Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain [1-7]. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants [8-11]. OSNs also exhibit odor-evoked responses with complex temporal dynamics [11], but the contribution of this activity to behavioral odor discrimination has received little attention [12]. Here, we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.     

Maxillary palp glomeruli and ipsilateral projections in the antennal lobe of<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The antennal lobe was examined by Golgi-silver impregnation to differentiate the glomeruli depending on the source and types of inputs. Thirty-five of the 43 ‘identified’ olfactory glomeruli were Golgi-silver impregnated in the present study. Seven glomeruli compared to three, reported previously, were found to be targets of maxillary palp chemosensory neurons. These include glomeruli VA3, VC2, VM5, VA7m/VA7l of the ventral antennal lobe and DC2, DC3, DM5 of the dorsal antennal lobe. The number of glomeruli receiving the maxillary palp sensory projections tallies with the number ofDrosophila olfactory receptors (seven) reported to be expressed exclusively in the maxillary palp. Twenty-eight Golgi-impregnated glomeruli were found to receive input from the antennal nerve. The ratio of glomeruli serving the maxillary palp to those serving the antenna (∼1:5) matches with the ratio ofDrosophila olfactory receptors expressed in these two olfactory organs respectively. In addition to glomerulus V, glomeruli VP1-3, VL1, VL2a/2p and VC3m/3l were found to receive ipsilateral projections. Thus, additional ipsilateral glomeruli have been identified.     

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.     

Spatial representation of alarm pheromone information in a secondary olfactory centre in the ant brain

Pheromones play major roles in intraspecific communication in many animals. Elaborated communication systems in eusocial insects provide excellent materials to study neural mechanisms for social pheromone processing. We previously reported that alarm pheromone information is processed in a specific cluster of glomeruli in the antennal lobe of the ant Camponotus obscuripes. However, representation of alarm pheromone information in a secondary olfactory centre is unknown in any animal. Olfactory information in the antennal lobe is transmitted to secondary olfactory centres, including the lateral horn, by projection neurons (PNs). In this study, we compared distributions of terminal boutons of alarm pheromone-sensitive and -insensitive PNs in the lateral horn of ants. Distributions of their dendrites largely overlapped, but there was a region where boutons of pheromone-sensitive PNs, but not those of pheromone-insensitive PNs, were significantly denser than in the rest of the lateral horn. Moreover, most of a major type of pheromone-sensitive efferent neurons from the lateral horn extended dendritic branches in this region, suggesting specialization of this region for alarm pheromone processing. This study is the first study to demonstrate the presence of specialized areas for the processing of a non-sexual, social pheromone in the secondary olfactory centre in any animal.     

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     

Glomerular interactions in olfactory processing channels of the antennal lobes

An open question in olfactory coding is the extent of interglomerular connectivity: do olfactory glomeruli and their neurons regulate the odorant responses of neurons innervating other glomeruli? In the olfactory system of the moth Manduca sexta, the response properties of different types of antennal olfactory receptor cells are known. Likewise, a subset of antennal lobe glomeruli has been functionally characterized and the olfactory tuning of their innervating neurons identified. This provides a unique opportunity to determine functional interactions between glomeruli of known input, specifically, (1) glomeruli processing plant odors and (2) glomeruli activated by antennal stimulation with pheromone components of conspecific females. Several studies describe reciprocal inhibitory effects between different types of pheromone-responsive projection neurons suggesting lateral inhibitory interactions between pheromone component-selective glomerular neural circuits. Furthermore, antennal lobe projection neurons that respond to host plant volatiles and innervate single, ordinary glomeruli are inhibited during antennal stimulation with the female’s sex pheromone. The studies demonstrate the existence of lateral inhibitory effects in response to behaviorally significant odorant stimuli and irrespective of glomerular location in the antennal lobe. Inhibitory interactions are present within and between olfactory subsystems (pheromonal and non-pheromonal subsystems), potentially to enhance contrast and strengthen odorant discrimination.     

Excitatory local circuits and their implications for olfactory processing in the fly antennal lobe

Conflicting views exist of how circuits of the antennal lobe, the insect equivalent of the olfactory bulb, translate input from olfactory receptor neurons (ORNs) into projection-neuron (PN) output. Synaptic connections between ORNs and PNs are one-to-one, yet PNs are more broadly tuned to odors than ORNs. The basis for this difference in receptive range remains unknown. Analyzing a Drosophila mutant lacking ORN input to one glomerulus, we show that some of the apparent complexity in the antennal lobe's output arises from lateral, interglomerular excitation of PNs. We describe a previously unidentified population of cholinergic local neurons (LNs) with multiglomerular processes. These excitatory LNs respond broadly to odors but exhibit little glomerular specificity in their synaptic output, suggesting that PNs are driven by a combination of glomerulus-specific ORN afferents and diffuse LN excitation. Lateral excitation may boost PN signals and enhance their transmission to third-order neurons in a mechanism akin to stochastic resonance.     

Model of cellular and network mechanisms for odor-evoked temporal patterning in the locust antennal lobe

Locust antennal lobe (AL) projection neurons (PNs) respond to olfactory stimuli with sequences of depolarizing and hyperpolarizing epochs, each lasting hundreds of milliseconds. A computer simulation of an AL network was used to test the hypothesis that slow inhibitory connections between local neurons (LNs) and PNs are responsible for temporal patterning. Activation of slow inhibitory receptors on PNs by the same GABAergic synapses that underlie fast oscillatory synchronization of PNs was sufficient to shape slow response modulations. This slow stimulus- and neuron-specific patterning of AL activity was resistant to blockade of fast inhibition. Fast and slow inhibitory mechanisms at synapses between LNs and PNs can thus form dynamical PN assemblies whose elements synchronize transiently and oscillate collectively, as observed not only in the locust AL, but also in the vertebrate olfactory bulb.     

Olfactory subsystems in the honeybee: sensory supply and sex specificity

The antennae of honeybee (Apis mellifera) workers and drones differ in various aspects. One striking difference is the presence of Sensilla basiconica in (female) workers and their absence in (male) drones. We investigate the axonal projection patterns of olfactory receptor neurons (ORNs) housed in S. basiconica in honeybee workers by using selective anterograde labeling with fluorescent tracers and confocal-microscopy analysis of axonal projections in antennal lobe glomeruli. Axons of S. basiconica-associated ORNs preferentially projected into a specific glomerular cluster in the antennal lobe, namely the sensory input-tract three (T3) cluster. T3-associated glomeruli had previously been shown to be innervated by uniglomerular projection (output) neurons of the medial antennal lobe tract (mALT). As the number of T3 glomeruli is reduced in drones, we wished to determine whether this was associated with the reduction of glomeruli innervated by medial-tract projection neurons. We retrogradely traced mALT projection neurons in drones and counted the innervated glomeruli. The number of mALT-associated glomeruli was strongly reduced in drones compared with workers. The preferential projections of S. basiconica-associated ORNs in T3 glomeruli together with the reduction of mALT-associated glomeruli support the presence of a female (worker)-specific olfactory subsystem that is partly innervated by ORNs from S. basiconica and is associated with the T3 cluster of glomeruli and mALT projection neurons. We propose that this olfactory subsystem supports parallel olfactory processing related to worker-specific olfactory tasks such as the coding of colony odors, colony pheromones and/or odorants associated with foraging on floral resources.     

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time¹. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)^2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning^4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs^6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode³. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings⁸. We provide details of our experimental setup and present representative recording traces for each of these techniques.     

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.     

Octopamine modulates activity of neural networks in the honey bee antennal lobe

Neuronal plasticity allows an animal to respond to environmental changes by modulating its response to stimuli. In the honey bee (Apis mellifera), the biogenic amine octopamine plays a crucial role in appetitive odor learning, but little is known about how octopamine affects the brain. We investigated its effect in the antennal lobe, the first olfactory center in the brain, using calcium imaging to record background activity and odor responses before and after octopamine application. We show that octopamine increases background activity in olfactory output neurons, while reducing average calcium levels. Odor responses were modulated both upwards and downwards, with more odor response increases in glomeruli with negative or weak odor responses. Importantly, the octopamine effect was variable across glomeruli, odorants, odorant concentrations and animals, suggesting that the octopaminergic network is shaped by plasticity depending on an individual animal’s history and possibly other factors. Using RNA interference, we show that the octopamine receptor AmOA1 (homolog of the Drosophila OAMB receptor) is involved in the octopamine effect. We propose a network model in which octopamine receptors are plastic in their density and located on a subpopulation of inhibitory neurons in a disinhibitory pathway. This would improve odor-coding of behaviorally relevant, previously experienced odors.     

Morphometric modeling of olfactory circuits in the insect antennal lobe: I. Simulations of spiking local interneurons.

Inhibitory local interneurons (LNs) play a critical role in shaping the output of olfactory glomeruli in both the olfactory bulb of vertebrates and the antennal lobe of insects and other invertebrates. In order to examine how the complex geometry of LNs may affect signaling in the antennal lobe, we constructed detailed multi-compartmental models of single LNs from the sphinx moth, Manduca sexta, using morphometric data from confocal-microscopic images. Simulations clearly revealed a directionality in LNs that impeded the propagation of injected currents from the sub-micron-diameter glomerular dendrites toward the much larger-diameter integrating segment (IS) in the coarse neuropil. Furthermore, the addition of randomly-firing synapses distributed across the LN dendrites (simulating the noisy baseline activity of afferent input recorded from LNs in the odor-free state) led to a significant depolarization of the LN. Thus the background activity typically recorded from LNs in vivo could influence synaptic integration and spike transformation in LNs through voltage-dependent mechanisms. Other model manipulations showed that active currents inserted into the IS can help synchronize the activation of inhibitory synapses in glomeruli across the antennal lobe. These data, therefore, support experimental findings suggesting that spiking inhibitory LNs can operate as multifunctional units under different ambient odor conditions. At low odor intensities, (i.e. subthreshold for IS spiking), they participate in local, mostly intra-glomerular processing. When activated by elevated odor concentrations, however, the same neurons will fire overshooting action potentials, resulting in the spread of inhibition more globally across the antennal lobe. Modulation of the passive and active properties of LNs may, therefore, be a deciding factor in defining the multi-glomerular representations of odors in the brain.     

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.     

A Complete Developmental Sequence of a Drosophila Neuronal Lineage as Revealed by Twin-Spot MARCM

Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.     

Spiking patterns and their functional implications in the antennal lobe of the tobacco hornworm Manduca sexta

Bursting as well as tonic firing patterns have been described in various sensory systems. In the olfactory system, spontaneous bursts have been observed in neurons distributed across several synaptic levels, from the periphery, to the olfactory bulb (OB) and to the olfactory cortex. Several in vitro studies indicate that spontaneous firing patterns may be viewed as "fingerprints" of different types of neurons that exhibit distinct functions in the OB. It is still not known, however, if and how neuronal burstiness is correlated with the coding of natural olfactory stimuli. We thus conducted an in vivo study to probe this question in the OB equivalent structure of insects, the antennal lobe (AL) of the tobacco hornworm Manduca sexta. We found that in the moth's AL, both projection (output) neurons (PNs) and local interneurons (LNs) are spontaneously active, but PNs tend to produce spike bursts while LNs fire more regularly. In addition, we found that the burstiness of PNs is correlated with the strength of their responses to odor stimulation--the more bursting the stronger their responses to odors. Moreover, the burstiness of PNs was also positively correlated with the spontaneous firing rate of these neurons, and pharmacological reduction of bursting resulted in a decrease of the neurons' responsiveness. These results suggest that neuronal burstiness reflects a physiological state of these neurons that is directly linked to their response characteristics.     

Neural Representations of Airflow in Drosophila Mushroom Body

The Drosophila mushroom body (MB) is a higher olfactory center where olfactory and other sensory information are thought to be associated. However, how MB neurons of Drosophila respond to sensory stimuli other than odor is not known. Here, we characterized the responses of MB neurons to a change in airflow, a stimulus associated with odor perception. In vivo calcium imaging from MB neurons revealed surprisingly strong and dynamic responses to an airflow stimulus. This response was dependent on the movement of the 3^rd antennal segment, suggesting that Johnston''s organ may be detecting the airflow. The calyx, the input region of the MB, responded homogeneously to airflow on. However, in the output lobes of the MB, different types of MB neurons responded with different patterns of activity to airflow on and off. Furthermore, detailed spatial analysis of the responses revealed that even within a lobe that is composed of a single type of MB neuron, there are subdivisions that respond differently to airflow on and off. These subdivisions within a single lobe were organized in a stereotypic manner across flies. For the first time, we show that changes in airflow affect MB neurons significantly and these effects are spatially organized into divisions smaller than previously defined MB neuron types.     

Model of transient oscillatory synchronization in the locust antennal lobe

Transient pairwise synchronization of locust antennal lobe (AL) projection neurons (PNs) occurs during odor responses. In a Hodgkin-Huxley-type model of the AL, interactions between excitatory PNs and inhibitory local neurons (LNs) created coherent network oscillations during odor stimulation. GABAergic interconnections between LNs led to competition among them such that different groups of LNs oscillated with periodic Ca(2+) spikes during different 50-250 ms temporal epochs, similar to those recorded in vivo. During these epochs, LN-evoked IPSPs caused phase-locked, population oscillations in sets of postsynaptic PNs. The model shows how alternations of the inhibitory drive can temporally encode sensory information in networks of neurons without precisely tuned intrinsic oscillatory properties.