Dendritic spines of striatal Medium Spiny Neurons (MSNs) receive converging dopaminergic and glutamatergic inputs. These spines undergo experience-dependent structural plasticity following repeated drug administration and during disease states like Huntington's and Parkinson's. Thus, understanding the molecular mechanisms leading to structural plasticity is an important step toward establishing a clear relationship between spine structure and function, and will ultimately contribute to understanding how changes in dendritic spine structure relate to behaviors or diseases. One major difficulty faced when studying MSN development is the lack of a detailed, standardized in vitro model system that produces MSNs with in vivo-like morphologies. For example, unlike cultured pyramidal neurons, MSNs grown in mono-cultures display stunted dendritic arborization and fail to develop a full cohort of mature dendritic spines. Here we report the generation of an embryonic mouse cortical-striatal co-culture that generates high cell yields from a single embryo. Unlike MSNs in striatal mono-culture, MSNs in co-culture develop in vivo-like morphologies and high densities of dendritic spines. Morphological identification of co-cultured MSNs expressing a soluble fluorescent protein can be confirmed by immunochemical detection of DARPP-32 (Dopamine and cyclic AMP regulated phospho. protein of 32. kDa). Additionally, co-cultured MSN spines contain PSD-95 puncta and are apposed to SV2 puncta, indicating the spines express synaptic machinery. Finally, whole-cell recordings of co-cultured MSNs exhibit higher mEPSC frequency compared to mono-cultured MSNs, suggesting that the spines are functionally mature. These studies establish that this co-culture system is suitable for studying the morphological and physiological development and function of MSN dendritic spines.
Bibliographical noteFunding Information:
We would like to thank Kacey Rajkovich and Arman Cicic for their efforts on data analysis and Teresa Nick for careful review of this manuscript. This project was supported by grants T32 GM008471, NIDA training grant T32 DA007234 and the Academic Health Center of the University of Minnesota (for RP), R01DA019666 (to MJT) and R01NS049178 (to LML). The funding agencies played no role in the design of the study or the writing of the manuscript. The mouse anti-SV2 monoclonal was developed by K.M. Buckley and obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.
- Dendritic spines
- Medium spiny neurons
- Primary cell culture