Title: Voltage imaging for revealing neuronal dynamics across scales

Abstract: Neurons transmit information by responding to and generating a rich repertoire of transmembrane voltage dynamics. These signals vary in amplitude across two orders of magnitude, ranging from 1 mV subthreshold events in an excitatory postsynaptic potential (EPSP) to 100 mV spikes in an action potential (AP). An efficient method to record neuronal voltage dynamics at millivolt resolution and with high throughput is therefore critical to understanding signal integration, measuring synaptic strength, and dissecting synaptic dysfunction in disease. In this context, voltage imaging has demonstrated great potential, as recent advancements in genetically encoded voltage indicators (GEVIs) have enabled single-trial imaging of spikes in the mouse brain. However, the limited signal-to-noise ratio (SNRs) of the extant indicators makes these experiments extremely challenging, and measuring unitary EPSPs in vivo has remained impossible. 
 
To address these challenges, I first developed ASAP5 as a GEVI with enhanced activation kinetics and responsivity near resting membrane potentials for improved detection of both spiking and subthreshold activity. ASAP5 reported action potentials (APs) in vivo with higher signal-to-noise ratios than previous GEVIs, and successfully detected graded and subthreshold responses to sensory stimuli in single 2-photon recording trials. In cultured rat or human neurons, somatic ASAP5 reported synaptic events propagating centripetally, and could detect ~1-mV voltage transients. By imaging spontaneous EPSPs throughout dendrites, we find that EPSP amplitudes decay exponentially during propagation, and that amplitude at the initiation site generally increases with distance from the soma. Inspired by molecular modeling, I next improved the responsivity of ASAP5, yielding ultrasensitive voltage indicators for subthreshold activity (ASAP5.7) and action potentials (ASAP5.8). ASAP5.7 showed 2.5-fold improvement over ASAP5 in detecting subthreshold activity, and achieved reliable EPSP recording in vivo. ASAP5.8 showed 4-fold improvement over ASAP5 in detecting action potentials, potentially allowing simultaneous recordings from hundreds of neurons in vivo in deep brain tissue. 
 
The superior sensitivity of ASAP5.7 enables multi-site recordings of EPSPs within single cells, in vivo. Leveraging this capability, I recorded ASAP5.7 fluorescence along dendritic trees in specific cell types in Drosophila to probe how neuronal morphology influences signal transmission and integration. These measurements revealed that some neurons are electrically compact, as voltage signals exhibited minimal signal decay and little delay as they flowed over the cell. However, other neurons were not electrically compact, as voltage changes decayed substantially, and were delayed, as they flowed over the surface of the cell. These findings align with finite-element modeling of voltage dynamics based on realistic neuron morphologies and provide direct evidence of voltage compartmentalization. Interestingly, one cell type displayed a high degree of compartmentalization and showed branch-specific visual tuning. 
 
Taken together, these results extend the applications of voltage imaging to the quantal response domain, paving the way for revealing principles of dendritic computation, quantifying synaptic plasticity, and high-throughput characterization of neuronal dysfunction in disease models.

Please contact Madelyn Bernstein for the Zoom link