"The main goal of the project is to develop new procedures to investigate single synaptic sites of the mammalian brain. For this purpose, synaptically connected pairs of molecular layer interneurons will be studied in cerebellar slices. Presynaptic calcium transients will be elicited at individual presumptive presynaptic release sites using targeted photorelease of the calcium precursor DM-nitrophen. Using this new approach several questions will be addressed including the degree of postsynaptic receptor saturation, the size and nature of the readily releasable pool of synaptic vesicles, and kinetics of vesicle recycling.This approach will be first developed for the GABAergic synapses between interneurons but will be later extended to study the glutamatergic synapses between granule cells and interneurons.Next we shall develop methods to selectively activate presynaptic ionotropic receptors by localized photolysis of precursors of neurotransmitters, while recording either the resulting current or the resulting calcium signal. In this manner we shall investigate, at the single site level, the presence and functional properties of presynaptic GABAA and glutamate receptors.Finally we will investigate the functional significance of presynaptic receptor signaling in vivo by using calcium imaging with genetically targeted calcium indicators. Using appropriate stimulation protocols we will determine whether these receptors can be activated either via autocrine or paracrine release of neurotransmitters."