Gap junction protein Innexin2 modulates the period of free-running rhythms in Drosophila melanogaster

Abstract

Summary A neuronal circuit of ∼150 neurons modulates rhythmic activity-rest behavior of Drosophila melanogaster . While it is known that coherent ∼24-hr rhythms in locomotion are brought about when 7 distinct neuronal clusters function as a network due to chemical communication amongst them, there are no reports of communication via electrical synapses made up of gap junctions. Here, we report that gap junction proteins, Innexins play crucial roles in determining the intrinsic period of activity-rest rhythms in flies. We show the presence of Innexin2 in the ventral lateral neurons, wherein RNAi-based knockdown of its expression slows down the speed of activity-rest rhythm along with alterations in the oscillation of a core-clock protein PERIOD and the output molecule pigment dispersing factor. Specifically disrupting the channel-forming ability of Innexin2 causes period lengthening, suggesting that Innexin2 may function as hemichannels or gap junctions in the clock circuit.

Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes

“…Our estimates of inter-clock connectivity will therefore not include these contralateral connections. Furthermore, recent work suggests that electrical synapses within the clock neuron network likely contribute to circadian timekeeping ( Ramakrishnan and Sheeba, 2021 ), but these are not visible in the hemibrain dataset ( Scheffer et al, 2020 ). In addition, neuropeptides can be released non-synaptically (e.g.…”

Synaptic connectome of theDrosophilacircadian clock

“…Additionally, the synaptic connectivity reported here is likely an underestimation due to several factors: 1) we did not explore connectivity via gap junctions, 2) approximately 30% of synapses are missing for photoreceptors (Dorkenwald et al ., 2023) and 3) we generally used a connectivity threshold of >4 synapses. Preliminary expression analysis using the single-cell transcriptomes of clock neurons suggests that gap junction genes are enriched in the clock network (data not shown) and they can influence activity-rest rhythms (Ramakrishnan and Sheeba, 2021). It remains to be seen which clock neurons are additionally coupled via gap junctions and how this electrical connectivity complements synaptic and peptidergic connectivity detailed here.…”

A new player in circadian networks: Role of electrical synapses in regulating functions of the circadian clock

“… Summary of the circadian phenotypes observed in Connexin36 mutant mice (left) and Innexin2 mutant Drosophila (right): (left) Electrophysiological recordings from SCN slices of Cx36 −/− mice show desynchronized firing of SCN neurons ( Long et al, 2005 ) , PER2-Luc rhythms in Cx36 −/− mice (red traces) oscillate with a longer free-running period compared to WT mice (black traces) over days ( Diemer et al, 2017 ), and the wheel-running activity of adult mice with Connexin36 mutation runs with a longer free-running period compared to WT mice under constant darkness ( Long et al, 2005 ; Diemer et al, 2017 ) (right). Knockdown of Innexin2 in clock neurons delays the phase of PER protein oscillation in major clock neuronal subsets in the Drosophila brain and lengthens the free-running period of activity-rest rhythms ( Ramakrishnan and Sheeba, 2021 ). Image created using BioRender.…”

“…A recent study from our group reports the role of specific gap junction proteins in the circadian circuit of Drosophila melanogaster . A genetic knockdown screen of all the eight Innexin genes in the clock neurons reveals the importance of Innexin1 and Innexin2 in modulating the free-running period ( Ramakrishnan and Sheeba, 2021 ). We show that the knockdown of gap junction genes Innexin2 and Innexin1 lengthens the free-running period of activity-rest rhythms ( Figure 2 ).…”

“…Innexin2 protein was shown to be present in the small and large ventral lateral neurons (s-LNv and l-LNv respectively). Knockdown of Innexin2 delays the molecular clocks in most circadian neurons ( Figure 2 ) and increases the levels and amplitude of oscillation of the circadian neuropeptide, Pigment Dispersing Factor (PDF), which could ultimately explain the lengthening of free-running period seen in behavioural rhythms ( Ramakrishnan and Sheeba, 2021 ). While this study provides a direct role for gap junction proteins in regulating a core clock property, much of the mechanism of how these proteins affect the molecular clock and the free-running period remains unknown.…”