Kamran Khodakhah

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 (KCa) 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 KCa channels may constitute a potential therapeutic target in EA2.

Active contribution of dendrites to the tonic and trimodal patterns of activity in cerebellar Purkinje neurons.

The cerebellum is responsible for coordination of movement and maintenance of balance. Cerebellar architecture is based on repeats of an anatomically well defined circuit. At the center of these functional circuits are Purkinje neurons, which form the sole output of the cerebellar cortex. It is proposed that coordination of movement is achieved by encoding timing signals in the rate of firing and pattern of activity of Purkinje cells. An understanding of cerebellar timing requires an appreciation of the intrinsic firing behavior of Purkinje cells and the extent to which their activity is regulated within the functional circuits. We have examined the spontaneous firing of Purkinje neurons in isolation from the rest of the cerebellar circuitry by blocking fast synaptic transmission in acutely prepared cerebellar slices. We find that, intrinsically, mature Purkinje cells show a complex pattern of activity in which they continuously cycle among tonically firing, bursting, and silent modes. This trimodal pattern of activity emerges as the cerebellum matures anatomically and functionally. Concurrent with the transformation of the immature tonically firing cells to those with the trimodal pattern of activity, the dendrites assume a prominent role in regulating the excitability of Purkinje cells. Thus, alterations in the rate and pattern of activity of Purkinje neurons are not solely the result of synaptic input but also arise as a consequence of the intrinsic properties of the cells.

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 (IH) 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 IH 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.

The neural substrates of Rapid-Onset Dystonia-Parkinsonism

Although dystonias are a common group of movement disorders, the mechanisms by which brain dysfunction results in dystonia are not understood. Rapid-onset Dystonia-Parkinsonism (RDP) is a hereditary dystonia caused by mutations in the ATP1A3 gene. Affected individuals can be free of symptoms for years, but rapidly develop persistent dystonia and Parkinsonism-like symptoms after a stressful experience. Using a mouse model, we found that an adverse interaction between the cerebellum and basal ganglia can account for the symptoms of these individuals. The primary instigator of dystonia was the cerebellum, whose aberrant activity altered basal ganglia function, which in turn caused dystonia. This adverse interaction between the cerebellum and basal ganglia was mediated through a di-synaptic thalamic pathway that, when severed, alleviated dystonia. Our results provide a unifying hypothesis for the involvement of cerebellum and basal ganglia in the generation of dystonia and suggest therapeutic strategies for the treatment of RDP.

The therapeutic mode of action of 4-aminopyridine in cerebellar ataxia

Episodic ataxia type 2 (EA2) is a hereditary cerebellar ataxia associated with mutations in the P/Q-type voltage-gated calcium (Ca2+) channels. Therapeutic approaches for treatment of EA2 are very limited. Presently, the potassium (K+) channel blocker 4-aminopyridine (4-AP) constitutes the most promising treatment, although its mechanism of action is not understood. Here we show that, in contrast to what is commonly believed, therapeutic concentrations of 4-AP do not increase the inhibitory drive of cerebellar Purkinje cells. Instead, 4-AP restores the severely diminished precision of pacemaking in Purkinje cells of EA2 mutant mice by prolonging the action potential and increasing the action potential afterhyperpolarization. Consistent with this mode of action, the therapeutic efficacy of 4-AP was comparable, and not additive, to chlorzoxazone, an activator of Ca2+-dependent K+ channels that also restores the precision of Purkinje cell pacemaking. The likely target of 4-AP at the concentrations used are the Kv1 family of K+ channels, possibly the Kv1.5 subtype. Because at higher concentrations 4-AP blocks a large array of K+ channels and is a proconvulsant, use of selective Kv1 channel blockers is likely to be a safer substitute for treatment of cerebellar ataxia.

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 (PO) 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 ≈35 µm decreasing to ≈3 µM 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.

Questioning the role of rebound firing in the cerebellum

A key component of recent theories on cerebellar function is rebound firing in neurons of the deep cerebellar nuclei (DCN). Despite the robustness of this phenomenon in vitro, in vivo studies have provided little evidence for its prevalence. We found that intact mouse or rat DCN neurons rarely showed rebound firing under physiological conditions in vitro or in vivo. These observations necessitate a critical re-evaluation of recent cerebellar models.

Short latency cerebellar modulation of the basal ganglia

The graceful, purposeful motion of our body is an engineering feat that remains unparalleled in robotic devices using advanced artificial intelligence. Much of the information required for complex movements is generated by the cerebellum and the basal ganglia in conjunction with the cortex. Cerebellum and basal ganglia have been thought to communicate with each other only through slow, multi-synaptic cortical loops, begging the question as to how they coordinate their outputs in real time. We found that the cerebellum rapidly modulates the activity of the striatum via a disynaptic pathway in mice. Under physiological conditions, this short latency pathway was capable of facilitating optimal motor control by allowing the basal ganglia to incorporate time-sensitive cerebellar information and by guiding the sign of cortico-striatal plasticity. Conversely, under pathological condition, this pathway relayed aberrant cerebellar activity to the basal ganglia to cause dystonia.

Fast activation and inactivation of inositol trisphosphate-evoked Ca2+ release in rat cerebellar Purkinje neurones.

1. Calcium release from stores via inositol trisphosphate (InsP3) activation of intracellular Ca2+ receptor‐channels is thought to have a role in regulating the excitability of cerebellar Purkinje neurones. The kinetic characteristics of InsP3 receptor activation in Purkinje neurones are reported here. 2. InsP3 was applied by flash photolysis of caged InsP3 during whole‐cell patch clamp. Ca2+ flux into the cytosol was measured with a low‐affinity fluorescent Ca2+ indicator and by activation of Ca(2+)‐dependent membrane conductance. 3. InsP3 produced Ca2+ release from stores with an initial well‐defined delay (mean, 85 ms at 10 microM InsP3), which decreased to less than 20 ms at high InsP3 concentrations. 4. The rate of rise of free [Ca2+], which provides a measure of Ca2+ efflux and InsP3 receptor activation, increased with increasing InsP3 concentration in each cell and had a high absolute value of up to 1400 microM s‐1 at 40 microM InsP3. The period of fast efflux was brief, inactivating in 25 ms at low and in 9 ms at high InsP3 concentration. 5. Peak free [Ca2+] was high (mean, 23 microM with a pulse of 40 microM InsP3) and increased with InsP3 concentration up to 80 microM InsP3 tested here. 6. Experiments with a flash‐released, stable 5‐thio‐InsP3 confirm that the low InsP3 sensitivity of Purkinje neurones does not result from metabolism of InsP3. 7. The low functional affinity and fast activation by InsP3 suggest a difference in InsP3 receptor properties from non‐neuronal cells tested in the same way. The large Ca2+ efflux and high peak [Ca2] probably result from high InsP3 receptor‐channel density. 8. Elevated cytosolic [Ca2+] produced by Ca2+ influx through plasmalemmal Ca2+ channels strongly suppressed InsP3‐evoked Ca2+ release from stores. Rapid termination of InsP3‐evoked efflux results mainly from inhibition by high [Ca2+]. 9. The fast InsP3 activation kinetics and rapid, strong inactivation by Ca2+ influx suggest that interactions between InsP3‐mediated and membrane Ca2+ signalling could occur on a time scale compatible with neuronal excitation.