Research reportGlutamatergic innervation of corticotropin-releasing hormone- and thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat
Introduction
The hypothalamic–pituitary–adrenal (HPA) and thyroid (HPT) axes are two major endocrine regulatory systems that are important for energy homeostasis [48]. These neuroendocrine systems are governed by the hypophysiotropic corticotropin-releasing hormone (CRH)- and thyrotropin-releasing hormone (TRH)-synthesizing neurons, both residing in the paraventricular nucleus of the hypothalamus (PVN) [36], [41], [56]. Modulation of basal hormone synthesis of these hypophysiotropic neurons and their response to specific stimuli require both hormonal and neuronal regulation. Thus, circulating levels of glucocorticoids and thyroid hormones exert negative feedback effects on the activity of hypophysiotropic CRH and TRH neurons, respectively [34], [49], and these neurons can be influenced by neuronal systems that establish synaptic formations with their cell bodies and dendrites. Suppression of CRH and TRH gene expression during fasting, for example, is partly attributable to inhibition of the release of α-melanocyte-stimulating hormone (α-MSH) from axons originating in the hypothalamic arcuate nucleus [16], [17]. Catecholamines, released from ascending, monosynaptic projections of the lower brainstem, are responsible for upregulation of TRH gene expression and release during cold stress [46], [59], and for the increase in CRH gene expression following endotoxin administration [15].
In addition to the peptidergic and catecholaminergic inputs to PVN neurons, the inhibitory and excitatory neurotransmitters, GABA and glutamate, may also contribute to the regulation of these systems [2], [20], [31], [40]. GABA-ergic axon terminals have been recently reported to densely innervate both hypophysiotropic CRH and TRH neurons [19], [43]. However, there is no morphological evidence as to whether these neurons also receive a glutamatergic innervation. Until the recent discovery of the vesicular glutamate transporters (VGLUT1–3), highly specific markers of glutamatergic neurons [3], [6], [22], [26], [53], [54], [57], there was no specific tool for the morphological analysis of glutamatergic neuronal systems.
Using antisera against VGLUT2, the most abundant VGLUT expressed in the hypothalamus, CRH, and proTRH (178–199), we have performed double-labeling immunocytochemistry at light and electron microscopic levels in order to study the glutamatergic innervation of the CRH- and TRH-synthesizing neurons in the PVN.
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Animals
The experiments were carried out on adult male Wistar rats, weighing 280–350 g, housed under standard environmental conditions (light between 06:00 and 18:00 h, temperature 22 ± 1 °C, rat chow and water ad libitum). All experimental protocols were reviewed and approved by the Animal Welfare Committee at the Institute of Experimental Medicine of the Hungarian Academy of Sciences and Tufts New England Medical Center.
Animal preparation for double-labeling immunocytochemistry at light and electron microscopic levels
Since our preliminary data indicated that colchicine pretreatment is necessary
VGLUT2-immunoreactive (IR) innervation of CRH-IR and proTRH-IR neurons in the PVN
Immunolabeling by both VGLUT2 antisera showed that VGLUT2-IR axons homogeneously and densely innervated all parvocellular subdivisions of the PVN, while a slightly lower density of labeled fibers was seen in the magnocellular division. VGLUT2-IR axon varicosities were frequently juxtaposed to the perikarya and proximal dendrites of all CRH-IR neurons located in the medial parvocellular subdivision of the PVN (Fig. 1, Fig. 2). Similarly, in each of the major parvocellular subdivisions where
Discussion
Until recently, the morphological examination of the glutamatergic system has been difficult due to the lack of marker molecules specific to the glutamatergic phenotype of neurons. Two highly homologous transmembrane proteins, vesicular glutamate transporter 1 and 2, have now been proven specific for glutamatergic neurons based on the following observations. First, both VGLUT1 and VGLUT2 specifically transport glutamate into synaptic vesicles [3], [6], [22], [25], [26], [53], [54], [57].
Acknowledgments
This work was supported by Sixth EC Framework Program (LSHM-CT-2003-503041), OTKA (T046492, T046574), Ministry of Education (01806/2002), NKFP1A-002-04, and NIH (DK-37021) grants. This publication reflects the author's views and not necessarily those of the EU. The information in this document is provided as is and no guarantee or warranty is given that the information is fit for any particular purpose. The user thereof uses the information at its sole risk and liability. We thank Drs. Paul E.
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