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Optoelectronic systems can exert precise control over targeted neurons and pathways throughout the brain in untethered animals, but similar technologies for the spinal cord are not well established. In the present study, we describe a system for ultrafast, wireless, closed-loop manipulation of targeted neurons and pathways across the entire dorsoventral spinal cord in untethered mice. We developed a soft stretchable carrier, integrating microscale light-emitting diodes (micro-LEDs), that conforms to the dura mater of the spinal cord. A coating of silicone-phosphor matrix over the micro-LEDs provides mechanical protection and light conversion for compatibility with a large library of opsins. A lightweight, head-mounted, wireless platform powers the micro-LEDs and performs low-latency, on-chip processing of sensed physiological signals to control photostimulation in a closed loop. We use the device to reveal the role of various neuronal subtypes, sensory pathways and supraspinal projections in the control of locomotion in healthy and spinal-cord injured mice., Optogenetics is applied to the entire mouse spinal cord.

Most studies investigating the impact of the cerebral cortex (CC) onto the cerebellum highlight the role of the pons, which provides the mossy fibers to the cerebellum. However, cerebro-cerebellar communication may also be mediated by the nuclei of the mesodiencephalic junction (MDJ) that project to the inferior olive (IO), which in turn provides the climbing fibers to the molecular layer. Here, we uncover the precise topographic relations of the inputs and outputs of the MDJ using multiple, classical, and transneuronal tracing methods as well as analyses of mesoscale cortical injections from Allen Mouse Brain. We show that the caudal parts of the CC predominantly project to the principal olive via the rostral MDJ and that the rostral parts of the CC predominantly project to the rostral medial accessory olive via the caudal MDJ. Moreover, using triple viral tracing technology, we show that the cerebellar nuclei directly innervate the neurons in the MDJ that receive input from CC and project to the IO. By unraveling these topographic and prominent, mono- and disynaptic projections through the MDJ, this work establishes that cerebro-cerebellar communication is not only mediated by the pontine mossy fiber system, but also by the climbing fiber system.

Metabolic acidosis, defined as a plasma or serum bicarbonate concentration, Graphical Abstract Graphical Abstract

Objective: While integrity of spinal pathways below injury is generally thought to be an important factor in the success-rate of neuromodulation strategies for spinal cord injury (SCI), it is still unclear how the integrity of these pathways conveying the effects of stimulation should be assessed. In one of our institutional case series of five patients receiving dorsal root ganglion (DRG)-stimulation for elicitation of immediate motor response in motor complete SCI, only two out of five patients presented as responders, showing immediate muscle activation upon DRG-stimulation. The current study focuses on post hoc clinical-neurophysiological tests performed within this patient series to illustrate their use for prediction of spinal pathway integrity, and presumably, responder-status. Materials and Methods: In a series of three nonresponders and two responders (all male, American Spinal Injury Association [ASIA] impairment scale [AIS] A/B), a test-battery consisting of questionnaires, clinical measurements, as well as a series of neurophysiological measurements was performed less than eight months after participation in the initial study. Results: Nonresponders presented with a complete absence of spasticity and absence of leg reflexes. Additionally, nonresponders presented with close to no compound muscle action potentials (CMAPs) or Hofmann(H)-reflexes. In contrast, both responders presented with clear spasticity, elicitable leg reflexes, CMAPs, H-reflexes, and sensory nerve action potentials, although not always consistent for all tested muscles. Conclusions: Post hoc neurophysiological measurements were limited in clearly separating responders from nonresponders. Clinically, complete absence of spasticity-related complaints in the nonresponders was a distinguishing factor between responders and nonresponders in this case series, which mimics prior reports of epidural electrical stimulation, potentially illustrating similarities in mechanisms of action between the two techniques. However, the problem remains that explicit use and report of preinclusion clinical-neurophysiological measurements is missing in SCI literature. Identifying proper ways to assess these criteria might therefore be unnecessarily difficult, especially for nonestablished neuromodulation techniques.

Over the past several decades, theories about cerebellar learning have evolved. A relatively simple view that highlighted the contribution of one major form of heterosynaptic plasticity to cerebellar motor learning has given way to a plethora of perspectives that suggest that many different forms of synaptic and non-synaptic plasticity, acting at various sites, can control multiple types of learning behaviour. However, there still seem to be contradictions between the various hypotheses with regard to the mechanisms underlying cerebellar learning. The challenge is therefore to reconcile these different views and unite them into a single overall concept. Here I review our current understanding of the changes in the activity of cerebellar Purkinje cells in different ‘microzones’ during various forms of learning. I describe an emerging model that indicates that the activity of each microzone is bound to either increase or decrease during the initial stages of learning, depending on the directional and temporal demands of its downstream circuitry and the behaviour involved.

Background and aims The objective of the present work was to determine to what extent sleep quality may mediate the association between chronodisruption (CD) and metabolic syndrome (MS), and between CD and body composition (BC). Methodology Cross-sectional study which included 300 adult health workers, 150 of whom were night shift workers and thereby exposed to CD. Diagnosis of MS was made based on Adult Treatment Panel III criteria. Sleep quality was measured using the Pittsburgh Sleep Quality Index. Body mass index (BMI), fat mass percentage, and visceral fat percentage were measured as indicators of body composition (BC). Data were analyzed using logistic, linear regression and structural equation models. Results The odds of health workers exposed to CD to suffer MS was 22.13 (IC95 8.68–66.07) when the model was adjusted for age, gender, physical activity and energy consumption. CD was also significantly associated with an increase in fat mass and visceral fat percentages, but not to BMI. Surprisingly, there was not enough evidence supporting the hypothesis that sleep quality contributes to the association between CD and MS or between CD and BC. Conclusions Sleep quality does not mediate the negative effects of CD on MS nor on BC.

Objectives: Current strategies for motor recovery after spinal cord injury (SCI) aim to facilitate motor performance through modulation of afferent input to the spinal cord using epidural electrical stimulation (EES). The dorsal root ganglion (DRG) itself, the first relay station of these afferent inputs, has not yet been targeted for this purpose. The current study aimed to determine whether DRG stimulation can facilitate clinically relevant motor response in motor complete SCI. Materials and Methods: Five patients with chronic motor complete SCI were implanted with DRG leads placed bilaterally on level L4 during five days. Based on personalized stimulation protocols, we aimed to evoke dynamic (phase 1) and isotonic (phase 2) motor responses in the bilateral quadriceps muscles. On days 1 and 5, EMG-measurements (root mean square [RMS] values) and clinical muscle force measurements (MRC scoring) were used to measure motor responses and their reproducibility. Results: In all patients, DRG-stimulation evoked significant phase 1 and phase 2 motor responses with an MRC ≥4 for all upper leg muscles (rectus femoris, vastus lateralis, vastus medialis, and biceps femoris) (p < 0.05 and p < 0.01, respectively), leading to a knee extension movement strong enough to facilitate assisted weight bearing. No significant differences in RMS values were observed between days 1 and 5 of the study, indicating that motor responses were reproducible. Conclusion: The current paper provides first evidence that bilateral L4 DRG stimulation can evoke reproducible motor responses in the upper leg, sufficient for assisted weight bearing in patients with chronic motor complete SCI. As such, a new target for SCI treatment has surfaced, using existing stimulation devices, making the technique directly clinically accessible.

Aim The primary aim of this study is to demonstrate that 7‐tesla functional magnetic resonance imaging (7T‐fMRI) can visualize the neural representations of the male pelvic floor in the whole brain of a single subject. Methods In total, 17 healthy male volunteers (age 20‐47) were scanned in a 7T‐MRI scanner (Philips Achieva). The scanning protocol consisted of two functional runs using a multiband echo planar imaging sequence and a T1‐weighted scan. The subjects executed two motor tasks, one involving consecutive pelvic floor muscle contractions (PFMC) and a control task with tongue movements. Results In single subjects, results of both tasks were visualized in the cortex, putamen, thalamus, and the cerebellum. Activation was seen during PFMC in the superomedial and inferolateral primary motor cortex (M1), supplementary motor area (SMA), insula, midcingulate gyrus (MCG), putamen, thalamus, and in the anterior and posterior lobes of the cerebellum. During tongue movement, activation was seen in the inferolateral M1, SMA, MCG, putamen, thalamus, and anterior and posterior lobes of the cerebellum. Tongue activation was found in the proximity of, but not overlapping with, the PFMC activation. Connectivity analysis demonstrated differences in neural networks involved in PFMC and tongue movement. Conclusion This study demonstrated that 7T‐fMRI can be used to visualize brain areas involved in pelvic floor control in the whole brain of single subjects and defined the specific brain areas involved in PFMC. Distinct differences between brain mechanisms controlling the pelvic floor and tongue movements were demonstrated using connectivity analysis.

Purkinje cells (PCs) in the cerebellar cortex can be divided into at least two main subpopulations: one subpopulation that prominently expresses ZebrinII (Z+), and shows a relatively low simple spike firing rate, and another that hardly expresses ZebrinII (Z–) and shows higher baseline firing rates. Likewise, the complex spike responses of PCs, which are evoked by climbing fiber inputs and thus reflect the activity of the inferior olive (IO), show the same dichotomy. However, it is not known whether the target neurons of PCs in the cerebellar nuclei (CN) maintain this bimodal distribution. Electrophysiological recordings in awake adult mice show that the rate of action potential firing of CN neurons that receive input from Z+ PCs was consistently lower than that of CN neurons innervated by Z– PCs. Similar in vivo recordings in juvenile and adolescent mice indicated that the firing frequency of CN neurons correlates to the ZebrinII identity of the PC afferents in adult, but not postnatal stages. Finally, the spontaneous action potential firing pattern of adult CN neurons recorded in vitro revealed no significant differences in intrinsic pacemaking activity between ZebrinII identities. Our findings indicate that all three main components of the olivocerebellar loop, i.e., PCs, IO neurons and CN neurons, operate at a higher rate in the Z– modules.

Significance Learning depends on synaptic plasticity. The signaling mechanisms that control induction of plasticity determine the learning rules at the specific synapse involved. Moreover, the relationship between the activity patterns of synaptic inputs and the type, direction, and level of plasticity induced may evolve during development. Here, we establish a key link between receptor activation presynaptic to cerebellar Purkinje cells, downstream signaling mechanisms, and the ability of adult animals to learn a cerebellar motor task., Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

Background: Epileptic (absence) seizures in the cerebral cortex can be stopped by pharmacological and optogenetic stimulation of the cerebellar nuclei (CN) neurons that innervate the thalamus. However, it is unclear how such stimulation can modify underlying thalamo-cortical oscillations. Hypothesis: Here we tested whether rhythmic synchronized thalamo-cortical activity during absence seizures can be desynchronized by single-pulse optogenetic stimulation of CN neurons to stop seizure activity. Methods: We performed simultaneous thalamic single-cell and electrocorticographical recordings in awake tottering mice, a genetic model of absence epilepsy, to investigate the rhythmicity and synchronicity. Furthermore, we tested interictally the impact of single-pulse optogenetic CN stimulation on thalamic and cortical recordings. Results: We show that thalamic firing is highly rhythmic and synchronized with cortical spike-and-wave discharges during absence seizures and that this phase-locked activity can be desynchronized upon single-pulse optogenetic stimulation of CN neurons. Notably, this stimulation of CN neurons was more effective in stopping seizures than direct, focal stimulation of groups of afferents innervating the thalamus. During interictal periods, CN stimulation evoked reliable but heterogeneous responses in thalamic cells in that they could show an increase or decrease in firing rate at various latencies, bi-phasic responses with an initial excitatory and subsequent inhibitory response, or no response at all. Conclusion: Our data indicate that stimulation of CN neurons and their fibers in thalamus evokes differential effects in its downstream pathways and desynchronizes phase-locked thalamic neuronal firing during seizures, revealing a neurobiological mechanism that may explain how cerebellar stimulation can stop seizures. (c) 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

The inferior olive (IO) is an evolutionarily conserved brain stem structure and its output activity plays a major role in the cerebellar computation necessary for controlling the temporal accuracy of motor behavior. The precise timing and synchronization of IO network activity has been attributed to the dendro-dendritic gap junctions mediating electrical coupling within the IO nucleus. Thus, the dendritic morphology and spatial arrangement of IO neurons governs how synchronized activity emerges in this nucleus. To date, IO neuron structural properties have been characterized in few studies and with small numbers of neurons; these investigations have described IO neurons as belonging to two morphologically distinct types, “curly” and “straight”. In this work we collect a large number of individual IO neuron morphologies visualized using different labeling techniques and present a thorough examination of their morphological properties and spatial arrangement within the olivary neuropil. Our results show that the extensive heterogeneity in IO neuron dendritic morphologies occupies a continuous range between the classically described “curly” and “straight” types, and that this continuum is well represented by a relatively simple measure of “straightness”. Furthermore, we find that IO neuron dendritic trees are often directionally oriented. Combined with an examination of cell body density distributions and dendritic orientation of adjacent IO neurons, our results suggest that the IO network may be organized into groups of densely coupled neurons interspersed with areas of weaker coupling. Electronic supplementary material The online version of this article (10.1007/s00429-019-01859-z) contains supplementary material, which is available to authorized users.

Key points Ventrolateral thalamus (VL) integrates information from cerebellar nuclei and motor cortical layer VI.Inputs from the cerebellar nuclei evoke large‐amplitude responses that depress upon repetitive stimulation while layer VI inputs from motor cortex induce small‐amplitude facilitating responses.We report that the spiking of VL neurons can be determined by the thalamic membrane potential, the frequency of cerebellar inputs and the duration of pauses after cerebellar high frequency stimulation.Inputs from motor cortical layer VI shift the VL membrane potential and modulate the VL spike output in response to cerebellar stimulation. These results help us to decipher how the cerebellar output is integrated in VL and modulated by motor cortical input. Abstract Orchestrating complex movements requires well‐timed interaction of cerebellar, thalamic and cerebral structures, but the mechanisms underlying the integration of cerebro‐cerebellar information in motor thalamus remain largely unknown. Here we investigated how excitatory inputs from cerebellar nuclei (CN) and primary motor cortex layer VI (M1‐L6) neurons may regulate the activity of neurons in the mouse ventrolateral (VL) thalamus. Using dual‐optical stimulation of the CN and M1‐L6 axons and in vitro whole‐cell recordings of the responses in VL neurons, we studied the individual responses as well as the effects of combined CN and M1‐L6 stimulation. Whereas CN inputs evoked large‐amplitude responses that were depressed upon repetitive stimulation, M1‐L6 inputs elicited small‐amplitude responses that were facilitated upon repetitive stimulation. Moreover, pauses in CN stimuli could directly affect VL spiking probability, an effect that was modulated by VL membrane potential. When CN and M1‐L6 pathways were co‐activated, motor cortical afferents increased the thalamic spike output in response to cerebellar stimulation, indicating that CN and M1 synergistically, yet differentially, control the membrane potential and spiking pattern of VL neurons., Key points Ventrolateral thalamus (VL) integrates information from cerebellar nuclei and motor cortical layer VI.Inputs from the cerebellar nuclei evoke large‐amplitude responses that depress upon repetitive stimulation while layer VI inputs from motor cortex induce small‐amplitude facilitating responses.We report that the spiking of VL neurons can be determined by the thalamic membrane potential, the frequency of cerebellar inputs and the duration of pauses after cerebellar high frequency stimulation.Inputs from motor cortical layer VI shift the VL membrane potential and modulate the VL spike output in response to cerebellar stimulation. These results help us to decipher how the cerebellar output is integrated in VL and modulated by motor cortical input.

Volitional suppression of responses to distracting external stimuli enables us to achieve our goals. This volitional inhibition of a specific behavior is supposed to be mainly mediated by the cerebral cortex. However, recent evidence supports the involvement of the cerebellum in this process. It is currently not known whether different parts of the cerebellar cortex play differential or synergistic roles in planning and execution of this behavior. Here, we measured Purkinje cell (PC) responses in the medial and lateral cerebellum in two rhesus macaques during a pro- and antisaccade task. During an antisaccade trial, non-human primates were instructed to make a saccadic eye movement away from a target, rather than towards it, as in prosaccade trials. Our data shows that the cerebellum plays an important role not only during execution of the saccades, but also during the volitional inhibition of eye movements towards the target. Simple Spike (SS) modulation during the instruction and execution period of pro- and antisaccades was prominent in PCs of both medial and lateral cerebellum. However, only the SS activity in the lateral cerebellar cortex contained information about trial identity and showed a stronger reciprocal interaction with complex spikes. Moreover, SS activity of different PC groups modulated bidirectionally in both regions, but the PCs that showed facilitating and suppressive activity were predominantly associated with instruction and execution, respectively. These findings show that different cerebellar regions and PC groups contribute to goal-directed behavior and volitional inhibition, but with different propensities, highlighting the rich repertoire of cerebellar control in executive functions.Significance StatementThe antisaccade task is commonly used in research and clinical evaluation as a test of volitional and flexible control of behavior. It requires volitional suppression of prosaccades, a function that has been attributed to the neocortex. However, recent findings indicate that cerebellum also contributes to this behavior. We recorded from neurons in the medial and lateral cerebellum to evaluate their responses in this task. We found that both regions significantly modulated their activity during this task, but only cells in the lateral cerebellum encoded the stimulus identity in each trial. These results indicate that the cerebellum actively contributes to the control of flexible behavior and that lateral and medial cerebellum play different roles during volitional eye movements.

Most studies investigating the impact of cerebral cortex (CC) onto the cerebellum highlight the role of the pontine mossy fibre system. However, cerebro-cerebellar communication may also be mediated by the olivary climbing fibres via a hub in the mesodiencephalic junction (MDJ). Here, we show that rostromedial and caudal parts of mouse CC predominantly project to the principal olive via the rostroventral MDJ and that more rostrolateral CC regions prominently project to the rostral medial accessory olive via the caudodorsal MDJ. Moreover, transneuronal tracing results show that the cerebellar nuclei innervate the olivary-projecting neurons in the MDJ that receive input from CC, and that they adhere to the same topographical relations. By unravelling these topographic and dense, mono- and disynaptic projections from the CC through the MDJ and inferior olive to the cerebellum, this work establishes that cerebro-cerebellar communication can be mediated by both the mossy fibre and climbing fibre system.

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic NMDA receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers nitric oxide signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-Purkinje cell (PF-PC) synapses. Consistent with this finding, the selective deletion of GCs NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.Significance StatementLearning depends on synaptic plasticity. The signaling mechanisms that control induction of plasticity determine the learning rules at the specific synapse involved. Moreover, the relationship between the activity patterns of synaptic inputs and the type, direction, and level of plasticity induced may evolve during development. Here, we establish a key link between NMDA receptor activation presynaptic to cerebellar Purkinje cells, downstream signaling mechanisms, and the ability of adult animals to learn a cerebellar motor task.

The cerebellum is crucial for various associative sensorimotor behaviors. Delay eyeblink conditioning (DEC) depends on the simplex lobule-interposed nucleus (IN) pathway, yet it is unclear how other cerebellar modules cooperate during this task. Here, we demonstrate the contribution of the vermis-fastigial nucleus (FN) pathway in controlling DEC. We found that task-related modulations in vermal Purkinje cells and FN neurons predict conditioned responses (CRs). Coactivation of the FN and the IN allows for the generation of proper motor commands for CRs, but only FN output fine-tunes unconditioned responses. The vermis-FN pathway launches its signal via the contralateral ventral medullary reticular nucleus, which converges with the command from the simplex-IN pathway onto facial motor neurons. We propose that the IN pathway specifically drives CRs, whereas the FN pathway modulates the amplitudes of eyelid closure during DEC. Thus, associative sensorimotor task optimization requires synergistic modulation of different olivocerebellar modules each provide unique contributions., Delay eyeblink conditioning depends on the simplex lobule-interposed nucleus pathway in the cerebellum. Here, the authors show that the vermis-fastigial nucleus-medullary reticular nucleus pathway modulates the conditioned and unconditioned eyelid closure during delay eyeblink conditioning.

Significance Coordinated activity of sensory and motor cortices is essential for adjusting movements based on sensory feedback. Sensory and motor cortices communicate directly as well as via the thalamus and also receive indirect input from the cerebellum. We show here that cerebellar activity can affect the amplitude and coherence of fast sensorimotor responses in the primary somatosensory and motor cortices upon whisker stimulation. The cerebellum can differentially alter sensory-induced theta- and gamma-band cortical coherences via a fast ascending pathway. In line with the functional heterogeneity of its modular organization, cerebellar impact is region-specific and tuned to ongoing motor responses. Our data highlight site-specific and context-dependent cerebello-cerebral interactions that can come into play during a plethora of sensorimotor functions., Activity of sensory and motor cortices is essential for sensorimotor integration. In particular, coherence between these areas may indicate binding of critical functions like perception, motor planning, action, or sleep. Evidence is accumulating that cerebellar output modulates cortical activity and coherence, but how, when, and where it does so is unclear. We studied activity in and coherence between S1 and M1 cortices during whisker stimulation in the absence and presence of optogenetic Purkinje cell stimulation in crus 1 and 2 of awake mice, eliciting strong simple spike rate modulation. Without Purkinje cell stimulation, whisker stimulation triggers fast responses in S1 and M1 involving transient coherence in a broad spectrum. Simultaneous stimulation of Purkinje cells and whiskers affects amplitude and kinetics of sensory responses in S1 and M1 and alters the estimated S1–M1 coherence in theta and gamma bands, allowing bidirectional control dependent on behavioral context. These effects are absent when Purkinje cell activation is delayed by 20 ms. Focal stimulation of Purkinje cells revealed site specificity, with cells in medial crus 2 showing the most prominent and selective impact on estimated coherence, i.e., a strong suppression in the gamma but not the theta band. Granger causality analyses and computational modeling of the involved networks suggest that Purkinje cells control S1–M1 phase consistency predominantly via ventrolateral thalamus and M1. Our results indicate that activity of sensorimotor cortices can be dynamically and functionally modulated by specific cerebellar inputs, highlighting a widespread role of the cerebellum in coordinating sensorimotor behavior.

Cacna1a encodes the pore-forming α1A subunit of CaV2.1 voltage-dependent calcium channels, which regulate neuronal excitability and synaptic transmission. Purkinje cells in the cortex of cerebellum abundantly express these CaV2.1 channels. Here, we show that homozygous tottering (tg) mice, which carry a loss-of-function Cacna1a mutation, exhibit severely impaired learning in Pavlovian eyeblink conditioning, which is a cerebellar-dependent learning task. Performance of reflexive eyeblinks is unaffected in tg mice. Transient seizure activity in tg mice further corrupted the amplitude of eyeblink conditioned responses. Our results indicate that normal calcium homeostasis is imperative for cerebellar learning and that the oscillatory state of the brain can affect the expression thereof.NEW & NOTEWORTHY In this study, we confirm the importance of normal calcium homeostasis in neurons for learning and memory formation. In a mouse model with a mutation in an essential calcium channel that is abundantly expressed in the cerebellum, we found severely impaired learning in eyeblink conditioning. Eyeblink conditioning is a cerebellar-dependent learning task. During brief periods of brain-wide oscillatory activity, as a result of the mutation, the expression of conditioned eyeblinks was even further disrupted.

FOXP2 has been identified as a gene related to speech in humans, based on rare mutations that yield significant impairments in speech at the level of both motor performance and language comprehension. Disruptions of the murine orthologue Foxp2 in mouse pups have been shown to interfere with production of ultrasonic vocalizations (USVs). However, it remains unclear which structures are responsible for these deficits. Here, we show that conditional knockout mice with selective Foxp2 deletions targeting the cerebral cortex, striatum or cerebellum, three key sites of motor control with robust neural gene expression, do not recapture the profile of pup USV deficits observed in mice with global disruptions of this gene. Moreover, we observed that global Foxp2 knockout pups show substantive reductions in USV production as well as an overproduction of short broadband noise “clicks”, which was not present in the brain region-specific knockouts. These data indicate that deficits of Foxp2 expression in the cortex, striatum or cerebellum cannot solely explain the disrupted vocalization behaviours in global Foxp2 knockouts. Our findings raise the possibility that the impact of Foxp2 disruption on USV is mediated at least in part by effects of this gene on the anatomical prerequisites for vocalizing.

The cerebellum is involved in cognition next to motor coordination. During complex tasks, climbing fiber input to the cerebellum can deliver seemingly opposite signals, covering both motor and non-motor functions. To elucidate this ambiguity, we hypothesized that climbing fiber activity represents the saliency of inputs leading to action-readiness. We addressed this hypothesis by recording Purkinje cell activity in lateral cerebellum of awake mice learning go/no-go decisions based on entrained saliency of different sensory stimuli. As training progressed, the timing of climbing fiber signals switched in a coordinated fashion with that of Purkinje cell simple spikes towards the moment of occurrence of the salient stimulus that required action. Trial-by-trial analysis indicated that emerging climbing fiber activity is not linked to individual motor responses or rewards per se, but rather reflects the saliency of a particular sensory stimulus that engages a general readiness to act, bridging the non-motor with the motor functions.In briefMice were trained to identify the saliency of different sensory inputs in that they had to learn to ignore a prominent sound cue and respond to a light tactile cue in a Go/No-Go licking task. As the mice learned to discriminate the two inputs and respond to the proper signal, the Purkinje cells in the lateral cerebellum switched their climbing fiber activity (i.e., complex spike activity) towards the moment of occurrence of the salient stimulus that required a response, while concomitantly shifting the phase of their simple spike modulation. Trial-by-trial analysis indicates that the emerging climbing fiber activity is not linked to the occurrence of the motor response or reward per se, but rather reflects the saliency of a particular sensory stimulus engaging a general readiness to act.

Significance The cerebellum has long been recognized as a partner of the cerebral cortex, and both have expanded greatly in human evolution. The thin cerebellar cortex is even more tightly folded than the cerebral cortex. By scanning a human cerebellum specimen at ultra-high magnetic fields, we were able to computationally reconstruct its surface down to the level of the smallest folds, revealing that the cerebellar cortex has almost 80% of the surface area of the cerebral cortex. By performing the same procedure on a monkey brain, we found that the surface area of the human cerebellum has expanded even more than that of the human cerebral cortex, suggesting a role in characteristically human behaviors, such as toolmaking and language., The surface of the human cerebellar cortex is much more tightly folded than the cerebral cortex. It was computationally reconstructed for the first time to the level of all individual folia from multicontrast high-resolution postmortem MRI scans. Its total shrinkage-corrected surface area (1,590 cm2) was larger than expected or previously reported, equal to 78% of the total surface area of the human neocortex. The unfolded and flattened surface comprised a narrow strip 10 cm wide but almost 1 m long. By applying the same methods to the neocortex and cerebellum of the macaque monkey, we found that its cerebellum was relatively much smaller, approximately 33% of the total surface area of its neocortex. This suggests a prominent role for the cerebellum in the evolution of distinctively human behaviors and cognition.

The cerebellar posterior vermis generates an estimation of our motion (translation) and orientation (tilt) in space using cues originating from semicircular canals and otolith organs. Theoretical work has laid out the basic computations necessary for this signal transformation, but details on the cellular loci and mechanisms responsible are lacking. Using a multicomponent modeling approach, we show that canal and otolith information are spatially and temporally matched in mouse posterior vermis Purkinje cells and that Purkinje cell responses combine translation and tilt information. Purkinje cell-specific inhibition of protein kinase C decreased and phase-shifted the translation component of Purkinje cell responses, but did not affect the tilt component. Our findings suggest that translation and tilt signals reach Purkinje cells via separate information pathways and that protein kinase C-dependent mechanisms regulate translation information processing in cerebellar cortex output neurons.

Significance Understanding mechanisms underlying learning and memory is crucial in view of tackling cognitive decline occurring during aging or following neurological disorders. The cerebellum offers an ideal system to achieve this goal because of the well-characterized forms of motor learning that it controls. It is so far unclear whether cerebellar memory processes depend on changes in perineuronal nets (PNNs). PNNs are assemblies of extracellular matrix molecules around neurons, which regulate neural plasticity. Here we demonstrate that during eyeblink conditioning (EBC), which is a form of cerebellar motor learning, PNNs in the mouse deep cerebellar nuclei are dynamically modulated, and PNN changes are essential for the formation and storage of EBC memories. Together, these results unveil an important mechanism controlling motor associative memories., Perineuronal nets (PNNs) are assemblies of extracellular matrix molecules, which surround the cell body and dendrites of many types of neuron and regulate neural plasticity. PNNs are prominently expressed around neurons of the deep cerebellar nuclei (DCN), but their role in adult cerebellar plasticity and behavior is far from clear. Here we show that PNNs in the mouse DCN are diminished during eyeblink conditioning (EBC), a form of associative motor learning that depends on DCN plasticity. When memories are fully acquired, PNNs are restored. Enzymatic digestion of PNNs in the DCN improves EBC learning, but intact PNNs are necessary for memory retention. At the structural level, PNN removal induces significant synaptic rearrangements in vivo, resulting in increased inhibition of DCN baseline activity in awake behaving mice. Together, these results demonstrate that PNNs are critical players in the regulation of cerebellar circuitry and function.

Orchestrating the ensemble of muscle contractions necessary for coordinated movements requires the interaction of cerebellar, thalamic and cerebral structures, but the mechanisms underlying the integration of information remain largely unknown. Here we investigated how excitatory inputs from cerebellar nuclei (CN) and primary motor cortex layer VI (M1 L6) neurons may regulate together the activity of neurons in the mouse ventrolateral (VL) thalamus. Using dual-optogenetic stimulation and whole-cell recordings in vitro we were able to specifically activate the CN and M1 pathways and study their differential impact. We found that VL spiking probability is effectively determined by a pause in CN stimuli, whereas VL membrane potential can be modulated subthreshold by M1 L6 input. Upon mild depolarization of the VL membrane potential, repetitive CN stimulation evokes at best single action potential firing, whereas more negative membrane potentials increase VL spiking probability. Moreover, whereas high-frequency cerebellar activity attenuates thalamic spiking, pauses in cerebellar activity re-activate thalamic spiking. In contrast, facilitating inputs from cerebral cortex modulate thalamo-cortical spike transfer via fluctuations in the membrane potential. The fine-tuning by cerebellar and cerebral activity allows the motor thalamus to operate as a low-pass filter for cerebellar activity, generating sparse but precisely timed outputs for the cerebral cortex.

Neurons store information by changing synaptic input weights. In addition, they can adjust their membrane excitability to alter spike output. Here, we demonstrate a role of such “intrinsic plasticity” in behavioral learning in a mouse model that allows us to detect specific consequences of absent excitability modulation. Mice with a Purkinje-cell–specific knockout (KO) of the calcium-activated K+ channel SK2 (L7-SK2) show intact vestibulo-ocular reflex (VOR) gain adaptation but impaired eyeblink conditioning (EBC), which relies on the ability to establish associations between stimuli, with the eyelid closure itself depending on a transient suppression of spike firing. In these mice, the intrinsic plasticity of Purkinje cells is prevented without affecting long-term depression or potentiation at their parallel fiber (PF) input. In contrast to the typical spike pattern of EBC-supporting zebrin-negative Purkinje cells, L7-SK2 neurons show reduced background spiking but enhanced excitability. Thus, SK2 plasticity and excitability modulation are essential for specific forms of motor learning., This study shows that cerebellum-dependent motor learning involves an adjustment of membrane excitability in Purkinje cells via modulation of SK2-type K+ channels; this "intrinsic plasticity" requirement can be separated from changes in synaptic strength.

The cerebellar vermis, long associated with axial motor control, has been implicated in a surprising range of neuropsychiatric disorders and cognitive and affective functions. Remarkably little is known, however, about the specific cell types and neural circuits responsible for these diverse functions. Here, using single-cell gene expression profiling and anatomical circuit analyses of vermis output neurons in the mouse fastigial (medial cerebellar) nucleus, we identify five major classes of glutamatergic projection neurons distinguished by gene expression, morphology, distribution, and input-output connectivity. Each fastigial cell type is connected with a specific set of Purkinje cells and inferior olive neurons and in turn innervates a distinct collection of downstream targets. Transsynaptic tracing indicates extensive disynaptic links with cognitive, affective, and motor forebrain circuits. These results indicate that diverse cerebellar vermis functions could be mediated by modular synaptic connections of distinct fastigial cell types with posturomotor, oromotor, positional-autonomic, orienting, and vigilance circuits.

Processing of genital sensations in the central nervous system of humans is still poorly understood. Current knowledge is mainly based on neuroimaging studies using electroencephalography (EEG), magneto-encephalography (MEG), and 1.5- or 3- Tesla (T) functional magnetic resonance imaging (fMRI), all of which suffer from limited spatial resolution and sensitivity, thereby relying on group analyses to reveal significant data. Here, we studied the impact of passive, yet non-arousing, tactile stimulation of the penile shaft using ultra-high field 7T fMRI. With this approach, penile stimulation evoked significant activations in distinct areas of the primary and secondary somatosensory cortices (S1 & S2), premotor cortex, insula, midcingulate gyrus, prefrontal cortex, thalamus and cerebellum, both at single subject and group level. Passive tactile stimulation of the feet, studied for control, also evoked significant activation in S1, S2, insula, thalamus and cerebellum, but predominantly, yet not exclusively, in areas that could be segregated from those associated with penile stimulation. Evaluation of the whole-brain activation patterns and connectivity analyses indicate that genital sensations following passive stimulation are, unlike those following feet stimulation, processed in both sensorimotor and affective regions.

Miniaturized fluorescence microscopes (miniscopes) have been instrumental to monitor neural activity during unrestrained behavior and their open-source versions have helped to distribute them at an affordable cost. Generally, the footprint and weight of open-source miniscopes is sacrificed for added functionality. Here, we present NINscope: a light-weight, small footprint, open-source miniscope that incorporates a high-sensitivity image sensor, an inertial measurement unit (IMU), and an LED driver for an external optogenetic probe. We highlight the advantages of NINscope by performing the first simultaneous cellular resolution (dual scope) recordings from cerebellum and cerebral cortex in unrestrained mice, revealing that the activity of both regions generally precedes the onset of behavioral acceleration. We further demonstrate the optogenetic stimulation capabilities of NINscope and show that cerebral cortical activity can be driven strongly by cerebellar stimulation. To validate the performance of our miniscope to image from deep-brain regions, we recorded in the dorsal striatum and, using the IMU to assess turning movements, replicate previous studies that show encoding of action space in this subcortical region. Finally, we combine optogenetic stimulation of distinct cortical regions projecting to the dorsal striatum, to probe functional connectivity. In combination with cross-platform control software, NINscope is a versatile addition to the expanding toolbox of open-source miniscopes and will aid multi-region circuit investigations during unrestrained behavior.

Highlights • Sleep deprivation directly after motor training impairs storage of memories. • Sleep deprivation directly after motor training also impairs retrieval of memories. • Sleeping longer directly following motor training facilitates memory formation. • Sleep may exert a subtle yet facilitatory role in consolidation of procedural memory., The relation between sleep and different forms of memory formation continues to be a relevant topic in our daily life. Sleep has been found to affect cerebellum-dependent procedural memory formation, but it remains to be elucidated to what extent the level of sleep deprivation directly after motor training also influences our ability to store and retrieve memories. Here, we studied the effect of disturbed sleep in mice during two different time-windows, one covering the first four hours following eyeblink conditioning (EBC) and another window following the next period of four hours. Compared to control mice with sleep ad libitum, the percentage of conditioned responses and their amplitude were impaired when mice were deprived of sleep directly after conditioning. This impairment was still significant when the learned EBC responses were extinguished and later reacquired. However, consolidation of eyeblink responses was not affected when mice were deprived later than four hours after acquisition, not even when tested during a different day-night cycle for control. Moreover, mice that slept longer directly following EBC showed a tendency for more conditioned responses. Our data indicate that consolidation of motor memories can benefit from sleep directly following memory formation.

Summary The cerebellum is involved in the control of voluntary and autonomic rhythmic behaviors, yet it is unclear to what extent it coordinates these in concert. We studied Purkinje cell activity during unperturbed and perturbed respiration in lobules simplex, crus 1, and crus 2. During unperturbed (eupneic) respiration, complex spike and simple spike activity encode the phase of ongoing sensorimotor processing. In contrast, when the respiratory cycle is perturbed by whisker stimulation, mice concomitantly protract their whiskers and advance their inspiration in a phase-dependent manner, preceded by increased simple spike activity. This phase advancement of respiration in response to whisker stimulation can be mimicked by optogenetic stimulation of Purkinje cells and prevented by cell-specific genetic modification of their AMPA receptors, hampering increased simple spike firing. Thus, the impact of Purkinje cell activity on respiratory control is context and phase dependent, highlighting a coordinating role for the cerebellar hemispheres in aligning autonomic and sensorimotor behaviors., Graphical Abstract, Highlights • During unperturbed respiration, Purkinje cells signal ongoing sensorimotor processing • After perturbation, mice advance their simple spike activity, whisking, and inspiration • Altering simple spike activity affects the impact of whisker stimulation on respiration • Cerebellar coordination of autonomic and sensorimotor behaviors is context dependent, Romano et al. show that cerebellar Purkinje cell activity follows the respiratory rhythm during rest. Triggered by sensory input, Purkinje cells can alter their activity and thereby accelerate the timing of the next inspiration. Concomitantly, they also augment whisker movements, highlighting a coordinating role in aligning autonomic and sensorimotor behaviors.

Substrates for neuron culture and implantation are required to be both biocompatible and display surface compositions that support cell attachment, growth, differentiation, and neural activity. Laminin, a naturally occurring extracellular matrix protein is the most widely used substrate for neuron culture and fulfills some of these requirements, however, it is expensive, unstable (compared to synthetic materials), and prone to batch-to-batch variation. This study uses a high-throughput polymer screening approach to identify synthetic polymers that supports the in vitro culture of primary mouse cerebellar neurons. This allows the identification of materials that enable primary cell attachment with high viability even under “serum-free” conditions, with materials that support both primary cells and neural progenitor cell attachment with high levels of neuronal biomarker expression, while promoting progenitor cell maturation to neurons.

The cerebellum drives motor coordination and sequencing of actions at the millisecond timescale through adaptive control of cerebellar nuclear output. Cerebellar nuclei integrate high-frequency information from both the cerebellar cortex and the two main excitatory inputs of the cerebellum: the mossy fibers and the climbing fiber collaterals. However, how nuclear cells process rate and timing of inputs carried by these inputs is still debated. Here, we investigate the influence of the cerebellar cortical output, the Purkinje cells, on identified cerebellar nuclei neuronsin vivoin male mice. Using transgenic mice expressing Channelrhodopsin2 specifically in Purkinje cells and tetrode recordings in the medial nucleus, we identified two main groups of neurons based on the waveform of their action potentials. These two groups of neurons coincide with glutamatergic and GABAergic neurons identified by optotagging after Chrimson expression inVGLUT2-creandGAD-cremice, respectively. The glutamatergic-like neurons fire at high rate and respond to both rate and timing of Purkinje cell population inputs, whereas GABAergic-like neurons only respond to the mean population firing rate of Purkinje cells at high frequencies. Moreover, synchronous activation of Purkinje cells can entrain the glutamatergic-like, but not the GABAergic-like, cells over a wide range of frequencies. Our results suggest that the downstream effect of synchronous and rhythmic Purkinje cell discharges depends on the type of cerebellar nuclei neurons targeted.SIGNIFICANCE STATEMENTMotor coordination and skilled movements are driven by the permanent discharge of neurons from the cerebellar nuclei that communicate cerebellar computation to other brain areas. Here, we set out to study how specific subtypes of cerebellar nuclear neurons of the medial nucleus are controlled by Purkinje cells, the sole output of the cerebellar cortex. We could isolate different subtypes of nuclear cell that differentially encode Purkinje cell inhibition. Purkinje cell stimulation entrains glutamatergic projection cells at their firing frequency, whereas GABAergic neurons are only inhibited. These differential coding strategies may favor temporal precision of cerebellar excitatory outputs associated with specific features of movement control while setting the global level of cerebellar activity through inhibition via rate coding mechanisms

Background and purpose Oncological neurosurgery relies heavily on making continuous, intra-operative tumor-brain delineations based on image-guidance. Limitations of currently available imaging techniques call for the development of real-time image-guided resection tools, which allow for reliable functional and anatomical information in an intra-operative setting. Functional ultrasound (fUS), is a new mobile neuro-imaging tool with unprecedented spatiotemporal resolution, which allows for the detection of small changes in blood dynamics that reflect changes in metabolic activity of activated neurons through neurovascular coupling. We have applied fUS during conventional awake brain surgery to determine its clinical potential for both intra-operative functional and vascular brain mapping, with the ultimate aim of achieving maximum safe tumor resection. Methods During awake brain surgery, fUS was used to image tumor vasculature and task-evoked brain activation with electrocortical stimulation mapping (ESM) as a gold standard. For functional imaging, patients were presented with motor, language or visual tasks, while the probe was placed over (ESM-defined) functional brain areas. For tumor vascular imaging, tumor tissue (pre-resection) and tumor resection cavity (post-resection) were imaged by moving the hand-held probe along a continuous trajectory over the regions of interest. Results A total of 10 patients were included, with predominantly intra-parenchymal frontal and temporal lobe tumors of both low and higher histopathological grades. fUS was able to detect (ESM-defined) functional areas deep inside the brain for a range of functional tasks including language processing. Brain tissue could be imaged at a spatial and temporal resolution of 300 μm and 1.5-2.0 ms respectively, revealing real-time tumor-specific, and healthy vascular characteristics. Conclusion The current study presents the potential of applying fUS during awake brain surgery. We illustrate the relevance of fUS for awake brain surgery based on its ability to capture both task-evoked functional cortical responses as well as differences in vascular characteristics between tumor and healthy tissue. As current neurosurgical practice is still pre-dominantly leaning on inherently limited pre-operative imaging techniques for tumor resection-guidance, fUS enters the scene as a promising alternative that is both anatomically and physiologically informative.

Coordination of bilateral movements is essential for a large variety of animal behaviors. The olivocerebellar system is critical for the control of movement, but its role in bilateral coordination has yet to be elucidated. Here, we examined whether Purkinje cells encode and influence synchronicity of left-right whisker movements. We found that complex spike activity is correlated with a prominent left-right symmetry of spontaneous whisker movements within parts, but not all, of Crus1 and Crus2. Optogenetic stimulation of climbing fibers in the areas with high and low correlations resulted in symmetric and asymmetric whisker movements, respectively. Moreover, when simple spike frequency prior to the complex spike was higher, the complex spike-related symmetric whisker protractions were larger. This finding alludes to a role for rebound potentiation in the cerebellar nuclei, which indeed turned out to be enhanced during symmetric protractions. The cerebellar nuclei neurons that received input from the Purkinje cells associated with symmetric whisker movements were found to project to the whisker regions on the contralateral side of the cerebellum, whereas those associated with asymmetric movements were not. Together, these data point towards the existence of modules on both sides of the cerebellar cortex that can differentially promote or reduce the symmetry of left and right movements in a context-dependent fashion.

Persistent and ramping neural activity in the frontal cortex anticipates specific movements1–6. Preparatory activity is distributed across several brain regions7,8, but it is unclear which brain areas are involved and how this activity is mediated by multi-regional interactions. The cerebellum is thought to be primarily involved in the short-timescale control of movement9–12; however, roles for this structure in cognitive processes have also been proposed13–16. In humans, cerebellar damage can cause defects in planning and working memory13. Here we show that persistent representation of information in the frontal cortex during motor planning is dependent on the cerebellum. Mice performed a sensory discrimination task in which they used short-term memory to plan a future directional movement. A transient perturbation in the medial deep cerebellar nucleus (fastigial nucleus) disrupted subsequent correct responses without hampering movement execution. Preparatory activity was observed in both the frontal cortex and the cerebellar nuclei, seconds before the onset of movement. The silencing of frontal cortex activity abolished preparatory activity in the cerebellar nuclei, and fastigial activity was necessary to maintain cortical preparatory activity. Fastigial output selectively targeted the behaviourally relevant part of the frontal cortex through the thalamus, thus closing a cortico-cerebellar loop. Our results support the view that persistent neural dynamics during motor planning is maintained by neural circuits that span multiple brain regions17, and that cerebellar computations extend beyond online motor control13–15,18. The cerebellum is critical for the coding of future movement in the frontal cortex.

Disruptions of the FOXP2 gene cause a speech and language disorder involving difficulties in sequencing orofacial movements. FOXP2 is expressed in cortico-striatal and cortico-cerebellar circuits important for fine motor skills, and affected individuals show abnormalities in these brain regions. We selectively disrupted Foxp2 in the cerebellar Purkinje cells, striatum or cortex of mice and assessed the effects on skilled motor behaviour using an operant lever-pressing task. Foxp2 loss in each region impacted behaviour differently, with striatal and Purkinje cell disruptions affecting the variability and the speed of lever-press sequences, respectively. Mice lacking Foxp2 in Purkinje cells showed a prominent phenotype involving slowed lever pressing as well as deficits in skilled locomotion. In vivo recordings from Purkinje cells uncovered an increased simple spike firing rate and decreased modulation of firing during limb movements. This was caused by increased intrinsic excitability rather than changes in excitatory or inhibitory inputs. Our findings show that Foxp2 can modulate different aspects of motor behaviour in distinct brain regions, and uncover an unknown role for Foxp2 in the modulation of Purkinje cell activity that severely impacts skilled movements.

The basal interstitial nucleus (BIN) in the white matter of the vestibulocerebellum has been defined more than three decades ago, but has since been largely ignored. It is still unclear which neurotransmitters are being used by BIN neurons, how these neurons are connected to the rest of the brain and what their activity patterns look like. Here, we studied BIN neurons in a range of mammals, including macaque, human, rat, mouse, rabbit and ferret, using tracing, immunohistological and electrophysiological approaches. We show that BIN neurons are GABAergic and glycinergic, that in primates they also express the marker for cholinergic neurons choline acetyl transferase (ChAT), that they project with beaded fibers to the glomeruli in the granular layer of the ipsilateral floccular complex, and that they are driven by excitation from the ipsilateral and contralateral medio-dorsal medullary gigantocellular reticular formation. Systematic analysis of co-distribution of the inhibitory synapse marker VIAAT, labeled BIN axons and Golgi cell marker mGluR2 indicate that BIN axon terminals complement Golgi cell axon terminals in glomeruli, accounting for a considerable proportion (> 20%) of the inhibitory terminals in the granule cell layer of the floccular complex. Together, these data show that BIN neurons represent a novel and relevant inhibitory input to the part of the vestibulocerebellum that controls compensatory and smooth pursuit eye movements. This article is protected by copyright. All rights reserved.

Summary The cerebellum plays a role in coordination of movements and non-motor functions. Cerebellar nuclei (CN) axons connect to various parts of the thalamo-cortical network, but detailed information on the characteristics of cerebello-thalamic connections is lacking. Here, we assessed the cerebellar input to the ventrolateral (VL), ventromedial (VM), and centrolateral (CL) thalamus. Confocal and electron microscopy showed an increased density and size of CN axon terminals in VL compared to VM or CL. Electrophysiological recordings in vitro revealed that optogenetic CN stimulation resulted in enhanced charge transfer and action potential firing in VL neurons compared to VM or CL neurons, despite that the paired-pulse ratio was not significantly different. Together, these findings indicate that the impact of CN input onto neurons of different thalamic nuclei varies substantially, which highlights the possibility that cerebellar output differentially controls various parts of the thalamo-cortical network., Graphical Abstract, Highlights • Cerebello-thalamic axons form terminals of varying size in distinct thalamic nuclei • Cerebello-thalamic responses vary in amplitude in distinct thalamic nuclei • Repetitive stimuli depress cerebello-thalamic responses in all thalamic nuclei, In this study, Gornati et al. demonstrate that the impact of cerebellar nuclei axons on thalamic neurons varies per thalamic region. These findings provide insights into how the versatile cerebellum can have a differential effect on the many brain regions that it connects to.

Among the many types of neurons expressing protein kinase C (PKC) enzymes, cerebellar Purkinje neurons are particularly reliant on appropriate PKC activity for maintaining homeostasis. The importance of PKC enzymes in Purkinje neuron health is apparent as mutations in PRKCG (encoding PKCγ) cause cerebellar ataxia. PRKCG has also been identified as an important node in ataxia gene networks more broadly, but the functional role of PKC in other forms of ataxia remains unexplored, and the mechanisms by which PKC isozymes regulate Purkinje neuron health are not well understood. Here, we investigated how PKC activity influences neurodegeneration in inherited ataxia. Using mouse models of spinocerebellar ataxia type 1 (SCA1) and 2 (SCA2) we identify an increase in PKC-mediated substrate phosphorylation in two different forms of inherited cerebellar ataxia. Normalizing PKC substrate phosphorylation in SCA1 and SCA2 mice accelerates degeneration, suggesting that the increased activity observed in these models is neuroprotective. We also find that increased phosphorylation of PKC targets limits Purkinje neuron membrane excitability, suggesting that PKC activity may support Purkinje neuron health by moderating excitability. These data suggest a functional role for PKC enzymes in ataxia gene networks, and demonstrate that increased PKC activity is a protective modifier of degeneration in inherited cerebellar ataxia.

Cerebellar plasticity is a critical mechanism for optimal feedback control. While Purkinje cell activity of the oculomotor vermis predicts eye movement speed and direction, more lateral areas of the cerebellum may play a role in more complex tasks, including decision-making. It is still under question how this motor-cognitive functional dichotomy between medial and lateral areas of the cerebellum plays a role in optimal feedback control. Here we show that elite athletes subjected to a trajectory prediction, go/no-go task manifest superior subsecond trajectory prediction accompanied by optimal eye movements and changes in cognitive load dynamics. Moreover, while interacting with the cerebral cortex, both the medial and lateral cerebellar networks are prominently activated during the fast feedback stage of the task, regardless of whether or not a motor response was required for the correct response. Our results show that cortico-cerebellar interactions are widespread during dynamic feedback and that experience can result in superior task-specific decision skills.

Background Cerebellar transcranial direct current stimulation has been reported to enhance motor associative learning and motor adaptation, holding promise for clinical application in patients with movement disorders. However, behavioral benefits from cerebellar tDCS have been inconsistent. Objective Identifying determinants of treatment success is necessary. BDNF Val66Met is a candidate determinant, because the polymorphism is associated with motor skill learning and BDNF is thought to mediate tDCS effects. Methods We undertook two cerebellar tDCS studies in subjects genotyped for BDNF Val66Met. Subjects performed an eyeblink conditioning task and received sham, anodal or cathodal tDCS (N = 117, between-subjects design) or a vestibulo-ocular reflex adaptation task and received sham and anodal tDCS (N = 51 subjects, within-subjects design). Performance was quantified as a learning parameter from 0 to 100%. We investigated (1) the distribution of the learning parameter with mixture modeling presented as the mean (M), standard deviation (S) and proportion (P) of the groups, and (2) the role of BDNF Val66Met and cerebellar tDCS using linear regression presented as the regression coefficients (B) and odds ratios (OR) with equally-tailed intervals (ETIs). Results For the eyeblink conditioning task, we found distinct groups of learners (MLearner = 67.2%; SLearner = 14.7%; PLearner = 61.6%) and non-learners (MNon-learner = 14.2%; SNon-learner = 8.0%; PNon-learner = 38.4%). Carriers of the BDNF Val66Met polymorphism were more likely to be learners (OR = 2.7 [1.2 6.2]). Within the group of learners, anodal tDCS supported eyeblink conditioning in BDNF Val66Met non-carriers (B = 11.9% 95%ETI = [0.8 23.0]%), but not in carriers (B = 1.0% 95%ETI = [-10.2 12.1]%). For the vestibulo-ocular reflex adaptation task, we found no effect of BDNF Val66Met (B = −2.0% 95%ETI = [-8.7 4.7]%) or anodal tDCS in either carriers (B = 3.4% 95%ETI = [-3.2 9.5]%) or non-carriers (B = 0.6% 95%ETI = [-3.4 4.8]%). Finally, we performed additional saccade and visuomotor adaptation experiments (N = 72) to investigate the general role of BDNF Val66Met in cerebellum-dependent learning and found no difference between carriers and non-carriers for both saccade (B = 1.0% 95%ETI = [-8.6 10.6]%) and visuomotor adaptation (B = 2.7% 95%ETI = [-2.5 7.9]%). Conclusions The specific role for BDNF Val66Met in eyeblink conditioning, but not vestibulo-ocular reflex adaptation, saccade adaptation or visuomotor adaptation could be related to dominance of the role of simple spike suppression of cerebellar Purkinje cells with a high baseline firing frequency in eyeblink conditioning. Susceptibility of non-carriers to anodal tDCS in eyeblink conditioning might be explained by a relatively larger effect of tDCS-induced subthreshold depolarization in this group, which might increase the spontaneous firing frequency up to the level of that of the carriers.

Skin biopsies from patients with neuropathic pain often show changes in epidermal innervation, although it remains to be elucidated to what extent such changes can be linked to a particular subgroup of nerve fibers and how these changes are correlated with pain intensity. Here, we investigated to what extent behavioral signs of hyperalgesia are correlated with immunohistochemical changes of peptidergic and non-peptidergic epidermal nerve fibers in a rat model of nerve injury-induced pain. Rats subjected to unilateral partial ligation of the sciatic nerve developed significant mechanical and thermal hyperalgesia as tested by the withdrawal responses of the ipsilateral footpad to von Frey hairs and hotplate stimulation. At day 14, epidermal nerve fiber density (IENFD) and total epidermal nerve fiber length/mm(2) (ENFL) were significantly and consistently reduced compared to the contralateral side, following testing and re-testing by two blinded observers. The expression of calcitonin gene-related peptide (CGRP), a marker for peptidergic nerve fibers, was not significantly changed on the ipsilateral side. In contrast, the expression of the P2X3 receptor, a marker for non-peptidergic nerve fibers, was not only significantly reduced but could also be correlated with behavioral hyperalgesia. When labeling both peptidergic and non-peptidergic nerve fibers with the pan-neuronal marker PGP9.5, the expression was significantly reduced, albeit without a significant correlation with behavioral hyperalgesia. In conjunction, our data suggest that the pathology of the P2X3 epidermal nerve fibers can be selectively linked to neuropathy, highlighting the possibility that it is the degeneration of these fibers that drives hyperalgesia. This article is protected by copyright. All rights reserved.

The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.

Compensatory eye movements elicited by head rotation, also known as vestibulo-ocular reflex (VOR), can be adapted with the use of visual feedback. The cerebellum is essential for this type of movement adaptation, but its neuronal correlates remain to be elucidated. Here we show that the direction of vestibular input determines the magnitude of eye movement adaptation induced by mismatched visual input in mice, with larger changes during contraversive head rotation. Moreover, the location of the neural correlate of this changed behaviour depends on the type of paradigm. Gain-increase paradigms induce increased simple spike (SS) activity in ipsilateral cerebellar Purkinje cells (PC), which is in line with eye movements triggered by optogenetic PC activation. In contrast, gain-decrease paradigms do not induce changes in SS activity, highlighting that the murine vestibulo-cerebellar cortical circuitry is optimally designed to enhance ipsiversive eye movements. This article is protected by copyright. All rights reserved

Classical delay eyeblink conditioning is likely the most commonly used paradigm to study cerebellar learning. As yet, few studies have focused on extinction and savings of conditioned eyeblink responses (CRs). Saving effects, which are reflected in a reacquisition after extinction that is faster than the initial acquisition, suggest that learned associations are at least partly preserved during extinction. In this study, we tested the hypothesis that acquisition-related plasticity is nihilated during extinction in the cerebellar cortex, but retained in the cerebellar nuclei, allowing for faster reacquisition. Changes of 7 T functional magnetic resonance imaging (fMRI) signals were investigated in the cerebellar cortex and nuclei of young and healthy human subjects. Main effects of acquisition, extinction, and reacquisition against rest were calculated in conditioned stimulus-only trials. First-level β values were determined for a spherical region of interest (ROI) around the acquisition peak voxel in lobule VI, and dentate and interposed nuclei ipsilateral to the unconditioned stimulus. In the cerebellar cortex and nuclei, fMRI signals were significantly lower in extinction compared to acquisition and reacquisition, but not significantly different between acquisition and reacquisition. These findings are consistent with the theory of bidirectional learning in both the cerebellar cortex and nuclei. It cannot explain, however, why conditioned responses reappear almost immediately in reacquisition following extinction. Although the present data do not exclude that part of the initial memory remains in the cerebellum in extinction, future studies should also explore changes in extracerebellar regions as a potential substrate of saving effects. Hum Brain Mapp, 2017. © 2017 Wiley Periodicals, Inc.