A brainstem integrator for self-location memory and positional homeostasis in zebrafish.

To track and control self-location, animals integrate their movements through space. Representations of self-location are observed in the mammalian hippocampal formation, but it is unknown if positional representations exist in more ancient brain regions, how they arise from integrated self-motion, and by what pathways they control locomotion. Here, in a head-fixed, fictive-swimming, virtual-reality preparation, we exposed larval zebrafish to a variety of involuntary displacements. They tracked these displacements and, many seconds later, moved toward their earlier location through corrective swimming ("positional homeostasis"). Whole-brain functional imaging revealed a network in the medulla that stores a memory of location and induces an error signal in the inferior olive to drive future corrective swimming. Optogenetically manipulating medullary integrator cells evoked displacement-memory behavior. Ablating them, or downstream olivary neurons, abolished displacement corrections. These results reveal a multiregional hindbrain circuit in vertebrates that integrates self-motion and stores self-location to control locomotor behavior.

A neural circuit state change underlying skilled movements.

In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.

Cerebellar Neurodynamics Predict Decision Timing and Outcome on the Single-Trial Level.

Goal-directed behavior requires the interaction of multiple brain regions. How these regions and their interactions with brain-wide activity drive action selection is less understood. We have investigated this question by combining whole-brain volumetric calcium imaging using light-field microscopy and an operant-conditioning task in larval zebrafish. We find global, recurring dynamics of brain states to exhibit pre-motor bifurcations toward mutually exclusive decision outcomes. These dynamics arise from a distributed network displaying trial-by-trial functional connectivity changes, especially between cerebellum and habenula, which correlate with decision outcome. Within this network the cerebellum shows particularly strong and predictive pre-motor activity (>10 s before movement initiation), mainly within the granule cells. Turn directions are determined by the difference neuroactivity between the ipsilateral and contralateral hemispheres, while the rate of bi-hemispheric population ramping quantitatively predicts decision time on the trial-by-trial level. Our results highlight a cognitive role of the cerebellum and its importance in motor planning.

Shared Cortex-Cerebellum Dynamics in the Execution and Learning of a Motor Task.

Throughout mammalian neocortex, layer 5 pyramidal (L5) cells project via the pons to a vast number of cerebellar granule cells (GrCs), forming a fundamental pathway. Yet, it is unknown how neuronal dynamics are transformed through the L5→GrC pathway. Here, by directly comparing premotor L5 and GrC activity during a forelimb movement task using dual-site two-photon Ca2+ imaging, we found that in expert mice, L5 and GrC dynamics were highly similar. L5 cells and GrCs shared a common set of task-encoding activity patterns, possessed similar diversity of responses, and exhibited high correlations comparable to local correlations among L5 cells. Chronic imaging revealed that these dynamics co-emerged in cortex and cerebellum over learning: as behavioral performance improved, initially dissimilar L5 cells and GrCs converged onto a shared, low-dimensional, task-encoding set of neural activity patterns. Thus, a key function of cortico-cerebellar communication is the propagation of shared dynamics that emerge during learning.

Histone bivalency regulates the timing of cerebellar granule cell development.

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

Adaptive Prediction for Social Contexts: The Cerebellar Contribution to Typical and Atypical Social Behaviors.

Social interactions involve processes ranging from face recognition to understanding others' intentions. To guide appropriate behavior in a given context, social interactions rely on accurately predicting the outcomes of one's actions and the thoughts of others. Because social interactions are inherently dynamic, these predictions must be continuously adapted. The neural correlates of social processing have largely focused on emotion, mentalizing, and reward networks, without integration of systems involved in prediction. The cerebellum forms predictive models to calibrate movements and adapt them to changing situations, and cerebellar predictive modeling is thought to extend to nonmotor behaviors. Primary cerebellar dysfunction can produce social deficits, and atypical cerebellar structure and function are reported in autism, which is characterized by social communication challenges and atypical predictive processing. We examine the evidence that cerebellar-mediated predictions and adaptation play important roles in social processes and argue that disruptions in these processes contribute to autism.

Current Principles of Motor Control, with Special Reference to Vertebrate Locomotion.

The vertebrate control of locomotion involves all levels of the nervous system from cortex to the spinal cord. Here, we aim to cover all main aspects of this complex behavior, from the operation of the microcircuits in the spinal cord to the systems and behavioral levels and extend from mammalian locomotion to the basic undulatory movements of lamprey and fish. The cellular basis of propulsion represents the core of the control system, and it involves the spinal central pattern generator networks (CPGs) controlling the timing of different muscles, the sensory compensation for perturbations, and the brain stem command systems controlling the level of activity of the CPGs and the speed of locomotion. The forebrain and in particular the basal ganglia are involved in determining which motor programs should be recruited at a given point of time and can both initiate and stop locomotor activity. The propulsive control system needs to be integrated with the postural control system to maintain body orientation. Moreover, the locomotor movements need to be steered so that the subject approaches the goal of the locomotor episode, or avoids colliding with elements in the environment or simply escapes at high speed. These different aspects will all be covered in the review.

A statistical method for image-mediated association studies discovers genes and pathways associated with four brain disorders.

Brain imaging and genomics are critical tools enabling characterization of the genetic basis of brain disorders. However, imaging large cohorts is expensive and may be unavailable for legacy datasets used for genome-wide association studies (GWASs). Using an integrated feature selection/aggregation model, we developed an image-mediated association study (IMAS), which utilizes borrowed imaging/genomics data to conduct association mapping in legacy GWAS cohorts. By leveraging the UK Biobank image-derived phenotypes (IDPs), the IMAS discovered genetic bases underlying four neuropsychiatric disorders and verified them by analyzing annotations, pathways, and expression quantitative trait loci (eQTLs). A cerebellar-mediated mechanism was identified to be common to the four disorders. Simulations show that, if the goal is identifying genetic risk, our IMAS is more powerful than a hypothetical protocol in which the imaging results were available in the GWAS dataset. This implies the feasibility of reanalyzing legacy GWAS datasets without conducting additional imaging, yielding cost savings for integrated analysis of genetics and imaging.

Cell division angle predicts the level of tissue mechanics that tune the amount of cerebellar folding.

Modeling has led to proposals that the amount of neural tissue folding is set by the level of differential expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here, we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a distinct critical period during which the folding amount diverges between the two strains. In this period, regional changes in the level of differential expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL varies regionally during the critical period alongside corresponding changes in wavelength. The number of SHH-expressing Purkinje cells predicts the folding amount, but the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential expansion and the thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential expansion along the perimeter and the thickness of the EGL are regionally tuned to set the amount and wavelength of folding.

Cerebellar granular neuron progenitors exit their germinative niche via BarH-like1 activity mediated partly by inhibition of T-cell factor.

Cerebellar granule neuron progenitors (GNPs) originate from the upper rhombic lip (URL), a germinative niche in which developmental defects produce human diseases. T-cell factor (TCF) responsiveness and Notch dependence are hallmarks of self-renewal in neural stem cells. TCF activity, together with transcripts encoding proneural gene repressors hairy and enhancer of split (Hes/Hey), are detected in the URL; however, their functions and regulatory modes are undeciphered. Here, we established amphibian as a pertinent model for studying vertebrate URL development. The amphibian long-lived URL is TCF active, whereas the external granular layer (EGL) is non-proliferative and expresses hes4 and hes5 genes. Using functional and transcriptomic approaches, we show that TCF activity is necessary for URL emergence and maintenance. We establish that the transcription factor Barhl1 controls GNP exit from the URL, acting partly through direct TCF inhibition. Identification of Barhl1 target genes suggests that, besides TCF, Barhl1 inhibits transcription of hes5 genes independently of Notch signaling. Observations in amniotes suggest a conserved role for Barhl in maintenance of the URL and/or EGL via co-regulation of TCF, Hes and Hey genes.

The olivary input to the cerebellum dissociates sensory events from movement plans.

Neurons in the inferior olive are thought to anatomically organize the Purkinje cells (P-cells) of the cerebellum into computational modules, but what is computed by each module? Here, we designed a saccade task in marmosets that dissociated sensory events from motor events and then recorded the complex and simple spikes of hundreds of P-cells. We found that when a visual target was presented at a random location, the olive reported the direction of that sensory event to one group of P-cells, but not to a second group. However, just before movement onset, it reported the direction of the planned movement to both groups, even if that movement was not toward the target. At the end of the movement if the subject experienced an error but chose to withhold the corrective movement, only the first group received information about the sensory prediction error. We organized the P-cells based on the information content of their olivary input and found that in the group that received sensory information, the simple spikes were suppressed during fixation, then produced a burst before saccade onset in a direction consistent with assisting the movement. In the second group, the simple spikes were not suppressed during fixation but burst near saccade deceleration in a direction consistent with stopping the movement. Thus, the olive differentiated the P-cells based on whether they would receive sensory or motor information, and this defined their contributions to control of movements as well as holding still.

Directed differentiation of human hindbrain neuroepithelial stem cells recapitulates cerebellar granule neurogenesis.

Cerebellar granule neurons (CGNs) are the most abundant neurons in the human brain. Dysregulation of their development underlies movement disorders and medulloblastomas. It is suspected that these disorders arise in progenitor states of the CGN lineage, for which human models are lacking. Here, we have differentiated human hindbrain neuroepithelial stem (hbNES) cells to CGNs in vitro using soluble growth factors, recapitulating key progenitor states in the lineage. We show that hbNES cells are not lineage committed and retain rhombomere 1 regional identity. Upon differentiation, hbNES cells transit through a rhombic lip (RL) progenitor state at day 7, demonstrating human specific sub-ventricular cell identities. This RL state is followed by an ATOH1+ CGN progenitor state at day 14. By the end of a 56-day differentiation procedure, we obtain functional neurons expressing CGN markers GABAARα6 and vGLUT2. We show that sonic hedgehog promotes GABAergic lineage specification and CGN progenitor proliferation. Our work presents a new model with which to study development and diseases of the CGN lineage in a human context.

Intrinsic circuitry of the rhombicbrain (central nervous system's intermediate sector) in a mammal.

The rhombicbrain (rhombencephalon or intermediate sector) is the vertebrate central nervous system part between the forebrain-midbrain (rostral sector) and spinal cord (caudal sector), and it has three main divisions: pons, cerebellum, and medulla. Using a data-driven approach, here we examine intrinsic rhombicbrain (intrarhombicbrain) network architecture that in rat consists of 52,670 possible axonal connections between 230 gray matter regions (115 bilaterally symmetrical pairs). Our analysis indicates that only 8,089 (15.4%) of these connections exist. Multiresolution consensus cluster analysis yields a nested hierarchy model of rhombicbrain subsystems that at the top level are associated with 1) the cerebellum and vestibular nuclei, 2) orofacial-pharyngeal-visceral integration, and 3) auditory connections; the bottom level has 68 clusters, ranging in size from 2 to 11 regions. The model provides a basis for functional hypothesis development and interrogation. More granular network analyses performed on the intrinsic connectivity of individual and combined main rhombicbrain divisions (pons, cerebellum, medulla, pons + cerebellum, and pons + medulla) demonstrate the mutability of network architecture in response to the addition or subtraction of connections. Clear differences between the structure-function network architecture of the rhombicbrain and forebrain-midbrain are discussed, with a stark comparison provided by the subsystem and small-world organization of the cerebellar cortex and cerebral cortex. Future analysis of the connections within and between the forebrain-midbrain and rhombicbrain will provide a model of brain neural network architecture in a mammal.

Neural evidence of switch processes during semantic and phonetic foraging in human memory.

Humans may retrieve words from memory by exploring and exploiting in "semantic space" similar to how nonhuman animals forage for resources in physical space. This has been studied using the verbal fluency test (VFT), in which participants generate words belonging to a semantic or phonetic category in a limited time. People produce bursts of related items during VFT, referred to as "clustering" and "switching." The strategic foraging model posits that cognitive search behavior is guided by a monitoring process which detects relevant declines in performance and then triggers the searcher to seek a new patch or cluster in memory after the current patch has been depleted. An alternative body of research proposes that this behavior can be explained by an undirected rather than strategic search process, such as random walks with or without random jumps to new parts of semantic space. This study contributes to this theoretical debate by testing for neural evidence of strategically timed switches during memory search. Thirty participants performed category and letter VFT during functional MRI. Responses were classified as cluster or switch events based on computational metrics of similarity and participant evaluations. Results showed greater hippocampal and posterior cerebellar activation during switching than clustering, even while controlling for interresponse times and linguistic distance. Furthermore, these regions exhibited ramping activity which increased during within-patch search leading up to switches. Findings support the strategic foraging model, clarifying how neural switch processes may guide memory search in a manner akin to foraging in patchy spatial environments.

Cerebellar control of a unitary head direction sense.

The head-direction (HD) system, a key neural circuit for navigation, consists of several anatomical structures containing neurons selective to the animal's head direction. HD cells exhibit ubiquitous temporal coordination across brain regions, independently of the animal's behavioral state or sensory inputs. Such temporal coordination mediates a single, stable, and persistent HD signal, which is essential for intact orientation. However, the mechanistic processes behind the temporal organization of HD cells are unknown. By manipulating the cerebellum, we identify pairs of HD cells recorded from two brain structures (anterodorsal thalamus and retrosplenial cortex) that lose their temporal coordination, specifically during the removal of the external sensory inputs. Further, we identify distinct cerebellar mechanisms that participate in the spatial stability of the HD signal depending on sensory signals. We show that while cerebellar protein phosphatase 2B-dependent mechanisms facilitate the anchoring of the HD signal on the external cues, the cerebellar protein kinase C-dependent mechanisms are required for the stability of the HD signal by self-motion cues. These results indicate that the cerebellum contributes to the preservation of a single and stable sense of direction.

Closed-Loop Optogenetic Perturbation of Macaque Oculomotor Cerebellum: Evidence for an Internal Saccade Model.

Internal models are essential for the production of accurate movements. The accuracy of saccadic eye movements is thought to be mediated by an internal model of oculomotor mechanics encoded in the cerebellum. The cerebellum may also be part of a feedback loop that predicts the displacement of the eyes and compares it to the desired displacement in real time to ensure that saccades land on target. To investigate the role of the cerebellum in these two aspects of saccade production, we delivered saccade-triggered light pulses to channelrhodopsin-2-expressing Purkinje cells in the oculomotor vermis (OMV) of two male macaque monkeys. Light pulses delivered during the acceleration phase of ipsiversive saccades slowed the deceleration phase. The long latency of these effects and their scaling with light pulse duration are consistent with an integration of neural signals at or downstream of the stimulation site. In contrast, light pulses delivered during contraversive saccades reduced saccade velocity at short latency and were followed by a compensatory reacceleration which caused gaze to land on or near the target. We conclude that the contribution of the OMV to saccade production depends on saccade direction; the ipsilateral OMV is part of a forward model that predicts eye displacement, whereas the contralateral OMV is part of an inverse model that creates the force required to move the eyes with optimal peak velocity for the intended displacement.

Developmentally Unique Cerebellar Processing Prioritizes Self- over Other-Generated Movements.

Animals must distinguish the sensory consequences of self-generated movements (reafference) from those of other-generated movements (exafference). Only self-generated movements entail the production of motor copies (i.e., corollary discharges), which are compared with reafference in the cerebellum to compute predictive or internal models of movement. Internal models emerge gradually over the first three postnatal weeks in rats through a process that is not yet fully understood. Previously, we demonstrated in postnatal day (P) 8 and P12 rats that precerebellar nuclei convey corollary discharge and reafference to the cerebellum during active (REM) sleep when pups produce limb twitches. Here, recording from a deep cerebellar nucleus (interpositus, IP) in P12 rats of both sexes, we compared reafferent and exafferent responses with twitches and limb stimulations, respectively. As expected, most IP units showed robust responses to twitches. However, in contrast with other sensory structures throughout the brain, relatively few IP units showed exafferent responses. Upon finding that exafferent responses occurred in pups under urethane anesthesia, we hypothesized that urethane inhibits cerebellar cortical cells, thereby disinhibiting exafferent responses in IP. In support of this hypothesis, ablating cortical tissue dorsal to IP mimicked the effects of urethane on exafference. Finally, the results suggest that twitch-related corollary discharge and reafference are conveyed simultaneously and in parallel to cerebellar cortex and IP. Based on these results, we propose that twitches provide opportunities for the nascent cerebellum to integrate somatotopically organized corollary discharge and reafference, thereby enabling the development of closed-loop circuits and, subsequently, internal models.

Structural and Functional Organization of Visual Responses in the Inferior Olive of Larval Zebrafish.

The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. Here, we investigate sensory representations in the inferior olive (IO) of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons, we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using two-photon imaging. We found that most IO neurons showed direction-selective and binocular responses to visual stimuli and that the functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between functional properties, soma location, and axonal projection patterns of IO neurons. Taken together, our results suggest that anatomically defined classes of IO neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.

SMARCA4 Loss and Mutated ß-Catenin Induce Proliferative Lesions in the Murine Embryonic Cerebellum.

Almost all medulloblastomas (MB) of the Wingless/Int-1 (WNT) type are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations. Additional alterations in SMARCA4 are detected in ∼20% of WNT MB, but their functional role is mostly unknown. We, therefore, amended previously described brain lipid binding protein (Blbp)-cre::Ctnnb1(ex3)fl/wt mice by the introduction of floxed Smarca4 alleles. Unexpectedly, mutated and thereby stabilized ß-catenin on its own induced severe developmental phenotypes in male and female Blbp-cre::Ctnnb1(ex3)fl/wt mice in our hands, including a thinned cerebral cortex, hydrocephalus, missing cerebellar layering, and cell accumulations in the brainstem and cerebellum. An additional loss of SMARCA4 even resulted in prenatal death for most mice. Respective Blbp-cre::Ctnnb1(ex3)fl/wt::Smarca4fl/rec mutants (male and female) developed large proliferative lesions in the cerebellum evolving from E13.5 to E16.5. Histological and molecular analysis of these lesions by DNA methylation profiling and single-cell RNA sequencing suggested an origin in early undifferentiated SOX2-positive cerebellar progenitors. Furthermore, upregulated WNT signaling, altered actin/cytoskeleton organization, and reduced neuronal differentiation were evident in mutant cells. In vitro, cells harboring alterations in both Ctnnb1 and Smarca4 were negatively selected and did not show tumorigenic potential after transplantation in adult female recipient mice. However, in cerebellar explant cultures, mutant cells displayed significantly increased proliferation, suggesting an important role of the embryonic microenvironment in the development of lesions. Altogether, these results represent an important first step toward the unraveling of tumorigenic mechanisms induced by aberrant WNT signaling and SMARCA4 deficiency.

Calcium-Dependent Regulation of Neuronal Excitability Is Rescued in Fragile X Syndrome by a Tat-Conjugated N-Terminal Fragment of FMRP.

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.