Two Mechanisms of Synaptic Vesicle Recycling in Rat Brain Nerve Terminals

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Abstract
KCl and 4‐aminopyridine (4‐AP) evoke glutamate release from rat brain cortical nerve terminals by voltage clamping or by Na+ channel‐generated repetitive action potentials, respectively. Stimulation by 4‐AP but not KCl is largely mediated by protein kinase C (PKC). To determine whether KCl and 4‐AP utilise the same mechanism to release glutamate, we correlated glutamate release with release of the hydrophobic synaptic vesicle (SV) marker FM2‐10. A strong correlation was observed for increasing concentrations of KCl 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 staurosporine, glutamate and FM2‐10 release also correlated. However, higher concentrations of 4‐AP or of 4‐AP plus PMA greatly increased glutamate release but did not further increase FM2‐10 release. This divergence suggests that 4‐AP recruits an additional mechanism of release during strong stimulation that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss‐and‐run, which is not detectable by styryl dyes. Our data suggest that glutamate release in nerve terminals occurs via two mechanisms: (1) complete SV fusion, which is PKC independent; and (2) a kiss‐and‐run‐like mechanism, which is PKC dependent. Recruitment of a second release mechanism may be a widespread means to facilitate neurotransmitter release in central neurons.