Vomeronasal sensory neurons from Sternotherus odoratus (stinkpot

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IntroductionThe accessory olfactory system (AOS) has been well

established as a primary detector for odor informationconcerning social organization and reproductive status(Halpern and Martínez-Marcos, 2003). In addition, in reptilianspecies such as the turtle, lizard and garter snake, the AOS alsoserves to detect prey items (Wang et al., 1997; Fadool et al.,2001; Vitt et al., 2003). The best-studied peripheral sensoryorgan for the AOS is the vomeronasal organ (VNO). Sexualdimorphism in the reptilian and amphibian VNO is found atboth the level of gross anatomy (Dawley, 1998) and the single

sensory neuron (Fadool et al., 2001). There are sexuallydistinct differences in vomeronasal sensory neuron (VSN)soma diameter as well as in the composition and kinetics ofvoltage-activated conductances (Fadool et al., 2001).

In terrestrial vertebrates, the VNO is a compartment of theolfactory system that is separate and distinct from the mainolfactory system (MOS). In reptiles, it is larger than the mainolfactory epithelium (MOE), the sensory organ for the MOS(Ernst et al., 1994; Murphy et al., 2001; Labra et al., 2005). Inmost animal classes, the VNO is thought to mediate primarilypheromonal signals, but not exclusively (Baxi et al., 2006).

The mammalian signal transduction apparatus utilizedby vomeronasal sensory neurons (VSNs) in thevomeronasal organ (VNO) has been richly explored, whilethat of reptiles, and in particular, the stinkpot or muskturtle Sternotherus odoratus, is less understood. Given thatthe turtle’s well-known reproductive and matingbehaviors are governed by chemical communication, 247patch-clamp recordings were made from male and femaleS. odoratus VSNs to study the chemosignal-activatedproperties as well as the second-messenger systemunderlying the receptor potential. Of the total neuronstested, 88 (35%) were responsive to at least one of fivecomplex natural chemicals, some of which demonstrated adegree of sexual dimorphism in response selectivity. Mostnotably, male VSNs responded to male urine with solelyoutward currents. Ruthenium Red, an IP3 receptor (IP3R)antagonist, failed to block chemosignal-activated currents,while the phospholipase C (PLC) inhibitor, U73122,abolished the chemosignal-activated current within 2·min,implicating the PLC system in the generation of a receptorpotential in the VNO of musk turtles. Dialysis of severalsecond messengers or their analogues failed to elicitcurrents in the whole-cell patch-clamp configuration,negating a direct gating of the transduction channel bycyclic adenosine monophosphate (cAMP), inositol 1,4,5-

trisphosphate (IP3), arachidonic acid (AA), ordiacylglycerol (DAG). Reversal potential analysis ofchemosignal-evoked currents demonstrated that inwardcurrents reversed at –5.7±7.8·mV (mean ± s.e.m.; N=10),while outward currents reversed at –28.2±2.4·mV (N=30).Measurements of conductance changes associated withoutward currents indicated that the outward currentrepresents a reduction of a steady state inward current bythe closure of an ion channel when the VSN is exposed toa chemical stimulus such as male urine. Chemosignal-activated currents were significantly reduced when apeptide mimicking a domain on canonical transientreceptor potential 2 (TRPC2), to which type 3 IP3 receptor(IP3R3) binds, was included in the recording pipette.Collectively these data suggest that there are multipletransduction cascades operational in the VSNs of S.odoratus, one of which may be mediated by a non-selectivecation conductance that is not gated by IP3 but may bemodulated by the interaction of its receptor with theTRPC2 channel.

Key words: olfaction, VNO, transduction, PLC, TRPC2, inositol1,4,5-trisphosphate receptor (IP3R), musk turtle, Sternotherusodoratus.

Summary

The Journal of Experimental Biology 209, 1914-1927Published by The Company of Biologists 2006doi:10.1242/jeb.02206

Vomeronasal sensory neurons from Sternotherus odoratus (stinkpot/musk turtle)respond to chemosignals via the phospholipase C system

Jessica H. Brann1,* and Debra A. Fadool1,2,†

The Florida State University, Department of Biological Science, Program in 1Neuroscience and2Molecular Biophysics, Biomedical Research Facility, Tallahassee, FL 32306, USA

*Present address: 920 Fairchild Center, MC 2439, Department of Biological Sciences, Columbia University, New York, NY 10027, USA†Author for correspondence (e-mail: [email protected])

Accepted 13 March 2006

THE JOURNAL OF EXPERIMENTAL BIOLOGY

1915Phospholipase C in musk turtle vomeronasal organ

Pheromones [originally described by Karlson and Lüscher(Karlson and Lüscher, 1959)] are substances that are secretedto the external environment by an individual and received bya second individual of the same species, in which they releasea specific reaction, for example, a definite behavior or adevelopmental process (Rodriguez et al., 2002). While theVNO is responsive to pheromones, it also detects a variety ofchemosignals that may not fit within the classical definition ofa pheromone. Often, the VNO is considered a detector ofnonvolatile odorants, but this assertion is incorrect, since theVNO detects volatile odorants as well (O’Connell andMeredith, 1984; Meredith, 1991; Meredith, 2001; Halpern andMartínez-Marcos, 2003; Trinh and Storm, 2003; Leinders-Zufall et al., 2004). In addition, the MOE is known to respondto some pheromones (Restrepo et al., 2004). Hence we will usethe term ‘chemosignal’ to incorporate any chemical receivedby the VNO and potentially able to elicit an electrical signalencoded by this organ for chemical communication.

Although a limited amount of published molecularchemoreceptor information is available for reptiles (i.e. Vogtet al., 2002), it is generally believed that the initial VNOtransduction event is governed by the binding ofchemosignals to G-protein coupled receptors (GPCRs), aswell described for rodents (Dulac and Axel, 1995; Herradaand Dulac, 1997; Matsunami and Buck, 1997; Ryba andTirindelli, 1997; Pantages and Dulac, 2000). Although thenucleotide sequence and expression patterns of thesereceptors have not yet been published for the reptilian VNO,we do know that in the common musk/stinkpot turtleSternotherus odoratus they must be coupled to thedownstream G-protein G�i, which has restricted expression inthe microvilli of the turtle VSNs, is expressed at greaterlevels in male versus female turtle VNO, and isdevelopmentally increased in the VNO coincident withreproductive maturity of the turtles (Murphy et al., 2001).More is known for the downstream signaling cascade foundin this species, in which there appears to be continuity withthat known for rodents. Rodent VSNs utilize phospholipaseC (PLC) signaling (Runnenburger et al., 2002) to cleavephosphatidyl-inositol bis-phosphate (PIP2) intodiacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3)second messengers (Berridge and Irvine, 1984). DAG or oneof its derivatives, arachidonic acid (AA), may activate the ionchannel responsible for carrying the chemosignal-activatedcurrent, the canonical transient receptor potential channel,TRPC2 (Spehr et al., 2002; Lucas et al., 2003). This channelis a six-transmembrane domain nonselective cation channeland is a member of the TRP ion channel superfamily (Minkeand Cook, 2002). TRPC2 is expressed in the microvilli ofrats, mice and turtles (Liman et al., 1999; Murphy et al., 2001;Leypold et al., 2002; Stowers et al., 2002). In both rodentsand turtles, TRPC2 shows a developmental pattern ofexpression, with an increased expression correlated to sexualmaturity (Murphy et al., 2001). TRPC2 and the type IIIinositol 1,4,5-trisphospate receptor (IP3R3) demonstrate co-localization in VSN microvilli and can be co-

immunoprecipitated from rodent VNO (Brann et al., 2002),but the functional relevance of this protein–proteininteraction is yet unknown.

Several questions remain unanswered, therefore, whendiscussing transduction in the VNO of S. odoratus anddownstream vertebrate VNO transduction in general. First, itis not clear whether PLC signaling is important to thegeneration of a receptor potential in the turtle VNO. Second,it is unknown if, and how, TRPC2 is activated in the turtle.Third, TRPC2 in mouse VSNs was shown to be activated byan analogue of diacylglycerol (DAG), independently of IP3

(Lucas et al., 2003), therefore it is unknown if a TRPC2/IP3R3complex is required for the final transduction event, or simplyprovides a form of modulation of the transduction current.Fourth, DAG (and its derivatives) has now been shown toactivate TRPC3 and TRPC6 (Chyb et al., 1999; Hofmann etal., 1999; Clapham et al., 2001; Venkatachalam et al., 2001;Albert and Large, 2003; Pocock et al., 2004), and may alsohave a role in the VNO (Spehr et al., 2002; Lucas et al., 2003).The dialysis of a natural DAG analogue (called SAG) intorodent VSNs, however, did not elicit a current withchemosignal-like kinetics. Nonetheless, the effectiveness ofSAG as an activator of the cation channel was severelydiminished in TRPC2-null mice, suggesting that TRPC2 is thechannel activated by SAG (Lucas et al., 2003). DAG has beenreported to not activate TRPC1, TRPC4 or TRPC5 (Hofmannet al., 1999; Venkatachalam et al., 2003). Together, thesestudies demonstrate a putative direct role of DAG in gatingTRPC2, but do not address possible modulation, amplificationor regulation of the primary cationic conductance by IP3Rsmediated by the protein–protein interaction between TRPC2and IP3R3.

Here, we explore currents elicited with several naturalchemosignals, including urine, a reproductive musk and a preyitem. The use of the PLC system in the transduction ofchemosignal information is examined pharmacologically, andreversal potential analysis is used to demonstrate the presenceof two separate ionic conductances found in the musk turtleVNO. Finally, the role of the signaling complex betweenTRPC2 and IP3R3 in the turtle is examined by disruption ofthe complex with a peptide inhibitor.

Materials and methodsAnimal collection, care, and maintenance

Sternotherus odoratus Latreille (stinkpot or common muskturtle) were hand-caught during the reproductive season (Aprilto September) at Indian Pines (Opelika, AL, USA) or LakeJackson (Tallahassee, FL, USA) with permission from theState of Alabama Department of Conservation and the State ofFlorida Fish and Wildlife Conservation Commission. Turtleswere housed for short periods, usually 1–2 months, in 10 gallon(38·l) aquaria with rocks for basking, maintained on a 15·h:9·hlight:dark cycle and fed a standard diet of catfish pellets(Florida Farm and Feed, Tallahassee, FL, USA). Turtles usedin the described experiments were adults (Ernst et al., 1994;


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Fadool et al., 2001). Mass and plastron length were noted onthe day of capture as well as on the day of experimental use.The plastron, or ventral shell, of a turtle is often used tomeasure growth or size of the animal, given the more variablecurvature of the dorsal carapace (Ernst et al., 1994). Allprocedures were performed in accordance with The FloridaState University Animal Care and Use Committee.

Solutions

Ca2+-free Ringer contained (in mmol·l–1): 116 NaCl, 4 KCl,1 MgCl2, 15 glucose, 10 Hepes, 10 EGTA, pH·7.4. Ringercontained (in mmol·l–1): 116 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2,15 glucose, 10 Hepes, 5 sodium pyruvate, pH·7.4. Pipettesolution contained (in mmol·l–1): 100 KCl, 20 NaCl, 10 Hepes,2 MgCl2, 1.1 EGTA, 0.8 CaCl2, 2.5 ATP, 0.5 GTP, pH·7.4.Phosphate-buffered saline (PBS) contained (in mmol·l–1): 137NaCl, 2.7 KCl, 10 Na2HPO4, 2 KH2PO4, pH·7.4. All chemicalswere obtained from either Sigma Chemical Company (StLouis, MO, USA) or Fisher Scientific (Fairlawn, NJ, USA).

Electrophysiology

Turtles were killed by administering a lethal intraperitonealdose of sodium pentabarbitol (Butler Company, Columbus, OH,USA), followed by decapitation. The VNO was quicklydissected, grossly cut into quarters, and incubated in L-cysteine-activated papain (Worthington Biochemical Corp., Lakewood,NJ, USA) in Ca2+-free Ringer for 15–18·min while gentlyrotated at 30·r.p.m. (Orbital Shaker, Model 3520, Lab-LineInstruments, Melrose Park, IL, USA) at room temperature (RT).Following enzymatic treatment, quarters could be held at 4°Cfor up to 8·h prior to physical dissociation. Sensory neuronswere physically dissociated by trituration using a size-gradedseries of fire-polished siliconized Pasteur pipettes and were thenplated on to 0.01% poly-D-lysine hydrobromide (Sigma) and0.1% laminin (BD Biosciences, San Jose, CA, USA) coatedCorning #25000 dishes (Fisher) (Leinders-Zufall et al., 1997).

Acutely isolated vomeronasal sensory neurons (VSNs) wereviewed at 40� magnification (Axiovert 135, Carl Zeiss,Thornwood, NY, USA) with Hoffman modulation contrastoptics for patch-clamp recording (Hamill et al., 1981). Patchpipettes were fabricated from Jencons borosilicate glass(catalog number M15/10; Jencons Limited, Bedfordshire, UK),fire-polished to approximately 1·�m (bubble number 5.0)(Mittman et al., 1987) and coated near the tip with beeswax toreduce the pipette capacitance. Pipette resistances werebetween 7 and 10·M�, which produced high-resistance seals(between 8 and 14·G�) by applying gentle suction to the lumenof the pipette upon contact with the cell. In all experiments,cells were voltage-clamped at a holding potential (Vh) of–60·mV, unless specified otherwise. Voltage- and chemicallyactivated currents were recorded in the whole-cellconfiguration using an integrating patch-clamp amplifier(Axopatch 200B, Axon Instruments, Union City, CA, USA).The analog output was filtered at 5·kHz and digitally sampledevery 0.5–4·ms. Data acquisition, storage and analysis of thedigitized recordings were carried out using pClamp8.0

J. H. Brann and D. A. Fadool

software (Axon Instruments) in conjunction with Origin 4.1(Microcal Software, Inc., Northampton, MA, USA), SigmaPlot8.0 and SigmaStat 3.0 (Systat Software, Point Richmond, CA,USA). Statistical tests are as noted, with statistical significancein all tests defined at the 95% confidence interval (Steel andTorrie, 1980).

Second messengers and pharmacological agents

Second messengers and non-permeant analogues wereintroduced via the pipette during whole-cell recording. Topromote seal formation and initial controlled recordingconditions, pipettes were tip-filled 1–2·mm with control patchsolution and then back-filled with messenger or analogue; amethod we have previously calibrated and applied (Fadool andAche, 1992). Cyclic adenosine monophosphate (cAMP; 0.5 or1·�mol·l–1; back-fill pipette; Sigma) and inositol 1,4,5-trisphosphate (IP3; 1–100·�mol l–1; back-fill pipette, Sigma)were made as a stock solution stored at –20°C and diluted inpipette solution daily. Arachidonic acid (AA; 10·�mol l–1;back-fill pipette; Calbiochem, San Diego, CA, USA), a non-cell permeable polyunsaturated fatty acid (PUFA) derivedfrom DAG by a DAG lipase or from phospholipids byphospholipase A2 (Spehr et al., 2002), was prepared fresh dailyin pipette solution. 1-Oleoyl-2-acetyl-sn-glycerol (OAG;100·�mol·l–1; both bath application and back-fill pipette tested;Calbiochem) and 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG;100·�mol·l–1; back-fill pipette; Calbiochem), analogues ofdiacylglycerol (DAG), were reconstituted daily in DMSO(final concentration �0.1%). Ruthenium Red (RR), shown toinhibit IP3-evoked currents (Fadool and Ache, 1992;Kashiwayanagi et al., 2000; Taniguchi et al., 2000), wasfreshly made daily in Ringer at a concentration of 1·mmol·l–1

and filtered (25·mm diameter, 0.2·�m pore size, Whatman,Clifton, NJ, USA) to avoid occlusion of the multi-barrel pipette(bath application; Sigma). U73122, an inhibitor ofphospholipase C, was reconstituted in DMSO (finalconcentration �0.1%) and stored at –20°C as a stock solution(50·�mol·l–1; bath application; BioMol, Plymouth Meeting,PA, USA). A synthesized peptide (10·�mol l–1) designed totarget and interrupt the interaction domain between TRPC2 andIP3R3 with the peptide sequence GSAGEGERVSYRLRVIK-ALVQRYIETARRE (905–934·mTRPC2) (Tang et al., 2001)was reconstituted in pipette solution and stored at –20°C untiluse (back-fill pipette; GeneMed Synthesis, Inc., South SanFrancisco, FL, USA). Based upon the mouse sequence forTRPC2 (GenBank accession no. AAD17196) (Vannier et al.,1999), we utilized web-based protein prediction software(called TMpred – Prediction of Transmembrane Regions andOrientation; http://www.ch.embnet.org/software/TMPRED_form.html,) to determine that the predicted binding would alignwith the intracellular C-terminal tail of the channel (Hofmannand Stoffel, 1993; Hirokawa et al., 1998).

Chemical stimulation

During the reproductive season (April–September), muskwas harvested from Sternotherus odoratus once a month by



gently milking the posterior reproductive musk gland. Musksamples obtained from a single animal ranged between 1–2·�l.Turtle urine was obtained post mortem by inserting a 27-gaugeneedle directly into the bladder. Catfish extract was preparedfrom a commercially available pellet (Florida Farm and Feed,Tallahassee, FL, USA). Pellets (2·g) were hydrated in 20·ml ofhigh-quality water, ground by mortar and pestle, and thenclarified by low-speed centrifugation for 5·min at roomtemperature. All samples were rapidly collected, placed on ice,and stored undiluted at –80°C until diluted in Ringer on theday of experimentation.

Chemosignals were spritzed (700·ms pulse) onto isolatedVSNs using custom-fabricated seven-barrel glassmicropipettes (1.2mm outer diameter, catalog no. 17-12-M;Frederick Haer, Bowdoinham, ME, USA) coupled to apressurized valve system (Picospritzer, General Valve,Fairfield, NJ, USA). Dilution of the chemical between themultibarrelled pipette and the cell surface, an average distanceof two cell diameters, was estimated to be approximately 9%on the basis of the calculated K+ permeability method(Firestein and Werblin, 1989). Chemical concentrations arereported as the pipette concentration and are not corrected forthis dilution. The pipette concentration of urine was 1:5 fromfull strength, between 1:100 and 1:300 for musk, and between1:100 and 1:1000 for catfish extract. All dilutions wereprepared daily in turtle Ringer, which served as the controlvehicle in all conditions. If a neuron responded to the control,it was assumed to be a mechanical stretch-activated response,and no further use was made of the cell. The peak magnitudeof a response was measured as the difference in current fromthe baseline prior to presentation of the chemical to the peakoutward- or inward-evoked current within 500·ms of valveactivation of the picospritzer. Zero current (no response) wasdefined as no observable deflection or a deflection that wasless than four times the total peak-to-peak (range) noise level(membrane plus equipment) under control baselineconditions.

ResultsMass and plastron length of Sternotherus odoratus

The mass of each turtle was recorded upon arrival in thelaboratory, and again on the date of use forelectrophysiological recording. As one metric of good animalhusbandry, it was noted that a majority of animals grew bothin plastron length and in mass over their time in captivity (datanot shown; usually 1–2 months). A variety of turtles, includingmusk turtles, are well-known to be sexually dimorphic in sizeas traditionally indexed by the length of the plastron (Ernst etal., 1994). Over our 4-year collection, female musk turtles hadlonger plastrons than males, reaching statistical significance inyears 2003 and 2004 [Fig.·1A, two-way ANOVA,Student–Neuman–Keuls (SNK) pairwise multiple comparisonbetween sex and year, P�0.05]. Using adult mass as a measureof dimorphism is not as reliable due to the fluctuation in themass of the female during egg production (Fig.·1B).

Chemically activated properties

247 vomeronasal sensory neurons (VSNs) isolated from atotal of 45 animals (31 females and 14 males) were tested fortheir ability to generate a current in response to stimulationwith up to five different natural chemosignals (see Materialsand methods; male musk, female musk, male urine, femaleurine and catfish extract). Each chemosignal was presented inthe absence of the others, and the order of presentation wasvaried. Current responses of both inward and outward polaritytypically rose to a maximum over several hundred millisecondsand subsequently declined to rest over a period of 3–4·s(Fig.·2). For all odor-exposure trials, neurons were clamped ata holding potential (Vh) of –60·mV, at which there is novoltage-gated channel activity. Eighty-eight of the 247 VSNs(35%) responded to the chemosignals presented (Fig.·3A). Ofthe 88 responsive VSNs, 40 (45%) responded to only one ofthe five chemicals presented in the array. The remaining VSNs(48 neurons, 55%) responded to between two and four of thefive presented chemicals. None of the 88 VSNs responded to

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Fig.·1. Plastron length and mass of Sternotherus odoratus, by year.(A) Adult female (filled circles) versus adult male (open squares)plastron length, S. odoratus versus collection year (asterisk denotessignificantly different between sexes in years shown; mean ± s.e.m.;two-way ANOVA, SNK pairwise multiple comparison between sexand year, P�0.05). (B) Mass of adult female (filled circles) and male(open squares) Sternotherus odoratus versus collection year (notsignificantly different; mean ± s.e.m.; two-way ANOVA, P�0.05). Nvalues are shown beside symbols.


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all five of the presented chemicals. The majority (64%) ofVSNs responding to the tested chemosignals were isolatedfrom female musk turtles due to animal collection bias, butfemales overall had a slightly lower response rate (defined bya response to at least one of the five chemicals presented; 34%)than did males (38%).

The recorded mean input resistance (RN) of 2.9±0.3·G�(N=11) of VSNs at rest was similar to that previously recorded(Fadool et al., 2001) and also underscores the importance of


small amplitude chemosignal-activated currents in thesespecialized sensory neurons. We have previously characterizedthe peak current response amplitudes for natural chemicalsusing an entropy (H metric) index from information theory thatquantified the breadth of tuning for these neurons (Fadool etal., 2001). This study now furthers our initial characterizationby exploring frequencies of response sorted to both sex andpolarity. Here we report that chemosignal-activated currentscan be of either polarity, depending on the cell and the

Fig.·2. Representative examples of chemosignal-activated current obtained with chemicalstimulation of vomeronasal sensory neurons(VSNs). (A) A male vomeronasal sensoryneuron (VSN) when stimulated with catfishextract exhibited an outward current, as definedby the polarity of the response obtained atholding potential Vh=–60·mV. The black barabove the trace denotes the time (700·ms) thatthe VSN was presented with chemosignaldelivered via a pressurized glass pipetteapproximately two cell-widths away from the VSN. (B) As in A but for a female VSN stimulated with female musk. Current record isrepresentative of an inward current, as defined by the polarity of the response obtained at Vh=–60·mV.

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Fig.·3. The percentage of cellsresponding with a given polarity to aparticular chemosignal is differentbetween male and femalevomeronasal sensory neurons(VSNs). (A) Histogram plot of thethree different response categories ofVSNs, separated on the basis of sex;male, gray; female, black. (B–F)Histogram plots of the percentage offemale and male VSNs respondingwith an outward (black bars) orinward (white bars) current to (B)male musk, (C) female musk, (D)male urine, (E) female urine, (F)catfish extract. *Significantlydifferent response frequency ofinward and outward current acrosssex by �2 analysis, P�0.05; NS, notsignificantly different. N values areshown beside bars.



chemosignal tested. Of the collective 165 responses generatedby the 88 VSNs responding to chemosignals, outward currentswere generated roughly twice as frequently as inward currents(107=outward responses; 58=inward responses; Fig.·3). Themajority of chemosignals tested could evoke both inward andoutward current dependent upon the individual VSN, butwithin a single VSN, a single chemosignal never elicited dualpolarity, recording at a fixed command potential (Vc)=–60·mV.Most notably, a similar number of VSNs from females (17cells, 30%) and males (6 cells, 20%) exhibited responses ofboth polarities when responsive to more than one chemosignal(data not shown). �2 analysis of the frequency pattern ofoutward and inward chemosignal-evoked current responsesbetween male and female neurons indicated that the responsepattern to male musk, male urine and catfish extract isstatistically different across sex (Fig.·3B,D,F). More femaleneurons responded to male musk than did male neurons, butthe response to female musk was not significantly differentacross sex (Fig.·3B,C; �2 analysis). Male urine evoked onlyoutward currents in male VSNs (significantly different �2

analysis; Fig.·3D) but the response to female urine was notsignificantly different across sex (Fig.·3E). The stimulus mostfrequently eliciting a current response in both male and femaleVSNs was catfish extract (males, 47%; females, 50%) andfemale urine (males, 47%; females 40%) (Fig.·3E,F).

Chemosignal responses were examined in the absence ofpharmacological agents for possible attenuation with repeatedstimulation over time. In Fig.·4A (top trace), an isolated VSNwas patch-clamped in the whole-cell configuration at a Vh of–60·mV. The VSN was then exposed to a 700·ms puff ofchemosignal, denoted by the black bar above the recording.The cell was then stimulated again at varying time intervals.The same chemosignal response 6·min after initialchemosignal exposure is shown in the bottom trace in Fig.·4A.As is shown in Fig.·4B, the normal chemosignal response wasnot altered over time (one-way repeated measures ANOVA,P�0.05).

Ruthenium Red fails to alter chemosignal-activated currents,but a phospholipase C inhibitor reduces the chemosignal-

activated current

Although IP3R3 is expressed in the VNO of Sternotherusodoratus (J. H. Brann, unpublished data), it is not clear if it isfound at the plasma membrane, as in olfactory cilia, or if it isrestricted to the endoplasmic reticulum (ER). If a plasmamembrane IP3R has a direct role in the generation of achemosignal-activated current, we reasoned that exposure toRuthenium Red (RR), an IP3R blocker, should reduce or alterthat current. RR has been used to block IP3-evoked currents inboth the vomeronasal system and in the main olfactory system(Fadool and Ache, 1992; Miyamoto et al., 1992; Taniguchi etal., 1995; Kashiwayanagi et al., 2000). In this set ofexperiments, a baseline response to a given chemosignal wasfirst obtained (defined as time 0, t0), and 1·mmol·1–1 RR wasadded to the bath surrounding the VSN. The chemosignalresponse was then recorded over the following 10·min (t10)

(Fig.·4C). Addition of RR to the bath did not alter thechemosignal response by 10·min (Fig.·4D; not significantlydifferent, two-way repeated measures ANOVA, P�0.05).

These experiments suggested that a plasma membraneIP3R did not serve as the main carrier of the chemosignal-activated current in musk turtle VSNs, but two questionsremained. First, it is not known whether the phospholipase C(PLC) system is required for the generation of chemosignal-activated currents in the musk turtle. Second, it is not clearwhat role, if any, the IP3R may have in modulating the ionchannel that is the primary carrier of the chemosignal-activated current. Therefore, a second set of VSNs wereexamined to investigate whether the chemosignal-activatedcurrent could be altered with the addition of a membrane-permeant phospholipase C inhibitor, U73122. Again, abaseline chemosignal response was obtained, 50·�mol·1–1

U73122 was added to the bath, and chemosignal-activatedcurrent was monitored for the following 10·min (Fig.·4E). Inthis case, drug treatment with U73122 did rapidly (within2·min) and significantly reduce the chemosignal-activatedcurrent, with the effect lasting for the duration of the test(Fig.·4F, significantly different, two-way repeated measuresANOVA, followed by SNK-pairwise multiple comparisonbetween treatment and time, P�0.05). The bath was notcontinually perfused, hence VSNs were not examined forwashout recovery of the chemosignal-activated current.Neither the lack of response to RR (Fig.·4C,D) nor thereduction in current induced by U73122 (Fig.·4E,F) wasdependent on current polarity, thus data were pooled forinward and outward current responses, and an example ofeach is demonstrated in the composition of this figure.

Second messenger analogues do not activate currents ofeither inward or outward polarity

Since the chemosignal-activated current was blocked byinhibiting phospholipase C, which would interfere with thegeneration of the two second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), we attemptedto elicit currents that mimicked the chemosignal-activatedcurrent by including various second messenger analogues inthe recording pipette while in the whole-cell configuration(Fig.·5). Fig.·5A is representative of recordings made at a Vh

of –60·mV (below the threshold for voltage-activation). When0.5 or 1·�mol·1–1 cAMP, 240·�mol·1–1 IP3 or 125·�mol·1–1

1-oleoyl-2-acetyl-sn-glycerol (OAG; an analogue of DAG)were included in the patch pipette, no deflections were seen(Fig.·5B–D). A polyunsaturated fatty acid (PUFA) derivativeof DAG, arachidonic acid (AA), was also included in therecording pipette, but failed to induce current fluctuationsother than rare rapid transients (Fig.·5E). An additional,naturally occurring DAG analogue, SAG, was also tested(Fig.·5F). SAG did not elicit a chemosignal-like response, butdid appear to make the membrane unstable, probably due toits incorporation into the membrane. SAG was tested not onlyin the whole-cell configuration, but also on inside-out patches(N=4; data not shown), and in combination with IP3 (N=5; data


1920

not shown), all of which failed to elicit chemosignal-likecurrents.

Reversal potential analysis supports a hypothesis of twochemosignal-activated conductances in the vomeronasal

organ of S. odoratus

Since analogues failed to elicit chemosignal-like current, we


tried to examine the ionic basis of the receptor potential as ameans to gather evidence for a particular channel type in theformation of the chemosignal response in VSNs. We thereforecharacterized chemosignal-activated currents by means ofreversal potential analysis. Representative examples of thereversal of an outward current (as defined by the polarity ofthe response at –60·mV) and an inward current are shown in

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Fig.·4. Ruthenium Red (RR), an antagonist of the IP3R, fails to block chemosignal-activated currents in S. odoratus vomeronasal sensory neurons(VSNs) while an inhibitor of phospholipase C (U73122) does block chemosignal-activated currents. (A) A representative example of the responseof a female VSN to catfish extract at 0·min (top trace) and 6·min later (bottom trace) without pharmacological perturbation. The chemosignalpresentation is shown as black bar above the response. This particular cell was used primarily for reversal potential analysis, hence the VSN hadbeen exposed to the chemical signal twelve times within the time span described. (B) Histogram plot of the normalized current magnitude(normalized to first exposure to chemosignal; t0) over time. Cells were sampled at varying time points (tn), such that not every cell was sampledfor each of the time points. The amplitude of the chemosignal-activated current was not altered over 12·min (one-way repeated measures ANOVA,P�0.05). (C) A representative example of the response of a female VSN to male urine at 0·min (top trace) and 6·min (bottom trace) following bathapplication of 1·mmol·1–1 RR. (D) Histogram plot of the normalized current magnitude (normalized to t0) over time. The chemosignal-activatedcurrent is not altered over 10·min (not significantly different by treatment or time, two-way repeated measures ANOVA, P�0.05) in response toRR treatment. The normal chemosignal response over time is denoted by the broken line. (E) A representative example of the response of a femaleVSN to catfish extract at 0·min (top trace) and 8·min (bottom trace) following bath application of 50·�mol·l–1 U73122. (F) As in D but for U73122treatment. Asterisks denote significant difference between treatments, two-way repeated measures ANOVA, followed by SNK pairwise multiplecomparison between treatment and time, P�0.05). N values are shown beside bars. (A,C,E) Broken line denotes baseline current.



Fig.·6A,B. The outward current depicted reversed at –20.8·mV,based upon a linear regression to the peak amplitude of thechemosignal-activated current at varying potentials(Fig.·6A,C). The inward current reversed at a similar potential,–27.9·mV (Fig.·6B,D). The average outward current reversedat –28.2±2.37·mV (means ± s.e.m.; N=30) and the inwardcurrent at –5.7±7.79·mV (N=10). The range of currentreversals sorted per chemosignal are plotted in Fig.·6E,F foroutward and inward polarity, respectively.

In addition to probing the reversal potential for thechemosignal response, we were interested in whetherchemosignal application was accompanied by conductancechanges in the VSN. If the chemosignal caused an ion channelto open, membrane resistance would decrease and conductancewould increase, according to Ohm’s Law and the calculatedtime constant. However, if an odorant caused an ion channelto close, exactly the opposite would occur, and conductance

would decrease. Conductance changes were measured byinjecting a series of five hyperpolarizing pulses fromVh=–60·mV to –90·mV (Vc) before, during and afterchemosignal stimulation eliciting outward currents (Fig.·7).Chemosignal-evoked inward currents were not tested. At rest,VSNs had a mean input resistance (RN) of 2.9±0.3·G� and amembrane time constant (�) of 17.7±3.2·ms (N=11). Analysisof the current at the peak of the chemosignal response, whencompared to the same hyperpolarizing step before and afterchemosignal exposure, revealed a significant reduction incurrent (from 10.9±0.8 to 6.6±0.9·pA) and conductance (from6.3±1.3 to 1.0±0.3·pS). In addition, a concomitant significantincrease in RN (5.7±0.9·G�) was seen as well as a significantdecrease in � to 4.3±1.2·ms (paired t-test, P�0.05; Fig.·7).

A peptide directed against the TRPC2-IP3R3 interactiondomain blocks the chemosignal-activated current

We previously demonstrated that the ion channels TRPC2and IP3R3 participate in a protein–protein interaction in theVNO of the rat (Brann et al., 2002). The following experimentswere designed to probe the question of a functional role of thisprotein–protein interaction, as well as to find possible commonsignal transduction mechanisms between the rat and the turtle.First, we sought to determine the functional role of the physicalinteraction between TRPC2 and IP3R3. A recent publicationusing GST-fusion proteins of portions of these two channelsdemonstrated that calmodulin (CaM) and IP3Rs share acommon binding site on the carboxyl terminal of all TRPCchannels (Tang et al., 2001). TRPC2 contains two such bindingdomains for CaM and all isoforms of the IP3R, the first beingamino acids 901–936, and the second being amino acids942–1072. IP3R3 also has a TRPC2 binding domain (aminoacids 669–698) (Tang et al., 2001). We generated a peptidemimicking the first binding domain in the C terminus ofTRPC2 (Fig.·8A), and included it in the patch pipette whilerecording from isolated VSNs in the whole-cell configurationand stimulating the VSN with a battery of differentchemosignals (see Materials and methods). In all experiments,the designation 0·min is defined as the time at which the VSNwas first exposed to a given chemosignal, or typically less than30·s after breakthrough to the whole-cell configuration. Atypical response from a VSN at 0·min is shown in the top traceof Fig.·8C. Inclusion of the peptide attenuated the chemosignalresponse within 5·min, with the effect lasting until 10·min(times later than 10·min were not tested) (Fig.·8B,C). Inclusionof boiled peptide in the recording pipette as a control did notalter the chemosignal response over 10·min (data not shown).Boiling results in denaturation of the peptide, and the peptidewould presumably fail to bind the IP3R, as has beendemonstrated previously using similar peptide blockingapproaches (Fadool and Ache, 1992; Holmes et al., 1996).

DiscussionPreviously we had demonstrated sexual dimorphism in

voltage-activated currents and somata size of VSNs isolated

Fig.·5. Dialysis of the second messengers cAMP, IP3, OAG,arachidonic acid (AA), and SAG in isolated S. odoratus vomeronasalsensory neurons (VSNs). Analogues were made as described (seeMaterials and methods) and back-filled in the recording pipette toallow diffusion upon attaining the whole-cell configuration. VSNswere voltage-clamped at Vh=–60·mV. (A) A representative exampleof a VSN that had not been dialyzed with a second messengeranalogue in order to illustrate stability of the patch. (B) 0.5 or1·�mol·l–1 cyclic adenosine monophosphate (cAMP) (N=27). (C)240·�mol·l–1 inositol 1,4,5-trisphosphate (IP3) (N=20). (D)125·�mol·l–1 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol (DAG), applied to either the bathsurrounding the VSN or within the recording pipette (N=15). Traceshown is an example of pipette perfusion. (E) 10·�mol·l–1 AA, apolyunsaturated fatty acid (PUFA) derivative of DAG (N=11). (F)10·mmol·l–1 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), a naturalanalogue of DAG (N=6).

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1922

from the musk turtle, S. odoratus. In accordance withpublished data on the life history of this turtle species (Ernstet al., 1994), the female turtles in our trapped sample hadlonger plastrons than males. Because the VNO size can varywith age, reproductive cycle, season or mating strategy


(Wilson and Raisman, 1980; Dawley and Crowder, 1995), itwould be of interest to look at this variable in future studieswhere the organ was not harvested for electrophysiologicalanalysis. In this study at the cellular level, we now demonstratethat chemosignal-activated currents show a degree of sexual

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Fig.·6. Analysis of chemosignal-activated reversal potentials supports a cationic receptor potential in vomeronasal sensory neurons (VSNs) ofS. odoratus. (A,B) Response of a female neuron to (A) catfish extract (1:10 dilution), (B) female musk (1:100 dilution). Broken horizontal linesindicate baseline currents. (C,D) Plotted current–voltage relation for the families of currents evoked in A and B, respectively. (E,F) Scatter plotof estimated reversal potentials derived in C and D for chemosignal-evoked outward (E) and inward (F) currents, respectively. For eachchemosignal, the vertical line depicts the mean reversal potential if more than one sample was obtained. The average reversal potential for theoutward current was –28.2±2.37·mV (N=30) and that for the inward current was –5.7±7.79·mV (N=10).



dimorphism. In particular, we found that male VSNs respondedmore readily to female musk, while female VSNs respondedmore readily to male musk; both chemosignals could eliciteither an inward or outward current, but not in the same VSN.While female urine and catfish extract elicited responses ofboth polarities equally well from both male and female VSNs,male VSNs responded to male urine with only an outwardcurrent. Current-clamp studies are needed to discern whetherthese chemosignals are altering action potential firingfrequency (Moss et al., 1997; Inamura and Kashiwayanagi,2000) and if there is evoked patterned activity that is sexspecific.

Through our single cell-electrophysiology analysis, wefound that several VSNs were found to respond to more thanone chemosignal with opposite polarities of response. Thismay provide evidence for at least two signaling cascades in asingle VSN. This assertion is supported by evidence that morethan one G-protein-coupled receptor may be expressed in asingle VSN (Martini et al., 2001; Spehr and Leinder-Zufall,

2005). Additionally, attempts to elicit chemosignal-activatedcurrents outside the mating season (April–September) failed(J.H.B., unpublished). One might conjecture that not only sizebut also components of the cellular function of the VNO (orcentral projections) may be seasonally regulated.

The fact that Ruthenium Red (RR) did not significantly alterchemosignal-activated currents may suggest that IP3R3 is notlocalized to the plasma membrane as has been demonstrated incilia of olfactory sensory neurons (Fadool and Ache, 1992;Restrepo et al., 1992; Cunningham et al., 1993; Honda et al.,1995; Cadiou et al., 2000). These data, however, fail to revealwhether IP3R3 is an important member of the signaltransduction scaffold. RR is classically used as a blocker of themitochondrial Ca2+ uniporter or of the ryanodine receptor(RyR) located on the ER. It has also been found, when appliedextracellularly, to block the IP3R localized to the dendrite ormicrovillus of an olfactory or vomeronasal sensory neuron,respectively (Fadool and Ache, 1992; Miyamoto et al., 1992;Taniguchi et al., 1995; Inamura et al., 1997; Taniguchi et al.,2000; Kashiwayanagi et al., 2000). An alternative possibilitythat must be considered is that the TRPC2/IP3R3 complex maypresent a configuration that is inaccessible for pharmacologicalinterception with RR.

This study provides the first evidence that dissociated VSNs

100 ms

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Fig.·7. Vomeronasal sensory neurons (VSNs) from S. odoratus exhibit a chemosignal-evoked conductance decrease linked with outward currents.A VSN from a female S. odoratus was held at –60·mV (Vh) with five hyperpolarizing steps to –90·mV (Vc) injected prior (black), during (gray)and following (black) chemosignal stimulation with catfish extract. Inward chemosignal-evoked conductance changes were not tested.

Fig.·8. A peptide directed against the interaction domain betweenTRPC2 and IP3R3 blocks the chemosignal-activated currents in S.odoratus vomeronasal sensory neurons (VSNs) when included in thepatch pipette in the whole-cell configuration. (A) Amino acidsequence of the synthesized peptide (derived from mTRPC2, aminoacids 905–934) (Tang et al., 2001). (B) Histogram plot of thechemosignal-activated current over 10·min when 10·�mol·l–1 peptideis included in the recording pipette. The normal chemosignal-activated current over time is denoted by a broken line (see Fig.·4).Current obtained for each cell at varying time points (tn) wasnormalized to its first exposure to chemosignal (t0) Asterisks denotesignificant differences between control and peptide, two-way repeatedmeasures ANOVA, followed by SNK pairwise multiple comparisonbetween treatment and time, P�0.05). (C) Representative example ofpeptide treatment. While in the whole-cell configuration, the VSN waspresented with a chemosignal, and a baseline (immediately after thewhole-cell configuration was obtained) recording was made (top). Thesame chemosignal-activated current is shown 10·min after peptideinfusion (bottom). Broken line denotes baseline current.

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GSAGEGERVSYRLRVIKALVQRYIETARRE


1924

from the semi-aquatic turtle, S. odoratus, utilize thephospholipase C (PLC) system in the detection of sociallyrelevant stimuli. A membrane-permeable inhibitor of PLC,U73122, significantly reduced the chemosignal-activatedcurrent. These data support the hypothesis that thephosphatidylinositol system underlies the chemosignalresponse in VSNs. However, it is not clear whether thederivatives of PIP2, DAG or IP3, have a direct role in gatingthe ion channels involved. In guinea pig ileal smooth musclecells, the activation of the muscarinic receptor-operatedcationic current (likely via a TRP channel), is dependent uponPLC, but is not dependent on either IP3 or DAG (Zholos et al.,2004). PIP2 may directly modulate the TRP channel inquestion, as is seen with TRPM5, TRPM7, TRPM7 andTRPV1 (Hilgemann et al., 2001; Liu and Qin, 2005; Chuanget al., 2001; Liu and Liman, 2003; Prescott and Julius, 2003).In these cases, PIP2 sometimes acts to suppress channelactivity. Interestingly, when mutations are made within thePIP2 binding domain on TRPV1, the channel becomes moresensitive to chemical or thermal stimuli (Prescott and Julius,2003). In the case of TRPM5 and TRPM8, PIP2 activates thechannel. PIP2 can reverse the desensitization of TRPM5 (Liuand Liman, 2003). Blocking PIP2 synthesis inhibits TRPM8activity (Liu and Qin, 2005). Therefore, PIP2 has the potentialto differentially and directly regulate several members of theTRP ion channel superfamily, and may be directly modulatingTRPC2 current in the VNO, either directly affecting theprimary receptor potential, or perhaps the formation of a signalcomplex with IP3R3.

Using our experimental design, dialysis with the secondmessengers cAMP, IP3 or DAG failed to elicit a response inisolated VSNs. Although cAMP did not have an effect, othercyclic nucleotides could be important in a transductionpathway in the musk turtle VNO (Taniguchi et al., 1996). Thelack of response with dialyzed messengers could be explainedin several ways. First, the whole-cell patch seal was formed onthe soma, not on the thin dendritic extension or microvilli.Thus it is possible that the messengers may not have reacheda critical concentration near the transduction apparatus in themicrovilli (Berghard and Buck, 1996; Ryba and Tirindelli,1997; Liman et al., 1999; Menco et al., 2001). Contrary to thisexplanation is that soma dialysis of second messengers by oneof the authors has been shown to evoke quite large currents inolfactory sensory neurons of lobster with outer dendrites(primary site of transduction) greater than 1000·�m from thecell body (Fadool and Ache, 1992). This space problem wasapparently alleviated in frog VSNs by using flash photolysisof caged IP3, and selective release of IP3 only near themicrovillus resulted in a series of transient inward currentswhen the membrane was held at –70·mV (Gjerstad et al.,2003). Second, the VSNs studied here were enzymaticallyisolated, and may have altered the ability of the VSN torespond to dialyzed second messenger; however, this is lesslikely because VSNs isolated in the same manner were shownto be responsive to chemosignals, and cells lacking microvilliwere not included in the current study. Third, PIP2 could


interact directly with TRPC2 in the musk turtle, bypassing theneed for either IP3 or DAG, as discussed above. Anotherpossibility is that DAG and IP3 must be presented in tandemfor effects to be detected; such synergism in the generation ofa non-selective cationic current has been demonstrated inrabbit portal vein myocytes (Albert and Large, 2003). Thishypothesis was informally tested here yet the combinedpresentation of IP3 and the DAG analogue, SAG, did not elicitcurrents of either inward or outward polarity. Finally,arachidonic acid, a polyunsaturated acid (PUFA) derived fromDAG previously reported to generate chemosignal-likecurrents in the rat vomeronasal system (Spehr et al., 2002), didnot elicit chemosignal-like currents in isolated S. odoratusVSNs but did appear to evoke rapid transients, the function ofwhich is unclear.

Two separate reversal potentials for inward and outwardcurrents were revealed. The majority of chemosignals, with theexception of male urine, were capable of eliciting bothpolarities of response; hence, most chemosignals did notappear to be stereotypically connected to a particular signaltransduction cascade. Male urine, however, may be operatingexclusively as a territorial cue, and the receptive pathway forsuch information may be stereotyped. The outward current,reversing at approximately –30·mV, may be due to the closureof an ion channel of unknown identity. Although a noveltransduction mechanism in the musk turtle VNO, such achannel operates in several sensory organs, including the visualsystem and the mouse VNO (Matesic and Liebman, 1987;Moss et al., 1997). The inward current reversed close to 0·mV,indicating that this current is due to a nonselective cationchannel, such as TRPC2. A good future directive would be toexplore whether the musk turtle VNO may express a Ca2+-activated non-selective (CaNS) cation channel like that foundin the hamster (Liman, 2003), or a Ca2+-activated chloridechannel, similar to that seen in olfactory sensory neurons thatwould contribute a late-phase amplification of the initial Ca2+

current (Kleene, 1993; Lowe and Gold, 1993; Paysan andBreer, 2001).

The interaction between TRPC channels and the IP3R hasbeen well established (Tang et al., 2001; Yuan et al., 2003;Brann et al., 2002). Here, we sought to demonstrate a role forthis interaction in the VNO of the musk turtle. Recent GST-fusion work has shown (Tang et al., 2001) that IP3R3 andcalmodulin bind to a conserved amino acid sequence found onmost TRPC channels, including mTRPC2 (mouse TRPC2;amino acids 901–936). mTRPC2 appears to be somewhatunique within the canonical TRP superfamily in that itpossesses an additional binding site (amino acids 944–1072)(Tang et al., 2001). We designed a peptide (see Materials andmethods) against this sequence to interrupt the binding ofIP3R3 to TRPC2 and analyzed its effect on the chemosignalresponse over time in musk turtle VSNs, and found that10·�mol·1–1 peptide nearly abolished the chemosignal-activated current by 10·min. The use of a peptide to disrupt theinteraction between the IP3R and a TRPC channel has beenshown previously; in Xenopus oocytes, suppression of the



mTRPC5 current was seen with preinjection of a peptidemimicking the IP3 binding domain of the IP3R (Kanki et al.,2001). These results indicate that there is a functional role forthe IP3R in the VNO of the musk turtle. Since dialysis of IP3

did not elicit a current alone, it is possible that the IP3R isserving to amplify the initial receptor potential generated byTRPC2, to achieve sensitivity to repeated stimulation withchemosignal, or to modulate seasonality in this species. It isalso possible that when IP3R/TRPC2 are in a complex, the IP3ligand could have less affinity or lack access to the receptor.This later possibility would be consistent with our data thatdemonstrate a lack of current after IP3 dialysis and failure ofan IP3R antagonist (RR) to significantly block chemosignal-activated current.

The experiments presented here demonstrate that the PLCsystem is required in VSNs from S. odoratus to detect a varietyof natural chemical cues, including urine and a reproductivemusk known to mediate mating behaviors/courtship (Eisner etal., 1977). This finding is consistent with studies in the rodent(Spehr et al., 2002; Lucas et al., 2003). In addition, it is likelythat the TRPC2-IP3R3 protein–protein interaction previouslydemonstrated in rodent (Brann et al., 2002) does have afunctional role in the chemosignal response of VSNs, asinterrupting the interaction resulted in loss of the chemosignalresponse. These data imply that the connection between theinternal Ca2+ stores is important to the maintenance of theresponse in the VNO, and may have a role in the ability of theVNO to respond to chemosignals repetitively without adecrease in action potential firing frequency, a property that isnot found in the main olfactory system (Liman and Corey,1996). Although second messenger dialysis failed to elicit achemosignal-like current, reversal potential analysis supportsthe existence of a conductance consistent with a nonspecificcation channel such as TRPC2, the expression of which wasfound in S. odoratus previously (Murphy et al., 2001). Onevery interesting result of these studies was that outwardcurrents (as defined at –60·mV) may be due to the closure ofan inwardly conducting ion channel. This is not the first reportof such a channel in the VNO (Moss et al., 1997; Moss et al.,1998), although it is the first report in the turtle.

List of abbreviationsAA arachidonic acidAOS accessory olfactory systemANOVA analysis of varianceCaM calmodulincAMP cyclic adenosine monophosphateCaNS Ca2+-activated non-selectiveDAG diacylglycerolER endoplasmic reticulumGPCR G-protein-coupled receptorIP3 inositol 1,4,5-trisphosphateIP3R IP3 receptorIP3R3 type 3 IP3 receptorMOE main olfactory epithelium

MOS main olfactory systemOAG 1-oleoyl-2-acetyl-sn-glycerolPBS phosphate buffered saline PIP2 phosphatidyl-inositol bis-phosphatePLC phospholipase CPUFA polyunsaturated fatty acidRN input resistanceRR Ruthenium RedRT room temperatureRyR ryanodine receptorSAG 1-stearoyl-2-arachidonoyl-sn-glycerolSNK Student–Newman–Keulst0 first exposure to chemosignaltn exposure to chemosignal at time ntp time pointTRPC2 canonical transient receptor potential 2Vc command potentialVh holding potentialVNO vomeronasal organVSN vomeronasal sensory neuronU73122 PLC inhibitor� time constant

J.H.B. was supported by a NIH/NIDCD Ruth L.Kirschstein National Research Service Award (NRSA) F31-DC006153 and by a NIH/NIDCD Predoctoral ChemosensoryTraining Grant T32-DC00044 to the Florida State University.We would like to thank Drs Francis Rose, Mary Mendoncaand Craig Guyer for the donation and collection of animals.We would also like to thank Mr Roger Thompson, Mr ChadThorson, and Ms Randa Perkins for invaluable fieldassistance.

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Vomeronasal sensory neurons from Sternotherus odoratus (stinkpot