Stereologic analysis of estrogen receptor alpha (ER alpha) expression in rat hypothalamus and its regulation by aging and estrogen

The estrogen receptor alpha (ERalpha) in the hypothalamus plays important roles in the regulation of reproductive development, physiology, and behavior. However, the expression of the ERalpha may change during aging or in response to varying estrogen levels. The present study measured changes in the numbers of ERalpha-expressing cells in specific hypothalamic and preoptic nuclei of ovariectomized female Sprague-Dawley rats at three ages (young [3-4 months], middle-aged [10-12 months], or old [24-26 months]) and with or without estrogen replacement. Numbers of ERalpha-immunoreactive neurons were quantified in four regions relevant to reproductive function: the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN), arcuate nucleus (ARH), and ventromedial nucleus (VMN), using an unbiased stereologic approach. In the AVPV and VMN, significant age-related increases in the numbers of ERalpha-expressing cells from the middle-aged to the old group were detected, and no differences were observed in the MPN and ARH, indicating that ERalpha neuron number is maintained or even elevated during aging. No significant effects of estrogen on ERalpha cell number were detected in any of the four regions studied. Therefore, ERalpha cell number in the rat hypothalamus and preoptic area changes with aging in a region-specific manner.     

Projections of the sexually dimorphic calcitonin gene-related peptide neurons of the preoptic area determined by retrograde tracing in the female rat

The medial preoptic area of the rat exhibits morphologic sex differences and is implicated in the control of sexually dimorphic behavior and function. Neurons expressing calcitonin gene-related peptide (CGRP) within the anteroventral periventricular (AVPV) and medial preoptic nucleus (MPN) of the medial preoptic area exhibit female-dominant sex differences in number through organizational and activational effects of gonadal steroids. The present study used retrograde tracing experiments to establish the projections of the AVPV and MPN CGRP neurons in the female rat. After the intraperitoneal administration of Fluoro-Gold to female rats (n = 5), we were unable to detect retrograde tracer in any CGRP-immunoreactive cells of the hypothalamus. Intracerebral injections of 50- to 100-nl volumes of Fluoro-Gold into the mediobasal hypothalamus resulted in up to 70% of CGRP neurons in the AVPV and MPN containing retrograde tracer. Similar large volume tracer depositions in the lateral septum, periaqueductal gray, two likely CGRP projection sites, resulted in no labeling of preoptic CGRP neurons. Experiments using small volume (30-nl) injections of Fluoro-Gold and green fluorescent microspheres at multiple sites in the mediobasal hypothalamus (n = 18) revealed that approximately 60% of AVPV and 30% of MPN neurons expressing CGRP were projecting to the region of the tuberal and ventral premammillary nuclei, with a minor projection to the dorsomedial nucleus. These findings demonstrate a major projection of the preoptic CGRP neurons to the posterior hypothalamus in the female rat and support further a functional role for these neurons in the sexually dimorphic regulation of reproductive functioning.     

The Anti-Dopaminergic Agent, Haloperidol, Antagonises the Feminising Effect of Neonatal Serotonin on Sexually Dimorphic Hypothalamic Nuclei and Tyrosine Hydroxylase Immunoreactive Neurones

There is a transient fall in hypothalamic serotonin (5‐hydroxytryptamine; 5‐HT) activity in the second week post partum in male but not female rats. When this fall is masked by administration of the 5‐HT₂ agonist (–) 2,5‐dimethoxy‐4‐iodophenyl]‐2‐aminopropane hydrochloride [(–)DOI], over days 8–16 post partum, males are feminised in adulthood. To investigate whether the effect of 5‐HT is mediated by dopamine and whether testosterone exerts its masculinising effect by reducing 5‐HT and dopamine activity, male pups were treated with (–)DOI alone or together with the dopamine antagonist, haloperidol, over days 8–16 post partum, whereas females were treated with testosterone propionate on day 2 post partum. In adulthood, the volumes of the anteroventral periventricular nucleus (AVPV), sexually dimorphic nucleus of the preoptic area (SDN‐POA) and arcuate nucleus (ARC) were determined, together with the number of tyrosine hydroxylase‐immunoreactive (TH‐ir) cells and fibres within them. The concentrations of 5‐HT, dopamine and their metabolites were also measured. (–)DOI treatment increased the volume of the AVPV, decreased that of the SDN‐POA and increased the number of TH‐ir cells in the AVPV. These feminising effects were antagonised by concurrent haloperidol treatment. Neonatal testosterone propionate masculinised the volumes of the female AVPV and SDN‐POA and reduced the number of TH‐ir cells in the AVPV. Dopamine and 5‐HT turnover in the AVPV was greater in female compared to male rats and neonatal testosterone propionate reduced dopamine concentration in the female AVPV. Neonatal (–)DOI had no effect on dopamine and 5‐HT activity in the AVPV but increased both in the ARC. The findings that TH‐ir neurone number and dopamine activity are greater in the female AVPV; the feminising effect of 5‐HT is prevented by a haloperidol; and the masculinising effect of testosterone propionate is accompanied by a decrease in TH‐ir neurone number and dopamine concentration in the female AVPV, suggest that dopamine is involved in hypothalamic sexual differentiation and may mediate the effect of 5‐HT.     

Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)-α and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10–20 h on oestrous. Brain sections were double-labelled to ERα or PR and tyrosine hydroxylase (TH). The number of TH-immunoreactive (ir) neurones did not vary significantly in any area evaluated. ERα expression in TH-ir neurones increased at 14 and 16 h in the rostral-ARC and dorsomedial-ARC, 14 h in the caudal-ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral-ARC, periventricular and AVPe. PR expression in TH-ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal-ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ERα and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.     

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos‐related antigen expression in tyrosine hydroxylase (TH)‐immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos‐related antigen in TH‐ir neurones of Pe and rostral‐, dorsomedial‐ and caudal‐ARC also decreased at 16.00 h. On dioestrus, 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos‐related antigen expression in TH‐ir neurones of Pe and rostral‐ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH‐ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.     

Central nesfatin‐1 influences the excitability of ghrelin‐responsive gastric distension neurons in the arcuate nucleus and reduces gastric motility in rats

Although the novel satiety peptide nesfatin‐1 has been shown to regulate gastric motility, the underlying mechanisms have yet to be elucidated. The study aimed to explore the effects of nesfatin‐1 on ghrelin‐responsive gastric distension (GD) neurons in the arcuate nucleus (Arc), and potential regulation mechanisms of gastric motility by the paraventricular nucleus (PVN). Single‐unit discharges in the Arc were recorded extracellularly, and gastric motility in conscious rats was monitored during the administration of nesfatin‐1 to the Arc or electrical stimulation of the PVN. Retrograde tracing and fluo‐immunohistochemistry staining were used to determine NUCB2/nesfatin‐1 neuronal projections. Nesfatin‐1 inhibited most of the ghrelin‐responsive GD‐excitatory neurons, but excited ghrelin‐responsive GD‐inhibitory neurons in the Arc. Gastric motility was significantly reduced by nesfatin‐1 administration to the Arc in a dose‐dependent manner. The firing activity in the Arc and changes to gastric motility were partly reduced by SHU9119, an antagonist of melanocortin 3/4 receptors. Electrical stimulation of PVN excited most of the ghrelin‐responsive GD neurons in the Arc and promoted gastric motility. Nonetheless, pretreatment with an anti‐NUCB2/nesfatin‐1 antibody in the Arc further increased the firing rate of most of the ghrelin‐responsive GD‐excitatory neurons and decreased the ghrelin‐responsive GD‐inhibitory neurons following electrical stimulation of the PVN. Gastric motility was enhanced by pretreatment with an anti‐NUCB2/nesfatin‐1 antibody in the Arc following PVN stimulation. Furthermore, NUCB2/nesfatin‐1/fluorogold double‐labeled neurons were detected in the PVN. These results suggest that nesfatin‐1 could serve as an inhibitory factor in the Arc to regulate gastric motility via the melanocortin pathway. The PVN could be involved in the regulation of the Arc in gastric activity.     

Distribution of prolactin receptor immunoreactivity in the brain of estrogen-treated,ovariectomized rats

Although there is extensive evidence for effects of prolactin (PRL) on the brain, knowledge about the PRL receptor (PRL-R) in the brain is limited. By using monoclonal antibodies raised against purified rat liver PRL-R, the distribution of PRL-R was investigated by immunohistochemistry in brains of the estrogen-treated ovariectomized (OVX+E) rat and the adult male rat. Immunohistochemistry was performed by using the avidin biotinylated horse radish peroxidase macromolecular complex method. In both male and OVX+E rats, strong immunostaining was detected in the choroid plexus of all cerebral ventricles. This immunostaining was localized predominately on epithelial cell membranes. In the OVX+E female rat, scattered immunoreactive perikarya were observed in the arcuate nucleus, periventricular hypothalamic nucleus, preoptic area, suprachiasmatic nucleus, and supraoptic nucleus of the hypothalamus. Immunostaining in hypothalamic nuclei was localized on neuronal cell bodies as well as on neuronal processes. In addition, there was extensive PRL-R immunoreactivity throughout the globus pallidus and ventral pallidum. Immunostaining in these striatal regions was not associated with neuronal cell bodies but appeared to be localized on processes or glial cells. In the male rat, less immunostaining was observed in the hypothalamus, and there was no immunostaining in the corpus striatum. No significant staining was observed in the cerebral cortex, thalamus, or hindbrain of either male or OVX+E rats. The implication of PRL-R existence in these brain regions remains to be investigated. J. Comp. Neurol. 394:462–474, 1998. © 1998 Wiley-Liss, Inc.     

Age‐related changes in hypothalamic androgen receptor and estrogen receptor α in male rats

The control of reproductive function involves actions of sex steroids upon their nuclear receptors in the hypothalamus and preoptic area (POA). Whether hypothalamic hormone receptors change their expression in aging male mammals has not been extensively pursued, although such changes may underlie functional losses in reproductive physiology occurring with aging. We performed a stereologic analysis of immunoreactive androgen receptor (AR) and estrogen receptor alpha (ERα) cells in three POA nuclei of male Sprague‐Dawley rats (anteroventral periventricular nucleus [AVPV], median preoptic area [MePO], and medial preoptic nucleus [MPN]), at young (3 months), middle‐aged (12 months), and old (20 months) ages. Serum testosterone and estradiol levels were assayed. Testosterone concentrations decreased significantly and progressively with aging. Estradiol concentrations were significantly higher in middle‐aged than either young or old rats. Stereologic analyses of the POA demonstrated that AR‐immunoreactive cell numbers and density in the AVPV, MePO, and MPN were significantly higher in old compared with young or middle‐aged rats. No change in the total number or density of ERα‐immunoreactive cells was detected with age, although when cells were subdivided by intensity of immunolabeling, the most heavily labeled ERα cells increased in number with aging in the AVPV and MePO, and in density in the AVPV. There are several interpretations to our finding of substantially increased AR cell numbers during aging, including a potential compensatory upregulation of the AR under diminished testosterone concentrations. These results provide further information about how the neural targets of steroid hormones change with advancing age. J. Comp. Neurol. 512:688–701, 2009. © 2008 Wiley‐Liss, Inc.     

Estrogen/progesterone treatment in adulthood affects the size of several components of the medial preoptic area in the male rat

The results of preliminary studies suggested that steroid and/or propylthiouracil (PTU) treatment of adult gonadectomized (Gxd) male rats significantly reduced the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA). Therefore, we designed a study to examine this effect in detail. Groups of adult rats were sham Gxd (intact) or Gxd, then treated with multiple injections of oil (males and females), or estrogen and progesterone (males). Gonadectomized estrogen/progesterone-treated males had a significantly smaller SDN-POA volume, smaller volume of the medial division of the medial preoptic nucleus (MPNm), smaller volume of the anteroventral MPNm (MPNav), and larger volume of the anteroventral periventricular nucleus (AVPv). The volume of the central division of the medial preoptic nucleus (MPNc) or of the suprachiasmatic nucleus was not affected. There were no differences between Gxd estrogen/progesterone-treated males vs the group that received PTU as well, indicating that the PTU treatment was unnecessary. The reduced volume of the SDN-POA was due to a reduced volume of the MPNav and of the portion of the SDN-POA located within the MPNm-exclusive of the MPNav and MPNc. In conclusion, estrogen/progesterone treatment in adulthood caused significant changes in the volume of several medial preoptic structures in two separate groups of Gxd males. Because the steroids produced no significant effects in intact males, testicular hormones appear to "protect" these structures from the effects of the estrogen/progesterone treatment.     

Distribution of leptin‐sensitive cells in the postnatal and adult mouse brain

Leptin plays a pivotal role in the regulation of energy homeostasis and neuroendocrine functions, and increasing evidence indicates that leptin acts on the brain to mediate many of these effects. Recent data have also suggested that leptin influences brain development during early postnatal life. Here we examined the distribution of cells that express mRNA encoding the long form of the leptin receptor (LepRb) in postnatal and adult mouse brains by using in situ hybridization. In both adults and neonates, LepRb mRNA was largely restricted to regions known to control energy balance. Labeled cells were found in the arcuate, ventromedial, and dorsomedial nuclei of the hypothalamus as well as in the lateral hypothalamic area. Heavily labeled cells were also found in the median preoptic and ventral premammillary nuclei, two hypothalamic nuclei that are known to control reproduction. Moreover, during postnatal and adult life, clearly labeled cells were found in extrahypothalamic autonomic control sites such as the nucleus of the tractus solitarius. Importantly, this receptor can induce intracellular signaling because peripheral injection of leptin caused STAT3 phosphorylation in most sites in which LepRb mRNA was expressed. LepRb mRNA was also transiently elevated in certain regions of the postnatal mouse brain, such as the cortex, hippocampus, and laterodorsal nucleus of the thalamus. Taken together, these observations are consistent with the proposed roles of leptin in feeding and neuroendocrine regulation. They also identify regions where LepRb mRNA is expressed during early postnatal life and suggest new roles for leptin in the nervous system during development. J. Comp. Neurol. 518:459–476, 2010. © 2009 Wiley‐Liss, Inc.     

Presence of vesicular glutamate transporter-2 in hypophysiotropic somatostatin but not growth hormone-releasing hormone neurons of the male rat

Recent evidence indicates that hypophysiotropic gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH) neurons of the adult male rat express mRNA and immunoreactivity for type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype. In the present study, we investigated the issue of whether these glutamatergic features are shared by growth hormone-releasing hormone (GHRH) neurons of the hypothalamic arcuate nucleus (ARH) and somatostatin (SS) neurons of the anterior periventricular nucleus (PVa), the two parvicellular neurosecretory systems that regulate anterior pituitary somatotrophs. Dual-label in situ hybridization studies revealed relatively few cells that expressed VGLUT2 mRNA in the ARH; the GHRH neurons were devoid of VGLUT2 hybridization signal. In contrast, VGLUT2 mRNA was expressed abundantly in the PVa; virtually all (97.5 +/- 0.4%) SS neurons showed labelling for VGLUT2 mRNA. In accordance with these hybridization results, dual-label immunofluorescent studies followed by confocal laser microscopic analysis of the median eminence established the absence of VGLUT2 immunoreactivity in GHRH terminals and its presence in many neurosecretory SS terminals. The GHRH terminals, in turn, were immunoreactive for the vesicular gamma-aminobutyric acid (GABA) transporter, used in these studies as a marker for GABA-ergic neuronal phenotype. Together, these results suggest the paradoxic cosecretion of the excitatory amino acid neurotransmitter glutamate with the inhibitory peptide SS and the cosecretion of the inhibitory amino acid neurotransmitter GABA with the stimulatory peptide GHRH. The mechanisms of action of intrinsic amino acids in hypophysiotropic neurosecretory systems require clarification.     

Reversal of the sexually dimorphic distribution of serotonin-immunoreactive fibers in the medial preoptic nucleus by treatment with perinatal androgen

A small, discrete nucleus at the rostral end of the third ventricle, the anteroventral periventricular nucleus (AVPv), has been reported to be involved in the control of gonadotropin release. Since monoaminergic neurotransmitter systems have also been implicated in this function we used an indirect immunohistochemical approach to examine the distribution of 3 monoaminergic neurotransmitter systems in this nucleus. Sections through the AVPv of both colchicine and non-colchicine-treated adult male and female Sprague-Dawley rats were processed for immunohistofluorescence with antisera directed against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), or serotonin (5-HT), and were subsequently counterstained with the fluorescent Nissl stain ethidium bromide. The distributions of TH-, DBH- and 5-HT-immunoreactive neural elements within the AVPv were evaluated and a comparison was made between males and females. In both sexes, few 5-HT-stained fibers were seen within the borders of the AVPv, in contrast to the relatively high 5-HT-stained fiber density of the surrounding region. A dramatic sexual dimorphism was found in the distribution of TH-immunoreactive fibers and cell bodies. Compared to males, the AVPv in the female contained 3-4 times as many TH-stained perikarya, and a 2- to 3-fold greater density of TH-stained fibers. A low to moderate density of DBH-immunoreactive fibers, and no DBH-stained cell bodies, were seen in the nucleus. A clear sex difference was not found in the density of DBH-stained fibers in the AVPv, indicating that the sexual dimorphism in TH-immunoreactive neural elements in this nucleus is due to a greater density of dopaminergic fibers and a greater number of dopaminergic cell bodies in the female. These results suggest that dopamine may participate in the control of gonadotropin secretion at the level of the AVPv.     

Distribution of catecholamine-containing neurons in the normal human hypothalamus

We have studied the distribution of catecholamine-containing neurons in the hypothalamus of 8 normal adult human brains, using Schmorl's stain for melanin and immunohistochemical staining for tyrosine hydroxylase (TH). TH immunoreactive perikarya were found in the wall of the third ventricle, in the areas in which dopaminergic neuroendocrine neurons are found in other primate species. Many of these neurons contained melanin pigment, and the percentage increased with age. Other melanin-pigmented neurons in the same distribution did not stain for TH, suggesting that postmortem TH immunostaining may not be sufficiently sensitive to visualize all catecholaminergic neurons. A separate group of larger TH-positive perikarya was seen in the lateral hypothalamic area. These may correspond to the incerto-hypothalamic dopamine neurons in other primate species. Only rare melanin-pigmented neurons were seen in this cell group, even at 66 years of age. Our data indicate that the hypothalamic neuroendocrine dopamine neurons in the human brain are distributed in a pattern similar to that in other primate species, and that both postmortem tyrosine hydroxylase and melanin staining provide an incomplete but representative sampling of the periventricular-arcuate cell group.     

Orexin-A regulates growth hormone-releasing hormone mRNA content in a nucleus-specific manner and somatostatin mRNA content in a growth hormone-dependent fashion in the rat hypothalamus

The orexins or hypocretins are two neuropeptides involved in the regulation of diverse biological processes such as feeding, sleep and neuroendocrine function. Recent findings suggest a possible functional interaction between orexins, somatostatin and growth hormone-releasing hormone (GHRH) in the rat hypothalamus. In order to understand the possible functional linkage between orexins and these neuropeptides, we determined the effects of intracerebroventricular orexin-A administration on hypothalamic somatostatin and GHRH mRNA levels. Furthermore, we examined whether growth hormone (GH) mediates these interactions by using two animal models that showed GH deficiency: hypophysectomized rats and dwarf Lewis rats. Using in situ hybridization, our data showed that GHRH mRNA levels in the paraventricular nucleus of the hypothalamus are decreased after orexin-A treatment, without changes in the arcuate nucleus of the hypothalamus. On the other hand, orexin-A treatment induces a GH-dependent stimulatory effect on somatostatin mRNA content in the periventricular nucleus of the hypothalamus. Finally, we demonstrated, for the first time, that hypophysectomized rats and dwarf Lewis rats, two classical models of GH deficiency with alterations in sleep patterns, showed a marked reduction in the GHRH mRNA levels in the paraventricular nucleus of the hypothalamus. These data improve our understanding of the interactions among the different systems involved in the control and pathophysiology of food intake, sleep and GH secretion.     

Projections of the medial preoptic nucleus: a Phaseolus vulgaris leucoagglutinin anterograde tract-tracing study in the rat

The projections of the medial preoptic nucleus (MPN) were examined by making injections of the anterogradely transported lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into the MPN and charting the distribution of labeled fibers. The evidence indicates that the MPN projects extensively to widely distributed regions in both the forebrain and brainstem, most of which also supply inputs to the nucleus. An important neuroendocrine role for the MPN is underscored by its extensive projections to almost all parts of the periventricular zone of the hypothalamus, including the anteroventral periventricular, anterior part of the periventricular, paraventricular (PVH), and arcuate nuclei, and a role in autonomic mechanisms is indicated by projections to such regions as the dorsal and lateral parvicellular parts of the PVH, the lateral parabrachial nucleus, and the nucleus of the solitary tract. Other projections of the MPN suggest participation in the initiation of specific motivated behaviors. For example, inputs to two nuclei of the medial zone of the hypothalamus, the ventromedial and dorsomedial nuclei, may be related to the control of reproductive and ingestive behaviors, respectively, although the possible functional significance of a strong projection to the ventral premammillary nucleus is presently unclear. The execution of these behaviors may involve activation of somatomotor regions via projections to the substantia innominata, zona incerta, ventral tegmental area, and pedunculopontine nucleus. Similarly, inputs to other regions that project directly to the spinal cord, such as the periaqueductal gray, the laterodorsal tegmental nucleus, certain medullary raphe nuclei, and the magnocellular reticular nucleus may also be involved in modulating somatic and/or autonomic reflexes. Finally, the MPN may influence a wide variety of physiological mechanisms and behaviors through its massive projections to areas like the ventral part of the lateral septal nucleus, the bed nucleus of the stria terminalis, the lateral hypothalamic area, the supramammillary nucleus, and the ventral tegmental area, all of which have extensive connections with regions along the medial forebrain bundle. Although the PHA-L method does not allow a clear demonstration of possible differential projections from each subdivision of the MPN, our results suggest that each of them does give rise to a unique pattern of outputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of mRNA for prolactin receptor (long form) in dopamine and pro-opiomelanocortin neurones in the arcuate nucleus of non-pregnant and lactating rats

Under most conditions, prolactin secretion from the pituitary gland is subject to negative-feedback regulation. Prolactin stimulates dopamine release from tuberoinfundibular (TIDA) neurones in the arcuate nucleus of the hypothalamus, which in turn suppresses the production of prolactin. However, during late pregnancy and continuing into lactation, this feedback mechanism becomes less responsive to prolactin and, as a result, a hyperprolactinaemic state develops. We investigated whether long-form prolactin receptor (PRL-R(L)) mRNA is present on TIDA neurones in nonpregnant and lactating rats. In addition, we examined whether PRL-R(L) mRNA is colocalized on hypothalamic pro-opiomelanocortin (POMC) neurones. Dual-label in situ hybridizations using an (35)S-labelled cRNA probe specific for long-form PRL-R, together with a digoxigenin-labelled RNA probe that encoded either tyrosine hydroxylase (TH) or POMC mRNA, were performed on brain sections. In both nonpregnant and lactating rats, the majority of TH mRNA-positive cells (> 90%) were found to express long-form PRL-R mRNA. In sections from nonpregnant rats, few non-TH positive cells expressed PRL-R(L) mRNA. By contrast, during lactation, the proportion of PRL-R(L) mRNA-positive cells that were not TH mRNA-positive increased to approximately 70%. Only a small number of neurones in this subpopulation of PRL-R(L) mRNA-positive neurones were found to be positive for POMC mRNA. These data show that the loss of responsiveness to prolactin occurring during lactation is not due to down regulation of long-form PRL-R gene expression on TIDA neurones. Moreover, the persistent expression of PRL-R(L) in arcuate neuroendocrine circuits suggests that PRL-R-mediated signalling continues to be important in these neurones during lactation.     

Distribution of serotonin immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko

The distribution of serotonin (5-hydroxytryptamine, 5-HT) in the forebrain and midbrain of the lizard Gekko gecko was studied by means of antibodies against serotonin. In the diencephalon, serotonin-immunoreactive (5-HTi) cell bodies were found in the hypothalamic periventricular organ and the ependymal wall of the infundibular recess. In the midbrain, 5-HTi cells were observed in the nucleus raphes superior and the lateral portion of the nucleus reticularis superior. In addition, 5-HTi cell bodies were found lateral to the ventral interpeduncular nucleus and around the ventral aspect of the medial longitudinal fasciculus. Serotonin-immunoreactive fibers and varicosities are present throughout the forebrain and the midbrain, but particularly in the nucleus accumbens, the septal area, the dorsal cortex, the dorsal thalamus, the lateral geniculate body, the ventromedial hypothalamic nucleus, the pretectal nucleus, and the basal optic nucleus. The medial habenular nucleus contains a dense 5-HTi plexus that shows a patchlike pattern. A laminar organization of 5-HTi fibers and varicosities is present in the midbrain tectum. When compared with data obtained in other vertebrates, the present study has confirmed that in the phylogenetic series fishes-amphibians-reptiles-birds-mammals there appears to be (1) a gradual decrease in the number of cerebrospinal-fluid-contacting serotoninergic cells in the hypothalamic periventricular layer and (2) a remarkable increase in number of serotoninergic cells in the midbrain tegmentum. As in mammals, a strong serotoninergic innervation of structures related to sensory, in particular visual, pathways could be recognized.