Alex C. Keene

Drosophila olfactory memory: single genes to complex neural circuits.

A central goal of neuroscience is to understand how neural circuits encode memory and guide behaviour. Studying simple, genetically tractable organisms, such as Drosophila melanogaster, can illuminate principles of neural circuit organization and function. Early genetic dissection of D. melanogaster olfactory memory focused on individual genes and molecules. These molecular tags subsequently revealed key neural circuits for memory. Recent advances in genetic technology have allowed us to manipulate and observe activity in these circuits, and even individual neurons, in live animals. The studies have transformed D. melanogaster from a useful organism for gene discovery to an ideal model to understand neural circuit function in memory.

A Genome-wide Drosophila Screen for Heat Nociception Identifies α2δ3 as an Evolutionarily Conserved Pain Gene

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

A Global In Vivo Drosophila RNAi Screen Identifies NOT3 as a Conserved Regulator of Heart Function

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

Diverse Odor-Conditioned Memories Require Uniquely Timed Dorsal Paired Medial Neuron Output

Amnesiac mutant flies have an olfactory memory defect. The amn gene encodes a homolog of vertebrate pituitary adenylate cyclase-activating peptide (PACAP), and it is strongly expressed in dorsal paired medial (DPM) neurons. DPM neurons ramify throughout the mushroom bodies in the adult fly brain, and they are required for stable memory. Here, we show that DPM neuron output is only required during the consolidation phase for middle-term odor memory and is dispensable during acquisition and recall. However, we found that DPM neuron output is required during acquisition of a benzaldehyde odor memory. We show that flies sense benzaldehyde by the classical olfactory and a noncanonical route. These results suggest that DPM neurons are required to consolidate memory and are differently involved in memory of a volatile that requires multisensory integration.

The cavefish genome reveals candidate genes for eye loss

Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction.

Evolutionary Convergence on Sleep Loss in Cavefish Populations

Patterns of sleep vary widely among species, but the functional and evolutionary principles responsible for this diversity remain unknown. The characin fish, Astyanax mexicanus, has eyed surface and numerous blind cave populations. The cave populations are largely independent in their origins, and the species is ideal for studying the genetic bases of convergent evolution. Here we show that this system is also uniquely valuable for the investigation of variability in patterns of sleep. We find that a clearly defined change in ecological conditions, from surface to cave, is correlated with a dramatic reduction in sleep in three independently derived cave populations of A. mexicanus. Analyses of surface × cave hybrids show that the alleles for reduced sleep in the Pachón and Tinaja cave populations are dominant in effect to the surface alleles. Genetic analysis of hybrids between surface and Pachón cavefish suggests that only a small number of loci with dominant effects are involved. Our results demonstrate that sleep is an evolutionarily labile phenotype, highly responsive to changes in ecological conditions. To our knowledge, this is the first example of a single species with a convergence on sleep loss exhibited by several independently evolved populations correlated with population-specific ecologies.

Clock and cycle Limit Starvation-Induced Sleep Loss in Drosophila

Neural systems controlling the vital functions of sleep and feeding in mammals are tightly interconnected: sleep deprivation promotes feeding, whereas starvation suppresses sleep. Here we show that starvation in Drosophila potently suppresses sleep, suggesting that these two homeostatically regulated behaviors are also integrated in flies. The sleep-suppressing effect of starvation is independent of the mushroom bodies, a previously identified sleep locus in the fly brain, and therefore is regulated by distinct neural circuitry. The circadian clock genes Clock (Clk) and cycle (cyc) are critical for proper sleep suppression during starvation. However, the sleep suppression is independent of light cues and of circadian rhythms as shown by the fact that starved period mutants sleep like wild-type flies. By selectively targeting subpopulations of Clk-expressing neurons, we localize the observed sleep phenotype to the dorsally located circadian neurons. These findings show that Clk and cyc act during starvation to modulate the conflict of whether flies sleep or search for food.

Taste-independent detection of the caloric content of sugar in Drosophila

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.

TrpA1 regulates thermal nociception in Drosophila.

Pain is a significant medical concern and represents a major unmet clinical need. The ability to perceive and react to tissue-damaging stimuli is essential in order to maintain bodily integrity in the face of environmental danger. To prevent damage the systems that detect noxious stimuli are therefore under strict evolutionary pressure. We developed a high-throughput behavioral method to identify genes contributing to thermal nociception in the fruit fly and have reported a large-scale screen that identified the Ca2+ channel straightjacket (stj) as a conserved regulator of thermal nociception. Here we present the minimal anatomical and neuronal requirements for Drosophila to avoid noxious heat in our novel behavioral paradigm. Bioinformatics analysis of our whole genome data set revealed 23 genes implicated in Ca2+ signaling that are required for noxious heat avoidance. One of these genes, the conserved thermoreceptor TrpA1, was confirmed as a bona fide “pain” gene in both adult and larval fly nociception paradigms. The nociceptive function of TrpA1 required expression within the Drosophila nervous system, specifically within nociceptive multi-dendritic (MD) sensory neurons. Therefore, our analysis identifies the channel TRPA1 as a conserved regulator of nociception.

Drosophila fatty acid taste signals through the PLC pathway in sugar-sensing neurons.

Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs) signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC) signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.