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Summary Animals exhibit innate and learned preferences for temperature and humidity—conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior., Graphical Abstract, Highlights • Two novel thermo- and/or hygrosensory glomeruli in the fly antennal lobe • First complete set of thermosensory and hygrosensory projection neurons • First connectome for a thermo- and hygrosensory neuropil • Third-order thermo- and hygrosensory neurons, including link to circadian clock, Marin et al. use connectomics and genetics for comprehensive identification of temperature and humidity sensory neurons in the Drosophila brain. They reconstruct all projections to higher brain areas and select higher-order targets, including the mushroom body lateral accessory calyx, linking thermosensation to memory and the circadian clock.

In this chapter we discuss methods that can be used to study apoptotic cell death in the Drosophila embryo, ovary, as well as in cultured cell lines. These methods assay various aspects of the cell death process, from mitochondrial changes to caspase activation and DNA cleavage. The assays are useful for examining apoptosis in normal development and in response to developmental perturbations and external stresses. These techniques include Acridine Orange staining, TUNEL, cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization.