Drosophila Antennal Lobe Research Articles

Glia control experience-dependent plasticity in an olfactory critical period.

Sensory experience during developmental critical periods has lifelong consequences for circuit function and behavior, but the molecular and cellular mechanisms through which experience causes these changes are not well understood. The Drosophila antennal lobe houses synapses between olfactory sensory neurons (OSNs) and downstream projection neurons (PNs) in stereotyped glomeruli. Many glomeruli exhibit structural plasticity in response to early-life odor exposure, indicating a general sensitivity of the fly olfactory circuitry to early sensory experience. We recently found that glia shape antennal lobe development in young adults, leading us to ask if glia also drive experience-dependent plasticity during this period. Here we define a critical period for structural and functional plasticity of OSN-PN synapses in the ethyl butyrate (EB)-sensitive glomerulus VM7. EB exposure for the first two days post-eclosion drives large-scale reductions in glomerular volume, presynapse number, and post-synaptic activity. Crucially, pruning during the critical period has long-term consequences for circuit function since both OSN-PN synapse number and spontaneous activity of PNs remain persistently decreased following early-life odor exposure. The highly conserved engulfment receptor Draper is required for this critical period plasticity as ensheathing glia upregulate Draper, invade the VM7 glomerulus, and phagocytose OSN presynaptic terminals in response to critical-period EB exposure. Loss of Draper fully suppresses the morphological and physiological consequences of critical period odor exposure, arguing that phagocytic glia engulf intact synaptic terminals. These data demonstrate experience-dependent pruning of synapses and argue that Drosophila olfactory circuitry is a powerful model for defining the function of glia in critical period plasticity.

Interactions between specialized gain control mechanisms in olfactory processing

Gain control is a process that adjusts a system's sensitivity when input levels change. Neural systems contain multiple mechanisms of gain control, but we do not understand why so many mechanisms are needed or how they interact. Here, we investigate these questions in the Drosophila antennal lobe, where we identify several types of inhibitory interneurons with specialized gain control functions. We find that some interneurons are nonspiking, with compartmentalized calcium signals, and they specialize in intra-glomerular gain control. Conversely, we find that other interneurons are recruited by strong and widespread network input; they specialize in global presynaptic gain control. Using computational modeling and optogenetic perturbations, we show how these mechanisms can work together to improve stimulus discrimination while also minimizing temporal distortions in network activity. Our results demonstrate how the robustness of neural network function can be increased by interactions among diverse and specialized mechanisms of gain control.

Heterogeneous receptor expression underlies non-uniform peptidergic modulation of olfaction in Drosophila

Sensory systems are dynamically adjusted according to the animal’s ongoing needs by neuromodulators, such as neuropeptides. Neuropeptides are often widely-distributed throughout sensory networks, but it is unclear whether such neuropeptides uniformly modulate network activity. Here, we leverage the Drosophila antennal lobe (AL) to resolve whether myoinhibitory peptide (MIP) uniformly modulates AL processing. Despite being uniformly distributed across the AL, MIP decreases olfactory input to some glomeruli, while increasing olfactory input to other glomeruli. We reveal that a heterogeneous ensemble of local interneurons (LNs) are the sole source of AL MIP, and show that differential expression of the inhibitory MIP receptor across glomeruli allows MIP to act on distinct intraglomerular substrates. Our findings demonstrate how even a seemingly simple case of modulation can have complex consequences on network processing by acting non-uniformly within different components of the overall network.

Early Draper-mediated glial refinement of neuropil architecture and synapse number in the Drosophila antennal lobe.

Glial phagocytic activity refines connectivity, though molecular mechanisms regulating this exquisitely sensitive process are incompletely defined. We developed the Drosophila antennal lobe as a model for identifying molecular mechanisms underlying glial refinement of neural circuits in the absence of injury. Antennal lobe organization is stereotyped and characterized by individual glomeruli comprised of unique olfactory receptor neuronal (ORN) populations. The antennal lobe interacts extensively with two glial subtypes: ensheathing glia wrap individual glomeruli, while astrocytes ramify considerably within them. Phagocytic roles for glia in the uninjured antennal lobe are largely unknown. Thus, we tested whether Draper regulates ORN terminal arbor size, shape, or presynaptic content in two representative glomeruli: VC1 and VM7. We find that glial Draper limits the size of individual glomeruli and restrains their presynaptic content. Moreover, glial refinement is apparent in young adults, a period of rapid terminal arbor and synapse growth, indicating that synapse addition and elimination occur simultaneously. Draper has been shown to be expressed in ensheathing glia; unexpectedly, we find it expressed at high levels in late pupal antennal lobe astrocytes. Surprisingly, Draper plays differential roles in ensheathing glia and astrocytes in VC1 and VM7. In VC1, ensheathing glial Draper plays a more significant role in shaping glomerular size and presynaptic content; while in VM7, astrocytic Draper plays the larger role. Together, these data indicate that astrocytes and ensheathing glia employ Draper to refine circuitry in the antennal lobe before the terminal arbors reach their mature form and argue for local heterogeneity of neuron-glia interactions.

Synaptic Development in Diverse Olfactory Neuron Classes Uses Distinct Temporal and Activity-Related Programs.

Developing neurons must meet core molecular, cellular, and temporal requirements to ensure the correct formation of synapses, resulting in functional circuits. However, because of the vast diversity in neuronal class and function, it is unclear whether or not all neurons use the same organizational mechanisms to form synaptic connections and achieve functional and morphologic maturation. Moreover, it remains unknown whether neurons united in a common goal and comprising the same sensory circuit develop on similar timescales and use identical molecular approaches to ensure the formation of the correct number of synapses. To begin to answer these questions, we took advantage of the Drosophila antennal lobe (AL), a model olfactory circuit with remarkable genetic access and synapse-level resolution. Using tissue-specific genetic labeling of active zones, we performed a quantitative analysis of synapse formation in multiple classes of neurons of both sexes throughout development and adulthood. We found that olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs) each have unique time courses of synaptic development, addition, and refinement, demonstrating that each class follows a distinct developmental program. This raised the possibility that these classes may also have distinct molecular requirements for synapse formation. We genetically altered neuronal activity in each neuronal subtype and observed differing effects on synapse number based on the neuronal class examined. Silencing neuronal activity in ORNs, PNs, and LNs impaired synaptic development but only in ORNs did enhancing neuronal activity influence synapse formation. ORNs and LNs demonstrated similar impairment of synaptic development with enhanced activity of a master kinase, GSK-3β, suggesting that neuronal activity and GSK-3β kinase activity function in a common pathway. ORNs also, however, demonstrated impaired synaptic development with GSK-3β loss-of-function, suggesting additional activity-independent roles in development. Ultimately, our results suggest that the requirements for synaptic development are not uniform across all neuronal classes with considerable diversity existing in both their developmental time frames and molecular requirements. These findings provide novel insights into the mechanisms of synaptic development and lay the foundation for future work determining their underlying etiologies.SIGNIFICANCE STATEMENT Distinct olfactory neuron classes in Drosophila develop a mature synaptic complement over unique timelines and using distinct activity-dependent and molecular programs, despite having the same generalized goal of olfactory sensation.

Parallel encoding of CO2 in attractive and aversive glomeruli by selective lateral signaling between olfactory afferents

We describe a novel form of selective crosstalk between specific classes of primary olfactory receptor neurons (ORNs) in the Drosophila antennal lobe. Neurotransmitter release from ORNs is driven by two distinct sources of excitation: direct activity derived from the odorant receptor and stimulus-selective lateral signals originating from stereotypic subsets of other ORNs. Consequently, the level of presynaptic neurotransmitter release from an ORN can be significantly dissociated from its firing rate. Stimulus-selective lateral signaling results in the distributed representation of CO2-a behaviorally important environmental cue that directly excites a single ORN class-in multiple olfactory glomeruli, each with distinct response dynamics. CO2-sensitive glomeruli coupled to behavioral attraction respond preferentially to fast changes in CO2 concentration, whereas those coupled to behavioral aversion more closely follow absolute levels of CO2. Behavioral responses to CO2 also depend on the temporal structure of the stimulus: flies walk upwind to fluctuating, but not sustained, pulses of CO2. Stimulus-selective lateral signaling generalizes to additional odors and glomeruli, revealing a subnetwork of lateral interactions between ORNs that reshapes the spatial and temporal structure of odor representations in a stimulus-specific manner.

Effects of Mono-2-ethylhexyl Phthalate on the Neural Transmission of PNs in Drosophila Antennal Lobe.

Long-term exposure to different types of chemicals is hazardous to human health. Di(2-ethylhexyl) phthalate (DEHP) could exert pleiotropic deleterious effects on nervous systems. Mono(2-ethylhexyl) phthalate (MEHP), as one of the most toxic metabolites of DEHP, may have similar effects on nervous systems. However, no effects of MEHP on neural circuits have been reported. To uncover the regulation of MEHP on neural transmission, the functional changes of neural excitability and synaptic plasticity of projection neurons (PNs) have been assessed. In the current study, we recorded the action potentials (APs), stimulate action potentials (sti-APs), mini excitement postsynaptic current (mEPSC), calcium currents, and sodium currents from PNs of isolated whole brain of Drosophila model utilizing patch clamp recordings. We found that MEHP-300 (at the concentration of 300 μM), but not MHEP-100 (at the concentration of 100 μM), significantly decreased the frequency and amplitude of APs. Besides, the amplitude and anti-amplitude of sti-APs were reduced with the application of MEHP-300. Meanwhile, MEHP-300 reduced the frequency of mEPSC, but not the amplitude. Furthermore, MEHP-300 reduced the peak current densities of sodium and calcium channels. Therefore, our results indicated that MEHP could alter the neural excitability and synaptic plasticity of PNs by inhibiting the ion channels activities, revealing the potential modulation of MEHP on neural transmission of PNs.

The Photoconvertible Fluorescent Probe, CaMPARI, Labels Active Neurons in Freely-Moving Intact Adult Fruit Flies.

Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI’s uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.

Interplay between axonal Wnt5-Vang and dendritic Wnt5-Drl/Ryk signaling controls glomerular patterning in the Drosophila antennal lobe.

Despite the importance of dendritic targeting in neural circuit assembly, the mechanisms by which it is controlled still remain incompletely understood. We previously showed that in the developing Drosophila antennal lobe, the Wnt5 protein forms a gradient that directs the ~45˚ rotation of a cluster of projection neuron (PN) dendrites, including the adjacent DA1 and VA1d dendrites. We report here that the Van Gogh (Vang) transmembrane planar cell polarity (PCP) protein is required for the rotation of the DA1/VA1d dendritic pair. Cell type-specific rescue and mosaic analyses showed that Vang functions in the olfactory receptor neurons (ORNs), suggesting a codependence of ORN axonal and PN dendritic targeting. Loss of Vang suppressed the repulsion of the VA1d dendrites by Wnt5, indicating that Wnt5 signals through Vang to direct the rotation of the DA1 and VA1d glomeruli. We observed that the Derailed (Drl)/Ryk atypical receptor tyrosine kinase is also required for the rotation of the DA1/VA1d dendritic pair. Antibody staining showed that Drl/Ryk is much more highly expressed by the DA1 dendrites than the adjacent VA1d dendrites. Mosaic and epistatic analyses showed that Drl/Ryk specifically functions in the DA1 dendrites in which it antagonizes the Wnt5-Vang repulsion and mediates the migration of the DA1 glomerulus towards Wnt5. Thus, the nascent DA1 and VA1d glomeruli appear to exhibit Drl/Ryk-dependent biphasic responses to Wnt5. Our work shows that the final patterning of the fly olfactory map is the result of an interplay between ORN axons and PN dendrites, wherein converging pre- and postsynaptic processes contribute key Wnt5 signaling components, allowing Wnt5 to orient the rotation of nascent synapses through a PCP mechanism.

A Population of Interneurons Signals Changes in the Basal Concentration of Serotonin and Mediates Gain Control in the Drosophila Antennal Lobe.

Serotonin (5-HT) represents a quintessential neuromodulator, having been identified in nearly all animal species [1] where it functions in cognition [2], motor control [3], and sensory processing [4]. In the olfactory circuits of flies and mice, serotonin indirectly inhibits odor responses in olfactory receptor neurons (ORNs) via GABAergic local interneurons (LNs) [5, 6]. However, the effects of 5-HT in olfaction are likely complicated, because multiple receptor subtypes are distributed throughout the olfactory bulb (OB) and antennal lobe (AL), the first layers of olfactory neuropil in mammals and insects, respectively [7]. For example, serotonin has a non-monotonic effect on odor responses in Drosophila projection neurons (PNs), where low concentrations suppress odor-evoked activity and higher concentrations boost PN responses [8]. Serotonin reaches the AL via the diffusion of paracrine 5-HT through the fly hemolymph [8] and by activation of the contralaterally projecting serotonin-immunoreactive deuterocerebral interneurons (CSDns): the only serotonergic cells that innervate the AL [9, 10]. Concentration-dependent effects could arise by either the expression of multiple 5-HT receptors (5-HTRs) on the same cells or by populations of neurons dedicated to detecting serotonin at different concentrations. Here, we identify a population of LNs that express 5-HT7Rs exclusively to detect basal concentrations of 5-HT. These LNs inhibit PNs via GABAB receptors and mediate subtractive gain control. LNs expressing 5-HT7Rs are broadly tuned to odors and target every glomerulus in the antennal lobe. Our results demonstrate that serotonergic modulation at low concentrations targets a specific population of LNs to globally downregulate PN odor responses in the AL.

A population of interneurons signals changes in the basal concentration of serotonin and mediates gain control in the Drosophila antennal lobe

A population of interneurons signals changes in the basal concentration of serotonin and mediates gain control in the Drosophila antennal lobe

Odor mixtures of opposing valence unveil inter-glomerular crosstalk in the Drosophila antennal lobe

Evaluating odor blends in sensory processing is a crucial step for signal recognition and execution of behavioral decisions. Using behavioral assays and 2-photon imaging, we have characterized the neural and behavioral correlates of mixture perception in the olfactory system of Drosophila. Mixtures of odors with opposing valences elicit strong inhibition in certain attractant-responsive input channels. This inhibition correlates with reduced behavioral attraction. We demonstrate that defined subsets of GABAergic interneurons provide the neuronal substrate of this computation at pre- and postsynaptic loci via GABAB- and GABAA receptors, respectively. Intriguingly, manipulation of single input channels by silencing and optogenetic activation unveils a glomerulus-specific crosstalk between the attractant- and repellent-responsive circuits. This inhibitory interaction biases the behavioral output. Such a form of selective lateral inhibition represents a crucial neuronal mechanism in the processing of conflicting sensory information.

Activity-Dependent Remodeling of Drosophila Olfactory Sensory Neuron Brain Innervation during an Early-Life Critical Period.

Critical periods are windows of development when the environment has a pronounced effect on brain circuitry. Models of neurodevelopmental disorders, including autism spectrum disorders, intellectual disabilities, and schizophrenia, are linked to disruption of critical period remodeling. Critical periods open with the onset of sensory experience; however, it remains unclear exactly how sensory input modifies brain circuits. Here, we examine olfactory sensory neuron (OSN) innervation of the Drosophila antennal lobe of both sexes as a genetic model of this question. We find that olfactory sensory experience during an early-use critical period drives loss of OSN innervation of antennal lobe glomeruli and subsequent axon retraction in a dose-dependent mechanism. This remodeling does not result from olfactory receptor loss or OSN degeneration, but rather from rapid synapse elimination and axon pruning in the target olfactory glomerulus. Removal of the odorant stimulus only during the critical period leads to OSN reinnervation, demonstrating that remodeling is transiently reversible. We find that this synaptic refinement requires the OSN-specific olfactory receptor and downstream activity. Conversely, blocking OSN synaptic output elevates glomeruli remodeling. We find that GABAergic neurotransmission has no detectable role, but that glutamatergic signaling via NMDA receptors is required for OSN synaptic refinement. Together, these results demonstrate that OSN inputs into the brain manifest robust, experience-dependent remodeling during an early-life critical period, which requires olfactory reception, OSN activity, and NMDA receptor signaling. This work reveals a pathway linking initial olfactory sensory experience to glutamatergic neurotransmission in the activity-dependent remodeling of brain neural circuitry in an early-use critical period.SIGNIFICANCE STATEMENT Neurodevelopmental disorders manifest symptoms at specific developmental milestones that suggest an intersection between early sensory experience and brain neural circuit remodeling. One classic example is Fragile X syndrome caused by loss of an RNA-binding translation regulator of activity-dependent synaptic refinement. As a model, Drosophila olfactory circuitry is well characterized, genetically tractable, and rapidly developing, and thus ideally suited to probe underlying mechanisms. Here, we find olfactory sensory neurons are dramatically remodeled by heightened sensory experience during an early-life critical period. We demonstrate removing the olfactory stimulus during the critical period can reverse the connectivity changes. We find that this remodeling requires neural activity and NMDA receptor-mediated glutamatergic transmission. This improved understanding may help us design treatments for neurodevelopmental disorders.

A resource for the Drosophila antennal lobe provided by the connectome of glomerulus VA1v.

Using FIB-SEM we report the entire synaptic connectome of glomerulus VA1v of the right antennal lobe in Drosophila melanogaster. Within the glomerulus we densely reconstructed all neurons, including hitherto elusive local interneurons. The fruitless-positive, sexually dimorphic VA1v included >11,140 presynaptic sites with ~38,050 postsynaptic dendrites. These connected input olfactory receptor neurons (ORNs, 51 ipsilateral, 56 contralateral), output projection neurons (18 PNs), and local interneurons (56 of >150 previously reported LNs). ORNs are predominantly presynaptic and PNs predominantly postsynaptic; newly reported LN circuits are largely an equal mixture and confer extensive synaptic reciprocity, except the newly reported LN2V with input from ORNs and outputs mostly to monoglomerular PNs, however. PNs were more numerous than previously reported from genetic screens, suggesting that the latter failed to reach saturation. We report a matrix of 192 bodies each having >50 connections; these form 88% of the glomerulus' pre/postsynaptic sites.

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.

Functional integration of a serotonergic neuron in the Drosophila antennal lobe.

Serotonin plays a critical role in regulating many behaviors that rely on olfaction and recently there has been great effort in determining how this molecule functions in vivo. However, it remains unknown how serotonergic neurons that innervate the first olfactory relay respond to odor stimulation and how they integrate synaptically into local circuits. We examined the sole pair of serotonergic neurons that innervates the Drosophila antennal lobe (the first olfactory relay) to characterize their physiology, connectivity, and contribution to pheromone processing. We report that nearly all odors inhibit these cells, likely through connections made reciprocally within the antennal lobe. Pharmacological and immunohistochemical analyses reveal that these neurons likely release acetylcholine in addition to serotonin and that exogenous and endogenous serotonin have opposing effects on olfactory responses. Finally, we show that activation of the entire serotonergic network, as opposed to only activation of those fibers innervating the antennal lobe, may be required for persistent serotonergic modulation of pheromone responses in the antennal lobe.

Hierarchical axon targeting of Drosophila olfactory receptor neurons specified by the proneural transcription factors Atonal and Amos.

Sensory information is spatially represented in the brain to form a neural map. It has been suggested that axon-axon interactions are important for neural map formation; however, the underlying mechanisms are not fully understood. We used the Drosophila antennal lobe, the first olfactory center in the brain, as a model for studying neural map formation. Olfactory receptor neurons (ORNs) expressing the same odorant receptor target their axons to a single glomerulus out of approximately 50 glomeruli in the antennal lobe. Previous studies have showed that the axons of Atonal ORNs, specified by Atonal, a basic helix-loop-helix (bHLH) transcription factor, pioneer antennal lobe formation; however, the details remain to be elucidated. Here, we show that genetic ablation of Atonal ORNs affects antennal lobe structure and axon targeting of Amos ORNs, another type of ORN specified by the bHLH transcription factor Amos. During development, Atonal ORNs reach the antennal lobe and form the axon commissure before Amos ORNs. We also found that N-cadherin knockdown specifically in Atonal ORNs disrupts the glomerular boundary in the whole antennal lobe. Our results suggest that Atonal ORNs function as pioneer axons. Thus, correct axon targeting of Atonal ORNs is essential for formation of the whole antennal lobe.

Projection neurons in Drosophila antennal lobes signal the acceleration of odor concentrations.

Temporal experience of odor gradients is important in spatial orientation of animals. The fruit fly Drosophila melanogaster exhibits robust odor-guided behaviors in an odor gradient field. In order to investigate how early olfactory circuits process temporal variation of olfactory stimuli, we subjected flies to precisely defined odor concentration waveforms and examined spike patterns of olfactory sensory neurons (OSNs) and projection neurons (PNs). We found a significant temporal transformation between OSN and PN spike patterns, manifested by the PN output strongly signaling the OSN spike rate and its rate of change. A simple two-dimensional model admitting the OSN spike rate and its rate of change as inputs closely predicted the PN output. When cascaded with the rate-of-change encoding by OSNs, PNs primarily signal the acceleration and the rate of change of dynamic odor stimuli to higher brain centers, thereby enabling animals to reliably respond to the onsets of odor concentrations.

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.

Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins.

Understanding information flow through neuronal circuits requires knowledge of their synaptic organization. In this study, we utilized fluorescent pre- and postsynaptic markers to map synaptic organization in the Drosophila antennal lobe, the first olfactory processing center. Olfactory receptor neurons (ORNs) produce a constant synaptic density across different glomeruli. Each ORN within a class contributes nearly identical active zone number. Active zones from ORNs, projection neurons (PNs), and local interneurons have distinct subglomerular and subcellular distributions. The correct number of ORN active zones and PN acetylcholine receptor clusters requires the Teneurins, conserved transmembrane proteins involved in neuromuscular synapse organization and synaptic partner matching. Ten-a acts in ORNs to organize presynaptic active zones via the spectrin cytoskeleton. Ten-m acts in PNs autonomously to regulate acetylcholine receptor cluster number and transsynaptically to regulate ORN active zone number. These studies advanced our ability to assess synaptic architecture in complex CNS circuits and their underlying molecular mechanisms.