Purkinje cells in eyeblink conditioning: a systematic review of methodological approaches in animal studies

Background Eyeblink conditioning is a canonical model of associative motor learning and has played a central role in theories of cerebellar plasticity. Purkinje cells occupy a central position in this circuit, receiving conditioned and unconditioned stimulus-related inputs and providing the sole output of the cerebellar cortex. However, the cellular mechanisms by which Purkinje cells contribute to the acquisition, timing, expression, and extinction of conditioned eyeblinks remain debated. Methods We systematically reviewed animal studies investigating Purkinje cell mechanisms in eyeblink conditioning to summarize the experimental approaches used in the field, extract qualitative and quantitative evidence, and identify theoretical trends and methodological limitations. A PubMed search identified 184 records, of which 74 met the inclusion criteria. Included studies were classified according to publication features, animal model, conditioning protocol, intervention type, Purkinje cell-related physiological or structural measurements, and proposed learning mechanism. Results Most studies either manipulated Purkinje cell function or measured Purkinje cell-associated physiological or structural properties, but only three combined both approaches, revealing a major separation between interventional and physiological evidence. Genetic interventions were predominantly performed in mice, whereas direct Purkinje cell physiological or structural measurement studies were more commonly based on electrophysiological recordings and showed greater model heterogeneity. Most conditioning protocols used parameters compatible with acquisition, although essential details such as interstimulus interval, intertrial interval, or trial number were incompletely reported in a subset of studies. When grouped by publication period, the reviewed studies showed a shift from LTD-centred explanations toward more diverse models that incorporate intrinsic excitability, Purkinje cell pause responses, inhibitory microcircuit mechanisms, cerebellar nuclear plasticity, and olivo-cerebellar feedback. Discussion These findings suggest that Purkinje cell contributions to eyeblink conditioning cannot be reduced to a single plasticity mechanism. Instead, the literature supports a multi-level model in which synaptic, intrinsic, and circuit mechanisms jointly shape learning, while also revealing substantial gaps in cross-species validation, reporting quality, and studies combining cell-specific intervention with direct physiological measurement.