Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency

Abstract

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage- and Ca(2+)-activated K(+) channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK(-/-)) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK(-/-) mice showed a dramatic reduction in spontaneous activity of the BK(-/-) cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia.

Fig. 1.

Analysis of BK channel expression in brain and footprint pattern of WT and BK^-/- mice. (a) Autoradiogram of brain sagittal sections from BK^+/+ and BK^-/- mice hybridized in situ with BK channel α subunit probe. (b) Immunohistochemical detection of BK channels in mouse cerebellar coronal sections: dense BKα immunostaining in the molecular layer (mo), PC somata (pc), and DCN: fastigial (f), interpositus (i), dentate (d) nucleus; weak staining in granule cells (gc) layer. BK^-/- sections incubated in parallel showed no staining. [Bars = 60 μm (on the left); 400 μm (on the right).] (c) Abnormal gait in BK^-/- mice. Footprints of 4-month-old WT and BK^-/- siblings (blue, forepaws; red, hindpaws). Statistics of stride length and paw abduction for males (M) and females (F). Three values were obtained from each run, excluding beginning and end; n = 6 WT and 7 BK^-/- per gender.

Fig. 2.

Motor impairment and abnormal conditioned eye blinking in BK^-/- mice. (a) Beam walking performance at regular (reg; i.e., smooth) beam and irregular (irreg; i.e., with steps) beam: distance moved, hindpaw slips, and falls. Means of two trials per beam, n = 6 per gender. (b) Accelerating rotarod performance: time to fall off the rotarod (mean value per trial), n = 6 per gender. (c) The conditioned eye-blink reflex, a cerebellum-specific form of motor learning, was abnormal in the BK^-/- mice. Statistical summary of conditioned eye blinking results from eight WT and eight BK^-/- mice. The conditioning phase includes 5 days followed by 3 days for extinction. All data are means ± SEM, *, P < 0.05; **, P < 0.01.

Fig. 3.

Reduction of fast AHP and severely reduced firing activity in BK^-/- PCs. (a Left) Effects of BK-channel blockade by IbTx (1 μM) on AHP and AP (Insets) in WT (blue) and BK^-/- (red) PCs. Records were obtained before (continuous trace) and 1 min after IbTx application (dashed). [Bars = 2ms/20 mV (AHPs), 0.5 ms/30 mV (APs)]. (Center) Time courses of IbTx effects on AHP in WT and BK^-/- PCs yielding the sample traces. (Right) Average time course of the AHP amplitudes of n = 5 WT and 3 BK^-/- PCs. (b) Whole-cell [intracellular (IC)] recordings from WT and BK^-/- PCs, showing spontaneous tonic and bursting firing patterns at fast and slow time scales. (Dashed lines, AP detection threshold for interspike interval distributions in Fig. 4a). (Bars = 200 ms/20 mV, 10 ms/20 mV.) (c) Distribution of trimodal firing patterns and difference in overall average firing rate between WT and BK^-/- PCs for whole-cell (IC) and cell-attached [extracellular (EC)] recordings, for the first 2 min of recording; n = 14–22 PCs per condition and genotype.

Fig. 4.

Spontanous discharge rates and depolarization block of BK^-/- cerebellar PC and increased depression at their DCN synapses. (a) Interspike interval (ISI) distributions for four typical PCs: tonic-firing (Upper) and burst-firing (Lower) WT and BK^-/- PCs. In each case, total AP number during a 15-s period is plotted. Note the fewer APs in BK^-/- compared with WT PCs, and longer ISI (lower discharge rate) in tonic-firing BK^-/- PCs. (b) Whole-cell recording from a silent BK^-/- PC with a depolarized resting potential (-46 mV). APs were evoked by hyperpolarizing the cell to -77 mV with DC current (-0.93 nA) before a depolarizing pulse (1.2 nA, 50 ms). (Bar = 20 mV, 1.7 nA.) (c) Enhanced paired-pulse depression in PC-DCN synapses. Significant differences were found for intervals shorter than 100 ms, n = 6 per genotype. (Left) Inhibitory postsynaptic currents (averages of 15 consecutive recordings) evoked by paired pulses (30-ms interval) in WT and BK^-/- slices. All data are means ± SEM, **, P < 0.01; *, P < 0.05.