Membranes from pituitary intermediate lobe cells enhance differentiation of fetal hypothalamic dopaminergic neurons in primary culture

Coculture of adult pituitary intermediate lobe (IL) cells, a target for hypothalamic dopaminergic neurons, with fetal rat hypothalamic cells accelerate differentiation of dopaminergic neurons. This involves long range diffusible as well as additional factors which may be membrane-bound. To determine whether IL membrane-bound factors contribute to the differentiating effect of IL cells, IL membranes were added to dispersed fetal hypothalamic neurons. This stimulated the outgrowth of dopaminergic neurites and elevated TH levels. Limited trypsin proteolysis of IL cell surface abolished the effect on TH levels. Addition of adenohypophyseal membranes was ineffective. Joint treatment with IL membranes, and medium conditioned (CM) over IL cells, produced the same effect on TH levels as did coculture with the same number of IL cells. The results demonstrate that IL cells express on their surface a membrane-bound factor promoting differentiation of fetal dopaminergic neurons in vitro; this factor acts in addition to diffusible activities.     

Immunohistochemical localization of gamma-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution of gamma-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigated using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Distinct populations of hypothalamic dopaminergic neurons exhibit differential responses to brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3)

We have previously demonstrated that differentiation of hypothalamic dopaminergic (DA) neurons can be induced in culture by their pituitary intermediate lobe target cells, through both membrane and diffusible factors. We also showed that subpopulations of DA neurons from the arcuate nucleus only, not the periventricular area, can respond to the target. Here we investigated the possibility that both neuronal subsets could also respond differentially to brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3). Addition of NT3, but not BDNF, enhanced growth and branching of neurites, tyrosine hydroxylase (TH) as well as increasing levels of cultured arcuate DA neurons. Conversely, BDNF, but not NT3, affected the same parameters in cultured periventricular DA neurons. The neurotrophins thus affect DA neurons in a structure and neuronal type-selective manner, since general neuronal markers were not affected by either neurotrophin. Neurotrophin effects were reversed by addition of specific antibodies directed against them or their respective receptors, TrkB or TrkC. By themselves, the antibodies inhibited development of DA neurons below that of control cultures, suggesting involvement of endogenous neurotrophins. BDNF and NT3 were indeed found in both arcuate and periventricular neurons and in the intermediate lobe. BDNF was always present as the mature peptide. The mature form of NT3 was only detected in the periventricular area; a precursor-like heavier form was present in all tissues studied. The present data suggest that NT3, but not BDNF, could participate in the differentiating action of intermediate lobe cells on arcuate DA neurons.     

Immunohistochemical localization of γ-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution on γ-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigaed using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Differentiation of rat hypothalamic dopaminergic neurons is stimulated in vitro by target cells: the melanotrophs

We have investigated in vitro the influence of pituitary intermediate lobe melanotrophs on the differentiation of their afferent hypothalamic dopaminergic neurons. The presence of melanotrophs in primary cultures of foetal hypothalamic neurons induces an increase of the number of dopaminergic neurons (while the total neuronal population remains unchanged) and induces a stimulation of their neuritic outgrowth. These effects are mediated by diffusible factors since they are reproduced by application of conditioned medium issued from co-cultures with intermediate lobe cells from newborn rats. Moreover, by immunoneutralization of α-melanocyte-stimulating hormone (αMSH) in the co-culture or conditioned medium, or by application of the peptide itself, we demonstrate that the neuritotrophic effect on dopaminergic neurons is mediated by αMSH, the main secretory product of melanotrophs, whereas the inductive effect on the number of dopaminergic neurons is attributable to another diffusible neurotrophic factor(s) present in foetal, but not adult, adenohypophysis. Similar effects are observed on cultures of newborn hypothalamic neurons. However, at this stage of neuronal development, αMSH also increases the number of dopaminergic neurons, which could be due to a change of neuronal receptivity. We show that the neuritotrophic influence of αMSH is restricted to the dopaminergic neurons connected to the melanotrophs, and that in addition, these neurons systematically co-express the tyrosine hydroxylase and glutamate decarboxylase as the neurons innervating the melanotrophs in situ. These findings indicate that the differentiation of dopaminergic hypothalamic neurons is influenced by the target cells, melanotrophs, and that this trophic influence implicates αMSH.     

Histochemical study of dopamine-degrading monoamine oxidase activity in dopaminergic neurons of rat brain

We examined whether dopamine-degrading activity of monoamine oxidase (MAO) is present in dopaminergic neurons of the rat brain. We employed a double-labeling procedure combining immunohistochemistry for tyrosine hydroxylase (TH) and enzyme histochemistry for MAO activity using dopamine as a substrate. The following dopaminergic cell groups were examined: A16 (glomerular layer of the olfactory bulb), A14 (hypothalamic periventricular region), A13 (zona incerta), A12 (arcuate nucleus), A11 (periventricular gray matter of the caudal thalamus), A10 (ventral tegmental area), A9 (substantia nigra pars compacta, SNC) and A8 (retrorubral nucleus). Although no MAO activity was detected in any of the TH-immunoreactive dopaminergic neurons, strong dopamine-degrading MAO activity was found in TH-positive neurons in the locus coeruleus (LC) (i.e., noradrenergic neurons). Our results indicate that dopamine-degrading MAO activity is very low in dopaminergic neurons compared to the MAO activity in LC noradrenergic neurons.     

The mammalian pituitary intermediate lobe: an update on innervation and regulation

The pituitary intermediate lobe (IL) in mammals is an area of uniform endocrine cells which synthesize and release specific peptide products of the proopiomelanocortin gene. The lobe receives direct synaptic connections onto the endocrine cells from hypothalamic dopaminergic neurons. This review updates information on the dopaminergic as well as the gamma-aminobutyric acid inhibitory neuroregulation for the IL. It also provides a discussion of stimulatory molecules which are likely to affect peptide release, particularly the neurotransmitter serotonin, which may be present via uptake into the dopaminergic nerve terminals. Other stimulatory molecules discussed which are likely to significantly affect peptide secretion are norepinephrine, corticotropin-releasing factor, and several opiate peptides. A new direction of study involves the potential interaction of neurotrophic factors, which are present in all areas of the pituitary, and may be suggested to have a supportive role for the neural elements of the IL. The endocrine cells of the IL and their direct hypothalamic innervation are considered to be an easily accessed peripheral model for study of both neural-endocrine and neurotrophic-target cell interactions.     

Role of dopamine in the regulation of proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus and pituitary gland of the female rat as studied by in situ hybridization.

The effects of the dopaminergic antagonist haloperidol (HAL) as well as the D2 dopamine receptor agonist bromocriptine (BRO) on proopiomelanocortin (POMC) mRNA levels in the female rat arcuate nucleus and pituitary were investigated by quantitative in situ hybridization. Since we had already shown that sex steroids could induce a decrease in POMC mRNA levels in the arcuate nucleus of castrated rats, the involvement of the dopaminergic system in the inhibitory effect of estradiol (E2) was also investigated. In situ hybridization was performed on paraformaldehyde-fixed cryostat sections through the arcuate nucleus and whole pituitary gland using a 35S-labelled cDNA probe encoding for POMC. In the arcuate nucleus of intact animals, a 14-day treatment with BRO increased by 54% the number of silver grains/unit of surface of labelled neurons while HAL decreased by 30% the value of this parameter. Hypophysectomy which induced a 20% decrease in the hybridization signal could not prevent the effects of BRO or HAL. Ovariectomy performed 14 days earlier increased by 20% the number of silver grains while a 14-day treatment of ovariectomized animals with E2 decreased the hybridization signal by 32%. On the other hand, the concomitant administration of HAL and E2 did not induce significant variations in POMC mRNA levels compared to those obtained following HAL administration, but slightly decreased the hybridization signal by 20% compared to that induced by E2 alone. In the intermediate lobe of the pituitary, BRO markedly depressed (30% of control values) and HAL increased by 50% the levels of POMC mRNA. The present data clearly demonstrate that POMC mRNA levels are differently regulated by dopamine in the intermediate lobe of the pituitary and the arcuate nucleus and that the effects of BRO and HAL on arcuate POMC mRNA are not mediated by the pituitary gland. They do not allow to draw any definite conclusion about the possible involvement of the dopaminergic system in the inhibitory role of E2 on POMC gene regulation.     

Resistance of hypothalamic dopaminergic neurons to neonatal 6-hydroxydopamine toxicity

We studied the effects of neonatal intracisternal administration of the 6-hydroxydopamine (6-OHDA) following desipramine pretreatment on dopaminergic (DA) neurons in the rat hypothalamus and substantia nigra by immunocytochemistry with an antiserum against tyrosine hydroxylase (TH). Neonatal intracisternal 6-OHDA injection induced almost complete loss of the TH-immunoreactivity in the substantia nigra and the caudate-putamen when examined at final (adult) stage. However, in this stage, no difference of TH-immunoreactivity was observed in hypothalamic DA neurons in the arcuate nucleus (A 12), peri ventricular area (A14), zona incerta (A 13), and posterior hypothalamic area (All). In the initial (neonatal) stage after the 6-OHDA injection, nigral DA neurons started to degenerate in 12 h and were almost completely destructed in 96 h, but hypothalamic DA neurons did not show any degenerative change at any time examined. The route of the injection (cistern, third ventricle or lateral ventricle) of the toxin did not influence the distribution of damage. These data show that 6-OHDA is not equally toxic to all brain DA neurons in neonates, and that all hypothalamic DA neuronal groups resist the toxicity of 6-OHDA, despite their anatomical and functional differences.     

Atrial natriuretic peptide-induced suppression of basal and dehydration-induced vasopressin secretion is not mediated by hypothalamic tuberohypophysial or tuberoinfundibular dopaminergic neurons

The purpose of this study was to examine the effects of atrial natriuretic peptide (ANP) on the secretion of vasopressin and the activities of hypothalamic tuberohypophysial and tuberoinfundibular dopaminergic neurons in normal and dehydrated male rats. Neuronal activity was estimated by measuring the concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and dopamine (DA) in brain and posterior pituitary regions containing terminals of tuberohypophysial (neural lobe; intermediate lobe) and tuberoinfundibular (median eminence) DA neurons. Intracerebroventricular (icv) administration of 20 micrograms ANP decreased basal arginine vasopressin concentrations in the plasma, but had no effect on the concentrations of DOPAC or DA in any region examined. Water deprivation caused a time-dependent increase in plasma arginine vasopressin concentrations, with maximal levels measured 2 days after removal of water bottles. Water deprivation had no effect on DOPAC concentrations in the neural lobe, intermediate lobe or median eminence, but increased DA concentrations in the neural lobe. ANP (20 micrograms/rat; icv) decreased arginine vasopressin concentrations in the plasma of water-deprived rats without altering DOPAC or DA concentrations in the neural lobe, intermediate lobe or median eminence. These results indicate that ANP-induced suppression of basal and dehydration-induced vasopressin secretion is not mediated by tuberohypophysial or tuberoinfundibular DA neurons.     

Immunohistochemistry of aromatic L-amino acid decarboxylase in the cat forebrain

The topographic distribution of aromatic L-amino acid decarboxylase (AADC)-immunoreactive (IR) neurons was investigated in the cat hypothalamus, limbic areas, and thalamus by using specific antiserum raised against porcine kidney AADC. The perikarya and main axons were mapped on an atlas in ten cross-sectional drawings from A8 to A16 of the Horsley Clarke stereotaxic plane. AADC-IR neurons were widely distributed in the anterior brain. They were identified in the posterior hypothalamic area, rostral arcuate nucleus of the hypothalamus, dorsal hypothalamic area, and periventricular complex of the hypothalamus, which contain tyrosine hydroxylase (TH)-IR cells and are known as A11 to A14 dopaminergic cell groups. AADC-IR perikarya were also found in the other hypothalamic areas where few or no TH-IR cells have been reported: the supramamillary nucleus, tuberomamillary nucleus, pre- and anterior mamillary nuclei, caudal arcuate nucleus, dorsal hypothalamic area immediately ventral to the mamillothalamic tract, anterior hypothalamic area, area of the tuber cinereum, retrochiasmatic area, preoptic area, suprachiasmatic and dorsal chiasmatic nuclei. We also identified them in the anterior commissure nucleus, bed nucleus of the stria terminalis, stria terminalis, medial and central amygdaloid nuclei, lateral septal nucleus, and nucleus of the diagonal band of Broca. AADC-IR neurons were localized in the ventromedial part of the thalamus, lateral posterior complex, paracentral nucleus and lateral dorsal nucleus of the thalamus, medial habenula, parafascicular nucleus, subparafascicular nucleus, and periaqueductal gray. Conversely, we detected only a few AADC-IR cells in the supraoptic nucleus whose rostral portion contains TH-IR perikarya. Comments are made on the relative localizations of the AADC-IR and TH-IR neurons, on species differences between the cat and rat, as well as on the possible physiological functions of the enzyme AADC.     

Pro-opiomelanocortin mRNA and peptide co-expression in the developing rat pituitary.

Pro-opiomelanocortin (POMC) is synthesized in both the pituitary gland and the brain. Various peptide products of this precursor, namely beta-endorphin, ACTH and alpha-MSH are co-localized in the anterior lobe corticotrophs, all intermediate lobe cells and in hypothalamic neurons. Messenger RNA (mRNA) for POMC has further been shown to exist in these tissues. In this study, we have shown that POMC mRNA, and peptide accumulation as detected by in situ hybridization and immunocytochemistry, respectively, occur simultaneously within the rat pituitary gland during ontogeny and that their maturation occurs in parallel during prenatal and early postnatal development.     

Presence of strong glucocorticoid receptor immunoreactivity within hypothalamic and hypophyseal cells containing pro-opiomelanocortic peptides.

The presence of nuclear glucocorticoid receptor immunoreactivity (GR IR) was studied in the adrenocorticotropin (ACTH), beta-Endorphin (beta-END) and alpha-melanocyte stimulating hormone (alpha-MSH) IR neuronal populations of the rat hypothalamus and hypophysis using double immunolabelling techniques. All the nuclei of the ACTH/beta-END/alpha-MSH IR neurons of the arcuate and periarcuate nuclei were strongly GR IR in the 48 h colchicine treated animal, but very few alpha-MSH-like IR perikarya located in the dorsal and lateral hypothalamus displayed nuclear GR IR. GR IR was present in the ACTH/beta-END corticotrophs and absent in the intermediate lobe of the hypophysis. The data provide morphological evidence for a glucocorticoid action through a nuclear GR in the arcuate ACTH/beta-END/alpha-MSH IR neurons and the ACTH/beta-END corticotrophs, whereas the alpha-MSH-like IR neurons of the lateral hypothalamus and the melanotropes of the intermediate lobe may not be directly affected by glucocorticoids under normal conditions.     

Hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) is not under dopaminergic control

A possible dopaminergic regulation of hypothalamic proopiomelanocortin (POMC)-containing neurons has been investigated in rats by means of in vivo and in vitro approaches. Acute or 3-weeks chronic in vivo treatments with the dopaminergic agonists apomorphine (1 mg/kg: s.c.) and 2-Br-alpha-ergocriptine (2.5 mg/kg; s.c.) or the dopaminergic antagonist haloperidol (0.15-3 mg/kg; i.p.) had no significant effect on the concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) in two hypothalamic regions: arcuate nucleus (AN) and dorsolateral area (DLH). In the same way, chronic administration of the dopaminergic agonists or antagonist did not induce any change in hypothalamic contents of beta-endorphin, another peptide derived from POMC. Reverse-phase high-performance liquid chromatographic analysis revealed that acetic acid extracts of AN and DLH both contained two major forms of alpha-MSH-like peptides: deacetylated alpha-MSH and authentic alpha-MSH. The ratio between these two forms was not altered after acute haloperidol treatment (3 mg/kg, i.p.). The possible effect of dopamine on the release of hypothalamic alpha-MSH was studied in vitro using perifused rat hypothalamic slices. Infusion of dopamine (10(-7)-10(-5)M) or its antagonist haloperidol (10(-5)M) had no effect on spontaneous alpha-MSH release from hypothalamic tissue. In addition, none of these drugs had any effect on potassium (50 mM)-induced alpha-MSH release. It is concluded that dopaminergic neurons are not involved in the regulation of synthesis, post-translational processing (acetylation) or release of hypothalamic alpha-MSH.     

Effects of coculture on the morphology of identified raphe and substantia nigra neurons from the embryonic rat brain

The substantia nigra (SN) is one of the earliest targets of the 5-HT neurons of the raphe nuclei (RN). To test the hypothesis that embryonic 5-HT and catecholamine neurons may influence the differentiation of their target cells or source neurons, we have produced dissociated cell cultures from embryonic day 14 (E14) rat rhombencephalon (containing the serotonergic RN) and mesencephalon (containing the dopaminergic substantia nigra, SN). These cells were grown for 6 days in vitro, either as single cultures (RN or SN) or cocultures (RN + SN). Effects of coculture on the morphological development of neurons immunoreactive (IR) for 5-HT or tyrosine hydroxylase (TH) were studied by measuring a series of morphological parameters related to size of the cell body and dendritic field, as well as to the complexity of neurites within this field, using computer-assisted morphometry. Increases in a number of these parameters were found in cocultures compared to single cultures for both types of monoamine neurons, but a greater number of parameters were increased for TH-IR cells, including size of the cell body. Although this might suggest that there was a greater effect of coculture on the TH-IR (dopaminergic) cells of the SN than on the 5-HT-IR cells of the RN, we must consider the fact that a significant population of TH-IR cells were present in single RN cultures, which contributed to the total population of TH-IR cells in cocultures. Indeed, when morphometric parameters for TH-IR cells in RN and SN single cultures were compared, it was found that TH-IR cells from the RN were generally larger and more complex than those from the SN. Therefore, an analysis was made of which parameters were significantly increased for TH-IR cells in cocultures compared to single cultures from both SN and RN. This was the case for two parameters: cell body size and absolute field area, indicating that these increases were probably due to the effects of coculture itself rather than to contamination by the larger and more complex TH-IR cells from the RN. It is impossible to ascertain, however, whether this effect was on cells from the RN, SN, or both. Coculture effects on 5-HT-IR cells were easier to analyze, since no such cells were found in single cultures of SN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Androgens stimulate the morphological maturation of embryonic hypothalamic aromatase-immunoreactive neurons in the mouse

Gonadal steroids play an important role as developmental factors for the rodent brain and are implicated in the sexual differentiation of neural structures. Estrogens have been linked to survival and plasticity of central neurons, thereby regulating the development of hypothalamic and limbic structures associated with reproductive functions. Besides estrogens, androgens also contribute actively to CNS maturation. We have shown recently that androgens stimulate the receptor-mediated functional differentiation of cultured hypothalamic aromatase-immunoreactive (Arom-IR) neurons by stimulating the expression of Arom, the key enzyme in estrogen formation. In the present study, we investigated whether androgens are capable of influencing morphological differentiation of hypothalamic Arom-IR neurons. Androgen treatment, unlike estrogen, stimulated the morphological differentiation of cultured embryonic hypothalamic Arom-IR cells by increasing neurite outgrowth and branching, soma size, and the number of stem processes. This effect was brain region- and transmitter phenotype-specific; neither cortical Arom-IR neurons nor hypothalamic GABAergic neurons responded to androgens. Moreover, morphogenetic effects depended on androgen receptor (AR) activation, since morphological changes were completely inhibited by flutamide. Double-labeling of hypothalamic Arom-IR neurons revealed a considerable number of cells coexpressing AR, whereas cortical Arom-IR cells did not label for AR. Our data demonstrate that androgens function as morphogenetic signals for developing hypothalamic Arom-IR cells, thus being potentially effective in influencing plasticity and synaptic connectivity of hypothalamic Arom-systems.     

A dopaminergic projection to the rat mammillary nuclei demonstrated by retrograde transport of wheat germ agglutinin-horseradish peroxidase and tyrosine hydroxylase immunohistochemistry.

The presence and distribution of dopaminergic neurons and terminals in the hypothalamus of the rat were studied by tyrosine hydroxylase (TH) immunohistochemistry. Strongly labelled TH-immunoreactive neurons were seen in the dorsomedial hypothalamic nucleus, periventricular region, zona incerta, arcuate nucleus, and supramammillary nucleus. A few TH-positive neurons were also identified in the dorsal and ventral premammillary nucleus, as well as the lateral hypothalamic area. TH-immunoreactive fibres and terminals were unevenly distributed in the mammillary nuclei; small, weakly labelled terminals were scattered in the medial mammillary nucleus, while large, strongly labelled, varicose terminals were densely concentrated in the internal part of the lateral mammillary nucleus. A few dorsoventrally oriented TH-positive axon bundles were also identified in the lateral mammillary nucleus. A dopaminergic projection to the mammillary nuclei from the supramammillary nucleus and lateral hypothalamic area was identified by double labelling with retrograde transport of wheat germ agglutinin-horseradish peroxidase and TH-immunohistochemistry. The lateral mammillary nucleus receives a weak dopaminergic projection from the medial, and stronger projections from the lateral, caudal supramammillary nucleus. The double-labelled neurons in the lateral supramammillary nucleus appear to encapsulate the caudal end of the mammillary nuclei. The medial mammillary nucleus receives a very light dopaminergic projection from the caudal lateral hypothalamic area. These results suggest that the supramammillary nucleus is the principal source of the dopaminergic input to the mammillary nuclei, establishing a local TH-pathway in the mammillary complex. The supramammillary cell groups are able to modulate the limbic system through its dopaminergic input to the mammillary nuclei as well as through its extensive dopaminergic projection to the lateral septal nucleus.     

Target-dependent synaptogenesis: Inductive effect of pituitary melanotrophs on their central innervation

The glandular activity of the vertebrate pituitary intermediate lobe (IL) is regulated by direct cellular innervation, in contrast with the purely humoral regulation of adjacent pituitary anterior lobe (AL). Thus in the rat IL, melanotrophs receive a dopaminergic and GABAergic innervation from the basal hypothalamus, which tonically inhibit their glandular activity. We studied this model of neuron–target interactions in cocultures in defined medium of fetal hypothalamic neurons with neonate pituitary glandular cells. In the cocultures with IL cells, neuroglandular contacts occurred after 4 days in vitro (DIV) but required another 8 DIV to exhibit ultrastructural and immunocytochemical features of fully differentiated functional synapses; by contrast, neuroneuronal synapses developed much faster and could already be detected after 4 DIV. In the cocultures with AL cells, neuroglandular contacts never mature in differentiated synapses. Confocal microscope observation revealed that dopaminergic neurons, which represented less than 1% of total neurons in the cocultures, established 50% of the synapses detected on the melanotrophs. These cells are thus able, contrary to the AL cells, to promote the establishment of functional synapses and, to some extent, to select their specific innervation. Synapse 27:267–277, 1997. © 1997 Wiley-Liss, Inc.     

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. © 1996 Wiley-Liss, Inc.     

Development of the neuronal system containing neuropeptide Y in the rat hypothalamus

In the rat hypothalamus, neuropeptide Y-containing neurons first appeared on day 14.5 of gestation in the arcuate nucleus and in the dorsolateral hypothalamic area. Until birth neuropeptide Y-containing cell bodies increased in number in the arcuate, dorsomedial-lateral and paraventricular nuclei, but disappeared thereafter, but some cells remaining in the arcuate nucleus. In animals treated neonatally with monosodium L-glutamate to destroy the arcuate nucleus, neuropeptide Y-immunoreactivity became evident in many cells scattered in the magnocellular paraventricular and dorsomedial-lateral hypothalamic nuclei on day 16 but not on days 60 and 12. These neuropeptide Y-immunoreactive neurons which appeared in the paraventricular nucleus were also vasopressin-positive. Neuropeptide Y fibers, on the contrary, remarkably diminished in number on day 16, particularly in the paraventricular and dorsomedial-lateral nuclei, and the medial preoptic area, but made a considerable recovery on days 60 and 120. Hence it is probable that, in normal ontogenetic progress, the development of the neuropeptide Y fibers in these areas is inhibitorily affected by that of arcuate neuropeptide Y neurons.