Functional Characterization of Individual Pre- and Postsynaptic Partners In the Drosophila Larval Central Nervous...

Connectome studies have greatly expanded understanding of synaptic connectivity in the nervous systems of various species. While characterization of synaptic partners using electron microscopy (EM) provides detailed anatomical information, functional aspects of neuronal networks require complementary approaches. Recent studies in Drosophila have revealed not only the complete connectome of the larval, as well as male and female adult central nervous system, but also the cellular components of neuronal networks that regulate specific behaviors, such as the larval nociceptive network. By the third instar larval stage, class IV dendritic arborization (cIVda) multidendritic sensory neurons (nociceptors) establish most synaptic contacts with Basin-4 interneurons. To assess the functional relevance of these anatomical connections, the genetically encoded calcium indicator CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) was employed in live, undissected third-instar larvae as an activity-dependent reporter to evaluate synaptic connectivity between cIVda neurons and Basin-4 interneurons. CaMPARI is a ratiometric fluorescent indicator whose emission spectrum changes in response to elevated intracellular calcium levels. Under baseline conditions, CaMPARI fluoresces green; in the presence of high calcium concentrations and upon exposure to photoconversion light (~400 nm), it irreversibly switches to red fluorescence. Because calcium influx into postsynaptic neurons is a hallmark of synaptic activation, CaMPARI photoconversion provides a readout of functional synaptic signaling. A step-by-step method is presented to immobilize third-instar larvae on a microscope slide for optogenetic activation of cIVda nociceptors using the red-shifted channelrhodopsin CsChrimson, combined with simultaneous CaMPARI photoconversion in Basin-4 neurons. Calcium-dependent photoconversion in Basin-4 neurons, despite internal movements and changes in focal plane, provides functional evidence of synaptic connectivity between these cells. This serves as proof-of-principle for the use of CaMPARI in combination with presynaptic optogenetic stimulation in intact, undissected Drosophila larvae.