Subcellular Localization of the P2X4 Receptor in Cochlear Sensory Hair Cells

Subcellular Localization of the P2X4 Receptor in Cochlear Sensory Hair Cells | 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 Research Article Subcellular Localization of the P2X4 Receptor in Cochlear Sensory Hair Cells Ziyin Silver Huang, Jacqueline M Ross, Shelly CY Lin, Kevin Roy, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4474581/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 May, 2025 Read the published version in Histochemistry and Cell Biology → Version 1 posted 11 You are reading this latest preprint version Abstract Our sense of hearing starts in the inner ear organ, the cochlea, which contains two types of auditory hair cells for signal transduction. Earlier research showed that the complex cochlear physiology is regulated in part by purinergic signalling through activations of purine mediated P2X, P2Y and adenosine receptors expressed in the cochlea. This study aims to extend our knowledge of purinergic signalling in the cochlea by comprehensively characterizing the expression of P2X 4 receptor subtype. Wistar rat cochlea (embryonic day 20.5–6 weeks, both sexes) were collected and the P2X 4 expression was examined by immunohistochemistry. Robust P2X 4 expression was found in the organ of Corti (OoC) in the inner hair cells (IHCs) and outer hair cells (OHCs), confirmed by double-labelling with HCs marker Myosin VIIa. In IHCs, a robust cytoplasmic P2X 4 expression occurred throughout the cell body, with the most intense signal at the medial side. In OHCs, P2X 4 formed puncta near the apical and basal ends of the cell body. Using markers for subcellular organelles, P2X4 immunoreactivity was associated mostly with the trans-Golgi network apparatus (27%) and early endosomes (26%) in IHC, and early endosomes (42.3%) and lysosomes (32.4%) in OHC in the mature cochlea. Taken together, these observations suggest unique roles for P2X 4 in mature IHCs and OHCs as a purinergic receptor subtype responsible for homeostatic regulation of hair cells and auditory sensory transduction. Cochlea sensory hair cell purinergic signalling P2X receptor P2X4 ATP Organ of Corti Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION According to the World Report on Hearing (2021), hearing loss affects 1.5B people globally and this number is expected to grow to 2.5B by 2050 (World Health Organization, 2021 ). The majority of cases are sensorineural hearing loss (SNHL), characterized by degenerative changes in the cochlea and the auditory nerve. There are few effective pharmacological treatments for SNHL and development of such treatment requires further understanding of cochlear physiology and pathophysiology at the cellular and molecular levels. The organ of Corti (OoC), the sensory apparatus within the cochlea, contains two types of auditory sensory cells, the inner hair cells (IHC) and outer hair cells (OHC). Hair cells (HCs) are so-called due to mechanosensory stereocilia located on the apical side of the cell supported by an actin-rich cuticular plate and are essential for sound transduction (Goodyear, Marcotti, Kros, & Richardson, 2005 ). Approximately 3500 IHCs are aligned as a single continuous row and 12,000 OHCs arranged in three rows more laterally in the human cochlea. In humans and animal models, loss of HCs, particularly OHC and loss of synapses and neurons innervating IHC have been observed as a common underlying pathology associated with SNHL (Liberman & Kujawa, 2017 -z. Wu, O'Malley, de Gruttola, & Liberman, 2020 ; P. Z. Wu et al., 2019 ). Purinergic signaling is involved in many cellular functions and pathologies in the inner ear and considered to be a potential therapeutic target for inner ear disorders. Purinergic signaling pathways are activated by extracellular nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine triphosphate (UTP), uridine diphosphate (UDP), and adenosine (Burnstock, 1997 ). There are two classes of purinergic receptors: P1 receptors (A 1 , A 2A , A 2B and A 3 ) and P2 receptors (P2X 1-7 , P2Y 1,2,4,6, 11-14 ) (Burnstock, 2007 ). Many of these purinoceptor subtypes have been identified in the cochlea (Köles, Szepesy, Berekméri, & Zelles, 2019 ; Vlajkovic & Thorne, 2022 ). P2X receptors form trimeric ligand-gated ion channels that are non-selectively permeable to cations (Na + , K + , Ca 2+ ) (Burnstock, 2007 ). Earlier studies had suggested a potential role for P2X receptors in the cochlea, such as regulation of afferent neuronal activity in response to agonists released in the perilymph (Robertson & Paki, 2002 ). More specific roles for P2X subtypes are emerging based on molecular and functional investigations; P2X 2 expressed in cells lining the endolymphatic compartment and in the stereocilia of OHC participate in the reduction of endocochlear potential during sound transduction and in modulating the sound sensitivity, respectively (Jarlebark, Housley, & Thorne, 2000 ; Morton-Jones et al., 2015 ; Z.-J. Wang & Neuhuber, 2003 ). P2X 1 and P2X 7 localized near synaptic terminals of the auditory neurons on IHCs and in the neurites of the SGN (P. Nikolic, Housley, Luo, Ryan, & Thorne, 2001 ; Predrag Nikolic, Housley, & Thorne, 2003 ) may play roles in Ca 2+ -dependent uncoupling of synapses known to occur at these postsynaptic terminals (Liberman & Kujawa, 2017 ). P2X 3 expressed in the developing spiral ganglion neurons regulate branching of afferent fibres (L.-C. Huang, Ryan, Cockayne, & Housley, 2006 ; Z. Wang et al., 2020 ). In contrast, molecular expression of P2X 4-6 has not been clearly demonstrated in the cochlea. P2X 4 has some unique features compared to other P2X subtypes. The human monomeric P2X 4 channel is sensitive to the extracellular Ca 2+ concentration with slow desensitization compared to P2X 1-3 isoforms requiring 4 sec at a 30µM concentration ATP (Hattori & Gouaux, 2012 ). Cytoplasmic expression of P2X 4 and localization to lysosomes have been reported (Huang et al., 2014 ; Murrell-Lagnado & Frick, 2019 ). Intracellular P2X 4 exhibit pH sensitivity (inactivated at low pH) and can be regulated by the pH within the lysosomal lumen (P. Huang et al., 2014 ; Ruth D. Murrell-Lagnado & Frick, 2019 ). In the guinea pig cochlea, functional expression of P2X 4 has been reported in the endothelial cells of the spiral ligament where it appears to regulate cochlear blood flow (Y. Wu et al., 2011 ) and in hair cells (Szücs et al., 2004 ), however, the detailed P2X 4 distribution in the cochlea still remains to be characterized. In the vestibular system of the inner ear, P2X 4 molecular and functional expression has recently been reported in vestibular supporting cells (Jeong et al., 2020 ). In this study, we investigated the distribution of P2X 4 in the developing and adult rat cochlea. Expression was confined to OHCs and IHCs, and located solely in the cytoplasm. Colocalization with markers of endoplasmic reticulum, Golgi apparatus and lysosomes suggests that P2X 4 is associated with different intracellular organelles (Golgi-ER in IHC and lysosomes in OHC) and may mediate calcium buffering within these intracellular organelles. METHOD AND MATERIALS Animals The use of animals for this project was approved by the University of Auckland Animal Ethics Committee (AEC002251). All animals were supplied by the Vernon Jensen Unit (VJU; The University of Auckland). Wistar rats of various ages and both sexes were used for this study; embryonic day 20.5, postnatal day 4 (P4), postnatal day 8 (P8), postnatal day 21 (P21), and 6-week-old (adult). Tissue preparation General chemicals were purchased from ThermoFisher Scientific (Auckland, New Zealand) unless otherwise specified. 4% w/v Paraformaldehyde (PFA, pH 7.4) was prepared with 0.1M Phosphate Buffer (PB, 24.6 mM NaH 2 PO 4 and 75.4 mM NaHPO 4 , pH 7.4) for fixation of all samples. P21 and adult rats were first anaesthetised and euthanized by drug overdose (Pentobarbital, ProVet NZ Pty Ltd), followed by perfusion of 0.1% w/v NaNO 2 in Phosphate-Buffered Saline (PBS, pH 7.4, Gibco), and 4% PFA through the left ventricle. The temporal bones were removed from the cranium and a small puncture was carefully made in the round window membrane to aid the penetration of PFA into the cochlea. The cochlea was then immersion fixed in 4% PFA at room temperature (RT) for 24 hours. Cochleae were washed with PBS three times (10 mins each). Adult cochlear tissues were decalcified by immersion in 4% w/v Ethylenediaminetetraacetic acid (EDTA, in 0.1PB, pH 7.4) at room temperature for up to 2 weeks with regular change of EDTA solution. Cochleae from Wistar rats younger than P8 were removed and immersed in PFA (4% PFA at room temperature (RT)) for 24 hours and further dissection was carried out without decalcification. For the organ of Corti (OoC) whole mount preparations, cochleae were micro-dissected in PBS and segments of OoC approximately equivalent to ½ turn, were taken from the apical, middle, and basal turn. For cryosectioning, cochleae were cryoprotected sequentially in 10% and 20% sucrose (w/v in PBS) for one hour at room temperature, then in 30% sucrose overnight at 4°C followed by embedding in Tissue-Tek Optimal Cutting Temperature Compound (OCT, ProSciTech, Australia) at -80°C. The tissue was cryosectioned at 20-30 mm (Leica, CM3050S) in the axial plane through the modiolus and cochlear ducts. Microdissected or cryosectioned tissue was stored in PBS for up to 1 week at 4°C before processing for immunohistochemistry. Immunohistochemistry The list of primary antibodies used in this study and dilutions are summarised in Table 1. A polyclonal primary antibody raised in rabbits against the C-terminal domain of the rat P2X 4 subunit (Alomone Inc., Jerusalem, Israel, catalogue no. APR-002) was used for detection of P2X 4 by immunohistochemistry. This antibody has been validated using P2X 4 knockout animals (Lalisse et al., 2018; Sim et al., 2006; Wyatt et al., 2014). Other antibodies used as cell-type specific markers and organelle markers are summarized in Table 1. Immunohistochemistry was performed following the protocol established previously (Fok et al., 2020; Han, Lin, Espinosa, Thorne, & Vlajkovic, 2019). Blocking solutions and antibody diluent solutions were prepared as following; blocking solution for whole mounts (10% v/v normal goat serum (NGS) and 2.5% (v/v) TritonX in PBS), blocking solution for cryosection (10% v/v NGS, 1% v/v TritonX in PBS), antibody diluent for wholemount (5% v/v NGS and 0.25% v/v TritonX in PBS) and antibody diluent for cryosection (5% v/v NGS and 0.1% v/v TritonX in PBS). Tissues were incubated for 2 hours in a blocking solution at room temperature. Tissues were then incubated in the diluted primary antibody overnight at 4 ֯ C°. For the anti-P2X 4 antibody control, the pre-absorbing peptide was added to the primary antibody solution in a 1mg-1mg ratio and sections or whole mounts were incubated for 2 hours following the manufacturer’s protocol (Alomone Labs, Israel). Tissues were washed 4 times at 1, 10, 15, 30 minutes intervals in PBS at RT followed by incubation with secondary antibodies overnight at 4°C in the dark. Secondary antibodies used were goat anti-rabbit Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488, and goat anti-mouse Alexa Fluor 647 (ThermoFisher Scientific, all used at 1:500 dilution in antibody diluent). The non-antibody labelling reagents Wheat Germ Agglutinin (WGA) and Phalloidin (Table 1) were included in the same mixture with the secondary antibody. From this step forward, tissues were covered to minimize light exposure. After incubation with secondary antibodies, the tissues were washed 4 times in PBS, incubated in DAPI (diluted in PBS, 0.02mg/ml) for an hour at room temperature, washed 4 times in PBS and mounted with coverslips on slides with CitiFluor AF1 mountant solution (Agar Scientific Ltd, UK). Slides were stored at 4°C in the dark until imaging. Confocal microscopy Fluorescently immunolabelled slides were imaged using a Zeiss LSM 800 Airyscan confocal microscope (Carl Zeiss GmbH, Jena, Germany) in the Biomedical Imaging Research Unit (BIRU) at The University of Auckland. Objective lenses used were 10x/0.45 NA Plan Apochromat, 20x/0.8 NA Plan Apochromat, 63x/1.4 NA Plan Apochromat oil immersion. Images were acquired at a pixel resolution of 0.18μm/pixel for 20x, and 0.035μm/pixel for 63x in Airyscan mode. The Z series were obtained using a 63x/1.4 NA oil immersion objective lens with a 0.5mm step size between optical sections. The top limit for the Z series was set at the level of the tip of the stereocilia of the hair cells, as visualized with phalloidin representing the most apical end of the cell body, and the bottom limit was set at the opposite end of the hair cell body at the position where the P2X 4 signal had just disappeared from the HCs. A typical Z-stack was 40mm thick. All of the images were acquired using ZEN 2.6 software (Carl Zeiss, Germany) and exported to TIFF as required for figure preparation or analysis. Image analysis and processing ImageJ (Schneider et al., 2012) was used for particle analysis and quantification of P2X 4 immunolabeling on individual cells from the z series images (see Supplementary material for details on methodology). An automated threshold was used to identify the particles of interest. The measurements were then carried out using the built-in Particle Analyzer. Parameters included “Count” and “Total Area”. “Count” represents the number of particles in each image and “Total Area” is the area represented by the sum of the particles in each image. ImageJ “line plot profile” analysis was performed to quantify the relative signal intensity within the hair cells in apical to basal direction. This function was also used to measure intensity at nine different locations in the hair cell across the apical-basal as well as medial-lateral directions and normalised relative to the total signal (Figure 3 & 4 and Supplementary Figure 1). The JACoP plugin (see Supplementary material for more detail) was used for colocalization analysis between P2X 4 and subcellular markers. Individual channels were separately processed by background subtraction and images were cropped so that the region of interest typically contained 8 cells. JACoP automatically calculates Manders’ colocalization coefficients (with and without a threshold) and Pearson’s correlation coefficient (Bolte & Cordelières, 2006; Dunn, Kamocka, & McDonald, 2011). The results were displayed as M1 and M2, each with the value range between 0-1.0, where M1 is defined as the ratio of the “summed intensities of pixels from the green channel for which the intensity in the red channel is above zero” to the “total intensity in the green channel”. M2 is identified as the same as the red and green reversed. High M1 and M2 coefficients indicate that a large proportion of one signal co-occurs with the other signal. Colocalization analyses were conducted for each subcellular marker separately, with 3 cochleae for each marker, and the mean and standard error of the mean (SEM) were calculated. Imaging processing was performed using Adobe Photoshop CC (version 19.1.3, Adobe system Incorporated) to prepare figures. RESULTS P2X 4 expression in the cochlea After testing the dilution range 1:50 to 1:2000, 1:1000 dilution was chosen to have the best signal to background ratio (data not shown) for the anti-P2X 4 antibody (Figure 1). High levels of expression of P2X 4 in the rat OoC were observed (Figure 1D, F), but less in the spiral ligament (Figure 1C, arrow) and spiral ganglion (Figure 1E, arrow) In the OoC, there was a relatively higher expression in IHC & and to a lesser extent in OHCs (Figure 1D, F-H). Immunolabeling of P2X4 throughout was abolished by preabsorbing P2X 4 antibody with excess peptide molecules in the controls (Figure 1I). The expression of P2X 4 was evident in the hair cells from E20.5 but was more prominent in the IHCs than OHCs. When compared the OoC at different rat ages,(E20.5, P4, P8, P21); Figure C-F), immature IHC at E20.5 expressed P2X4 above the background, but the signal was relatively weak. At P4 and P8, expression of P2X 4 was clearly evident in IHCs and OHCs. By P21, a week after the hearing onset, P2X 4 exhibited a similar expression pattern to the adult cochlea with strong expression of P2X 4 in the IHCs. At P8, P2X 4 expression was prominent in IHC & OHC (Figure 2A-B). Some cells lining the cochlear scala tympani and vestibuli also expressed detectable levels of P2X 4 (Figure 2A-B, arrows). It is not possible to identify these cells but they appear morphologically very similar to Iba1-expressing macrophages observed in the postnatal mouse cochlea (Kishimoto, Okano, Nishimura, Motohashi, & Omori, 2019). To confirm the identity of cells expressing P2X 4 , two cell-type specific markers were used: myosin VIIa, which is consistently expressed in IHC and OHCs (Jung et al., 2019; Xiong et al., 2019) and Sox2, which is a transcription factor expressed in nuclei of all types of supporting cells (Smeti et al., 2011). P2X 4 -labelled cells co-expressed myosin VIIa, confirming these to be the IHCs and OHCs (Figure 2G), while P2X 4 was not observed in cells expressing Sox2 (Figure 2H). Polarity of P2X 4 subcellular distribution within IHC and OHC in adult rat cochlea We next investigated the subcellular localizations of IHCs and OHCs in the adult rat cochlea to correlate the distribution of P2X 4 with the distinct functional domains of IHC and OHC. Analysis of z-stack images of OoC enabled compartmentalisation of the hair cells into four different sub-domains from the apical surface to the basal pole of the cell; sub-cuticular (Figure 3A), cytoplasmic (Figure 3B), nuclear (Figure 3C) and subnuclear zones (Figure 3D). At the sub-cuticular plate level of IHC, P2X 4 immunolabeling appeared as bright irregular clusters in the cell cytoplasm immediately underneath the cuticular plate (Figure 3A). In the supranuclear cytoplasm, between the cuticular plate and nucleus, there were similar clusters of P2X 4 immunolabelling, but these appeared larger and brighter (Figure 3B). At the nucleus and sub-nucleus levels, the cytoplasmic immunolabeling for P2X 4 appeared brightest and the most abundant (Figure 3C, D). This pattern is also evident in the 3D re-constructions (Figure 3E). Orthogonal views of the images were also generated with ImageJ where the stack of images was viewed in a XY, YZ, XZ planes, where the X, Y, Z planes correspond to the left and right (Le-R), medial and lateral (M-L) and the apical and basal (A-B), respectively (Figure 3F). Orthogonal visualization confirmed more intense P2X 4 immunolabeling along the medial side of the IHCs (Figure 3F’’, arrow). This corresponds to the large, patchy signal appearance in the 3D re-construction (Figure 3E). More intense signal was also observed at the apical part of the image (Figure 3F’’’). Signal distribution for P2X 4 along the apical-basal and medial-lateral axes of the cells was quantified using ImageJ (Supplementary Figure 4) to confirm these visual observations that the P2X 4 expression was more concentrated at the basal end of the IHCs (Figure 3G) and at the medial side of the IHCs (Figure 3H). Similar analyses in the OHCs (Figure 4) showed P2X 4 immunolabeling at all four levels predominately in the cell cytoplasm, however the characteristic pattern of P2X 4 localisation was quite different from that observed in IHCs. At the sub-cuticular plate level, the P2X 4 expression appeared to be more concentrated than observed in IHC (Figure 4A). Interestingly, the cluster of P2X 4 labelling often appeared immediately underneath the ‘cuticular-free zone’, a small region on the lateral aspect of the cell that does not stain with phalloidin (Figure 4 arrow). At the cytoplasmic level, regions of P2X4 appeared more scattered, but some medium-sized clusters were observed (Figure 4B). At the nucleus level, the P2X 4 immunoreactivity was less obvious (Figure 4C), but more intense in the basal sub-nucleus level of OHC (Figure 4D). When reconstructed in 3D, a prominent cluster of P2X 4 immunolabeling was observed at the apical part of the cytoplasm, and it was not as homogenously distributed through the whole cell compared to P2X 4 immunolabeling in IHC (Figure 4E). Examined using the orthogonal view, the most intense signal for P2X 4 (Figure 4F’’, asterisks), appearing as a prominent cluster, was observed at the lateral side of each cell underneath the CP free zone (Figure 4E). Z-stack images obtained for the OHCs were quantified using ImageJ (See supplementary figure 5 for details) to confirm that the P2X 4 expression was more concentrated at both the apical and the basal end of the OHCs (Figure 4G). The gradient in OHC was very subtle in the medial to lateral direction (Figure 4L-M), compared to the clear trend observed for IHC (Figure 3H). Immunolabeling for of P2X 4 appeared as more discrete dots in the OHCs compared to the IHCs, allowing additional “particle analysis” (Figure 5A & B; see Supplementary Figure 3 for more detail). The total area occupied by P2X 4 immunolabeling was the greatest in the sub-cuticular zone, compared to the three other zones (Figure 5C). In contrast, the total amount of staining was the highest in the cytoplasmic zone compared to the other three zones (Figure 5D). Localization of P2X 4 to subcellular organelles within IHCs and OHCs. The P2X 4 immunostaining was in clusters and appeared to be vesiculated. To determine if these were associated with other membranous intracellular organelles, we looked at co-localisation of P2X 4 with endosomes, lysosomes, Golgi bodies and mitochondria using immunohistochemistry (Table 1). Early endosomes are derived from the plasma membrane (Gindhart & Weber, 2009) and distinguished from late endosomes and other vesicles by the expression of early endosome antigen 1 (EEA-1) (Patki et al., 1997), including in IHCs and OHCs (Schug et al., 2006). Endosomes and the Golgi apparatus are part of the intracellular protein transportation and recycling pathway. EEA-1 labelling in IHCs had a diffuse appearance, with vesicular labelling more concentrated in the apical part of the cell (Figure 6A). In OHCs, EEA-1 labelled vesicles appeared throughout (Figure 6B). There was some co-labelling between EEA-1 and P2X 4 (Figure 6A, B arrow) in both IHC and OHC, with qualitatively more co-occurrence observed in OHC. To quantify the co-localization of EEA-1 with P2X 4 , the JACoP plugin (Bolte & Cordelières, 2006) in ImageJ was used (see Supplementary Figure 3 for details). Z-stack images covering either entire OHCs or IHCs were selected for analysis. JACoP quantifies the co-occurrence of P2X 4 and EEA-1 as two “Mander’s coefficients” calculated as M1 and M2 coefficients with a value range between 0-1.0. M1 represents the proportion of EEA-1 co-localized with P2X 4 signal over the total P2X 4 signal. M2 represents the proportion of the EEA-1 co-localized with P2X 4 over the total signal of EEA-1. The average M1 values for each organelle marker in IHC and OHC are summarised in Figure 6J & K. Taking the same approach, we analysed the co-occurrence of P2X 4 with LAMP-1, GM130, Tom20 and Wheat Germ Agglutinin (WGA). LAMP-1 is a protein found on lysosomes and lysosome-endosome fusion vesicles and is commonly used as a marker for lysosomes (Huotari & Helenius, 2011). Lysosomes are distributed throughout the cell in the IHCs and OHCs, but large lysosomes are often found at the apical, lateral side of the cell (Spicer, Thomopoulos, & Schulte, 1999). OHCs have a greater number of lysosomes compared to IHCs (Spicer, Thomopoulos, & Schulte, 1998; Wiwatpanit et al., 2018). LAMP-1 labelling in IHCs had a more diffuse appearance with lower signal levels, and minimally co-occurred with P2X 4 (Figure 6C) where the OHCs had a vesicular appearance (Figure 6D). There was a clear overlap of the P2X 4 immunolabelling and LAMP-1 in OHC (Figure 6D, Table 2). GM130 is a marker for Golgi matrix protein of 130kDa, which typically targets the cis-component of Golgi (Nakamura, Lowe, Levine, Rabouille, & Warren, 1997). The Golgi apparatus is located mainly around the apical part of the cytoplasm in HCs (Schug et al., 2006; Spicer et al., 1998, 1999). In the rat cochlea, cytoplasmic expression of GM130 was observed in the IHCs and OHCs with vesicular, string-like structures (Figure 6E, F), consistent with previous reports (Schug et al., 2006). Notably, the co-occurrence of the GM130 and P2X 4 in both the IHCs and OHCs was minimal (Figure 6E, F), 11.7% ± 2.4%, and in the OHCs, 27% ± 2%. TOM20 is a protein expressed on the mitochondrial outer membrane (Balaker, Ishiyama, Lopez, Ishiyama, & Ishiyama, 2013) and was used here as the marker for mitochondria. There was some overlap of TOM20 and P2X 4 signal in the IHCs (Figure 6G, arrow). However, there was little co-localization between P2X 4 and TOM20 in both OHCs and IHC (Figure 6H). TOM20 was co-occurred with P2X 4 in the IHCs 13.7% ± 2.5% and in OHCs 11.3% ± 1.2%. Finally, WGA is naturally occurring molecule known to bind to glycoproteins found in the cell membrane, and fluorescent conjugates are commonly used as a marker for cell membrane (Emde, Heinen, Gödecke, & Bottermann, 2014). The WGA labelled the OHC membrane but did not stain IHC, similar to a previous study (Gil-Loyzaga & Brownell, 1988). Therefore, the association with the IHC membrane was inconclusive and therefore not included in this study. We observed the minimal overlap between WGA and P2X 4 in the OHCs(Figure 6I). WGA was co-localized with P2X 4 in OHCs 4.3% ± 0.2% (Figure 6K). In summary, in IHCs, EEA-1 and GM130 have the highest percentage of co-localization with P2X 4 , at 26% and 27%, respectively, compared to other organelle markers, suggesting cytoplasmic P2X 4 were likely associated with endosomes and Golgi apparatus. The co-localization pattern in OHCs was slightly different from that with IHCs; EEA-1 and LAMP-1 have a higher percentage of co-localization with P2X 4 at 42.3% and 32.%, respectively. This suggests that P2X 4 associate with endosomes and lysosomes in OHCs (Figure 6 K&L, Table 2). DISCUSSION In this study, we have comprehensively mapped the expression of P2X 4 in the Wistar rat cochlea using immunohistochemistry and reported the expression in the IHCs and OHCs of the Wistar rat cochlea for the first time. The minimal expression of P2X 4 labelling in the stria vascularis was unexpected, as the literature suggested that P2X 4 receptors are expressed in the endothelial cells of spiral ligament capillaries in the lateral wall of the guinea pig cochlea (Y. Wu et al., 2011). This discrepancy might occur because different antibody from Abcam (UK) was used, which is no longer available on the manufacturer website. The expression of P2X 4 in sensory HCs was observed uniformly throughout the apical, middle and basal turn of the cochlea (Supplementary Figure 4). P2X 4 immunolcalisation was observed in a small population of cells in the spiral ligament and the spiral ganglia, however, the frequency was very low in the adult cochlea. It is important to note that the P2X 4 subunit can form heteromeric channels with P2X 1, 5, 6, and 7 subunits. P2X 4 immunolocalisationin the OoC in our study is distinctively different from what has been reported for P2X 1 (Xiang, Bo, & Burnstock, 1999), P2X 7 (Predrag Nikolic et al., 2003) and P2X 2 (Jarlebark et al., 2000; J. C. C. Wang et al., 2003), which might suggest that P2X 4 has different roles compared to other isoforms. The most intense P2X 4 expression was observed in the IHCs and OHCs, where P2X 4 was predominately localised in the cytoplasm with distinct polarity in the subcellular distribution of the receptor protein. Using organelle markers, we show that the majority of cytoplasmic P2X 4 was co-localized with vesiculated structures, particularly early endosomes and Golgi (trans-Golgi network), which combined represented 53% of the co-localisation for IHCs and 63.2 % for OHCs. Additional association with lysosomes occurred in OHCs only. There was little evidence of P2X 4 expression in the cell plasma membrane. The cytoplasm of IHCs and OHCs are enriched with endosomes (Spicer et al., 1998, 1999). This P2X 4 localisation may represent a pool of P2X 4, which will become inserted into the membrane under certain conditions, or it reflects continual membrane-cytoplasm cycling. Cytoplasmic P2X 4 has been reported in many tissues, including alveolar epithelium, and neurons (Bobanovic, Royle, & Murrel-Lagnoado, 2002; Qureshi, Paramasivam, Yu, & Murrell-Lagnado, 2007; Stokes, Layhadi, Bibic, Dhuna, & Fountain, 2017). In the ocular lens, cytoplasmic P2X 4 becomes more associated with the cell membrane under osmotic stress (Suzuki-Kerr, Lim, Vlajkovic, & Donaldson, 2009), supporting the notion that cytoplasmic vesicles containing P2X 4 are dynamic. Interestingly, the distribution of cytoplasmic P2X 4 showed polarity within the cell. The apical cell domain of both OHCs and IHCs are in contact with potassium-rich endolymphatic fluid, whereas the basal-lateral domain is in contact with sodium-rich perilymph and has the pre-synaptic clefts for synaptic transmission. A large proportion of cytoplasmic P2X 4 immunolabeling in the IHCs occurred on the apical and basal ends, in proximity to the synaptic cleft, which may suggest physiological P2X 4 roles in regulating pre-synaptic function. In addition, the cytoplasmic labelling in the IHCs was concentrated adjacent to the medial side of the lateral membrane. The medial side of IHCs is adjacent to the inner border cells, which abundantly express connexin 26 and 30, and while their primary role is communication between supporting cells as gap junctions, they also exist as connexin hemichannels (Taylor, Jagger, & Forge, 2012; Zhao, Yu, & Fleming, 2005). It is interesting to speculate that P2X 4 may be activated in a paracrine manner by connexin hemichannel-mediated ATP released from the inner border cells. Such a gradient of P2X 4 distribution in the IHCs was not observed in younger animals (P4-P8), suggesting that P2X 4 signalling may be established in mature IHCs. OHCs are the other type of sensory epithelial cells in the cochlea, however, they have distinct functional role than IHCs as part of the ‘cochlear amplifier’ by contracting and elongating in response to sound (Pickles, 1998). The robust expression of P2X 4 was found in OHCs mainly in the cytoplasmic space near the apical membrane and also towards the basal membrane. This was less evident in young animals, suggesting critical roles for P2X 4 in more mature OHCs. The large proportion (63.2%) of cytoplasmic P2X 4 in OHCs co-occurred with trans-Golgi network similar to the IHCs, and this may represent the dynamic cycling pool of P2X 4 receptors moving to and from the plasma membrane. Given the close proximity of vesicular P2X 4 to the apical and basal membranes, we may speculate ATP released from Deiters cells underneath OHCs which express connexin 26 and 30, proteins capable of forming hemi-channels and gap-junctions (Hosoya et al., 2021; Taylor et al., 2012; Zhao et al., 2005). In addition to the robust basal expression, both qualitative and quantitative analysis showed a robust P2X 4 expression at the apical sub-cuticular level of the OHCs, where they exhibited very characteristic appearance of “plaque” or “cluster” of vesicles. These were often found immediately underneath the CP free zone. While only 11.7% of P2X 4 co-localized in IHCs with the lysosome marker, a greater proportion (32.4%) of P2X 4 co-localized with lysosome marker in OHCs, showing differences between IHCs and OHCs. One possibility for lysosomal localization of P2X 4 is a part of protein cycling; the late-endosome will fuse with lysosome during protein degradation, some of which may correspond to P2X 4 receptors trafficking en route for degradation. Alternatively, P2X 4 has been suggested to play a role as a lysosomal ionic channel based on the observation in cultured neurons (R. D. Murrell-Lagnado, 2018; Ruth D. Murrell-Lagnado & Frick, 2019). Lysosomal P2X 4 receptor activation is influenced by pH within the lysosome lumen in cell culture and induces membrane fusion (Cao et al., 2015). In the cochlea, lysosomal dysfunction has been reported to lead to cellular toxicity in OHCs but not in IHCs (Wiwatpanit et al., 2018). It would be interesting to explore the difference in lysosomal physiology between IHCs and OHCs, and how P2X 4 in OHC lysosomes may be involved in such a process. Understanding the role of P2X 4 will require further investigation into the physiological activation of P2X 4 , including pharmacological manipulations. P2X 4 receptor signalling and its intracellular roles in the cochlea likely contribute to the sensory cell physiology and pathophysiology. Abbreviations ADP – adenosine 5’-diphosphate ATP – adenosine 5’-triphosphate BDNF – Brain-derived neurotrophic factors Ca 2+ – calcium ion dB – decibels EP – endocochlear potential IHCs – inner hair cells JACoP – Just Another Co-localization Plugin (ImageJ plugin) K + – potassium ion kHz – Kilohertz mM – micromolar Mo – modiolus OHCs – outer hair cells OoC – organ of Corti ROI – Region of interest SGN – spiral ganglion neuron SNHL – Sensorineural hearing loss SV – stria vascularis UDP – uridine diphosphate UTP – uridine triphosphate v/v – volume per volume w/v – weight per volume WGA – wheat germ agglutin WHO – World Health Organization Declarations ACKKNOWLEDGEMENTS This work was supported by the Auckland Medical Research Foundation (New Zealand) and Eisdell Moore Centre (New Zealand). Embryonic cochlea issue (E20) was supplied as a by-product from another AEC-approved study conducted by Dr. Rashika Karunasinghe (Department of Physiology, the University of Auckland, AEC 1977). CONFLICTS OF INTERESTS The authors declare that they have no conflict of interest. AUTHOR CONTRIBUTIONS HSK conceptualized and designed the study. ZH performed the majority of experiments, followed by HSK performing parts of the experiment. KR contributed to experiments in Figure 2. Technical protocols for optimization of the experiment, image acquisition and image analyses were designed and optimized by ZH, HSK, JMR, and SCY, followed by ZH conducting the data analysis. HSK and ZH wrote the draft of the manuscript. SMV and PRT were co-supervisors to ZH during her postgraduate degree and provided scientific guidance to the manuscript. ZH and HSK prepared figures. All authors have reviewed and approved the manuscript. References Balaker, A. E., Ishiyama, P., Lopez, I. A., Ishiyama, G., & Ishiyama, A. (2013). Immunocytochemical localization of the translocase of the outer mitochondrial membrane (Tom20) in the human cochlea. The Anatomical Record, 296 (2), 326-332. Bobanovic, L. 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Cho, H. (2011). Synthesis and biological evaluation of novel thiazolidinedione analogues as 15-hydroxyprostaglandin dehydrogenase inhibitors. Journal of medicinal chemistry, 54 (14), 5260-5264. https://dx.doi.org/10.1021/jm200390u Wyatt, L. R., Finn, D. A., Khoja, S., Yardley, M. M., Asatryan, L., Alkana, R. L., & Davies, D. L. (2014). Contribution of P2X4 receptors to ethanol intake in male C57BL/6 mice. Neurochemical research, 39 (6), 1127-1139. 10.1007/s11064-014-1271-9 Xiang, Z., Bo, X., & Burnstock, G. (1999). P2X receptor immunoreactivity in the rat cochlea, vestibular ganglion and cochlear nucleus. Hear Res, 128 (1), 190-196. https://doi.org/10.1016/S0378-5955(98)00208-1 Xiong, H., Chen, S., Lai, L., Yang, H., Xu, Y., Pang, J., . . . Zheng, Y. (2019). Modulation of miR-34a/SIRT1 signaling protects cochlear hair cells against oxidative stress and delays age-related hearing loss through coordinated regulation of mitophagy and mitochondrial biogenesis. Neurobiology of Aging, 79 , 30-42. Zhao, H.-B., Yu, N., & Fleming, C. R. (2005). Gap junctional hemichannel-mediated ATP release and hearing controls in the inner ear. Proceedings of the National Academy of Sciences, 102 (51), 18724-18729. Tables Table 1. Antibodies used in this study. Relevant information was gathered from datasheets for each antibody. Antibody Company/ catalogue No. Epitope Dilution Reactivity Marker for Reference Anti-P2X 4 , Rabbit IgG polyclonal Alomone APR-002 C-terminus of mouse P2X4 isoform 1:1000 Mouse, rat and human Anti-SOX2, Mouse IgG monoclonal Santa Cruz Biotechnology Inc. Sc-365823 Human SOX2 amino acids 170-201 1:100 Mouse, rat and human Cochlear supporting cell nucleus (Oesterle et al., 2007) (Smeti et al., 2011) Anti-Myosin VIIa, Mouse IgG monoclonal Santa Cruz Biotechnology Inc. sc-74516 N-terminus of human Myosin VIIa 1:50 Mouse, rat and human Inner hair cells & Outer hair cells (Xiong et al., 2019) (Jung et al., 2019) Anti-LAMP-1, Mouse IgG monoclonal Santa Cruz Biotechnology Inc. sc-20011 Adherent spleen cells of human origin 1:200 Mouse, rat and human Lysosomes (Spangenberg et al., 2019) (Oh et al., 2020) Anti-EEA-1, Mouse IgG monoclonal Santa Cruz Biotechnology Inc. sc-137130 N-terminus of human EEA1 1:200 Mouse, rat, human and monkey Endosomes (Kuszczyk et al., 2013) (Men et al., 2019) Anti-GM130, Mouse IgG monoclonal BD Biosciences 610822 Rat GM130 aa. 869-982 1:200 Human dog and mouse Golgi (Dandoy-Dron et al., 2003) (Zheng et al., 2010) Anti-TOM20, Mouse IgG monoclonal Santa Cruz Biotechnology Inc. sc-17764 Human Tom20 1:200 Mouse, rat and human Mitochondria (Balaker et al., 2012) (Xiong et al., 2019) WGA Alexa 647 Conjugate Thermofisher Wheat Germ Agglutinin (WGA) 1:300 All Plasma membrane (Graveleau et al., 2005) (Cui et al., 2020) Phalloidin Alexa 488 Thermofisher Phalloidin 1:500 All Cytoskeletal actin Table 2 is not available with this version. Additional Declarations No competing interests reported. Supplementary Files HuangetalSupplementarymaterialv3.docx Cite Share Download PDF Status: Published Journal Publication published 20 May, 2025 Read the published version in Histochemistry and Cell Biology → Version 1 posted Editorial decision: Revision requested 09 Sep, 2024 Reviews received at journal 09 Sep, 2024 Reviewers agreed at journal 27 Aug, 2024 Reviews received at journal 17 Jun, 2024 Reviewers agreed at journal 27 May, 2024 Reviews received at journal 27 May, 2024 Reviewers agreed at journal 26 May, 2024 Reviewers invited by journal 25 May, 2024 Editor assigned by journal 25 May, 2024 Submission checks completed at journal 25 May, 2024 First submitted to journal 24 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4474581","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308869471,"identity":"c727c937-d24d-4411-93d2-03a6b3b39df7","order_by":0,"name":"Ziyin Silver Huang","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Ziyin","middleName":"Silver","lastName":"Huang","suffix":""},{"id":308869473,"identity":"c7a24840-f687-4d79-9cda-d8cd89931d40","order_by":1,"name":"Jacqueline M Ross","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Jacqueline","middleName":"M","lastName":"Ross","suffix":""},{"id":308869474,"identity":"852807bf-2111-4ebf-9a88-9952b2ee4e9f","order_by":2,"name":"Shelly CY Lin","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Shelly","middleName":"CY","lastName":"Lin","suffix":""},{"id":308869475,"identity":"8416cd6c-a209-4431-b22d-7fcf59182bc4","order_by":3,"name":"Kevin Roy","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Roy","suffix":""},{"id":308869476,"identity":"8cda8621-17aa-4688-a2ea-16bd2df63173","order_by":4,"name":"Srdjan M Vlajkovic","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Srdjan","middleName":"M","lastName":"Vlajkovic","suffix":""},{"id":308869477,"identity":"5dbac71c-1415-44f1-8c2c-df6d1c79fe52","order_by":5,"name":"Peter R. 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(A) \u003c/strong\u003eSchematic drawing of OoC cryosection. \u003cstrong\u003e(B-I)\u003c/strong\u003e cryosections (B-E) and OoC wholemounts (F-I) were prepared from adult Wistar rat cochlea and labelled with anti-P2X\u003csub\u003e4\u003c/sub\u003e antibody (\u003cem\u003ered\u003c/em\u003e), phalloidin (\u003cem\u003egreen\u003c/em\u003e) and DAPI (\u003cem\u003eblue\u003c/em\u003e). (I) P2X\u003csub\u003e4\u003c/sub\u003e antibodies pre-absorbed with excess control peptide\u0026nbsp;. Representative image from n=6 cochlea. Scale bar 200 µm (B), 20 µm (C, E, F, I), and 10 µm (D, G, H). \u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/069c34c318370d5b43008e15.png"},{"id":57869551,"identity":"b6fdc8e3-969c-41ec-b775-f6984394b5a0","added_by":"auto","created_at":"2024-06-06 16:51:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3065711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopmental expression of P2X\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e in IHCs and OHCs.\u003c/strong\u003e \u003cstrong\u003e(A-F) \u003c/strong\u003eCryosection (A-B) and OoC whole mount preparation (C-F) of Wistar rat cochleae at P8 (A-B, E), E20.5 (C), P4 (D), P21 (F) labelled with anti-P2X\u003csub\u003e4\u003c/sub\u003e antibody (\u003cem\u003ered\u003c/em\u003e) and phalloiding (\u003cem\u003egreen\u003c/em\u003e). \u003cstrong\u003e(G-H)\u003c/strong\u003e Adult cochlea whole mounts were labelled with anti-P2X\u003csub\u003e4\u003c/sub\u003e (\u003cem\u003ered\u003c/em\u003e) and co-labelled with anti-MyosinVIIa (G, \u003cem\u003egreen\u003c/em\u003e) or anti-SOX2 (H, \u003cem\u003egreen\u003c/em\u003e) antibodies and DAPI (\u003cem\u003eblue\u003c/em\u003e) in the OoC of the adult rat cochlea. Scale bar 50 µm (A) and 10 µm (B-H).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/181b4ff80a92fd4127d50d80.png"},{"id":57869681,"identity":"b98f9b26-aebc-4789-aafe-13f535f5c9c7","added_by":"auto","created_at":"2024-06-06 16:59:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":905251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular distribution of P2X\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e in IHC. (A-D)\u003c/strong\u003e The P2X\u003csub\u003e4\u003c/sub\u003e expression in the IHCs compared at different focal planes visualized by optical slicing along the z-axis. The axis on the left represents the images taken from the apical [A] to the basal [B] region of the cells. \u003cstrong\u003e(E)\u003c/strong\u003e The 3D reconstruction of the IHCs. The axis on the left bottom corner represents the cell orientation. \u003cstrong\u003e(F)\u003c/strong\u003e The orthogonal views of the IHCs with three planes shown XY plane (F’), YZ plane (F’’), XZ plane (F’’’). (Arrow) The bright P2X\u003csub\u003e4\u003c/sub\u003e signal at the medial end of the IHCs. Axis in the figure are: [A] apical [B] basal [L] lateral (towards lateral wall side) [M] medial (towards modiolus side) [Le] left [R] right. Scale bars = 10mm. \u003cstrong\u003e(G \u0026amp; H)\u003c/strong\u003e P2X\u003csub\u003e4\u003c/sub\u003e normalized signal intensity (Y-axis) from 3 cochlea along the apical-basal axis (G) and medial-lateral axis (H). See Supplementary figure 5 and 6 for more information on the quantification. Axis in the figure are: [A] apical [B] basal [L] lateral (towards lateral wall side) [M] medial (towards modiolus side) [Le] left [R] right.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/414faae93b63018e9193f812.png"},{"id":57869549,"identity":"18077db2-4532-4b7c-83e8-ac08dc725d9b","added_by":"auto","created_at":"2024-06-06 16:51:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1012138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular distribution of P2X\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e in OHC. (A, B, C, D)\u003c/strong\u003e The P2X\u003csub\u003e4\u003c/sub\u003e expression in the OHCs compared at different focal planes visualized by optical slicing along the z-axis. The axis on the left represents the images taken from the apical [A] to the basal [B] part of the cells. (E) The 3D reconstruction of the OHCs. The axis on the left bottom corner represents the cell orientation. (F) The orthogonal views of the OHCs with three planes shown as XY plane (F’), YZ plane (F’’), XZ plane (F’’’). (*) The prominent cluster of P2X\u003csub\u003e4\u003c/sub\u003e vesicles in the apical-lateral end of the OHCs. Axis in the figure are: [A] apical [B] basal [L] lateral (towards lateral wall side) [M] medial (towards modiolus side) [Le] left [R] right. Scale bars = 10mm. \u003cstrong\u003e(G \u0026amp; H)\u003c/strong\u003e P2X\u003csub\u003e4\u003c/sub\u003e normalized signal intensity (Y-axis) in 3 cochleae along the apical-basal axis (G) and along medial-lateral axis (H). See Supplementary figure 5 and 6 for more information on the quantification. Axis in the figure are: [A] apical [B] basal [L] lateral (towards lateral wall side) [M] medial (towards modiolus side) [Le] left [R] right.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/3667efb3e270725ce1ea982f.png"},{"id":57869682,"identity":"820d5b94-29df-44a6-af10-b7021ffdb754","added_by":"auto","created_at":"2024-06-06 16:59:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":211612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParticle analysis of P2X\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e immunolabeling in OHCs. (A)\u003c/strong\u003e Schematic example of the selection of the cell and the particle analysis example. Each Z-stack of OHCs contains 70-90 optical sections depending on the plane of focus. Each slide in the Z-stack is displayed with particle counts and total area. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic OHCs with the zone division. \u003cstrong\u003e(C)\u003c/strong\u003e The summary graph of the P2X\u003csub\u003e4\u003c/sub\u003e expression average total area and \u003cstrong\u003e(D)\u003c/strong\u003e normalized particle count. Axis in the figure is: [L] lateral (towards lateral wall side) [M] medial (towards modiolus side) [Le] left [R] right. Nine cells from three adult Wistar rats cochlea were subjected to the particle analysis.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/6be2fc2ecfd277ddd50a5a1f.png"},{"id":57869555,"identity":"eb186068-84a4-44cd-b4af-cfcd6d0e020f","added_by":"auto","created_at":"2024-06-06 16:51:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1589305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-localization of P2X4 in IHCs and OHCs with organelle markers. (A-J) \u003c/strong\u003eOoC preparation from adult Wistar rats were immunolabelled with an anti-P2X\u003csub\u003e4\u003c/sub\u003e antibody (\u003cem\u003ered\u003c/em\u003e) and one of the organelle markers (\u003cem\u003egreen\u003c/em\u003e); anti-EEA-1 (A-B), anti-LAMP-1(C-D), anti-GM130 (E-F), anti-TOM20 (G-H), WGA (I). High-resolution images were taken at IHC (A, C, E, G), and OHC (B, D, F, H, I). Open arrows indicate a double-positive signal, while closed arrows indicate a single-positive marker signal. Scale bars = 10μm. \u003cstrong\u003e(J-K)\u003c/strong\u003e Quantification of the co-localization of P2X\u003csub\u003e4\u003c/sub\u003e and organelle markers in IHCs (J) and OHCs (K) using JACoP ImageJ Plugin and Mander’s coefficient (value range 0-1.0) has been summarised. n=3 cochleae were examined.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/b720cdd053539110c4984eac.png"},{"id":83460041,"identity":"7210c642-63a0-4638-b5ae-f7bb7fa7b932","added_by":"auto","created_at":"2025-05-26 16:09:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20375818,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/e96c5989-afaf-41d8-942b-3e2a54c9b03a.pdf"},{"id":57869552,"identity":"79344c99-ff2b-4d66-b075-f00650b27fc3","added_by":"auto","created_at":"2024-06-06 16:51:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4984638,"visible":true,"origin":"","legend":"","description":"","filename":"HuangetalSupplementarymaterialv3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4474581/v1/3c4ebfe1e00813845c078986.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Subcellular Localization of the P2X4 Receptor in Cochlear Sensory Hair Cells","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAccording to the World Report on Hearing (2021), hearing loss affects 1.5B people globally and this number is expected to grow to 2.5B by 2050 (World Health Organization, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The majority of cases are sensorineural hearing loss (SNHL), characterized by degenerative changes in the cochlea and the auditory nerve. There are few effective pharmacological treatments for SNHL and development of such treatment requires further understanding of cochlear physiology and pathophysiology at the cellular and molecular levels. The organ of Corti (OoC), the sensory apparatus within the cochlea, contains two types of auditory sensory cells, the inner hair cells (IHC) and outer hair cells (OHC). Hair cells (HCs) are so-called due to mechanosensory stereocilia located on the apical side of the cell supported by an actin-rich cuticular plate and are essential for sound transduction (Goodyear, Marcotti, Kros, \u0026amp; Richardson, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Approximately 3500 IHCs are aligned as a single continuous row and 12,000 OHCs arranged in three rows more laterally in the human cochlea. In humans and animal models, loss of HCs, particularly OHC and loss of synapses and neurons innervating IHC have been observed as a common underlying pathology associated with SNHL (Liberman \u0026amp; Kujawa, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e-z. Wu, O'Malley, de Gruttola, \u0026amp; Liberman, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; P. Z. Wu et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePurinergic signaling is involved in many cellular functions and pathologies in the inner ear and considered to be a potential therapeutic target for inner ear disorders. Purinergic signaling pathways are activated by extracellular nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine triphosphate (UTP), uridine diphosphate (UDP), and adenosine (Burnstock, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). There are two classes of purinergic receptors: P1 receptors (A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2A\u003c/sub\u003e, A\u003csub\u003e2B\u003c/sub\u003e and A\u003csub\u003e3\u003c/sub\u003e) and P2 receptors (P2X\u003csub\u003e1-7\u003c/sub\u003e, P2Y\u003csub\u003e1,2,4,6, 11-14\u003c/sub\u003e) (Burnstock, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Many of these purinoceptor subtypes have been identified in the cochlea (K\u0026ouml;les, Szepesy, Berekm\u0026eacute;ri, \u0026amp; Zelles, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vlajkovic \u0026amp; Thorne, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). P2X receptors form trimeric ligand-gated ion channels that are non-selectively permeable to cations (Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e) (Burnstock, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Earlier studies had suggested a potential role for P2X receptors in the cochlea, such as regulation of afferent neuronal activity in response to agonists released in the perilymph (Robertson \u0026amp; Paki, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). More specific roles for P2X subtypes are emerging based on molecular and functional investigations; P2X\u003csub\u003e2\u003c/sub\u003e expressed in cells lining the endolymphatic compartment and in the stereocilia of OHC participate in the reduction of endocochlear potential during sound transduction and in modulating the sound sensitivity, respectively (Jarlebark, Housley, \u0026amp; Thorne, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Morton-Jones et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Z.-J. Wang \u0026amp; Neuhuber, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). P2X\u003csub\u003e1\u003c/sub\u003e and P2X\u003csub\u003e7\u003c/sub\u003e localized near synaptic terminals of the auditory neurons on IHCs and in the neurites of the SGN (P. Nikolic, Housley, Luo, Ryan, \u0026amp; Thorne, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Predrag Nikolic, Housley, \u0026amp; Thorne, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) may play roles in Ca\u003csup\u003e2+\u003c/sup\u003e-dependent uncoupling of synapses known to occur at these postsynaptic terminals (Liberman \u0026amp; Kujawa, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). P2X\u003csub\u003e3\u003c/sub\u003e expressed in the developing spiral ganglion neurons regulate branching of afferent fibres (L.-C. Huang, Ryan, Cockayne, \u0026amp; Housley, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Z. Wang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, molecular expression of P2X\u003csub\u003e4-6\u003c/sub\u003e has not been clearly demonstrated in the cochlea. P2X\u003csub\u003e4\u003c/sub\u003e has some unique features compared to other P2X subtypes. The human monomeric P2X\u003csub\u003e4\u003c/sub\u003e channel is sensitive to the extracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration with slow desensitization compared to P2X\u003csub\u003e1-3\u003c/sub\u003e isoforms requiring 4 sec at a 30\u0026micro;M concentration ATP (Hattori \u0026amp; Gouaux, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Cytoplasmic expression of P2X\u003csub\u003e4\u003c/sub\u003e and localization to lysosomes have been reported (Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Murrell-Lagnado \u0026amp; Frick, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Intracellular P2X\u003csub\u003e4\u003c/sub\u003e exhibit pH sensitivity (inactivated at low pH) and can be regulated by the pH within the lysosomal lumen (P. Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ruth D. Murrell-Lagnado \u0026amp; Frick, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the guinea pig cochlea, functional expression of P2X\u003csub\u003e4\u003c/sub\u003e has been reported in the endothelial cells of the spiral ligament where it appears to regulate cochlear blood flow (Y. Wu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and in hair cells (Sz\u0026uuml;cs et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), however, the detailed P2X\u003csub\u003e4\u003c/sub\u003e distribution in the cochlea still remains to be characterized. In the vestibular system of the inner ear, P2X\u003csub\u003e4\u003c/sub\u003e molecular and functional expression has recently been reported in vestibular supporting cells (Jeong et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the distribution of P2X\u003csub\u003e4\u003c/sub\u003e in the developing and adult rat cochlea. Expression was confined to OHCs and IHCs, and located solely in the cytoplasm. Colocalization with markers of endoplasmic reticulum, Golgi apparatus and lysosomes suggests that P2X\u003csub\u003e4\u003c/sub\u003e is associated with different intracellular organelles (Golgi-ER in IHC and lysosomes in OHC) and may mediate calcium buffering within these intracellular organelles.\u003c/p\u003e"},{"header":"METHOD AND MATERIALS","content":"\u003ch2\u003eAnimals\u003c/h2\u003e\n\u003cp\u003eThe use of animals for this project was approved by the University of Auckland Animal Ethics Committee (AEC002251). All animals were supplied by the Vernon Jensen Unit (VJU; The University of Auckland). Wistar rats of various ages and both sexes were used for this study; embryonic day 20.5, postnatal day 4 (P4), postnatal day 8 (P8), postnatal day 21 (P21), and 6-week-old (adult). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eTissue preparation\u003c/h2\u003e\n\u003cp\u003eGeneral chemicals were purchased from ThermoFisher Scientific (Auckland, New Zealand) unless otherwise specified.\u0026nbsp;4% w/v Paraformaldehyde (PFA, pH 7.4)\u0026nbsp;was prepared with\u0026nbsp;0.1M Phosphate Buffer (PB, 24.6 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and 75.4 mM NaHPO\u003csub\u003e4\u003c/sub\u003e, pH 7.4) for fixation of all samples. P21 and adult rats were first anaesthetised and euthanized by drug overdose (Pentobarbital, ProVet NZ Pty Ltd), followed by perfusion of 0.1% w/v NaNO\u003csub\u003e2\u003c/sub\u003e in Phosphate-Buffered Saline (PBS, pH 7.4, Gibco), and 4% PFA through the left ventricle. The temporal bones were removed from the cranium and a small puncture was carefully made in the round window membrane to aid the penetration of PFA into the cochlea. The cochlea was then immersion fixed in 4% PFA at room temperature (RT) for 24 hours. Cochleae were washed with PBS three times (10 mins each). Adult cochlear tissues were decalcified by immersion in 4% w/v Ethylenediaminetetraacetic acid (EDTA, in 0.1PB, pH 7.4) at room temperature for up to 2 weeks with regular change of EDTA solution. Cochleae from Wistar rats younger than P8 were removed and immersed in PFA (4% PFA at room temperature (RT)) for 24 hours and further dissection was carried out without decalcification. For the organ of Corti (OoC) whole mount preparations, cochleae were micro-dissected in PBS and segments of OoC approximately equivalent to \u0026frac12; turn, were taken from the apical, middle, and basal turn. For cryosectioning, cochleae were cryoprotected sequentially in 10% and 20% sucrose (w/v in PBS) for one hour at room temperature, then in 30% sucrose overnight at 4\u0026deg;C followed by embedding in Tissue-Tek Optimal Cutting Temperature Compound (OCT, ProSciTech, Australia) at -80\u0026deg;C. The tissue was cryosectioned at 20-30 mm (Leica, CM3050S) in the axial plane through the modiolus and cochlear ducts. Microdissected or cryosectioned tissue was stored in PBS for up to 1 week at 4\u0026deg;C before processing for immunohistochemistry.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\n\u003cp\u003eThe list of primary antibodies used in this study and dilutions are summarised in Table 1. A polyclonal primary antibody raised in rabbits against the C-terminal domain of the rat P2X\u003csub\u003e4\u003c/sub\u003e subunit (Alomone Inc., Jerusalem, Israel, catalogue no. APR-002) was used for detection of P2X\u003csub\u003e4\u003c/sub\u003e by immunohistochemistry. This antibody has been validated using P2X\u003csub\u003e4\u003c/sub\u003e knockout animals\u0026nbsp;(Lalisse et al., 2018; Sim et al., 2006; Wyatt et al., 2014). Other antibodies used as cell-type specific markers and organelle markers are summarized in Table 1. Immunohistochemistry was performed following the protocol established previously\u0026nbsp;(Fok et al., 2020; Han, Lin, Espinosa, Thorne, \u0026amp; Vlajkovic, 2019). Blocking solutions and antibody diluent solutions were prepared as following; blocking solution for whole mounts \u0026nbsp;(10% v/v\u0026nbsp;normal\u0026nbsp;goat serum (NGS) and 2.5% (v/v) TritonX in PBS), blocking solution for cryosection (10% v/v NGS, 1% v/v TritonX in PBS), antibody diluent for wholemount (5% v/v NGS and 0.25% v/v TritonX in PBS) and antibody diluent for cryosection (5% v/v NGS and 0.1% v/v TritonX in PBS). Tissues were incubated for 2 hours in a blocking solution at room temperature. Tissues were then incubated in the diluted primary antibody overnight at 4\u003cspan dir=\"RTL\"\u003e֯\u003c/span\u003eC\u0026deg;. For the anti-P2X\u003csub\u003e4\u003c/sub\u003e antibody control, the pre-absorbing peptide was added to the primary antibody solution in a 1mg-1mg ratio and sections or whole mounts were incubated for 2 hours following the manufacturer\u0026rsquo;s protocol (Alomone Labs, Israel). Tissues were washed 4 times at 1, 10, 15, 30 minutes intervals in PBS at RT followed by incubation with secondary antibodies overnight at 4\u0026deg;C in the dark.\u0026nbsp;Secondary antibodies used were goat anti-rabbit Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488, and goat anti-mouse Alexa Fluor 647 (ThermoFisher Scientific, all used at 1:500 dilution in antibody diluent).\u0026nbsp;The non-antibody labelling reagents Wheat Germ Agglutinin (WGA) and Phalloidin (Table 1) were included in the same mixture with the secondary antibody. From this step forward, tissues were covered to minimize light exposure. After incubation with secondary antibodies, the tissues were washed 4 times in PBS, incubated in DAPI (diluted in PBS, 0.02mg/ml) for an hour at room temperature, washed 4 times in PBS and mounted with coverslips on slides with CitiFluor AF1 mountant solution (Agar Scientific Ltd, UK). Slides were stored at 4\u0026deg;C in the dark until imaging. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConfocal microscopy\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eFluorescently immunolabelled slides were imaged using a Zeiss LSM 800 Airyscan confocal microscope (Carl Zeiss GmbH, Jena, Germany) in the Biomedical Imaging Research Unit (BIRU) at The University of Auckland. Objective lenses used were 10x/0.45 NA Plan Apochromat, 20x/0.8 NA Plan Apochromat, 63x/1.4 NA Plan Apochromat oil immersion. Images were acquired at a pixel resolution of 0.18\u0026mu;m/pixel for 20x, and 0.035\u0026mu;m/pixel for 63x in Airyscan mode.\u0026nbsp;The Z series were obtained using a 63x/1.4 NA oil immersion objective lens with a 0.5mm step size between optical sections. The top limit for the Z series was set at the level of the tip of the stereocilia of the hair cells, as visualized with phalloidin representing the most apical end of the cell body, and the bottom limit was set at the opposite end of the hair cell body at the position where the P2X\u003csub\u003e4\u003c/sub\u003e signal had just disappeared from the HCs. A typical Z-stack was 40mm thick. All of the images were acquired using ZEN 2.6 software (Carl Zeiss, Germany) and exported to TIFF as required for figure preparation or analysis.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eImage analysis and processing\u003c/h2\u003e\n\u003cp\u003eImageJ (Schneider et al., 2012) was used for particle analysis and quantification of P2X\u003csub\u003e4\u003c/sub\u003e immunolabeling on individual cells from the z series images (see Supplementary material for details on methodology). An automated threshold was used to identify the particles of interest. The measurements were then carried out using the built-in Particle Analyzer. Parameters included \u0026ldquo;Count\u0026rdquo; and \u0026ldquo;Total Area\u0026rdquo;. \u0026ldquo;Count\u0026rdquo; represents the number of particles in each image and \u0026ldquo;Total Area\u0026rdquo; is the area represented by the sum of the particles in each image.\u0026nbsp; ImageJ \u0026ldquo;line plot profile\u0026rdquo; analysis was performed to quantify the relative signal intensity within the hair cells in apical to basal direction. This function was also used to measure intensity at nine different locations in the hair cell across the apical-basal as well as medial-lateral directions and normalised relative to the total signal (Figure 3 \u0026amp; 4 and Supplementary Figure 1).\u0026nbsp;The JACoP plugin (see Supplementary material for more detail) was used\u0026nbsp;for colocalization analysis between P2X\u003csub\u003e4\u003c/sub\u003e and subcellular markers. Individual channels were separately processed by background subtraction and images were cropped so that the region of interest typically contained 8 cells. JACoP automatically calculates Manders\u0026rsquo; colocalization coefficients (with and without a threshold) and Pearson\u0026rsquo;s correlation coefficient (Bolte \u0026amp; Cordeli\u0026egrave;res, 2006; Dunn, Kamocka, \u0026amp; McDonald, 2011). The results were displayed as M1 and M2, each with the value range between 0-1.0, where M1 is defined as the ratio of the \u0026ldquo;summed intensities of pixels from the green channel for which the intensity in the red channel is above zero\u0026rdquo; to the \u0026ldquo;total intensity in the green channel\u0026rdquo;. M2 is identified as the same as the red and green reversed. High M1 and M2 coefficients indicate that a large proportion of one signal co-occurs with the other signal. Colocalization analyses were conducted for each subcellular marker separately, with 3 cochleae for each marker, and the mean and standard error of the mean (SEM) were calculated. Imaging processing was performed using Adobe Photoshop CC (version 19.1.3, Adobe system Incorporated) to prepare figures.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003ch2\u003eP2X\u003csub\u003e4\u003c/sub\u003e expression in the cochlea\u003c/h2\u003e\n\u003cp\u003eAfter testing the dilution range 1:50 to 1:2000, 1:1000 dilution was chosen to have the best signal to background ratio (data not shown) for the anti-P2X\u003csub\u003e4\u003c/sub\u003e antibody (Figure 1). High levels of expression of P2X\u003csub\u003e4\u003c/sub\u003e in the rat OoC were observed (Figure 1D, F), but less in the spiral ligament (Figure 1C, arrow) \u0026nbsp;and spiral ganglion (Figure 1E, arrow) In the OoC, there was a relatively higher expression in IHC \u0026amp; and to a lesser extent in OHCs (Figure 1D, F-H). Immunolabeling of P2X4 throughout was abolished by preabsorbing P2X\u003csub\u003e4\u003c/sub\u003e antibody with excess peptide molecules in the controls (Figure 1I). The expression of P2X\u003csub\u003e4\u003c/sub\u003e was evident in the hair cells from E20.5 but was more prominent in the IHCs than OHCs. When compared the OoC at different rat ages,(E20.5, P4, P8, P21); Figure C-F), immature IHC at E20.5 expressed P2X4 above the background, but the signal was relatively weak. At P4 and P8, expression of P2X\u003csub\u003e4\u003c/sub\u003e was clearly evident in IHCs and OHCs. By P21, a week after the hearing onset, P2X\u003csub\u003e4\u003c/sub\u003e exhibited a similar expression pattern to the adult cochlea with strong expression of P2X\u003csub\u003e4\u003c/sub\u003e in the IHCs. At P8, P2X\u003csub\u003e4\u003c/sub\u003e expression was prominent in IHC \u0026amp; OHC (Figure 2A-B). Some cells lining the cochlear scala tympani and vestibuli also expressed detectable levels of P2X\u003csub\u003e4\u003c/sub\u003e (Figure 2A-B, arrows). It is not possible to identify these cells but they appear morphologically very similar to Iba1-expressing macrophages observed in the postnatal mouse cochlea (Kishimoto, Okano, Nishimura, Motohashi, \u0026amp; Omori, 2019). To confirm the identity of cells expressing P2X\u003csub\u003e4\u003c/sub\u003e, two cell-type specific markers were used: myosin VIIa, which is consistently expressed in IHC and OHCs\u0026nbsp;(Jung et al., 2019; Xiong et al., 2019)\u0026nbsp;and Sox2, which is a transcription factor expressed in nuclei of all types of supporting cells\u0026nbsp;(Smeti et al., 2011). P2X\u003csub\u003e4\u003c/sub\u003e-labelled cells co-expressed myosin VIIa, confirming these to be the IHCs and OHCs (Figure 2G), while P2X\u003csub\u003e4\u003c/sub\u003e was not observed in cells expressing Sox2 (Figure 2H). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePolarity of P2X\u003csub\u003e4\u003c/sub\u003e subcellular distribution within IHC and OHC in adult rat cochlea\u003c/h2\u003e\n\u003cp\u003eWe next investigated the subcellular localizations of IHCs and OHCs in the adult rat cochlea to correlate the distribution of P2X\u003csub\u003e4\u003c/sub\u003e with the distinct functional domains of IHC and OHC. Analysis of z-stack images of OoC enabled compartmentalisation of the hair cells into four different sub-domains from the apical surface to the basal pole of the cell; sub-cuticular (Figure 3A), cytoplasmic (Figure 3B), nuclear (Figure 3C) and subnuclear zones (Figure 3D). At the sub-cuticular plate level of IHC, P2X\u003csub\u003e4\u003c/sub\u003e immunolabeling appeared as bright irregular clusters in the cell cytoplasm immediately underneath the cuticular plate (Figure 3A). In the supranuclear cytoplasm, between the cuticular plate and nucleus, there were similar clusters of P2X\u003csub\u003e4\u003c/sub\u003e immunolabelling, but these appeared larger and brighter (Figure 3B). At the nucleus and sub-nucleus levels, the cytoplasmic immunolabeling for P2X\u003csub\u003e4\u003c/sub\u003e appeared brightest and the most abundant (Figure 3C, D). This pattern is also evident in the 3D re-constructions (Figure 3E). Orthogonal views of the images were also generated with ImageJ where the stack of images was viewed in a XY, YZ, XZ planes, where the X, Y, Z planes correspond to the left and right (Le-R), medial and lateral (M-L) and the apical and basal (A-B), respectively (Figure 3F). Orthogonal visualization confirmed more intense P2X\u003csub\u003e4\u003c/sub\u003e immunolabeling along the medial side of the IHCs (Figure 3F\u0026rsquo;\u0026rsquo;, arrow). This corresponds to the large, patchy signal appearance in the 3D re-construction (Figure 3E). More intense signal was also observed at the apical part of the image (Figure 3F\u0026rsquo;\u0026rsquo;\u0026rsquo;). Signal distribution for P2X\u003csub\u003e4\u003c/sub\u003e along the apical-basal and medial-lateral axes of the cells was quantified using ImageJ (Supplementary Figure 4) to confirm these visual observations that the P2X\u003csub\u003e4\u003c/sub\u003e expression was more concentrated at the basal end of the IHCs (Figure 3G) and at the medial side of the IHCs (Figure 3H).\u003c/p\u003e\n\u003cp\u003eSimilar analyses in the OHCs (Figure 4) showed P2X\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eimmunolabeling at all four levels predominately in the cell cytoplasm, however the characteristic pattern of P2X\u003csub\u003e4\u003c/sub\u003e localisation was quite different from that observed in IHCs. At the sub-cuticular plate level, the P2X\u003csub\u003e4\u003c/sub\u003e expression appeared to be more concentrated than observed in IHC (Figure 4A). Interestingly, the cluster of P2X\u003csub\u003e4\u003c/sub\u003e labelling often appeared immediately underneath the \u0026lsquo;cuticular-free zone\u0026rsquo;, a small region on the lateral aspect of the cell that does not stain with phalloidin (Figure 4 arrow). At the cytoplasmic level, regions of P2X4 appeared more scattered, but some medium-sized clusters were observed (Figure 4B). At the nucleus level, the P2X\u003csub\u003e4\u003c/sub\u003e immunoreactivity was less obvious (Figure 4C), but more intense in the basal sub-nucleus level of OHC (Figure 4D). When reconstructed in 3D, a prominent cluster of P2X\u003csub\u003e4\u003c/sub\u003e immunolabeling was observed at the apical part of the cytoplasm, and it was not as homogenously distributed through the whole cell compared to P2X\u003csub\u003e4\u003c/sub\u003e immunolabeling in IHC (Figure 4E). Examined using the orthogonal view, the most intense signal for P2X\u003csub\u003e4\u003c/sub\u003e (Figure 4F\u0026rsquo;\u0026rsquo;, asterisks), appearing as a prominent cluster, was observed at the lateral side of each cell underneath the CP free zone (Figure 4E). Z-stack images obtained for the OHCs were quantified using ImageJ (See supplementary figure 5 for details) to confirm that the P2X\u003csub\u003e4\u003c/sub\u003e expression was more concentrated at both the apical and the basal end of the OHCs (Figure 4G). The gradient in OHC was very subtle in the medial to lateral direction (Figure 4L-M), compared to the clear trend observed for IHC (Figure 3H). Immunolabeling for of P2X\u003csub\u003e4\u003c/sub\u003e appeared as more discrete dots in the OHCs compared to the IHCs, allowing additional \u0026ldquo;particle analysis\u0026rdquo; (Figure 5A \u0026amp; B; see Supplementary Figure 3 for more detail). The total area occupied by P2X\u003csub\u003e4\u003c/sub\u003e immunolabeling was the greatest in the sub-cuticular zone, compared to the three other zones (Figure 5C). In contrast, the total amount of staining was the highest in the cytoplasmic zone compared to the other three zones (Figure 5D). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eLocalization of P2X\u003csub\u003e4\u003c/sub\u003e to subcellular organelles within IHCs and OHCs.\u003c/h2\u003e\n\u003cp\u003eThe P2X\u003csub\u003e4\u003c/sub\u003e immunostaining was in clusters and appeared to be vesiculated. To determine if these were associated with other membranous intracellular organelles, we looked at co-localisation of P2X\u003csub\u003e4\u003c/sub\u003e with endosomes, lysosomes, Golgi bodies and mitochondria using immunohistochemistry (Table 1). Early endosomes are derived from the plasma membrane (Gindhart \u0026amp; Weber, 2009) and distinguished from late endosomes and other vesicles by the expression of early endosome antigen 1 (EEA-1) (Patki et al., 1997), including in IHCs and OHCs (Schug et al., 2006). Endosomes and the Golgi apparatus are part of the intracellular protein transportation and recycling pathway. EEA-1 labelling in IHCs had a diffuse appearance, with vesicular labelling more concentrated in the apical part of the cell (Figure 6A). In OHCs, EEA-1 labelled vesicles appeared throughout (Figure 6B). There was some co-labelling between EEA-1 and P2X\u003csub\u003e4\u003c/sub\u003e (Figure 6A, B arrow) in both IHC and OHC, with qualitatively more co-occurrence observed in OHC. To quantify the co-localization of EEA-1 with P2X\u003csub\u003e4\u003c/sub\u003e, the JACoP plugin\u0026nbsp;(Bolte \u0026amp; Cordeli\u0026egrave;res, 2006)\u0026nbsp;in ImageJ was used (see Supplementary Figure 3 for details). Z-stack images covering either entire OHCs or IHCs were selected for analysis. JACoP quantifies the co-occurrence of P2X\u003csub\u003e4\u003c/sub\u003e and EEA-1 as two \u0026ldquo;Mander\u0026rsquo;s coefficients\u0026rdquo; calculated as M1 and M2 coefficients with a value range between 0-1.0. M1 represents the proportion of EEA-1 co-localized with P2X\u003csub\u003e4\u003c/sub\u003e signal over the total P2X\u003csub\u003e4\u003c/sub\u003e signal. M2 represents the proportion of the EEA-1 co-localized with P2X\u003csub\u003e4\u003c/sub\u003e over the total signal of EEA-1. The average M1 values for each organelle marker in IHC and OHC are summarised in Figure 6J \u0026amp; K. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaking the same approach, we analysed the co-occurrence of P2X\u003csub\u003e4\u003c/sub\u003e with LAMP-1, GM130, Tom20 and Wheat Germ Agglutinin (WGA). LAMP-1 is a protein found on lysosomes and lysosome-endosome fusion vesicles and is commonly used as a marker for lysosomes (Huotari \u0026amp; Helenius, 2011). \u0026nbsp;Lysosomes are distributed throughout the cell in the IHCs and OHCs, but large lysosomes are often found at the apical, lateral side of the cell (Spicer, Thomopoulos, \u0026amp; Schulte, 1999). OHCs have a greater number of lysosomes compared to IHCs (Spicer, Thomopoulos, \u0026amp; Schulte, 1998; Wiwatpanit et al., 2018). LAMP-1 labelling in IHCs had a more diffuse appearance with lower signal levels, and minimally co-occurred with P2X\u003csub\u003e4\u003c/sub\u003e (Figure 6C) where the OHCs had a vesicular appearance (Figure 6D). There was a clear overlap of the P2X\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eimmunolabelling and LAMP-1 in OHC (Figure 6D, Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGM130\u0026nbsp;is a marker for\u0026nbsp;Golgi matrix protein of 130kDa,\u0026nbsp;which typically targets the cis-component of Golgi\u0026nbsp;(Nakamura, Lowe, Levine, Rabouille, \u0026amp; Warren, 1997). The Golgi apparatus is located mainly around the apical part of the cytoplasm in HCs\u0026nbsp;(Schug et al., 2006; Spicer et al., 1998, 1999). In the rat cochlea, cytoplasmic expression of GM130 was observed in the IHCs and OHCs with vesicular, string-like structures (Figure 6E, F), consistent with previous reports\u0026nbsp;(Schug et al., 2006). Notably, the co-occurrence of the GM130 and P2X\u003csub\u003e4\u003c/sub\u003e in both the IHCs and OHCs was minimal (Figure 6E, F), 11.7% \u0026plusmn; 2.4%, and in the OHCs, 27% \u0026plusmn; 2%.\u0026nbsp;TOM20 is a protein expressed on the mitochondrial outer membrane\u0026nbsp;(Balaker, Ishiyama, Lopez, Ishiyama, \u0026amp; Ishiyama, 2013)\u0026nbsp;and was used here as the marker for mitochondria. There was some overlap of TOM20 and P2X\u003csub\u003e4\u003c/sub\u003e signal in the IHCs (Figure 6G, arrow). However, there was little co-localization between P2X\u003csub\u003e4\u003c/sub\u003e and TOM20 in both OHCs and IHC (Figure 6H).\u0026nbsp;TOM20 was co-occurred with P2X\u003csub\u003e4\u003c/sub\u003e in the IHCs\u0026nbsp;13.7% \u0026plusmn; 2.5% and in OHCs 11.3% \u0026plusmn; 1.2%. Finally,\u0026nbsp;WGA is naturally occurring molecule known to bind to glycoproteins found in the cell membrane, and fluorescent conjugates are commonly used as a marker for cell membrane\u0026nbsp;(Emde, Heinen, G\u0026ouml;decke, \u0026amp; Bottermann, 2014). The WGA labelled the OHC membrane but did not stain IHC, similar to a previous study\u0026nbsp;(Gil-Loyzaga \u0026amp; Brownell, 1988). Therefore, the association with the IHC membrane was inconclusive and therefore not included in this study. We observed the minimal overlap between WGA and P2X\u003csub\u003e4\u003c/sub\u003e in the OHCs(Figure 6I).\u0026nbsp;WGA was co-localized with P2X\u003csub\u003e4\u003c/sub\u003e in OHCs 4.3% \u0026plusmn; 0.2% (Figure 6K).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, in IHCs, EEA-1 and GM130 have the highest percentage of co-localization with P2X\u003csub\u003e4\u003c/sub\u003e, at 26% and 27%, respectively, compared to other organelle markers, suggesting cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e were likely associated with endosomes and Golgi apparatus. The co-localization pattern in OHCs was slightly different from that with IHCs; EEA-1 and LAMP-1 have a higher percentage of co-localization with P2X\u003csub\u003e4\u003c/sub\u003e at 42.3% and 32.%, respectively. This suggests that P2X\u003csub\u003e4\u003c/sub\u003e associate with endosomes and lysosomes in OHCs (Figure 6 K\u0026amp;L, Table 2).\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we have comprehensively mapped the expression of P2X\u003csub\u003e4\u003c/sub\u003e in the Wistar rat cochlea using immunohistochemistry and reported the expression in the IHCs and OHCs of the Wistar rat cochlea for the first time. The minimal expression of P2X\u003csub\u003e4\u003c/sub\u003e labelling in the stria vascularis was unexpected, as the literature suggested that P2X\u003csub\u003e4\u003c/sub\u003e receptors are expressed in the endothelial cells of spiral ligament capillaries in the lateral wall of the guinea pig cochlea (Y. Wu et al., 2011). This discrepancy might occur because different antibody from Abcam (UK) was used, which is no longer available on the manufacturer website. The expression of P2X\u003csub\u003e4\u003c/sub\u003e in sensory HCs was observed uniformly throughout the apical, middle and basal turn of the cochlea (Supplementary Figure 4). P2X\u003csub\u003e4\u003c/sub\u003e immunolcalisation was observed in a small population of cells in the spiral ligament and the spiral ganglia, however, the frequency was very low in the adult cochlea.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is important to note that the P2X\u003csub\u003e4\u003c/sub\u003e subunit can form heteromeric channels with P2X\u003csub\u003e1, 5, 6, and 7\u003c/sub\u003e subunits. P2X\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eimmunolocalisationin the OoC in our study \u0026nbsp;is distinctively different from what has been reported for P2X\u003csub\u003e1\u003c/sub\u003e (Xiang, Bo, \u0026amp; Burnstock, 1999), P2X\u003csub\u003e7\u0026nbsp;\u003c/sub\u003e(Predrag Nikolic et al., 2003)\u0026nbsp;and P2X\u003csub\u003e2\u003c/sub\u003e (Jarlebark et al., 2000; J. C. C. Wang et al., 2003), which might suggest that P2X\u003csub\u003e4\u003c/sub\u003e has different roles compared to other isoforms. The most intense P2X\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eexpression was observed in the IHCs and OHCs, where P2X\u003csub\u003e4\u003c/sub\u003e was predominately localised in the cytoplasm with distinct polarity in the subcellular distribution of the receptor protein. Using organelle markers, we show that the majority of cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e was co-localized with vesiculated structures, particularly early endosomes and Golgi (trans-Golgi network), which combined represented 53% of the co-localisation for IHCs and 63.2 % for OHCs. Additional association with lysosomes occurred in OHCs only. There was little evidence of P2X\u003csub\u003e4\u003c/sub\u003e expression in the cell plasma membrane.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cytoplasm of IHCs and OHCs are enriched with endosomes\u0026nbsp;(Spicer et al., 1998, 1999).\u0026nbsp;This P2X\u003csub\u003e4\u003c/sub\u003e localisation may represent a pool of P2X\u003csub\u003e4,\u003c/sub\u003e which will become inserted into the membrane under certain conditions, or it reflects continual membrane-cytoplasm cycling. Cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e has been reported in many tissues, including alveolar epithelium, and neurons (Bobanovic, Royle, \u0026amp; Murrel-Lagnoado, 2002; Qureshi, Paramasivam, Yu, \u0026amp; Murrell-Lagnado, 2007; Stokes, Layhadi, Bibic, Dhuna, \u0026amp; Fountain, 2017). In the ocular lens, cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e becomes more associated with the cell membrane under osmotic stress (Suzuki-Kerr, Lim, Vlajkovic, \u0026amp; Donaldson, 2009), supporting the notion that cytoplasmic vesicles containing P2X\u003csub\u003e4\u003c/sub\u003e are dynamic. Interestingly, the distribution of cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e showed polarity within the cell. The apical cell domain of both OHCs and IHCs are in contact with potassium-rich endolymphatic fluid, whereas the basal-lateral domain is in contact with sodium-rich perilymph and has the pre-synaptic clefts for synaptic transmission. A large proportion of cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e immunolabeling in the IHCs occurred on the apical and basal ends, in proximity to the synaptic cleft, which may suggest physiological P2X\u003csub\u003e4\u003c/sub\u003e roles in regulating pre-synaptic function. In addition, the cytoplasmic labelling in the IHCs was concentrated adjacent to the medial side of the lateral membrane. The medial side of IHCs is adjacent to the inner border cells, which abundantly express connexin 26 and 30, and while their primary role is communication between supporting cells as gap junctions, they also exist as connexin hemichannels \u0026nbsp;(Taylor, Jagger, \u0026amp; Forge, 2012; Zhao, Yu, \u0026amp; Fleming, 2005). It is interesting to speculate that P2X\u003csub\u003e4\u003c/sub\u003e may be activated in a paracrine manner by connexin hemichannel-mediated ATP released from the inner border cells. Such a gradient of P2X\u003csub\u003e4\u003c/sub\u003e distribution in the IHCs was not observed in younger animals (P4-P8), suggesting that P2X\u003csub\u003e4\u003c/sub\u003e signalling may be established in mature IHCs. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOHCs are the other type of sensory epithelial cells in the cochlea, however, they have distinct functional role than IHCs as part of the \u0026lsquo;cochlear amplifier\u0026rsquo; by contracting and elongating in response to sound\u0026nbsp;(Pickles, 1998). The robust expression of P2X\u003csub\u003e4\u003c/sub\u003e was found in OHCs mainly in the cytoplasmic space near the apical membrane and also towards the basal membrane. This \u0026nbsp;was less evident in young animals, suggesting critical roles for P2X\u003csub\u003e4\u003c/sub\u003e in more mature OHCs. The large proportion (63.2%) of cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e in OHCs co-occurred with trans-Golgi network similar to the IHCs, and this may represent the dynamic cycling pool of P2X\u003csub\u003e4\u003c/sub\u003e receptors moving to and from the plasma membrane. Given the close proximity of vesicular P2X\u003csub\u003e4\u003c/sub\u003e to the apical and basal membranes, we may speculate ATP released from Deiters cells underneath OHCs which express connexin 26 and 30, proteins capable of forming hemi-channels and gap-junctions (Hosoya et al., 2021; Taylor et al., 2012; Zhao et al., 2005). In addition to the robust basal expression, both qualitative and quantitative analysis showed a robust P2X\u003csub\u003e4\u003c/sub\u003e expression at the apical sub-cuticular level of the OHCs, where they exhibited very characteristic appearance of \u0026ldquo;plaque\u0026rdquo; or \u0026ldquo;cluster\u0026rdquo; of vesicles. These were often found immediately underneath the CP free zone. While only 11.7% of P2X\u003csub\u003e4\u003c/sub\u003e co-localized in IHCs with the lysosome marker, a greater proportion (32.4%) of P2X\u003csub\u003e4\u003c/sub\u003e co-localized with lysosome marker in OHCs, showing differences between IHCs and OHCs. One possibility for lysosomal localization of P2X\u003csub\u003e4\u003c/sub\u003e is a part of protein cycling; the late-endosome will fuse with lysosome during protein degradation, some of which may correspond to P2X\u003csub\u003e4\u003c/sub\u003e receptors trafficking \u003cem\u003een route\u003c/em\u003e for degradation. Alternatively, P2X\u003csub\u003e4\u003c/sub\u003e has been suggested to play a role as a lysosomal ionic channel based on the observation in cultured neurons (R. D. Murrell-Lagnado, 2018; Ruth D. Murrell-Lagnado \u0026amp; Frick, 2019). Lysosomal P2X\u003csub\u003e4\u003c/sub\u003e receptor activation is influenced by pH within the lysosome lumen in cell culture and induces membrane fusion (Cao et al., 2015). In the cochlea, lysosomal dysfunction has been reported to lead to cellular toxicity in OHCs but not in IHCs (Wiwatpanit et al., 2018). It would be interesting to explore the difference in lysosomal physiology between IHCs and OHCs, and how P2X\u003csub\u003e4\u003c/sub\u003e in OHC lysosomes may be involved in such a process. Understanding the role of P2X\u003csub\u003e4\u003c/sub\u003e will require further investigation into the physiological activation of P2X\u003csub\u003e4\u003c/sub\u003e, including pharmacological manipulations. P2X\u003csub\u003e4\u0026nbsp;\u003c/sub\u003ereceptor signalling and its intracellular roles in the cochlea likely contribute to the sensory cell physiology and pathophysiology.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eADP \u0026ndash; adenosine 5\u0026rsquo;-diphosphate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eATP \u0026ndash; adenosine 5\u0026rsquo;-triphosphate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBDNF \u0026ndash; Brain-derived neurotrophic factors\u003c/p\u003e\n\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e \u0026ndash; calcium ion\u0026nbsp;\u003c/p\u003e\n\u003cp\u003edB \u0026ndash; decibels\u003c/p\u003e\n\u003cp\u003eEP \u0026ndash; endocochlear potential\u003c/p\u003e\n\u003cp\u003eIHCs \u0026ndash; inner hair cells\u003c/p\u003e\n\u003cp\u003eJACoP \u0026ndash; Just Another Co-localization Plugin (ImageJ plugin)\u003c/p\u003e\n\u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e \u0026ndash; potassium ion\u003c/p\u003e\n\u003cp\u003ekHz \u0026ndash; Kilohertz\u003c/p\u003e\n\u003cp\u003emM \u0026ndash; micromolar\u003c/p\u003e\n\u003cp\u003eMo \u0026ndash; modiolus\u003c/p\u003e\n\u003cp\u003eOHCs \u0026ndash; outer hair cells\u003c/p\u003e\n\u003cp\u003eOoC \u0026ndash; organ of Corti\u003c/p\u003e\n\u003cp\u003eROI \u0026ndash; Region of interest\u003c/p\u003e\n\u003cp\u003eSGN \u0026ndash; spiral ganglion neuron\u003c/p\u003e\n\u003cp\u003eSNHL \u0026ndash; Sensorineural hearing loss\u003c/p\u003e\n\u003cp\u003eSV \u0026ndash; stria vascularis\u003c/p\u003e\n\u003cp\u003eUDP \u0026ndash; uridine diphosphate\u003c/p\u003e\n\u003cp\u003eUTP \u0026ndash; uridine triphosphate\u003c/p\u003e\n\u003cp\u003ev/v \u0026ndash; volume per volume\u003c/p\u003e\n\u003cp\u003ew/v \u0026ndash; weight per volume\u003c/p\u003e\n\u003cp\u003eWGA \u0026ndash; wheat germ agglutin\u003c/p\u003e\n\u003cp\u003eWHO \u0026ndash; World Health Organization\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eACKKNOWLEDGEMENTS\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Auckland Medical Research Foundation (New Zealand) and Eisdell Moore Centre (New Zealand). Embryonic cochlea issue (E20) was supplied as a by-product from another AEC-approved study conducted by Dr. Rashika Karunasinghe (Department of Physiology, the University of Auckland, AEC 1977). \u003cstrong\u003e\u003cu\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ch2\u003eCONFLICTS OF INTERESTS\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\n\u003cp\u003eHSK conceptualized and designed the study. ZH \u0026nbsp;performed the majority of experiments, followed by HSK performing parts of the experiment. KR contributed to experiments in Figure 2. Technical protocols for optimization of the experiment, image acquisition and image analyses were designed and optimized by ZH, HSK, JMR, and SCY, followed by ZH conducting the data analysis. HSK and ZH wrote the draft of the manuscript. SMV and PRT were co-supervisors to ZH during her postgraduate degree and provided scientific guidance to the manuscript. ZH and HSK prepared figures. All authors have reviewed and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBalaker, A. E., Ishiyama, P., Lopez, I. A., Ishiyama, G., \u0026amp; Ishiyama, A. (2013). Immunocytochemical localization of the translocase of the outer mitochondrial membrane (Tom20) in the human cochlea. \u003cem\u003eThe Anatomical Record, 296\u003c/em\u003e(2), 326-332.\u003c/li\u003e\n\u003cli\u003eBobanovic, L. K., Royle, S. J., \u0026amp; Murrel-Lagnoado, R. D. (2002). P2X Receptor Trafficking in Neurons Is Subunit Specific. \u003cem\u003eThe Journal of Neuroscience, 22\u003c/em\u003e(12), 4814-4824.\u003c/li\u003e\n\u003cli\u003eBolte, S., \u0026amp; Cordeli\u0026egrave;res, F. P. (2006). 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P2X receptor immunoreactivity in the rat cochlea, vestibular ganglion and cochlear nucleus. \u003cem\u003eHear Res, 128\u003c/em\u003e(1), 190-196. https://doi.org/10.1016/S0378-5955(98)00208-1\u003c/li\u003e\n\u003cli\u003eXiong, H., Chen, S., Lai, L., Yang, H., Xu, Y., Pang, J., . . . Zheng, Y. (2019). Modulation of miR-34a/SIRT1 signaling protects cochlear hair cells against oxidative stress and delays age-related hearing loss through coordinated regulation of mitophagy and mitochondrial biogenesis. \u003cem\u003eNeurobiology of Aging, 79\u003c/em\u003e, 30-42.\u003c/li\u003e\n\u003cli\u003eZhao, H.-B., Yu, N., \u0026amp; Fleming, C. R. (2005). Gap junctional hemichannel-mediated ATP release and hearing controls in the inner ear. \u003cem\u003eProceedings of the National Academy of Sciences, 102\u003c/em\u003e(51), 18724-18729.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Antibodies used in this study. Relevant information was gathered from datasheets for each antibody.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"694\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompany/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ecatalogue No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEpitope\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDilution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eReactivity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMarker for\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-P2X\u003csub\u003e4\u003c/sub\u003e,\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRabbit IgG polyclonal\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eAlomone\u003c/p\u003e\n \u003cp\u003eAPR-002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eC-terminus of mouse P2X4 isoform\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eMouse, rat and human\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-SOX2,\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMouse IgG monoclonal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology Inc.\u003c/p\u003e\n \u003cp\u003eSc-365823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eHuman SOX2 amino acids 170-201\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eMouse, rat and human\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003eCochlear supporting cell nucleus\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e(Oesterle\u0026nbsp;et al., 2007)\u003c/p\u003e\n \u003cp\u003e(Smeti\u0026nbsp;et al., 2011)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-Myosin VIIa, Mouse IgG monoclonal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology Inc.\u003c/p\u003e\n \u003cp\u003esc-74516\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eN-terminus of human Myosin VIIa\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eMouse, rat and human\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003eInner\u0026nbsp;hair cells\u003c/p\u003e\n \u003cp\u003e\u0026amp;\u003c/p\u003e\n \u003cp\u003eOuter hair cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e(Xiong et al., 2019)\u003c/p\u003e\n \u003cp\u003e(Jung et al., 2019)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-LAMP-1, Mouse IgG monoclonal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology Inc.\u003c/p\u003e\n \u003cp\u003esc-20011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eAdherent spleen cells of human origin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eMouse, rat and human\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003eLysosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e(Spangenberg et al., 2019)\u003c/p\u003e\n \u003cp\u003e(Oh et al., 2020)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-EEA-1, Mouse IgG monoclonal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology Inc.\u003c/p\u003e\n \u003cp\u003esc-137130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eN-terminus of human EEA1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eMouse, rat, human and monkey\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003eEndosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e(Kuszczyk et al., 2013)\u003c/p\u003e\n \u003cp\u003e(Men et al., 2019)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-GM130, Mouse IgG monoclonal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eBD Biosciences\u003c/p\u003e\n \u003cp\u003e610822\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eRat GM130 aa. 869-982\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eHuman dog and mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003eGolgi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e(Dandoy-Dron et al., 2003)\u003c/p\u003e\n \u003cp\u003e(Zheng et al., 2010)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-TOM20, Mouse IgG monoclonal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology Inc.\u003c/p\u003e\n \u003cp\u003esc-17764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eHuman Tom20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eMouse, rat and human\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003eMitochondria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e(Balaker et al., 2012)\u003c/p\u003e\n \u003cp\u003e(Xiong et al., 2019)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWGA Alexa 647\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConjugate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eThermofisher\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003eWheat\u0026nbsp;Germ Agglutinin (WGA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eAll\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003ePlasma membrane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e(Graveleau et al., 2005)\u003c/p\u003e\n \u003cp\u003e(Cui et al., 2020)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.956834532374101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhalloidin Alexa 488\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.841726618705035%\" valign=\"top\"\u003e\n \u003cp\u003eThermofisher\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.964028776978417%\" valign=\"top\"\u003e\n \u003cp\u003ePhalloidin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.928057553956835%\" valign=\"top\"\u003e\n \u003cp\u003e1:500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.661870503597122%\" valign=\"top\"\u003e\n \u003cp\u003eAll\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.525179856115107%\" valign=\"top\"\u003e\n \u003cp\u003eCytoskeletal actin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.12230215827338%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\n\u003cdiv id=\"_com_1\" language=\"JavaScript\"\u003e\u003c/div\u003e\u003cp\u003eTable 2 is not available with this version.\n\u003c/p\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":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cochlea, sensory hair cell, purinergic signalling, P2X receptor, P2X4, ATP, Organ of Corti","lastPublishedDoi":"10.21203/rs.3.rs-4474581/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4474581/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOur sense of hearing starts in the inner ear organ, the cochlea, which contains two types of auditory hair cells for signal transduction. Earlier research showed that the complex cochlear physiology is regulated in part by purinergic signalling through activations of purine mediated P2X, P2Y and adenosine receptors expressed in the cochlea. This study aims to extend our knowledge of purinergic signalling in the cochlea by comprehensively characterizing the expression of P2X\u003csub\u003e4\u003c/sub\u003e receptor subtype. Wistar rat cochlea (embryonic day 20.5\u0026ndash;6 weeks, both sexes) were collected and the P2X\u003csub\u003e4\u003c/sub\u003e expression was examined by immunohistochemistry. Robust P2X\u003csub\u003e4\u003c/sub\u003e expression was found in the organ of Corti (OoC) in the inner hair cells (IHCs) and outer hair cells (OHCs), confirmed by double-labelling with HCs marker Myosin VIIa. In IHCs, a robust cytoplasmic P2X\u003csub\u003e4\u003c/sub\u003e expression occurred throughout the cell body, with the most intense signal at the medial side. In OHCs, P2X\u003csub\u003e4\u003c/sub\u003e formed puncta near the apical and basal ends of the cell body. Using markers for subcellular organelles, P2X4 immunoreactivity was associated mostly with the trans-Golgi network apparatus (27%) and early endosomes (26%) in IHC, and early endosomes (42.3%) and lysosomes (32.4%) in OHC in the mature cochlea. Taken together, these observations suggest unique roles for P2X\u003csub\u003e4\u003c/sub\u003e in mature IHCs and OHCs as a purinergic receptor subtype responsible for homeostatic regulation of hair cells and auditory sensory transduction.\u003c/p\u003e","manuscriptTitle":"Subcellular Localization of the P2X4 Receptor in Cochlear Sensory Hair Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-06 16:50:57","doi":"10.21203/rs.3.rs-4474581/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-10T00:23:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-09T17:01:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121798070675426145212454988153667835598","date":"2024-08-27T11:26:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-17T13:26:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50215698736584840820138835521888851492","date":"2024-05-27T14:06:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-27T12:36:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144817257377561152194073007094253290725","date":"2024-05-26T19:05:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-26T01:59:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-25T13:29:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-25T09:48:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Histochemistry and Cell Biology","date":"2024-05-24T23:23:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"deb3ee9a-1eea-48d0-97d4-b221bf7bed48","owner":[],"postedDate":"June 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-26T16:01:47+00:00","versionOfRecord":{"articleIdentity":"rs-4474581","link":"https://doi.org/10.1007/s00418-025-02386-1","journal":{"identity":"histochemistry-and-cell-biology","isVorOnly":false,"title":"Histochemistry and Cell Biology"},"publishedOn":"2025-05-20 15:57:48","publishedOnDateReadable":"May 20th, 2025"},"versionCreatedAt":"2024-06-06 16:50:57","video":"","vorDoi":"10.1007/s00418-025-02386-1","vorDoiUrl":"https://doi.org/10.1007/s00418-025-02386-1","workflowStages":[]},"version":"v1","identity":"rs-4474581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4474581","identity":"rs-4474581","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}