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Elucidation of the mechanisms involved in neurodegenerative diseases of the cerebellum has been hampered by the lack of robust single cell models to study Purkinje neurons and replicate at the same time in vivo features. Cerebellar Purkinje neurons are difficult to grow in dispersed cell culture, and only limited work has been done using rat cells. We developed a refined protocol for growing rat Purkinje neurons from embryonic and postnatal tissue ex vivo that results in well-developed, mature, functional, and synaptically active neurons. The rat Purkinje neurons generated are responsive to paracrine factors and genetic manipulation, allowing great experimental flexibility at the single-cell level. This ex vivo model can be used to investigate disease mechanisms that disturb Purkinje neuron morphology, function, and communication in high- and low-throughput screening formats.

Background: Elucidation of the mechanisms involved in neurodegenerative disease has been hampered by the lack of robust cellular models that replicate in vivo features. Methods: We developed a refined protocol for generation of age-dependent, well-developed, and synaptically active rat Purkinje neurons in a multilayer cell network culture that can be used to study neurodegenerative diseases such as paraneoplastic cerebellar degeneration (PCD). Result: Independent of the age of the tissue from which the culture was driven, the properties of the generated rat Purkinje neuron culture are complex and robust, allowing great experimental flexibility. Using our model, we found that the application of Yo antibodies, which are associated with PCD, alters the structure of the dendritic arbour of cultured Purkinje neurons within 48h. The numbers of dendrites per branch-order, the branch-order in itself, and the dendritic length were reduced by treatment with anti-Yo. Conclusion: The here presented in vitro model is flexible and can be used to investigate disease mechanisms that disturb Purkinje neuron function and communication in a multilayer network. It proved that anti-Yo has a functional role in the pathogenesis of PCD.

Improved understanding of the mechanisms involved in neurodegenerative disease has been hampered by the lack of robust cellular models that faithfully replicate in vivo features. Here, we present a refined protocol for generating age-dependent, well-developed and synaptically active rat Purkinje neurons in a 3D cell network culture which are responsive to a disease inducer. Using our model, we found that the application of autoantibody Yo, a paraneoplastic cerebellar degeneration (PCD) inducer, alters the structure of the dendritic arbour of cultured Purkinje neurons. The numbers of dendrites per branch-order, the branch-order in itself and the dendritic length were reduced by anti-Yo, proving a functional role for anti-Yo in the pathogenesis of PCD. Our new ex-vivo model is flexible and can be used to investigate disease mechanisms that disturb Purkinje neuron function and communication in 3D. Since it is possible to use the approach in a multi-well format, this method also has high-throughput screening potential.

Improved understanding of the mechanisms involved in neurodegenerative disease has been hampered by the lack of robust cellular models that faithfully replicate in vivo features. Here, we present a refined protocol for generating age-dependent, well-developed and synaptically active rat Purkinje neurons, responsive to paracrine factors and supporting a 3D cell network. Our model provides high experimental flexibility, high-throughput screening capabilities and reliability to elucidate Purkinje neuron function, communication and neurodegenerative mechanisms.