Effects of reduced vesicular filling on synaptic transmission in rat hippocampal neurones

1. The consequence of reduced uptake of neurotransmitters into synaptic ves icles on synaptic transmission was examined in rat hippocampal slices and c ulture using bafilomycin A1 (Baf), a potent and specific blocker of the vac uolar-type (V-type) ATPase, which eliminates the driving force for the upta ke of both glutamate and GABA into synaptic vesicles. 2. After incubation with Baf, both the amplitude and frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were reduced in the sl ice preparation. Similar effects were seen with glutamatergic miniature exc itatory postsynaptic currents (mEPSCs) and GABAergic mIPSCs from cultured n eurons. This result indicates that vesicular content is reduced by Baf. The dramatic reduction in the frequency of mPSCs could result either from the exocytosis of empty vesicles or from a mechanism which prevents the exocyto sis of depleted vesicles. 3. Vesicle cycling was directly examined using confocal imaging with FM 1-4 3. In the presence of Baf, vesicles could still be endocytosed and they wer e released at the same probability as from control untreated synapses. 4. Prolonged high-frequency electrical stimulation of synapses in culture f ailed to alter the amplitude of mEPSCs, suggesting that the filling of vesi cles is rapid compared to the rate of vesicle recycling during repetitive s ynaptic stimulation. 5. Profound release of glutamate with alpha-latrotoxin did cause a small, b ut reproducible, reduction in quantal size. 6. These results indicate that decreasing the amount of glutamate and GABA in synaptic vesicles reduces quantal size. Furthermore, the probability of vesicle exocytosis appears to be entirely independent of the state of filli ng of the vesicle. However, even during high-frequency action potential-evo ked release of glutamate, quantal size remained unchanged.