P2X receptor immunoreactivity in the rat cochlea, vestibular ganglionand cochlear nucleus

Zhenghua Xiang b, Xuenong Bo a, Geo¡rey Burnstock a;*a Autonomic Neuroscience Institute, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK

b Department of Histology and Embryology, Second Military Medical University, Shanghai, China

Received 23 May 1998; received in revised form 26 October 1998; accepted 7 November 1998

Abstract

P2X receptors have been reported to be involved in neurotransmission in both central and peripheral nerves. In the present study,polyclonal antibodies against P2X1, P2X2, P2X3, P2X4, P2X5, and P2X6 were used to study the distribution of P2X receptors in ratcochlea and vestibulocochlear nerve pathways. It was found that in the vestibular ganglion all six types of antibodies stained theneurons to different intensities. The strongest signal was obtained with the P2X2 antibodies, followed by P2X3 antibodies. The otherfour antibodies produced weak signals, of approximately the same intensity. In the spiral ganglion, the six types of antibodies alsostained almost all neurons. However, the rank order of intensity was different from that in the vestibular ganglion: the strongestsignal was still obtained with P2X2 antibodies, followed by P2X4, P2X1, and P2X3 antibodies. The immunolabelling was muchweaker with P2X5 and P2X6 antibodies compared with the other four types of antibodies. In the cochlea, besides the spiral ganglionneurons, other tissues such as stria vascularis, the organ of Corti and the tectorial membrane were labelled intensively with P2X2

antibodies only. High density P2X2 immunoreactivity was also observed in the vestibulocochlear nerve fibres. In the cochlearnucleus, neurons and nerve fibres were stained with the P2X2 antibodies, as were the neurons in the trapezoid body. These resultssuggested that P2 receptors, especially the P2X2 receptors, may play important roles in the signal transduction involved in theperception of sound and balance. z 1999 Elsevier Science B.V. All rights reserved.

Key words: P2X receptor; P2X purinoceptor; ATP; Spiral ganglion; Cochlea; Vestibular ganglion; Vestibulocochlear nerve

1. Introduction

In recent years the biological e¡ects of extracellularpurine nucleotides have been studied in many kinds ofcells and tissues (Burnstock, 1997). There is abundantevidence indicating that ATP might function as a neu-rotransmitter or a neuromodulator in both central andperipheral nervous systems. A current area of intenseinterest is the involvement of ATP in sensory nocicep-tion (Burnstock and Wood, 1996; Burnstock, 1996),especially since the cloning of P2X3 receptors from dor-sal root ganglion (Chen et al., 1995). In special sensoryorgans, the e¡ects of ATP on hair cells in the cochlea

have been intensively studied (see Thorne and Housley,1996; Housley, 1998). In outer hair cells of guinea-pigand chick, ATP was reported to increase intracellularcalcium concentration which could be separated intotwo components: one rapid rise was due to calciumin£ux, and one slow and sustained rise was due to cal-cium release from an internal store (Ashmore and Oh-mori, 1990; Shigemoto and Ohmori, 1990). An electro-physiological study showed that ATP induced aninward current through a large cation channel in theouter hair cells (Nakagawa et al., 1990). Similar re-sponses were also recorded in inner hair cells, suggest-ing the presence of two types of ATP receptors (Suga-sawa et al., 1996a). Further studies demonstrated thatATP-induced responses were not limited to hair cells,but were also observed in the supporting cells on theorgan of Corti (Ashmore and Ohmori, 1990; Dulon et

0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 2 0 8 - 1

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* Corresponding author. Tel. : +44 (171) 830 2948;Fax: +44 (171) 830 2949; E-mail: g.burnstock@ucl.ac.uk

Hearing Research 128 (1999) 190^196

al., 1993; Sugasawa et al., 1996b) and the non-sensoryepithelial cells of the cochlear lateral wall (Ikeda et al.,1995; Suzuki et al., 1995).

In other parts of the nerve pathways of the auditoryand vestibular systems, studies of ATP-induced re-sponses are less than that on hair cells. In guinea-pigspiral ganglion cells ATP was reported to cause an in-crease in intracellular calcium (Cho et al., 1997). In ratmedial vestibular nucleus, a subpopulation of neuronswas shown to express P2X and P2Y receptors (Chessellet al., 1997). Autoradiographic localisation with P2 re-ceptor radioligands [35S]ATPKS and [3H]K,L-methyleneATP showed that their speci¢c binding sites have het-erogeneous distribution in the guinea-pig inner ear(Matchett et al., 1995). Using molecular biology techni-ques, the P2X2 receptor and its three splice variantshave been identi¢ed in the rat cochlea (Housley et al.,1995; Braëndle et al., 1997) and spiral ganglion neurons(Salih et al., 1998). Three variants of P2X2 receptorswere also isolated from the guinea-pig organ of CorticDNA library (Parker et al., 1998). The P2X2 receptormRNA transcripts have been localised in various tissuesin the rat inner ear (Housley et al., 1998). The evidenceabove indicates that P2 receptors may be involved inmany steps of signal transduction in the auditory andvestibular systems. So far, seven P2X and about tenP2Y receptor subtypes have been cloned. There is notmuch information about which subtypes of P2 recep-tors are present in the auditory and vestibular nervoussystems except for the reports on P2X2 receptors in thecochlea. Recently, our collaborators in Roche Bio-science (USA) have provided us with polyclonal anti-bodies against P2X1 to P2X6 receptors. In the presentstudy, we have used these antibodies to localise thesereceptors in the rat inner ear and the vestibulocochlearpathways. Such information should provide a guidancefor further functional studies on the role of ATP in theauditory and vestibular systems.

2. Methods

2.1. Tissue preparation

Six Wistar rats (200^250 g) were killed by asphyxia-tion with CO2. The inner ear, vestibular ganglion, andbrain stem were dissected out immediately and ¢xed in4% paraformaldehyde and 0.2% saturated picric acid in0.1 M phosphate bu¡ered saline (PBS, pH 7.2) for 24 h.The inner ear blocks were then transferred to 0.1 MEDTA and kept in this solution for 5^7 days to decal-cify the bony labyrinth. After that the blocks were im-mersed in 20% sucrose in 0.05 M PBS until they sank tothe bottom. Thereafter the blocks were rapidly frozenby immersion in 370³C isopentane and cut into sec-tions of 10 Wm thickness in a cryostat. Sections were

mounted on gelatine-coated slides and dried at roomtemperature.

2.2. Immunohistochemistry

The immunogens used for production of polyclonalantibodies were synthetic peptides corresponding to thecarboxy termini of the cloned rat P2X receptors, cova-lently linked to keyhole limpet haemocyanin. The pep-tide sequences are as follows: P2X1 : amino acids 385^399, ATSSTLGLQENMRTS; P2X2 : amino acids 458^472, QQDSTSTDPKGLAQL; P2X3 : amino acids 383^397, VEKQSTDSGAYSIGH; P2X4 : amino acids 374^388, YVEDYEQGLSGEMNQ; P2X5 : amino acids437^451, RENAIVNVKQSQILH; P2X6 : amino acids357^371, EAGFYWRTKYEEARA. The polyclonalantibodies were raised by multiple monthly injectionsof New Zealand rabbits with the peptides. The speci¢c-ity of the antisera was veri¢ed by immunoblotting withmembrane preparations from CHO K1 cells expressingthe cloned P2X receptors and with native tissues likeurinary bladder and dorsal root ganglion. IgG fractionswere isolated from the immune sera and the pre-im-mune sera using chromatography on DEAE A¤-Gelblue gel (Bio-Rad, Hemel Hempstead, UK).

Sections which had been treated as described abovewere incubated in 0.5% H2O2 and 50% methanol for20 min to block the endogenous peroxidase. The sec-tions were then pre-incubated in 10% normal horse se-rum in PBS for 30 min at room temperature, followedby incubation with the primary antibodies diluted 1:500to 1:1000 in 10% normal horse serum, 0.1% TritonX-100 and 0.1% sodium azide in PBS overnight. Sub-sequently the sections were incubated with biotinylateddonkey anti-rabbit IgG (Jackson ImmunoResearchLab., West Grove, PA, USA) diluted 1:500 in 0.1%Triton X-100 and PBS for 1 h, and then with ExtrA-vidin peroxidase diluted 1:1500 in PBS for 1 h at roomtemperature. For colour reaction a solution containing0.05% 3,3P-diaminobenzidine (DAB), 0.04% nickel am-monium sulfate, 0.2% L-D-glucose, 0.004% ammoniumnitrate, and 1.2 U/ml glucose oxidase in 0.01 M PBSwere applied. Sections were washed three times with0.1 M PBS after each of the above steps except pre-incubation. Finally the DAB end-products in a groupof sections were enhanced with the silver-gold intensi¢-cation method according to the protocol of Liposits etal. (1984).

The control experiments were carried out with theprimary antibodies pre-absorbed with the peptides forimmunising the rabbits, the primary antibody omitted,or the primary antibody replaced with the preimmunerabbit sera. In the experiment with the silver-gold in-tensi¢cation method, the sections for control were notincubated with DAB solution or the sections from theabove-mentioned control experiments were used.

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3. Results

3.1. Vestibular ganglion

All six P2X receptor antibodies stained the neuronsin the vestibular ganglion, but to di¡erent intensity. Thestrongest signals were seen on the sections stained withP2X2 receptor antibody (Fig. 1B). Both the neuronalcell bodies and nerve ¢bres were stained intensely,with almost all the neurons in this ganglion beingP2X2-immunoreactivity (-ir) positive. The intensity of

P2X3-ir was lower than that of P2X2-ir (Fig. 1C), andthe intensity varied greatly in individual neurons. Thesmall- and medium-diameter neurons were stained moreheavily than the large-diameter ones. Nerve ¢bres withP2X3-ir were also observed; however, the density ofP2X3-ir positive nerve ¢bres was less than that withP2X2-ir. The intensity of immunostaining with P2X1,P2X4, P2X5, and P2X6 receptor antibodies was sim-ilar, and was much lower than that with P2X2 andP2X3 receptor antibodies (Fig. 1A, D, E, F), withonly a few neurons showing strong signals. The posi-

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Fig. 1. Distribution of P2X receptor immunoreactivity in rat vestibular ganglion. A: P2X1 ; B: P2X2 ; C: P2X3 ; D: P2X4 ; E: P2X5 ; F: P2X6.Scale bar = 50 Wm.

Z. Xiang et al. / Hearing Research 128 (1999) 190^196192

tive neurons were mainly small-diameter ones. Somenerve ¢bres with faint P2X1, P2X4, P2X5, or P2X6

receptor-ir were visible in the ganglion (Fig. 1A, D,E, F).

3.2. Spiral ganglion

In spiral ganglion, neurons were also stained by allsix P2X receptor antibodies to di¡erent intensity. All ofthe neurons were positive with the six P2X receptor-ir.As that in the vestibular ganglion, the strongest immu-

nostaining signals were obtained with the P2X2 anti-body (Fig. 2B). Some P2X2-ir positive nerve ¢breswere also observed. The second strongest immunostain-ing signals in spiral ganglion were P2X4 receptor-ir,which intensity was almost similar to that of P2X2-ir.The third in the rank of immunostaining intensity wasthat of P2X1-ir (Fig. 2A), followed by P2X3-ir (Fig.2C). The intensity of immunostaining with P2X5 andP2X6 antibodies was quite similar, and was much weak-er than that of the other four P2X receptor subtypes(Fig. 2E, F).

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Fig. 2. Distribution of P2X receptor immunoreactivity in rat spiral ganglion. A: P2X1 ; B: P2X2 ; C: P2X3 ; D: P2X4 ; E: P2X5 ; F: P2X6.Scale bar = 50 Wm.

Z. Xiang et al. / Hearing Research 128 (1999) 190^196 193

3.3. Other cells in the cochlea

Many other types of cells in the organ of Corti andthe cells lining the endolymphatic compartment wereP2X2-ir positive (Fig. 3A). In the stria vascularis, im-munostaining was very intense and it seems almost allcells were P2X2-ir positive. P2X2-ir positive nerve ¢breswere found in the connective tissue in osseous spirallamina, which connects the organ of Corti. No positivereactions were found with the other ¢ve purinoceptorsubtype antibodies.

3.4. Cochlear nucleus and trapezoid body

In the cochlear nucleus neurons and nerve ¢bres withstrong P2X2-ir signals were observed (Fig. 3B). Thevestibulocochlear nerve bundle was stained heavilywith P2X2 antibody as well (Fig. 3C). In the trapezoidbody many perikarya and some ¢bres with P2X2-irwere found (Fig. 3D). No positive immunoreactivitywas found in the cochlear nucleus and trapezoid bodywith other P2X antibodies. In vestibular nucleus, only afew nerve ¢bres were P2X2 receptor-ir positive.

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Fig. 3. Distribution of P2X2 receptor immunoreactivity in rat inner ear and auditory nerve pathway. A: Inner ear (SG: spiral ganglia; TM:tectorial membrane; SV: stria vascularis; scale bar=200 Wm); B: cochlear nucleus and trigeminal spinal tract (CN: cochlear nucleus; TST: tri-geminal spinal tract; scale bar=100 Wm); C: vestibulocochlear nerve (scale bar = 200 Wm); D: trapezoid body (scale bar = 200 Wm).

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4. Discussion

The results in the present study have demonstratedthat P2X receptors have a wide distribution in the innerear and the vestibulocochlear pathways. In both thevestibular and spiral ganglia, all six P2X receptor sub-types were expressed. However, there are signi¢cantdi¡erences in the level of expression for di¡erent recep-tor subtypes. In the vestibular ganglion P2X2 and P2X3

receptors appear to be dominant, while in the spiralganglion P2X1, P2X2, P2X3, and P2X4 were all highlyexpressed. The expression of several receptor subtypesin single neurons may indicate that the native P2X re-ceptors are heteromultimers. Similar expression of mul-ti-receptor subtypes in single neurons have been ob-served in other ganglia like dorsal root ganglion,trigeminal ganglion, nodose ganglion, superior cervicalganglion, and coeliac ganglion (Xiang et al., 1998).Although clear evidence for the composition of nativeP2X receptors is still not available, some reports indi-cate that these receptors exist as heteromultimers (Lewiset al., 1995; Radford et al., 1997). Detailed electrophy-siological and pharmacological studies on P2X recep-tors in the neurons in vestibular and spiral ganglion arerequired. High level expression of P2X3 receptors inneurons in both vestibular and spiral ganglia indicatesthat this receptor subtype is not only involved in noci-ception but also in signal transduction mechanisms forthe perception of other sensory stimuli.

In a recent study with in situ reverse transcriptase-polymerase chain reaction (RT-PCR), it was reportedthat P2X2 receptor cDNA was detected in 49% of thespiral ganglion neurons (Salih et al., 1998). However, inour present study almost all spiral ganglion neuronswere stained intensely with P2X2 receptor antibodies.Such discrepancy might be due to the method of sil-ver-gold enhancement of the immunostaining signalsbeing more sensitive than in situ RT-PCR.

Although neurons in the vestibular and spiral gan-glion express all six P2X receptor subtypes, only P2X2

receptor-ir was observed in other tissues in the innerear. Due to the fragility of the organ of Corti, wewere unable to obtain an intact structure on the cryo-stat section. However, strong P2X2 receptor-ir was ob-served on the scattered hair cells and the supportingcells, which was in agreement with the results obtainedwith RT-PCR (Housley et al., 1995; Braëndle et al.,1997) and in situ hybridisation (Housley et al., 1998).In pharmacological studies, it was found that P2X re-ceptors on guinea-pig outer hair cells do not desensitise(Nakagawa et al., 1990; Housley et al., 1992), which isa typical feature of the cloned P2X2 receptor (Brake etal., 1994). In a recent study on the outer hair cells fromneonatal mouse, the ATP-activated inward currentshowed a certain degree of desensitisation (Glowatzkiet al., 1997). However, when a high concentration of

ATP (300 WM) was applied for a longer period (1 min),desensitisation could occur (Braëndle et al., 1997). Bythe way, both P2X2 and its splice variant P2X2ÿ2

have been identi¢ed in the organ of Corti of rat, anddesensitisation is almost complete for P2X2ÿ2 (Braëndleet al., 1997). It is possible that the ATP-induced inwardcurrent recorded by Glowatzki et al. (1997) could be amixed response mediated by both P2X2 and P2X2ÿ2.

In other non-sensory epithelial cells lining the endo-lymphatic compartment, strong P2X2 receptor-ir wasobserved. Although ATP-induced Ca2� in£ux hasbeen reported in these cells, there is no informationabout which P2X subtype is involved (Ikeda et al.,1995). The stria vascularis is another structure heavilylabelled by P2X2 antibodies. A splicing variant of theP2X2 (P2X2ÿ1) receptor has been identi¢ed in this tissue(Housley et al., 1995). However, the antibodies we usedin the present study should not recognise the splicingvariant because the C-terminus of P2X2 has been re-placed with a very short C-terminus in P2X2ÿ1 whichdoes not contain the peptide sequence that was used toimmunise the rabbits. Therefore, it is possible that bothP2X2 and P2X2ÿ1 are present in the stria vascularis. Itis not known whether P2X2ÿ1 can form a functionalreceptor or not.

Functional studies on P2X receptors in the centralnervous pathways of the vestibulocochlear system arescarce. One report showed that K,L-methylene ATPincreased the spontaneous ¢ring rate in about 35% ofthe neurons in the rat medial vestibular nucleus, whichcould be blocked by suramin and pyridoxal-phosphate-6-azophenyl-2P,4P-disulfonic acid (PPADS) (Chessell etal., 1997). The results indicate the presence of P2X1 orP2X3 receptors because only these two subtypes areresponsive to K,L-methylene ATP and can be blockedby suramin and PPADS. However, these P2X receptorsin the vestibular nucleus were not identi¢ed by ourpolyclonal antibodies. It is possible that another un-cloned P2X receptor subtype exists in the central ner-vous system, which shares some pharmacological fea-tures with P2X1 and P2X3. In the cochlear nucleus,P2X2 receptor-ir positive neurons and nerve ¢breswere identi¢ed, which indicates that P2X2 receptorsare also involved in auditory signal transduction inthe second order of neurons apart from the ¢rst-orderneurons in the spiral ganglion. This is further supportedby the observation of P2X2 receptor-ir positive neuronsand nerve ¢bres in the trapezoid body.

In conclusion, the results in the present study haveshown that neurons in vestibular and spiral ganglia ex-press P2X1 to P2X6 receptors, with the P2X2 receptorthe most dominant. P2X2 receptor-ir was also observedin the sensory and non-sensory epithelial cells in theorgan of Corti and stria vascularis. Neurons and nerve¢bres in the cochlear nucleus were labelled with P2X2

antibodies. These results indicate that P2X receptors,

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especially the P2X2 subtype, are involved in the percep-tion and transduction of auditory and balance signals.

Acknowledgments

We gratefully acknowledge the support from RocheBioscience in Palo Alto, USA. The editorial work ofMr. R. Jordan in the preparation of the manuscript isgreatly appreciated. The work was also supported bythe British Heart Foundation.

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