RESUMO
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. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.
Assuntos
Senescência Celular/fisiologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Potenciais de Ação , Vias Aferentes/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Hormônio do Crescimento/fisiologia , Hormônio Liberador de Hormônio do Crescimento/genética , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismoRESUMO
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. TH immunoreactivity occurred in 36-35% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice and these proportions increased to 40 and 45% in 8-month-old mice. NT immunoreactivity was present in 14 and 24% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice up to 28 and 26% in 8-month-old mice. Thus, co-localization of peptides and enzyme in GHRH-eGFP neurons displays a sexual dimorphism at 3-month of age for NT, and at 8-month for TH, while the total number of GHRH-eGFP neurons does not exhibit gender difference at either age. In summary, it appears that changes in co-localized (and presumably co-released) peptides, rather than GHRH per se, may contribute to the changes in sexually dimorphic GH secretion with aging in the mouse.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Galanina/metabolismo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Neurônios/metabolismo , Neurotensina/metabolismo , Envelhecimento , Animais , Mapeamento Encefálico/métodos , Feminino , Proteínas de Fluorescência Verde , Masculino , Camundongos , Camundongos Transgênicos , Fatores Sexuais , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
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. 26% in male GHRH-eGFP mice (P < 0.02). This sex difference in sst2A distribution on GHRH neurons may play an important role in the sexually differentiated pattern of GH secretion.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/análise , Hipotálamo/química , Receptores de Somatostatina/análise , Caracteres Sexuais , Animais , Feminino , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Somatostatina/genéticaRESUMO
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. Immunohistochemical experiments with wild-type and MCH gene-null mice demonstrated genuine expression of MGOP confined to the MCH-containing neurons in the lateral hypothalamus area and the presence of an 'MGOP-like' antigen in periventricular nucleus and arcuate nucleus neurons and their area of projection. This suggested a colocalization in somatostatin (SRIF) hypophysiotropic neurons. Further characterization, using SRIF gene-null mice and Western blot analysis with recombinant proteins, revealed that the MGOP-like product was pro-SRIF1-64. The role of pro-SRIF1-64 on fetal hypothalamic neurons was evaluated and a strong tonic inhibitory effect on SRIF secretion was found. These results (i) indicate that MGOP expression is restricted to the MCH neurons in the lateral hypothalamus and that MGOP-like immunoreactivity outside this system corresponds to pro-SRIF1-64, and (ii) provide the first evidence for a negative feedback regulation by pro-SRIF1-64 on SRIF secretion, suggesting new mechanisms by which the pro-region of a neuropeptide precursor may control the regulated secretion of a neuropeptide derived from the same precursor.
Assuntos
Hipotálamo/química , Proteínas do Tecido Nervoso/análise , Precursores de Proteínas/fisiologia , Somatostatina/antagonistas & inibidores , Somatostatina/fisiologia , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Precursores de Proteínas/farmacologia , Ratos , Ratos Wistar , Somatostatina/metabolismo , Somatostatina/farmacologiaRESUMO
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. In parallel, spontaneous postsynaptic currents were recorded as an index of neuronal connectivity. Neurotrophin-3 (NT3) was expressed in a much larger population of neurons than brain-derived neurotrophic factor (BDNF) at both culture times. At 6 DIV, synaptic currents were scarce and expression of the neurotrophin receptors trkB and trkC was found in a small proportion of neurons only. These parameters increased markedly between 6 and 14 DIV, and also upon addition of neurotrophins. The most striking consequence of arcuate neuron maturation in vitro between 6 and 14 DIV was a marked phenotypic specification affecting somatostatin, neuropeptide Y and pro-opiomelanocortin, the three major neuropeptides expressed in the cultures. NT3, but not BDNF, was able to reproduce maturation-related phenotypic specification in 6 DIV arcuate cultures. Maturation-dependent phenotypic specification was less marked in periventricular cultures; in that case BDNF, not NT3 had a slight effect on phenotype specification. It is concluded that NT3 plays a selective role in phenotypic specification of neuropeptides in the arcuate region, whereas other maturation parameters (neurotrophin receptor expression and/or synaptogenesis) can be potentiated by either neurotrophin in both structures.