Electrophysiological Validation of Monosynaptic Connectivity between Premotor Interneurons and the aCC Motoneuron in the Drosophila Larval CNS

Abstract

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. JN-RM-2463-21 R1 4 2 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.

A premotor microcircuit to generate behavior-specific muscle activation patterns in Drosophila larvae

“…An organism as simple as Drosophila larva, uses these PMN-PMN motifs to (i) exert antagonistic effects on downstream MNs, (ii) prevent premature activation of MNs, (iii) restrict duration of excitatory inputs onto MNs, and (iv) shut down the MNs once their activity is no longer needed. Our work supports the notion that EM reconstruction of neuroanatomical connectome is necessary but not sufficient to fully understand how neural circuits works [50]. The discovery of bi-modal and dedicated PMNs clearly demonstrate that a given neuroanatomical circuit may not necessarily act the same way during different behaviors.…”

Balance of activity during a critical period tunes a developing network

“…Motoneuron activity reflects the integration of the excitatory and inhibitory premotor synaptic drive to which the cell is exposed. We therefore tested the effect of embryonic exposure to PTX on the synaptic drive provided by two identified premotor interneurons – one exciter and one inhibitor – which are monosynaptically connected to aCC ( Giachello et al, 2022 ). Specifically, we quantified the strength of cholinergic excitor A18a (a.k.a.…”

“…However, the effects of activity perturbation on the specific cellular components of the locomotor network have not been identified. Here, we make use of the output of the connectome project to focus attention on excitatory cholinergic A18a ( Hasegawa et al, 2016 ) and inhibitory GABAergic A31k ( Schneider-Mizell et al, 2016 ) interneurons, as well as the aCC motoneuron which these interneurons are monosynaptically connected to ( Giachello et al, 2022 ). Specifically, we perform patch-clamp experiments on aCC to show how synaptic connectivity of these single-cell types (A18a or A31k) is influenced following activity perturbation during the embryonic CP.…”

Critical periods in Drosophila neural network development: Importance to network tuning and therapeutic potential

“…The Drosophila larval connectome provides great promise for identifying those components of the larval CPG that are altered following activity perturbation during a CP. We have recently validated monosynaptic connectivity, that had been predicted by the connectome, for four identified premotor interneurons (INs) with the aCC motoneuron (Zarin et al, 2019;Giachello et al, 2022). These cells are the cholinergic excitors A27h and A18a, and the GABAergic inhibitors A23a and A31K.…”