Abstract
Primary cultures of murine striatal neurons are widely used to explore cellular mechanisms in neurobiology, including brain diseases. Here we describe a detailed and standardized protocol to dissect and culture embryonic murine striatal neurons GABA-positive/DARPP-32-positive for 12 days in vitro, when they show good neuronal cell connectivity and the presence of dendritic spines, which reflects the maturation of the network.
Keywords: Primary cultures, Striatum dissection, Striatal neurons, Medium spiny neurons
Background
Striatum is a critical component of the motor and reward systems and dysfunction of striatal neurons can lead to a variety of neuronal disorders, from obsessive-compulsive-like behaviors (Welch et al., 2007) to neurodegeneration, as observed in Huntington’s disease (Reiner et al., 1988). Therefore, a well-stablished striatal culture may be a great value as a model for studying these and other conditions. The major neuronal subtype in adult striatum is medium spiny projection neurons (MSNs), which constitutes approximately 95% of all striatal neurons and use the inhibitory transmitter gamma-aminobutyric acid (GABA). They are characterized by medium-sized cell bodies, complex dendritic arbors and a high density of dendritic spines that receive both glutamatergic and dopaminergic inputs. The remaining 5% of neurons are composed by GABAergic aspiny interneurons (Graveland and DiFiglia, 1985; Matamales et al., 2009). The following protocol describes the preparation of primary cultures of mouse embryonic striatal neurons with a low percentage of astrocytes, which proliferation is prevented by the mitosis inhibitor 5-fluoro-2’-deoxyuridine (5-FdU). After 12 days in vitro (DIV) the striatal neurons obtained by the described protocol are expected to be immunolabeled for microtubule-associated protein 2 (MAP2) or NeuN, GABA and a large percentage for dopamine- and cAMP-regulated phosphoprotein (DARPP-32).
Materials and Reagents
Equipment
Software
Procedure
Note: Except for the coverslips washing (described in the following Procedure A, before poly-D-lysine coating) and brain dissection, all the procedures must be done in a sterile environment using a flow chamber.
Data analysis
For striatal cultures characterization, 6 to 8 images per individual experiment were randomly taken with confocal microscope for each antibody labeled. GABA- and DARPP-32-positive cells were counted using Fiji software (ImageJ, National Institute of Health, USA) with Cell Counter plugin, and the percentage were calculated in relative to MAP2-positive cells.
Recipes
Acknowledgments
We would like to thank Carina Maranga (CNC, University of Coimbra, Coimbra, Portugal) for assisting in filming brain dissection. This work was supported by European community FEDER fund through the ‘Programa Operacional Factores de Competitividade’ – COMPETE 2020; and national funds by ‘Fundação para a Ciência e a Tecnologia’ (FCT), Portugal [project ref. POCI-01-0145-FEDER-007440]. FVB/NJ mice used in these primary cultures were supported by a project financed by Teva Pharmaceutical Industries Ltd. The authors declare that they have no financial or competing conflict of interests.
References
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