An electron microscopic, immunogold analysis of glutamate and glutamine in terminals of rat spinocerebellar fibers

Abstract

A semiquantitative, electron microscopic immunocytochemical procedure based on the use of colloidal gold particles as markers was employed to analyze the subcellular distribution of glutamate and glutamine, a major glutamate precursor, in a subpopulation of spinocerebellar mossy fiber terminals. These terminals were identified by anterograde transport of a horseradish peroxidase-wheat germ agglutinin conjugate, injected in the thoracic spinal cord. Gold particles signalling glutamate-like immunoreactivity were enriched over clusters of synaptic vesicles relative to organelle-free cytoplasmic matrix, and there was a strong positive correlation between gold particle and synaptic vesicle densities (correlation coefficient 0.94). Gold particles indicating glutamine-like immunoreactivity showed a much weaker correlation with vesicle density (correlation coefficient 0.36) and were about equally concentrated over cytoplasmic matrix as over clusters of synaptic vesicles. Compared with the mossy fibers, the putative GABAergic Golgi cell terminals exhibited a lower level of glutamate-like immunoreactivity, which was very weakly correlated with the vesicle density (correlation coefficient 0.27). The level of glutamine-like immunoreactivity in the Golgi cell terminals was similar to that in mossy fibers, but much lower than that in glial cells. The anterogradely labelled mossy fiber terminals were not enriched in immunoreactivities for aspartate or GABA. These results suggest that the level and subcellular distribution of glutamate in presumed glutamatergic terminals differs from that in terminals in which glutamate only serves metabolic or precursor roles, and that these differences can be exploited in immunocytochemical studies aimed at identifying glutamate-using neurons. In contrast, glutamine immunocytochemistry does not seem to be generally useful in this regard.

Pre-embedding Staining for GAD67 Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

“…In this study, the presence of peroxidase reaction product in GAD 67 -labeled nerve terminals did not appear to cause any significant quenching of immunogold labeling for GABA, as has been reported in other studies (e.g., Ji et al 1991) because there was statistically higher GABA immunogold labeling over peroxidase-containing (i.e., GAD 67 -positive) nerve terminals. Moreover, our quantitative results support the general observation of Maxwell et al (1989) that GAD-positive nerve terminals were more heavily labeled for GABA than GAD-negative terminals.…”

Cervicothalamic tract terminals are enriched in glutamate-like immunoreactivity: an electron microscopic double-labeling study in the cat

“…One of the routes through which transmitter Glu may be formed is through deamidation of Gln (Fonnum, 1984;McGeer and McGeer, 1989), and it appears reasonable that high Glu:Gln ratios should be present in terminals that release and have a high turnover rate of Glu. Indeed, in recent studies on the cerebellar cortex (Ottersen et al, 1991;Zhang et al, 199 l), high Glu:Gln ratios were found in parallel and mossy fiber terminals whereas in comparison to these terminals the ratios in other tissue compartments were low or moderate. Concerning Gln-LI in the VPL, it would be helpful to have more complete immunogold data on types of structures other than those given in the present report before further conclusions are made.…”

“…The explanation for different concentrations of Glu in glutamatergic terminals is not clear. In Glu immunogold-labeled preparations, a relationship between the density of gold particles and the packing density of synaptic vesicles has been observed in individual terminals or among terminals of the same type (Montero and Wenthold, 1989;Ottersen et al, 1990b;Ji et al, 1991). The higher packing density of vesicles in RS terminals as compared to CTT and RL terminals (Blomqvist et al, 1985b) could thus explain the higher content of Glu-LI in RS terminals.…”

“…The shape of the curve is probably sensitive to even small differences in incubation parameters inasmuch as it has shown some variation among different studies (cf. Bramham et al, 1990;Ottersen et al, 1991).…”

Synaptic Vesicular Localization and Exocytosis ofl-Aspartate in Excitatory Nerve Terminals: A Quantitative Immunogold Analysis in Rat Hippocampus

“…When the rats went into a coma, they were given an injection of pentobarbital (100 mg / kg, i.p.) and fixed by perf usion through the heart with glutaraldehyde and formaldehyde (see above) (Ji et al, 1991). Blood samples for glucose analysis were taken immediately before the fixation.…”