Long-Lasting Response Changes in Deep Cerebellar Nuclei in vivo Correlate With Low-Frequency Oscillations

Abstract

The deep cerebellar nuclei (DCN) have been suggested to play a critical role in sensorimotor learning and some forms of long-term synaptic plasticity observed in vitro have been proposed as a possible substrate. However, till now it was not clear whether and how DCN neuron responses manifest long-lasting changes in vivo. Here, we have characterized DCN unit responses to tactile stimulation of the facial area in anesthetized mice and evaluated the changes induced by theta-sensory stimulation (TSS), a 4 Hz stimulation pattern that is known to induce plasticity in the cerebellar cortex in vivo. DCN units responded to tactile stimulation generating bursts and pauses, which reflected combinations of excitatory inputs most likely relayed by mossy fiber collaterals, inhibitory inputs relayed by Purkinje cells, and intrinsic rebound firing. Interestingly, initial bursts and pauses were often followed by stimulus-induced oscillations in the peri-stimulus time histograms (PSTH). TSS induced long-lasting changes in DCN unit responses. Spike-related potentiation and suppression (SR-P and SR-S), either in units initiating the response with bursts or pauses, were correlated with stimulus-induced oscillations. Fitting with resonant functions suggested the existence of peaks in the theta-band (burst SR-P at 9 Hz, pause SR-S at 5 Hz). Optogenetic stimulation of the cerebellar cortex altered stimulus-induced oscillations suggesting that Purkinje cells play a critical role in the circuits controlling DCN oscillations and plasticity. This observation complements those reported before on the granular and molecular layers supporting the generation of multiple distributed plasticities in the cerebellum following naturally patterned sensory entrainment. The unique dependency of DCN plasticity on circuit oscillations discloses a potential relationship between cerebellar learning and activity patterns generated in the cerebellar network.

Keywords: cerebellum; deep cerebellar nuclei; in vivo electrophysiology; oscillations; plasticity.

FIGURE 1

Extracellular recordings from DCN units in vivo. (A) Schematic representation of the main pathways activated by air puff stimulation of the peri-oral region in mice: the trigemino-cerebellar (solid red line) and thalamo-cortical-pontine (dashed red line) pathways. The region in the gray box is expanded at the right to show the main circuit elements relevant to DCN neuron activity. MI, primary motor cortex; SI, primary sensory cortex; Th, thalamus, PN, pontine nuclei; TN, trigeminal nucleus, DCN, deep cerebellar nuclei; GrC, granule cells; MLIs, molecular layer interneurons; PC, Purkinje cell; IO, inferior olive; pf, parallel fibers; mf, mossy fibers; cf, climbing fiber; t.s., tactile stimulation. MEA, multi-electrode array (Eckhorn matrix, see section “Materials and Methods” for details). (B) Toluidine blue stained coronal cerebellar slice showing the electric lesion (arrow) made by the recording electrode in the fastigial nucleus. (C) Two single-unit recordings showing a burst and a pause in response to tactile stimulation (arrow). The raster plots show the spike discharge during ∼2 s recordings and its change caused by tactile stimulation in 300 consecutive trials.

FIGURE 2

Bursts and pauses in DCN unit responses to tactile stimulation. (A) Example of PSTHs obtained from DCN units showing different responses to tactile stimulation (arrows): double burst (5 ms-bin), burst-pause (5 ms-bin), and pause-burst (15 ms-bin). Red dashed lines show the basal discharge frequency. The scale bars in the inset on top are 5 sp/bin and 25 ms. (B) In the pool of responses starting with a burst, two groups were discriminated using cluster analysis (k-means) on peak latency and burst duration. This results in the identification of early and late peaks, whose latencies are compatible with inputs from the trigeminal and cortical pathways conveying sensory stimuli to the cerebellum (cfr. Figure 1A). (C) Characterization of pause-burst responses. A positive correlation was found between rebound-peak amplitude and pause depth [R² = 0.87, Fisher’s F-test p(F) = 0.001, n = 8].

FIGURE 3

Pharmacological and optogenetic manipulations of burst and pause responses. (A) Example of PSTHs from a DCN unit showing a burst as initial response (left), that was prevented (right) by excitatory transmission blockers (NBQX and D-APV) injection in the nucleus (red dashed lines show the basal discharge frequency). The gray dashed rectangles show the areas that are overlapped in the inset. The histogram shows the % of the response, whether PSTH peaks or pauses, left after blockers injection, compared to control (n = 4 for both; paired Student’s t-test; ^∗p < 0.05). (B) Example of PSTHs from a DCN unit showing burst-pause response before (left) and during (right) optogenetic stimulation of the molecular layer (the blue rectangle showing the time and duration of laser activation; red dashed lines show the basal discharge frequency). The histograms on the right show the percent change on the peak amplitude of the excitatory response and on the pause area (obtained by multiplying pause depth and duration) during optogenetic stimulation compared to control, in the single units recorded. The gray shadows show the average % change observed at the same time points in the stability controls (see section “Materials and Methods”). Note that peak amplitude is not affected, while the pause is significantly modified.

FIGURE 4

Stimulus-induced oscillations in DCN units. (A) PSTH obtained from a DCN unit showing low frequency oscillation following a burst-pause response. (B) Autocorrelogram obtained from the unit shown in (A) (oscillation frequency 3.15 Hz; magnitude 0.16). (C) The magnitude and frequency of oscillations deriving from the autocorrelation analysis shown in (B) were plotted for each unit. The k-means clustering revealed two groups of data, characterized by high frequency – low magnitude oscillations (black symbols) and low frequency – high magnitude oscillations (green and red symbols, for pause-first and burst-first responses respectively). The units in which the TSS was delivered are represented as circles, while those in which the TSS was not delivered are represented as squares. Gray filled symbols are used for the units in which pharmacology was applied, while blue-filled circles are used for the units in the optogenetics experiments. The arrow indicates the unit shown in (A,B). (D) Relationship between stimulus-induced oscillation frequency and spontaneous firing frequency for the low frequency – high magnitude oscillation units in (C). The linear fitting shows no evident trend [R² = 0.03, Fisher’s F-test p(F) = 0.89]. (E) Relationship between magnitude of stimulus-induced oscillations and spontaneous firing frequency for the low frequency – high magnitude oscillation units in (C). The linear fitting suggests a positive trend [R² = 0.25, Fisher’s F-test p(F) < 0.08]. In (C–E), the data points are divided into burst-first and pause-first units, and circles represent the units that were further used for plasticity induction (see Figure 5, 6). (F) The histogram shows the percent change in stimulus-induced oscillation frequency during optogenetics in the same units reported in (C–E). The gray shadow shows the average percent change observed at the same time points in the stability controls (see section “Materials and Methods”).

FIGURE 5

Long-lasting changes induced by TSS in DCN unit responses. (A) Example of PSTHs illustrating the peak changes (SR-P or SR-S) induced by TSS in DCN units of the burst-first category. The histogram shows the average percent changes in PSTH peak amplitude for all the units showing SR-P, SR-S, or stability controls (no TSS; paired Student’s t-test; ^∗p < 0.05, ^∗∗p < 0.01). (B) Example of PSTHs illustrating the pause changes (SR-P or SR-S) induced by TSS in DCN units of the pause-first category. The histogram shows the average percent changes in PSTH peak amplitude for all the units showing SR-P, SR-S, or stability controls (no TSS; paired Student’s t-test; ^∗∗p < 0.01). (C) Average time-course of peak (in burst-first units, left) and pause (in pause-first units, right) amplitude percent changes normalized to the control period before TSS (dashed line) in the units showing SR-P, SR-S and in the stability group (TSS not delivered). Note that the response changes for peaks and pauses differed significantly from the stability controls in the first 15 min after the TSS.

FIGURE 6

Frequency-dependence of long-lasting changes after TSS. (A) The plot shows the distribution of peak amplitude changes after TSS in burst-first units with respect to stimulus induced oscillation frequency. The Lorentzian fitting [R² = 0.83; Fisher’s F-test p(F) = 0.01] shows a peak at 9.2 Hz. (B) The plot shows the distribution of pause amplitude changes after TSS in pause-first units with respect to stimulus induced oscillation frequency. The Lorentzian fitting [R² = 0.78; Fisher’s F-test p(F) = 0.02] shows a peak at 5.5 Hz. Both in (A,B), open symbols identify the same low-frequency units reported in Figure 4 and filled symbols are the average values ( ± SEM) of high frequency oscillation units. Both in (A,B), the gray area shows the 95% confidence interval of the fitting.

FIGURE 7

The impact of optogenetic stimulation on long-lasting response changes. (A) The plot shows the Lorentzian fitting as Figure 6A, with the gray area showing the 95% confidence interval. Note that the data-points representing burst-first units in which the TSS was paired with optogenetics fall far outside the confidence interval. (B) The plot shows the Lorentzian fitting as in Figure 6B, with the gray area showing the 95% confidence interval, extrapolated beyond the last point of control to compare new data. Note that the data-points representing pause-first units in which the TSS was paired with optogenetics fall far outside the confidence interval (except for the two points representing the units in which optogenetics did not show any effect; crossed circles). (C) The histogram shows the average distance of the optogenetics data points from the fitting curves in (A,B). The gray shadow shows the average distance of control data in Figure 6 from the same fitting curves.