doi: 10.3389/fncir.2013.00133.
eCollection 2013.
Strength and timing of motor responses mediated by rebound firing in the cerebellar nuclei after Purkinje cell activation
Affiliations
- PMID: 23970855
- PMCID: PMC3748751
- DOI: 10.3389/fncir.2013.00133
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Strength and timing of motor responses mediated by rebound firing in the cerebellar nuclei after Purkinje cell activation
Laurens Witter et al.
Front Neural Circuits.
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Abstract
The cerebellum refines the accuracy and timing of motor performance. How it encodes information to perform these functions is a major topic of interest. We performed whole cell and extracellular recordings of Purkinje cells (PCs) and cerebellar nuclei neurons (CNs) in vivo, while activating PCs with light in transgenic mice. We show for the first time that graded activation of PCs translates into proportional CN inhibition and induces rebound activity in CNs, which is followed by graded motor contractions timed to the cessation of the stimulus. Moreover, activation of PC ensembles led to disinhibition of climbing fiber activity, which coincided with rebound activity in CNs. Our data indicate that cessation of concerted activity in ensembles of PCs can regulate both timing and strength of movements via control of rebound activity in CNs.
Keywords: Purkinje cells; cerebellar nuclei; motor control; olivo-cerebellar network; rebound.
Figures
PC-specific expression of ChR2(H134R)-eYFP under control of L7-pcp2. (A) Coronal section of the cerebellum of an L7-ChR2(H134R)-eYFP mice. PC and molecular layers show dense expression of the ChR2-eYFP fusion protein (eYFP: yellow). (B) Detail of a sagittal section of an L7-ChR2(H134R)-eYFP mouse. ChR2-eYFP protein expression was restricted to PC membranes. PC somata are indicated with arrowheads. Note that MLIs are visible as small dark exclusions in the PC arborizations of the molecular layer. The neuronal expression of ChR2-eYFP fusion protein was found only in PCs of the cerebellum, but not in other neuronal structures in the rest of the brain. (C) Detail of PC axons innervating the cerebellar nuclei. CNs are marked with ‘*’. Counterstain in (A) with DAPI (pink).
Graded whole field light stimulation by three blue light emitting diodes (LEDs) positioned around the cerebellum of mice controlled by a custom-built linear LED driver. (A) Overview of the experimental set-up. Whole cell and extracellular recordings were made from Purkinje cells (PCs), cerebellar nuclei neurons (CNs), molecular layer interneurons (MLIs) and granule cells. At the same time, bilateral electroencephalogram (EEG) was recorded from motor cortex, referenced on the right parietal cortex. (B) Circuit diagram of one channel of the LED driver. A 10-turn dial permitted setting of light-intensity during experiments. A TTL input can be used to trigger the light from an external source. V-Max, V-Min, and A-Lim (measuring from calibration voltage or current) are used to limit the voltage and current through the LED and to calibrate the 10-turn dial. Up to three LEDs can be connected in parallel on a single channel. (C) LED power is a linear function of the dial setting in the range between 20 and 75%.
Whole cell in vivo recordings of PCs during optogenetic stimulation. (A) Latency to the first simple spike (SS) and the increase in firing frequency during the light stimuli at different intensities. (B,C) Light stimulation (465 nm, 1000 ms, denoted by the blue bars below the traces) of the cerebellum at weak (left panels) and strong (right panels) light intensities. Both in the upstate (B) and downstate (C) PCs show graded increases of SSs and CSs during stimulation. Note, ChR2 (H134R) has slow kinetics; at light-offset, the cell remains depolarized for a few milliseconds before it settles back down to a baseline state (arrow). Red lines indicate the subthreshold membrane potentials before and during the light stimulation. Asterisks indicate CSs.
Whole cell and extracellular in vivo recordings of CNs before, during, and after optogenetic activation of PCs. (A) Brief 1 ms activation of PCs (blue bar) was sufficient to evoke IPSPs in CNs (onset is indicated with an arrow). The inset shows the average trace. (B) CN responses (top panel) at various holding potentials (lower panel) used to determine the reversal potential of the evoked events in CNs (stimulus in blue). The dashed numbered lines indicate the peaks of the first (1) and second (2) induced postsynaptic event, respectively. (C) IV curves of all CN neurons tested (individual CN neurons are indicated with different colors, squares indicate responses to the first peak, triangles to the second). The reversal potential (Erev = −76.42 ± 8.66 mV) is in agreement with a GABAA-mediated current. (D) Graded PC stimulation (blue bar) [ranging from 0.3 to 6.2 dial setting (Figure 2C)] evoked a graded response in CNs. Black lines indicate raw data, red lines Gaussian convolved traces. (E) Histograms of the latency of the first spike in CNs after light-driven PC mediated inhibition. The distribution shows a long tail for both weak and strong stimulation intensities. (F) The normalized firing rate (firing rate during stimulation or rebound divided by the prestimulus firing rate) of the CN shown in (D) during light stimulus (blue), 100 ms post light stimulus (green dots), or 200 ms post light stimulus (orange dots). (G) Summary as in (F), but for all cells. Different neurons are color coded over the three panels. (H) The maximal stimulation intensities from the panels in (F) are plotted to show that inhibition during the stimulation is necessary for rebound to occur.
Timed motor responses in awake mice during optogenetic activation of PCs. (A) For the behavioral assay head-fixed mice were placed on a transparent disc that could freely rotate. The optic fiber was placed on the brain surface of lobules V and VI (left) for optogenetic stimulation. Light was delivered to the brain via a LED coupled to the optic fiber. Right: Bottom view of a mouse responding to optogenetic activation of PCs (250 ms, ~5 mW/mm2) with a twitch of its tail and hind legs after stimulus offset. Camera frames were acquired at 100 Hz. Differences between two frames at the stimulus offset ("pre," cyan) and 200 ms post-offset ("post," red) show relative position change between the two time points. (B) Individual behavioral responses (gray traces), response corresponding to twitch shown in (A) [red trace, one frame chosen at offset (pre), and one 200 ms post-offset (post)] and mean behavioral response (black trace) following a 250 ms light stimulus. (C) Behavioral responses were graded with increases in light intensity. Estimated power densities are shown at a depth of the PC monolayer (~120 μm). (D) Normalized behavioral response plotted vs. power density showing a linear correlation (R2 = 0.9993, slope = 0.19). (E) Raster plot showing individual behavioral onsets relative to the stimulus offset (time = 0). Inset: box plots (three mice indicated by different colors) of behavioral onsets relative to the stimulus offset (whiskers indicate distance from 25 to 75% interquartile ranges to furthest observations, center mark represents the median). (F) The onset of behavior shifted with an increase in stimulus duration, such that the relative delay to a behavioral response onset relative to stimulus offset was maintained. Note that behavioral responses can be elicited by stimuli with durations of 25 ms. (G) Time from stimulus onset to behavioral onset plotted against stimulus duration followed a linear relationship (R2 = 0.9999 and slope = 0.96) demonstrating that the onset of behavioral responses shifts relative to the stimulus duration. (H) The interstimulus interval did not have an effect on strength of the behavioral response (r = −0.071, p = 0.49). (I) and (J) Simultaneous recordings of CNs, bihemispheric EEG, and EMG to 500 ms (I) and 1000 ms (J) light stimulation of PCs in anesthetized mice. For clarity, the stimulation period has been truncated and only the last 45 ms of the stimulus is shown in the blue box. Vertical scale bars apply to both EEG traces and EMG traces in (I) and (J). Top panels, average Gaussian-convoluted spike train of all CNs. Middle panels, left and right EEG. Bottom panels, rectified, differentiated and again rectified EMG responses. The vertical dotted lines indicate the location of the positive (P1 to P3) and negative (N1 to N2) deflections in the EEG signals. Note that the onset of the EMG response occurs before the first response peak in the EEG, while the EMG signal itself is preceded by CN activity.
Optogenetic stimulation of PCs elicits an increase in CS activity, which is most likely a network effect. (A) Light stimulation (blue bar) evokes an increase in CS activity even when the SS increase is prevented by intracellular current injection via the patch electrode. Additionally, a CS was observed after stimulus offset. Notice the depolarized membrane potentials after stimulus offset indicating slow inactivation of the ChR2 (H134R) channel (arrows). (B) MLI activity is not directly increased in response to PC stimulation but after >50 ms delay. This activation is likely due to the recorded CS increase (A) that leads to MLI activation through glutamate spillover. The increase in MLI firing frequency outlasted the light stimulus [see also Gaussian-convoluted trace in red; Putative MLIs, N = 3; baseline firing rate 50 ms before light stimulation: 1.69 ± 9.54, firing rate <50 ms after strong light stimulation: 1.93 ± 8.97, t(123) = −0.421, p = 0.675] similar to what we see for CS activity (A). (C) A voltage and subsequent current clamp recording (D) of a single representative CN during light stimulation of PCs. (C) Voltage clamp recordings of CNs reveal several large, summating EPSCs present during and directly after the light stimulus (arrows), which may be evoked by the increased climbing fiber activity (A). (D) In current clamp, the inhibition from firing during PC stimulation (blue bar) and the biphasic rebound activity after the inhibition is visible. Note, the timing of the CS activity (A, C) of PCs after offset of the light stimulus precedes the break in the CN rebound (D, see also Gaussian-convoluted trace in red). (E) Example complex spike from the trace in (A) (1), example EPSCs from the trace in (C) (2 and 3), and magnification of rebound firing in (D) (4).
Voltage clamp recordings of three CNs during light stimulation of PCs (blue bar, the time during the stimulus is not completely shown; notice the break between the blue bars). Three representative CNs are shown. Per neuron we show three overlays of each four traces (so, a total of 12 traces per cell). (A) CNs react with an outward current in response to the light stimulus. In addition, there are several large, summating EPSCs present during and directly after the light stimulus, probably induced by climbing fiber activity. (B) There were no differences in the distributions of various EPSC kinetics before (green) and after (red) stimulation onset. Also, the (C) number of events, (D) amplitude of the excitatory postsynaptic potentials, (E) the area, (F) the decay and (G) rise time do not differ before and after stimulation onset, suggesting that the mossy and climbing fiber inputs share similar kinetics. The significance of pairwise comparisons is listed for each panel separately.
References
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- Aizenman C. D., Linden D. J. (1999). Regulation of the rebound depolarization and spontaneous firing patterns of deep nuclear neurons in slices of rat cerebellum. J. Neurophysiol. 82, 1697–1709 - PubMed
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