Vesicular Glutamate Transporter 2 Immunoreactivity Research Articles

Expression of Glutaminase and Vesicular Glutamate Transporter Type 2 Immunoreactivity in Rat Sacral Dorsal Root Ganglia Following a Surgical Tail Incision.

Glutamate is an excitatory neurotransmitter, released by primary sensory peripheral nerve and spinal synaptic terminals during nociceptive (pain) signaling. The primary source of neurotransmitter, glutamate, is provided from its synthetic enzyme, glutaminase (GLS). Neurotransmitter glutamate is packaged into synaptic vesicles in nociceptive neurons by the vesicular glutamate transporter 2 (VGluT2). Little is known, however, what effect a surgical incision has on GLS and VGluT2 in primary afferent neurons. In this study, an aseptic, midline incision in the proximal one-third of the rat-tail was examined to determine whether sacral dorsal root ganglia innervate the area of surgical incision, utilizing the retrograde tracer Fluoro-Gold™. Subsequently, the amount of VGluT2 and GLS immunoreactivity (IR) in sacral dorsal root ganglia (DRG) was evaluated using immunofluorescence with image analysis and Western immunoblotting with density analysis. GLS messenger RNA (mRNA) changes were evaluated using real-time reverse transcriptase polymerase chain reaction (RT2-PCR). Our findings revealed that sacral-1 (S1) DRG neurons innervate the area of surgical incision. Both GLS and VGluT2-ir are elevated post-surgical incision in S1 DRG neurons for up to 72 hours, while GLS mRNA levels rapidly decreased post-incision and remain depressed for at least 96 hours. Following a surgical incision of the tail, sacral DRGs rapidly deplete their available supply of GLS mRNA and alter their production of the synthetic enzyme, GLS and the vesicular transporter, VGluT2. The rapid use of GLS mRNA and subsequent elevation of GLS protein, along with VGluT2 protein may result in both increased glutamate production and release at peripheral and central processes contributing to primary and secondary sensitization, respectively.

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.

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat.

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO2 and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.

Glutamatergic neurons in the lateral periaqueductal gray innervate neurokinin-1 receptor-expressing neurons in the ventrolateral medulla of the rat

The neural pathways underlying the respiratory responses elicited by electrical or chemical stimulation of the lateral part of the periaqueductal gray (lPAG) remain unsettled. In the present study, we examined the lPAG projection to neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the ventrolateral medulla (VLM) which have been implicated in the control of respiration. After biotinylated dextranamine (BDA) injection into the lPAG, NK1R-ir neurons in the rostral VLM were embedded in the plexus of BDA-labeled fibers. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites and additionally with somata of the NK1R-ir neurons. Using retrograde tracing combined with in situ hybridization, we demonstrated that the vast majority of the lPAG neurons projecting to the rostral VLM were positive for vesicular glutamate transporter 2 (VGLUT2) mRNA, but not for glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde tracing and immunohistochemistry, we further demonstrated that the lPAG axon terminals with VGLUT2 immunoreactivity made close apposition with the NK1R-ir neuronal profiles in the rostral VLM. These data suggest that lPAG neurons exert an excitatory influence on NK1R-expressing neurons in the rostral VLM for the control of respiration.

Glutamatergic Neurons of the Rat Epicardial Ganglia

Some epicardial ganglion neurons may have a sensory function, transmitting information from the heart surface to the ganglia. In dorsal root ganglia, all somatic and visceral sensory neurons are glutamatergic, therefore, we asked if glutamatergic markers could identify sensory neurons in the rat epicardial ganglia. We examined vesicular glutamate transporter 2 (VGLUT2) and glutaminase (GLS) immunoreactivity in the rat epicardial ganglia. Tissue sections of epicardiac fat and atria were processed for VGLUT2 or GLS immunoreactivity along with peripherin. Immunoreactive (ir) VGLUT2 and GLS neurons were present in epicardiac ganglia and contacted by VGLUT2 and GLS ir nerve fibers. VGLUT2 and GLS nerve fibers occurred in adjacent atrial myocardium. Cell bodies and nerve fibers were peripherin ir. These results indicate that many rat epicardiac ganglion neurons are glutamatergic in nature. They contain GLS, enzyme for glutamate production, and VGLUT2, transporter for glutamate storage. Investigation of the glutamatergic phenotype of epicardiac ganglion cells may help in understanding the function of epicardial neurons in physiological and pathological conditions.Grant Funding Source : NIH AR047410

Projection neurons from the central nucleus of the amygdala to the nucleus pontis oralis

The present retrograde labeling study was designed to determine the presence and pattern of projections from individual subdivisions of the central nucleus of the amygdala (CNA) to the nucleus pontis oralis (NPO), which is a critical brainstem site involved in the generation and maintenance of active (REM) sleep. Projections from the CNA were labeled with the retrograde tracer cholera toxin B-subunit (CTB), which was injected, unilaterally, via microiontophoresis, into the NPO. Sections of the amygdala were immunostained in order to identify CTB-labeled CNA neurons and CNA neurons that contained CTB plus the vesicular glutamate transporter 2 (VGLUT2), which is a marker for glutamatergic neurons. Histological analyses revealed that retrogradely labeled neurons that project to the NPO were localized, ipsilaterally, within the medial, lateral, and capsular subdivisions of the CNA. In addition, a substantial proportion (24%) of all retrogradely labeled CNA neurons also exhibited VGLUT2 immunoreactivity. The present study demonstrates that glutamatergic neurons, which are present within various subdivisions of the CNA, project directly to the NPO. These data lend credence to the hypothesis that NPO neurons that are involved in the control of active sleep are activated by glutamatergic projections from the amygdala.

Glutamatergic innervation of growth hormone-releasing hormone-containing neurons in the hypothalamic arcuate nucleus and somatostatin-containing neurons in the anterior periventricular nucleus of the rat

Growth hormone-releasing hormone (GHRH) and somatostatin are the two main hypothalamic neurohormones, which stimulate or inhibit directly hypophysial growth hormone (GH) release. Majority of the GHRH neurons projecting to the median eminence is situated in the arcuate nucleus and the somatostatin neurons in the anterior periventricular nucleus. Data suggest that the excitatory amino acid glutamate may play an important role in the control of hypothalamic neuroendocrine neurons and processes including the control of GH. There is a dense plexus of glutamatergic fibres in the hypothalamic arcuate and anterior periventricular nucleus. The aim of the present studies was to examine the relationship of these fibres to the GHRH neurons in the arcuate nucleus and to somatostatin neurons in the anterior periventricular nucleus. Double-labelling immuno-electron microscopy was used. Glutamatergic structures were identified by the presence of vesicular glutamate transporter 2 (VGluT2) (a selective marker of glutamatergic elements) immunoreactivity. A significant number of VGluT2-immunoreactive boutons was observed to make asymmetric type of synapses with GHRH-immunostained nerve cells in the arcuate and with somatostatin neurons in the anterior periventricular nucleus. A subpopulation of somatostatin-immunoreactive neurons displayed also VGluT2 immunoreactivity. Our findings provide direct neuromorphological evidence for the view that the action of glutamate on GH release is exerted, at least partly, directly on GHRH and somatostatin neurons releasing these neurohormones into the hypophysial portal blood.

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.

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies.

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.

Glutamatergic pathways from the Kölliker–Fuse nucleus to the phrenic nucleus in the rat

After ipsilateral injections of biotinylated dextran amine (BDA) into the Kölliker–Fuse (KF) nucleus and cholera toxin B subunit (CTb) into the ventral horn in C4 to C5 segments of the spinal cord, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the rostral ventral respiratory group (rVRG) region ipsilateral to the injection sites. After ipsilateral injections of BDA into the KF and Fluoro-Gold (FG) into the ventral horn in C4 to C5 segments of the spinal cord, BDA-labeled axons were found to make asymmetrical synapses with the somata and dendrites of FG-labeled neurons within the neuropil of the rVRG region. Using retrograde tracing combined with immunohistochemistry for phosphate-activated glutaminase (PAG), we observed that as many as 72% of the rVRG neurons projecting to the PhN were immunoreactive for PAG and that approximately 62% and 75% of the KF neurons projecting respectively to the rVRG region and PhN contain PAG immunoreactivity. Using anterograde tracing combined with immunohistochemistry for vesicular glutamate transporter 2 (VGluT2), we further demonstrated that the KF axon terminals in the rVRG and PhN regions as well as the rVRG axon terminals in the PhN region contain VGluT2 immunoreactivity. The present results suggest that the glutamatergic pathways from the KF to the PhN directly and indirectly via the rVRG region may exist and underlie the inspiratory responses that are elicited by activation of the KF neurons.

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.