Keeping track of time: An interaction of mossy fibers and climbing fibers.

Precisely tracking time over second-long timescales is important for accurate anticipation and consequential actions, yet the neurobiological underpinnings remain unknown. In this issue of Neuron, Garcia-Garcia and colleagues1 show that computations in the cerebellum resulting from interactions between the mossy fiber and climbing fiber pathways contribute to long-interval learning during operant conditioning.

Plasticity mechanisms of genetically distinct Purkinje cells.

Despite its uniform appearance, the cerebellar cortex is highly heterogeneous in terms of structure, genetics and physiology. Purkinje cells (PCs), the principal and sole output neurons of the cerebellar cortex, can be categorized into multiple populations that differentially express molecular markers and display distinctive physiological features. Such features include action potential rate, but also their propensity for synaptic and intrinsic plasticity. However, the precise molecular and genetic factors that correlate with the differential physiological properties of PCs remain elusive. In this article, we provide a detailed overview of the cellular mechanisms that regulate PC activity and plasticity. We further perform a pathway analysis to highlight how molecular characteristics of specific PC populations may influence their physiology and plasticity mechanisms.

Disynaptic Inhibitory Cerebellar Control Over Caudal Medial Accessory Olive.

The olivocerebellar system, which is critical for sensorimotor performance and learning, functions through modules with feedback loops. The main feedback to the inferior olive comes from the cerebellar nuclei (CN), which are predominantly GABAergic and contralateral. However, for the subnucleus d of the caudomedial accessory olive (cdMAO), a crucial region for oculomotor and upper body movements, the source of GABAergic input has yet to be identified. Here, we demonstrate the existence of a disynaptic inhibitory projection from the medial CN (MCN) to the cdMAO via the superior colliculus (SC) by exploiting retrograde, anterograde, and transsynaptic viral tracing at the light microscopic level as well as anterograde classical and viral tracing combined with immunocytochemistry at the electron microscopic level. Retrograde tracing in Gad2-Cre mice reveals that the cdMAO receives GABAergic input from the contralateral SC. Anterograde transsynaptic tracing uncovered that the SC neurons receiving input from the contralateral MCN provide predominantly inhibitory projections to contralateral cdMAO, ipsilateral to the MCN. Following ultrastructural analysis of the monosynaptic projection about half of the SC terminals within the contralateral cdMAO are GABAergic. The disynaptic GABAergic projection from the MCN to the ipsilateral cdMAO mirrors that of the monosynaptic excitatory projection from the MCN to the contralateral cdMAO. Thus, while completing the map of inhibitory inputs to the olivary subnuclei, we established that the MCN inhibits the cdMAO via the contralateral SC, highlighting a potential push-pull mechanism in directional gaze control that appears unique in terms of laterality and polarity among olivocerebellar modules.

Synaptic mechanisms for associative learning in the cerebellar nuclei.

Associative learning during delay eyeblink conditioning (EBC) depends on an intact cerebellum. However, the relative contribution of changes in the cerebellar nuclei to learning remains a subject of ongoing debate. In particular, little is known about the changes in synaptic inputs to cerebellar nuclei neurons that take place during EBC and how they shape the membrane potential of these neurons. Here, we probed the ability of these inputs to support associative learning in mice, and investigated structural and cell-physiological changes within the cerebellar nuclei during learning. We find that optogenetic stimulation of mossy fiber afferents to the anterior interposed nucleus (AIP) can substitute for a conditioned stimulus and is sufficient to elicit conditioned responses (CRs) that are adaptively well-timed. Further, EBC induces structural changes in mossy fiber and inhibitory inputs, but not in climbing fiber inputs, and it leads to changes in subthreshold processing of AIP neurons that correlate with conditioned eyelid movements. The changes in synaptic and spiking activity that precede the CRs allow for a decoder to distinguish trials with a CR. Our data reveal how structural and physiological modifications of synaptic inputs to cerebellar nuclei neurons can facilitate learning.

Cerebellar nuclei: Associative motor learning in zebrafish.

Cerebellar output neurons integrate strong inhibitory input and weaker excitatory input during the control of spontaneous and learned movements. A new study sheds light on how those inputs are integrated during associative swimming in zebrafish larvae.

In vivo whole-cell recording from morphologically identified mouse superior colliculus neurons.

In vivo whole-cell recording, when combined with morphological characterization after biocytin labeling, is a powerful technique to study subthreshold synaptic processing in cell-type-identified neuronal populations. Here, we describe steps for performing whole-cell recordings in the superior colliculus of urethane-anesthetized mice, a major visual processing region in the rodent brain. We detail two types of visual stimulation techniques: full-field light-emitting diode (LED) flashes and visual stimuli shown on monitors. While we focus on superior colliculus, this protocol is applicable to other brain areas.

Cerebellar plasticity and associative memories are controlled by perineuronal nets.

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.

Action perception recruits the cerebellum and is impaired in patients with spinocerebellar ataxia.

Our cerebellum has been proposed to generate prediction signals that may help us plan and execute our motor programmes. However, to what extent our cerebellum is also actively involved in perceiving the action of others remains to be elucidated. Using functional MRI, we show here that observing goal-directed hand actions of others bilaterally recruits lobules VI, VIIb and VIIIa in the cerebellar hemispheres. Moreover, whereas healthy subjects (n = 31) were found to be able to discriminate subtle differences in the kinematics of observed limb movements of others, patients suffering from spinocerebellar ataxia type 6 (SCA6; n = 21) were severely impaired in performing such tasks. Our data suggest that the human cerebellum is actively involved in perceiving the kinematics of the hand actions of others and that SCA6 patients' deficits include a difficulty in perceiving the actions of other individuals. This finding alerts us to the fact that cerebellar disorders can alter social cognition.

Functional Architecture and Encoding of Tactile Sensorimotor Behavior in Rat Posterior Parietal Cortex.

The posterior parietal cortex (PPC) in rodents is reciprocally connected to primary somatosensory and vibrissal motor cortices. The PPC neuronal circuitry could thus encode and potentially integrate incoming somatosensory information and whisker motor output. However, the information encoded across PPC layers during refined sensorimotor behavior remains largely unknown. To uncover the sensorimotor features represented in PPC during voluntary whisking and object touch, we performed loose-patch single-unit recordings and extracellular recordings of ensemble activity, covering all layers of PPC in anesthetized and awake, behaving male rats. First, using single-cell receptive field mapping, we revealed the presence of coarse somatotopy along the mediolateral axis in PPC. Second, we found that spiking activity was modulated during exploratory whisking in layers 2-4 and layer 6, but not in layer 5 of awake, behaving rats. Population spiking activity preceded actual movement, and whisker trajectory endpoints could be decoded by population spiking, suggesting that PPC is involved in movement planning. Finally, population spiking activity further increased in response to active whisker touch but only in PPC layers 2-4. Thus, we find layer-specific processing, which emphasizes the computational role of PPC during whisker sensorimotor behavior.SIGNIFICANCE STATEMENT The posterior parietal cortex (PPC) is thought to merge information on motor output and sensory input to orchestrate interaction with the environment, but the function of different PPC microcircuit components is poorly understood. We recorded neuronal activity in rat PPC during sensorimotor behavior involving motor and sensory pathways. We uncovered that PPC layers have dedicated function: motor and sensory information is merged in layers 2-4; layer 6 predominantly represents motor information. Collectively, PPC activity predicts future motor output, thus entailing a motor plan. Our results are important for understanding how PPC computationally processes motor output and sensory input. This understanding may facilitate decoding of brain activity when using brain-machine interfaces to overcome loss of function after, for instance, spinal cord injury.

Early Trajectory Prediction in Elite Athletes.

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.

An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum.

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.

Impaired Spatio-Temporal Predictive Motor Timing Associated with Spinocerebellar Ataxia Type 6.

Many daily life activities demand precise integration of spatial and temporal information of sensory inputs followed by appropriate motor actions. This type of integration is carried out in part by the cerebellum, which has been postulated to play a central role in learning and timing of movements. Cerebellar damage due to atrophy or lesions may compromise forward-model processing, in which both spatial and temporal cues are used to achieve prediction for future motor states. In the present study we sought to further investigate the cerebellar contribution to predictive and reactive motor timing, as well as to learning of sequential order and temporal intervals in these tasks. We tested patients with spinocerebellar ataxia type 6 (SCA6) and healthy controls for two related motor tasks; one requiring spatio-temporal prediction of dynamic visual stimuli and another one requiring reactive timing only. We found that healthy controls established spatio-temporal prediction in their responses with high temporal precision, which was absent in the cerebellar patients. SCA6 patients showed lower predictive motor timing, coinciding with a reduced number of correct responses during the 'anticipatory' period on the task. Moreover, on the task utilizing reactive motor timing functions, control participants showed both sequence order and temporal interval learning, whereas patients only showed sequence order learning. These results suggest that SCA6 affects predictive motor timing and temporal interval learning. Our results support and highlight cerebellar contribution to timing and argue for cerebellar engagement during spatio-temporal prediction of upcoming events.

Juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons.

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post hoc identification and morphological reconstruction.