Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals

KCl and 4-aminopyridine (4-AP) evoke glutamate release from rat brain corti cal nerve terminals by voltage clamping or by Na+ channel-generated repetit ive action potentials, respectively. Stimulation by 4-AP but not KCI is lar gely mediated by protein kinase C (PKC), To determine whether KCI and 4-AP utilise the same mechanism to release glutamate, we correlated glutamate re lease with release of the hydrophobic synaptic vesicle (SV) marker FM2-10. A strong correlation was observed for increasing concentrations of KCI and after application of phorbol 12-myristate 13-acetate (PMA) or staurosporine . The parallel increase in exocytosis measured by two approaches suggested it occurred by a PKC-independent mechanism involving complete fusion of SVs with the plasma membrane. At low concentrations of 4-AP, alone or with sta urosporine, glutamate and FM2-10 release also correlated. However, higher c oncentrations of 4-AP or of 4-AP plus PMA greatly increased glutamate relea se but did not further increase FM2-10 release. This divergence suggests th at 4-AP recruits an additional mechanism of release during strong stimulati on that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss-and-run, which is not detectabl e by styryl dyes. Our data suggest that glutamate release in nerve terminal s occurs via two mechanisms: (I) complete SV fusion, which is PKC independe nt; and (2) a kiss-and-run-like mechanism, which is PKC dependent. Recruitm ent of a second release mechanism may be a widespread means to facilitate n eurotransmitter release in central neurons.