostained with rabbit anti-GnRH using the peroxidase anti-peroxidase technique with diaminobenzidine as the chromogen. Neuroendocrine GnRH neurons were easily identified in tissue sections as brown, immunostained cell bodies containing more than three distinct, dark blue, tracer-filled lysosomes. Neuronal counts from each complete series of sections were compiled by anatomical region, and the percentages of GnRH and neuroendocrine GnRH neurons determined. The highest proportion of neuroendocrine GnRH neurons (with projections to the median eminence) occurred in the ventral hypothalamic tract, especially in its medial third (71%), and in the supraoptic decussation just anterior to it. Proportions decreased moving laterally into the middle third (58%) and lateral third (25%) of the ventral hypothalamic tract. Further anterior and lateral, progressively smaller but significant neuroendocrine GnRH contributions were found in the supraoptic nucleus (57%) and lateral hypothalamus (33%), and in the medial preoptic area (26%). Although the medial preoptic area contained a greater percentage of the total GnRH-immunoreactive cell bodies (36%) than the ventral hypothalamic tract (27%), as a whole, the ventral hypothalamic tract contained 60% of the neuroendocrine GnRH neurons compared to only 25% from the medial preoptic area. Large numbers of GnRH cell bodies found in the diagonal band of Broca near the organum vasculosum of the lamina terminalis were not retrogradely labeled. GnRH neurons were not observed in the arcuate nucleus, the few in the paraventricular nucleus were not neuroendocrine, and the contribution from the periventricular zone was negligible. Our results here are the first to identify the neurons giving rise to the neuroendocrine GnRH system in juvenile monkeys. The data indicate that more GnRH neurons close to the infundibulum serve a neuroendocrine (perhaps hypophysiotropic) role than do those in more anterior areas. Furthermore, they suggest that the ventral hypothalamic tract is the most important, and perhaps most influential, neuroendocrine GnRH cell group in primates. The data substantiate the observed autonomy of the medial basal hypothalamus in controlling gonadotropin secretion and menstrual cyclicity in these animals. However, they also infer that perhaps 60% of the GnRH neurons do not project to the primate median eminence, and thus may serve other non-neuroendocrine functions.     

Activation of Nesfatin‐1‐Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin‐1 in Rats

Peripheral anorectic hormones, such as glucagon‐like peptide (GLP)‐1, cholecystokinin (CCK)‐8 and leptin, suppress food intake. The newly‐identified anorectic neuropeptide, nesfatin‐1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP‐1 and CCK‐8 and co‐administrations of GLP‐1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin‐1‐immunoreactive (‐IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP‐1 (100 μg/kg) caused significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK‐8 (50 μg/kg) resulted in marked increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP‐1 (33 μg/kg) or leptin. However, co‐administration of GLP‐1 (33 μg/kg) and leptin resulted in significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP‐1, CCK‐8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin‐1. These results indicate that nesfatin‐1‐expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP‐1 and leptin in addition to CCK‐8, and also suppress food intake in rats.     

Oestradiol Microimplants in the Ventromedial Preoptic Area Inhibit Secretion of Luteinizing Hormone via Dopamine Neurones in Anoestrous Ewes

Oestradiol exerts a season-specific negative feedback effect on the GnRH/LH neurosecretory system of the Suffolk ewe. This neuroendocrine suppression is mediated in part by dopamine A15 neurones, but these neurones do not possess the oestrogen receptor. Based on indirect evidence, we hypothesized that oestrogen receptor-containing neurones in the ventromedial preoptic area (vmPOA) may be the initial step in a neuronal system whereby oestradiol suppresses GnRH secretion during the non-breeding season. To test this, three experiments were conducted using ovariectomized ewes receiving either empty or oestradiol-containing bilateral microimplants directed at the vmPOA or s.c. subcutaneous oestradiol-containing implants. In the first experiment, LH pulse frequency was measured on days 0, 1, 7 and 14 of treatment during seasonal anoestrus. In vmPOA oestradiol and s.c. oestradiol groups only, LH pulse frequency was suppressed on days 7 and 14, with maximal suppression evident by day 7. In the second experiment, this protocol was repeated during the breeding season, with LH pulses examined on days 0 and 7; LH pulse frequency did not change in any group. The third experiment tested if the effect of vmPOA oestradiol during anoestrus could be overcome by an injection of the dopamine-D2 receptor antagonist (-)-sulpiride. The vmPOA microimplants and s.c. oestradiol implants again suppressed LH pulse frequency and this was reversed by sulpiride in vmPOA oestradiol ewes. We conclude that oestradiol acts on cells in the vmPOA to stimulate a system involving dopamine neurones that inhibits GnRH/LH pulsatility in the anoestrous ewe.     

Evidence for Expression of Galanin Receptor Gal-R1 mRNA in Certain Gonadotropin Releasing Hormone Neurones of the Rostral Preoptic Area

Previous studies have shown that galanin plays an important role in the regulation of gonadotropin releasing hormone (GnRH) release. At present, it is not known if this role is exerted by direct or indirect interactions between galanin producing neurones and GnRH neurones. The objective of this study was to determine whether or not GnRH neurones could express galanin receptor Gal-R1 mRNA. Dual in-situ hybridization experiments were carried out with digoxigenin-labelled cRNA probes encoding GnRH in combination with 35S-labelled riboprobe encoding the galanin receptor Gal-R1. In order to detect possible variations in the expression of the Gal-R1 mRNA under different physiological conditions, male rats, intact female rats throughout the phases of the oestrous cycle, ovariectomized (OVX) and steroid-treated rats were analysed. The results show that many cells expressing Gal-R1 mRNA were present throughout the preoptic area. Gal-R1 mRNA-expressing cells were observed in very close proximity with GnRH neurones. In the female rat, some GnRH neurones located in the rostral preoptic area/vascular organ of the lamina terminalis expressed Gal-R1 mRNA. These double-labelled cells were observed at all times of the oestrous cycle, except during diestrus at 08.00 h and pro-oestrus at 18.00 h. Conspicuously, at oestrus 1800 h, we found that 21.6% of rostral GnRH neurones expressed the Gal-R1 mRNA. In addition, dual-labelled GnRH neurones were seen in OVX animals but not in oestrogen plus progesterone-treated ones. In the male rat, colocalization of GnRH mRNA and Gal-R1 receptor mRNA was not observed. In the medial preoptic area, no double-labelled GnRH neurones were detected regardless of the endocrine conditions. These results suggest that, in addition to a possible indirect action of galanin on GnRH cells via neurones located at close proximity, the effects of galanin on GnRH can be mediated by direct activation of galanin receptors in rostral GnRH neurones. This study also shows that expression of Gal-R1 mRNA in GnRH cells is influenced by the levels of circulating gonadal steroids.     

Further Evidence that Most Luteinizing Hormone-Releasing Hormone Neurons are not Directly Estrogen-Responsive: Simultaneous Localization of Luteinizing Hormone-Releasing Hormone and Estrogen Receptor Immunoreactivity in the Guinea-Pig Brain

Gonadotropin secretion from the pituitary is regulated in large part by steroid action on the brain. An important question concerns whether luteinizing hormone-releasing hormone (LHRH) neurons themselves transduce steroid signals, or whether, alternatively, steroid-sensitive interneuronal populations regulate their activity. A previous study in the rat employing steroid autoradiography combined with LHRH immunocytochemistry revealed that only an exceedingly small percentage of LHRH-immunoreactive (ir) neurons was estrogen concentrating. This study has examined the relationship of estrogen receptive and LHRH-ir cells in the male and female guinea-pig brain with double label immunocytochemistry. Since estrogen receptor-ir, as demonstrated with antibody H222, is known to be confined predominantly to the cell nucleus, whereas LHRH-ir is localized mainly in the cytoplasm, single cells can be double-labeled. Diaminobenzidine tetrahydrochloride was used for localization of LHRH-ir while nickel-enhanced diaminobenzidine tetrahydrochloride was used for localization of estrogen receptor-ir. The results revealed that there were many brain nuclei that contained both LHRH and estrogen receptor-positive cells, including the preventricular periventricular nucleus, the anterior subcompact nucleus of the medial preoptic nucleus (MPNa), the remainder of the medial preoptic nucleus, the retrochiasmatic area, and the anterior, dorsomedial, ventrolateral and arcuate nuclei. However, of a total of 2,604 LHRH-ir cells that were examined, we observed only 5 double-labeled cells (<0.2%). The double-labeled cells were not restricted to a single nucleus; they were present in the MPNa, the retrochiasmatic area and the arcuate nucleus. Moreover, at the light microscopic level, LHRH cells quite frequently appeared to be apposed to estrogen receptor-positive cells (8.8% in the female), especially in the MPNa. These results lend further support to the notion that estrogen-mediated regulation of the LHRH system is achieved primarily through estrogen receptive interneurons. However, due to the existence of LHRH-LHRH synaptic interactions, the possibility also exists that a small population of estrogen-sensitive LHRH neurons could contribute to generalized activation of the LHRH system.     

Interaction between Dopamine‐ and Isotocin‐Containing Neurones in the Preoptic Area of the Catfish,Clarias batrachus: Role in the Regulation of Luteinising Hormone Cells

Apart from gonadotrophin‐releasing hormone (GnRH) and dopamine (DA), oxytocin has emerged as an important endogenous agent that regulates reproduction. Although the interaction between these factors has been extensively studied in mammals, parallel information in teleosts is much limited. We studied the organisation of tyrosine hydroxylase (TH; a marker for dopamine) and isotocin neurones in the preoptic area (POA) and hypothalamus of the catfish, Clarias batrachus and its implication in the regulation of luteinising hormone (LH) cells in the pituitary. Nucleus preopticus periventricularis (NPP), a major dopaminergic centre in the brain, consists of anterior (NPPa) and posterior (NPPp) subdivisions. Using retrograde neuronal tracing, we found that majority of the DA neurones in NPPa, but none from NPPp, project to the pituitary. The nucleus preopticus (NPO) of C. batrachus contains a conspicuous assemblage of large isotocin‐positive neurones. It consists of a paraventricular subdivision (NPOpv) located on either side of the third ventricle and lies roughly sandwiched between the dopaminergic neurones of NPPa and NPPp. An additional subset of isotocin neurones was located above the optic chiasm in the supraoptic subdivision of the NPO (NPOso). Isotocin‐containing neurones in both the subdivisions of NPO were densely innervated by DA fibres. Superfusion of the POA‐containing brain slices with DA D₁‐like receptor agonist (SKF‐38393) resulted in significant increase in isotocin immunoreactivity in the NPOpv neurones; NPOso neurones did not respond. However, treatment with DA D₂‐like receptor agonist (quinpirole) reduced isotocin immunoreactivity in the NPOso, but not in the NPOpv. Thus, DA appears to differentially regulate the components of isotocinergic system. Isotocin fibres extend to the pituitary and terminate on LH cells and the superfused pituitary slices treated with isotocin caused significant reduction in LHβ‐immunoreactivity. An elaborate interplay between the DA and isotocin systems appears to be an important component of the LH regulatory system.     

Increased Fos Expression in Preoptic Calcitonin Gene-Related Peptide (CGRP) Neurones following Mating but not the Luteinizing Hormone Surge in Female Rats

The functional relationship between sexually dimorphic neural populations and sex differences in reproductive functioning is unclear. The present study has investigated the function of the sexually dimorphic, estrogen-receptive, calcitonin gene-related peptide (CGRP) neurones in the female preoptic area by examining patterns of Fos immunoreactivity within these cells in relation to the luteinizing hormone surge and lordosis behaviour. In the first experiment, ovariectomized rats were treated with estradiol alone or estradiol plus progesterone to induce the luteinizing hormone surge. The percentage of CGRP neurones with Fos-positive nuclei was not different in estradiol alone (18 ± 4%) and estradiol/progesterone-treated (24 ± 3%) rats although the number of Fos-immunoreactive cells in the medial preoptic nucleus was increased 2-fold (P<0.01) in estrogen/progesterone-treated rats and 40 ± 5% of luteinizing hormone-releasing hormone neurones were found to express Fos in this group. In the second experiment, ovariectomized rats were treated with estradiol and progesterone and either, mated with a single male or placed in an empty cage, for 30 min. The number of Fos-immunoreactive cells in the medial preoptic nucleus was increased 4-fold in mated rats (P<0.01) and the percentage of CGRP neurones with Fos-positive nuclei increased from 24 ± 3% to 38 ± 2% (P<0.01) in mated animals. No differences were detected in the number of luteinizing hormone-releasing hormone neurones with Fos-positive nuclei in mated and non-mated animals. These results suggest that a sub-population of CGRP neurones in the medial preoptic nucleus may express Fos on a constitutive basis in steroid-treated animals and that, while not altered in relation to the luteinizing hormone surge, Fos expression by these cells is increased following mating. Although the precise role of these CGRP neurones has yet to be ascertained, the present experiments provide direct evidence of a functional relationship between a specific sexually dimorphic neural population and a component of sexually differentiated reproductive functioning.     

The Luteinising Hormone Surge‐Generating System is Functional in Male Goats as in Females: Involvement of Kisspeptin Neurones in the Medial Preoptic Area

A luteinising hormone (LH) surge is fundamental to the induction of ovulation in mammalian females. The administration of a preovulatory level of oestrogen evokes an LH surge in ovariectomised females, whereas the response to oestrogen in castrated males differs among species; namely, the LH surge‐generating system is sexually differentiated in some species (e.g. rodents and sheep) but not in others (e.g. primates). In the present study, we aimed to determine whether there is a functional LH surge‐generating system in male goats, and whether hypothalamic kisspeptin neurones in male goats are involved in the regulation of surge‐like LH secretion. By i.v. infusion of oestradiol (E₂; 6 μg/h) for 16 h, a surge‐like LH increase occurred in both castrated male and ovariectomised female goats, although the mean peak LH concentration was lower and the mean peak of the LH surge was later in males compared to females. Dual staining with KISS1 in situ hybridisation and c‐Fos immunohistochemistry revealed that E₂ treatment significantly increased c‐Fos expression in the medial preoptic area (mPOA) KISS1 cells in castrated males, as well as ovariectomised females. By contrast, dual‐labelled cells were scarcely detected in the arcuate nucleus (ARC) after E₂ treatment in both sexes. These data suggest that kisspeptin neurones in the mPOA, but not those in the ARC, are involved in the induction of surge‐like LH secretion in both male and female goats. In summary, our data show that the mechanism that initiates the LH surge in response to oestrogen, the mPOA kisspeptin neurones, is functional in male goats. Thus, sexual differentiation of the LH surge‐generating system would not be applicable to goats.     

Corticosterone Exerts Site-Specific and State-Dependent Effects in Prefrontal Cortex and Amygdala on Regulation of Adrenocorticotropic Hormone, Insulin and Fat Depots

Chronic stress stimulates corticosterone secretion and recruits brain pathways that regulate energy balance (caloric acquisition and deposition) and facilitate hypothalamic-pituitary-adrenal responsiveness to new stressors. We implanted corticosterone or cholesterol bilaterally either near the central nucleus of the amygdala (CeA) or in the prefrontal cortex to determine whether high concentrations of the steroid act at either site, with or without chronic stress. Rats were adrenalectomized and treated systemically with low doses of corticosterone. Half were maintained at room temperature and the other half were exposed to 5 degrees C cold for 5 days before all rats were restrained. There was limited diffusion of corticosterone from brain implants. Corticosterone in prefrontal cortex, but not CeA, decreased plasma insulin and adrenocorticotropic hormone (ACTH) responses to acute restraint in both control and chronically cold stressed rats. Corticosterone implants near CeA decreased the weight of fat depots only in cold; corticosterone implants in prefrontal cortex were ineffective. We conclude that (i) corticosterone inhibits insulin and ACTH secretion by an action in prefrontal cortex but not CeA; (ii) high concentrations of corticosterone secreted during chronic stress alter metabolism through (autonomic) outputs of the CeA and prefrontal cortex in site- and variable-specific fashion; and (iii) the amygdala is a component of a stress-recruited, state-dependent pathway.     

Progesterone Receptor-Containing Neurons in the Guinea-Pig Mediobasal Hypothalamus have Axonal Projections to the Medial Preoptic Area

The location and number of progesterone receptor-containing neurons in the mediobasal hypothalamus that project to the medial preoptic area were determined by combining retrograde fluorescent tract tracing with progesterone receptor immunocytochemistry. Injections of the retrograde tract tracer Fluoro-gold were made in the preoptic area of female guinea-pigs ovariectomized and primed with estradiol. After 5 days survival to allow for retrograde transport, tissue sections were incubated with monoclonal antibodies to the progesterone receptor to detect the presence of progesterone receptor-immunoreactive neurons. Cell bodies were labelled with Fluoro-gold throughout the arcuate nucleus. These neurons were not concentrated in any particular area of the nucleus but were diffusely distributed bilaterally. Retrogradely-labelled neurons were also observed in the ventrolateral and ventromedial nuclei mainly contralateral to the injection site. Progesterone receptor immunofluorescence labelled a subpopulation (7% to 10%) of these retrogradely-labelled cells particularly in the arcuate nucleus, including the median eminence. The double-labelled cells were more numerous in the anterior two-thirds of the arcuate nucleus. Although our estimates of the proportion of hypothalamic progesterone receptor-immunoreactive neurons that sent axons directly to the medial preoptic area were low, (about 0.35%), these neurons may be part of a neural circuit involved in the regulation of reproductive processes.     

Immunocytochemical Localization of Progesterone Receptors in Galanin Neurons in the Guinea Pig Hypothalamus

A double-label immunocytochemical technique was used to determine whether progesterone receptor-containing neurons in the female guinea-pig hypothalamus also contained galanin. Adult ovariectomized guinea-pigs were primed by estradiol to induce progesterone receptors and injected intracerebroventricularly with colchicine to visualize galanin-immunopositive neurons. A small proportion of progesterone receptor-containing perikarya in the medial preoptic area and the mediobasal hypothalamus were found to be immunoreactive for galanin. The medial preoptic, periventricular and arcuate nuclei showed the greatest concentration of double-labelled cells. Galanin varicosities appeared in close proximity to neurons with progesterone receptor-containing nuclei. These results provide neuroanatomical evidence that a subset of hypothalamic galanin-immunoreactive neurons is directly regulated by progesterone.     

Efferent Projections from the Ovarian Steroid Receptor-Containing Area of the Ventrolateral Hypothalamus in Female Guinea Pigs

The ventrolateral hypothalamus (VLH) in female guinea pigs includes a subset of neurons which contain estrogen and progestin receptors, and which are implicated in the regulation of female sexual behavior by steroid hormones. However, little is known about where these neurons project, and consequently which other brain areas are involved in sexual behavior in female guinea pigs. The anterograde tracer Phaseolus vulgaris -Leucoagglutinin was used to label efferents from the ovarian steroid receptor-containing part of the VLH. To identify the correct placement of the tracer specifically within the group of neurons containing estrogen receptors, medial hypothalamic sections were also immunostained for estrogen receptors. Forebrain areas receiving dense projections from the ventrolateral hypothalamus included the bed nucleus of the stria terminalis, medial preoptic area, anterior hypothalamic area, anterior ventromedial hypothalamus, and caudal ventrolateral hypothalamus. The midbrain central gray was also heavily labeled. Moderate innervation was observed in the forebrain in the basolateral amygdala, medial preoptic nucleus, lateroanterior hypothalamic nucleus, dorsal hypothalamic areas, posterior hypothalamus, zona incerta, and in the midbrain interspersed among the central and lateral tegmental tracts. The major efferent pathways from the VLH appeared to travel rostrally through the mediobasal hypothalamus and preoptic area, and caudally via the medial thalamic nuclei and periventricular fiber system. These findings are similar to those of previous studies tracing the efferents from the ventromedial nucleus in rats and from the lateral hypothalamus in guinea pigs. Many of these areas that receive input from the steroid receptor rich area within the VLH are likely to be involved in the regulation of female sexual behavior.     

Postnatal development and sex difference in neurons containing estrogen receptor-α immunoreactivity in the preoptic brain,the diencephalon,and the amygdala in the rat

Estrogen has been considered as a key substance that induces sexual differentiation of the brain during fetal and neonatal life in the rat. Thus, to define the brain regions involved in the brain sexual differentiation, we examined the regions where the estrogen receptor (ER) is located in the developing rat brain. We examined immunohistochemical distribution of the cells containing estrogen receptor-α (ER-α) in the preoptic region, the diencephalon, and the amygdala in male and female rats on postnatal days 1–35 (PD1–PD35). The antibody used recognizes ER-α equally well for both occupied and unoccupied forms. ER-α immunostaining was restricted to the cell nuclei of specific cell groups. In PD1 rats, ER-α-immunoreactive (ER-IR) signals were detected in the lateral septum, the organum vasculosum lamina terminalis, the medial preoptic nucleus (MPN), the median preoptic nucleus, the bed nucleus of the stria terminalis, the hypothalamic periventricular nucleus, the lateral habenula, the posterodorsal part of the medial amygdala nucleus, the posterior part of the cortical amygdala nucleus, the hypothalamic ventromedial nucleus (VMH), the hypothalamic arcuate nucleus, and the posterior hypothalamic periventricular nucleus. The distribution pattern of ER-IR cells in the newborn rat was much the same as that in the adult in the preoptic-hypothalamic and amygdala regions. Moreover, the signals in the MPN and the VMH were stronger in the female than in the male, perhaps reflecting the ability of estrogen generated by aromatization of testosterone in the male to down-regulate the ER signal. Thus, the brain regions showing sex differences may be sites of sexual differentiation of the brain by aromatizable androgen during the neonatal period. J. Comp. Neurol. 389:81–93, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of Prolactin‐Releasing Peptide in the Activation of Oxytocin Neurones in Response to Food Intake

Food intake activates neurones expressing prolactin‐releasing peptide (PrRP) in the medulla oblongata and oxytocin neurones in the hypothalamus. Both PrRP and oxytocin have been shown to have an anorexic action. In the present study, we investigated whether the activation of oxytocin neurones following food intake is mediated by PrRP. We first examined the expression of PrRP receptors (also known as GPR10) in rats. Immunoreactivity of PrRP receptors was observed in oxytocin neurones and in vasopressin neurones in the paraventricular and supraoptic nuclei of the hypothalamus and in the bed nucleus of the stria terminalis. Application of PrRP to isolated supraoptic nuclei facilitated the release of oxytocin and vasopressin. In mice, re‐feeding increased the expression of Fos protein in oxytocin neurones of the hypothalamus and bed nucleus of the stria terminalis. The increased expression of Fos protein in oxytocin neurones following re‐feeding or i.p. administration of cholecystokinin octapeptide (CCK), a peripheral satiety factor, was impaired in PrRP‐deficient mice. CCK‐induced oxytocin increase in plasma was also impaired in PrRP‐deficient mice. Furthermore, oxytocin receptor‐deficient mice showed an increased meal size, as reported in PrRP‐deficient mice and in CCK_A receptor‐deficient mice. These findings suggest that PrRP mediates, at least in part, the activation of oxytocin neurones in response to food intake, and that the CCK–PrRP–oxytocin pathway plays an important role in the control of the termination of each meal.     

Evidence for a projection from the lateral preoptic area and substantia innominata to the ‘mesencephalic locomotor region’ in the rat

A series of anatomical and electrophysiological experiments have been carried out to examine the organization of a direct projection from the substantia innominata and the lateral preoptic area of the hypothalamus, referred to collectively as the subpallidal region, to the pedunculopontine nucleus and adjacent parts of the dorsal midbrain in the adult rat. In the first series of experiments, the retrogradely transported fluorescent tracer SITS, which does not appear to be taken up by fibers-of-passage, was injected into the pedunculopontine nucleus, and the distribution of labeled neurons was plotted in the substantia innominata and lateral preoptic area, as well as in adjacent regions including the medial preoptic area, the bed nucleus of the stria terminalis and parvocellular parts of the paraventricular nucleus. Then, the anterogradely transported lectin PHA-L, which also does not appear to be taken up in effective amounts by fibers-of-passage, was injected into parts of the substantia innominata and lateral preoptic area that project directly to the pedunculopontine nucleus. These experiments demonstrated that fibers from both regions descend through the medial forebrain bundle and give rise in the pedunculopontine nucleus to a terminal field that contains many structures with the appearance of terminal boutons. They also indicated that individual fibers from the subpallidal region innervate both the pedunculopontine nucleus and adjacent parts of the central gray, and that the pathway innervates areas along the length of the medial forebrain bundle on its way to the dorsal midbrain. In a third series of experiments the retrogradely transported fluorescent tracer True Blue was injected into upper thoracic levels of the spinal cord, and it was found that the region of the pedunculopontine nucleus that receives the densest input from the subpallidal region contained many retrogradely labeled neurons on both sides of the brain. And finally, a series of electrophysiological experiments demonstrated that single-pulse stimulation of the substantia innominata and the lateral preoptic area altered the firing rate of a majority of the neurons in and around the pedunculopontine nucleus, and that excitatory and inhibitory responses occurred about equally. These results clearly suggest that the subpallidal region projects directly to the pedunculopontine nucleus and adjacent regions including the central gray and the superior colliculus.(ABSTRACT TRUNCATED AT 400 WORDS)