Representation of natural stimuli in the rodent main olfactory bulb

Natural odorants are complex mixtures of diverse chemical compounds. Monomolecular odorants are represented in the main olfactory bulb by distinct spatial patterns of activated glomeruli. However, it remains unclear how individual compounds contribute to population representations of natural stimuli, which appear to be unexpectedly sparse. We combined gas chromatography and intrinsic signal imaging to visualize glomerular responses to natural stimuli and their fractionated components. While whole stimuli activated up to 20 visible glomeruli, each fractionated component activated only one or few glomeruli, and most glomeruli were activated by only one component. Thus, responses to complex mixtures reflected activation by multiple components, with each contributing only a small part of the overall representation. We conclude that the population response to a complex stimulus is largely the sum of the responses to its individual components, and activation of an individual glomerulus independently signals the presence of a specific component.     

Calcium responses to pheromones and plant odours in the antennal lobe of the male and female moth Heliothis virescens

In male moths, the primary olfactory integration centre, the antennal lobe, consists of two systems. The macroglomerular complex processes pheromone information, while the ordinary glomeruli process plant odour information. Females lack a macroglomerular complex. We measured the spatial representation of odours using in-vivo optical recording. We found that: (1) pheromone substances elicited activity exclusively in the MGC. No response was found in female antennal lobes. (2) Plant odours elicited combinatorial activity patterns in the ordinary glomeruli in both males and females. No response was found in the MGC of male moths. (3) A clean air puff often led to activity, in both males and females, suggesting that mechano-sensory information is also processed in the antennal lobe. (4) With an interstimulus interval of 5 or 10 s, strongly activated glomeruli were able to follow the temporal structure of the stimulus, while others lost their phase-locking. Some glomeruli showed "off" responses. These properties were odour dependent. This confirms and extends previous studies, showing the functional significance of the two subsystems for processing olfactory information. Pheromones are coded in a combinatorial manner within the macroglomerular complex, with each glomerulus corresponding to one information channel. Plant odours are coded in an across-glomeruli code in the ordinary glomeruli.     

Sequence matters – selective adaptation in electroantennographic response to binary odour mixtures by the Colorado potato beetle

Chemically mediated behaviour of insects is often strongly affected by mixtures of odour stimuli and their temporal characteristics. Both sensory transduction and central processing of odour mixtures can give rise to several different kinds of interaction, which can influence how individual components are perceived and processed. In particular, odour mixtures have been examined in model experiments as premixed binary mixtures in comparison with pure odour stimuli. Only in few experiments, the influence of the temporal structure of odour mixtures on odour perception has been taken into account. Natural odour stimuli often have a pulsed structure and may in general be superimposed on a background of irrelevant or interfering compounds, which can fluctuate with different frequencies, depending on their source. To achieve a better representation of these natural conditions, our odour mixing experiments apply a new kind of stimulation protocol: odours were not premixed but superimposed with a specific time pattern; one odour stimulus was presented as a longer persisting background and the second stimulus was a superimposed short test signal. To gain an overview of odour interaction patterns in the Colorado potato beetle by causing adaptation of one receptor population at naturally occurring levels of concentration and time intervals, electroantennographic recordings were made on excised antennae. A matrix of 12 stimulus compounds led to 132 pairs of compounds tested, each in the role of background and test stimulus. In 64 cases, the interaction was significantly different, when the role of background and stimulus was exchanged. Interaction patterns ranging from no interference (independence) to suppression were found and assigned to four clearly distinguishable types. We discuss that the observed effects of the presentation sequence in odour mixtures may contribute to the mechanisms of olfactory pattern recognition and olfactory contrast perception by insects.     

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.     

Dose-response characteristics of glomerular activity in the moth antennal lobe

Odours are represented as unique combinations of activated glomeruli in the antennal lobes of insects. Receptor neurons arborizing in the glomeruli are not only qualitatively selective, but in addition respond to variations in stimulus concentration. As each glomerulus likely represents a single receptor neuron type, optical recordings of calcium changes in insect antennal lobes show how concentration variations affect a large population of afferents. We measured the glomerular responses in the moth Spodoptera littoralis to different concentrations of plant-related odorants. Localized calcium responses were shown to correspond to individual glomeruli. We found that the dynamic range of glomerular responses spanned 3-4 log units of concentration and the most strongly responding glomeruli often reached a plateau at high stimulus doses. Further, we showed that the single most active glomerulus was often not the same across concentrations. However, if the principal glomerulus moved, it was generally to an adjacent or proximal glomerulus. As concentration increased, a higher number of glomeruli became activated. Correlations of glomerular representations of the same compound at different doses decreased as the difference in concentration increased. Moreover, representations evoked by different odorants were more correlated at high than at low doses, which means that the uniqueness of activity patterns decreased with increasing concentration. Thus, if odours are coded as spatial patterns of glomerular activity, as has been suggested, these olfactory codes are not persistent across concentrations.     

Perceptual interactions between characteristic notes smelled above aqueous solutions of odorant mixtures

Twenty-two experienced panelists rated odor intensity of aqueous solutions of citral, octen-1-ol-3, and hexanal. The panel assessed unmixed components and mixtures (9 binary and 4 ternary). In sensory sessions dedicated to mixtures (n = 6), evaluation was focused on one target odor, presented at a fixed concentration. All components had lower odor intensity on mixed presentations. In many cases, information obtained from simpler systems was not extended to complex mixtures. In a mixture, the competition between odorant molecules on qualitative aspects (dominance/suppression) imbalanced components contribution, anticipated from the quantitative distribution. Hexanal appeared to be the potentially weaker odorant in paired combinations, whereas octen-1-ol-3 had a lower relative impact on ternary systems. Suppression of the odor of octen-1-ol-3 and a concomitant increase in the odor of hexanal was common to all ternary mixtures. Reciprocal inhibition of octen-1-ol-3 and citral odors through perceptual interactions was suspected. Mutual suppression is suspected to have eased the perception of hexanal intensity.     

Topological reorganization of odor representations in the olfactory bulb

Odors are initially represented in the olfactory bulb (OB) by patterns of sensory input across the array of glomeruli. Although activated glomeruli are often widely distributed, glomeruli responding to stimuli sharing molecular features tend to be loosely clustered and thus establish a fractured chemotopic map. Neuronal circuits in the OB transform glomerular patterns of sensory input into spatiotemporal patterns of output activity and thereby extract information about a stimulus. It is, however, unknown whether the chemotopic spatial organization of glomerular inputs is maintained during these computations. To explore this issue, we measured spatiotemporal patterns of odor-evoked activity across thousands of individual neurons in the zebrafish OB by temporally deconvolved two-photon Ca²⁺ imaging. Mitral cells and interneurons were distinguished by transgenic markers and exhibited different response selectivities. Shortly after response onset, activity patterns exhibited foci of activity associated with certain chemical features throughout all layers. During the subsequent few hundred milliseconds, however, MC activity was locally sparsened within the initial foci in an odor-specific manner. As a consequence, chemotopic maps disappeared and activity patterns became more informative about precise odor identity. Hence, chemotopic maps of glomerular input activity are initially transmitted to OB outputs, but not maintained during pattern processing. Nevertheless, transient chemotopic maps may support neuronal computations by establishing important synaptic interactions within the circuit. These results provide insights into the functional topology of neural activity patterns and its potential role in circuit function.     

Representation of primary plant odorants in the antennal lobe of the moth Heliothis virescens using calcium imaging

The primary olfactory centre, the antennal lobe of Heliothis virescens moths, contains 62 glomeruli which process plant odour information and four male-specific glomeruli which form the macroglomerular complex, involved in processing information about pheromone and interspecific signals. Using calcium imaging, we recorded the spatio-temporal activity pattern of the glomeruli in the anterior antennal lobe during stimulation with odorants produced by plants or insects. Each odorant elicited specific excitatory responses in one or a few glomeruli: the major pheromone component did so exclusively in the large glomerulus of the macroglomerular complex and the plant odours exclusively in the ordinary glomeruli. Eight glomeruli, with corresponding plant odour responses and positions, were identified within each sex. Glomeruli responded specifically to linalool, beta-ocimene/beta-myrcene or germacrene D/alpha-farnesene. Responses to two essential plant oils covered the response areas of their major constituents, as well as activating additional glomeruli. Stronger activation in the AL due to increased odour concentration was expressed as increased response strength within the odorant-specific glomeruli as well as recruitment of less sensitive glomeruli.     

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.     

Neurophysiological and behavioural evidence for an olfactory function for the dorsal organ and a gustatory one for the terminal organ in Drosophila melanogaster larvae

Multicellular electrophysiological responses from the dorsal organ on the cephalic lobes of third instar Drosophila melanogaster larvae (wild-type Canton S) stimulated with a cold-trapped banana volatile extract showed that this structure has an olfactory function in the fruit fly. Responses of the dorsal organ were also recorded to constituents of the banana volatile extract as they eluted from a gas chromatographic column (GC-coupled dorsal organ electrophysiology). The active chemostimulants were identified as 2-heptanone, isoamyl alcohol, hexyl acetate, hexanol and hexyl butyrate by gas chromatography-coupled mass spectrometry. Applying the same recording system to the terminal organ sensilla, no responses were obtained to either the banana volatile bouquet or its constituents. By contrast, high frequency multicellular responses were recorded in response to touching the terminal organ with the gustatory stimuli KCl and grapefruit juice; responses were absent on similar stimulation of the dorsal organ with either NaCl or KCl. This suggests a role for olfaction by the dorsal organ and for gustation by the terminal organ in Drosophila larvae.In a 7-mm high wind tunnel with a thin 1.2% agar floor, the Drosophila larvae showed odour-conditioned upwind responses in an air stream of 0.1 m/s bearing banana volatiles. Drosophila larvae responded best to the odour of cut bananas. A 1:1 mixture of the banana odour constituents 2-heptanone and hexanol (at either 50 or 100 &mgr;g source dose each) proved as attractive as the known larval attractants propionic acid and isoamyl acetate on their own at 100 &mgr;g, whereas hexanol and 2-heptanone on their own at a 100 &mgr;g source dose were less attractive. The stronger behavioural response to the banana volatile bouquet and to the binary mixture serves to underline the multireceptor nature of the dorsal organ response to food odour in Drosophila.     

Maturation of odor representation in the honeybee antennal lobe

The antennal lobe (AL) is the first center for processing odors in the insect brain, as is the olfactory bulb (OB) in vertebrates. Both the AL and the OB have a characteristic glomerular structure; odors sensed by olfactory receptor neurons are represented by patterns of glomerular activity. Little is known about when and how an odor begins to be perceived in a developing brain. We address this question by using calcium imaging to monitor odor-evoked neural activity in the ALs of bees of different ages. We find that odor-evoked neural activity already occurs in the ALs of bees as young as 1 or 2 days. In young bees, the responses to odors are relatively weak and restricted to a small number of glomeruli. However, different odors already evoke responses in different combinations of glomeruli. In mature bees, the responses are stronger and are evident in more glomeruli, but continue to have distinct odor-dependent signatures. Our findings indicate that the specific glomerular patterns for odors are conserved during the development, and that odor representations are fully developed in the AL during the first 2 weeks following emergence.     

Representation of binary pheromone blends by glomerulus-specific olfactory projection neurons

An outstanding challenge in olfactory neurobiology is to explain how glomerular networks encode information about stimulus mixtures, which are typical of natural olfactory stimuli. In the moth Manduca sexta, a species-specific blend of two sex-pheromone components is required for reproductive signaling. Each component stimulates a different population of olfactory receptor cells that in turn target two identified glomeruli in the macroglomerular complex of the males antennal lobe. Using intracellular recording and staining, we examined how responses of projection neurons innervating these glomeruli are modulated by changes in the level and ratio of the two essential components in stimulus blends. Compared to projection neurons specific for one component, projection neurons that integrated information about the blend (received excitatory input from one component and inhibitory input from the other) showed enhanced ability to track a train of stimulus pulses. The precision of stimulus-pulse tracking was furthermore optimized at a synthetic blend ratio that mimics the physiological response to an extract of the females pheromone gland. Optimal responsiveness of a projection neuron to repetitive stimulus pulses therefore appears to depend not only on stimulus intensity but also on the relative strength of the two opposing synaptic inputs that are integrated by macroglomerular complex projection neurons.     

Multiple inhibitory pathways shape odor-evoked responses in lobster olfactory projection neurons

This study characterizes odor-evoked responses of the glomerular output neurons of the spiny lobster olfactory lobe, and implicates previously identified γ-aminobutyric acid (GABA)- and histamine-mediated inhibitory pathways in shaping these responses. Odor-evoked responses were more complex than electrically evoked responses, with up to three distinct components: a brief, short-latency (fast) depolarization, a longer-duration, longer-latency (slow) depolarization, and a slow hyperpolarization. Seventy-seven percent of all responses contained the hyperpolarization, while only 31% and 23% contained the fast and slow depolarizations, respectively. The broader tuning of the hyperpolarization relative to the other two components suggests that the hyperpolarization mediates lateral inhibitory interactions across olfactory glomeruli. Perfusing the brain with the GABA-receptor antagonist picrotoxin increased the amplitude of the hyperpolarization, while the histamine-receptor antagonist cimetidine decreased the hyperpolarization in some instances but increased it in others. Pharmacological enhancement or suppression of the hyperpolarization could mask or unmask, respectively, the slow depolarization. Both antagonists could also cause the appearance of the fast depolarization when it was not apparent prior to treatment. We conclude that GABA- and histamine-mediated inhibition contributes to the overall pattern of the response of projection neurons to odors by regulating the relative strength of these three distinct types of input. Accepted: 17 September 1997     

Pheromones and signature mixtures: defining species-wide signals and variable cues for identity in both invertebrates and vertebrates

Pheromones have been found in species in almost every part of the animal kingdom, including mammals. Pheromones (a molecule or defined combination of molecules) are species-wide signals which elicit innate responses (though responses can be conditional on development as well as context, experience, and internal state). In contrast, signature mixtures, in invertebrates and vertebrates, are variable subsets of molecules of an animal’s chemical profile which are learnt by other animals, allowing them to distinguish individuals or colonies. All signature mixtures, and almost all pheromones, whatever the size of molecules, are detected by olfaction (as defined by receptor families and glomerular processing), in mammals by the main olfactory system or vomeronasal system or both. There is convergence on a glomerular organization of olfaction. The processing of all signature mixtures, and most pheromones, is combinatorial across a number of glomeruli, even for some sex pheromones which appear to have ‘labeled lines’. Narrowly specific pheromone receptors are found, but are not a prerequisite for a molecule to be a pheromone. A small minority of pheromones act directly on target tissues (allohormone pheromones) or are detected by non-glomerular chemoreceptors, such as taste. The proposed definitions for pheromone and signature mixture are based on the heuristic value of separating these kinds of chemical information. In contrast to a species-wide pheromone, there is no single signature mixture to find, as signature mixtures are a ‘receiver-side’ phenomenon and it is the differences in signature mixtures which allow animals to distinguish each other.     

Chemotopy of amino acids on the olfactory bulb predicts olfactory discrimination capabilities of zebrafish Danio rerio

Amino acids reliably evoke strong responses in fish olfactory system. The molecular olfactory receptors (ORs) are located in the membrane of cilia and microvilli of the olfactory receptor neurons (ORNs). Axons of ORNs converge on specific olfactory bulb (OB) glomeruli and the neural responses of ORNs expressing single Ors activate glomerular activity patterns typical for each amino acid. Chemically similar amino acids activate more similar glomerular activity patterns then chemically different amino acids. Differential glomerular activity patterns are the structural basis for amino acid perception and discrimination. We studied olfactory discrimination in zebrafish Danio rerio (Hamilton 1822) by conditioning them to respond to each of the following amino acids: L-Ala, L-Val, L-Leu, L-Arg, and L-Phe. Subsequently, zebrafish were tested for food searching activities with 18 nonconditioned amino acids. The food searching activity during 90 s of the test period was significantly greater after stimulation with the conditioned stimulus than with the nonconditioned amino acid. Zebrafish were able to discriminate all the tested amino acids except L-Ile from L-Val and L-Phe from L-Tyr. We conclude that zebrafish have difficulties discriminating amino acid odorants that evoke highly similar chemotopic patterns of activity in the OB.     

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.     

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.     

Predator odour per se does not frighten domestic horses

Horses frequently react nervously when passing animal production farms and other places with distinctive smells, leading riders to believe that horses are innately frightened by certain odours. In three experiments, we investigated how horses respond to (1) urine from wolves and lions, (2) blood from slaughtered conspecifics and fur-derived wolf odour, and (3) a sudden auditory stimulus in either presence or absence of fur-derived wolf odour. The experiments were carried out under standardised conditions using a total of 45 naïve, 2-year-old horses. In the first two experiments we found that horses showed significant changes in behaviour (Experiments 1 and 2: increased sniffing; Experiment 2 only: increased vigilance, decreased eating, and more behavioural shifts), but no increase in heart rate compared to controls when exposed to predator odours and conspecific blood in a known test environment. However, the third experiment showed that exposure to a combination of wolf odour and a sudden stimulus (sound of a moving plastic bag) caused significantly increased heart rate responses and a tendency to a longer latency to resume feeding, compared to control horses exposed to the sudden stimulus without the wolf odour. The results indicate that predator odour per se does not frighten horses but it may cause an increased level of vigilance. The presence of predator odour may, however, cause an increased heart rate response if horses are presented to an additional fear-eliciting stimulus. This strategy may be adaptive in the wild where equids share habitats with their predators, and have to trade-off time and energy spent on anti-predation responses against time allocated to essential non-defensive activities.     

Component information is preserved in glomerular responses to binary odor mixtures in the moth Spodoptera littoralis

Natural odors are often complex mixtures of different compounds. These mixtures can be perceived to have qualities that are different from their components. Moreover, components can be difficult to distinguish within a blend, even if those components are identifiable when presented individually. Thus, odor components can interact along the olfactory pathway in a nonlinear fashion such that the mixture is not perceived simply as the sum of its components. Here we investigated odor-evoked changes in Ca2+ concentration to binary blends of plant-related substances in individually identified glomeruli in the moth Spodoptera littoralis. We used a wide range of blend ratios and a range of concentrations below the level at which glomerular responses become saturated. We found no statistically significant cases where the mixture response was greater than both component responses at the same total concentration (synergistic interactions) and no statistically significant cases where the mixture response was less than either component presented individually (suppressive interactions). Therefore, we conclude that, for the plant mixtures studied, information of their components is preserved in the neural representations encoded at the first stage of olfactory processing in this moth species.     

Olfactory coding in the insect brain: molecular receptive ranges, spatial and temporal coding

Odor information is coded in the insect brain in a sequence of steps, ranging from the receptor cells, via the neural network in the antennal lobe, to higher order brain centers, among which the mushroom bodies and the lateral horn are the most prominent. Across all of these processing steps, coding logic is combinatorial, in the sense that information is represented as patterns of activity across a population of neurons, rather than in individual neurons. Because different neurons are located in different places, such a coding logic is often termed spatial, and can be visualized with optical imaging techniques. We employ in vivo calcium imaging in order to record odor‐evoked activity patterns in olfactory receptor neurons, different populations of local neurons in the antennal lobes, projection neurons linking antennal lobes to the mushroom bodies, and the intrinsic cells of the mushroom bodies themselves, the Kenyon cells. These studies confirm the combinatorial nature of coding at all of these stages. However, the transmission of odor‐evoked activity patterns from projection neuron dendrites via their axon terminals onto Kenyon cells is accompanied by a progressive sparsening of the population code. Activity patterns also show characteristic temporal properties. While a part of the temporal response properties reflect the physical sequence of odor filaments, another part is generated by local neuron networks. In honeybees, γ‐aminobutyric acid (GABA)‐ergic and histaminergic neurons both contribute inhibitory networks to the antennal lobe. Interestingly, temporal properties differ markedly in different brain areas. In particular, in the antennal lobe odor‐evoked activity develops over slow time courses, while responses in Kenyon cells are phasic and transient. The termination of an odor stimulus is reflected by a decrease in activity within most glomeruli of the antennal lobe and an off‐response in some glomeruli, while in the mushroom bodies about half of the odor‐activated Kenyon cells also exhibit off‐responses.