Journal of neuroscience methods · 2025
A robust method for primary cerebellar culture and genetic manipulation of Purkinje cells from postnatal mice.
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Lu Heiling, Stephani Friederike, Aulia Myrna, Mientjes Edwin, Schonewille Martijn, Osório Catarina
Аннотация
BACKGROUND: Primary cerebellar cultures are a powerful system for studying genes and pathways involved in development and disease. However, maintaining the survival of Purkinje cells in vitro remains challenging. These neurons represent a small fraction of the cerebellar population and are highly sensitive to culture conditions, making genetic manipulation difficult. Existing protocols often use more than one culture medium, vary in the developmental stage of the cerebellar tissue used, and may require supplementary support cell cultures.
NEW METHOD: We describe a robust method for dissociated cerebellar cultures that supports Purkinje cell development and enables efficient genetic manipulation using two techniques.
RESULTS: Our protocol supports the development of Purkinje cells from embryonic day 18 and postnatal day 1 mouse cerebellar tissue, with P1-derived neurons more closely resembling their in vivo counterparts. Genetic manipulation was achieved via cell-specific adeno-associated viral transduction and non-cell-specific antisense oligonucleotide transfection. Functional neuronal activity was confirmed using calcium imaging with Fluo-4 AM.
COMPARISON WITH EXISTING METHODS: Our approach unifies key aspects of existing protocols. It uses a single-medium composition, eliminating the need for separate seeding and maintenance media. Culturing each cerebellum individually preserves the intrinsic number of supporting cells, eliminating the need for parallel cultures. Using postnatal tissue also reduces animal usage. Additionally, gene expression can be successfully modulated using antisense oligonucleotides.
CONCLUSIONS: This protocol provides an efficient framework for the culture, development, and genetic manipulation of Purkinje cells, facilitating their application in molecular, cellular, and functional studies of cerebellar development and disease models.
NEW METHOD: We describe a robust method for dissociated cerebellar cultures that supports Purkinje cell development and enables efficient genetic manipulation using two techniques.
RESULTS: Our protocol supports the development of Purkinje cells from embryonic day 18 and postnatal day 1 mouse cerebellar tissue, with P1-derived neurons more closely resembling their in vivo counterparts. Genetic manipulation was achieved via cell-specific adeno-associated viral transduction and non-cell-specific antisense oligonucleotide transfection. Functional neuronal activity was confirmed using calcium imaging with Fluo-4 AM.
COMPARISON WITH EXISTING METHODS: Our approach unifies key aspects of existing protocols. It uses a single-medium composition, eliminating the need for separate seeding and maintenance media. Culturing each cerebellum individually preserves the intrinsic number of supporting cells, eliminating the need for parallel cultures. Using postnatal tissue also reduces animal usage. Additionally, gene expression can be successfully modulated using antisense oligonucleotides.
CONCLUSIONS: This protocol provides an efficient framework for the culture, development, and genetic manipulation of Purkinje cells, facilitating their application in molecular, cellular, and functional studies of cerebellar development and disease models.