Encoding of whisker input by cerebellar Purkinje cells

Abstract

The cerebellar cortex is crucial for sensorimotor integration. Sensorimotor inputs converge on cerebellar Purkinje cells via two afferent pathways: the climbing fibre pathway triggering complex spikes, and the mossy fibre–parallel fibre pathway, modulating the simple spike activities of Purkinje cells. We used, for the first time, the mouse whisker system as a model system to study the encoding of somatosensory input by Purkinje cells.We show that most Purkinje cells in ipsilateral crus 1 and crus 2 of awake mice respond to whisker stimulation with complex spike and/or simple spike responses. Single-whisker stimulation in anaesthetised mice revealed that the receptive fields of complex spike and simple spike responses were strikingly different. Complex spike responses, which proved to be sensitive to the amplitude, speed and direction of whisker movement, were evoked by only one or a few whiskers. Simple spike responses, which were not affected by the direction of movement, could be evoked by many individual whiskers. The receptive fields of Purkinje cells were largely intermingled, and we suggest that this facilitates the rapid integration of sensory inputs from different sources. Furthermore, we describe that individual Purkinje cells, at least under anaesthesia, may be bound in two functional ensembles based on the receptive fields and the synchrony of the complex spike and simple spike responses. The ‘complex spike ensembles’ were oriented in the sagittal plane, following the anatomical organization of the climbing fibres, while the ‘simple spike ensembles’ were oriented in the transversal plane, as are the beams of parallel fibres.

Figure 1. Multiple single-unit recordings of Purkinje cells in mouse crus 1 and crus 2 in vivo

A, summary of the main neuronal pathways involved in the integration of tactile input from the whiskers and the coordination of whisker movements (see also Kleinfeld et al. 1999). The cerebellum is centrally located in both the sensory and the motor pathways. B, overview of the recording setup. A set of quartz–platinum recording electrodes placed in crus 1 and crus 2 can be seen in the craniotomy. In the upper part of the photograph are the aluminum guide tubes, allowing the electrodes to be placed individually with an inter-electrode distance of 305 μm. C, raw trace of an extracellular recording from a Purkinje cell in vivo. Purkinje cells produce two kinds of spikes: infrequent complex spikes (*) and frequent simple spikes (•). D, overlay of 312 traces aligned on a complex spike. Simple spike firing is absent for several milliseconds following a complex spike – the ‘complex spike pause’– which is the hallmark of a Purkinje cell single-unit recording. E, complex spike-triggered simple spike histogram. Following a complex spike (at t= 0 ms), simple spikes are completely absent for 5 ms. During the first 20 ms following a complex spike, only very few simple spikes were present. In many Purkinje cells, as in this example, the complex spike pause is followed by a transient increase in simple spike firing rate. The data in the panels C, D and E originate from the same Purkinje cell recording.

Figure 7. Receptive fields of Purkinje cells in anaesthetised mice

A, the follicles of the large vibrissae are ordered in a grid on the mystacial pad. For the experiments presented in this figure, we confined ourselves to the 14 whiskers depicted here. ‘Rows’ are lines of whiskers ordered in the rostro-caudal plane, ‘arcs’ are lines of whiskers ordered in the dorso-ventral plane. B, during a recording of a Purkinje cell, we tested one by one which of the whiskers elicited a complex spike response. For each responsive whisker, we tested whether the neighbouring whiskers also elicited complex spike responses. We discriminated between direct neighbours and neighbours two or three whiskers away, as well as between neighbours in the same row and in the same arc. C, as for B but for the simple spike responses. D, fraction of Purkinje cells in which a given whisker could elicit a complex spike response. For whiskers in arc ‘3’ we did not have enough data. E, as for D but for the simple spike responses. F, for each of the 28 Purkinje cells tested, the whisker that elicited the largest complex spike response is depicted as a coloured circle on the approximate location, as projected to the brain surface, in crus 1 or crus 2. Colour coding is the same as in A and D. (Near) overlapping locations have been displaced minimally to increase visibility. Purkinje cells that did not have a complex spike response to any of the whiskers tested are not shown. G, as for F but for the simple spike responses. Here, we illustrated the early-negative responses. H, surface-projected locations of Purkinje cells showing a complex spike response to whisker stimulation (filled symbols). Purkinje cells that did not show a complex spike response are indicated by open symbols. Circles: single-whisker stimulation (C2); bars: multiple-whisker stimulation (C-row). Most complex spike responses were found centrally in crus 1 (see also Supplemental Fig. 2). I, as for H but for the simple spike responses. The sagittal and transversal axes are indicated by R (rostral) and C (caudal), and by M (medial) and L (lateral), respectively.

Figure 4. Single-whisker stimulation affects both complex spike and simple spike firing in anaesthetised mice

A, schematic drawing of the organization of the mouse mystacial pad, showing the relative positions of the whiskers used in this study. B, upper trace: programmed trajectory of the C2 whisker, which was attached to a piezo drive. Lower trace: extracellular recording of a Purkinje cell, showing both complex and simple spikes. Note that complex spikes (*) occur shortly after the start of the whisker movement. C, whisker movement tracked with a high-speed CCD camera (sample frequency, 1.0 kHz). Time scale as in D–I. D, raster plot showing the timing of complex spike firing. During each trial, we stimulated whisker C2 according to trajectory depicted in C, but in a random direction. E, as in D but for the simple spikes. Note that the average firing frequency showed some long-term changes, possibly related to variations in the state of anaesthesia (see also Supplemental Fig. 1). The simple spike response was, however, present in periods with higher as well as with lower basal firing rate (data not shown). F, peri-stimulus histogram of the complex spike firing in a representative experiment following stimulation of whisker C2 (777 trials). The largest response was during the movement from the resting position to the extreme position (1). In some experiments, including this one, a second peak was observed around 100 ms later (2). The backward movement evoked only a very small response (3). G, as in F but for the simple spikes. In this experiment, an early-positive simple spike response was present following the forward movements (1), followed by an early-negative response (2). The backward movements triggered an early-negative response (3). H, average peri-stimulus histogram of 7 Purkinje cells showing complex spike responses to C2 whisker stimulation. Only the initial complex spike response to the forward movement is consistent over all experiments (cf. arrow 1 in F). I, average peri-stimulus histogram of 15 Purkinje cells showing simple spike responses to C2 whisker stimulation. Simple spike responses occurred both in response to the forward and to the backward movement, and consisted of two phases: an early and a late phase. Each phase, in turn, had an initial positive and a later negative simple spike modulation. The late-negative simple spike modulation was not observed in single-whisker stimulation trials (see Table 1).

Figure 2. Whisker stimulation triggers Purkinje cell responses in awake mice

A, photograph of the whiskers of a mouse. The whiskers located outside the focal plane were cut. The movement of the whiskers along the green line was tracked at 1 kHz. Whisker position was defined as the intersection point of the green line and the whisker. In this experiment, 6 whiskers could be tracked. B, top: positions of the 6 whiskers indicated in A. The colours of the traces correspond to those of the circles in A. The ‘yellow’ whisker moved out of the field of view during air puffs. The timing of the air puffs is indicated by the two black lines. Bottom: the corresponding electrophysiological recording of a Purkinje cell. Complex spikes are indicated by an asterisk. Raster plots of the time stamps of complex spikes (C) and simple spikes (D) during 556 trials. Time t= 0.0 s indicates the onset of the air puffs. Peri-stimulus histograms of the complex spikes (E) and simple spikes (F). In this Purkinje cell, the complex spike response was mono-phasic, while the simple spike response was tri-phasic: an early-positive response (1) was followed by an early-negative response (2). Finally, there was a late-positive response (3). The data in panels A–F originate from the same experiment. G, average peri-stimulus of histogram of all 16 Purkinje cells showing a complex spike response to air puff whisker stimulation. Inset: of the 24 Purkinje cells recorded in crus 1 and crus 2, 16 showed a complex spike response to whisker stimulation. H, average peri-stimulus of histogram of all 18 Purkinje cells showing a simple spike response to air puff whisker stimulation. Inset: of the 24 Purkinje cells recorded in crus 1 and crus 2, 18 showed a simple spike response to whisker stimulation.

Figure 3. Sensory induced simple spike inhibition can be independent of climbing fibre activity in awake mice

A, peri-stimulus histograms of two representative Purkinje cells. Both Purkinje cells responded to repeated air puff stimulation (at t= 0.0 ms) with complex spike (blue) and simple spike (red) responses. The left Purkinje cell received 338 stimuli, the right Purkinje cell 490. They were recorded from the same mouse and for a large part simultaneously. B, raster plots of simple spike times of all trials with a complex spike response, i.e. with a complex spike during the responsive period (cf. A). C, raster plot of an equal amount of trials as in B, but now from trials without a complex spike response. It can be seen that the simple spike inhibition seen in B is still present in the left Purkinje cell, but not in the right Purkinje cell. Thus, the complex spike pause cannot be the sole cause of sensory-induced simple spike inhibition in the left Purkinje cell. D, peri-stimulus histograms of the simple spike times, comparing the trials with (filled boxes) and without (open boxes) a complex spike response. In 6 out of 8 Purkinje cells, the whisker stimulation-induced simple spike inhibition was not correlated to the complex spike response, when studied at a trial-by-trial base (left column), as it was in the other 2 Purkinje cells (right column).

Figure 5. Direction selectivity of Purkinje cell responses in anaesthetised mice

A, complex spike responses depended on the direction of whisker movement. Eight peri-stimulus histograms show the complex spike responses for each direction of a typical Purkinje cell. For this Purkinje cell, stimulation of whisker C2 in the dorsal direction had the largest impact. The polar plot shows the number of complex spikes during the response period (the light blue bars in the peri-stimulus histograms) per direction (light blue line), as well as the complex spikes during the same time interval before the onset of the whisker movement (dark blue line). B, the dorso-caudal direction was most often found to be the ‘favourite’ direction of the Purkinje cells. The arrow indicates the number of Purkinje cells for which each direction evoked the largest complex spike response. C, while complex spike responses depended on the direction of the movement, simple spike responses did not. This proved to be true for all four kinds of simple spike responses: early-positive, early-negative, late-positive and late-negative responses. An ‘octogonality’ value (see Methods section) of 1.0 implies no direction selectivity at all. For this analysis, we measured complex spike responses from 15 Purkinje cells, and simple spike responses from 7, 9, 7 and 5 Purkinje cells for the early-positive, early-negative, late-positive and late-negative responses, respectively.

Figure 6. Complex spikes encode the amplitude and speed of whisker movement in anaesthetised mice

A, complex spike peri-stimulus histograms of a representative Purkinje cell recording during which the amplitude and time to maximal deflection of the whisker movement were systematically varied. Going from left to right, each next column represents data obtained with a doubled stimulus amplitude. Going from top to bottom, each next row represents data obtained with stimuli using half the time to reach the maximal deflection. Consequently, the diagonals (from bottom-left to top-right) have the same angular speed (ω). The green lines show the programmed whisker trajectories. In each histogram, the red line depicts the threshold (upper border of the 99% confidence interval). In the histograms marked with a coloured background, the response threshold was exceeded. From this experiment, it can be concluded that complex spike firing depends on a velocity threshold rather than an amplitude threshold. B, for shorter time intervals, the increase in time to maximal angular velocity (ω_max) is linear with the increase in latency time. For longer time intervals, the latency does not increase linearly anymore. This indicates that for relatively fast movements, the complex spikes encode the maximal angular velocity, but for very long-lasting movements, complex spikes also occur before the maximal angular velocity is reached. C, doubling both the amplitude of a movement and the time to complete that movement does not result in a change in ω_max. Accordingly, the number of complex spikes evoked was unchanged (left bar). Doubling the amplitude, but keeping the time interval constant, was more effective in evoking complex spikes (middle bar) than halving the time, but maintaining the same amplitude (right bar). The analysis shown in B and C is based on the data from 7 Purkinje cells.

Figure 8. Anatomical connections to crus 1 and crus 2

A, in 5 animals, we made in total 7 injections with retrograde tracers (4 gold–lectin and 3 cholera toxin b subunit (CTb)). The injection areas (which were ∼500 μm deep) are shown on a cross-section of the cerebellum. B, enlarged image of the right hemisphere. For each injection spot, we established in which sagittal zone(s) it was located on the basis of staining in the inferior olive. The grey lines indicate the most likely locations of the sagittal zones in crus 1 and crus 2. C and D, two representative images of the staining pattern in the inferior olive. The white lines delineate the contralateral inferior olive, the coloured lines indicate the stained area. Note that the colour coding corresponds to that of the injection sites in panels A and B. Gold–lectin staining can be recognized as bright spots, while the CTb staining has a reddish appearance. DAO, dorsal accessory olive; MAO, medial accessory olive; PO, principal olive. Microscopic images of the staining in the ipsilateral trigeminal nuclei: E shows a stained area in the ventral part of the principal trigeminal nucleus (Pr5). F shows staining in the dorsal part of Pr5. In addition, a small spot in the trigeminal motor nucleus (M5) was found. G depicts staining in the ventral part of the spinal trigeminal nucleus pars interpolaris (Sp5i). Staining in the dorsal part of Sp5i can be seen in H. I and J, microscopic images of the staining in the contralateral pontine nuclei (Pn) (RtTg, reticulo-tegmental nucleus). I comes from the same animal as E and G, J comes from the same animal as F and H. The scale bars represent 200 μm in C–J.

Figure 9. Synchronous firing in anaesthetised mice

A, raster plot showing the occurrence of complex spikes in 7 Purkinje cells recorded simultaneously. Several Purkinje cell pairs showed a marked correlation in complex spike firing. Two pairs have been highlighted here: PC1 vs. PC5 and PC4 vs. PC7. Complex spikes that occurred within 10 ms of each other are marked red or green, respectively. Complex spikes that occurred within 100 ms of each other are marked dark red and dark green, respectively. B, relative locations of the recording electrodes used for the recordings in A. The inter-electrode distance is 305 μm (heart-to-heart) in the x- and y-direction. C, cross-correlogram of the complex spikes of PC1 vs. those of PC5 (2 ms bins). Inset: enlargement of the middle part (from −500 ms to +500 ms). The black line indicates the average number of complex spikes during the interval from −3 to −2 s. The green line indicates the threshold (mean + 5 s.d.). D, as for C but now for the pair PC4 vs. PC7. E, the synchrony indices for the two pairs shown in C and D and for the pair PC1 vs. PC3 (that did not show any correlation in complex spike firing). F, in total, 295 pairs of Purkinje cells were compared. In 42% of these, the patterns of complex spike firing were significantly correlated. About half of these pairs showed synchronous firing within 10 ms, with a part showing complex spike synchrony within 5 or even within 2 ms. G, the timing of the maximal peak in the cross-correlogram (y-axis) increased with larger (Euclidean) distances between the two Purkinje cells of each pair. Black line: linear regression line. H, as for G but now for the difference in depth. Purkinje cells that are located at a deeper location tended to fire before more superficial Purkinje cells. *P < 0.05 (linear regression). I, synchronous simple spike firing in a Purkinje cell pair of which both Purkinje cells responded to stimulation of the same whisker (10 ms bins). J, the centre part of the histogram in I, but now with 2 ms bins (green line). The red line indicates the histogram of the same experiment, but without the 500 ms following each whisker stimulation. Note that the y-axis is normalized to correct for changes in absolute number of simple spikes between both conditions. K, of all 20 Purkinje cell pairs of which both cells responded with a simple spike response to stimulation of the same whisker(s), 9 pairs showed simple spike synchrony (right). Of the 45 other pairs tested, only 5 showed simple spike synchrony (left). P < 0.01 (Fisher's exact test)

Figure 10. Functional ensembles have an increased tendency to fire complex spikes in synchrony in anaesthetised mice

A, joined peri-stimulus histogram (JPSTH) of two Purkinje cells that both showed a strong complex spike response upon whisker stimulation. The whisker movement is shown in green. The JPSTH of the complex spikes of the two simultaneously recorded Purkinje cells (upper right) is composed of a trial-by-trial correlation of the raster plots of the two Purkinje cells. The peri-stimulus histograms of the two Purkinje cells are also shown. The histogram in the lower left corner is the histogram over the bins on the 45 deg line in the JPSTH, normalized for the firing rate per bin. It can be seen that the degree of synchrony does not vary to a large extent over the period, which indicates that the complex spike synchrony is not significantly larger during whisker movement than during periods of rest. B, cross-correlograms of the Purkinje cell pair depicted in A. The green line is the cross-correlogram of the whole recording, the black line is constructed with the omission of the 500 ms following the onset of stimulation (see the black and blue lines in the histogram in the lower left corner in A). The y-axis has been corrected for the different absolute numbers of complex spikes for the two conditions. C, synchrony indices for the whole trace (green line) and for the trace without the 500 ms following movement onset (black line). For comparison, the brown line shows the synchrony indices after a random shuffling of the inter-spike intervals (ISIs). The data in the panels A, B and C originate from the same Purkinje cell pair. D, Purkinje cells that both show a strong modulation in complex spike firing (>5 s.d. of baseline rate, ‘strong co-modulation’) have a strongly increased chance to fire in a correlated or even a synchronous manner. Purkinje cell pairs of which both Purkinje cells showed a significant complex spike response to stimulation of the same whisker(s) (>3 s.d., but not both >5 s.d., ‘weak co-modulation’) did show an increase in correlated firing, but hardly of truly synchronous firing. *P < 0.01 (as compared to fraction of non-co-modulating Purkinje cell pairs, Fisher's exact test). E, Purkinje cell pairs showing strong complex spike co-modulation were located in a sagittal band. Each line connects the surface-projected locations of two Purkinje cells showing strong complex spike co-modulation. F, as for E but for the simple spikes. Simple spike co-modulation is predominantly found along the transversal axis.