Cerebellar plasticity underlies motor learning. However, how the cerebellum operates to enable learned changes in motor output is largely unknown. We developed a sensory-driven adaptation protocol for reflexive whisker protraction and recorded Purkinje cell activity from crus 1 and 2 of awake mice. Before training, simple spikes of individual Purkinje cells correlated during reflexive protraction with the whisker position without lead or lag. After training, simple spikes and whisker protractions were both enhanced with the spiking activity now leading behavioral responses. Neuronal and behavioral changes did not occur in two cell-specific mouse models with impaired long-term potentiation at their parallel fiber to Purkinje cell synapses. Consistent with cerebellar plasticity rules, increased simple spike activity was prominent in cells with low complex spike response probability. Thus, potentiation at parallel fiber to Purkinje cell synapses may contribute to reflex adaptation and enable expression of cerebellar learning through increases in simple spike activity.

In the cerebellar network, a precise relationship between plasticity and neuronal discharge has been predicted. However, the potential generation of persistent changes in Purkinje cell (PC) spike discharge as a consequence of plasticity following natural stimulation patterns has not been clearly determined. Here, we show that facial tactile stimuli organized in theta-patterns can induce stereotyped N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA-A) receptor-dependent changes in PCs and molecular layer interneurons (MLIs) firing: invariably, all PCs showed a long-lasting increase (Spike-Related Potentiation or SR-P) and MLIs a long-lasting decrease (Spike-Related Suppression or SR-S) in baseline activity and spike response probability. These observations suggests that tactile sensory stimulation engages multiple long-term plastic changes that are distributed along the mossy fiber-parallel fiber (MF-PF) pathway and operate synergistically to potentiate spike generation in PCs. In contrast, theta-pattern electrical stimulation (ES) of PFs indistinctly induced SR-P and SR-S both in PCs and MLIs, suggesting that tactile sensory stimulation preordinates plasticity upstream of the PF-PC synapse. All these effects occurred in the absence of complex spike changes, supporting the theoretical prediction that PC activity is potentiated when the MF-PF system is activated in the absence of conjunctive climbing fiber (CF) activity.

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.

1 2 Cerebellar plasticity underlies motor learning. However, how the cerebellum operates to enable 3 learned changes in motor output is largely unknown. We developed a sensory-driven adaptation 4 protocol for reflexive whisker protraction and recorded Purkinje cell activity from crus 1 and 2 of 5 awake mice. Before training, simple spikes of individual Purkinje cells correlated during reflexive 6 protraction with the whisker position without lead or lag. After training, simple spikes and whisker 7 protractions were both enhanced with the spiking activity now leading the behavioral response. 8 Neuronal and behavior changes did not occur in two cell-specific mouse models with impaired 9 long-term potentiation at parallel fiber to Purkinje cell synapses. Consistent with cerebellar 10 plasticity rules, increased simple spike activity was prominent in cells with low complex spike 11 response probability. Thus, potentiation at parallel fiber to Purkinje cell synapses may contribute 12 to reflex adaptation and enable expression of cerebellar learning through increases in simple spike 13 activity. 14 3 Impact statement 15 Romano et al. show that expression of cerebellar whisker learning can be 16 mediated by increases in simple spike activity, depending on LTP 17 induction at parallel fiber to Purkinje cell synapses. 18 4

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In the published article, there was an error in affiliation 2. Instead of "Brain Connectivity Center, C. Mondino National Neurological Institute, Pavia, Italy," it should be "IRCCS Mondino Foundation, Pavia, Italy." The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

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