Glutaminase 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.

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

Glutaminase Immunoreactivity and Enzyme Activity Is Increased in the Rat Dorsal Root Ganglion Following Peripheral Inflammation

Following inflammation, primary sensory neurons in the dorsal root ganglion (DRG) alter the production of several proteins. Most DRG neurons are glutamatergic, using glutaminase as the enzyme for glutamate production, but little is known about glutaminase following inflammation. In the present study, adjuvant-induced arthritis (AIA) was produced in rats with complete Freund's adjuvant into the hindpaw. At 7 days of AIA, DRG were examined with glutaminase immunohistochemistry, Western blot immunoreactivity, and enzyme activity. Image analysis revealed that glutaminase was elevated most in small-sized neurons (21%) (P < 0.05). Western blot analysis revealed a 19% increase (P < 0.05) in total glutaminase and 21% in mitochondrial glutaminase (P < 0.05). Glutaminase enzyme activity was elevated 29% (P < 0.001) from 2.20 to 2.83 moles/kg/hr. Elevated glutaminase in primary sensory neurons could lead to increased glutamate production in spinal primary afferent terminals contributing to central sensitization or in the peripheral process contributing to peripheral sensitization.

Adolescent anabolic androgenic steroids reorganize the glutamatergic neural circuitry in the hypothalamus

Chronic treatment with anabolic–androgenic steroids (AAS) during adolescence alters the activity of various neurotransmitter systems in male Syrian hamsters ( Mesocricetus auratus). The present study was conducted to determine whether glutamatergic cells in the lateral anterior hypothalamus (LAH), a sub-region of the anterior hypothalamus, have lasting activation following adolescent AAS exposure, and to examine AAS-induced alterations in the connections between the LAH and the ventrolateral hypothalamus (VLH) governed by glutamate. Hamsters were administered AAS during adolescence and then examined for changes in FOS (protein product of the immediate early gene c-fos) and phosphate activated glutaminase (PAG; the rate-limiting enzyme in the synthesis of glutamate) immunoreactivity (FOS/PAG-IR) using double-label immunohistochemistry. In a second experiment, a retrograde tracing study was conducted using a red fluorescent tracer microinjected into the VLH. Then brains were processes for PAG immunofluorescence and examined for AAS-induced changes in the number of PAG positive cells containing the tracer (PAG/Tracer) in the LAH. When compared to oil-treated controls, AAS-treated hamsters showed significant increases in PAG-IR and FOS/PAG-IR in the LAH, decreases in afferent innervation from the LAH to the VLH and decreases in the total number of glutamate cells in the LAH projecting to the VLH. Together with previous research from our lab showing increased AAS-induced expression of PAG in the AH and increased glutamate receptor expression in the VLH, the current results suggest that adolescent AAS exposure leads to alterations in the function and expression of the glutamatergic system as well as changes in hypothalamic neural connections. In addition, the current results further strengthen the notion that a specific nucleus in the AH, the LAH is a critical hypothalamic sub-region particularly sensitive to AAS-induced neurodevelopmental effects.

Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (∼96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled. The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved the synthesis of transmitter glutamate may be less uniform than previously expected.

Phosphate activated glutaminase is concentrated in mitochondria of sensory hair cells in rat inner ear: a high resolution immunogold study.

Glutamate has been implicated in signal transmission between sensory hair cells and afferent fibers in the inner ear. However, the mechanisms responsible for glutamate replenishment in these cells are not known. Here we provide evidence that phosphate activated glutaminase, which is thought to be the predominant glutamate-synthesizing enzyme in the brain, is concentrated in all types of hair cell in the organ of Corti and vestibular epithelium. By use of two different antibodies (directed to the N and C terminus, respectively) it was shown that glutaminase is largely restricted to mitochondria and that part of the enzyme pool is associated with the inner membrane of this organelle. Quantitative analysis of immunogold labelled Lowicryl sections revealed that the level of glutaminase immunoreactivity in mitochondria of supporting cells is less than 15% of that in hair cell mitochondria. Using triple labelling for glutaminase, glutamate, and glutamine, evidence was provided of a positive correlation between the glutamate/glutamine ratio and the level of glutaminase immunoreactivity, suggesting that the glutaminase antibodies identify a functional enzyme pool. Our results strengthen the idea that glutamate is a hair cell transmitter and indicate that the sensory epithelia in the inner ear show a metabolic compartmentation analogous to that in the brain.

Phosphate-activated glutaminase immunoreactivity in brainstem respiratory neurons

The aim of this study was to determine if immunoreactivity for phosphate activated glutaminase (PAG), an enzyme involved in the biosynthesis of glutamate and a putative marker for neurons that use glutamate as a neurotransmitter, is present within respiratory neurons in the ventrolateral medulla oblongata. Intracellular recordings were obtained from neurons in the ventrolateral medulla of adult anaesthetised Sprague-Dawley rats. Neurons with a respiratory-related modulation of their membrane potential were filled with Neurobiotin (Vector, CA). After histochemical processing, sections of brainstem were examined by fluorescence and light microscopy. Some PAG immunoreactivity was found in all of the four types of respiratory neurons examined. PAG immunoreactivity was graded as strong or weak. (1) Of six inspiratory neurons in the rostral ventral respiratory group five were strongly PAG immunoreactive and one was weakly PAG immunoreactive. (2) Of six expiratory neurons in the caudal ventral respiratory group five were strongly PAG immunoreactive while one was weak. (3) Seven motoneurons in the nucleus ambiguus were all strongly PAG immunoreactive. (4) Five neurons in the Bötzinger area were examined. Four were weakly PAG immunoreactive while one contained strong PAG immunoreactivity. These data demonstrate a heterogeneity of PAG immunoreactivity amongst brainstem respiratory neurons.

Glutamine cycle enzymes in the crayfish giant nerve fiber: Implications for axon‐to‐glia signaling

Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis.

Expression and role of ionotropic glutamate receptors in pancreatic islet cells

Although the excitatory amino acid glutamate and its receptors play crucial roles in many functions of the central nervous system (CNS), their presence in the peripheral tissues has remained unclear. In the present study, we have identified kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) receptor subtype mRNAs in pancreatic islets, using reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) measurements and electrophysiological recordings indicate that kainate, AMPA, and NMDA all elicit increases of [Ca2+]i in single pancreatic beta-cells and depolarize them. In addition, kainate and AMPA stimulate insulin secretion from isolated pancreatic islets, whereas NMDA does not. Also, immunocytochemical study shows the presence of intense glutaminase immunoreactivity in pancreatic alpha-cells and intrapancreatic ganglia, a finding compatible with the possibility that glutamate is released from alpha-cells as well as from neurons. Because the inhibitory amino acid gamma-amino butyric acid (GABA) is present in beta-cells as well as in neurons and inhibits glucagon secretion from alpha-cells, the present study suggests that glutamate and GABA are coordinated in the regulation of hormone secretion in pancreatic islets.

Glutamate-synthesizing enzymes in gabaergic neurons of the neocortex: A double immunofluorescence study in the rat

Glutamate is the immediate precursor of the inhibitory transmitter GABA, and considered to be supplied from α-ketoglutarate through a transamination reaction or from glutamine through a glutaminase reaction. In the present study, the localization of aspartate aminotransferase and glutaminase in GABAergic neurons was investigated in the rat neocortex by a double immunofluorescence method. Immunoreactivities for both soluble and mitochondrial aspartate aminotransferases were detected in more than 90% of GABA-positive neurons, whereas glutaminase immunoreactivity was not found in GABApositive neurons. All neocortical neurons with soluble aspartate aminotransferase immunoreactivity were immunopositive for GABA, but none for glutaminase. Neurons with mitochondrial aspartate aminotransferase immunoreactivity showed either glutaminase or GABA immunoreactivity. Under confocal laser scan microscopy, immunoreactivity for soluble aspartate aminotransferase was observed in many axons and axon terminals showing immunoreactivity for glutamic acid decarboxylase, whereas immunoreactivity for mitochondrial aspartate aminotransferase was seen in only a few axons displaying immunoreactivity for glutamic acid decarboxylase. The present results indicate that soluble aspartate aminotransferase is selectively localized to cell bodies and axon terminals of GABAergic non-pyramidal neurons in the cerebral neocortex. This suggests that glutamate is supplied from α-ketoglutarate via transamination and works as the immediate precursor for GABA in axon terminals of GABAergic neurons. The absence of glutaminase immunoreactivity in GABAergic neurons indicates that glutamine is a “metabolically remote” precursor for GABA. Mitochondrial aspartate aminotransferase was located in perikarya, rather than in axon terminals of GABAergic neurons, suggesting a transmitter-irrelevant role of this enzyme in neurons.

Distribution of phosphate-activated glutaminase-like immunoreactivity in the retina of rodents.

The distribution of phosphate-activated glutaminase-like immunoreactivity was examined in the retinas of rodents. Intense glutaminase immunoreactivity was observed in many neuronal perikarya in the ganglion cell layer and inner nuclear layer including those of ganglion, bipolar and amacrine cells and possibly horizontal cells. Almost all bipolar cells containing protein kinase C were immunoreactive for glutaminase, suggesting that the majority of glutaminase immunoreactive bipolar cells were of the ON type. Intense glutaminase immunoreactivity was also found in the neuropil of the inner and outer plexiform layers and around the outer limiting membrane. Weak to moderate immunoreactivity was seen in the outer nuclear layer and photoreceptor inner and outer segments. Under electron microscopy, glutaminase immunoreactivity was seen in bipolar cell axons and amacrine cell processes in the inner plexiform layer. In the outer plexiform layer, immunoreactivity was found in the Müller cell processes, but not in the photoreceptor cell terminals. These results indicate that ganglion cells and ON type bipolar cells use glutaminase to produce transmitter glutamate and suggest glutaminase has additional roles in Müller cells. A population of amacrine cells and horizontal cells showed immunoreactivity to glutaminase.

Phosphate-activated glutaminase immunoreactive synapses in the intermediolateral nucleus of rat thoracic spinal cord

A monoclonal antibody against phosphate-activated glutaminase was used to identify glutamatergic neuronal components in the intermediolateral nucleus of the thoracic spinal cord of the rat. Under electron microscopy of the intermediolateral nucleus, most glutaminase immunoreactivity was detected in the axoplasm surrounding spherical synaptic vesicles in the presynaptic axon varicosities which formed asymmetric synapses with small dendrites and occasionally with neuronal cell bodies. About 40% of axon varicosities within the intermediolateral nucleus and 49% of the axon varicosities forming asymmetric synaptic contacts showed glutaminase immunoreactivity. Glutaminase immunoreactivity was further seen in mitochondria of neuronal perikarya and dendrites in the intermediolateral nucleus, and occasionally in the cytoplasm of the dendrites and glial processes in the vicinity of glutaminase-immunoreactive axon varicosities. By the combined method of immunocytochemistry and retrograde axonal transport, glutaminase-immunoreactive axons were shown to make direct synaptic contacts with the preganglionic sympathetic neurons, which were retrogradely labeled by injection of horseradish peroxidase conjugated with choleratoxin B subunit into the superior cervical ganglion. The present results indicate that glutaminase-containing axons are the major synaptic inputs to inter-mediolateral nucleus neurons including preganglionic sympathetic ones, suggesting that glutamate is used as the neurotransmitter to control those neurons in the intermediolateral nucleus.

Paucity of glutaminase‐immunoreactive nonpyramidal neurons in the rat cerebral cortex

Glutaminase has been considered to be a synthesizing enzyme of transmitter glutamate in pyramidal neurons of the cerebral cortex. In the present study, an attempt was made to examine with a double immunofluorescence method whether or not nonpyramidal neurons of the cerebral cortex are immunoreactive for glutaminase. Glutaminase was stained with mouse anti-glutaminase IgM and FITC-labeled anti-[mouse IgM] antibody. In the same section, parvalbumin (PA), calbindin (CB), choline acetyltransferase (CAT), vasoactive intestinal polypeptide (VIP), corticotropin releasing factor (CRF), cholecystokinin (CCK), somatostatin (SS), or neuropeptide Y (NPY) was visualized as a marker for nonpyramidal neurons with an antibody to each substance, biotinylated secondary antibody and Texas Red-labeled avidin. Virtually no glutaminase immunoreactivity was seen in PA-, CB-, CAT-, VIP-, CRF-, CCK-, SS-, or NPY-immunoreactive neuronal perikarya in the neocortex and mesocortex (cingulate and retrosplenial cortices), although it was detected in a few PA-, CB-, VIP-, CCK-, SS-, or NPY-immunoreactive nonpyramidal neurons in the piriform, entorhinal, and hippocampal cortices. PA- and CB-positive neurons have been reported to constitute the major population of GABAergic neurons in the cerebral cortex. Thus, the present results, together with the previous reports, suggest that most GABAergic, cholinergic and peptidergic nonpyramidal neurons in the neo- and mesocortex do not contain glutaminase.

Mosaic distribution of phosphate-activated glutaminase-like immunoreactivity in the rat striatum

The dorsal and ventral striatum of mammals has been known to be organized in a mosaic manner, referred to as “patches” and “matrix” of the caudatoputamen. 12,17,32 The present study was primarily attempted in order to reveal the relationship of glutamatergic neuronal components to the mosaic organization in the rat striatum by using a monoclonal antibody to phosphate-activated glutaminase, a major synthetic enzyme of transmitter glutamate. Antibodies against glutamate decarboxylase and choline acetyltransferase were also used as the markers for GABAergic and cholinergic neuronal components, respectively. Glutaminase immunoreactivity was seen in a number of large- and a few medium-sized neurons in the caudatoputamen, nucleus accumbens and olfactory tubercle. The large neurons with glutaminase immunoreactivity were immunoreactive for choline acetyltransferase. A mosaic distribution of glutaminase immunoreactivity was observed in the neuropil of the caudatoputamen and nucleus accumbens; glutaminase immunoreactivity was particularly marked in the neuropil of island-like patchy areas although it was seen throughout the neuropil of the nuclei. In the caudatoputamen, island-like areas with marked glutaminase immunoreactivity exhibited less marked choline acetyltransferase immunoreactivity than the surrounding background region, and were thus considered to correspond to the patches. The mosaic distribution of glutamate decarboxylase immunoreactivity in the caudatoputamen seemed identical with that of glutaminase immunoreactivity. However, in the nucleus accumbens, the mosaic pattern of neuropil labeling for glutaminase was neither consistent with that for glutamate decarboxylase nor that for choline acetyltransferase, suggesting the presence of non-GABAergic glutaminase-containing nerve terminals in the nucleus. In an attempt to clarify the origin of neuropil labeling for glutaminase in the striatum, lesions were made in the regions sending projection fibers to the caudatoputamen and nucleus accumbens. After placing lesions in the cerebral cortex, glutaminase immunoreactivity was decreased in neuropil of the caudatoputamen, but the mosaic pattern remained. Lesions which were placed in the intralaminar thalamic nuclei, amygdaloid body, globus pallidus or substantia nigra produced no substantial change in glutaminase immunoreactivity in the caudatoputamen and nucleus accumbens. After injection of kainic acid into the caudatoputamen or nucleus accumbens, glutaminase immunoreactivity in the neuropil of the affected regions was decreased to lose the mosaic pattern, indicating that neuronal components with glutaminase immunoreactivity in the neuropil of the patches were mainly of intrinsic origin. In summary, possible axon terminals containing glutaminase were observed with mosaic patterns in the caudatoputamen and nucleus accumbens, in which large cholinergic and medium-sized non-cholinergic neurons were immunoreactive for glutaminase. In the caudatoputamen, glutaminase immunoreactivity in neuropil was more marked in the patches than in the matrix. The main source of glutaminase-containing axons in the patches or matrix was indicated to be non-cholinergic medium-sized neurons within the patches or glutamatergic neurons in the cerebral cortex, respectively. In the nucleus accumbens, glutaminase-containing axons appeared to be mainly derived from medium-sized intrinsic, putatively glutamatergic neurons.

Enhancement of glutaminase-like immunoreactivity in rat brain by an irreversible inhibitor of the enzyme

Changes of glutaminase immunoreactivity in rat brain were examined after intracranial injection of 6-diazo-5-oxo- l-norleucine (DON), an irreversible inhibitor of glutaminase. When 1 M DON was injected into the lateral ventricle, a half-lethal dose was 7.5–10 μmol. After intraventricular injection of 2–7.5 μmol DON, glutaminase immunoreactivity was dose dependently enhanced with the maximum enhancement 3–5 days after the injection. The enhanced glutaminase immunoreactivity was recognized by enlarged granular immunodeposits in both perikarya and neuropil in many regions, such as the hippocampus, thalamus, hypothalamus, periaqueductal gray, and some brain stem, cerebellar, and spinal cord regions. Intrathalamic injection of 0.2 μmol DON enhanced glutaminase immunoreactivity in many neuronal perikarya in the thalamus and in some perikarya in layer VI of the cerebral cortex. Intrastriatal injection of the same dose of DON enhanced glutaminase immunoreactivity in neuropil of the caudoputamen and in many neuronal perikarya of the intralaminar thalamic nuclei. These results suggested that DON induced a new massive synthesis of glutaminase in the affected neurons.

Identification of mitochondrial and non‐mitochondrial glutaminase within select neurons and glia of rat forebrain by electron microscopic immunocytochemistry

Antibodies against the mitochondrial enzyme glutaminase (EC 3.5.1.2), have been used in previous immunocytochemical studies to help identify glutamate-releasing neurons among all glutamate-containing neurons. The studies were based on the idea that glutaminase is enriched within the releasable "transmitter" pools of glutamate. However, evidence is also available to suggest that the enzyme does not occur exclusively within glutamate-releasing neurons. Thus we sought to determine whether glutaminase was immunocytochemically detectable within presynaptic terminals forming asymmetric (putatively excitatory) synapses or, alternatively, occurs in association with mitochondria throughout the cell. For this purpose, we examined the cellular and subcellular distribution of glutaminase- immunoreactivity in neocortical (visual and somatosensory) areas known to contain glutamatergic perikarya. This localization was compared with the distribution in striatal (caudate-putamen and nucleus accumbens) regions recognized to contain high densities of glutamatergic terminals but fewer, if any, glutamatergic perikarya. Glutaminase-immunoreactive perikarya were numerous within the infragranular laminae of neocortex (approximately 1 per 1,000 microns 2 tissue area) but sparse within the caudate-putamen nuclei and accumbens nuclei (less than 1 per 20,000 microns 2.). In addition, heterogeneous distribution of small (less than 1 microns) punctate immunoreactive structures was notable. Relatively high densities of these punctate structures occurred within the supragranular laminae of neocortex, dorsolateral quadrant of the caudate-putamen nuclei, and surrounding certain groups of myelinated fiber bundles throughout the striatum. Electron microscopy revealed diffusely distributed peroxidase immunoreactivity in a select population of dendritic spines, glial processes, and axons. Eight percent of all synapses within the supra-granular laminae were formed by terminals labeled for glutaminase. These principally formed asymmetric junctions on spiny processes. When tissue was incubated with the antibody in the presence of a permeabilizing agent, Photo-flo, high levels of glutaminase immunoreactivity was detectable by electron microscopy within select mitochondria of neocortical (4%) and striatal (8%) perikarya and dendrites, while the diffuse distribution of immunoreactivity within axons and glia was greatly diminished. The differential ultrastructural conditions provide direct demonstration that glutaminase in brain occurs in at least two forms discriminable by their diffuse distribution within non-mitochondrial cytoplasm versus discrete localization within mitochondria. The morphological characteristics of synapses formed by axons exhibiting diffuse distributions of glutaminase immunoreactivity are consistent with the idea that glutaminase-enriched terminals mediate excitatory chemical transmission via the release of glutamate. Because glia containing glutaminase occur juxtaposed to the asymmetric junctions, the glia may utilize neuronally released glutamate for energy metabolism.

Colocalization of fixative‐modified glutamate and glutaminase but not GAD in rubrospinal neurons

In an attempt to identify putative neurotransmitters of rubrospinal neurons, immunocytochemical procedures were utilized in combination with retrograde tracing techniques in 15 adult male rats. Following injections of horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) into the spinal cord, midbrain sections were processed with a combined procedure that allowed visualization of both the retrograde tracer and one or more antigens including glutamate, glutaminase, and glutamatic acid decarboxylase (GAD). Initial colocalization studies demonstrated that glutamatelike and glutaminaselike immunoreactivities were cocontained within the same neurons. Following injections of HRP or WGA-HRP into the spinal cord approximately 53% of retrogradely labeled neurons contained glutamate immunoreactivity. Triple-labeling experiments indicated that glutamatelike immunoreactivity was colocalized with glutaminase immunoreactivity in retrogradely labeled rubrospinal neurons. Retrogradely labeled neurons did not contain GAD immunoreactivity. Moreover, triple labeling experiments verified that glutamatelike immunoreactive retrogradely labeled cells did not cocontain GAD immunoreactivity. These studies demonstrate that glutamate and its synthesizing enzyme, glutaminase, are present in some rubrospinal neurons and raise the possibility that a component of the rubrospinal projection may be glutamatergic. GAD, on the other hand, is not present in rubrospinal neurons. This finding supports the hypothesis that GABAergic neurons play a role as interneurons in the red nucleus.

Development of aspartate aminotransferase and glutaminase immunoreactivity in the rat auditory nerve

Aspartate aminotransferase and glutaminase immunoreactive labeling of the auditory nerve has previously been reported. In the present study, the development of these immunoreactivities was examined in the auditory nerve of the rat, at ages ranging from 17 days gestation to four postnatal weeks. Cells and processes were examined in the cochlea, and fibers and terminals in the cochlear nucleus. In the cochlea, immunoreactive labeling with antisera to both enzymes was first seen at 20 gestational days, in spiral ganglion cells. It was not until two postnatal weeks, however, that this immunoreactive labeling was first seen in primary afferent terminals around spherical cells in the anteroventral cochlear nucleus. This correlates with the establishment of mature synaptic connections and function.

Localization of elevated glutaminase immunoreactivity in small DRG neurons

Glutamate has long been considered to be a neurotransmitter candidate in vertebrate spinal sensory nerve cells. We report here the first immunohistochemical evidence in support of this hypothesis. We find that up to 30% of the moderately small dorsal root ganglion neurons in the rat contain elevated levels of glutaminase immunoreactivity. This enzyme, which mediates the synthesis of glutamate from glutamine, is not found at these high levels in large diameter neurons of the same ganglia. In contrast, another enzyme associated with glutamate metabolism, aspartate aminotransferase, is rather uniformly distributed within neurons of the sensory ganglia. These data define a subpopulation of sensory neurons which appear to contain an elevated capacity to synthesize glutamate through the glutamine cycle and suggest that glutaminase immunoreactivity may be an indicator of glutamatergic function in some nerve cells.