Synaptic development in semi-dissociated cultures of rat retina

Cultured neurons provide a simpler and more accessible environment to study the synaptic physiology. However, it is not clear if development of synapses in culture is similar to that in the in vivo condition. We studied the developmental sequence and morphological differentiation of chemical synapses in semi-dissociated rat retinal cultures that consisted of dissociated neurons as well as undissociated retinal aggregates. Synapses were quantified by synaptophysin immunoreactive puncta. During second week of in vitro development the average number of chemical synapses on the cell body decreased while that on the neurites increased significantly. Conventional synapses appeared both in aggregate and in dissociated neurons, with the developmental profile similar to that reported for in vivo retina. In contrast, the development of ribbon synapses was adversely affected by the in vitro microenvironment as suggested by following observations. The ribbon synapses were more frequently found in aggregate than in dissociated neurons, and were not associated with dyadic or triadic synaptic arrangement. The photoreceptor ribbons did not contact a postsynaptic process while bipolar ribbons made single (monadic) synapses. Further, photoreceptor ribbons in dissociated neurons were late to form and took more time to mature as compared to those in the aggregate cultures. Most of the rod bipolar cells, identified by their immunoreactivity to protein kinase C (PKC), had three or more neurites. Unlike in the in vivo retina, the dissociated rod bipolar cells did not show any PKC immunoreactive varicosities, suggesting that they failed to develop a well-differentiated synaptic terminal. Interestingly, we did not find any parvalbumin positive AII amacrine cells that are normally postsynaptic to rod bipolar cells. These results show that the conventional synapses of retina, which are similar to chemical synapses in other parts of the brain, develop normally both in aggregate and dissociated neurons. However, the highly specialized ribbon synapses have more stringent developmental requirements, and their normal development may require the presence of postsynaptic neurons in their close vicinity.