Discrimination training with multimodal stimuli changes activity in the mushroom body of the hawkmoth Manduca sexta

Background

The mushroom bodies of the insect brain play an important role in olfactory processing, associative learning and memory. The mushroom bodies show odor-specific spatial patterns of activity and are also influenced by visual stimuli.

Methodology/Principal Findings

Functional imaging was used to investigate changes in the in vivo responses of the mushroom body of the hawkmoth Manduca sexta during multimodal discrimination training. A visual and an odour stimulus were presented either together or individually. Initially, mushroom body activation patterns were identical to the odour stimulus and the multimodal stimulus. After training, however, the mushroom body response to the rewarded multimodal stimulus was significantly lower than the response to the unrewarded unimodal odour stimulus, indicating that the coding of the stimuli had changed as a result of training. The opposite pattern was seen when only the unimodal odour stimulus was rewarded. In this case, the mushroom body was more strongly activated by the multimodal stimuli after training. When no stimuli were rewarded, the mushroom body activity decreased for both the multimodal and unimodal odour stimuli. There was no measurable response to the unimodal visual stimulus in any of the experiments. These results can be explained using a connectionist model where the mushroom body is assumed to be excited by olfactory stimulus components, and suppressed by multimodal configurations.

Conclusions

Discrimination training with multimodal stimuli consisting of visual and odour cues leads to stimulus specific changes in the in vivo responses of the mushroom body of the hawkmoth.     

Modelling efficiency in insect olfactory information processing

The olfactory system of insects is essential for the search of food and mates, and weak signals can be detected, amplified and discriminated in a fluctuating environment. The olfactory system also allows for learning and recall of odour memories. Based on anatomical, physiological, and behavioural data from the olfactory system of insects, we have developed a cross-scale dynamical neural network model to simulate the presentation, amplification and discrimination of host plant odours and sex pheromones. In particular, we model how the spatial and temporal patterns of the odour information emerging in the glomeruli of the antennal lobe (AL) rely on the glomerular morphology, the connectivity and the complex dynamics of the AL circuits. We study how weak signals can be amplified, how different odours can be discriminated, based on stochastic (resonance) dynamics and the connectivity of the network. We further investigate the spatial and temporal coding of sex pheromone components and plant volatile compounds, in relation to the glomerular structure, arborizing patterns of the projection neurons (PNs) and timing patterns of the neuronal spiking activity.     

Perception of plant volatile blends by herbivorous insects--finding the right mix

Volatile plant secondary metabolites are detected by the highly sensitive olfactory system employed by insects to locate suitable plants as hosts and to avoid unsuitable hosts. Perception of these compounds depends on olfactory receptor neurones (ORNs) in sensillae, mostly on the insect antennae, which can recognise individual molecular structures. Perception of blends of plant volatiles plays a pivotal role in host recognition, non-host avoidance and ensuing behavioural responses as different responses can occur to a whole blend compared to individual components. There are emergent properties of blend perception because components of the host blend may not be recognised as host when perceived outside the context of that blend. Often there is redundancy in the composition of blends recognised as host because certain compounds can be substituted by others. Fine spatio-temporal resolution of the synchronous firing of ORNs tuned to specific compounds enables insects to pick out relevant host odour cues against high background noise and with ephemeral exposure to the volatiles at varying concentrations. This task is challenging as they usually rely on ubiquitous plant volatiles and not those taxonomically characteristic of host plants. However, such an odour coding system has the advantage of providing flexibility; it allows for adaptation to changing environments by alterations in signal processing while maintaining the same peripheral olfactory receptors.     

Olfactory perception in honeybees: Concatenated and mixed odorant stimuli,concentration, and exposure effects

Summary Here we present results obtained from 7 different series of experiments, all employing odor conditioning of proboscis extension in worker honeybees and each designed to address a particular question involving olfactory perception. The questions relate to: temporal complexity of odor cues; effects of concentration, suppression, and/or potentiation in mixture perception; acquisition and extinction rates, as well as levels of generalization associated with aliphatic compounds that have the same functional groups or same alkyl radical length; and the effects of continuous exposure to odorants in the first several days of adult life on various learning and discrimination tasks involving olfactory perception. From the data obtained in these experiments we were able to conclude the following: First, worker honeybees have a limited ability to perceive complex temporal odor-quality patterns in olfactory stimuli — they learn to associate the quality of only the last part of the stimulus with a sucrose reward. Second, we confirm that citral is qualitatively different in several perceptual contexts involving odor learning and conditioning and our results help elucidate the nature of these differences as they relate to learning, discrimination, mixture perception, and continuous exposure to particular odorants. Third, we appear to have uncovered some important perceptual differences between functional groups attached to the first as opposed to the second carbon atom of alkyl radicals. Finally, we failed to uncover any significant effects relating to continuous exposure to odorants during the first several days of a worker's adult life, despite evidence that considerable sensory development takes place during this period. Thus ontogenetic changes to the peripheral system due to environmental effects appear to leave basic perceptual systems unaltered.     

Identification of plant odours activating receptor neurones in the weevil<Emphasis Type="Italic"> Pissodes notatus</Emphasis> F. (Coleoptera,Curculionidae)

Plants release complex mixtures of volatiles important in the interaction with insects and other organisms. In the search for compounds that contribute to the perception of odour quality in the weevil Pissodes notatus, single olfactory receptor neurones on the antennae were screened for sensitivity to naturally produced plant volatiles by the use of gas chromatography linked to single cell recordings. We here present 60 olfactory neurones responding to 25 of the numerous compounds released by host and non-host plants. All the neurones show high selectivity and are classified into 12 distinct types. The two most abundant types respond to alpha-pinene, beta-pinene, and 3-carene ( n=17), and to isopinocamphone and pinocamphone ( n=17), respectively. Other neurone types respond to limonene ( n=9), beta-phellandrene ( n=3), and fenchone ( n=4). Responses to beta-caryophyllene ( n=1) and to ethanol ( n=4) are also shown. Except for two pairs, the neurone types do not show overlap of the molecular receptive range. The active compounds are present in the host, Pinus pinaster, as well as in non-hosts, supporting the idea that plant odour quality is mediated by the ratio of the compounds rather than specific odorants.     

Odour representation in honeybee olfactory glomeruli shows slow temporal dynamics: an optical recording study using a voltage-sensitive dye

Stimulation with odours has been shown to elicit characteristic patterns of activated glomeruli in the antennal lobe (AL) of honeybees. In this study we show that these patterns are dynamic in a time window of 2-3 s after stimulus onset. We measured changes in the averaged membrane potential of all cells in the glomerular neuropil by optical imaging of the voltage-sensitive dye RH795 using a slow scan CCD camera (3 frames/s). The four substances 1-hexanol, hexanal, citral and clove-oil as well as the binary mixtures hexanol+hexanal and hexanol+citral were used as stimuli (2 s stimulus duration). We found that: (1) every odour elicited an odour-specific activity pattern, and conversely every glomerulus had a characteristic odour response profile; (2) some glomeruli had a tonic, some a phasic-tonic, and some a slow phasic response pattern; (3) the difference between the glomerular response patterns increased within 2 s of stimulus presentation, which suggests that odour representations became more characteristic over stimulus time; and (4) the responses to odorant mixtures were complex and glomerulus-dependent: some responses correspond to the sum of the compounds' responses, some to the response of one of the components.     

Patterned response to odor in single neurones of goldfish olfactory bulb: influence of odor quality and other stimulus parameters

Responses of 75 single units in the goldfish olfactory bulb were analyzed in detail for their relationship to the time-course of the change in odor concentration during each odor stimulus. Odor stimuli were controlled for rise time, duration, and peak concentration by an apparatus developed for the purpose. This apparatus enabled aqueous odor stimuli to be interposed into a constant water stream without changes in flow rate. The time-course of the concentration change within the olfactory sac was inferred from conductivity measurements at the incurrent and excurrent nostrils. Temporal patterns of firing rate elicited by stimuli with relatively slow rising and falling phases could be quite complex combinations of excitation and suppression. Different temporal patterns were produced by different substances at a single concentration in most units. Statistical measures of the temporal pattern of response for a small number of cells at a given concentration were more characteristic of the stimulus substance than any of three measures of magnitude of response. The temporal patterns change when the peak concentration, duration, and rise time of the stimuli are varied. The nature of these changes suggests that the different patterns are due primarily to the combined influence of two factors: (a) a stimulus whose concentration varies over time and (b) a relationship between concentration and impulse frequency which varies from unit to unit. Some units produce patterns suggestive of influence by neural events of long time constant. The importance of temporal patterns in odor quality and odor intensity coding is discussed.     

Dynamic representation of odours by oscillating neural assemblies

Stimulus evoked oscillatory synchronization of neural assemblies has been most clearly documented in the olfactory and visual systems. Recent results with the olfactory system of locusts show that information about odour identity is contained in spatial and temporal aspects of an oscillatory population response. This suggests that brain oscillations may reflect a common reference for messages encoded in time. Although stimulus-evoked oscillatory phenomena are reliable, their roles in perception, memory and pattern recognition remain to be demonstrated. Using honey bees, we demonstrated that odour encoding involves, as in locusts, the oscillatory synchronization of assemblies of neurons, and that this synchronization is, here also, selectively abolished by the GABA receptor antagonist picrotoxin. In collaboration with Dr Brian Smith's laboratory, we showed, using a behavioural learning paradigm, that picrotoxin-induced desynchronization impairs the discrimination of molecularly similar odourants, but not that of dissimilar odours. It appears, therefore, that oscillatory synchronization of neuronal assemblies is relevant, and essential for fine odour discrimination. Finally, experiments with locust mushroom body neurons, two synapses downstream from the antennal lobe, indicate that their responses to odours become less specific when antennal lobe neurons are desynchronized by picrotoxin injection. These results suggest that oscillatory synchronization and the kind of temporal encoding it affords provide an additional dimension by which the brain can segment spatially overlapping stimulus representations.     

Stimulus and network dynamics collide in a ratiometric model of the antennal lobe macroglomerular complex

Time is considered to be an important encoding dimension in olfaction, as neural populations generate odour-specific spatiotemporal responses to constant stimuli. However, during pheromone mediated anemotactic search insects must discriminate specific ratios of blend components from rapidly time varying input. The dynamics intrinsic to olfactory processing and those of naturalistic stimuli can therefore potentially collide, thereby confounding ratiometric information. In this paper we use a computational model of the macroglomerular complex of the insect antennal lobe to study the impact on ratiometric information of this potential collision between network and stimulus dynamics. We show that the model exhibits two different dynamical regimes depending upon the connectivity pattern between inhibitory interneurons (that we refer to as fixed point attractor and limit cycle attractor), which both generate ratio-specific trajectories in the projection neuron output population that are reminiscent of temporal patterning and periodic hyperpolarisation observed in olfactory antennal lobe neurons. We compare the performance of the two corresponding population codes for reporting ratiometric blend information to higher centres of the insect brain. Our key finding is that whilst the dynamically rich limit cycle attractor spatiotemporal code is faster and more efficient in transmitting blend information under certain conditions it is also more prone to interference between network and stimulus dynamics, thus degrading ratiometric information under naturalistic input conditions. Our results suggest that rich intrinsically generated network dynamics can provide a powerful means of encoding multidimensional stimuli with high accuracy and efficiency, but only when isolated from stimulus dynamics. This interference between temporal dynamics of the stimulus and temporal patterns of neural activity constitutes a real challenge that must be successfully solved by the nervous system when faced with naturalistic input.     

Primary events in odour detection

An analysis of the interaction between stimulus molecules and the olfactory receptor cell membrane is presented. The model is based upon a sequence of events, i.e. stimulus delivery at the olfactory epithelium, absorption of molecules in the mucus layer, diffusion of the molecules towards the receptor cells and molecule-receptor cell membrane interaction. The mathematical analysis considers the situation during electrophysiological experiments, where an odour puff is delivered at an exposed olfactory mucosa. Such a situation resembles sniffing of odour samples. The analysis is discussed in relation to experimental evidence.     

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal´s dynamic behavioral reaction.     

Cross-modal identification: Effects of contingent changes in the stimulus series

Two computer controlled experiments in an olfactory cross-modal matching task, using two-component odour mixtures matched against bar diagrams, were designed so that stimulus presentation was contingent upon the recent performance of the subject; stimuli that were relatively poorly (in experiment 1) or well (in experiment 2) matched were more frequently presented. Analysis shows that the autoregressive structure of the performance is modified by such contingent presentation and that there is a weak relationship between transmitted information in matching and the time series structure of the matching errors. It is suggested that the process is nonlinear.     

The role of glomeruli in the neural representation of odours: results from optical recording studies

Odours are received by olfactory receptors, which send their axons to the first sensory neuropils, the antennal lobes (in insects) or the olfactory bulb (in vertebrates). From here, processed olfactory information is relayed to higher-order brain centres. A striking similarity in olfactory systems across animal phyla is the presence of glomeruli in this first sensory neuropil. Various experiments have shown that odours elicit a mosaic of activated glomeruli, suggesting that odour quality is coded in an 'across-glomeruli' activity code. In recent years, studies using optical recording techniques have greatly improved our understanding of the resulting 'across-glomeruli pattern', making it possible to simultaneously measure responses in several, often identifiable, glomeruli. For the honeybee Apis mellifera, a functional atlas of odour representation is being created: in this atlas, the glomeruli that are activated by different odorants are named. However, several limitations remain to be investigated. In this paper, we review what optical recording of odour-evoked glomerular activity patterns has revealed so far, and discuss the necessary next steps, with emphasis on the honeybee.     

Mixture and odorant processing in the olfactory systems of insects: a comparative perspective

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.     

Cross-modal interactions between olfaction and touch

We report two experiments designed to investigate the nature of any cross-modal interactions between olfactory and tactile information processing. In Experiment 1, we assessed the influence of olfactory cues on the tactile perception of fabric softness using computer-controlled stimulus presentation. The results showed that participants rated fabric swatches as feeling significantly softer when presented with a lemon odor than when presented with an animal-like odor, demonstrating that olfactory cues can modulate tactile perception. In Experiment 2, we assessed whether this modulatory effect varied as a function of the particular odors being used and/or of the spatial coincidence between the olfactory and tactile stimuli. The results replicated those reported in Experiment 1 thus further supporting the claim that people's rating of tactile stimuli can be modulated by the presence of an odor. Taken together, the results of the two experiments reported here support the existence of a cross-modal interaction between olfaction and touch.     

Olfactory responses to attractants and repellents in tsetse

The aims of this study were to investigate how antennal olfactory cells of tsetse (Diptera: Glossinidae) code odour quality and how they are able to discriminate between attractive and repellent odours. For Glossina pallidipes Austen, a survey is presented of the cells' responses to attractive (1-octen-3-ol, acetone, 3-methylphenol, carbon dioxide) and repellent stimuli (2-methoxyphenol, acetophenone, lactic acid, naphthalene). In addition, the responses of these cells to binary mixtures and the dose-response curves of 1-octen-3-ol, 3-methylphenol, 2-methoxyphenol and acetophenone are presented. A minority of the cells responded to one attractant or repellent only, whereas the vast majority were excited by more than one of the attractive and/or repellent stimuli. It is proposed that the peripheral olfactory cells of tsetse discriminate between different compounds via an across-fibre pattern coding, in which the cells that specifically code for attractants or repellents may play a substantial role in composing a unique excitation pattern that informs the central nervous system about the specificity of odours.     

Odour concentration affects odour identity in honeybees

The fact that most types of sensory stimuli occur naturally over a large range of intensities is a challenge to early sensory processing. Sensory mechanisms appear to be optimized to extract perceptually significant stimulus fluctuations that can be analysed in a manner largely independent of the absolute stimulus intensity. This general principle may not, however, extend to olfaction; many studies have suggested that olfactory stimuli are not perceptually invariant with respect to odour intensity. For many animals, absolute odour intensity may be a feature in itself, such that it forms a part of odour identity and thus plays an important role in discrimination alongside other odour properties such as the molecular identity of the odorant. The experiments with honeybees reported here show a departure from odour-concentration invariance and are consistent with a lower-concentration regime in which odour concentration contributes to overall odour identity and a higher-concentration regime in which it may not. We argue that this could be a natural consequence of odour coding and suggest how an 'intensity feature' might be useful to the honeybee in natural odour detection and discrimination.     

A combined perceptual, physico-chemical, and imaging approach to 'odour-distances' suggests a categorizing function of the Drosophila antennal lobe

How do physico-chemical stimulus features, perception, and physiology relate? Given the multi-layered and parallel architecture of brains, the question specifically is where physiological activity patterns correspond to stimulus features and/or perception. Perceived distances between six odour pairs are defined behaviourally from four independent odour recognition tasks. We find that, in register with the physico-chemical distances of these odours, perceived distances for 3-octanol and n-amylacetate are consistently smallest in all four tasks, while the other five odour pairs are about equally distinct. Optical imaging in the antennal lobe, using a calcium sensor transgenically expressed in only first-order sensory or only second-order olfactory projection neurons, reveals that 3-octanol and n-amylacetate are distinctly represented in sensory neurons, but appear merged in projection neurons. These results may suggest that within-antennal lobe processing funnels sensory signals into behaviourally meaningful categories, in register with the physico-chemical relatedness of the odours.     

Plant odour plumes as mediators of plant–insect interactions

Insect olfactory orientation along odour plumes has been studied intensively with respect to pheromonal communication, whereas little knowledge is available on how plant odour plumes (POPs) affect olfactory searching by an insect for its host plants. The primary objective of this review is to examine the role of POPs in the attraction of insects. First, we consider parameters of an odour source and the environment which determine the size, shape and structure of an odour plume, and we apply that knowledge to POPs. Second, we compare characteristics of insect pheromonal plumes and POPs. We propose a ‘POP concept’ for the olfactory orientation of insects to plants. We suggest that: (i) an insect recognises a POP by means of plant volatile components that are encountered in concentrations higher than a threshold detection limit and that occur in a qualitative and quantitative blend indicating a resource; (ii) perception of the fine structure of a POP enables an insect to distinguish a POP from an unspecific odorous background and other interfering plumes; and (iii) an insect can follow several POPs to their sources, and may leave the track of one POP and switch to another one if this conveys a signal with higher reliability or indicates a more suitable resource. The POP concept proposed here may be a useful tool for research in olfactory‐mediated plant–insect interactions.     

Temporal Features of Spike Trains in the Moth Antennal Lobe Revealed by a Comparative Time-Frequency Analysis

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.