RESEARCH ARTICLE Ammonia sensing by neuroepithelial cells and ventilatory responses to ammonia in rainbow trout

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1 2678 The Journl of Experimentl Biology 214, Pulished y The Compny of Biologists Ltd doi:1.1242/je RESEARCH ARTICLE Ammoni sensing y neuroepithelil cells nd ventiltory responses to mmoni in rinow trout Li Zhng 1, Colin A. Nurse 1, Michel G. Jonz 2 nd Chris M. Wood 1 1 Deprtment of Biology, McMster University, 128 Min Street West, Hmilton, ON, Cnd, L8S 4K1 nd 2 Deprtment of Biology, University of Ottw, 3 Mrie Curie, Ottw, ON, Cnd, K1N 6N5 Author for correspondence (mrshnzl@gmil.com) Accepted 1 My 211 SUMMARY Ammoni, the third respirtory gs in teleost fish, cts s n cute stimulnt to ventiltion in mmoniotelic rinow trout. We investigted whether this sensitivity is mintined in trout chroniclly exposed (1 months) to high environmentl mmoni [HEA, 25 mmol l 1 (NH 4 ) 2 SO 4 ] in the wter, nd whether gill neuroepithelil cells (NECs) re involved in mmoni sensing. Hyperventiltion ws induced oth y cute externl (NH 4 ) 2 SO 4 exposure [25 or 5 mmol l 1 (NH 4 ) 2 SO 4 ] nd y intr-rteril (NH 4 ) 2 SO 4 injection (58 mmol kg 1 of mmoni) in control trout, ut these responses were olished in chronic HEA nimls. Hyperventiltion in response to cute mmoni exposure persisted fter ilterl removl of ech of the four gill rch pirs seprtely or fter comined removl of rches III nd IV, ut ws delyed y removl of gill rch I, nd eliminted y comined removl of rches I nd II. NECs, identified y immunoleling ginst 5-HT, were minly orgnized in two lines long the filment epithelium in ll four gill rches. In control trout, NECs were slightly smller ut more undnt on rches I nd II thn on rches III nd IV. Chronic HEA exposure reduced the density of the NECs on ll four rches, nd their size on rches I nd II only. Fur- 2 fluorescence imging ws used to mesure intrcellulr free clcium ion concentrtion ([C 2 ] i ) responses in single NECs in short-term (24 48 h) culture in vitro. [C 2 ] i ws elevted to comprle extent y perfusion of 3 mmol l 1 KCl nd 1 mmol l 1 NH 4 Cl, nd these [C 2 ] i responses presented in two different forms, suggesting tht mmoni my e sensed y multiple mechnisms. The [C 2 ] i responses to high mmoni were ttenuted in NECs isolted from trout chroniclly exposed to HEA, especilly in ones from gill rch I, ut responses to high K were unchnged. We conclude tht the hyperventiltory response to mmoni is lost fter chronic wterorne HEA exposure, nd tht NECs, especilly the ones locted in gill rches I nd II, re proly mmoni chemoreceptors tht prticipte in ventiltory modultion in trout. Key words: mmoni, NH 3, NH 4, fish, neuroepithelil cell, chemoreceptors, ventiltion, cclimtion, cid se sttus. INTRODUCTION Ammoni is the third importnt respirtory gs fter oxygen nd cron dioxide in mmoniotelic teleosts. (Note: throughout this pper, the term mmoni is used to refer to totl NH 3 NH 4, wheres NH 3 nd NH 4 refer to the individul components of mmoni gs nd mmonium ion, respectively.) Ammoni is the mjor nitrogenous wste (pproximte 7%) produced from the ctolism of dietry nd structurl proteins in the liver, muscle nd other tissues in mmoniotelic teleosts. To void highly toxic effects, mmoni is excreted continully from the gills to the wter t rte of out 1% of the rte of CO 2 excretion (Rndll, 199; Rndll nd Ip, 26). Unlike O 2 nd CO 2, which hve een widely ccepted s the importnt drivers to ventiltion in fish for mny yers (Perry nd Wood, 1989; Gilmour, 21), mmoni ws not clerly reveled s potentil stimulnt to ventiltion until recently. McKenzie nd collegues reported tht the injection of n NH 4 HCO 3 solution into the dorsl ort of trout cused mrked hyperventiltion, ut s lood [HCO 3 ] nd CO 2 tension (P CO2 ) levels lso incresed, the responses could not e ttriuted specificlly to mmoni (McKenzie et l., 1993). Our recent study (Zhng nd Wood, 29) followed McKenzie nd collegues ide nd elevted plsm mmoni y numer of different tretments to directly investigte whether mmoni cn ct s ventiltory stimulnt. Increses in plsm totl mmoni concentrtion ([T mm ]) y vriety of methods lwys resulted in hyperventiltion, nd this occurred even in circumstnces where there ws no chnge in lood P CO2, O 2 tension (P O2 ) or cid se sttus. In fish, elevted internl [T mm ] occurs fter feeding (e.g. Wicks nd Rndll, 22; Bucking nd Wood, 28), exhustive exercise (e.g. Wood, 1988; Wng et l., 1994) or exposure to high environmentl mmoni (HEA) (Wilson nd Tylor, 1992; Knoph, 1996; Nwt et l., 27), suggesting the possile function of mmoni to induce hyperventiltion in these circumstnces. For the first two circumstnces (fter feeding, fter exercise), this stimultion could e dptive to improve O 2 nd CO 2 exchnge (nd possily mmoni excretion) during specific dynmic ction or excess postexercise O 2 consumption, or even during the stress ssocited with short-term mmoni pulses in the wter (Zhng nd Wood, 29). However, it is difficult to see how this hyperventiltory response would remin dptive during chronic HEA, ecuse ventiltion in fish is costly process (e.g. Jones, 1971). We therefore hypothesized tht the response would e ttenuted or lost during chronic HEA; the first ojective of our study ws to test this hypothesis, nd to see whether the sme ttenution occurred in response to intrvsculr mmoni injection.

2 Ammoni sensing nd ventiltory responses in trout 2679 The hyperventiltory response to mmoni in trout suggests the presence of mmoni-sensing chemoreceptors. There is now undnt evidence tht O 2 nd CO 2 /ph sensors occur on the gills, with prticulr focus on the first pir of gill rches (gill rch I) (Smith nd Jones, 1978; Gilmour, 21; Milsom nd Burleson, 27). Mny recent studies indicte tht the rnchil neuroepithelil cells (NECs) re the ctul chemoreceptors for these two respirtory gses in lood nd/or wter (Jonz et l., 24; Jonz nd Nurse, 26; Vulesevic et l., 26; Milsom nd Burleson, 27; Coolidge et l., 28; Qin et l., 21). Indeed, the receptors on gill rches I nd II (emryonic rches III nd IV) re thought to represent the phylogenetic ntecedents of the mmmlin crotid nd ortic odies, respectively (Milsom nd Burleson, 27). Our second ojective ws therefore to evlute whether the hyperventiltory response to cute mmoni exposure ws ltered y selective removl (y ligtion) of different pirs of rches. We hypothesized tht if there ws n effect, it would e most pronounced for the first nd possily second pir (gill rches I nd II). Assuming positive results with the first two hypotheses, our third ojective ws to investigte whether rnchil NECs were involved in the oserved responses to mmoni. NECs in fish resemle the type I glomus cells of the crotid ody, which re recognized to ct s oth O 2 nd CO 2 sensors in mmmls (Gonzlez et l., 1994; Lhiri nd Forster, 23). NECs were immunofluorescently leled y ntiser ginst serotonin (5-HT). We hypothesized tht the loss of the cute hyperventiltory response to mmoni fter chronic exposure to HEA would e ccompnied y chnges in the size, density or distriution of NECs on gill rches if these cells were involved in mmoni sensing. Such chnges hve een seen in zerfish sujected to chronic hypoxi (Jonz et l., 24) nd hyperoxi (Vulesevic et l., 26). By using ptch-clmp techniques, Jonz et l. (Jonz et l., 24) nd Burleson et l. (Burleson et l., 26) demonstrted tht NECs of oth zerfish nd ctfish ct s O 2 chemoreceptors, s hypoxi cused inhiition of their ckground K currents. More recently, Qin et l. (Qin et l., 21) showed tht the NECs of the zerfish gill respond to incresing CO 2 levels in the sme wy s to hypoxi, demonstrting tht NECs re imodl sensors of O 2 nd CO 2. It ws proposed tht the demonstrted inhiition of ckground K current nd resulting prtil cell depolriztion cused y hypoxi or hypercpni would led to voltge-gted C 2 influx, susequent neurotrnsmitter relese nd fferent nerve ctivtion, though these steps hve not yet een directly proven for teleost NECs. Furthermore, there hs so fr een no study on the responses of teleost NECs to mmoni. However, Rndll nd Ip (Rndll nd Ip, 26) hve suggested tht the NECs could lso medite mmoni-induced hyperventiltion ecuse the K chnnels re permele to NH 4 (most vlues of the permeility rtio of NH 4 /K in K chnnels re in the rnge.1.3) (Choe et l., 2), nd NECs therefore my sense mmoni when the ckground K current is inhiited y NH 4. With this ckground in mind, our finl ojective ws to ssess whether [C 2 ] i in rnchil NECs from trout gills is sensitive to mmoni t physiologiclly relistic levels. NECs were isolted from control trout nd trout chroniclly exposed to HEA (chronic HEA trout), nd their [C 2 ] i responses to 1 mmol l 1 NH 4 Cl nd 3 mmol l 1 KCl (s positive control) were exmined using Fur- 2 fluorescence imging methods (Willims et l., 1985). We hypothesized tht if NECs serve s mmoni sensors t relistic levels, their [C 2 ] i would chnge, nd this response would e ttenuted in NECs isolted from trout chroniclly exposed to HEA. MATERIALS AND METHODS Fish husndry nd HEA exposure All procedures were pproved y the McMster University Animl Reserch Ethics Bord nd re in ccordnce with the Guidelines of the Cndin Council on Animl Cre. Rinow trout (Oncorhynchus mykiss, Wlum; 5 1 or 25 5 g) were otined from Humer Springs Trout Htchery (Orngeville, ON, Cnd) nd then cclimted to lortory conditions for more thn 4 weeks efore experimenttion. The trout were held in flowing dechlorinted Hmilton (ON, Cnd) tp wter (concentrtion in mmol l 1 : N.6, Cl.7, K.5, C 2 1., Mg 2.1; titrtion lklinity 1.9 mequiv l 1 ; hrdness, 14 mg l 1 s CCO 3 equivlents; ph , 12±1 C). The fish were fed commercil trout food (crude protein 41%; crohydrtes 3%; crude ft 11%; Mrtin Mills, Elmir, ON, Cnd) t rtion of 2% ody mss every 3 dys. All the fish were fsted t lest 5 dys efore experimenttion, to minimize the influence of feeding on mmoni metolism. In chronic HEA tretments, 2 lrge trout (25 5 g) nd 5 smll trout (5 1 g) were held in seprte tnks contining 8 nd 2 l dechlorinted Hmilton tp wter, respectively. (NH 4 ) 2 SO 4 stock solution (djusted to ph 7.8) ws dded to the tnks to chieve nominl HEA concentrtion of 25 mmol l 1 (i.e. 5 mmol l 1 mmoni). Fish were fed (1% ody mss) every 3 dys. Two-thirds of the wter ws renewed 24 h fter feeding nd n pproprite mount of (NH 4 ) 2 SO 4 ws dded to mintin the correct HEA concentrtion. In the control tretment, fish were held under the sme conditions s for the mmoni-exposed ones, ut without the ddition of (NH 4 ) 2 SO 4 to the wter. The cclimtion lsted 1 3 months, nd mmoni concentrtions were checked regulrly y ssy (Verdouw et l., 1978) to ensure tht they remined within ±15% of nominl vlues. Nitrite concentrtions were checked y test strips (Aqurium Phrmceuticls, Chlfont, PA, USA) during chronic HEA exposure, nd no elevtion ws found (lower limit of detection,.5 mg l 1 ). Ventiltory responses to wterorne mmoni Experiments were performed on lrge trout tht hd een held under control conditions or chroniclly exposed to HEA. Trout were nesthetized nd irrigted with 8 mg l 1 tricine methnesulfonte (MS-222, Syndel Lortories Ltd, Vncouver, BC, Cnd; djusted to ph 7.8 with NOH) in tp wter on n operting tle. Buccl ctheters (flred tuing, Cly-Adms PE9, Sprks, MD, USA) were implnted through hole drilled in the roof of the mouth (see Holeton nd Rndll, 1967), in order to monitor ventiltion. Dorsl ortic ctheters were implnted s descried elsewhere (Soivio et l., 1975), nd filled with Cortlnd sline (in mmol l 1 : 124 NCl, 5.1 KCl, 1.6 CCl 2,.9 MgSO 4, 11.9 NHCO 3, 3. NH 2 PO 4, 5.6 glucose) (Wolf, 1963), for lood smpling with miniml disturnce. Trout were then plced individully in drkened Plexigls oxes (4.5 l volume) served with constnt ertion nd flowing wter (.4 l min 1 ) nd llowed to recover for 24 h efore experimenttion. The fish tht hd een chroniclly exposed to HEA were lwys kept in wter contining 25 mmol l 1 (NH 4 ) 2 SO 4 (ph 7.8) during surgery nd recovery. During experimenttion, control trout were continuously exposed to clen wter for 3 min, 25 mmol l 1 (NH 4 ) 2 SO 4 (i.e. 5 mmol l 1 mmoni) for 6 min, nd 5 mmol l 1 (NH 4 ) 2 SO 4 (i.e. 1 mmol l 1 mmoni) for 6 min y dding pproprite mounts of (NH 4 ) 2 SO 4 stock to the closed 4.5 l oxes t 3 nd 9 min from the strt. Chronic HEA trout were continuously exposed to 25 mmol l 1 (NH 4 ) 2 SO 4 for 3 min nd 5 mmol l 1 (NH 4 ) 2 SO 4 for 6 min. (NH 4 ) 2 SO 4 ws used ecuse the sulfte ion ws found to hve no effect on ventiltion in our previous study (Zhng nd Wood, 29).

3 268 L. Zhng nd others Ventiltion ws mesured immeditely efore nd t 1 min intervls during exposure. After exposure, trout were kept in their originl wter for 2 h of recovery. Then the procedures were repeted, with the ddition of lood smpling. Dorsl rteril lood smples (6 ml ech, with sline replcement) were drwn immeditely vi the ctheters efore the end of ech exposure to different (NH 4 ) 2 SO 4 concentrtion. At the end of the experiments, trout were killed y n overdose of ph-djusted MS-222. Ventiltory response to intrvsculr mmoni injection nd chronic HEA trout were implnted with dorsl ortic nd uccl ctheters using the sme methods s ove. After 24 h recovery in the drkened Plexigls oxes, the fish were injected with 4.13±.1 ml kg 1 of either Cortlnd sline or 7 mmol l 1 (NH 4 ) 2 SO 4 (ph 7.8) vi the dorsl ortic cnnul over 5 min period. The injected dose ws therefore out 58 mmol kg 1 of mmoni. This injection rte did not cuse ny ehviorl response or irrittion. Ventiltion ws mesured immeditely efore nd 2 min fter the 5 min injection period. The role of different gill rches in the ventiltory responses to wterorne mmoni Individul pirs of rches (rches I, II, III nd IV seprtely, s well s rches I nd II simultneously, nd rches III nd IV simultneously) were functionlly removed from control trout efore experimenttion. Fish were nesthetized nd irrigted with 8 mg l 1 MS-222 on n operting tle. The two ends of oth rches (i.e. ilterlly) in pir were then tightly tied y no. 2- surgicl silk sutures (Ethicon Inc., Somerville, NJ, USA). After recovery in the holding tnk for 24 h, trout in which the ligtions were ilterlly successful, s demonstrted y ovious cogultion or complete loss of lood in the gill filments of oth rches, were chosen for experimenttion. After the recovery period, the ehvior of trout ppered norml. These fish were then nesthetized gin, fitted with uccl ctheters only, nd llowed to recover for further 24 h in flowing clen wter. Therefter, ventiltion ws monitored for 3 min in clen wter, followed y 6 min in the presence of HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ]. A seprte group of fish, with ll gill rches intct, were put through n identicl protocol nd served s controls. Identifiction nd quntifiction of NECs y immunofluorescence nd chronic HEA smll trout were killed y n overdose of ph-djusted MS-222. Gill tissues were prepred nd immunoleled s descried previously (Jonz nd Nurse, 23). In rief, gill rches were immeditely removed, wshed in cold PBS (in mmol l 1 : 137 NCl, 15.2 N 2 HPO 4, 2.7 KCl, 1.5 KH 2 PO 4 ; ph 7.8) to remove mucus, nd fixed y immersion in 4% prformldehyde in PBS t 4 C overnight. Arches were then rinsed with PBS 3 times nd permeilized in.5% Triton X-1 in PBS (PBS-TX) t 4 C for h. NECs of the gill filments were identified y immunoleling with ntiser ginst 5-HT. Arches were incuted in the primry ntiody, 1:25 rit nti-5-ht ntiody, t 4 C overnight. After rinse with PBS-TX, rches were incuted in the secondry ntiody, 1:5 got nti-rit ntiserum, conjugted with fluorescein isothiocynte (FITC; Jckson ImmunoReserch Lortories Inc., West Grove, PA, USA) t room temperture in the drk for 1 h. Arches were rinsed in PBS nd the gill filments were removed nd mounted on glss slides in Vectshield (Vector Lortories Inc., Burlingme, CA, USA) to reduce photo-leching. The slides were exmined under n upright Olympus BX 6 microscope equipped with epifluorescence (Crson Group Inc., Mrkhm, ON, Cnd) nd the imges were cptured using CCD cmer nd nlyzed y imging nlysis softwre (Northern Eclipse, Empix Imging Inc., Cheektowg, NY, USA). Four filments were selected rndomly from specific rches in ech of eight trout. The densities of NECs in the filments were clculted y counting NECs t different plnes of focus in the segments of the filments ner the tips (excluding the tips themselves) nd were descried s the numer per mm of filment length. The sizes of five individul NECs in ech of the four filments were nlyzed y converting the pixels of their projection re to mm 2 using Adoe Photoshop CS (Adoe Systems Incorported, Sn Jose, CA, USA). [C 2 ] i responses of NECs NECs were isolted from smll trout tht hd een held under control conditions or chroniclly exposed to HEA. Methods were modified from previous studies (Kelly et l., 2; Jonz et l., 24). Specific gill rches were excised, then rinsed with PBS (composition s ove). The gill filments were cut off the gill rs nd plced into 15 ml centrifuge tue contining 2 ml of wsh solution (sterilized penicillin 2 i.u. l 1, streptomycin 2 mg ml 1 nd gentmicin 4 mg ml 1 in sterilized PBS) on ice for 15 min, twice. Then the tissues were plced in plstic culture dish filled with 2 ml of.25% trypsin/edta t room temperture. After digestion in trypsin/edta for 45 min t room temperture, the filment tissues were torn prt y two pirs of flme-sterilized forceps until few whole filment tips could e seen. The tissue suspension ws trnsferred to 15 ml centrifuge tue nd triturted rpidly 2 times y plstic pipet to continue dissocition. Fetl clf serum (.2 ml, FCS; Invitrogen, Grnd Islnd, NY, USA) ws dded nd triturted riefly to stop the trypsin rection. After removl of the undissocited tissue y pssge through 1 mm cell striner (BD Flcon, Bedford, MA, USA), the cell suspension ws centrifuged t 5g for 5 min t 4 C. The superntnt ws spirted nd the pellet ws resuspended in 2 ml of rinse solution (5% FCS in PBS) nd centrifuged gin, s ove. The pellet ws then resuspended in 2 ml L-15 medi (Invitrogen; contining sterilized penicillin 2 i.u. ml 1 nd streptomycin 2 mg ml 1 ) y triturting gently. A few drops of the cell suspension were lyered on the centrl wells of modified 35 mm polystyrene dishes (Nunc, Roskilde, Denmrk). The dishes hd een modified y drilling 1 mm dimeter round centrl hole in the ottom, nd psting glss coverslip (VMR, Rdnor, PA, USA) to the underside with Sylgrd (Pisley Products Inc., Scrorough, ON, Cnd). Before use, the modified dishes were UV sterilized, nd coted with poly L-lysine (.1 mg ml 1, Sigm, St Louis, MO, USA) nd Mtri-Gel (Collortive Reserch, Bedford, MA, USA). Cells were then kept in n 18 C incutor for h. L-15 medi (2 ml) ws dded to the dishes 15 h fter seeding. The mjority of cells in the dishes were pvement cells (PVCs), ecuse the mjor proportion of gill cells isolted in this mnner re PVCs (~8%) nd mitochondri-rich cells (~1%) ut mitochondri-rich cells cnnot e seeded using this method (Kelly et l., 2). NECs were identified y stining with 2 mg ml 1 Neutrl Red (Sigm) s in previous studies (Jonz et l., 24), nd verified y immunoleling with 5-HT ntiserum (Jonz nd Nurse, 23). PVCs were identified s the predominnt unstined cells. [C 2 ] i ws monitored y the fluorescent C 2 indictor Fur- 2/AM (Invitrogen). Rtiometric C 2 imging ws performed using Nikon Eclipse TE2-U inverted microscope (Nikon, Mississug, ON, Cnd) equipped with Lmd DG-4 ultr high-speed wvelength chnger (Sutter Instrument Co., Novto, CA,

4 Ammoni sensing nd ventiltory responses in trout 2681 USA; exposure time 1 ms), Nikon S-Fluor 4 oil-immersion ojective lens with numericl perture of 1.3, nd Hmmtsu OCRCA-ET digitl CCD cmer (Hmmtsu, Sewickley, PA, USA). Dul imges (34 nd 38 nm excittion nd 51 nm emission) were collected, nd pseudocolor rtiometric dt were otined using Simple PCI softwre version 5.3 (Hmmtsu). The imging system ws stndrdized with Fur-2 clcium-imging clirtion kit (Invitrogen) through procedure descried previously (Buttigieg et l., 28). Cells ttched to the dish were loded with 1 mmol l 1 Fur- 2/AM (in Cortlnd sline) for 4 min t room temperture nd susequently wshed in Cortlnd sline erted with.3% CO 2 nd 99.7% O 2 gs mixture for 15 min to remove free dye. During the experiment, cells were continuously perfused with the gsequilirted sline. High K (3 mmol l 1 ) nd high NH 4 sline (1 mmol l 1 ), which were mde y equimolr sustitution of KCl or NH 4 Cl for NCl in the Cortlnd sline so s not to chnge the chloride concentrtion, were sustituted for control Cortlnd sline for 15 s intervls t certin time points. Anlyticl techniques Ventiltion ws mesured s descried efore (Zhng nd Wood, 29). This system recorded ventiltion rte (f V, reths min 1 ) nd the uccl pressure mplitude ( P uccl, mmhg), s n index of stroke volume. f V ws clculted s the frequency of reths in 1 min t the designted time. P uccl ws clculted s the men vlue of 1 mesurements of mplitude (rndomly selected from periods of norml rething, omitting episodes of coughing or disturnce) t the designted time. Arteril lood smples (3 ml) were nlyzed immeditely for rteril lood ph (ph), O 2 tension (P O2 ) nd O 2 content (C O2 ), nd frozen for lter hemogloin (H) nlysis. The remining lood (3 ml) ws centrifuged t 9 g for 3 s to the seprte plsm; 1 ml plsm ws used for the nlysis of totl CO 2 nd the reminder ws frozen in liquid nitrogen for lter nlysis of plsm [T mm ], [N ] nd [Cl ]. All these steps were finished within 2 min of lood smpling. The whole lood ph nd P O2 were mesured in 12 C thermostticlly controlled chmers using Rdiometer GK241C glss comintion electrode coupled to PHM82 stndrd ph meter (Rdiometer Ltd, Copenhgen, Denmrk), nd polrogrphic oxygen electrode coupled to polrogrphic mplifier (Model 19, A-M Systems, Everett, WA, USA), respectively. C O2 ws mesured in duplicte on 2 ml smples using lood oxygen content nlyzer (Oxycon TM, Cmeron Instrument Compny, Port Arnss, TX, USA). Blood (5 ml) ws trnsferred to ullet tue with smll mount of lithium heprin (Sigm) nd stored t 2 C for H mesurement. Whole lood H ws lter ssyed using Drkin s regent (Sigm) nd n LKB 454 UV/visile spectrophotometer (LKB-Biochrom, Cmridge, UK), nd mesured ginst clirtion series of H stndrds from ovine lood (Sigm). Plsm [T mm ] ws mesured using the sme spectrophotometer with commercil kit (Richem, Sn Diego, CA, USA) sed on the glutmte dehydrogense/nad method. Plsm [N ] nd [Cl ] were mesured using Vrin Spectr-22FS flme tomic sorption spectrometer (Vrin, Mulgrve, Austrli) nd coulometric chloride titrtor (CMT1, Rdiometer), respectively. Plsm totl CO 2 ws mesured in duplicte on 5 ml smples using Corning model 965 CO 2 nlyzer (Lowell, MA, USA). Plsm P CO2 nd [HCO 3 ] were clculted using the Henderson Hssellch eqution with plsm pk vlues nd CO 2 soluility coefficients for trout plsm t 12 C from Boutilier et l. (Boutilier et l., 1984). Plsm [NH 3 ] ws clculted using the Henderson Hssellch eqution with pk vlues for trout plsm t 12 C from Cmeron nd Heisler (Cmeron nd Heisler, 1983). Wter mmoni levels were checked y the colorimetric ssy of Verdouw et l. (Verdouw et l., 1978). Sttistics Dt re routinely expressed s mens ± 1 s.e.m. (N), where N is the numer of fish in tretment men (except for NEC [C 2 ] i mesurements, where N is the numer of NEC cells). A one-wy ANOVA followed y Tukey s test ws pplied to compre: (1) the density nd size of NECs mong the four gill rches; (2) [C 2 ] i in NECs efore nd fter high K or high NH 4 perfusion; nd (3) f V, P uccl, nd lood nd plsm vriles in different (NH 4 ) 2 SO 4 exposures in oth control fish nd those chroniclly exposed to HEA. A one-wy repeted mesures (RM)ANOVA followed y Dunnett s test ws pplied to compre the f V nd P uccl during (NH 4 ) 2 SO 4 exposures ck to initil control vlues. A Student s two-tiled pired t-test ws pplied to compre f V nd P uccl mens efore nd fter (NH 4 ) 2 SO 4 exposures in the fish with specific gill rches removed, nd to compre f V nd P uccl efore nd fter intrvsculr (NH 4 ) 2 SO 4 injections in oth control nd chronic HEA trout. A significnce level of P<.5 ws employed throughout. All sttisticl tests were run using SigmStt (v. 3.1; Systt Softwre, Sn Jose, CA, USA). RESULTS Ventiltory responses to wterorne mmoni In the sence of lood smpling The P uccl nd f V remined constnt t 1.5±.2 mmhg nd 66±6 reths min 1 in control trout exposed to clen wter during the initil 3 min (Fig. 1A,C). After cute exposure to 25 nd 5 mmol l 1 (NH 4 ) 2 SO 4, P uccl incresed continuously throughout the exposure periods, up to 17% of the initil mplitude t the end (Fig. 1A), wheres f V kept constnt (Fig. 1C). After pooling P uccl nd f V t different times in ech (NH 4 ) 2 SO 4 exposure, the three P uccl mens were significntly different from ech other (Fig. 1B), ut the three f V mens were similr (Fig. 1D). In chronic HEA trout, P uccl nd f V were kept constnt t 1.3±.2 mmhg nd 6±3 reths min 1 during exposure to 25 mmol l 1 (NH 4 ) 2 SO 4 (Fig. 1A,C). This ws in fct the chronic HEA medium, nd the vlues were not significntly different from those of the control group in clen wter, so fter 1 month continuous HEA exposure, the hyperventiltory response hd een lost. After pooling vlues t different times in ech (NH 4 ) 2 SO 4 exposure, P uccl ws comprle to the vlue in control trout exposed to clen wter nd significntly less thn tht in control trout exposed to 25 mmol l 1 (NH 4 ) 2 SO 4 (Fig. 1B). The f V ws not significntly different from the men rte in control trout in clen wter or in 25 mmol l 1 (NH 4 ) 2 SO 4 (Fig. 1D). After 5 mmol l 1 (NH 4 ) 2 SO 4 exposure, P uccl in chronic HEA trout remined constnt (Fig. 1A) ut f V ws reduced grdully, to 52±2 reths min 1 t the end (Fig. 1C). After pooling, this decrese ws gin significnt reltive to control trout. The mens of P uccl nd f V etween 25 nd 5 mmol l 1 (NH 4 ) 2 SO 4 exposure tretments were not significntly different in chronic HEA trout (Fig. 1B,D), gin emphsizing the loss of the hyperventiltory response s result of long-term exposure. With lood smpling When the fish were lood smpled, there were some differences in solute vlues, ut sic ptterns remined qulittively similr. Pooled vlues t different times in ech (NH 4 ) 2 SO 4 exposure re

5 2682 L. Zhng nd others ΔP uccl (mmhg) f V (reths min 1 ) A (NH 4 ) 2 SO 4 C HEA 25 μmol l 1 (NH 4 ) 2 SO 4 5 μmol l 1 (NH 4 ) 2 SO 4 B D d,c,d c,d,,c,,,c 25 μmol l 1 (NH 4 ) 2 SO 4 5 μmol l 1 (NH 4 ) 2 SO 4,,, c c Fig. 1. Ventiltory responses to elevted wterorne mmoni (NH 4 ) 2 SO 4 exposure in control trout nd in trout chroniclly exposed to high environmentl mmoni (HEA) [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months. A nd C show the chnges of ventiltory mplitude ( P uccl ) nd frequency (f V ) over time during the exposure periods. B nd D show the men vlues fter pooling P uccl nd f V mesurements t different times in ech (NH 4 ) 2 SO 4 exposure period from the dt reported in A nd C. In ddition, B nd D lso summrize comprle results in trout sujected to lood smpling during the experimentl periods, s control with lood smpling (/BS) nd chronic HEA exposure with lood smpling (HEA/BS). N 5. In A nd C, sterisks indicted significnt increses reltive to the dt in the (NH 4 ) 2 SO 4 exposure period (P<.5). In B nd D, mens not shring the sme letter re significntly different from one nother (P<.5). 4 (NH 4 ) 2 SO 4 25 μmol l 1 (NH 4 ) 2 SO 4 5 μmol l 1 (NH 4 ) 2 SO Time (min) /BS HEA HEA/BS shown (Fig. 1B,D). In control trout, P uccl ws higher nd f V ws lower thn in undistured fish. As in non-lood-smpled fish, P uccl further incresed fter high (NH 4 ) 2 SO 4 exposure, ut the increse in f V ws not significnt. In chronic HEA trout sujected to lood smpling, the seline P uccl t 25 mmol l 1 (NH 4 ) 2 SO 4 ws unchnged, ut f V ws reduced reltive to non-lood-smpled fish (Fig. 1D). After cute exposure to 5 mmol l 1 (NH 4 ) 2 SO 4, neither P uccl nor f V chnged, gin demonstrting the loss of the hyperventiltory response fter chronic HEA exposure (Fig. 1D). Some rteril lood vriles were lso chnged significntly fter cute exposure of control fish to 25 nd 5 mmol l 1 (NH 4 ) 2 SO 4 (Tle 1). In prticulr, ph, [T mm ] nd [NH 3 ] ll incresed progressively. Although plsm [HCO 3 ] did not chnge, P CO2 decresed significntly coincident with the hyperventiltion. However, contrry to expecttions, oth lood C O2 nd P O2 lso decresed progressively. As there ws smll ut significnt fll in H, proly due to repetitive lood smpling, this could hve cused the decrese in C O2. However, when the dt were expressed s C O2 /H, the decrese remined significnt, indicting true drop in H O 2 sturtion (Tle 1). Plsm [N ] nd [Cl ] remined constnt. In chronic HEA trout, mny rteril lood vriles were significntly different from those in control trout (Tle 1). In prticulr, in chronic HEA trout smpled under long-term exposure conditions [25 mmol l 1 (NH 4 ) 2 SO 4 ] reltive to control trout smpled under control conditions, rteril ph, [T mm ] nd [NH 3 ] were ll significntly higher, P CO2 ws significntly lower, while C O2, P O2 nd C O2 /H were comprle, indicting tht these lst vriles were restored during chronic HEA exposure. [Cl ] were comprle ut [N ] were significntly lower. Notly, [T mm ] nd [NH 3 ] were 3- to 4-fold higher in these chronic HEA fish thn in control trout exposed to the sme mmoni level [25 mmol l 1 (NH 4 ) 2 SO 4 ]. Also notle is the fct tht in chronic HEA fish, none of the mesured vriles chnged significntly when the mmoni exposure ws cutely chnged from 25 to 5 mmol l 1 (NH 4 ) 2 SO 4 (Tle 1). Ventiltory response to intrvsculr mmoni injection In control trout, the initil selines of P uccl nd f V were 1.5±.2 mmhg nd 53±2 reths min 1 (Fig. 2). f V nd P uccl did not chnge fter injection of sline, ut oth incresed significntly (y pired t-test) fter intrvsculr injection of out 58 mmol kg 1 of mmoni [s 7 mmol l 1 (NH 4 ) 2 SO 4 ]. The response ws immedite. In trout chroniclly exposed to HEA, the initil selines of P uccl nd f V were 1.6±.2 mmhg nd 51±2 reths min 1, comprle to vlues in control trout. However, there were no significnt ventiltory chnges fter injection of (NH 4 ) 2 SO 4 or sline (Fig. 2). Thus, the loss of the ventiltory response s result of chronic wterorne HEA exposure is lso seen when the hyperventiltory stimulus is presented only to the loodstrem. The role of different gill rches in the ventiltory response to wterorne mmoni All trout with ligted gill rches ppered helthy nd rethed in norml fshion. P uccl nd f V in control fish without gill ligtions were 1.5±.2 mmhg nd 64±5 reths min 1 (Fig. 3A,C). P uccl nd f V in trout with the vrious pirs of gill rches removed were comprle to control, except for the f V in fish where oth rches I nd II hd een ligted, which ws 71±3 reths min 1. After cute exposure to wterorne mmoni [25 mmol l 1 (NH 4 ) 2 SO 4 ], P uccl incresed grdully nd significntly over the ensuing 6 min in ll control nimls, with the first significnt increse t out 1 min, wheres f V did not chnge (Fig. 3A,C). Hyperventiltion lso occurred in response to 25 mmol l 1 (NH 4 ) 2 SO 4 in trout sujected to seprte removl of gill rches I, II, III or IV, or comined removl of gill rches III nd IV. However, in trout lcking gill rch I, the increse in P uccl ws delyed until 4 min (Fig. 3A). Furthermore, in trout in which oth rches I nd II were removed, there ws no hyperventiltory response to cute wterorne mmoni exposure, with oth P uccl nd f V remining constnt throughout the 6 min experimentl period. This difference

6 Ammoni sensing nd ventiltory responses in trout 2683 Tle 1. Arteril lood vriles in control nd chronic high environmentl mmoni (HEA) trout Wter (NH 4 ) 2 SO 4 (mmol l 1 ) Chronic HEA ph 7.83± ±.3, 7.89± ± ±.2 [T mm ](mmol l 1 ) 78.1± ± ±97.1 d ±5.3 c 816.3±73.3 d [NH 3 ] (mmol l 1 ).84± ± ±1.28 d 5 5.7±.86 c 1.73±.95 d [HCO 3 ] (mmol l 1 ) 5.56± ±.74, 4.31±.48, ±.71, 3.76±.48 P O 2 (mmhg) 1.82± ± ± ± ±.13 C O 2 (ml 1 ml 1 ) 11.± ± ± ± ±1.2, P O 2 (mmhg) 75.8± ±6.9, 68.9±3.5, ± ±4.2, H (g 1 ml 1 ) 8.95± ±.59, 7.89±.63, ± ±.47 C O 2 /H (ml g 1 ) 1.27± ± ± ± ±.12 [Cl ] (mmol l 1 ) 125.4± ± ±1.4, ± ±1.9 [N ] (mmol l 1 ) 162.4± ± ± ± ±2.9 Arteril lood ph (ph), O 2 content (C O 2 ), O 2 tension (P O 2 ), hemogloin (H), C O2 /H rtio, plsm totl mmoni ([T mm]), non-ionized mmoni ([NH 3 ]), [HCO 3 ], CO 2 tension (P CO 2 ), [N ] nd [Cl ] in control trout nd trout chroniclly exposed to HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months. Vlues were recorded from fish in their cclimtion wter [either or 25 mmol l 1 (NH 4 ) 2 SO 4 ] nd fter 6 min exposure to higher wterorne mmoni concentrtions [25 nd 5 mmol l 1 (NH 4 ) 2 SO 4 ]. Vlues re presented s mens ± s.e.m. (N 5). Mens not shring the sme letters re significntly different (P<.5) from one nother. ws lso clerly seen when the dt were pooled cross the period (Fig. 3B,D). Identifiction nd quntifiction of NECs y immunofluorescence There were no ovious differences in rrngement or stining in 5- HT-immunorective NECs mong different gill rches. Therefore, the immunofluorescence results of rch I re shown s representtives in Fig. 4. In oth control nd chronic HEA trout, NECs were dispersed within the primry filment epithelium ut not in the secondry respirtory lmelle. NECs were minly orgnized in two lines in the filment epithelium. Although the rrngement of NECs did not chnge in trout chroniclly exposed to HEA, the density nd size of NECs were oth reduced. In control trout, the density of NECs ws comprle mong the four gill rches, ut slightly higher in rches I nd II (19±3 nd 16±5 cells mm 1 filment) thn in rches III nd IV (11±4 nd 13±3 cells mm 1 filment) (Fig. 5A). Chronic exposure to HEA cused significnt 9% reduction in NEC density in rches I nd II, ut not in rches III nd IV. Furthermore, in control fish, NECs were smller in rches I nd II thn in rches III nd IV (76±2 versus 87±3 mm 2), wheres in chronic HEA fish, the NECs were comprle in size in the four rches (69±2 mm 2 ) ut significntly smller y out 15% thn in control trout (Fig. 5B). [C 2 ] i responses of NECs NECs in culture were identified s cells tht were prtilly colored red, s result of the stining of intrcellulr vesicles with Neutrl Red, s descried previously (Jonz et l., 24). Bseline [C 2 ] i in NECs ws out 1 nmol l 1. Of the 43 NECs tht responded to high K (3 mmol l 1 ) with rise in [C 2 ] i, 39 lso responded to high NH 4 (1 mmol l 1 ) perfusion. In contrst, the Neutrl Rednegtive PVCs, which were predominnt mong the dispersed cell popultion, never exhiited ny ovious [C 2 ] i responses to either high K or high NH 4 perfusion (Fig. 6A). In NECs, [C 2 ] i lwys incresed nd recovered rpidly fter the high K perfusion wheres [C 2 ] i responses to high NH 4 perfusion presented two ptterns (Fig. 6A,C). There were 19 cells (11 from control nd 8 from chronic HEA trout) tht presented slow increse followed y slow recovery of [C 2 ] i (type A, in Fig. 6A). And there were 2 cells (15 from control nd 5 from chronic HEA fish) tht presented rpid increse nd recovery followed y susequent long-term elevtion of [C 2 ] i (type B, in Fig. 6C). Becuse the second elevtion of [C 2 ] i in type B responses ws usully prolonged nd sometimes persisted eyond the end of the recording period, the first rpid elevtions of [C 2 ] i were chosen to quntify responses in type B cells. In NECs from oth control nd chronic HEA fish, the ckground [C 2 ] i ws similr, t 1±6 nmol l 1, regrdless of whether the

7 2684 L. Zhng nd others ΔP uccl (mmhg) f V (reths min 1 ) A B Sline Before injection After injection (NH 4 ) 2 SO 4 Sline (NH 4 ) 2 SO 4 HEA Fig. 2. Ventiltory responses to dorsl ortic injection of sline or 58 mmol kg 1 of mmoni [s 7 mmol l 1 (NH 4 ) 2 SO 4 ] in control trout nd in trout chroniclly exposed to HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months. A nd B show the chnges of ventiltory mplitude ( P uccl ) nd frequency (f V ) efore nd fter injection, respectively. N 5. Asterisks indicted significnt increses (Student s pired t-test) in response to injection (P<.5). NECs exhiited type A or type B responses (Fig. 6B,D). After high K perfusion, the [C 2 ] i incresed to comprle levels in NECs from control nd chronic HEA fish. However, fter high NH 4 perfusion, the [C 2 ] i incresed to significntly higher levels in NECs from control trout thn in those from chronic HEA trout. This pttern of differentil effect on the responses to high NH 4 versus high K ws seen in NECs exhiiting oth type A (Fig. 6B) nd type B [C 2 ] i responses (Fig. 6D). There were no ovious differences in the frequency of type A versus type B responses when the dt were nlyzed ccording to the origins of the NECs from different gill rches, or different chronic exposure conditions. However, it ws notle tht the [C 2 ] i response to high NH 4 perfusion ws significntly lower in NECs from gill rch I of HEA fish (Fig. 7). DISCUSSION Overview After cute exposure to elevted wterorne mmoni [either 25 or 5 mmol l 1 (NH 4 ) 2 SO 4 ], rinow trout exhiited hyperventiltory response consisting of mrked increses in mplitude ( P uccl ) ut no chnge in rething frequency (f V ). However, fter chronic exposure to wterorne HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months, ventiltion hd returned to norml nd there ws no longer ny cute stimultory effect of higher wterorne levels of mmoni [5 mmol l 1 (NH 4 ) 2 SO 4 ]. Thus, in confirmtion of our first hypothesis, the fish hd cclimted physiologiclly, nd the stimultory effect of mmoni on ventiltion ws ttenuted or olished, phenomenon tht is likely dptive in the light of the high cost of ventiltion in fish. The sme ttenution occurred in the responses to intrvsculr mmoni injections (58 mmol kg 1 ), suggesting tht sensitivity to internl nd externl mmoni ws ffected in similr mnner. The cute hyperventiltory response to wterorne mmoni ws delyed y removl of gill rch I, nd eliminted y removl of oth gill rches I nd II. These findings support our second hypothesis tht the phylogenetic ntecedents of the mmmlin crotid nd ortic odies re involved in mmoni sensing in fish. In ccord with our third hypothesis, chronic HEA exposure reduced the density of NECs on gill rches I nd II, nd their size on ll four gill rches, therey providing indirect evidence of their involvement in mmoni sensing. More direct evidence nd confirmtion of our finl hypothesis ws provided y Fur-2 imging of isolted NECs. These experiments demonstrted tht their [C 2 ] i surges in response to physiologicl levels of mmoni, nd tht these responses re ttenuted in NECs isolted from fish tht hve een chroniclly exposed to HEA. The hyperventiltory response, its loss during chronic HEA exposure, nd cclimtion to mmoni In greement with mny previous studies (see Introduction), cute elevtion of wter mmoni cused hyperventiltion in trout. In our study, only ventiltory mplitude ( P uccl ) incresed in response to cute wterorne mmoni, wheres f V remined unchnged (Figs 1 nd 3). Interestingly, in oth our previous (Zhng nd Wood, 29) nd present studies (Fig. 2), intrvsculr mmoni injections cused smll elevtions in f V in ddition to lrge elevtions of P uccl, suggesting tht either the threshold or the sensors for the rething rte response my e different from those for the rething mplitude response. Estimtes of the cost of ventiltion in fish re generlly high, rnging from 1% to 43% of resting metolism, with mny in the 5 15% rnge (see Jones, 1971). Therefore while hyperventiltion in response to n cute mmoni signl my e eneficil for severl resons (see Introduction), chroniclly elevted ventiltion in the fce of persistent wterorne HEA would likely e mldptive. Our finding tht hyperventiltion disppered fter chronic HEA exposure (1 months) is in ccord with one previous 28 dy study, where trout ppered to hyperventilte (sed on f V counts only) during the initil 7 dys nd recovered fterwrds (Lng et l., 1987). Notly, in the present study, the hyperventiltory responses to oth further elevtions in wterorne mmoni (Fig. 1) nd intrvsculr injections of mmoni (Fig. 2) were lso lost, suggesting mjor lunting of oth externl nd internl sensitivities. In our previous study (Zhng nd Wood, 29), the ventiltory index ( P uccl f V ) ws positively correlted with the rteril plsm mmoni prmeters ([T mm ] nd [NH 3 ]) in 24 h intrvsculr mmoni infusion experiments. In the present study, the ventiltory index in chronic HEA trout exposed to 25 mmol l 1 (NH 4 ) 2 SO 4 ws only 81% of tht in control trout exposed to clen wter, or 65% of tht in control trout exposed to the sme medi (Fig. 1). At 5 mmol l 1 (NH 4 ) 2 SO 4, the ventiltory index of chronic HEA trout dropped to 48% of the comprle vlue in control trout. These differences occurred despite the fct tht the rteril plsm mmoni prmeters ([T mm ] nd [NH 3 ]) were significntly higher in chronic HEA trout thn in control trout under ll test conditions (Tle 1). These results suggest tht rinow trout cclimte to chronic HEA in physiologicl sense; toxicologicl cclimtion to mmoni hs een estlished previously (Render nd Stickney, 1979; Alster et l., 1979).

8 Ammoni sensing nd ventiltory responses in trout 2685 ΔP uccl (mmhg) f V (reths min 1 ) A C Arch I - Arch II Arch III Arch IV Arch I & II Arch III Arch I Arch IV Arch III & IV Arch I & II Arch III & IV Arch II 4 Pre Pre Pre Time (min) B D 4 25 μmol l 1 (NH 4 ) 2 SO 4 I II III IV I & II III & IV Fig. 3. Ventiltory responses to elevted wterorne mmoni [25 mmol l 1 (NH 4 ) 2 SO 4 ] exposure in trout sujected to removl of specific gill rch pirs y ligtion. trout hd ll gill rches intct. A nd C show the chnges of ventiltory mplitude ( P uccl ) nd frequency (f V ) t different times in ech exposure period. B nd D show the men vlues fter pooling P uccl nd f V mesurements t different times in ech (NH 4 ) 2 SO 4 exposure period from the dt reported in A nd C. N 5. Asterisks indicte significnt increses (Student s pired t-test) reltive to the dt efore (Pre) (NH 4 ) 2 SO 4 exposure (P<.5). Ventiltion ws not the only prmeter to chnge s result of cclimtion. Acute exposure to 25 mmol l 1 (NH 4 ) 2 SO 4 in control trout rised plsm [NH 3 ] nd [T mm ], nd lowered C O2 nd C O2 /H, with lrger ltertions fter exposure to 5 mmol l 1 (NH 4 ) 2 SO 4 (Tle 1). After chronic HEA exposure, the chnges in rteril C O2 nd C O2 /H hd een corrected despite even greter elevtions in [T mm ] nd [NH 3 ], s well s increses in ph, nd decreses in P CO2 nd plsm [N ] (Tle 1). The chnges of C O2 nd C O2 /H re of prticulr interest, nd rise the possiility tht mmoni my ct in prt y reducing rteril H O 2 sturtion, therey eliciting the O 2 drive to ventiltion (Smith nd Jones, 1981). However, there ws no indiction of methemogloinemi ccording to the color of the lood. The mechnism y which mmoni lters H O 2 inding is worthy of future study. The loction of mmoni sensors in trout gills An importnt im of this study ws to find out the loctions of the mmoni sensors involved in hyperventiltion. The gill rch removl experiments provided n nswer t corse level. Hyperventiltion in response to (NH 4 ) 2 SO 4 injections ws immedite (see lso Zhng nd Wood, 29), ut took out 1 min in response to cute wterorne (NH 4 ) 2 SO 4 exposure (Figs 1 nd 3). The response ws delyed until 3 4 min in trout lcking rch I, nd eliminted in trout without rches I nd II (Fig. 3), suggesting tht the mmoni sensors re concentrted in these two rches. In the light of the 3 4 min dely in response seen fter rch I removl, we speculte tht rch I receptors my minly sense the externl wterorne mmoni (reltively fst response), wheres rch II receptors my sense the internl plsm mmoni (much slower response). In the former cse, it will tke short ut finite time for wterorne mmoni to diffuse cross the epithelil nd mucus rrier nd rise the mmoni level t externl NECs sufficiently to trigger the hyperventiltory response. In the ltter cse, the much longer dely would e due to the time needed for the process of mmoni uptke from the wter to rise plsm mmoni to the threshold level needed to trigger the internl NECs tht sense plsm mmoni levels. At finer level, the density of NECs decresed in rches I nd II (Fig. 5A), nd the [C 2 ] i response to high NH 4 ws clerly ttenuted in NECs from rch I (Fig. 7) s result of chronic HEA exposure, tretment tht lso eliminted the hyperventiltory response to high mmoni (Figs 1 nd 2). In teleosts, gill rch I is innervted y rnch of the glossophryngel nerve (IX) nd ll four rches re innervted y rnches of the vgus nerve (X) (Milsom nd Burleson, 27). In phylogenetic terms, NECs in rch I (emryonic rch III) pper to e the ntecedents of the mmmlin crotid odies, while the NECs of rch II (emryonic rch IV) re the ntecedents of the ortic odies. However, the reson why only gill rches I nd II, nd not rches III nd IV, pper to function in mmoni sensing remins uncler, ecuse 5-HT-positive NECs were distriuted in ll of the four gill rches without lrge differences in numer or size (Fig. 5), which grees with previous studies in zerfish (Jonz nd Nurse, 23). Furthermore, NECs from ll four rches exhiited [C 2 ] i responses to high NH 4 (Fig. 7). Gill rch I is now widely recognized s n importnt site for sensors of qutic respirtory gs tensions. For exmple, hypoxi nd hypercpni oth produce reflex rdycrdi in fish; the receptors ssocited with these responses re restricted to gill rch I nd pper to minly sense wter gs tensions in mny species including rinow trout (Dxoeck nd Holeton, 1978; Smith nd Jones, 1978; Smith nd Jones, 1981; Perry nd Reid, 22). However, the sensors for the ventiltory response to hypoxi pper to e more diffuse nd vrile, sensing oth externl nd internl

9 2686 L. Zhng nd others NEC density (cells mm 1 filment) NEC size (μm 2 ) A B c c,, c HEA,, c 2 Arch I Arch II Arch III Arch IV Fig. 4. Serotonin (5-HT)-immunorective neuroepithelil cells (NECs) in filments of gill rch I from (A) control trout nd (B) trout chroniclly exposed to HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months. F nd L indicte the primry filment nd the secondry respirtory lmelle, respectively. Br indictes 1 mm. gs levels (Dxoeck nd Holeton, 1978). For exmple, in the ctfish, the ventiltory response to hypoxi rises only from receptors confined to the gill rches (Burleson nd Smtresk, 199), ut in mny fish, totl gill denervtion fils to eliminte the hypoxic ventiltory response (Sunders nd Sutterlin, 1971; Sundin et l., 1998) nd the remining receptors pper to occur t extrrnchil sites including the orornchil cvity (Milsom et l., 22). It is possile ut unlikely tht the pseudornch is involved in mmoni sensing. The pseudornch is served with rteril lood from the first efferent gill rtery ut is not exposed to the externl environment. Both older nd more recent reserch suggests tht the pseudornch my not function in gs sensing. For exmple, ilterl de-fferentition of the pseudornch hd no effect on the ventiltory responses to hypoxi or hyperoxi (Rndll nd Jones, 1973) nd hypoxic relese of ctecholmines from chromffin cells medited y O 2 chemoreceptors did not involve the pseudornch in trout (Reid nd Perry, 23). Indeed, only one or two 5-HTcontining cells resemling O 2 -sensitive gill NECs were found in ech pseudornch filment in zerfish (Jonz nd Nurse, 23). Morphologicl chnges in NECs during chronic HEA While the detrimentl effects of very high mmoni on gill histology (e.g. lmellr fusion, epithelil lifting nd hemorrhge) hve een well documented (Smrt, 1976; Mlltt, 1985; Frncis et l., 2; Spencer et l., 28), this ppers to e the first study exmining the effects of chronic sulethl HEA exposure on the rnchil Fig. 5. The (A) density nd (B) size of NECs in the filments of different gill rches from control trout nd from trout chroniclly exposed to HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months. N 8. Mens not shring the sme letter re significntly different from one nother (P<.5). NECs. Both the cute ventiltory responses to mmoni (Figs 1 nd 2) nd the morphometry of the NECs were ltered, with significnt reductions in NEC numer (density) in rches I nd II (Fig. 5A), nd reductions in their size (Fig. 5B) in ll four rches. The response ppers to e prtilly, ut not completely, nlogous to the findings of Vulesevic et l. (Vulesevic et l., 26) on zerfish, who showed significnt 35% decrese in NEC density fter hyperoxi cclimtion for 28 dys, with lunted sensitivity to susequent cute hypoxic or hypercpnic chllenge. Both studies suggest tht the sensitivity of the ventiltory responses is influenced y the numer of NECs. The difference, however, is tht cute hyperoxi normlly inhiits ventiltion wheres cute mmoni normlly stimultes ventiltion. Further complicting the sitution is the fct tht Vulesevic et l. (Vulesevic et l., 26) nd Jonz et l. (Jonz et l., 24) oserved 15% increse in size nd no chnge in the numer (density) of NECs in zerfish fter prolonged cclimtion to hypoxi, ut no chnge in the hyperventiltory response to cute hypoxi. Clerly there is much more to lern out the morphologicl nd functionl plsticity of rnchil NECs. NECs re proly mmoni chemoreceptors An importnt finding of the present study is tht not only do NECs respond to mmoni in fish ut lso they respond with mrked increse in [C 2 ] i, consistent with mmoni cusing clcium influx. By nlogy to investigtions on glomus cells of the mmmlin crotid ody, previous studies on fish NECs sujected to hypoxic (Jonz et l., 24) or hypercpnic stimuli (Qin et l., 21) hve speculted ut not demonstrted tht this [C 2 ] i surge occurs. In this scheme, the pproprite stimulus inhiits ckground K current, cusing memrne depolriztion, which triggers voltge-

10 Ammoni sensing nd ventiltory responses in trout 2687 [C 2 ]i (nmol l 1 ) A High K High NH 4 Type A C High K High NH 4 NEC PVC NEC B D HEA c,c,c c Fig. 6. Two types of [C 2 ] i response in NECs to the perfusion of high K (3 mmol l 1 ) or high NH 4 (1 mmol l 1 ) Cortlnd sline. (A) A typicl type A slow chnge of [C 2 ] i during nd fter the high NH 4 perfusion period in representtive NEC, s well s in pvement cell (PVC). PVCs never responded. (C) A typicl type B chnge (oth fst nd slow) of [C 2 ] i during nd fter the high NH 4 perfusion period in representtive NEC. (B) A summry of ckground nd pek [C 2 ] i of type A responses in NECs from control trout (N 11) nd from trout (N 8) chroniclly exposed to HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months. (D) A summry of ckground nd pek [C 2 ] i of type B responses in NECs from control trout (N 15) nd from trout (N 5) chroniclly exposed to HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 months. Mens not shring the sme letter re significntly different from one nother (P<.5). Type B Time (s) Bckground High K High NH 4 gted C 2 influx, susequent neurotrnsmitter relese nd fferent nerve ctivtion (Lhiri nd DeLney, 1975; Peers, 199; Peers, 199; Buckler nd Vughn-Jones, 1994; Buckler nd Vughn- Jones, 1994; Dsso et l., 2; Zhng nd Nurse, 24). If these ides re correct, the sensing of mmoni y fish NECs would intersect with the hypoxi- nd hypercpni-sensing pthwys. Thus, the elevtion of [C 2 ] i reveled in the present study would e due to chnges in the K current in NECs. Two of the four known clsses of K chnnels (Hille, 21), the ckground (or lek) K chnnels nd voltge-dependent K chnnels, hve een evluted in fish NECs. Becuse the depolriztions induced y oth hypoxi nd hypercpni re locked y the presence of quinidine, non-specific locker of ckground K chnnels, ut not y severl lockers of voltge-dependent K chnnels, it hs een proposed tht these stimuli cuse memrne depolriztion y inhiiting ckground K chnnels (Jonz et l., 24; Qin et l., 21). In turn, the elevtion of [C 2 ] i in NECs would led to the susequent relese of neurotrnsmitter for the ventiltory modultion, with 5-HT eing the prime cndidte (Fritsche et l., 1992; Burleson nd Milsom, 1995; Sundin et l., 1998; Jonz nd Nurse, 23; Coolidge et l., 28). Ctecholmines, ACh nd ATP (co-stored with monomines in vesicles) my lso ply importnt roles s likely neurotrnsmitters in NEC physiology (Burleson nd Milsom, 1995; Coolidge et l., 28; Nurse, 21). It ws interesting tht there were two types of [C 2 ] i response to high NH 4 perfusion; type A showing only slow response, nd type B showing fst plus slow response (Fig. 6). Although the slow responses in the two types were not the sme (peked increse in type A, prolonged increse in type B), oth of them occurred fter NH 4 hd een removed from the perfusion medi, nd were long lsting. It is well known tht mmoni exposure to cells results in ph i disturnce. Indeed, this is the sis of the clssic mmonium prepulse technique (Roos nd Boron, 1981), with n lklosis during the mmoni loding period, nd n cidosis fter mmoni wshout, reflecting the more rpid flux of NH 3 thn of NH 4 or H. The recovery of cells from cidifiction is long process. The temporl consistency of the slow [C 2 ] i response nd cidosis to NH 4 perfusion suggests tht intrcellulr cidifiction my e cndidte mechnism for how some NECs sense mmoni. In recent study on zerfish NECs, cronic nhydrse ws shown to enhnce the electrophysiologicl response to hypercpni (Qin et l., 21). This phenomenon suggests tht intrcellulr cidifiction is one of the mechnisms for sensing CO 2, s in mmmlin type I glomus cells (Putnm et l., 24; Lhiri nd Forster, 23; Qin et l., 21). In future investigtions, it will e [C 2 ]i (nmol l 1 ) Arch I Arch II /3 mmol l 1 K HEA/3 mmol l 1 K /1 mmol l 1 NH 4 HEA/1 mmol l 1 NH 4 Arch III Arch IV Fig. 7. [C 2 ] i responses to perfusion of high K (3 mmol l 1 ) or high NH 4 (1 mmol l 1 ) Cortlnd sline in NECs from different gill rches of control trout nd of trout chroniclly exposed to HEA [25 mmol l 1 (NH 4 ) 2 SO 4 ] for 1 month. Type A nd type B responses hve een comined. N vlues for NECs in gill rches I, II, III nd IV re 17, 3, 5 nd 1 in the control trout nd 5, 5, 1 nd 2 in the HEA trout, respectively. Asterisk highlights significnt reduction in the response to high NH 4 (1 mmol l 1 ) only on gill rch I (P<.5).