GABA expression in the mammalian taste bud functions as a route of inhibitory cell-to-cell communication

Abstract

Recent advances have underscored cell-to-cell communication as an important component of the operation of taste buds with individual taste receptor cells (TRCs) communicating with one another by means of a number of neurotransmitters and neuropeptides, although functional roles are not yet understood. Here, we characterize the presence, distribution pattern, phenotype, and functional consequences of a previously undescribed inhibitory route within the taste bud mediated by the classic neurotransmitter GABA and its receptors. By using immunocytochemistry, subsets of TRCs within rat taste buds were identified as expressing GABA, and its synthetic enzyme glutamate decarboxylase (GAD). GAD expression was verified with Western blotting. Immunofluorescent studies revealed complex coexpression patterns of GAD with the TRC protein markers gustducin, neural cell adhesion molecule, protein gene product 9.5, and synaptosomal-associated protein of 25 kDa that collectively outline hardwired signaling pathways of GABAergic TRCs. RT-PCR and immunocytochemistry demonstrated that both GABA(A) and GABA(B) receptors are expressed in the taste bud. The later was observed in a subset TRCs paracrine to GAD-expressing TRCs. Physiological effects of GABA were examined by patch clamp recordings. GABA and the GABA(A) agonists muscimol and isoguvacine enhanced isolated chloride currents in a dose-dependent manner. Also, GABA and the GABA(B) agonist baclofen both elicited increases of the inwardly rectifying potassium currents that could be blocked by the GABA(B) receptor antagonist CGP 35348 and the G protein blocker GDP-betaS. Collectively, these data suggest that GABAergic TRCs are able to shape the final chemosensory output of the bud by means of processes of cell-to-cell modulation.

Fig. 1.

TRCs express GABA and its synthetic enzyme GAD. Representative GABA-immunopositive TRCs from FOL (Left) and CV (Center); and GAD65/67-immunopositive TRCs (Right) overlaid on a bright field image from CV are illustrated. (Scale bars, 10 μm.) (Lower) A Western blotting using a GAD 65/67 antibody on proteins extracted from either PT, AT, or positive controls CTX or CE confirm its regional expression in the tongue.

Fig. 2.

TRCs express the α1 subunit of the GABA_A receptor. PCR on cDNA reverse-transcribed from lingual epithelium with primer sets specific to the α1 subunit of the GABA_A receptor or to β-actin demonstrated products of the expected size (511 bp; 132 bp), but not to the α3 subunit (325 bp). Immunocytochemistry using a GABA_A receptor α1 subunit specific antibody demonstrated immunopositive TRCs within the taste bud. (Scale bars, 30 μm.) Western blotting on protein extracted from CTX, CE, CV, FOL, and AT demonstrated the presence of α1 protein in positive control and taste tissue, whereas α3 protein was not detected from taste tissue.

Fig. 3.

The application of exogenous GABA to TRCs results in an enhancement of a chloride current. Whole cell patch clamp data from a representative cell (Upper). Application of GABA reversibly increases the isolated chloride current. This enhancement is concentration dependent (Lower) with a half maximal effect occurring at 16-μM GABA. The number of cells contributing to each point is indicated.

Fig. 4.

The GABA_A receptor agonists muscimol and isoguavacine mimic the effect of GABA on isolated chloride currents. (A) Data from a representative cell demonstrate that the application of muscimol increased the outwardly rectifying portion of isolated chloride current (□), recorded from a dissociated TRC when compared with preapplication magnitude (■). This enhancement was reversible with washout of muscimol from the bathing solution (▣). The enhancement of chloride currents by muscimol was dose-dependent (B). Over 4 tested concentrations, the enhancement increased with increasing concentration to a plateau value of ≈130%. Above each data point, the number of responding cells over the number of tested cells is indicated. Data are mean and SE when compared with pretreatment values. The mean and SE of the pretreatment values are indicated by an open triangle. (C) Isoguavacine similarly enhanced the outwardly rectifying portion of the chloride current (□) when compared with control values (■) in a reversible manner (■). (D) The effect was dose-dependent with a magnitude of effect (≈140%), comparable with that produced by muscimol or GABA. The number of responding cells over the number of tested cells for each tested concentration is indicated.

Fig. 5.

A subset of rat TRCs expressed the GABA_B receptor as demonstrated by RT-PCR and immunocytochemistry. (Top) Immunocytochemistry using an antibody specific to the R1 subunit of the GABA_B receptor demonstrated a subset of immunopositive TRCs in the taste bud. (Middle) PCR on cDNA reverse-transcribed from RNA extracted from lingual epithelium with primer sets specific to either the R1 or R2 subunits of the GABA_B receptor demonstrated products of the expected size (R1 209 bp; R2 466 bp); cDNA reverse-transcribed from CTX served as positive control. (Bottom) Nonoverlapping GAD65/67-immunoreactivity with GABA_B receptor R1 subunit immunoreactivity in rat posterior TRCs. (Scale bars, 30 μm.)

Fig. 6.

Application of exogenous GABA enhanced the magnitude of K_IR in a subset of TRCs. (A) Whole-cell currents from a representative cell demonstrate that control currents (■) were enhanced by application of 1 mM GABA (□). (B) The effect of GABA on K_IR is mimicked by the GABA_B subtype receptor agonist Baclofen. (C) Of the cells that responded to Baclofen (19 n of 59 tested cells), the current was increased to ≈130% of the control (preapplication) values. (D) The response to Baclofen was abolished when cells were pretreated with the GABA_B antagonist CGP 35348. Although slightly >30% of cells were responsive to Baclofen, only 1 cell of 10 tested Baclofen-responding cells responded to Baclofen in the presence of CGP 35348. Similarly, using the G protein antagonist GDP-βS, none of 19 tested baclofen-responding cells responded.