Presence of vesicular glutamate transporter-2 in hypophysiotropic somatostatin but not growth hormone-releasing hormone neurons of the male rat Erik Hrabovszky, Gergely F. Turi and Zsolt Liposits Laboratory of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony u. 43., Budapest, 1083 Hungary Keywords: arcuate nucleus, confocal microscopy, in situ hybridization, median eminence, periventricular nucleus Abstract Recent evidence indicates that hypophysiotropic gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH) neurons of the adult male rat express mRNA and immunoreactivity for type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype. In the present study, we investigated the issue of whether these glutamatergic features are shared by growth hormone-releasing hormone (GHRH) neurons of the hypothalamic arcuate nucleus (ARH) and somatostatin (SS) neurons of the anterior periventricular nucleus (PVa), the two parvicellular neurosecretory systems that regulate anterior pituitary somatotrophs. Dual-label in situ hybridization studies revealed relatively few cells that expressed VGLUT2 mRNA in the ARH; the GHRH neurons were devoid of VGLUT2 hybridization signal. In contrast, VGLUT2 mRNA was expressed abundantly in the PVa; virtually all (97.5 ± 0.4%) SS neurons showed labelling for VGLUT2 mRNA. In accordance with these hybridization results, dual-label immunofluorescent studies followed by confocal laser microscopic analysis of the median eminence established the absence of VGLUT2 immunoreactivity in GHRH terminals and its presence in many neurosecretory SS terminals. The GHRH terminals, in turn, were immunoreactive for the vesicular c-aminobutyric acid (GABA) transporter, used in these studies as a marker for GABA-ergic neuronal phenotype. Together, these results suggest the paradoxic cosecretion of the excitatory amino acid neurotransmitter glutamate with the inhibitory peptide SS and the cosecretion of the inhibitory amino acid neurotransmitter GABA with the stimulatory peptide GHRH. The mechanisms of action of intrinsic amino acids in hypophysiotropic neurosecretory systems require clarification. Introduction l-Glutamate is the major excitatory neurotransmitter in the central nervous system (Monaghan et al., 1989; Collingridge & Singer, 1990; Headley & Grillner, 1990), whereas c-aminobutyric acid (GABA) functions as the primary mediator of inhibitory synaptic transmission (Decavel & Van den Pol, 1990). The palisade zone of the hypothalamic median eminence (ME) represents the termination field for hypophysiotropic axons which secrete releasing and release- inhibiting hormones into the pericapillary space of the hypophysial portal system. This region receives a dense GABA-ergic innervation (Meister & Hokfelt, 1988); moreover, the marker enzyme for GABA, glutamic acid decarboxylase, has been localized specifically to growth hormone-releasing hormone (GHRH)-, tyrosine hydroxylase (dopam- inergic marker)-, neurotensin- and galanin-immunoreactive (IR), but not to somatostatin (SS)-IR hypophysiotropic axon terminals (Meister & Hokfelt, 1988). The hypothalamus also hosts a large number of glutamatergic cell bodies that express the mRNA for type-2 vesicular glutamate transporter (VGLUT2) selectively (Herzog et al., 2001; Takamori et al., 2001; Lin et al., 2003). A subset of these VGLUT2-IR neurons are located in hypophysiotropic regions and innervate the ME (Lin et al., 2003; Varoqui et al., 2002); recent studies have shown that glutamatergic fibres of the ME are partly identical with hypophysi- otropic gonadotropin-releasing hormone (GnRH; Hrabovszky et al., 2004b)-, thyrotropin-releasing hormone (TRH; Hrabovszky et al., 2005)- and corticotropin-releasing hormone (CRH; Hrabovszky et al., 2005)-IR terminals. The putative cosecretion of glutamate from additional hypophys- iotropic neurosecretory systems, including GHRH and SS neurons, has not been addressed. The synthesis of growth hormone (GH) and its release from the somatotrophs is under the dual control of the stimulatory GHRH and the inhibitory SS; these two peptide neuro- hormones are synthesized in neuronal perikarya located in the arcuate and the anterior periventricular nuclei (ARH; PVa), respectively (Tannenbaum et al., 1990). In the present studies, we used dual-label in situ hybridization histochemistry (ISHH) to address the expression of VGLUT2 mRNA in the perikaryon of hypophysiotropic GHRH and ⁄ or SS neurons. In addition, we conducted dual-label immunoflu- orescent studies to investigate VGLUT2 immunoreactivity in GHRH and ⁄ or SS axon terminals of the ME. Based on a previous report indicating that hypophysiotropic GHRH neurons might be GABAer- gic (Meister & Hokfelt, 1988), instead of glutamatergic, we also examined the putative occurrence of the vesicular GABA transporter (VGAT) in GHRH terminals. Although VGAT is used in common for vesicular neurotransmitter uptake by glycinergic and GABAergic inhibitory neurons, it represents an authentic GABA marker within hypophysiotropic GHRH terminals which originate in the ARH, a Correspondence: Dr Erik Hrabovszky, as above. E-mail: hrabovszky@koki.hu Received 21 October 2004, revised 26 January 2005, accepted 2 February 2005 European Journal of Neuroscience, Vol. 21, pp. 2120–2126, 2005 ª Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2005.04076.x