Immunocytochemical methods for peptides and serotonin have greatly advanced the study of neurones in which these substances are likely to be transmitters. Such direct techniques have not so far been available for the amino acid transmitter candidates. We report here the selective immunocytochemical visualization of the putative transmitters glutamate (Glu) and gamma-aminobutyrate (GABA) by the use of antibodies raised against the amino acids coupled to bovine serum albumin (BSA) with glutaraldehyde (GA). The tissue localizations of Glu-like and GABA-like immunoreactivities (Glu-LI and GABA-LI) matched those of specific uptake sites for Glu and GABA, and, in the case of GABA-LI, also that of the specific marker enzyme glutamic acid decarboxylase (GAD). Thus, GABA-LI was located in what are believed to be GABAergic inhibitory neurones, whereas Glu-LI was concentrated in excitatory, possibly glutamatergic neurones. Preliminary electron microscopic observations suggest that the transmitter amino acids are significantly concentrated in synaptic vesicles.

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The immunogold labeling for glutamate and glutamine was studied at the electron microscopic level in hippocampal slice cultures following inhibition of L-glutamine synthetase [L-glutamate :ammonia ligase (ADP-forming) ; EC 6 .3 .1 .2] . In control cultures, glutamate-like immunoreactivity was highest in terminals, intermediate in pyramidal cell bodies, and low in glial cells . Glutamine-like immunoreactivity was high in glial cells, intermediate in pyramidal cell bodies, and low in terminals . After inhibition of glutamine synthetase with L-methionine sulfoximine, glutamate-like immunoreactivity was reduced by 52% in terminals and increased nearly fourfold in glia . Glutamine-like immunoreactivity was reduced by 66% in glia following L-methionine sulfoximine, but changed little in other compartments . In cultures that were treated with both L-methionine sulfoximine and glutamine (1 .0 mM), glutamate-like immunoreactivity was maintained at control levels in terminals, whereas in glia glutamate-like immunoreactivity was increased and glutamine-like immunoreactivity was decreased to a similar extent as in cultures treated with L-methionine sulfoximine alone . We conclude that (a) glutamate accumulates in glia when the flux through glutamine synthetase is blocked, emphasizing the importance of this pathway for the handling of glutamate ; and (b) glutamine is necessary for the maintenance of a normal level of glutamate in terminals, and neither reuptake nor de novo synthesis through pathways other than the glutaminase reaction is sufficient . Abbreviations used : GLN-LT, glutamine-like immunoreactivity ;Glu-LI, glutamate-like immunoreactivity; MSO, i .-methionine sulfoximine.

The laminar staining of the rat hippocampal region with the Timm sulphide silver method is from studies on adult rats known to depend on the various fibersystems terminating in these laminae. In order to illustrate the development of these fibersystems the laminar differentiation of the Timm staining of fascia dentata, hippocampus and subiculum is presented for rats between 1 and 31 days old. Corresponding changes in cytoarchitectonics as revealed by thionin staining are briefly demonstrated. Even on the first postnatal day there are indications of the mature, laminar staining pattern, and between three and nine days all the laminae corresponding to the terminal fields of the major afferent and intrinsic systems appear. After 12 days only minor additions to the laminar pattern develop, but there are adjustments of absolute and relative dimensions of layers and fields, and also the staining densities of individual laminae change. These observations are in good correlation with the available information on both hippocampal neurogenesis and cytodifferentiation, and the few fiber tracing studies performed on the developing hippocampal region. Compared to the latter, which ideally marks only one system or one lamina per animal, the Timm method provides an excellent means for getting an overview of the general developmental situation at the different ages. Thus developmental gradients along septotemporal, medio-lateral and basal-apical axes are found; which should be heeded in future studies on hippocampal synaptogenesis. The observations are discussed in relation to current models for neuronal growth and formation of nervous connections.

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