Allan M. Wong

Two-Photon Calcium Imaging Reveals an Odor-Evoked Map of Activity in the Fly Brain

An understanding of the logic of odor perception requires a functional analysis of odor-evoked patterns of activity in neural assemblies in the brain. We have developed a sensitive imaging system in the Drosophila brain that couples two-photon microscopy with the specific expression of the calcium-sensitive fluorescent protein, G-CaMP. At natural odor concentration, each odor elicits a distinct and sparse spatial pattern of activity in the antennal lobe that is conserved in different flies. Patterns of glomerular activity are similar upon imaging of sensory and projection neurons, suggesting the faithful transmission of sensory input to higher brain centers. Finally, we demonstrate that the response pattern of a given glomerulus is a function of the specificity of a single odorant receptor. The development of this imaging system affords an opportunity to monitor activity in defined neurons throughout the fly brain with high sensitivity and excellent spatial resolution.

Spatial Representation of the Glomerular Map in the Drosophila Protocerebrum

In the fruit fly, Drosophila, olfactory sensory neurons expressing a given receptor project to spatially invariant loci in the antennal lobe to create a topographic map of receptor activation. We have asked how the map in the antennal lobe is represented in higher sensory centers in the brain. Random labeling of individual projection neurons using the FLP-out technique reveals that projection neurons that innervate the same glomerulus exhibit strikingly similar axonal topography, whereas neurons from different glomeruli display very different patterns of projection in the protocerebrum. These results demonstrate that a topographic map of olfactory information is retained in higher brain centers, but the character of the map differs from that of the antennal lobe, affording an opportunity for integration of olfactory sensory input.

A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila

All animals exhibit innate behaviours in response to specific sensory stimuli that are likely to result from the activation of developmentally programmed neural circuits. Here we observe that Drosophila exhibit robust avoidance to odours released by stressed flies. Gas chromatography and mass spectrometry identifies one component of this ‘Drosophila stress odorant (dSO)’ as CO2. CO2 elicits avoidance behaviour, at levels as low as 0.1%. We used two-photon imaging with the Ca2+-sensitive fluorescent protein G-CaMP to map the primary sensory neurons governing avoidance to CO2. CO2 activates only a single glomerulus in the antennal lobe, the V glomerulus; moreover, this glomerulus is not activated by any of 26 other odorants tested. Inhibition of synaptic transmission in sensory neurons that innervate the V glomerulus, using a temperature-sensitive Shibire gene (Shits), blocks the avoidance response to CO2. Inhibition of synaptic release in the vast majority of other olfactory receptor neurons has no effect on this behaviour. These data demonstrate that the activation of a single population of sensory neurons innervating one glomerulus is responsible for an innate avoidance behaviour in Drosophila.

The Drosophila pheromone cVA activates a sexually dimorphic neural circuit

Courtship is an innate sexually dimorphic behaviour that can be observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate this behaviour are developmentally programmed. In Drosophila, courtship involves a complex yet stereotyped array of dimorphic behaviours that are regulated by FruM, a male-specific isoform of the fruitless gene. FruM is expressed in about 2,000 neurons in the fly brain, including three subpopulations of olfactory sensory neurons and projection neurons (PNs). One set of Fru+ olfactory neurons expresses the odorant receptor Or67d and responds to the male-specific pheromone cis-vaccenyl acetate (cVA). These neurons converge on the DA1 glomerulus in the antennal lobe. In males, activation of Or67d+ neurons by cVA inhibits courtship of other males, whereas in females their activation promotes receptivity to other males. These observations pose the question of how a single pheromone acting through the same set of sensory neurons can elicit different behaviours in male and female flies. Anatomical or functional dimorphisms in this neural circuit might be responsible for the dimorphic behaviour. We therefore developed a neural tracing procedure that employs two-photon laser scanning microscopy to activate the photoactivatable green fluorescent protein. Here we show, using this technique, that the projections from the DA1 glomerulus to the protocerebrum are sexually dimorphic. We observe a male-specific axonal arbor in the lateral horn whose elaboration requires the expression of the transcription factor FruM in DA1 projection neurons and other Fru+ cells. The observation that cVA activates a sexually dimorphic circuit in the protocerebrum suggests a mechanism by which a single pheromone can elicit different behaviours in males and in females.

Distinct sensory representations of wind and near-field sound in the Drosophila brain

Behavioural responses to wind are thought to have a critical role in controlling the dispersal and population genetics of wild Drosophila species, as well as their navigation in flight, but their underlying neurobiological basis is unknown. We show that Drosophila melanogaster, like wild-caught Drosophila strains, exhibits robust wind-induced suppression of locomotion in response to air currents delivered at speeds normally encountered in nature. Here we identify wind-sensitive neurons in Johnston’s organ, an antennal mechanosensory structure previously implicated in near-field sound detection (reviewed in refs 5 and 6). Using enhancer trap lines targeted to different subsets of Johnston’s organ neurons, and a genetically encoded calcium indicator, we show that wind and near-field sound (courtship song) activate distinct populations of Johnston’s organ neurons, which project to different regions of the antennal and mechanosensory motor centre in the central brain. Selective genetic ablation of wind-sensitive Johnston’s organ neurons in the antenna abolishes wind-induced suppression of locomotion behaviour, without impairing hearing. Moreover, different neuronal subsets within the wind-sensitive population respond to different directions of arista deflection caused by air flow and project to different regions of the antennal and mechanosensory motor centre, providing a rudimentary map of wind direction in the brain. Importantly, sound- and wind-sensitive Johnston’s organ neurons exhibit different intrinsic response properties: the former are phasically activated by small, bi-directional, displacements of the aristae, whereas the latter are tonically activated by unidirectional, static deflections of larger magnitude. These different intrinsic properties are well suited to the detection of oscillatory pulses of near-field sound and laminar air flow, respectively. These data identify wind-sensitive neurons in Johnston’s organ, a structure that has been primarily associated with hearing, and reveal how the brain can distinguish different types of air particle movements using a common sensory organ.

Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo

Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons in mice that express the G-protein-coupled receptor MRGPRB4 (refs 5, 6). These neurons exclusively innervate hairy skin with large terminal arborizations that resemble the receptive fields of C-tactile (CT) afferents in humans. Unlike other molecularly defined mechanosensory C-fibre subtypes, MRGPRB4+ neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in the spinal projections of these neurons during stimulation of the periphery in intact mice. Here we show that MRGPRB4+ neurons are activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C fibres expressing MRGPRD was activated by pinching but not by stroking, consistent with previous physiological and behavioural data. Pharmacogenetic activation of Mrgprb4-expressing neurons in freely behaving mice promoted conditioned place preference, indicating that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MRGPRB4 neurons during natural behaviours, and provide a general approach to the functional characterization of genetically identified subsets of somatosensory neurons in vivo.

CNK, a RAF-Binding Multidomain Protein Required for RAS Signaling

Kinase suppressor of ras (ksr) is required for efficient signal transmission within the RAS/MAPK cascade. A screen for mutations that modify a ksr-dependent phenotype identified a novel gene, connector enhancer of ksr (cnk), that functions upstream or in parallel to RAF in the RAS pathway. cnk encodes a protein containing several protein-protein interaction domains, suggesting that it brings different signaling molecules together. CNK is required in multiple receptor tyrosine kinase pathways where it appears to be a tyrosine phosphorylation target. Finally, CNK physically interacts with RAF and appears to localize to cell-cell contact regions. Together, these findings suggest that CNK is a novel component of a RAS-dependent signaling pathway that regulates RAF function and/or targets RAF to a specific subcellular compartment upon RAS activation.

Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship

Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.

A connectome of a learning and memory center in the adult Drosophila brain

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB’s α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall. DOI: http://dx.doi.org/10.7554/eLife.26975.001

CisOrtho: a program pipeline for genome-wide identification of transcription factor target genes using phylogenetic footprinting.

BackgroundAll known genomes code for a large number of transcription factors. It is important to develop methods that will reveal how these transcription factors act on a genome wide level, that is, through what target genes they exert their function.ResultsWe describe here a program pipeline aimed at identifying transcription factor target genes in whole genomes. Starting from a consensus binding site, represented as a weight matrix, potential sites in a pre-filtered genome are identified and then further filtered by assessing conservation of the putative site in the genome of a related species, a process called phylogenetic footprinting. CisOrtho has been successfully used to identify targets for two homeodomain transcription factors in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.ConclusionsCisOrtho will identify targets of other nematode transcription factors whose DNA binding specificity is known and can be easily adapted to search other genomes for transcription factor targets.