Timing in the cerebellum during motor learning: from neuron to athlete to patient

The cerebellum is involved in the encoding and integration of spatial and temporal information. Although these processes are crucial to our survival, the neuronal mechanisms the cerebellum utilizes to carry out this type of spatio-temporal processing, as well as the precise cerebellar contribution to temporal aspects of various behaviors, remain to be clarified. The research in this doctoral thesis describes findings from experiments done in rodents and humans, with a particular focus on cerebellar timing mechanisms. In rodents, neuronal circuit mechanisms underlying eyeblink conditioning are investigated. The contribution of pontocerebellar mossy fibers and perineuronal nets in the cerebellar nuclei to associative learning are examined. In humans, cerebellar involvement in action observation and spatio-temporal trajectory prediction are investigated in spinocerebellar ataxia type 6 (SCA6) patients, healthy controls and baseball athletes. Together, these studies demonstrate that the cerebellum is important for temporal processes during a range of behaviors, while revealing the neuronal circuit responsible

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.</p

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.</p

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

Semi-automatic standardized analysis method to objectively evaluate near-infrared fluorescent dyes in image-guided surgery

Significance: Near-infrared fluorescence imaging still lacks a standardized, objective method to evaluate fluorescent dye efficacy in oncological surgical applications. This results in difficulties in translation between preclinical to clinical studies with fluorescent dyes and in the reproduction of results between studies, which in turn hampers further clinical translation of novel fluorescent dyes. Aim: Our aim is to develop and evaluate a semi-automatic standardized method to objectively assess fluorescent signals in resected tissue. Approach: A standardized imaging procedure was designed and quantitative analysis methods were developed to evaluate non-targeted and tumor-targeted fluorescent dyes. The developed analysis methods included manual selection of region of interest (ROI) on white light images, automated fluorescence signal ROI selection, and automatic quantitative image analysis. The proposed analysis method was then compared with a conventional analysis method, where fluorescence signal ROIs were manually selected on fluorescence images. Dice similarity coefficients and intraclass correlation coefficients were calculated to determine the inter- and intraobserver variabilities of the ROI selections and the determined signal- and tumor-to-background ratios. Results: The proposed non-targeted fluorescent dyes analysis method showed statistically significantly improved variabilities after application on indocyanine green specimens. For specimens with the targeted dye SGM-101, the variability of the background ROI selection was statistically significantly improved by implementing the proposed method. Conclusion: Semi-automatic methods for standardized quantitative analysis of fluorescence images were successfully developed and showed promising results to further improve the reproducibility and standardization of clinical studies evaluating fluorescent dyes.</p

Semi-automatic standardized analysis method to objectively evaluate near-infrared fluorescent dyes in image-guided surgery

Significance: Near-infrared fluorescence imaging still lacks a standardized, objective method to evaluate fluorescent dye efficacy in oncological surgical applications. This results in difficulties in translation between preclinical to clinical studies with fluorescent dyes and in the reproduction of results between studies, which in turn hampers further clinical translation of novel fluorescent dyes. Aim: Our aim is to develop and evaluate a semi-automatic standardized method to objectively assess fluorescent signals in resected tissue. Approach: A standardized imaging procedure was designed and quantitative analysis methods were developed to evaluate non-targeted and tumor-targeted fluorescent dyes. The developed analysis methods included manual selection of region of interest (ROI) on white light images, automated fluorescence signal ROI selection, and automatic quantitative image analysis. The proposed analysis method was then compared with a conventional analysis method, where fluorescence signal ROIs were manually selected on fluorescence images. Dice similarity coefficients and intraclass correlation coefficients were calculated to determine the inter- and intraobserver variabilities of the ROI selections and the determined signal- and tumor-to-background ratios. Results: The proposed non-targeted fluorescent dyes analysis method showed statistically significantly improved variabilities after application on indocyanine green specimens. For specimens with the targeted dye SGM-101, the variability of the background ROI selection was statistically significantly improved by implementing the proposed method. Conclusion: Semi-automatic methods for standardized quantitative analysis of fluorescence images were successfully developed and showed promising results to further improve the reproducibility and standardization of clinical studies evaluating fluorescent dyes.</p

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

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

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

<i>In vivo</i> 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.</p