Model-driven analysis of eyeblink classical conditioning reveals the underlying structure of cerebellar plasticity and neuronal activity

The cerebellum plays a critical role in sensorimotor control. However, how the specific circuits and plastic mechanisms of the cerebellum are engaged in closed-loop processing is still unclear. We developed an artificial sensorimotor control system embedding a detailed spiking cerebellar microcircuit with three bidirectional plasticity sites. This proved able to reproduce a cerebellar-driven associative paradigm, the eyeblink classical conditioning (EBCC), in which a precise time relationship between an unconditioned stimulus (US) and a conditioned stimulus (CS) is established. We challenged the spiking model to fit an experimental data set from human subjects. Two subsequent sessions of EBCC acquisition and extinction were recorded and transcranial magnetic stimulation (TMS) was applied on the cerebellum to alter circuit function and plasticity. Evolutionary algorithms were used to find the near-optimal model parameters to reproduce the behaviors of subjects in the different sessions of the protocol. The main finding is that the optimized cerebellar model was able to learn to anticipate (predict) conditioned responses with accurate timing and success rate, demonstrating fast acquisition, memory stabilization, rapid extinction, and faster reacquisition as in EBCC in humans. The firing of Purkinje cells (PCs) and deep cerebellar nuclei (DCN) changed during learning under the control of synaptic plasticity, which evolved at different rates, with a faster acquisition in the cerebellar cortex than in DCN synapses. Eventually, a reduced PC activity released DCN discharge just after the CS, precisely anticipating the US and causing the eyeblink. Moreover, a specific alteration in cortical plasticity explained the EBCC changes induced by cerebellar TMS in humans. In this paper, for the first time, it is shown how closed-loop simulations, using detailed cerebellar microcircuit models, can be successfully used to fit real experimental data sets. Thus, the changes of the model parameters in the different sessions of the protocol unveil how implicit microcircuit mechanisms can generate normal and altered associative behaviors

Trial-by-Trial Coding of Instructive Signals in the Cerebellum: Insights From Eyeblink Conditioning in Mice

The cerebellum is an area of the brain that plays a crucial role in the learning of motor skills. This process involves climbing fibers, which provide teaching signals to Purkinje cells in the cerebellar cortex when perturbations occur during a movement. However, controversy has arisen over climbing fibers contribution to cerebellar learning. This is because climbing-fiber signals are described as all-or-nothing : they fire a single burst of action potentials in response to all supra-threshold stimuli, regardless of their strength. On the contrary, motor learning is not all-or-nothing: the amount of learning is driven by the strength of perturbations. In this dissertation, I describe the experiments that I performed to unravel how climbing fibers may encode the strength of teaching signals. In Chapter 2, I present my behavioral studies in mice, which involved a simple cerebellar-dependent motor learning task, eyeblink conditioning. I show that mice take into account the strength of unexpected perturbations to adapt their movements trial-by-trial. In Chapter 3, I present a review of the previous literature and provide a hypothesis on how climbing fibers can encode the strength of teaching signals in a single trial. In Chapter 4, I present the findings of my in vivo two-photon calcium imaging experiments, which suggest climbing-fiber signals may not be all-or-nothing at the post-synaptic level. Finally, in Chapter 5 I describe the different mechanisms that we discovered for coding the intensity of teaching signals by Purkinje cells in the cerebellum of awake mice

Manipulating the Perineuronal Net in the Deep Cerebellar Nucleus

Perineuronal nets (PNN) are a type of specialized extracellular matrix in the central nervous system. The PNN forms during postnatal development but the ontogeny of the PNN has yet to be elucidated. Studying the PNN in the rat brain may allow us to further understand the PNN’s role in development, learning, and memory. The PNN is fully developed in the deep cerebellar nuclei (DCN) of rats by post-natal day 18. By using enzymatic digestion of the PNN with chondroitinase ABC (ChABC), we studied how digestion of the PNN affects cerebellar-dependent eyeblink conditioning (EBC) and performed electrophysiological recordings from DCN neurons. In vivo degradation of the PNN resulted in differences in EBC amplitude and area. Female animals in the vehicle group demonstrated higher levels of conditioning as well as higher post-probe conditioned responses compared to males in that group, differences not present in the ChABC group. In vitro, DCN neurons with disrupted PNNs following exposure to ChABC had altered membrane properties, fewer rebound spikes, and decreased intrinsic excitability. Doxycycline, an antibiotic, can inhibit endogenous enzymes that digest the PNN. Rats given doxycycline had higher PNN staining in the DCN compared to vehicle. Animals receiving doxycycline prior to behavior have a smaller eyeblink area in comparison to the vehicle group. However, these rats also had more unconditioned responses, suggesting in addition to preventing the PNN from being remodeled, doxycycline may cause non-associative effects. This study further elucidates the role of the PNN in cerebellar learning

Dynamic modulation of activity in cerebellar nuclei neurons during pavlovian eyeblink conditioning in mice

While research on the cerebellar cortex is crystallizing our understanding of its function in learning behavior, many questions surrounding its downstream targets remain. Here, we evaluate the dynamics of cerebellar interpositus nucleus (IpN) neurons over the course of Pavlovian eyeblink conditioning. A diverse range of learning-induced neuronal responses was observed, including increases and decreases in activity during the generation of conditioned blinks. Trial-bytrial correlational analysis and optogenetic manipulation demonstrate that facilitation in the IpN drives the eyelid movements. Adaptive facilitatory responses are often preceded by acquired transient inhibition of IpN activity that, based on latency and effect, appear to be driven by complex spikes in cerebellar cortical Purkinje cells. Likewise, during reflexive blinks to periocular stimulation, IpN cells show excitation-suppression patterns that suggest a contribution of climbing fibers and their collaterals. These findings highlight the integrative properties of subcortical neurons at the cerebellar output stage mediating conditioned behavior

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.</p