Sexually dimorphic distribution of sst2A receptors on growth hormone-releasing hormone neurones in mice: modulation by gonadal steroids.

The ultradian pulsatile pattern of growth hormone (GH) secretion is markedly sexually dimorphic in rodents as in primates, but the neuroanatomical mechanisms of this phenomenon are not clear. In the arcuate nucleus of the hypothalamus, GH-releasing hormone (GHRH) neurones receive somatostatinergic inputs through the sst2A receptor (sst2A-R) and the percentage of GHRH neurones bearing sst2A-R is higher in female than in male GHRH-enhanced green fluorescent protein (eGFP) mice. In the present study, we hypothesised that sst2A-R expression on GHRH neurones is modulated by gonadal steroids and constitutes a mechanism for sexually differentiated GH secretion.

Preserved memory capacities in aged Lou/C/Jall rats.

Although memory impairments are a hallmark of aging, the degree of deficit varies across animal models, and is likely to reflect different states of deterioration in metabolic and endocrinological properties. This study investigated memory-related processes in young (3-4 months) and old (24 months) Sprague-Dawley rats (SD), which develop age-linked pathologies such as obesity or insulin-resistance and Lou/C/Jall rats, which do not develop such impairments. In short- and long-term memory recognition tasks, old Lou/C/Jall rats were never impaired whereas old SD rats were deficient at 1 and 24h latencies.

Selective alteration at the growth-hormone- releasing-hormone nerve terminals during aging in GHRH-green fluorescent protein mice.

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods.

Multiple co-localizations in arcuate GHRH-eGFP neurons in the mouse hypothalamus.

In the present work, we took advantage of a recently described model of GHRH-enhanced green fluorescent protein (eGFP) transgenic mice to evaluate the extent of co-localization of GHRH neurons with galanin (GAL), neurotensin (NT) and tyrosine hydroxylase (TH) in 3- and 8-month-old male and female mice. The total number of GHRH-eGFP neurons along the rostro-caudal axis of the arcuate nucleus did not differ according to gender or age. GAL-immunoreactivity was present in 40-44% of 3-month-old GHRH-eGFP neurons in male and female arcuate nucleus, respectively, but only 25-22% in 8-month-old mice.

Sexually dimorphic distribution of sst2A somatostatin receptors on growth hormone-releasing hormone neurons in mice.

The pulsatile pattern of GH secretion exhibits sexual dimorphism that is likely to depend on somatostatin (SRIH) effects on somatoliberin (GHRH) neurons in the hypothalamus. Using transgenic GHRH-enhanced green fluorescent protein (eGFP) mice, no difference in the total number of GHRH-eGFP neurons or change in somatostatin receptor sst2 and SRIH mRNA levels in ventromedial hypothalamic nucleus-arcuate nucleus and periventricular nucleus regions and GHRH mRNA levels in the ventromedial hypothalamic-arcuate region were observed between male and female mice. However, the percentage of GHRH-eGFP neurons bearing sst2A receptors reached 78% in female vs.

Characterization of MCH-gene-overprinted-polypeptide-immunoreactive material in hypothalamus reveals an inhibitory role of pro-somatostatin1-64 on somatostatin secretion.

The melanin-concentrating hormone (MCH) gene encodes two proteins, pro-MCH and MCH-gene-overprinted polypeptide (MGOP), produced through alternative splicing of the primary transcript. Our initial purpose was to characterize the MGOP-immunoreactive material. First, MGOP mRNA was clearly found in rat and mouse hypothalami but Western blot analysis failed to unambiguously identify MGOP in protein extracts.

The neurotrophins NT3 and BDNF induce selective specification of neuropeptide coexpression and neuronal connectivity in arcuate and periventricular hypothalamic neurons in vitro.

Little is known on the influence of epigenetic factors in the developing hypothalamus, a region particularly involved in neuroendocrine regulation and rich in neuropeptides. The present study evaluated the effects of neurotrophins and neuronal activity on neuronal differentiation in hypothalamic cultures sampled from either arcuate or anterior periventricular regions of 17-day-old Sprague-Dawley fetuses. Expression of neuropeptides, tyrosine hydroxylase, neurotrophins and neurotrophin receptors was tested on young (6 days in vitro, DIV) and more mature (14 DIV) cultured neurons by multiple reverse transcription polymerase chain reaction on single cells.

Brain-derived neurotrophic factor but not neurotrophin-3 enhances differentiation of somatostatin neurons in hypothalamic cultures.

The present work investigated whether neurotrophins could differentially affect in vitro growth and maturation of two related subsets of hypothalamic neurons, hypophysiotropic somatostatin (SRIH) neurons projecting from the periventricular area and arcuate SRIH interneurons. For this purpose, the hypothalamus of 17-day-old rat fetuses was sampled and separated into a ventral and a dorsal fragment containing respectively periventricular and arcuate regions. Each fragment was dissociated and seeded separately in defined medium.

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.

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).

Somatostatin receptor subtypes sst1 and sst2 elicit opposite effects on the response to glutamate of mouse hypothalamic neurones: an electrophysiological and single cell RT-PCR study.

We have previously shown that somatostatin can either enhance or decrease AMPA/kainate receptor-mediated responses to glutamate in mouse-dissociated hypothalamic neurones grown in vitro. To investigate whether this effect is due to differential activation of somatostatin (SRIF) receptor subtypes, we compared modulation of the response to glutamate by SRIF with that induced by CH-275 and octreotide, two selective agonists of sst1 and sst2/sst5 receptors, respectively. Somatostatin either significantly decreased (49%) or increased (30%) peak currents induced by glutamate, and was ineffective in the remaining cells.

Profiles of amyloid precursor and presenilin 2-like proteins are correlated during development of the mouse hypothalamus.

The amyloid precursor protein (APP) and APP-like (APLP) material, as visualized with the Mab22C11 antibody, have previously been shown to be associated with radial glia in hypothalamus, which are known to promote neurite outgrowth. By Northern blot analysis, APP 695 mRNA levels increased steadily over hypothalamic development, APP 770 mRNA was transiently expressed at 12 days postnatally, and APLP mRNA was only weakly expressed in the hypothalamus. The developmental pattern of APP moeities in mouse hypothalamus and in fetal hypothalamic neurons in culture was compared with a presenilin 2 (PS2) related protein using an antibody developed against the N-terminal part of PS2.

Polysialylated neural cell adhesion is involved in target-induced morphological differentiation of arcuate dopaminergic neurons.

We have previously shown that the morphological and biochemical maturation of developing rat hypothalamic dopaminergic neurons is accelerated when they are cocultivated with pituitary intermediate lobe cells, one of their targets. Only two subsets of hypothalamic dopaminergic neurons (arcuate, A12, and periventricular, A14, nuclei) may project to the pars intermedia. In order to determine whether the two populations are equally responsive to coculture conditions, we microdissected the hypothalamus of 17-day-old rat fetuses in two fragments containing cell bodies from the A12 and from the A14 regions, prepared neuronal cultures from both portions and incubated them separately with intermediate lobe cells.

Distinct patterns of expression and physiological effects of sst1 and sst2 receptor subtypes in mouse hypothalamic neurons and astrocytes in culture.

Somatostatin (SRIF) receptor subtypes (sst) were characterized in hypothalamic neurons and astrocytes by quantitative reverse transcription-polymerase chain reaction and radioreceptor assays using [125I-Tyr0,D-Trp8]SRIF-14 as a ligand in ionic conditions discriminating between SRIF-1 (sst2, -3, and -5 receptors) and SRIF-2 (sst1 and -4 receptors) binding sites. In neurons, sstl mRNA levels were twofold higher than those of sst2, and sst3-5 expression was only minor. Astrocytes expressed 10-fold less sst mRNAs than neurons, which corresponded mostly (80%) to sst2.

Differential expression of somatostatin receptors by quantitative PCR in the rat brain.

Five subtypes of somatostatin receptors (sst) have recently been cloned and reported to be expressed in rat brain. However, conventional mRNA measurement techniques do not allow to accurately compare the levels of expression of the 5 sst. Thus, we established a quantitative reverse transcriptase polymerase chain reaction method for the 5 sst.

Modulation by somatostatin of glutamate sensitivity during development of mouse hypothalamic neurons in vitro.

Glutamate sensitivity development and interactions of somatostatin (SRIF) with AMPA/Kainate receptor-mediated glutamate responses were studied in dissociated hypothalamic neurons from 16-day-old mouse embryos grown in vitro. Only 18% of functionally innervated cells could be found at 6-9 DIV whereas the percentage of innervated neurons progressively increased thereafter to reach 100% at 19-22 DIV. The glutamate sensitivity, estimated from glutamate-induced peak inward current, was very low at 6-9 DIV, sharply increased at 11-14 DIV and developed at a low increase rate thereafter.

Molecular pharmacology of somatostatin receptors.

Somatostatin was discovered for its ability to inhibit growth hormone (GH) secretion. Later, it was found to be widely distributed in other brain regions, in which it fulfills a neuromodulatory role, and in several organs of the gastrointestinal tract where it can act as a paracrine factor or as a true circulating factor. In mammals, two molecules of 14 (somatostatin 14) and 28 (somatostatin 28) amino acids are the only biologically active members of the family.

Selective patterns of expression of G protein alpha subunits during in vitro development of hypothalamic neurons.

Expression of different alpha subunits of G proteins was studied in hypothalamic primary cultures grown in defined medium and enriched in either neurons or glial (astrocyte) cells. In parallel, the cellular distribution of Gi, Gs, and GoA subunits was visualized by in situ hybridization. Immunoblots using specific antisera and hybridization of mRNAs with specific oligonucleotide probes allowed us to characterize Gs, Gi2, and GoA as major neuronal G proteins in the hypothalamus, whereas the glial cells expressed mostly Gs, Gi2, and GoB forms.

Decreased choline acetyltransferase activity in nerve growth factor-transgenic mice during brain development.

Activity of the synthetic enzyme for acetylcholine, choline acetyltransferase was investigated during development and in adult nerve growth factor-transgenic mice. A conspicuous reduction of choline acetyltransferase activity was observed in the anterior brain of nerve growth factor-transgenic embryos from embryonic days 13 to 16 (E13 to E16). Choline acetyltransferase activity levels subsequently resumed to normal levels, with the exception of a 15% increase in the adult hippocampus.

Coculture of rat melanotrophs with hypothalamic cells enhances differentiation of dopaminergic neurons.

The interaction between a glandular target and hypothalamic dopaminergic neurons during development was studied by cocultivating dispersed fetal hypothalamic and adult intermediate lobe rat cells in serum-free medium. In such conditions, hypothalamic neurons aggregated around intermediate lobe cells and were interconnected by well-developed neurites. They could be grown in coculture at lower density than alone.

Ontogeny of thyrotropin-releasing hormone biosynthesis and release in hypothalamic neurons.

Thyrotropin-releasing hormone (TRH) is expressed at early postmitotic stages of hypothalamic neuron development, in the mouse and rat, as revealed by the presence of the mature peptide, of pro-TRH mRNAs, and of large precursor forms. This indicates a coordinate expression of several genes encoding, respectively, pro-TRH, its processing enzymes, and the cell machinery for intracellular transport, sorting, and release of TRH. During development, an acceleration of pro-TRH processing is revealed by an increased proportion of the mature peptide.

Differential expression and subcellular localization of secretogranin II and synaptophysin during early development of mouse hypothalamic neurons in culture.

Mature neurons contain two distinct regulated secretory pathways, characterized electron microscopically by so-called large dense core vesicles and small synaptic vesicles, respectively. Each vesicle type is characterized by vesicle-specific proteins, such as the granins (chromogranins/secretogranins) for the matrix of large dense core vesicles and synaptophysin for the membrane of small synaptic vesicles. So far, no data exist on the biogenesis of these two distinct vesicle types during neuronal development.

Alpha 1-adrenergic receptor coupling with phospholipase-C is negatively regulated by protein kinase-C in primary cultures of hypothalamic neurons and glial cells.

In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages.

Subcellular distribution of clathrin in cultured hypothalamic neurons.

The subcellular distribution of clathrin has been examined in developing hypothalamic neurons cultured in a chemically defined medium up to synapse formation (12-13 days in vitro) and exposed, or not, to a depolarizing concentration of KCl (60 mM for 3 min) followed, or not, by a return to control KCl concentration (3 mM KCl for 3 min). Previous studies have shown that such treatments induce in synaptic boutons a rapid vesicle depletion followed by massive restoration. Using an enzyme immunoassay, we have compared the relative proportion of assembled and unassembled pools of clathrin as a function of exposure to depolarizing or repolarizing concentrations of KCl.