Metabolic compartmentation of glutamate and glutamine: Morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum

An electron microscopic, double-labelling immunocytochemical procedure was used to assess the level of fixed glutamate and glutamine in different cell profiles in ultrathin sections of rat cerebellar cortex. The procedure was based on sequential immunolabelling with two rabbit antisera, using gold particles of different sizes as markers and formaldehyde vapour as a means to avoid interference between the two incubations. Model sections containing a series of known concentrations of the respective amino acids (aldehyde--fixed to rat brain protein) were incubated together with the tissue material. These revealed a close to linear relationship between gold particle density and antigen concentration throughout the range of biological relevance. The ratio between the density of the two categories of gold particles was calculated for the individual profile types. This ratio showed a 20-fold variation, with the highest glutamate/glutamine ratios obtained for putative excitatory terminals (terminals of parallel fibres in the outer part of the molecular layer, followed by mossy and climbing fibre boutons) and the lowest for glial cells (Bergmann glia, astrocytes in the granule cell layer, and oligodendrocytes). Granule cell bodies and dendrites, and cell bodies and processes of putative GABAergic cells (Purkinje, basket and Golgi cells) displayed intermediate ratios. The ratios corresponded to millimolar ratios (mM fixed glutamate/mM fixed glutamine) ranging from 4.5 to 0.2, tentatively assessed by adjusting for differences in labelling efficiency of the two antigens. Our results show that the compartmentation of glutamate and glutamine, an issue previously addressed mainly in the test tube, can be studied in morphologically intact preparations at a resolution that matches the complexity of CNS tissue. The data indicate that glutamate is effectively converted to glutamine in all categories of glial cells, and that glutamate synthesis prevails in each of the three types of excitatory terminals in the cerebellar cortex. Terminals of putative GABAergic cells form a distinct low glutamate/low glutamine compartment.