Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice

摘要

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca²⁺ transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.Key points class="unordered" style="list-style-type:disc"> Acute inhibition of purinergic receptors with a selective P2X3 antagonist prevents transmission of information from taste buds to sensory nerves. The P2X3 antagonist has no effect on taste-evoked release of ATP, confirming the effect is postsynaptic. The results confirm previous results with P2X2/3 double knockout mice that ATP is required for transmission of all taste qualities, including sour and salty. Previously, ATP was confirmed to be required for bitter, sweet and umami tastes, but was questioned for salty and sour tastes due to pleomorphic deficits in the double knockout mice. The geniculate ganglion in mouse contains two populations of ganglion cells with different subunit composition of P2X2 and P2X3 receptors making them differently susceptible to pharmacological block and, presumably, desensitization. class="head no_bottom_margin" id="__sec2title">IntroductionTaste buds are unique among the special sensory end organs in utilizing ATP as the primary transmitter that links activation of receptor cells to excitation of afferent nerve fibres. Taste stimuli evoke release of ATP from taste receptor cells (Huang et al. 2007; Romanov et al. ; Murata et al. ; Taruno et al. ), which then activates gustatory afferent fibres expressing the ionotropic purinoceptors composed of P2X2 and/or P2X3 subunits (Bo et al. ). The released ATP is then degraded by a specific ectoATPase, NTPDase2, expressed on the membranes of glial-like support cells (type I cells) in the taste bud (Bartel et al. ; Vandenbeuch et al. a).Evidence for the essential role of ATP in taste function is based largely on recordings from mice lacking both P2X2 and P2X3 subunits: P2X2/P2X3 double knockout (DKO) mice (Cockayne et al. ; Finger et al. ). These mice lack gustatory nerve responses to all taste stimuli (Finger et al. ), which suggests that all taste qualities require functional homotrimeric P2X2, P2X3 and/or heterotrimeric P2X2/3 receptors to communicate with nerve fibres. However, to date, ATP release has been detected only from type II taste cells, those that possess the receptors and transduction machinery for sweet, bitter and umami taste stimuli (Huang et al. 2007; Romanov et al. ; Murata et al. ). Several investigators have failed to detect ATP release from type III cells (Huang et al. 2007, b; Murata et al. ), which are required for sour (Huang et al. , b) and possibly salty (Oka et al. ) transduction although these cells do release both serotonin and GABA (Huang et al. href="#b22" rid="b22" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434896915">2009). Hence, it is controversial whether sour and salty stimuli utilize ATP as a taste transmitter, despite the lack of responses to these stimuli in the P2X2/P2X3 DKO mice.Further confounding interpretation of the role of ATP as a taste transmitter are recent experiments on P2X2/P2X3 DKO mice, which show that taste buds in these KOs fail to release ATP normally in response to taste stimuli (Huang et al. href="#b53" rid="b53" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434896896">2011b). Thus, the absence of taste responses may be due to reduced ATP release in addition to the lack of postsynaptic P2X receptors. Such a failure to release ATP could result in pleiotropic developmental defects unrelated to the role of P2X receptors on the afferent fibres. As conditional KOs of P2X2 and P2X3 subunits are not available, we have adopted a pharmacological approach to resolve whether postsynaptic P2X receptors are required for transmission of all taste qualities to the primary afferent fibres innervating the taste buds. We utilize the selective, membrane permeant P2X3 antagonist, AF-353, to block the afferent purinergic receptor responses to ATP chemically rather than genomically. When tested in vitro, AF-353 potently and non-competitively blocks recombinant human P2X purinoceptors that contain P2X3 subunits, i.e. P2X3 homotrimers and P2X2/3 heterotrimers, with respective IC50 values of approximately 3–10 nm and 20–100 nm (Gever et al. href="#b13" rid="b13" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434896854">2010). In the taste system, while 50–60% of the geniculate ganglion neurons express the P2X2 subunit, nearly all of the ganglion cells express the P2X3 subunit (Ishida et al. href="#b27" rid="b27" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434896910">2009). Further, gustatory afferent fibres innervating fungiform papillae from rats and mice apparently co-express both P2X2 and P2X3 subunits (Ishida et al. href="#b27" rid="b27" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434896876">2009) leaving them potentially sensitive to AF-353. Our Ca²⁺ imaging results on isolated geniculate ganglion neurons demonstrate that ATP responses are mediated exclusively by receptors containing P2X2 and P2X3 subunits and confirm the selectivity of AF-353 for P2X3 over P2X2 receptors. Furthermore, we demonstrate that AF-353 has no effect on the taste-evoked release of ATP. Finally, chorda tympani nerve recordings and brief access lickometer assays confirm our earlier studies with P2X2/P2X3 DKO mice and show that all taste qualities are affected in a dose-dependent manner by pharmacological inhibition of the P2X3 and P2X2/3 receptors.