Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat.

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus.

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water-electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.