Neuronal sandwiches: A method for rapid and controlled initiation of synapses

Synapse formation is a fast, dynamic process that involves the assembly of many molecules following axodendritic contact. Neuronal cultures are often used to study the insertion of fluorescently tagged pre- and postsynaptic molecules in vitro. However, this task still remains challenging, since the time-point and location of newly forming synapses are largely unpredictable and rely on random contact events. We developed a technique that controls the time-point of interaction between axons and dendrites, and thus the onset of synapse formation. Dissociated hippocampal neurons were cultivated on two different coverslips, allowing for the separate outgrowth of axonal networks and of neurons with sparsely innervated dendrites. Pre- and postsynaptic partners were brought in contact as coverslips were merged. Time-lapse imaging showed clustering of GFP/PSD-95 in postsynaptic neurons within 1–3 h, indicating the rapid formation of new synaptic sites. Localization of DsRed, as a control protein, remained unchanged. Imaging of neuronal activity using calcium sensitive dyes revealed that in a number of cases neurons of the pre- and postsynaptic layer were synchronously active, suggesting the functionality of newly formed synapses across layers. Therefore, our new method is a valuable tool to control synapse formation and for investigating the temporal role of signaling molecules during this process.