Glutaminase-like Immunoreactivity Research Articles

Glutamic acid decarboxylase-like immunoreactive axon terminals in synaptic contact with mesencephalic trigeminal nucleus neurons in the rat

The purpose of the present study was to obtain reliable evidence for the presence of γ-aminobutyric acid-ergic (GABAergic) synapses upon mesencephalic trigeminal nucleus (MTN) neurons in the rat. For confocal laser-scanning microscopy, phosphate-activated glutaminase-like immunoreactivity (-IR) of MTN neurons was visualized with red fluorescence of Texas Red, while glutamic acid decarboxylase (GAD)-IR of GABA axons was visualized with green fluorescence of dichlorotriazinyl aminofluorescein. Many GAD-axon terminals were in close apposition to the cell bodies of MTN neurons. For electron microscopy, MTN neurons were labeled with wheat germ agglutinin-horseradish peroxidase injected into the masseter nerve, while axon terminals were labeled with GAD-IR. GAD-axon terminals were in symmetric synaptic contact with the cell bodies of MTN neurons. Primary proprioceptive neurons in the orofacial regions might be regulated post-synaptically by GABA 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.

Glutaminase-like immunoreactivity in rat spinomesencephalic tract cells

Retrograde transport of the fluorescent tracer Fluorogold was used in combination with immunohistochemical staining for the enzyme glutaminase to identify putative glutamatergic neurons belonging to the rat spinomesencephalic tract. Glutaminase-like staining in spinal projection neurons suggests that the relay of nociceptive information from the spinal cord to midbrain may involve the excitatory amino acid glutamate.

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.

Glutaminase-like immunoreactivity in the intermediolateral nucleus of the thoracic spinal cord of the rat

Glutaminase-like immunoreactivity in the intermediolateral nucleus of the thoracic spinal cord of the rat

Glutaminase-like immunoreactivity is exhibited in pyramidal neurons, but not in most non-pyramidal neurons in the cerebral cortex of the rat

Glutaminase-like immunoreactivity is exhibited in pyramidal neurons, but not in most non-pyramidal neurons in the cerebral cortex of the rat

Heterogeneity of neuropil labeling with glutaminase-like immunoreactivity in rat neostriatum

Heterogeneity of neuropil labeling with glutaminase-like immunoreactivity in rat neostriatum

Heterogeneity of neuropil labeling with glutaminase-like immunoreactivity in rat neostriatum : Takeshi Kaneko, Akitoshi Hanazawa, Ryuichi Shigemoto, Kazuo Itoh and Noboru Mizuno, Department of Anatomy (1st Division), Faculty of Medicine, Kyoto University, Kyoto 606, Japan

Heterogeneity of neuropil labeling with glutaminase-like immunoreactivity in rat neostriatum : Takeshi Kaneko, Akitoshi Hanazawa, Ryuichi Shigemoto, Kazuo Itoh and Noboru Mizuno, Department of Anatomy (1st Division), Faculty of Medicine, Kyoto University, Kyoto 606, Japan

Immunohistochemical demonstration of glutaminase in catecholaminergic and serotoninergic neurons of rat brain

The presence of phosphate-activated glutaminase in monoaminergic neurons was shown in the rat brain by a double-staining method using a mouse monoclonal anti-glutaminase antibody combined with rabbit antisera against tyrosine hydroxylase, dopamine-ß-hydroxylase,phenylethanolamine-N-methyltransferase or serotonin. Glutaminase-like immunoreactivity was detected within perikarya of many monoaminergic neurons in the substantia nigra pars compacta, locus ceruleus, raphe nuclei, etc. Possible functional significance of glutaminase in monoamine neurons was discussed.

Glutaminase-like immunoreactivity in the lower brainstem and cerebellum of the adult rat

Distribution of putative glutamatergic neurons in the lower brainstem and cerebellum of the rat was examined immunocytochemically by using a monoclonal antibody against phosphate-activated glutaminase, which has been proposed to be a major synthetic enzyme of transmitter glutamate and so may serve as a marker for glutamatergic neurons in the central nervous system. Intensely-immunolabeled neuronal cell bodies were densely distributed in the main precerebellar nuclei sending mossy fibers to the cerebellum; in the pontine nuclei, pontine tegmental reticular nucleus of Bechterew, external cuneate nucleus, and lateral reticular nucleus of the medulla oblongata. Phosphate-activated glutaminase-immunoreactive granular deposits were densely seen in the brachium pontis and restiform body, suggesting the immunolabeling of mossy fibers of passage. In the cerebellum, neuropil within the granule cell layer of the cerebellar cortex displayed intense phosphate-activated glutaminase-immunoreactivity, and that within the deep cerebellar nuclei showed moderate immunoreactivity. These results indicate that many mossy fiber terminals originate from phosphate-activated glutaminase-containing neurons and utilize phosphate-activated glutaminase for the synthesis of transmitter glutamate. Intensely-immunostained neuronal cell bodies were further observed in other regions which have been reported to contain neurons sending mossy fibers to the cerebellum; in the dorsal part of the principal sensory trigeminal nucleus, dorsomedial part of the oral subnucleus of the spinal trigeminal nucleus, interpolar subnucleus of the spinal trigeminal nucleus, paratrigeminal nucleus, supragenual nucleus, regions dorsal to the abducens nucleus and genu of the facial nerve, superior and medial vestibular nuclei, cell groups f, x and y, hypoglossal prepositus nucleus, intercalated nucleus, nucleus of Roller, reticular regions intercalated between the motor trigeminal and principal sensory trigeminal nuclei, linear nucleus, and gigantocellular and paramedian reticular formation. Neuronal cell bodies with intense phosphate-activated glutaminase-immunoreactivity were also found in other brainstem regions, such as the paracochlear glial substance, posterior ventral cochlear nucleus, and cell group e. Although it is still controversial whether all glutamatergic neurons use phosphate-activated glutaminase in a transmitter-related process and whether phosphate-activated glutaminase is involved in other metabolism-related processes, the neurons showing intense phosphate-activated glutaminase-immuno-reactivity in the present study were suggested to be putative glutamatergic neurons.

Localization of glutamate in trigeminothalamic projection neurons: a combined retrograde transport-immunohistochemical study.

Trigeminothalamic projection neurons are important components of the pathways for conscious perception of pain, temperature, and tactile sensation from the orofacial region. The neurotransmitters utilized by trigeminal neurons projecting to the thalamus are unknown. By use of a monoclonal antibody specific for fixative-modified glutamate and a polyclonal antiserum against glutaminase, we recently identified neurons in the trigeminal sensory complex that contain glutamate-like immunoreactivity (Glu-LI) and glutaminase-like immunoreactivity. In the present study, we utilized combined retrograde transport-immunohistochemical techniques to localize putative glutamatergic trigeminothalamic neurons. Following injection of the retrograde tracer, wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP), into the ventroposterior medial thalamus (VPM), the number of neuronal profiles that were double-labeled with WGA:HRP and Glu-LI was greatest in principal sensory nucleus (Pr5), followed by subnuclei interpolaris (Sp5I) and caudalis (Sp5C). The average percentages of projection neurons double-labeled with Glu-LI were approximately 60-70% in Pr5 and Sp5I and 40% in Sp5C. The majority of double-labeled profiles in Sp5C were located in the magnocellular layer, as opposed to the marginal and substantia gelatinosa layers. A large injection site that spread into the intralaminar thalamic nuclei and nucleus submedius--areas implicated in the processing of nociceptive information--resulted in an increase in the ratio of single-labeled to double-labeled projection profiles in Sp5C. These results suggest that glutamate may be the neurotransmitter for a majority of trigeminothalamic projection neurons located in Sp5I and Pr5. However, on the basis of anatomical association, glutamate does not appear to be the major transmitter for neurons in Sp5C that forward nociceptive information to the thalamus.

Phosphate activated glutaminase-like immunoreactivity in the nervous system from different species and in different neuronal cell types and in astrocytes

Using antibodies against pig brain phosphate activated glutaminase, the enzyme appears to be rather conservative as we have observed immuno- staining in the brain from all species investegated [pig, cow, rabbit, rat, mouse, man, fish (cod and salmon) and bird (chicken)]. In addition, phosphate activated glutaminase from cultured mouse cerebral cortex inter- neurons (mainly GABA-ergic), cerebellar granule cells (glutamatergic) and astrocytes stained in an analogous manner. However, no phosphate activated glutaminase-like immunostaining was found in lobster ganglion. yeast and E. coli. Using the Western blotting technique, phosphate activated glutaminase from dodecyl sulfate treated samples from all the above mentioned preparations revealed a MW close to 64 K d. The MW is similar to the MW of the subunit of phosphate activated glutaminase in a highly purified pig brain preparation, The Western blotting technique seems to be well suited to identify phosphate activated glutaminase-like immunoreactivity in different tissues.

Immunohistochemical localization of glutamate, glutaminase and aspartate aminotransferase in neurons of the pontine nuclei of the rat

The pontine nuclei form the key relay nuclei in the cerebropontocerebellar pathway. Although a great deal of information is available regarding the anatomy of this region, the identity of the neurotransmitter(s) contained in the neurons of the pontine gray are not known. The aim of the present investigation is to utilize immunohistochemical techniques to determine whether glutamate, a putative excitatory transmitter, and the enzymes responsible for its metabolism, are found in pontine neurons. Both glutaminase, an enzyme which converts glutamine to glutamate, and aspartate aminotransferase, an enzyme which is involved in the interconversion between glutamate and aspartate, have been proposed to be markers of neurons which use excitatory amino acids as neurotransmitters. The present study utilizes a monoclonal antibody against carbodiimide-fixed glutamate and polyclonal antisera against glutaminase and aspartate aminotransferase in conjunction with the indirect peroxidase technique or the peroxidase-labeled biotin-avidin procedure to localize glutamatergic neurons in the pontine nuclei of the rat. Numerous neurons in all subdivisions of the pontine nuclei were found to contain carbodiimide-fixed glutamate-like immunoreactivity, glutaminase-like immunoreactivity or aspartate aminotransferase-like immunoreactivity. Horseradish peroxidase was injected into the cerebellum of four rats for use with a combined retrograde transport-immunohistochemical procedure. Double-labeled neurons were observed in all subdivisions of the pontine nuclei, indicating that pontine neurons which contain glutamate-like immunoreactivity project to the cerebellum. Based on the hypothesis that increased levels of glutamate, glutaminase and aspartate aminotransferase reflect a transmitter role for glutamate, the present data raise the possibility that glutamate may be a major neurotransmitter of pontocerebellar fibers.

Localization of glutaminase-like and aspartate aminotransferase-like immunoreactivity in neurons of cerebral neocortex

The distribution of glutaminase (GLNase)- and aspartate aminotransferase (AATase)-immunoreactive cells was examined in the cerebral neocortex of rat and guinea pig and in the somatic sensorimotor and primary visual cortex of the Macaca fascicularis monkey. These enzymes are involved in the metabolism of glutamate and aspartate, two amino acids thought to be excitatory amino acid transmitters for cortical neurons. In each of the species examined a large percentage of layer V and VI pyramidal neurons have pronounced glutaminase-like immunoreactivity (GLNase IR). In contrast, neurons in layers I, II, and IV show little GLNase IR. Layer III in the rat and guinea pig contains only a few, densely labeled GLNase-like-immunoreactive (GLNase-Ir) pyramidal neurons, whereas in the monkey the number of GLNase-Ir cells in layer III varies between cytoarchitectonic fields. Area 3b of the primary somatic sensory cortex and area 17 (primary visual cortex) contain few GLNase-Ir cells in layer III. However, layer III contains moderate numbers of GLNase IR in cells in areas 3a, 1, 2, 5, and in the primary motor cortex. Within the motor cortex the largest pyramidal ("Betz") cells are not labeled. In marked contrast to the results with antibody to GLNase, antibody to AATase labels cells that appear nonpyramidal in form, and these cells are in all cortical layers in each of the species examined. This distribution is roughly similar throughout all areas of rodent neocortex, but in monkey visual cortex AATase-immunoreactive neurons are more numerous in layers II-III, IVc, and VI. When combined with the findings of other studies, our results suggest that GLNase IR marks pyramidal neurons that use an excitatory amino acid transmitter. Antibody to AATase appears to mark intrinsic cortical neurons. The AATase immunoreactivity of these cells could indicate that they use an excitatory amino acid transmitter. However, their form and distribution in cortex suggest that this antibody labels GABAergic neurons.

Immunocytochemical localization of glutaminase-like and aspartate aminotransferase-like immunoreactivities in the rat and guinea pig hippocampus

There is considerable evidence that pathways of the hippocampus use an excitatory amino acids as transmitter. We have attempted to immunocytochemically identify excitatory amino acid neurons in the hippocampus of the rat and guinea pig using antiserum to glutaminase and antiserum to aspartate aminotransferase, which have been proposed as markers for aspartergic/glutamergic neurons. Glutaminase-like immunoreactivity was seen in granule cells in the dentate gyrus and fibers and puncta associated with the mossy fiber pathway in the hilus and stratum lucidum of the hippocampus. At the ultrastructural level, glutaminase-like immunoreactivity was observed in mossy fiber terminals in the stratum lucidum. Glutaminase-like immunoreactivity was also seen in pyramidal cells in regio inferior and regio superior and in cells in layer two of the entorhinal cortex. Schaffer collateral terminals, commissural fiber terminals and perforant pathway terminals were not seen at the light microscopic level. Glutaminase-like immunoreactivity is thus found in the cell bodies of proposed excitatory amino acid neurons of hippocampal pathways, but does not appear to label all terminals. Aspartate aminotransferase-like immunoreactivity was not seen in any cells, fibers or terminals in the rat or guinea pig hippocampus.

Glutaminase-like immunoreactivity in the organ of Corti of guinea pig.

The distribution of glutaminase (GLNase)-like immunoreactivity (IR) in the normal and surgically de-efferented organ of Corti of guinea pig was studied. Primary antisera were against phosphate-dependent GLNase from rat kidney. Indirect immunocytochemical techniques were used; IR was visualized in cryostat sections through immunofluorescence, and through immunofluorescence or with horseradish peroxidase reaction product in surface preparations. Standard microscopy and video-enhanced light microscopy with asymmetric illumination contrast were used. GLNase-like IR was found at inner hair cells (IHCs) in the normal and in the de-efferented organ of Corti, in the tunnel spiral bundle, in tunnel-crossing fibers, in endings high up on outer hair cells (OHCs), in outer spiral bundles, in puncta close to OHCs, and in large, efferent endings at OHC bases. There was no GLNase-like IR at OHCs in the de-efferented organ of Corti. It is concluded that GLNase-like IR is present in auditory nerve dendrites at IHCs and in olivocochlear efferents of the medial system, and that future studies are needed to determine whether also the lateral system of olivocochlear efferents contains GLNase-like IR. A diagram is included depicting the relation between OHCs and efferent nerve endings along the cochlear spiral, showing that in the apicalmost 3/4 turn of the spiral OHCs have no efferent endings.

Immunocytochemical localization of glutaminase-like immunoreactivity in the auditory nerve

The immunocytochemical localization of glutaminase, which we have proposed as a marker for excitatory amino acid neurotransmitters was determined in the guinea pig auditory nerve. Glutaminase-like immunoreactivity was seen in auditory nerve terminals in the cochlear nucleus and in the cell bodies of the auditory nerve in the cochlea. This staining was seen in type I and not type II spiral ganglion cells. Glutaminase-like immunoreactivity was also observed in granule cells in the cochlear nucleus.