Connectomic Analysis of the Drosophila Lateral Neuron Clock Cells Reveals the Synaptic Basis of Functional Pacemaker Classes

Abstract

The circadian clock orchestrates daily changes in physiology and behavior to ensure internal temporal order and optimal timing across the day. In animals, a central brain clock orchestrates circadian rhythms throughout the body and is characterized by a remarkable resilience that depends on synaptic connections between constituent neurons. The clock neuron network of Drosophila, which is built of many fewer neurons yet shares network motifs with clock networks in the mammalian brain, offers a powerful model for understanding the network properties of circadian timekeeping. Here we report an assessment of synaptic connectivity within a clock network, focusing on the critical LN clock neuron classes. Our results reveal that previously identified anatomical and functional subclasses of LNs represent distinct connectomic types with distinct synaptic output pathways. Moreover, we identify a small number of clock cell subtypes representing highly synaptically coupled nodes within the fly’s clock neuron network, and suggest that neurons lacking molecular timekeeping likely play integral roles within the circadian timekeeping network. To our knowledge, this represents the first comprehensive connectomic analysis of a neural network capable of driving endogenous circadian rhythms. The organizational principles uncovered in this study provide remarkable insights into clock network organization.

Neural connectivity molecules best identify the heterogeneous clock and dopaminergic cell types in theDrosophilaadult brain

“…In this context, Waddell and colleagues have identified 20 PAM-γ5 subtypes by anatomy (Otto et al, 2020), and it would not be surprising if many of these anatomical subtypes are also discrete by transcriptional profiling. Consistent with this notion are recent anatomy and connectome analyses of the 6 LNd clock neurons: there are 4 different groups, which precisely match our previous classification based on gene expression (Ma et al, 2021;Shafer et al, 2022;Yao et al, 2016). This complexity of the adult fly brain transcriptomes is not predicted by previous results.…”

Neuronal correlates of time integration into memories

“…Importantly, cAMP and calcium are increased in DN1p neurons in response to PDF application. PDF acutely depolarizes and increases action potential firing rates of DN1p neurons (25,26), proving that DN1p neurons are direct downstream targets of PDF signalling (24,(27)(28)(29). To anatomically confirm these findings of overlap between PDF and DN1p neurons, we used the GRASP technique which allows to label potential synaptic contacts by reconstituted GFP fluorescence (30).…”

Endocrine cybernetics: neuropeptides as molecular switches in behavioural decisions

“…With analysis of conventional synapses, it is possible to determine how identified peptidergic neurons connect to other neurons, but the resulting connectome is likely to predominantly represent signalling with SMNs. Thus, the connections of a set of identified peptidergic neurosecretory cells and associated neurons in the brain of the early Drosophila larva [ 194 , 333 ], and a partial connectome of the adult clock neurons, many of which are peptidergic [ 334 , 335 ] tell us little about peptidergic signalling. Yet, these studies are vital since they inform us that peptidergic neurons do signal quite extensively also with SMNs.…”

“…Thus, not only LK and AstA-expressing neurons, but also ones signalling with AstC, CRZ, DH31, DH44, Hugin-pyrokinin, myoinhibitory peptide, natalisin and NPF seem to be intermediate size neurons with multiple inputs and in a position to modulate circuits that affect behaviour ( table 2 and references therein). For analysis of executive neuromodulation by neuropeptides and co-transmission with SMNs, the antennal lobe/olfactory system and the clock circuits are excellent established models, especially since many of the intrinsic neurons are peptidergic [ 85 , 116 – 118 , 335 ], and their connectomes are available [ 75 , 334 , 339 ].…”

“…royalsocietypublishing.org/journal/rsob Open Biol. 12: 220174 especially since many of the intrinsic neurons are peptidergic [85,[116][117][118]335], and their connectomes are available [75,334,339].…”