Circadian programming of the ellipsoid body sleep homeostat in Drosophila

Homeostatic and circadian processes collaborate to appropriately time and consolidate sleep and wake. To understand how these processes are integrated, we scheduled brief sleep deprivation at different times of day in Drosophila and find elevated morning rebound compared to evening. These effects depend on discrete morning and evening clock neurons, independent of their roles in circadian locomotor activity. In the R5 ellipsoid body sleep homeostat, we identified elevated morning expression of activity dependent and presynaptic gene expression as well as the presynaptic protein BRUCHPILOT consistent with regulation by clock circuits. These neurons also display elevated calcium levels in response to sleep loss in the morning, but not the evening consistent with the observed time-dependent sleep rebound. These studies reveal the circuit and molecular mechanisms by which discrete circadian clock neurons program a homeostatic sleep center.

Thermoresponsive motor behavior is mediated by ring neuron circuits in the central complex of Drosophila

Insects are ectothermal animals that are constrained in their survival and reproduction by external temperature fluctuations which require either active avoidance of or movement towards a given heat source. In Drosophila, different thermoreceptors and neurons have been identified that mediate temperature sensation to maintain the animal’s thermal preference. However, less is known how thermosensory information is integrated to gate thermoresponsive motor behavior. Here we use transsynaptic tracing together with calcium imaging, electrophysiology and thermogenetic manipulations in freely moving Drosophila exposed to elevated temperature and identify different functions of ellipsoid body ring neurons, R1-R4, in thermoresponsive motor behavior. Our results show that warming of the external surroundings elicits calcium influx specifically in R2-R4 but not in R1, which evokes threshold-dependent neural activity in the outer layer ring neurons. In contrast to R2, R3 and R4d neurons, thermogenetic inactivation of R4m and R1 neurons expressing the temperature-sensitive mutant allele of dynamin, shibireTS, results in impaired thermoresponsive motor behavior at elevated 31 °C. trans-Tango mediated transsynaptic tracing together with physiological and behavioral analyses indicate that integrated sensory information of warming is registered by neural activity of R4m as input layer of the ellipsoid body ring neuropil and relayed on to R1 output neurons that gate an adaptive motor response. Together these findings imply that segregated activities of central complex ring neurons mediate sensory-motor transformation of external temperature changes and gate thermoresponsive motor behavior in Drosophila.

Neuronal constituents and putative interactions within the Drosophila ellipsoid body neuropil

The central complex (CX) is a midline-situated collection of neuropil compartments in the arthropod central brain, implicated in higher-order processes such as goal-directed navigation. Here, we provide a systematic genetic-neuroanatomical analysis of the ellipsoid body (EB), a compartment which represents a major afferent portal of the Drosophila CX. The neuropil volume of the EB, along with its prominent input compartment, called the bulb, is subdivided into precisely tessellated domains, distinguishable based on intensity of the global marker DN-cadherin. EB tangential elements (so-called ring neurons), most of which are derived from the DALv2 neuroblast lineage, interconnect the bulb and EB domains in a topographically-organized fashion. Using the DN-cadherin domains as a framework, we first characterized the bulb-EB connectivity by Gal4 driver lines expressed in different DALv2 ring neuron (R-neuron) subclasses. We identified 11 subclasses, 6 of which correspond to previously described projection patterns, and 5 novel patterns. These subclasses both spatially (based on EB innervation pattern) and numerically (cell counts) summate to the total EB volume and R-neuron cell number, suggesting that our compilation of R-neuron subclasses approaches completion. EB columnar elements, as well as non-DALv2 derived extrinsic ring neurons (ExR-neurons), were also incorporated into this anatomical framework. Finally, we addressed the connectivity between R-neurons and their targets, using the anterograde trans-synaptic labeling method, trans-Tango. This study demonstrates putative interactions of R-neuron subclasses and reveals general principles of information flow within the EB network. Our work will facilitate the generation and testing of hypotheses regarding circuit interactions within the EB and the rest of the CX.

A circadian output circuit controls sleep-wake arousal threshold in Drosophila

SummaryThe Drosophila core circadian circuit contains distinct groups of interacting neurons that give rise to diurnal sleep-wake patterns. Previous work showed that a subset of Dorsal Neurons 1 (DN1s) are sleep-promoting through their inhibition of activity-promoting circadian pacemakers. Here we show that these anterior-projecting DNs (APDNs) also “exit” the circadian circuitry and communicate with the homeostatic sleep center in higher brain regions to regulate sleep and sleep-wake arousal threshold. These APDNs connect to a small discrete subset of tubercular-bulbar neurons, which are connected in turn to specific sleep-centric Ellipsoid Body (EB)-Ring neurons of the central complex. Remarkably, activation of the APDNs produces sleep-like oscillations in the EB and also raises the arousal threshold, which requires neurotransmission throughout the circuit. The data indicate that this APDN-TuBusup-EB circuit temporally regulates sleep-wake arousal threshold in addition to the previously defined role of the TuBu-EB circuit in vision, navigation and attention.

The laminar organization of the Drosophila ellipsoid body is semaphorin-dependent and prevents the formation of ectopic synaptic connections

The ellipsoid body (EB) in the Drosophila brain is a central complex (CX) substructure that harbors circumferentially laminated ring (R) neuron axons and mediates multifaceted sensory integration and motor coordination functions. However, what regulates R axon lamination and how lamination affects R neuron function remain unknown. We show here that the EB is sequentially innervated by small-field and large-field neurons and that early developing EB neurons play an important regulatory role in EB laminae formation. The transmembrane proteins semaphorin-1a (Sema-1a) and plexin A function together to regulate R axon lamination. R neurons recruit both GABA and GABA-A receptors to their axon terminals in the EB, and optogenetic stimulation coupled with electrophysiological recordings show that Sema-1a-dependent R axon lamination is required for preventing the spread of synaptic inhibition between adjacent EB lamina. These results provide direct evidence that EB lamination is critical for local pre-synaptic inhibitory circuit organization.