How the Olfactory Bulb Got Its Glomeruli’ May Be More Complex, and Rough Organization Is Specified Genetically More Revealing, Than Has Been Supposed

PerSPeCTIveS

cess) effectively determines the identity of the opinion sensory neuron, dictating what odours will activate it and where its axons will project. How the olfactory bulb got its These data gave rise to an ancillary principle of olfactory wiring for which there is glomeruli: a just so story? less direct support, but which has nonetheless become generally accepted. This principle states that there is a stereotypical, Dong-Jing Zou, Alexander Chesler and Stuart Firestein animal-invariant, topographical map of the Abstract | The nearly 2,000 glomeruli that cover the surface of the olfactory bulb glomeruli. Each glomerulus has a position and identity that is endowed by the odor- are so distinctive that they were noted specifically in the earliest of Cajal’s ant receptor expressed by the innervating catalogues. They have variously been considered a functional unit, an organizational neurons. A corollary of this principle is unit and a crucial component of the olfactory coding circuit. Despite their central that the cues for determining the location position in olfactory processing, the development of the glomeruli has only recently of a glomerulus and signals that enable the begun to be investigated with new and powerful genetic tools. Some unexpected axon to locate a specific glomerulus are almost entirely genetically determined — findings have been made that may lead to a new understanding of the processes largely by the choice of receptor gene. This involved in wiring sensory regions of the brain. It may no longer be sufficient to is a significant departure from the strategy simply invoke genes, spikes and their interplay in the construction of brain circuits. used by other sensory systems, in which a The story of ‘how the olfactory bulb got its glomeruli’ may be more complex, and rough organization is specified genetically more revealing, than has been supposed. during early development and then refined postnatally by experience16–18. Although the organization of the glomer- The mammalian olfactory nerve is composed The axons of cells expressing one of the more uli suggests that they have a crucial early of the axons of the primary olfactory sensory than 1,000 odorant receptor genes were seen functional role in the transformation of a neurons (OSNs), the cell bodies of which to travel from distant portions of the olfactory chemical stimulus into an olfactory percept, are located in the olfactory epithelium. The epithelium and converge with astonishing new findings from several laboratories have first synapse made by these axons occurs in precision in one or two of the 1,800 olfactory cast some doubt on this tidy scheme and a glomerulus, a specialized structure in the glomeruli (FIG. 1). In adult mice, the axons alternative views are emerging. These new olfactory bulb. Each glomerulus is devoid of all cells expressing the same receptor, data require us to reconsider our ideas about of cell somas and consists of incoming OSN and only those axons, converge into a single both olfactory bulb development and the axons, the dendrites of both second-order glomerulus7. Furthermore, when the entire ways in which information is passed from the projection neurons and local interneurons, receptor was replaced or parts of its sequence periphery to the first central synapse. It now and the processes of astrocytes. The glomer- were changed, the axons were redirected to seems that the glomerular layout can vary, uli are among the structures of the nervous other glomeruli6,8–11. Thus, the receptor itself even between genetically identical individu- system containing the highest density of syn- was identified as a crucial component of the als, and that neural activity may have a sig- apses. For many years glomeruli have been axon-guidance process. The importance of nificant role in determining the position and referred to and characterized as functional this finding has yet to be fully appreciated — composition of a glomerulus. Perhaps more units of olfactory processing1–3. it is one of the first instances in which G pro- importantly, this activity is not necessarily The olfactory system was the first neural tein-coupled receptors (GPCRs) have been electrical, differing from the classical view system in which the wiring diagram was implicated in the process of axon guidance. that axons ‘fire together and wire together’. In determined by molecular, rather than physi- One attribute of this relationship between this Perspective article, we outline the recent ological, techniques. These studies revealed receptor expression and axon target is espe- progress in understanding the development the remarkable organization of the glomeru- cially important. There are over 1,000 odor- of the mammalian glomeruli, with a particular layer of the mouse olfactory bulb. Early ant receptor genes in the mouse genome; lar focus on the roles of signalling. We pro- in situ hybridization studies indicated that however, it seems that an individual sensory pose that glomeruli provide a developmental each glomerulus contained a population of neuron chooses only one of these genes solution to the problem of organizing 1,000 axons from OSNs expressing the same odor- (indeed only one allele of one gene) for trans- populations of axons, rather than acting ant receptor4,5, and later pioneering gene lation into protein12,13 (however, this conclu- mainly as functional units. Furthermore, we targeting studies visualized the glomeruli sion has been questioned by some; see REFS suggest that activity of the odorant receptor- with unprecedented resolution and further 14,15). It has been proposed that the choice mediated signalling cascade has a crucial role substantiated this cardinal wiring principle6. of odorant receptor (a still mysterious pro- in this organizational process.

Background Cl– channels. unlike many neurons, OSNs single glomerulus. mitral and tufted cells also The molecular era in research of the olfac- maintain a high intracellular Cl– concentra- have lateral dendrites that extend great dis- tory system began with the publication in tion, and opening these channels produces tances around the olfactory bulb and synapse 1991 of the landmark discovery by Buck and a Cl– efflux, further depolarizing the cell extensively with a population of interneurons Axel of a large family of GPCRs expressed membrane and generating action potentials known as granule cells (FIG. 3). The axons of by mammalian OSNs19. This finding allowed that are propagated down the axon to the mitral cells, fasciculating as the lateral olfac- the use of molecular and genetic tools to synapses in the glomerulus20 (FIG. 2). tory tract, project to the piriform cortex, the decipher how we detect and discriminate presumed site of higher olfactory processing. between an enormous number and variety Glomerular circuitry. The glomeruli, Tufted cells seem to project both within the of chemical compounds. After more than a located on the surface of the olfactory bulb olfactory bulb and to the anterior olfactory decade of intense investigation the periph- just under the layer of incoming axons, are nucleus. Their axons have been shown to eral mechanisms used by OSNs to generate bounded by glial cells and by periglomerular connect the glomeruli that are innervated by odour-dependent signals are now generally cells, a diverse population of mostly inhibi- an identical population of sensory cell axons, well understood20. tory interneurons that extend neurites (that either within the same bulb or between the is, both dendrites and axons) into multiple bulbs on each side of a mouse21–23. A common second messenger transduction glomeruli in a local neighbourhood. The In the glomerular neuropil there are cascade. Binding of odorant compounds to glomerulus is also innervated by the apical three basic synaptic circuits. Axodendritic odorant receptors initiates a transduction dendrites of mitral and tufted cells. mitral synapses connect the axons of OSNs with cascade involving a G protein and activa- cells lie mainly in a thin layer ~400–500 the dendrites of mitral or tufted cells and tion of adenylyl cyclase 3 (AC3), a potent μm deep in the olfactory bulb, whereas periglomerular cells. This is a site of tremen- catalyst that produces the second messen- tufted cells lie in the external plexiform dous convergence: up to 25,000 OSN axons ger cyclic AmP (cAmP). cAmP binds to a layer between the mitral cell layer and the form synapses on the apical dendrites of up cyclic nucleotide-gated (CNG) ion chan- glomeruli. These two populations of cells are to 100 mitral and tufted cells (in rabbits24). nel, increasing its open time and allowing both pyramidal-like excitatory projection These OSNs are the sole source of primary the influx of cations, which depolarize the neurons. The apical dendrite of a mitral or sensory information to the glomerulus cell membrane. Ca2+ entering through this tufted cell branches extensively once it enters and, effectively, to the brain. Reciprocal channel can also activate Ca2+-dependent the glomerulus, but it innervates only a dendrodentritic synapses between mitral or tufted cell dendrites and periglomerular cell abdendrites provide a second source of input to periglomerular cells. Finally, inhibitory A synapses between the axons of periglomeru- LL lar cells and mitral or tufted cell dendrites mediate feedback or feedforward inhibition (FIG. 3). Recently, an excitatory synapse from OE OB a periglomerular cell to a mitral cell has been identified25, although its role in processing is as yet unclear. M71L What do the glomeruli do? The glomerulus seems properly positioned cdAmyl acetate Peanut butter to act as a functional unit in which sensory input signals converge and activate circuitry that shapes the signal before transmitting it to higher centres. One might expect the output from the glomerulus to cortical areas to be information rich, given that there is only one more synapse in the cortex at which to transform this message into at least an elementary percept. However, how this Figure 1 | Axonal sorting into glomeruli. a | The projection of axons from olfactory sensory neurons Nature Reviews | Neuroscience information is encoded by the glomeruli (OSNs) expressing a single odorant receptor. The photograph shows a whole-mount preparation of remains controversial and largely obscure. mouse olfactory epithelium (Oe) and olfactory bulb (OB). A population of cells expressing the odorant There are several ways to approach this receptor M71 and the marker lacZ is shown in blue. Axons from cells distributed around the epithelial question. As outlined below, the functional sheet fasciculate and enter the bulb through the cribiform plate (indicated by the arrowhead), where data mostly suggest that odour space — the they crawl over the surface of the OB and converge into a glomerulus (indicated by the arrow) that is collection of molecules capable of eliciting only ~50 μm in diameter. b | A top view of the four glomeruli (indicated by arrows) formed by axons expressing the M71 receptor. c,d | Odour stimulation results in patterns of glomerular activation that an olfactory percept — is topographically reflect the convergence of axons expressing the same odorant receptor. Dark patches show optical mapped onto the glomeruli. However, exam- imaging of intrinsic signals of neuronal activity in response to the odorants amyl acetate (c) and peanut ining glomerular development for clues butter (d) in the olfactory bulb of a rat. A, anterior; L, lateral. Parts a and b are reproduced, with permis- to its function (a strategy suggested, if not sion, from REF. 9  (2004) Cell Press . Parts c and d are reproduced, with permission, from REF. 91 pioneered, by Cajal26) leads to a somewhat  (1999) Cell Press. different conclusion.

Odorant Na+ Ca2+ than a requirement for olfactory informa- compound CNG Cl– tion coding. Odorant receptors with similar Plasma channel channel sequences are clustered in the genome and membrane are often expressed in overlapping patterns OR AC3 in the olfactory epithelium, and the axons Gγ from OSNs that express these receptors typically project to neighbouring glomeruli30. Cytosol Gβ Given the primary role that the odorant Gα Cl– receptor is thought to have in axonal projec- GTP tion, it is hardly surprising that neighbour- ATP Kinases cAMP ing glomeruli are innervated by axons from OSNs expressing similar odorant receptors, Transcription factors as these axons would be likely to respond to the same extracellular guidance cues. Growth cone Gene transcription As highly similar receptors are also likely to bind similar odorants (aldehydes versus Figure 2 | The odorant transduction pathway. Binding of odorant compounds to an odorant recep- ketones, for example), this could lead to tor (Or) initiates a transduction cascade involving a G protein and activationNature ofRe adenylateviews | Neur cyclaseoscienc 3e (AC3), which in turn produces the second messenger cyclic AMP. cAMP binds to a cyclic nucleotide- what looks like a grossly functional topog- gated (CNG) channel and results in the influx of cations (Na+ and Ca2+), which depolarize the cell mem- raphy in the olfactory bulb. However, the brane. Ca2+ can also activate a Ca2+-dependent Cl– channel. Olfactory sensory neurons (OSNs) maintain topography observed at the level of glomer- a high intracellular Cl– concentration, such that this channel supports an efflux of negatively charged uli has not yet been seen at any higher brain Cl–, producing a further depolarization of the cell membrane. Notably, the elevated levels of cAMP in centre, not even at the level of mitral cell the soma have a crucial role in regulating the phosphorylation of proteins and the transcription of somas. Glomerular organization may there- genes important for growth and survival of the axons of OSNs. The receptor79,80 and other essential fore be primarily a developmental solution components of the signalling cascade have been detected in the OSN axons, indicating a possible role to organizing 1,000 populations of axons, for the Or-mediated cAMP cascade in the activity of growth cones. and only secondarily important in olfactory coding. Although these are not mutually Investigations on functional aspects have molecules with very different structures can exclusive roles, the developmental func- given rise to the notion of a topography of give rise to a similar percept, whereas those tion has received much less attention and glomerular activation patterns in which a with similar structures may smell different. deserves to be considered more fully. To single odour or class of odours gives rise to a The nature of the olfactory stimulus, which determine its relative importance we need repeatable pattern of odour-evoked activity is non-topographical and varies along mul- to understand the processes that determine in specific glomeruli located in particular tiple dimensions (including charge, volatil- the projection of OSN axons and their relations to each other. For example, alde- ity, atomic composition, molecular weight coalescence into glomeruli. hyde-containing odours activate subsets of and aromaticity), thus does not require or glomeruli in a particular region of the olfac- particularly lend itself to topographical Hard-wired glomerular organization tory bulb, whereas esters activate another representations in the brain. Indeed, several Early evidence for genetically determined region2,3. using various recording proce- recent physiological studies cast doubt on stereotypical positioning of the glomeruli dures — including intrinsic imaging, live the concept of a functional glomerular map came from the aforementioned gene-targeting Ca2+ imaging, imaging of activity-dependent organized in a simple topographical manner. experiments. Once it was recognized that the fluorophores and measurement of 2-deoxy- One study found that neighbouring glomer- odorant receptor had a crucial role in deter- glucose activity — odour-dependent activity uli were as diverse in their odorant response mining an axon’s destination in the olfactory maps of the glomeruli have been produced2,3 spectra as distant glomeruli. The similarity bulb it seemed likely that there would be a (FIG. 1c,d). As expected, each glomerulus is of any two glomeruli in their odour sensi- strong genetic influence on glomerular target- activated by a subset of odours and most tivity had almost no correlation with their ing. many glomeruli have formed by birth, odours activate a subset of glomeruli in a proximity27. In addition, reports suggest that well before the olfactory epithelium would concentration-dependent manner. However, neighbouring mitral cells show a very diver- come into contact with anything but uterine it is unknown whether the odour-evoked gent sensitivity to odorants, as opposed to the ‘odours’, which suggests that an environ- activity patterns have a function in olfac- highly similar odour response profiles that mental influence is unlikely. Furthermore, tory processing — that is, whether an odour would be expected from an odour-driven many genes encoding odorant receptors are is identified by the particular region of the topographic organization of glomeruli28,29. expressed embryonically, before synaptic olfactory bulb that is activated and whether Taken together, these results cast doubt on connections are made in the glomerulus31. there is an odour-driven topographic the existence of a fine-scale chemotopic However, in the early papers showing the organization to the glomerular layout. glomerular map. convergence of axons expressing the same In the visual and the auditory systems, If the patterns of odour-evoked glomeru- odorant receptor, mombaerts was careful to stimuli are spatially well defined and have lar activation do not represent a functional point out that the receptor may be only part an inherent continuous nature. Topographic map in the sense of a topographical identi- of the story, clearly having an instructional maps corresponding to these peripheral stim- fication of odour quality, what role or roles role in axon guidance but not likely to be uli are well preserved at each level of infor- do glomeruli have in olfaction? One pos- the sole determinant in establishing a map32. mation processing in the brain. By contrast, sibility is that glomerular organization is the Nevertheless, until recently it was generally olfactory stimuli are extremely divergent. result of developmental processes, rather accepted that the organization of the olfactory

system at the first synapse is largely, if not results in a population of cells that send their glomerulus that never occurs under normal entirely, determined genetically, and that it axons to a glomerulus distinct from that circumstances it is unlikely that it was there is stereotypical. Neural activity, in particular, targeted by cells expressing endogenous m71 as a target before the new axons arrived. was thought to be of little importance. or mOR2310. As this is a completely novel Further support for the lack of a pre-existing Several experiments supported this view. All OSNs have the same second messen- ac ger cascade leading to neuronal activation Glomerulus ONL (see earlier). Therefore, knocking out any component of the cascade would silence the neuron while allowing receptor expression GL to occur normally. Transgenic mice lacking the G protein subunit Golf or the CNG channel subunit CNGA2 were generated: in both EPL cases the resulting wiring appeared mostly normal33–36. The olfactory bulb was smaller and cell numbers were reduced, but all the ONL MCL cells expressing a particular receptor contin- GL ued to send their axons to the same specific EPL GCL glomeruli. This suggested that neural activ- MCL GCL ity was not crucial for axon guidance or targeting. However, supernumerary glomeruli were found for some olfactory receptors in the CNGA2-knockout mice36. Furthermore, bd LOT only one receptor population was examined in the Golf knockouts and this was examined only at very early postnatal stages33. Despite these caveats, popular opinion remained in Dendrodendritic Dendrodendritic favour of activity-independent mechanisms. This hypothesis left the lingering question of how the receptor guides an axon to the correct glomerulus. Are there chemical cues that determine position on the surface of the olfactory bulb? Do cues from mitral Axodendritic Axodendritic and tufted cells or periglomerular cells in Figure 3 | The olfactory glomerulus. a | The anatomy of the olfactory bulb, shown in cross section. The axons of olfactory sensory neurons (OSNs) cover the outermost layer of the bulb, forming the the glomeruli attract the correct axons? In olfactory nerve layer (ONL). These axons then enter the glomeruli, whereNa turtheye Re branchviews |and Neur formoscienc syne- several lines of transgenic animals, dele- apses with second-order projection neurons in the glomerular layer (GL; shown in blue). The olfactory tions of transcription factors resulted in bulb has a laminar structure with interneuron cell bodies around and beneath the glomeruli, and a thin the loss of either mitral or tufted cells or band of mitral cell bodies (that is, the mitral cell layer (MCL); shown in red). In the centre of the olfactory periglomerular cells. Although the layered bulb lies the granule cell layer (GCL), which contains migrating immature interneurons (shown in structure of the olfactory bulbs was dis- green). Between the glomerular layer and the mitral cell layer lies the external plexiform layer (ePL), rupted, the axons of neurons expressing where extensive interaction occurs between the apical dendrites of granule cells and the lateral den- the same receptor still coalesced37. An even drites of mitral cells. b | A confocal image of a glomerulus from a mouse in which the gene encoding more striking finding suggests that olfactory the rat I7 receptor was targeted into the mouse M71 locus. The image indicates that the expression of axons can sort and form aggregates in the an ectopic olfactory receptor is sufficient to instruct the formation of functional neural circuitry. OSN axons are shown in orange and mitral and tufted cells are shown in green. Arrows point to the apical absence of the olfactory bulb entirely38. dendrites of mitral or tufted cells. c | The synaptic architecture of a single glomerulus. The axons of the These experiments raise more fundamen- OSN (shown in dark blue) enter the glomerulus. The glomeruli are bounded by glial cells (not shown) tal questions. Are glomeruli present in the and by periglomerular cells (shown in light blue), which extend neurites into multiple glomeruli within olfactory bulb before axonal innervation or a local neighbourhood. Also extending into the glomerulus are the apical dendrites of the mitral cells do they emerge as the result of innervation? (shown in red; for simplicity tufted cells are not shown). each mitral cell innervates only a single That is, are there targets for axons to grow glomerulus with its apical dendrites and also extends lateral dendrites around the oflactory bulb that to, or does the coalescence of the axons form synapse extensively with a population of interneurons known as granule cells (shown in green). Finally, the glomeruli? How do odorant receptors the axons of mitral cells, fasciculating as the lateral olfactory tract (LOT), project to the piriform cortex, instruct axons to fasciculate with others of which is the presumed site of higher olfactory processing. d | In the glomerular neuropil there are three the same identity and arrive at a glomerular basic synaptic circuits: axodendritic synapses that connect the axons of OSNs to the dendrites of mitral cells and periglomerular cells; reciprocal dendrodentritic synapses between the dendrites position? Are glomeruli fully formed during of mitral cells and the dendrites of periglomerular cells, providing a second source of input to peri- development? Are their positions invariant glomerular cells; and inhibitory axodendritic synapses connecting axons of periglomerular cells to the and stereotypical? Swapping receptors by, dendrites of mitral cells, which mediate either feedback or feedforward inhibition. In addition, in the for example, targeting the gene encoding external plexiform layer, reciprocal dendrodendritic synapses are observed between mitral cell den- the mOR23 (also known as OlFR16) recep- drites and granule cell dendrites. The extensive interactions of these synapses are thought to be cru- tor into the locus of the gene encoding the cial for controlling the output of mitral cells. Part a is courtesy of Matt valley. Part b is reproduced, with m71 receptor (also known as OlFR151) permission, from REF. 21  (2002) Macmillan Publishers Ltd. All rights reserved.

glomerulus target comes from another not homogenously innervated45,46, even in sufficient to alter developmental outcomes, crucial experiment. Replacing the odor- animals more than 1 year old46. The axons regardless of electrical activity. This may ant receptor m71 with the β2-adrenergic of neurons expressing different receptors explain how the importance of neural activ- receptor (β2AR), a GPCR that is otherwise become commingled in the absence of AC3 ity in glomerular formation was missed in unrelated to odorant receptors, results in (REF. 47). Therefore, the absence of AC3 the early experiments that focused on axons forming a new ‘β-adrenergic’ glomer- severely disrupted the normal development electrical activity. ulus10. Clearly there could not have been a and maturation of the glomerular population. It is now clear that, although the odor- β-adrenergic glomerulus waiting for these ant receptors have a crucial instructive role axons. These results, plus data from several neural activity has a role: a solution? in axon guidance, additional factors down- recent papers that will be described later, In both the AC3-knockout and the CNG- stream of the receptors are likely to be equally have forced the field to rethink these issues. knockout mice the OSNs are electrically important. What these factors are remains silent — the CNG channel either cannot largely unknown. cAmP is a ubiquitous intra- neural activity has a role: a conundrum be activated by odorants or is missing in cellular messenger that has effects ranging One of the earliest clear indications that these cells. The crucial difference between from biochemical regulation to gene expres- there was an activity-dependent contribu- these mice is that cAmP is still generated sion. Furthermore, cAmP may also regulate tion to glomerular formation surfaced unex- in the CNG-knockout mice. Thus it seems the activity of OSN axon growth cones. There pectedly from work undertaken to provide that narrowly defining ‘activity’ as electrical are therefore multiple candidates, and it will further support to the genetic model39. using activity may not be correct. Instead, activity take some time to unravel this story. Several well-established lines of gene-targeted mice, should also include the production of the downstream molecules are likely to have a we investigated glomerular formation in signalling molecule cAmP. role. In particular, cAmP could be crucial for early postnatal life (5–25 days) and observed But what about the Golf-knockout mice regulating the expression of guidance factors that in many cases there were two or more described earlier? As the G protein acts and their receptors such as neuropilin 1 glomeruli where adult animals have only one upstream of AC3 in the signalling cascade, (REFS 45,50). One intriguing idea is that the each in the lateral and medial olfactory bulb. there should be no production of cAmP in odorant receptor chosen for expression some- Furthermore, the glomeruli of animals less these animals, and yet the olfactory bulb how helps determine the expression than ~10 days old often contained mixed seems to develop normally. However, odor- profile of downstream guidance genes. populations of axons. These heterogeneous ant receptors can couple through Gs, an For example, several recent studies found (REF. 48) glomeruli disappeared by 60 days of age isoform of Golf, to activate AC3 . that Ephrins and their Eph receptors, the and this process of glomerular refinement Gs is expressed at high levels by immature Nephrins (also known as Kirrels), and BIG2 seemed to be activity dependent. In animals OSNs during early development, and is (also known as CNTN4 or contactin 4) have 49 51–53 in which one nostril was experimentally later replaced by Golf . Although the reason mosaic expression patterns . These mol- closed, glomerular refinement on the ipsilat- for this switch is obscure the effect is that ecules are expressed only in those sensory eral side of the olfactory bulb was disturbed. knocking out Golf does not prevent cAmP neurons that seem to correlate with particular Similarly, in a line of transgenic mice in signalling activity in early development, dur- receptor types, whereas variable expression which neurons initially innervate the inap- ing the time that axons are coursing to the levels are observed in different glomeruli. propriate region in the olfactory bulb, the olfactory bulb and coalescing into glomeruli. Furthermore, the differential expression levels elimination of these ectopic axonal projec- A series of studies using transgenic and of these molecules can influence the position tions seems to require sensory activity40. retroviral techniques to alter gene expres- and composition of glomeruli in the bulb. As Recently, odour conditioning was shown sion have now shown that levels of G pro- the expression of these molecules is regulated to accelerate the pace of glomerular refine- tein activity and AC3 production of cAmP by sensory input52,53, investigating their pos- ment41. In addition, inhibiting neurotrans- are crucial for the proper development of sible roles in the process of glomerular refine- mitter release and suppressing spontaneous glomeruli into well-defined homogeneous ment, as described previously39,40, would be OSN firing were shown to influence structures47,50. Odorant receptors that can- of interest. It is also possible that the odorant glomerular formation and maintenance42. not couple to the G protein fail to induce receptor gene locus carries information sepa- These findings presented a puzzle, as glomeruli whereas ectopic expression of rate from the receptor coding region and that genetic lesions of the transduction pathway non-olfactory GPCRs, such as the β2AR, some axonal behaviour is controlled by locus should have produced roughly the same phe- that couple to the stimulatory pathway are choice46,54. notype as nostril closure — that is, reduced sufficient to induce glomeruli10,47. Expression We also subscribe to the idea that the activity in the olfactory sensory epithelium. of constitutively activated G protein mutants glomerulus is not a pre-existing struc- However, the axons of OSNs that lack a is also sufficient to induce glomerular for- ture but is induced by the convergence of functional CNG channel, and which are thus mation, demonstrating that activation of the similar axons. Several experiments show electrically inactive, nonetheless coalesce cAmP pathway alone can drive the process that numerous factors can affect the posi- normally into glomeruli35,36. Studies using independent of the odorant receptors47,50. tion of a glomerulus, running counter to the a recently generated transgenic mouse in These results suggest two important prevailing notion that glomerular position is which AC3 was deleted provided a possible changes in our view of development and stereotypic and nearly invariant. Indeed, the solution43. In these mice, the glomerular wiring in the olfactory system. First we note most experimentally sensitive attribute of a layer of the olfactory bulb was seriously dis- that the common concept of neural activity, glomerulus seems to be its position. Slight rupted44. There were much fewer glomeruli typically summarized in the phrase ‘axons variations in the amino acid sequence of an and the shapes and sizes of those remaining that fire together wire together’, may need odorant receptor can result in new glomer- were more variable than normal. On closer to be expanded. In the case of biochemical uli10. Remarkably, transgenic expression of examination the remaining glomeruli were activity of OSNs, the production of cAmP is odorant receptors can also result in significant

55–58 movement of glomerular position . Almost Box 1 | Stages of glomerular formation all of the genes so far implicated in guidance of OSN axons affect the position of glomeruli The formation of the glomeruli requires a series of hierarchical decisions to take place, as illustrated in the figure. On the left-hand side of the figure are schematic illustrations of a mouse head split rather than glomerular composition per se. along the midline, and the right-hand side illustrates events taking place in the olfactory epithelium Glomerular position has been treated as the (OE) or olfactory blub (OB). crucial phenotypic feature in the development of the olfactory bulb. However, it now seems odorant receptor choice Soon after gaining neuronal identity, progenitor cells in the OE choose to express a single odorant to be the most malleable of properties, leading receptor (OR) (see part a of the figure; each colour represents a different OR choice). These cells to the suggestion that timing, axonal segrega- are located near the basal layer and have not yet extended dendrites or axons. The position of tion, coalescence and convergence are the the progenitor in one of several epithelial subdomains restricts the choice to a subpopulation of the pivotal and developmentally more interesting 1,000 or more OR genes. How a single OR gene is chosen remains elusive. NC, nasal cavity. parameters to consider. osN axon targeting If changes in the state of the primary In the OB, olfactory sensory neuron (OSN) axons expressing the same receptor initially grow to a sensory neuron can alter the destination of broad but restricted region and do not yet penetrate deeply into the glomerular layer (see part b of its axon there is no need to posit a predeter- the figure). Convergence of receptor identical axons is not yet apparent and glomerular structures mined target on the surface of the olfactory are not visible85–88. The dashed circle in the figure indicates the area where gomeruli will emerge. bulb. A glomerulus may not exist until its Axons from ventrally positioned OSNs project to the ventral bulb, whereas those from dorsally induction by the coalescence of a group of situated cells project to the dorsal bulb. Interestingly, in the dorsal epithelium OSNs expressing two 54 axons, after which subsequent maturation classes of ORs are intermingled, but their axons project to separate subdomains in the dorsal bulb . processes refine the structure. It has been How these arrangements are achieved remains elusive. Axon guidance molecules are likely to have important roles in OSN axon targeting. The activation of a particular OR locus may also be involved suggested that the role of the receptor is to in determining the position of axonal projections. The transcription machinery associated with the allow axons to recognize one another and OR locus activation may not only ensure which OR is expressed, but also regulate the expression of to promote fasciculation of like axons, form- various guidance molecules. ing ever larger bundles that crawl over the 9 Glomerular formation surface of the bulb . Although it is unlikely During glomerular formation, the accumulation and convergence of axons that have identical that the receptors themselves are both nec- receptors becomes apparent (see part c of the figure). OSN axons fasciculate and penetrate into essary and sufficient for such processes, the glomerular layer, make branches and establish synaptic contacts with bulbar neurons. At this the receptor (or perhaps more precisely stage of glomerular formation, heterogeneous glomeruli innervated by multiple axonal populations the choice to express the receptor) clearly are often seen. endows the neuron’s axon with an identity. The OR itself is crucially involved in glomerular formation, probably through its activation of the When an as-yet-unknown cue, perhaps the cyclic AMP-dependent signalling cascade, its regulation of the differential expression of adhesion number of fibres, a regional localization cue, molecules and perhaps an unidentified mechanism of homotypical interaction. an intracellular event (such as the switch Glomerular maturation In ever-increasing numbers, identical axons coalesce into a single homogeneous glomerulus (see from Gs to Golf) or just time, arrests the axon’s progress, a glomerulus is formed. part d of the figure). The refinement and maintenance of homogeneous glomeruli requires sensory 39,42,89,90 One unaddressed caveat to a model that input that may function by regulating the dynamics of OSN turnover and that may regulate relies solely on olfactory receptor signalling the differential expression of adhesion molecules. — indeed the caveat that made the purely a OB OE Supporting cell genetic model so attractive — is that it is dif- OR choice ficult to imagine how the signalling of 1,000 OSNs OR+, Gs+ receptors through a common pathway could NC create so many unique axonal identities. OE However, the work of many laboratory groups Progenitor cell over several years has identified a series of b guidance and adhesion molecules that are OSN axon targeting thought to function at different levels of the Guidance molecules OR signalling cascade process59–67. It is becoming clear that axon OB guidance and glomerular formation occur through several continuous developmental stages during which the odorant receptor has c Glomerular formation an important, but not absolute, role. OR signalling cascade Adhesion molecules Conclusions and future perspectives Synaptogenesis Determining the position of a glomerulus may be seen as a series of hierarchical decisions (BOX 1). Odorant receptor expression d Glomerular maturation is regionally restricted in the olfactory Sensory input epithelium such that a given receptor gene Synaptic transmission is expressed only by cells in one of a few major regions. That regionality is somewhat carried over into the olfactory bulb, where Nature Reviews | Neuroscience

axons originating from cells in the dorsal This leads to a new set of interest- mechanisms may prevail. A more compre- zone of the epithelium are restricted to the ing questions. What are the functions of hensive view of piriform cortex organization dorsal zone of the bulb, and so forth. Some the odorant receptor and its downstream and processing than is currently available will biochemical differences have been detected signalling molecules at the terminals of be crucial to understanding the nature of the in the zones; for example, olfactory axon OSN axons, where they have recently been signal that is being passed on, and therefore cell-adhesion molecule (OCAm; also known identified79,80? How do these axons act on how much of the glomerular topography is as NCAm2) is expressed in all zones except second-order neurons such as periglomeru- retained. Answers to these difficult questions the dorsal zone68,69, whereas OmACS (acyl- lar and mitral and/or tufted cells to produce may not come easily or quickly, and it may CoA synthetase medium-chain family mem- the circuitry of the glomerulus? How does be that we will need to maintain a culture of ber 4; also known as ACSm4) and NQO1 the identity of the glomerulus emerge and what Keats called ‘negative capability’ — the (NAD(P)H dehydrogenase, quinone 1) are become finalized? In many cases, axons from ability to be in a state of mystery without detected only in the dorsal zone70,71. Thus, OSNs expressing the same odorant receptors irritability — investigating and discussing all chemical cues probably provide a first layer innervate two glomeruli, one on the lateral ideas with equanimity. of regional restriction on where axons may and the other on the medial side of each What the findings reviewed here impress go, independent of neural activity. olfactory bulb. What, if any, is the functional on us is that the function of glomeruli, as with In these large zones the actual position significance of this pattern of innervation? all neural structures, cannot be investigated in of a glomerulus may be variable and may As it is known that odorant receptor genes isolation from their development. The many depend on various factors, even extending to are turned on and off during the lifespan possible roles of glomeruli — not just as func- rearing conditions. One reason that glomer- of an animal31, how are the ‘old’ glomeruli tional units, but also as a developmental solu- ular positions initially seemed invariant may removed, and how are new ones established? tion to managing 1,000 populations of axons be that the experiments were all of necessity If the population of olfactory neurons is — must be considered. It has long been said carried out in highly interbred animals and continually regenerating, how are glomeruli that in the brain ‘structure is function’. One under the strictly controlled rearing condi- maintained? These fundamental questions might extend that to ‘development is structure tions typical of laboratory animal facilities. remain to be satisfactorily answered. is function’. Indeed, even in highly inbred littermates Finally, perhaps the most provocative Dong-Jing Zou, Alexander Chesler and Stuart Firestein one finds significant local permutations question dividing the field is whether there are at the Department of Biological Sciences, Columbia in the relative positions of neighbouring is a glomerular map that acts to encode University, 923 Fairchild Center M.C. 2438 New York, glomeruli72,73. We have yet to visualize the odours at this very first synapse and, if so, NY 10027, USA Correspondence to S.F. olfactory bulb of a wild mouse. 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