Functional Microstructure of Ca_V-Mediated Calcium Signaling in the Axon Initial Segment

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (Ca_V). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of Ca_V2 and Ca_V3 channels that differentially localized to discrete regions. Ca_V3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas Ca_V2 localized to non-hotspot regions. Thus, different pools of Ca_Vs appear to play distinct roles in AIS function.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (Ca_V). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of Ca_Vs. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: Ca_V3 generates hotspot regions of calcium influx coupled to calcium stores, whereas Ca_V2 channels underlie diffuse calcium influx between hotspots. Therefore, different Ca_V classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes.