Coexpression of vesicular glutamate transporter-3 and gamma-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column

Markers of serotonergic, gamma-aminobutyric acid (GABA)-ergic (glutamic acid decarboxylase, 67 kDa isoform; GAD-67), and glutamatergic transmission (vesicular glutamate transporter 3; VGLUT3) have been detected in presumed sympathetic premotor neurons of the medullary raphe, a region that controls sympathetic tone to brown fat, skin blood vessels, and heart. In this study, the degree of coexpression of these markers was examined in raphe neurons by simultaneous histological detection of tryptophan hydroxylase (TrpOH) immunoreactivity with GAD-67 mRNA and VGLUT3 mRNA. Over half (52%) of the VGLUT3 mRNA-positive neurons expressed one or both of the other markers. The proportion of VGLUT3 neurons containing at least one of the other two markers was even higher (89%) for VGLUT3 spinally projecting neurons. VGLUT3 neurons containing markers for both serotonin and GABA were especially numerous (50-72%, depending on rostrocaudal level) within the marginal layer of raphe pallidus and the parapyramidal region. The dual GABAergic and glutamatergic nature of some bulbospinal raphe neurons was suggested by the presence of nerve terminals immunoreactive (ir) for both VGLUT3 and GABA in the intermediolateral cell column (IML) as detected by electron microscopy. VGLUT3-ir terminals formed approximately equal numbers of symmetric and asymmetric synapses onto presumed preganglionic neurons (nitric oxide synthase-ir profiles) or GABA-ir dendrites in IML, and terminals immunoreactive for both VGLUT3 and GABA always formed symmetric synapses. These data suggest that medullary raphe VGLUT3 neurons could inhibit sympathetic outflow and that their spinal targets include both preganglionic neurons and GABAergic interneurons.     

Vesicular glutamate transporter DNPI/VGLUT2 mRNA is present in C1 and several other groups of brainstem catecholaminergic neurons

The mouse glutamate vesicular transporter VGLUT2 has recently been characterized. The rat homolog of VGLUT2, differentiation-associated Na(+)/P(i) cotransporter (DNPI), was examined using a digoxigenin-labeled DNPI/VGLUT2 cRNA probe in the present study to determine which, if any, of the various groups of pontine or medullary monoaminergic neurons express DNPI/VGLUT2 mRNA and, thus, are potentially glutamatergic. DNPI/VGLUT2 mRNA was widely distributed within the brainstem and seemed exclusively neuronal. By using a double in situ hybridization method, the presence of the mRNA for DNPI/VGLUT2 and glutamic acid decarboxylase (GAD)-67 was mutually exclusive. By combining DNPI/VGLUT2 mRNA detection and conventional immunohistochemistry, DNPI/VGLUT2 mRNA was undetectable in lower brainstem cholinergic and serotonergic cells, but it was present in several tyrosine hydroxylase-immunoreactive (TH-ir) cell groups. DNPI/VGLUT2 mRNA was detected in most of the adrenergic neurons of the C1, C2, and C3 groups (75-80% of TH-ir neurons), in the A2 noradrenergic group (80%), and in vast numbers of area postrema cells. Within the A1 region, many fewer TH-ir cells contained DNPI/VGLUT2 (16%). Finally, DNPI/VGLUT2 mRNA was undetectable in the pontine noradrenergic cell groups (A5 and A6/locus coeruleus). In conclusion, the general pattern of DNPI/VGLUT2 expression and its exclusion from GABAergic, cholinergic, and serotonergic neurons supports the notion that DNPI/VGLUT2 mRNA identifies a subset of glutamatergic neurons in the lower brainstem. Within this region several catecholaminergic cell groups appear to be glutamatergic, including but not limited to the adrenergic cell groups C1-C3. Based on the present evidence, the noradrenergic cell groups of the pons (A5 and A6) do not contain either known vesicular glutamate transporter and are most likely not glutamatergic.     

Expression of GABAergic and glutamatergic phenotypic markers in hypothalamic proopiomelanocortin neurons

Hypothalamic proopiomelanocortin (POMC) neurons have traditionally been defined by their peptide transmitters, which are important regulators of energy balance and reward. Recent work shows that POMC neurons can also release the amino acid transmitters γ‐aminobutyric acid (GABA) and glutamate, although studying GABAergic and glutamatergic populations of POMC neurons has been hindered by the difficulty in reliably identifying amino acid (AA) transmitter phenotypes. In the present study, fluorescent in situ hybridization and immunohistochemistry were used to identify POMC neurons and to detect the presence of mRNA for the transporters responsible for packaging either GABA (vesicular GABA transporter [vGAT]) or glutamate (vesicular glutamate transporter [vGLUT]) into vesicles, as well as the enzymes responsible for GABA synthesis, glutamic acid decarboxylase (GAD)65 and GAD67. Approximately 7% of POMC neurons expressed vGlut2 and the highest percentage of vGlut2‐positive POMC cells were located in the rostral arcuate nucleus. Despite the reports of GABA release from POMC neurons, vGat was not detected in POMC neurons, although Gad65 and Gad67 were present in ～40% of POMC neurons. Approximately half of the vGlut2‐expressing POMC cells also expressed Gad65. Markers of neurotransmitter phenotype were better detected by using in situ hybridization techniques rather than transgenic expression of fluorophores under the control of the vGat or Gad67 promoters. It is now clear that the expression of markers of AA phenotype provides a useful means to identify distinct subpopulations of POMC neurons. Additionally, the method described will be useful to explore the possibility that plasticity of AA phenotype is an important aspect of POMC neuron function. J. Comp. Neurol. 520:3863–3876, 2012. © 2012 Wiley Periodicals, Inc.     

Age‐dependent changes in amino acid phenotype and the role of glutamate release from hypothalamic proopiomelanocortin neurons

Hypothalamic proopiomelanocortin (POMC) neurons are important regulators of energy balance. Recent studies indicate that in addition to their peptides, POMC neurons can release either the amino acid (AA) transmitter gamma‐aminobutyric acid (GABA) or glutamate. A small subset of POMC neurons appears to have a dual AA phenotype based on coexpression of mRNA for the vesicular glutamate transporter (vGlut2) and the GABA synthetic enzyme Gad67. To determine whether the colocalization of GABAergic and glutamatergic markers may be indicative of a switch in AA transmitter phenotype, fluorescent in situ hybridization was used to detect vGlut2 and Gad mRNA in POMC neurons during early postnatal development. The percentage of POMC neurons expressing vGlut2 mRNA in POMC neurons progressively decreased from ～40% at day 1 to less than 10% by 8 weeks of age, whereas Gad67 was only expressed in ～10% of POMC neurons at day 1 and increased until ～45% of POMC neurons coexpressed Gad67 at 8 weeks of age. To determine whether the expression of vGlut2 may play a role in energy balance regulation, genetic deletion of vGlut2 in POMC neurons was accomplished using Cre‐lox technology. Male, but not female, mice lacking vGlut2 in POMC neurons were unable to maintain energy balance to the same extent as control mice when fed a high‐fat diet. Altogether, the results indicate that POMC neurons are largely glutamatergic early in life and that the release of glutamate from these cells is involved in sex‐ and diet‐specific regulation of energy balance. J. Comp. Neurol. 524:1222–1235, 2016. © 2015 Wiley Periodicals, Inc.     

Glutamatergic pallidothalamic projections and their implications in the pathophysiology of Parkinson's disease

GABAergic projections emitted from the entopeduncular nucleus (ENT) and the substantia nigra pars reticulata (SNr) innervate different thalamic nuclei and they are known to be hyperactive after dopaminergic depletion. Here we show that isoform 2 of the vesicular glutamate transporter (VGLUT2) is expressed by neurons in the ENT nucleus but not in the SNr. Indeed, dual in situ hybridization demonstrated that the ENT nucleus contains two different subpopulations of projection neurons, one single-expressing GAD65/67 mRNAs and another one that co-expresses either of the GAD isoforms together with VGLUT2 mRNA. Unilateral dopaminergic depletion induced marked changes in pallidothalamic-projecting neuron gene expression, resulting in increased expression of GAD65/67 mRNAs together with a clear down-regulation of VGLUT2 mRNA expression. Our results indicate that the increased thalamic inhibition typical of dopamine depletion might be explained by a synergistic effect of increased GABA outflow coupled to decreased glutamate levels, both neurotransmitters coming from ENT neurons.     

Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers

Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions.     

Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2

Initially recognized for their importance in control of appetite, orexins (also called hypocretins) are neuropeptides that are also involved in regulating sleep, arousal, and cardiovascular function. Loss of orexin appears to be the primary cause of narcolepsy. Cells expressing the orexins are restricted to a discrete region of the hypothalamus, but their terminal projections are widely distributed throughout the brain. With the diversity of function and broad distribution of orexin terminals, it is not known whether the orexin cells constitute a homogeneous population. Because orexins produce neuroexcitatory effects, we hypothesized that orexin-containing neurons are glutamatergic. In the present study we used digoxigenin-labeled cRNA probes for the vesicular glutamate transporters, VGLUT1 and VGLUT2, for in situ hybridization studies in combination with immunohistochemical detection of orexin cell bodies in the hypothalamus. In general, cells in the hypothalamus expressed low levels of the vesicular glutamate transporters relative to other areas of the forebrain, such as the cortex and thalamus. Light labeling for VGLUT2 mRNA was detected in about 50% of the orexin-immunoreactive neurons, and a much smaller percentage (approximately 13%) of orexin-immunoreactive cells was found to express VGLUT1. Despite the fact that intense labeling for GAD67 mRNA was found in a large number of cells throughout the hypothalamus, none of the orexin-immunoreactive cells was found to be GABAergic. These findings, showing that many of the orexin neurons are glutamatergic, are consistent with the neuroexcitatory effects of orexin but suggest that another neurochemical phenotype may define the remaining subset of orexin neurons.     

Expression of glutamate and inhibitory amino acid vesicular transporters in the rodent auditory brainstem

Glutamate is the main excitatory neurotransmitter in the auditory system, but associations between glutamatergic neuronal populations and the distribution of their synaptic terminations have been difficult. Different subsets of glutamatergic terminals employ one of three vesicular glutamate transporters (VGLUT) to load synaptic vesicles. Recently, VGLUT1 and VGLUT2 terminals were found to have different patterns of organization in the inferior colliculus, suggesting that there are different types of glutamatergic neurons in the brainstem auditory system with projections to the colliculus. To positively identify VGLUT-expressing neurons as well as inhibitory neurons in the auditory brainstem, we used in situ hybridization to identify the mRNA for VGLUT1, VGLUT2, and VIAAT (the vesicular inhibitory amino acid transporter used by GABAergic and glycinergic terminals). Similar expression patterns were found in subsets of glutamatergic and inhibitory neurons in the auditory brainstem and thalamus of adult rats and mice. Four patterns of gene expression were seen in individual neurons. 1) VGLUT2 expressed alone was the prevalent pattern. 2) VGLUT1 coexpressed with VGLUT2 was seen in scattered neurons in most nuclei but was common in the medial geniculate body and ventral cochlear nucleus. 3) VGLUT1 expressed alone was found only in granule cells. 4) VIAAT expression was common in most nuclei but dominated in some. These data show that the expression of the VGLUT1/2 and VIAAT genes can identify different subsets of auditory neurons. This may facilitate the identification of different components in auditory circuits.     

GABAergic cell types in the superficial layers of the mouse superior colliculus

To begin to unravel the complexities of GABAergic circuits in the superior colliculus (SC), we utilized mouse lines that express green fluorescent protein (GFP) in cells that contain the 67 kDa isoform of glutamic acid decarboxylase (GAD67-GFP), or Cre-recombinase in cells that contain glutamic acid decarboxylase (GAD; GAD2-cre). We used Cre-dependent virus injections in GAD2-Cre mice and tracer injections in GAD67-GFP mice, as well as immunocytochemical staining for gamma amino butyric acid (GABA) and parvalbumin (PV) to characterize GABAergic cells that project to the pretectum (PT), ventral lateral geniculate nucleus (vLGN) or parabigeminal nucleus (PBG), and interneurons in the stratum griseum superficiale (SGS) that do not project outside the SC. We found that approximately 30% of SGS neurons in the mouse are GABAergic. Of these GABAergic neurons, we identified three categories of potential interneurons in the GAD67-GFP line (GABA+GFP ~45%, GABA+GFP + PV ~15%, and GABA+PV ~10%). GABAergic cells that did not contain GFP or PV were identified as potential projection neurons (GABA only ~30%). We found that GABAergic neurons that project to the PBG are primarily located in the SGS and exhibit narrow field vertical, stellate, and horizontal dendritic morphologies, while GABAergic neurons that project to the PT and vLGN are primarily located in layers ventral to the SGS. In addition, we examined GABA and GAD67-containing elements of the mouse SGS using electron microscopy to further delineate the relationship between GABAergic circuits and retinotectal input. Approximately 30% of retinotectal synaptic targets are the presynaptic dendrites of GABAergic interneurons, and GAD67-GFP interneurons are a source of these presynaptic dendrites.     

Complementary distribution of glutamatergic cerebellar and GABAergic basal ganglia afferents to the rat motor thalamic nuclei

Motor thalamic nuclei, ventral anterior (VA), ventral lateral (VL) and ventral medial (VM) nuclei, receive massive glutamatergic and GABAergic afferents from the cerebellum and basal ganglia, respectively. In the present study, these afferents were characterized with immunoreactivities for glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular glutamate transporter (VGluT)2, and examined by combining immunocytochemistry with the anterograde axonal labeling and neuronal depletion methods in the rat brain. VGluT2 immunoreactivity was intense in the caudodorsal portion of the VA-VL, whereas GAD67 immunoreactivity was abundant in the VM and rostroventral portion of the VA-VL. The rostroventral VA-VL and VM contained two types of GAD67-immunopositive varicosities (large and small), but the caudodorsal VA-VL comprised small ones alone. VGluT2-immunopositive varicosities were much larger in the caudodorsal VA-VL than those in the rostroventral VA-VL and VM. When anterograde tracers were injected into the basal ganglia output nuclei, the vast majority of labeled axon varicosities were large and distributed in the rostroventral VA-VL and VM, showing immunoreactivity for GAD67, but not for VGluT2. Only the large GAD67-immunopositive varicosities were mostly abolished by kainic acid depletion of substantia nigra neurons. In contrast, large to giant axon varicosities derived from the deep cerebellar nuclei were distributed mostly in the caudodorsal VA-VL, displaying VGluT2 immunoreactivity. The VGluT2-positive varicosities disappeared from the core portion of the caudodorsal VA-VL by depletion of cerebellar nucleus neurons. Thus, complementary distributions of large VGluT2- and GAD67-positive terminals in the motor thalamic nuclei are considered to reflect glutamatergic cerebellar and GABAergic basal ganglia afferents, respectively.     

GABA and glutamate in mating-activated cells in the preoptic area and medial amygdala of male gerbils

The posterodorsal medial amygdala (MeApd), the posterodorsal preoptic nucleus (PdPN), and the medial cell group of the sexually dimorphic preoptic area (mSDA) contain cells that are activated specifically at ejaculation as assessed by Fos expression. The mSDA also expresses Fos early in the mating context. Because little is known about the neurotransmitters of these activated cells, the possibility that they use gamma-aminobutyric acid (GABA) or glutamate was assessed. Putative glutamatergic cells were visualized with immunocytochemistry (ICC) for glutamate and its neuron-specific transporter. Their distributions were compared with those of GABAergic cells visualized with ICC for the 67-kDa form of glutamic acid decarboxylase (GAD(67)) and in situ hybridization for GAD(67) messenger RNA (mRNA). Colocalization of Fos and GAD(67) mRNA in recently mated males indicated that half of the activated cells in the PdPN, mSDA, and lateral MeApd are GABAergic. Colocalization of Fos and glutamate suggested that a quarter of the activated mSDA and lateral MeApd cells are glutamatergic. The PdPN does not appear to have glutamatergic cells. In the lateral MeApd, the percentage of activated cells that are GABAergic (45%) matches the percentage that project to the principal part of the bed nucleus of the stria terminalis (BST; 43%), and the percentage likely to be glutamatergic (27%) matches the percentage projecting to the mSDA (27%). The latter could help to trigger ejaculation. The distribution of GABAergic and putative glutamatergic cells in the caudal preoptic area, caudal BST, and medial amygdala of male gerbils is also described.     

Density of glutamic acid decarboxylase 67 messenger RNA-containing neurons that express the N-methyl-D-aspartate receptor subunit NR2A in the anterior cingulate cortex in schizophrenia and bipolar disorder

BACKGROUND: Disturbances of gamma-aminobutyric acid interneurons in the cerebral cortex contribute to the pathophysiology of schizophrenia and bipolar disorder. The activity of these neurons is, in turn, modulated by glutamatergic inputs furnished by pyramidal neurons. OBJECTIVE: To test the hypothesis that glutamatergic inputs onto gamma-aminobutyric acid interneurons via the N-methyl-d-aspartate (NMDA) receptor are altered in the anterior cingulate cortex in schizophrenia and bipolar disorder. DESIGN: A double in situ hybridization technique was used to simultaneously label the messenger RNA (mRNA) for the NMDA NR(2A) subunit with (35)sulfur and the mRNA for the 67-kDa isoform of the gamma-aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (GAD(67)) with digoxigenin. SETTING: Postmortem human brain studies. PARTICIPANTS: We studied 17 subjects with schizophrenia, 17 subjects with bipolar disorder, and 17 normal control subjects. RESULTS: The density of all GAD(67) mRNA-containing neurons was decreased by 53% and 28%, in layers 2 and 5, respectively, in subjects with schizophrenia, whereas in subjects with bipolar disorder there was a 35% reduction in layer 2 only. For GAD(67) mRNA-containing neurons that co-expressed NR(2A)mRNA, their numerical density was decreased by 73% and 52%, in layers 2 and 5, respectively, in subjects with schizophrenia and by 60% in layer 2 in those with bipolar disorder. In the schizophrenia group, the density of the GAD(67)mRNA-containing neurons that did not co-express NR(2A)mRNA was also decreased by 42% in layer 2. In both disease groups, the expression level of NR(2A)mRNA in GAD(67) mRNA-containing cells was unaltered. CONCLUSIONS: The density of gamma-aminobutyric acid interneurons that express the NMDA NR(2A)subunit appears to be decreased in schizophrenia and bipolar disorder. Future studies will address whether subpopulations of these neurons may be differentially affected in the 2 conditions.     

Differential hippocampal expression of glutamic acid decarboxylase 65 and 67 messenger RNA in bipolar disorder and schizophrenia

BACKGROUND: Expression of messenger RNA (mRNA) for the gamma-aminobutyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase (GAD), in the prefrontal cortex and the number of GABAergic neurons in the hippocampus are reduced in schizophrenia and bipolar disorder. We tested the hypothesis that the expression of the 2 isoforms, one 65 kd (GAD(65)) and the other 67 kd (GAD(67)), is differentially affected in the hippocampus in schizophrenia and bipolar disorder. METHODS: Hippocampal sections from 15 subjects in 3 groups (control subjects and subjects with schizophrenia and bipolar disorder) were studied using an in situ hybridization protocol with sulfur 35-labeled complementary riboprobes for GAD(65) and GAD(67) mRNA. Emulsion-dipped slides were analyzed for the density of GAD mRNA-positive neurons in 4 sectors of the hippocampus and for the cellular expression level of both GAD mRNAs. RESULTS: The density of GAD(65) and GAD(67) mRNA-positive neurons was decreased by 45% and 43%, respectively, in subjects with bipolar disorder, but only 14% and 4%, respectively, in subjects with schizophrenia. The decreased density of GAD(65) mRNA-positive neurons in subjects with bipolar disorder was significant in sectors CA2/3 and dentate gyrus, and that of GAD(67) mRNA-positive neurons was significant in CA4, but not other hippocampal sectors. Cellular GAD(65) mRNA expression was significantly decreased in subjects with bipolar disorder, particularly in CA4, but not in schizophrenic subjects. Cellular GAD(67) mRNA expression was normal in both groups. CONCLUSION: We have found a region-specific deficit of GAD(65) and GAD(67) mRNA expression in bipolar disorder.     

Presence of vesicular glutamate transporter-2 in hypophysiotropic somatostatin but not growth hormone-releasing hormone neurons of the male rat

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 gamma-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.     

Distribution of vesicular glutamate transporter mRNA in rat hypothalamus

Two isoforms of the vesicular glutamate transporter, VGLUT1 and VGLUT2, were recently cloned and biophysically characterized. Both VGLUT1 and VGLUT2 specifically transport glutamate into synaptic vesicles, making them definitive markers for neurons using glutamate as a neurotransmitter. The present study takes advantage of the specificity of the vesicular transporters to afford the first detailed map of putative glutamatergic neurons in the rat hypothalamus. In situ hybridization analysis was used to map hypothalamic distributions of VGLUT1 and VGLUT2 mRNAs. VGLUT2 is clearly the predominant vesicular transporter mRNA found in the hypothalamus; rich expression can be documented in regions regulating energy balance (ventromedial hypothalamus), neuroendocrine function (preoptic nuclei), autonomic tone (posterior hypothalamus), and behavioral/homeostatic integration (lateral hypothalamus, mammillary nuclei). Expression of VGLUT1 is decidedly more circumspect and is confined to relatively weak labeling in lateral hypothalamic regions, neuroendocrine nuclei, and the suprachiasmatic nucleus. Importantly, dual-label analysis revealed no incidence of colocalization of VGLUT1 or VGLUT2 mRNAs in glutamic acid decarboxylase (GAD) 65-positive neurons, indicating that GABA neurons do not express either transporter. Our data support a major role for hypothalamic glutamatergic neurons in regulation of all aspects of hypothalamic function.     

Localization of cells preferentially expressing GAD(67) with negligible GAD(65) transcripts in the rat hippocampus. A double in situ hybridization study.

Two major forms of glutamic acid decarboxylase (GAD) are present in the mammalian brain, a 65-kDa isoform (GAD(65)) and a 67-kDa isoform (GAD(67)), and it is usually assumed that all GABAergic neurons contain both. The two forms have not yet been colocalized to the same neurons, because the GAD(65) protein is found almost exclusively in axon terminals, while GAD(67) is found predominantly in the cell body. Using double in situ hybridization (DISH) with both radioactive [35S] and non-radioactive (digoxigenin, DIG) probes, the distributions of GAD(65) and GAD(67) mRNA have been simultaneously examined in the rat hippocampus. The results suggest that [35S] radioprobes are slightly more sensitive than DIG probes, and that the reversal of labels is necessary in DISH studies to determine whether a neuronal subtype which expresses only one isoform of GAD may be present. The data indicate that the majority of cells (90%) showing labeling were labeled for both GAD(65) and GAD(67) mRNA. In sectors CA1 and CA3 approximately 5-10% of the cells positive for GAD(67) showed little or no detectable GAD(65) mRNA. In the hilus, however, GAD(65) levels were higher, and all cells seem to express both GAD(65) and GAD(67) mRNA. Taken together, these results support the view that most GABAergic neurons in the hippocampus express both GAD(65) and GAD(67). However, it appears that some interneurons in the CA subfields differ from "classic" GABAergic interneurons by preferentially expressing the 67-kDa isoform of GAD under baseline conditions, with GAD(65) mRNA levels very low or absent.     

Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons

The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non‐GABAergic phenotype, while they express well‐established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (–) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV⁺/NOS^?/GAD67⁺, PV⁺/NOS⁺/GAD67⁺, PV⁺/NOS^?/GAD67^?, and PV^?/NOS⁺/GAD67^?. Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67^? IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67⁺ IC neurons remained unchanged in all subdivisions. The NDs of PV⁺/NOS^?/GAD67⁺ neurons and PV^?/NOS⁺/GAD67^? neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV⁺/NOS⁺/GAD67⁺ neurons and PV⁺/NOS^?/GAD67^? neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67⁺ IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868–884, 2017. © 2016 Wiley Periodicals, Inc.