Grueneberg Ganglion Cells Mediate Alarm Pheromone Detection in ...
Grueneberg Ganglion Cells Mediate Alarm Pheromone Detection in ...
Grueneberg Ganglion Cells Mediate Alarm Pheromone Detection in ...
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REPORTS<strong>Grueneberg</strong> <strong>Ganglion</strong> <strong>Cells</strong> <strong>Mediate</strong><strong>Alarm</strong> <strong>Pheromone</strong> <strong>Detection</strong> <strong>in</strong> MiceJulien Brechbühl, Magali Klaey, Marie-Christ<strong>in</strong>e Broillet*<strong>Alarm</strong> pheromones (APs) are widely used throughout the plant and animal k<strong>in</strong>gdoms. Species suchas fish, <strong>in</strong>sects, and mammals signal danger to conspecifics by releas<strong>in</strong>g volatile alarm molecules.Thus far, neither the chemicals, their bodily source, nor the sensory system <strong>in</strong>volved <strong>in</strong> theirdetection have been isolated or identified <strong>in</strong> mammals. We found that APs are recognized by the<strong>Grueneberg</strong> ganglion (GG), a recently discovered olfactory subsystem. We showed with electronmicroscopy that GG neurons bear primary cilia, with cell bodies ensheathed by glial cells. APsevoked calcium responses <strong>in</strong> GG neurons <strong>in</strong> vitro and <strong>in</strong>duced freez<strong>in</strong>g behavior <strong>in</strong> vivo, whichcompletely disappeared when the GG degenerated after axotomy. We conclude that mice detectAPs through the activation of olfactory GG neurons.Department of Pharmacology and Toxicology, University ofLausanne, Bugnon 27, CH-1005 Lausanne, Switzerland.*To whom correspondence should be addressed. E-mail:mbroille@unil.chCORRECTED 20 NOVEMBER 2009; SEE LAST PAGEIn a threaten<strong>in</strong>g situation, plants and animalswarn conspecifics by secret<strong>in</strong>g alarm pheromones(APs) (1–4). These substances signal<strong>in</strong>jury, distress, or the presence of predators. Inanimals, the response of conspecifics to thesesignals is usually to freeze, attack, or disperse(5–7). Mammalian APs have not been identifiedyet; they may be low-molecular-weight volatilesubstances (8, 9), and it rema<strong>in</strong>s unknown whethertheir detection by conspecifics is mediated bya specialized olfactory subsystem.In mammals, the most recently discoveredolfactory subsystem (10–14), the so-called<strong>Grueneberg</strong> ganglion (GG) (15), is present atthe tip of the nose, close to the open<strong>in</strong>g of thenaris, and is easy to observe <strong>in</strong> one particulargene-targeted mouse stra<strong>in</strong> called OMP-GFP(olfactory marker prote<strong>in</strong>–green fluorescent prote<strong>in</strong>)(Fig. 1A). In these mice, a GFP-positive,arrow-shaped neuronal structure 750 to 1000 mm<strong>in</strong> length l<strong>in</strong>es both sides of the nasal septumand comprises 300 to 500 cells (Fig. 1B). Eachcell sends out a s<strong>in</strong>gle axon, and axons fasciculateimmediately (Fig. 1C) as they projectcaudally along the dorsal roof of the nasal cavityto the necklace complex <strong>in</strong> the olfactory bulb(10–12). The GG starts develop<strong>in</strong>g around embryonicday 16 and appears to be complete atbirth (10–14).Olfactory neurons sense the external chemicalenvironment through the presence, at theend of their s<strong>in</strong>gle dendrite, of cilia or microvilli<strong>in</strong> direct contact with the airspace of the nasalcavity. In contrast, under light or confocal microscopy,the mouse GG looks like a densely packedpopulation of round cell bodies lack<strong>in</strong>g thesetypical olfactory neuronal features or support<strong>in</strong>gglial cells (Fig. 1D) (11–14). GG cells express,<strong>in</strong> addition to OMP, molecular olfactorycomponents. Indeed, extensive reverse transcriptionpolymerase cha<strong>in</strong> reaction and <strong>in</strong> situhybridization analyses identified one vomeronasalorgan (VNO) receptor, a dist<strong>in</strong>ct subtypeof the V2R class, V2R83; and heterotrimericGTP-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>s (G prote<strong>in</strong>s) G ao and G ai2(16, 17). This suggests that the GG is <strong>in</strong> fact anolfactory subsystem with specific chemosensoryfunctions.To resolve the apparent contradiction betweenthe lack of cilia or dendrites and a possibleolfactory function of GG cells, we looked at thestructure and cellular organization of the mouseGG by perform<strong>in</strong>g scann<strong>in</strong>g electron microscopy(SEM) on coronal slices (Fig. 1, E to G). Wefound clusters of round GG cells <strong>in</strong> a fibroblastmeshwork between the nasal septum and a kerat<strong>in</strong>izedepithelium (KE) (Fig. 1, E and F) (13).With this technical approach, four to eight ciliaryprocesses were observed per GG cell (Fig. 1G).They were further characterized by transmissionelectron microscopy (TEM) (figs. S1 to S3) to benonmotile primary cilia accord<strong>in</strong>g to their microtubularcharacteristics (fig. S1, B and C) (18, 19).Thirty to forty primary cilia were present per GGcell, profoundly <strong>in</strong>vag<strong>in</strong>ated <strong>in</strong>to the cytoplasm(5 mm) (fig. S1B) and grouped <strong>in</strong>to three to fourpacks of 8 to 12 per cell (figs. S1C and S3). Analyz<strong>in</strong>gserial ultrath<strong>in</strong> sections, we estimated boththeir length (15 mm) and their diameter (0.2 mm).Primary cilia were observed <strong>in</strong> the extracellularmatrix (fig. S1, E to G) but not with<strong>in</strong> the leakyKE, and they did not cross the kerat<strong>in</strong> layers toFig. 1. Ciliary processes are present on the mouse GG cells. (A) Sagittal section through the nasalcavity of an OMP-GFP mouse: Neurons from the ma<strong>in</strong> olfactory epithelium (MOE), septal organ(SO), VNO, and GG (white rectangle) project to the olfactory bulb (OB). (B) Dorsal view of the GG,an arrow-shaped parallel organ on both sides of the nasal septum (Se). (C) High-power view fromone GG [rectangle <strong>in</strong> (A)]. (D) Maximum-<strong>in</strong>tensity projection (12 mm) of a GG cell cluster byconfocal microscopy. (E) SEM micrograph of a GG coronal section [l<strong>in</strong>e <strong>in</strong> (B)]. Clusters of GG cells(GC) are shown, located along the Se underneath the KE l<strong>in</strong><strong>in</strong>g the nasal cavity (NC) [squaredetailed <strong>in</strong> (F)]. (F) Close-up view of the GG cells <strong>in</strong> a fibroblast meshwork (Fb) [square detailed <strong>in</strong>(G)]. (G) GG cell (false color green) with its axon (false color red) and th<strong>in</strong> ciliary processes (falsecolor blue). Scale bars, 1 mm <strong>in</strong> (A); 0.1 mm <strong>in</strong> (B); 0.25 mm <strong>in</strong> (C); 25 mm <strong>in</strong> (D); 20 mm <strong>in</strong> (E) and(F); and 5 mm <strong>in</strong> (G).Downloaded from www.sciencemag.org on April 18, 2010109222 AUGUST 2008 VOL 321 SCIENCE www.sciencemag.org
each the nasal cavity. Immunosta<strong>in</strong><strong>in</strong>g experimentsconfirmed the presence of multiple ciliaon GG cells (fig. S1, H to K).We tried to resolve the difference between thenumber of cilia observed with SEM versus TEM(4 to 8 versus 30 to 40) and found another cellpopulation that trapped most cilia <strong>in</strong>side the ganglionstructure (fig. S1, D and K). These cells havea triangular shape with th<strong>in</strong> cytoplasmic extensions(Fig. 2A). Immunosta<strong>in</strong><strong>in</strong>g experiments demonstratedthe glial orig<strong>in</strong> of the wrapp<strong>in</strong>g cells bypositive sta<strong>in</strong><strong>in</strong>g for S100b (Fig. 2, B to D) andglial fibrillary acidic prote<strong>in</strong> (fig. S4, A to C). TheGFP-positive cells expressed the olfactory markerG ai2 (Fig. 2, E to G) and the neuronal marker bIIItubul<strong>in</strong>(fig. S4, D to F). Thus, a GG comprises twoREPORTSdifferent cell populations: glial cells and neurons(GFP-positive cells), which bear multiple primarycilia, putative sites of chemosensory transduction.By systematic scann<strong>in</strong>g of the nasal cavity(fig. S5), we found that the KE cover<strong>in</strong>g the GGwas already present at birth (mice aged 30 m<strong>in</strong>);no cilia were found protrud<strong>in</strong>g <strong>in</strong>to the airspaceof the nasal cavity. The aspect of KE did notFig. 2. The GG is composed of cells of different orig<strong>in</strong>s. (A) TEMmicrographconfirm<strong>in</strong>g the presence of ensheath<strong>in</strong>g cells (EC, false color blue) <strong>in</strong>dividuallypackag<strong>in</strong>g GG cells (GC). An EC had a triangular cell body, a roundnucleus,andlongarmscircl<strong>in</strong>gGGcells.(B to G) High-power views of glialor neuronal prote<strong>in</strong> immunoreactivity. GG cells were recognized by theirOMP-GFP expression [(B) and (E)]. (C) Glial marker S100b expressed <strong>in</strong> cellssurround<strong>in</strong>g the GG cells. (D) Merge of (B) and (C). White arrowheads <strong>in</strong>dicateGFP-negative glial cells [(B) to (D)]. (F) G ai2 sta<strong>in</strong><strong>in</strong>g <strong>in</strong> GG cells. (G) Merge of(E) and (F), where colocalization appears <strong>in</strong> yellow. Scale bars, 2 mm<strong>in</strong>(A)and20 mm <strong>in</strong> (B) to (G).Downloaded from www.sciencemag.org on April 18, 2010Fig. 3. APs activate GG cells. (A) Coronal slice from the GG region. (B)Uptake of Fura-2AM <strong>in</strong>to GG cells with<strong>in</strong> the white rectangle from (A),measured at 380 nm before (B) and at the peak of the <strong>in</strong>tracellular calcium<strong>in</strong>crease <strong>in</strong>duced by a control pulse of KCl (C). (D to E) Lack of calciumtransients after the perfusion of mouse milk (Milk), nipple wash (NW), amix of odors and known mouse pheromones (Mix), or mouse ur<strong>in</strong>e. (F) APsbut not general pheromones (GP) <strong>in</strong>duced a reversible calcium transient.(G) AP response was abolished only after depletion of <strong>in</strong>tracellular calciumstores by thapsigarg<strong>in</strong> (Th, 20 mM) <strong>in</strong> divalent-free medium. Fluorescence<strong>in</strong>tensity ratio (DF) = F340/F380. Perfusion times are <strong>in</strong>dicated byhorizontal bars <strong>in</strong> (D) to (G). Scale bars, 20 mm <strong>in</strong> (A) and 10 mm <strong>in</strong> (B)and (C).www.sciencemag.org SCIENCE VOL 321 22 AUGUST 2008 1093
REPORTSchange with ag<strong>in</strong>g (fig. S5, B to D). To assesshow potential chemosensory stimuli could reachand activate GG cells, we performed standardsk<strong>in</strong> permeability assays us<strong>in</strong>g Toluid<strong>in</strong>e blue(fig. S6, A to C) and Lucifer yellow (fig. S6, Dand E). In contrast to the control (the back sk<strong>in</strong> ofthe same animal) (fig. S6D), the KE cover<strong>in</strong>g theGG cells was found to be permeable by hydrophilicsubstances, suggest<strong>in</strong>g that water-solublechemostimuli have access to the outside zone ofthe GG cell clusters, where primary cilia are located(fig. S6E). A similar morphological situationisobserved<strong>in</strong>Caenorhabditiselegans, <strong>in</strong>which the cilia of the amphid chemosensory neurons(AWA, AWB, and AWC), located under thecuticle, sense volatile and hydrophilic odors viadirect diffusion (20).Focus<strong>in</strong>g on its potential water-soluble quality,we used calcium imag<strong>in</strong>g on coronal slices toidentify the GG chemostimulus (Fig. 3). Tissueslices were <strong>in</strong>cubated <strong>in</strong> Fura-2 acetoxymethylester (AM), a calcium-sensitive dye. GG cellswere identified by the <strong>in</strong>tr<strong>in</strong>sic green fluorescenceof GFP <strong>in</strong> their cell bodies (Fig. 3A), andthe uptake of the dye was confirmed by fluorescencemeasurements (Fig. 3B). Chemical stimuliwere delivered <strong>in</strong> oxycarbonated artificial cerebrosp<strong>in</strong>alfluid (ACSF) cont<strong>in</strong>uously perfused onthe tissue slices <strong>in</strong> the imag<strong>in</strong>g chamber. Cellviability was assessed by pulses of KCl (Fig. 3, Cto F). Various hypotheses have been made concern<strong>in</strong>gthe possible GG function, the mostfavorite be<strong>in</strong>g its role <strong>in</strong> mother-pup recognition(10, 11, 14) because of its presence at birth (11)and axonal wir<strong>in</strong>g to the necklace glomeruli ofthe olfactory bulb implicated <strong>in</strong> newborn suckl<strong>in</strong>gbehavior (21). However, we did not recordany calcium <strong>in</strong>crease with mouse milk (n =24cells, 4 mice) or mammary secretions (n =29cells, 5 mice) from lactat<strong>in</strong>g female mice (Fig.3D). A mix of odors and known isolated mousepheromones (22) (n = 43 cells, 15 mice) ormouse ur<strong>in</strong>e as the major source of pheromones(n = 29 cells, 9 mice) did not enhance fluorescence(Fig. 3E). Look<strong>in</strong>g for other volatile andwater-soluble stimuli to test on GG cells, wenoticed that the <strong>in</strong>traspecies danger signals, theso-called APs, released by stressed animals areknown to have these physical characteristics (8, 9).We collected APs dur<strong>in</strong>g kill<strong>in</strong>g of mice withCO 2 , which is known to be a major stress factor(23). The perfusion of APs <strong>in</strong>duced a reversiblefluorescence enhancement <strong>in</strong> almost all testedGG cells from newborn and adult mice (n =39out of 41 cells, 7 mice; Fig. 3F). As a control,general pheromones (GPs) collected from miceFig. 4. AP sens<strong>in</strong>g depends on a functional GG. (A and B) APs <strong>in</strong> ACSF solution (white bars, ACSF;gray bars, ACSF + AP) <strong>in</strong>duced a significant <strong>in</strong>crease <strong>in</strong> freez<strong>in</strong>g behavior and a decrease <strong>in</strong> walk<strong>in</strong>gdistance <strong>in</strong> control (Ctrl) but not <strong>in</strong> axotomized (Axo) OMP-GFP mice. (C) Whole-mount dorsal viewsof the GG region from Ctrl and Axo mice. (D) Representative track<strong>in</strong>g of the walk<strong>in</strong>g distancecovered by mice <strong>in</strong> the test chamber <strong>in</strong> the absence (white square) or presence (gray square) of APs<strong>in</strong> the ACSF conta<strong>in</strong>er (square). Red dots <strong>in</strong>dicate the position of the mice at the beg<strong>in</strong>n<strong>in</strong>g of the3-m<strong>in</strong> test. (E) Olfactory function <strong>in</strong> f<strong>in</strong>d<strong>in</strong>g the cookie was similar <strong>in</strong> both experimental groups. In(A), (B), and (E), error bars <strong>in</strong>dicate the SEM of n = 5 to 6 observations, *P < 0.05; ns, notsignificant. Scale bars, 500 mm <strong>in</strong> (C).freely behav<strong>in</strong>g and communicat<strong>in</strong>g, when nostress was applied, did not <strong>in</strong>duce any calcium<strong>in</strong>crease (n = 25 cells, 5 mice; Fig. 3F) and neitherdid CO 2 itself (24)(n = 26 cells, 5 mice) or otherhypothesized stimuli (10–14) such as acidification(pH = 6) (n = 19 cells, 3 mice) or temperaturechanges (from 37° to 4°C) (n = 5 cells, 2 mice)(fig. S7).We <strong>in</strong>vestigated the orig<strong>in</strong> of the calcium<strong>in</strong>crease observed <strong>in</strong> the presence of APs andfound that it could be recorded <strong>in</strong> divalent-freeACSF solution (n = 26 cells, 6 mice; Fig. 3G),<strong>in</strong>dicat<strong>in</strong>g that it was generated preferentially byCa 2+ released from <strong>in</strong>tracellular stores and notonly by external calcium <strong>in</strong>flux. Depletion of Ca 2+stores by thapsigarg<strong>in</strong> (20 mM), an <strong>in</strong>hibitor ofthe microsomal Ca 2+ -ATPases (25), abolishedthe response to APs (n = 8 cells, 2 mice; Fig. 3G).At the end of these experiments, perfusion ofACSF conta<strong>in</strong><strong>in</strong>g calcium (2 mM) <strong>in</strong>duced anovershoot of the <strong>in</strong>tracellular calcium concentration,confirm<strong>in</strong>g the previous open<strong>in</strong>g of storeoperatedcalcium channels. Thus, contrary toolfactory neurons, GG cells, with their primarycilia localized <strong>in</strong> an extracellular matrix made oftype IV collagen and not <strong>in</strong> a mucus layer (fig.S1), can act <strong>in</strong>dependently from the calcium concentrationpresent <strong>in</strong> their environment.We next addressed the question of the behavioralrelevance of AP sens<strong>in</strong>g by GG cells.The isolated nature of the GG makes it suitablefor lesion experiments, allow<strong>in</strong>g axonal projectionbundles of GG cells to be sectioned (axotomy)(11). After surgery, pups were given back to theirmothers and mother-pup recognition was notaffected by the procedure. Thirty days after thelesion, we tested whether APs able to activate acalcium <strong>in</strong>crease <strong>in</strong> GG cells (ACSF + AP) alsomodified mice behavior <strong>in</strong> vivo (Fig. 4). APs <strong>in</strong>ducea typical freez<strong>in</strong>g reaction <strong>in</strong> rodents (26).Such a reaction was observed <strong>in</strong> control mice(n = 6) placed <strong>in</strong> a closed Plexiglas conta<strong>in</strong>er <strong>in</strong>the presence of ACSF + AP (Fig. 4, A and B, andmovie S1). After axonal lesions (Axo mice, n =5;Fig. 4C), this behavior was replaced by explor<strong>in</strong>gactivity. These Axo mice were not affected anymoreby the presence of APs <strong>in</strong> their environment(Fig. 4D and movie S2). Control and lesioned micewere equally efficient at f<strong>in</strong>d<strong>in</strong>g a buried cookie<strong>in</strong> the bedd<strong>in</strong>g (Fig. 4E).Pheromonal communication plays an importantrole <strong>in</strong> social <strong>in</strong>teractions among <strong>in</strong>dividualsof the same species, affect<strong>in</strong>g, <strong>in</strong> particular, sexual,territorial, and maternal behaviors (27). Wemorphologically characterized the GG and identifiedit as the olfactory subsystem mediat<strong>in</strong>g APdetection <strong>in</strong> neonate and adult mice. To performthis primordial function, the GG has a specializedand basic morphology: a ganglion protected fromthe external world by water-permeable kerat<strong>in</strong>.The GG acts as a warn<strong>in</strong>g system dedicated to therecognition of short-lived molecules encod<strong>in</strong>gdanger. These molecules require immediate attention.The unusual location of the GG at the tipof the nose, far from the ma<strong>in</strong> olfactory system,Downloaded from www.sciencemag.org on April 18, 2010109422 AUGUST 2008 VOL 321 SCIENCE www.sciencemag.org
CORRECTIONS & CLARIFICATIONSERRATUMPost date 20 November 2009Reports: “<strong>Grueneberg</strong> ganglion cells mediate alarm pheromone detection <strong>in</strong> mice“ byJ. Brechbühl et al. (22 August 2008, p. 1092). The next-to-last sentence of the text read “Thepresence of a GG has been identified <strong>in</strong> all mammalian species looked at so far, <strong>in</strong>clud<strong>in</strong>ghumans (15, 30).” Instead, it should read “The presence of a GG has been identified <strong>in</strong> allmammalian orders looked at so far, <strong>in</strong>clud<strong>in</strong>g human embryos (15, 30).”Downloaded from www.sciencemag.org on April 18, 2010www.sciencemag.org SCIENCE ERRATUM POST DATE 20 NOVEMBER 2009 1