Growth Hormone-Releasing Hormone (GHRH) Neurons in GHRH-Enhanced Green Fluorescent Protein Transgenic Mice: A Ventral Hypothalamic Network

Abstract

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.

Dual-Level Afferent Control of Growth Hormone-Releasing Hormone (GHRH) Neurons in GHRH–Green Fluorescent Protein Transgenic Mice

“…Female (13.6 Ϯ 0.3 weeks, n ϭ 79) and male (13.6 Ϯ 0.3 weeks, n ϭ 87) GHRH-GFP mice (Balthasar et al, 2003) were anesthetized by isofluorane inhalation, killed by decapitation, and brains quickly removed into cold (0 -2°C) solution 1 [containing the following (in mM): 92 NMDG-Cl, 2. In some experiments, vaginal smears were obtained from isoflouraneanesthetized female mice by flushing the vagina 2-3 times with 35 l of 0.9% saline.…”

“…Thus, other factors are playing major roles in the physiological organization of GHRH neuronal activity and its gender difference. Here, we studied how identified GHRH neurons worked and responded to the muscarinic agonist, carbachol, in acute brain slices from both adult male and female GHRH-green fluorescent protein (GFP) mice (Balthasar et al, 2003). Strikingly, carbachol revealed a gender difference in the control of GHRH neuronal activity, which involved fine tuning of afferent inputs at both short and long distances from the GHRH neurons.…”

Notch/Rbpjκ signaling regulates progenitor maintenance and differentiation of hypothalamic arcuate neurons

“…The in situ probes used were against Hes1, Hes5, Notch2 (Akazawa et al, 1992), Mash1 (Carninci et al, 2003), Notch1 (a gift from Dr Andy Groves, Baylor College of Medicine, Houston, TX, USA), Nkx2.1 (a gift from Dr Lori Sussel, Columbia University, New York, NY, USA), Rax (a gift from Dr Seth Blackshaw, Johns Hopkins University, Baltimore, MD, USA) , Pomc (a gift from Dr Malcom Low, University of Michigan, Ann Arbor, MI, USA) (Japón et al, 1994), Ghrh (a gift from Dr Paul Le Tissier, MRC NIMR, London, UK) (Balthasar et al, 2003;Le Tissier et al, 2005). Probes were linearized and transcribed with polymerase in the presence of digoxigenin-labeled nucleotides.…”

An updated view of hypothalamic–vascular–pituitary unit function and plasticity

“…Using genetically-modified mouse models with GHRH neurons marked with green fluorescent protein 118 122 ) and peripheral tissues (such as, ghrelin) 117 .…”