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The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca(2+) signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca(2+)-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca(2+)/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca(2+) signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca(2+)-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca(2+)-signaling networks in the olfactory system of the rat.
The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.
The ability of an animal to detect, discriminate, and respond to odors depends on the functions of its olfactory receptor neurons (ORNs). The extent to which each ORN, upon activation, contributes to chemotaxis is not well understood. We hypothesized that strong activation of each ORN elicits a different behavioral response in the Drosophila melanogaster larva by differentially affecting the composition of its navigational behavior. To test this hypothesis, we exposed Drosophila larvae to specific odorants to analyze the effect of individual ORN activity on chemotaxis. We used two different behavioral paradigms to analyze the chemotaxis response of larvae to odorants. When tested with five different odorants that elicit strong physiological responses from single ORNs, larval behavioral responses toward each odorant differed in the strength of attraction as well as in the composition of discrete navigational elements, such as runs and turns. Further, behavioral responses to odorants did not correlate with either the strength of odor gradients tested or the sensitivity of each ORN to its cognate odorant. Finally, we provide evidence that wild-type larvae with all ORNs intact exhibit higher behavioral variance than mutant larvae that have only a single pair of functional ORNs. We conclude that individual ORNs contribute differently to the olfactory circuit that instructs chemotactic responses. Our results, along with recent studies from other groups, suggest that ORNs are functionally nonequivalent units. These results have implications for understanding peripheral odor coding.
Olfactory sensory neurons choose to express a single odorant receptor (OR) from a large gene repertoire and extend axons to reproducible, OR-specific locations within the olfactory bulb. This developmental process produces a topographically organized map of odorant experience in the brain. The axon guidance mechanisms that generate this pattern of connectivity, as well as those that coordinate OR choice and axonal guidance receptor expression, are incompletely understood. We applied the powerful approach of single-cell RNA-seq on newly born olfactory sensory neurons (OSNs) in young zebrafish larvae to address these issues. Expression profiles were generated for 56 individual Olfactory Marker Protein (OMP) positive sensory neurons by single-cell (SC) RNA-seq. We show that just as in mouse OSNs, mature zebrafish OSNs typically express a single predominant OR transcript. Our previous work suggests that OSN targeting is related to the OR clade from which a sensory neuron chooses to express its odorant receptor. We categorized each of the mature cells based on the clade of their predominantly expressed OR. Transcripts expressed at higher levels in each of three clade-related categories were identified using Penalized Linear Discriminant Analysis (PLDA). A genome-wide approach was used to identify membrane-associated proteins that are most likely to have guidance-related activity. We found that OSNs that choose to express an OR from a particular clade also express specific subsets of potential axon guidance genes and transcription factors. We validated our identification of candidate axon guidance genes for one clade of OSNs using bulk RNA-seq from a subset of transgene-labeled neurons that project to a single protoglomerulus. The differential expression patterns of selected candidate guidance genes were confirmed using fluorescent in situ hybridization. Most importantly, we observed axonal mistargeting in knockouts of three candidate axonal guidance genes identified in this analysis: nrp1a, nrp1b, and robo2. In each case, targeting errors were detected in the subset of axons that normally express these transcripts at high levels, and not in the axons that express them at low levels. Our findings demonstrate that specific, functional, axonal guidance related genes are expressed in subsets of OSNs that that can be categorized by their patterns of OR expression.
In the Drosophila antenna, different subtypes of olfactory receptor neurons (ORNs) housed in the same sensory hair (sensillum) can inhibit each other non-synaptically. However, the mechanisms underlying this underexplored form of lateral inhibition remain unclear. Here we use recordings from pairs of sensilla impaled by the same tungsten electrode to demonstrate that direct electrical ("ephaptic") interactions mediate lateral inhibition between ORNs. Intriguingly, within individual sensilla, we find that ephaptic lateral inhibition is asymmetric such that one ORN exerts greater influence onto its neighbor. Serial block-face scanning electron microscopy of genetically identified ORNs and circuit modeling indicate that asymmetric lateral inhibition reflects a surprisingly simple mechanism: the physically larger ORN in a pair corresponds to the dominant neuron in ephaptic interactions. Thus, morphometric differences between compartmentalized ORNs account for highly specialized inhibitory interactions that govern information processing at the earliest stages of olfactory coding.
During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to generate defined motor outputs, or behavior. This integration requires a developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown. Here, we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless (fru). The first mode involves a developmental program that coordinately restricts to appropriate ORNs the expression of fru and two olfactory receptors (Or47b and Ir84a) involved in sex-specific behaviors. This regulation requires the chromatin modulatory protein Alhambra (Alh). The second mode relies on the signaling from the olfactory receptors through CamK and histone acetyl transferase p300/CBP to maintain ORN-specific fru expression. Our results highlight two feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs. Such a dual mechanism of fru regulation in ORNs might be a trait of neurons driving plastic aspects of sex-specific behaviors.
In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a G protein-coupled receptor-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kgamma and cloned a gene for a PI3K with amino acid homology with PI3Kbeta. The lobster olfactory PI3K co-immunoprecipitates with the G protein alpha and beta subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kgamma and beta isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction.
Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/- ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/- mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.
We show that lobster olfactory receptor neurons (ORNs), much like their vertebrate counterparts, generate a transient elevation of intracellular calcium (Ca(i)) in response to odorant activation that can be used to monitor ensemble ORN activity. This is done in antennal slice preparation in situ maintaining the polarity of the cells and the normal micro-environment of the olfactory cilia. The Ca(i) signal is ligand-specific and increases in a dose-dependent manner in response to odorant stimulation. Saturating stimulation elicits a robust increase of up to 1 μM free Ca(i) within 1-2s of stimulation. The odor-induced Ca(i) response closely follows the discharge pattern of extracellular spikes elicited by odorant application, with the maximal rise in Ca(i) matching the peak of the spike generation. The Ca(i) signal can be used to track neuronal activity in a functional subpopulation of rhythmically active ORNs and discriminate it from that of neighboring tonically active ORNs. Being able to record from many ORNs simultaneously over an extended period of time not only allows more accurate estimates of neuronal population activity but also dramatically improves the ability to identify potential new functional subpopulations of ORNs, especially those with more subtle differences in responsiveness, ligand specificity, and/or transduction mechanisms.
Gene expression is highly regulated to functionally diversify cells. Genes that cooperate in the same physiological processes occasionally reside within nearby regions in a chromosome. Olfactory marker protein (OMP) is highly expressed in mature olfactory receptor neurons (ORNs), but its physiological roles are not fully understood. According to the genomic map, the OMP gene is located within an intron of the calcium-dependent protease, calpain 5 (CAPN5); in other words, the OMP gene is a nested intronic gene. Thus, we attempted to investigate the gene expression and protein distribution of CAPN5 in the olfactory epithelium compared with that in the central nervous system (CNS). By performing reverse-transcriptase PCR and in situ hybridization, we confirmed that CAPN5 mRNA was expressed in the olfactory epithelium. We then performed immunohistological investigations using sliced preparations obtained from mice expressing GFP under OMP promoter activity. The detected GFP fluorescence was restricted to the knob, soma and axon bundles of the ORNs, while CAPN5 immunoreactivity (CAPN5-IR) was ubiquitously detected in the olfactory epithelial layer and lamina propria; signals were strongly detected in the supporting cells within the epithelium. In the CNS, CAPN5 signals were widely detected and were especially strong in the hippocampal formation and the piriform cortex as previously indicated. Therefore, these data indicate that ORNs express OMP but not CAPN5 from CAPN5 gene expression even though they are localized in the same genomic locus. The mechanisms by which the OMP promoter is regulated require detailed investigations.
At least two different G-protein-mediated transduction cascades, the adenylate cyclase and phospholipase C (PLC) pathway, process chemosensory stimuli for various species. In squid olfactory receptor neurons (ORNs), physiological studies indicate that both pathways may be present; however, confirmation of the transduction molecules at the protein level is absent. Here we provide evidence that the G-proteins involved in both adenylate cyclase and PLC pathways are present in squid ORNs (Lolliguncula brevis). We used immunoblotting to show that Galpha(olf), Galpha(q), and a downstream effector, enzyme PLC140, are present in the squid olfactory epithelium (OE). To localize these proteins to one or more of the five morphological cell types described for squid OE, paraformaldehyde-fixed olfactory organs were cryosectioned (10 microm), double-labeled for Galpha(olf), Galpha(q), or PLC140, and imaged. Analysis of serial sections from entire olfactory organs for epithelial area and patterns of immunofluorescence revealed a region of highest immunoreactivity at the anterior half of the organ. At the cellular level, type 1 cells could not be distinguished morphologically and were not included in the analysis. The three labeling patterns observed in type 2 cells were Galpha(q) alone, PLC140 alone, and colocalization of Galpha(q) and PLC140. Subsets of cell types 3, 4, and 5 showed colocalization of Galpha(olf) with Galpha(q) but not with PLC140. These data suggest that the PLC pathway predominates in type 2 cells; however, coexpression of Galpha(olf) with Galpha(q) in cell types 3, 4, and 5 suggests that both pathways may participate in olfactory transduction in non-type 2 squid ORNs.
Odorants binding to olfactory receptor neurons (ORNs) trigger bursts of action potentials, providing the brain with its only experience of the olfactory environment. Our recordings made in vivo from locust ORNs showed that odor-elicited firing patterns comprise four distinct response motifs, each defined by a reliable temporal profile. Different odorants could elicit different response motifs from a given ORN, a property we term motif switching. Further, each motif undergoes its own form of sensory adaptation when activated by repeated plume-like odor pulses. A computational model constrained by our recordings revealed that organizing responses into multiple motifs provides substantial benefits for classifying odors and processing complex odor plumes: each motif contributes uniquely to encode the plume's composition and structure. Multiple motifs and motif switching further improve odor classification by expanding coding dimensionality. Our model demonstrated that these response features could provide benefits for olfactory navigation, including determining the distance to an odor source.
A developmental program of epigenetic repression prepares each mammalian olfactory sensory neuron (OSN) to strongly express one allele from just one of hundreds of odorant receptor (OR) genes, but what completes this process of OR gene choice by driving the expression of this allele is incompletely understood. Conditional deletion experiments in mice demonstrate that Lhx2 is necessary for normal expression frequencies of nearly all ORs and all trace amine-associated receptors, irrespective of whether the deletion of Lhx2 is initiated in immature or mature OSNs. Given previous evidence that Lhx2 binds OR gene control elements, these findings indicate that Lhx2 is directly involved in driving OR expression. The data also support the conclusion that OR expression is necessary to allow immature OSNs to complete differentiation and become mature. In contrast to the robust effects of conditional deletion of Lhx2, the loss of Emx2 has much smaller effects and more often causes increased expression frequencies. Lhx2:Emx2 double mutants show opposing effects on Olfr15 expression that reveal independent effects of these two transcription factors. While Lhx2 is necessary for OR expression that supports OR gene choice, Emx2 can act differently; perhaps by helping to control the availability of OR genes for expression.
Neuropeptide S (NPS) is a newly identified neuromodulator located in the brainstem and regulates various biological functions by selectively activating the NPS receptors (NPSR). High level expression of NPSR mRNA in the olfactory cortex suggests that NPS-NPSR system might be involved in the regulation of olfactory function. The present study was undertaken to investigate the effects of intracerebroventricular (i.c.v.) injection of NPS or co-injection of NPSR antagonist on the olfactory behaviors, food intake, and c-Fos expression in olfactory cortex in mice. In addition, dual-immunofluorescence was employed to identify NPS-induced Fos immunereactive (-ir) neurons that also bear NPSR. NPS (0.1-1 nmol) i.c.v. injection significantly reduced the latency to find the buried food, and increased olfactory differentiation of different odors and the total sniffing time spent in olfactory habituation/dishabituation tasks. NPS facilitated olfactory ability most at the dose of 0.5 nmol, which could be blocked by co-injection of 40 nmol NPSR antagonist [D-Val(5)]NPS. NPS administration dose-dependently inhibited food intake in fasted mice. Ex-vivo c-Fos and NPSR immunohistochemistry in the olfactory cortex revealed that, as compared with vehicle-treated mice, NPS markedly enhanced c-Fos expression in the anterior olfactory nucleus (AON), piriform cortex (Pir), ventral tenia tecta (VTT), the anterior cortical amygdaloid nucleus (ACo) and lateral entorhinal cortex (LEnt). The percentage of Fos-ir neurons that also express NPSR were 88.5% and 98.1% in the AON and Pir, respectively. The present findings demonstrated that NPS, via selective activation of the neurons bearing NPSR in the olfactory cortex, facilitates olfactory function in mice.
Two large families of olfactory receptors, the Odorant Receptors (ORs) and Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odorant binding "tuning" OrX receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco. The situation for IRs is more complex. Different tuning IrX receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a co-receptor, but its function has not been systematically examined. Surprisingly, recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses. In contrast, amine responses persist in the absence of Ir76b or Ir25a in ac1 sensilla but are lost in a double mutant. We show that responses mediated by acid-sensing neurons do not require Ir76b, despite their expression of this co-receptor. Our study also demonstrates that one population of coeloconic olfactory neurons exhibits Ir76b/Ir25a-dependent and Orco-dependent responses to distinct odorants. Together, our data establish the role of Ir76b as a bona fide co-receptor, which acts in partnership with Ir25a. Given that these co-receptors are among the most highly conserved olfactory receptors and are often co-expressed in chemosensory neurons, our data suggest Ir76b and Ir25a also work in tandem in other insects.
In olfactory receptor neurons (ORNs) of aquatic animals amino acids have been shown to be potent stimuli. Here we report on calcium imaging experiments in slices of the olfactory mucosa of Xenopus laevis tadpoles. We were able to determine the response profiles of 283 ORNs to 19 amino acids, where one profile comprises the responses of one ORN to 19 amino acids. 204 out of the 283 response profiles differed from each other. 36 response spectra occurred more than once, i.e., there were 36 classes of ORNs identically responding to the 19 amino acids. The number of ORNs that formed a class ranged from 2 to 13. Shape and duration of amino acid-elicited [Ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid. Different amino acids, however, in some cases led to clearly distinguishable calcium responses in individual ORNs. Furthermore, ORNs clearly appeared to gain selectivity over time, i.e., ORNs of later developmental stages responded to less amino acids than ORNs of earlier stages. We discuss the narrowing of ORN selectivity over stages in the context of expression of olfactory receptors.
Individual receptor neurons in the peripheral olfactory organ extend long axons into the olfactory bulb forming synapses with projection neurons in spherical neuropil regions, called glomeruli. Generally, odor map formation and odor processing in all vertebrates is based on the assumption that receptor neuron axons exclusively connect to a single glomerulus without any axonal branching. We comparatively tested this hypothesis in multiple fish and amphibian species (both sexes) by applying sparse cell electroporation to trace single olfactory receptor neuron axons. Sea lamprey (jawless fish) and zebrafish (bony fish) support the unbranched axon concept, with 94% of axons terminating in single glomeruli. Contrastingly, axonal projections of the axolotl (salamander) branch extensively before entering up to six distinct glomeruli. Receptor neuron axons labeled in frog species (Pipidae, Bufonidae, Hylidae, and Dendrobatidae) predominantly bifurcate before entering a glomerulus and 59 and 50% connect to multiple glomeruli in larval and postmetamorphotic animals, respectively. Independent of developmental stage, lifestyle, and adaptations to specific habitats, it seems to be a common feature of amphibian olfactory receptor neuron axons to frequently bifurcate and connect to multiple glomeruli. Our study challenges the unbranched axon concept as a universal vertebrate feature and it is conceivable that also later diverging vertebrates deviate from it. We propose that this unusual wiring logic evolved around the divergence of the terrestrial tetrapod lineage from its aquatic ancestors and could be the basis of an alternative way of odor processing.
Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency. Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements. In this report, we show that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected. These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information.
Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of Drosophila olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.
One of the fundamental questions in olfaction is whether olfactory receptor neurons (ORNs) behave as independent entities within the olfactory epithelium. On the basis that mature ORNs express multiple connexins, I postulated that gap junctional communication modulates olfactory responses in the periphery and that disruption of gap junctions in ORNs reduces olfactory sensitivity. The data collected from characterizing connexin 43 (Cx43) dominant negative transgenic mice OlfDNCX, and from calcium imaging of wild type mice (WT) support my hypothesis.
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