Expression of the P2X1 Receptor Converges onto the Type II Spiral Ganglion Neurons in the Mature Rat Cochlea

Expression of the P2X1 Receptor Converges onto the Type II Spiral Ganglion Neurons in the Mature Rat Cochlea | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Expression of the P2X1 Receptor Converges onto the Type II Spiral Ganglion Neurons in the Mature Rat Cochlea Prakansha N Kumar, Srdjan M Vlajkovic, Peter R Thorne, Haruna Suzuki-Kerr This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6272432/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jan, 2026 Read the published version in Purinergic Signalling → Version 1 posted 13 You are reading this latest preprint version Abstract Our sense of hearing commences in the cochlea, the peripheral sensory organ for hearing. Spiral ganglion neurons (SGN) in the cochlea are primary auditory neurons responsible for auditory neurotransmission. There are two classes of SGNs: type I SGNs, which make up 90-95% of the SGN population, and type II SGNs, which make up the remainder. Previous studies have shown that SGNs express a combination of purinergic (P2X, P2Y and adenosine) receptors at the mRNA and protein levels. In this study, we have focused on P2X1 receptor to characterize its expression pattern in Wistar rat cochlea at postnatal day 8 and in adult (6–8-week-old) rats of both genders using immunohistochemistry. Our results show differential expression of P2X1 in approximately ~10% of the SGN population. In these neurons, P2X1 was co-expressed with peripherin-1, an established type II SGN marker. These results imply the role of P2X1 receptor signalling in auditory neurotransmission that drives medial olivocochlear reflex suppression of the cochlear amplifier. Cochlea adult rat spiral ganglion neurons P2X1 receptor immunohistochemistry auditory neurotransmission Figures Figure 1 Figure 2 Introduction The cochlea, a ~ 1 cm organ deeply embedded in the temporal bone, is our peripheral organ for hearing. The human cochlea contains approximately 3400 inner hair cells (IHCs) and 14,000 outer hair cells (OHCs) across the 34 mm length of the basilar membrane (1), and 29,000–35,000 spiral ganglion neurons (SGN) (2). Electrical signals from IHCs are transmitted through SGNs to the central auditory pathways in the brain. There are two broad classes of SGNs in the cochlea: type I and type II SGNs (3). The cell bodies of both types of SGNs are located in Rosenthal’s canal in the central bony modiolus of the cochlea. Of the total SGN population, 90–95% are type I SGN. Type I SGNs are myelinated bipolar neurons with large cell bodies and axonal diameters, allowing rapid propagation of action potentials. These neurons are the primary auditory neurons that play a crucial role in transmitting sound information from the cochlea to the brain (4). The dendrite from each Type I SGN forms a synaptic connection with only one IHC. Each IHC is innervated by 20 to 30 Type I SGNs. Functional observations of different thresholds of Type I SGNs, and recent RNA-sequencing-based evidence point out that at least three subclasses of Type I exist in the mammalian cochlea (5–7). On the other hand, type II SGNs make up only the remaining 5–10% of the total SGN population. Type II SGNs are unipolar neurons, and their thin unmyelinated dendrites branch to innervate 5–30 OHCs, usually in the same row (8). Their cell bodies are distributed throughout the spiral ganglion, with fewer found in the most apical region (9). Type II SGNs have been suggested to be involved in the olivocochlear efferent reflex, which modulates cochlear amplification (10). P2X receptors are ATP-gated ion channels often localised in pre- or post-synaptic terminals, where ATP functions as a neurotransmitter (11). Previous studies have described the expression of P2X1 in the spiral limbus, Reisner’s membrane and SGNs in the cochlea of juvenile (P0-P6) and adult Wistar rats (12, 13). Nikolic et al. (2001) demonstrated P2X1 immunoreactivity in both afferent and efferent neurite projections and the soma of SGNs in the developing rat cochlea with a developmental decline from P10 onwards (12). Here, we report that P2X1 expression remains in SGNs after P10 in Wistar rat cochlea. P2X1 immunoreactivity was observed in a subpopulation of SGNs likely representing Type II neurons, but was mostly absent or from Type I neurons. These findings imply differential roles of P2X1 receptor signalling in the developing and mature rat cochlea. Method Animal studies were approved by the Animal Ethics Committee at the University of Auckland (AEC approval No. 002251). Otherwise stated, all the chemicals were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Wistar rats of both sexes were used in equal proportions in this study. P8 Wistar rat pups were decapitated using surgical scissors, and 6–8-week-old Wistar rats were euthanized with an overdose of pentobarbital (90 mg/kg, ProVet NZ Pty Ltd, New Zealand) followed by cardiac perfusion with flushing solution (0.1M phosphate-buffered saline (PBS) with NaNO 2 ) and 4% w/v paraformaldehyde (PFA). Temporal bones were extracted and immersed in 4% PFA for 24 hours at room temperature (RT). Cochleae from 6-week-old rats were decalcified by immersion in 4% w/v Ethylenediaminetetraacetic Acid (EDTA) in PBS at the RT for two weeks, while this step was unnecessary for P8 cochlea. For the Organ of Corti (OoC), whole mounts and cryosections (at 30 µm thickness) were used for immunochemistry following our standard protocol (14), which includes incubation with blocking solution (2.5% v/v Triton X-100 in 0.1M PBS and 10% v/v Normal Goat Serum (NGS) or donkey serum (DS) for whole mounts, and 1% Triton X-100 v/v in 0.1M PBS and 10% v/v/ NGS/DS for cryosections). All antibodies were diluted in antibody solution (0.25% Triton X-100 v/v in 0.1M PBS and 10% v/v/ NGS/DS for whole mounts, 0.1% Triton X-100 v/v in 0.1M PBS and 10% v/v/ NGS/DS for cryosections). The primary antibodies used were: rabbit polyclonal anti-P2X1 antibody (Alomone Labs, Jerusalem, Israel, Catalog# APR-001, 1:500 dilution), mouse anti-β III tubulin (IgG2a monoclonal, BioLegend, San Diego, CA, catalog No. 801213, 1:1000 dilution), goat polyclonal anti-peripherin-1 antibody (Everest Biotech, Upper Heyford, UK, catalog No. EB12405, 1:1000 dilution). For the peptide preabsorption control, P2X1 receptor blocking peptide (Alomone Labs, Jerusalem, Israel, Catalog# BLP-PR001) was mixed with the primary antibody in a 1 mg to 1 mg ratio and incubated for one hour at RT following the manufacturer’s protocol. Secondary antibodies used were: goat polyclonal anti-rabbit Alexa 594, goat polyclonal anti-mouse Alexa 488, donkey polyclonal anti-rabbit DyLight 594, donkey polyclonal anti-goat Dylight 488 (ThermoFisher, all used at 1:1000 dilutions). 4′,6-diamidino-2-phenylindole (DAPI, 300 nM final concentration) was added to the secondary antibody mixture. Between each incubation, tissues were washed with 0.1M PBS at RT four times (for 3 min, 5 min, 10 min and 20 min). The tissues were mounted with CitiFluorTM AF1 (Science Services, Germany) and imaged with confocal microscopy (Olympus FV1000, Tokyo, Japan). Z-stacks were obtained with 20x objective lens and 60x objective lens at 0.263µm/pixels and 0.24µm/pixels. Image analysis was carried out using FIJI (Image J). Total SGNs were calculated based on anti-β III tubulin immunolabelling. SGNs were counted when the soma contained 50% or more of the cytoplasm. To estimate the density of SGNs, the area of Rosenthal’s canal was estimated using the ‘free-hand’ tool to select the region of interest (FIJI) and the SGN density was expressed as the cell count per 100 µm 3 to give a number of cells/100µm 3 as previously described (9)(Table 1 ). Table 1 Quantification of the population of SGNs stained with anti-P2X 1 antibody. Density (SGNs/ (100um) 3 ) Adult (n = 4) P8 (n = 4) Apical Middle Basal Apical Middle Basal Strongly P2X 1 positive number/(100µm) 3 72 ± 30 (12.5%) 29 ± 7 (9.44%) 40 ± 9.0 (14.2%) 77 ± 2.5 (15.7%) 71 ± 3.0 (14.2%) 100 ± 24 (23.3%) Total SGN number/(100µm) 3 575 ± 180 307 ± 46 280 ± 120 490 ± 40 500 ± 34 430 ± 35 Results The protein expression pattern of the P2X1 receptor was investigated in the cochleae of developing and adult Wistar rats using the commercially available anti-P2X1 antibody (Alomone, #APR-001). The antibody was raised against the intracellular C-terminal domain of rat P2X1 (residues 383–399) and has been used in over 70 published articles, including testing in gene knockout tissues of the arterial smooth muscle (15) and male reproductive systems (16). In the adult Wistar rat cochlea, a subpopulation of SGNs remained strongly immunolabelled with anti-P2X1 antibody (Fig. 1 A, B & D). P2X1 immunolabeling was observed in the regions where SGN processes reached the OoC (Fig. 1 B), which was attributed to SGN processes extending towards OHCs at the higher resolution (Fig. 1 C & F). Control tissues pre-absorbed with the corresponding peptide did not show immunolabelling (Fig. 1 D). To visualize SGN, an anti-βIII tubulin antibody was used as a pan-SGN marker in the cochlea (17) (Fig. 1 E, Fig. 2 A-C). P2X1-immunolabelled SGNs were sparsely distributed and tended to have smaller soma sizes than unlabelled SGNs (Fig. 1 E, Fig. 2 A-C). A similar expression pattern was observed in P8 cochlea (data not shown). By labelling a whole mount preparation and examining Z-stacks counterstained with DAPI and phalloidin, the P2X1 immunolabeling was strong in the dendritic processes at the base of the OoC (Fig. 1 F, *), extending over the tunnel or Corti (Fig. 1 F & 1 F’, arrows) towards OHCs (Fig. 1 F). P2X1 expression in a subset of SGNs was observed in the apical, mid and basal turns of the cochlea (Fig. 2 A-C). The number of P2X1 positive SGNs was counted separately in apical, middle and basal turns of the adult cochlea and P8 cochlea to estimate the cell density (Table 1 ). More SGNs were observed in the apical turn (575 ± 180 SGNs/(100µm) 3 ) than in the basal turn (280 ± 120 SGNs/(100µm) 3 ) of the adult rat cochlea, consistent with the density pattern observed in cats (18). The proportion of strongly P2X1 positive SGNs ranged between 9.4%-14.2% in the adult rat cochlea and between 14.2 and 23.3% in the P8 cochlea (Table 1 ). This is slightly higher than expected from the ported proportion of type II SGNs relative to the total number of SGNs (9). We then tested if the subpopulation of P2X1-positive SGNs were type II SGNs by co-immunolabeling with the peripherin-1 antibody. Peripherin-1 is an intermediate filament protein selectively expressed by type II SGNs in the postnatal and adult cochlea (19, 20). The anti-peripherin 1 antibody (Everest Biotech, # EB12405) has been validated in peripherin-1-knockout 129Sv/C57BL/6 mice (21, 22). In the adult cochlea, SGNs labelled with anti-peripherin-1 antibody were also co-labelled with P2X1 (Fig. 2 D), which was confirmed by quantitative analysis (Table 2 ). At a higher resolution, both peripherin-1 and P2X1 appeared to be co-expressed in the soma of type II SGNs albeit with slightly different patterns; P2X1 appeared more punctate closer to the nucleus while peripherin-1 immunolabeling was distributed throughout the cytoplasm (Fig. 2 E). The high degree of overlap between P2X1 and peripherin-1 immunolabelling was also observed in the P8 cochlea (Fig. 2 F, G, Table 2 ) with near-100% co-expression. Table 2 Co-labelling of SGNs with anti-P2X1 antibody and anti-Peripherin-1 antibody SGN Counts Adult (n = 4) P8 (n = 4) Apical Mid Basal Apical Mid Basal Total number of SGNs (n = 4 cochlea) 333 410 101 501 480 153 P2X 1 positive 29 32 11 56 53 22 Peripherin-1 positive 45 41 1 68 53 24 Peripherin-1 & P2X1 positive 29 32 1 56 53 22 Discussion In the present study, P2X1 protein expression in the adult and P8 Wistar rat spiral ganglion was mostly confined to type II SGNs, as evidenced by co-expression with peripherin-1. A previous study by Nikolic et al. (2001) demonstrated a robust P2X1 immunolabeling in the majority of SGNs in the Wistar rat cochlea between E16 – P6. However, with further differentiation and maturation of the SGNs, P2X1 expression declines in type I SGNs but remains prominently expressed in type II SGNs in the mature cochlea. Cytoplasmic expression of P2X receptors is commonly observed(23); P2X1 in the soma of type II SGN may represent P2X1 synthesized in the endoplasmic reticulum (ER) and in the process of being trafficked along dendritic processes. Near-100% co-expression of P2X1 and peripherin-1 likely demonstrates the utility of anti-P2X1 antibody as an alternative marker for type II SGNs in the mature cochlea. This brief report describes an interesting finding, given that much remains to be unveiled about the roles of type II SGNs in regulating cochlear amplification by OHCs. In addition to forming afferent synaptic connections with the OHCs, type II SGN axons make synaptic connections with interneurons in the anterior-ventricular, posterior-ventricular and dorsal cochlea nucleus (3, 24). From the posterior-ventricular cochlear nucleus, these interneurons then project to the medial olivocochlear neurons in the trapezoid body that send contralateral and ipsilateral medial-olivocochlear efferent back to OHCs in the cochlea (24). The medial-olivocochlear efferent fibers release acetylcholine at the efferent OHC synapse, thereby hyperpolarizing OHCs and reducing their electromotility. Hence, the activation of the type II SGNs stimulates the medial-olivocochlear efferent serving as the feedback loop to suppress OHC responses and cochlear amplification in response to sound. It was demonstrated that the knock-out of type II SGN processes abolished OHC suppression by medial-olivocochlear efferents (22). The role of P2X1 receptors in this process remains to be evaluated in gene knockout and functional studies. In addition, ATP signalling in Type II SGNs has been postulated as the molecular mechanism of the ‘trauma detector’ in the cochlea which is activated when OHCs are damaged (25). Liu et al. (22) used Sprague-Dawley rat pups (P7-10) to demonstrate that insult to OHC causes ATP-dependent response in type II SGNs, which was abolished by a broad-spectrum purinergic blocker pyridoxalphosphate-6-azophenyl-2', 4'-sulfonic acid (PPADS) (25). Type II afferents were identified as the cochlear nociceptors, with a role to avoid further damage to the inner ear. Further studies are required to confirm the functional expression of P2X1 in the presynaptic terminals of the type II SGNs at the SGN-OHC synapses. In addition, it would be interesting to investigate P2X1 expression in the presynaptic terminal of type II SGNs in the brain to understand the ATP interplay with other neurotransmitters. Purinergic receptors (P2X, P2Y, and adenosine receptors) are broadly expressed in the cochlea (26) and vestibular system (27), and SGNs express a broad range of purinergic receptor subtypes. All P2X receptors identified so far are expressed in the SGNs at different developmental stages, and some (e.g. P2X 7 and P2X 2 ) remain in more mature SGNs (26). This report demonstrates P2X1 receptor expression in type II SGNs of the mature rat cochlea, a unique expression pattern that has not been reported for other P2X receptor subtypes. These details are required to understand the roles of P2X receptor signalling in cochlear amplification by the OHCs and response to damage. Conclusion This study demonstrated P2X1 receptor expression in type II SGN cell bodies and dendrites in adult (6-8-week-old) and juvenile (P8) Wistar rat cochlea. The specific localisation of the P2X1 receptors in type II SGN suggests their role in the modulation of efferent olivocochlear feedback to OHCs and a possible role as trauma detectors in OHCs. Declarations Statements and Declarations: Authors do not have any financial or non-financial interests that are directly or indirectly related to the work submitted for publication. Funding Declaration: This research was supported by funding from Auckland Medical Research Foundation (Auckland, New Zealand) and Eisdell Moore Centre (Auckland, New Zealand). Author Contribution H.S-K. and P.K. wrote the main manuscript text and prepared figures 1-2. S.M.V. and P.R.T. provided scientific input. All authors reviewed the manuscript. Acknowledgement Confocal microscopy was performed in the Biomedical Imaging Research Unit at The University of Auckland, and we thank Ms Jacqueline Ross and other staff at the Unit for their technical support and guidance. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6272432","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":435525733,"identity":"c2c0171e-082d-4a70-af75-1ec15d8ef669","order_by":0,"name":"Prakansha N Kumar","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Prakansha","middleName":"N","lastName":"Kumar","suffix":""},{"id":435525737,"identity":"e4c037bd-5fa1-440b-b021-3f143b77b8aa","order_by":1,"name":"Srdjan M Vlajkovic","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Srdjan","middleName":"M","lastName":"Vlajkovic","suffix":""},{"id":435525740,"identity":"359312e0-9ab5-4603-8e5d-7cfcec733a07","order_by":2,"name":"Peter R Thorne","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"R","lastName":"Thorne","suffix":""},{"id":435525742,"identity":"85de9305-3230-40a6-b13e-d52fa360a8c7","order_by":3,"name":"Haruna Suzuki-Kerr","email":"data:image/png;base64,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","orcid":"","institution":"University of Auckland","correspondingAuthor":true,"prefix":"","firstName":"Haruna","middleName":"","lastName":"Suzuki-Kerr","suffix":""}],"badges":[],"createdAt":"2025-03-20 21:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6272432/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6272432/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11302-026-10129-7","type":"published","date":"2026-01-24T15:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79572250,"identity":"8ce2aed0-b0ae-4151-a98d-9748fbf585a8","added_by":"auto","created_at":"2025-03-31 10:53:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2X1 expression in the adult Wistar rat cochlea. \u003c/strong\u003eCryosections (A-B, E) and whole mount (C-D, F) from adult (6-8 week-old) Wistar rat cochleae were immunolabelled with anti-βIII tubulin (green) and anti-P2X1 antibody (red), and counterstained with DAPI (blue). (A-B) Overviews of the rat cochlear cryosections showing P2X1 expression in the organ of Corti (OoC) and the spiral ganglion neurons (SGN). (C-D) 6–8-week-old rat cochlea whole mount OoC preparation showing P2X1 immunolabelling focusing on the SGN processes underneath the inner and outer hair cells (C) and the corresponding region in P2X1 peptide block control (D). (E) Rosenthal’s canal of cryosection from (B). (F - F’) 6–8-week-old OoC rat cochlea whole mount z-stack images showing tunnel crossing fibres labelled with anti-P2X1 antibody (arrowheads) and P2X1 immunolabelling at the base of the OoC (*). Scale bars, 50 mm (A), 40 mm (B-E), 20 mm (F) Representative images from n = 4 cochleae from four different animals.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6272432/v1/8f27cc0a11b7ca18a17bbe46.png"},{"id":79572249,"identity":"4a08d7eb-1ee9-4e0c-be79-19a93ca53ec9","added_by":"auto","created_at":"2025-03-31 10:53:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":428790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-expression of P2X1 and peripherin-1 in Wistar rat cochlea. \u003c/strong\u003e(A-C) Cryosections from adult (6-8 week-old) Wistar rat cochleae were immunolabelled with anti-βIII tubulin (green) and anti-P2X1 antibody (red), and counterstained with DAPI (blue). (F) White arrows indicate SGNs with strong P2X1 signals relative to other SGNs. Images were taken of the basal (A), mid- (B) and apical (C) regions of the cochlea. (D-G) Cryosections from 6–8-week-old Wistar rat cochlea (D-E) and P8 cochlea (F-G) were co-labelled with anti-peripherin-1 antibody (\u003cem\u003egreen\u003c/em\u003e), and anti-P2X1 antibody (\u003cem\u003ered\u003c/em\u003e). Representative images from n = 4 cochlea from four different animals.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6272432/v1/072e1242a4ebcd0e7cb82ab2.png"},{"id":101151746,"identity":"540ece14-2882-440a-8743-784861471985","added_by":"auto","created_at":"2026-01-26 16:04:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1454337,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6272432/v1/acda95b3-4f1e-4f7c-957e-0031893f1706.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression of the P2X1 Receptor Converges onto the Type II Spiral Ganglion Neurons in the Mature Rat Cochlea","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cochlea, a\u0026thinsp;~\u0026thinsp;1 cm organ deeply embedded in the temporal bone, is our peripheral organ for hearing. The human cochlea contains approximately 3400 inner hair cells (IHCs) and 14,000 outer hair cells (OHCs) across the 34 mm length of the basilar membrane (1), and 29,000\u0026ndash;35,000 spiral ganglion neurons (SGN) (2). Electrical signals from IHCs are transmitted through SGNs to the central auditory pathways in the brain. There are two broad classes of SGNs in the cochlea: type I and type II SGNs (3). The cell bodies of both types of SGNs are located in Rosenthal\u0026rsquo;s canal in the central bony modiolus of the cochlea. Of the total SGN population, 90\u0026ndash;95% are type I SGN. Type I SGNs are myelinated bipolar neurons with large cell bodies and axonal diameters, allowing rapid propagation of action potentials. These neurons are the primary auditory neurons that play a crucial role in transmitting sound information from the cochlea to the brain (4). The dendrite from each Type I SGN forms a synaptic connection with only one IHC. Each IHC is innervated by 20 to 30 Type I SGNs. Functional observations of different thresholds of Type I SGNs, and recent RNA-sequencing-based evidence point out that at least three subclasses of Type I exist in the mammalian cochlea (5\u0026ndash;7). On the other hand, type II SGNs make up only the remaining 5\u0026ndash;10% of the total SGN population. Type II SGNs are unipolar neurons, and their thin unmyelinated dendrites branch to innervate 5\u0026ndash;30 OHCs, usually in the same row (8). Their cell bodies are distributed throughout the spiral ganglion, with fewer found in the most apical region (9). Type II SGNs have been suggested to be involved in the olivocochlear efferent reflex, which modulates cochlear amplification (10).\u003c/p\u003e \u003cp\u003eP2X receptors are ATP-gated ion channels often localised in pre- or post-synaptic terminals, where ATP functions as a neurotransmitter (11). Previous studies have described the expression of P2X1 in the spiral limbus, Reisner\u0026rsquo;s membrane and SGNs in the cochlea of juvenile (P0-P6) and adult Wistar rats (12, 13). Nikolic et al. (2001) demonstrated P2X1 immunoreactivity in both afferent and efferent neurite projections and the soma of SGNs in the developing rat cochlea with a developmental decline from P10 onwards (12). Here, we report that P2X1 expression remains in SGNs after P10 in Wistar rat cochlea. P2X1 immunoreactivity was observed in a subpopulation of SGNs likely representing Type II neurons, but was mostly absent or from Type I neurons. These findings imply differential roles of P2X1 receptor signalling in the developing and mature rat cochlea.\u003c/p\u003e "},{"header":"Method","content":" \u003cp\u003e Animal studies were approved by the Animal Ethics Committee at the University of Auckland (AEC approval No. 002251). Otherwise stated, all the chemicals were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Wistar rats of both sexes were used in equal proportions in this study. P8 Wistar rat pups were decapitated using surgical scissors, and 6\u0026ndash;8-week-old Wistar rats were euthanized with an overdose of pentobarbital (90 mg/kg, ProVet NZ Pty Ltd, New Zealand) followed by cardiac perfusion with flushing solution (0.1M phosphate-buffered saline (PBS) with NaNO\u003csub\u003e2\u003c/sub\u003e) and 4% w/v paraformaldehyde (PFA). Temporal bones were extracted and immersed in 4% PFA for 24 hours at room temperature (RT). Cochleae from 6-week-old rats were decalcified by immersion in 4% w/v Ethylenediaminetetraacetic Acid (EDTA) in PBS at the RT for two weeks, while this step was unnecessary for P8 cochlea. For the Organ of Corti (OoC), whole mounts and cryosections (at 30 \u0026micro;m thickness) were used for immunochemistry following our standard protocol (14), which includes incubation with blocking solution (2.5% v/v Triton X-100 in 0.1M PBS and 10% v/v Normal Goat Serum (NGS) or donkey serum (DS) for whole mounts, and 1% Triton X-100 v/v in 0.1M PBS and 10% v/v/ NGS/DS for cryosections). All antibodies were diluted in antibody solution (0.25% Triton X-100 v/v in 0.1M PBS and 10% v/v/ NGS/DS for whole mounts, 0.1% Triton X-100 v/v in 0.1M PBS and 10% v/v/ NGS/DS for cryosections). The primary antibodies used were: rabbit polyclonal anti-P2X1 antibody (Alomone Labs, Jerusalem, Israel, Catalog# APR-001, 1:500 dilution), mouse anti-β III tubulin (IgG2a monoclonal, BioLegend, San Diego, CA, catalog No. 801213, 1:1000 dilution), goat polyclonal anti-peripherin-1 antibody (Everest Biotech, Upper Heyford, UK, catalog No. EB12405, 1:1000 dilution). For the peptide preabsorption control, P2X1 receptor blocking peptide (Alomone Labs, Jerusalem, Israel, Catalog# BLP-PR001) was mixed with the primary antibody in a 1 mg to 1 mg ratio and incubated for one hour at RT following the manufacturer\u0026rsquo;s protocol. Secondary antibodies used were: goat polyclonal anti-rabbit Alexa 594, goat polyclonal anti-mouse Alexa 488, donkey polyclonal anti-rabbit DyLight 594, donkey polyclonal anti-goat Dylight 488 (ThermoFisher, all used at 1:1000 dilutions). 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, 300 nM final concentration) was added to the secondary antibody mixture. Between each incubation, tissues were washed with 0.1M PBS at RT four times (for 3 min, 5 min, 10 min and 20 min). The tissues were mounted with CitiFluorTM AF1 (Science Services, Germany) and imaged with confocal microscopy (Olympus FV1000, Tokyo, Japan). Z-stacks were obtained with 20x objective lens and 60x objective lens at 0.263\u0026micro;m/pixels and 0.24\u0026micro;m/pixels. Image analysis was carried out using FIJI (Image J). Total SGNs were calculated based on anti-β III tubulin immunolabelling. SGNs were counted when the soma contained 50% or more of the cytoplasm. To estimate the density of SGNs, the area of Rosenthal\u0026rsquo;s canal was estimated using the \u0026lsquo;free-hand\u0026rsquo; tool to select the region of interest (FIJI) and the SGN density was expressed as the cell count per 100 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e to give a number of cells/100\u0026micro;m\u003csup\u003e3\u003c/sup\u003e as previously described (9)(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantification of the population of SGNs stained with anti-P2X\u003csub\u003e1\u003c/sub\u003e antibody.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDensity\u003c/p\u003e \u003cp\u003e(SGNs/ (100um)\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eAdult (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eP8 (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMiddle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBasal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eApical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMiddle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBasal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStrongly P2X\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epositive\u003c/b\u003e\u003c/p\u003e \u003cp\u003enumber/(100\u0026micro;m)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e72\u0026thinsp;\u0026plusmn;\u0026thinsp;30 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;7 (9.44%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0 (14.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 (15.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e71\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 (14.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;24 (23.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal SGN\u003c/b\u003e\u003c/p\u003e \u003cp\u003enumber/(100\u0026micro;m)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e575\u0026thinsp;\u0026plusmn;\u0026thinsp;180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e307\u0026thinsp;\u0026plusmn;\u0026thinsp;46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e280\u0026thinsp;\u0026plusmn;\u0026thinsp;120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e490\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e500\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e430\u0026thinsp;\u0026plusmn;\u0026thinsp;35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe protein expression pattern of the P2X1 receptor was investigated in the cochleae of developing and adult Wistar rats using the commercially available anti-P2X1 antibody (Alomone, #APR-001). The antibody was raised against the intracellular C-terminal domain of rat P2X1 (residues 383\u0026ndash;399) and has been used in over 70 published articles, including testing in gene knockout tissues of the arterial smooth muscle (15) and male reproductive systems (16). In the adult Wistar rat cochlea, a subpopulation of SGNs remained strongly immunolabelled with anti-P2X1 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B \u0026amp; D). P2X1 immunolabeling was observed in the regions where SGN processes reached the OoC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which was attributed to SGN processes extending towards OHCs at the higher resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u0026amp; F). Control tissues pre-absorbed with the corresponding peptide did not show immunolabelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To visualize SGN, an anti-βIII tubulin antibody was used as a pan-SGN marker in the cochlea (17) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). P2X1-immunolabelled SGNs were sparsely distributed and tended to have smaller soma sizes than unlabelled SGNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). A similar expression pattern was observed in P8 cochlea (data not shown). By labelling a whole mount preparation and examining Z-stacks counterstained with DAPI and phalloidin, the P2X1 immunolabeling was strong in the dendritic processes at the base of the OoC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, *), extending over the tunnel or Corti (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026rsquo;, arrows) towards OHCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eP2X1 expression in a subset of SGNs was observed in the apical, mid and basal turns of the cochlea (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). The number of P2X1 positive SGNs was counted separately in apical, middle and basal turns of the adult cochlea and P8 cochlea to estimate the cell density (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). More SGNs were observed in the apical turn (575\u0026thinsp;\u0026plusmn;\u0026thinsp;180 SGNs/(100\u0026micro;m)\u003csup\u003e3\u003c/sup\u003e) than in the basal turn (280\u0026thinsp;\u0026plusmn;\u0026thinsp;120 SGNs/(100\u0026micro;m)\u003csup\u003e3\u003c/sup\u003e) of the adult rat cochlea, consistent with the density pattern observed in cats (18). The proportion of strongly P2X1 positive SGNs ranged between 9.4%-14.2% in the adult rat cochlea and between 14.2 and 23.3% in the P8 cochlea (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is slightly higher than expected from the ported proportion of type II SGNs relative to the total number of SGNs (9). We then tested if the subpopulation of P2X1-positive SGNs were type II SGNs by co-immunolabeling with the peripherin-1 antibody. Peripherin-1 is an intermediate filament protein selectively expressed by type II SGNs in the postnatal and adult cochlea (19, 20). The anti-peripherin 1 antibody (Everest Biotech, # EB12405) has been validated in peripherin-1-knockout 129Sv/C57BL/6 mice (21, 22). In the adult cochlea, SGNs labelled with anti-peripherin-1 antibody were also co-labelled with P2X1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), which was confirmed by quantitative analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At a higher resolution, both peripherin-1 and P2X1 appeared to be co-expressed in the soma of type II SGNs albeit with slightly different patterns; P2X1 appeared more punctate closer to the nucleus while peripherin-1 immunolabeling was distributed throughout the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The high degree of overlap between P2X1 and peripherin-1 immunolabelling was also observed in the P8 cochlea (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) with near-100% co-expression.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCo-labelling of SGNs with anti-P2X1 antibody and anti-Peripherin-1 antibody\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSGN Counts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eAdult (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eP8 (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBasal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eApical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBasal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal number of SGNs\u003c/b\u003e (n\u0026thinsp;=\u0026thinsp;4 cochlea)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP2X\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epositive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePeripherin-1 positive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePeripherin-1 \u0026amp; P2X1 positive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, P2X1 protein expression in the adult and P8 Wistar rat spiral ganglion was mostly confined to type II SGNs, as evidenced by co-expression with peripherin-1. A previous study by Nikolic et al. (2001) demonstrated a robust P2X1 immunolabeling in the majority of SGNs in the Wistar rat cochlea between E16 \u0026ndash; P6. However, with further differentiation and maturation of the SGNs, P2X1 expression declines in type I SGNs but remains prominently expressed in type II SGNs in the mature cochlea. Cytoplasmic expression of P2X receptors is commonly observed(23); P2X1 in the soma of type II SGN may represent P2X1 synthesized in the endoplasmic reticulum (ER) and in the process of being trafficked along dendritic processes. Near-100% co-expression of P2X1 and peripherin-1 likely demonstrates the utility of anti-P2X1 antibody as an alternative marker for type II SGNs in the mature cochlea.\u003c/p\u003e \u003cp\u003eThis brief report describes an interesting finding, given that much remains to be unveiled about the roles of type II SGNs in regulating cochlear amplification by OHCs. In addition to forming afferent synaptic connections with the OHCs, type II SGN axons make synaptic connections with interneurons in the anterior-ventricular, posterior-ventricular and dorsal cochlea nucleus (3, 24). From the posterior-ventricular cochlear nucleus, these interneurons then project to the medial olivocochlear neurons in the trapezoid body that send contralateral and ipsilateral medial-olivocochlear efferent back to OHCs in the cochlea (24). The medial-olivocochlear efferent fibers release acetylcholine at the efferent OHC synapse, thereby hyperpolarizing OHCs and reducing their electromotility. Hence, the activation of the type II SGNs stimulates the medial-olivocochlear efferent serving as the feedback loop to suppress OHC responses and cochlear amplification in response to sound. It was demonstrated that the knock-out of type II SGN processes abolished OHC suppression by medial-olivocochlear efferents (22). The role of P2X1 receptors in this process remains to be evaluated in gene knockout and functional studies.\u003c/p\u003e \u003cp\u003eIn addition, ATP signalling in Type II SGNs has been postulated as the molecular mechanism of the \u0026lsquo;trauma detector\u0026rsquo; in the cochlea which is activated when OHCs are damaged (25). Liu et al. (22) used Sprague-Dawley rat pups (P7-10) to demonstrate that insult to OHC causes ATP-dependent response in type II SGNs, which was abolished by a broad-spectrum purinergic blocker pyridoxalphosphate-6-azophenyl-2', 4'-sulfonic acid (PPADS) (25). Type II afferents were identified as the cochlear nociceptors, with a role to avoid further damage to the inner ear. Further studies are required to confirm the functional expression of P2X1 in the presynaptic terminals of the type II SGNs at the SGN-OHC synapses. In addition, it would be interesting to investigate P2X1 expression in the presynaptic terminal of type II SGNs in the brain to understand the ATP interplay with other neurotransmitters.\u003c/p\u003e \u003cp\u003ePurinergic receptors (P2X, P2Y, and adenosine receptors) are broadly expressed in the cochlea (26) and vestibular system (27), and SGNs express a broad range of purinergic receptor subtypes. All P2X receptors identified so far are expressed in the SGNs at different developmental stages, and some (e.g. P2X\u003csub\u003e7\u003c/sub\u003e and P2X\u003csub\u003e2\u003c/sub\u003e) remain in more mature SGNs (26). This report demonstrates P2X1 receptor expression in type II SGNs of the mature rat cochlea, a unique expression pattern that has not been reported for other P2X receptor subtypes. These details are required to understand the roles of P2X receptor signalling in cochlear amplification by the OHCs and response to damage.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated P2X1 receptor expression in type II SGN cell bodies and dendrites in adult (6-8-week-old) and juvenile (P8) Wistar rat cochlea. The specific localisation of the P2X1 receptors in type II SGN suggests their role in the modulation of efferent olivocochlear feedback to OHCs and a possible role as trauma detectors in OHCs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eStatements and Declarations:\u003c/h2\u003e \u003cp\u003eAuthors do not have any financial or non-financial interests that are directly or indirectly related to the work submitted for publication.\u003c/p\u003e\u003ch2\u003eFunding Declaration:\u003c/h2\u003e \u003cp\u003eThis research was supported by funding from Auckland Medical Research Foundation (Auckland, New Zealand) and Eisdell Moore Centre (Auckland, New Zealand).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.S-K. and P.K. wrote the main manuscript text and prepared figures 1-2. S.M.V. and P.R.T. provided scientific input. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eConfocal microscopy was performed in the Biomedical Imaging Research Unit at The University of Auckland, and we thank Ms Jacqueline Ross and other staff at the Unit for their technical support and guidance.\u003c/p\u003e\u003ch2\u003eAvailability of data and material:\u003c/h2\u003e \u003cp\u003eData are available upon request to the corresponding author.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026Uacute;lehlov\u0026aacute; L, Voldřich L, Janisch R. Correlative study of sensory cell density and cochlear length in humans. Hearing research. 1987;28(2-3):149-51.\u003c/li\u003e\n\u003cli\u003eOtte J, Schuknecht HF, Kerr AG. Ganglion cell populations in normal and pathological human cochleae. Implications for cochlear implantation. The Laryngoscope. 1978;88(8):1231-46.\u003c/li\u003e\n\u003cli\u003eNayagam BA, Muniak MA, Ryugo DK. The spiral ganglion: Connecting the peripheral and central auditory systems. Hearing Research. 2011;278(1):2-20.\u003c/li\u003e\n\u003cli\u003eWichmann C. Molecularly and structurally distinct synapses mediate reliable encoding and processing of auditory information. Hearing Research. 2015;330:178-90.\u003c/li\u003e\n\u003cli\u003ePetitpre C, Wu H, Sharma A, Tokarska A, Fontanet P, Wang Y, et al. Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system. Nature communications. 2018;9(1):3691.\u003c/li\u003e\n\u003cli\u003eShrestha BR, Chia C, Wu L, Kujawa SG, Liberman MC, Goodrich LV. Sensory Neuron Diversity in the Inner Ear Is Shaped by Activity. Cell. 2018;174(5):1229-46.e17.\u003c/li\u003e\n\u003cli\u003eSun S, Babola T, Pregernig G, So KS, Nguyen M, Su S-SM, et al. Hair Cell Mechanotransduction Regulates Spontaneous Activity and Spiral Ganglion Subtype Specification in the Auditory System. Cell. 2018;174(5):1247-63.e15.\u003c/li\u003e\n\u003cli\u003eZhang KD, Coate TM, editors. Recent advances in the development and function of type II spiral ganglion neurons in the mammalian inner ear. Seminars in cell \u0026amp; developmental biology; 2017: Elsevier.\u003c/li\u003e\n\u003cli\u003eBarclay M, Ryan AF, Housley GD. Type I vs type II spiral ganglion neurons exhibit differential survival and neuritogenesis during cochlear development. Neural development. 2011;6:33.\u003c/li\u003e\n\u003cli\u003eFroud KE, Wong ACY, Cederholm JME, Klugmann M, Sandow SL, Julien J-P, et al. Type II spiral ganglion afferent neurons drive medial olivocochlear reflex suppression of the cochlear amplifier. Nature communications. 2015;6:7115.\u003c/li\u003e\n\u003cli\u003eBurnstock G. Historical review: ATP as a neurotransmitter. Trends in pharmacological sciences. 2006;27(3):166-76.\u003c/li\u003e\n\u003cli\u003eNikolic P, Housley GD, Luo L, Ryan AF, Thorne PR. Transient expression of P2X(1) receptor subunits of ATP-gated ion channels in the developing rat cochlea. Brain research Developmental brain research. 2001;126(2):173-82.\u003c/li\u003e\n\u003cli\u003eXiang Z, Bo X, Burnstock G. P2X receptor immunoreactivity in the rat cochlea, vestibular ganglion and cochlear nucleus. Hearing research. 1999;128(1-2):190-6.\u003c/li\u003e\n\u003cli\u003eLin SCY, Thorne PR, Housley GD, Vlajkovic SM. Resistance to neomycin ototoxicity in the extreme basal (hook) region of the mouse cochlea. Histochemistry \u0026amp; Cell Biology. 2018;150(3):281-9.\u003c/li\u003e\n\u003cli\u003eVial C, Evans RJ. P2X1 receptor-deficient mice establish the native P2X receptor and a P2Y6-like receptor in arteries. Molecular pharmacology. 2002;62(6):1438-45.\u003c/li\u003e\n\u003cli\u003eWhite CW, Choong Y-T, Short JL, Exintaris B, Malone DT, Allen AM, et al. Male contraception via simultaneous knockout of \u0026alpha;1A-adrenoceptors and P2X1-purinoceptors in mice. 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International journal of developmental neuroscience. 1993;11(4):507-12.\u003c/li\u003e\n\u003cli\u003eCederholm JM, Parley KE, Perera CJ, von Jonquieres G, Pinyon JL, Julien J-P, et al. Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice. Frontiers in Neurology. 2022;13:962227.\u003c/li\u003e\n\u003cli\u003eFroud KE, Wong ACY, Cederholm JM, Klugmann M, Sandow SL, Julien J-P, et al. Type II spiral ganglion afferent neurons drive medial olivocochlear reflex suppression of the cochlear amplifier. Nature communications. 2015;6(1):7115.\u003c/li\u003e\n\u003cli\u003eDutton JL, Poronnik P, Li GH, Holding CA, Worthington RA, Vandenberg RJ, et al. P2X(1) receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras. Neuropharmacology. 2000;39(11):2054-66.\u003c/li\u003e\n\u003cli\u003eLopez-Poveda EA. Olivocochlear efferents in animals and humans: from anatomy to clinical relevance. Frontiers in neurology. 2018;9:197.\u003c/li\u003e\n\u003cli\u003eLiu C, Glowatzki E, Fuchs PA. Unmyelinated type II afferent neurons report cochlear damage. Proceedings of the National Academy of Sciences. 2015;112(47):14723-7.\u003c/li\u003e\n\u003cli\u003eVlajkovic SM, Thorne PR. Purinergic signalling in the cochlea. International Journal of Molecular Sciences. 2022;23(23):14874.\u003c/li\u003e\n\u003cli\u003eKim SH, Choi JY. Purinergic signaling in the peripheral vestibular system. Purinergic Signalling. 2022;18(2):165-76.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cochlea, adult rat, spiral ganglion neurons, P2X1 receptor, immunohistochemistry, auditory neurotransmission","lastPublishedDoi":"10.21203/rs.3.rs-6272432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6272432/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Our sense of hearing commences in the cochlea, the peripheral sensory organ for hearing. Spiral ganglion neurons (SGN) in the cochlea are primary auditory neurons responsible for auditory neurotransmission. There are two classes of SGNs: type I SGNs, which make up 90-95% of the SGN population, and type II SGNs, which make up the remainder. Previous studies have shown that SGNs express a combination of purinergic (P2X, P2Y and adenosine) receptors at the mRNA and protein levels. In this study, we have focused on P2X1 receptor to characterize its expression pattern in Wistar rat cochlea at postnatal day 8 and in adult (6–8-week-old) rats of both genders using immunohistochemistry. Our results show differential expression of P2X1 in approximately ~10% of the SGN population. In these neurons, P2X1 was co-expressed with peripherin-1, an established type II SGN marker. These results imply the role of P2X1 receptor signalling in auditory neurotransmission that drives medial olivocochlear reflex suppression of the cochlear amplifier.","manuscriptTitle":"Expression of the P2X1 Receptor Converges onto the Type II Spiral Ganglion Neurons in the Mature Rat Cochlea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 10:52:56","doi":"10.21203/rs.3.rs-6272432/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-24T20:16:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-24T20:07:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T13:37:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T11:26:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-13T20:00:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76715053754706989730667100568898104750","date":"2025-03-28T20:09:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310042530159287293866019562368768037152","date":"2025-03-21T14:14:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286921980781242687641618614675673660982","date":"2025-03-21T13:45:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41954664758769004676959441883685247148","date":"2025-03-21T11:58:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T11:48:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-21T04:38:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-21T04:38:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Purinergic Signalling","date":"2025-03-20T20:54:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"deb3ee9a-1eea-48d0-97d4-b221bf7bed48","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:01:08+00:00","versionOfRecord":{"articleIdentity":"rs-6272432","link":"https://doi.org/10.1007/s11302-026-10129-7","journal":{"identity":"purinergic-signalling","isVorOnly":false,"title":"Purinergic Signalling"},"publishedOn":"2026-01-24 15:57:54","publishedOnDateReadable":"January 24th, 2026"},"versionCreatedAt":"2025-03-31 10:52:56","video":"","vorDoi":"10.1007/s11302-026-10129-7","vorDoiUrl":"https://doi.org/10.1007/s11302-026-10129-7","workflowStages":[]},"version":"v1","identity":"rs-6272432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6272432","identity":"rs-6272432","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}