4.- the cell biology of smell

8/6/2019 4.- The Cell Biology of Smell

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The Rockefeller University Press $30.00 J. Cell Biol. Vol. 191 No. 3 443452

www.jcb.org/cgi/doi/10.1083/jcb.201008163 JCB 443

JCB: Review

Correspondence to John Ngai: [email protected] used in this paper: CNG, cyclic nucleotide gated; OSN, ol actorysensory neuron.

Introduction

A major challenge o sensory neurobiologyand neurosciencein generalis to understand the pathways that bridge an exter nal stimulus to its representation in the brain as a percept, andhow such pathways ultimately drive behaviors. The rst step o

this process involves the primary sensory neurons, which detectphysical stimuli through a variety o highly specialized recep tors. The perception o a smell begins with the activation o receptors expressed by the ol actory sensory neurons (OSNs),which reside in the main ol actory epithelium lining the nasalcavity (Fig. 1 A). Each OSN extends a single dendrite to thelumenal sur ace o the epithelium, rom which immotile ciliaextend to catch inhaled odorants rom the air. These ol actorysensory cilia are enriched in the odorant receptors and other sig naling components that mediate the initial transduction eventsin the cell (Firestein, 2001). At the other end o the neuron, asingle, unbranched axon projects to the ol actory bulb, a special ization o the orebrain that serves as the rst relay station in

this neural pathway. The axons o OSNs in the periphery togethercomprise the ol actory nerve. Once the axons reach the ol ac tory bulb, they make synapses with the dendrites o projectionneurons, within discrete structures known as glomeruli. In themouse, there are 510 million OSNs in the ol actory epithelium

and 1,800 glomeruli in each ol actory bulb, which translatesto an 103 old convergence o primary sensory axons ontoeach ol actory glomerulus (Firestein, 2001). This convergencelies at the heart o the coding strategy or ol actory sensoryin ormation. In parallel to the main ol actory epithelium, thevomeronasal organan anatomical specialization o the nose interrestrial vertebrates that is separate rom the main ol actoryepitheliumsenses nonvolatile chemical stimuli, includingpheromones (Dulac and Torello, 2003; Mombaerts, 2004). Thepresent review ocuses on the main ol actory epithelium and themultiple roles that the OR amily o odorant receptors play,not only as detectors o volatile chemicals in the environment,but also as regulators o key developmental decisions made bydi erentiating OSNs.

The nose, its receptors, and connections

to the brain

A large multigene amily o ol actory speci c G proteincoupledreceptors (GPCRs) was initially identi ed in the rat (Buck andAxel, 1991) and belongs to what is now re erred to as the OR

amily o odorant receptors (Mombaerts, 2004). The predictedstructure o these receptors exhibits a seven transmembrane do

main topology, and their sequences place them in the rhodopsinclass o GPCRs. The size o the OR gene amily in mammals isextremely large and ranges rom 700 genes in humans (abouthal o which are unctional) to over 1,200 genes in rodents(about two thirds o which are unctional; Mombaerts, 2004;Nei et al., 2008). In the sh, the size o the OR repertoire ap pears to be much smaller, containing only 40140 intact genesdepending on the species (Alioto and Ngai, 2005; Niimura andNei, 2005). The ORs exhibit extensive sequence diversity withintheir transmembrane domainsthe presumed sites o ligandbinding in this class o GPCR. Thus, the OR amily has evolvedto detect a wide range o chemical structures present in the ani mals environment. In addition to the ORs, members o the

much smaller trace amineassociated receptor (TAAR) amilyare expressed in OSNs o the main ol actory epithelium and arethought to mediate the reception o amine cues (Liberles andBuck, 2006). Finally, the neurons o the vomeronasal epitheliumexpress receptors rom three unrelated GPCR amilies, the V1R,

The olfactory system detects and discriminates myriadchemical structures across a wide range of concentrations.To meet this task, the system utilizes a large family of G proteincoupled receptorsthe odorant receptors which are the chemical sensors underlying the perceptionof smell. Interestingly, the odorant receptors are also involvedin a number of developmental decisions, including theregulation of their own expression and the patterning of

the olfactory sensory neurons synaptic connections inthe brain. This review will focus on the diverse roles of theodorant receptor in the function and development of theolfactory system.

Review series

The cell biology of smellShannon DeMaria and John Ngai

Department of Molecular and Cell Biology, Helen Wills Neuroscience Institute and Functional Genomics Laboratory, University of California, Berkeley, Berkeley, CA 9

2010 DeMaria and Ngai This article is distributed under the terms o an AttributionNoncommercialShare AlikeNo Mirror Sites license or the rst six months a ter the pub-lication date (see http://www.rupress.org/terms). A ter six months it is available undera Creative Commons License (AttributionNoncommercialShare Alike 3.0 Unported license,as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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JCB VOLUME 191 NUMBER 3 2010444

V2R, and ormyl peptide like receptors (Mombaerts, 2004;Yang et al., 2005; Rivire et al., 2009).

Upon binding its cognate odor ligand, the activated OR (andpresumably also TAAR) couples through G ol , a G s iso orm en riched in OSNs (Belluscio et al., 1998). Activated G ol in turn ac tivates type III adenylyl cyclase (Wong et al., 2000), which catalyzesthe production o cAMP. The increase in intracellular cAMP gatesor opens a cyclic nucleotide gated (CNG) channel, leading to an in fux o sodium and calcium ions and depolarization o the neuron(Brunet et al., 1996). This initial depolarization is urther ampli edby the subsequent activation o calcium activated chloride chan nels and, owing to the low concentration o extracellular Cl in themucus bathing the ol actory cilia, the e fux o Cl rom the cell(Stephan et al., 2009; Fig. 2 A). The odor induced depolarization inthe ol actory cilia spreads throughout the neuron, resulting in theopening o voltage sensitive ion channels in the sensory neuronsaxon hillock, the ring o action potentials, and the release o neuro transmitter at the synaptic terminal in the ol actory bulb.

To determine the identity o an odorant stimulus, the ner vous system must discern which o the 1,000 ORs has been

activated. Two organizing principles o the peripheral ol actorysystem underlie the encoding o this in ormation. First, each OSNin the ol actory epithelium expresses just one allele o a singleOR (Chess et al., 1994; Serizawa et al., 2003; Lewcock and Reed,2004; Fig. 1 B)a phenomenon re erred to as the one receptor,one neuron rule. In this way, the spectrum o odorous com pounds to which an individual OSN can respond (its receptive

eld) is a direct unction o the ligand tuning properties o itssingularly expressed OR. How, then, is activity rom a particularclass o OSNs (here de ned by the OR that it expresses) distin guished rom activity o all the other OSN classes, and by exten sion, rom activity o other ORs? This second level o processingrelies upon the projection o axons rom OSNs expressing the

same OR to common glomeruli in the ol actory bulb (Fig. 1 B).Interestingly, the projection o OSNs to the ol actory bulb com prises a discontinuous map; neurons expressing a given OR, al though distributed broadly in the peripheral sensory epithelium(Ressler et al., 1993; Vassar et al., 1993), converge to discreteglomeruli in the ol actory bulb in a spatially invariant pattern(Ressler et al., 1994; Vassar et al., 1994; Mombaerts et al., 1996;Mori et al., 1999). This sensory map displays a mirror symmetry,such that OR speci c neurons typically innervate one glomerulusin the ol actory bulbs medial hemisphere and another glomer ulus in the lateral hemisphere (Ressler et al., 1994; Vassar et al.,1994; Mombaerts et al., 1996; Nagao et al., 2000). The dendriteso the projection neurons o the ol actory bulbthe mitral andtu ted cellsin turn innervate the glomeruli and carry this in or mation via their axons to the ol actory cortex. The spatial patternso activity elicited in the ol actory bulb appear to be representednot as a corresponding spatial map in the ol actory cortex, butrather in a sparse and distributed manner at this level (Poo andIsaacson, 2009; Stettler and Axel, 2009).

The odorant receptor: chemical sensor

par excellence

Since their discovery in 1991, the ORs have been widely as sumed to comprise a amily o odorant receptors based on their

Figure 1. Anatomy of the rodent peripheral olfactory system.(A) Sche-matic representation o a parasagittal section through adult mouse head.Axons o the OSNs in the main ol actory epithelium comprise the ol ac-tory nerve and innervate the ol actory bulb. Vomeronasal sensory neuronsproject their axons via a separate tract, the vomeronasal nerve, to inner-vate the accessory ol actory bulb. (B) Each OSN o the main ol actoryepithelium expresses only one odorant receptor gene (OR A, OR B, OR C,etc.) out o a repertoire o over 1,000 genes. Neurons expressing a givenOR are organized into broad zones along the dorsalventral axis o theol actory epithelium (OE) and converge to a common glomerulus at cor-responding dorsalventral zones in the ol actory bulb (OB). Each glomerulusthus receives innervation rom sensory neurons expressing a single odorantreceptor, providing the anatomical basis o the ol actory sensory map.



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445The cell biology of smell DeMaria and Ngai

screen in a high throughput ashion a large array o receptorsagainst a similarly large panel o candidate odorantsarguablya necessity i we are to understand how this amily o 1,000receptors is used to discriminate among the vast range o chemicals in odor space. One approach to achieve sur ace ex pression o ORs in cultured cells utilizes receptor chimeras inwhich an N terminal peptide rom rhodopsin is used to the ORN terminus, acilitating cell sur ace expression and character ization o some (but not all) ORs tested in heterologous cells(Krautwurst et al., 1998). A more e ective and generally appli cable solution was ound by Matsunami and colleagues, who

ound that coexpression o ORs with the ol actory speci cchaperones RTP1 and REEP allows a large number o ORsto be unctionally expressed in heterologous cells (Saitoet al., 2004, 2009; Matsunami et al., 2009). Together, thesevarious strategies have yielded insights into the speci cities o individual ORs, with several principles emerging. For exam ple, while showing a pre erence or compounds with certainmolecular eatures (e.g., naliphatic aldehydes over alcohols),an individual OR can be broadly tuned or other characteris

tics, such as carbon chain length (Zhao et al., 1998; Aranedaet al., 2000; Kajiya et al., 2001; Aba y et al., 2006; Repicky andLuetje, 2009; Saito et al., 2009). These observations provide themolecular underpinnings o a combinatorial code in which theidentity o an individual odorant is encoded by the particularsubset o receptors that it activates (Fig. 3). Variations in theligands structural eatures (e.g., in carbon chain length, or thenature o a unctional group) would elicit activity in a di erentsubset o receptors and give rise to the perception o a di erentsmell (Repicky and Luetje, 2009; Saito et al., 2009). Given thecombinatorial nature o the code and the large number o di

erent ORs ound in a given species, the number o di erentperceived odors is vast.

The ability to characterize the unctional properties o ORs has allowed interesting insights into the mechanisms o human ol action. For example, it has long been known thatsome humans can detect the steroid androstenone (a com pound ound in human sweat), whereas others cannot. Theandrostenone detectors can be urther divided, with onegroup describing the odor as musky and even pleasant and theother nding it distinctly unpleasant, akin to the smell o sweaty socks. What underlies these di erences in human per ception? A solution was ound through an elegant combina tion o human genetics and unctional characterization o speci c OR genes (Keller et al., 2007). Two single nucleotidepolymorphisms (SNPs) associated with androstenone non detectors were identi ed within the coding sequences o anOR gene. Functional expression in heterologous cells showedthat the OR is activated by androstenone, whereas the double SNP variant is not. The detection and perception o andro stenone can be predicted based on this polymorphic OR:individuals homozygous or the wild type allele tend to per ceive androstenone as unpleasant, whereas those possessingone or no unctional alleles perceive androstenone as less un pleasant or undetectable (Keller et al., 2007). These resultshighlight the role that a single OR receptor can play in humanol actory perception.

numbers, sequence diversity, and patterns o expression in OSNs.Although work over the last two decades has unequivocallysupported this view, nding the ligands or these receptors (aprocess o ten re erred to as de orphaning) has been surpris ingly di cult. A major impediment to identi ying ligands orthese chemosensory receptors has been the di culty in obtain ing cell sur ace expression o cloned receptors in heterologouscells (Touhara, 2007). One approachobviating the problems

ound in heterologous cell expressionhas been to study theproperties o virally transduced or even endogenous ORs inOSNs in vivo, thereby allowing them to be tested in their na tive cellular environment (Zhao et al., 1998; Malnic et al.,1999; Touhara et al., 1999) and providing a rich source o in

ormation on OR ligand tuning properties. Indeed, the rst ORto be unctionally characterized was de orphaned by virallytransduced expression in vivo (Zhao et al., 1998). However,such approaches are cumbersome and preclude the ability to

Figure 2. Signal transduction in the OSN.(A) Representation o the re-ceptors, enzymes, and ion channelspresent in the ol actory ciliathattransduce activity o the odorant receptor (OR) into changes in membranepotential and gene expression. Binding o an odorant to its cognate ORresults in the activation o heterotrimeric G protein (Gol plus G ). Ac-tivated G ol in turn activates type III adenylyl cyclase (AC3), leadingto the production o cyclic AMP (cAMP) rom ATP. cAMP gates or opensthe cyclic nucleotide-gated (CNG) ion channel, leading to the infux oNa + and Ca 2+, depolarizing the cell. This initial depolarization is ampli-

ed through the activation o a Ca2+-dependent Cl channel. In addition,cAMP activates protein kinase A (PKA), which can regulate other intra-cellular events, including transcription o cAMP-regulated genes. (B) Events inthe nucleus o OSNs important or establishing and maintaining sensoryneuron identity. Selection o a particular OR gene by the cell is thoughtto occur via interaction o a cis-regulatory locus control region with the

proximal promoter o a single OR gene within a cluster o OR genes. Thischoice is stabilizedand the expression rom all other OR genes in thegenome is silencedby an OR-dependent eedback loop, which ensuresthe expression o a single OR per sensory neuron. The mechanism under-lying OR-mediated, OR gene silencing is at present not understood. OR-mediated activity also leads to transcriptional regulation o cAMP responseelement binding protein (CREB)dependent gene expression via CREBsphosphorylation by PKA.



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in which the OR gene choice had already been made wouldshow a grossly perturbed pattern o OR gene expression be cause all cells o the animal would have inherited the structuralrearrangement that led to expression o that one OR allele. Thisidea was ruled out by the observation that mice cloned rom asingle, mature OSN in act express the ull complement o ORswith no apparent perturbations (Eggan et al., 2004; Li et al.,2004). Thus, the mechanism by which an OSN chooses an ORgene to express does not involve irreversible structural changesin genomic DNA.

How, then, is an OR gene selected or expression? Giventhat OR genes are ound in clusters on multiple chromosomes(Mombaerts, 2004; Niimura and Nei, 2005), perhaps local se quence elements regulate the expression o genes within eachcluster. In one model, a locus control region (LCR) governs ORgene expression as part o a hierarchy o cis acting regulatoryelements. In support o this model, an 2 kb sequence residing75 kb upstream o a cluster o OR genes on mouse chromosome14termed the H region because o its homology to a similarsequence in the human genomewas shown to unction as an

enhancer o OR gene expression in transgenic mice made withyeast arti cial chromosome (YAC) constructs with inserts su ciently large to contain the H region together with the proximalOR gene sequences themselves (Serizawa et al., 2003). The Hregion has the hallmarks o an enhancer: moving it closer to anOR gene increases the number o cells expressing that gene, anddeletion o the H region rom the YAC transgene results inhighly reduced expression o transgenic ORs (Serizawa et al.,2003). Cis acting enhancers have also been identi ed in andaround OR gene clusters in the zebra sh genome (Nishizumiet al., 2007); although these elements show no obvious sequencesimilarity to the mammalian H region, their presence supportsthe notion that each OR gene cluster is regulated by one or more

LCR. Interestingly, only a single OR is expressed per cell roman H regioncontaining YAC transgene carrying a cluster o ORgenes (Serizawa et al., 2003). Similar to the mechanism used toassure the expression o a single red or green opsin gene in thecone photoreceptor cell (Cook and Desplan, 2001), LCRs asso ciated with OR clusters could account or the initial selection o a single OR gene by the OSN via a stable intrachromosomal inter action between the H region and the chosen ORs proximalpromoter (Serizawa et al., 2003; Fig. 2 B). In an intriguing vari ation o this model, it was suggested that the H region might

unction as a master regulator o all OR genes in the genome,serving as both a cis and trans chromosomal regulatory element(Lomvardas et al., 2006). Indeed, experiments using chromo some con ormation capture demonstrated physical associationsbetween the H region and OR genes on other chromosomes(Lomvardas et al., 2006). This was a tantalizing observation, asan exclusive association o a single OR genes proximal pro moter with one alleles H region could help explain singularityo OR expression. However, subsequent studies showed thatgenetic ablation o the H region resulted in the loss o expres sion o only the most proximal OR genes in the neighboringOR gene cluster on chromosome 14, with normal expression o more distant genes in the cluster, as well as genes on other chromo somes (Fuss et al., 2007; Nishizumi et al., 2007). Thus, it is

Odorant receptor gene expression:

the problem o choosing one and orsaking

all others

During development, each di erentiating OSN must select andexpress a single receptor at the exclusion o all other receptorsin the genome. How is this singularity in OR gene expressioninitially established and subsequently maintained over the li e time o the cell? Several models have been advanced to explainhow a single OR gene is expressed in each OSN. One idea pos its somatic DNA rearrangements as a way o irreversibly select ing a single OR gene or expression while simultaneouslyrepressing other genes in the repertoire. For example, DNA re combination events may be required to place the coding se quence adjacent to a promoter sequence or into a transcriptionallyactive locus, similar to the selection o a single antigen receptorgene via V(D)J recombination by cells in the immune system orantigenic variation o sur ace coat proteins in trypanosomes(Borst, 2002). I this were the case, mice cloned rom an OSN

Figure 3. Combinatorial coding of olfactory information.Graphic repre-sentation o the ol actory receptor combinatorial code. In this hypotheticalexample, the responses o ve odorant receptors to seven odorants (ag)are shown, with the magnitudes o responses proportional to the sizes othe circles. Refecting unctional studies on individual odorant receptors,some receptors are more narrowly tuned than others, and individual odor-ants can activate di erent subsets (and numbers) o receptors. The patterno receptor activation elicited by a particular compound is thought to rep-resent that compounds chemical identity.



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in lieu o an intact OR ails to suppress the expression o otherOR genes (Imai et al., 2006). These observations leave us witha bit o a puzzle: the OR is required or silencing, but this pro cess does not appear to operate through the expected G proteinsignaling pathway. Perhaps other, noncanonical, G proteinindependent mechanisms are at play. It is also possible that theinactivated mutant receptor used in the above mentioned study(Imai et al., 2006) retains a level o intrinsic activityrefectingthe receptors equilibrium between inactive and active states inthe absence o bound agonist (Rosenbaum et al., 2009)to re press expression rom other OR gene loci. Accordingly, OR mediated gene silencing may well depend on intrinsic ORactivity, which could be transduced not through G ol /cAMP mediated signaling, but via G signaling, the other arm o the

likely that the H regionand similarsequences ound within other OR geneclustersact in cis to regulate the expressiono OR genes in their immediate chromo somal vicinity. In a hierarchical mechanism,the selection o a single OR by a cell couldcome about through the initial restrictionto a single LCR, which in turn stochasticallymakes a stable interaction with a single ORgene within its associated cluster.

The odorant receptor:

en orcer o the one

receptor, one neuron rule

Once an OR has been chosen by an OSN, itis stably expressed or the li etime o thecell, at the exclusion o all other OR genesin the genome. What is the mechanism be hind this exquisite mode o gene regula tion? An important clue came rom the

observation that individual sensory neu rons can in rare instances sequentially ex press multiple OR genes, with such geneswitching events occurring more re quently when the initial OR gene expressedby the cell is a pseudogene (Serizawa et al.,2003; Lewcock and Reed, 2004; Shykindet al., 2004). These observations support amodel in which a unctional OR protein,once selected and expressed, silences theexpression o other OR genes in the ge nome (Serizawa et al., 2004; Shykind,2005). In a negative eedback mechanism,

OR dependent gene silencing prevents geneswitching and ensures the stable expressiono a single OR in each OSN (Fig. 2 B). Thereare two obvious consequences to this ar rangement. First, i an OR pseudogene isinitially selected or expression, the gener ation o a unctional OSN is ensured byswitching to another OR genean impor tant quality control/quality assurance mecha nism considering that a signi cant ractiono the OR gene repertoire comprises pseudogenes (2530% inrodents and 50% in humans; Mombaerts, 2004; Niimura andNei, 2005). Second, once a unctional OR is selected or expres sion, gene silencing sa eguards the unctional identity o theOSN over the li etime o the cell.

What are the intracellular signaling mechanisms mediat ing OR dependent gene silencing? Several lines o evidencesuggest that this eedback mechanism does not depend on ORactivity (Imai et al., 2006). First, expression o multiple ORs percell is repressed by transgenic expression o a mutant OR inwhich a conserved receptor activation moti is mutated, render ing the receptor unresponsive to odorant stimulation (Imai et al.,2006). Second, a constitutively active G s mutant (which is ex pected to bypass or substitute or an active receptor) expressed

Glossary

Axon growth cone.The leading, motile structure o a neurons growing axon; the growth conesenses gradients o axon guidance cues via transmembrane receptors expressed on the plasmamembrane. These interactions can cause local changes in actin polymerization and depolymer-ization, resulting in directional changes in the growth cone trajectory.

Axon guidance cue.A secreted, cell sur ace, or extracellular matrix protein that infuences thegrowth o extending axons toward (attractive) or away rom (repulsive) the source o the cue.These cues, which include proteins such as the semaphorins, netrins, ephrins, and slits, unctionby interacting with their cognate receptors on the axon growth cone.

Glomerulus.Functional unit o the ol actory bulb that segregates and organizes the synaptic in-puts rom the OSNs with their partner output neurons (the mitral cells), which in turn carry in or-mation rom the ol actory bulb to higher cortical centers.

GPCR.G proteincoupled receptor; representing a large super amily o receptors with a character-istic seven-transmembrane topology and whose intracellular signals are transduced by hetero-trimeric G proteins; includes receptors or hormones, neurotransmitters, visual stimuli, andchemosensory (ol action and taste) stimuli.

LCR.Locus control region; a class o transcriptional regulatory element that can infuence thetranscription o genes within a large stretch o genomic DNA. Regulation o transcription byLCRs is thought to involve a physical interaction o the LCR and the target genes proximal pro-moter sequencea regulatory DNA sequence ound immediately adjacent to the protein-codingportion o a gene.

Odor. Scent or smell; natural sources o odors are o ten complex mixtures o many compounds,

some o which (but not necessarily all) can contribute to the perception o the particular smell.Odorant. A compound that elicits the perception o smell.

Odorant receptors. Receptors expressed by OSNs and belonging to the GPCR super amily, re-sponsible primarily or receiving chemosensory or chemical cues rom the environment. Thereare multiple amilies o odorant receptors, which include the OR (the largest amily), TAAR, V1R,V2R, and ormyl peptide-like receptors.

Olfaction.The sense o smell.

Olfactory bulb.A specialized structure o the orebrain, which receives direct input rom theOSNs in the nose.

Olfactory cortex.Collectively re ers to the ve brain regions that receive direct input rom theol actory bulb: the piri orm cortex, anterior ol actory nucleus, ol actory tubercle, entorhinal cortex,and amygdala. The regions o the ol actory cortex are responsible or the perception o smelland or generating odor-evoked behaviors.

Olfactory epithelium.The sensory epithelium that resides in the nose and contains the primarysensory neurons (the OSNs) that are responsible or detecting chemical stimuli romthe environment.

Olfactory sensory neuron.The primary sensory neuron o the ol actory system, responsible orreceiving and transducing chemical in ormation rom the environment. The ol actory sensoryneuron is where the ol actory system meets the outside chemical world.

Projection neuron.The mitral or tu ted cell in the case o the ol actory bulb; these neurons receivein ormation rom the OSNs in the ol actory epithelium, and relay or project this in ormation tothe next level in the ol actory cortex.



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Loss o unction mutations either in Robo2 (the receptor) orSlit1 (the ligand) result in ectopic projections o OSN axonsoriginating rom the dorsomedial epithelium into the ventralol actory bulb (Nguyen Ba Charvet et al., 2008). In addition, thechemorepellent Semaphorin3F is released by the axon termi nals o dorsally projecting OSNs (which are the rst to inner vate the bulb) and prevents later born ventrally derived axons

rom entering the dorsal ol actory bulb via its receptor, Neuro pilin2, which is expressed in a high ventral to low dorsal gradi ent by OSNs in the ol actory epithelium (Takeuchi et al., 2010).Thus, complementary chemorepellent gradients o Slit1 andSemaphorin3F infuence positioning o OSN projections alongthe dorsalventral axis (Fig. 4 A).

The projection o OSNs expressing a particular OR to thelateral versus medial ol actory bulb creates a mirror symmetryo innervation in this structure (Ressler et al., 1994; Vassar et al.,1994; Mombaerts et al., 1996; Nagao et al., 2000). What infuences

heterotrimeric G protein signaling cascade. Future studies willhope ully soon identi y the intracellular signaling pathwaysresponsible or OR dependent gene silencing, which is critical

or maintaining the stable expression o a single OR gene andthere ore the unctional identity o the OSN.

Wiring up the ol actory sensory map

Patterning o connections by odorant receptor

independent mechanisms. In other sensory systems, neu ronal activityhere de ned as activity leading to changes inmembrane potential o the sensory neuronplays a role inthe re nement o synaptic connections during development andthe modi cation o such connections underlying experience dependent plasticity (Fox and Wong, 2005). What role does suchactivity play in the establishment o the precise targeting o OSNs in the ol actory bulb? Early studies using gene knock outs o the ol actory speci c CNG channelthe ion channelresponsible or transducing OR driven increases in cAMP ac cumulation into membrane depolarization (Fig. 2 A)showedthat targeting and convergence o OSN axons to the appropriate

regions o the ol actory bulb can occur in the absence o stimulus evoked activity (Brunet et al., 1996; Zheng et al., 2000).

The projection o axons rom OSNs expressing speci cORs to spatially invariant glomeruli in the ol actory bulb sug gests that the targeting o sensory axons in the ol actory bulbdepends at least in part on spatially restricted guidance cues inthe target tissue and along the axonal trajectory. How is this eato connectivity achieved during development? In one model, ahierarchy o cues guides axons o neurons expressing the sameodorant receptor rst to the general vicinity o their targets, andthen to their singular target glomerulus (Lin and Ngai, 1999;St John et al., 2002). Regarding the initial steps o axon path

nding, it is instructive to consider this problem in terms o the

ol actory bulbs dorsalventral, mediallateral, and anteriorposterior axes, given that the spatial identity o each glomeruluson the sur ace o this three dimensional structure can be de nedby its coordinates along these three principal axes. As we willdiscuss in the remaining sections, targeting o OSN axons topositions along the dorsalventral and mediallateral axes oc curs primarily via mechanisms independent o OR mediatedactivity, whereas projection along the anteriorposterior axisand axon convergence both depend on OR activity.

Neurons expressing a speci c odorant receptor are segre gated within circumscribed zones in the epithelium along thedorsomedialventrolateral axis (corresponding more or less tothe dorsalventral axis; Ressler et al., 1993; Vassar et al., 1993;Miyamichi et al., 2005). While axons arising rom each zoneproject to a corresponding dorsalventral zone in the ol actorybulb (Mori et al., 1999; Miyamichi et al., 2005) within theselatter zones odorant receptor speci c axons converge to ormdiscrete glomeruli. How is this initial level o dorsalventralsegregation established? The repulsive axon guidance cue, Slit1,and its receptor, Robo2, play a role in segregating OSN axonsalong the dorsalventral axis o the ol actory bulb. Slit1 is ex pressed in the ventral ol actory bulb, and its receptor Robo2 isexpressed in OSNs in a high dorsomedial to low ventrolateralgradient across the ol actory epithelium (Cho et al., 2007).

Figure 4. Targeting of OSN axons to the olfactory bulb.The projection oOSNs in the ol actory epithelium (OE) to their target glomeruli in the ol actory bulb (OB) can be considered along the bulbs three principal axes.(A) Zone-to-zone projection along the dorsalventral axis is shaped in part bycomplementary gradients o the chemorepellent molecules Slit1 and Sema-phorin3F (Sema3F) and their receptors Robo2 and Neuropilin2 (Nrp2),respectively. (B) Innervation o the lateral ol actory bulb is dependent onIGF signaling, which may unction to counteract a de ault tendency o alol actory neurons to project medially. (C) Projection o ol actory sensoraxons along the ol actory bulbs anteriorposterior axis depends not on theposition o the cell in the OE, but rather on the level o intracellular cAMPwhich in turn regulates the expression o the axon guidance receptorNeuropilin1 (Nrp1). By modulating the expression levels o axon guidancereceptors such as Nrp1, the sensory axons are either more or less sensitiveto guidance cues ound in the OB or along the projection pathway.



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o the receptor (i.e., activity in an unliganded state) could set thegain level or sensitivity o the cell to respond to what we mightconsider more traditional axon guidance cues. Indeed, cAMP(as well as cGMP) can modulate the growth cones responseto axon guidance cues (Song and Poo, 2001). In this scenario,OSNs project to di erent positions along the anteriorposterioraxis o the bulb according to the particular ORs level o intrin sic activity (or in the case o the 2 adrenergic receptor swap,the 2 adrenergic receptors intrinsic activity).

Support or this latter model has come rom experimentsin the mouse showing that perturbations in cAMP signalingcause anteriorposterior shi ts in OSN axon targeting (Imai et al.,2006). A decrease in cAMP mediated signaling caused by trans genic expression o either an inactive mutant OR or a dominantnegative protein kinase A (PKA) mutant results in an anteriorshi t in the target glomerulus (Imai et al., 2006). Conversely,transgenic expression o constitutively active G s or constitu tively active PKA causes a posterior shi t in innervation (Imai et al.,2006). Intriguingly, transcription o speci c genes in OSNscorrelates with the level o cAMP signaling (Imai et al., 2006).

Notable among these is the gene encoding Neuropilin1, a receptoror the repulsive axon guidance cue Semaphorin3A (Imai et al.,

2006); OSNs expressing high levels o Neuropilin1 project tothe posterior ol actory bulb, whereas OSNs expressing low lev els project anteriorly. Thus, a model emerges in which the positiono a given OSNs target glomerulus along the ol actory bulbsanteriorposterior axis is in part determined by the OSNs sen sitivity to axon guidance cues such as Semaphorin3A (Fig. 4 C).This sensitivity in turn is determined by the level o intrinsic ac tivity mani ested by the particular OR expressed by the OSN,which determinesvia cAMP/PKA signalingthe level o ex pression o the Semaphorin3A receptor, Neuropilin1. In thisway, OSN axons are sorted along the anteriorposterior axis o

the ol actory bulb not based on their originating positions in theol actory epithelium (as we discussed or dorsalventral andmediallateral positioning), but rather based on the intrinsic ac tivity o the OR expressed by each neuron. Interestingly, axonsdestined or the anterior versus posterior ol actory bulb are pre sorted in the ol actory nerve be ore they enter the ol actorybulb, and Semaphorin3ANeuropilin1 signaling is involved inthis presorting process (Imai et al., 2009).

The nal step: convergence o like ol actory

axons is infuenced by OR-mediated

neuronal activity

How do OSNs expressing the same OR that are distributedbroadly in the ol actory epithelium converge upon a commonglomerulus in each bulb hemisphere? OSN axons can convergeto ectopic sites in mice missing their ol actory bulbs due toeither surgical or genetic manipulations (Bul one et al., 1998;St John et al., 2003; Chehrehasa et al., 2006; Ardiles et al.,2007), suggesting that convergence refects a process intrinsicto the OSNs, independent o their cellular targets in the ol ac tory bulb. Insight into the mechanism underlying convergencewas provided by a study showing that the homophilic cell adhe sion molecules, Kirrel2 and Kirrel3, as well as the repulsivemolecules EphrinA5 and EphA5 (a ligandreceptor pair), are

the choice o an extending OSN axon to project laterally versusmedially? Neurons located in medial or lateral positions o theol actory epithelium innervate the medial or lateral ol actorybulb, respectively (Levai et al., 2003). Gene knockout studies inthe mouse demonstrated that insulin like growth actor (IGF)signaling plays an important role in the choice o OSNs to inner vate the lateral versus medial ol actory bulb (Scolnick et al.,2008). Mice homozygous or a knockout o the IGF receptor,IGF1R, demonstrate a loss o innervation o the lateral ol actorybulb; axons normally destined or this bulb hemisphere reroutedto more medial locations, whereas the positions o medial glo meruli appear unperturbed (Scolnick et al., 2008). IGF can serveas a chemoattractant or OSN axon growth cones in culture, andthis activity is dependent on PI3 kinase (Scolnick et al., 2008),a downstream target o IGF1R and a mediator o the growthcones response to multiple axon guidance cues (Song and Poo,2001). However, the absence o any clear cut mediallateralgradients o IGF1 and IGF2 (the two IGF ligands) either alongthe ol actory axon trajectory or in the ol actory bulb makes itdi cult to explain how IGF signaling promotes innervation o

the lateral ol actory bulb. One possibility is that IGF bindingproteins (E stratiadis, 1998) bind to and mask the IGFs alongthe ol actory projection, thereby altering their spatial distribu tion o available IGF ligands. Another possibility is that growthtoward the medial ol actory bulb is the de ault or all ol actoryaxons, regardless o their point o origin in the ol actory epithe lium. Axons originating rom the lateral ol actory epitheliumwould there ore require a counterbalancing lateral attraction inorder to extend into the lateral hemisphere o the ol actory bulb;IGF ligands could serve as this attractive cue (Fig. 4 B). What ever the case, it appears that IGF participates in concert withother guidance cues to direct OSN axons to the medial or lateralol actory bulb.

Patterning o connections by odorant receptor

dependent mechanisms. In contrast to the mechanismsunderlying the patterning o OSN axon projections along thedorsalventral and mediallateral axes in the ol actory bulb, tar geting along the anteriorposterior axis is infuenced by the ORitsel . This was rst demonstrated through gene knock in ex periments in which the protein coding region o one OR genewas replaced with that o another OR. In these receptor swapexperiments, neurons expressing the modi ed allele projectedtheir axons to an ectopic glomerulus close tobut distinct romthe target glomerulus o the substituted receptor (Mombaertset al., 1996; Wang et al., 1998), indicating that the OR is justone determinant o the nal position o the glomerulus. Twomodels can be entertained to explain these observations.In the rst, the OR receives and transduces axon guidance cuesat the growth cone, thereby serving a dual unction as chemo sensory receptor and axon guidance receptor. Consistentwith this notion, OR protein has been observed in OSN axonterminals (Barnea et al., 2004), although their role as axon guid ance receptors has yet to be demonstrated. This model alsoseems implausible in light o the demonstration that a receptorswap with the 2 adrenergic receptor coding sequence in placeo the OR results in targeting o axons to glomeruli in the ol ac tory bulb (Feinstein et al., 2004). Alternatively, intrinsic activity



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dispersed locations in the sensory epithelium to discrete and in variant glomeruli in the ol actory bulb represents a major eat o axon guidance and synaptic speci city. Future research in thisdomain should help to unravel the intertwined roles o classicalaxon guidance cues and their receptors (not to mention novelplayers that may well be involved), neuronal activity, and theORs themselves in establishing and maintaining the ol actorysensory map in the ol actory bulb.

This work was funded by grants from the National Institute on Deafness aOther Communication Disorders (DC002253 and DC007325).

Other reviews in this series are: The cell biology of hearing (Schwandet al. 2010. J. Cell Biol . doi:10.1083/jcb.201001138), The cell biologyof taste (Chaudhari and Roper. 2010. J. Cell Biol . doi: 10.1083/jcb.201003144), The cell biology of vision (Sung and Chuang. 2010. J. Cell Biol .doi: 10.1083/jcb.201006020), and The cell biology of touch (Lumpkin et al.2010. J. Cell Biol . doi: 10.1083/jcb.201006074).

Submitted: 27 August 2010 Accepted: 11 October 2010

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expressed in complementary patterns by OSNs, such that cellsexpressing high levels o Kirrel2 express low levels o Kirrel3,and vice versa; a similar pattern is observed or EphrinA5 andEphA5 (Serizawa et al., 2006). Moreover, expression o each o these (and other) molecules at high, low, or intermediate levelscorrelates with the particular OR they express (Serizawa et al.,2006; Kaneko Goto et al., 2008). Additional investigationsusing the CNG channel knockout mouse urther demonstratedthat expression levels o Kirrel2Kirrel3 and EphrinA5EphA5are dependent on neuronal activity, and CNG channel gain o

unction in OSNs results in the ormation o ectopic glomeruliadjacent to the original targets (Serizawa et al., 2006). Together,these results suggest a mechanism in which homophilic Kirrel2Kirrel2 or Kirrel3Kirrel3 interactions play a role in promotingconvergence o like axons, whereas repulsive EphrinA5EphA5signaling unctions to segregate dissimilar axons. It seems un likely, however, that interactions o a small number o cell adhe sion or cell repulsion molecules can alone account or thesorting and segregation o >1,000 OR speci c axon types.Rather, such interactions may represent the nal step in a hier

archy o mechanisms used to sort OSN axons as they innervatetheir target glomeruli. It is worth noting that or some OR expressing neurons, the initial projection is made to a smallnumber o adjacent glomeruli and is re ned postnatally to a sin gle glomerular target; this re nement depends on CNG channeldependent neuronal activity (Zou et al., 2004). Postnatalre nement o glomerular innervation may refect the nal stepsin a developmental process shaped by OR mediated experi ence dependent plasticity; it will be interesting to determinewhether this process involves the activity dependent expressiono cell sur ace molecules such as Kirrel2/3 and Ephrin/Eph.

Conclusions

In addition to providing the basis or the recognition o diversechemical stimuli in the ol actory sensory world, the OR playscritical roles in sa eguarding the stability o odorant receptorgene choice and the wiring o OSN axons in the ol actory bulb.These latter duties o the OR ensure that the signals it initiatesupon odorant activation are transmitted in a stable and coherent

ashion to the next relay stations in the ol actory sensory path way. A number o exciting challenges remain or the uture.For example, deciphering and understanding the molecularprinciples underlying the odor codei.e., how an animalscomplete repertoire o ORs is used to detect the vast array o chemicals in odor spaceis now possible with the latest ad vances in high throughput cell based screening and computa tional modeling o the OR proteins. Future research should alsoelucidate the molecular details o how the OR exerts its infu ence on the developmental decisions underlying OR gene regu lation and OSN axon targeting and convergence. In terms o theregulation o OR gene expression, although we have some in triguing glimpses into the complexities behind the one receptor,one neuron rule, mechanistically this mode o gene expressionremains a mystery. It will be ascinating to discover the geneticand epigenetic mechanisms that act to select a particular ORgene or expression and subsequently silence expression romall other OR gene loci. Finally, the targeting o OSNs rom

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4.- the cell biology of smell

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