Neurons of the inferior olive respond to broad classes of sensory input while subject to homeostatic control

Abstract

Key points: Purkinje cells in the cerebellum integrate input from sensory organs with that from premotor centres. Purkinje cells use a variety of sensory inputs relaying information from the environment to modify motor control. Here we investigated to what extent the climbing fibre inputs to Purkinje cells signal mono- or multi-sensory information, and to what extent this signalling is subject to recent history of activity. We show that individual climbing fibres convey multiple types of sensory information, together providing a rich mosaic projection pattern of sensory signals across the cerebellar cortex. Moreover, firing probability of climbing fibres following sensory stimulation depends strongly on the recent history of activity, showing a tendency to homeostatic dampening.

Abstract: Cerebellar Purkinje cells integrate sensory information with motor efference copies to adapt movements to behavioural and environmental requirements. They produce complex spikes that are triggered by the activity of climbing fibres originating in neurons of the inferior olive. These complex spikes can shape the onset, amplitude and direction of movements and the adaptation of such movements to sensory feedback. Clusters of nearby inferior olive neurons project to parasagittally aligned stripes of Purkinje cells, referred to as 'microzones'. It is currently unclear to what extent individual Purkinje cells within a single microzone integrate climbing fibre inputs from multiple sources of different sensory origins, and to what extent sensory-evoked climbing fibre responses depend on the strength and recent history of activation. Here we imaged complex spike responses in cerebellar lobule crus 1 to various types of sensory stimulation in awake mice. We find that different sensory modalities and receptive fields have a mild, but consistent, tendency to converge on individual Purkinje cells, with climbing fibres showing some degree of input-specificity. Purkinje cells encoding the same stimulus show increased events with coherent complex spike firing and tend to lie close together. Moreover, whereas complex spike firing is only mildly affected by variations in stimulus strength, it depends strongly on the recent history of climbing fibre activity. Our data point towards a mechanism in the olivo-cerebellar system that regulates complex spike firing during mono- or multi-sensory stimulation around a relatively low set-point, highlighting an integrative coding scheme of complex spike firing under homeostatic control.

Keywords: Purkinje cell; cerebellum; climbing fibre; homeostatic mechanisms; inferior olive; sensory integration.

Figure 1. Sensory pathways carrying facial input to the cerebellar cortex

A, scheme of the main routes conveying facial tactile input via the climbing fibre pathway to cerebellar Purkinje cells (PC). Climbing fibres, which cause complex spike firing in Purkinje cells, exclusively originate from the inferior olive. The inferior olive, in turn, is directly innervated by neurons from the trigeminal nuclei as well as indirectly via thalamo‐cortical pathways that project to the inferior olive mainly via the nuclei of the mesodiencephalic junction (MDJ). The MDJ itself also receives direct input from the trigeminal nuclei. See main text for references. B, in vivo two‐photon Ca²⁺ imaging was performed to characterize Purkinje cell complex spike responses to sensory stimulation in the medial part of crus 1. Purkinje cells were detected using independent component analysis and the position of a Purkinje cell dendrite (yellow area on the right) within a field of view is shown in the inset. At the end of each recording session, the brain was removed and the location of the dye injection in medial crus 1 was confirmed through ex vivo epifluorescence imaging (black circle). The white rectangle indicates the approximate recording location. C, complex spikes that were triggered by climbing fibre activity were retrieved from fluorescence traces of individual Purkinje cells. A representative trace obtained from the Purkinje cell dendrite illustrated in B is shown together with the detected complex spikes (grey lines). The light blue episode is enlarged in D. Complex spikes were detected by the combination of a threshold and a template matching algorithm. Only events with a sharp rising phase were accepted as complex spikes. In the 60 s interval shown in C, there was one event with a slower rise time (see arrow in D), as indicated at a larger time scale in E. The events in E are scaled to peak.

Figure 2. Whisker stimulation evokes complex spike responses in cerebellar crus 1

A, complex spikes elicit large increases in the Ca²⁺ concentration within Purkinje cell dendrites that can be resolved using in vivo two‐photon microscopy in combination with a fluorescent Ca²⁺ indicator. An example of a field of view with 19 identified Purkinje cell dendrites located in the medial part of crus 1 is shown with each individual dendrite denoted by a number and a unique colour. This recording was made in an awake mouse. B, fluorescence traces of each of these dendrites show distinct Ca²⁺ transient events, which are to a large extent associated with air puff stimulation of the facial whiskers (times of stimulation indicated by vertical lines). The boxed area is enlarged in E. C, summed fluorescence trace composed of all 19 individual traces emphasizing the participation of many Purkinje cells to the stimulus‐triggered responses. D, after complex spike extraction, a clear relationship between stimulus and activity was observed as illustrated by summing, for each frame, the number of complex spikes observed over all dendrites. The vertical scale bar corresponds to the simultaneous activity of five Purkinje cells. E, enlargement of the boxed area in B. The air puffs were delivered once every second. F, peri‐stimulus time histogram of a Purkinje cell dendrite (marked as number 7 in A and B) in response to air puff stimulation to the ipsilateral whiskers (154 trials). The bin size (40 ms) corresponds to the acquisition frame rate (25 Hz). G, normalized stacked line graph of the Purkinje cells in this field of view showing that every cell contributed to the overall response. The Purkinje cells are ranked by their maximal response and the data are normalized so that the top line reflects the average frequency per bin. Cell no. 5 (dashed line) had a relatively poor signal‐to‐noise ratio during later parts of the recording (see Fig. 3), but it had nevertheless a complex spike response profile that was indistinguishable from the other cells. The colours match those in A and B. Inset: in total, 102 out of 117 cells analysed (87%) were responsive to whisker air puff stimulation (peak response exceeded average + 3 SD of the pre‐stimulus interval). H, median fluorescence traces of the trials with (red) and without (black) complex spikes fired during the first 200 ms after air puff onset. In the absence of complex spike firing, only a very small increase in fluorescence was observed, indicating that most of the change in fluorescence was associated with complex spike firing. Note the longer time scale than in F and G. The lines indicate the medians and the shaded areas the interquartile ranges of the 19 Purkinje cells in this field of view.

Figure 3. Complex spike detection

A, from the same experiment as illustrated in Fig. 2, we randomly selected four Purkinje cells of which all trials with (top) and without (bottom) a complex spike within 200 ms of stimulus onset are shown. The signals are scaled to the maximum of the median complex spike responses. The fat coloured lines indicate the medians, with the same colour code as in Fig. 2. Trials without a complex spike response can still show increased fluorescence, but the kinetics of the non‐complex spike response differed from those of the complex spike responses. Note that the raw trace of cell 5 had a period with increased noise levels, making it the least reliable cell of this recording. Nevertheless, cell 5 had a complex spike response profile that was very similar to those of the other cells (see dashed line in Fig. 2 G). Cells (in other recordings) that had a signal‐to‐noise ratio worse than for cell 5 of this recording were excluded from analysis. B, to better characterize rise times, we made recordings with a smaller field of view, but a higher temporal resolution (100 Hz). Shown are 60 s traces of 12 simultaneously recorded Purkinje cells. C, of four randomly selected Purkinje cells, the first 100 events were plotted and superimposed (D). The coloured lines in D indicate the medians. Virtually all events had a rise time of 10 or 20 ms (one or two frames).

Figure 4. Purkinje cells in crus 1 respond to various types of sensory stimulation

A, tactile stimuli were presented to four facial regions in awake mice: the whisker pad (WP), the upper lip (UL), the lower lip (LL) and the cheek (Ch). Each stimulus was delivered with a piezo‐actuator that made a muted, yet audible sound which was also delivered without touch [‘sound only (SO)’]. A blue light flash generated by an LED was used as the visual stimulus. These experiments were performed in awake mice. B, to avoid interference of adjacent areas, we applied gentle touches (0.686 mN). The complex spike response ratio was much reduced relative to the strong air puff stimulation to all ipsilateral whiskers illustrated in Fig. 2. A histogram of the peak responses (expressed as Z value) of all responses to either of the four tactile stimuli demonstrates that the response strength is a continuum, showing the lack of a clear separation between ‘responsive’ and ‘non‐responsive’ Purkinje cells (998 stimulus conditions in 282 Purkinje cells). We considered Purkinje cells that showed a peak response above Z = 3 as ‘significantly responsive’ (represented with black bars), but we provide most of the analyses also for the population as a whole (e.g. Fig. 5). C, peri‐stimulus time histograms (PSTHs) of a representative Purkinje cell. The shades of grey indicate 1, 2 and 3 SD around the average. Each stimulus was repeated 154 times at 1 Hz. D, for every stimulus condition, we averaged the PSTHs for all Purkinje cells that were significantly responsive to that particular stimulus [coloured lines; medians (interquartile range)]. These were contrasted to the averaged PSTH of the other Purkinje cells (black lines). The pie charts represent the fraction of Purkinje cells significantly responsive to a particular stimulus. See also Table 1. E, peak responses of the significantly responding Purkinje cells were lowest for sound‐only and for upper lip stimulation. ^* P < 0.05; ^** P < 0.01 (post hoc tests after Kruskal–Wallis test). F, as expected for complex spike responses to weak stimulation, the latencies were relatively long and variable, but consistent across types of stimulation. Only visual stimulation (LED) had a remarkably longer latency time. ^*** P < 0.001 (post hoc tests after Kruskal–Wallis test for LED vs. whisker pad, upper lip, lower lip and cheek and P < 0.05 compared to sound only).

Figure 5. Variations in response strength

A, stacked line plots illustrating the peri‐stimulus time histograms of all Purkinje cells recorded under either of the six indicated stimulus conditions. The cells are ordered based upon their peak responses (calculated as Z value) during the 200 ms interval following stimulus onset, with the cell with the lowest response at the bottom of each graph. The grey lines indicate cells with a peak response deemed not significant (Z < 3) and the coloured lines indicate the significant responses (Z > 3). The graphs are normalized so that the upper line depicts the average of all cells. As shown in Fig. 4 B, we cannot discriminate between responsive and non‐responsive cells in an all‐or‐none fashion. Instead, the cells form a continuum that ranges from non‐responsive to highly responsive. As this way of plotting relies on the numerical average, a skewed distribution can put emphasis on a relatively small group of cells. The Purkinje cell responses are therefore also compared using cumulative histograms (B) using the same colour scheme as in A. From this representation, it is confirmed that whisker pad and lower lip stimulation yield the strongest responses, while visual stimulation (LED) recruits a few cells with a relatively strong response, increasing the numerical average (see A). Here, we tested the complete distributions (^*** P < 0.001; Kruskal–Wallis test). Pair‐wise comparisons of all stimulus conditions are presented in Fig. 6.

Figure 6. Convergence of sensory input on Purkinje cells

A, to test whether sensory inputs converge on individual Purkinje cells in awake mice, we made pair‐wise comparisons of the response amplitudes to two different stimuli per Purkinje cell (scatter plots). For all possible combinations, we found a positive slope of the linear regression analysis. For the majority of combinations, the correlation between response strengths was highly significant: ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001 (Pearson correlation with Benjamini–Hochberg correction for multiple comparisons). Only upper lip vs. sound only and upper lip vs. visual stimulation were not significantly correlated. For this analysis, we included all Purkinje cells, whether they had a statistically significant response or not. The red dotted lines indicate a Z‐score of 3, which we set as the threshold for significance (cf. Fig. 4 B). They grey arrows indicate the fraction of observations above and below the unity line (grey dotted line). The relative strengths of each stimulus combination were compared in a pairwise fashion (Wilcoxon tests with Benjamini–Hochberg correction for multiple comparisons): ^& P < 0.05, ^&& P < 0.01 and ^&&& P < 0.001. B, we performed a similar analysis focusing only on statistically significant responses (Venn diagrams). Again, all combinations had a positive Z score (as evaluated by a bootstrap method; see Methods), indicating more than expected convergence. The diameter of each circle indicates the fraction of Purkinje cells showing a significant response to that particular, colour‐coded stimulus. The size of the bar represents the Z score of the overlapping fraction. C, the same for the combinations of three tactile stimuli. Overall, sensory streams tended to converge, rather than diverge, on Purkinje cells. ^# P < 0.10; ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001 (Z test with Benjamini–Hochberg correction).

Figure 7. Sound‐only stimulation systematically recruited fewer complex spikes than tactile stimulation

Fluorescence traces of 26 Purkinje cells in a field of view during whisker pad (A) and sound‐only (B) stimulation. The moments of stimulation (at 1 Hz) are indicated by the vertical lines. Note that the sound‐only stimulation involved the sound of the mechanical device delivering tactile stimuli. Overall, whisker pad stimulation triggered more complex spike responses than sound‐only stimulation, as illustrated by the sum of the events (C). The boxed part (10 s) is enlarged in D. E, peri‐stimulus time histograms (PSTHs) of four randomly selected Purkinje cells from the experiment illustrated in A and B. In each, the response to whisker pad stimulation was stronger than to sound‐only stimulation, which is also reflected in the median of the PSTHs of all 26 Purkinje cells (F) and the median difference between whisker pad and sound‐only stimulation (G). The shaded areas indicate the interquartile range.

Figure 8. Purkinje cell excitation depends partially, but not completely, on generic sensory input

For the 188 Purkinje cells that received all four tactile stimuli, we calculated the full (A) and partial (B) correlation between the peak responses (in Z scores) of the four different tactile stimuli. This largely confirms the pair‐wise correlations illustrated in Fig. 6 A. Note, however, that the partial correlations are less pronounced than the full correlations, suggesting the existence of a common component reflecting general excitability, not specific for stimulus location. C, principal component analysis confirmed that a part of the observed variance can indeed be explained by a common factor, as the first principal component of the experimental data is significantly larger than that of bootstrapped data. Error bars indicate the 1–99% confidence interval. The inset shows the relative contributions of the different stimuli to the first principal component. The relatively weak stimuli (lower lip and cheek) were more in tune with the general excitability than the stronger stimuli (whisker pad and upper lip). D, scatter plot showing the correlation between average response strength to the four tactile stimuli vs. the four response strengths per Purkinje cell. The Purkinje cells on the left were insensitive to whatever tactile stimulus we presented, while those on the right were sensitive to any tactile stimulus, but showed a bias towards one or a few stimulus locations. This bias becomes more obvious when plotting the minimum and maximum response per Purkinje cell (E). Inset: a strong correlation between the minimum and maximum response strength per Purkinje cell. r values come from Spearman's correlation test. Similar plots were made comparing the sound‐only (F) and LED (G) stimulation vs. the four tactile stimuli. ^* P < 0.05 and ^*** P < 0.001.

Figure 9. Stimulus strength has only a minor impact on complex spike responsiveness

A, movements of all large facial whiskers were performed using a piezo‐actuator at three different speeds (weak: 1 mm displacement in 62 ms; moderate: 2 mm displacement in 31 ms; strong: 4 mm displacement in 16 ms). The stimulus sequence was randomly permuted. The recordings were made in awake mice. B, field of view with 24 identified and colour‐coded Purkinje cells (left) and their corresponding fluorescence traces (right). Stimuli were presented every 2 s and in between trials the laser illumination was briefly blocked to avoid photobleaching. Note that the periods without laser illumination are not drawn to scale. The vertical shaded areas indicate stimulus duration (which was inverse with the stimulus strength). C, summed fluorescence trace composed of all 24 individual traces showing that not all trials evoked ensemble‐wide responses. Some spontaneous, inter‐trial activity was also observed. D, median number of complex spikes per frame (of 40 ms) per trial (shaded areas: interquartile ranges) for the three stimulus strengths show little difference for the weak and moderate stimulation. The time course and amplitude (1–4 mm) of the three stimuli is shown schematically at the bottom of the graph. Strong stimulation elicited about 30% more complex spikes, as evident from the peak responses for each stimulus intensity. E, box plots showing the response strength for the 209 significantly responsive Purkinje cells (out of 340 Purkinje cells that were measured in this way). F, response rates for all Purkinje cells that showed an increase in response rate with increased stimulus intensity. Only a few cells stand out in that they show a strong response that consistently increases with stimulus strength (lines on top). G, the same for the Purkinje cells that showed a decrease in response strength with increasing stimulus intensity. H, histogram of the differences in peak responses between the strong and the weak stimuli of all 340 recorded Purkinje cells. W = weak; M = moderate; S = Strong; ^*** P < 0.001 (Friedman's test).

Figure 10. Purkinje cells encoding the same stimulus tend to be spatially grouped

Schematic drawing of a field of view with 26 Purkinje cells organized in the medio‐lateral direction of crus 1 in an awake mouse. The colour of each Purkinje cell corresponds to the maximal response to whisker pad (A) or upper lip (B) stimulation. Purkinje cells with a filled soma had a peak response with a Z score > 3 and were considered to be statistically significant, in contrast to those with an open soma. Responsive and non‐responsive cells are generally intermingled, but a group of ‘strong responders’ can be observed for whisker pad stimulation (red rectangle). C, the anecdotal data in A suggest the presence of clusters of Purkinje cells encoding specific stimuli. For this to be the case, one would expect that neighbouring Purkinje cells have roughly similar response strengths. We found that this assumption does not hold as the differences in response strengths of neighbours could not be discriminated from randomly selected cells in the same recording if all Purkinje cells are considered (compared with bootstrap analysis based upon randomly chosen cell pairs within each field of view: all P > 0.8; Z test). Data are represented in violin plots, with the grey lines indicating the 10^th, 25^th, 50^th, 75^th and 90^th percentiles. D, when considering only the Purkinje cells with statistically significant responses, spatial grouping does occur. For each stimulus type, the black portion of the left bar indicates the fraction of Purkinje cells showing a significant response to that stimulus. The filled portion of the right bar indicates the fraction of the neighbours (always on the medial side) of these significantly responsive Purkinje cells that were also significantly responsive. As can be seen, this fraction is always substantially larger than the fraction of significantly responsive Purkinje cells, indicating a tendency of similar Purkinje cells to group together. Statistical significance was tested by comparing the fraction of Purkinje cells with statistically significant responses and the fraction of neighbours of Purkinje cells with statistically significant responses that showed statistically significant responses as well (after correction for border effects) using Fisher's exact test and after Benjamini–Hochberg correction for multiple comparisons: ^* P < 0.05 and ^*** P < 0.001.

Figure 11. Purkinje cell responses to single whisker stimulation show weak clustering

A, to investigate smaller receptive fields, we sequentially stimulated five of the large facial whiskers. To avoid interference with other whiskers during active movement, we performed these experiments under ketamine/xylazine anaesthesia. Most Purkinje cells, if responsive to single‐whisker stimulation, responded only to one of the five whiskers (B). This is illustrated by five peri‐stimulus time histograms (PSTHs) from a single, representative Purkinje cell. This particular cell was sensitive to stimulation of the C3 whisker only. The average and 3 SD of the baseline firing are indicated (dashed line and grey area). C, Purkinje cells that responded to more than one whisker were typically responsive to the more anterior whiskers (see also Table 5). The widths of the lines indicate the Z value of the occurrence of multiple responses per cell. D, two recording spots, in close proximity in crus 1 of the same animal, with the identified Purkinje cell dendritic trees. For each dendrite, the colour indicates the whisker(s) to which it was responsive (see legend below with grey denoting the absence of a statistically significant response). E, for each of the two recording sites, the medians of the responsive and the non‐responsive Purkinje cells are indicated (to the C3 whisker in the left panel and to the C2 whisker in the right panel). Note that only a single cell was responsive to C2 stimulation in recording spot 2. The shades indicate interquartile ranges. F, linear regression revealed that Purkinje cells that were surrounded by other Purkinje cells responsive to the same whisker (same colour code as in A) tended to show stronger responses to stimulation of that whisker than Purkinje cells that were more isolated. The x‐axis represents the fraction of Purkinje cells responsive to the particular whisker within the respective field of view. r = +0.52, P < 0.001.

Figure 12. Purkinje cells encoding the same response are closer together

A, to compare the extent of synchronous firing between the medio‐lateral and the antero‐posterior axes, we made recordings with a larger field of view. Identified Purkinje cell dendrites in a representative field of view colour‐coded according to their membership of one of the two cluster identified with meta k means clustering (Dunn index = 0.85). The dendrites indicated in grey could not be contributing to either of the two groups. B, heat map of the pair‐wise comparisons of the correlation between firing of the dendrites shown in A. Although two clusters were identified, it is clear that under our recording conditions, synchronous firing is not strictly related to a single micro‐zone. C, raw traces of the neurons indicated in A and B. D, as most of the variation was along the medio‐lateral axis, we continued with smaller fields of view oriented along the medio‐lateral axis. Representative field of view with segmented PC dendrites. Non‐responsive cells are depicted in shades of blue and responsive cells in shades of red during whisker pad stimulation. E, for each pair of Purkinje cells we calculated the correlation coefficient (r) during 1 Hz whisker pad stimulation. The pairs of two Purkinje cells that were both responsive to whisker pad stimulation had on average a higher level of synchrony than the pairs connecting a responsive and a non‐responsive Purkinje cell (P < 0.001; two‐dimensional Kolmogorov–Smirnov test). The pairs consisting of two non‐responsive Purkinje cells were excluded from this analysis. F, interestingly, even in the absence of sensory stimulation, the pairs of Purkinje cells that were both responsive to whisker pad stimulation maintained a higher level of synchrony than ‘heterogeneous pairs’. Thus, Purkinje cells with the same receptive field tended to fire more synchronously, even in the absence of stimulation. This analysis was expanded in the presence (G) and absence (H) of sensory stimulation for six different types of stimulation and illustrated as the median r value per distance category (six bin values of equal distance at a log scale). The shaded areas represent the interquartile ranges. ^* P < 0.05 and ^*** P < 0.001 (Kolmogorov–Smirnov tests).

Figure 13. Purkinje cells encode strong and weak sensory stimulation via synchronous firing

A, aggregate peri‐stimulus time histograms (PSTHs) show that coherent firing of complex spikes predominantly occurs following sensory stimulation. For each field of view, we calculated the number of complex spikes occurring per frame, summing those of all Purkinje cells in that field of view. Subsequently, we made aggregate PSTHs where the colour of each bin refers to the number of dendrites simultaneously active. The black fields indicate frames in which only a single dendrite was active. In this field of view, 17 Purkinje cells were measured. Of these, 17 (100%) reacted to air puff, 12 (71%) to whisker pad, 3 (18%) to upper lip, 4 (24%) to lower lip and 1 (6%) to cheek stimulation. B, based on a Poisson distribution of complex spikes over all dendrites and bins, one would expect between 0 and 3 simultaneously active dendrites (grey bars). The red bars indicate events involving more dendrites simultaneously than expected from a random distribution. Thus, the sparse firing as expected by chance is relatively constant throughout the trials, but the simultaneous activity of multiple dendrites is strongly enhanced following sensory stimulation. C, direct overlay of the aggregate PSTHs in response to air puff and lower lip stimulation showing that the strong response found after air puff stimulation comes at the expense of intertrial complex spikes (152 trials per condition). D, for equally long recordings in the presence of different types of stimulation, equal complex spike frequencies were observed as during spontaneous activity (F _2.544,20.348 = 2.561, P = 0.091, repeated‐measures ANOVA), indicating that sensory stimulation results in a temporal re‐ordering of complex spikes, rather than to the production of more complex spikes.

Figure 14. Sensory stimulation results in a temporal re‐ordering of complex spikes

A, the temporal distribution of complex spikes was compared in a pair‐wise fashion between sessions with sensory stimulation and sessions without. For this analysis, we included only Purkinje cells that displayed a statistically significant response to the stimulus involved (n = 102 for air puff, n = 45 for whisker pad and n = 27 for visual stimulation). The spontaneous recordings were analysed by creating post hoc pseudo‐stimuli at the same 1 Hz frequency as during sensory stimulation. Shown are the medians of the peri‐stimulus time histograms. The shaded areas indicate the interquartile ranges. B, the reduction in baseline firing, measured during the interval −500 to −250 ms, was significant in all cases (Wilcoxon matched‐pairs test after Benjamini–Hochberg correction for multiple comparisons). C, the larger the response amplitude, the stronger the reduction in inter‐trial firing (Pearson correlation tests after Benjamini–Hochberg correction for multiple comparisons). This analysis was performed on all Purkinje cells (n = 117 for air puff and whisker pad stimulation and n = 60 for LED stimulation; dotted lines mark the criterion for statistical significance at Z = 3). Note the differences in the x‐axis scaling with the air puff evoking relatively stronger responses. ^* P < 0.05; ^*** P ≤ 0.001.