Level Of Glutamate Immunoreactivity Research Articles

Assessment of glutamate loss from the ganglion cell layer of young DBA/2J mice with glaucoma

To determine whether glutamate contents are decreased in the ganglion cell layer (GCL) of retinas of DBA/2J mice with glaucoma, compared with unaffected control mice. 20 eyes from DBA/2J mice (9-week-old mice [n = 8] and 4- [4], 6- [4], and 12-month-old [4] mice) and 17 eyes from control CD-1 (7) and C57/BL6 (10) mice of similar age. After euthanasia, the eyes were rapidly dissected and fixed. Serial 0.5-microm sections were prepared from eyecups and stained with toluidine blue (to identify damaged cells) or immunogold (to localize glutamate). Microscopic images were captured digitally for comparison; immunostaining densities were assessed via special software. In the GCL of control mice, few cells appeared damaged; large amounts of glutamate were detected in 83 +/- 8.3% of cells. In DBA/2J mice > or = 9 weeks of age, damaged neurons were observed in retinal sections; the level of glutamate immunoreactivity was high in a few cells near areas of damage (13 +/- 3.2%) and in many cells in less-damaged regions of the same sections (82 +/- 4.2%). Many neurons with low amounts of glutamate in damaged regions did not appear damaged histologically. In retinas of young DBA/2J mice, damaged and undamaged GCL cells had decreased levels of immunostaining for glutamate, compared with less-damaged adjacent regions or retinas from control mice. The loss of neuronal glutamate in damaged retinal regions suggests that glutamate is contributing to early retinal damage prior to changes in intraocular pressure.

Phenotype of cerebellar glutamatergic neurons is altered in stargazer mutant mice lacking brain‐derived neurotrophic factor mRNA expression

Brain-derived neurotrophic factor (BDNF) influences neuronal survival, differentiation, and maturation. More recently, its role in synapse formation and plasticity has also emerged. In the cerebellum of the spontaneous recessive mutant mouse stargazer (stg) there is a specific and pronounced deficit in BDNF mRNA expression. BDNF protein levels in the cerebellum as a whole are reduced by 70%, while in the granule cells (GCs) there is a selective and near total reduction in BDNF mRNA expression. Recently, we published data demonstrating that inhibitory neurons in the cerebella of stgs have significantly reduced levels (approximately 50%) of gamma-aminobutyric acid (GABA) and fewer, smaller inhibitory synapses compared to wildtype (WT) controls. Our current investigations indicate that the stargazer mutation has an even more pronounced effect on the phenotype of glutamatergic neurons in the cerebellum. There is a profound decrease in the levels of glutamate-immunoreactivity (up to 77%) in stg compared to WT controls. The distribution profile of presynaptic vesicles is also markedly different: stgs have proportionally fewer docked vesicles and fewer vesicles located adjacent to the active zone ready to dock than WTs. Furthermore, the thickness of the postsynaptic density (PSD) at mossy fiber-granule cell (MF-GC) and parallel fiber-Purkinje cell (PF-PC) synapses is severely reduced (up to 33% less than WT controls). The number and length of excitatory synapses, however, appear to be relatively unchanged. It is possible that at least some of theses changes in phenotype are directly attributable to the lack of BDNF in the cerebellum of the stg mutant.

Distribution of glutamate receptor subunits at neurochemically characterized synapses in the entopeduncular nucleus and subthalamic nucleus of the rat

Glutamatergic neurotransmission in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia is critical in the expression of basal ganglia function, and increased glutamate transmission in these nuclei has been implicated in the pathology of Parkinson's disease. In order to determine the precise spatial relationship of subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors to nerve terminals enriched in glutamate or γ-aminobutyric acid (GABA) in one of the output nuclei, the entopeduncular nucleus (EP), and the STN, postembedding immunolabelling for glutamate receptor subunits and for glutamate and GABA was carried out in the rat. Immunolabelling for the AMPA glutamate receptor subunits 1, 2/3, and 4 (GluR1, GluR2/3, and GluR4) and the NMDA receptor subunit 1 (NR1) was localized predominantly within asymmetrical synapses in both the EP and STN. Quantitative analysis revealed that, on average for the whole population, each of the receptor subunits was evenly distributed along the synaptic specialization. Multiple AMPA receptor subunits and the GluR2/3 and NMDA (NR1) subunits were co-localized within individual synapses. The combination of immunolabelling for glutamate and GABA with the receptor immunolabelling revealed that the majority of axon terminals presynaptic to the receptor-immunoreactive synapses were enriched in glutamate immunoreactivity and were GABA-immunonegative. However, at some NR1- and GluR2/3-positive synapses, the level of glutamate immunoreactivity was low in the presynaptic terminal and, in the STN, some of them were GABA-immunopositive. It is concluded that glutamatergic transmission at individual synapses of different origins in the EP and STN is mediated by a combination of AMPA and NMDA glutamate receptors. J. Comp. Neurol. 397:403–420, 1998. © 1998 Wiley-Liss, Inc.

Distribution of glutamate receptor subunits at neurochemically characterized synapses in the entopeduncular nucleus and subthalamic nucleus of the rat

Glutamatergic neurotransmission in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia is critical in the expression of basal ganglia function, and increased glutamate transmission in these nuclei has been implicated in the pathology of Parkinson's disease. In order to determine the precise spatial relationship of subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors to nerve terminals enriched in glutamate or gamma-aminobutyric acid (GABA) in one of the output nuclei, the entopeduncular nucleus (EP), and the STN, postembedding immunolabelling for glutamate receptor subunits and for glutamate and GABA was carried out in the rat. Immunolabelling for the AMPA glutamate receptor subunits 1, 2/3, and 4 (GluR1, GluR2/3, and GluR4) and the NMDA receptor subunit 1 (NR1) was localized predominantly within asymmetrical synapses in both the EP and STN. Quantitative analysis revealed that, on average for the whole population, each of the receptor subunits was evenly distributed along the synaptic specialization. Multiple AMPA receptor subunits and the GluR2/3 and NMDA (NR1) subunits were co-localized within individual synapses. The combination of immunolabelling for glutamate and GABA with the receptor immunolabelling revealed that the majority of axon terminals presynaptic to the receptor-immunoreactive synapses were enriched in glutamate immunoreactivity and were GABA-immunonegative. However, at some NR1- and GluR2/3-positive synapses, the level of glutamate immunoreactivity was low in the presynaptic terminal and, in the STN, some of them were GABA-immunopositive. It is concluded that glutamatergic transmission at individual synapses of different origins in the EP and STN is mediated by a combination ofAMPA and NMDA glutamate receptors.

Glutamate immunoreactivity is enriched over pinealocytes of the gerbil pineal gland.

Mammalian pinealocytes have been shown to contain synaptic-like microvesicles with putative secretory functions. As a first step to elucidate the possibility that pinealocyte microvesicles store messenger molecules, such as neuroactive amino acids, we have studied the distributional pattern of glutamate immunoreactivity in the pineal gland of the Mongolian gerbil (Meriones unguiculatus) at both light- and electron-microscopic levels. In semithin sections of plastic-embedded pineals, strong glutamate immunoreactivity could be detected in pinealocytes throughout the pineal gland. The density of glutamate immunolabeling in pinealocytes varied among individual cells and was mostly paralleled by the density of immunostaining for synaptophysin, a major integral membrane protein of synaptic and synaptic-like vesicles. Postembedding immunogold staining of ultrathin pineal sections revealed that gold particles were enriched over pinealocytes. In particular, a high degree of immunoreactivity was associated with accumulations of microvesicles that filled dilated process terminals of pinealocytes. A positive correlation between the number of gold particles and the packing density of microvesicles was found in three out of four process terminals analyzed. However, the level of glutamate immunoreactivity in pinealocyte process endings was lower than in presumed glutamatergic nerve terminals of the cerebellum and posterior pituitary. The present results provide some evidence for a microvesicular compartmentation of glutamate in pinealocytes. Our findings thus lend support to the hypothesis that glutamate serves as an intrapineal signal molecule of physiological relevance to the neuroendocrine functions of the gland.

Immunocytochemical evidence that neurotensin is present in glutamatergic neurons in the superficial dorsal horn of the rat

In order to determine whether glutamate is enriched in neurotensin-containing axons in the superficial dorsal horn of the rat spinal cord, we have carried out preembedding immunocytochemistry with an antiserum to neurotensin and then used a postembedding immunogold method with antiserum to glutamate. The immunogold label (corresponding to glutamate-like immunoreactivity) over 40 neurotensin-immunoreactive boutons in laminae I and II of the lumbar dorsal horn was compared with that over nearby axons that formed asymmetrical or symmetrical synapses. In addition, for 20 of these boutons, the labeling was compared with that over mossy and parallel fiber terminals (both of which are thought to use glutamate as a transmitter) from sections of cerebellum that had been processed together with those of spinal cord. Glutamate-like immunoreactivity was consistently high over neurotensin-immunoreactive boutons relative to most surrounding profiles. Immunostaining over these boutons was slightly (11%) lower than that over matched terminals that formed asymmetrical synapses, but considerably higher than that over the terminals that formed symmetrical synapses. The level of glutamate immunoreactivity in neurotensin-immunoreactive boutons in dorsal horn was similar to that in cerebellar parallel fiber terminals, but significantly lower than that in mossy fiber terminals. These results suggest that glutamate is a transmitter used by neurotensin-immunoreactive axons in the dorsal horn, and since these axons are thought to be largely or entirely derived from neurotensin-containing neurons in laminae I-III, they provide immunocytochemical evidence for a population of excitatory glutamatergic neurons in this region.

Demonstration of glutamate-like immunoreactivity at rat neuromuscular junctions by quantitative electron microscopic immunocytochemistry

Motor nerve terminals and adjacent structures in the extensor digitorum longus and soleus muscles of young adult rats were examined for their content of glutamate by means of quantitative, electron microscopic immunocytochemistry employing colloidal gold particles as markers. The level of glutamate immunoreactivity was stronger in the extensor digitorum longus terminals than in the soleus terminals. In both muscles the glutamate immunolabelling was stronger in the nerve terminals than in the synaptic clefts and the postsynaptic tissue separating the secondary clefts, but the differences were larger in the extensor digitorum longus than in the soleus muscle. The myofibrils of the soleus muscle were more densely labelled than those in the extensor digitorum longus muscle. The level of immunoreactivity was high in the Schwann cells of both muscles. By comparing the labelling intensity of motor nerve terminals with that of muscle fibres and hippocampal mossy fibres (compartments that have been analysed previously with respect to their glutamate content), the mean concentration of fixed glutamate in the extensor digitorum terminals was estimated to be in the range of 10-20 mmol/l. An association of glutamate immunoreactivity with synaptic vesicles was demonstrated in the most strongly labelled terminals. Whether these epitopes were localized in the interior of the vesicles or at their external surface could not be resolved with the present technique. These data indicate that motor nerve terminals contain glutamate, and that the enrichment of this amino acid is more pronounced in the terminals of the extensor digitorum longus muscle (a fast muscle) than in those of the soleus muscle (a slow muscle). A possible modulatory or trophic role of glutamate in the mammalian neuromuscular junction should be considered.