Optogenetic control of phosphoinositide at mouse calyx of Held synapse
Final Report Abstract
PartI: Despite extensive efforts over the years in the presynaptic field trying to understand the molecular mechanisms that underlie synaptic transmission, our knowledge of how phospholipids regulate synaptic transmission remains very limited. The major obstacle is due to the lack of technique that allows spatiotemporal control of lipid composition at the active zone. The project aims to establish an optogenetic tool at calyx of Held synapse that allows readily control the phospholipid composition with light, and further study the consequence after phospholipid depletion. Using RNA virus sequence P2A that allows bi-/tri-cistronic expression, we found different gene orientation largely affected the overall protein expression. Secondly, we found that the best time window to target gene into calyx of Held synapse using in utero electroporation is embryonic stage (E10.5-11). Thirdly, a mixture protein expression in both pre- and post-synaptic neuron was observed when using focal electroporation to deliver gene into mature calyx of Held synapse (P16-18). Thus, we concluded using adeno-associated virus-based gene delivery might yield higher specificity and better protein expression level at presynaptic terminal. This project is still ongoing and the electrophysiological results are expected to provide more insights into the functional role of phosphoinositide in synaptic transmission and synaptic plasticity. PartII: P/Q-type calcium channel (Cav2.1) is the major channel that mediates Ca2+ influx during action potentials (APs) to trigger neurotransmitter release from presynaptic terminals. Repetitive activity induces its Ca2+-dependent facilitation (CDF) via binding of calmodulin superfamily proteins with the IQ-like motif (isoleucine (I) and methionine (M)) on the cytoplasmic c-terminus of Cav2.1. However, whether and how CDF contributes to short-term synaptic plasticity remains highly controversial. By recording from the calyx of Held terminal in IQ-like motif point mutation knock-in mice (Cav2.1 IM-AA KI), we found activity-dependent CDF is completely abolished, resulting in profound reductions in the extent of short-term facilitation and depression (STF and STD) in different Ca2+ concentrations. These results indicate that CDF of Ca2+ channel dictates the polarity and magnitude of presynaptic plasticity in central synapses.
