PP2B-Dependent Cerebellar Plasticity Sets the Amplitude of the Vestibulo-ocular Reflex during Juvenile Development.

Throughout life, the cerebellum plays a central role in the coordination and optimization of movements, using cellular plasticity to adapt a range of behaviors. Whether these plasticity processes establish a fixed setpoint during development, or continuously adjust behaviors throughout life, is currently unclear. Here, by spatiotemporally manipulating the activity of protein phosphatase 2B (PP2B), an enzyme critical for cerebellar plasticity in male and female mice, we examined the consequences of disrupted plasticity on the performance and adaptation of the vestibulo-ocular reflex (VOR). We find that, in contrast to Purkinje cell (PC)-specific deletion starting early postnatally, acute pharmacological as well as adult-onset genetic deletion of PP2B affects all forms of VOR adaptation but not the level of VOR itself. Next, we show that PC-specific genetic deletion of PP2B in juvenile mice leads to a progressive loss of the protein PP2B and a concurrent change in the VOR, in addition to the loss of adaptive abilities. Finally, re-expressing PP2B in adult mice that lack PP2B expression from early development rescues VOR adaptation but does not affect the performance of the reflex. Together, our results indicate that chronic or acute, genetic, or pharmacological block of PP2B disrupts the adaptation of the VOR. In contrast, only the absence of plasticity during cerebellar development affects the setpoint of VOR, an effect that cannot be corrected after maturation of the cerebellum. These findings suggest that PP2B-dependent cerebellar plasticity is required during a specific period to achieve the correct setpoint of the VOR.

Pre-ataxic loss of intrinsic plasticity and motor learning in a mouse model of SCA1.

Spinocerebellar ataxias are neurodegenerative diseases, the hallmark symptom of which is the development of ataxia due to cerebellar dysfunction. Purkinje cells, the principal neurons of the cerebellar cortex, are the main cells affected in these disorders, but the sequence of pathological events leading to their dysfunction is poorly understood. Understanding the origins of Purkinje cells dysfunction before it manifests is imperative to interpret the functional and behavioural consequences of cerebellar-related disorders, providing an optimal timeline for therapeutic interventions. Here, we report the cascade of events leading to Purkinje cells dysfunction before the onset of ataxia in a mouse model of spinocerebellar ataxia 1 (SCA1). Spatiotemporal characterization of the ATXN1[82Q] SCA1 mouse model revealed high levels of the mutant ATXN1[82Q] weeks before the onset of ataxia. The expression of the toxic protein first caused a reduction of Purkinje cells intrinsic excitability, which was followed by atrophy of Purkinje cells dendrite arborization and aberrant glutamatergic signalling, finally leading to disruption of Purkinje cells innervation of climbing fibres and loss of intrinsic plasticity of Purkinje cells. Functionally, we found that deficits in eyeblink conditioning, a form of cerebellum-dependent motor learning, precede the onset of ataxia, matching the timeline of climbing fibre degeneration and reduced intrinsic plasticity. Together, our results suggest that abnormal synaptic signalling and intrinsic plasticity during the pre-ataxia stage of spinocerebellar ataxias underlie an aberrant cerebellar circuitry that anticipates the full extent of the disease severity. Furthermore, our work indicates the potential for eyeblink conditioning to be used as a sensitive tool to detect early cerebellar dysfunction as a sign of future disease.

Sphingolipid metabolism governs Purkinje cell patterned degeneration in Atxn1[82Q]/+ mice.

Patterned degeneration of Purkinje cells (PCs) can be observed in a wide range of neuropathologies, but mechanisms behind nonrandom cerebellar neurodegeneration remain unclear. Sphingolipid metabolism dyshomeostasis typically leads to PC neurodegeneration; hence, we questioned whether local sphingolipid balance underlies regional sensitivity to pathological insults. Here, we investigated the regional compartmentalization of sphingolipids and their related enzymes in the cerebellar cortex in healthy and pathological conditions. Analysis in wild-type animals revealed higher sphingosine kinase 1 (Sphk1) levels in the flocculonodular cerebellum, while sphingosine-1-phosphate (S1P) levels were higher in the anterior cerebellum. Next, we investigated a model for spinocerebellar ataxia type 1 (SCA1) driven by the transgenic expression of the expanded Ataxin 1 protein with 82 glutamine (82Q), exhibiting severe PC degeneration in the anterior cerebellum while the flocculonodular region is preserved. In Atxn1[82Q]/+ mice, we found that levels of Sphk1 and Sphk2 were region-specific decreased and S1P levels increased, particularly in the anterior cerebellum. To determine if there is a causal link between sphingolipid levels and neurodegeneration, we deleted the Sphk1 gene in Atxn1[82Q]/+ mice. Analysis of Atxn1[82Q]/+; Sphk1-/- mice confirmed a neuroprotective effect, rescuing a subset of PCs in the anterior cerebellum, in domains reminiscent of the modules defined by AldolaseC expression. Finally, we showed that Sphk1 deletion acts on the ATXN1[82Q] protein expression and prevents PC degeneration. Taken together, our results demonstrate that there are regional differences in sphingolipid metabolism and that this metabolism is directly involved in PC degeneration in Atxn1[82Q]/+ mice.

NMDARs in granule cells contribute to parallel fiber-Purkinje cell synaptic plasticity and motor learning.

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.

SK2 channels in cerebellar Purkinje cells contribute to excitability modulation in motor-learning-specific memory traces.

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.

Protein Phosphatase 2B Dual Function Facilitates Synaptic Integrity and Motor Learning.

Protein phosphatase 2B (PP2B) is critical for synaptic plasticity and learning, but the molecular mechanisms involved remain unclear. Here we identified different types of proteins that interact with PP2B, including various structural proteins of the postsynaptic densities (PSDs) of Purkinje cells (PCs) in mice. Deleting PP2B reduced expression of PSD proteins and the relative thickness of PSD at the parallel fiber to PC synapses, whereas reexpression of inactive PP2B partly restored the impaired distribution of nanoclusters of PSD proteins, together indicating a structural role of PP2B. In contrast, lateral mobility of surface glutamate receptors solely depended on PP2B phosphatase activity. Finally, the level of motor learning covaried with both the enzymatic and nonenzymatic functions of PP2B. Thus, PP2B controls synaptic function and learning both through its action as a phosphatase and as a structural protein that facilitates synapse integrity.SIGNIFICANCE STATEMENT Phosphatases are generally considered to serve their critical role in learning and memory through their enzymatic operations. Here, we show that protein phosphatase 2B (PP2B) interacts with structural proteins at the synapses of cerebellar Purkinje cells. Differentially manipulating the enzymatic and structural domains of PP2B leads to different phenotypes in cerebellar learning. We propose that PP2B is crucial for cerebellar learning via two complementary actions, an enzymatic and a structural operation.

Correction to: Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper.

In the original version of this paper, the Title should have been written with "A Consensus paper" to read "Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper".

Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper [corrected].

The compartmentalization of the cerebellum into modules is often used to discuss its function. What, exactly, can be considered a module, how do they operate, can they be subdivided and do they act individually or in concert are only some of the key questions discussed in this consensus paper. Experts studying cerebellar compartmentalization give their insights on the structure and function of cerebellar modules, with the aim of providing an up-to-date review of the extensive literature on this subject. Starting with an historical perspective indicating that the basis of the modular organization is formed by matching olivocorticonuclear connectivity, this is followed by consideration of anatomical and chemical modular boundaries, revealing a relation between anatomical, chemical, and physiological borders. In addition, the question is asked what the smallest operational unit of the cerebellum might be. Furthermore, it has become clear that chemical diversity of Purkinje cells also results in diversity of information processing between cerebellar modules. An additional important consideration is the relation between modular compartmentalization and the organization of the mossy fiber system, resulting in the concept of modular plasticity. Finally, examination of cerebellar output patterns suggesting cooperation between modules and recent work on modular aspects of emotional behavior are discussed. Despite the general consensus that the cerebellum has a modular organization, many questions remain. The authors hope that this joint review will inspire future cerebellar research so that we are better able to understand how this brain structure makes its vital contribution to behavior in its most general form.

Mechanisms underlying vestibulo-cerebellar motor learning in mice depend on movement direction.

KEY POINTS: Directionality, inherent to movements, has behavioural and neuronal correlates. Direction of vestibular stimulation determines motor learning efficiency. Vestibulo-ocular reflex gain-increase correlates with Purkinje cell simple spike potentiation. The locus of neural correlates for vestibulo-ocular reflex adaptation is paradigm specific. ABSTRACT: 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, although its neuronal correlates remain to be clarified. In the present study, 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. By contrast, gain-decrease paradigms do not induce changes in SS activity, indicating that the murine vestibulo-cerebellar cortical circuitry is optimally designed to enhance ipsiversive eye movements.

Reversibility of neuropathology and motor deficits in an inducible mouse model for FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of the fragile X-premutation, who have an expanded CGG repeat in the 5'-UTR of the FMR1 gene. FXTAS is characterized by progressive development of intention tremor, ataxia, parkinsonism and neuropsychological problems. The disease is thought to be caused by a toxic RNA gain-of-function mechanism, and the major hallmark of the disease is ubiquitin-positive intranuclear inclusions in neurons and astrocytes. We have developed a new transgenic mouse model in which we can induce expression of an expanded repeat in the brain upon doxycycline (dox) exposure (i.e. Tet-On mice). This Tet-On model makes use of the PrP-rtTA driver and allows us to study disease progression and possibilities of reversibility. In these mice, 8 weeks of dox exposure was sufficient to induce the formation of ubiquitin-positive intranuclear inclusions, which also stain positive for the RAN translation product FMRpolyG. Formation of these inclusions is reversible after stopping expression of the expanded CGG RNA at an early developmental stage. Furthermore, we observed a deficit in the compensatory eye movements of mice with inclusions, a functional phenotype that could be reduced by stopping expression of the expanded CGG RNA early in the disease development. Taken together, this study shows, for the first time, the potential of disease reversibility and suggests that early intervention might be beneficial for FXTAS patients.

Raising cytosolic Cl- in cerebellar granule cells affects their excitability and vestibulo-ocular learning.

Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo-ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K-Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABA(A)-receptor Cl(-) channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl(-)](i) roughly two-fold. The reduced Cl(-) gradient nearly abolished GABA-induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long-term phase learning of the vestibulo-ocular reflex, whereas baseline performance, short-term gain-decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning.

Spatiotemporal firing patterns in the cerebellum.

Neurons are generally considered to communicate information by increasing or decreasing their firing rate. However, in principle, they could in addition convey messages by using specific spatiotemporal patterns of spiking activities and silent intervals. Here, we review expanding lines of evidence that such spatiotemporal coding occurs in the cerebellum, and that the olivocerebellar system is optimally designed to generate and employ precise patterns of complex spikes and simple spikes during the acquisition and consolidation of motor skills. These spatiotemporal patterns may complement rate coding, thus enabling precise control of motor and cognitive processing at a high spatiotemporal resolution by fine-tuning sensorimotor integration and coordination.

T-type channel blockade impairs long-term potentiation at the parallel fiber-Purkinje cell synapse and cerebellar learning.

CaV3.1 T-type channels are abundant at the cerebellar synapse between parallel fibers and Purkinje cells where they contribute to synaptic depolarization. So far, no specific physiological function has been attributed to these channels neither as charge carriers nor more specifically as Ca(2+) carriers. Here we analyze their incidence on synaptic plasticity, motor behavior, and cerebellar motor learning, comparing WT animals and mice where T-type channel function has been abolished either by gene deletion or by acute pharmacological blockade. At the cellular level, we show that CaV3.1 channels are required for long-term potentiation at parallel fiber-Purkinje cell synapses. Moreover, basal simple spike discharge of the Purkinje cell in KO mice is modified. Acute or chronic T-type current blockade results in impaired motor performance in particular when a good body balance is required. Because motor behavior integrates reflexes and past memories of learned behavior, this suggests impaired learning. Indeed, subjecting the KO mice to a vestibulo-ocular reflex phase reversal test reveals impaired cerebellum-dependent motor learning. These data identify a role of low-voltage activated calcium channels in synaptic plasticity and establish a role for CaV3.1 channels in cerebellar learning.

Differential Purkinje cell simple spike activity and pausing behavior related to cerebellar modules.

The massive computational capacity of the cerebellar cortex is conveyed by Purkinje cells onto cerebellar and vestibular nuclei neurons through their GABAergic, inhibitory output. This implies that pauses in Purkinje cell simple spike activity are potentially instrumental in cerebellar information processing, but their occurrence and extent are still heavily debated. The cerebellar cortex, although often treated as such, is not homogeneous. Cerebellar modules with distinct anatomical connectivity and gene expression have been described, and Purkinje cells in these modules also differ in firing rate of simple and complex spikes. In this study we systematically correlate, in awake mice, the pausing in simple spike activity of Purkinje cells recorded throughout the entire cerebellum, with their location in terms of lobule, transverse zone, and zebrin-identified cerebellar module. A subset of Purkinje cells displayed long (>500-ms) pauses, but we found that their occurrence correlated with tissue damage and lower temperature. In contrast to long pauses, short pauses (<500 ms) and the shape of the interspike interval (ISI) distributions can differ between Purkinje cells of different lobules and cerebellar modules. In fact, the ISI distributions can differ both between and within populations of Purkinje cells with the same zebrin identity, and these differences are at least in part caused by differential synaptic inputs. Our results suggest that long pauses are rare but that there are differences related to shorter intersimple spike intervals between and within specific subsets of Purkinje cells, indicating a potential further segregation in the activity of cerebellar Purkinje cells.

Aberrant outputs of cerebellar nuclei and targeted rescue of social deficits in an autism mouse model.

The cerebellum is heavily connected with other brain regions, sub-serving not only motor but also non-motor functions. Genetic mutations leading to cerebellar dysfunction are associated with mental diseases, but cerebellar outputs have not been systematically studied in this context. Here, we present three dimensional distributions of 50,168 target neurons of cerebellar nuclei (CN) from wild-type mice and Nlgn3R451C mutant mice, a mouse model for autism. Our results derived from 36 target nuclei show that the projections from CN to thalamus, midbrain and brainstem are differentially affected by Nlgn3R451C mutation. Importantly, Nlgn3R451C mutation altered the innervation power of CN→zona incerta (ZI) pathway, and chemogenetic inhibition of a neuronal subpopulation in the ZI that receives inputs from the CN rescues social defects in Nlgn3R451C mice. Our study highlights potential role of cerebellar outputs in the pathogenesis of autism and provides potential new therapeutic strategy for this disease.

Genetic dissection of the function of hindbrain axonal commissures.

In Bilateria, many axons cross the midline of the central nervous system, forming well-defined commissures. Whereas in mammals the functions of commissures in the forebrain and in the visual system are well established, functions at other axial levels are less clearly understood. Here, we have dissected the function of several hindbrain commissures using genetic methods. By taking advantage of multiple Cre transgenic lines, we have induced site-specific deletions of the Robo3 receptor. These lines developed with the disruption of specific commissures in the sensory, motor, and sensorimotor systems, resulting in severe and permanent functional deficits. We show that mice with severely reduced commissures in rhombomeres 5 and 3 have abnormal lateral eye movements and auditory brainstem responses, respectively, whereas mice with a primarily uncrossed climbing fiber/Purkinje cell projection are strongly ataxic. Surprisingly, although rerouted axons remain ipsilateral, they still project to their appropriate neuronal targets. Moreover, some Cre;Robo3 lines represent potential models that can be used to study human syndromes, including horizontal gaze palsy with progressive scoliosis (HGPPS). To our knowledge, this study is one of the first to link defects in commissural axon guidance with specific cellular and behavioral phenotypes.

Lobule-Related Action Potential Shape- and History-Dependent Current Integration in Purkinje Cells of Adult and Developing Mice.

Purkinje cells (PCs) are the principal cells of the cerebellar cortex and form a central element in the modular organization of the cerebellum. Differentiation of PCs based on gene expression profiles revealed two subpopulations with distinct connectivity, action potential firing and learning-induced activity changes. However, which basal cell physiological features underlie the differences between these subpopulations and to what extent they integrate input differentially remains largely unclear. Here, we investigate the cellular electrophysiological properties of PC subpopulation in adult and juvenile mice. We found that multiple fundamental cell physiological properties, including membrane resistance and various aspects of the action potential shape, differ between PCs from anterior and nodular lobules. Moreover, the two PC subpopulations also differed in the integration of negative and positive current steps as well as in size of the hyperpolarization-activated current. A comparative analysis in juvenile mice confirmed that most of these lobule-specific differences are already present at pre-weaning ages. Finally, we found that current integration in PCs is input history-dependent for both positive and negative currents, but this is not a distinctive feature between anterior and nodular PCs. Our results support the concept of a fundamental differentiation of PCs subpopulations in terms of cell physiological properties and current integration, yet reveals that history-dependent input processing is consistent across PC subtypes.

Purkinje cell microzones mediate distinct kinematics of a single movement.

The classification of neuronal subpopulations has significantly advanced, yet its relevance for behavior remains unclear. The highly organized flocculus of the cerebellum, known to fine-tune multi-axial eye movements, is an ideal substrate for the study of potential functions of neuronal subpopulations. Here, we demonstrate that its recently identified subpopulations of 9+ and 9- Purkinje cells exhibit an intermediate Aldolase C expression and electrophysiological profile, providing evidence for a graded continuum of intrinsic properties among PC subpopulations. By identifying and utilizing two Cre-lines that genetically target these floccular domains, we show with high spatial specificity that these subpopulations of Purkinje cells participate in separate micromodules with topographically organized connections. Finally, optogenetic excitation of the respective subpopulations results in movements around the same axis in space, yet with distinct kinematic profiles. These results indicate that Purkinje cell subpopulations integrate in discrete circuits and mediate particular parameters of single movements.

Purkinje-cell-specific MeCP2 deficiency leads to motor deficits and autistic-like behavior due to aberrations in PTP1B-TrkB-SK signaling.

Patients with Rett syndrome suffer from a loss-of-function mutation of the Mecp2 gene, which results in various symptoms including autistic traits and motor deficits. Deletion of Mecp2 in the brain mimics part of these symptoms, but the specific function of methyl-CpG-binding protein 2 (MeCP2) in the cerebellum remains to be elucidated. Here, we demonstrate that Mecp2 deletion in Purkinje cells (PCs) reduces their intrinsic excitability through a signaling pathway comprising the small-conductance calcium-activated potassium channel PTP1B and TrkB, the receptor of brain-derived neurotrophic factor. Aberration of this cascade, in turn, leads to autistic-like behaviors as well as reduced vestibulocerebellar motor learning. Interestingly, increasing activity of TrkB in PCs is sufficient to rescue PC dysfunction and abnormal motor and non-motor behaviors caused by Mecp2 deficiency. Our findings highlight how PC dysfunction may contribute to Rett syndrome, providing insight into the underlying mechanism and paving the way for rational therapeutic designs.