Eph Receptor Effector Ephexin Mediates Olfactory Dendrite Targeting in Drosophila

Deciphering the mechanisms of sensory neural map formation is a central aim in neurosciences. Failure to form a correct map frequently leads to defects in sensory processing and perception. The olfactory map develops in subsequent steps initially forming a rough and later a precise map of glomeruli in the antennal lobe (AL), mainly consisting of olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites. The mechanisms underpinning the later stage of class‐specific glomerulus formation are not understood. Recent studies have shown that the important guidance molecule Eph and its ligand ephrin play a role in class‐specific PN targeting. Here, we reveal aspects of the mechanism downstream of Eph signaling during olfactory map formation. We show that the Eph‐specific RhoGEF Ephexin (Exn) is required to fine tune PN dendrite patterning within specific glomeruli. We provide the first report showing an in vivo neurite guidance defect in an exn mutant. Interestingly, the quality of the phenotypes is different between eph and exn mutants; while loss of Eph leads to strong misprojections of DM3/Or47a neurons along the medial–lateral axis of the antennal lobe (AL), loss of Exn induces ventral ectopic innervation of a neighboring glomerulus. Genetic interaction experiments suggest that differential signaling of the small GTPases Rac1 and Cdc42 mediated by Exn‐dependent and ‐independent Eph signaling fine tunes spatial targeting of PN dendrites within the olfactory map. We propose that their distinct activities on the actin cytoskeleton are required for precise navigation of PN dendrites within the olfactory map. Taken together, our results suggest that the precise connectivity of an individual neuron can depend on different modes of signaling downstream of a single guidance receptor. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2018     

Representation of the glomerular olfactory map in the Drosophila brain

We explored how the odor map in the Drosophila antennal lobe is represented in higher olfactory centers, the mushroom body and lateral horn. Systematic single-cell tracing of projection neurons (PNs) that send dendrites to specific glomeruli in the antennal lobe revealed their stereotypical axon branching patterns and terminal fields in the lateral horn. PNs with similar axon terminal fields tend to receive input from neighboring glomeruli. The glomerular classes of individual PNs could be accurately predicted based solely on their axon projection patterns. The sum of these patterns defines an "axon map" in higher olfactory centers reflecting which olfactory receptors provide input. This map is characterized by spatial convergence and divergence of PN axons, allowing integration of olfactory information.     

Temporally staggered glomerulus development in the moth Manduca sexta

Glomeruli, neuropilar structures composed of olfactory receptor neuron (ORN) axon terminals and central neuron dendrites, are a common feature of olfactory systems. Typically, ORN axons segregate into glomeruli based on odor specificity, making glomeruli the basic unit for initial processing of odorant information. Developmentally, glomeruli arise from protoglomeruli, loose clusters of ORN axons that gradually synapse onto dendrites. Previous work in the moth Manduca sexta demonstrated that protoglomeruli develop in a wave across the antennal lobe (AL) during stage 5 of the 18 stages of metamorphic adult development. However, ORN axons from the distal segments of the antenna arrive at the AL for several more days. We report that protoglomeruli present at stage 5 account for only approximately two or three of adult glomeruli with the number of structures increasing over subsequent stages. How do these later arriving axons incorporate into glomeruli? Examining the dendritic projections of a unique serotonin-containing neuron into glomeruli at later stages revealed glomeruli with immature dendritic arbors intermingled among more mature glomeruli. Labeling ORN axons that originate in proximal segments of the antenna suggested that early-arriving axons target a limited number of glomeruli. We conclude that AL glomeruli form over an extended time period, possibly as a result of ORNs expressing new odorant receptors arriving from distal antennal segments.     

Secreted semaphorins from degenerating larval ORN axons direct adult projection neuron dendrite targeting

During assembly of the Drosophila olfactory circuit, projection neuron (PN) dendrites prepattern the developing antennal lobe before the arrival of axons from their presynaptic partners, the adult olfactory receptor neurons (ORNs). We previously found that levels of transmembrane Semaphorin-1a, which acts as a receptor, instruct PN dendrite targeting along the dorsolateral-ventromedial axis. Here we show that two secreted semaphorins, Sema-2a and Sema-2b, provide spatial cues for PN dendrite targeting. Sema-2a and Sema-2b proteins are distributed in gradients opposing the Sema-1a protein gradient, and Sema-1a binds to Sema-2a-expressing cells. In Sema-2a and Sema-2b double mutants, PN dendrites that normally target dorsolaterally in the antennal lobe mistarget ventromedially, phenocopying cell-autonomous Sema-1a removal from these PNs. Cell ablation, cell-specific knockdown, and rescue experiments indicate that secreted semaphorins from degenerating larval ORN axons direct dendrite targeting. Thus, a degenerating brain structure instructs the wiring of a developing circuit through the repulsive action of secreted semaphorins.     

Graded expression of semaphorin-1a cell-autonomously directs dendritic targeting of olfactory projection neurons

Gradients of axon guidance molecules instruct the formation of continuous neural maps, such as the retinotopic map in the vertebrate visual system. Here we show that molecular gradients can also instruct the formation of a discrete neural map. In the fly olfactory system, axons of 50 classes of olfactory receptor neurons (ORNs) and dendrites of 50 classes of projection neurons (PNs) form one-to-one connections at discrete units called glomeruli. We provide expression, loss- and gain-of-function data to demonstrate that the levels of transmembrane Semaphorin-1a (Sema-1a), acting cell-autonomously as a receptor or part of a receptor complex, direct the dendritic targeting of PNs along the dorsolateral to ventromedial axis of the antennal lobe. Sema-1a also regulates PN axon targeting in higher olfactory centers. Thus, graded expression of Sema-1a contributes to connection specificity from ORNs to PNs and then to higher brain centers, ensuring proper representation of olfactory information in the brain.     

Activation of glial FGFRs is essential in glial migration, proliferation, and survival and in glia-neuron signaling during olfactory system development

Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.     

Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions

Axon-axon interactions have been implicated in neural circuit assembly, but the underlying mechanisms are poorly understood. Here, we show that in the Drosophila antennal lobe, early-arriving axons of olfactory receptor neurons (ORNs) from the antenna are required for the proper targeting of late-arriving ORN axons from the maxillary palp (MP). Semaphorin-1a is required for targeting of all MP but only half of the antennal ORN classes examined. Sema-1a acts nonautonomously to control ORN axon-axon interactions, in contrast to its cell-autonomous function in olfactory projection neurons. Phenotypic and genetic interaction analyses implicate PlexinA as the Sema-1a receptor in ORN targeting. Sema-1a on antennal ORN axons is required for correct targeting of MP axons within the antennal lobe, while interactions amongst MP axons facilitate their entry into the antennal lobe. We propose that Sema-1a/PlexinA-mediated repulsion provides a mechanism by which early-arriving ORN axons constrain the target choices of late-arriving axons.     

Comprehensive maps of Drosophila higher olfactory centers: spatially segregated fruit and pheromone representation

In Drosophila, approximately 50 classes of olfactory receptor neurons (ORNs) send axons to 50 corresponding glomeruli in the antennal lobe. Uniglomerular projection neurons (PNs) relay olfactory information to the mushroom body (MB) and lateral horn (LH). Here, we combine single-cell labeling and image registration to create high-resolution, quantitative maps of the MB and LH for 35 input PN channels and several groups of LH neurons. We find (1) PN inputs to the MB are stereotyped as previously shown for the LH; (2) PN partners of ORNs from different sensillar groups are clustered in the LH; (3) fruit odors are represented mostly in the posterior-dorsal LH, whereas candidate pheromone-responsive PNs project to the anterior-ventral LH; (4) dendrites of single LH neurons each overlap with specific subsets of PN axons. Our results suggest that the LH is organized according to biological values of olfactory input.     

Afferent induction of olfactory glomeruli requires N-cadherin

Drosophila olfactory receptor neurons (ORNs) elaborate a precise internal representation of the external olfactory world in the antennal lobe (AL), a structure analagous to the vertebrate olfactory bulb. ORNs expressing the same odorant receptor innervate common targets in a highly organized neuropilar structure inside the AL, the glomerulus. During normal development, ORNs target to specific regions of the AL and segregate into subclass-specific aggregates called protoglomeruli prior to extensive intermingling with target dendrites to form mature glomeruli. Using a panel of ORN subclass-specific markers, we demonstrate that in the adult AL, N-cadherin (N-cad) mutant ORN terminals remain segregated from dendrites of target neurons. N-cad plays a crucial role in protoglomerulus formation but is largely dispensible for targeting to the appropriate region of the AL. We propose that N-cad, a homophilic cell adhesion molecule, acts in a permissive fashion to promote subclass-specific sorting of ORN axon terminals into protoglomeruli.     

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.     

Developmentally programmed remodeling of the Drosophila olfactory circuit

Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input by synapsing with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Here we show that embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to innervate a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons.     

Homeostatic matching and nonlinear amplification at identified central synapses

Here we describe the properties of a synapse in the Drosophila antennal lobe and show how they can explain certain sensory computations in this brain region. The synapse between olfactory receptor neurons (ORNs) and projection neurons (PNs) is very strong, reflecting a large number of release sites and high release probability. This is likely one reason why weak ORN odor responses are amplified in PNs. Furthermore, the amplitude of unitary synaptic currents in a PN is matched to the size of its dendritic arbor. This matching may compensate for a lower input resistance of larger dendrites to produce uniform depolarization across PN types. Consistent with this idea, a genetic manipulation that lowers input resistance increases unitary synaptic currents. Finally, strong stimuli produce short-term depression at this synapse. This helps explain why PN odor responses are transient, and why strong ORN odor responses are not amplified as powerfully as weak responses.     

Pheromone-sensitive glomeruli in the primary olfactory centre of ants

Tremendous evolutional success and the ecological dominance of social insects, including ants, termites and social bees, are due to their efficient social organizations and their underlying communication systems. Functional division into reproductive and sterile castes, cooperation in defending the nest, rearing the young and gathering food are all regulated by communication by means of various kinds of pheromones. No brain structures specifically involved in the processing of non-sexual pheromone have been physiologically identified in any social insects. By use of intracellular recording and staining techniques, we studied responses of projection neurons of the antennal lobe (primary olfactory centre) of ants to alarm pheromone, which plays predominant roles in colony defence. Among 23 alarm pheromone-sensitive projection neurons recorded and stained in this study, eight were uniglomerular projection neurons with dendrites in one glomerulus, a structural unit of the antennal lobe, and the remaining 15 were multiglomerular projection neurons with dendrites in multiple glomeruli. Notably, all alarm pheromone-sensitive uniglomerular projection neurons had dendrites in one of five 'alarm pheromone-sensitive (AS)' glomeruli that form a cluster in the dorsalmost part of the antennal lobe. All alarm pheromone-sensitive multiglomerular projection neurons had dendrites in some of the AS glomeruli as well as in glomeruli in the anterodorsal area of the antennal lobe. The results suggest that components of alarm pheromone are processed in a specific cluster of glomeruli in the antennal lobe of ants.     

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.     

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete‐expressing local interneurons in development of the adult olfactory circuitry. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Physiological mismatching between neurons innervating olfactory glomeruli in a moth

The primary olfactory centres of most vertebrates and most neopteran insects are characterized by the presence of spherical neuropils, glomeruli, where synaptic interactions between olfactory receptor neurons and second-order neurons take place. In the neopteran insect taxa investigated so far, receptor neurons of a specific physiological identity target one glomerulus and thus bestow a functional identity on the glomerulus. In moths, input from pheromone-specific receptor neurons is received in a male-specific structure of the antennal lobe, called the macroglomerular complex (MGC), which consists of a number of specialized glomeruli. Each glomerulus of the complex receives a set of peripheral sensory afferents that encode one of several compounds involved in sexual communication. The complex is also innervated by dendritic branches of antennal lobe output neurons called projection neurons, which transfer information from the antennal lobe to higher centres of the brain. A hypothesis stemming from earlier work on moths claims that the receptor neuron innervation pattern of the MGC should be reflected in the pattern of dendrites of projection neurons invading the different MGC glomeruli. In this study we show that in the noctuid moth Trichoplusia ni, as in several other noctuid moth species, this hypothesis does not hold. The degree of matching between axon terminals of receptor neurons and the dendritic branches of identified projection neurons that express similar physiological specificity is very low.     

Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology,calcium imaging and confocal microscopy

The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known.Abbreviations ACT antenno-cerebralis-tract - AL antennal lobe - AP action potential - l-ACT lateral ACT - LN local neuron - LPL lateral protocerebral lobe - m-ACT medial ACT - MB mushroom body - OSN olfactory sensory neuron - PN projection neuron - T1 tract 1 of the antennal nerve