Simple spike patterns and synaptic mechanisms encoding sensory and motor signals in Purkinje cells and the cerebellar nuclei.

Whisker stimulation in awake mice evokes transient suppression of simple spike probability in crus I/II Purkinje cells. Here, we investigated how simple spike suppression arises synaptically, what it encodes, and how it affects cerebellar output. In vitro, monosynaptic parallel fiber (PF)-excitatory postsynaptic currents (EPSCs) facilitated strongly, whereas disynaptic inhibitory postsynaptic currents (IPSCs) remained stable, maximizing relative inhibitory strength at the onset of PF activity. Short-term plasticity thus favors the inhibition of Purkinje spikes before PFs facilitate. In vivo, whisker stimulation evoked a 2-6 ms synchronous spike suppression, just 6-8 ms (∼4 synaptic delays) after sensory onset, whereas active whisker movements elicited broadly timed spike rate increases that did not modulate sensory-evoked suppression. Firing in the cerebellar nuclei (CbN) inversely correlated with disinhibition from sensory-evoked simple spike suppressions but was decoupled from slow, non-synchronous movement-associated elevations of Purkinje firing rates. Synchrony thus allows the CbN to high-pass filter Purkinje inputs, facilitating sensory-evoked cerebellar outputs that can drive movements.

Simple and complex spike responses of mouse cerebellar Purkinje neurons to regular trains and omissions of somatosensory stimuli.

Cerebellar Purkinje neurons help compute absolute subsecond timing, but how their firing is affected during repetitive sensory stimulation with consistent subsecond intervals remains unaddressed. Here, we investigated how simple and complex spikes of Purkinje cells change during regular application of air puffs (3.3 Hz for ∼4 min) to the whisker pad of awake, head-fixed female mice. Complex spike responses fell into two categories: those in which firing rates increased (at ∼50 ms) and then fell [complex spike elevated (CxSE) cells] and those in which firing rates decreased (at ∼70 ms) and then rose [complex spike reduced (CxSR) cells]. Both groups had indistinguishable rates of basal complex (∼1.7 Hz) and simple (∼75 Hz) spikes and initially responded to puffs with a well-timed sensory response, consisting of a short-latency (∼15 ms), transient (4 ms) suppression of simple spikes. CxSE more than CxSR cells, however, also showed a longer-latency increase in simple spike rate, previously shown to reflect motor command signals. With repeated puffs, basal simple spike rates dropped greatly in CxSR but not CxSE cells; complex spike rates remained constant, but their temporal precision rose in CxSR cells and fell in CxSE cells. Also over time, transient simple spike suppression gradually disappeared in CxSE cells, suggesting habituation, but remained stable in CxSR cells, suggesting reliable transmission of sensory stimuli. During stimulus omissions, both categories of cells showed complex spike suppression with different latencies. The data indicate two modes by which Purkinje cells transmit regular repetitive stimuli, distinguishable by their climbing fiber signals.NEW & NOTEWORTHY Responses of cerebellar Purkinje cells in awake mice form two categories defined by complex spiking during regular trains of brief, somatosensory stimuli. Cells in which complex spike probability first increases or decreases show simple spike suppressions that habituate or persist, respectively. Stimulus omissions alter complex spiking. The results provide evidence for differential suppression of olivary cells during sensory stimulation and omissions and illustrate that climbing fiber innervation defines Purkinje cell responses to repetitive stimuli.

Cerebellar modulation of synaptic input to freezing-related neurons in the periaqueductal gray.

Innate defensive behaviors, such as freezing, are adaptive for avoiding predation. Freezing-related midbrain regions project to the cerebellum, which is known to regulate rapid sensorimotor integration, raising the question of cerebellar contributions to freezing. Here, we find that neurons of the mouse medial (fastigial) cerebellar nuclei (mCbN), which fire spontaneously with wide dynamic ranges, send glutamatergic projections to the ventrolateral periaqueductal gray (vlPAG), which contains diverse cell types. In freely moving mice, optogenetically stimulating glutamatergic vlPAG neurons that express Chx10 reliably induces freezing. In vlPAG slices, mCbN terminals excite ~20% of neurons positive for Chx10 or GAD2 and ~70% of dopaminergic TH-positive neurons. Stimulating either mCbN afferents or TH neurons augments IPSCs and suppresses EPSCs in Chx10 neurons by activating postsynaptic D2 receptors. The results suggest that mCbN activity regulates dopaminergic modulation of the vlPAG, favoring inhibition of Chx10 neurons. Suppression of cerebellar output may therefore facilitate freezing.

Effects of FGF14 and NaVß4 deletion on transient and resurgent Na current in cerebellar Purkinje neurons.

Voltage-gated Na channels of Purkinje cells are specialized to maintain high availability during high-frequency repetitive firing. They enter fast-inactivated states relatively slowly and undergo a voltage-dependent open-channel block by an intracellular protein (or proteins) that prevents stable fast inactivation and generates resurgent Na current. These properties depend on the pore-forming α subunits, as well as modulatory subunits within the Na channel complex. The identity of the factors responsible for open-channel block remains a question. Here we investigate the effects of genetic mutation of two Na channel auxiliary subunits highly expressed in Purkinje cells, NaVß4 and FGF14, on modulating Na channel blocked as well as inactivated states. We find that although both NaVß4 and the FGF14 splice variant FGF14-1a contain sequences that can generate resurgent-like currents when applied to Na channels in peptide form, deletion of either protein, or both proteins simultaneously, does not eliminate resurgent current in acutely dissociated Purkinje cell bodies. Loss of FGF14 expression does, however, reduce resurgent current amplitude and leads to an acceleration and stabilization of inactivation that is not reversed by application of the site-3 toxin, anemone toxin II (ATX). Tetrodotoxin (TTX) sensitivity is higher for resurgent than transient components of Na current, and loss of FGF14 preferentially affects a highly TTX-sensitive subset of Purkinje α subunits. The data suggest that NaV1.6 channels, which are known to generate the majority of Purkinje cell resurgent current, bind TTX with high affinity and are modulated by FGF14 to facilitate open-channel block.

Sensorimotor Integration and Amplification of Reflexive Whisking by Well-Timed Spiking in the Cerebellar Corticonuclear Circuit.

To test how cerebellar crus I/II Purkinje cells and their targets in the lateral cerebellar nuclei (CbN) integrate sensory and motor-related inputs and contribute to reflexive movements, we recorded extracellularly in awake, head-fixed mice during non-contact whisking. Ipsilateral or contralateral air puffs elicited changes in population Purkinje simple spike rates that matched whisking kinematics (∼1 Hz/1° protraction). Responses remained relatively unaffected when ipsilateral sensory feedback was removed by lidocaine but were reduced by optogenetically inhibiting the reticular nuclei. Optogenetically silencing cerebellar output suppressed movements. During puff-evoked whisks, both Purkinje and CbN cells generated well-timed spikes in sequential 2- to 4-ms windows at response onset, such that they alternately elevated their firing rates just before protraction. With spontaneous whisks, which were smaller than puff-evoked whisks, well-timed spikes were absent and CbN cells were inhibited. Thus, sensory input can facilitate millisecond-scale, well-timed spiking in Purkinje and CbN cells and amplify reflexive whisker movements.