Vesicular Glutamate Transporter 2 mRNA Research Articles

CPEB2-activated axonal translation of VGLUT2 mRNA promotes glutamatergic transmission and presynaptic plasticity

BackgroundLocal translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity.MethodsBehavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles.ResultsElectrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles.ConclusionsWe identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

Immunoreactivity of Vesicular Glutamate Transporter 2 Corresponds to Cytochrome Oxidase-Rich Subcompartments in the Visual Cortex of Squirrel Monkeys.

Cytochrome oxidase (CO) histochemistry has been used to reveal the cytoarchitecture of the primate brain, including blobs/puffs/patches in the striate cortex (V1), and thick, thin and pale stripes in the middle layer of the secondary visual cortex (V2). It has been suggested that CO activity is coupled with the spiking activity of neurons, implying that neurons in these CO-rich subcompartments are more active than surrounding regions. However, we have discussed possibility that CO histochemistry represents the distribution of thalamo-cortical afferent terminals that generally use vesicular glutamate transporter 2 (VGLUT2) as their main glutamate transporter, and not the activity of cortical neurons. In this study, we systematically compared the labeling patterns observed between CO histochemistry and immunohistochemistry (IHC) for VGLUT2 from the system to microarchitecture levels in the visual cortex of squirrel monkeys. The two staining patterns bore striking similarities at all levels of the visual cortex, including the honeycomb structure of V1 layer 3Bβ (Brodmann's layer 4A), the patchy architecture in the deep layers of V1, the superficial blobs of V1, and the V2 stripes. The microarchitecture was more evident in VGLUT2 IHC, as expected. VGLUT2 protein expression that produced specific IHC labeling is thought to originate from the thalamus since the lateral geniculate nucleus (LGN) and the pulvinar complex both show high expression levels of VGLUT2 mRNA, but cortical neurons do not. These observations support our theory that the subcompartments revealed by CO histochemistry represent the distribution of thalamo-cortical afferent terminals in the primate visual cortex.

Malocclusion Generates Anxiety-Like Behavior Through a Putative Lateral Habenula-Mesencephalic Trigeminal Nucleus Pathway.

Malocclusion is an important risk factor for temporomandibular disorder (TMD), a series of disorders characterized by dysfunction in the orofacial region involving the temporomandibular joint (TMJ) and jaw muscles. We recently showed that experimental unilateral anterior crossbite (UAC) produced masseter hyperactivity through a circuit involving the periodontal proprioception, trigeminal mesencephalic nucleus (Vme), and trigeminal motor nucleus (Vmo). Anxiety is a common complication in patients with TMD. The lateral habenula (LHb) is involved in emotional modulation and has direct projections to the Vme. Therefore, the present research examined whether UAC facilitates excitatory input from the LHb to the Vme and, subsequently, anxiety-like behaviors in rats. The LHb activation was evaluated by the electrophysiological recording, assessment of vesicular glutamate transporter-2 (VGLUT2) mRNA expression, and measurement of anxiety-like behaviors. The effects of LHb activity on Vme were evaluated by electrophysiological recording from Vme neurons and local changes in VGLUT2 protein density. UAC produced anxiety in modeled rats and increased neuronal activity in the LHb. VGLUT2 mRNA expression was also increased in the LHb. Further, VGLUT2-positive boutons were observed in close apposite upon parvalbumin (PV)-labeled Vme neurons. VGLUT2 protein expression was also increased in the Vme. Significantly, injection of VGLUT2-targeted shRNA into the LHb reduced the expression of VGLUT2 protein in the Vme, attenuated UAC-associated anxiety-like behaviors, and attenuated electrophysiological changes in the Vme neurons. In conclusion, we show that UAC activates the LHb neurons as well as the periodontal proprioceptive pathway to provide excitatory input to the Vme and produce anxiety in rats. These findings provide a rationale for suppressing activity of the LHb to attenuate both the physical and psychological effects of TMD.

Neurokinin-1 Receptor-Immunopositive Neurons in the Medullary Dorsal Horn Provide Collateral Axons to both the Thalamus and Parabrachial Nucleus in Rats.

It has been suggested that the trigemino-thalamic and trigemino-parabrachial projection neurons in the medullary dorsal horn (MDH) are highly implicated in the sensory-discriminative and emotional/affective aspects of orofacial pain, respectively. In previous studies, some neurons were reported to send projections to both the thalamus and parabrachial nucleus by way of collaterals in the MDH. However, little is known about the chemoarchitecture of this group of neurons. Thus, in the present study, we determined whether the neurokinin-1 (NK-1) receptor, which is crucial for primary orofacial pain signaling, was expressed in MDH neurons co-innervating the thalamus and parabrachial nucleus. Vesicular glutamate transporter 2 (VGLUT2) mRNA, a biomarker for the subgroup of glutamatergic neurons closely related to pain sensation, was assessed in trigemino-parabrachial projection neurons in the MDH. After stereotactic injection of fluorogold (FG) and cholera toxin subunit B (CTB) into the ventral posteromedial thalamic nucleus (VPM) and parabrachial nucleus (PBN), respectively, triple labeling with fluorescence dyes for FG, CTB and NK-1 receptor (NK-1R) revealed that approximately 76 % of the total FG/CTB dually labeled neurons were detected as NK-1R-immunopositive, and more than 94 % of the triple-labeled neurons were distributed in lamina I. In addition, by FG retrograde tract-tracing combined with fluorescence in situ hybridization (FISH) for VGLUT2 mRNA, 54, 48 and 70 % of FG-labeled neurons in laminae I, II and III, respectively, of the MDH co-expressed FG and VGLUT2 mRNA. Thus, most of the MDH neurons co-innervating the thalamus and PBN were glutamatergic. Most MDH neurons providing the collateral axons to both the thalamus and parabrachial nucleus in rats were NK-1R-immunopositive and expressed VGLUT2 mRNA. NK-1R and VGLUT2 in MDH neurons may be involved in both sensory-discriminative and emotional/affective aspects of orofacial pain processing.

The isthmic nuclei providing parallel feedback connections to the avian tectum have different neurochemical identities: Expression of glutamatergic and cholinergic markers in the chick (Gallus gallus).

Retinal inputs to the optic tectum (TeO) triggered by moving stimuli elicit synchronized feedback signals from two isthmic nuclei: the isthmi parvocelullaris (Ipc) and isthmi semilunaris (SLu). Both of these nuclei send columnar axon terminals back to the same tectal position receiving the retinal input. The feedback signals from the Ipc seem to act as an attentional spotlight by selectively boosting the propagation of retinal inputs from the tectum to higher visual areas. Although Ipc and SLu nuclei are widely considered cholinergic because of their immunoreactivity for choline acetyltransferase (ChAT), contradictory findings, including the expression of the vesicular glutamate transporter 2 (VGluT2) mRNA in Ipc neurons, have raised doubts about the purely cholinergic nature of this nucleus. In this study, in chicks, we revise the neurochemical identity of the isthmic nuclei by using in situ hybridization assays for VGluT2 along with three cholinergic markers: the vesicular acetylcholine transporter (VAChT), the high-affinity choline transporter (CHT1) and ChAT. We found that neurons in the SLu showed strong mRNA expression of all three cholinergic markers, whereas the expression of VAChT mRNA in the Ipc was undetectable in our essays. Instead, Ipc neurons exhibited a strong expression of VGluT2 mRNA. Immunohistochemistry assays showed VGluT2 immunoreactivity in the TeO codistributing with anterogradely labeled Ipc axon-terminal boutons, further supporting a glutamatergic function for the Ipc nucleus. Therefore, our results strongly suggest that, in the chick, whereas the feedback from the SLu to the TeO is indeed cholinergic, the feedback from the Ipc has a marked glutamatergic component.

Glutamatergic and dopaminergic neurons in the mouse ventral tegmental area.

The ventral tegmental area (VTA) comprises dopamine (DA), γ-aminobutyric acid (GABA) and glutamate (Glu) neurons. Some rat VTA Glu neurons, expressing vesicular glutamate transporter 2 (VGluT2), co-express tyrosine hydroxylase (TH). While transgenic mice are now being used in attempts to determine the role of VGluT2/TH neurons in reward and neuronal signaling, such neurons have not been characterized in mouse tissue. By cellular detection of VGluT2 mRNA and TH immunoreactivity (TH-IR), we determined the cellular expression of VGluT2 mRNA within VTA TH-IR neurons in the mouse. We found that some mouse VGluT2 neurons coexpressed TH-IR, but their frequency was lower than in the rat. To determine whether low expression of TH mRNA or TH-IR accounts for this low frequency, we evaluated VTA cellular coexpression of TH transcripts and TH protein. Within the medial aspects of the VTA, some neurons expressed TH mRNA but lacked TH-IR; among them a subset coexpressed VGluT2 mRNA. To determine if lack of VTA TH-IR was due to TH trafficking, we tagged VTA TH neurons by Cre-inducible expression of mCherry in TH::Cre mice. By dual immunofluorescence, we detected axons containing mCherry, but lacking TH-IR, in the lateral habenula, indicating that low frequency of VGluT2 mRNA (+)/TH-IR (+) neurons in the mouse is due to lack of synthesis of TH protein, rather than TH protein trafficking. In conclusion, VGluT2 neurons are present in the rat and mouse VTA, but they differ in the populations of VGluT2/TH and TH neurons. Under normal conditions, the translation of TH protein is suppressed in the mouse mesohabenular TH neurons.

Distribution of vesicular glutamate transporter 2 in auditory and song control brain regions in the adult zebra finch (Taeniopygia guttata)

The songbird brain has a system of interconnected nuclei that are specialized for singing and song learning. Wada et al. (2004; J. Comp. Neurol. 476:44-64) found a unique distribution of the mRNAs for glutamate receptor subunits in the song control brain areas of songbirds. In conjunction with data from electrophysiological studies, these finding indicate a role for the glutamatergic neurons and circuits in the song system. This study examines vesicular glutamate transporter 2 (VGLUT2) mRNA and protein expression in the zebra finch brain, particularly in auditory areas and song nuclei. In situ hybridization assays for VGLUT2 mRNA revealed high levels of expression in the ascending auditory nuclei (magnocellular, angular, and laminar nuclei; dorsal part of the lateral mesencephalic nucleus; ovoidal nucleus), high or moderate levels of expression in the telencephalic auditory areas (cudomedial mesopallium, field L, caudomedial nidopallium), and expression in the song nuclei (HVC, lateral magnocellular nucleus of the anterior nidopallium, robust nucleus of the arcopallium), where levels of expression were greater than in the surrounding brain subdivisions. Area X did not show expression of VGLUT2 mRNA. Nuclei in the descending motor pathway (dorsomedial nucleus of the intercollicular complex, retroambigual nucleus, tracheosyringeal motor nucleus of the hypoglossal nerve) expressed VGLUT2 mRNA. The target nuclei of VGLUT2 mRNA-expressing nuclei showed immunoreactivity for VGLUT2 as well as hybridization signals for the mRNA of glutamate receptor subunits. The present findings demonstrate the origins and targets of glutamatergic neurons and indicate a central role for glutamatergic circuits in the auditory and song systems in songbirds.

Brainstem origins of glutamatergic innervation of the rat hypothalamic paraventricular nucleus

Multiple lines of evidence document a role for glutamatergic input to the hypothalamic paraventricular nucleus (PVH) in stress-induced activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. However, the neuroanatomical origins of the glutamatergic input have yet to be definitively determined. We have previously shown that vesicular glutamate transporter 2 (VGLUT2) is the predominant VGLUT isoform expressed in the basal forebrain and brainstem, including PVH-projecting regions, and that the PVH is preferentially innervated by VGLUT2-immunoreactive terminals/boutons. The present study employed a dual-labeling approach, combining immunolabeling for a retrograde tract tracer, Fluoro-Gold (FG), with in situ hybridization for VGLUT2 mRNA, to map the brainstem and caudal forebrain distribution of glutamatergic PVH-projecting neurons. The present report presents evidence for substantial dual labeling in the periaqueductal gray, caudal portions of the zona incerta and subparafascicular nucleus, and the lateral parabrachial nucleus. The current data also suggest that relatively few PVH-projecting neurons in ascending raphe nuclei, nucleus of the solitary tract, or ventrolateral medulla are VGLUT2 positive. The data reveal multiple brainstem origins of glutamatergic input to PVH that are positioned to play a role in transducing a diverse range of stressful stimuli.

Mesocorticolimbic Glutamatergic Pathway

The mesocorticolimbic dopamine (DA) system plays important roles in reward, motivation, learning, memory, and movement. This system arises from the A10 region, comprising the ventral tegmental area and three adjacent midline nuclei (caudal linear nucleus, interfascicular nucleus, and rostral linear nucleus of the raphe). DAergic and GABAergic neurons are intermingled in this region with recently discovered glutamatergic neurons expressing the vesicular glutamate transporter 2 (VGluT2). Here, we show by in situ hybridization and immunohistochemistry that there are two subpopulations of neurons expressing VGluT2 mRNA in the A10 region: (1) a major subpopulation that expresses VGluT2 but lacks tyrosine hydroxylase (TH; VGluT2-only neurons), present in each nucleus of the A10 region, and (2) a smaller subpopulation that coexpresses VGluT2 and TH (VGluT2-TH neurons). By quantitative real-time PCR, we determined the mRNA copy numbers encoding VGluT2 or TH in samples of individual microdissected TH immunoreactive (IR) neurons. Data from both in situ hybridization and from mRNA quantification showed that VGluT2 mRNA is not present in every TH-IR neuron, but restricted to a subset of TH-IR neurons located in the medial portion of the A10 region. By integration of tract tracing, in situ hybridization, and immunohistochemistry, we found that VGluT2-only neurons and VGluT2-TH neurons each innervate both the prefrontal cortex and the nucleus accumbens. These findings establish that in addition to the well-recognized mesocorticolimbic DA-only and GABA-only pathways, there exist parallel mesocorticolimbic glutamate-only and glutamate-DA pathways.

VGLUT2 expression in primary afferent neurons is essential for normal acute pain and injury-induced heat hypersensitivity

Dorsal root ganglia (DRG) neurons, including the nociceptors that detect painful thermal, mechanical, and chemical stimuli, transmit information to spinal cord neurons via glutamatergic and peptidergic neurotransmitters. However, the specific contribution of glutamate to pain generated by distinct sensory modalities or injuries is not known. Here we generated mice in which the vesicular glutamate transporter 2 (VGLUT2) is ablated selectively from DRG neurons. We report that conditional knockout (cKO) of the Slc17a6 gene encoding VGLUT2 from the great majority of nociceptors profoundly decreased VGLUT2 mRNA and protein in these neurons, and reduced firing of lamina I spinal cord neurons in response to noxious heat and mechanical stimulation. In behavioral assays, cKO mice showed decreased responsiveness to acute noxious heat, mechanical, and chemical (capsaicin) stimuli, but responded normally to cold stimulation and in the formalin test. Strikingly, although tissue injury-induced heat hyperalgesia was lost in the cKO mice, mechanical hypersensitivity developed normally. In a model of nerve injury-induced neuropathic pain, the magnitude of heat hypersensitivity was diminished in cKO mice, but both the mechanical allodynia and the microgliosis generated by nerve injury were intact. These findings suggest that VGLUT2 expression in nociceptors is essential for normal perception of acute pain and heat hyperalgesia, and that heat and mechanical hypersensitivity induced by peripheral injury rely on distinct (VGLUT2 dependent and VGLUT2 independent, respectively) primary afferent mechanisms and pathways.

Localization of Vesicular Glutamate Transporter 2 mRNA in the Dorsal Root Ganglion of the Pigeon (Columba Livia)

Our previous study showed localization of glutamate receptor 1 (GluR1) mRNA in neurons of the pigeon spinal cord, suggesting glutamatergic input from intrinsic and extrinsic origins. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) mRNA to confirm an extrinsic origin of glutamatergic neurons in the dorsal root ganglion (DRG). GluR1 and GluR2 mRNAs were examined in DRG and spinal cord to seek projection regions from VGLUT2 mRNA-expressing neurons. VGLUT2 mRNA was expressed in most DRG neurons and labelling intensity varied from weakly to intensely. Intense VGLUT2 mRNA expression was mainly seen in medium to large neurons. GluR1 and GluR2 mRNAs were expressed in the dorsal horn and GluR2 mRNA signal was also seen in the marginal nucleus. The results suggest that the pigeon DRG has an extrinsic glutamatergic origin that project to the dorsal horn and marginal nucleus of the spinal cord.

Distribution of vesicular glutamate transporter 2 and glutamate receptor 1 and 2 mRNA in the pigeon retina

Glutamate is an excitatory neurotransmitter in the central and peripheral nervous systems of the vertebrate. The previous studies show the presence of mRNAs of AMPA-type glutamate receptors, GluR1 and GluR2, in the optic tectum of the pigeon, suggesting glutamatergic input from the retina. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) and GluR1 and GluR2 to confirm source of glutamatergic neurons in the pigeon retina by in situ hybridization histochemistry. VGLUT2 mRNA expressed in the inner nuclear layer and ganglion cells, while GluR1 and GluR2 mRNAs were observed in the inner nuclear layer, ganglion cells, and superficial layers of the optic tectum. The results suggest that photoreceptor cells, bipolar cells and ganglion cells are glutamatergic in the avian retina as in mammals.

Amino acid transporter (VIAAT, VGLUT2) and chloride cotransporter (KCC1, KCC2 and NKCC1) expression in the vestibular nuclei of intact and labyrinthectomized rat

We report the first investigation of whether unilateral labyrinthectomy in adult rats affects the expression of two amino acid transporters, vesicular glutamate transporter 2 (VGLUT2) and vesicular inhibitory amino acid transporter (VIAAT) and of chloride cotransporters (KCC1, KCC2 and NKCC1) in the intact and deafferented medial vestibular nuclei (MVN). In situ hybridization with specific radioactive oligonucleotide probes and immunofluorescent methods were used in normal and unilaterally labyrinthectomized rats at various times following the lesion: 5 h, and 1, 3 and 8 days. In normal animals, several brainstem regions including the lateral, medial, superior and inferior vestibular nuclei contained VGLUT2, VIAAT and KCC2 mRNA. In contrast, no or a very faint labeling was observed with KCC1 and NKCC1 probes. In unilaterally lesioned rats, there was no asymmetry between the two MVN with any of the oligonucleotide probes at any time after the lesion. Similarly, there were no differences in the intensity of MVN labeling between controls and lesioned animals. Finally, no over-expression of the cotransporter KCC1 and NKCC1 was found in ipsilateral or controlateral MVN in lesioned rats at any time. Immunohistochemical experiments gave similar conclusions. Our findings suggest that the recovery of the resting discharge of the deafferented MVN neurons, and consequently the functional compensation of the deficits, are not dependent on changes in the expression of amino acid transporters (VIAAT, VGLUT2), and chloride cotransporters (KCC1, KCC2 and NKCC1) or on their mRNAs.

Peripheral chemoreceptor inputs to retrotrapezoid nucleus (RTN) CO2-sensitive neurons in rats.

The rat retrotrapezoid nucleus (RTN) contains pH-sensitive neurons that are putative central chemoreceptors. Here, we examined whether these neurons respond to peripheral chemoreceptor stimulation and whether the input is direct from the solitary tract nucleus (NTS) or indirect via the respiratory network. A dense neuronal projection from commissural NTS (commNTS) to RTN was revealed using the anterograde tracer biotinylated dextran amine (BDA). Within RTN, 51% of BDA-labelled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2) but only 5% contained glutamic acid decarboxylase-67 (GAD67). Awake rats were exposed to hypoxia (n = 6) or normoxia (n = 5) 1 week after injection of the retrograde tracer cholera toxin B (CTB) into RTN. Hypoxia-activated neurons were identified by the presence of Fos-immunoreactive nuclei. CommNTS neurons immunoreactive for both Fos and CTB were found only in hypoxia-treated rats. VGLUT2 mRNA was detected in 92 +/- 13% of these neurons whereas only 12 +/- 9% contained GAD67 mRNA. In urethane-chloralose-anaesthetized rats, bilateral inhibition of the RTN with muscimol eliminated the phrenic nerve discharge (PND) at rest, during hyperoxic hypercapnia (10% CO(2)), and during peripheral chemoreceptor stimulation (hypoxia and/or i.v. sodium cyanide, NaCN). RTN CO(2)-activated neurons were recorded extracellularly in anaesthetized intact or vagotomized rats. These neurons were strongly activated by hypoxia (10-15% O(2); 30 s) or by NaCN. Hypoxia and NaCN were ineffective in rats with carotid chemoreceptor denervation. Bilateral injection of muscimol into the ventral respiratory column 1.5 mm caudal to RTN eliminated PND and the respiratory modulation of RTN neurons. Muscimol did not change the threshold and sensitivity of RTN neurons to hyperoxic hypercapnia nor their activation by peripheral chemoreceptor stimulation. In conclusion, RTN neurons respond to brain P(CO(2)) presumably via their intrinsic chemosensitivity and to carotid chemoreceptor activation via a direct glutamatergic pathway from commNTS that bypasses the respiratory network. RTN neurons probably contribute a portion of the chemical drive to breathe.

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water–electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.

Expression and immunohistochemical localization of vesicular glutamate transporter 2 in the migratory pathway from the rat olfactory placode.

The localization of vesicular glutamate transporter 2 (VGLUT2) was examined by immunohistochemistry and in situ hybridization histochemistry in the developing rat olfactory region with special relation to the spatiotemporal location of NCAM, a neural cell adhesion molecule expressed in differentiated neurons, and the calcium-binding protein calbindin D-28k, a marker of neurons migrating from the vomeronasal organ anlage (Y. Toba et al. (2001) J. Neuroendocrinol., 13, 683-694). Both VGLUT2 and NCAM immunoreactivities were first detected at embryonic day 11.5 (E11.5) in the neuronal cell mass beneath the telencephalic vesicle. After E12.5, VGLUT2-immunoreactive cells were detected in the migratory pathways from both medial and lateral olfactory pits, anlagen of the vomeronasal organ and olfactory epithelium. Between E15.5 and E19.5, moderate to intense VGLUT2 immunoreactivity was observed in cell clusters situated along NCAM-bearing vomeronasal nerves, and frequently colocalized with calbindin D-28k immunoreactivity. Using in situ hybridization histochemistry, VGLUT2 mRNA signals were detected in the clustered cells as well as in cells of the vomeronasal and olfactory epithelium. After E20.5, migrating cells gradually decreased in number and VGLUT2 immunoreactivity attenuated in the clustered cells, although calbindin D-28k immunoreactivity in these residual cells was still intense. The presence of intense VGLUT2 immunoreactivity in neurons actively migrating from the olfactory placode suggests that this transporter is involved in the migratory process of these neurons.

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.

Distribution of vesicular glutamate transporter-2 messenger ribonucleic Acid and protein in the septum-hypothalamus of the rat.

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.

Inspiratory augmenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes.

Many of the inspiratory augmenting (I-AUG) neurons of the rostral ventral respiratory group (rVRG) are premotor neurons that excite phrenic motor neurons during inspiration, probably by releasing glutamate. In the present study, we demonstrate that these neurons are indeed glutamatergic, in that their cell bodies contain vesicular glutamate transporter-2 (VGLUT2) mRNA and spinal terminals from neurons in the region of the rVRG contain VGLUT2 protein. We also demonstrate by using parallel in situ hybridization and immunocytochemical evidence that most rVRG inspiratory premotor neurons are enkephalinergic. After iontophoretic deposits of biotinylated dextran amine (BDA) in the area of the rVRG, many BDA-labeled terminals in the ventral horn of cervical spinal cord (C4-C5) were immunoreactive for enkephalin and VGLUT2. Injections of Fluoro-Gold amidst phrenic motor neurons in C4-C5 labeled neurons in the area of the rVRG that contained both VGLUT2 mRNA and preproenkephalin (PPE) mRNA as revealed by double in situ hybridization. Thirty-eight bulbospinal I-AUG neurons were recorded in the rVRG and filled with biotinamide by using the juxtacellular labeling technique. Every biotinamide-filled cell tested was positively labeled for VGLUT2 mRNA (n = 14), and most of the cells tested in a separate population exhibited PPE mRNA (16/18). We conclude that most of the phrenic inspiratory premotor neurons of the rVRG are glutamatergic neurons that may also release enkephalins.