Electrophysiological validation of monosynaptic connectivity between premotor interneurons and the aCC motoneuron in the Drosophila larval CNS.

The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour-de-force, the data remain under-utilised, partly due to a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioural assays and/or GRASP or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell (aCC) motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favourable alternative to GRASP. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotors they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects.SIGNIFICANCE STATEMENTThe Drosophila connectome project aims to provide a complete description of connectivity between neurons in an organism that presents experimental advantages over other models. It has reconstructed over 80 percent of the fly larva's synaptic connections by manual identification of anatomical landmarks present in serial section transmission electron microscopy (ssTEM) volumes of the larval CNS. We use a highly reliable electrophysiological approach to verify these connections, so provide useful insight into the accuracy of work based on ssTEM. We also present a novel imaging tool for validating excitatory monosynaptic connections between cells, and show that several genetic driver lines designed to target neurons of the larval connectome exhibit non-specific and/or unreliable expression.