Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, we reveal that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. We characterise IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. We further demonstrate that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. Our work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour.

Visual systems transduce, process and transmit light-dependent environmental cues. Computation of visual features depends on photoreceptor neuron types (PR) present, organization of the eye and wiring of the underlying neural circuit. Here, we describe the circuit architecture of the visual system of Drosophila larvae by mapping the synaptic wiring diagram and neurotransmitters. By contacting different targets, the two larval PR-subtypes create two converging pathways potentially underlying the computation of ambient light intensity and temporal light changes already within this first visual processing center. Locally processed visual information then signals via dedicated projection interneurons to higher brain areas including the lateral horn and mushroom body. The stratified structure of the larval optic neuropil (LON) suggests common organizational principles with the adult fly and vertebrate visual systems. The complete synaptic wiring diagram of the LON paves the way to understanding how circuits with reduced numerical complexity control wide ranges of behaviors.

Animals possess highly specialized sensory neurons that are capable of perceiving information about a continuously changing environment. Measuring neuronal activity in these cells is an important step toward understanding the basic coding principles, as well as the spatial and temporal dynamics, of sensory systems. The fruit fly Drosophila melanogaster is widely used as a genetic model organism to uncover basic mechanisms of taste coding and odor perception [1][2][3][4][5][6][7] . Recent insights into the molecular and functional mechanisms of sensory perception in Drosophila have been achieved by the use of single-sensillum recordings of adult taste or olfactory sensilla 1,4,[8][9][10][11][12][13] . This method enables sensitive measurements of action potential patterns and shows whether responses are excitatory or inhibitory, but drawing final conclusions about the individual firing neuron is difficult, as each sensillum contains two to four neurons 14 . In the larva, multicellular electrophysiological measurements of the taste system have been performed, but this method is technically challenging and it is not clear how many taste neurons are housed in which sensillum 15 .Microfluidic devices have been used previously to image command interneurons (AVA) and sensory neurons (ASH) in the nematode Caenorhabditis elegans and for studies of axotomy in peripheral sensory neurons of Drosophila larvae [16][17][18] . To implement a similar strategy for Drosophila larval chemosensation, we developed a simple and widely applicable microfluidic chip. We used this custom-made microfluidic device to deliver precise and temporally restricted tastant stimulation in the first published physiological characterization of individual larval gustatory receptor neurons (GRNs) expressing the calcium sensor GCaMP5 (ref. 19). Development of the protocolThe design of the chip is simple. It contains a single channel that is connected to a chamber in which the sample can be mounted to bring it into contact with fluids in the channel. The chamber was originally designed to exactly fit the head of a third-instar Drosophila larva; however, it is also suitable for the proboscis or the leg of an adult fly. The channel for delivering stimulants is relatively broad, designed at a width of 1 mm to reduce flow pressure on the sample. We use a single-channel system in which changes of liquids are performed by the tubing system outside the chip, allowing a relatively slow flow rate inside the chip. Here we provide information required for the design and production of this microfluidic device ( Fig. 1; Supplementary Data), as well as a detailed protocol for live imaging to acquire GCaMP-based calcium imaging (Figs. 2 and 3). By introducing the larval head, or the adult proboscis or leg, into the chamber, neuronal activity can be recorded while chemosensory neurons are in contact with the solution pumped through the channel (Figs. 2 and 3).Overview of the procedure An overview of the workflow involved in implementing the procedure is provided in Figu...

Taste perception is thought to involve the encoding of appetitive and aversive chemical cues in food through a limited number of sensory pathways. Through expression analysis of the complete repertoire of Drosophila Ionotropic Receptors (IRs), a sensory subfamily of ionotropic glutamate receptors, we reveal that the majority of IRs is expressed in diverse peripheral neuron populations across gustatory organs in both larvae and adults, implying numerous roles in taste-evoked behaviours. We characterise Ir56d, which labels two anatomically-distinct classes of neurons in the proboscis: one represents a subset of sugar-and fatty acid-sensing neurons, while the other responds to carbonated solutions and fatty acids. Mutational analysis shows that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological activation by carbonation and fatty acids, but not sucrose. We further demonstrate that carbonation is behaviourally attractive to flies (in an IR56d-dependent manner), but in a distinct way to other appetitive stimuli. Our work provides a valuable toolkit for investigating the taste functions of IRs, defines a molecular basis of carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory receptors in distinct neuron types to coordinate behaviour.Classic models of gustatory perception in mammals highlight the existence of a small number of taste classes signalling nutritive content (e.g., sugars and amino acids) or toxicity (e.g., bitter) that determine -through activation of hard-wired neural circuits -behavioural acceptance or rejection of food 1, 2 . Different classes of tastants are recognised by discrete sensory channels that express distinct, and relatively small, receptor families. For example, detection of all sugars depends upon a single heterodimeric GPCR complex (T1R2/T1R3), while "bitter" cells -which detect an enormous diversity of noxious compounds -co-express a few dozen GPCRs of the T2R family 1, 2 . Such models have been pervasive in interpreting how gustatory perception occurs in other animals, including insects, where analogous segregated sensory pathways for sweet and bitter compounds have been defined [3][4][5][6] . However, in contrast to mammals, where taste -mediated by lingual taste buds -informs only feeding decisions, insect gustation occurs in multiple sensory appendages, including the proboscis, legs, wings and sexual organs, and controls diverse behaviours, such as foraging, feeding, sexual/social recognition and oviposition [3][4][5][6] . In addition to stereotyped appetitive and aversive feeding responses to sweet and bitter compounds respectively, insects display behavioural reactions to many other types of chemicals, including salt 7 , water 8 , carbonation (i.e., aqueous CO 2 ) 9 , organic and inorganic acids 10,11 , and pheromonal cuticular hydrocarbons 12 . The wide-ranging roles of the insect gustatory system are concordantly reflected in the underlying molecular receptors that mediate...

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