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 carboxyterminal 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 I neurons described in previous Golgi studies. The findings of this investigation, as well as previous studies using the same Glu-1 antibody, suggest that the immunoreactive substances recognized by this antibody in aldehyde-fixed brain are alpha- and beta-tubulin with a carboxy-terminal glutamate, although the possibility exists that some of the staining represents the distribution of a form of fixative-modified glutamate. This tubulin-like immunoreactivity is co-localized with glutamate-like immunoreactivity and is associated with putative excitatory neurons in the hippocampus and amygdala.