Electrophysiological validation of premotor interneurons monosynaptically connected to the aCC motoneuron in theDrosophilalarval CNS

Abstract

Mapping the wired connectivity of a nervous system is a prerequisite for full understanding of function. In this respect, such endeavours can be likened to genome sequencing projects. These projects similarly produce impressive amounts of data which, whilst a technical tour-de-force, remain under-utilised without validation. Validation of neuron synaptic connectivity requires electrophysiology which has the necessary temporal and spatial resolution to map synaptic connectivity. However, this technique is not common and requires extensive equipment and training to master, particularly when applied to the small CNS of the Drosophila larva. Thus, validation of connectivity in this CNS has been more reliant on behavioural analyses and, in particular, activity imaging using the calcium-sensor GCaMP. Whilst both techniques are powerful, they each have significant limitations for this purpose. Here we use electrophysiology to validate an array of driver lines reported to label specific premotor interneurons that the Drosophila connectome project suggests are monosynaptically connected to an identified motoneuron termed the anterior corner cell (aCC).Our results validate this proposition for four selected lines. Thus, in addition to validating the connectome with respect to these four premotor interneurons, our study highlights the need to functionally validate driver lines prior to use.

The Drosophila Larval Locomotor Circuit Provides a Model to Understand Neural Circuit Development and Function

“…Hasegawa et al, used GFP reconstitution across synaptic partners (GRASP) to predict monosynaptic connections between A18b3 and aCC/RP2, but reconstructions performed by Zarin et al, only showed synapses between A18b3 and MN19-Ib, MN5-Ib (LO1), MN6/7-Ib (RP3), MN14-Ib (RP1), and MN30-Ib (RP4). Electrophysiology supports Zarin et al, as depolarising A18b3 does not change the membrane potential recorded in aCC ( Giachello et al, 2020 ). This highlights the importance of using the same TERPS (electrophysiology) experiments that validated the connection between A27h and aCC ( Giachello et al, 2020 ), to address controversy and validate synapses posed by TEM reconstruction(s).…”

“…Electrophysiology supports Zarin et al, as depolarising A18b3 does not change the membrane potential recorded in aCC ( Giachello et al, 2020 ). This highlights the importance of using the same TERPS (electrophysiology) experiments that validated the connection between A27h and aCC ( Giachello et al, 2020 ), to address controversy and validate synapses posed by TEM reconstruction(s). A18b3 signals (via relevant MNs) to coactive muscle group “F2” [muscles 3, 4, 5, 9, 12, 13, 18, 19, 25, 26, and 29 ( Zarin et al, 2019 )], which is (mostly) active earlier in segmental contraction than group F3.…”

“…Indeed, dual electrophysiological recordings showed that current injection into A27h, depolarises aCC ( Fushiki et al, 2016 ). More recent work employed tetrodotoxin-engineered resistance for probing synapses [TERPS, ( Zhang and Gaudry, 2018 )] to show that this occurs via a monosynaptic connection ( Giachello et al, 2020 ). A27h also connects to the RP5 MN (MNISNb/d, Fushiki et al, 2016 ), and according to later reconstruction of all 236 premotor interneurons, to several others: MN-20Ib; MN12-III (V-MN); MN14Ib (RP1); MN26-Ib; MN27-Ib; MN15/16-Ib (MN-VO4/5); MN15/16/17-Ib (MN-VO4-6) and MN28-Ib ( Zarin et al, 2019 ).…”

“…The latter lacks ganglionic structure and exhibits a high internal pressure that is problematic for dissection. This is significant, as electrophysiology plays an important role in characterising neurons by describing ionic and synaptic currents ( Baines and Bate, 1998 ), and validating synaptic connectivity ( Giachello et al, 2020 ). Finally, while the nematode worm is the only model organism to have an established and complete neural connectome ( White et al, 1986 ), Drosophila is catching up quickly.…”

Drosophila parabss Flies as a Screening Model for Traditional Medicine: Anticonvulsant Effects of Annona senegalensis

“…Drosophila could provide an early neurophysiological characterization of the mechanisms of action of new candidate compounds with potential translational anti-seizure effects ( 25 – 27 , 47 , 48 ). Specific neurons whose neurotransmitter identity and connectivity are known could be patch-clamped in para bss and the effect of the drug tested ( 49 ). The contribution of inherited or de novo mutations in the etiology of epilepsy, including focal epilepsy ( 6 , 50 ), is growing.…”