To survive, animals must learn to control their movements with millisecond-level precision, and adjust the kinematics if conditions, or task requirements, change. Here, we examine adaptive timing of motor output in mice, using a simple eyelid conditioning task. Mice were trained to blink in response to a light stimulus that was always followed by a corneal air-puff at a constant time interval. Different mice were trained with different intervals of time separating the onset of the light and the air-puff. As in previous work in other animal species, mice learned to control the speed of the blink, such that the time of maximum eyelid closure matched the interval used during training. However, we found that the time of maximum eyelid speed was always in the first 100 ms after movement onset and did not scale with the training interval, indicating that adaptive timing is not accomplished by slowing down (or speeding up) the eyelid movement uniformly throughout the duration of the blink. A new analysis, specifically designed to examine the kinematics of blinks in single trials, revealed that the underlying control signal responsible for the eyelid movement is made up of oscillatory bursts that are time-locked to the light stimulus at the beginning of the blink, becoming desynchronized later on. Furthermore, mice learn to blink at different speeds and time the movement appropriately by adjusting the amplitude, but not the frequency of the bursts in the eyelid oscillation.

While research on the cerebellar cortex is crystallizing our understanding of its function in learning behavior, many questions surrounding its downstream targets remain. Here, we evaluate the dynamics of cerebellar interpositus nucleus (IpN) neurons over the course of Pavlovian eyeblink conditioning. A diverse range of learning-induced neuronal responses was observed, including increases and decreases in activity during the generation of conditioned blinks. Trial-by-trial correlational analysis and optogenetic manipulation demonstrate that facilitation in the IpN drives the eyelid movements. Adaptive facilitatory responses are often preceded by acquired transient inhibition of IpN activity that, based on latency and effect, appear to be driven by complex spikes in cerebellar cortical Purkinje cells. Likewise, during reflexive blinks to periocular stimulation, IpN cells show excitation-suppression patterns that suggest a contribution of climbing fibers and their collaterals. These findings highlight the integrative properties of subcortical neurons at the cerebellar output stage mediating conditioned behavior.

The role of the cerebellum in predictive motor control and coordination has been thoroughly studied during movements of a single body part. In the real world, however, actions are often more complex. Here, we show that a small area in the rostral anterior interpositus nucleus (rAIN) of the mouse cerebellum is responsible for generating a predictive motor synergy that serves to protect the eye by precisely coordinating muscles of the eyelid, neck, and forelimb. Within the rAIN region, we discovered a new functional category of neurons with unique properties specialized for control of motor synergies. These neurons integrated inhibitory cutaneous inputs from multiple parts of the body, and their activity was correlated with the vigor of the defensive motor synergy on a trial-by-trial basis. We propose that some regions of the cerebellum are organized in poly-somatotopic "action maps" to reduce dimensionality and simplify motor control during ethologically relevant behaviors.

The brain is constantly monitoring its own performance, using error signals to trigger mechanisms of plasticity that help improve future behavior. Indeed, adaptive changes in behavior have been observed after a single error trial in many learning tasks, including cerebellum-dependent eyeblink conditioning. Here, we demonstrate that the plasticity underlying single-trial learning during eyeblink conditioning in mice is bidirectionally regulated by positive and negative prediction errors, has an ephemeral effect on behavior (decays in <1 min), and can be triggered in the absence of errors in performance. We suggest that these three properties of single-trial learning may be particularly useful for driving mechanisms of motor adaptation that can achieve optimal performance in the face of environmental disturbances with a fast timescale.

Climbing fiber inputs to Purkinje cells are thought to be involved in generating the instructive signals that drive cerebellar learning. To investigate how these instructive signals are encoded, we recorded the activity of individual climbing fibers during cerebellum-dependent eyeblink conditioning in mice. We found that climbing fibers signaled both the unexpected delivery and the unexpected omission of the periocular airpuff that served as the instructive signal for eyeblink conditioning. In addition, we observed that climbing fibers activated by periocular airpuffs also responded to stimuli from other sensory modalities if those stimuli were novel or if they predicted that the periocular airpuff was about to be presented. This pattern of climbing fiber activity is markedly similar to the responses of dopamine neurons during reinforcement learning, which have been shown to encode a particular type of instructive signal known as a temporal difference prediction error.

Climbing fibers (CFs) are thought to contribute to cerebellar plasticity and learning by triggering a large influx of dendritic calcium in the postsynaptic Purkinje cell (PC) to signal the occurrence of an unexpected sensory event. However, CFs fire about once per second whether or not an event occurs, raising the question of how sensory-driven signals might be distinguished from a background of ongoing spontaneous activity. Here, we report that in PC dendrites of awake mice, CF-triggered calcium signals are enhanced when the trigger is a sensory event. In addition, we show that a large fraction of the total enhancement in each PC dendrite can be accounted for by an additional boost of calcium provided by sensory activation of a non-CF input. We suggest that sensory stimulation may modulate dendritic voltage and calcium concentration in PCs to increase the strength of plasticity signals during cerebellar learning.

Transcranial direct-current stimulation (tDCS) is a non-invasive brain stimulation technique consisting in the application of weak electric currents on the scalp. Although previous studies have demonstrated the clinical value of tDCS for modulating sensory, motor, and cognitive functions, there are still huge gaps in the knowledge of the underlying physiological mechanisms. To define the immediate impact as well as the after effects of tDCS on sensory processing, we first performed electrophysiological recordings in primary somatosensory cortex (S1) of alert mice during and after administration of S1-tDCS, and followed up with immunohistochemical analysis of the stimulated brain regions. During the application of cathodal and anodal transcranial currents we observed polarity-specific bidirectional changes in the N1 component of the sensory-evoked potentials (SEPs) and associated gamma oscillations. On the other hand, 20 min of cathodal stimulation produced significant after-effects including a decreased SEP amplitude for up to 30 min, a power reduction in the 20-80 Hz range and a decrease in gamma event related synchronization (ERS). In contrast, no significant changes in SEP amplitude or power analysis were observed after anodal stimulation except for a significant increase in gamma ERS after tDCS cessation. The polarity-specific differences of these after effects were corroborated by immunohistochemical analysis, which revealed an unbalance of GAD 65-67 immunoreactivity between the stimulated versus non-stimulated S1 region only after cathodal tDCS. These results highlight the differences between immediate and after effects of tDCS, as well as the asymmetric after effects induced by anodal and cathodal stimulation.

Rett syndrome is a devastating childhood neurological disorder caused by mutations in MECP2. Of the many symptoms, motor deterioration is a significant problem for patients. In mice, deleting Mecp2 from the cortex or basal ganglia causes motor dysfunction, hypoactivity, and tremor, which are abnormalities observed in patients. Little is known about the function of Mecp2 in the cerebellum, a brain region critical for motor function. Here we show that deleting Mecp2 from the cerebellum, but not from its neuronal subtypes, causes a delay in motor learning that is overcome by additional training. We observed irregular firing rates of Purkinje cells and altered heterochromatin architecture within the cerebellum of knockout mice. These findings demonstrate that the motor deficits present in Rett syndrome arise, in part, from cerebellar dysfunction. For Rett syndrome and other neurodevelopmental disorders, our results highlight the importance of understanding which brain regions contribute to disease phenotypes.

High-density probes allow electrophysiological recordings from many neurons simultaneously across entire brain circuits but fail to determine each recorded neuron's cell type. Here, we develop a strategy to identify cell types from extracellular recordings in awake animals, opening avenues to unveil the computational roles of neurons with distinct functional, molecular, and anatomical properties. We combine optogenetic activation and pharmacology using the cerebellum as a testbed to generate a curated ground-truth library of electrophysiological properties for Purkinje cells, molecular layer interneurons, Golgi cells, and mossy fibers. We train a semi-supervised deep-learning classifier that predicts cell types with greater than 95% accuracy based on waveform, discharge statistics, and layer of the recorded neuron. The classifier's predictions agree with expert classification on recordings using different probes, in different laboratories, from functionally distinct cerebellar regions, and across animal species. Our approach provides a general blueprint for cell-type identification from extracellular recordings across the brain.

The climbing fiber input to Purkinje cells acts as a teaching signal by triggering a massive influx of dendritic calcium that marks the occurrence of instructive stimuli during cerebellar learning. Here, we challenge the view that these calcium spikes are all-or-none and only signal whether the instructive stimulus has occurred, without providing parametric information about its features. We imaged ensembles of Purkinje cell dendrites in awake mice and measured their calcium responses to periocular airpuffs that serve as instructive stimuli during cerebellar-dependent eyeblink conditioning. Information about airpuff duration and pressure was encoded probabilistically across repeated trials, and in two additional signals in single trials: the synchrony of calcium spikes in the Purkinje cell population, and the amplitude of the calcium spikes, which was modulated by a non-climbing fiber pathway. These results indicate that calcium-based teaching signals in Purkinje cells contain analog information that encodes the strength of instructive stimuli trial-by-trial.

Cerebellar inhibitory interneurons are important regulators of neural circuit activity for diverse motor and nonmotor functions. The molecular layer interneurons (MLIs), consisting of basket cells (BCs) and stellate cells (SCs), provide dendritic and somatic inhibitory synapses onto Purkinje cells, respectively. They are sequentially generated in an inside-out pattern from Pax2+ immature interneurons, which migrate from the prospective white matter to the ML of the cortex. However, little is known about how MLI subtype identities and pool sizes are determined, nor are their contributions to motor learning well understood. Here, we show that GABAergic progenitors fated to generate both BCs and SCs respond to the Sonic hedgehog (Shh) signal. Conditional abrogation of Shh signaling of either sex inhibited proliferation of GABAergic progenitors and reduced the number of Pax2+ cells, whereas persistent Shh pathway activation increased their numbers. These changes, however, did not affect early born BC numbers but selectively altered the SC pool size. Moreover, genetic depletion of GABAergic progenitors when BCs are actively generated also resulted in a specific reduction of SCs, suggesting that the specification of MLI subtypes is independent of Shh signaling and their birth order and likely occurs after Pax2+ cells settle into their laminar positions in an inside-out sequence. Mutant mice with reduced SC numbers displayed decreased dendritic inhibitory synapses and neurotransmission onto Purkinje cells, resulting in an impaired acquisition of eyeblink conditioning. These findings also reveal an essential role of Shh signaling-dependent SCs in regulating inhibitory dendritic synapses and motor learning.SIGNIFICANCE STATEMENT The cerebellar circuit that enables fine motor learning involves MLIs of BCs and SCs, which provide dendritic and somatic inhibitory synapses onto Purkinje cells. Little is known about how their identities and numbers are determined, nor are their specific contributions to motor learning well understood. We show that MLI subtypes are specified independent of Shh signaling and their birth orders but appear to occur in their terminal laminar positions according to the inside-out sequence. This finding challenges the current view that MLI subtypes are specified sequentially at the progenitor level. We also demonstrate that dendritic inhibition by Shh signaling-dependent SC pool is necessary for motor learning.

The brain generates negative prediction error (NPE) signals to trigger extinction, a type of inhibitory learning that is responsible for suppressing learned behaviors when they are no longer useful. Neurons encoding NPE have been reported in multiple brain regions. Here, we use an optogenetic approach to demonstrate that GABAergic cerebello-olivary neurons can generate a powerful NPE signal, capable of causing extinction of conditioned motor responses on its own.

Transcranial direct-current stimulation (tDCS) is a promising non-invasive neuromodulatory technique being proposed for treating neurologic disorders. However, there is a lack of knowledge about how externally applied currents affect neuronal spiking activity in cerebellar circuits in vivo . In this study, we observe a heterogeneous polarity modulation of the firing rate of Purkinje cells (PC) and non-PC in the mouse cerebellar cortex. Using a combination of juxtacellular labeling and high-density Neuropixels recordings, we demonstrate that the apparently heterogeneous effects of tDCS on PC activity can be fully explained by taking into account the somatodendritic orientation relative to the electric field. Our findings emphasize the importance of considering neuronal orientation and morphological aspects to increase the predictive power of tDCS computational models and optimize desired effects in basic and clinical human applications.