A new co-culture method utilizing a dual compartment cell-allocation apparatus for observation and identification of...

A new co-culture method for observation and identification of synapse formation utilizing dissociated retinal and tectal neurons from 7-day-old chick embryos was developed using a glass apparatus which we named 'Dual Compartment Cell-Allocation Apparatus.' This apparatus enabled us to culture these two types of cells by forming two parallel lanes, each with a dimension of 200 microns by 15 mm and with an interval of 200 microns separating the two lanes, on a collagen-coated cover slip. Infiltration and/or migration of retinal cells into the tectal cell lane was minimal with only 0.15% identified and confirmed with the fluorescence-dye labeling method. Growth of neurites connecting the two lanes was observed within 48 h of culture in a serum-free medium. Double-immunostaining of the culture preparations with anti-neurofilament and anti-synaptophysin antibodies revealed the presence of synaptophysin-hot spots on neurofilament-positive processes connecting the retinal neurons with the tectal neurons, thus confirming the formation of synapses between the neurons of the retinal lane and the neurons of the tectal lane. The dual compartment cell-allocation apparatus has proven itself to be advantageous and useful for studying the formation of synapses between two types of neurons from different origin.