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

Abstract

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.

Fig. 1.

Block of SK channels increases the firing rate of Purkinje neurons. Activity of individual Purkinje neurons in rat cerebellar slices was recorded extracellularly using a differential amplifier. A–C, The records show the average firing rate and the effect of apamin. Samples of the extracellular records in the presence (top trace) and absence (bottom trace) of apamin are shown atbottom left of each panel. The relative occurrences of the interspike intervals are shown at the bottom right of each panel. A, Example of a tonic firing cell. Apamin made the cell burst at random and increased the firing rate. The lower interspike interval histogram shows the longer intervals corresponding to the intraburst intervals seen in the presence of apamin. Such long intervals were always seen when apamin was applied, but given their very low occurrence, they are omitted in the subsequent figures. B, Example of a cell that burst at random. Apamin exaggerated the bursts and increased the firing rate.C, Example of a cell with the trimodal pattern of activity. Apamin increased the firing rate and shortened the pattern period. D, The predominant and maximum firing rates were estimated from interspike histograms. The predominant firing rate is defined as the firing rate most often observed (the peak of the histogram). The maximum firing rate was defined as the fastest firing rate that was observed 5% of time relative to the predominant firing rate. The data reported are average values for five cells (mean ± SEM). The mean values obtained in apamin were significantly different from those obtained under control conditions (*p < 0.01, **p < 0.02; determined by one-way ANOVA).

Fig. 2.

Block of SK channels increases the firing rate of tonically firing Purkinje neurons in the absence of synaptic input.A, Average firing rate from a neuron in a mouse cerebellar slice in the presence of fast excitatory and inhibitory synaptic blockers (5 mm kynurenate and 100 μmpicrotoxin). The tonic activity of the cells changed to random bursting when 100 nm apamin was bath applied. B, Sample extracellular records from the neuron described inA in control solution (top trace) and in the presence of apamin (bottom trace). C, Relative distribution of interspike intervals for the cell described inA under control conditions (c) and in apamin (a). D, The average predominant and maximum firing rates for Purkinje neurons that fired tonically under control conditions (mean ± SEM,n = 7). *Statistical significance versus control conditions at p < 0.01 determined by one-way ANOVA.

Fig. 3.

Block of SK channels increases the firing rate but maintains the trimodal pattern in cells that have the trimodal pattern of activity. A, Average firing rate from a neuron in a mouse cerebellar slice in the presence of synaptic blockers. In the absence of apamin, the cell exhibited a trimodal pattern of activity with a cycle duration of ∼5 min. The cycle duration shortened and the firing rate increased in the presence of apamin. B, Sample extracellular records from the neuron described inA in the presence of apamin. A single cycle of the trimodal pattern of activity is shown. A tonic firing phase is followed by periods of bursting. The interburst intervals gradually increase until the cell stops firing. After a period of silence, the pattern resumed with the start of another tonic firing phase. C, Distribution of interspike intervals for the cell described inA under control conditions (c) and in apamin (a). D, The average predominant and maximum firing rates for Purkinje neurons that exhibited the trimodal pattern of activity (mean ± SEM,n = 7). *Statistical significance versus control conditions at p < 0.01 determined by one-way ANOVA. E, The average duration of a single cycle of the trimodal pattern of activity (pattern period) in the presence and absence of 100 nm apamin (mean ± SEM,n = 7). *Statistical significance versus control conditions at p < 0.01 by one-way ANOVA.F, Histograms show the average duration of the tonic phase of firing, the bursting phase, and the silent period in the presence and absence of apamin in cells with the trimodal pattern of activity (mean ± SEM, n = 7). *Statistical significance versus control conditions at p < 0.01 determined by one-way ANOVA.

Fig. 4.

SK channels affect the action potential afterhyperpolarization in Purkinje neurons. A whole-cell current-clamp recording from a Purkinje neuron in a mouse cerebellar slice.A, Average firing rate of the current-clamped cell (I = 0) shows that apamin increased the firing rate and caused the cell to burst at random. B, Recordings of membrane potential from the neuron described in A under control conditions (top trace) and in the presence of apamin (bottom trace). C, Average action potentials from the neuron described in A recorded in control medium (1) and in the presence of apamin while the cell was firing tonically (2) or bursting (3). Each trace is the average of 30 seconds of firing. Numbersat the top of the trace in Aindicate the times at which action potentials were averaged. Action potentials have been truncated to show the afterhyperpolarization more clearly. The full action potentials are shown in theinset.

Fig. 5.

Silent periods during the trimodal pattern of activity are associated with long hyperpolarizations. The trimodal pattern of activity was recorded in the perforated whole-cell current-clamp configuration (I = 0) in the presence of apamin. Two silent hyperpolarized periods flank the tonic and bursting modes.

Fig. 6.

SK channels play an essential functional role in adult Purkinje neurons. Activity of individual Purkinje neurons was recorded using extracellular electrodes in cerebellar slices obtained from adult (> 3 months old) rats and mice. A, Firing rate of a Purkinje neuron in an adult rat cerebellar slice in the absence of synaptic blockers. The temperature of the solution bathing the slice is shown in the top trace. Apamin increased the firing rate and shortened the pattern duration. The cell stopped firing when the temperature was allowed to drop to room temperature in the presence of apamin. B, Relative occurrence of interspike intervals for the cell described in A in control solution (c) and in the presence of apamin (a). C, Average of predominant (open bars) and maximum (hatched bars) firing rate for adult Purkinje neurons in the absence of synaptic blockers (n = 4; 3 rats and 1 mouse). *Statistical significance versus control conditions atp < 0.01 determined by one-way ANOVA.D, Firing rate of a Purkinje neuron in an adult mouse cerebellar slice in the presence of synaptic blockers. The cell stopped firing when the temperature was reduced to 27°C and resumed firing when it was increased back to 35°C. Bath application of apamin increased the firing rate and reduced the pattern duration. Apamin did not prevent the cell from becoming quiescent when temperature was reduced a second time. E, Relative occurrence of interspike intervals for the cell described in D in control solution (c) and in the presence of apamin (a). F, Average of predominant (open bars) and maximum (hatched bars) firing rate for adult Purkinje neurons in the presence of synaptic blockers (n = 6; 3 each of rats and mice). *Statistical significance versus control conditions atp < 0.01 determined by one-way ANOVA.G, Firing rate of a Purkinje neuron in an adult rat cerebellar slice in the presence of synaptic blockers. Application of 30 nm scyllatoxin increased the firing rate and made the pattern period shorter. H, Relative occurrence of interspike intervals for the cell described in G in control solution (c) and in the presence of scyllatoxin (s).

Fig. 7.

SK channels are not saturated during the spontaneous activity of Purkinje cells. A, Bath application of 10 μm EBIO to a tonic firing cell reversibly reduced the firing rate. B, Relative occurrence of interspike intervals for the cell described inA under control conditions (c) and in the presence of EBIO (e). Bath application of EBIO to a cell with the trimodal pattern of activity reduced the firing rate and made the cell fire tonically. The effects of EBIO were reversible.D, Relative occurrence of interspike intervals for the cell described in C under control conditions (c) or in EBIO (e).

Fig. 8.

Dendritic SK channels contribute to spontaneous firing in Purkinje neurons. Apamin (100 nm) was perfused locally onto the dendrites of mouse Purkinje neurons to assess the contribution of dendritic SK channels to spontaneous firing. Recordings were made in the presence of synaptic blockers.Diagrams at the top of each panelshow the placement of the perfusion and recording pipettes relative to the Purkinje neuron and the approximate area of the dendrites exposed to apamin. A, Firing rate of a Purkinje neuron that fired tonically in the absence of apamin (black trace). Apamin was applied selectively to the distal half (1/2 D) of the dendritic tree (red trace) and then to the whole cell (WC) (green trace).B, Firing rate of a Purkinje neuron that exhibited a trimodal pattern of activity in the absence of apamin (black trace). Apamin was applied selectively to the distal two-thirds (2/3 D) of the dendritic tree (blue trace) and then to the whole cell (green trace).C, The average increase in firing rate induced by apamin in neurons with the trimodal pattern of activity is compared with the average decrease in the pattern period. Averages are shown for apamin applied locally to the distal half and distal two-thirds of the dendritic tree and to the whole cell. Results were obtained from neurons in the presence of synaptic blockers and are reported as mean ± SEM. Numbers in the parenthesesdenote the number of experiments.