Faculty Bibliography

BACKGROUND: Food intake of the adult fruit fly Drosophila melanogaster, an intermittent feeder, is attributed to several behavioral elements including foraging, feeding initiation and termination, and food ingestion. Despite the development of various feeding assays in fruit flies, how each of these behavioral elements, particularly food ingestion, is regulated remains largely uncharacterized. RESULTS: To this end, we have developed a manual feeding (MAFE) assay that specifically measures food ingestion of an individual fly completely independent of the other behavioral elements. This assay reliably recapitulates the effects of known feeding modulators, and offers temporal resolution in the scale of seconds. Using this assay, we find that fruit flies can rapidly assess the nutritional value of sugars within 20-30 s, and increase the ingestion of nutritive sugars after prolonged periods of starvation. Two candidate nutrient sensors, SLC5A11 and Gr43a, are required for discriminating the nutritive sugars, D-glucose and D-fructose, from their non-nutritive enantiomers, respectively. This suggests that differential sensing mechanisms play a key role in determining food nutritional value. CONCLUSIONS: Taken together, our MAFE assay offers a platform to specifically examine the regulation of food ingestion with excellent temporal resolution, and identifies a fast-acting neural mechanism that assesses food nutritional value and modulates food intake.

Sugars in the natural environment can be detected through taste-dependent and taste-independent modalities. Taste-dependent modalities consist mainly of peripheral chemosensory neurons such as sweet taste receptors, which primarily detect the orosensory value of sugar (i.e. sweetness). Evidence of a taste-independent modality - a post-ingestive sugar sensor - that detects the nutritional value of sugar has been shown in insects and mammals. However, the identity of the post-ingestive sugar sensor and the mechanism by which animals respond to the nutritional content of sugar independently of orosensory value is not currently understood. Here, we show that six neurosecretory cells in the Drosophila brain that produce Diuretic hormone 44 (Dh44), a homologue of the mammalian corticotropin- releasing hormone (CRH), were activated by nutritive sugars that are present in the hemolymph and not by nonnutritive sugars. Dh44 neuronal cell bodies are located primarily in the pars intercerebralis and extend their dendrites to the dorsal region of the subesophageal ganglion zone (SEZ), and project their axons along the esophagus to innervate the gut. Flies in which the activity of these neurons or the expression of the Dh44 gene was disrupted failed to select nutritive sugars over nonnutritive ones after periods of starvation. Manipulation of the function of Dh44 receptors had a similar effect. Notably, artificial activation of Dh44 receptor-1 neurons dramatically increased the rate of proboscis extension reflex (PER) responses, promoting food intake, and excretion. Conversely, reduced Dh44 activity led to decreased excretion. Together, we propose that the Dh44 system directs the detection, ingestion, and digestion of nutritive sugar through a positive feedback loop to continue consumption of nutritive sugar

Animals can detect and consume nutritive sugars without the influence of taste. However, the identity of the taste-independent nutrient sensor and the mechanism by which animals respond to the nutritional value of sugar are unclear. Here, we report that six neurosecretory cells in the Drosophila brain that produce Diuretic hormone 44 (Dh44), a homolog of the mammalian corticotropin-releasing hormone (CRH), were specifically activated by nutritive sugars. Flies in which the activity of these neurons or the expression of Dh44 was disrupted failed to select nutritive sugars. Manipulation of the function of Dh44 receptors had a similar effect. Notably, artificial activation of Dh44 receptor-1 neurons resulted in proboscis extensions and frequent episodes of excretion. Conversely, reduced Dh44 activity led to decreased excretion. Together, these actions facilitate ingestion and digestion of nutritive foods. We propose that the Dh44 system directs the detection and consumption of nutritive sugars through a positive feedback loop.

Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs-including GCaMP3, GCaMP5 and GCaMP6-can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.

Drosophila olfactory sensory neurons express either odorant receptors or ionotropic glutamate receptors (IRs). The sensory neurons that express IR64a, a member of the IR family, send axonal projections to either the DC4 or DP1m glomeruli in the antennal lobe. DC4 neurons respond specifically to acids/protons, whereas DP1m neurons respond to a broad spectrum of odorants. The molecular composition of IR64a-containing receptor complexes in either DC4 or DP1m neurons is not known, however. Here, we immunoprecipitated the IR64a protein from lysates of fly antennal tissue and identified IR8a as a receptor subunit physically associated with IR64a by mass spectrometry. IR8a mutants and flies in which IR8a was knocked down by RNAi in IR64a+ neurons exhibited defects in acid-evoked physiological and behavioral responses. Furthermore, we found that the loss of IR8a caused a significant reduction in IR64a protein levels. When expressed in Xenopus oocytes, IR64a and IR8a formed a functional ion channel that allowed ligand-evoked cation currents. These findings provide direct evidence that IR8a is a subunit that forms a functional olfactory receptor with IR64a in vivo to mediate odor detection.

Animals can determine the nutritional value of sugar without the influence of taste. We examined a Drosophila mutant that is insensitive to the nutritional value of sugars, responding only to the concentration (that is, sweetness). The affected gene encodes a sodium/solute co-transporter-like protein, designated SLC5A11 (or cupcake), which is structurally similar to mammalian sodium/glucose co-transporters that transport sugar across the intestinal and renal lumen. However, SLC5A11 was prominently expressed in 10-13 pairs of R4 neurons of the ellipsoid body in the brain and functioned in these neurons for selecting appropriate foods.

Animals across various phyla exhibit odor-evoked innate attraction behavior that is developmentally programmed. The mechanism underlying such behavior remains unclear because the odorants that elicit robust attraction responses and the neuronal circuits that mediate this behavior have not been identified. Here, we describe a functionally segregated population of olfactory sensory neurons (OSNs) and projection neurons (PNs) in Drosophila melanogaster that are highly specific to ammonia and amines, which act as potent attractants. The OSNs express IR92a, a member of the chemosensory ionotropic receptor (IR) family and project to a pair of glomeruli in the antennal lobe, termed VM1. In vivo calcium-imaging experiments showed that the OSNs and PNs innervating VM1 were activated by ammonia and amines but not by nonamine odorants. Flies in which the IR92a(+) neurons or IR92a gene was inactivated had impaired amine-evoked physiological and behavioral responses. Tracing neuronal pathways to higher brain centers showed that VM1-PN axonal projections within the lateral horn are topographically segregated from those of V-PN and DC4-PN, which mediate innate avoidance behavior to carbon dioxide and acidity, respectively, suggesting that these sensory stimuli of opposing valence are represented in spatially distinct neuroanatomic loci within the lateral horn. These experiments identified the neurons and their cognate receptor for amine detection, and mapped amine attractive olfactory inputs to higher brain centers. This labeled-line mode of amine coding appears to be hardwired to attraction behavior.

Alarm pheromones are important semiochemicals used by many animal species to alert conspecifics or other related species of impending danger. In this review, we describe recent developments in our understanding of the neural mechanisms underlying the ability of fruit flies, zebrafish and mice to mediate the detection of alarm pheromones. Specifically, alarm pheromones are detected in these species through specialized olfactory subsystems that are unique to the chemosensitive receptors, second messenger-signaling and physiology. Thus, the alarm pheromones appears to be detected by signaling mechanisms that are distinct from those seen in the canonical olfactory system.

Feeding behavior is influenced primarily by two factors: nutritional needs and food palatability. However, the role of food deprivation and metabolic needs in the selection of appropriate food is poorly understood. Here, we show that the fruit fly, Drosophila melanogaster, selects calorie-rich foods following prolonged food deprivation in the absence of taste-receptor signaling. Flies mutant for the sugar receptors Gr5a and Gr64a cannot detect the taste of sugar, but still consumed sugar over plain agar after 15 h of starvation. Similarly, pox-neuro mutants that are insensitive to the taste of sugar preferentially consumed sugar over plain agar upon starvation. Moreover, when given a choice between metabolizable sugar (sucrose or d-glucose) and nonmetabolizable (zero-calorie) sugar (sucralose or l-glucose), starved Gr5a; Gr64a double mutants preferred metabolizable sugars. These findings suggest the existence of a taste-independent metabolic sensor that functions in food selection. The preference for calorie-rich food correlates with a decrease in the two main hemolymph sugars, trehalose and glucose, and in glycogen stores, indicating that this sensor is triggered when the internal energy sources are depleted. Thus, the need to replenish depleted energy stores during periods of starvation may be met through the activity of a taste-independent metabolic sensing pathway