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First published online 28 February 2007
doi: 10.1242/dev.002279

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P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea

¹ Department of Physiology, University of Auckland, Private Bag 92019, Auckland, New Zealand.
² Centre for Auditory Research, University College London, London, UK.
³ Audiology Discipline, University of Auckland, Private Bag 92019, Auckland, New Zealand.
⁴ Department of Physiology, University College Medical School, University College London, London, UK.
⁵ Departments of Surgery, University of California, San Diego, 9500 Gilman Drive, La Jolla, San Diego, CA 92093, USA.
⁶ Departments of Neuroscience, University of California, San Diego, 9500 Gilman Drive, La Jolla, San Diego, CA 92093, USA.

View larger version (50K): [in this window] [in a new window]   Fig. 1. Immature and mature configurations of the afferent innervation of the cochlea by the intrinsic SGN. (Top) In the early postnatal (P3) rat cochlea, type I and type II SGN innervation is mismatched with target hair cells. (Bottom) By the onset of hearing (around P11), several type I SGN neurites (blue) exclusively innervate individual inner hair cells (IHC) with pruning of the synaptic processes to a few puncta. By contrast, the considerably less numerous type II SGN neurites (red) drop their innervation of the IHC and provide extensive en passant innervation of multiple outer hair cells (OHC) through the outer spiral bundles (osb). rf, radial fibers. Spiral ganglion neurite outgrowth is promoted by neurotrophins, particularly BDNF, which is a paracrine factor secreted by the hair cells. Here we provide evidence that extracellular ATP signaling acts through a P2X2/3 heteromeric receptor to inhibit this neurotrophic support.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Specificity of P2X primer and probe sets and standard curve analysis. (A) Real-time PCR amplification plot showing specificity of P2X receptor primer/probe sets with their target templates. Seven successful amplifications are shown, with absence of amplification of mismatched templates (see B). (B) Grid representation of primer and probe specificity showing CT values for on-target cDNA amplification for all seven P2X receptors. Note the absence of non-specific amplification. (C) An example of a standard curve for P2X1 receptor cDNA amplification. Note the linearity to <10 copies. (D) Standard curve analysis of the P2X receptor targets showing linearity of all seven dilution series. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSE, neuron-specific enolase. Copy range used for the standard curves is estimated from the serial dilutions of template cDNAs.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Analysis of P2X receptor expression in neonatal rat SGN. (A) Isolation of a single SGN from a P4 rat cochlear slice using a micropipette. (B) Example of an inward current response to the P2X3 and P2X2/3 receptor agonist ,ßMeATP (100 µM) in a SGN using whole-cell voltage clamp (holding potential -60 mV). (C) Block of the ATP response (100 µM, 5 seconds of focal application) by the P2X2/3 receptor-specific antagonist A-317491 (500 nM, bath superfusion). (D) Real-time PCR amplification plot showing detection of P2X3 cDNA in a sample of individual SGN. (E) Average transcript copy number for each P2X receptor subunit and the housekeeping genes Nse and Gapdh in individual neurons. Note that the P2X3 transcript number was twice that of P2X2 (*P<0.01; P2X3 transcript number was significantly greater than the other P2X subunits; GAPDH transcript copy number was significantly greater than NSE transcript copy number). (F) Relative distribution of P2X receptor subunits in the population of SGN. This plot shows the normalized mRNA transcript copy number for each of the seven candidate P2X receptor subunits expressed by individual SGN. (G) Immunofluorescence labeling of P2X2, P2X3 and P2X4 in neonatal (P4) spiral ganglion tissue.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Variation in expression of P2X2 splice variants in SGN. (A) Schematic of the three P2X2 isoforms expressed in SGN, showing their C-terminal region variation. ECD, extracellular domain; TM, transmembrane domain. (B) End-point single-cell RT-PCR analysis of a sample of six SGN showing the expression of P2X2 isoforms in individual cells (lanes 1-6). Amplicon sizes: P2X2-1=499 bp; P2X2-2=292 bp; P2X2-3=480 bp. P2X2-3 was the most prominent isoform (4/6 cells). The agarose gel also includes two positive controls of cDNA from whole spiral ganglion (lane 9) and whole cochlea (lane 10). Lane 7 (-) control for no template. Lane 8 (-) bath sample processed for RT-PCR. (C) Analysis of P2X2 splice variant combinations detected in the 31 single-cell RT-PCR experiments. P2X2-3 was the dominant isoform either expressed alone or with one or more of the other isoforms (23/31 neurons).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Pharmacology of the recombinant P2X2-3/3 receptor. (A) Injection of P2X2-3 and P2X3 mRNAs in a ratio of 1:2 into Xenopus oocytes resulted in expression of ATP-gated inward currents with a broad sensitivity to ATP agonists. (B) Concentration-response curves for ATP at pH 7.5 show an EC50 of 0.4 µM for these ATP-gated ion channels. Acidification to pH 6.5 produced a leftward shift in the concentration-response curve, to reduce activation thresholds to <10 nM ATP. This is attributable to positive allosteric modulation by protons acting through the P2X2 subunit.

View larger version (117K): [in this window] [in a new window]   Fig. 6. Developmental profile of P2X receptor subunit expression in rat spiral ganglion tissue from P0-P14, determined by end-point RT-PCR. Note the downregulation of the P2X3 transcript by P14.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Purinergic receptor antagonism of BDNF-dependent spiral ganglion neurite development. Cultures of P4 spiral ganglion explants were treated for three days with medium (control) or with addition of BDNF at 1 ng/ml or 10 ng/ml. The effect of P2X receptor activation on BDNF-dependent neurite development was assessed with ATPS (100 µM) and ,ßMeATP (100 µM). (A-F) Immunofluorescence images of spiral ganglion explants labeled for neurofilament. BDNF at 10 ng/ml. Scale bars: 500 µm. (G,H) Experiments comparing BDNF-induced neurite growth with and without ATPS (n=12-20 explants per group). (I,J) Experiments comparing BDNF-induced neurite growth with and without ,ßMeATP (n=12 explants per group). *P<0.05; **P<0.01.

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