Preparation of Coverslips for Neuronal Cultures

Abstract

INTRODUCTIONThis protocol describes methods for preparing glass coverslips to use with three different types of dissociated neuronal cultures: mass culture, microisland culture, and macroisland culture. In mass culture, the entire glass coverslip is precoated with poly-D-lysine and laminin and then plated with astrocytes followed by dissociated neurons. In this system, the plated neurons tend to form an extensive and widespread network of synapses. However, depending on the origin of the neurons, proximity might not be a good predictor of the presence of synapses between any two cells. Microisland culture aims to increase the probability of identifying neurons synaptically connected to each other; its basic principle is to restrict spatially the choice of connections. This is done by plating neurons on astrocyte-seeded collagen dots on a background of a nonadhesive agarose substrate. Synaptic interactions between dorsal horn (DH) neurons or between dorsal-root ganglion (DRG) and DH neurons on the microislands are reliably found using this system, whereas they are much harder to find in mass culture. The macroisland approach is similar, but generates larger islands. It was developed specifically to improve neuronal survival in DRG/DH cocultures while still providing a high probability of connection between DRG and DH neurons. For DH-DH synapses however, microislands are still the best system.