A conditional strategy for cell-type specific labeling of endogenous excitatory synapses in Drosophila reveals subsynaptic architecture

Chemical neurotransmission occurs at specialized contacts where presynaptic neurotransmitter release machinery apposes clusters of postsynaptic neurotransmitter receptors and signaling molecules. A complex program underlies recruitment of pre- and postsynaptic proteins to sites of neuronal connection and enables the correct three-dimensional synaptic organization that underlies circuit processing and computation. To better study the developmental events of synaptogenesis in individual neurons, we need cell-type specific strategies to visualize the individual proteins at their endogenous levels at synapses. Though such strategies exist for a variety of presynaptic proteins, postsynaptic proteins remain less studied due to a paucity of reagents that allow visualization of endogenous individual postsynapses in a cell-type specific manner. To study excitatory postsynapses, we engineered dlg1[4K], a conditional, epitope-tagged marker of the excitatory postsynaptic density in Drosophila. In combination with binary expression systems, dlg1[4K] effectively labels postsynaptic regions at both peripheral neuromuscular and central synapses in larvae and adults. Using dlg1[4K], we find distinct rules govern the postsynaptic organization of different adult neuron classes, that multiple binary expression systems can concurrently label pre- and postsynaptic regions of synapses in a cell-type-specific manner, and for the first time, visualize neuronal DLG1 at the neuromuscular junction. These results validate a novel strategy for conditional postsynaptic labeling without the caveats of overexpression and demonstrate new principles of subsynaptic organization. The use of dlg1[4K] marks a notable advancement in studying cell-type specific synaptic organization in Drosophila and the first example of a general postsynaptic marker to complement existing presynaptic strategies.