Distinct Kv channel subtypes contribute to differences in spike signaling properties in the axon initial segment and presynaptic boutons of cerebellar interneurons

Abstract

The discrete arrangement of voltage-gated K(+) (Kv) channels in axons may impart functional advantages in action potential (AP) signaling yet, in compact cell types, the organization of Kv channels is poorly understood. We find that in cerebellar stellate cell interneurons of mice, the composition and influence of Kv channels populating the axon is diverse and depends on location allowing axonal compartments to differentially control APs in a local manner. Kv1 channels determine AP repolarization at the spike initiation site but not at more distal sites, limiting the expression of use-dependent spike broadening to the most proximal axon region, likely a key attribute informing spiking phenotype. Local control of AP repolarization at presynaptic boutons depends on Kv3 channels keeping APs brief, thus limiting Ca(2+) influx and synaptic strength. These observations suggest that AP repolarization is tuned by the local influence of distinct Kv channel types, and this organization enhances the functional segregation of axonal compartments.

Figure 1.

Two-photon VSD imaging in SC interneurons. A, Raw traces show AP-evoked voltage transients recorded in an axon of an SC during point measurement of fluorescence. Following acquisition, traces were digitally filtered off-line and aligned to remove any jitter using the peak of APs recorded at the soma with electrophysiology. Trials were averaged and photobleaching subtracted using a trend line extrapolated from a slope fit during the baseline period indicated by the dashed line. Inset shows that responses were stable throughout the recording. B₁–B₃, On the upper left, APs recorded at the soma using electrophysiology prior to and after repatching with an internal solution containing VSD. In a second cell shown on the right, somatic APs recorded immediately after whole-cell break-in and following prolonged dialysis with an internal solution containing VSD. Below are summary graphs showing that somatic AP duration and amplitude as well as holding current were unchanged during VSD dialysis. R_s remained ±15% of initial value, measured by bridge balance, throughout the experiment. C, Two examples of AP-like voltage transients recorded using VSD imaging at the somata of voltage-clamped SCs. The voltage-clamp command waveform, simulating an AP, is shown at the top. D, The half-widths of the AP-like voltage transients were nearly identical to the command waveforms. Below, voltage transients are superimposed with the command waveforms. E, AP-like voltage transients recorded from the soma of a voltage-clamped SC. The half-width of the second command waveform (AP2; red) was prolonged by 1.23 compared with the first waveform (AP1; black). F, AP-like voltage transients from the example on the left including response SEM. G, Summary plots showing that small changes in command waveform duration are accurately reported using VSD recording; *p < 0.05 with paired t test. H, Linearity of the VSD optical response, measured at the soma, for AP-like voltage commands of varying width. All points were significantly different from one another; *p < 0.05 by one-way ANOVA. Unity is indicated by the dashed line. I, A 2P fluorescence image of a cerebellar SC filled with the volume indicator dye Alexa 594 (60 μm) via the whole-cell somatic pipette. In the magnified view, a fluorescently coated second pipette (BSA-Alexa 594) is used for loose-seal, cell-attached recording from an axonal bouton. J, Simultaneous current-clamp and cell-attached recordings of aligned and average APs from the soma and bouton of the SC shown on the left. Dashed lines indicate the peak-to-peak measurements used to assess AP duration. K, Simultaneous current-clamp and cell-attached recordings from SC soma. Arrows indicate the half-duration for APs measured with each recording configuration. L, Summary data of AP half-widths from simultaneous current-clamp and cell-attached recordings. At boutons, similar AP half-width values were obtained with electrical recording and VSD imaging.

Figure 2.

AP repolarization is determined by distinct K_v channel subtypes in axonal subregions. A, A 2P fluorescence image of an SC filled with the green volume indicator dye Alexa 488 (20 μm). An axonal bouton and the AIS, targeted for voltage imaging by inclusion of the red VSD di-2-AN(F)EPPTEA (30 μm), are shown in the magnified views. B, Immunohistochemical labeling for AnkG (red) in the molecular layer of the cerebellum. An axon protruding from an SC, filled with LY (green) during whole-cell recording, is labeled for AnkG (indicated by white arrows). C, Comparison of AP latency for spikes simultaneously recorded in the soma with electrophysiology (black) and at the AIS (15 μm) with 2P VSD imaging (red). APs were elicited by somatic current injection. These traces are the average of multiple trials and have been normalized to the peak of the AP to facilitate comparison. D, Group data (n = 61 cells) showing the onset latency of the axonal AP relative to the somatic spike (0 μm). On the y-axis are latency differences from simultaneous electrophysiological and 2P VSD measurements of somatic APs. The red bar indicates the average position of the AIS, determined using AnkG immunolabeling. E, For the SC shown on the left, APs recorded at the soma with electrophysiology and at the AIS and a bouton using 2P VSD imaging. Each trace is the average of multiple trials. F, Superimposed APs, recorded at the AIS in control (black) and following bath application of either DTX (100 nm) or BDS-I (1 μm). The peak amplitudes of the averaged APs are normalized to facilitate comparison. G, APs recorded from axonal boutons in control (black) and following application of either DTX or BDS-I. H, AP repolarization at the AIS was prolonged by DTX but not by BDS-I. Data are mean ± SEM; *p < 0.05 by one-way ANOVA. I, Pharmacological profile of AP repolarization at presynaptic boutons. Data are mean ± SEM; *p < 0.05 by one-way ANOVA.

Figure 3.

Local determination of AP repolarization in axons. A, APs recorded at a bouton in a background of 500 μm TEA (black) and following application of 100 nm DTX. Traces are the average of many trials and are shown normalized to the peak of the AP to facilitate comparison. B, Summary of DTX-induced AP widening in TEA at boutons. Data are mean ± SEM; *p < 0.05 by unpaired t test. C, In a background of TEA, lack of dependence of DTX-induced AP widening at a bouton with distance from the axon hillock. Fit of the linear regression is indicated by the dashed line. D, Diagram depicting the experimental configuration. AP recording sites included both the AIS and an axonal bouton located in the same field of view to eliminate need for objective refocusing. Local photolysis of RuBi-4AP (bath applied at 150 μm) was limited to the AIS. Uncaging pulse trains (highlighted in red) were repeated five times at 0.33 Hz before each imaging trial. An imaging trial (highlighted in blue) consisted of five APs stimulated at 0.33 Hz. Measurements of AP waveform were made iteratively between these two sites in control and following RuBi-4AP uncaging. E, APs recorded at the AIS in control (black) and immediately following local photolysis of RuBi-4AP at the AIS. In the same cell, APs were also recorded at a bouton following RuBi-4AP photolysis at the AIS. F, Effect of AIS-directed RuBi-4AP photolysis on AP duration for spikes recorded at the AIS. In addition, control experiments show that laser pulses alone have no effect on AP repolarization and that RuBi-4AP has no basal effect on AP duration without laser-induced uncaging. Data are mean ± SEM; *p < 0.05 by one-way ANOVA. G, Effect of AIS-directed RuBi-4AP photolysis on AP repolarization at the AIS and boutons in basal conditions and in a background of TEA (500 μm). Data are mean ± SEM; *p < 0.05 by paired t test. The distance of the bouton recording position relative to the hillock is indicated below in parenthesis. H, Recording configuration showing that a short region of an axon branch was targeted for local Rubi-4AP photolysis. VSD responses were recorded in alternating trials from boutons located in either the targeted region or on a distal axon branch. I, APs recorded in control (black) and following Rubi-4AP photolysis (red) at bouton locations. J, AP widening, induced at target boutons by Rubi-4AP photolysis, was highly reduced in boutons located on distal axon branches. Data are mean ± SEM; *p < 0.05 by paired t test.

Figure 4.

Activity-dependent broadening of APs in the AIS. A, A high-frequency train of APs (40 Hz) elicited by a series of brief current injections at the soma. B, The first (black) and the twentieth (red) APs in a spike train are shown superimposed for recordings made at the AIS in basal conditions and in DTX (200 nm). APs in each condition, averaged over many trials, were obtained from two different cells. C, Superimposed APs recorded at a bouton in basal conditions or with TEA (500 μm) in the bath. Averaged APs in each condition are from two different cells. D, Summary showing the effect of repeated firing on AP duration for spikes recorded at the AIS or boutons. Data are mean ± SEM; *p < 0.05 by one-way ANOVA. E, Plot of the increase in AP duration during repeated spiking (40 Hz) in the AIS and boutons. Significant differences in AP duration with spike number, compared with the first AP at either the AIS or boutons; *p < 0.05 by one-way ANOVA.

Figure 5.

K_v1 channels inform spike rate and pattern. A, Maximum sustained AP firing induced by prolonged somatic current injection in control, TEA (500 μm) and, following wash, DTX (100 nm). B, The average steady-state spiking frequency, recorded during maximum sustained AP firing, in TEA, or DTX. Data are mean ± SEM; *p < 0.05 by unpaired t test. C, ISF plotted in AP sequence for the trials illustrated on the left. A decrease in ISF is indicative of spike-frequency accommodation. D, Summary plots showing ISF for SCs recorded in control and following TEA or DTX. APs, normalized for total spike number, were binned according to their relative position in the spike train. Data are mean ± SEM with significance differences between controls and matched pharmacological conditions, *p < 0.05 by two-way ANOVA. Cont., control.

Figure 6.

K_v1 channels in the AIS contribute to spiking phenotype. A₁, Images of an SC with uncaging locations demarcated by points in the magnified views for either the AIS (red) or a dendrite (blue). A₂, Diagram depicting the experimental configuration. Local photolysis of RuBi-4AP (300 μm) directly preceded somatic current injection (25 ms). Uncaging pulses, highlighted in red, are indicated by filled circles. B, Sustained firing, induced by somatic current injection, in control and immediately following RuBi-4AP photolysis at either the AIS or along a dendritic segment as illustrated in the images shown above. C, The average steady-state spiking frequency is reduced when photolysis of RuBi-4AP is directed immediately adjacent to the AIS. Moving the location of uncaging a short-distance lateral to the orientation of the axon demonstrates that area affected by photolyzed RuBi-4AP occurs in a spatially restricted manner. Data are mean ± SEM; *p < 0.05 by one-way ANOVA. D, Summary plots showing ISF for spike trains measured in control and immediately after photolysis of RuBi-4AP at the AIS. Data are mean ± SEM; *p < 0.05 by two-way ANOVA. In the bottom plot, laser pulses alone have no effect on ISF. For these plots, APs were normalized for total number and then binned based on their relative position in the spike train. E, Spiking measurements obtained following RuBi-4AP uncaging at two locations in the same cell including the AIS and at a dendritic (Dend.) site. Steady-state firing was induced by somatic current injection. Data are mean ± SEM; *p < 0.05 by one-way ANOVA in top plot, two-way ANOVA in the bottom plot. F, Diagram depicting the experimental recording configuration used for extracellular PF stimulation with a resulting postsynaptic SC response, recorded in control, shown below. G, Spike firing induced by repetitive PF stimulation (Stim.) in control and following AIS-targeted Rubi-4AP photolysis. An expanded view is shown with stimulus artifacts blanked for clarity. H, Summary graph showing AIS-directed Rubi-4AP photolysis reduces spiking. Data are mean ± SEM; *p < 0.05 by paired t test. Cont., control.

Figure 7.

Control of AP-evoked Ca²⁺ entry and neurotransmission is determined by K_v3 channels. A, An AP (black), recorded from an SC bouton, was used as the voltage command for a kinetic model of Ca_v channel gating. Ca_v channel open probability (P_o) was integrated and is shown below. AP duration was prolonged (red) simulating the loss of K_v3-mediated repolarization resulting in a proportional increase in Ca_v channel opening. B, Simultaneous recording of spike waveform using VSD imaging and the resulting AP-evoked Ca²⁺ transient recorded with the green Ca²⁺ indicator dye, OGB-1. C, APs and the resulting AP-evoked Ca²⁺ transients simultaneously recorded in a bouton in control and in TEA (500 μm). D, In a paired recording from synaptically connected SCs, APs evoked by on-cell stimulation of a presynaptic SC (SC Pre.) evoke GABA_A receptor-mediated IPSCs in a voltage-clamped postsynaptic SC (SC Post.). Averaged postsynaptic responses are shown in control and following bath application of TEA (500 μm). E, Average AP-evoked IPSC from a paired SC recording in control and BDS-I (1 μm). F, Spontaneous mIPSCs (0.5 μm TTX) in control and in TEA (500 μm). Cumulative probability of mIPSC amplitudes is shown in the histogram on the right. Superimposed in the inset are the averaged and aligned mIPSCs from each condition. G, A recording from synaptically connected SCs with AP-evoked IPSCs in control and DTX (100 nm). H, In a background of TEA (500 μm), AP-evoked IPSCs from a paired SC recording with responses in control and DTX (100 nm). I, Summary data from synaptically connected SC recordings showing the effect of K_v channel blockers on the amplitude of AP-evoked IPSCs. Data are mean ± SEM; *p < 0.05 by one-way ANOVA. Cont., control.