Different Purkinje cell pathologies cause specific patterns of progressive gait ataxia in mice.

Gait ataxia is one of the most common and impactful consequences of cerebellar dysfunction. Purkinje cells, the sole output neurons of the cerebellar cortex, are often involved in the underlying pathology, but their specific functions during locomotor control in health and disease remain obfuscated. We aimed to describe the effect of gradual adult-onset Purkinje cell degeneration on gaiting patterns in mice, and to determine whether two different mechanisms that both lead to Purkinje cell degeneration cause different patterns in the development of gait ataxia. Using the ErasmusLadder together with a newly developed limb detection algorithm and machine learning-based classification, we subjected mice to a challenging locomotor task with detailed analysis of single limb parameters, intralimb coordination and whole-body movement. We tested two Purkinje cell-specific mouse models, one involving stochastic cell death due to impaired DNA repair mechanisms (Pcp2-Ercc1-/-), the other carrying the mutation that causes spinocerebellar ataxia type 1 (Pcp2-ATXN1[82Q]). Both mouse models showed progressive gaiting deficits, but the sequence with which gaiting parameters deteriorated was different between mouse lines. Our longitudinal approach revealed that gradual loss of Purkinje cell function can lead to a complex pattern of loss of function over time, and that this pattern depends on the specifics of the pathological mechanisms involved. We hypothesize that this variability will also be present in disease progression in patients, and that our findings will facilitate the study of therapeutic interventions in mice, as subtle changes in locomotor abilities can be quantified by our methods.

Swept-3-D Ultrasound Imaging of the Mouse Brain Using a Continuously Moving 1-D-Array-Part II: Functional Imaging.

Functional ultrasound (fUS) using a 1-D-array transducer normally is insufficient to capture volumetric functional activity due to being restricted to imaging a single brain slice at a time. Typically, for volumetric fUS, functional recordings are repeated many times as the transducer is moved to a new location after each recording, resulting in a nonunique average mapping of the brain response and long scan times. Our objective was to perform volumetric 3-D fUS in an efficient and cost-effective manner. This was achieved by mounting a 1-D-array transducer to a high-precision motorized linear stage and continuously translating over the mouse brain in a sweeping manner. We show how the speed at which the 1-D-array is translated over the brain affects the sampling of the hemodynamic response (HR) during visual stimulation as well as the quality of the resulting power Doppler image (PDI). Functional activation maps were compared between stationary recordings, where only one functional slice is obtained for every recording, and our swept-3-D method, where volumetric fUS was achieved in a single functional recording. The results show that the activation maps obtained with our method closely resemble those obtained during a stationary recording for that same location, while our method is not restricted to functional imaging of a single slice. Lastly, a mouse brain subvolume of ~6 mm is scanned at a volume rate of 1.5 s per volume, with a functional PDI reconstructed every [Formula: see text], highlighting swept-3-D's potential for volumetric fUS. Our method provides an affordable alternative to volumetric fUS using 2-D-matrix transducers, with a high SNR due to using a fully sampled 1-D-array transducer, and without the need to repeat functional measurements for every 2-D slice, as is most often the case when using a 1-D-array. This places our swept-3-D method as a potentially valuable addition to conventional 2-D fUS, especially when investigating whole-brain functional connectivity, or when shorter recording durations are desired.

The integrated brain network that controls respiration.

Respiration is a brain function on which our lives essentially depend. Control of respiration ensures that the frequency and depth of breathing adapt continuously to metabolic needs. In addition, the respiratory control network of the brain has to organize muscular synergies that integrate ventilation with posture and body movement. Finally, respiration is coupled to cardiovascular function and emotion. Here, we argue that the brain can handle this all by integrating a brainstem central pattern generator circuit in a larger network that also comprises the cerebellum. Although currently not generally recognized as a respiratory control center, the cerebellum is well known for its coordinating and modulating role in motor behavior, as well as for its role in the autonomic nervous system. In this review, we discuss the role of brain regions involved in the control of respiration, and their anatomical and functional interactions. We discuss how sensory feedback can result in adaptation of respiration, and how these mechanisms can be compromised by various neurological and psychological disorders. Finally, we demonstrate how the respiratory pattern generators are part of a larger and integrated network of respiratory brain regions.

A Systematic Review of Direct Outputs from the Cerebellum to the Brainstem and Diencephalon in Mammals.

The cerebellum is involved in many motor, autonomic and cognitive functions, and new tasks that have a cerebellar contribution are discovered on a regular basis. Simultaneously, our insight into the functional compartmentalization of the cerebellum has markedly improved. Additionally, studies on cerebellar output pathways have seen a renaissance due to the development of viral tracing techniques. To create an overview of the current state of our understanding of cerebellar efferents, we undertook a systematic review of all studies on monosynaptic projections from the cerebellum to the brainstem and the diencephalon in mammals. This revealed that important projections from the cerebellum, to the motor nuclei, cerebral cortex, and basal ganglia, are predominantly di- or polysynaptic, rather than monosynaptic. Strikingly, most target areas receive cerebellar input from all three cerebellar nuclei, showing a convergence of cerebellar information at the output level. Overall, there appeared to be a large level of agreement between studies on different species as well as on the use of different types of neural tracers, making the emerging picture of the cerebellar output areas a solid one. Finally, we discuss how this cerebellar output network is affected by a range of diseases and syndromes, with also non-cerebellar diseases having impact on cerebellar output areas.

Purkinje cells translate subjective salience into readiness to act and choice performance.

The brain selectively allocates attention from a continuous stream of sensory input. This process is typically attributed to computations in distinct regions of the forebrain and midbrain. Here, we explore whether cerebellar Purkinje cells encode information about the selection of sensory inputs and could thereby contribute to non-motor forms of learning. We show that complex spikes of individual Purkinje cells change the sensory modality they encode to reflect changes in the perceived salience of sensory input. Comparisons with mouse models deficient in cerebellar plasticity suggest that changes in complex spike activity instruct potentiation of Purkinje cells simple spike firing, which is required for efficient learning. Our findings suggest that during learning, climbing fibers do not directly guide motor output, but rather contribute to a general readiness to act via changes in simple spike activity, thereby bridging the sequence from non-motor to motor functions.

Region-specific preservation of Purkinje cell morphology and motor behavior in the ATXN1[82Q] mouse model of spinocerebellar ataxia 1.

Purkinje cells are the primary processing units of the cerebellar cortex and display molecular heterogeneity that aligns with differences in physiological properties, projection patterns, and susceptibility to disease. In particular, multiple mouse models that feature Purkinje cell degeneration are characterized by incomplete and patterned Purkinje cell degeneration, suggestive of relative sparing of Purkinje cell subpopulations, such as those expressing Aldolase C/zebrinII (AldoC) or residing in the vestibulo-cerebellum. Here, we investigated a well-characterized Purkinje cell-specific mouse model for spinocerebellar ataxia type 1 (SCA1) that expresses human ATXN1 with a polyQ expansion (82Q). Our pathological analysis confirms previous findings that Purkinje cells of the vestibulo-cerebellum, i.e., the flocculonodular lobes, and crus I are relatively spared from key pathological hallmarks: somatodendritic atrophy, and the appearance of p62/SQSTM1-positive inclusions. However, immunohistological analysis of transgene expression revealed that spared Purkinje cells do not express mutant ATXN1 protein, indicating the sparing of Purkinje cells can be explained by an absence of transgene expression. Additionally, we found that Purkinje cells in other cerebellar lobules that typically express AldoC, not only display severe pathology but also show loss of AldoC expression. The relatively preserved flocculonodular lobes and crus I showed a substantial fraction of Purkinje cells that expressed the mutant protein and displayed pathology as well as loss of AldoC expression. Despite considerable pathology in these lobules, behavioral analyses demonstrated a relative sparing of related functions, suggestive of sufficient functional cerebellar reserve. Together, the data indicate that mutant ATXN1 affects both AldoC-positive and AldoC-negative Purkinje cells and disrupts normal parasagittal AldoC expression in Purkinje cells. Our results show that, in a mouse model otherwise characterized by widespread Purkinje cell degeneration, sparing of specific subpopulations is sufficient to maintain normal performance of specific behaviors within the context of the functional, modular map of the cerebellum.

Cerebellar Purkinje cells can differentially modulate coherence between sensory and motor cortex depending on region and behavior.

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.

Lack of a Clear Behavioral Phenotype in an Inducible FXTAS Mouse Model Despite the Presence of Neuronal FMRpolyG-Positive Aggregates.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a rare neurodegenerative disorder caused by a 55-200 CGG repeat expansion in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. FXTAS is characterized by progressive cerebellar ataxia, Parkinsonism, intention tremors and cognitive decline. The main neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in neurons and astrocytes throughout the brain. The molecular pathology of FXTAS involves the presence of 2 to 8-fold elevated levels of FMR1 mRNA, and of a repeat-associated non-AUG (RAN) translated polyglycine peptide (FMRpolyG). Increased levels of FMR1 mRNA containing an expanded CGG repeat can result in cellular toxicity by an RNA gain-of-function mechanism. The increased levels of CGG repeat-expanded FMR1 transcripts may create RNA foci that sequester important cellular proteins, including RNA-binding proteins and FMRpolyG, in intranuclear inclusions. To date, it is unclear whether the FMRpolyG-positive intranuclear inclusions are a cause or a consequence of FXTAS disease pathology. In this report we studied the relation between the presence of neuronal intranuclear inclusions and behavioral deficits using an inducible mouse model for FXTAS. Neuronal intranuclear inclusions were observed 4 weeks after dox-induction. After 12 weeks, high numbers of FMRpolyG-positive intranuclear inclusions could be detected in the hippocampus and striatum, but no clear signs of behavioral deficits related to these specific brain regions were found. In conclusion, the observations in our inducible mouse model for FXTAS suggest a lack of correlation between the presence of intranuclear FMRpolyG-positive aggregates in brain regions and specific behavioral phenotypes.

WhiskEras: A New Algorithm for Accurate Whisker Tracking.

Rodents engage in active touch using their facial whiskers: they explore their environment by making rapid back-and-forth movements. The fast nature of whisker movements, during which whiskers often cross each other, makes it notoriously difficult to track individual whiskers of the intact whisker field. We present here a novel algorithm, WhiskEras, for tracking of whisker movements in high-speed videos of untrimmed mice, without requiring labeled data. WhiskEras consists of a pipeline of image-processing steps: first, the points that form the whisker centerlines are detected with sub-pixel accuracy. Then, these points are clustered in order to distinguish individual whiskers. Subsequently, the whiskers are parameterized so that a single whisker can be described by four parameters. The last step consists of tracking individual whiskers over time. We describe that WhiskEras performs better than other whisker-tracking algorithms on several metrics. On our four video segments, WhiskEras detected more whiskers per frame than the Biotact Whisker Tracking Tool. The signal-to-noise ratio of the output of WhiskEras was higher than that of Janelia Whisk. As a result, the correlation between reflexive whisker movements and cerebellar Purkinje cell activity appeared to be stronger than previously found using other tracking algorithms. We conclude that WhiskEras facilitates the study of sensorimotor integration by markedly improving the accuracy of whisker tracking in untrimmed mice.

Functional Convergence of Autonomic and Sensorimotor Processing in the Lateral Cerebellum.

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.

Generation of an Atxn2-CAG100 knock-in mouse reveals N-acetylaspartate production deficit due to early Nat8l dysregulation.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion mutations in the ATXN2 gene, mainly affecting motor neurons in the spinal cord and Purkinje neurons in the cerebellum. While the large expansions were shown to cause SCA2, the intermediate length expansions lead to increased risk for several atrophic processes including amyotrophic lateral sclerosis and Parkinson variants, e.g. progressive supranuclear palsy. Intense efforts to pioneer a neuroprotective therapy for SCA2 require longitudinal monitoring of patients and identification of crucial molecular pathways. The ataxin-2 (ATXN2) protein is mainly involved in RNA translation control and regulation of nutrient metabolism during stress periods. The preferential mRNA targets of ATXN2 are yet to be determined. In order to understand the molecular disease mechanism throughout different prognostic stages, we generated an Atxn2-CAG100-knock-in (KIN) mouse model of SCA2 with intact murine ATXN2 expression regulation. Its characterization revealed somatic mosaicism of the expansion, with shortened lifespan, a progressive spatio-temporal pattern of pathology with subsequent phenotypes, and anomalies of brain metabolites such as N-acetylaspartate (NAA), all of which mirror faithfully the findings in SCA2 patients. Novel molecular analyses from stages before the onset of motor deficits revealed a strong selective effect of ATXN2 on Nat8l mRNA which encodes the enzyme responsible for NAA synthesis. This metabolite is a prominent energy store of the brain and a well-established marker for neuronal health. Overall, we present a novel authentic rodent model of SCA2, where in vivo magnetic resonance imaging was feasible to monitor progression and where the definition of earliest transcriptional abnormalities was possible. We believe that this model will not only reveal crucial insights regarding the pathomechanism of SCA2 and other ATXN2-associated disorders, but will also aid in developing gene-targeted therapies and disease prevention.

Quasiperiodic rhythms of the inferior olive.

Inferior olivary activity causes both short-term and long-term changes in cerebellar output underlying motor performance and motor learning. Many of its neurons engage in coherent subthreshold oscillations and are extensively coupled via gap junctions. Studies in reduced preparations suggest that these properties promote rhythmic, synchronized output. However, the interaction of these properties with torrential synaptic inputs in awake behaving animals is not well understood. Here we combine electrophysiological recordings in awake mice with a realistic tissue-scale computational model of the inferior olive to study the relative impact of intrinsic and extrinsic mechanisms governing its activity. Our data and model suggest that if subthreshold oscillations are present in the awake state, the period of these oscillations will be transient and variable. Accordingly, by using different temporal patterns of sensory stimulation, we found that complex spike rhythmicity was readily evoked but limited to short intervals of no more than a few hundred milliseconds and that the periodicity of this rhythmic activity was not fixed but dynamically related to the synaptic input to the inferior olive as well as to motor output. In contrast, in the long-term, the average olivary spiking activity was not affected by the strength and duration of the sensory stimulation, while the level of gap junctional coupling determined the stiffness of the rhythmic activity in the olivary network during its dynamic response to sensory modulation. Thus, interactions between intrinsic properties and extrinsic inputs can explain the variations of spiking activity of olivary neurons, providing a temporal framework for the creation of both the short-term and long-term changes in cerebellar output.

Neurons of the inferior olive respond to broad classes of sensory input while subject to homeostatic control.

KEY POINTS: Purkinje cells in the cerebellum integrate input from sensory organs with that from premotor centres. Purkinje cells use a variety of sensory inputs relaying information from the environment to modify motor control. Here we investigated to what extent the climbing fibre inputs to Purkinje cells signal mono- or multi-sensory information, and to what extent this signalling is subject to recent history of activity. We show that individual climbing fibres convey multiple types of sensory information, together providing a rich mosaic projection pattern of sensory signals across the cerebellar cortex. Moreover, firing probability of climbing fibres following sensory stimulation depends strongly on the recent history of activity, showing a tendency to homeostatic dampening. ABSTRACT: Cerebellar Purkinje cells integrate sensory information with motor efference copies to adapt movements to behavioural and environmental requirements. They produce complex spikes that are triggered by the activity of climbing fibres originating in neurons of the inferior olive. These complex spikes can shape the onset, amplitude and direction of movements and the adaptation of such movements to sensory feedback. Clusters of nearby inferior olive neurons project to parasagittally aligned stripes of Purkinje cells, referred to as 'microzones'. It is currently unclear to what extent individual Purkinje cells within a single microzone integrate climbing fibre inputs from multiple sources of different sensory origins, and to what extent sensory-evoked climbing fibre responses depend on the strength and recent history of activation. Here we imaged complex spike responses in cerebellar lobule crus 1 to various types of sensory stimulation in awake mice. We find that different sensory modalities and receptive fields have a mild, but consistent, tendency to converge on individual Purkinje cells, with climbing fibres showing some degree of input-specificity. Purkinje cells encoding the same stimulus show increased events with coherent complex spike firing and tend to lie close together. Moreover, whereas complex spike firing is only mildly affected by variations in stimulus strength, it depends strongly on the recent history of climbing fibre activity. Our data point towards a mechanism in the olivo-cerebellar system that regulates complex spike firing during mono- or multi-sensory stimulation around a relatively low set-point, highlighting an integrative coding scheme of complex spike firing under homeostatic control.

Corrigendum: Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy.

[This corrects the article DOI: 10.3389/fncel.2017.00346.].

Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy.

Absence epilepsy is characterized by the occurrence of generalized spike and wave discharges (GSWDs) in electrocorticographical (ECoG) recordings representing oscillatory activity in thalamocortical networks. The oscillatory nature of GSWDs has been shown to be reflected in the simple spike activity of cerebellar Purkinje cells and in the activity of their target neurons in the cerebellar nuclei, but it is unclear to what extent complex spike activity is implicated in generalized epilepsy. Purkinje cell complex spike firing is elicited by climbing fiber activation and reflects action potential firing in the inferior olive. Here, we investigated to what extent modulation of complex spike firing is reflected in the temporal patterns of seizures. Extracellular single-unit recordings in awake, head-restrained homozygous tottering mice, which suffer from a mutation in the voltage-gated CaV2.1 calcium channel, revealed that a substantial proportion of Purkinje cells (26%) showed increased complex spike activity and rhythmicity during GSWDs. Moreover, Purkinje cells, recorded either electrophysiologically or by using Ca2+-imaging, showed a significant increase in complex spike synchronicity for both adjacent and remote Purkinje cells during ictal events. These seizure-related changes in firing frequency, rhythmicity and synchronicity were most prominent in the lateral cerebellum, a region known to receive cerebral input via the inferior olive. These data indicate profound and widespread changes in olivary firing that are most likely induced by seizure-related activity changes in the thalamocortical network, thereby highlighting the possibility that olivary neurons can compensate for pathological brain-state changes by dampening oscillations.

Cerebellar control of gait and interlimb coordination.

Synaptic and intrinsic processing in Purkinje cells, interneurons and granule cells of the cerebellar cortex have been shown to underlie various relatively simple, single-joint, reflex types of motor learning, including eyeblink conditioning and adaptation of the vestibulo-ocular reflex. However, to what extent these processes contribute to more complex, multi-joint motor behaviors, such as locomotion performance and adaptation during obstacle crossing, is not well understood. Here, we investigated these functions using the Erasmus Ladder in cell-specific mouse mutant lines that suffer from impaired Purkinje cell output (Pcd), Purkinje cell potentiation (L7-Pp2b), molecular layer interneuron output (L7-Δγ2), and granule cell output (α6-Cacna1a). We found that locomotion performance was severely impaired with small steps and long step times in Pcd and L7-Pp2b mice, whereas it was mildly altered in L7-Δγ2 and not significantly affected in α6-Cacna1a mice. Locomotion adaptation triggered by pairing obstacle appearances with preceding tones at fixed time intervals was impaired in all four mouse lines, in that they all showed inaccurate and inconsistent adaptive walking patterns. Furthermore, all mutants exhibited altered front-hind and left-right interlimb coordination during both performance and adaptation, and inconsistent walking stepping patterns while crossing obstacles. Instead, motivation and avoidance behavior were not compromised in any of the mutants during the Erasmus Ladder task. Our findings indicate that cell type-specific abnormalities in cerebellar microcircuitry can translate into pronounced impairments in locomotion performance and adaptation as well as interlimb coordination, highlighting the general role of the cerebellar cortex in spatiotemporal control of complex multi-joint movements.

Cerebellar modules operate at different frequencies.

Due to the uniform cyto-architecture of the cerebellar cortex, its overall physiological characteristics have traditionally been considered to be homogeneous. In this study, we show in awake mice at rest that spiking activity of Purkinje cells, the sole output cells of the cerebellar cortex, differs between cerebellar modules and correlates with their expression of the glycolytic enzyme aldolase C or zebrin. Simple spike and complex spike frequencies were significantly higher in Purkinje cells located in zebrin-negative than zebrin-positive modules. The difference in simple spike frequency persisted when the synaptic input to, but not intrinsic activity of, Purkinje cells was manipulated. Blocking TRPC3, the effector channel of a cascade of proteins that have zebrin-like distribution patterns, attenuated the simple spike frequency difference. Our results indicate that zebrin-discriminated cerebellar modules operate at different frequencies, which depend on activation of TRPC3, and that this property is relevant for all cerebellar functions.DOI: http://dx.doi.org/10.7554/eLife.02536.001.

Cerebellar potentiation and learning a whisker-based object localization task with a time response window.

Whisker-based object localization requires activation and plasticity of somatosensory and motor cortex. These parts of the cerebral cortex receive strong projections from the cerebellum via the thalamus, but it is unclear whether and to what extent cerebellar processing may contribute to such a sensorimotor task. Here, we subjected knock-out mice, which suffer from impaired intrinsic plasticity in their Purkinje cells and long-term potentiation at their parallel fiber-to-Purkinje cell synapses (L7-PP2B), to an object localization task with a time response window (RW). Water-deprived animals had to learn to localize an object with their whiskers, and based upon this location they were trained to lick within a particular period ("go" trial) or refrain from licking ("no-go" trial). L7-PP2B mice were not ataxic and showed proper basic motor performance during whisking and licking, but were severely impaired in learning this task compared with wild-type littermates. Significantly fewer L7-PP2B mice were able to learn the task at long RWs. Those L7-PP2B mice that eventually learned the task made unstable progress, were significantly slower in learning, and showed deficiencies in temporal tuning. These differences became greater as the RW became narrower. Trained wild-type mice, but not L7-PP2B mice, showed a net increase in simple spikes and complex spikes of their Purkinje cells during the task. We conclude that cerebellar processing, and potentiation in particular, can contribute to learning a whisker-based object localization task when timing is relevant. This study points toward a relevant role of cerebellum-cerebrum interaction in a sophisticated cognitive task requiring strict temporal processing.