Expression patterns of the transcription factors Fezf1, Fezf2, and Bcl11b in the olfactory organs of turtle embryos.

Many tetrapod vertebrates have two distinct olfactory organs, the olfactory epithelium (OE) and vomeronasal organ (VNO). In turtles, the olfactory organ consists of two types of sensory epithelia, the upper chamber epithelium (UCE; corresponding to the OE) and the lower chamber epithelium (LCE; corresponding to the VNO). In many turtle species, the UCE contains ciliated olfactory receptor cells (ORCs) and the LCE contains microvillous ORCs. To date, several transcription factors involved in the development of the OE and VNO have been identified in mammals. Fez family zinc-finger protein 1 and 2 (Fezf1 and 2) are expressed in the OE and VNO, respectively, of mouse embryos, and are involved in the development and maintenance of ORCs. B-cell lymphoma/leukemia 11B (Bcl11b) is expressed in the mouse embryo OE except the dorsomedial parts of the nasal cavity, and regulates the expression of odorant receptors in the ORCs. In this study, we examined the expression of Fezf1, Fezf2, and Bcl11b in the olfactory organs of embryos in three turtle species, Pelodiscus sinensis, Trachemys scripta elegans, and Centrochelys sulcata, to evaluate their involvement in the development of reptile olfactory organs. In all three turtle species, Bcl11b was expressed in the UCE, Fezf2 in the LCE, and Fezf1 in both the UCE and LCE. These results imply that the roles of the transcription factors Fezf1, Fezf2, and Bcl11b in olfactory organ development are conserved among mammals and turtles.

Semiochemical responsive olfactory sensory neurons are sexually dimorphic and plastic.

Understanding how genes and experience work in concert to generate phenotypic variability will provide a better understanding of individuality. Here, we considered this in the main olfactory epithelium, a chemosensory structure with over a thousand distinct cell types in mice. We identified a subpopulation of olfactory sensory neurons, defined by receptor expression, whose abundances were sexually dimorphic. This subpopulation of olfactory sensory neurons was over-represented in sex-separated mice and robustly responsive to sex-specific semiochemicals. Sex-combined housing led to an attenuation of the dimorphic representations. Single-cell sequencing analysis revealed an axis of activity-dependent gene expression amongst a subset of the dimorphic OSN populations. Finally, the pro-apoptotic gene Baxwas necessary to generate the dimorphic representations. Altogether, our results suggest a role of experience and activity in influencing homeostatic mechanisms to generate a robust sexually dimorphic phenotype in the main olfactory epithelium.

Widespread receptor-driven modulation in peripheral olfactory coding.

Olfactory responses to single odors have been well characterized but in reality we are continually presented with complex mixtures of odors. We performed high-throughput analysis of single-cell responses to odor blends using Swept Confocally Aligned Planar Excitation (SCAPE) microscopy of intact mouse olfactory epithelium, imaging ~10,000 olfactory sensory neurons in parallel. In large numbers of responding cells, mixtures of odors did not elicit a simple sum of the responses to individual components of the blend. Instead, many neurons exhibited either antagonism or enhancement of their response in the presence of another odor. All eight odors tested acted as both agonists and antagonists at different receptors. We propose that this peripheral modulation of responses increases the capacity of the olfactory system to distinguish complex odor mixtures.

Bilateral and unilateral odor processing and odor perception.

Imagine smelling a novel perfume with only one nostril and then smelling it again with the other nostril. Clearly, you can tell that it is the same perfume both times. This simple experiment demonstrates that odor information is shared across both hemispheres to enable perceptual unity. In many sensory systems, perceptual unity is believed to be mediated by inter-hemispheric connections between iso-functional cortical regions. However, in the olfactory system, the underlying neural mechanisms that enable this coordination are unclear because the two olfactory cortices are not topographically organized and do not seem to have homotypic inter-hemispheric mapping. This review presents recent advances in determining which aspects of odor information are processed unilaterally or bilaterally, and how odor information is shared across the two hemispheres. We argue that understanding the mechanisms of inter-hemispheric coordination can provide valuable insights that are hard to achieve when focusing on one hemisphere alone.

The Zonal Organization of Odorant Receptor Gene Choice in the Main Olfactory Epithelium of the Mouse.

A mature olfactory sensory neuron (OSN) of the main olfactory epithelium (MOE) typically expresses one allele of one odorant receptor (OR) gene. It is widely thought that the great majority of the 1,141 intact mouse OR genes are expressed in one of four MOE zones (or bands or stripes), which are largely non-overlapping. Here, we develop a multiplex method to map, in 3D and MOE-wide, the expression areas of multiple OR genes in individual, non-genetically modified mice by three-color fluorescence in situ hybridization, semi-automated image segmentation, and 3D reconstruction. We classify the expression areas of 68 OR genes into 9 zones. These zones are highly overlapping and strikingly complex when viewed in 3D reconstructions. There could well be more zones. We propose that zones reflect distinct OSN types that are each restricted in their choice to a subset of the OR gene repertoire.

Single-cell analysis of olfactory neurogenesis and differentiation in adult humans.

The presence of active neurogenic niches in adult humans is controversial. We focused attention to the human olfactory neuroepithelium, an extracranial site supplying input to the olfactory bulbs of the brain. Using single-cell RNA sequencing analyzing 28,726 cells, we identified neural stem cell and neural progenitor cell pools and neurons. Additionally, we detailed the expression of 140 olfactory receptors. These data from the olfactory neuroepithelium niche provide evidence that neuron production may continue for decades in humans.

Respiratory syncytial virus tropism for olfactory sensory neurons in mice.

The olfactory mucosa, where the first step of odor detection occurs, is a privileged pathway for environmental toxicants and pathogens toward the central nervous system. Indeed, some pathogens can infect olfactory sensory neurons including their axons projecting to the olfactory bulb allowing them to bypass the blood-brain barrier and reach the central nervous system (CNS) through the so-called olfactory pathway. The respiratory syncytial virus (RSV) is a major respiratory tract pathogen but there is growing evidence that RSV may lead to CNS impairments. However, the mechanisms involved in RSV entering into the CNS have been poorly described. In this study, we wanted to explore the capacity of RSV to reach the CNS via the olfactory pathway and to better characterize RSV cellular tropism in the nasal cavity. We first explored the distribution of RSV infectious sites in the nasal cavity by in vivo bioluminescence imaging and a tissue clearing protocol combined with deep-tissue imaging and 3D image analyses. This whole tissue characterization was confirmed with immunohistochemistry and molecular biology approaches. Together, our results provide a novel 3D atlas of mouse nasal cavity anatomy and show that RSV can infect olfactory sensory neurons giving access to the central nervous system by entering the olfactory bulb. Cover Image for this issue: doi: 10.1111/jnc.14765.

Therapeutic potential of ectopic olfactory and taste receptors.

Olfactory and taste receptors are expressed primarily in the nasal olfactory epithelium and gustatory taste bud cells, where they transmit real-time sensory signals to the brain. However, they are also expressed in multiple extra-nasal and extra-oral tissues, being implicated in diverse biological processes including sperm chemotaxis, muscle regeneration, bronchoconstriction and bronchodilatation, inflammation, appetite regulation and energy metabolism. Elucidation of the physiological roles of these ectopic receptors is revealing potential therapeutic and diagnostic applications in conditions including wounds, hair loss, asthma, obesity and cancers. This Review outlines current understanding of the diverse functions of ectopic olfactory and taste receptors and assesses their potential to be therapeutically exploited.

A Near-Complete Spatial Map of Olfactory Receptors in the Mouse Main Olfactory Epithelium.

Different regions of the mammalian nose smell different odors. In the mouse olfactory system, spatially regulated expression of >1000 olfactory receptors (ORs) along the dorsomedial-ventrolateral (DV) axis forms a topological map in the main olfactory epithelium (MOE). However, the locations of most ORs along the DV axis are currently unknown. By sequencing mRNA of 12 isolated MOE pieces, we mapped out the DV locations-as quantified by "zone indices" on a scale of 1-5-of 1033 OR genes with an estimated error of 0.3 zone indices. Our map covered 81% of all intact OR genes and 99.4% of the total OR mRNA abundance. Spatial regulation tended to vary gradually along chromosomes. We further identified putative non-OR genes that may exhibit spatial expression along the DV axis.

The mechanisms underlying olfactory deficits in apolipoprotein E-deficient mice: focus on olfactory epithelium and olfactory bulb.

Apolipoprotein E (ApoE) is highly expressed in the central nervous system including the olfactory epithelium (OE) and olfactory bulb (OB). ApoE induction is beneficial for Alzheimer's disease (AD) treatment, whereas ApoE deficiency results in impaired olfaction, but the timing and underlying molecular and cellular mechanisms of these effects remain unclear. Uncovering the mechanisms underlying olfactory dysfunction in ApoE-deficient mice might provide a potential avenue for the early diagnosis of AD. We used an ApoE knockout (ApoE-/-) mouse model and a cookie-finding test to reveal an olfactory deficit in 3- to 5-month-old, but not 1- to 2-month-old, ApoE-/- mice. Electrophysiological experiments indicated a significant decline in the electroolfactogram (EOG) amplitude, which was associated with an increase in rise time in ApoE-/- mice. Knockout mice also exhibited compromised olfactory adaptation, as well as a reduced number of mature olfactory sensory neurons in the OE. Local field potential recording in the OB showed that gamma oscillation power was enhanced, which might be attributed to an increase in GABAergic inhibition mediated by parvalbumin-expressing (PV) interneurons. This study demonstrates the critical involvement of ApoE in olfactory information processing in the OE and OB. ApoE deficiency results in olfaction deficits in mice as young as 3 months old, which has implications for AD pathogenesis.

Simultaneous Loss of NCKX4 and CNG Channel Desensitization Impairs Olfactory Sensitivity.

In vertebrate olfactory sensory neurons (OSNs), Ca2+ plays key roles in both mediating and regulating the olfactory response. Ca2+ enters OSN cilia during the response through the olfactory cyclic nucleotide-gated (CNG) channel and stimulates a depolarizing chloride current by opening the olfactory Ca2+-activated chloride channel to amplify the response. Ca2+ also exerts negative regulation on the olfactory transduction cascade, through mechanisms that include reducing the CNG current by desensitizing the CNG channel via Ca2+/calmodulin (CaM), to reduce the response. Ca2+ is removed from the cilia primarily by the K+-dependent Na+/Ca2+ exchanger 4 (NCKX4), and the removal of Ca2+ leads to closure of the chloride channel and response termination. In this study, we investigate how two mechanisms conventionally considered negative regulatory mechanisms of olfactory transduction, Ca2+ removal by NCKX4, and desensitization of the CNG channel by Ca2+/CaM, interact to regulate the olfactory response. We performed electro-olfactogram (EOG) recordings on the double-mutant mice, NCKX4-/-;CNGB1ΔCaM, which are simultaneously lacking NCKX4 (NCKX4-/-) and Ca2+/CaM-mediated CNG channel desensitization (CNGB1ΔCaM). Despite exhibiting alterations in various response attributes, including termination kinetics and adaption properties, OSNs in either NCKX4-/- mice or CNGB1ΔCaM mice show normal resting sensitivity, as determined by their unchanged EOG response amplitude. We found that OSNs in NCKX4-/-;CNGB1ΔCaM mice displayed markedly reduced EOG amplitude accompanied by alterations in other response attributes. This study suggests that what are conventionally considered negative regulatory mechanisms of olfactory transduction also play a role in setting the resting sensitivity in OSNs. SIGNIFICANCE STATEMENT: Sensory receptor cells maintain high sensitivity at rest. Although the mechanisms responsible for setting the resting sensitivity of sensory receptor cells are not well understood, it has generally been assumed that the sensitivity is set primarily by how effectively the components in the activation cascade of sensory transduction can be stimulated. Our findings in mouse olfactory sensory neurons suggest that mechanisms that are primarily responsible for terminating the olfactory response are also critical for proper resting sensitivity.

Engraftment and regenerative effects of bone marrow stromal cell transplantation on damaged rat olfactory mucosa.

To develop a new therapeutic method to treat olfactory deficits, we investigated the engraftment and regenerative effects of transplanted bone marrow stromal cells (BMSCs) on damaged rat olfactory mucosa. To induce olfactory nerve degeneration, one side of the olfactory mucosa of Sprague-Dawley rats was damaged via Triton X-100 irrigation. Phosphate-buffered saline containing syngeneic BMSCs was injected into the olfactory mucosa for transplantation. PKH fluorescent cell dye labeling of BMSCs was used to monitor the transplanted cells. After transplantation of BMSCs, the thickness and regeneration of olfactory mucosa were analyzed using hematoxylin-eosin (H&E) staining. S100 immunohistochemical staining was used to measure nerve sheath regeneration. The increase in NGF (nerve growth factor) level in the olfactory mucosa was measured by Western blot analysis. Transplanted bone marrow stromal cells were engrafted to the lamia propria of damaged mucosa. The mean time for normalization of thickness and morphological recovery of the olfactory mucosa was 4 weeks in the therapeutic group and 9 weeks in the control group. S100 immunoreactivity was higher on the BMSC-treated side than on the control side. During regeneration, the expression of NGF increased in the olfactory mucosa of the experimental group. Based on these results, BMSC transplantation accelerated regeneration of olfactory mucosa damaged by Triton X-100, and NGF may be essential to this regenerative process.

Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis.

The sense of smell allows chemicals to be perceived as diverse scents. We used single-neuron RNA sequencing to explore the developmental mechanisms that shape this ability as nasal olfactory neurons mature in mice. Most mature neurons expressed only one of the ~1000 odorant receptor genes (Olfrs) available, and at a high level. However, many immature neurons expressed low levels of multiple Olfrs. Coexpressed Olfrs localized to overlapping zones of the nasal epithelium, suggesting regional biases, but not to single genomic loci. A single immature neuron could express Olfrs from up to seven different chromosomes. The mature state in which expression of Olfr genes is restricted to one per neuron emerges over a developmental progression that appears to be independent of neuronal activity involving sensory transduction molecules.

Lesion of the olfactory epithelium accelerates prion neuroinvasion and disease onset when prion replication is restricted to neurons.

Natural prion diseases of ruminants are moderately contagious and while the gastrointestinal tract is the primary site of prion agent entry, other mucosae may be entry sites in a subset of infections. In the current study we examined prion neuroinvasion and disease induction following disruption of the olfactory epithelium in the nasal mucosa since this site contains environmentally exposed olfactory sensory neurons that project directly into the central nervous system. Here we provide evidence for accelerated prion neuroinvasion and clinical onset from the olfactory mucosa after disruption and regeneration of the olfactory epithelium and when prion replication is restricted to neurons. In transgenic mice with neuron restricted replication of prions, there was a reduction in survival when the olfactory epithelium was disrupted prior to intranasal inoculation and there was >25% decrease in the prion incubation period. In a second model, the neurotropic DY strain of transmissible mink encephalopathy was not pathogenic in hamsters by the nasal route, but 50% of animals exhibited brain infection and/or disease when the olfactory epithelium was disrupted prior to intranasal inoculation. A time course analysis of prion deposition in the brain following loss of the olfactory epithelium in models of neuron-restricted prion replication suggests that neuroinvasion from the olfactory mucosa is via the olfactory nerve or brain stem associated cranial nerves. We propose that induction of neurogenesis after damage to the olfactory epithelium can lead to prion infection of immature olfactory sensory neurons and accelerate prion spread to the brain.

NaV1.5 sodium channel window currents contribute to spontaneous firing in olfactory sensory neurons.

Olfactory sensory neurons (OSNs) fire spontaneously as well as in response to odor; both forms of firing are physiologically important. We studied voltage-gated Na(+) channels in OSNs to assess their role in spontaneous activity. Whole cell patch-clamp recordings from OSNs demonstrated both tetrodotoxin-sensitive and tetrodotoxin-resistant components of Na(+) current. RT-PCR showed mRNAs for five of the nine different Na(+) channel α-subunits in olfactory tissue; only one was tetrodotoxin resistant, the so-called cardiac subtype NaV1.5. Immunohistochemical analysis indicated that NaV1.5 is present in the apical knob of OSN dendrites but not in the axon. The NaV1.5 channels in OSNs exhibited two important features: 1) a half-inactivation potential near -100 mV, well below the resting potential, and 2) a window current centered near the resting potential. The negative half-inactivation potential renders most NaV1.5 channels in OSNs inactivated at the resting potential, while the window current indicates that the minor fraction of noninactivated NaV1.5 channels have a small probability of opening spontaneously at the resting potential. When the tetrodotoxin-sensitive Na(+) channels were blocked by nanomolar tetrodotoxin at the resting potential, spontaneous firing was suppressed as expected. Furthermore, selectively blocking NaV1.5 channels with Zn(2+) in the absence of tetrodotoxin also suppressed spontaneous firing, indicating that NaV1.5 channels are required for spontaneous activity despite resting inactivation. We propose that window currents produced by noninactivated NaV1.5 channels are one source of the generator potentials that trigger spontaneous firing, while the upstroke and propagation of action potentials in OSNs are borne by the tetrodotoxin-sensitive Na(+) channel subtypes.

Innate immune responses and neuroepithelial degeneration and regeneration in the mouse olfactory mucosa induced by intranasal administration of Poly(I:C).

The pathogenesis of postviral olfactory disorder (PVOD) has not been fully elucidated. We investigated morphological changes and innate immune responses in the mouse olfactory mucosa induced by intranasal administration of polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA. Mice received three administrations of saline with or without Poly(I:C), once every 24 h. The olfactory mucosa was harvested at various intervals after the first administration (8 h, 3, 9 and 24 days). In the Poly(I:C) group, the number of apoptotic cells in the olfactory neuroepithelium had increased at 8 h. At 9 days, the olfactory neuroepithelium had severely degenerated and behavioral tests demonstrated that the mice showed signs of olfactory deterioration. At 24 days, the structure of the neuroepithelium had regenerated almost completely. Regarding the innate immune responses, many neutrophils had infiltrated the olfactory neuroepithelium at 8 h and had exuded into the nasal cavity by 3 days. Macrophages had also infiltrated the olfactory neuroepithelium at 8 h although to a lesser extent, but they still remained in the neuroepithelium at 24 days. Poly(I:C)-induced neuroepithelial damage was significantly inhibited by a neutrophil elastase inhibitor and was suppressed in neutropenic model mice. These findings suggest that the secondary damage caused by the neutrophil-mediated innate immune response plays an important role in the pathogenesis of PVOD.

Immunohistochemical characterization of the crypt neurons in the olfactory epithelium of adult zebrafish.

The fish sensory epithelium contains three types of sensory cells denominated ciliated, microvillous, and crypt neurons. Each one differs from the other in its morphological, ultrastructural and molecular features, as well as in their projections to the central nervous system. Crypt neurons are present in both bony and cartilaginous fish and can be identified on the basis of their morphology and the expression of some specific proteins and genes. In this study we have investigated the morphology of crypt neurons, as well as the occurrence and co-localization of S100 protein, calretinin and TRPV4, three proposed markers for crypt cells, in the olfactory epithelium of adult zebrafish (Danio rerio) using double immunofluorescence associated to laser confocal microscopy. A sparse population of superficial S100 protein positive cells was detected being identified as crypt neurons. The calretinin immunoreactive cells were more abundant, occasionally resembling the morphology of the crypt cells but never displaying co-localization of both proteins. The TRPV4 positive cells differed in morphology from crypt cells, thus excluding the occurrence of TRPV4 in those cells. These results demonstrate that only S100 protein immunoreactivity can be used to identify crypt cells. Because some calretinin positive cells showed localization and morphology similar to the crypt cells of the sensory epithelium, the occurrence of two subtypes of crypt cells, S100 protein and calretinin positive, cannot be excluded. The significance of these findings remains to be elucidated.

Olfaction: anatomy, physiology, and disease.

The olfactory system is an essential part of human physiology, with a rich evolutionary history. Although humans are less dependent on chemosensory input than are other mammals (Niimura 2009, Hum. Genomics 4:107-118), olfactory function still plays a critical role in health and behavior. The detection of hazards in the environment, generating feelings of pleasure, promoting adequate nutrition, influencing sexuality, and maintenance of mood are described roles of the olfactory system, while other novel functions are being elucidated. A growing body of evidence has implicated a role for olfaction in such diverse physiologic processes as kin recognition and mating (Jacob et al. 2002a, Nat. Genet. 30:175-179; Horth 2007, Genomics 90:159-175; Havlicek and Roberts 2009, Psychoneuroendocrinology 34:497-512), pheromone detection (Jacob et al. 200b, Horm. Behav. 42:274-283; Wyart et al. 2007, J. Neurosci. 27:1261-1265), mother-infant bonding (Doucet et al. 2009, PLoS One 4:e7579), food preferences (Mennella et al. 2001, Pediatrics 107:E88), central nervous system physiology (Welge-Lüssen 2009, B-ENT 5:129-132), and even longevity (Murphy 2009, JAMA 288:2307-2312). The olfactory system, although phylogenetically ancient, has historically received less attention than other special senses, perhaps due to challenges related to its study in humans. In this article, we review the anatomic pathways of olfaction, from peripheral nasal airflow leading to odorant detection, to epithelial recognition of these odorants and related signal transduction, and finally to central processing. Olfactory dysfunction, which can be defined as conductive, sensorineural, or central (typically related to neurodegenerative disorders), is a clinically significant problem, with a high burden on quality of life that is likely to grow in prevalence due to demographic shifts and increased environmental exposures.

Kinetics of chemically mediated neurodegeneration/neuroregeneration of mouse olfactory epithelium: monitoring by hyperlayer sedimentation field flow fractionation.

The increase in the incidence of neurodegenerative diseases linked to aging or injury needs to be addressed in research into neuroprotective or neuroregenerative therapies, and requires the development of specific biological models. To achieve this goal we propose (1) the use of the mouse olfactory epithelium as a biological support which specifically exhibits a regenerative or a self-renewing capacity and during the lifetime necessitates the presence of neural stem cells, and (2) the use of an intraperitoneal injection of 2,6-dichlorobenzonitrile (diclobenil) as a chemical inducer of neurodegeneration in olfactory epithelium by selectively killing mature cells. We developed a biological model to follow the processes of neurodegeneration (chemically induced) and neuroregeneration (self-renewal of olfactory epithelium). The purpose of this study was to develop a method to monitor quickly neurodegeneration/neuroregeneration processes in order to further screen protective and regenerative therapies. For this purpose, we used the sedimentation field flow fractionation elution of olfactory epithelium. We obtained specific elution profiles and retention parameters allowing the monitoring of the induction and kinetics of biological processes. The use of insulin-like growth factor 1α as a neuroprotective agent in an innovative nebulization protocol showed sedimentation field flow fractionation to be a simple, fast and low-cost method to monitor such a biological event on the scale of an entire organism.