Functional microstructure of Ca_V-mediated calcium signaling in the axon initial segment

Abstract

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). 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 utilize conventional 2-photon laser scanning and diffraction-limited, high-speed spot 2-photon imaging to resolve action potential-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 CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-dependent stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.

Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

“…Within the AIS, we show that calcium influx occurs both before and after the peak of the AP in K1422Eexpressing neurons (Figure 5B-D). This contrasts with typical conditions where sodium and calcium influx are separated on the rising and falling phases of the AP, respectively (Filipis and Canepari, 2021;Lipkin et al, 2021, but see Hanemaaijer et al, 2020. This additional calcium influx could have myriad effects on AIS function, as many AIS components are regulated directly by calcium or through calcium/calmodulin interactions (Wen and Levitan, 2002;Kim et al, 2004;Swain et al, 2015;Clarkson et al, 2017).…”

“…Two-Photon Imaging. Imaging was performed as described previously (Lipkin et al, 2021). A Coherent Ultra II laser was tuned to 810 nm and epifluorescence and transfluorescence were collected through a 60x, 1.0 NA objective and a 1.4 NA oil immersion condenser, respectively (Olympus).…”

“…Data were then smoothed using a 40-point binomial filter in IgorPro for analysis. Calcium transients were normalized to saturating conditions (∆G/Gsat) as previously described (Lipkin et al, 2021). Transient onset was defined as the time at which signals exceeded root-mean-squared noise levels of the 20 ms period preceding AP onset.…”

“…To test whether calcium influx through NaV1.2 channels can be observed in intact neurons, we imaged AP-evoked calcium transients in the AIS with high spatiotemporal precision using 2-photon pointscan imaging. Under normal conditions, calcium influx occurs during AP repolarization in all regions of the axon, including the AIS, and can be separated temporally from sodium influx occurring during AP depolarization (Geiger and Jonas 2000;Ritzau-Jost et al 2014;Rowan, Tranquil, and Christie 2014;Filipis and Canepari 2021;Lipkin et al 2021; but see Hanemaaijer et al 2020). But in cells expressing K1422E channels, calcium influx may also occur during AP depolarization in regions enriched with NaV1.2 channels.…”

Analysis of the effect of the scorpion toxin AaH-II on action potential generation in the axon initial segment

“…When analysing Ca 2+ influx associated with the AP, we found a significant increase after local delivery of AaH-II, that was however substantially smaller with respect to the increase in Na + influx. It is known that a large component of Ca 2+ influx in the AIS of L5 pyramidal neurons is mediated by voltage-gated Ca 2+ channels [27], but it is also known that Na v 1.2 is permeable to Ca 2+ [25]. Thus, we hypothesised that the additional Ca 2+ in the presence of the toxin could be mediated by Na v 1.2.…”