Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia

Glutamate is transported into synaptic vesicles by vesicular glutamate transporter (VGLUT) proteins. Three different VGLUTs, VGLUT1, VGLUT2, and VGLUT3, have recently been characterized, and they are considered to represent the most specific marker so far for neurons using glutamate as transmitter. We analyzed the cellular localization of VGLUT1-3 in the rat spinal cord and dorsal root ganglia (DRGs) in control rats and after dorsal rhizotomy. Using in situ hybridization, VGLUT1 mRNA containing neurons were shown in the dorsomedial part of the intermediate zone, whereas VGLUT2 mRNA-expressing neurons were present in the entire intermediate zone, both populations most likely representing interneurons. VGLUT3 mRNA could not be detected in the spinal cord. In the ventral horn, a dense plexus of VGLUT1-immunoreactive (ir) nerve terminals was present, with large varicosities abutting on presumed motoneurons. In the dorsal horn a similarly dense plexus was seen, except in laminae I and II. A very dense plexus of VGLUT2-ir fibers was distributed in the entire gray matter of the spinal cord, with many fibers lying close to presumed motoneurons. Few VGLUT3-ir fibers were distributed in the white and gray matter, including lamina IX. However, a dense VGLUT3-ir plexus was seen in the sympathetic intermedio-lateral column (IML). Multiple-labeling immunohistochemistry revealed that the VGLUT1-, VGLUT2-, and VAChT-containing varicosities in lamina IX all represent separate entities. There was no colocalization of VGLUT3 with VAChT or 5-HT in varicose fibers of the ventral horn, but some VGLUT3-ir fibers in the IML were 5-HT-positive. Lesioning of the dorsal roots resulted in an almost complete disappearance of VGLUT1-ir fibers around motoneurons and a less pronounced decrease in the remaining gray matter, whereas the density of VGLUT2- and VAChT-ir fibers appeared unaltered after lesion. Many VGLUT1-ir neurons were observed in DRGs; they were almost all large and did not colocalize calcitonin gene-related peptide (CGRP), and there was no overlap between these markers in fibers in the superficial dorsal horn. VGLUT2 was, at most, seen in a few DRG neurons. Taken together, these results suggest that the VGLUTs mRNAs are present in distinct subsets of neuronal populations at the spinal level. VGLUT1 is mainly present in primary afferents from large, CGRP-negative DRG neurons, VGLUT2 has mainly a local origin, and VGLUT3 fibers probably have a supraspinal origin.     

Distinct glutamatergic and GABAergic subsets of hypothalamic pro‐opiomelanocortin neurons revealed by in situ hybridization in male rats and mice

Pro‐opiomelanocortin (POMC) and agouti‐related protein (AGRP) neurons in the hypothalamus regulate various aspects of energy homeostasis and metabolism. POMC and AGRP neurons, respectively, agonize and antagonize melanocortin receptors on their common downstream neurons. However, it is unknown whether they also reciprocally stimulate and inhibit the same neurons by amino acid transmitters. Whereas AGRP neurons are mostly GABAergic, surprisingly, only a small population of POMC neurons has been found to be glutamatergic, and a significantly larger subpopulation to be GABAergic. To further examine amino acid phenotypes of POMC neurons, we studied mRNA expression for the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), and the GABA synthetic enzyme, glutamic acid decarboxylase 67 (GAD67), in POMC neurons of both rats and mice by using in situ hybridization techniques. In rats, approximately 58% of POMC neurons were labeled for VGLUT2 and 37% for GAD67 mRNA. In mice, approximately 43% of POMC neurons contained VGLUT2, and 54% contained GAD67 mRNA. In both species, a prominent mediolateral distribution pattern was observed at rostral and mid levels of the POMC cell group with VGLUT2–POMC neurons dominating in lateral portions and GAD67–POMC neurons in medial portions. These data demonstrate that both glutamatergic and GABAergic cells are present in comparably significant numbers among POMC neurons. Their glutamatergic or GABAergic phenotype may represent a major functional division within the POMC cell group. J. Comp. Neurol. 521:3287–3302, 2013. © 2013 Wiley Periodicals, Inc.     

Somatostatin varicosities contain the vesicular GABA transporter and contact orexin neurons in the hypothalamus

Somatostatin (SST) is a neuropeptide with known inhibitory actions in the hypothalamus, where it inhibits release of growth hormone‐releasing hormone (GHRH), while also influencing the sleep–wake cycle. Here we investigated in the rat whether SST neurons might additionally release GABA (gamma‐aminobutyric acid) or glutamate in different regions and whether they might contact orexin neurons that play an important role in the maintenance of wakefulness. In dual‐immunostained sections viewed by epifluorescence microscopy, we examined if SST varicosities were immunopositive for the vesicular transporter for GABA (VGAT) or glutamate (VGLUT2) in the posterolateral hypothalamus and neighboring arcuate nucleus and median eminence. Of the SST varicosities in the posterolateral hypothalamus, 18% were immunopositive for VGAT, whereas ≤ 1% were immunopositive for VGLUT2. In the arcuate and median eminence, 26 and 64% were VGAT+ and < 3% VGLUT2 + , respectively. In triple‐immunostained sections viewed by epifluorescence and confocal microscopy, SST varicosities were seen in contact with orexin somata, and of these varicosities, a significant proportion (23%) contained VGAT along with synaptophysin, the presynaptic marker for small synaptic vesicles, and a similar proportion (25%) abutted puncta that were immunostained for gephyrin, the postsynaptic marker for GABAergic synapses. Our results indicate that a significant proportion of SST varicosities in the hypothalamus have the capacity to release GABA, to form inhibitory synapses upon orexin neurons, and accordingly through their peptide and/or amino acid, to inhibit orexin neurons, as well as GHRH neurons. Thus while regulating GHRH release, SST neurons could serve to attenuate arousal and permit progression through the sleep cycle.     

Evidence for estrogenic regulation of gonadotropin-releasing hormone neurons by glutamatergic neurons in the ewe brain: An immunohistochemical study using an antibody against vesicular glutamate transporter-2

Gonadotropin-releasing hormone (GnRH) secretion is controlled by various factors, including the excitatory neurotransmitter glutamate. Estrogen (E) regulates GnRH secretion by means of E-responsive cells in the brain that relay the feedback effects to the preoptic area (POA). We used an antibody to vesicular glutamate transporter 2 (VGluT2) to label glutamatergic neurons in the areas of the ewe brain that control GnRH secretion. VGluT2-immunoreactive cells were observed in the arcuate nucleus (ARC)/ventromedial hypothalamic nucleus (VMH) complex, POA, bed nucleus of stria terminalis (BnST), and A1 and A2 cell groups in the brainstem. In three ewes, E receptor-alpha was detected in 52-61% of glutamatergic neurons in ARC/VMH, 37-52% of neurons in the POA, and 37-58% of neurons in the BnST. E injection (i.m. or i.v.) increased the percentage of glutamatergic cells that expressed Fos protein in the ARC (P < 0.01 and P < 0.001, respectively). In six ewes, injection of the retrograde tracer Fluoro-Gold into the POA labeled cells in the ARC and 6-29% of these were also VGluT2-immunoreactive. Double-labeling of varicosities in the POA showed colocalization of VGluT2 in 12.5 +/- 3% of dopamine beta-hydroxylase-immunoreactive terminals, indicating that a subset of glutamatergic inputs could arise from brainstem noradrenergic neurons cells. In the POA, 60% of GnRH neurons had close appositions that were VGluT2-immunoreactive. We conclude that E-responsive glutamatergic neurons arising from the brainstem, the BnST, and ARC/VMH provide input to the POA and may be involved in the regulation of GnRH secretion.     

Identification of neurones in the female rat hypothalamus that express oestrogen receptor-alpha and vesicular glutamate transporter-2

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)-alpha and ER-beta. Evidence suggests that oestrogen-receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin-releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor-expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter-2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERalpha mRNA-(35S-labelling) and VGLUT2 mRNA-(digoxigenin-labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERalpha mRNA are consistent with previous data in the literature. Dual-labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 +/- 3.4% of the ERalpha mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERalpha expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen-receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones.     

Hippocampal distribution of vesicular glutamate transporter 1 in patients with temporal lobe epilepsy

Purpose:   Vesicular glutamate transporters (VGLUTs) are responsible for loading synaptic vesicles with glutamate, determining the phenotype of glutamatergic neurons, and have been implicated in the regulation of quantal size and presynaptic plasticity. We analyzed VGLUT subtype expression in normal human hippocampus and tested the hypothesis that alterations in VGLUT expression may contribute to long-term changes in glutamatergic transmission reported in patients with temporal lobe epilepsy (TLE).
Methods:   VGLUT immunohistochemistry, immunofluorescence, in situ hybridization, Western blotting, and quantitative polymerase chain reaction (qPCR) were performed on biopsies from TLE patients without (non-HS) and with hippocampal sclerosis (HS) and compared to autopsy controls and rat hippocampus. VGLUT1 expression was compared with synaptophysin, neuropeptide Y (NPY), and Timm's staining.
Results:   VGLUT1 was the predominant VGLUT in human hippocampus and appeared to be localized to presynaptic glutamatergic terminals. In non-HS hippocampi, VGLUT1 protein levels were increased compared to control and HS hippocampi in all subfields. In HS hippocampi VGLUT1 expression was decreased in subfields with severe neuronal loss, but strongly up-regulated in the dentate gyrus, characterized by mossy fiber sprouting.
Discussion:   VGLUT1 is used as marker for glutamatergic synapses in the human hippocampus. In HS hippocampi VGLUT1 up-regulation in the dentate gyrus probably marks new glutamatergic synapses formed by mossy fiber sprouting. Our data indicate that non-HS patients have an increased capacity to store glutamate in vesicles, most likely due to an increase in translational processes or upregulation of VGLUT1 in synapses from afferent neurons outside the hippocampus. This up-regulation may increase glutamatergic transmission, and thus contribute to increased extracellular glutamate levels and hyperexcitability.     

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.     

Sensorimotor gating, working and social memory deficits in mice with reduced expression of the vesicular glutamate transporter VGLUT1

Glutamate is the main excitatory neurotransmitter in the central nervous system. A hypoglutamatergic state is believed to play an important role in the pathophysiology of schizophrenia. The release of glutamate in the brain is modulated by a class of vesicular glutamate transporters, VGLUT1-3. Among them, VGLUT1 represents the isoform predominantly expressed in the neocortex and hippocampus. Here we investigated the potential involvement of VGLUT1 deficiency in generating schizophrenia-like abnormalities by testing mice with diminished expression of VGLUT1 in several behavioural tests relevant for schizophrenia. We found behavioural alterations in these mice resembling correlates of schizophrenia, such as working- and social memory impairments and deficits in prepulse inhibition (PPI) of the acoustic startle reflex (ASR), but normal locomotor behaviour under basal conditions. Our data may be important for a better understanding of the contribution of reduced VGLUT1-mediated presynaptic glutamatergic neurotransmission in the generation of several behavioural abnormalities associated with schizophrenia.     

Expression of vesicular glutamate transporter 1 immunoreactivity in peripheral and central endings of trigeminal mesencephalic nucleus neurons in the rat

The major neuronal components of the trigeminal mesencephalic nucleus (Vmes) are primary afferent neurons that convey proprioceptive information from the cranioorofacial regions. In the present study, we examined expression of vesicular glutamate transporters (VGLUTs), VGLUT1 and VGLUT2, in the primary afferent neurons of the Vmes (Vmes neurons) in neonatal and adult rats. VGLUT1 immunoreactivity was detected in the cell bodies of Vmes neurons in neonatal rats younger than 11 days old, but not in older rats. However, in situ hybridization signals for VGLUT1 mRNA were detected in both neonatal and adult rats. No VGLUT2 immunoreactivity was detected in Vmes neurons of neonatal or adult rats. VGLUT1 immunoreactivity was also seen in the peripheral sensory endings on the equatorial regions of intrafusal fibers of muscle spindles in the masseter muscles in both neonatal and adult rats. In adult rats injected with cholera toxin B subunit (CTb) into the masseter nerve, central axon terminals of Vmes neurons were identified on masseter motoneurons within the trigeminal motor nucleus (Vm) by transganglionically and retrogradely transported CTb. VGLUT1-immunopositive axon terminals in close apposition to CTb-labeled Vm motoneurons were also detected by dual-immunofluorescence histochemistry for VGLUT1/CTb. Electron microscopy after dual immunolabeling for VGLUT1/CTb by the VGLUT1/immunoperoxidase and CTb/immunogold-silver methods further revealed synaptic contact of VGLUT1- and CTb-immunopositive axon terminals upon CTb-labeled neuronal profiles within the Vm. These data indicate that VGLUT1 is expressed in both the central axon terminals and the peripheral sensory endings of Vmes neurons, although no VGLUT1 immunoreactivity was detectable in the cell bodies of Vmes neurons in adult rats.     

Changes in vesicular transporters for gamma-aminobutyric acid and glutamate reveal vulnerability and reorganization of hippocampal neurons following pilocarpine-induced seizures

The reorganizations of the overall intrinsic glutamatergic and gamma-aminobutyric acid (GABA)-ergic hippocampal networks as well as the time course of these reorganizations during development of pilocarpine-induced temporal lobe epilepsy were studied with in situ hybridization and immunohistochemistry experiments for the vesicular glutamate transporter 1 (VGLUT1) and the vesicular GABA transporter (VGAT). These transporters are particularly interesting as specific markers for glutamatergic and GABAergic neurons, respectively, whose expression levels could reflect the demand for synaptic transmission and their average activity. We report that 1) concomitantly with the loss of some subpopulations of VGAT-containing neurons, there was an up-regulation of VGAT synthesis in all remaining GABA neurons as early as 1 week after pilocarpine injection. This enhanced synthesis is characterized by marked increases in the relative amount of VGAT mRNAs in interneurons associated with increased intensity of axon terminal labeling for VGAT in all hippocampal layers. 2) There was a striking loss of mossy cells during the latent period, demonstrated by a long-term decrease of VGLUT1 mRNA-containing hilar neurons and associated loss of VGLUT1-containing terminals in the dentate gyrus inner molecular layer. 3) There were aberrant VGLUT1-containing terminals at the chronic stage resulting from axonal sprouting of granule and pyramidal cells. This is illustrated by a recovery of VGLUT1 immunoreactivity in the inner molecular layer and an increased VGLUT1 immunolabeling in the CA1-CA3 dendritic layers. These data indicate that an increased activity of remaining GABAergic interneurons occurs during the latent period, in parallel with the loss of vulnerable glutamatergic and GABAergic neurons preceding the reorganization of glutamatergic networks.     

Plasma membrane and vesicular glutamate transporter expression in chromaffin cells of bovine adrenal medulla

The study of the functional expression of glutamate signaling molecules in peripheral tissues has received relatively little attention. However, evidence is increasing for a role of glutamate as an extracellular signal mediator in endocrine systems, in addition to having an excitatory amino acid neurotransmitter role in the CNS. Chromaffin cells are good models of catecholaminergic neurons, in which previous work from our group demonstrated the existence of both functional glutamate receptors and specific exocytotic and nonexocytotic glutamate release. In this work, the presence of specific plasma membrane (EAATs) and vesicular glutamate (VGLUTs) transporters has been investigated by using confocal microscopy, flow cytometric analysis, Western blot, and qRT-PCR techniques. We found specific expression of EAAT3, EAAT2, VGLUT1, and VGLUT3 in about 95%, 65%, 55%, and 25%, respectively, of the whole chromaffin cell population. However, chromaffin cells do not express VGLUT2 and have a very low expression of EAAT1. VGLUTs are localized mainly in the membrane fraction, and EAATs share their subcellular location between membrane and cytosolic fractions. Their estimated molecular weights were about 70 kDa for EAAT2, about 65 kDa for EAAT3, about 50 kDa for VGLUT1, and about 60 kDa for VGLUT3. RT-qPCR techniques confirm the expression of these glutamate transporters at the mRNA level and show a different regulation by cytokines and glucocorticoids between VGLUT1 and -3 and EAAT2 and -3 subfamilies. These interesting results support the participation of these glutamate transporters in the process of glutamate release in chromaffin cells and in the regulation of their neurosecretory function in adrenal medulla.     

Neuronal activity regulates glutamate transporter dynamics in developing astrocytes

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT-1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. Green fluorescent protein (GFP)-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse three-dimensional confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses.     

The dorsomedial hypothalamic nucleus is not necessary for food-anticipatory circadian rhythms of behavior, temperature or clock gene expression in mice

Circadian rhythms in mammals are regulated by a light-entrainable circadian pacemaker in the hypothalamic suprachiasmatic nucleus and food-entrainable oscillators located elsewhere in the brain and body. The dorsomedial hypothalamic nucleus (DMH) has been proposed to be the site of oscillators driving food-anticipatory circadian rhythms, but this is controversial. To further evaluate this hypothesis, we measured clock gene, temperature and activity rhythms in intact and DMH-ablated mice. A single 4-h midday feeding after an overnight fast induced mPer1 and mPer2 mRNA expression in the DMH, arcuate nucleus, nucleus of the solitary tract and area postrema, and reset daily rhythms of mPer1 , mPer2 and mBMAL1 in the DMH, arcuate and neocortex. These rhythms persisted during 2 days of food deprivation after 12 days of scheduled daytime feeding. Acute induction of DMH mPer1 and mPer2 was N -methyl- d -aspartate (NMDA) receptor-dependent, whereas rhythmic expression after 6 days of restricted feeding was not. Thermal DMH lesions did not affect acute induction or rhythmic expression of clock genes in other brain regions in response to scheduled daytime feeding. DMH lesions attenuated mean daily activity levels and nocturnality but did not affect food-anticipatory rhythms of activity and body temperature in either light–dark or constant darkness. These results confirm that the DMH and other brain regions express circadian clock gene rhythms sensitive to daytime feeding schedules, but do not support the hypothesis that DMH oscillations drive food-anticipatory behavioral or temperature rhythms.     

Increased expression of the neuronal glutamate transporter (EAAT3/EAAC1) in hippocampal and neocortical epilepsy

PURPOSE: To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy. METHODS: The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis. RESULTS: The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with non-dysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue. CONCLUSIONS: Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.     

Expression of the mRNAs encoding for the vesicular glutamate transporters 1 and 2 in the rat thalamus

Vesicular glutamate transporters (VGLUTs) are responsible for glutamate trafficking and for the subsequent regulated release of this excitatory neurotransmitter at the synapse. Three isoforms of the VGLUT have been identified, now known as VGLUT1, VGLUT2, and VGLUT3. Both VGLUT1 and VGLUT2 have been considered definitive markers of glutamatergic neurons, whereas VGLUT3 is expressed in nonglutamatergic neurons such as cholinergic striatal interneurons. It is widely believed that VGLUT1 and VGLUT2 are expressed in a complementary manner at the cortical and thalamic levels, suggesting that these glutamatergic neurons fulfill different physiological functions. In the present work, we analyzed the pattern of VGLUT1 and VGLUT2 mRNA expression at the thalamic level by using single and dual in situ hybridization. In accordance with current beliefs, we found significant expression of VGLUT2 mRNA in all the thalamic nuclei, while moderate expression of VGLUT1 mRNA was consistently found in both the principal relay and the association thalamic nuclei. Interestingly, individual neurons within these nuclei coexpressed both VGLUT1 and VGLUT2 mRNAs, suggesting that these individual thalamic neurons may have different ways of trafficking glutamate. These results call for a reappraisal of the previously held concept regarding the mutually exclusive distribution of VGLUT transporters in the central nervous system.     

Vesicular glutamate transporter 3 immunoreactivity is present in cholinergic basal forebrain neurons projecting to the basolateral amygdala in rat

The basal forebrain (BF) plays a role in behavioral and cortical arousal, attention, learning, and memory. It has been suggested that cholinergic BF neurons co-release glutamate, and some cholinergic BF neurons have been reported to contain vesicular glutamate transporter 3 (VGLUT3). We examined the distribution and projections of BF cholinergic neurons containing VGLUT3, by using dual-label immunofluorescence for choline acetyltransferase (ChAT) and VGLUT3, in situ hybridization, and retrograde tracing. Neurons immunoreactive (+) or containing mRNAs for both ChAT and VGLUT3 were mainly localized to the ventral pallidum and more caudal BF regions; the co-immunoreactive neurons represented 31% of cholinergic neurons in the ventral pallidum and 5-9% more caudally. Examination of cholinergic axon terminals in known target areas of BF projections indicated that the basolateral amygdaloid nucleus contained numerous terminals co-immunoreactive for ChAT and VGLUT3, whereas sampled areas of the olfactory bulb, neocortex, hippocampus, reticular thalamic nucleus, and interpeduncular nucleus were devoid of double-labeled terminals. The basolateral amygdala is innervated by cholinergic BF neurons lacking low-affinity p75 nerve growth factor receptors; many ChAT+VGLUT3+ BF neurons were immunonegative to this receptor. Twenty-five to 79% of ChAT+VGLUT3+ neurons in different BF regions were retrogradely labeled from the basolateral amygdala, up to 52% (ventral pallidum) of the retrogradely labeled ChAT+ neurons were VGLUT3+, and the largest number of amygdala-projecting ChAT+VGluT3+ neurons was found in the ventral pallidum. These findings indicate that BF cholinergic neurons containing VGLUT3 project to the basolateral amygdala and suggest that these neurons might have the capacity to release both acetylcholine and glutamate.