James E. Madl

Monoclonal antibody specific for carbodiimide-fixed glutamate: immunocytochemical localization in the rat CNS.

Glutamate is widely distributed in the central nervous system (CNS) and is present in greater amounts than any other putative neurotransmitter. To study its distribution in the CNS, a monoclonal antibody was raised against gamma-L-glutamyl-L-glutamic acid (gamma-Glu-Glu) conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehydeborohydride. By use of this antibody, indirect immunoperoxidase staining was observed in CNS tissue fixed with carbodiimide to form gamma-Glu-Glu from glutamate and post-fixed with glutaraldehyde or paraformaldehyde. In contrast, immunoreactivity was quite low in tissues fixed only with glutaraldehyde. Absorption controls indicated that the staining of carbodiimide-fixed tissue could be inhibited by micromolar concentrations of gamma-Glu-Glu but not by other small molecules. Using ELISA, the antibody reacted strongly with the gamma-Glu-Glu/KLH conjugate used to immunize the mouse, but not with other small molecules conjugated to KLH. The reactivity of the antibody with the gamma-Glu-Glu/KLH conjugate on ELISA was inhibited by free gamma-Glu-Glu in micromolar concentrations, but not by similar dipeptides or amino acids. Dense immunocytochemical staining was observed in cortical pyramidal cells, cerebellar granule cells, and the cochlear nuclei. Staining with this monoclonal antibody correlated well with other methods of localizing glutamate in the CNS.

Localization of glutamate, glutaminase, aspartate and aspartate aminotransferase in the rat midbrain periaqueductal gray

SummaryGlutamate and aspartate are putative excitatory neurotransmitters in the central nervous system. The present study utilized novel monoclonal antibodies against fixative-modified glutamate and aspartate and polyclonal antisera against the amino acid synthesizing enzymes, glutaminase and aspartate aminotransferase, to analyze the distribution of these amino acids in the rodent midbrain periaqueductal gray. Glutamate-, aspartate-, glutaminase- and aspartate aminotransferase-like immunoreactive neurons, fibers and processes are present throughout the rostrocaudal length of the periaqueductal gray. Glutamate- and glutaminase-like immunoreactive neurons displayed a similar homogeneous pattern of distribution, being localized predominantly to the lateral and dorsal subdivisions of the periaqueductal gray. Co-localization experiments suggest that glutamate and glutaminase are in fact co-contained within the same PAG neurons. Aspartate aminotransferase-like immunoreactive neurons were distributed in a pattern similar to glutamate and glutaminase with the exception that fewer cells were stained in the dorsocaudal and the rostral third of the PAG. Aspartate-like immunoreactive neurons were less numerous than glutamate-like immunoreactive cells and were located in the lateral aspect of the PAG. These results demonstrate a specific and distinct distribution of glutamate and aspartate immunoreactive neurons and support recent data suggesting that glutamate and aspartate serve as excitatory neurotransmitters in the PAG.

Co-localization of glutamate and tubulin in putative excitatory neurons of the hippocampus and amygdala: an immunohistochemical study using monoclonal antibodies.

Tubulin and glutamate were immunohistochemically localized in the hippocampus and amygdala of rats using monoclonal antibodies to gamma-Glu-Glu (Glu-1) and glutaraldehyde-fixed glutamate (Glu-2), respectively. Glu-2 was shown to be selectively immunoreactive for glutaraldehyde-fixed Glu using enzyme-linked immunoassays and inhibition enzyme-linked immunoassays. Glu-1 was immunoreactive with tubulin on immunoblots of brain homogenates. However, only tubulin with a glutamate carboxy-terminal appeared to be immunoreactive with Glu-1 since tubulin from Chinese hamster ovary cells was not immunoreactive on immunoblots unless the tubulin was first treated with carboxypeptidase. Intense immunocytochemical staining by Glu-1 of hippocampus and amygdala was co-localized in the same neurons as the immunocytochemical staining for glutaraldehyde-fixed Glu produced by Glu-2. The distribution of immunostaining in the brain by Glu-1 was very similar to the distribution of immunostaining produced by Glu-2. The major difference was that glutamate-like immunoreactivity, visualized by Glu-2 staining, was intense in the nuclei of neurons, while nuclei were unstained by Glu-1. The distribution of immunostaining by these monoclonal antibodies was very similar to that reported in previous studies using other antibodies to Glu. All granule cells in the area dentata of the hippocampus exhibited intense immunoreactivity with both antibodies. Immunoreactivity was also observed in the stratum lucidum of CA3, the zone of termination of mossy fiber axons of granule cells. The majority of pyramidal cells in CA1, and many pyramidal cells in CA3 of the hippocampus were immunoreactive. In addition, it appeared that all of the pyramidal cells in the subiculum exhibited immunoreactivity. Light, diffuse immunoreactivity was observed in the neuropil of the hippocampus and subiculum. Most perikarya in the amygdala were characterized by light to moderate Glu-1 immunoreactivity and moderate to intense Glu-2 immunoreactivity. Fairly intense Glu-1 and Glu-2 immunoreactivity was seen in some neurons of the lateral nucleus, basolateral nucleus, lateral subdivision of the central nucleus, and the periamygdaloid cortex. The morphology of immunostained neurons in the lateral and basolateral nuclei indicates that the majority of these cells correspond to the pyramidal class 1 neurons described in previous Golgi studies.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Immunocytochemical localization of glutamate-, glutaminase- and aspartate aminotransferase-like immunoreactivity in the rat deep cerebellar nuclei

Although the anatomy and the connectivity of the deep cerebellar nuclei have been well documented, little is known about the neurotransmitter systems mediating cerebellar efferent pathways. The present study utilizes immunohistochemical procedures in conjunction with a novel monoclonal antibody specific for carbodiimide-fixed glutamate and polyclonal antisera against glutaminase (GLNase) and aspartate aminotransferase (AATase) to examine the presence of putative excitatory amino acid transmitters in neurons of the deep cerebellar nuclei. Carbodiimide-fixed glutamate-like, GLNase-like and AATase-like immunoreactivities were observed in neurons of the lateral, posterior interpositus, anterior interpositus and medial deep cerebellar nuclei. More neurons were stained with AATase antiserum than with the GLNase antiserum or the monoclonal antibody. These results suggest glutamate, GLNase and AATase are present in neurons of the deep cerebellar nuclei and raise the possibility that glutamate may be an excitatory transmitter in these structures.

Immunocytochemical localization of glutamate dehydrogenase in mitochondria of the cerebellum: an ultrastructural study using a monoclonal antibody

Monoclonal antibodies to glutamate dehydrogenase (GDH) were produced and shown to have high degrees of specificity using immunoblots and ELISA. Immunocytochemical staining of electron microscopic preparations revealed selective intense staining of mitochondria in Bergmann glia, oligodendrocytes and astrocytes in the cerebellum of the rat. Differential intensity of staining among mitochondria within individual glial cells and between glial cells was observed and may provide an anatomical means of detecting differences in glutamate metabolism.