Review article e-Neuroforum 2010 · 1:9–16 M. Spehr DOI 10.1007/s13295-010-0002-1 Institute for Biology II/Dept. Chemosensation,Sammelbau © Springer-Verlag 2010 Biologie, 42D/R253, RWTH Aachen, Aachen out social signals Chemical communication and the vomeronasal organ

“Odors have a power of persuasion stronger Not too long ago, it was believed that Subsystem organization of than that of words, appearances, emotions, the mammalian had only the mammalian nose or will. The persuasive power of an odor two anatomical and functional divisions: a cannot be fended off, it enters into us like main and an accessory (vomeronasal) ol- Nearly two decades ago, the discovery of breath into our lungs, it fills us up, imbues factory system. The main olfactory system the odorant receptor (OR) multigene fam- us totally. There is no remedy for it.” – In was thought to predominantly detect gen- ily in rodents by Linda B. Buck and Rich- his novel Perfume: The Story of a Murder- eral environmental odors (to probe, e.g., ard Axel [7] marked the beginning of the er, Patrick Süskind [65] skillfully couches the type and quality of foods), where- molecular era in olfactory research. Since the profound emotional depth that can be as the accessory olfactory system and its that watershed event for understanding ol- evoked by odor . Understand- peripheral sensory structure – the vom- factory function, the past years have seen ing how the mammalian nose detects and eronasal organ (VNO) – was considered an explosion of studies on the neurobiol- simultaneously discriminates thousands to play a critical role in the detection and ogy of the main olfactory system. of different scents, how odor information communication of social chemosignals The main olfactory neuroepithelium is decoded in different areas of the brain, (pheromones) that impact stereotyped lines the dorsocaudal regions of the na- and how perception of a specific odor fre- social and sexual behaviors or hormonal sal septum and the endoturbinates within quently becomes a trigger of long forgot- responses among conspecifics. It has now the nasal cavity of most mammals. Here, ten memories – both pleasant and repul- become clear that the organization of the the ‘classical’/canonical olfactory sensory sive – is one of the most fascinating areas of smell is much more complex, re- (OSNs) express a single type of in modern sensory . There- vealing a diversity of subsystems that was receptor from a massive gene repertoire of fore, the nose has become a busy place for not anticipated even a few years ago [1, OR genes (>1000 functional OR genes in these days, with a multi- 45]. With this newfound appreciation of rodents; ~350 in ). In an individ- tude of recent discoveries now adding to functional diversity, several new and ex- ual OSN, this monogenic (indeed, mono- an emerging conceptual view of the ar- citing questions now rank high on the re- allelic) receptor gene expression is tightly chitecture of the olfactory system that ap- search agenda of (chemo)sensory neuro- regulated by the gene products – the OR pears, at least in part, fundamentally dif- scientists: Which different receptor struc- proteins – themselves, which exert a nega- ferent from previous conceptions. tures have evolved to confer and main- tive feedback effect on OR gene choice [60, The theoretical range of olfactory stimu- tain a sufficient degree of selec- 33]. ORs share various hallmarks of typi- li is virtually infinite. To meet the bewil- tivity for each subsystem? Which signal- cal G protein-coupled receptors (GPCRs), dering complexity of such structurally ing strategies are implemented by the dif- e.g., seven putative membrane-spanning diverse chemical signals, several distinct ferent cell populations? What function- α-helices and the highly conserved DRY populations of sensory cells have evolved al logic underpins the anatomical segre- amino acid motif [43]. A group of hyper- within the mammalian nose. Each sen- gation of the different subsystems in the variable residues within transmembrane sory cell type is identified by a unique re- nasal cavity? How is subsystem-specif- domains 3–6 likely builds the OR ligand ceptor expression profile and characteris- ic (parallel) information segregated, pro- binding pocket. It took seven years from tic central projection patterns. This cellu- cessed, and integrated by higher-order publication of the OR discovery for the lar diversity has given rise to the organi- brain centers? first unambiguous OR-ligand pair to be re- zational concept of olfactory subsystems ported [72]. Though some ORs have since – or noses within noses [45] – each dedi- been matched to cognate ligands, the great cated to a particular role in chemosensa- majority of mammalian OR genes are yet tion (. Fig. 1). to be deorphanized. Numerous laborato-

e-Neuroforum 1 · 2010 |  Review article

role in chemosensory signaling remains to be determined. Both TAARs and ORs are typical rho- dopsin-like class A GPCRs that signal via a G protein-mediated cAMP-dependent transduction pathway [46]. A third group of neurons in the main olfactory epitheli- um, however, is likely to transduce olfac- tory stimuli independent of OR or TAAR expression and cAMP signaling. As a com- mon molecular marker, these neurons ex- press an orphan receptor guanylyl cyclase (GC), denoted as GC-D [15]. About 0.1% of OSNs share expression of GC-D in con- cert with other proteins reminiscent of a cGMP-mediated transduction cascade (e.g., phosphodiesterase PDE2 and the CNG channel subunit A3 [23, 41]. Anoth- er distinctive feature of GC-D-expressing OSNs is their clustered distribution with- in rather dorsal areas of the main olfacto- Fig. 1 8 Schematic drawing of the subsystem organization of the rodent ol- factory system. Peripheral sensory structures consist of at least four anatom- ry epithelium. What, if any, chemosenso- ically separated tissues: the main olfactory epithelium (MOE), the septal or- ry role is fulfilled by these cells? Ever since gan of Masera (SOM), the Grueneberg ganglion (GG) and the vomeronasal their discovery, the common structure of organ (VNO). Sensory neurons located in either the MOE, the SOM or the GG receptor GC proteins [16] – an extracel- project their axons to glomeruli within the main olfactory bulb (OB), where- lular peptide binding domain coupled to as vomeronasal neurons make synaptic contacts with mitral cell in the accessory olfactory bulb (AOB). In the MOE, sensory neurons can be di- an intracellular catalytic domain by a sin- vided into at least three subpopulations according to individual receptor ex- gle transmembrane α-helix – has fueled pression profiles. The great majority of neurons express one member of the speculation about GC-D-positive OSNs as large superfamily of canonical odorant receptors (ORs). Smaller groups of peptide sensors that regulate intracellular sensory cells are characterized by the expression of either a receptor guanyl- cGMP levels. At present, a conclusive pic- yl cyclase D (GC-D) or trace amine-associated receptors (TAARs) ture of how these cells are functionally in- volved in olfaction is lacking. In 2007, two parallel studies suggested different and ries have been puzzled by inherent diffi- of action potentials [14]. Convergent OR- somewhat controversial functions for GC- culties in recombinant OR expression as specific axonal projection patterns to a D-expressing OSNs. Hu et al. [21] provid- these receptor proteins are frequently re- few distinct glomeruli in the main olfac- ed evidence for CO2-mediated, carbonic tained in ER/Golgi membranes and hardly tory bulb confer an ‘OR identity’ to each anhydrase type II (CAII)-dependent Ca2+ translocate to the plasma membrane [40]. glomerulus and, thus, underlie processing signals in GC-D-expressing neurons. By All ORs deorphaned to date detect volatile of odor information by odotopic activity contrast, functional data from both wild- odorants of diverse chemical classes and ‘maps’ [44]. type and gene-targeted mice strongly sup- are broadly tuned to multiple stimuli. Vice Aside from canonical ORs, a second port a role of GC-D-positive OSNs as sen- versa, different receptors can respond to family of chemosensory GPCRs in the sitive and selective sensors for two natri- the same odor molecule. Thus, odor infor- main olfactory epithelium was identified uretic peptides – uroguanylin and gua- mation is encoded by combinatorial acti- in 2006 by Linda B. Buck’s laboratory [34]. nylin [31]. Are these findings necessari- vation of multiple ORs [6, 37]. Highly en- In a broad screen of OSN-enriched mu- ly contradictory? The pharmacological riched in the apical ciliary compartments rine cDNA, members of the trace amine- profiles of both CO2- and peptide-depen- of OSNs (the site of odor interaction), OR associated receptor (TAAR) family were dent Ca2+ responses in GC-D-expressing activation triggers a complex biochemi- found selectively expressed by sparse, neurons indicate that both chemosignals cal signaling cascade leading to adenylate nonoverlapping subset of OSNs. Further- could share a final common transduction cyclase activity and transiently increased more, Liberles and Buck reported that pathway. Future studies on this still enig- cAMP levels. Opening of cyclic nucleo- TAAR and OR expression appear mutu- matic OSN subpopulation will eventually tide-gated (CNG) channels and succes- ally exclusive, thus, suggesting a distinct elucidate whether both pathways can be sive activation of Ca2+-gated Cl− channels olfactory function for TAAR-expressing combined in an integrated signaling mod- results in a depolarizing receptor poten- OSNs. However, their exact functional el or if either mechanism serves a predom- tial that is transformed into axonal trains inant physiological function.

10 | e-Neuroforum 1 · 2010 Abstract

In addition to the main olfactory ep- cells share the characteristic expression of e-Neuroforum 2010 · 1:9–16 ithelium and its cellular heterogeneity, the olfactory marker protein (OMP; [38]) DOI 10.1007/s13295-010-0002-1 © Springer-Verlag 2010 many mammals possess at least three fur- with canonical OSNs, TAAR- and GC-D- ther olfactory tissues – the VNO, the Gru- expressing neurons of the main olfactory M. Spehr eneberg ganglion (GG), and the septal or- system, vomeronasal neurons, and senso- Sniffing out social signals. gan of Masera (SOM) – adding a whole ry cells of the SOM. By contrast, GG cells Chemical communication new layer of complexity to an already seem to lack direct access to the lumen of and the vomeronasal organ complex organization (. Fig. 1). Com- the nasal cavity. In light-microscopic im- pared to our detailed knowledge of the ages, GG cells show no prominent cellular Abstract main olfactory system and, to a lesser ex- processes such as dendrites, cilia, or mi- In most mammals, conspecific chemical com- tent, the vomeronasal system, our func- crovilli. Using scanning electron micros- munication strategies control complex social tional understanding of both the GG and copy, however, Brechbühl et al. [3] recent- and sexual behavior. Just a few years ago, our the SOM is still in its infancy. ly demonstrated that mouse GG neurons concept of how the olfactory system is orga- Located near the entrances to the na- bear primary cilia that can be accessed by nized to ensure faithful transmission of so- sopalatine ducts [53], the rodent SOM is a water-soluble chemostimuli via a water- cial information built on the rather simplis- tic assumption that two fundamentally dif- small, relatively flat, isolated patch of neu- permeable keratin layer. The authors of ferent classes of stimuli – ‘general’ odors ver- roepithelium that is composed of rough- the same study also reported transient cy- sus ‘pheromones’ – are exclusively detected ly 10,000 ciliated sensory neurons that ap- tosolic Ca2+ elevations in GG neurons in by either of two sensory structures: the main pear to largely resemble canonical OSNs response to chemical cues that are secret- olfactory epithelium or the vomeronasal or- of the main epithelium with respect to ed under stress to signal danger to conspe- gan. A number of exciting recent findings, however, revealed a much more complex OR and downstream signaling protein cifics. The molecular nature of such ‘alarm and functionally diverse organizational struc- expression. Interestingly, the vast major- pheromones,’ however, has not been iden- ture of the . At least four ana- ity of SOM neurons (>90%) choose one tified. tomically segregated olfactory subsystems, member of a group of only nine ORs for some remarkably heterogeneous in their cel- monogenic expression [67]. An uncon- The vomeronasal organ – a key lular composition, detect distinct, but partial- ventionally abundant receptor, SR1, is ex- player in chemical communication ly overlapping populations of sensory stimuli. and social interaction Discerning how subsystem-specific receptor pressed in ~50% of SOM neurons and has architectures and signaling pathways orches- recently been physiologically scrutinized trate the coding logic of social chemosignals, in great detail in gene-targeted mice [19]. Probably the most well characterized ol- will ultimately shed new light on the neuro- These experiments revealed an unusu- factory subsystem division is between the physiological basis of social behavior. ally broad odor response profile over a main olfactory and vomeronasal systems. wide concentration range in SR1 express- The latter’s peripheral sensory structure, Keywords Olfaction · Social chemosignals · Sensory neu- ing neurons. Given the observed corre- the VNO – first described in 1813 by Lud- rophysiology · Homeostatic plasticity · Che- lation between SR1-dependent broad re- vig L. Jacobson [22] – consists of two bi- moreceptor sponsiveness and mechanosensitivity [17, laterally symmetrical blind-ended cigar- 18] the authors discuss the hypothesis that shaped tubes which lie within the vomer the SOM could function as a strategically bone at the anterior nasal septum. Vom- placed outpost of the main olfactory epi- eronasal sensory neurons (VSNs) reside thelium that might signal general changes medially in a crescent-shaped sensory in airflow and/or odor environment and, neuroepithelium. Each bipolar VSN ex- thus, prime the main system for overall tends a single apical that ends sensitivity adjustment. in a microvillous knob which is bathed in Similar to the history of the septal or- mucus secreted by vomeronasal glands. gan, the Grueneberg ganglion [19] made a Upon intimate contact with a pheromone comeback in chemosensory research ac- source, stimuli access the VNO lumen via tivity just a few years ago. GG cell bodies autonomically controlled pulsative vas- are bilaterally located at the dorsal tips of cular contractions of a large lateral blood each nasal cavity, in close proximity to the vessel [27]. This mechanism enables the opening of the naris. Each ganglion com- VNO to take up relatively nonvolatile cues prises 300–500 cells that project single ax- from urine deposits, vaginal secretions, ons along the dorsal roof of the nasal cav- scent gland secretions, or saliva [30]. ity to dorsocaudal regions of the main ol- The rodent vomeronasal system is or- factory bulb. This area somewhat overlaps ganized in at least a bipartite manner. Two with the bulb region harboring the so- topographically segregated VSN subpop- called necklace glomeruli, which receive ulations express distinct repertoires of re- input from GC-D expressing neurons. GG ceptors and other putative signaling mol-

e-Neuroforum 1 · 2010 | 11 Review article

Fig. 2 8 Tissue architecture and putative signal transduction pathways in the mouse VNO. a) Confocal photomicrograph of a coronal VNO cryosection. Mature VSNs are labeled by fluorescence of a green fluorescent protein (GFP) expressed under the control of the regulatory sequences of the olfactory marker protein (omp) gene (OMP-GFP). SE, sensory epithelium; L, lumen; BV, blood vessel. b) Schematic view of the VNO sensory neuroepithelium that is divided into an apical (AL) and basal layer (BL). c) Putative signaling cascades implemented in VSNs. Volatile urinary compounds (e.g., 2-heptanone or 2,5-dimethyl pyr- azine) activate V1R- and Gαi2-expressing neurons, whereas V2R- and Gαo-expressing neurons are likely activated by non-vol- atile social cues such as major urinary proteins (MUPs), exocrine-gland-secreting peptides (ESPs) or major histocompatibili- ty complex (MHC) class I peptides (e.g., SYFPEITHI). Sulfated steroids (e.g., corticosterone 21-sulfate), rather non-volatile stress signals, have also been described as vomeronasal stimuli. However, the receptors detecting these ligands are still unknown. FPR-rs-expressing VSNs are activated by disease-associated ligands like the formylated peptide fMLF. Current downstream signaling models hypothesize G-protein-dependent activation of phospholipase C (PLC), cleavage phosphatidylinositol-4,5- 2+ bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), and final gating of a Ca current that is, at least for some stimuli, critically dependent on the TRPC2 ion channel subunit. A number of interactions, however, are still elu- 2+ sive. Future investigations will have to pinpoint the exact function of, e.g., Gαi2 and Gαo subunits, the recently described Ca - − 2+ gated Cl channel, IP3 or polyunsaturated fatty acids (PUFAs) that have also been shown to induce a Ca responses in VSNs

ecules (. Fig. 2). VSNs located in the other , and, notably, se- Today, we believe that both V1Rs and apical layer of the sensory epithelium ex- vere behavioral deficits observed in mice V2Rs are activated by a structurally di- press Gαi2 in concert with one member of deficient for a gene cluster that encodes verse group of semiochemicals that have a multigene family that encodes 137 intact for 16 V1R proteins [12]. frequently been collectively referred to as G protein-coupled receptors (GPCRs) Neurons of the basal Gαo-positive zone pheromones – a term whose definition is – the V1Rs [13, 54]. Like OR genes, their express members of an unrelated class C currently in flux [5, 64]. Pheromonal cues coding regions show no introns, they are GPCR family – the V2Rs [39, 56]. The are typically embedded in complex bodi- located in genomic clusters and expressed hallmark of all ~120 apparently function- ly secretions such as urine or sweat and in a tightly controlled monoallelic fashion al V2R receptor proteins [70] is a large hy- range from small volatile molecules ([29, [55]. However, OR and V1R genes share no drophobic amino (N)-terminal extracel- 51] to steroids [47], complex peptides [27, significant sequence homology. With one lular domain, sharing sequence similarity 30, 32], and proteins [9]. A blunt categori- prominent exception (V1Rb2; [2]), all ver- with metabotropic glutamate, Ca2+-sens- zation of the VNO as a specialized pher- tebrate V1R proteins still represent orphan ing, and sweet/umami-sensing T1R omone detector and the main olfacto- receptors whose putative chemosensory receptors. Based on this observation, this ry system as a general sensor of ‘conven- function is only indirectly inferred from extracellular domain has been proposed tional’ odors would, however, be simplis- their tissue distribution, expression pat- to form the V2R ligand binding site [43]. tic [50, 62]. tern, organizational commonalities with

12 | e-Neuroforum 1 · 2010 Important aspects of V1/2R function and downstream signal transduction pathways remain elusive. Based on layer- specific coexpression, a role of Gαi2 and Gαo in V1R- and V2R-mediated signaling pathways, respectively, represents an at- tractive model. However, functional evi- dence supporting this hypothesis is lack- ing. Knockout models failed to demon- strate a critical role of Gαi2 and Gαo sub- units in pheromone sensing [48, 66]. Far better consensus is achieved on a role of phospholipase C (PLC). Inositol-1,4,5-tri- sphosphate (IP3), diacylglycerol (DAG) as well as polyunsaturated fatty acids (PU- FAs) have all been implicated in gating a Ca2+ permeable transduction channel [63]. Efforts to identify this channel have focused on a distinct transient receptor potential channel subunit, TRPC2 [35]. TRPC2-/- mice show severe defects in so- cial and sexual behaviors. Yet, there are significant differences between TRPC2 deletion and surgical VNO ablation [25]. A DAG-activated TRPC2-dependent cur- rent [36] not only appears to be activat- ed as a downstream effector in VSN sig- naling, but also functions in vomeronasal sensory adaptation and gain control [61]. Likely, this current also provides the ini- tial Ca2+ influx that has recently been pro- posed to trigger a Ca2+-activated Cl–cur- rent that could boost membrane depolar- ization via Cl- efflux [69]. Compared to the many substantial ad- vances in our understanding of canonical OSN signaling, our present conception of sensory signaling in the VNO is still frag- Fig. 3 8 Illustration of the VNO expression profiling approach used to uncover ion channel proteins mentary. Given the prime biological im- involved in homeostatic VSN plasticity and activity-dependent maintenance of output stability. Top) Schematic diagram illustrating the experimental strategy used for pheromone exposure (left) and portance of intraspecific social commu- stimulus deprivation (right) of singly housed C57Bl/6 mice. Middle) Typical VNO preparations used for nication, current research in my laborato- gene expression profiling (left), patch-clamp recordings in acute tissue slices (middle), and cryosec- ry focuses on the basic physiological con- tions for immunochemistry (right). Bottom) Western blot analysis demonstrating increased levels of cepts underlying chemical communica- vomeronasal ERG1a expression in stimulated versus deprived mice. Blots were additionally probed tion in conspecific mammals. Thus, in the for β-tubulin as loading control (left). IR-DIC photomicrograph shows a patch pipette used for dye (Al- exa®488) loading of VSNs during recordings (middle). The molecular identity of the can later long term, we aim to gain detailed func- be determined by post-hoc immunocytochemistry against layer-specific marker proteins (e.g. V2R2; tional insight into the neuronal mecha- right) nisms that link chemosensation and so- cial behaviors. sal transcripts that are regulated in an ac- by means of RT-PCR and immunocyto- Homeostatic plasticity in tivity-dependent fashion. We designed an chemistry, respectively [20]. We initially vomeronasal neurons expression profiling paradigm that inte- hypothesized that, analog to non-synaptic grates various levels of analysis (. Fig. 3) homeostatic plasticity in many brain ar- To better understand the complex mech- by combining microarray-based quantita- eas primarily associated with learning and anisms involved in pheromone sensing, tive determination of activity-dependent memory [11, 68], the dynamic range and we recently engaged in a high-through- ‘transcriptomes’ with ‘manual’ confirma- stability of VSN input-output relation- put whole-genome search for vomerona- tion of candidate signaling genes/proteins ships is constantly adjusted within mean-

e-Neuroforum 1 · 2010 | 13 Review article

chemistry and three-dimensional recon- struction of fluorescently labeled neurons, we demonstrated that basal VSNs exhibit a fast ERG-mediated K+ current that is sig- nificantly reduced after stimulus depriva- tion. When dissecting the ‘internal anato- my’ [1] of basal VSN spikes using the ac- tion potential (AP) clamp technique, our recordings reveal that ERG currents are critically involved in VSN spike repolar- ization. Consequently, AP discharge in basal VSNs is substantially impaired after ERG channel inhibition. Together, our findings illustrate that, by regulating the expression level of ERG K+ channels, basal VSNs are equipped to dynamically control/extend the range of their individual stimulus-response func- tion. This novel example of homeostatic plasticity in the periphery of the accessory olfactory system is ideally suited to adjust VSNs to a target output range in a layer- specific and use-dependent manner [20].

A third family of vomeronasal 2+ Fig. 4 8 Formyl-peptides evoke Ca signals in VSN dendritic tips. Top) chemoreceptors Merged macroscopic bright field and fluorescence images of the hemisect- ed rostral head of an OMP-GFP mouse, illustrating the en face confocal Ca2+ imaging approach recently established in our laboratory. Bottom) High Given the increasing diversity of neuro- magnification confocal section of the dendritic surface of fluo-4/AM-loaded nal subpopulations in the main olfactory 2+ VSNs (left). Representative original recordings of cytosolic Ca signals in re- epithelium, it is tempting to hypothesize sponse to fMLF (900 nM), 2-heptanone (1 µM), diluted urine (1:500), and el- evated extracellular K+ (40 mM; right) that potentially still unidentified sensory cell populations and corresponding che- moreceptors might also exist in the VNO. In close collaboration with the laboratory ingful firing rate limits in response to al- icantly up-regulated in stimulated mice. of Ivan Rodriguez (University of Geneva), tered sensory input. One mechanism that Characterized by rather unconventional we therefore designed a screening strate- ensures such homeostatic plasticity on a gating kinetics (slow activation, fast inac- gy for putative mouse receptors which we longer time scale, i.e., hours to days [71], tivation), hERG, the homolog of expected to share the hallmarks of all pre- is compensatory feedback regulation of de mERG1 and founding family member, has viously identified chemosensory GPCRs, novo protein synthesis. This concept pro- been intensely investigated because of the i.e., (a) showing a seven-transmembrane vided the temporal framework for inves- severe cardiac phenotype (long QT syn- topology, (b) displaying a punctate expres- tigating the consequences of stimulus de- drome 2) caused by channel mutations [57, sion pattern in the vomeronasal neuroepi- privation in the mouse VNO (. Fig. 3). 59]. By contrast, the physiological roles of thelium, (c) excluding any coexpression of In this context, modulation of voltage- ERG channels in the nervous system are other olfactory groups, (d) gated K+ channel gene expression – ma- poorly understood [58]. Using immuno- excluding cotranscription of other mem- jor determinants of membrane excitability chemistry, we showed that ERG1 channel bers of their own receptor family, (e) be- – represents a key molecular mechanism proteins are selectively expressed in basal ing located/enriched at the VSN dendritic to orchestrate the output of an individu- V2R-positive VSNs and substantially reg- tips, and (f) triggering neuronal activity in al neuron [28]. By systematically compar- ulated upon pheromone exposure/depri- response to biologically relevant stimuli. ing vomeronasal K+ channel transcrip- vation. However, a key issue in interpret- Screening mouse vomeronasal tissue tion levels in male mice strongly exposed ing these findings is to resolve whether the for the expression of ~100 candidate GP- to rich sources of pheromonal cues ver- observed expression changes are reflected CRs by RT-PCR [52], we identified five sus stimulus-deprived animals, we iden- in VSN physiology. By combining whole- non-V1/2R GPCR genes [criterion (a)], tified a member of the ether-à-go-go re- cell patch-clamp recordings from iden- all members of the formyl peptide recep- lated gene (ERG) K+ channel subfamily, tified VSNs in acute vomeronasal slice tor (FPR)-like genes (Fpr-rs1, rs3, rs4, rs6 mERG1, as both consistently and signif- preparations with post-hoc immunocyto- and rs7). Quantitative PCR experiments

14 | e-Neuroforum 1 · 2010 showed that the presence of these tran- nal projections to the accessory olfactory 2. Boschat C, Pelofi C, Randin O et al. (2002) Phero- mone detection mediated by a V1r vomeronasal scripts indeed turned out highly VNO- bulb are left intact. Strikingly, we record- receptor. Nat Neurosci 5:1261–1262 specific. In situ analysis of FPR-rs1-7, as ed exquisitely sensitive and concentration 3. Brechbuhl J, Klaey M, Broillet MC (2008) Gruene- well as immunocytochemical localization dependent responses to overlapping sets berg ganglion cells mediate alarm pheromone de- tection in mice. Science 321:1092–1095 studies of FPR-rs3, revealed both strong of FPR-rs agonists [criterion (f)], strong- 4. Breer H, Fleischer J, Strotmann J (2006) The sense and punctate gene expression in the VNO ly suggesting that Fpr-rs-expressing VSNs of smell: multiple olfactory subsystems. Cell Mol sensory epithelium [criterion (b)], as well represent a previously unrecognized type Life Sci 63:1465–1475 5. Brennan PA, Zufall F (2006) Pheromonal communi- as protein translocation to the microvil- of chemosensory neuron. cation in vertebrates. Nature 444:308–315 lous dendritic endings of VSNs [criterion What might be the physiological func- 6. Buck LB (2000) The molecular architecture of (e)]. Furthermore, we demonstrated both tion of these cells? It has been known for odor and pheromone sensing in mammals. Cell 100:611–618 Fpr-rs expression in VSN subsets that do quite some time that mice use the olfacto- 7. Buck L, Axel R (1991) A novel multigene family not coexpress other known vomeronasal ry system to discern pathogenicity or the may encode odorant receptors: a molecular basis receptors [criterion (c)] and monogen- health status of a conspecific [24]. How- for odor recognition. Cell 65:175–187 8. Casella R, Shariat SF, Monoski MA et al. (2002) Uri- ic Fpr-rs expression within these neurons ever, no olfactory subsystem dedicated to nary levels of urokinase-type plasminogen activa- [criterion (d)]. Intriguingly, with the ex- the identification of pathogens, or patho- tor and its receptor in the detection of bladder car- ception of FPR-rs1, FPR-rs proteins are genic states, has yet been identified in cinoma. Cancer 95:2494–2499 9. Chamero P, Marton TF, Logan DW et al. (2007) restricted to VSNs found localized in the mammals [45]. Since FPR-rs agonists are Identification of protein pheromones that pro- apical Gαi2-expressing layer of the neuro- found in bodily secretions at various stag- mote aggressive behaviour. Nature 450:899–902 epithelium. es of diseases [8], our results could pro- 10. Chromek M, Slamova Z, Bergman P et al. (2006) The antimicrobial peptide cathelicidin protects the Immune cells such as granulocytes or vide the link to understand how animals urinary tract against invasive bacterial infection. macrophages express FPR1 and FPR-rs2, identify pathogens or unhealthy potential Nat Med 12:636–641 two family members not transcribed in partners. 11. Davis GW (2006) Homeostatic control of neural ac- tivity: from phenomenology to molecular design. VSNs. These receptors show broad tun- Annu Rev Neurosci 29:307–323 ing profiles with ligand spectra rather de- Corresponding address 12. Del Punta K, Leinders-Zufall T, Rodriguez I et al. fined by immunological function than Prof. Dr. rer. nat. M. Spehr (2002) Deficient pheromone responses in mice Institute for Biology II/Dept. lacking a cluster of vomeronasal receptor genes. by structural properties [10, 42]. These Nature 419:70–74 Chemosensation,Sammelbau Biologie, 42D/ FPR agonists include peptides and lipids 13. Dulac C, Axel R (1995) A novel family of genes en- R253, RWTH Aachen derived from pathogens (such as fMLF, coding putative pheromone receptors in mam- Worringer Weg 1, 52074 Aachen mals. Cell 83:195–206 the prototypical formylated peptide re- [email protected] 14. Firestein S (2001) How the olfactory system makes leased by gram negative bacteria), or in- sense of scents. Nature 413:211–218 volved in acute inflammatory respons- M. Spehr Marc Spehr: born August 7th 1973; received 15. Fulle HJ, Vassar R, Foster DC et al. (1995) A recep- his graduate and doctoral degree in biology (Dip- tor guanylyl cyclase expressed specifically in ol- es (such as the antimicrobial compounds lom (1999), Dr. rer. nat. (2002)) from the Ruhr-Univer- factory sensory neurons. Proc Natl Acad Sci U S A CRAMP and Lipoxin A4). However, no li- sity Bochum (Prof. Dr. Hanns Hatt, Department of Cel- 92:3571–3575 gands were described for FPR-rs3, rs4, rs6, lular Physiology). Postdoctoral training (until 2006) as 16. Gibson AD, Garbers DL (2000) Guanylyl cyclases as an Emmy Noether research fellow in the groups of Prof. a family of putative odorant receptors. Annu Rev or rs7 receptors. When we engineered hu- Dr. Frank Zufall and Prof. Dr. Trese Leinders-Zufall (De- Neurosci 23:417–439 man embryonic kidney (HEK) cells to ex- partment of Anatomy and Neurobiology, School of 17. Grosmaitre X, Santarelli LC, Tan J et al. (2007) Du- press recombinant FPR-rs1–7 proteins, we Medicine, University of Maryland, Baltimore, MD, USA). al functions of mammalian olfactory sensory neu- 2006 – 2009: Independent Junior Research Group rons as odor detectors and mechanical sensors. were able to examine receptor activation. Leader funded by the DFG Emmy Noether-Program Nat Neurosci 10:348–354 Surprisingly, VSN-specific FPR-rs pro- (Department of Cellular Physiology, Ruhr-University 18. Grosmaitre X, Fuss SH, Lee AC et al (2009) SR1, a teins display distinct, but overlapping li- Bochum). In 2008, appointed member of the Acade- mouse odorant receptor with an unusually broad my of Sciences NRW (Junges Kolleg). At present, Marc response profile. J Neurosci 29:14545–14552 gand profiles that, to a large extent, resem- Spehr is a W2 Lichtenberg-Professor of the Volkswagen 19. Gruneberg H (1973) A ganglion probably belong- ble those agonist spectra of immune-cell Foundation at RWTH-Aachen University (Institute for ing to the N. terminalis system in the nasal mucosa FPR proteins. Biology II; chair: Prof. Dr. Hermann Wagner). of the mouse. Z Anat Entwicklungsgesch 140:39– 52 Are these results transferable to the in 20. Hagendorf S, Fluegge D, Engelhardt C, Spehr M Acknowledgement. Work in the author’s laborato- (2009) Homeostatic control of sensory output in vivo situation, i.e., do these different dis- ry is generously supported by the Emmy Noether- ease- and inflammation-associated com- basal vomeronasal neurons: activity-dependent Program of the Deutsche Forschungsgemeinschaft expression of ether-a-go-go-related gene potassi- pounds actually activate vomeronasal neu- (SP724/2–1), the Mercator Foundation (Junges Kol- um channels. J Neurosci 29:206–221 leg), and within the funding initiative Lichtenberg Pro- 21. Hu J, Zhong C, Ding C et al. (2007) Detection of rons? To address this question, we estab- fessorships of the Volkswagen Foundation. lished an in situ approach that combined near-atmospheric concentrations of CO2 by an ol- factory subsystem in the mouse. Science 317:953– whole mount vomeronasal preparations Conflict of interest. No statement made. 957 with dendritic Ca2+ imaging in the intact 22. Jacobson L (1813) Anatomisk Beskriv- neuroepithelium (. Fig. 4). This way, we else over et nyt Organ i Huusdyrenes Næse. Veterinær=Selskapets Skrifter [in Danish] 2:209– were able to record responses from in- References 246 dividual VSN knobs while the dendritic tips are kept covered by mucus and both 1. Bean BP (2007) The in mammalian central neurons. Nat Rev Neurosci 8:451–465 the epithelial structure and the VSN axo-

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