Demonstration of a releasable pool of glutamate in cerebellar mossy and parallel fibre terminals by means of light and electron microscopic immunocytochemistry.

The chemical substance(s) responsible for the fast signalling in the mossy fibre to granule cell synapses in the cerebellum has not been identified, although recent studies suggest that glutamate is a strong candidate. In the present investigation, this issue was explored by means of a quantitative electron microscopic immunocytochemical procedure. Ultrathin sections of plastic-embedded rat cerebella were treated with an antiserum specific for glutaraldehyde-fixed glutamate, followed by a secondary antibody coupled to colloidal gold particles. The gold particle density over mossy fibre terminals was assessed in tissue that had been rapidly fixed by perfusion, as well as in tissue that had been incubated in artificial cerebrospinal fluid in vitro before immersion fixation. In both preparations the mossy fibres appeared as the most intensely glutamate-immunoreactive profile type in the cerebellar cortex, and the parallel fibre terminals were also strongly labelled. Corresponding results were obtained at the light microscopic level. Most of the immunoreactivity in the mossy and parallel fibre terminals could be depleted in a Ca(+)-dependent manner by depolarization with a high K+ concentration. These data suggest that the mossy and parallel fibre terminals contain a glutamate pool that behaves as a transmitter pool.