Decreases in the precision of Purkinje cell pacemaking cause cerebellar dysfunction and ataxia.

Episodic ataxia type-2 (EA2) is caused by mutations in P/Q-type voltage-gated calcium channels that are expressed at high densities in cerebellar Purkinje cells. Because P/Q channels support neurotransmitter release at many synapses, it is believed that ataxia is caused by impaired synaptic transmission. Here we show that in ataxic P/Q channel mutant mice, the precision of Purkinje cell pacemaking is lost such that there is a significant degradation of the synaptic information encoded in their activity. The irregular pacemaking is caused by reduced activation of calcium-activated potassium (K(Ca)) channels and was reversed by pharmacologically increasing their activity with 1-ethyl-2-benzimidazolinone (EBIO). Moreover, chronic in vivo perfusion of EBIO into the cerebellum of ataxic mice significantly improved motor performance. Our data support the hypothesis that the precision of intrinsic pacemaking in Purkinje cells is essential for motor coordination and suggest that K(Ca) channels may constitute a potential therapeutic target in EA2.

Calcium-activated potassium channels are selectively coupled to P/Q-type calcium channels in cerebellar Purkinje neurons.

Cerebellar Purkinje neurons fire spontaneously in the absence of synaptic transmission. P/Q-type voltage-gated calcium channels and calcium-activated potassium channels are required for normal spontaneous activity. Blocking P/Q-type calcium channels paradoxically mimics the effects of blocking calcium-activated potassium channels. Thus, an important function of the P/Q-type calcium channels is to provide calcium for activation of calcium-activated potassium channels. Purkinje neurons express several classes of voltage-gated calcium channels, and the P/Q- and T-type channels make comparable contributions to total calcium entry after an action potential. Here we demonstrate that calcium-activated potassium channels are activated exclusively by calcium entering through P/Q-type voltage-gated calcium channels. This selective coupling is maintained even when calcium flux through voltage-gated channels is increased by increasing the extracellular calcium concentration. Small decreases in P/Q current density are likely to alter spontaneous activity of Purkinje neurons via decreased recruitment of calcium-activated potassium channels. In both human and murine animal models, mutations that decrease P/Q current density in Purkinje neurons also cause cerebellar ataxia. Alterations in the spontaneous activity of Purkinje neurons may be an important contributing factor to the ataxia in these subjects.

Dendritic control of spontaneous bursting in cerebellar Purkinje cells.

We investigated the mechanisms that contribute to spontaneous regular bursting in adult Purkinje neurons in acutely prepared cerebellar slices. Bursts consisted of 3-20 spikes and showed a stereotypic waveform. Each burst developed with an increase in firing rate and was terminated by a more rapid increase in firing rate and a decrease in spike height. Whole-cell current-clamp recordings showed that each burst ended with a rapid depolarization followed by a hyperpolarization. Dual dendritic and somatic extracellular recordings revealed that each burst was terminated by a dendritic calcium spike. The contributions of T- and P/Q-type calcium current, large (BK) and small (SK) conductance calcium-activated potassium currents, and hyperpolarization-activated (I(H)) current to bursting were investigated with specific channel blockers. None of the currents, except for P/Q, were required to sustain spontaneous bursting or the stereotypic burst waveform. T-type calcium, BK, and SK channels contributed to interspike and interburst intervals. The effect of T-type calcium channel block was more pronounced after BK channel block and vice versa, indicating that these two currents interact to regulate burst firing. Block of I(H) current had no effect on bursting. Partial block of P/Q-type calcium channels concurrently eliminated dendritic calcium spikes and caused a switch from regular bursting to tonic firing or irregular bursting. Dendritic calcium spikes persisted in the presence of tetrodotoxin, indicating that their initiation did not require somatic sodium spikes. Our results demonstrate an important role for dendritic conductances in burst firing in intact Purkinje neurons.

Somatic and dendritic small-conductance calcium-activated potassium channels regulate the output of cerebellar Purkinje neurons.

Cerebellar Purkinje neurons provide the sole output of the cerebellar cortex and play a crucial role in motor coordination and maintenance of balance. They are spontaneously active, and it is thought that they encode timing signals in the rate and pattern of their activity. An understanding of factors that control their excitability is important for delineating their computational role in the cerebellum. We evaluated the role of small-conductance calcium-activated potassium (SK) channels in the regulation of activity of mouse and rat Purkinje neurons. We find that somatic SK channels effectively limit the maximum firing rate of Purkinje neurons; when SK channels are blocked by the specific antagonists apamin or scyllatoxin, cells fire spontaneously at rates as high as 500 spikes per second. Dendritic SK channels, however, control primarily the extent to which dendrites contribute to the firing rate of Purkinje cells. Given their presence in the dendrites, it is likely that SK channels in the proximal dendrites govern the efficacy of dendrosomatic electrical coupling. When studied under physiological conditions, it is found that SK channels play the same role in controlling the excitability of adult Purkinje neurons as they do in young cells.

Characterization of large conductance Ca2+-activated K+ channels in cerebellar Purkinje neurons.

We investigated the role of large conductance, calcium-activated potassium channels (BK channels) in regulation of the excitability of cerebellar Purkinje neurons. Block of BK channels by iberiotoxin reduced the afterhyperpolarization of spontaneous action potentials in Purkinje neurons in acutely prepared cerebellar slices. To establish the conditions required for activation of BK channels in Purkinje neurons, the dependence of BK channel open probability on calcium concentration and membrane voltage were investigated in excised patches from soma of acutely prepared Purkinje cells. Single channel currents were studied under conditions designed to select for potassium currents and in which voltage-activated currents were largely inactivated. Micromolar calcium concentrations activated channels with a mean single channel conductance of 266 pS. BK channels were activated by both calcium and membrane depolarization, and showed no sign of inactivation. At a given calcium concentration, depolarization over a 60-mV range increased the mean open probability (P(O)) from < 0.1 to > 0.8. Increasing the calcium concentration shifted the voltage required for half maximal activation to more hyperpolarized potentials. The apparent affinity of the channels for calcium increased with depolarization. At -60 mV the apparent affinity was approximately 35 micro m decreasing to approximately 3 micro M at +40 mV. These results suggest that BK channels are unlikely to be activated at resting membrane potentials and calcium concentrations. We tested the hypothesis that Purkinje cell BK channels may be activated by calcium entry during individual action potentials. Significant BK channel activation could be detected when brief action potential-like depolarizations were applied to patches under conditions in which the sole source of calcium was flux across the plasma membrane via the endogenous voltage-gated calcium channels. It is proposed that BK channels regulate the excitability of Purkinje cells by contributing to afterhyperpolarizations and perhaps by shaping individual action potentials.