Phosphodiesterase 5A regulates the vomeronasal pump in mice.

The vomeronasal organ (VNO) is a specialized chemoreceptive structure in many vertebrates that detects chemical stimuli, mostly pheromones, which often elicit innate behaviors such as mating and aggression. Previous studies in rodents have demonstrated that chemical stimuli are actively transported to the VNO via a blood vessel-based pumping mechanism, and this pumping mechanism is necessary for vomeronasal stimulation in behaving animals. However, the molecular mechanisms that regulate the vomeronasal pump remain mostly unknown. In this study, we observed a high level of expression of phosphodiesterase 5A (PDE5A) in the vomeronasal blood vessel of mice. We provided evidence to support the potential role of PDE5A in vomeronasal pump regulation. Local application of PDE5A inhibitors-sildenafil or tadalafil-to the vomeronasal organ (VNO) reduced stimulus delivery into the VNO, decreased the pheromone-induced activity of vomeronasal sensory neurons, and attenuated male-male aggressive behaviors. PDE5A is well known to play a role in regulating blood vessel tone in several organs. Our study advances our understanding of the molecular regulation of the vomeronasal pump.

Quantifying Peripheral Modulation of Olfaction by Trigeminal Agonists.

In the mammalian nose, two chemosensory systems, the trigeminal and the olfactory mediate the detection of volatile chemicals. Most odorants are able to activate the trigeminal system, and vice versa, most trigeminal agonists activate the olfactory system as well. Although these two systems constitute two separate sensory modalities, trigeminal activation modulates the neural representation of an odor. The mechanisms behind the modulation of olfactory response by trigeminal activation are still poorly understood. We addressed this question by looking at the olfactory epithelium (OE), where olfactory sensory neurons (OSNs) and trigeminal sensory fibers co-localize and where the olfactory signal is generated. Our study was conducted in a mouse model. Both sexes, males and females, were included. We characterize the trigeminal activation in response to five different odorants by measuring intracellular Ca2+ changes from primary cultures of trigeminal neurons (TGNs). We also measured responses from mice lacking TRPA1 and TRPV1 channels known to mediate some trigeminal responses. Next, we tested how trigeminal activation affects the olfactory response in the olfactory epithelium using electro-olfactogram (EOG) recordings from wild-type (WT) and TRPA1/V1-knock out (KO) mice. The trigeminal modulation of the olfactory response was determined by measuring responses to the odorant, 2-phenylethanol (PEA), an odorant with little trigeminal potency after stimulation with a trigeminal agonist. Trigeminal agonists induced a decrease in the EOG response to PEA, which depended on the level of TRPA1 and TRPV1 activation induced by the trigeminal agonist. This suggests that trigeminal activation can alter odorant responses even at the earliest stage of the olfactory sensory transduction.SIGNIFICANCE STATEMENT Most odorants reaching the olfactory epithelium (OE) can simultaneously activate olfactory and trigeminal systems. Although these two systems constitute two separate sensory modalities, trigeminal activation can alter odor perception. Here, we analyzed the trigeminal activity induced by different odorants proposing an objective quantification of their trigeminal potency independent from human perception. We show that trigeminal activation by odorants reduces the olfactory response in the olfactory epithelium and that such modulation correlates with the trigeminal potency of the trigeminal agonist. These results show that the trigeminal system impacts the olfactory response from its earliest stage.

Paradoxical electro-olfactogram responses in TMEM16B knock-out mice.

The Ca2+-activated Cl¯ channel TMEM16B carries up to 90% of the transduction current evoked by odorant stimulation in olfactory sensory neurons and control the number of action potential firing and therefore the length of the train of action potentials. A loss of function approach revealed that TMEM16B is required for olfactory-driven behaviors such as tracking unfamiliar odors. Here, we used the electro-olfactogram (EOG) technique to investigate the contribution of TMEM16B to odorant transduction in the whole olfactory epithelium. Surprisingly, we found that EOG responses from Tmem16b knock out mice have a bigger amplitude compared to those of wild type. Moreover, the kinetics of EOG responses is faster in absence of TMEM16B, while the ability to adapt to repeated stimulation is altered in knock out mice. The larger EOG responses in Tmem16b knock out may be the results of the removal of the clamping and/or shunting action of the Ca2+-activated Cl¯ currents leading to the paradox of having smaller transduction current but larger generator potential.

Ca2+-activated Cl- current ensures robust and reliable signal amplification in vertebrate olfactory receptor neurons.

Activation of most primary sensory neurons results in transduction currents that are carried by cations. One notable exception is the vertebrate olfactory receptor neuron (ORN), where the transduction current is carried largely by the anion [Formula: see text] However, it remains unclear why ORNs use an anionic current for signal amplification. We have sought to provide clarification on this topic by studying the so far neglected dynamics of [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] in the small space of olfactory cilia during an odorant response. Using computational modeling and simulations we compared the outcomes of signal amplification based on either [Formula: see text] or [Formula: see text] currents. We found that amplification produced by [Formula: see text] influx instead of a [Formula: see text] efflux is problematic for several reasons: First, the [Formula: see text] current amplitude varies greatly, depending on mucosal ion concentration changes. Second, a [Formula: see text] current leads to a large increase in the ciliary [Formula: see text] concentration during an odorant response. This increase inhibits and even reverses [Formula: see text] clearance by [Formula: see text] exchange, which is essential for response termination. Finally, a [Formula: see text] current increases the ciliary osmotic pressure, which could cause swelling to damage the cilia. By contrast, a transduction pathway based on [Formula: see text] efflux circumvents these problems and renders the odorant response robust and reliable.

The functional relevance of olfactory marker protein in the vertebrate olfactory system: a never-ending story.

Olfactory marker protein (OMP) was first described as a protein expressed in olfactory receptor neurons (ORNs) in the nasal cavity. In particular, OMP, a small cytoplasmic protein, marks mature ORNs and is also expressed in the neurons of other nasal chemosensory systems: the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. While its expression pattern was more easily established, OMP's function remained relatively vague. To date, most of the work to understand OMP's role has been done using mice lacking OMP. This mostly phenomenological work has shown that OMP is involved in sharpening the odorant response profile and in quickening odorant response kinetics of ORNs and that it contributes to targeting of ORN axons to the olfactory bulb to refine the glomerular response map. Increasing evidence shows that OMP acts at the early stages of olfactory transduction by modulating the kinetics of cAMP, the second messenger of olfactory transduction. However, how this occurs at a mechanistic level is not understood, and it might also not be the only mechanism underlying all the changes observed in mice lacking OMP. Recently, OMP has been detected outside the nose, including the brain and other organs. Although no obvious logic has become apparent regarding the underlying commonality between nasal and extranasal expression of OMP, a broader approach to diverse cellular systems might help unravel OMP's functions and mechanisms of action inside and outside the nose.

Cilia- and Flagella-Associated Protein 69 Regulates Olfactory Transduction Kinetics in Mice.

Animals detect odorous chemicals through specialized olfactory sensory neurons (OSNs) that transduce odorants into neural electrical signals. We identified a novel and evolutionarily conserved protein, cilia- and flagella-associated protein 69 (CFAP69), in mice that regulates olfactory transduction kinetics. In the olfactory epithelium, CFAP69 is enriched in OSN cilia, where olfactory transduction occurs. Bioinformatic analysis suggests that a large portion of CFAP69 can form Armadillo-type α-helical repeats, which may mediate protein-protein interactions. OSNs lacking CFAP69, remarkably, displayed faster kinetics in both the on and off phases of electrophysiological responses at both the neuronal ensemble level as observed by electroolfactogram and the single-cell level as observed by single-cell suction pipette recordings. In single-cell analysis, OSNs lacking CFAP69 showed faster response integration and were able to fire APs more faithfully to repeated odor stimuli. Furthermore, both male and female mutant mice that specifically lack CFAP69 in OSNs exhibited attenuated performance in a buried food pellet test when a background of the same odor to the food pellet was present even though they should have better temporal resolution of coding olfactory stimulation at the peripheral. Therefore, the role of CFAP69 in the olfactory system seems to be to allow the olfactory transduction machinery to work at a precisely regulated range of response kinetics for robust olfactory behavior.SIGNIFICANCE STATEMENT Sensory receptor cells are generally thought to evolve to respond to sensory cues as fast as they can. This idea is consistent with mutational analyses in various sensory systems, where mutations of sensory receptor cells often resulted in reduced response size and slowed response kinetics. Contrary to this idea, we have found that there is a kinetic "damper" present in the olfactory transduction cascade of the mouse that slows down the response kinetics and, by doing so, it reduces the peripheral temporal resolution in coding odor stimuli and allows for robust olfactory behavior. This study should trigger a rethinking of the significance of the intrinsic speed of sensory transduction and the pattern of the peripheral coding of sensory stimuli.

Non-synaptic inhibition between grouped neurons in an olfactory circuit.

Diverse sensory organs, including mammalian taste buds and insect chemosensory sensilla, show a marked compartmentalization of receptor cells; however, the functional impact of this organization remains unclear. Here we show that compartmentalized Drosophila olfactory receptor neurons (ORNs) communicate with each other directly. The sustained response of one ORN is inhibited by the transient activation of a neighbouring ORN. Mechanistically, such lateral inhibition does not depend on synapses and is probably mediated by ephaptic coupling. Moreover, lateral inhibition in the periphery can modulate olfactory behaviour. Together, the results show that integration of olfactory information can occur via lateral interactions between ORNs. Inhibition of a sustained response by a transient response may provide a means of encoding salience. Finally, a CO(2)-sensitive ORN in the malaria mosquito Anopheles can also be inhibited by excitation of an adjacent ORN, suggesting a broad occurrence of lateral inhibition in insects and possible applications in insect control.

The Odorant Receptor-Dependent Role of Olfactory Marker Protein in Olfactory Receptor Neurons.

Olfactory receptor neurons (ORNs) in the nasal cavity detect and transduce odorants into action potentials to be conveyed to the olfactory bulb. Odorants are delivered to ORNs via the inhaled air at breathing frequencies that can vary from 2 to 10 Hz in the mouse. Thus olfactory transduction should occur at sufficient speed such that it can accommodate repetitive and frequent stimulation. Activation of odorant receptors (ORs) leads to adenylyl cyclase III activation, cAMP increase, and opening of cyclic nucleotide-gated channels. This makes the kinetic regulation of cAMP one of the important determinants for the response time course. We addressed the dynamic regulation of cAMP during the odorant response and examined how basal levels of cAMP are controlled. The latter is particularly relevant as basal cAMP depends on the basal activity of the expressed OR and thus varies across ORNs. We found that olfactory marker protein (OMP), a protein expressed in mature ORNs, controls both basal and odorant-induced cAMP levels in an OR-dependent manner. Lack of OMP increases basal cAMP, thus abolishing differences in basal cAMP levels between ORNs expressing different ORs. Moreover, OMP speeds up signal transduction for ORNs to better synchronize their output with high-frequency stimulation and to perceive brief stimuli. Last, OMP also steepens the dose-response relation to improve concentration coding although at the cost of losing responses to weak stimuli. We conclude that OMP plays a key regulatory role in ORN physiology by controlling multiple facets of the odorant response.

Heterotrimeric G protein subunit Gγ13 is critical to olfaction.

The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G-protein consisting of α, ß, and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its ß and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We, and others, have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knock-out mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice's olfactory epithelium. Intriguingly, Gαolf, ß1 subunits, Ric-8B and CEP290 proteins, were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G-protein with Gß1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G-protein's ßγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs.

Evaluation of the role of g protein-coupled receptor kinase 3 in desensitization of mouse odorant receptors in a Mammalian cell line and in olfactory sensory neurons.

Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated.

Short-term consumption of highly processed diets varying in macronutrient content impair the sense of smell and brain metabolism in mice.

OBJECTIVE: Food processing greatly contributed to increased food safety, diversity, and accessibility. However, the prevalence of highly palatable and highly processed food in our modern diet has exacerbated obesity rates and contributed to a global health crisis. While accumulating evidence suggests that chronic consumption of such foods is detrimental to sensory and neural physiology, it is unclear whether its short-term intake has adverse effects. Here, we assessed how short-term consumption (<2 months) of three diets varying in composition and macronutrient content influence olfaction and brain metabolism in mice. METHODS: The diets tested included a grain-based standard chow diet (CHOW; 54% carbohydrate, 32% protein, 14% fat; #8604 Teklad Rodent diet , Envigo Inc.), a highly processed control diet (hpCTR; 70% carbohydrate, 20% protein, 10% fat; #D12450B, Research Diets Inc.), and a highly processed high-fat diet (hpHFD; 20% carbohydrate, 20% protein, 60% fat; #D12492, Research Diets Inc.). We performed behavioral and metabolic phenotyping, electro-olfactogram (EOG) recordings, brain glucose metabolism imaging, and mitochondrial respirometry in different brain regions. We also performed RNA-sequencing (RNA-seq) in the nose and across several brain regions, and conducted differential expression analysis, gene ontology, and network analysis. RESULTS: We show that short-term consumption of the two highly processed diets, but not the grain-based diet, regardless of macronutrient content, adversely affects odor-guided behaviors, physiological responses to odorants, transcriptional profiles in the olfactory mucosa and brain regions, and brain glucose metabolism and mitochondrial respiration. CONCLUSIONS: Even short periods of highly processed food consumption are sufficient to cause early olfactory and brain abnormalities, which has the potential to alter food choices and influence the risk of developing metabolic disease.

Signaling by olfactory receptor neurons near threshold.

An important contributing factor for the high sensitivity of sensory systems is the exquisite sensitivity of the sensory receptor cells. We report here the signaling threshold of the olfactory receptor neuron (ORN). We first obtained a best estimate of the size of the physiological electrical response successfully triggered by a single odorant-binding event on a frog ORN, which was ∼0.034 pA and had an associated transduction domain spanning only a tiny fraction of the length of an ORN cilium. We also estimated the receptor-current threshold for an ORN to fire action potentials in response to an odorant pulse, which was ∼1.2 pA. Thus, it takes about 35 odorant-binding events successfully triggering transduction during a brief odorant pulse in order for an ORN to signal to the brain.

The Role of the Stimulus in Olfactory Plasticity.

Plasticity, the term we use to describe the ability of a nervous system to change with experience, is the evolutionary adaptation that freed animal behavior from the confines of genetic determinism. This capacity, which increases with brain complexity, is nowhere more evident than in vertebrates, especially mammals. Though the scientific study of brain plasticity dates back at least to the mid-19th century, the last several decades have seen unprecedented advances in the field afforded by new technologies. Olfaction is one system that has garnered particular attention in this realm because it is the only sensory modality with a lifelong supply of new neurons, from two niches no less! Here, we review some of the classical and contemporary literature dealing with the role of the stimulus or lack thereof in olfactory plasticity. We have restricted our comments to studies in mammals that have used dual tools of the field: stimulus deprivation and stimulus enrichment. The former manipulation has been implemented most frequently by unilateral naris occlusion and, thus, we have limited our comments to research using this technique. The work reviewed on deprivation provides substantial evidence of activity-dependent processes in both developing and adult mammals at multiple levels of the system from olfactory sensory neurons through to olfactory cortical areas. However, more recent evidence on the effects of deprivation also establishes several compensatory processes with mechanisms at every level of the system, whose function seems to be the restoration of information flow in the face of an impoverished signal. The results of sensory enrichment are more tentative, not least because of the actual manipulation: What odor or odors? At what concentrations? On what schedule? All of these have frequently not been sufficiently rationalized or characterized. Perhaps it is not surprising, then, that discrepant results are common in sensory enrichment studies. Despite this problem, evidence has accumulated that even passively encountered odors can "teach" olfactory cortical areas to better detect, discriminate, and more efficiently encode them for future encounters. We discuss these and other less-established roles for the stimulus in olfactory plasticity, culminating in our recommended "aspirations" for the field going forward.

ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification.

For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calcium-activated chloride channel.

A 3D transcriptomics atlas of the mouse nose sheds light on the anatomical logic of smell.

The sense of smell helps us navigate the environment, but its molecular architecture and underlying logic remain understudied. The spatial location of odorant receptor genes (Olfrs) in the nose is thought to be independent of the structural diversity of the odorants they detect. Using spatial transcriptomics, we create a genome-wide 3D atlas of the mouse olfactory mucosa (OM). Topographic maps of genes differentially expressed in space reveal that both Olfrs and non-Olfrs are distributed in a continuous and overlapping fashion over at least five broad zones in the OM. The spatial locations of Olfrs correlate with the mucus solubility of the odorants they recognize, providing direct evidence for the chromatographic theory of olfaction. This resource resolves the molecular architecture of the mouse OM and will inform future studies on mechanisms underlying Olfr gene choice, axonal pathfinding, patterning of the nervous system, and basic logic for the peripheral representation of smell.

Olfactory receptor neuron responses coding for rapid odour sampling.

Vertebrate olfactory receptor neurons (ORNs) are stimulated in a rhythmic manner in vivo, driven by delivery of odorants to the nasal cavity carried by the inhaled air, making olfaction a sense where animals can control the frequency of stimulus delivery. How ORNs encode repeated stimulation at resting, low breathing frequencies and at increased sniffing frequencies is not known, nor is it known if the olfactory transduction cascade is accurate and fast enough to follow high frequency stimulation. We investigated mouse olfactory responses to stimulus frequencies mimicking odorant exposure during low (2Hz) and high (5Hz) frequency sniffing. ORNs reliably follow low frequency stimulations with high fidelity by generating bursts of action potentials at each stimulation at intermediate odorant concentrations, but fail to do so at high odorant concentrations. Higher stimulus frequencies across all odorant concentrations reduced the likelihood of action potential generation, increased the latency of response, and decreased there liability of encoding the onset of stimulation. Thus an increase in stimulus frequency degrades and at high odorant concentrations entirely prevents action potential generation in individual ORNs, causing reduced signalling to the olfactory bulb. These results demonstrate that ORNs do not simply relay timing and concentration of an odorous stimulus, but also process and modulate the stimulus in a frequency-dependent manner which is controlled by the chosen sniffing rate.

Mechanisms of chloride uptake in frog olfactory receptor neurons.

Odorant stimulation of olfactory receptor neurons (ORNs) leads to the activation of a Ca(2+) permeable cyclic nucleotide-gated (CNG) channel followed by opening of an excitatory Ca(2+)-activated Cl(-) channel, which carries about 70% of the odorant-induced receptor current. This requires ORNs to have a [Cl(-)](i) above the electrochemical equilibrium to render this anionic current excitatory. In mammalian ORNs, the Na(+)-K(+)-2Cl(-) co-transporter 1 (NKCC1) has been characterized as the principal mechanism by which these neurons actively accumulate Cl(-). To determine if NKCC activity is needed in amphibian olfactory transduction, and to characterize its cellular location, we used the suction pipette technique to record from Rana pipiens ORNs. Application of bumetanide, an NKCC blocker, produced a 50% decrease of the odorant-induced current. Similar effects were observed when [Cl(-)](i) was decreased by bathing ORNs in low Cl(-) solution. Both manipulations reduced only the Cl(-) component of the current. Application of bumetanide only to the ORN cell body and not to the cilia decreased the current by again about 50%. The results show that NKCC is required for amphibian olfactory transduction, and suggest that the co-transporter is located basolaterally at the cell body although its presence at the cilia could not be discarded.

Sensory Transduction in Photoreceptors and Olfactory Sensory Neurons: Common Features and Distinct Characteristics.

The past decades have seen tremendous progress in our understanding of the function of photoreceptors and olfactory sensory neurons, uncovering the mechanisms that determine their properties and, ultimately, our ability to see and smell. This progress has been driven to a large degree by the powerful combination of physiological experimental tools and genetic manipulations, which has enabled us to identify the main molecular players in the transduction cascades of these sensory neurons, how their properties affect the detection and discrimination of stimuli, and how diseases affect our senses of vision and smell. This review summarizes some of the common and unique features of photoreceptors and olfactory sensory neurons that make these cells so exciting to study.

Odor sampling strategies in mice with genetically altered olfactory responses.

Peripheral sensory cells and the central neuronal circuits that monitor environmental changes to drive behaviors should be adapted to match the behaviorally relevant kinetics of incoming stimuli, be it the detection of sound frequencies, the speed of moving objects or local temperature changes. Detection of odorants begins with the activation of olfactory receptor neurons in the nasal cavity following inhalation of air and airborne odorants carried therein. Thus, olfactory receptor neurons are stimulated in a rhythmic and repeated fashion that is determined by the breathing or sniffing frequency that can be controlled and altered by the animal. This raises the question of how the response kinetics of olfactory receptor neurons are matched to the imposed stimulation frequency and if, vice versa, the kinetics of olfactory receptor neuron responses determine the sniffing frequency. We addressed this question by using a mouse model that lacks the K+-dependent Na+/Ca2+ exchanger 4 (NCKX4), which results in markedly slowed response termination of olfactory receptor neuron responses and hence changes the temporal response kinetics of these neurons. We monitored sniffing behaviors of freely moving wildtype and NCKX4 knockout mice while they performed olfactory Go/NoGo discrimination tasks. Knockout mice performed with similar or, surprisingly, better accuracy compared to wildtype mice, but chose, depending on the task, different odorant sampling durations depending on the behavioral demands of the odorant identification task. Similarly, depending on the demands of the behavioral task, knockout mice displayed a lower basal breathing frequency prior to odorant sampling, a possible mechanism to increase the dynamic range for changes in sniffing frequency during odorant sampling. Overall, changes in sniffing behavior between wildtype and NCKX4 knockout mice were subtle, suggesting that, at least for the particular odorant-driven task we used, slowed response termination of the odorant-induced receptor neuron response either has a limited detrimental effect on odorant-driven behavior or mice are able to compensate via an as yet unknown mechanism.

Insulin-Dependent Maturation of Newly Generated Olfactory Sensory Neurons after Injury.

Loss of olfactory sensory neurons (OSNs) after injury to the olfactory epithelium (OE) triggers the generation of OSNs that are incorporated into olfactory circuits to restore olfactory sensory perception. This study addresses how insulin receptor-mediated signaling affects the functional recovery of OSNs after OE injury. Insulin levels were reduced in mice by ablating the pancreatic ß cells via streptozotocin (STZ) injections. These STZ-induced diabetic and control mice were then intraperitoneally injected with the olfactotoxic drug methimazole to selectively ablate OSNs. The OE of diabetic and control mice regenerated similarly until day 14 after injury. Thereafter, the OE of diabetic mice contained fewer mature and more apoptotic OSNs than control mice. Functionally, diabetic mice showed reduced electro-olfactogram (EOG) responses and their olfactory bulbs (OBs) had fewer c-Fos-active cells following odor stimulation, as well as performed worse in an odor-guided task compared with control mice. Insulin administered intranasally during days 8-13 after injury was sufficient to rescue recovery of OSNs in diabetic mice compared with control levels, while insulin administration between days 1 and 6 did not. During this critical time window on days 8-13 after injury, insulin receptors are highly expressed and intranasal application of an insulin receptor antagonist inhibits regeneration. Furthermore, an insulin-enriched environment could facilitate regeneration even in non-diabetic mice. These results indicate that insulin facilitates the regeneration of OSNs after injury and suggest a critical stage during recovery (8-13 d after injury) during which the maturation of newly generated OSNs is highly dependent on and promoted by insulin.