Comparative ultrastructure of the olfactory system in the East African root rat (Tachyoryctes splendens) and the naked mole rat (Heterocephalus glaber).

The ultrastructure of the olfactory system of most fossorial rodents remains largely unexplored. This study sought to investigate the functional structure of the olfactory mucosa and olfactory bulb of two species of fossorial rodents that have distinct behaviour and ecology, the East African root rat (RR) and the naked mole rat (NMR). Transmission electron microscopy and scanning electron microscopy were employed. The basic ultrastructural design of the olfactory system of the two species was largely comparable. In both species, the olfactory mucosa comprised an olfactory epithelium and an underlying lamina propria. The olfactory epithelium revealed olfactory knobs, cilia and microvilli apically and sustentancular cells, olfactory receptor neurons and basal cells in the upper, middle and basal zones, respectively. The lamina propria was constituted by Bowman's glands, olfactory nerve bundles and vasculature supported by loose connective tissue. Within the olfactory bulb, intracellular and extracellular structures including cell organelles, axons and dendrites were elucidated. Notable species differences were observed in the basal zone of the olfactory epithelium and on the luminal surface of the olfactory mucosa. The basal zone of the olfactory epithelium of the RR consisted of a single layer of flattened electron-dense horizontal basal cells while the NMR had juxtaposed electron-dense and electron-lucent heterogenous cells, an occurrence seen as being indicative of quiescent and highly proliferative states of the olfactory epithelia in the two species, respectively. The olfactory epithelial surface of the NMR comprised an elaborate cilia network that intertwined extensively forming loop-like structures whereas in the RR, the surface was rugged and consisted of finger-like processes and irregular masses. With gross and histological studies showing significant differences in the olfactory structures of the two species, these findings are a further manifestation that the olfactory system of the RR and the NMR have evolved differently to reflect their varied olfactory functional needs.

A cilia-bound unconventional secretory pathway for Drosophila odorant receptors.

BACKGROUND: Post-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway. RESULTS: In the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity. CONCLUSIONS: Our results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons.

Distinct information conveyed to the olfactory bulb by feedforward input from the nose and feedback from the cortex.

Sensory systems are organized hierarchically, but feedback projections frequently disrupt this order. In the olfactory bulb (OB), cortical feedback projections numerically match sensory inputs. To unravel information carried by these two streams, we imaged the activity of olfactory sensory neurons (OSNs) and cortical axons in the mouse OB using calcium indicators, multiphoton microscopy, and diverse olfactory stimuli. Here, we show that odorant mixtures of increasing complexity evoke progressively denser OSN activity, yet cortical feedback activity is of similar sparsity for all stimuli. Also, representations of complex mixtures are similar in OSNs but are decorrelated in cortical axons. While OSN responses to increasing odorant concentrations exhibit a sigmoidal relationship, cortical axonal responses are complex and nonmonotonic, which can be explained by a model with activity-dependent feedback inhibition in the cortex. Our study indicates that early-stage olfactory circuits have access to local feedforward signals and global, efficiently formatted information about odor scenes through cortical feedback.

Activity-dependent formation of the topographic map and the critical period in the development of mammalian olfactory system.

Neural activity influences every aspect of nervous system development. In olfactory systems, sensory neurons expressing the same odorant receptor project their axons to stereotypically positioned glomeruli, forming a spatial map of odorant receptors in the olfactory bulb. As individual odors activate unique combinations of glomeruli, this map forms the basis for encoding olfactory information. The establishment of this stereotypical olfactory map requires coordinated regulation of axon guidance molecules instructed by spontaneous activity. Recent studies show that sensory experiences also modify innervation patterns in the olfactory bulb, especially during a critical period of the olfactory system development. This review examines evidence in the field to suggest potential mechanisms by which various aspects of neural activity regulate axon targeting. We also discuss the precise functions served by neural plasticity during the critical period.

Epigenetic programming of stochastic olfactory receptor choice.

The mammalian sense of smell relies upon a vast array of receptor proteins to detect odorant compounds present in the environment. The proper deployment of these receptor proteins in olfactory sensory neurons is orchestrated by a suite of epigenetic processes that remodel the olfactory genes in differentiating neuronal progenitors. The goal of this review is to elucidate the central role of gene regulatory processes acting in neuronal progenitors of olfactory sensory neurons that lead to a singular expression of an odorant receptor in mature olfactory sensory neurons. We begin by describing the principal features of odorant receptor gene expression in mature olfactory sensory neurons. Next, we delineate our current understanding of how these features emerge from multiple gene regulatory mechanisms acting in neuronal progenitors. Finally, we close by discussing the key gaps in our understanding of how these regulatory mechanisms work and how they interact with each other over the course of differentiation.

CD20/MS4A1 is a mammalian olfactory receptor expressed in a subset of olfactory sensory neurons that mediates innate avoidance of predators.

The mammalian olfactory system detects and discriminates between millions of odorants to elicit appropriate behavioral responses. While much has been learned about how olfactory sensory neurons detect odorants and signal their presence, how specific innate, unlearned behaviors are initiated in response to ethologically relevant odors remains poorly understood. Here, we show that the 4-transmembrane protein CD20, also known as MS4A1, is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium of the murine nasal cavity and functions as a mammalian olfactory receptor that recognizes compounds produced by mouse predators. While wildtype mice avoid these predator odorants, mice genetically deleted of CD20 do not appropriately respond. Together, this work reveals a CD20-mediated odor-sensing mechanism in the mammalian olfactory system that triggers innate behaviors critical for organismal survival.

Experience-dependent glial pruning of synaptic glomeruli during the critical period.

Critical periods are temporally-restricted, early-life windows when sensory experience remodels synaptic connectivity to optimize environmental input. In the Drosophila juvenile brain, critical period experience drives synapse elimination, which is transiently reversible. Within olfactory sensory neuron (OSN) classes synapsing onto single projection neurons extending to brain learning/memory centers, we find glia mediate experience-dependent pruning of OSN synaptic glomeruli downstream of critical period odorant exposure. We find glial projections infiltrate brain neuropil in response to critical period experience, and use Draper (MEGF10) engulfment receptors to prune synaptic glomeruli. Downstream, we find antagonistic Basket (JNK) and Puckered (DUSP) signaling is required for the experience-dependent translocation of activated Basket into glial nuclei. Dependent on this signaling, we find critical period experience drives expression of the F-actin linking signaling scaffold Cheerio (FLNA), which is absolutely essential for the synaptic glomeruli pruning. We find Cheerio mediates experience-dependent regulation of the glial F-actin cytoskeleton for critical period remodeling. These results define a sequential pathway for experience-dependent brain synaptic glomeruli pruning in a strictly-defined critical period; input experience drives neuropil infiltration of glial projections, Draper/MEGF10 receptors activate a Basket/JNK signaling cascade for transcriptional activation, and Cheerio/FLNA induction regulates the glial actin cytoskeleton to mediate targeted synapse phagocytosis.

Insertion of a neomycin selection cassette in the Amigo1 locus alters gene expression in the olfactory epithelium leading to region-specific defects in olfactory receptor neuron development.

During development of the nervous system, neurons connect to one another in a precisely organized manner. Sensory systems provide a good example of this organization, whereby the composition of the outside world is represented in the brain by neuronal maps. Establishing correct patterns of neural circuitry is crucial, as inaccurate map formation can lead to severe disruptions in sensory processing. In rodents, olfactory stimuli modulate a wide variety of behaviors essential for survival. The formation of the olfactory glomerular map is dependent on molecular cues that guide olfactory receptor neuron axons to broad regions of the olfactory bulb and on cell adhesion molecules that promote axonal sorting into specific synaptic units in this structure. Here, we demonstrate that the cell adhesion molecule Amigo1 is expressed in a subpopulation of olfactory receptor neurons, and we investigate its role in the precise targeting of olfactory receptor neuron axons to the olfactory bulb using a genetic loss-of-function approach in mice. While ablation of Amigo1 did not lead to alterations in olfactory sensory neuron axonal targeting, our experiments revealed that the presence of a neomycin resistance selection cassette in the Amigo1 locus can lead to off-target effects that are not due to loss of Amigo1 expression, including unexpected altered gene expression in olfactory receptor neurons and reduced glomerular size in the ventral region of the olfactory bulb. Our results demonstrate that insertion of a neomycin selection cassette into the mouse genome can have specific deleterious effects on the development of the olfactory system and highlight the importance of removing antibiotic resistance cassettes from genetic loss-of-function mouse models when studying olfactory system development.

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period.

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.

Transcriptomic Signatures of Neuronally Derived Extracellular Vesicles Reveal the Presence of Olfactory Receptors in Clinical Samples from Traumatic Brain Injury Patients.

Traumatic brain injury (TBI) is defined as an injury to the brain by external forces which can lead to cellular damage and the disruption of normal central nervous system functions. The recently approved blood-based biomarkers GFAP and UCH-L1 can only detect injuries which are detectable on CT, and are not sensitive enough to diagnose milder injuries or concussion. Exosomes are small microvesicles which are released from the cell as a part of extracellular communication in normal as well as diseased states. The objective of this study was to identify the messenger RNA content of the exosomes released by injured neurons to identify new potential blood-based biomarkers for TBI. Human severe traumatic brain injury samples were used for this study. RNA was isolated from neuronal exosomes and total transcriptomic sequencing was performed. RNA sequencing data from neuronal exosomes isolated from serum showed mRNA transcripts of several neuronal genes. In particular, mRNAs of several olfactory receptor genes were present at elevated concentrations in the neuronal exosomes. Some of these genes were OR10A6, OR14A2, OR6F1, OR1B1, and OR1L1. RNA sequencing data from exosomes isolated from CSF showed a similar elevation of these olfactory receptors. We further validated the expression of these samples in serum samples of mild TBI patients, and a similar up-regulation of these olfactory receptors was observed. The data from these experiments suggest that damage to the neurons in the olfactory neuroepithelium as well as in the brain following a TBI may cause the release of mRNA from these receptors in the exosomes. Hence, olfactory receptors can be further explored as biomarkers for the diagnosis of TBI.

S100Z is expressed in a lateral subpopulation of olfactory receptor neurons in the main olfactory system of Xenopus laevis.

In contrast to other S100 protein members, the function of S100 calcium-binding protein Z (S100Z) remains largely uncharacterized. It is expressed in the olfactory epithelium of fish, and it is closely associated with the vomeronasal organ (VNO) in mammals. In this study, we analyzed the expression pattern of S100Z in the olfactory system of the anuran amphibian Xenopus laevis. Using immunohistochemistry in whole mount and slice preparations of the larval olfactory system, we found exclusive S100Z expression in a subpopulation of olfactory receptor neurons (ORNs) of the main olfactory epithelium (MOE). S100Z expression was not co-localized with TP63 and cytokeratin type II, ruling out basal cell and supporting cell identity. The distribution of S100Z-expressing ORNs was laterally biased, and their average number was significantly increased in the lateral half of the olfactory epithelium. The axons of S100Z-positive neurons projected exclusively into the lateral and intermediate glomerular clusters of the main olfactory bulb (OB). Even after metamorphic restructuring of the olfactory system, S100Z expression was restricted to a neuronal subpopulation of the MOE, which was then located in the newly formed middle cavity. An axonal projection into the ventro-lateral OB persisted also in postmetamorphic frogs. In summary, S100Z is exclusively associated with the main olfactory system in the amphibian Xenopus and not with the VNO as in mammals, despite the presence of a separate accessory olfactory system in both classes.

Molecular mechanisms of differentiation and class choice of olfactory sensory neurons.

The sense of smell is intricately linked to essential animal behaviors necessary for individual survival and species preservation. During vertebrate evolution, odorant receptors (ORs), responsible for detecting odor molecules, have evolved to adapt to changing environments, transitioning from aquatic to terrestrial habitats and accommodating increasing complex chemical environments. These evolutionary pressures have given rise to the largest gene family in vertebrate genomes. Vertebrate ORs are phylogenetically divided into two major classes; class I and class II. Class I OR genes, initially identified in fish and frog, have persisted across vertebrate species. On the other hand, class II OR genes are unique to terrestrial animals, accounting for ~90% of mammalian OR genes. In mice, each olfactory sensory neuron (OSN) expresses a single functional allele of a single OR gene from either the class I or class II OR repertoire. This one neuron-one receptor rule is established through two sequential steps: specification of OR class and subsequent exclusive OR expression from the corresponding OR class. Consequently, OSNs acquire diverse neuronal identities during the process of OSN differentiation, enabling animals to detect a wide array of odor molecules. This review provides an overview of the OSN differentiation process through which OSN diversity is achieved, primarily using the mouse as a model animal.

An expanded neurogenetic toolkit to decode olfaction in the African malaria mosquito Anopheles gambiae.

Anopheles gambiae uses its sense of smell to hunt humans. We report a two-step method yielding cell-type-specific driver lines for enhanced neuroanatomical and functional studies of its olfactory system. We first integrated a driver-responder-marker (DRM) system cassette consisting of a linked T2A-QF2 driver, QUAS-GFP responder, and a gut-specific transgenesis marker into four chemoreceptor genes (Ir25a, Ir76b, Gr22, and orco) using CRISPR-Cas9-mediated homology-directed repair. The DRM system facilitated rapid selection of in-frame integrations via screening for GFP+ olfactory sensory neurons (OSNs) in G1 larval progeny, even at genomic loci such as orco where we found the transgenesis marker was not visible. Next, we converted these DRM integrations into T2A-QF2 driver-marker lines by Cre-loxP excision of the GFP responder, making them suitable for binary use in transcuticular calcium imaging. These cell-type-specific driver lines tiling key OSN subsets will support systematic efforts to decode olfaction in this prolific malaria vector.

A noncoding role of coding mRNA in monogenic olfactory receptor choice.

In a recent study, Pourmorady and colleagues uncovered a noncoding role for olfactory receptor (OR)-coding mRNA in mediating nuclear architecture and singular OR choice. The OR mRNAs reinforce the prevailing enhancer hub and inhibit other competitors, facilitating transition from polygenic to singular OR expression.

Calcium imaging of adult olfactory epithelium reveals amines as important odor class in fish.

The odor space of aquatic organisms is by necessity quite different from that of air-breathing animals. The recognized odor classes in teleost fish include amino acids, bile acids, reproductive hormones, nucleotides, and a limited number of polyamines. Conversely, a significant portion of the fish olfactory receptor repertoire is composed of trace amine-associated receptors, generally assumed to be responsible for detecting amines. Zebrafish possess over one hundred of these receptors, but the responses of olfactory sensory neurons to amines have not been known so far. Here we examined odor responses of zebrafish olfactory epithelial explants at the cellular level, employing calcium imaging. We report that amines elicit strong responses in olfactory sensory neurons, with a time course characteristically different from that of ATP-responsive (basal) cells. A quantitative analysis of the laminar height distribution shows amine-responsive cells undistinguishable from ciliated neurons positive for olfactory marker protein. This distribution is significantly different from those measured for microvillous neurons positive for transient receptor potential channel 2 and basal cells positive for proliferating cell nuclear antigen. Our results suggest amines as an important odor class for teleost fish.

Somatic ion channels and action potentials in olfactory receptor cells and vomeronasal receptor cells.

Olfactory receptor cells are primary sensory neurons that catch odor molecules in the olfactory system, and vomeronasal receptor cells catch pheromones in the vomeronasal system. When odor or pheromone molecules bind to receptor proteins expressed on the membrane of the olfactory cilia or vomeronasal microvilli, receptor potentials are generated in their receptor cells. This initial excitation is transmitted to the soma via dendrites, and action potentials are generated in the soma and/or axon and transmitted to the central nervous system. Thus, olfactory and vomeronasal receptor cells play an important role in converting chemical signals into electrical signals. In this review, the electrophysiological characteristics of ion channels in the somatic membrane of olfactory receptor cells and vomeronasal receptor cells in various species are described and the differences between the action potential dynamics of olfactory receptor cells and vomeronasal receptor cells are compared.

A single mutation in the mosquito (Aedes aegypti) olfactory receptor 8 causes loss of function to 1-octen-3-ol.

The host-seeking behavior of mosquitoes have long been established to be primarily odor-mediated. In this process, olfactory receptors (Ors) play a critical role. 1-Octen-3-ol is a common volatile compound that is attractive to hematophagous arthropods such as mosquitos. The olfactory receptor 8 (AaOr8) on the tip of the stylet and maxillary palp of Aedes aegypti is tuned to 1-octen-3-ol, which is required for mosquitoes to quickly find blood vessels from a vertebrate host. However, little is known about the interaction of AaOr8 with 1-octen-3-ol which was studied in vivo and in silico in this study. The molecular binding poses and energies between ligands and the receptor were investigated. Three mutants of AaOr8 were cloned and compared with in vivo calcium imaging utilizing heterologous expression systems. As a result, our findings imply that a genetic disruption including targeted modification of Ors genes may be used to reduce mosquito bites.

Females smell differently: characteristics and significance of the most common olfactory sensilla of female silkmoths.

In the silkmoth Bombyx mori, the role of male sensilla trichodea in pheromone detection is well established. Here we study the corresponding female sensilla, which contain two olfactory sensory neurons (OSNs) and come in two lengths, each representing a single physiological type. Only OSNs in medium trichoids respond to the scent of mulberry, the silkworm's exclusive host plant, and are more sensitive in mated females, suggesting a role in oviposition. In long trichoids, one OSN is tuned to (+)-linalool and the other to benzaldehyde and isovaleric acid, both odours emitted by silkworm faeces. While the significance of (+)-linalool detection remains unclear, isovaleric acid repels mated females and may therefore play a role in avoiding crowded oviposition sites. When we examined the underlying molecular components of neurons in female trichoids, we found non-canonical co-expression of Ir8a, the co-receptor for acid responses, and ORco, the co-receptor of odorant receptors, in long trichoids, and the unexpected expression of a specific odorant receptor in both trichoid sensillum types. In addition to elucidating the function of female trichoids, our results suggest that some accepted organizational principles of the insect olfactory system may not apply to the predominant sensilla on the antenna of female B. mori.

Olfactory system structure and function in newly hatched and adult locusts.

An important question in neuroscience is how sensory systems change as animals grow and interact with the environment. Exploring sensory systems in animals as they develop can reveal how networks of neurons process information as the neurons themselves grow and the needs of the animal change. Here we compared the structure and function of peripheral parts of the olfactory pathway in newly hatched and adult locusts. We found that populations of olfactory sensory neurons (OSNs) in hatchlings and adults responded with similar tunings to a panel of odors. The morphologies of local neurons (LNs) and projection neurons (PNs) in the antennal lobes (ALs) were very similar in both age groups, though they were smaller in hatchlings, they were proportional to overall brain size. The odor evoked responses of LNs and PNs were also very similar in both age groups, characterized by complex patterns of activity including oscillatory synchronization. Notably, in hatchlings, spontaneous and odor-evoked firing rates of PNs were lower, and LFP oscillations were lower in frequency, than in the adult. Hatchlings have smaller antennae with fewer OSNs; removing antennal segments from adults also reduced LFP oscillation frequency. Thus, consistent with earlier computational models, the developmental increase in frequency is due to increasing intensity of input to the oscillation circuitry. Overall, our results show that locusts hatch with a fully formed olfactory system that structurally and functionally matches that of the adult, despite its small size and lack of prior experience with olfactory stimuli.

Post-exposure intranasal IFNα suppresses replication and neuroinvasion of Venezuelan Equine Encephalitis virus within olfactory sensory neurons.

BACKGROUND: Venezuelan Equine Encephalitis virus (VEEV) may enter the central nervous system (CNS) within olfactory sensory neurons (OSN) that originate in the nasal cavity after intranasal exposure. While it is known that VEEV has evolved several mechanisms to inhibit type I interferon (IFN) signaling within infected cells, whether this inhibits virologic control during neuroinvasion along OSN has not been studied. METHODS: We utilized an established murine model of intranasal infection with VEEV and a repository of scRNAseq data from IFN-treated OSN to assess the cellular targets and IFN signaling responses after VEEV exposure. RESULTS: We found that immature OSN, which express higher levels of the VEEV receptor LDLRAD3 than mature OSN, are the first cells infected by VEEV. Despite rapid VEEV neuroinvasion after intranasal exposure, olfactory neuroepithelium (ONE) and olfactory bulb (OB) IFN responses, as assessed by evaluation of expression of interferon signaling genes (ISG), are delayed for up to 48 h during VEEV neuroinvasion, representing a potential therapeutic window. Indeed, a single intranasal dose of recombinant IFNα triggers early ISG expression in both the nasal cavity and OB. When administered at the time of or early after infection, IFNα treatment delayed onset of sequelae associated with encephalitis and extended survival by several days. VEEV replication after IFN treatment was also transiently suppressed in the ONE, which inhibited subsequent invasion into the CNS. CONCLUSIONS: Our results demonstrate a critical and promising first evaluation of intranasal IFNα for the treatment of human encephalitic alphavirus exposures.