Roles of odorant receptors during olfactory glomerular map formation

The organization of the olfactory glomerular map involves the convergence of olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) into glomeruli in the olfactory bulb (OB). A remarkable feature of the olfactory glomerular map formation is that the identity of OR instructs the topography of the bulb, resulting in thousands of discrete glomeruli in mice. Several lines of evidence indicate that ORs control the expression levels of various kinds of transmembrane proteins to form glomeruli at appropriate regions of the OB. In this review, we will discuss how the OR identity is decoded by OSNs into gene expression through intracellular regulatory mechanisms.


| INTRODUCTION
Olfaction is essential for the survival and reproduction of animals in their natural environments.Since the discovery of odorant receptor (OR) genes by Buck and Axel (1991), extensive research has been dedicated to understanding the mechanisms of odor detection and its neural representation in the brain.Odorants in the environment are detected by ORs that are expressed on olfactory sensory neurons (OSNs) within the olfactory epithelium (OE) of the nasal cavity.The binding of an odorant to ORs on cilia surfaces activates the G protein-adenylyl cyclase type 3 (AC3) pathway, elevating cAMP levels, which subsequently opens cyclic nucleotide-gated (CNG) channels, resulting in the depolarization of OSNs (Bradley et al., 2005;Breer et al., 1990) (Figure 1b).In mice, ORs comprise the largest family of G protein-coupled receptors with >1000 genes (Zhang & Firestein, 2002).Individual OSNs express only one functional OR gene and axons from OSNs expressing a given OR converge onto a specific pair of glomeruli at stereotyped locations in the olfactory bulb (OB) (Figure 1a).The principal output neurons in the OB, mitral/ tufted (M/T) cells, extend a single apical dendrite into a glomerulus where they receive inputs from OSNs expressing the corresponding OR.Thus, each glomerulus is regarded as a functional unit dedicated to a specific OR.Notably, individual ORs can be activated by multiple odorants, and individual odorants can activate multiple OR species with differential magnitudes (Malnic et al., 1999).Consequently, odor information is topographically represented as a pattern of activated glomeruli in the OB (Figure 1c).Previous studies have shown that changes in the number, organization, or location of glomeruli lead to impairments in odor discrimination abilities, indicating the pivotal role of the glomerular map in odor information processing (Fleischmann et al., 2008;Lorenzon et al., 2015).
The development of functional neural circuits emerges from the interplay between genetic programming and electrical neural activity.In the mouse olfactory system, global targeting of OSN axons is directed by a genetically determined process, and glomerular formation is regulated by neural activity.The global targeting can be classified into two orthogonal axes: anterior-posterior (A-P) and dorsal-ventral (D-V).The D-V positioning of glomeruli correlates topographically with the OSN cell bodies' locations (Astic et al., 1987;Miyamichi et al., 2005;Ressler et al., 1994;Vassar et al., 1994).Positional information within the OE regulates both OR gene selection and the expression of axon guidance molecules that dictate the D-V positioning of glomeruli (Cho et al., 2007;Takeuchi et al., 2010).Conversely, A-P projection is independent of OSN cell positions but relies on the expressed OR type.
Ligand-independent OR-derived cAMP signals have been shown to regulate axon guidance molecules that guide global OSN axon targeting along the A-P axis (Imai et al., 2006;Nakashima et al., 2013).Beyond A-P projection, ORs also regulate glomerular formation in an activity-dependent manner (Mobley et al., 2010;Yu et al., 2004;Zheng et al., 2000).OR expression induces patterns of spontaneous neural activity and regulates the expression of axon sorting molecules necessary for glomerular formation (Nakashima et al., 2019;Serizawa et al., 2006).Therefore, ORs fulfill multiple roles in shaping the olfactory glomerular map by regulating OSN gene expression programs.But how is OR identity translated into gene expression patterns of axon guidance and sorting molecules?This review will summarize our current understanding of OR-directed glomerular map formation and discuss cellular mechanisms that yield diverse gene expression patterns corresponding to OR types in OSNs.

| Axon guidance molecules in anterior-posterior axon targeting
The instructive role of the OR in global targeting was initially demonstrated in an OR coding-swap experiment, where substituting the M12 OR gene with P2 repositioned the glomeruli posteriorly compared to endogenous P2 glomeruli (Mombaerts et al., 1996).Subsequent experiments with various G-protein coupled receptors (GPCRs), including non-OR GPCRs, corroborated the significance of ORderived signals in A-P global targeting (Feinstein et al., 2004;Katidou et al., 2018;Nakashima et al., 2013;Wang et al., 1998).Intriguingly, in a series of GPCR swap experiments, replacing an OR with a β2-adrenergic receptor (β2-AR) or melanocortin 4 receptor (Mc4r) resulted in the formation of a functional glomerulus, whereas substitution with a vomeronasal receptor, which does not bind to Gi and Gq, but not to Gs, did not form a glomerulus (Feinstein et al., 2004;Katidou et al., 2018).Further, mutation in DRY motif that is essential for OR-Gs coupling failed to form glomerular structures (Imai et al., 2006).This suggests that activation of the G proteins-AC3 pathway is critical for maintaining glomerular structures.Further manipulation of the olfactory transduction pathway signaling levels indicated that OR-derived cAMP levels govern the A-P topography of the OB (Figure 2a) (Imai et al., 2006;Nakashima et al., 2013).Moreover, disrupting the A-P topography through AC3 KO altered the expression of axon guidance molecules (e.g., Nrp1, PlxnA1) that show graded expression in the OB (Col et al., 2007;Imai et al., 2009;Nakashima et al., 2013).Nrp1 protein levels measured at OSN axon termini revealed an anterior-low/posterior-high gradient in the OB.Genetic manipulation of Nrp1 expression in OSNs exhibited glomerular relocations along the A-P axis.Additionally, the A-P topography of the glomerular map was significantly disrupted in mice lacking Nrp1 and its repulsive ligand, Sema3A (Assens et al., 2016;Imai et al., 2009;Zapiec et al., 2016).These findings suggest that OR-derived cAMP signals are translated into expression levels of axon guidance molecules that direct axon targeting (Nakashima et al., 2013).A detailed histological analysis revealed that map order emerges before OSN axons reach their target.Nrp1 and Sema3A are expressed complementarily in OSNs, and axons positive for Nrp1 and Sema3A are presorted within the axon bundles (Imai et al., 2009).The specific KO of Nrp1 or Sema3A in OSNs not only disrupted axon sorting within the bundle but also induced an anterior shift of glomeruli in the OB.Therefore, pretarget axon sorting within the bundle likely plays a role in olfactory map formation along the A-P axis.However, the positions of glomeruli for some ORs did not show a consistent shift in Nrp1 KO, leaving the detailed role of Nrp1 in topographic targeting along the A-P axis still under debate (Assens et al., 2016;Zapiec et al., 2016).It is also noteworthy that Sema3A is also abundantly expressed in olfactory ensheathing glial cells (Schwarting et al., 2000), which migrate alongside OSN axons from the OE and localize to the anterior part of the OB during development.This glia-derived Sema3A may explain the less severe phenotype observed in OSN-specific Sema3A KO compared to the total KO and might serve as a landmark for determining the absolute A-P position of the glomeruli.

| OR-derived basal activity
The genetic KO of AC3 and transgenic manipulations of Gs protein have elucidated that Gs-AC3 signaling downstream of ORs is crucial for axon targeting (Chesler et al., 2007;Imai et al., 2006;Nakashima et al., 2013;Zou et al., 2007).Given that CNGA2 deficiency or naris occlusion does not impact the expression of A-P targeting molecules (Nakashima et al., 2013), it suggests that ORs may regulate A-P targeting in a ligand-independent fashion.The question that arises is what type of OR activity is responsible for this regulation, and how is it generated?
It is generally accepted that GPCRs, including ORs, exist in two different conformational states: active and inactive (Seifert & Wenzel-Seifert, 2002).Agonists stabilize receptors in an active conformation, while inverse agonists maintain them in an inactive state.This F I G U R E 2 Odorant receptor (OR)-derived basal cAMP levels regulate axon targeting along the A-P axis.(a) Olfactory sensory neurons with higher basal levels of cAMP direct their axons to the posterior part of the olfactory bulb (OB), while those with lower levels project to the anterior OB.(b) G-protein-coupled receptors (GPCRs) can exist in active or inactive states.In the absence of ligands, GPCRs can spontaneously toggle between these states, leading to agonistindependent basal activity of cAMP.(c) A model is proposed where each OR generates a unique basal cAMP level, distinct from others, which is translated into specific expression levels of A-P targeting molecules.(modified from Takeuchi & Sakano, 2014).conventional signaling mechanism permits the receptor to produce its "textbook" cellular responses.However, in vitro studies have shown that GPCRs can spontaneously alternate between active and inactive states in the absence of ligands, resulting in some degree of basal signaling activities (Figure 2b).The agonist-independent activity of GPCRs was once considered insignificant background noise, and its functional relevance has yet to be fully understood.We hypothesized that this ligand-independent OR activity plays a part in the A-P targeting of OSN axons.
To prove this hypothesis, we utilized the β2-AR as a model system to provide causal proof that basal receptor signaling directs axon targeting.As mentioned in the preceding section, β2-AR serves as a surrogate for ORs in instructing OSN axonal projection (Feinstein et al., 2004).While ORs themselves have been poorly characterized, β2-AR benefits from being extensively studied for distinct receptor functions via in vitro studies (Ballesteros et al., 2001;O'Dowd et al., 1988;Savarese & Fraser, 1992).Moreover, structural studies have provided three-dimensional insights, allowing us to interpret the effects of β2-AR mutations through structure-based analysis (Rasmussen, Choi, et al., 2011;Rasmussen, DeVree, et al., 2011).Consequently, the comprehensive understanding of β2-AR signaling enabled us to directly demonstrate whether basal GPCR activity influences axon targeting.Among the β2-AR mutations we analyzed, some influenced both agonist-independent and -dependent activities, while others selectively modified basal signaling alone.These mutations are located intracellularly, distant from the transmembrane ligand binding pocket.This spatial separation suggests that the basal activity generation is regulated independently through a specific allosteric site.Such mutations could alter the spontaneous conformational transitions and, hence, the agonist-independent receptor activity.We generated transgenic mouse lines expressing β2-AR under the control of an OR promoter and analyzed the effects of wild-type and basal activitymutant β2-ARs on axon targeting of OSNs.The results demonstrated that β2-AR mutants with reduced basal activity shifted glomerulus locations anteriorly, while a mutant with increased activity shifted them posteriorly.Furthermore, the expression levels of A-P targeting molecules, Nrp1 and PlxnA1 changed in accordance with the basal activity levels (Figure 2c) (Nakashima et al., 2013).Collectively, this research indicates that GPCR basal activity is a pivotal signal in modulating A-P targeting molecules, thereby determining the axonal projection patterns of OSNs.This is corroborated by the measurement of the basal activity of natural ORs, where ORs extracted from the anterior OB exhibited lower agonist-independent activity than those from the posterior OB (Nakashima et al., 2013).A recent study extensively examined the connection between OR protein sequences and glomerular positions within the OB at a repertoire level.By combining sequential sectioning with target-capture sequencing, they spatially mapped 966 ORs, constituting 86% of the mouse genome's ORs (Zhu et al., 2022).This comparison between anterior and posterior OR groups highlighted key amino acid residues, such as those in intracellular loop 3, which could potentially influence G-protein interactions with ORs.A parallel investigation by the Greer laboratory has demonstrated that each OSN can be distinguished by its expression profiles of axon guidance molecules, with this unique transcriptional program being sufficient to predict glomerular locations in the OB (Wang et al., 2022).Hence, the agonist-independent basal activity, inherently defined by OR proteins, appears to be a significant determinant of A-P targeting (Figure 2c).Factors such as promoter activity, protein stability, and membrane transport may also contribute to the overall basal activity levels of endogenous ORs.

| A combinatorial molecular code of axon sorting molecules
During embryonic development, an initial topographical map is formed by a combination of spatial cues and ligand-independent ORderived basal activity.Once OSN axons reach their proximate targets in the OB, further refinement of the glomerular map occurs, involving the fasciculation of similar OSN axons and the segregation of dissimilar ones.Point mutations introduced into OR sequences have led to the formation of additional glomeruli, underscoring the role of ORs in this process (Feinstein & Mombaerts, 2004;Ishii et al., 2001).
Investigating the control mechanisms behind OR-specific axon sorting, we identified a set of genes with expression profiles correlated to specific ORs.In transgenic mice designed to express a particular OR predominantly, we identified genes coding for homophilic adhesion molecules, such as Kirrel2 and Kirrel3 (Serizawa et al., 2006).
The mosaic gain or loss of function of these genes resulted in duplicated glomeruli even though the expressed ORs were the same, suggesting that Kirrel2 and Kirrel3 are crucial in the fasciculation process of like OSN axons.Notably, upregulated Kirrel2/3 levels in OSNs that naturally express these genes also led to duplicated glomeruli, highlighting that OSN axons discern not only the type but also the expression levels of axon sorting molecules.This contributes to increasing variations of the molecular code for glomerular segregation.Additionally, repulsive molecules like ephrinAs and EphA receptors are expressed in an OR-specific and complementary manner across different OSN subsets (Serizawa et al., 2006).The interactions between subsets-one high in ephrinA and low in EphA receptor, and vice versa-may be instrumental in segregating nonlike OSN axons.Although correlated with OR types, the expression patterns of these molecules are not identical (Ihara et al., 2016).
Their unique expression profiles imply that a combinatorial code of adhesive and repulsive molecules imparts OR identity to OSN axons during glomerular formation.Axonal shafts expressing the same OR are bundled together before reaching their target glomerulus.The molecular code would be utilized not only for terminal axon-axon interaction but also for shaft-shaft interaction for zippering axons to efficiently converge onto the same glomeruli.Since such shaft-shaft fasciculation is thought to depend on the balance between axonaxon adhesion forces and the mechanical longitudinal tension of axons (Breau & Trembleau, 2023), It is interesting to investigate the mechanical tension of OSN axons during development.
To date, several studies identified multiple axon-sorting molecules that are expressed in OSNs (Ihara et al., 2016;Kaneko-Goto et al., 2008;Martinez et al., 2023;Mountoufaris et al., 2017;Nakashima et al., 2019;Williams et al., 2011).The exact number of molecules implicated in this process is still unclear, but a handful of transmembrane proteins have been implicated.

| Patterns of spontaneous activity link OR types and the molecular codes
As mentioned above, OR molecules control the expression of various axon-sorting molecules, which serve to regulate glomerular formation through their adhesive or repulsive interactions (Takeuchi & Sakano, 2014).The expression of these molecules is activitydependent, consistent with the observation that glomerular formation is likewise activity-dependent.It is anticipated that neural activity serves as a bridge between OR types and the combinatorial expression of axon-sorting molecules.The mechanism by which neural activity establishes this link was previously unclear.Through calcium imaging of OSNs, we have discerned that different ORs elicit distinct temporal patterns of spontaneous neural activity (Nakashima et al., 2019).Furthermore, optogenetically differentiated activity patterns prompted specific expressions of corresponding axon-sorting molecules; for instance, short, high-frequency burst patterns particularly induced the expression of the axon-sorting molecule Kirrel2, whereas longer, lower-frequency bursts promoted the expression of other molecules like PCDH10, also a participant in glomerular formation (Nakashima et al., 2019).A significant breakthrough is the discovery that it is the unique temporal characteristics of spike patterns, rather than mere neural activity, that provide the instructive signals for inducing OR-specific expression profiles of axon-sorting molecules.This discovery proposes a novel activity-dependent mechanism, which is different from the prevailing Hebbian model of plasticity that requires correlated activity.In summary, this research unveils an activity-dependent mechanism where spontaneous spiking patterns instructively regulate the expression of axon guidance molecules, thus conferring OR identity to OSN axons for glomerular convergence (Figure 3).However, it remains to be done how many variations there are in neural activity patterns of OSNs and how OSNs translate different temporal patterns of calcium dynamics into different gene expression patterns.

| DEVELOPMENTAL TIMING-DEPENDENT REGULATION OF GLOMERULAR MAP FORMATION
The OR-derived cAMP signaling cascade is pivotal in establishing the olfactory glomerular map, as evidenced by the alteration in the expression of axon-guidance and axon-sorting molecules, such as Nrp1, PlxnA1, and Kirrel2/3, upon disruption of the AC3 gene.Thus, this signaling pathway regulates gene expression, thereby controlling global axon targeting and glomerular segregation, two processes essential for establishing the olfactory glomerular map.The question then arises: how are A-P targeting and glomerular formation differentially regulated during olfactory map development?Temporal segregation of two distinct cAMP signals from ORs has been proposed by prior studies as a mechanism enabling the separate regulation of axon targeting and glomerular formation in OSNs.Gene expression analyses revealed that A-P targeting molecules, such as Nrp1, are present from embryonic day (E) 13.5, while axon-sorting molecules like Kirrel2 are detected primarily in late embryogenesis, post E17.5.Correspondingly, the signaling protein Gs is expressed by E13.5, which is prior to the expression of its closely related homolog Golf, detected from E17.5 onwards.Loss-of-function studies have demonstrated that KO of Gs influences the expression of A-P targeting molecules exclusively, whereas Golf KO impacts axon sorting molecules without affecting A-P targeting molecules (Nakashima et al., 2013).In summary, differences in expression onset allow stimulatory G-proteins to sequentially orchestrate the formation of the glomerular map, governing both axon targeting and segregation.Early G-protein signaling F I G U R E 3 Cell-type specific patterned activity regulates odorant receptor (OR)-specific expression of axon sorting molecules.Distinct spontaneous activity patterns of olfactory sensory neurons, dictated by their respective ORs, lead to unique expression profiles of axonsorting molecules.These differential expressions orchestrate a combination of axon-sorting molecules for each OR type, crucial for the segregation of glomeruli.OE, olfactory epithelium.
regulates A-P targeting of OSNs, subsequently facilitating local glomerular segregation (Figure 4a).Furthermore, biochemical assays have indicated that Golf exhibits a lower affinity for GDP and is more rapidly deactivated by its high GTPase activity compared to Gs (Liu et al., 2001;Seifert et al., 1998).This suggests that Gs mediates ligand-independent activity more effectively than Golf (Nakashima et al., 2013), potentially explaining its specific role in the transcriptional regulation of A-P targeting molecules.

| GLOMERULAR MAP MAINTENANCE BEYOND THE CRITICAL PERIOD
The olfactory system serves as a model for studying the development and maintenance of neural maps.Unlike the other sensory systems, the mammalian olfactory system can regenerate OSNs and integrate new neurons into established circuits (Graziadei & Monti Graziadei, 1985).Newly generated OSNs rapidly generate functional synapses to activate OB postsynaptic neurons (Browne et al., 2022).
Despite continuous neuronal turnover, OSNs consistently rewire to target specific glomeruli within the OB.Emerging research posits a critical developmental period for olfactory map formation; disturbances in initial map organization lead to permanent disorganization of map topography thereafter.Ma et al. showed that perturbing OSN activity at perinatal stages prevented glomerular targeting (Ma et al., 2014), and Tsai and Barnea found that ectopic receptor expression prior to map establishment caused axon rerouting, whereas after map establishment, it did not (Tsai & Barnea, 2014).Additionally, the olfactory topographic map fails to recover adequately after widespread loss of OSNs in adulthood (Costanzo, 2000;Gogos et al., 2000;Murai et al., 2016).These findings collectively suggest that the maintenance of the glomerular map is contingent upon an accurately formed initial map during a perinatal critical window.
The incorporation of nascent neurons is mediated by interactions between existing and newly generated OSNs that express the same ORs.While these pioneer-follower interactions are known to be governed by the expressed ORs (Tsai & Barnea, 2014) (Figure 4b), the detailed molecular mechanisms remain to be fully delineated.Interestingly, the maintenance of the glomerular map in adulthood is not solely dependent on the OSNs.The preservation of the glomerular map in adulthood also appears to be influenced by the synaptic partner.Removal of the M/T cells during the perinatal period did not affect OSN fasciculation, but in adulthood, it disrupts the axonal projection pattern of newly generated OSNs (Sánchez-Guardado et al., 2024).The findings imply that M/T cells are not indispensable for forming the glomerular map but are crucial for maintaining the glomerular map.The maintenance of the glomerular map in adulthood appears to be achieved through a combination of homotypic axonaxon interactions between OSNs expressing the same OR and an M/T cell-dependent mechanism.Understanding how these two mechanisms work together to maintain the glomerular map will provide valuable insights into the plasticity and regeneration of the olfactory system.Elucidating the molecular details of how the newborn OSN axons are integrated into the existing map will shed light not only on the plasticity of olfactory circuits but also on potential therapeutic interventions for circuits disrupted by trauma.

| CONCLUSIONS
The intricate glomerular map that allows odor information processing depends critically on signaling by ORs themselves (Dorrego-Rivas & Grubb, 2022;Francia & Lodovichi, 2021;Mori & Sakano, 2011).Independent of odor stimulation, ORs manifest ligand-independent activity, setting baseline cAMP accumulation and specific patterns of spontaneous neural activity.This intrinsic signaling is fundamental to establishing glomerular maps in the bulb through dual pathways: the modulation of axon guidance molecule expression by basal cAMP levels that direct axonal projection along the A-P axis; and the refinement of glomerular structures by distinct temporal patterns of spontaneous activity defined by OR types.In addition to the OR downstream signals mentioned above, a recent study suggests the importance of the ER stress signal as an intrinsic factor that translates OR identity into axonal projection patterns (Shayya et al., 2022).Differences in OR amino acid sequences lead to variations in ER stress levels and ultimately translate into different expression patterns of axon guidance molecules.The regulation of olfactory map formation through multiple OR-derived signaling mechanisms may provide robustness to this system.Thus, intrinsic OR-derived signals are vital for directing the self-organized assembly of the olfactory glomerular map.Conversely, extrinsic signals seem to refine the subtler aspects of circuitry and plasticity, rather than directing topography of the glomerular map.Odor exposure influences structural remodeling, evident in size and number of glomeruli, hastened refinement, and altered cell survival rates.Spontaneous activity regulates axon sorting molecules like Kirrel2 and PCDH10, yet some analyses suggest that odor-evoked activity also modifies their expressions (Nakashima et al., 2013;Tsukahara et al., 2021).It has been shown that the expression of axon-sorting molecules is modulated according to environmental changes (Tsukahara et al., 2021).Activity-dependent axon-sorting molecules may thus also contribute to odor-induced structural changes, inviting further research into these dynamics and their functional consequences.This review focuses on the self-organization of OSN axons to form the olfactory map and the mechanisms involved in this process.
Several studies have suggested that the map formation is not entirely a self-organizing process but is influenced by surrounding cues.Zamparo et al. showed that the PEBP1 protein, expressed in the OB, binds to OR proteins on OSN axon terminals and causes the axons to change direction (Zamparo et al., 2019).These findings reveal that OSN axonal projections and map formation are regulated by both autonomous mechanisms and external factors, such as chemical signals from the OB.
The extraordinary wiring specificity of the OSN has long intrigued developmental neuroscientists and has been the subject of debate as to how the OR directs glomerular formation (Dorrego-Rivas & Grubb, 2022;Takeuchi & Sakano, 2014;Zamparo et al., 2019).The findings obtained by olfactory circuit formation studies over the past few decades provide important insights that are not specific to the olfactory system but can be extended to other brain regions.Neural activity influences a multitude of developmental and plasticity processes, such as cell-type specification, dendritic branching, synaptic maturation, and the underpinnings of learning and memory, through a complex program of gene regulation (Greer & Greenberg, 2008;Spitzer, 2006;West & Greenberg, 2011).Although several hundreds of genes have been identified as activity-dependent genes (Flavell & Greenberg, 2008;Lee et al., 2017;Tyssowski et al., 2018), their regulatory mechanisms and functions are not fully understood.Through our exploration of olfactory glomerular map formation, we have unveiled that varying temporal patterns of neural activity are translated into discrete gene expression profiles.This strategy endows neurons with the capacity to generate a diversity of gene expressions with a single second messenger, likely calcium ions.Additionally, the principle of combinatorial molecular coding enables the representation of diverse information with a minimal molecular repertoire.The insights offer profound implications for our understanding of the complex neural circuit architecture and the mechanisms underlying information processing in the central nervous system.

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I G U R E 1 The mouse olfactory system.(a) In mice, each olfactory sensory neuron (OSN) exclusively expresses one type of odorant receptor (OR) gene.Axons from OSNs with the same OR converge at a specific site within the olfactory bulb (OB), forming a glomerular structure.(b) The olfactory signal transduction pathway is illustrated schematically.Odorant binding to ORs activates cAMP synthesis through the G proteinadenylyl cyclase 3 (AC3) pathway.An increase in cAMP levels opens cyclic nucleotide-gated (CNG) channels, leading to influx of Na + and Ca 2+ .The increased Ca 2+ then activates the chloride (Cl À ) channel, resulted in an amplification of cell membrane depolarization.(c) Odor signals received in the olfactory epithelium (OE) are represented as a topographic map of activated glomeruli within the OB (modified from Takeuchi & Sakano, 2014).D, dorsal; V, ventral; A, anterior; P, posterior.

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I G U R E 4 Stepwise development of the olfactory glomerular map.(a) The regulation of both A-P-targeting and glomerular segregation molecules mediated by OR-derived signals.In immature olfactory sensory neurons (OSNs), each OR generates a unique level of baseline cAMP via agonist-independent activation of the G protein-AC3 pathway.In mature OSNs, OR-derived signals generate different temporal patterns of neuronal activity, which set expression levels of glomerular segregation molecules, such as Kirrel2/3 and ephrinA/EphA receptors.(b) During the embryonic stage, OSN axons are organized along the A-P axis according to the basal cAMP levels dictated by their respective ORs (left).Prior to birth, neighboring glomerular structures intermingle, and discrete glomeruli form during the perinatal stage.OR-correlated neuronal activity instructs the formation of glomeruli by regulating the expression of glomerular segregation molecules (middle).After the perinatal stage, axons from newly generated OSNs are incorporated into preexisting connections via homotypic interactions between pioneer-follower OSNs.OR, odorant receptor; AC, adenylyl cyclase; PKA, protein kinase A; CREB, cAMP responsive element binding protein; PDE, phosphodiesterase.