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1.  System identification of Drosophila olfactory sensory neurons 
The lack of a deeper understanding of how olfactory sensory neurons (OSNs) encode odors has hindered the progress in understanding the olfactory signal processing in higher brain centers. Here we employ methods of system identification to investigate the encoding of time-varying odor stimuli and their representation for further processing in the spike domain by Drosophila OSNs. In order to apply system identification techniques, we built a novel low-turbulence odor delivery system that allowed us to deliver airborne stimuli in a precise and reproducible fashion. The system provides a 1% tolerance in stimulus reproducibility and an exact control of odor concentration and concentration gradient on a millisecond time scale. Using this novel setup, we recorded and analyzed the in-vivo response of OSNs to a wide range of time-varying odor waveforms. We report for the first time that across trials the response of OR59b OSNs is very precise and reproducible. Further, we empirically show that the response of an OSN depends not only on the concentration, but also on the rate of change of the odor concentration. Moreover, we demonstrate that a two-dimensional (2D) Encoding Manifold in a concentration-concentration gradient space provides a quantitative description of the neuron’s response. We then use the white noise system identification methodology to construct one-dimensional (1D) and two-dimensional (2D) Linear-Nonlinear-Poisson (LNP) cascade models of the sensory neuron for a fixed mean odor concentration and fixed contrast. We show that in terms of predicting the intensity rate of the spike train, the 2D LNP model performs on par with the 1D LNP model, with a root mean-square error (RMSE) increase of about 5 to 10%. Surprisingly, we find that for a fixed contrast of the white noise odor waveforms, the nonlinear block of each of the two models changes with the mean input concentration. The shape of the nonlinearities of both the 1D and the 2D LNP model appears to be, for a fixed mean of the odor waveform, independent of the stimulus contrast. This suggests that white noise system identification of Or59b OSNs only depends on the first moment of the odor concentration. Finally, by comparing the 2D Encoding Manifold and the 2D LNP model, we demonstrate that the OSN identification results depend on the particular type of the employed test odor waveforms. This suggests an adaptive neural encoding model for Or59b OSNs that changes its nonlinearity in response to the odor concentration waveforms.
doi:10.1007/s10827-010-0265-0
PMCID: PMC3736744  PMID: 20730480
System identification; Olfactory sensory neurons; White noise analysis; I/O modeling
2.  A Large-Scale Analysis of Odor Coding in the Olfactory Epithelium 
The Journal of Neuroscience  2011;31(25):9179-9191.
Mammals can perceive and discriminate myriad volatile chemicals as having a distinct odor. Odorants are initially detected by odorant receptors (ORs) on olfactory sensory neurons (OSNs) in the nose. In the mouse, each OSN expresses one of ∼1000 different OR genes. Although OSNs and their expressed ORs constitute the fundamental units of sensory input to the brain, a comprehensive understanding of how they encode odor identities is still lacking. To gain a broader and more detailed understanding of odorant recognition and odor coding at this level, we tested the responses of 3000 mouse OSNs to 125 odorants with diverse structures and perceived odors. These studies revealed extraordinary diversity, but also bias, in odorant recognition by the OSN, and thus OR, repertoire. They indicate that most OSNs are narrowly tuned to detect a subset of odorants with related structures and often related odors, but that the repertoire also includes broadly tuned components. Strikingly, the vast majority of odorants activated a unique set of OSNs, usually two or more in combination. The resulting combinatorial codes varied in size among odorants and sometimes contained both narrowly and broadly tuned components. While many OSNs recognized multiple odorants, some appeared specific for a given pheromone or other animal-associated compound, or for one or more odorants with a particular odor quality, raising the possibility that signals derived from some OSNs and ORs might elicit an innate behavior or convey a specific odor quality.
doi:10.1523/JNEUROSCI.1282-11.2011
PMCID: PMC3758579  PMID: 21697369
3.  Learned odor discrimination in Drosophila without distinct combinatorial odor maps in the antennal lobe 
Current biology : CB  2008;18(21):1668-1674.
A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant receptor gene converge onto the same glomeruli in the brain (1–7). Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants (8–11). OSNs also exhibit odor-evoked responses with complex temporal dynamics (11), but the contribution of this activity to behavioral odor discrimination has received little attention (12). Here we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture, and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration indicating that they encode odorant identity. Therefore flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.
doi:10.1016/j.cub.2008.08.071
PMCID: PMC2602956  PMID: 18951022
4.  Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity 
Insect olfactory sensory neurons (OSN) express a diverse array of receptors from different protein families, i.e. ionotropic receptors (IR), gustatory receptors (GR) and odorant receptors (OR). It is well known that insects are exposed to a plethora of odor molecules that vary widely in both space and time under turbulent natural conditions. In addition to divergent ligand specificities, these different receptors might also provide an increased range of temporal dynamics and sensitivities for the olfactory system. To test this, we challenged different Drosophila OSNs with both varying stimulus durations (10–2000 ms), and repeated stimulus pulses of key ligands at various frequencies (1–10 Hz). Our results show that OR-expressing OSNs responded faster and with higher sensitivity to short stimulations as compared to IR- and Gr21a-expressing OSNs. In addition, OR-expressing OSNs could respond to repeated stimulations of excitatory ligands up to 5 Hz, while IR-expressing OSNs required ~5x longer stimulations and/or higher concentrations to respond to similar stimulus durations and frequencies. Nevertheless, IR-expressing OSNs did not exhibit adaptation to longer stimulations, unlike OR- and Gr21a-OSNs. Both OR- and IR-expressing OSNs were also unable to resolve repeated pulses of inhibitory ligands as fast as excitatory ligands. These differences were independent of the peri-receptor environment in which the receptors were expressed and suggest that the receptor expressed by a given OSN affects both its sensitivity and its response to transient, intermittent chemical stimuli. OR-expressing OSNs are better at resolving low dose, intermittent stimuli, while IR-expressing OSNs respond more accurately to long-lasting odor pulses. This diversity increases the capacity of the insect olfactory system to respond to the diverse spatiotemporal signals in the natural environment.
doi:10.3389/fncel.2012.00054
PMCID: PMC3499765  PMID: 23162431
odorant receptors; ionotropic receptors; pulse resolution; single sensillum recording
5.  Modeling Peripheral Olfactory Coding in Drosophila Larvae 
PLoS ONE  2011;6(8):e22996.
The Drosophila larva possesses just 21 unique and identifiable pairs of olfactory sensory neurons (OSNs), enabling investigation of the contribution of individual OSN classes to the peripheral olfactory code. We combined electrophysiological and computational modeling to explore the nature of the peripheral olfactory code in situ. We recorded firing responses of 19/21 OSNs to a panel of 19 odors. This was achieved by creating larvae expressing just one functioning class of odorant receptor, and hence OSN. Odor response profiles of each OSN class were highly specific and unique. However many OSN-odor pairs yielded variable responses, some of which were statistically indistinguishable from background activity. We used these electrophysiological data, incorporating both responses and spontaneous firing activity, to develop a Bayesian decoding model of olfactory processing. The model was able to accurately predict odor identity from raw OSN responses; prediction accuracy ranged from 12%–77% (mean for all odors 45.2%) but was always significantly above chance (5.6%). However, there was no correlation between prediction accuracy for a given odor and the strength of responses of wild-type larvae to the same odor in a behavioral assay. We also used the model to predict the ability of the code to discriminate between pairs of odors. Some of these predictions were supported in a behavioral discrimination (masking) assay but others were not. We conclude that our model of the peripheral code represents basic features of odor detection and discrimination, yielding insights into the information available to higher processing structures in the brain.
doi:10.1371/journal.pone.0022996
PMCID: PMC3153476  PMID: 21857978
6.  Spatiotemporal Alterations in Primary Odorant Representations in Olfactory Marker Protein Knockout Mice 
PLoS ONE  2013;8(4):e61431.
Olfactory marker protein (OMP) is highly and selectively expressed in primary olfactory sensory neurons (OSNs) across species, but its physiological function remains unclear. Previous studies in the olfactory epithelium suggest that it accelerates the neural response to odorants and may modulate the odorant-selectivity of OSNs. Here we used a line of gene-targeted mice that express the fluorescent exocytosis indicator synaptopHluorin in place of OMP to compare spatiotemporal patterns of odorant-evoked neurotransmitter release from OSNs in adult mice that were heterozygous for OMP or OMP-null. We found that these patterns, which constitute the primary neural representation of each odorant, developed more slowly during the odorant presentation in OMP knockout mice but eventually reached the same magnitude as in heterozygous mice. In the olfactory bulb, each glomerulus receives synaptic input from a subpopulation of OSNs that all express the same odor receptor and thus typically respond to a specific subset of odorants. We observed that in OMP knockout mice, OSNs innervating a given glomerulus typically responded to a broader range of odorants than in OMP heterozygous mice and thus each odorant evoked synaptic input to a larger number of glomeruli. In an olfactory habituation task, OMP knockout mice behaved differently than wild-type mice, exhibiting a delay in their onset to investigate an odor stimulus during its first presentation and less habituation to that stimulus over repeated presentations. These results suggest that the actions of OMP in olfactory transduction carry through to the primary sensory representations of olfactory stimuli in adult mice in vivo.
doi:10.1371/journal.pone.0061431
PMCID: PMC3632605  PMID: 23630588
7.  Early survival factor deprivation in the olfactory epithelium enhances activity-driven survival 
The neuronal olfactory epithelium undergoes permanent renewal because of environmental aggression. This renewal is partly regulated by factors modulating the level of neuronal apoptosis. Among them, we had previously characterized endothelin as neuroprotective. In this study, we explored the effect of cell survival factor deprivation in the olfactory epithelium by intranasal delivery of endothelin receptors antagonists to rat pups. This treatment induced an overall increase of apoptosis in the olfactory epithelium. The responses to odorants recorded by electroolfactogram were decreased in treated animal, a result consistent with a loss of olfactory sensory neurons (OSNs). However, the treated animal performed better in an olfactory orientation test based on maternal odor compared to non-treated littermates. This improved performance could be due to activity-dependent neuronal survival of OSNs in the context of increased apoptosis level. In order to demonstrate it, we odorized pups with octanal, a known ligand for the rI7 olfactory receptor (Olr226). We quantified the number of OSN expressing rI7 by RT-qPCR and whole mount in situ hybridization. While this number was reduced by the survival factor removal treatment, this reduction was abolished by the presence of its ligand. This improved survival was optimal for low concentration of odorant and was specific for rI7-expressing OSNs. Meanwhile, the number of rI7-expressing OSNs was not affected by the odorization in non-treated littermates; showing that the activity-dependant survival of OSNs did not affect the OSN population during the 10 days of odorization in control conditions. Overall, our study shows that when apoptosis is promoted in the olfactory mucosa, the activity-dependent neuronal plasticity allows faster tuning of the olfactory sensory neuron population toward detection of environmental odorants.
doi:10.3389/fncel.2013.00271
PMCID: PMC3870945  PMID: 24399931
Olfaction; activity-dependent plasticity; EOG; U131; WISH
8.  Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae 
The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.
doi:10.3791/1725
PMCID: PMC2830253  PMID: 20164822
9.  Large-Scale Investigation of the Olfactory Receptor Space Using a Microfluidic Microwell Array 
Lab on a chip  2010;10(9):1120-1127.
The mammalian olfactory system is able to discriminate among tens of thousands of odorant molecules. In mice, each odorant is sensed by a small subset of the approximately 1,000 odorant receptor (OR) types, with one OR gene expressed by each olfactory sensory neuron (OSN). However, the sum of the large repertoire of OR/OSN types and difficulties with heterologous expression have made it almost impossible to analyze odorant responsiveness across all OR/OSN types. We have developed a microfluidic approach that allowed us to screen over 20,000 single cells at once in microwells. By using calcium imaging, we were able to detect and analyze odorant responses of about 2,900 OSNs simultaneously. Importantly, this technique allows for both the detection of rare responding OSNs as well as the identification of OSN populations broadly responsive to odorants of unrelated structures. This technique is generally applicable for screening large numbers of single cells and should help to characterize rare cell behaviors in fields such as toxicology, pharmacology, and cancer research.
doi:10.1039/b920585c
PMCID: PMC3135426  PMID: 20390129
10.  Odor Information Processing by the Olfactory Bulb Analyzed in Gene-Targeted Mice 
Neuron  2010;65(6):912-926.
SUMMARY
In mammals, olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) gene project with precise stereotypy onto mitral/tufted (M/T) cells in the main olfactory bulb (MOB). It remains challenging to understand how incoming olfactory signals are transformed into outputs of M/T cells. By recording from OSNs expressing mouse I7 receptor and their postsynaptic neurons in the bulb, we found that I7 OSNs and their corresponding M/T cells exhibit similarly selective tuning profiles at low concentrations. Increasing the concentration significantly reduces response selectivity for both OSNs and M/T cells, although the tuning curve of M/T cells remains comparatively narrow. By contrast, interneurons in the MOB are broadly tuned, and blocking GABAergic neurotransmission reduces selectivity of M/T cells at high odorant concentrations. Our results indicate that olfactory information carried by an OR is channeled to its corresponding M/T cells and support the role of lateral inhibition via interneurons in sharpening the tuning of M/T cells.
doi:10.1016/j.neuron.2010.02.011
PMCID: PMC2901914  PMID: 20346765
11.  Odor-Specific, Olfactory Marker Protein-Mediated Sparsening of Primary Olfactory Input to the Brain after Odor Exposure 
The Journal of Neuroscience  2013;33(15):6594-6602.
Long-term plasticity in sensory systems is usually conceptualized as changing the interpretation of the brain of sensory information, not an alteration of how the sensor itself responds to external stimuli. However, here we demonstrate that, in the adult mouse olfactory system, a 1-week-long exposure to an artificially odorized environment narrows the range of odorants that can induce neurotransmitter release from olfactory sensory neurons (OSNs) and reduces the total transmitter release from responsive neurons. In animals heterozygous for the olfactory marker protein (OMP), this adaptive plasticity was strongest in the populations of OSNs that originally responded to the exposure odorant (an ester) and also observed in the responses to a similar odorant (another ester) but had no effect on the responses to odorants dissimilar to the exposure odorant (a ketone and an aldehyde). In contrast, in OMP knock-out mice, odorant exposure reduced the number and amplitude of OSN responses evoked by all four types of odorants equally. The effect of this plasticity is to preferentially sparsen the primary neural representations of common olfactory stimuli, which has the computational benefit of increasing the number of distinct sensory patterns that could be represented in the circuit and might thus underlie the improvements in olfactory discrimination often observed after odorant exposure (Mandairon et al., 2006a). The absence of odorant specificity in this adaptive plasticity in OMP knock-out mice suggests a potential role for this protein in adaptively reshaping OSN responses to function in different environments.
doi:10.1523/JNEUROSCI.1442-12.2013
PMCID: PMC3865540  PMID: 23575856
12.  G-protein coupled Receptor Kinase 2 is required for rhythmic olfactory responses in Drosophila 
Current biology : CB  2008;18(11):787-794.
Summary
Background
The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), which suggests that odorant receptors(ORs) or OR-dependent processes are under clock control. Since responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, we tested whether regulators of ORs were under circadian clock control.
Results
The levels of G-protein coupled receptor kinase 2 (Gprk2) mRNA and protein cycle in a circadian clock-dependent manner with a peak around mid-night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc01 flies elicits constant high amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk mutants produce constant low amplitude EAG responses. Odorant receptors (ORs) accumulate to high levels in the dendrites of OSNs around mid-night, and this dendritic localization of ORs is enhanced by Gprk2 at times when ORs are primarily localized in the cell body.
Conclusion
These results support a model in which circadian clock-dependent rhythms in Gprk2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of Gprks in desensitizing activated receptors, and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity.
doi:10.1016/j.cub.2008.04.062
PMCID: PMC2474769  PMID: 18499458
13.  Functional recovery of odor representations in regenerated sensory inputs to the olfactory bulb 
The olfactory system has a unique capacity for recovery from peripheral damage. After injury to the olfactory epithelium (OE), olfactory sensory neurons (OSNs) regenerate and re-converge on target glomeruli of the olfactory bulb (OB). Thus far, this process has been described anatomically for only a few defined populations of OSNs. Here we characterize this regeneration at a functional level by assessing how odor representations carried by OSN inputs to the OB recover after massive loss and regeneration of the sensory neuron population. We used chronic imaging of mice expressing synaptopHluorin in OSNs to monitor odor representations in the dorsal OB before lesion by the olfactotoxin methyl bromide and after a 12 week recovery period. Methyl bromide eliminated functional inputs to the OB, and these inputs recovered to near-normal levels of response magnitude within 12 weeks. We also found that the functional topography of odor representations recovered after lesion, with odorants evoking OSN input to glomerular foci within the same functional domains as before lesion. At a finer spatial scale, however, we found evidence for mistargeting of regenerated OSN axons onto OB targets, with odorants evoking synaptopHluorin signals in small foci that did not conform to a typical glomerular structure but whose distribution was nonetheless odorant-specific. These results indicate that OSNs have a robust ability to reestablish functional inputs to the OB and that the mechanisms underlying the topography of bulbar reinnervation during development persist in the adult and allow primary sensory representations to be largely restored after massive sensory neuron loss.
doi:10.3389/fncir.2013.00207
PMCID: PMC3882662  PMID: 24431990
olfactory bulb; regeneration; sensory neurons; synaptopHluorin; axon targeting
14.  Odor identity influences tracking of temporally patterned plumes in Drosophila 
BMC Neuroscience  2011;12:62.
Background
Turbulent fluid landscapes impose temporal patterning upon chemical signals, and the dynamical neuronal responses to patterned input vary across the olfactory receptor repertoire in flies, moths, and locusts. Sensory transformations exhibit low pass filtering that ultimately results in perceptual fusion of temporally transient sensory signals. For example, humans perceive a sufficiently fast flickering light as continuous, but the frequency threshold at which this fusion occurs varies with wavelength. Although the summed frequency sensitivity of the fly antenna has been examined to a considerable extent, it is unknown how intermittent odor signals are integrated to influence plume tracking behavior independent of wind cues, and whether temporal fusion for behavioral tracking might vary according to the odor encountered.
Results
Here we have adopted a virtual reality flight simulator to study the dynamics of plume tracking under different experimental conditions. Flies tethered in a magnetic field actively track continuous (non-intermittent) plumes of vinegar, banana, or ethyl butyrate with equal precision. However, pulsing these plumes at varying frequency reveals that the threshold rate, above which flies track the plume as if it were continuous, is unique for each odorant tested. Thus, the capability of a fly to navigate an intermittent plume depends on the particular odorant being tracked during flight. Finally, we measured antennal field potential responses to an intermittent plume, found that receptor dynamics track the temporal pattern of the odor stimulus and therefore do not limit the observed behavioral temporal fusion limits.
Conclusions
This study explores the flies' ability to track odor plumes that are temporally intermittent. We were surprised to find that the perceptual critical fusion limit, determined behaviorally, is strongly dependent on odor identity. Antennal field potential recordings indicate that peripheral processing of temporal cues faithfully follow rapid odor transients above the rates that can be resolved behaviorally. These results indicate that (1) higher order circuits create a perceptually continuous signal from an intermittent sensory one, and that (2) this transformation varies with odorant rather than being constrained by sensory-motor integration, thus (3) offering an entry point for examining the mechanisms of rapid olfactory decision making in an ecological context.
doi:10.1186/1471-2202-12-62
PMCID: PMC3145592  PMID: 21708035
15.  Postnatal Experience Modulates Functional Properties of Mouse Olfactory Sensory Neurons 
The European journal of neuroscience  2012;36(4):2452-2460.
Early experience considerably modulates the organization and function of all sensory systems. In the mammalian olfactory system, deprivation of the sensory inputs via neonatal, unilateral naris closure has been shown to induce structural, molecular, and functional changes from the olfactory epithelium to the olfactory bulb and cortex. However, it remains unknown how early experience shapes functional properties of individual olfactory sensory neurons (OSNs), the primary odor detectors in the nose. To address this question, we examined odorant response properties of mouse OSNs in both the closed and open nostril after four weeks of unilateral naris closure with age-matched untreated animals as control. Using patch-clamp technique on genetically-tagged OSNs with defined odorant receptors (ORs), we found that sensory deprivation increased the sensitivity of MOR23 neurons in the closed side while overexposure caused the opposite effect in the open side. We next analyzed the response properties including rise time, decay time, and adaptation induced by repeated stimulation in MOR23 and M71 neurons. Even though these two types of neurons showed distinct properties in dynamic range and response kinetics, sensory deprivation significantly slowed down the decay phase of odorant-induced transduction events in both types. Using western blotting and antibody staining, we confirmed upregulation of several signaling proteins in the closed side as compared with the open side. This study suggests that early experience modulates functional properties of OSNs, probably via modifying the signal transduction cascade.
doi:10.1111/j.1460-9568.2012.08170.x
PMCID: PMC3424323  PMID: 22703547
naris-closure; experience-dependent plasticity; olfactory signal transduction; patch clamp; and gene-targeting
16.  Response enhancement of olfactory sensory neurons-based biosensors for odorant detection*  
This paper presents a novel strategy for the response enhancement of olfactory sensory neurons (OSNs)-based biosensors by monitoring the enhancive responses of OSNs to odorants. An OSNs-based biosensor was developed on the basis of the light addressable potentiometric sensor (LAPS), in which rat OSNs were cultured on the surface of LAPS chip and served as sensing elements. LY294002, the specific inhibitor of phosphatidylinositol 3-kinase (PI3K), was used to enhance the responses of OSNs to odorants. The responses of OSNs to odorants with and without the treatment of LY294002 were recorded by LAPS. The results show that the enhancive effect of LY294002 was recorded efficiently by LAPS and the responses of this OSNs-LAPS hybrid biosensor were enhanced by LY294002 by about 1.5-fold. We conclude that this method can enhance the responses of OSNs-LAPS hybrid biosensors, which may provide a novel strategy for the bioelectrical signal monitor of OSNs in biosensors. It is also suggested that this strategy may be applicable to other kinds of OSNs-based biosensors for cellular activity detection, such as microelectrode array (MEA) and field effect transistor (FET).
doi:10.1631/jzus.B0820220
PMCID: PMC2666205  PMID: 19353747
Olfactory sensory neurons (OSNs); Response enhancement; Light addressable potentiometric sensor (LAPS); Olfactory-based biosensor
17.  Topological Reorganization of Odor Representations in the Olfactory Bulb 
PLoS Biology  2007;5(7):e178.
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.
Author Summary
Many sensory brain areas contain topographic maps where the physical location of neuronal activity contains information about a stimulus feature. In the first central processing center of the olfactory pathway, the olfactory bulb, chemically distinct odors often elicit spatially segregated input activity so that general chemical features are initially represented in a topographic fashion. It is, however, unclear whether this “chemotopic” organization of odor representations is maintained at subsequent stages of odor processing. To address this question, we visualized activity patterns across thousands of individual neurons in the intact olfactory bulb of zebrafish over time using two-photon calcium imaging. Our results demonstrate that odor-evoked activity across the output neurons of the olfactory bulb is chemotopically organized shortly after stimulus onset but becomes more widely distributed during the subsequent few hundred milliseconds of the response. This reorganization of olfactory bulb output activity is most likely mediated by inhibitory feedback and reduces the redundancy in activity patterns evoked by related stimuli. These results indicate that topographically organized activity maps in the olfactory bulb are not maintained during information processing, but contribute to the function of local circuits.
Two-photon calcium imaging in the zebrafish olfactory bulb reveals that mitral cells show more selective responses to odors than interneurons, and odor-evoked firing patterns of populations of mitral cells evolve over hundreds of milliseconds to become more distinct for different odors, thus providing more information about odor identity.
doi:10.1371/journal.pbio.0050178
PMCID: PMC1904499  PMID: 17608564
18.  The Na+/Ca2+ exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response 
Nature Neuroscience  2011;15(1):131-137.
Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. Here, we identify NCKX4, a potassium – dependent Na+/Ca2+ exchanger, to be necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4−/− mouse OSNs display substantially prolonged responses and stronger adaptation. Single – cell electrophysiological analyses demonstrate that the majority of Na+ – dependent Ca2+ exchange in OSNs relevant to sensory transduction is due to NCKX4 and that Nckx4−/− mouse OSNs are deficient in encoding action potentials upon repeated stimulation. Olfactory – specific Nckx4 knockout mice have a reduced ability to locate an odorous source and lower body weights. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception.
doi:10.1038/nn.2943
PMCID: PMC3245797  PMID: 22057188
19.  Olfactory marker protein is critical for functional maturation of olfactory sensory neurons and development of mother preference 
Survival of many altricial animals critically depends on the sense of smell. Curiously, the olfactory system is rather immature at birth and undergoes a maturation process, which is poorly understood. Using patch clamp technique on mouse olfactory sensory neurons (OSNs) with a defined odorant receptor (OR), we demonstrate that OSNs exhibit functional maturation during the first month of postnatal life by developing faster response kinetics, higher sensitivity, and most intriguingly, higher selectivity. OSNs expressing the receptor MOR23 are relatively broadly tuned in neonates and become selective detectors for the cognate odorant within two weeks. Remarkably, these changes are prevented by genetic ablation of olfactory marker protein (OMP), which is exclusively expressed in mature OSNs. Biochemical and pharmacological evidence supports that alteration in odorant-induced phosphorylation of signaling proteins underlie some of the OMP−/− phenotypes. Furthermore, in a novel behavioral assay in which the mouse pups are given a choice between the biological mother and another unfamiliar lactating female, wild-type pups prefer the biological mother, while OMP knockout pups fail to show preference. These results reveal that OSNs undergo an OMP-dependant functional maturation process that coincides with early development of the smell function, which is essential for pups to form preference for their mother.
doi:10.1523/JNEUROSCI.5067-10.2011
PMCID: PMC3084592  PMID: 21414919
olfactory sensory neurons; olfactory marker protein; odorant receptors; functional maturation; maternal preference
20.  State-Dependent Sculpting of Olfactory Sensory Neurons Attributed to Sensory Enrichment, Odor Deprivation, and Aging 
Neuroscience letters  2010;483(2):90-95.
Gene-targeted deletion of the predominant Shaker potassium channel, Kv1.3, in the mitral cells of the olfactory bulb, decreases the number of presynaptic, odorant receptor (OR)-identified olfactory sensory neurons (OSNs) in the main olfactory epithelium (MOE) and alters the nature of their postsynaptic connections to mitral cell targets. The current study examined whether OSN density was state-dependent by examining the impact of 1) odor enrichment, 2) sensory deprivation, and 3) aging upon the number of P2- or M72-expressing neurons. Histological approaches were used to quantify the number of OSNs across entire epithelia for wildtype (WT) vs. Kv1.3-null (KO) mice bred onto an ORtauLacZ reporter background. Following either odor-enrichment or early unilateral naris-occlusion, the number of M72-expressing OSNs was significantly decreased in WT mice, but was unchanged in KO animals. Following naris-occlusion, the number of P2-expressing OSNs was decreased regardless of genotype. Animals that were reared to 2 years of age demonstrated loss of both P2- and M72-expressing OSNs in WT mice and a concomitant loss of only M72-expressing neurons in KO mice. These findings suggest that voltage-gated activity of the mitral cells is important for OSN plasticity, and can prevent neuronal loss via sensory- and OR-dependent mechanisms.
doi:10.1016/j.neulet.2010.07.059
PMCID: PMC3090668  PMID: 20691762
potassium channel; odor enrichment; naris-occlusion; aging; odorant receptor
21.  Plant odorants interfere with detection of sex pheromone signals by male Heliothis virescens 
In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.
doi:10.3389/fncel.2012.00042
PMCID: PMC3465774  PMID: 23060749
pheromone detection; antennal lobe; pheromone receptor; pheromone binding protein; olfaction
22.  Expression Patterns of Odorant Receptors and Response Properties of Olfactory Sensory Neurons in Aged Mice 
Chemical Senses  2009;34(8):695-703.
The sense of smell deteriorates in normal aging, but the underling mechanisms are still elusive. Here we investigated age-related alterations in expression patterns of odorant receptor (OR) genes and functional properties of olfactory sensory neurons (OSNs)—2 critical factors that define the odor detection threshold in the olfactory epithelium. Using in situ hybridization for 9 representative OR genes, we compared the cell densities of each OR in coronal nose sections at different ages (3–27 months). The cell density for different ORs peaked at different time points and a decline was observed for 6 of 9 ORs at advanced ages. Using patch clamp recordings, we then examined the odorant responses of individual OSNs coexpressing a defined OR (MOR23) and green fluorescent protein. The MOR23 neurons recorded from aged animals maintained a similar sensitivity and dynamic range in response to the cognate odorant (lyral) as those from younger mice. The results indicate that although the cell densities of OSNs expressing certain types of ORs decline at advanced ages, individual OSNs can retain their sensitivity. The implications of these findings in age-related olfactory deterioration are discussed.
doi:10.1093/chemse/bjp056
PMCID: PMC2745353  PMID: 19759360
aging; main olfactory epithelium; odorant responses; olfactory receptor; olfactory sensory neurons
23.  Multilevel control of run orientation in Drosophila larval chemotaxis 
Chemotaxis is a powerful paradigm to study how orientation behavior is driven by sensory stimulation. Drosophila larvae navigate odor gradients by controlling the duration of their runs and the direction of their turns. Straight runs and wide-amplitude turns represent two extremes of a behavioral continuum. Here we establish that, on average, runs curl toward the direction of higher odor concentrations. We find that the orientation and strength of the local odor gradient perpendicular to the direction of motion modulates the orientation of individual runs in a gradual manner. We discuss how this error-correction mechanism, called weathervaning, contributes to larval chemotaxis. We use larvae with a genetically modified olfactory system to demonstrate that unilateral function restricted to a single olfactory sensory neuron (OSN) is sufficient to direct weathervaning. Our finding that bilateral sensing is not necessary to control weathervaning highlights the role of temporal sampling. A correlational analysis between sensory inputs and behavioral outputs suggests that weathervaning results from low-amplitude head casts implemented without interruption of the run. In addition, we report the involvement of a sensorimotor memory arising from previous reorientation events. Together, our results indicate that larval chemotaxis combines concurrent orientation strategies that involve complex computations on different timescales.
doi:10.3389/fnbeh.2014.00038
PMCID: PMC3923145  PMID: 24592220
active sensing; chemotaxis; Drosophila larva; sensorimotor integration; stereo-olfaction; weathervaning
24.  Phosphorylation via PKC Regulates the Function of the Drosophila Odorant Co-Receptor 
Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the Gq protein signaling cascade reduces the odor response (Kain et al., 2008). We investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo we combined single sensillum electrophysiological recordings with microinjection of agents affecting PLC and PKC function and observed an altered response of olfactory sensory neurons (OSNs) to odorant stimulation. Injection of the PLC inhibitor U73122 or the PKC inhibitor Gö6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. We conclude that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response.
doi:10.3389/fncel.2011.00005
PMCID: PMC3118453  PMID: 21720521
insect odorant receptor; Drosophila; Or83b; orco; G protein; cAMP; phosphorylation; single sensillum recording
25.  Rapid Encoding and Perception of Novel Odors in the Rat 
PLoS Biology  2008;6(4):e82.
To gain insight into which parameters of neural activity are important in shaping the perception of odors, we combined a behavioral measure of odor perception with optical imaging of odor representations at the level of receptor neuron input to the rat olfactory bulb. Instead of the typical test of an animal's ability to discriminate two familiar odorants by exhibiting an operant response, we used a spontaneously expressed response to a novel odorant—exploratory sniffing—as a measure of odor perception. This assay allowed us to measure the speed with which rats perform spontaneous odor discriminations. With this paradigm, rats discriminated and began responding to a novel odorant in as little as 140 ms. This time is comparable to that measured in earlier studies using operant behavioral readouts after extensive training. In a subset of these trials, we simultaneously imaged receptor neuron input to the dorsal olfactory bulb with near-millisecond temporal resolution as the animal sampled and then responded to the novel odorant. The imaging data revealed that the bulk of the discrimination time can be attributed to the peripheral events underlying odorant detection: receptor input arrives at the olfactory bulb 100–150 ms after inhalation begins, leaving only 50–100 ms for central processing and response initiation. In most trials, odor discrimination had occurred even before the initial barrage of receptor neuron firing had ceased and before spatial maps of activity across glomeruli had fully developed. These results suggest a coding strategy in which the earliest-activated glomeruli play a major role in the initial perception of odor quality, and place constraints on coding and processing schemes based on simple changes in spike rate.
Author Summary
Olfactory stimuli elicit temporally complex patterns of activity across groups of receptor neurons as well as across central neurons. It remains unclear which parameters among these complex activity patterns are important in shaping odor perception. To address this issue, we imaged from the olfactory bulb of awake rats as they detected and responded to odorants. We used a spontaneously expressed response to novel odorants—exploratory sniffing—as a behavioral measure of odor perception. This assay allowed us to measure the speed with which rats perform simple odor discriminations by monitoring changes in respiration. Rats discriminated a novel odorant from a learned one in as little as 140 ms. Simultaneously imaging the sensory input to the olfactory bulb carried by receptor neurons revealed that the bulk of the response time is due to the peripheral events underlying odorant detection (inhalation and receptor neuron activation), leaving only 50–100 ms for central processing and response initiation. In most trials, responses to a novel odorant began before the initial barrage of input had ceased and before spatial patterns of input to the bulb had fully developed. These results suggest a coding strategy in which the earliest inputs play a major role in the initial perception of odor quality and place constraints on coding schemes based on simple changes in firing rate.
Imaging the olfactory bulb of awake rats reveals that odor discrimination occurs about 100 ms after sensory input reaches the brain, sharply limiting the role that spike rate and temporal integration can play in coding odor identity.
doi:10.1371/journal.pbio.0060082
PMCID: PMC2288628  PMID: 18399719

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