Evidence that climbing fibers control an intrinsic spike generator in cerebellar Purkinje cells

Abstract

It is well established that the climbing fiber (CF) input to a cerebellar Purkinje cell (PC) can exert a controlling influence on the background simple spike (SS) activity of the cell, in that repetitive stimulation of CFs causes a decrease in SS activity, and removal or inactivation of CFs is followed by a rise in activity. In the present study, the effects of inactivation of CFs in the short term and longer term (hours) were investigated in anesthetized rats to determine how the CFs control the PC SS activity. Inactivation of the CF input to a PC was accomplished by either reversibly inactivating with lignocaine or by microlesioning the inferior olive. Consistent with previous findings, CF removal caused a transformation of the PC firing pattern, with SSs discharging more regularly and rising to an exceptionally high level. In cases in which CF activity resumed, SS rate declined to control levels within a few seconds. However, with sustained CF inactivation (30 min to 5 hr), SS activity continues to rise progressively and develops an oscillating firing pattern, consisting of alternating bursts of high-frequency discharge at up to 100-150 Hz followed by 10-20 sec periods of electrical quiescence. No accompanying changes in the threshold for evoking SSs via the parallel fibers were seen to accompany the increases in tonic SS activity. We conclude that the increase in SS activity that follows CF inactivation could be caused by the removal of an inhibitory action that CFs exert on the intrinsic pacemaker present in PCs under normal conditions.

Figure 1.

Effects of CNQX on PC responsiveness to PF stimulation before and after the infusion of 50 μm CNQX. A, Arrow indicates an SS evoked with latency of 3.5 msec by PF stimulation (triangle). B, C, Shortly after the addition of CNQX. Note the disappearance of the evoked SS. D, After the effect of CNQX has worn off, evoked SS activity returns. Spontaneously occurring SS indicated by asterisk. Each trace is an average of 10 records.

Figure 2.

Effects of 50 μm CNQX on SS rates. A, No change in SS rate occurred for 12 of 18 PCs. B, Example PC that displayed a 20-30% reduction in discharge rate (5 of 18). C, A 50% decrease in SS rate (1 of 18). Bars in A-C represent period of CNQX infusion. Once PF stimulation could no longer evoke SSs, infusion was suspended. D, Comparison of SS rate on 18 PCs before, during, and after CNQX infusion. Post-CNQX values were measured during a 1-5 min time window after infusion, during which no SSs could be evoked. Values are mean ± SEM. Difference was not significant.

Figure 3.

Identification of responses mediated by the stimulation of the CFs via the IO. A, Superimposed evoked responses recorded on the surface of the cerebellar cortex. CF field potential was recorded below, just above, and at 2× threshold (threshold = 12.5 μA). B, Example of an evoked CS generated by IO stimulation. CS is marked by a dot at the initial response. A spontaneously occurring SS (asterisk) can be seen before the CS. Triangle refers to stimulus artifact.

Figure 4.

The effects of CF inactivation on SS rate. A, Short-term CF inactivation achieved with 5% lignocaine. B, SS discharge of 17 short-term inactivated cells at various time points after CF removal. Each data point represents group mean ± SEM. Dotted lines represent time of CF removal (time 0) and CF return (+CF). C, SS rate of seven long-term inactivated PCs at various time points after CF removal. Each data point represents group mean ± SEM. Dotted line represents time of CF removal. D, Long-term CF inactivation in which cell has developed oscillatory activity. CF removal was achieved with 10% lignocaine. E, PC that has been inactivated for 2 hr. Here the cell has entered its oscillatory mode, with activity fluctuating between periods of intense firing and quiescence. Bin widths, 1 sec.

Figure 5.

Recording of PC activity during CF inactivation with 5% lignocaine. Ai, Aii, Example of control spike trains, demonstrating the occurrence of both SSs and CSs. Bi—Biii, PC activity 1, 2, and 3 min after its CF has been removed. Note the disappearance of the CSs and the increased firing rate of the SSs. Ci—Ciii, Records of PC activity after CF inactivation, at 1, 2, and 3 min after the first reappearance of the CS. The SS rate progressively decreases with increasing CS discharge. The dots indicate the initial spike of the CS. Insets beside Ai, Ci-Ciii display the CS waveform on an expanded time scale. Calibration: 2 msec, 0.1 mV.

Figure 6.

ISI histograms of short- and long-term inactivated PCs. CVs under the various conditions are given. Ai, Aii, ISI histograms of all short-term inactivated PCs (n = 17). Ai, ISI histogram of SS activity before CF inactivation. Aii, When the CF was removed, the firing pattern of the PC became more regular, reflected in the narrow symmetric distribution of the ISI. Bi, Bii, ISI histograms of all long-term inactivated PCs (n = 7). Bi, Pre-CF removal; Bii, post-CF removal. Note the shift in ISI distribution after CF removal. The CVs were significantly decreased for both short- and long-term inactivated cells after CF removal.

Figure 7.

Correlation demonstrating the relationship of the change in SS rate after CF inactivation and the background discharge rate of the same cell for all 24 PCs studied. Correlation coefficient (r²) = 0.0055.

Figure 8.

A, Effect of long-term CF inactivation on SS activity, followed by a period of CF stimulation. CF inactivation was achieved with a DC lesion. After a 2 hr inactivation period, the CF was stimulated electrically at 1 Hz. The period of stimulation is indicated by the length of the bar. Stimulation of the CF resulted in a return of SS activity toward its preinactivated level. B, Expanded time base of the CF stimulation period. C, ISI histogram of a PC whose CF has been removed, then stimulated after 2 hr inactivation period. Ci, ISI of the control period. Cii, ISI of period of inactivation. Ciii, ISI after CF stimulation restored SS firing frequency to control levels.

Figure 9.

Plot demonstrating the threshold for evoking a SS by direct stimulation of the PFs, before and after the removal of the CF input. Time point 0 refers to the time of CF removal.