Characterization and Coding of Behaviorally Significant Odor Mixtures

Animals typically perceive natural odor cues in their olfactory environment as a complex mixture of chemically diverse components. In insects, the initial representation of an odor mixture occurs in the first olfactory center of the brain, the antennal lobe (AL). The contribution of single neurons to the processing of complex mixtures in insects, and in particular moths, is still largely unknown. Using a novel multicomponent stimulus system to equilibrate component and mixture concentrations according to vapor pressure, we performed intracellular recordings of projection and interneurons in an attempt to quantitatively characterize mixture representation and integration properties of single AL neurons in the moth. We found that the fine spatiotemporal representation of 2–7 component mixtures among single neurons in the AL revealed a highly combinatorial, non-linear process for coding host mixtures presumably shaped by the AL network: 82% of mixture responding projection neurons and local interneurons showed non-linear spike frequencies in response to a defined host odor mixture, exhibiting an array of interactions including suppression, hypoadditivity, and synergism. Our results indicate that odor mixtures are represented by each cell as a unique combinatorial representation, and there is no general rule by which the network computes the mixture in comparison to single components. On the single neuron level, we show that those differences manifest in a variety of parameters, including the spatial location, frequency, latency, and temporal pattern of the response kinetics.

The discrimination of complex sensory stimuli in a noisy environment is an immense computational task. Sensory systems often encode stimulus features in a spatiotemporal fashion through the complex firing patterns of individual neurons. To identify these temporal features, we have developed an analysis that allows the comparison of statistically significant features of spike trains localized over multiple scales of time-frequency resolution. Our approach provides an original way to utilize the discrete wavelet transform to process instantaneous rate functions derived from spike trains, and select relevant wavelet coefficients through statistical analysis. Our method uncovered localized features within olfactory projection neuron (PN) responses in the moth antennal lobe coding for the presence of an odor mixture and the concentration of single component odorants, but not for compound identities. We found that odor mixtures evoked earlier responses in biphasic response type PNs compared to single components, which led to differences in the instantaneous firing rate functions with their signal power spread across multiple frequency bands (ranging from 0 to 45.71 Hz) during a time window immediately preceding behavioral response latencies observed in insects. Odor concentrations were coded in excited response type PNs both in low frequency band differences (2.86 to 5.71 Hz) during the stimulus and in the odor trace after stimulus offset in low (0 to 2.86 Hz) and high (22.86 to 45.71 Hz) frequency bands. These high frequency differences in both types of PNs could have particular relevance for recruiting cellular activity in higher brain centers such as mushroom body Kenyon cells. In contrast, neurons in the specialized pheromone-responsive area of the moth antennal lobe exhibited few stimulus-dependent differences in temporal response features. These results provide interesting insights on early insect olfactory processing and introduce a novel comparative approach for spike train analysis applicable to a variety of neuronal data sets.

Drosophila senses diverse kinds of olfactory stimuli in the external environment. These stimuli are often not pure chemicals, but rather mixtures of many odorants. The fly olfactory system, however, needs to extract the requisite information from this complex stimulus. This is done by converting odor representations into meaningful, discrete categories in the perceptual space. The first olfactory relay system in the Drosophila brain is the antennal lobe (AL) which consists of olfactory sensory neurons (OSNs), projection neurons (PNs) and local interneurons (LNs), and these three populations of neurons interact and process the odor stimuli. To observe any transformations of odor representations in terms of odor classification, linearly varying odor stimuli were presented. This was achieved using binary mixtures such that one odor was "morphed" into another. Three binary mixtures were made from combinations of three distinct odorants ,i.e., 4-methylcyclohexanol (MCH), 3-Octanol (3-Oct) and Pentyl acetate (PA). In the first step, I determined and confirmed that each single odor-evoked distinct odor representations across its different concentrations in the AL, especially in the OSNs. A second requirement was to determine the degree of similarity between the three odors in both OSNs and PNs. From their odor representations, it was found that PA was more similar to 3-Oct compared to MCH in OSNs. Moreover, the similarity between PA and 3-Oct became even stronger in PNs. After determining the prerequisites of using PA, 3-Oct and MCH for binary mixtures, odor-evoked Ca2+ activity patterns for the three different types of binary mixtures were determined in both OSNs and PNs. Each binary odor mixture showed a different kind of odor classification. The MCH/3-Oct binary mixtures showed no odor classification in OSNs, but in PNs two distinct categories were observed. However, the same could not be found in either of the other two odor mixtures. For MCH/PA binary mixtures, no odor classification was found in OSNs. But in PNs, all mixtures having greater than 0% PA got categorized and distinctly separated from the 100% MCH-0% PA. This was referred to as odor dominance of PA. The last odor mixture was of the similar odor pair, 3-Oct/PA, in which all the intermediate mixtures formed one cluster in both OSNs and PNs. This was due to the high degree of similarity already present between the individual odors in OSNs and PNs. The glomeruli in the AL are interconnected by a population of GABAergic LNs and they are known from previous studies to be involved in odor processing. GABA binds to two kinds of receptors: GABAA and GABAB. GABAA receptors are known to be involved in the early phase of the odor processing and they can be pharmacologically blocked by picrotoxin. Therefore, fly brains were subjected to picrotoxin solution and its effects were observed on the three types of odor classification found with the combination of the selected odors. GABAergic inhibition mediated via GABAA receptors was found to be required for the odor classification of binary mixtures of MCH/3-Oct. However, no changes were found due to picrotoxin on MCH/PA and 3-Oct/PA binary mixtures. In conclusion, different kinds of odor classification could be observed for different odors in the AL of the fly. GABAergic inhibition has been found to play a role in mediating odor classification of different stimuli. However, not every odor classification requires GABAergic inhibition, as was observed in the case of MCH/PA binary mixtures, thereby also suggesting an existence of other possible mechanisms for odor classification.

Natural olfactory stimuli are often complex mixtures of volatiles, of which the identities and ratios of constituents are important for odor-mediated behaviors. Despite this importance, the mechanism by which the olfactory system processes this complex information remains an area of active study. In this review, we describe recent progress in how odorants and mixtures are processed in the brain of insects. We use a comparative approach toward contrasting olfactory coding and the behavioral efficacy of mixtures in different insect species, and organize these topics around four sections: (1) Examples of the behavioral efficacy of odor mixtures and the olfactory environment; (2) mixture processing in the periphery; (3) mixture coding in the antennal lobe; and (4) evolutionary implications and adaptations for olfactory processing. We also include pertinent background information about the processing of individual odorants and comparative differences in wiring and anatomy, as these topics have been richly investigated and inform the processing of mixtures in the insect olfactory system. Finally, we describe exciting studies that have begun to elucidate the role of the processing of complex olfactory information in evolution and speciation.

In colonies of eusocial Hymenoptera cooperation is organized through social odors, and particularly ants rely on a sophisticated odor communication system. Neuronal information about odors is represented in spatial activity patterns in the primary olfactory neuropile of the insect brain, the antennal lobe (AL), which is analog to the vertebrate olfactory bulb. The olfactory system is characterized by neuroanatomical compartmentalization, yet the functional significance of this organization is unclear. Using two-photon calcium imaging, we investigated the neuronal representation of multicomponent colony odors, which the ants assess to discriminate friends (nestmates) from foes (nonnestmates). In the carpenter ant Camponotus floridanus, colony odors elicited spatial activity patterns distributed across different AL compartments. Activity patterns in response to nestmate and nonnestmate colony odors were overlapping. This was expected since both consist of the same components at differing ratios. Colony odors change over time and the nervous system has to constantly adjust for this (template reformation). Measured activity patterns were variable, and variability was higher in response to repeated nestmate than to repeated nonnestmate colony odor stimulation. Variable activity patterns may indicate neuronal plasticity within the olfactory system, which is necessary for template reformation. Our results indicate that information about colony odors is processed in parallel in different neuroanatomical compartments, using the computational power of the whole AL network. Parallel processing might be advantageous, allowing reliable discrimination of highly complex social odors.

Insects use information about CO2 to perform vital tasks such as locating food sources. In certain moths, CO2 is involved in oviposition behavior. The labial palps of adult moths that feed as adults have a pit organ containing sensory receptor cells that project into the antennal lobes, the sites of primary processing of olfactory information in the brain. In the moth Manduca sexta and certain other species of Lepidoptera, these receptor cells in the labial-palp pit organ have been shown to be tuned to CO2, and their axons project to a single, identified glomerulus in the antennal lobe, the labial-palp pit organ glomerulus. At present, however, nothing is known about the function of this glomerulus or how CO2 information is processed centrally. We used intracellular recording and staining to reveal projection (output) neurons in the antennal lobes that respond to CO2 and innervate the labial-palp pit organ glomerulus. Our results demonstrate that this glomerulus is the site of first-order processing of sensory information about ambient CO2. We found three functional types of CO2-responsive neurons (with their cell bodies in the antennal lobe or the protocerebrum) that provide output from the antennal lobe to higher centers in the brain. Some physiological characteristics of those neurons are described.

Neurons in the silkmoth antennal lobe (AL) are well characterized in terms of their morphology and odor-evoked firing activity. However, their intrinsic electrical properties including voltage-gated ionic currents and synaptic connectivity remain unclear. To address this, whole cell current- and voltage-clamp recordings were made from second-order projection neurons (PNs) and two morphological types of local interneurons (LNs) in the silkmoth AL. The two morphological types of LNs exhibited distinct physiological properties. One morphological type of LN showed a spiking response with a voltage-gated sodium channel gene expression, whereas the other type of LN was nonspiking without a voltage-gated sodium channel gene expression. Voltage-clamp experiments also revealed that both of two types of LNs as well as PNs possessed two types of voltage-gated potassium channels and calcium channels. In dual whole cell recordings of spiking LNs and PNs, activation of the PN elicited depolarization responses in the paired spiking LN, whereas activation of the spiking LN induced no substantial responses in the paired PN. However, simultaneous recording of a nonspiking LN and a PN showed that activation of the nonspiking LN induced hyperpolarization responses in the PN. We also observed bidirectional synaptic transmission via both chemical and electrical coupling in the pairs of spiking LNs. Thus our results indicate that there were two distinct types of LNs in the silkmoth AL, and their functional connectivity to PNs was substantially different. We propose distinct functional roles for these two different types of LNs in shaping odor-evoked firing activity in PNs.

A multitude of potential neurotransmitters and neuromodulators, including peptides, have been detected in the antennal lobe (AL), the first synaptic relay of the central olfactory pathway in the insect brain. However, the functional role of neuropeptides in this system has yet to be revealed. An important prerequisite to understanding the role of neuropeptides is to match the functionally different cell types in the AL with their peptide profiles by using electrophysiological recordings combined with immunocytochemical studies and/or single-cell mass spectrometry. The olfactory system of Periplaneta americana is particularly well suited to accomplish this goal because several physiologically distinct neuron types can be unequivocally identified. With the aim to analyze the neuropeptide inventory of the P. americana AL, this study is an essential step in this direction. First, we systematically analyzed different parts of the AL by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to obtain the complete set of neuropeptides present. Altogether, 56 ion signals could be assigned to products of 10 neuropeptide genes (allatostatins A, B, C, SIFamide, allatotropin, FMRFamide-related peptides [myosuppressin, short neuropeptides F, extended FMRFamides], crustacean cardioactive peptide, tachykinin-related peptides). In a second step, a combination of immunocytochemistry and mass spectrometric profiling of defined AL compartments was used to reveal the spatial distribution of neuropeptide-containing cells. Finally, we demonstrated the feasibility of MALDI-TOF mass spectrometric profiling of single AL neurons, which is an important precondition for combining electrophysiology with peptide profiling at the single-cell level.

An important component in understanding central olfactory processing and coding in the insect brain relates to the characterization of the functional divisions between morphologically distinct types of projection neurons (PN). Using calcium imaging, we investigated how the identity, concentration and mixtures of odors are represented in axon terminals (boutons) of two types of PNs – lPN and mPN. In lPN boutons we found less concentration dependence, narrow tuning profiles at a high concentration, which may be optimized for fine, concentration-invariant odor discrimination. In mPN boutons, however, we found clear rising concentration dependence, broader tuning profiles at a high concentration, which may be optimized for concentration coding. In addition, we found more mixture suppression in lPNs than in mPNs, indicating lPNs better adaptation for synthetic mixture processing. These results suggest a functional division of odor processing in both PN types.

Associative memory in the Mushroom Body of the fruit fly brain depends on the encoding and processing of odorants in the first three stages of the Early Olfactory System: the Antenna, the Antennal Lobe and the Mushroom Body Calyx. The Kenyon Cells (KCs) of the Calyx provide the Mushroom Body compartments the identity of pure and odorant mixtures encoded as a train of spikes. Characterizing the code underlying the KC spike trains is a major challenge in neuroscience. To address this challenge we start by explicitly modeling the space of odorants using constructs of both semantic and syntactic information. Odorant semantics concerns the identity of odorants while odorant syntactics pertains to their concentration amplitude. These odorant attributes are multiplicatively coupled in the process of olfactory transduction. A key question that early olfactory systems must address is how to disentangle the odorant semantic information from the odorant syntactic information. To address the untanglement we devised an Odorant Encoding Machine (OEM) modeling the first three stages of early olfactory processing in the fruit fly brain. Each processing stage is modeled by Divisive Normalization Processors (DNPs). DNPs are spatio-temporal models of canonical computation of brain circuits. The end-to-end OEM is constructed as cascaded DNPs. By extensively modeling and characterizing the processing of pure and odorant mixtures in the Calyx, we seek to answer the question of its functional significance. We demonstrate that the DNP circuits in the OEM combinedly reduce the variability of the Calyx response to odorant concentration, thereby separating odorant semantic information from syntactic information. We then advance a code, called first spike sequence code, that the KCs make available at the output of the Calyx. We show that the semantics of odorants can be represented by this code in the spike domain and is ready for easy memory access in the Mushroom Body compartments.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions. Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP and Cameleon, the bioluminescent calcium indicator GFP-aequorin, or a reporter of synaptic transmission, synapto-pHluorin has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS, LexA/LexAop, Q system) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed. Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

The neurobiology of insect olfaction: Sensory processing in a comparative context

Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons.

Honey bees have a rich repertoire of olfactory learning behaviors, and they therefore are an excellent model to study plasticity in olfactory circuits. Recent behavioral, physiological, and molecular evidence suggested that the antennal lobe, the first relay of the olfactory system in insects and analog to the olfactory bulb in vertebrates, is involved in associative and nonassociative olfactory learning. Here we use calcium imaging to reveal how responses across antennal lobe projection neurons change after association of an input odor with appetitive reinforcement. After appetitive conditioning to 1-hexanol, the representation of an odor mixture containing 1-hexanol becomes more similar to this odor and less similar to the background odor acetophenone. We then apply computational modeling to investigate how changes in synaptic connectivity can account for the observed plasticity. Our study suggests that experience-dependent modulation of inhibitory interactions in the antennal lobe aids perception of salient odor components mixed with behaviorally irrelevant background odors.

BackgroundOptical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system.MethodologyWe visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx.ConclusionsThis study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.