Mature Olfactory Sensory Neurons Research Articles

Lectin histochemistry of the olfactory mucosa of Korean native cattle, Bos taurus coreanae.

The olfactory mucosa (OM) is crucial for odorant perception in the main olfactory system. The terminal carbohydrates of glycoconjugates influence chemoreception in the olfactory epithelium (OE). The histological characteristics and glycoconjugate composition of the OM of Korean native cattle (Hanwoo, Bos taurus coreae) were examined to characterize their morphology and possible functions during postnatal development. The OM of neonate and adult Korean native cattle was evaluated using histological, immunohistochemical, and lectin histochemical methods. Histologically, the OM in both neonates and adults consists of the olfactory epithelium and the lamina propria. Additionally, using periodic acid Schiff and Alcian blue (pH 2.5), the mucus specificity of the Bowman's gland duct and acini in the lamina propria was determined. Immunohistochemistry demonstrated that mature and immature olfactory sensory neurons of OEs express the olfactory marker protein and growth associated protein-43, respectively. Lectin histochemistry indicated that numerous glycoconjugates, including as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose groups, were expressed at varied levels in the different cell types in the OMs of neonates and adults at varying levels. According to our observations, the cattle possessed a well-developed olfactory system, and the expression patterns of glycoconjugates in neonatal and adult OMs varied considerably. This is the first study to describe the morphological assessment of the OM of Korean native cattle with a focus on lectin histochemistry. The findings suggest that glycoconjugates may play a role in olfactory chemoreception, and that their labeling properties may be closely related to OM development and maturity.

Protective Effect of Insulin in Mouse Nasal Mucus Against Olfactory Epithelium Injury

Insulin is present in nasal mucus and plays an important role in the survival and activity of individual olfactory sensory neurons (OSNs) via insulin receptor-mediated signaling. However, it is unclear whether insulin acts prophylactically against olfactotoxic drug-induced olfactory epithelium (OE) injury, and whether the degree of damage is affected by the concentration of insulin in the nasal mucus. The apoptosis-inducing drug methimazole was administered to the nasal mucus of diabetic and normal mice along with different concentrations of insulin. Immunohistochemical analysis was used to assess the relationship between damage to the OE and the mucus insulin concentration and the protective effect of insulin administration against eosinophilic cationic protein (ECP)-induced OE injury. Diabetic mice had lower concentrations of insulin in their nasal mucus than normal mice (diabetic vs. normal mice, p < 0.001). Methimazole administration reduced the number of OSNs in normal mice and had a more marked effect in diabetic mice. However, unilateral insulin administration prevented the methimazole-induced reduction in the number of OSNs on the ipsilateral side but not on the contralateral side (OSNs; Insulin vs. contralateral side, p < 0.001). Furthermore, intranasal ECP administration damaged the OE by inducing apoptosis (OSNs; ECP vs. contralateral side, p < 0.001), but this damage was largely prevented by insulin administration (OSNs; Insulin + ECP vs. contralateral side, p = 0.36), which maintained the number of mature OSNs. The severity of methimazole-induced damage to the OE is related to the insulin concentration in the nasal mucus (Correlation between the insulin concentration in nasal mucus and the numbers of OSNs, R2 = 0.91, p < 0.001), which may imply that nasal insulin protects OSNs and that insulin administration might lead to the development of new therapeutic agents for ECP-induced OE injury.

Heterogeneous distribution of mature olfactory sensory neurons in human olfactory epithelium.

The olfactory cleft (OC) comprising the olfactory epithelium (OE) is the most important anatomical location for olfactory function. Endoscopic sinus surgery (ESS) is used to treat diseases related to the OC and improve olfactory dysfunction. However, iatrogenic OE injury occasionally occurs. Comprehensive knowledge of the olfactory region is required to avoid damage to the OE during endoscopic procedures. Immunohistochemistry was performed on olfactory mucosa obtained from the unaffected side of olfactory neuroblastoma surgical specimens. The OE was defined as the epithelium containing mature olfactory sensory neurons (OSNs). The distribution and cell kinetics of the OE were examined. The OE was selectively localized to the anterior two-thirds of the superior turbinate (ST) and in the nasal septum (NS) just opposite to the ST; the OE was not detected within the mucosa of the superior meatus. The density of mature OSNs was high at the ethmoid tegmen but gradually decreased with distance from the ethmoid tegmen. The extent of cell death and proliferation was relatively even across the OE. Analysis of airflow profiles revealed that resection of inferior ST does not decrease airflow to the OC. The results indicate that the distribution and degree of differentiation of mature OSNs are heterogenous throughout the OE. Epithelial resection of the anterior or superior ST has the potential to damage olfactory function. Resection of the inferior or posterior ST or widening of the superior meatus is a safer alternative that does not damage mature OSNs or alter airflow to the OC.

Olfactory epithelium and ontogeny of the nasal chambers in the bowhead whale (Balaena mysticetus).

In a species of baleen whale, we identify olfactory epithelium that suggests a functional sense of smell and document the ontogeny of the surrounding olfactory anatomy. Whales must surface to breathe, thereby providing an opportunity to detect airborne odorants. Although many toothed whales (odontocetes) lack olfactory anatomy, baleen whales (mysticetes) have retained theirs. Here, we investigate fetal and postnatal specimens of bowhead whales (Balaena mysticetus). Computed tomography (CT) reveals the presence of nasal passages and nasal chambers with simple ethmoturbinates through ontogeny. Additionally, we describe the dorsal nasal meatuses and olfactory bulb chambers. The cribriform plate has foramina that communicate with the nasal chambers. We show this anatomy within the context of the whole prenatal and postnatal skull. We document the tunnel for the ethmoidal nerve (ethmoid foramen) and the rostrolateral recess of the nasal chamber, which appears postnatally. Bilateral symmetry was apparent in the postnatal nasal chambers. No such symmetry was found prenatally, possibly due to tissue deformation. No nasal air sacs were found in fetal development. Olfactory epithelium, identified histologically, covers at least part of the ethmoturbinates. We identify olfactory epithelium using six explicit criteria of mammalian olfactory epithelium. Immunohistochemistry revealed the presence of olfactory marker protein (OMP), which is only found in mature olfactory sensory neurons. Although it seems that these neurons are scarce in bowhead whales compared to typical terrestrial mammals, our results suggest that bowhead whales have a functional sense of smell, which they may use to find prey.

Functional validation of epitope-tagged ATF5 knock-in mice generated by improved genome editing of oviductal nucleic acid delivery (i-GONAD).

Activating transcription factor 5 (ATF5) is a stress-responsive transcription factor that belongs to the cAMP response element-binding protein (CREB)/ATF family, and is essential for the differentiation and survival of sensory neurons in murine olfactory organs. However, the study of associated proteins and target genes for ATF5 has been hampered due to the limited availability of immunoprecipitation-grade ATF5 antibodies. To overcome this issue, we generated hemagglutinin (HA)-tag knock-in mice for ATF5 using CRISPR/Cas9-mediated genome editing with one-step electroporation in oviducts (i-GONAD). ATF5-HA fusion proteins were detected in the nuclei of immature and some mature olfactory and vomeronasal sensory neurons in the main olfactory epithelium and vomeronasal organ, respectively, as endogenous ATF5 proteins were expressed, and some ATF5-HA proteins were found to be phosphorylated. Chromatin immunoprecipitation (ChIP) experiments revealed that ATF5-HA bound to the CCAAT/enhancer-binding protein (C/EBP)-ATF response element site in the promotor region of receptor transporting protein 1 (Rtp1), a chaperone gene responsible for proper olfactory receptor expression. These knock-in mice may be used to examine the expression, localization, and protein-protein/-DNA interactions of endogenous ATF5 and, ultimately, the function of ATF5 in vivo.

Expansion of murine and human olfactory epithelium/mucosa colonies and generation of mature olfactory sensory neurons under chemically defined conditions.

Olfactory dysfunctions, including hyposmia and anosmia, affect ~100 million people around the world and the underlying causes are not fully understood. Degeneration of olfactory sensory neurons and incapacity of globose basal cells to generate olfactory sensory neurons are found in elder people and patients with smell disorders. Thus, olfactory stem cell may function as a promising tool to replace inactivated globose basal cells and to generate sensory neurons.Methods: We established clonal expansion of cells from the murine olfactory epithelium as well as colony growth from human olfactory mucosa using Matrigel-based three-dimensional system. These colonies were characterized by immunostaining against olfactory epithelium cellular markers and by calcium imaging of responses to odors. Chemical addition was optimized to promote Lgr5 expression, colony growth and sensory neuron generation, tested by quantitative PCR and immunostaining against progenitor and neuronal markers. The differential transcriptomes in multiple signaling pathways between colonies under different base media and chemical cocktails were determined by RNA-Seq.Results: In defined culture media, we found that VPA and CHIR99021 induced the highest Lgr5 expression level, while LY411575 resulted in the most abundant yield of OMP+ mature sensory neurons in murine colonies. Different base culture media with drug cocktails led to apparent morphological alteration from filled to cystic appearance, accompanied with massive transcriptional changes in multiple signaling pathways. Generation of sensory neurons in human colonies was affected through TGF-β signaling, while Lgr5 expression and cell proliferation was regulated by VPA.Conclusion: Our findings suggest that targeting expansion of olfactory epithelium/mucosa colonies in vitro potentially results in discovery of new source to cell replacement-based therapy against smell loss.

Age-Dependent Activation and Neuronal Differentiation of Lgr5+ Basal Cells in Injured Olfactory Epithelium via Notch Signaling Pathway.

Aging is an important factor affecting function of smell, leading to the degeneration of mature olfactory sensory neurons and inducing the occurrence of smell loss. The mammalian olfactory epithelium (OE) can regenerate when subjected to chemical assaults. However, this capacity is not limitless. Inactivation of globose basal cells and failure to generate sensory neurons are the main obstacles to prevent the OE regeneration. Here, we found the significant attenuation in mature sensory neuronal generation and apparent transcriptional alternation in the OE from aged mice compared with young ones. The recruitment of leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)-positive cells in injured OE was weakened in aged mice, and more Lgr5+ cells remained quiescence in aged OE postinjury. Lineage-traced progenies from Lgr5+ cells were significantly fewer in the OE with aging. Moreover, Notch activation enhanced the neuronal regeneration in aged OE, making the regenerative capacity of aged OE comparable with that of young animals after injury. The growth and morphology of three-dimensional (3D)-cultured organoids from the OE of young and aged mice varied and was modulated by small molecules regulating the Notch signaling pathway. Thus, we concluded that activation of Lgr5+ cells in injured OE was age dependent and Notch activation could enhance the capacity of neuronal generation from Lgr5+ cells in aged OE after injury.

Zone-specific damage of the olfactory epithelium under protein restriction

Oxidative stress causes tissue damage, affecting age-related pathologies. Protein restriction (PR) provides a powerful intervention strategy for reducing oxidative stress, which may have a positive effect on individual organs. However, it is unknown whether PR intervention influences the olfactory system. Here, we investigated how 10 months of PR could affect the cell dynamics of the olfactory epithelium (OE) in mice. We found that PR reduced age-related loss of outer hair cells in the cochlea, providing preventive effects against age-related hearing loss. In contrast, PR resulted in reduced mature olfactory sensory neurons (OSNs), increased proliferative basal cells, and increased apoptotic OSNs in zone 1 (the only area containing neurons expressing NQO1 [quinone dehydrogenase 1]) of the OE in comparison with animals given a control diet. Substantial oxidative stress occurred in NQO1-positive cells and induced apoptotic OSNs in zone 1. These results indicate that in contrast to the positive effect on the auditory system, PR induces oxidative stress and structurally and functionally negative effects on OSNs in zone 1, which is probably involved in the bioactivation of NQO1.

A murine model of eosinophilic chronic rhinosinusitis using the topical application of a vitamin D3 analog.

Eosinophilic chronic rhinosinusitis (ECRS) is a chronic inflammatory disease, characterized by eosinophilic infiltration, T-helper type 2 (Th2-type) response, and olfactory dysfunction. A master regulator of Th2-type inflammation, thymic stromal lymphopoietin (TSLP), is important for basophil activation. TSLP-elicited basophils are a key factor in the pathogenesis of ECRS. In order to elucidate the mechanisms of ECRS in humans, we aimed to establish a murine model of ECRS based on TSLP production in response to the topical application of MC903 (a vitamin D3 analog) and the subsequent TSLP-induced basophil activation. Histological analyses were performed to assess immune cell infiltration into the nasal mucosa and to explore the impact of eosinophilic inflammation on the olfactory epithelium. The status of Th2-type inflammation was evaluated by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). Eosinophils, basophils, and M2 macrophages increased significantly in the nasal mucosa of the mice treated with MC903 and ovalbumin (OVA), compared to those treated with OVA alone or the controls. Quantitative real-time PCR and ELISA revealed elevated expression of interleukin (IL)-4, IL-5, IL-13, TSLP, the chemokine CCL11, and CCL24 in the nasal mucosa of the ECRS mice. In parallel, thinned olfactory epithelium and decreased mature olfactory sensory neurons were observed in the ECRS mice. Our model of ECRS displayed Th2-type inflammation in the sinonasal region, including both eosinophil infiltration and basophil infiltration. Additionally, olfactory epithelium turned out to be affected by eosinophilic inflammation. These features are consistent with the characteristics of the human ECRS.

Olfactory Stimulation Regulates the Birth of Neurons That Express Specific Odorant Receptors

SUMMARYIn mammals, olfactory sensory neurons (OSNs) are born throughout life, ostensibly solely to replace damaged OSNs. During differentiation, each OSN precursor “chooses,” out of hundreds of possibilities, a single odorant receptor (OR) gene, which defines the identity of the mature OSN. The relative neurogenesis rates of the hundreds of distinct OSN “subtypes” are thought to be constant, as they are determined by a stochastic process in which each OR is chosen with a fixed probability. Here, using histological, single-cell, and targeted affinity purification approaches, we show that closing one nostril in mice selectively reduces the number of newly generated OSNs of specific subtypes. Moreover, these reductions depend on an animal’s age and/or environment. Stimulation-dependent changes in the number of new OSNs are not attributable to altered rates of cell survival but rather production. Our findings indicate that the relative birth rates of distinct OSN subtypes depend on olfactory experience.

The role of the NMD factor UPF3B in olfactory sensory neurons.

The UPF3B-dependent branch of the nonsense-mediated RNA decay (NMD) pathway is critical for human cognition. Here, we examined the role of UPF3B in the olfactory system. Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. UPF3B also regulates the expression of a large cadre of antimicrobial genes in OSNs, and promotes the selection of specific olfactory receptor (Olfr) genes for expression in mature OSNs (mOSNs). RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b-null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. Together, our data provides a valuable resource for the olfactory field and insights into the roles of NMD in vivo.

Severe Acute Respiratory Syndrome Coronavirus 2 Infects and Damages the Mature and Immature Olfactory Sensory Neurons of Hamsters.

BackgroundCoronavirus Disease 2019 (COVID-19) is primarily an acute respiratory tract infection. Distinctively, a substantial proportion of COVID-19 patients develop olfactory dysfunction of uncertain underlying mechanism which can be severe and prolonged. The roles of inflammatory obstruction of the olfactory clefts leading to conductive impairment, inflammatory cytokines affecting olfactory neuronal function, destruction of olfactory neurons or their supporting cells, and direct invasion of olfactory bulbs, in causing olfactory dysfunction are uncertain.MethodsIn this study, we investigated the location for the pathogenesis of SARS-CoV-2 from the olfactory epithelium (OE) of the nasopharynx to the olfactory bulb of golden Syrian hamsters.ResultsAfter intranasal inoculation with SARS-CoV-2, inflammatory cell infiltration and proinflammatory cytokine/chemokine responses were detected in the nasal turbinate tissues which peaked between 2 to 4 days post-infection with the highest viral load detected at day 2 post-infection. Besides the nasopharyngeal pseudo-columnar ciliated respiratory epithelial cells, SARS-CoV-2 viral antigens were also detected in the more superficial mature olfactory sensory neurons labeled by olfactory marker protein (OMP), the less mature olfactory neurons labelled by Tuj1 at more basal position, and the sustentacular cells which provide metabolic and physical support for the olfactory neurons, resulting in apoptosis and severe destruction of the OE. During the whole course of infection, SARS-CoV-2 viral antigens were not detected in the olfactory bulb.ConclusionsBesides acute inflammation at OE, infection of mature and immature olfactory neurons, and the supporting sustentacular cells by SARS-CoV-2 may contribute to the unique olfactory dysfunction of COVID-19 which is not reported with SARS-CoV.

Immunocytochemical Localization of Olfactory-signaling Molecules in Human and Rat Spermatozoa.

Expression of olfactory receptors (ORs) in non-olfactory tissues has been widely reported over the last 20 years. Olfactory marker protein (OMP) is highly expressed in mature olfactory sensory neurons (mOSNs) of the olfactory epithelium. It is involved in the olfactory signal transduction pathway, which is mediated by well-conserved components, including ORs, olfactory G protein (Golf), and adenylyl cyclase 3 (AC3). OMP is widely expressed in non-olfactory tissues with an apparent preference for motile cells. We hypothesized that OMP is expressed in compartment-specific locations and co-localize with an OR, Golf, and AC3 in rat epididymal and human-ejaculated spermatozoa. We used immunocytochemistry to examine the expression patterns of OMP and OR6B2 (human OR, served as positive olfactory control) in experimentally induced modes of activation and determine whether there are any observable differences in proteins expression during the post-ejaculatory stages of spermatozoal functional maturation. We found that OMP was expressed in compartment-specific locations in human and rat spermatozoa. OMP was co-expressed with Golf and AC3 in rat spermatozoa and with OR6B2 in all three modes of activation (control, activated, and hyperactivated), and the mode of activation changed the co-expression pattern in acrosomal-reacted human spermatozoa. These observations suggest that OMP expression is a reliable indicator of OR-mediated chemoreception, may be used to identify ectopically expressed ORs, and could participate in second messenger signaling cascades that mediate fertility.

TRPM5-expressing Microvillous Cells Regulate Region-specific Cell Proliferation and Apoptosis During Chemical Exposure

The mammalian main olfactory epithelium (MOE) is exposed to a wide spectrum of external chemicals during respiration and relies on adaptive plasticity to maintain its structural and functional integrity. We previously reported that the chemo-responsive and cholinergic transient receptor potential channel M5 (TRPM5)-expressing-microvillous cells (MCs) in the MOE are required for maintaining odor-evoked electrophysiological responses and olfactory-guided behavior during two-week exposure to an inhaled chemical mixture. Here, we investigated the underlying factors by assessing the potential modulatory effects of TRPM5-MCs on MOE morphology and cell proliferation and apoptosis, which are important for MOE maintenance. In the posterior MOE of TRPM5-GFP mice, we found that two-week chemical exposure induced a significant increase in Ki67-expressing proliferating basal stem cells without a significant reduction in the thickness of the whole epithelium or mature olfactory sensory neuron (OSN) layer. This adaptive increase in stem cell proliferation was missing in chemical-exposed transcription factor Skn-1a knockout (Skn-1a−/−) mice lacking TRPM5-MCs. In addition, a greater number of isolated OSNs from chemical-exposed Skn-1a−/− mice displayed unhealthily high levels of resting intracellular Ca2+. Intriguingly, in the anterior MOE where we found a higher density of TRPM5-MCs, chemical-exposed TRPM5-GFP mice exhibited a time-dependent increase in apoptosis and a loss of mature OSNs without a significant increase in proliferation or neurogenesis to compensate for OSN loss. Together, our data suggest that TRPM5-MC-dependent region-specific upregulation of cell proliferation in the majority of the MOE during chemical exposure contributes to the adaptive maintenance of OSNs and olfactory function.

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.

A Group of Olfactory Receptor Alleles that Encode Full Length Proteins are Down-Regulated as Olfactory Sensory Neurons Mature

The family of olfactory receptors (ORs) subserves the sense of smell and includes both functional alleles and pseudogenes, the latter identified by mutations resulting in frame shift or premature truncation. During neuronal differentiation, nonfunctional ORs are expressed initially but then are switched out, and/or the olfactory sensory neurons (OSNs) expressing them die. We carried out a transcriptomic analysis of FACS-isolated cells from ΔSox2-eGFP, Neurog1-eGFP BAC and ΔOMP-eGFP strains of uninjured and olfactory bulbectomized transgenic mice that correspond to distinct stages in the progression from globose basal cell stem cells to fully mature OSNs. We analyzed the expression pattern of 1094 unique receptors across this progression and found that the vast majority were characterized by a typical and expected pattern of expression; i.e., levels of OR mRNA peaking in mature OSNs. However, 43 ORs, including several known pseudogenes, were different, such that mRNA expression declined in the mature OSNs relative to earlier stages. Protein and promoter sequence analysis of the atypical group did not uncover any obvious differences between them and more typical ORs. Nonetheless, the pattern of expression suggests that atypical ORs may be non-functional despite the lack of any obvious abnormality in the sequence analyses.

Olig2 regulates terminal differentiation and maturation of peripheral olfactory sensory neurons.

The bHLH transcription factor Olig2 is required for sequential cell fate determination of both motor neurons and oligodendrocytes and for progenitor proliferation in the central nervous system. However, the role of Olig2 in peripheral sensory neurogenesis remains unknown. We report that Olig2 is transiently expressed in the newly differentiated olfactory sensory neurons (OSNs) and is down-regulated in the mature OSNs in mice from early gestation to adulthood. Genetic fate mapping demonstrates that Olig2-expressing cells solely give rise to OSNs in the peripheral olfactory system. Olig2 depletion does not affect the proliferation of peripheral olfactory progenitors and the fate determination of OSNs, sustentacular cells, and the olfactory ensheathing cells. However, the terminal differentiation and maturation of OSNs are compromised in either Olig2 single or Olig1/Olig2 double knockout mice, associated with significantly diminished expression of multiple OSN maturation and odorant signaling genes, including Omp, Gnal, Adcy3, and Olfr15. We further demonstrate that Olig2 binds to the E-box in the Omp promoter region to regulate its expression. Taken together, our results reveal a distinctly novel function of Olig2 in the periphery nervous system to regulate the terminal differentiation and maturation of olfactory sensory neurons.

Sequential Maturation of Olfactory Sensory Neurons in the Mature Olfactory Epithelium.

The formation of the olfactory nerve and olfactory bulb (OB) glomeruli begins embryonically in mice. However, the development of the olfactory system continues throughout life with the addition of new olfactory sensory neurons (OSNs) in the olfactory epithelium (OE). Much attention has been given to the perinatal innervation of the OB by OSN axons, but in the young adult the process of OSN maturation and axon targeting to the OB remains controversial. To address this gap in understanding, we used BrdU to label late-born OSNs in young adult mice at postnatal day 25 (P25-born OSNs) and timed their molecular maturation following basal cell division. We show that OSNs in young adults undergo a sequential molecular development with the expression of GAP 43 (growth-associated protein 43) > AC3 (adenylyl cyclase 3) > OMP (olfactory marker protein), consecutively, in a time frame of ∼8 d. To assess OSN axon development, we implemented an in vivo fate-mapping strategy to label P25-born OSNs with ZsGreen. Using sampling intervals of 24 h, we demonstrate the progressive extension of OSN axons in the OE, through the foramen of the cribriform plate, and onto the surface of the OB. OSN axons reached the OB and began to target and robustly innervate specific glomeruli ∼10 d following basal cell division, a time point at which OMP expression becomes evident. Our data demonstrate a sequential process of correlated axon extension and molecular maturation that is similar to that seen in the neonate, but on a slightly longer timescale and with regional differences in the OE.

Loss of BMI1 in mature olfactory sensory neurons leads to increased olfactory basal cell proliferation

Damage to olfactory sensory neurons (OSNs), situated within the neuroepithelium of the olfactory cleft, may be associated with anosmia. Although their direct contact with the nasal airspace make OSNs vulnerable to injury and death, multiple mechanisms maintain epithelium integrity and olfactory function. We hypothesized that BMI1, a polycomb protein found to be enriched in OSNs, may function in neuroprotection. Here, we explored BMI1 function in a mouse model. Utilizing a mouse genetic approach to delete Bmi1 selectively in mature OSNs, we investigated changes in OE homeostasis by performing immunohistochemical, biochemical, and functional assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunostaining, and electro-olfactograms were used to compare gene expression, cell composition, and olfactory function in OSN-specific BMI1 knockout mice (n = 3 to 5) and controls. Chromatin studies were also performed to identify protein-DNA interactions between BMI1 and its target genes (n = 3). OSN-specific BMI1 knockout led to increased neuron death and basal cell activation. Chromatin studies suggested a mechanism of increased neurodegeneration due to de-repression of a pro-apoptosis gene, p19ARF. Despite the increased turnover, we found that olfactory neuroepithelium thickness and olfactory function remained intact. Our studies also revealed the presence of additional polycomb group proteins that may compensate for the loss of BMI1 in mature OSNs. The olfactory neuroepithelium employs multiple mechanisms to maintain epithelial homeostasis. Our findings provide evidence that in a mouse model of BMI1 deletion, the overall integrity and function of the olfactory neuroepithelium are not compromised, despite increased neuronal turnover, reflecting a remarkable reparative capacity to sustain a critical sensory system.

1804-P: Characterizing Central Olfactory Circuits Modulating Energy Homeostasis

Olfactory inputs are important for hedonic evaluation of food, resulting in food choice and possible consumption. Olfactory acuity is determined by the feeding status, with hunger enhancing smell and food seeking behavior, whereas satiety suppresses olfaction and promotes energy expenditure. Efforts to understand the cellular basis of food intake and energy expenditure revealed the crucial role of the hypothalamus as a master regulator of whole-body energy homeostasis, integrating internal and external stimuli to modulate energy intake and expenditure accordingly. However, how the hypothalamus adjusts circuits regulating energy homeostasis depending on external stimuli, such as smell, remains an intriguing mystery. Our previous work revealed that ablation of olfactory sensory neurons (OSNs) stimulates hypothalamic-driven autonomic tone and promotes catabolic pathways, enhancing thermogenesis in adipose tissue. These findings unravel a new function for the olfactory system in controlling energy homeostasis in response to sensory signals. Here, we analyzed neural circuits connecting olfaction and hypothalamus and determined the role of olfactory inputs on energy homeostasis. To identify neural circuits linking olfactory and hypothalamic neurons, we conducted anterograde viral tracing from mitral cell layer in the main olfactory bulb (MOB) in Tbx21-Cre mice and retrograde viral tracing from pro-opiomelanoctin (POMC)- and agouti-related protein (AgRP)-expressing hypothalamic neurons in POMC-Cre and AgRP-IRES-Cre mice using Cre-dependent helper virus and modified rabies virus. Additionally, we used chemical ablation of mature OSNs and chemogenetic silencing of mitral cell layer neurons to determine the effect of olfaction on energy homeostasis. Our data suggest that piriform cortex (PC)-amygdala (Amy)-arcuate nucleus of hypothalamus (ARC) is one of the plausible circuits responsible for transferring signals from MOB to the hypothalamus. Disclosure C. Riera: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-15-INI-12)