Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation

Abstract

Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, K(Ca)3.1) by local, activity-dependent calcium (Ca(2+)) influx at nodes of Ranvier via a T-type voltage-gated Ca(2+) current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells.

Graphical abstract

Figure 1

Highly Reliable Spike Propagation along Purkinje Cell Axons Is Secured by Node of Ranvier IK Channels (A) Schematic of experimental configuration. (B) Simultaneous whole-cell somatic and cell-attached axonal recordings of a spontaneously firing Purkinje cell in response to a somatic current ramp injection (2 nA). ^∗Differentially propagated spike. (C) Maximal firing frequencies at soma (292 ± 19 Hz, n = 19), main axon (257 ± 17 Hz, n = 9, includes data from Monsivais et al., 2005), and axon collateral (253 ± 14 Hz, n = 19). (D) Local drug application (green bars) to branchpoint during whole-cell somatic recording (black) and cell-attached axonal recordings (blue) downstream of the targeted branchpoint. Baseline firing rate is indicated above somatic traces. (E) Averaged whole-cell somatic and cell-attached spikes before (blue), during (green), and after (gray) drug application. (F) Spike amplitude before (blue, a1) and during (green, a2) drug application. (G) Data summary of axonal spike suppression. p < 0.0001 for TRAM 1 μM, 0 Ca²⁺, Ni²⁺, p < 0.005 TRAM 500 nM, p < 0.002 TTX. TEA, HBS not significantly different from baseline (Student’s t test). Error bars, ±SEM.

Figure 2

Expression of K_Ca3.1 in Purkinje Cell Axons (A) Immunolabeling of K_Ca3.1 in cerebellar vermis (overview, left), PC soma, and dendrites (middle) and the center of the granule cell layer, which typically corresponds to the anatomical location where Purkinje cell axons have their first axonal branchpoint (70–100 μm) (right, arrowhead: potential axonal branchpoint). (B) Co-immunolabeling of calbindin and K_Ca3.1 at PC somata and dendrites. Left, calbindin; Middle, K_Ca3.1; Right, merge. Maximum intensity projection. (C) K_Ca3.1 staining along calbindin-positive axons in the granule cell layer. Maximum intensity projection. (D) Calbindin-positive axonal branchpoint in the granule cell layer. Single optical plane, image deconvolved.

Figure 3

Local, Activity-Dependent Ca²⁺ Influx at Nodes of Ranvier (A) Two-photon image of cerebellar Purkinje cell indicating line scan locations. (B) Ca²⁺ transients (top, line scans) at locations shown in (A) during current-evoked spike trains (bottom). AIS, axon initial segment. BP1 and BP2, first and second axonal branchpoint. (C) Frame-scan time series of BP1 during spike train. Green, axon morphology. (D) Spike train-evoked ΔF/F at ROIs shown in (C) (red boxes). Normalized integrated ΔF/F (ΔF/F^∗s) against distance from an axonal branchpoint (bottom, n = 13 neurons). (E) Soma, AIS, and first BP ΔF/F in response to 500-ms somatic voltage steps (voltage clamp) in bath-applied TTX (0.5 μM). (F) Pooled data for max ΔF/F versus somatic command potential (soma, black; AIS, red; BP1, blue. n = 6 neurons). (G) Activity-dependent changes in ΔF/F upon somatic current injection. Baseline holding current: 0 pA. (H) Summary data for the change in ΔF/F^∗s during silence and activity (n = 5 cells). (I) Ca²⁺ influx at the axon initial segment, first branchpoint and presynaptic boutons before and after bath application of 0 mM extracellular Ca²⁺ (n = 4), Agatoxin (AgaTX, n = 4, AIS = 3), and Mibefradil (Mibef, n = 9). Error bars, ±SEM.

Figure 4

K_Ca3.1 Sets Node of Ranvier Membrane Potential and Preserves Nodal Excitability (A) Data obtained from a PC multicompartmental model. NoR (red, sixth node, 1,820 μm from soma) and somatic (gray) APs (Vm, top row) during spontaneous firing in a Purkinje cell model with and without NoR K_Ca3.1 channels. Left column: AP propagation is sustained by nodal gK_Ca3.1 (0.05 S/cm²) and juxtaparanodal (JP) delayed rectifier K⁺ channels (0.002 S/cm²). Right column: lack of axonal K_Ca3.1 causes depolarization block at the NoR. Second row: Ca²⁺ current at the NoR. Third row: total K⁺ current at the JP and NoR, respectively, for each model. Fourth row: fractional resurgent Na_v states during spontaneous firing of each respective model. Bottom row: local Ca²⁺ concentration. (B) Activity-dependent axonal Ca²⁺ diffusion in the model axon. (C) Somatic recording from a spontaneously firing Purkinje cell during local application of 1 μM TRAM-34 to the AIS. TRAM-34 causes a depolarizing shift in Vm and a reduction of firing rate. (D) Example APs (left) before (black) and during TRAM-34 (blue) application illustrate TRAM-34-induced change in Vm. Average second derivative of somatic APs (middle). Arrowhead indicates first peak originating in the axon. Summary data show a TRAM-induced reduction in the amplitude of the first axonal peak of the second derivative of the somatic action potential (right). Horizontal bars indicate average ±SEM (n = 15), p < 0.0005.