Optical analysis of nanoscale physiology at presynaptic terminals

Abstract

Synaptic transmission forms the basis of neural communication, yet much of its fundamental mechanisms remains to be elucidated. In particular, evoked neurotransmitter release (which occurs in response to an action potential) and spontaneous release (which occurs independent of action potentials) have been found to be functionally segregated, yet their spatial segregation remains incompletely understood. We first validate that iGluSnFR, a glutamate sensing fluorescent probe, and GCaMP8s-Syb2, which can detect presynaptic calcium (Ca2+) signals, has single synapse level resolution. We utilize photobleaching as a use-dependent inhibitor of fluorescence detection, and we find that it has a differential effect on evoked and spontaneous glutamate release as measured by iGluSnFR. This supports the hypothesis that evoked release occurs in diffusion-restricted “nanocolumns”. On the other hand, the resistance of spontaneous release to being photobleached suggests that it occurs over a more diffuse and dispersed area across the postsynaptic organization.

We apply this approach to presynaptic Ca2+ signals, and we find we can detect three distinct Ca2+ signals: evoked presynaptic Ca2+ transients (ePreCTs), spontaneous presynaptic Ca2+ transients (sPreCTs), and baseline Ca2+ signals. We find that all three signals derive from separate Ca2+ sources, occur in spatially distinct domains within the presynaptic terminal, and enact unique effects on different modes of neurotransmission. These results open a novel approach to examining nanoscale level physiology, and ultimately relate spatially non-overlapping signals to their functional consequences.

Finally, we use the previously validated optical tools to measure the effects of brain derived neurotrophic factor (BDNF) at the single synapse level. We find that exogenous application of BDNF selectively increases evoked glutamate release without affecting spontaneous glutamate release. This differential effect is recapitulated by chronic deletion of the TrkB receptor, which is the high affinity receptor for BDNF, and this effect is mediated by scaling of presynaptic voltage-gated Ca2+ channels. Overall, work presented in this thesis contributes to overall understanding of synaptic physiology at the nano-scale level, from their spatial organization to interaction with neurotrophic factors.