Distribution of Vesicular Glutamate Transporter-2 Messenger Ribonucleic Acid and Protein in the Septum-Hypothalamus of the Rat
Abstractx}-z:3, 百拇医药
The excitatory neurotransmitter glutamate is involved in the control of most, perhaps all, neuroendocrine systems, yet the sites of glutamatergic neurons and their processes are unknown. Here, we used in situ hybridization and immunohistochemistry for the neuron-specific vesicular glutamate transporter-2 (VGLUT2) to identify the neurons in female rats that synthesize the neurotransmitter glutamate as well as their projections throughout the septum-hypothalamus. The results show that glutamatergic neurons are present in the septum-diagonal band complex and throughout the hypothalamus. The preoptic area and ventromedial and dorsomedial nuclei are particularly rich in glutamatergic neurons, followed by the supraoptic, paraventricular, and arcuate nuclei, whereas the suprachiasmatic nucleus does not express detectable amounts of VGLUT2 mRNA. Immunoreactive neurites are seen in very high densities in all regions analyzed, particularly in the preoptic region, followed by the ventromedial, dorsomedial, and arcuate nuclei as well as the external layer of the median eminence, whereas the mammillary complex does not exhibit VGLUT2 immunoreactivity. Many VGLUT2 immunoreactive fibers also contained synaptophysin, suggesting that the transporter is indeed localized to presynaptic terminals. Together, the results identify glutamatergic cell bodies throughout the septum-hypothalamus in region-specific patterns and show that glutamatergic nerve terminals are present in very large numbers such that most neurons in these brain regions can receive glutamatergic input. We examined the GnRH system as an example of a typical neuroendocrine system and could show that the GnRH perikarya are closely apposed by many VGLUT2-immunoreactive boutons, some of which also contained synaptophysin. The presence of VGLUT2 mRNA-containing cells in specific nuclei of the hypothalamus indicates that many neuroendocrine neurons coexpress glutamate as neurotransmitter, in addition to neuropeptides. These systems include the oxytocin, vasopressin, or CRH neurons as well as many others in the periventricular and mediobasal hypothalamus. The presence of VGLUT2 mRNA in steroid-sensitive regions of the hypothalamus, such as the anteroventral periventricular, paraventricular, or ventromedial nuclei indicates that gonadal and adrenal steroid can directly alter the functions of these glutamatergic neurons.
Introductiond(9o, 百拇医药
GLUTAMATE IS regarded as the most abundant excitatory neurotransmitter in the hypothalamus that is involved in the regulation of most, if not all, neuroendocrine systems (1, 2). Thus, glutamate stimulates the release of GnRH, CRH, GHRH, or oxytocin and vasopressin by activating a variety of postsynaptic receptors (for review, see Refs. 3 and 4). However, due to the lack of specific markers, the localization of neurons that synthesize glutamate as a neurotransmitter has been ambiguous. This is because glutamate is used in many metabolic pathways and in the synthesis of most proteins, and thus, this amino acid is present in every cell of the body. Although most glutamate used for metabolic purposes is derived from the breakdown of glucose to pyruvate, recent studies have shown that glutamate used for neurotransmission is preferentially derived from glutamine, which is synthesized in glial cells. Glutamine, which does not activate postsynaptic glutamate receptors, is released by the glial cells and taken up by nerve terminals for conversion to neurotransmitter glutamate (5). Once released at a presynaptic terminal, glutamate is taken back up into the nerve terminal by specific glutamate transporters. Many glutamate transporters have been identified; however, none of them had been unique for neurons. Recently, several vesicular Na+-dependent phosphate transporter genes have been cloned (6, 7) and shown to be uniquely expressed in glutamatergic neurons. These proteins transport glutamate into secretory vesicles with the same kinetics as those previously described for synaptic vesicles, and based on this function, these transporters have been called vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3) (8, 9, 10, 11). To date, they are the most specific markers to identify glutamatergic neurons. Previous studies have shown that the VGLUT1 protein is preferentially located in the cortex and hippocampus (12, 13, 14), whereas VGLUT2 mRNA and protein are abundant in the thalamus and hypothalamus (15, 16, 17), and VGLUT3 is most prominent in the striatum (11). However, the location of the cells that produce the transporters is unknown, as immunohistochemical approaches have identified only axon terminals, but not perikarya. In the present study we used in situ hybridization to localize VGLUT2 mRNA and immunohistochemistry to localize VGLUT2 protein in the rat hypothalamus and, by inference, identify and localize glutamatergic perikarya and neurites. Because work by others and our preliminary studies suggested that VGLUT1 and VGLUT3 mRNAs and proteins are either absent in the hypothalamus or expressed only at very low levels, the distribution of these transporters was not evaluated.
Materials and Methodsns}(^, 百拇医药
Animalsns}(^, 百拇医药
Young adult (60 to 120 d old), regularly cycling, female Sprague Dawley rats were purchased from Zivic Laboratories (Porterville, PA) and housed in the central animal facility of the University of Kentucky Medical Center in a temperature- and light-controlled environment. All procedures involving animals were conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the animal care and use committee of University of Kentucky.ns}(^, 百拇医药
Tissue preparation for in situ hybridizationns}(^, 百拇医药
Deeply anesthetized female rats were decapitated, and the brains were rapidly removed and frozen on dry ice. Twelve-micron-thick coronal sections were prepared through the medial septum-diagonal band complex to the mammary bodies, mounted onto positively charged SuperFrost Plus slides (CMS, Houston, TX), and stored at -80 C until use. Immediately before in situ hybridization, the slides were equilibrated to room temperature, fixed for 5 min in 4% paraformaldehyde, followed by a 2-min rinse in 0.1 M sodium phosphate buffer (pH 7.4). The slides were acetylated for 10 min in 0.1 M triethanolamine (pH 8.0) and 0.25% acetic anhydride, then dehydrated and air-dried.
Riboprobe preparationi*cjerv, 百拇医药
The partial cDNA sequence of rat VGluT2 was obtained by RT-PCR from rat brain total RNA using two oligonucleotide primers (forward primer, 5'-cgaaagatcatgaactgcggg-3'; reverse primer, 5'-gcccaaggttgcttctctcc-3') and cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, CA). The cDNA sequence corresponds to nucleotides 1615–1979 of GenBank entry 271235 (submitted by Drs. H. Mashima and T. Kojima).i*cjerv, 百拇医药
cRNA riboprobe was synthesized by in vitro transcription in the presence of HindIII-linearized VGluT2 plasmid, 35S-labeled UTP, and T7 RNA polymerase.i*cjerv, 百拇医药
In situ hybridizationi*cjerv, 百拇医药
The above brain sections were hybridized overnight at 55 C with the 35S-labeled cRNA riboprobe (1 x 106 cpm/section) diluted in hybridization cocktail containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 300 mM NaCl, 50% (vol/vol) deionized formamide, 10% (vol/vol) dextran sulfate, 1x Denhardt’s solution, and 100 mM dithiothreitol. After 16 h, sections were rinsed twice for 10 min each time in 2x SSC (1x SSC = 0.25 M NaCl and 0.015 M sodium citrate, pH 7.2) containing 10 mM dithiothreitol at 22 C, treated with ribonuclease A (20 µg/ml) for 30 min at 37 C, washed with 1x SSC for 15 min at room temperature, and washed twice with 0.1x SSC at 63 C for 30 min each time. Finally, the sections were rinsed in 0.1x SSC at room temperature, quickly dehydrated, and air-dried. Slides were exposed to BIO-MAX MR x-ray film (Kodak, Rochester, NY) for 1–2 wk.
Tissue preparation for immunohistochemistryq@, 百拇医药
For immunohistochemical experiments, 10 animals were perfusion-fixed under anesthesia with PBS (0.1 M, pH 7.4), followed by 4% paraformaldehyde and 7.5% saturated picric acid in PBS, and the brains were removed and postfixed in the above fixative overnight at 4 C. Serial 40-µm coronal Vibratome (St. Louis, MO) sections were collected from the hypothalamus, infiltrated with cryoprotectant (18), and stored at -20 C. Five animals received an intracerebroventricular injection of colchicine (100 µg/10 µl/animal) 2 d before perfusion fixation.q@, 百拇医药
Immunocytochemistryq@, 百拇医药
Vibratome sections were equilibrated to room temperature, washed in Tris-HCl buffer (0.05 M; pH 7.6), and treated with the blocking buffer (10% normal horse serum, 0.2% Triton X-100, and 0.1% sodium azide in Tris-HCl) for 1 h at room temperature. Sections were then incubated overnight at room temperature in affinity-purified rabbit or guinea pig anti-VGLUT2 antibody (1:1000). After several washes with Tris-HCl buffer, sections were incubated 1 h in affinity-purified, biotinylated donkey antirabbit IgG, or antiguinea pig antibody (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA), washed again in Tris-HCl buffer, and exposed for 1 h to avidin-biotin peroxidase complex (Elite, Vector Laboratories, Inc., Burlingame, CA). After two 10-min washes in Tris-HCl buffer, sections were stained for 10 min in Tris-HCl buffer containing 50 mg diaminobenzidine and 5 µl H2O2 (30%). For dual labeling studies, sections were exposed overnight to rabbit anti-VGLUT2 and monoclonal antisynaptophysin antibody (Sigma-Aldrich, St. Louis, MO; 1:1,000). After two 10-min washes, sections were incubated in affinity-purified, cross-absorbed second antibodies that were labeled with Texas Red or fluorescein isothiocyanate (1:100; Jackson ImmunoResearch Laboratories). For triple labeling studies, sections were exposed overnight to rabbit anti-VGLUT2, monoclonal antisynaptophysin, and guinea pig anti-GnRH (GP6–4; 1:1,000); washed twice for 10 min each time; and incubated in affinity-purified, cross-absorbed second antibodies that were labeled with Texas Red, fluorescein isothiocyanate, or Cy5 (1:100; Jackson ImmunoResearch Laboratories). Fluorescent-labeled sections were examined with a confocal microscope (Leica Corp., Rockleigh, NJ) using sequential scanning settings.
Specificity controls included absorption with antigen (VGLUT2 and GnRH peptides), omission of the primary antibodies, and extensive tests of cross-reactivities of the second antibodies. All control experiments resulted in the absence of staining.(7u, 百拇医药
VGLUT2 antibodies(7u, 百拇医药
Antibodies were raised is rabbits and guinea pig against a synthetic decapeptide unique to the C terminus of VGLUT2. The peptide was coupled to hemocyanin via glutaraldehyde, and 100 µg of this antigen were injected per immunization cycle. Resulting antibodies were affinity-purified and tested for their specificity by Western blot analysis using cortical and thalamic/hypothalamic tissue, immunohistochemistry, and absorption with the synthetic antigen peptide. An example of a Western blot is shown in Fig. 1.(7u, 百拇医药
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Figure 1. Western blot of cortical and hypothalamic/thalamic tissue, showing the presence of VGLUT2 immunoreactivity in hypothalamic, but not cortical, tissue fractions.
Analysis[[{2)@, 百拇医药
Every 10th section of five animals was analyzed, and the labeling intensities in the different anatomical structures were ranked on a subjective graded scale ranging from "not detected" to +++ (very intense labeling).[[{2)@, 百拇医药
Results[[{2)@, 百拇医药
In situ hybridization[[{2)@, 百拇医药
Autoradiographic detection of VGLUT2 mRNA revealed that in the septum many cells in the lambdoid zone exhibited strong hybridization signal throughout the rostro-caudal extent of this structure, whereas in the lateral septum only relatively few cells were labeled in the ventral portion (Fig. 2A). In the medial septum, most heavily labeled cells were located in the lateral aspects of this nucleus, and this band of cells extended through the vertical limb of the diagonal band into the horizontal limb. This distribution pattern continued caudally to the organum vasculosum of the lamina terminalis (OVLT), which is very heavily labeled with the VGLUT2 cRNA probe (Fig. 2, B and C).
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Figure 2. Distribution of VGLUT2 mRNA in the septum and hypothalamus from rostral (A) to caudal (J). Bar, 1 mm.z?}0:, http://www.100md.com
At the level of the preoptic region, the anteroventral periventricular nucleus (AVPV) and the anteroventral preoptic nuclei were most heavily labeled, followed by the median preoptic, anterodorsal, and medial preoptic area, especially its medial part (Fig. 2D). The central part and the ventral aspects of the medial preoptic nucleus, medial part, were strongly labeled, whereas further caudally the remaining portion of the medial preoptic nucleus and the lateral preoptic area were sparsely labeled (Fig. 2, E and F). At this level, VGLUT2 mRNA-containing cells continued to be present in the horizontal diagonal band and ventral portion of the lateral septum.z?}0:, http://www.100md.com
At the anterior level, the supraoptic and paraventricular nuclei were moderately labeled. In the paraventricular nucleus, both magnocellular and parvocellular portions showed hybridization signal that was strongest in the medial and dorsal parvocellular part (Fig. 2G). The periventricular nucleus was only lightly labeled. The lateroanterior hypothalamic nucleus contained many intensely labeled cells, and slightly fewer cells were seen in the anterior hypothalamic area, ventrolateral hypothalamic nucleus, and lateral hypothalamic area (Fig. 2H).
At the tuberal level, the posterior periventricular nucleus was unlabeled, whereas the arcuate nucleus contained moderate levels of hybridization signal throughout its rostrocaudal extent. The ventromedial nucleus was heavily labeled, whereas the dorsomedial nucleus exhibited slightly lighter signal (Fig. 2I). The lateral and dorsal hypothalamic areas contained patches of moderately labeled cells.a/;g, 百拇医药
The mammillary complex contained large amounts of VGLUT2 mRNA. Thus, both the dorsal and ventral premammillary nuclei, the median portion of the medial mammillary nucleus, as well as the supramammillary nucleus were very heavily labeled in contrast to the medial and lateral portions, which contained only moderate amounts of signal (Fig. 2J). The lateral hypothalamus contained patches of moderately labeled cells. The results are summarized in Table 1.a/;g, 百拇医药
fig.ommitteedfig.ommitteeda/;g, 百拇医药
Table 1. Distribution of VGLUT2 mRNA and protein in the septum-hypothalamus as revealed by in situ hybridization and immunohistochemistry
Immunohistochemistrym-, http://www.100md.com
Cell bodies.m-, http://www.100md.com
VGLUT2-immunoreactive cell bodies were seen only after colchicine treatment, and even after this treatment, the number of cells was relatively small. In general, the shapes and sizes of immunoreactive cells varied from large, multipolar neurons in, for example, the diagonal band to small, fusiform cells in the preoptic region.m-, http://www.100md.com
Beginning rostrally, a few immunoreactive cells were seen in the ventral part of the lateral septum, medial septum, and diagonal band (Fig. 3A). In contrast, numerous positive cells surrounded the organum vasculosum of the lamina terminalis in a distribution pattern reminiscent of a triangle with the base formed by the ventral surface of the brain and the apex reaching into the vertical limb of the diagonal band (Fig. 3B).m-, http://www.100md.com
fig.ommitteedfig.ommitteedm-, http://www.100md.com
Figure 3. Examples of VGLUT2 immunohistochemistry showing labeled neurons in the lateral septum (A), OVLT (B), anterior periventricular nucleus (C), median preoptic nucleus (D), SON (E and F), PVN (G and H), ventromedial nucleus (I), and arcuate nucleus (J). B, C, D, E, and G, Colchicinized animals. Bar, 50 µm (A–D, I, and J), 100 µm (E and F), and 200 µm (G and H).
In the preoptic region, many positive cells were seen in the periventricular preoptic nucleus, median preoptic nucleus, and anteroventral preoptic nuclei where they were highly concentrated next to the optic chiasm (Fig. 3, C and D). The medial preoptic complex contained slightly fewer immunoreactive cells in the anterodorsal and medial preoptic subunits, and only a few cells were seen in the lateral preoptic area.2tr], 百拇医药
At the anterior level, a few positive cells were detected in the periventricular nucleus, the caudal portion of the medial preoptic nucleus, and the anterior hypothalamic nucleus. Here, a strand of labeled cells appeared to be aligned in a ventrolateral orientation next to the lateral hypothalamus that extended from the fornix to the supraoptic nucleus. In contrast, most neurons of the supraoptic and the parvo- as well as magnocellular paraventricular nuclei contained immunoreactive VGLUT2 (Fig. 3, E–H). The suprachiasmatic nucleus was unlabeled, as was the lateral hypothalamus.
In the tuberal region many immunoreactive cells were present in the dorsal hypothalamic area, and fewer and less densely stained cells were seen in the dorsomedial, ventromedial, and arcuate nuclei. Only a few cells were identified in the lateral hypothalamus. In the mammillary complex, labeled cells were seen only in the lateral and medial mammillary nuclei.+h+0?, http://www.100md.com
Fibers.+h+0?, http://www.100md.com
VGLUT2-immunoreactive fibers had a punctuate appearance and were present in very large numbers throughout the septum-hypothalamus. At the light microscopic level, it appeared that immunoreactive puncta were juxtaposed to almost every neuron in the brain regions examined. There were, however, small regional differences in the number of immunoreactive puncta. Thus, the medial septum, most parts of the lateral septum, and the vertical limb of the diagonal band contained fewer VGLUT2-immunoreactive puncta compared with the horizontal limb of the diagonal band, the ventral part of the lateral septum, as well as the lambdoid region (Fig. 3A). The preoptic area was very heavily labeled, especially the periventricular preoptic nucleus, the anteroventral periventricular nucleus, and the region surrounding the organum vasculosum of the lamina terminalis (Fig. 3B). In the anterior hypothalamus, the supraoptic, paraventricular, and suprachiasmatic nuclei contained less abundant VGLUT2-containing puncta compared with the anterior hypothalamic nucleus. In the tuberal region, immunoreactive puncta were seen in high density in the ventromedial, dorsomedial, and arcuate nuclei as well as in the external layer of the median eminence and the posterior pituitary (Fig. 3, I and J). The mammillary complex did not contain VGLUT2-immunoreactive puncta, whereas the lateral hypothalamus contained a comparatively moderate number of VGLUT2-immunoreactive fibers throughout its extent. The results are summarized in Table 1.
Dual immunohistochemistry for VGLUT2 and synaptophysin showed that many VGLUT2-immunoreactive puncta were colocalized with synaptophysin immunoreactivity. Examples of colocalizations of VGLUT2 and synaptophysin in the ventromedial nucleus and the median eminence are shown in Fig. 4, A–F.2vmhu, 百拇医药
fig.ommitteedfig.ommitteed2vmhu, 百拇医药
Figure 4. Examples of immunohistochemical dual labeling for VGLUT2 (A and D) and synaptophysin (B and E) and well as overlays of both images (C and F) taken from the ventromedial nucleus (A–C) and the median eminence (D–F), showing that most glutamatergic boutons also contain the presynaptic marker synaptophysin. Such colocalizations appear in yellow in C and F (arrowheads). Bar, 20 µm.2vmhu, 百拇医药
To determine whether VGLUT2-immunoreactive fibers are juxtaposed to neuroendocrine neurons, immunohistochemical triple labeling for GnRH, VGLUT2, and synaptophysin was employed. The results show that GnRH neurons are contacted by several VGLUT2-immunoreactive boutons, many of which also contained synaptophysin (Fig. 5, A–D).
fig.ommitteedfig.ommitteedit'\, 百拇医药
Figure 5. Example of immunohistochemical triple labeling for VGLUT 2 (A), synaptophysin (B), and GnRH (C) as well as an overlay of the three images (D), showing that many glutamatergic boutons are juxtaposed to the GnRH neuron, and some of these terminals contain synaptophysin (arrowheads). Bar, 20 µm.it'\, 百拇医药
Discussionit'\, 百拇医药
In the present study a VGLUT2 cRNA probe and specific antibodies directed against the transporter protein were used to identify glutamatergic neurons in the septum-hypothalamus. Based upon the results of recent studies it appears that VGLUTs are indeed highly specific markers for excitatory neurons that use glutamate as neurotransmitter (19). Thus, the different VGLUT proteins transport glutamate with similar kinetic properties and amino acid specificities compared with synaptic vesicles in a highly specific fashion, in that they do not interact with related amino acids such as L- or D-aspartate, glycine or -aminobutyric acid. VGLUT proteins are present in the areas of the brain that are known to be glutamatergic, and VGLUTs are localized to synaptic vesicles, as has been shown with electron microscopic immunohistochemistry and by differential centrifugation followed by Western blot analysis (8, 10). In addition, we observed colocalization of VGLUT2 protein with synaptophysin, which further indicates that this transporter is preferentially directed to presynaptic terminals.
The results of the present study confirm and extend the data reported by Kaneko et al. (16) and show that VGLUT2 protein is present in numerous neurites in all regions of the septum and hypothalamus, which suggests that glutamate-containing presynaptic terminals are, from an anatomical point of view, in a position to affect almost every neuron in these brain regions. These data thus support the hypothesis that glutamate is the major excitatory neurotransmitter in the hypothalamus and participates in the regulation of probably all neuroendocrine systems (1). As a typical example of a neuroendocrine system, we examined GnRH perikarya in the medial septum-diagonal band and showed that these neurons were closely apposed by many VGLUT2-immunoreactive boutons, many of which contained synaptophysin. In addition to numerous axo-somatic contacts between VGLUT2-immunoreactive terminals and their target neurons, we identified VGLUT 2 protein in axon terminals in the external layer of the median eminence, which is the site where all hypophysiotropic hormones are released into the primary plexus of the hypothalamo-hypohyseal vasculature (20). The presence of VGLUT2 in this layer of the median eminence suggests that glutamate can stimulate the release of neurohormones from neighboring axon terminals into the blood, thus exerting a final control over coordinated neurohormone release. This view is supported by the findings that many neuroendocrine axon terminals in the median eminence contain glutamate receptor subunits, such as the N-methyl-D,L-aspartate or kainate subunits (21, 22, 23). Thus, glutamate released from the VGLUT2-positive axon terminals could bind to and activate these ion channels, which would cause the release of neurohormones. Furthermore, glutamate released in the external layer of the median eminence could enter the fenestrated capillaries of the primary plexus and act on the anterior pituitary cells directly. Such a direct stimulatory action of glutamate has been demonstrated for GH- and PRL-secreting cells, which release their hormone in response to the addition of glutamate in vitro (24, 25).
An examination of the distribution of VGLUT2 mRNA indicates that many neuroendocrine neurons probably coexpress glutamate in addition to neuropeptides. Thus, the magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) nuclei, which synthesize oxytocin or vasopressin, also express VGLUT2 mRNA and protein, as do certain cells in the parvocellular paraventricular nucleus that house the neurons that synthesize CRH. Previous studies have shown that many neurosecretory neurons in these nuclei are sensitive to glutamate (26, 27), and it was thought that most excitatory input originates from intrahypothalamic sites near the SON or PVN (28). The possibility that glutamatergic neurons could also reside inside the SON and PVN was first substantiated by electrophysiological studies that measured fast excitatory postsynaptic potentials in isolated nuclei that were blocked by glutamate receptor antagonists (29). The results of the present study confirm the existence of such intranuclear glutamatergic neurons as well as a strong glutamatergic innervation of these neurosecretory neurons, and they raise the possibility that, in fact, certain oxytocin-, vasopressin-, or CRH-synthesizing neurons are also glutamatergic. This view is supported by the finding that the size and location of the VGLUT2-immunoreactive neurons correspond well to the magnocellular oxytocin- and vasopressin-positive neurons in the supraoptic and paraventricular nuclei as well as to the CRH neurons in the parvocellular paraventricular nucleus. Similarly, the arcuate nucleus, which houses a large number of neuroendocrine cells that synthesize, among many others, somatostatin, dopamine, or neuropeptide Y, contains many VGLUT2 mRNA-expressing neurons, and, based upon the numbers and location of the glutamatergic neuron, it can be expected that many of the aminergic and peptidergic neurons in the arcuate nucleus coexpress glutamate as neurotransmitter, as has been shown for the catecholaminergic neurons in the brainstem (30). Future dual labeling experiments are needed to determine the exact phenotype of the glutamatergic neurons in these brain regions.
A comparison of the distribution of VGLUT2 mRNA with the distribution of the different ionotropic glutamate receptor subunit mRNAs suggests that many glutamatergic cells are themselves regulated by glutamate (31). For instance, in the ventromedial nucleus most neurons express VGLUT2 mRNA as well as large amounts of N-methyl-D,L-aspartate, -amino-3 hydroxy-5 methyl-4 isoxazole proprionic acid, and kainate subunit mRNAs. Similarly in the preoptic region, the AVPV and the median preoptic nuclei express most ionotropic glutamate receptor subunits as well as VGLUT2 mRNA. An exception to this scheme appears to be the suprachiasmatic nucleus, which expresses all known ionotropic glutamate receptor subunits (31), but not VGLUT2 mRNA. This finding indicates that the suprachiasmatic nucleus receives its extensive glutamatergic input from the retina and other brain regions and not from intranuclear glutamatergic neurons, and that the output of this nucleus is mediated by neurotransmitters other than glutamate.
Up until now no data have been available on the promotor region of the VGLUT2 gene and the regulation of VGLUT2 expression. However, based upon the distribution of steroid hormone receptor protein (32) and mRNAs (33) and VGLU2 mRNA in the hypothalamus, we predict that VGLUT2 expression is, at least in certain hypothalamic regions, regulated by gonadal and adrenal steroids. For instance, many neurons in the AVPV, arcuate nucleus, and ventromedial nucleus contain estrogen receptor-, and it is known that estradiol regulates the synthesis of many neurotransmitters/hormones as well as receptor proteins in these cells. Similarly, the neurons in the supraoptic and paraventricular nucleus express estrogen receptor-ß (34), and because many of these neurons are in the same location as GLUT2-containing cells, it is likely that steroid hormones could regulate the glutamate transporter expression.|u8cz7e, 百拇医药
In summary, the results show that glutamatergic neurons as identified by the presence of VGLUT2 mRNA and VGLUT2 protein are widely distributed throughout the septum-hypothalamus in a distinct pattern, suggesting that glutamatergic neurons participate in the control of probably all neuroendocrine systems and that many glutamatergic cells probably express other neuropeptides or monoamines. Detailed studies are needed to determine the precise phenotypes of the glutamatergic neurons as well as their regulation by neurotransmitters and steroid hormones.
received August 29, 2002.l, 百拇医药
Accepted for publication October 31, 2002.l, 百拇医药
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The excitatory neurotransmitter glutamate is involved in the control of most, perhaps all, neuroendocrine systems, yet the sites of glutamatergic neurons and their processes are unknown. Here, we used in situ hybridization and immunohistochemistry for the neuron-specific vesicular glutamate transporter-2 (VGLUT2) to identify the neurons in female rats that synthesize the neurotransmitter glutamate as well as their projections throughout the septum-hypothalamus. The results show that glutamatergic neurons are present in the septum-diagonal band complex and throughout the hypothalamus. The preoptic area and ventromedial and dorsomedial nuclei are particularly rich in glutamatergic neurons, followed by the supraoptic, paraventricular, and arcuate nuclei, whereas the suprachiasmatic nucleus does not express detectable amounts of VGLUT2 mRNA. Immunoreactive neurites are seen in very high densities in all regions analyzed, particularly in the preoptic region, followed by the ventromedial, dorsomedial, and arcuate nuclei as well as the external layer of the median eminence, whereas the mammillary complex does not exhibit VGLUT2 immunoreactivity. Many VGLUT2 immunoreactive fibers also contained synaptophysin, suggesting that the transporter is indeed localized to presynaptic terminals. Together, the results identify glutamatergic cell bodies throughout the septum-hypothalamus in region-specific patterns and show that glutamatergic nerve terminals are present in very large numbers such that most neurons in these brain regions can receive glutamatergic input. We examined the GnRH system as an example of a typical neuroendocrine system and could show that the GnRH perikarya are closely apposed by many VGLUT2-immunoreactive boutons, some of which also contained synaptophysin. The presence of VGLUT2 mRNA-containing cells in specific nuclei of the hypothalamus indicates that many neuroendocrine neurons coexpress glutamate as neurotransmitter, in addition to neuropeptides. These systems include the oxytocin, vasopressin, or CRH neurons as well as many others in the periventricular and mediobasal hypothalamus. The presence of VGLUT2 mRNA in steroid-sensitive regions of the hypothalamus, such as the anteroventral periventricular, paraventricular, or ventromedial nuclei indicates that gonadal and adrenal steroid can directly alter the functions of these glutamatergic neurons.
Introductiond(9o, 百拇医药
GLUTAMATE IS regarded as the most abundant excitatory neurotransmitter in the hypothalamus that is involved in the regulation of most, if not all, neuroendocrine systems (1, 2). Thus, glutamate stimulates the release of GnRH, CRH, GHRH, or oxytocin and vasopressin by activating a variety of postsynaptic receptors (for review, see Refs. 3 and 4). However, due to the lack of specific markers, the localization of neurons that synthesize glutamate as a neurotransmitter has been ambiguous. This is because glutamate is used in many metabolic pathways and in the synthesis of most proteins, and thus, this amino acid is present in every cell of the body. Although most glutamate used for metabolic purposes is derived from the breakdown of glucose to pyruvate, recent studies have shown that glutamate used for neurotransmission is preferentially derived from glutamine, which is synthesized in glial cells. Glutamine, which does not activate postsynaptic glutamate receptors, is released by the glial cells and taken up by nerve terminals for conversion to neurotransmitter glutamate (5). Once released at a presynaptic terminal, glutamate is taken back up into the nerve terminal by specific glutamate transporters. Many glutamate transporters have been identified; however, none of them had been unique for neurons. Recently, several vesicular Na+-dependent phosphate transporter genes have been cloned (6, 7) and shown to be uniquely expressed in glutamatergic neurons. These proteins transport glutamate into secretory vesicles with the same kinetics as those previously described for synaptic vesicles, and based on this function, these transporters have been called vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3) (8, 9, 10, 11). To date, they are the most specific markers to identify glutamatergic neurons. Previous studies have shown that the VGLUT1 protein is preferentially located in the cortex and hippocampus (12, 13, 14), whereas VGLUT2 mRNA and protein are abundant in the thalamus and hypothalamus (15, 16, 17), and VGLUT3 is most prominent in the striatum (11). However, the location of the cells that produce the transporters is unknown, as immunohistochemical approaches have identified only axon terminals, but not perikarya. In the present study we used in situ hybridization to localize VGLUT2 mRNA and immunohistochemistry to localize VGLUT2 protein in the rat hypothalamus and, by inference, identify and localize glutamatergic perikarya and neurites. Because work by others and our preliminary studies suggested that VGLUT1 and VGLUT3 mRNAs and proteins are either absent in the hypothalamus or expressed only at very low levels, the distribution of these transporters was not evaluated.
Materials and Methodsns}(^, 百拇医药
Animalsns}(^, 百拇医药
Young adult (60 to 120 d old), regularly cycling, female Sprague Dawley rats were purchased from Zivic Laboratories (Porterville, PA) and housed in the central animal facility of the University of Kentucky Medical Center in a temperature- and light-controlled environment. All procedures involving animals were conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the animal care and use committee of University of Kentucky.ns}(^, 百拇医药
Tissue preparation for in situ hybridizationns}(^, 百拇医药
Deeply anesthetized female rats were decapitated, and the brains were rapidly removed and frozen on dry ice. Twelve-micron-thick coronal sections were prepared through the medial septum-diagonal band complex to the mammary bodies, mounted onto positively charged SuperFrost Plus slides (CMS, Houston, TX), and stored at -80 C until use. Immediately before in situ hybridization, the slides were equilibrated to room temperature, fixed for 5 min in 4% paraformaldehyde, followed by a 2-min rinse in 0.1 M sodium phosphate buffer (pH 7.4). The slides were acetylated for 10 min in 0.1 M triethanolamine (pH 8.0) and 0.25% acetic anhydride, then dehydrated and air-dried.
Riboprobe preparationi*cjerv, 百拇医药
The partial cDNA sequence of rat VGluT2 was obtained by RT-PCR from rat brain total RNA using two oligonucleotide primers (forward primer, 5'-cgaaagatcatgaactgcggg-3'; reverse primer, 5'-gcccaaggttgcttctctcc-3') and cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, CA). The cDNA sequence corresponds to nucleotides 1615–1979 of GenBank entry 271235 (submitted by Drs. H. Mashima and T. Kojima).i*cjerv, 百拇医药
cRNA riboprobe was synthesized by in vitro transcription in the presence of HindIII-linearized VGluT2 plasmid, 35S-labeled UTP, and T7 RNA polymerase.i*cjerv, 百拇医药
In situ hybridizationi*cjerv, 百拇医药
The above brain sections were hybridized overnight at 55 C with the 35S-labeled cRNA riboprobe (1 x 106 cpm/section) diluted in hybridization cocktail containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 300 mM NaCl, 50% (vol/vol) deionized formamide, 10% (vol/vol) dextran sulfate, 1x Denhardt’s solution, and 100 mM dithiothreitol. After 16 h, sections were rinsed twice for 10 min each time in 2x SSC (1x SSC = 0.25 M NaCl and 0.015 M sodium citrate, pH 7.2) containing 10 mM dithiothreitol at 22 C, treated with ribonuclease A (20 µg/ml) for 30 min at 37 C, washed with 1x SSC for 15 min at room temperature, and washed twice with 0.1x SSC at 63 C for 30 min each time. Finally, the sections were rinsed in 0.1x SSC at room temperature, quickly dehydrated, and air-dried. Slides were exposed to BIO-MAX MR x-ray film (Kodak, Rochester, NY) for 1–2 wk.
Tissue preparation for immunohistochemistryq@, 百拇医药
For immunohistochemical experiments, 10 animals were perfusion-fixed under anesthesia with PBS (0.1 M, pH 7.4), followed by 4% paraformaldehyde and 7.5% saturated picric acid in PBS, and the brains were removed and postfixed in the above fixative overnight at 4 C. Serial 40-µm coronal Vibratome (St. Louis, MO) sections were collected from the hypothalamus, infiltrated with cryoprotectant (18), and stored at -20 C. Five animals received an intracerebroventricular injection of colchicine (100 µg/10 µl/animal) 2 d before perfusion fixation.q@, 百拇医药
Immunocytochemistryq@, 百拇医药
Vibratome sections were equilibrated to room temperature, washed in Tris-HCl buffer (0.05 M; pH 7.6), and treated with the blocking buffer (10% normal horse serum, 0.2% Triton X-100, and 0.1% sodium azide in Tris-HCl) for 1 h at room temperature. Sections were then incubated overnight at room temperature in affinity-purified rabbit or guinea pig anti-VGLUT2 antibody (1:1000). After several washes with Tris-HCl buffer, sections were incubated 1 h in affinity-purified, biotinylated donkey antirabbit IgG, or antiguinea pig antibody (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA), washed again in Tris-HCl buffer, and exposed for 1 h to avidin-biotin peroxidase complex (Elite, Vector Laboratories, Inc., Burlingame, CA). After two 10-min washes in Tris-HCl buffer, sections were stained for 10 min in Tris-HCl buffer containing 50 mg diaminobenzidine and 5 µl H2O2 (30%). For dual labeling studies, sections were exposed overnight to rabbit anti-VGLUT2 and monoclonal antisynaptophysin antibody (Sigma-Aldrich, St. Louis, MO; 1:1,000). After two 10-min washes, sections were incubated in affinity-purified, cross-absorbed second antibodies that were labeled with Texas Red or fluorescein isothiocyanate (1:100; Jackson ImmunoResearch Laboratories). For triple labeling studies, sections were exposed overnight to rabbit anti-VGLUT2, monoclonal antisynaptophysin, and guinea pig anti-GnRH (GP6–4; 1:1,000); washed twice for 10 min each time; and incubated in affinity-purified, cross-absorbed second antibodies that were labeled with Texas Red, fluorescein isothiocyanate, or Cy5 (1:100; Jackson ImmunoResearch Laboratories). Fluorescent-labeled sections were examined with a confocal microscope (Leica Corp., Rockleigh, NJ) using sequential scanning settings.
Specificity controls included absorption with antigen (VGLUT2 and GnRH peptides), omission of the primary antibodies, and extensive tests of cross-reactivities of the second antibodies. All control experiments resulted in the absence of staining.(7u, 百拇医药
VGLUT2 antibodies(7u, 百拇医药
Antibodies were raised is rabbits and guinea pig against a synthetic decapeptide unique to the C terminus of VGLUT2. The peptide was coupled to hemocyanin via glutaraldehyde, and 100 µg of this antigen were injected per immunization cycle. Resulting antibodies were affinity-purified and tested for their specificity by Western blot analysis using cortical and thalamic/hypothalamic tissue, immunohistochemistry, and absorption with the synthetic antigen peptide. An example of a Western blot is shown in Fig. 1.(7u, 百拇医药
fig.ommitteedfig.ommitteed(7u, 百拇医药
Figure 1. Western blot of cortical and hypothalamic/thalamic tissue, showing the presence of VGLUT2 immunoreactivity in hypothalamic, but not cortical, tissue fractions.
Analysis[[{2)@, 百拇医药
Every 10th section of five animals was analyzed, and the labeling intensities in the different anatomical structures were ranked on a subjective graded scale ranging from "not detected" to +++ (very intense labeling).[[{2)@, 百拇医药
Results[[{2)@, 百拇医药
In situ hybridization[[{2)@, 百拇医药
Autoradiographic detection of VGLUT2 mRNA revealed that in the septum many cells in the lambdoid zone exhibited strong hybridization signal throughout the rostro-caudal extent of this structure, whereas in the lateral septum only relatively few cells were labeled in the ventral portion (Fig. 2A). In the medial septum, most heavily labeled cells were located in the lateral aspects of this nucleus, and this band of cells extended through the vertical limb of the diagonal band into the horizontal limb. This distribution pattern continued caudally to the organum vasculosum of the lamina terminalis (OVLT), which is very heavily labeled with the VGLUT2 cRNA probe (Fig. 2, B and C).
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Figure 2. Distribution of VGLUT2 mRNA in the septum and hypothalamus from rostral (A) to caudal (J). Bar, 1 mm.z?}0:, http://www.100md.com
At the level of the preoptic region, the anteroventral periventricular nucleus (AVPV) and the anteroventral preoptic nuclei were most heavily labeled, followed by the median preoptic, anterodorsal, and medial preoptic area, especially its medial part (Fig. 2D). The central part and the ventral aspects of the medial preoptic nucleus, medial part, were strongly labeled, whereas further caudally the remaining portion of the medial preoptic nucleus and the lateral preoptic area were sparsely labeled (Fig. 2, E and F). At this level, VGLUT2 mRNA-containing cells continued to be present in the horizontal diagonal band and ventral portion of the lateral septum.z?}0:, http://www.100md.com
At the anterior level, the supraoptic and paraventricular nuclei were moderately labeled. In the paraventricular nucleus, both magnocellular and parvocellular portions showed hybridization signal that was strongest in the medial and dorsal parvocellular part (Fig. 2G). The periventricular nucleus was only lightly labeled. The lateroanterior hypothalamic nucleus contained many intensely labeled cells, and slightly fewer cells were seen in the anterior hypothalamic area, ventrolateral hypothalamic nucleus, and lateral hypothalamic area (Fig. 2H).
At the tuberal level, the posterior periventricular nucleus was unlabeled, whereas the arcuate nucleus contained moderate levels of hybridization signal throughout its rostrocaudal extent. The ventromedial nucleus was heavily labeled, whereas the dorsomedial nucleus exhibited slightly lighter signal (Fig. 2I). The lateral and dorsal hypothalamic areas contained patches of moderately labeled cells.a/;g, 百拇医药
The mammillary complex contained large amounts of VGLUT2 mRNA. Thus, both the dorsal and ventral premammillary nuclei, the median portion of the medial mammillary nucleus, as well as the supramammillary nucleus were very heavily labeled in contrast to the medial and lateral portions, which contained only moderate amounts of signal (Fig. 2J). The lateral hypothalamus contained patches of moderately labeled cells. The results are summarized in Table 1.a/;g, 百拇医药
fig.ommitteedfig.ommitteeda/;g, 百拇医药
Table 1. Distribution of VGLUT2 mRNA and protein in the septum-hypothalamus as revealed by in situ hybridization and immunohistochemistry
Immunohistochemistrym-, http://www.100md.com
Cell bodies.m-, http://www.100md.com
VGLUT2-immunoreactive cell bodies were seen only after colchicine treatment, and even after this treatment, the number of cells was relatively small. In general, the shapes and sizes of immunoreactive cells varied from large, multipolar neurons in, for example, the diagonal band to small, fusiform cells in the preoptic region.m-, http://www.100md.com
Beginning rostrally, a few immunoreactive cells were seen in the ventral part of the lateral septum, medial septum, and diagonal band (Fig. 3A). In contrast, numerous positive cells surrounded the organum vasculosum of the lamina terminalis in a distribution pattern reminiscent of a triangle with the base formed by the ventral surface of the brain and the apex reaching into the vertical limb of the diagonal band (Fig. 3B).m-, http://www.100md.com
fig.ommitteedfig.ommitteedm-, http://www.100md.com
Figure 3. Examples of VGLUT2 immunohistochemistry showing labeled neurons in the lateral septum (A), OVLT (B), anterior periventricular nucleus (C), median preoptic nucleus (D), SON (E and F), PVN (G and H), ventromedial nucleus (I), and arcuate nucleus (J). B, C, D, E, and G, Colchicinized animals. Bar, 50 µm (A–D, I, and J), 100 µm (E and F), and 200 µm (G and H).
In the preoptic region, many positive cells were seen in the periventricular preoptic nucleus, median preoptic nucleus, and anteroventral preoptic nuclei where they were highly concentrated next to the optic chiasm (Fig. 3, C and D). The medial preoptic complex contained slightly fewer immunoreactive cells in the anterodorsal and medial preoptic subunits, and only a few cells were seen in the lateral preoptic area.2tr], 百拇医药
At the anterior level, a few positive cells were detected in the periventricular nucleus, the caudal portion of the medial preoptic nucleus, and the anterior hypothalamic nucleus. Here, a strand of labeled cells appeared to be aligned in a ventrolateral orientation next to the lateral hypothalamus that extended from the fornix to the supraoptic nucleus. In contrast, most neurons of the supraoptic and the parvo- as well as magnocellular paraventricular nuclei contained immunoreactive VGLUT2 (Fig. 3, E–H). The suprachiasmatic nucleus was unlabeled, as was the lateral hypothalamus.
In the tuberal region many immunoreactive cells were present in the dorsal hypothalamic area, and fewer and less densely stained cells were seen in the dorsomedial, ventromedial, and arcuate nuclei. Only a few cells were identified in the lateral hypothalamus. In the mammillary complex, labeled cells were seen only in the lateral and medial mammillary nuclei.+h+0?, http://www.100md.com
Fibers.+h+0?, http://www.100md.com
VGLUT2-immunoreactive fibers had a punctuate appearance and were present in very large numbers throughout the septum-hypothalamus. At the light microscopic level, it appeared that immunoreactive puncta were juxtaposed to almost every neuron in the brain regions examined. There were, however, small regional differences in the number of immunoreactive puncta. Thus, the medial septum, most parts of the lateral septum, and the vertical limb of the diagonal band contained fewer VGLUT2-immunoreactive puncta compared with the horizontal limb of the diagonal band, the ventral part of the lateral septum, as well as the lambdoid region (Fig. 3A). The preoptic area was very heavily labeled, especially the periventricular preoptic nucleus, the anteroventral periventricular nucleus, and the region surrounding the organum vasculosum of the lamina terminalis (Fig. 3B). In the anterior hypothalamus, the supraoptic, paraventricular, and suprachiasmatic nuclei contained less abundant VGLUT2-containing puncta compared with the anterior hypothalamic nucleus. In the tuberal region, immunoreactive puncta were seen in high density in the ventromedial, dorsomedial, and arcuate nuclei as well as in the external layer of the median eminence and the posterior pituitary (Fig. 3, I and J). The mammillary complex did not contain VGLUT2-immunoreactive puncta, whereas the lateral hypothalamus contained a comparatively moderate number of VGLUT2-immunoreactive fibers throughout its extent. The results are summarized in Table 1.
Dual immunohistochemistry for VGLUT2 and synaptophysin showed that many VGLUT2-immunoreactive puncta were colocalized with synaptophysin immunoreactivity. Examples of colocalizations of VGLUT2 and synaptophysin in the ventromedial nucleus and the median eminence are shown in Fig. 4, A–F.2vmhu, 百拇医药
fig.ommitteedfig.ommitteed2vmhu, 百拇医药
Figure 4. Examples of immunohistochemical dual labeling for VGLUT2 (A and D) and synaptophysin (B and E) and well as overlays of both images (C and F) taken from the ventromedial nucleus (A–C) and the median eminence (D–F), showing that most glutamatergic boutons also contain the presynaptic marker synaptophysin. Such colocalizations appear in yellow in C and F (arrowheads). Bar, 20 µm.2vmhu, 百拇医药
To determine whether VGLUT2-immunoreactive fibers are juxtaposed to neuroendocrine neurons, immunohistochemical triple labeling for GnRH, VGLUT2, and synaptophysin was employed. The results show that GnRH neurons are contacted by several VGLUT2-immunoreactive boutons, many of which also contained synaptophysin (Fig. 5, A–D).
fig.ommitteedfig.ommitteedit'\, 百拇医药
Figure 5. Example of immunohistochemical triple labeling for VGLUT 2 (A), synaptophysin (B), and GnRH (C) as well as an overlay of the three images (D), showing that many glutamatergic boutons are juxtaposed to the GnRH neuron, and some of these terminals contain synaptophysin (arrowheads). Bar, 20 µm.it'\, 百拇医药
Discussionit'\, 百拇医药
In the present study a VGLUT2 cRNA probe and specific antibodies directed against the transporter protein were used to identify glutamatergic neurons in the septum-hypothalamus. Based upon the results of recent studies it appears that VGLUTs are indeed highly specific markers for excitatory neurons that use glutamate as neurotransmitter (19). Thus, the different VGLUT proteins transport glutamate with similar kinetic properties and amino acid specificities compared with synaptic vesicles in a highly specific fashion, in that they do not interact with related amino acids such as L- or D-aspartate, glycine or -aminobutyric acid. VGLUT proteins are present in the areas of the brain that are known to be glutamatergic, and VGLUTs are localized to synaptic vesicles, as has been shown with electron microscopic immunohistochemistry and by differential centrifugation followed by Western blot analysis (8, 10). In addition, we observed colocalization of VGLUT2 protein with synaptophysin, which further indicates that this transporter is preferentially directed to presynaptic terminals.
The results of the present study confirm and extend the data reported by Kaneko et al. (16) and show that VGLUT2 protein is present in numerous neurites in all regions of the septum and hypothalamus, which suggests that glutamate-containing presynaptic terminals are, from an anatomical point of view, in a position to affect almost every neuron in these brain regions. These data thus support the hypothesis that glutamate is the major excitatory neurotransmitter in the hypothalamus and participates in the regulation of probably all neuroendocrine systems (1). As a typical example of a neuroendocrine system, we examined GnRH perikarya in the medial septum-diagonal band and showed that these neurons were closely apposed by many VGLUT2-immunoreactive boutons, many of which contained synaptophysin. In addition to numerous axo-somatic contacts between VGLUT2-immunoreactive terminals and their target neurons, we identified VGLUT 2 protein in axon terminals in the external layer of the median eminence, which is the site where all hypophysiotropic hormones are released into the primary plexus of the hypothalamo-hypohyseal vasculature (20). The presence of VGLUT2 in this layer of the median eminence suggests that glutamate can stimulate the release of neurohormones from neighboring axon terminals into the blood, thus exerting a final control over coordinated neurohormone release. This view is supported by the findings that many neuroendocrine axon terminals in the median eminence contain glutamate receptor subunits, such as the N-methyl-D,L-aspartate or kainate subunits (21, 22, 23). Thus, glutamate released from the VGLUT2-positive axon terminals could bind to and activate these ion channels, which would cause the release of neurohormones. Furthermore, glutamate released in the external layer of the median eminence could enter the fenestrated capillaries of the primary plexus and act on the anterior pituitary cells directly. Such a direct stimulatory action of glutamate has been demonstrated for GH- and PRL-secreting cells, which release their hormone in response to the addition of glutamate in vitro (24, 25).
An examination of the distribution of VGLUT2 mRNA indicates that many neuroendocrine neurons probably coexpress glutamate in addition to neuropeptides. Thus, the magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) nuclei, which synthesize oxytocin or vasopressin, also express VGLUT2 mRNA and protein, as do certain cells in the parvocellular paraventricular nucleus that house the neurons that synthesize CRH. Previous studies have shown that many neurosecretory neurons in these nuclei are sensitive to glutamate (26, 27), and it was thought that most excitatory input originates from intrahypothalamic sites near the SON or PVN (28). The possibility that glutamatergic neurons could also reside inside the SON and PVN was first substantiated by electrophysiological studies that measured fast excitatory postsynaptic potentials in isolated nuclei that were blocked by glutamate receptor antagonists (29). The results of the present study confirm the existence of such intranuclear glutamatergic neurons as well as a strong glutamatergic innervation of these neurosecretory neurons, and they raise the possibility that, in fact, certain oxytocin-, vasopressin-, or CRH-synthesizing neurons are also glutamatergic. This view is supported by the finding that the size and location of the VGLUT2-immunoreactive neurons correspond well to the magnocellular oxytocin- and vasopressin-positive neurons in the supraoptic and paraventricular nuclei as well as to the CRH neurons in the parvocellular paraventricular nucleus. Similarly, the arcuate nucleus, which houses a large number of neuroendocrine cells that synthesize, among many others, somatostatin, dopamine, or neuropeptide Y, contains many VGLUT2 mRNA-expressing neurons, and, based upon the numbers and location of the glutamatergic neuron, it can be expected that many of the aminergic and peptidergic neurons in the arcuate nucleus coexpress glutamate as neurotransmitter, as has been shown for the catecholaminergic neurons in the brainstem (30). Future dual labeling experiments are needed to determine the exact phenotype of the glutamatergic neurons in these brain regions.
A comparison of the distribution of VGLUT2 mRNA with the distribution of the different ionotropic glutamate receptor subunit mRNAs suggests that many glutamatergic cells are themselves regulated by glutamate (31). For instance, in the ventromedial nucleus most neurons express VGLUT2 mRNA as well as large amounts of N-methyl-D,L-aspartate, -amino-3 hydroxy-5 methyl-4 isoxazole proprionic acid, and kainate subunit mRNAs. Similarly in the preoptic region, the AVPV and the median preoptic nuclei express most ionotropic glutamate receptor subunits as well as VGLUT2 mRNA. An exception to this scheme appears to be the suprachiasmatic nucleus, which expresses all known ionotropic glutamate receptor subunits (31), but not VGLUT2 mRNA. This finding indicates that the suprachiasmatic nucleus receives its extensive glutamatergic input from the retina and other brain regions and not from intranuclear glutamatergic neurons, and that the output of this nucleus is mediated by neurotransmitters other than glutamate.
Up until now no data have been available on the promotor region of the VGLUT2 gene and the regulation of VGLUT2 expression. However, based upon the distribution of steroid hormone receptor protein (32) and mRNAs (33) and VGLU2 mRNA in the hypothalamus, we predict that VGLUT2 expression is, at least in certain hypothalamic regions, regulated by gonadal and adrenal steroids. For instance, many neurons in the AVPV, arcuate nucleus, and ventromedial nucleus contain estrogen receptor-, and it is known that estradiol regulates the synthesis of many neurotransmitters/hormones as well as receptor proteins in these cells. Similarly, the neurons in the supraoptic and paraventricular nucleus express estrogen receptor-ß (34), and because many of these neurons are in the same location as GLUT2-containing cells, it is likely that steroid hormones could regulate the glutamate transporter expression.|u8cz7e, 百拇医药
In summary, the results show that glutamatergic neurons as identified by the presence of VGLUT2 mRNA and VGLUT2 protein are widely distributed throughout the septum-hypothalamus in a distinct pattern, suggesting that glutamatergic neurons participate in the control of probably all neuroendocrine systems and that many glutamatergic cells probably express other neuropeptides or monoamines. Detailed studies are needed to determine the precise phenotypes of the glutamatergic neurons as well as their regulation by neurotransmitters and steroid hormones.
received August 29, 2002.l, 百拇医药
Accepted for publication October 31, 2002.l, 百拇医药
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