Neural circuit mechanisms for pattern detection and feature combination in olfactory cortex

Odors are initially encoded in the brain as a set of distinct physicochemical characteristics but are ultimately perceived as a unified sensory object--a "smell." It remains unclear how chemical features encoded by diverse odorant receptors and segregated glomeruli in the main olfactory bulb (MOB) are assembled into integrated cortical representations. Combining patterned optical microstimulation of MOB with in vivo electrophysiological recordings in anterior piriform cortex (PCx), we assessed how cortical neurons decode complex activity patterns distributed across MOB glomeruli. PCx firing was insensitive to single-glomerulus photostimulation. Instead, individual cells reported higher-order combinations of coactive glomeruli resembling odor-evoked sensory maps. Intracellular recordings revealed a corresponding circuit architecture providing each cortical neuron with weak synaptic input from a distinct subpopulation of MOB glomeruli. PCx neurons thus detect specific glomerular ensembles, providing an explicit neural representation of chemical feature combinations that are the hallmark of complex odor stimuli.     

Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity and specificity of receptor-defined glomeruli

Odorant identity is represented in the olfactory bulb (OB) by the glomerular activity pattern, which reflects a combination of activated odorant receptors (ORs) in the olfactory epithelium. To elucidate this neuronal circuit at the molecular level, we established a functional OR identification strategy based on glomerular activity by combining in vivo Ca(2+) imaging, retrograde dye labeling, and single-cell RT-PCR. Spatial and functional mapping of OR-defined glomeruli revealed that the glomerular positional relationship varied considerably between individual animals, resulting in different OR maps in the OB. Notably, OR-defined glomeruli exhibited different ligand spectra and far higher sensitivity compared to the in vitro pharmacological properties of corresponding ORs. Moreover, we found that the olfactory mucus was an important factor in the regulation of in vivo odorant responsiveness. Our results provide a methodology to examine in vivo glomerular responses at the receptor level and further help address the long-standing issues of olfactory sensitivity and specificity under physiological conditions.     

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.     

Odorant representations are modulated by intra- but not interglomerular presynaptic inhibition of olfactory sensory neurons

Input to the central nervous system from olfactory sensory neurons (OSNs) is modulated presynaptically. We investigated the functional organization of this inhibition and its role in odor coding by imaging neurotransmitter release from OSNs in slices and in vivo in mice expressing synaptopHluorin, an optical indicator of vesicle exocytosis. Release from OSNs was strongly suppressed by heterosynaptic, intraglomerular inhibition. In contrast, inhibitory connections between glomeruli mediated only weak lateral inhibition of OSN inputs in slices and did not do so in response to odorant stimulation in vivo. Blocking presynaptic inhibition in vivo increased the amplitude of odorant-evoked input to glomeruli but had little effect on spatial patterns of glomerular input. Thus, intraglomerular inhibition limits the strength of olfactory input to the CNS, whereas interglomerular inhibition plays little or no role. This organization allows for control of input sensitivity while maintaining the spatial maps of glomerular activity thought to encode odorant identity.     

Functional development of the olfactory system in zebrafish

The olfactory system has become a popular model to study the function of neuronal circuits and the molecular and cellular mechanisms underlying the development of neurons and their connections. An excellent model to combine studies of function and development is the zebrafish because it not only permits sophisticated molecular and genetic analyses of development, but also functional measurements of neuronal activity patterns in the intact brain. This article reviews insights into the functional development of the olfactory system that have been obtained in zebrafish. The focus is on the specification of olfactory sensory neurons (OSNs), the mechanisms controlling odorant receptor expression and OSN identity, the pathfinding of OSN axons towards target glomeruli in the olfactory bulb (OB), the development of glomeruli and functional topographic maps in the OB, and the development of inhibitory interneurons in the OB.     

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.     

Dishevelled Proteins Are Associated with Olfactory Sensory Neuron Presynaptic Terminals

Olfactory sensory neurons (OSNs) project their axons from the olfactory epithelium toward the olfactory bulb (OB) in a heterogeneous and unsorted arrangement. However, as the axons approach the glomerular layer of the OB, axons from OSNs expressing the same odorant receptor (OR) sort and converge to form molecularly homogeneous glomeruli. Axon guidance cues, cell adhesion molecules, and OR induced activity have been implicated in the final targeting of OSN axons to specific glomeruli. Less understood, and often controversial, are the mechanisms used by OSN axons to initially navigate from the OE toward the OB. We previously demonstrated a role for Wnt and Frizzled (Fz) molecules in OSN axon extension and organization within the olfactory nerve. Building on that we now turned our attention to the downstream signaling cascades from Wnt-Fz interactions. Dishevelled (Dvl) is a key molecule downstream of Fz receptors. Three isoforms of Dvl with specific as well as overlapping functions are found in mammals. Here, we show that Dvl-1 expression is restricted to OSNs in the dorsal recess of the nasal cavity, and labels a unique subpopulation of glomeruli. Dvl-2 and Dvl-3 have a widespread distribution in both the OE and OB. Both Dvl-1 and Dvl-2 are associated with intra-glomerular pre-synaptic OSN terminals, suggesting a role in synapse formation/stabilization. Moreover, because Dvl proteins were observed in all OSN axons, we hypothesize that they are important determinants of OSN cell differentiation and axon extension.     

Principles of glomerular organization in the human olfactory bulb--implications for odor processing

Olfactory sensory neurons (OSN) in mice express only 1 of a possible 1,100 odor receptors (OR) and axons from OSNs expressing the same odor receptor converge into approximately 2 of the 1,800 glomeruli in each olfactory bulb (OB) in mice; this yields a convergence ratio that approximates 2:1, 2 glomeruli/OR. Because humans express only 350 intact ORs, we examined human OBs to determine if the glomerular convergence ratio of 2:1 established in mice was applicable to humans. Unexpectedly, the average number of human OB glomeruli is >5,500 yielding a convergence ratio of approximately 16:1. The data suggest that the initial coding of odor information in the human OB may differ from the models developed for rodents and that recruitment of additional glomeruli for subpopulations of ORs may contribute to more robust odor representation.     

An olfactory neuronal network for vapor recognition in an artificial nose

Odorant sensitivity and discrimination in the olfactory system appear to involve extensive neural processing of the primary sensory inputs from the olfactory epithelium. To test formally the functional consequences of such processing, we implemented in an artificial chemosensing system a new analytical approach that is based directly on neural circuits of the vertebrate olfactory system. An array of fiber-optic chemosensors, constructed with response properties similar to those of olfactory sensory neurons, provide time-varying inputs to a computer simulation of the olfactory bulb (OB). The OB simulation produces spatiotemporal patterns of neuronal firing that vary with vapor type. These patterns are then recognized by a delay line neural network (DLNN). In the final output of these two processing steps, vapor identity is encoded by the spatial patterning of activity across units in the DLNN, and vapor intensity is encoded by response latency. The OB-DLNN combination thus separates identity and intensity information into two distinct codes carried by the same output units, enabling discrimination among organic vapors over a range of input signal intensities. In addition to providing a well-defined system for investigating olfactory information processing, this biologically based neuronal network performs better than standard feed-forward neural networks in discriminating vapors when small amounts of training data are used. Received: 30 June 1997 / Accepted in revised form: 12 January 1998     

Analysis of training-induced changes in ethyl acetate odor maps using a new computational tool to map the glomerular layer of the olfactory bulb

Odor quality is thought to be encoded by the activation of partially overlapping subsets of glomeruli in the olfactory bulb (odor maps). Mouse genetic studies have demonstrated that olfactory sensory neurons (OSNs) expressing a particular olfactory receptor target their axons to a few individual glomeruli in the bulb. While the specific targeting of OSN axons provides a molecular underpinning for the odor maps, much remains to be understood about the relationship between the functional and molecular maps. In this article, we ask the question whether intensive training of mice in a go/no-go operant conditioning odor discrimination task affects odor maps measured by determining c-fos up-regulation in periglomerular cells. Data analysis is performed using a newly developed suite of computational tools designed to systematically map functional and molecular features of glomeruli in the adult mouse olfactory bulb. This suite provides the necessary tools to process high-resolution digital images, map labeled glomeruli, visualize odor maps, and facilitate statistical analysis of patterns of identified glomeruli in the olfactory bulb. The software generates odor maps (density plots) based on glomerular activity, density, or area. We find that training up-regulates the number of glomeruli that become c-fos positive after stimulation with ethyl acetate.     

Pheromonal and host-odor processing in the insect antennal lobe: how different?

In the olfactory bulb of vertebrates and the antennal lobe of insects, precise connections between sensory receptor cells and olfactory glomeruli form the basis of a highly organized chemotopic map at the first stage of central processing in the brain. Beyond this basic level of organization, the olfactory system is typically separated into two subsystems: a 'main' olfactory pathway that detects and processes information about most environmental odorants, and an 'accessory' olfactory pathway that is devoted to information about social signals such as sex pheromones. A growing number of studies show, however, that it is not always possible to draw clear functional distinctions between the two subsystems. These findings have led some to speculate that the organizational principles by which olfactory stimuli are represented across glomeruli may be more similar in these two olfactory subsystems than previously thought.     

Optical imaging of odorant representations in the mammalian olfactory bulb.

We adapted the technique of intrinsic signal imaging to visualize how odorant concentration and structure are represented spatially in the rat olfactory bulb. Most odorants activated one or more glomeruli in the imaged region of the bulb; these optically imaged responses reflected the excitation of underlying neurons. Odorant-evoked patterns were similar across animals and symmetrical in the two bulbs of the same animal. The variable sensitivity of individual glomeruli produced distinct maps for different odorant concentrations. Using a series of homologous aldehydes, we found that glomeruli were tuned to detect particular molecular features and that maps of similar molecules were highly correlated. These characteristics suggest that odorants and their concentrations can be encoded by distinct spatial patterns of glomerular activation.     

Slits and Robo-2 regulate the coalescence of subsets of olfactory sensory neuron axons within the ventral region of the olfactory bulb

Olfactory sensory neurons (OSNs) project their axons to second-order neurons in the olfactory bulb (OB) to form a precise glomerular map and these stereotypic connections are crucial for accurate odorant information processing by animals. To form these connections, olfactory sensory neuron (OSN) axons respond to axon guidance molecules that direct their growth and coalescence. We have previously implicated the axon guidance receptor Robo-2 in the accurate coalescence of OSN axons within the dorsal region of the OB (Cho et al., 2011). Herein, we have examined whether Robo-2 and its ligands, the Slits, contribute to the formation of an accurate glomerular map within more ventral regions of the OB. We have ablated expression of Robo-2 in OSNs and assessed the targeting accuracy of axons expressing either the P2 or MOR28 olfactory receptors, which innervate two different regions of the ventral OB. We show that P2-positive axons, which express Robo-2, coalesce into glomeruli more ventrally and form additional glomeruli in the OB of robo-2^lox/lox;OMP-Cre mice. We also demonstrate that Robo-2-mediated targeting of P2 axons along the dorsoventral axis of the OB is controlled by Slit-1 and Slit-3 expression. Interestingly, although MOR28-positive OSNs only express low levels of Robo-2, a reduced number of MOR28-positive glomeruli is observed in the OB of robo-2^lox/lox;OMP-Cre mice. Taken together, our results demonstrate that Slits and Robo-2 are required for the formation of an accurate glomerular map in the ventral region of the OB.     

Functional topography of connections linking mirror-symmetric maps in the mouse olfactory bulb

In rodents, each main olfactory bulb contains two mirror-symmetric glomerular maps, a feature not found in the initial topographic maps of other sensory systems. Targeting tracer injections to identified glomeruli revealed that isofunctional odor columns-translaminar assemblies connected to a given glomerulus-were specifically and reciprocally interconnected through a mutually inhibitory circuit with exquisite topographic specificity. Thus, instead of containing two mirror-symmetric maps, we propose that the olfactory bulb contains a single integrated map in which isofunctional odor columns are connected through an intrabulbar link, analogous to the specific horizontal connections linking iso-orientation columns in primary visual cortex.     

Faster, deeper, better: the impact of sniffing modulation on bulbar olfactory processing

A key feature of mammalian olfactory perception is that sensory input is intimately related to respiration. Different authors have considered respiratory dynamics not only as a simple vector for odor molecules but also as an integral part of olfactory perception. Thus, rats adapt their sniffing strategy, both in frequency and flow rate, when performing odor-related tasks. The question of how frequency and flow rate jointly impact the spatio-temporal representation of odor in the olfactory bulb (OB) has not yet been answered. In the present paper, we addressed this question using a simulated nasal airflow protocol on anesthetized rats combined with voltage-sensitive dye imaging (VSDi) of odor-evoked OB glomerular maps. Glomerular responses displayed a tonic component during odor stimulation with a superimposed phasic component phase-locked to the sampling pattern. We showed that a high sniffing frequency (10 Hz) retained the ability to shape OB activity and that the tonic and phasic components of the VSDi responses were dependent on flow rate and inspiration volume, respectively. Both sniffing parameters jointly affected OB responses to odor such that the reduced activity level induced by a frequency increase was compensated by an increased flow rate.     

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.     

Olfactory activation patterns in the antennal lobe of the sphinx moth,<Emphasis Type="Italic"> Manduca sexta</Emphasis>

The sphinx moth Manduca sexta is a well-studied insect with regard to central olfactory functions. Until now, the innervation patterns of olfactory receptor neurons into the array of olfactory glomeruli in the antennal lobe have, however, been unclear. Using optical imaging to visualize calcium dynamics within the antennal lobe we demonstrate specific patterns elicited by sex pheromone components and plant-derived odours. These patterns mainly reflect receptor neuron activity. Within the male-specific macroglomerular complex the two major pheromone components evoke stereotyped activity in either of two macroglomerular complex glomeruli. Based on previous knowledge of output neuron specificity, our results suggest a matching of information between input and output in the macroglomerular complex. Plant odours evoked activity in the sexually isomorphic glomeruli. Two major results were obtained: (1). terpenes and aromatic compounds activate different clusters of glomeruli with only minor overlapping, and (2). the position of certain key glomeruli is fixed in both males and females, which suggests that host-plant related odorants are processed in a similar way in both sexes.     

Spontaneous high-gamma band activity reflects functional organization of auditory cortex in the awake macaque

In the absence of sensory stimuli, spontaneous activity in the brain has been shown to exhibit organization at multiple spatiotemporal scales. In the macaque auditory cortex, responses to acoustic stimuli are tonotopically organized within multiple, adjacent frequency maps aligned in a caudorostral direction on the supratemporal plane (STP) of the lateral sulcus. Here, we used chronic microelectrocorticography to investigate the correspondence between sensory maps and spontaneous neural fluctuations in the auditory cortex. We first mapped tonotopic organization across 96 electrodes spanning approximately two centimeters along the primary and higher auditory cortex. In separate sessions, we then observed that spontaneous activity at the same sites exhibited spatial covariation that reflected the tonotopic map of the STP. This observation demonstrates a close relationship between functional organization and spontaneous neural activity in the sensory cortex of the awake monkey.     

How do features of sensory representations develop?

Sensory representations in the brainstem and cortex have a number of features that support the idea that neural activity patterns are important in their development. Many of these features vary across species in ways that could result from perturbances in the balance of the effects of activity patterns and position-dependent gene expression. (1) Most notably, disruptions or septa in sensory maps often reflect actual discontinuities in the receptor sheet, and the discontinuities may be reflected in a series of interconnected maps. Species with different disruption patterns in sensory sheets have different matching disruption patterns in the sensory maps and variant individuals and strains of the same species have matching variations in the receptor disruption patterns and their sensory maps. (2) In addition, mutations that misdirect some of the retinal afferents from one side of the brain to the other create new sensory maps that preserve continuities in the altered pattern of input, while creating new structural discontinuities. (3) Furthermore, functionally different classes of afferents that are mixed in the receptor sheet often segregate to activate separate populations of target cells. (4) Finally, early developing portions of receptor sheets may gain more than their share of territory in sensory maps. These and other variable features of sensory maps are most readily accommodated by theories that involve roles for instruction by evoked and spontaneous neural activity patterns.