Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophila's advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.     

Voltage-activated and odor-modulated conductances in olfactory neurons of Drosophila melanogaster

Voltage-activated currents and odor-modulated conductances were studied in cells in semi-intact Drosophila third antennal segments (the main olfactory organ) using patch-clamp techniques. All neurons expressed outward currents, and most expressed labile fast transient inward currents with kinetics similar to Na⁺ currents in other systems. Action potentials were detected as bipolar capacitative current transients in cell-attached or loose patches from the soma of both odor-sensitive (97%) and insensitive neurons. A mixture of odorants from five chemical classes caused an increase (∼70%), decrease (∼10%), or no effect on firing frequency in pharate adult neurons. The development of chemosensitivity was examined and odor-induced changes in action potential firing frequency were recorded in pupal antennal neurons as early as P8, a stage after completion of sensillar development. The character of odor-induced responses was more profound and complex later in development; small, tonic increases in firing frequency were observed at pupal stages P8 through P11(ii), while in older pupae and young adults ∼25% of the increased responses were phasic-tonic. The apical dendrite was the site of odor modulation in ∼90% and 100% of responsive adult and early pupal neurons, respectively. Whole-cell recordings revealed that apparent nonselective cation and chloride conductances were modulated by a mixture of odorants in separate antennal neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 123–137, 1997.     

Retinoid receptor signaling in postmitotic motor neurons regulates rostrocaudal positional identity and axonal projection pattern

The identity of motor neurons diverges markedly at different rostrocaudal levels of the spinal cord, but the signals that specify their fate remain poorly defined. We show that retinoid receptor activation in newly generated spinal motor neurons has a crucial role in specifying motor neuron columnar subtypes. Blockade of retinoid receptor signaling in brachial motor neurons inhibits lateral motor column differentiation and converts many of these neurons to thoracic columnar subtypes. Conversely, expression of a constitutively active retinoid receptor derivative impairs the differentiation of thoracic motor neuron columnar subtypes. These findings provide evidence for a regionally restricted role for retinoid signaling in the postmitotic specification of motor neuron columnar identity.     

Endosomal entry regulates Notch receptor activation in Drosophila melanogaster

Signaling through the transmembrane receptor Notch is widely used throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we systematically assessed Notch localization, processing, and signaling in a comprehensive set of Drosophila melanogaster mutants that block access of cargo to different endocytic compartments. We find that gamma-secretase cleavage and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. In mutants that block endosomal entry, we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data show that endosomal access of the Notch receptor is critical to achieve physiological levels of signaling and further suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.     

Diverse functions of N-cadherin in dendritic and axonal terminal arborization of olfactory projection neurons

The cadherin superfamily of cell adhesion molecules have been proposed to play important roles in determining synaptic specificity in developing nervous systems. We examine the function of N-cadherin in Drosophila second order olfactory projection neurons (PNs), each of which must selectively target their dendrites to one of approximately 50 glomeruli. Our results do not support an instructive role for N-cadherin in selecting dendritic targets; rather, N-cadherin is essential for PNs to restrict their dendrites to single glomeruli. Mosaic analyses suggest that N-cadherin mediates dendro-dendritic interactions between PNs and thus contributes to refinement of PN dendrites to single glomeruli. N-cadherin is also essential for the development of PN axon terminal arbors in two distinct central targets: regulating branch stability in the lateral horn and restricting high-order branching in the mushroom body. Although the N-cadherin locus potentially encodes eight alternatively spliced isoforms, transgenic expression of one isoform is sufficient to rescue all phenotypes.     

The optic lobe projection pattern of polarization-sensitive photoreceptor cells in Drosophila melanogaster

Summary Histological staining of wild-type and sevenless transgenic Drosophila melanogaster bearing Rh3-lacZ fusion genes permits the selective visualization of polarization-sensitive R7 and R8 photoreceptor cells located along the dorsal anterior eye margin. Diffusion of -galactosidase throughout these cells reveals that they project long axons to the two most peripheral synaptic target rows of the dorsal posterior medulla, defining a specialized marginal zone of this optic lobe. Comparison of the staining patterns of marginal and nonmarginal Rh3-lacZ-expressing photoreceptor cells in the same histological preparations suggests that the marginal cells possess morphologically specialized axons and synaptic terminals. These findings are discussed with reference to the neuroanatomy of the corresponding dorsal marginal eye and optic lobe regions of the larger dipterans Musca and Calliphora, and in relation to the ability of Drosophila to orient to polarized light.     

The role of mitochondria in shaping odor responses in Drosophila melanogaster olfactory sensory neurons

Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing also Ca²⁺. Here, we investigate if and how odor-induced Ca²⁺ signals in Drosophila melanogaster OSNs are controlled by intracellular Ca²⁺ stores, especially by mitochondria. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs we performed Ca²⁺ imaging to determine the role of Ca²⁺ influx and efflux pathways in OSN mitochondria. The results indicate that mitochondria participate in shaping the OR responses. The major players of this modulation are the mitochondrial Ca²⁺ uniporter and the mitochondrial permeability transition pore. Intriguingly, OR-induced Ca²⁺ signals were only mildly affected by modulating the Ca²⁺ management of the endoplasmic reticulum.     

A tyramine receptor gene mutation causes a defective olfactory behavior in Drosophila melanogaster

We characterized molecular profiles of a new olfactory mutant line, honoka (hono), which was found among 500 viable P-element insertion lines screened first by 5-bromo-4-chloro-3-indrolyl-beta-D-galactopyranoside (X-gal) staining on the third segment of the antenna, and then by behavioral assays to several pure chemicals.The behavioral responses of hono mutants to repellents such as ethyl acetate (EA), benzaldehyde (BZ) and 4-methylcycrohexanol (MCH), were reduced compared with those of a control strain. The location of the P-element insertion was determined to be about 100bp) upstream of the first exon of the tyramine receptor gene. The level of 3.6kb tyramine receptor mRNA expression was reduced in hono compared with that of wild-type flies. The tyramine receptor cDNA hybridized to the chromosomal division 79C-D, the same locus as the P-element insertion point in hono, and not to 99A-B, previously reported by Arakawa et al. (1990. Neuron 2, 343-354).Electrophysiological responses to octopamine and tyramine were examined by measuring the excitatory junctional potential (EJP) amplitude from larval body-wall muscles of the hono mutant. The hono was impaired with responding to tyramine, while displaying normal response to octopamine. These results indicate that tyramine has a functional role in the Drosophila olfactory system as a neurotransmitter or a neuromodulator, and hono is the first tyramine receptor mutant. This study provides the first step toward understanding of the molecular genetics of tyramine-mediated neural functions in Drosophila.     

Environmental temperature modulates olfactory reception in Drosophila melanogaster

Sensory systems, including the olfactory system, are able to adapt to changing environmental conditions. In nature, changes in temperature modify the volatility and concentration of odorants in the air. If the olfactory system does not adapt to these changes, it could relay wrong information about the distance to or direction of odor sources. Recent behavioral studies in Drosophila melanogaster showed olfactory acclimation to temperature. In this report, we investigated if temperature affects olfaction at the level of the receptors themselves. With this aim, we performed electroantennograms (EAGs) and single sensillum recordings (SSRs) to measure the response to several odorants in flies that had been submitted to temperature treatments. In response to all tested odorants, the amplitude of the EAGs increased in flies that had been exposed to a higher temperature and decreased after cold treatment, revealing that at least part of the reported change in olfactory perception happens at reception level. SSRs of odorant stimulated basiconic sensilla ab2 and ab3 showed some changes in the number of spikes after heat or cold treatment. However, the number and shape of spontaneous action potentials were unaffected, suggesting that the observed changes related specifically to the olfactory function of the neurons.     

Go contributes to olfactory reception in Drosophila melanogaster

Background  

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology and function as ligand-gated cation channels. Consequently, the involvement of cyclic nucleotides and G proteins in insect odor reception is controversial. Since the heterotrimeric G_oα subunit is expressed in Drosophila olfactory receptor neurons, we reasoned that G_o acts together with insect odorant receptor cation channels to mediate odor-induced physiological responses.     

Experimental evolution of olfactory memory in Drosophila melanogaster

In order to address the nature of genetic variation in learning performance, we investigated the response to classical olfactory conditioning in "high-learning" Drosophila melanogaster lines previously subject to selection for the ability to learn an association between the flavor of an oviposition medium and bitter taste. In a T-maze choice test, the seven high-learning lines were better at avoiding an odor previously associated with aversive mechanical shock than were five unselected "low-learning" lines originating from the same natural population. Thus, the evolved improvement in learning ability of high-learning lines generalized to another aversion learning task involving a different aversive stimulus (shock instead of bitter taste) and a different behavioral context than that used to impose selection. In this olfactory shock task, the high-learning lines showed improvements in the learning rate as well as in two forms of consolidated memory: anesthesia-resistant memory and long-term memory. Thus, genetic variation underlying the experimental evolution of learning performance in the high-learning lines affected several phases of memory formation in the course of olfactory aversive learning. However, the two forms of consolidated memory were negatively correlated among replicate high-learning lines, which is consistent with a recent hypothesis that these two forms of consolidated memory are antagonistic.     

Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam

Different classes of olfactory receptor neurons (ORNs) in Drosophila innervate distinct targets, or glomeruli, in the antennal lobe of the brain. Here we demonstrate that specific ORN classes require the cell surface protein Dscam (Down Syndrome Cell Adhesion Molecule) to synapse in the correct glomeruli. Dscam mutant ORNs frequently terminated in ectopic sites both within and outside the antennal lobe. The morphology of Dscam mutant axon terminals in either ectopic or cognate targets was abnormal. Target specificity for other ORNs was not altered in Dscam mutants, suggesting that different ORNs use different strategies to regulate wiring. Multiple forms of Dscam RNA were detected in the developing antenna, and Dscam protein was localized to developing ORN axons. We propose a role for Dscam protein diversity in regulating ORN target specificity.     

Signal peptides and signal peptidase in Drosophila melanogaster

Translation of poly(A)-containing RNA from the female fat body of Drosophila melanogaster in a rabbit reticulocyte cell-free system results in the synthesis of previtellogenin polypeptides (PVs) having higher apparent molecular weights (46,000 and 45,000) than the forms seen after an in vivo pulse labeling. However, when this RNA is translated in the presence of EDTA-stripped microsomal membranes from the dog pancreas, vitellogenin precursors are produced that, upon SDS- polyacrylamide gel electrophoresis, comigrate with the in vivo forms (apparent molecular weights, 45,000 and 44,000). These processed forms are sequestered within the microsomal lumen, as evidenced by their insensitivity to trypsin digestion. Neither processing nor sequestration occur posttranslationally. In addition, a microsomal membrane fraction derived from Drosophila embryos is able to cotranslationally process the PVs as well as a murine pre-light chain IgG. These observations support a signal-mediated mode of secretion in Drosophila, and suggest that signal sequence recognition and signal peptidase activities are conserved even between mammalian and insect systems.     

Semaphorin-1a controls receptor neuron-specific axonal convergence in the primary olfactory center of Drosophila

In the olfactory system of Drosophila, 50 functional classes of sensory receptor neurons (ORNs) project in a highly organized fashion into the CNS, where they sort out from one another and converge into distinct synaptic glomeruli. We identified the transmembrane molecule Semaphorin-1a (Sema-1a) as an essential component to ensure glomerulus-specific axon segregation. Removal of sema-1a in ORNs does not affect the pathfinding toward their target area but disrupts local axonal convergence into a single glomerulus, resulting in two distinct targeting phenotypes: axons either intermingle with adjacent ORN classes or segregate according to their odorant receptor identity into ectopic sites. Differential Sema-1a expression can be detected among neighboring glomeruli, and mosaic analyses show that sema-1a functions nonautonomously in ORN axon sorting. These findings provide insights into the mechanism by which afferent interactions lead to synaptic specificity in the olfactory system.     

Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude

Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency.Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements.In this report, we show that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected.These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information.     

Female nutritional status determines the magnitude and sign of responses to a male ejaculate signal in Drosophila melanogaster

Ejaculate chemicals transferred from males to females during mating cause significant changes in female behaviour and physiology, but the causes of phenotypic variation in these responses is little understood. We tested here the effect of adult female nutrition on the response of female Drosophila melanogaster to a specific ejaculate component, the sex peptide (SP), which is of interest because of its effects on female egg laying, sexual receptivity, feeding rate, immune responses and potential role in mediating sexual conflict. We exposed adult females to five different diets and kept them continuously with males that did or did not transfer SP. Diet altered the presence, magnitude and sign of the effects of SP on different phenotypic traits (egg laying, receptivity and lifespan) and different traits responded in different ways. This showed that the set of responses to mating can be uncoupled and can vary independently in different environments. Importantly, diet also significantly affected whether exposure to SP transferring males was beneficial or costly to females, with beneficial effects occurring more often than expected. Hence, the food environment can also shape significantly the strength and direction of selection on mating responses.