Distinct populations of Purkinje cells (PCs) with unique molecular and connectivity features are at the core of the modular organization of the cerebellum. Previously, we showed that firing activity of Purkinje cells differs between ZebrinII-positive (Z+) and -negative (Z−) cerebellar modules (Zhou et al., 2014; Wu et al., 2019). Here, we investigate the timing and extent of PC differentiation during development in mice. We found that several features of PCs, including activity levels, dendritic arborisation, axonal shape and climbing fiber input, develop differentially between nodular and anterior PC populations. Although all PCs show a particularly rapid development in the second postnatal week, anterior PCs typically have a prolonged physiological and dendritic maturation. In line herewith, younger mice exhibit attenuated anterior-dependent eyeblink conditioning, but faster nodular-dependent compensatory eye movement adaptation. Our results indicate that specific cerebellar regions have unique developmental timelines which match with their related, specific forms of cerebellum-dependent behaviors.

Here we investigate stimulus generalization in a cerebellar learning paradigm, called eyeblink conditioning. Mice were conditioned to close their eyes in response to a 10 kHz tone by repeatedly pairing this tone with an air puff to the eye 250 ms after tone onset. After ten consecutive days of training, when mice showed reliable conditioned eyelid responses to the 10 kHz tone, we started to expose them to tones with other frequencies, ranging from 2 to 20 kHz. We found that mice had a strong generalization gradient, whereby the probability and amplitude of conditioned eyelid responses gradually decreases depending on the dissimilarity with the 10 kHz tone. Tones with frequencies closest to 10kHz evoked the most and largest conditioned eyelid responses and each step away from the 10 kHz tone resulted in fewer and smaller conditioned responses. In addition, we found that tones with lower frequencies resulted in conditioned responses that peaked earlier after tone onset compared to those to tones with higher frequencies. Together, our data show prominent generalization patterns in cerebellar learning. Since the known function of cerebellum is rapidly expanding from pure motor control to domains that include cognition, reward-learning, fear-learning, social function, and even addiction, our data implies generalization controlled by cerebellum in all these domains. Significance Statement: Conditioned stimuli that resemble each other will evoke a rather similar behavioral response. Here, we investigate this phenomenon of stimulus generalization using Pavlovian eyeblink conditioning to probe cerebellar function. Cerebellum is a brain structure whose known function is rapidly expanding from pure motor control to domains that include cognition, reward-learning, fear-learning, social function, and even addiction. Since we found a strong generalization of the auditory stimulus in eyeblink conditioning, our data implies an important role for generalization in motor and non-motor domains. Methods SubjectsWe used 14 wild-type C57Bl/6 mice, 7 of which were male and 7 were female. Mice were between 11-16 weeks old at the start of the experiment. All mice were housed individually during the experiment with food and water ad libitum in a 12:12 light/dark cycle. Experiments were performed during the light phase. All experiments were performed in accordance with the European Communities Council Directive. Protocols were reviewed and approved by the Erasmus Laboratory Animal Science Center (work protocol nr. 15-273-138; project license nr. AVD101002015273) Auditory Brainstem Responses Since C57Bl/6 mice are prone for developing hearing problems, we recorded prior to the start of eyeblink conditioning training the auditory brainstem responses (ABRs) to obtain hearing level thresholds. Mice were anesthetized with a ketamine/xylazine mixture at a dose of 100/10 mg/kg body weight, administered intraperitoneally (ketamine: Alfasan, Woerden, NL; xylazine: Sedazine®, AST Farma, Oudewater, NL). After this, they were placed in a soundand ligh...

Spinocerebellar ataxias are neurodegenerative diseases the hallmark symptom of which is the development of ataxia due to cerebellar dysfunction. Purkinje cells (PCs), 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 PC 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 PC dysfunction before the onset of ataxia in a mouse model of spinocerebellar ataxia 1. Spatiotemporal characterisation 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 PC intrinsic excitability, which was followed by atrophy of PC dendrite arborisation and aberrant glutamatergic signalling, finally leading to disruption of PC innervation of climbing fibers (CFs) and loss of intrinsic plasticity of PCs. Functionally, we found that deficits in eyeblink conditioning, a form of cerebellum-dependent motor learning, precede the onset of ataxia, matching the timeline of CF 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.