Activity-Dependent Plasticity of Spike Pauses in Cerebellar Purkinje Cells

Abstract

The plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst-pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2(-/-) mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity-by altering the Purkinje cell output-may be crucial to cerebellar information storage and learning.

Figure 1. Intrinsic plasticity is absent from SK2^−/− mice

(A) Top traces: in WT mice an increase in the spike count is triggered by a 5Hz-injection of depolarizing currents. Bottom traces: no excitability change is seen in SK2^−/− Purkinje cells. (B) Time graph showing that intrinsic plasticity is triggered in WT (n=7), but not in SK2^−/− Purkinje cells (n=7). The arrow indicates the time point of tetanization. (C) Golgi staining of Purkinje cells in WT (left) and SK2^−/− mice (right). Scale bar: 50µm. (D) WT (n=10) and SK2^−/− Purkinje cells (n=7) did not differ in dendritic complexity (Sholl analysis; left), the cumulative length of the dendrite (middle), or the length of the primary dendrite (right). Values are shown as mean ± SEM. *p<0.05; **p<0.01.

Figure 2. Plasticity of complex spike pauses is mediated by SK2 channel downregulation

(A) Typical trace showing simple spike firing evoked by depolarizing current injection and a complex spike triggered by CF stimulation. The pause is measured as the interval between the complex spike onset and the next subsequent simple spike (grey area). (B) Top: typical traces (green) show that 5Hz-injection of depolarizing currents causes a shortening of the spike pause. In contrast, pause duration remains stable in control recordings (black traces). Bottom: time graph showing that the IP protocol shortens the pause duration (n=14), while the pause remains unaltered in non-tetanized cells (n=8). (C) In SK2^−/− Purkinje cells, no pause plasticity is observed (n=6). (D) Wash-in of the SK channel inhibitor apamin (10nM) shortens the pause duration (n=6). (E) The IP protocol does not trigger pause plasticity in the presence of apamin (10nM) in the bath (n=6). Arrows indicate the time point of tetanization. Values are shown as mean ± SEM. **p<0.01.

Figure 3. Intrinsic plasticity has a larger effect on the pause than on general excitability

(A) Average distribution of ISI values following the complex spike (post-CS) before (black line; shaded grey) and after (green line) application of the IP protocol. The ISI duration is normalized to pre-complex spike (pre-CS) ISI values monitored before tetanization. Arrowheads outline tails of the distribution affected by the IP protocol. The dots below indicate the averaged pause duration for each cell before (filled dots) and after tetanization (empty dots). (B) Kurtosis values of post-CS ISI values from each cell and their group average before and after tetanization (n=14; **p=0.009, Wilcoxon sign-ranked test). (C) Cumulative distribution of pooled post-CS ISI values before (black line; shaded grey) and after (green line) application of the IP protocol. The ISI values were rescaled (compared to the distribution in A) by normalization in each individual cell to median pre-CS ISI values, and monitored before (n=1095 ISIs) and after tetanization (n=1081 ISIs) to assess changes in the tails. (D) Maximal ISIs obtained from each individual cell, normalized as in (C) (n=14; *p=0.011; Wilcoxon sign-ranked test). (E) Changes in pause duration plotted against changes in pre-CS ISI values for tetanized (green; n=14) and non-tetanized cells (black; n=8); the grey dashed lines indicate a null variation after the protocol, the black dashed line indicates equal variation in ISI and pause. (F) Comparison between the variation in the median pre-CS ISI and the variation in the pause in tetanized (left) and control cells (right; n=14; *p=0.040, paired t-test). (G) Pause duration plotted against pre-CS ISI duration at increasing amplitude of injected current (n=10 cells); each line represents an individual cell; 5–9 steps (single measurement) per cell. (H) Intrinsic plasticity shortens the duration of the pause, but not the duration of subsequent ISIs (normalized values). Duration of the ISIs following the complex spike (pause = ISI #1) expressed as % of baseline after normalization to the average ISI (calculated from the eight ISIs preceding the complex spike). Only the pause was significantly shortened in tetanized cells (n=14; compared to control cells, n=8; repeated measure ANOVA; group effect p=0.0121, ISI effect p=0.829, ISI×group interaction p=0.00030; **Tukey’s post-hoc comparisons: p=0.000326, pause in tetanized vs non-tetanized cells). The data shown in (A–F) and (H) were obtained from the same recordings that are shown in Fig. 2B. Values are shown as mean ± SEM. *p<0.05; **p<0.01.

Figure 4. Intrinsic plasticity reduces the amplitude of the complex spike AHP

(A) Typical traces showing complex spike AHPs before and after tetanization. The AHP amplitude is reduced in tetanized cells (top) but not in control cells (bottom). (B) Time graph showing that the IP protocol reduces the AHP amplitude (n=8), which remains unaltered in control cells (n=10). The arrow indicates the time point of tetanization. Values are shown as mean ± SEM. *p<0.05; **p<0.01.