The lateral posterior clock neurons of Drosophila melanogaster express three neuropeptides and have multiple connections within the circadian clock network and beyond

Abstract

Drosophila's lateral posterior neurons (LPNs) belong to a small group of circadian clock neurons that is so far not characterized in detail. Thanks to a new highly specific split-Gal4 line, here we describe LPNs' morphology in fine detail, their synaptic connections, daily bimodal expression of neuropeptides, and propose a putative role of this cluster in controlling daily activity and sleep patterns. We found that the three LPNs are heterogeneous. Two of the neurons with similar morphology arborize in the superior medial and lateral protocerebrum and most likely promote sleep. One unique, possibly wakefulness-promoting, neuron with wider arborizations extends from the superior lateral protocerebrum toward the anterior optic tubercle. Both LPN types exhibit manifold connections with the other circadian clock neurons, especially with those that control the flies' morning and evening activity (M-and E-neurons, respectively). In addition, they form synaptic connections with neurons of the mushroom bodies, the fanshaped body, and with many additional still unidentified neurons. We found that both LPN types rhythmically express three neuropeptides, Allostatin A, Allostatin C, and Diuretic Hormone 31 with maxima in the morning and the evening. The three LPN neuropeptides may, furthermore, signal to the insect hormonal center in the pars intercerebralis and contribute to rhythmic modulation of metabolism, feeding, and reproduction.We discuss our findings in the light of anatomical details gained by the recently published hemibrain of a single female fly on the electron microscopic level and of previous functional studies concerning the LPN.

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

“…Representatives of all classes of clock neurons, with the exception of the DN2 and DN3 classes, are identified in the annotation of the hemibrain volume used here (Figure 1A and B; please see Table 1 for the naming scheme used here for the clock neurons identified in the dataset with their corresponding unique body IDs). The patterns of connectivity among some of these neurons have recently been briefly described (11, 13). Scheffer and colleagues (15) define synaptic strength within the hemibrain volume based on the number of synaptic connections formed between neurons, defining connections consisting of only one or two chemical synapses as weak and subject to error, three to nine synapses as medium strength, and of ten or more synapses as strong.…”

“…For example, the strong targets of E1 outputs preferentially form strong connections back onto E1 LNds (Figure 7E). For all four groups of LN, strong outputs recurring to the clock network form either strong or medium strength synapses on the LPNs, implicating this class as a particularly rich hub for polysynaptic inter-clock connectivity (Figures 6E, 7E, 8E, 9E, and 10C; (13)).…”

“…Most clock neuron pairs form either no synapses or weak synaptic connections (Figure 1C). However, there are a few clear exceptions: a single LNd (LNd6) and the 5 th s-LNv form strong connections with one another and with members of the DN1 group (Figure 1C; see also Reinhard and colleagues (13)) and stand out among the clock neurons as hubs of inter-clock synaptic connectivity.…”

“…The current annotation of the hemibrain data set identifies all the expected lateral neuron (LN) classes: four l-LNvs, four s-LNvs, the 5th-s-LNv, and six LNds ((15); Figure 1B and C). In addition, all of the LPN and DN1as have been identified, as have seven out of the ~15 expected DN1ps (13,15). However, some clock neurons have not yet been unequivocally identified within the hemibrain data set: approximately half of the DN1ps and all of the DN2s and DN3s.…”

“…Furthermore, genetic tools for the specific manipulation of anatomical subclasses of the fly have made it possible to characterize the functional roles of these subclasses in the production of endogenous circadian rhythms in sleep and activity (10, 11). Though transgenic markers of synaptic connectivity and live-imaging-based assessments of functional connectivity have been used to address neural connectivity within the clock neuron network (e.g., (12, 13), a systematic accounting of synaptic connectivity within this network is lacking. Such an accounting would provide valuable insights into the network structure of neural circadian timekeeping in the brain.…”