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A series of biochemical determinations was performed on five cellular fractions obtained from the cerebellum of 8- to 14-day-old mice. Cerebellar tissue was dissociated by mild trypsinization and mechanical agitation. The dissociated cellular material was separated into five fractions using a series of continuous density gradients formed with Percoll. Three of the fractions were comprised primarily of cell bodies. One of these was dominated by cells having the size and morphological appearance of granule cells, and based on several criteria the other two were were enriched in astrocyte cell bodies. Morphological analysis indicated that the remaining two fractions were enriched, respectively, in nerve terminals and large nucleated cell bodies. The uptake of 12 amino acids and 4 other metabolites by these cellular fractions was examined, and Km and Vmax values were determined for 10 of the compounds studied. High affinity transport carriers (Km approximately 1 to 20 microM) for most of the compounds studied were evident in neuronal and astrocyte-enriched fractions; however, for glutamate and gamma-aminobutyric acid (GABA) additional carriers with higher substrate affinities (Km approximately 0.1 to 0.3 microM) were evident in the astrocyte-enriched fraction. The fraction enriched in granule cell bodies was distinguished by an exceptionally low uptake of GABA and citrate, and a comparatively low uptake of beta-alanine, taurine, alpha-ketoglutarate, and glutamate. An analysis of the content of nine amino acids in the five fractions revealed that only glutamate, aspartate, and GABA were concentrated in the fraction enriched in nerve terminals. GABA was concentrated also in the fraction enriched in large cell bodies and was present at a low concentration in the fraction enriched in granule cell bodies. The other amino acids measured were distributed nearly evenly among the five fractions. Several differences in metabolic activity among the five fractions were observed. Radiolabel from several precursors was incorporated into GABA preferentially in the fractions enriched in large cell bodies and nerve terminals. In contrast, the accumulation of label in glutamine occurred preferentially in the fractions enriched in astrocytes and granule cell bodies. Labeling of alanine from [14C]pyruvate and of proline from [14C]ornithine was most prominent in the fractions enriched in astrocytes and granule cell bodies.

When rabbits are injected with tissue homogenates of white matter from bovine corpus callosum, an antiserum is produced which reacts with the surface membrane of 20-30% of all cells obtained by trypsin-dissociation of cerebellum from 10-day-old mice. The antigen or set of antigens recognized by this antiserum is detectable on embryonic, early postnatal, and adult mouse brain, but not on liver, spleen, kidney, thymus and sperm. The antigen is expressed in different regions of the brain and also, in decreased amounts, on retina. In histological sections of cerebellum from 21-day-old mice the antigen is predominantly localized in white matter tracts. Whereas nervous tissue from chicken and rabbit does not carry detectable levels of the antigen, rat, bovine and human brains are antigen-positive.

Glial cell-enriched and -depleted populations isolated from 10-day-old mouse cerebella have been grown in vitro. There are marked differences in the cellular morphology between these two populations. The glial cell-enriched populations are very heterogeneous with respect to cell size, morphology and processes, whereas the glial cell-depleted populations are very homogeneous, containing a cell type with a small cell body and predominantly bipolar processes. Further characterization of the cell types has been affected using antiserum prepared against GFA protein and tubulin. The immunocytochemical localization of these proteins clearly identifies astrocytes and neurons. The glial cell enriched populations contain several types of astrocytes and neurons in addition to cells of non-ectodermal origin, whereas the glial cell-depleted populations contain predominantly a single neuronal cell type, the granule cells.

Preparative amounts of populations enriched and depleted in glial cells have been isolated from 10-day-old mouse cerebella. Discontinuous density bovine serum albumin (BSA) gradients provide two distinct populations: the one derived from the 10-15% BSA interface is enriched in cyclic nucleotide phosphohydrolase (CNPase) activity, and a percentage of S-100, GFA protein, and NS-1 antigen-positive cells; the other, located as the 15-31% interface, contains a cell population depleted in these compounds. This report also describes the first use of flow microfluorimetry and electronic cell sorting techniques for the analysis and isolation of cell populations derived from the mammalian central nervous system. Glial cell-enriched and -depleted populations obtained by BSA density gradient centrifugation were analyzed for their forward angle light scattering properties and their capacity to bind anti-corpus anti-serum to the cell surface. The glial cell-enriched fraction shows a different frequency distribution of light scattering than the glial-depleted fraction. Anti-corpus callosum antiserum binds preferentially to the glial cell-rich fraction. Cells can be sorted into corpus callosum antigen-positive and -negative cell fractions and recovered with a viability of more than 95% as judged by trypan blue exclusion. Anti-corpus callosum-positive sorted cells are enriched in CNPase activity, S-100, and glial fibrillary acidic proteins.

In order to provide evidence relevant to the hypothesis that nonsynaptically derived alpha-ketoglutarate serves as a metabolic precursor of the neurotransmitter pools of glutamate and GABA the uptake and metabolism of alpha-ketoglutarate by nerve terminal enriched material was studied and compared to corresponding data for glutamine. Both alpha-ketoglutarate and glutamine were transported across the cell membrane by high affinity and low affinity carriers. Under conditions prevailing in vivo alpha-ketoglutarate probably is transported primarily by its high affinity carrier, whereas gluatmine should be transported primarily by one or more low affinity carriers. Based upon reciprocal uptake inhibition experiments glutamine appeared to be transported by the alanine preferring system, and to a lesser extent by the basic amino acid and large neutral amino acid carriers. A comparison of the rate of uptake by different cellular preparations enriched in either nerve terminals or cell bodies indicated that alpha-ketoglutarate is transported selectively by nerve terminals. Both substrates were rapidly converted to glutamate; however, glutamine was more readily metabolized to GABA. The results of our study are consistent with the concept that both glutamine and alpha-ketoglutarate derived from extra-neuronal sources are taken up by nerve terminals and utilized to replenish the neurotransmitter pools of glutamate and GABA.