Spatial mismatch between the Na+ flux and spike initiation in axon initial segment

Abstract

It is widely believed that, in cortical pyramidal cells, action potentials (APs) initiate in the distal portion of axon initial segment (AIS) because that is where Na(+) channel density is highest. To investigate the relationship between the density of Na(+) channels and the spatiotemporal pattern of AP initiation, we simultaneously recorded Na(+) flux and action currents along the proximal axonal length. We found that functional Na(+) channel density is approximately four times lower in the AP trigger zone than in the middle of the AIS, where it is highest. Computational analysis of AP initiation revealed a paradoxical mismatch between the AP threshold and Na(+) channel density, which could be explained by the lopsided capacitive load imposed on the proximal end of the AIS by the somatodendritic compartment. Favorable conditions for AP initiation are therefore achieved in the distal AIS portion, close to the edge of myelin, where the current source-load ratio is highest. Our findings suggest that cable properties play a central role in determining where the AP starts, such that small plastic changes in the local AIS Na(+) channel density could have a large influence on neuronal excitability as a whole.

Fig. 1.

Distribution of AP evoked Na⁺ flux along the soma–axon axis. (A, Top) Representative neuron as seen during the imaging experiment with the NeuroCCD-SMQ camera. The rectangle and arrows indicate the regions from where fluorescence measurements were obtained. Red line indicates the axonal region where fast-increasing fluorescent signals were detected. (Middle) Averaged ΔF transients (n = 60) elicited by a single AP in the indicated region in the soma and in the 5-µm-long axonal segments. (Bottom) Total SBFI fluorescence measured along the soma–axon axis. The half-difference of the somatic and axonal fluorescence (dashed lines) was assigned as zero point for the axonal length measurements. (B) AP evoked Na⁺ flux in the soma and axon of a layer 5 pyramidal neuron. Dots are averaged peak ΔF values from 1-µm-long pixels in the soma and along the axon. Dashed lines are linear fits to the data; continuous lines designate the slope of Na⁺ influx as a function of distance in the soma (black) and in the incrementing, steady, and decreasing flux subsegments of the AIS (red; S₁, S₂, and S₃, respectively). (C) In a model with Na⁺ channel distribution based on measured Na⁺ fluxes, AP initiates in the distal AIS (S₃), outside of the maximal Na⁺ influx subsegment, S₂. Voltage–distance plots along the soma–axon axis shown at time intervals of 50 µs. Green arrows indicate positions of maximum voltage. Relative S₁, S₂, and S₃ position are color-coded as blue, red, and green, respectively.

Fig. 2.

Spatial mismatch between the Na⁺ flux and AP initiation. (A) Averaged action currents (n = 100–200) and Na⁺ transients (n = 25) elicited by a single AP at the axonal regions indicated by arrows. The regions are color coded as follows: red, 17 μm from the edge of the soma, as determined from IR differential interference contrast image; yellow, 30 μm; pink, 41 μm; green, 44 μm; blue, 46 μm; and brown, 58 μm. Physiologically, the distal AIS boundary was identified as the location where rapid AP-evoked Na⁺ elevation in fluorescence recording became undetectable (43 μm). Note that, at distances beyond this location, in the presumed myelinated region of the axon, Na⁺ transient amplitudes were smaller and their peaks were progressively delayed. (B) Spatiotemporal pattern of AP initiation. (Upper) Normalized action currents recorded from the same locations as in A to demonstrate the difference in the delay of their onset. (Lower) Delay of AP initiation plotted against distance from the edge of the soma. Each dot corresponds to the mean delay to the onset of the somatic AP (n = 10–40) at a given location, measured at half maximal amplitude. Note that AP initiates in a region located at the distance of 36 to 41 μm from the soma and propagates in both directions with apparent conduction velocity of ∼0.4 m/s. (C) Peak fluorescence change: distance plot along the soma–axon axis. Relative S₂ and S₃ position are color-coded as red and green, respectively. Dots are averaged ΔF values from each 5-µm-long segment along the axon during the time interval 2 to 10 ms following the peak of the AP; dashed lines are linear fits to the data. Pink arrow indicates the amplitude of the relative AP-evoked Na⁺ flux at the distance of 38 μm from the soma, in the center of the trigger zone, which was ∼25% of the mean S₂ value.

Fig. 3.

Implications of depolarizing current source position within the AIS for AP generation. (A) Effect of varying Na⁺ channel density in the S₁, S₂, and S₃ subsegments on AP current threshold. In a model with Na⁺ channels present in only one of the three AIS subsegments, increasing gNa in S₃ is most effective in lowering the amount of current the somatic electrode must deliver to elicit the AP. (B) Distal Na⁺ channels are most effective in shifting AP initiation to the axon. Voltage-distance plots along the soma–axon axis shown for gNa of 750 pS/µm² in each of the respective subsegments, at a time when the voltage reaches −30 mV. Note that, even with a threefold S₁:soma gNa ratio, the AP initiates simultaneously in the soma and in the axon.

Fig. 4.

AP generation critically depends on electrical isolation of the trigger zone from the soma. (A) Effect of varying S₁ length on maximal rate of rise of the S₂ AP. In a model in which only S₂ contains Na⁺ channels whereas the rest of the neuronal membrane is passive, the amplitude and rate of rise of APs elicited in S₂ by just superthreshold somatic depolarizing current increased with S₁ length. (Left) Voltage (gray), dV/dt (black), and current traces for S₁ lengths of 5, 40, and 80 μm. (Right) Maximal rate of rise of the S₂ AP increased exponentially as a function of the S₁ length, reflecting a difference in isolation of S₂ from somatodendritic capacitive load. (B) Effect of varying S₁ length and resistance on AP initiation. (Left) In a model as in A, brief current steps in the soma with amplitude of less than 0.8 nA fail to elicit an AP when S₁ is short (2 µm). Current threshold decreases to less than 0.7 nA for S₁ length of 40 µm. A 20-fold increase in axoplasm resistivity in S₁ rescues AP initiation even in the models with the shortest S₁ (2 µm). (Right) Varying the S₁ length has a biphasic effect on current threshold. Keeping the S₁ axoplasmic resistance constant by adjusting the axoplasmic resistivity causes the AP current threshold to increase linearly with increase in S₁ length. The circles and the square indicate the S₁ lengths and thresholds for the traces on the left.