Veit Grabe

A Conserved Dedicated Olfactory Circuit for Detecting Harmful Microbes in Drosophila

Flies, like all animals, need to find suitable and safe food. Because the principal food source for Drosophila melanogaster is yeast growing on fermenting fruit, flies need to distinguish fruit with safe yeast from yeast covered with toxic microbes. We identify a functionally segregated olfactory circuit in flies that is activated exclusively by geosmin. This microbial odorant constitutes an ecologically relevant stimulus that alerts flies to the presence of harmful microbes. Geosmin activates only a single class of sensory neurons expressing the olfactory receptor Or56a. These neurons target the DA2 glomerulus and connect to projection neurons that respond exclusively to geosmin. Activation of DA2 is sufficient and necessary for aversion, overrides input from other olfactory pathways, and inhibits positive chemotaxis, oviposition, and feeding. The geosmin detection system is a conserved feature in the genus Drosophila that provides flies with a sensitive, specific means of identifying unsuitable feeding and breeding sites.

A digital in vivo 3D atlas of the antennal lobe of Drosophila melanogaster

As a model for primary olfactory perception, the antennal lobe (AL) of Drosophila melanogaster is among the most thoroughly investigated and well‐understood neuronal structures. Most studies investigating the functional properties and neuronal wiring of the AL are conducted in vivo, although so far the AL morphology has been mainly analyzed in vitro. Identifying the morphological subunits of the AL—the olfactory glomeruli—is usually done using in vitro AL atlases. However, the dissection and fixation procedure causes not only strong volumetric but also geometrical modifications; the result is unpredictable dislocation and a distortion of the AL glomeruli between the in vitro and in vivo brains. Hence, to characterize these artifacts, which are caused by in vitro processing, and to reliably identify glomeruli for in vivo applications, we generated a transgenic fly that expresses the red fluorescent protein DsRed directly fused to the presynaptic protein n‐synaptobrevin, under the control of the pan‐neuronal promotor elav to label the neuropil in the live animal. Using this fly line, we generated a digital 3D atlas of the live Drosophila AL; this atlas, the first of its kind, provides an excellent geometric match for in vivo studies. We verified the identity of 63% of AL glomeruli by mapping the projections of 34 GAL4‐lines of individual chemosensory receptor genes. Moreover, we characterized the innervation patterns of the two most frequently used GAL4‐lines in olfactory research: Orco‐ and GH146‐GAL4. The new in vivo AL atlas will be accessible online to the neuroscience community. J. Comp. Neurol. 523:530–544, 2015.

Love makes smell blind: mating suppresses pheromone attraction in Drosophila females via Or65a olfactory neurons

In Drosophila, the male sex pheromone cis-vaccenyl acetate (cVA) elicits aggregation and courtship, through the odorant receptor Or67d. Long-lasting exposure to cVA suppresses male courtship, via a second channel, Or65a. In females, the role of Or65a has not been studied. We show that, shortly after mating, Drosophila females are no longer attracted to cVA and that activation of olfactory sensory neurons (OSNs) expressing Or65a generates this behavioral switch: when silencing Or65a, mated females remain responsive to cVA. Neurons expressing Or67d converge into the DA1 glomerulus in the antennal lobe, where they synapse onto projection neurons (PNs), that connect to higher neural circuits generating the attraction response to cVA. Functional imaging of these PNs shows that the DA1 glomerulus is inhibited by simultaneous activation of Or65a OSNs, which leads to a suppression of the attraction response to cVA. The behavioral role of postmating cVA exposure is substantiated by the observation that matings with starved males, which produce less cVA, do not alter the female response. Moreover, exposure to synthetic cVA abolishes attraction and decreases sexual receptivity in unmated females. Taken together, Or65a mediates an aversive effect of cVA and may accordingly regulate remating, through concurrent behavioral modulation in males and females.

Elucidating the Neuronal Architecture of Olfactory Glomeruli in the Drosophila Antennal Lobe

Olfactory glomeruli are morphologically conserved spherical compartments of the olfactory system, distinguishable solely by their chemosensory repertoire, anatomical position, and volume. Little is known, however, about their numerical neuronal composition. We therefore characterized their neuronal architecture and correlated these anatomical features with their functional properties in Drosophila melanogaster. We quantitatively mapped all olfactory sensory neurons (OSNs) innervating each glomerulus, including sexually dimorphic distributions. Our data reveal the impact of OSN number on glomerular dimensions and demonstrate yet unknown sex-specific differences in several glomeruli. Moreover, we quantified uniglomerular projection neurons for each glomerulus, which unraveled a glomerulus-specific numerical innervation. Correlation between morphological features and functional specificity showed that glomeruli innervated by narrowly tuned OSNs seem to possess a larger number of projection neurons and are involved in less lateral processing than glomeruli targeted by broadly tuned OSNs. Our study demonstrates that the neuronal architecture of each glomerulus encoding crucial odors is unique.

Feeding regulates sex pheromone attraction and courtship in Drosophila females.

In Drosophila melanogaster, gender-specific behavioural responses to the male-produced sex pheromone cis-vaccenyl acetate (cVA) rely on sexually dimorphic, third-order neural circuits. We show that nutritional state in female flies modulates cVA perception in first-order olfactory neurons. Starvation increases, and feeding reduces attraction to food odour, in both sexes. Adding cVA to food odour, however, maintains attraction in fed females, while it has no effect in males. Upregulation of sensitivity and behavioural responsiveness to cVA in fed females is paralleled by a strong increase in receptivity to male courtship. Functional imaging of the antennal lobe (AL), the olfactory centre in the insect brain, shows that olfactory input to DA1 and VM2 glomeruli is also modulated by starvation. Knocking down insulin receptors in neurons converging onto the DA1 glomerulus suggests that insulin-signalling partly controls pheromone perception in the AL, and adjusts cVA attraction according to nutritional state and sexual receptivity in Drosophila females.

Fundamental principles of the olfactory code

Sensory coding represents a basic principle of all phyla in nature: species attempt to perceive their natural surroundings and to make sense of them. Ultimately, sensory coding is the only way to allow a species to make the kinds of crucial decisions that lead to a behavioral response. In this manner, animals are able to detect numerous parameters, ranging from temperature and humidity to light and sound to volatile or non-volatile chemicals. Most of these environmental cues represent a clearly defined stimulus array that can be described along a single physical parameter, such as wavelength or frequency; odorants, in contrast, cannot. The odor space encompasses an enormous and nearly infinite number of diverse stimuli that cannot be classified according to their positions along a single dimension. Hence, the olfactory system has to encode and translate the vast odor array into an accurate neural map in the brain. In this review, we will outline the relevant steps of the olfactory code and describe its progress along the olfactory pathway, i.e., from the peripheral olfactory organs to the first olfactory center in the brain and then to the higher processing areas where the odor perception takes place, enabling an organism to make odor-guided decisions. We will focus mainly on studies from the vinegar fly Drosophila melanogaster, but we will also indicate similarities to and differences from the olfactory system of other invertebrate species as well as of the vertebrate world.

Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendages

Optical imaging of gene expression by RNA-fluorescent in situ hybridisation (FISH) in whole-mount sensory appendages of insects is often impeded by their highly pigmented cuticle. Since most chemical bleaching agents are incompatible with imaging fluorescent-labelled nucleotides, we developed a RNA interference-based method for clearing cuticular pigmentation that allows imaging of fluorescent mRNA in whole-mount appendages of insects. Silencing key genes of the tyrosine-derived pigmentation pathway by injecting dsRNA of laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi, Phaedon cochleariae) resulted in clearance of the highly pigmented cuticle and in significant decreased light absorbance. Intact chemosensory appendages (palps, antennae and legs) from RNAi-cleared individuals were used to image expression and spatial distribution of antisense mRNA of two chemosensory genes (gustatory receptor, odorant-binding protein) via RNA FISH and confocal laser scanning microscopy. Imaging of these genes did neither work for RNAi-controls (dsGfp) due to retained pigmentation, nor for FISH-controls using sense mRNA. Furthermore, we show that several chemical bleaching agents are not feasible with FISH, either due to significant degradation of polynucleotides, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents allowing fluorescence imaging in whole-mount appendages and organs.

Silencing cuticular pigmentation genes enables RNA FISH in intact insect appendages

ABSTRACT Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. As most chemical bleaching agents are incompatible with FISH, we developed an RNA interference (RNAi)-based method for clearing cuticular pigmentation which enables the use of whole-mount body appendages for RNA FISH (termed RNA-i-FISH). Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi and Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, legs) from RNAi-cleared individuals were used to image the expression and spatial distribution of antisense mRNA of two chemosensory genes encoding gustatory receptor and odorant-binding protein. Imaging did not work for RNAi controls because the pigmentation was retained, or for FISH controls (sense mRNA). Several bleaching agents were incompatible with FISH, because of degradation of RNA, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents, enabling FISH in intact insect appendages. Summary: Clearing cuticular pigmentation in beetles by gene silencing enables the use of intact appendages for fluorescence in situ hybridisation.

Calcium imaging revealed no modulatory effect on odor-evoked responses of the Drosophila antennal lobe by two populations of inhibitory local interneurons

Although we have considerable knowledge about how odors are represented in the antennal lobe (AL), the insects’ analogue to the olfactory bulb, we still do not fully understand how the different neurons in the AL network contribute to the olfactory code. In Drosophila melanogaster we can selectively manipulate specific neuronal populations to elucidate their function in odor processing. Here we silenced the synaptic transmission of two distinct subpopulations of multiglomerular GABAergic local interneurons (LN1 and LN2) using shibire (shits) and analyzed their impact on odor-induced glomerular activity at the AL input and output level. We verified that the employed shits construct effectively blocked synaptic transmission to the AL when expressed in olfactory sensory neurons. Notably, selective silencing of both LN populations did not significantly affect the odor-evoked activity patterns in the AL. Neither the glomerular input nor the glomerular output activity was modulated in comparison to the parental controls. We therefore conclude that these LN subpopulations, which cover one third of the total LN number, are not predominantly involved in odor identity coding per se. As suggested by their broad innervation patterns and contribution to long-term adaptation, they might contribute to AL–computation on a global and longer time scale.