Immunocytochemical analysis of P2X2 in rat circumvallate taste buds

Abstract

Background: Our laboratory has shown that classical synapses and synaptic proteins are associated with Type III cells. Yet it is generally accepted that Type II cells transduce bitter, sweet and umami stimuli. No classical synapses, however, have been found associated with Type II cells. Recent studies indicate that the ionotropic purinergic receptors P2X2/P2X3 are present in rodent taste buds. Taste nerve processes express the ionotropic purinergic receptors (P2X2/P2X3). P2X2/P2X3(Dbl-/-) mice are not responsive to sweet, umami and bitter stimuli, and it has been proposed that ATP acts as a neurotransmitter in taste buds. The goal of the present study is to learn more about the nature of purinergic contacts in rat circumvallate taste buds by examining immunoreactivity to antisera directed against the purinergic receptor P2X2.

Results: P2X2-like immunoreactivity is present in intragemmal nerve processes in rat circumvallate taste buds. Intense immunoreactivity can also be seen in the subgemmal nerve plexuses located below the basal lamina. The P2X2 immunoreactive nerve processes also display syntaxin-1-LIR. The immunoreactive nerves are in close contact with the IP(3)R3-LIR Type II cells and syntaxin-1-LIR and/or 5-HT-LIR Type III cells. Taste cell synapses are observed only from Type III taste cells onto P2X2-LIR nerve processes. Unusually large, "atypical" mitochondria in the Type II taste cells are found only at close appositions with P2X2-LIR nerve processes. P2X2 immunogold particles are concentrated at the membranes of nerve processes at close appositions with taste cells.

Conclusions: Based on our immunofluorescence and immunoelectron microscopical studies we believe that both perigemmal and most all intragemmal nerve processes display P2X2-LIR. Moreover, colloidal gold immunoelectron microscopy indicates that P2X2-LIR in nerve processes is concentrated at sites of close apposition with Type II cells. This supports the hypothesis that ATP may be a key neurotransmitter in taste transduction and that Type II cells release ATP, activating P2X2 receptors in nerve processes.

Figure 1

Double-label confocal images showing P2X2- with IP₃R3-LIR in rat circumvallate taste buds. A: P2X2-LIR is present in nerve processes. Intense immunoreactivity can be seen in the nerve plexus near the basal lamina (BL). B: Type II taste cells displaying IP₃R3-LIR. C. Double-label image showing P2X2-LIR nerve processes in close apposition with IP₃R3-LIR taste cells (C1 and C2). C3 indicates an IP₃R3-LIR cell that is enveloped by a P2X2-LIR nerve process. TP: taste pore, Scale bar = 20 μm.

Figure 2

Double-label confocal images showing P2X2- with syntaxin-1- or 5-HT-LIR in rat circumvallate taste buds. A: P2X2-LIR nerve processes extend from basal lamina to taste pore. B: A subset of taste cells (arrowheads) and nerve processes in taste buds displaying syntaxin-1-LIR. C: P2X2-LIR nerve processes also express syntaxin-1 (arrow). D: P2X2-LIR. E: 5-HT is present in a subset of taste cells (arrowheads). No colocalization occurs between P2X2- and 5-HT-LIR in the taste buds, however, P2X2 immunoreactive nerve processes are in close apposition with 5-HT-LIR cells (e.g., rectangular box). TP: taste pore; BL: basal lamina. Scale bars = 20 μm.

Figure 3

A DAB immunoelectron micrograph demonstrates that P2X2-LIR is present in nerve processes in a transverse section of a taste bud. The P2X2 immunoreactive nerve processes (arrows) are in close apposition with Type II (II) and Type III (III) taste cells. Inset: A P2X2-LIR nerve process (N) closely contacts a Type III taste cell (III); its nucleus contains characteristic electron-dense patches of heterochromatin (arrowhead). Scale bar = 2 μm; Scale bar = 0.5 μm in Inset.

Figure 4

A DAB immunoelectron image showing a Type III cell (III) with a synaptic contact (S) onto a P2X2 immunoreactive nerve process (N). Numerous small clear synaptic vesicles (arrowheads) are present in presynaptic zone. Scale bar = 0.5 μm.

Figure 5

DAB immunoelectron micrographs showing Type II cells (TC) with “atypical” mitochondria located only at close appositions with P2X2 immunoreactive nerve processes. A: A P2X2-LIR nerve process (N) is partially enveloped by a large “atypical” mitochondrion (arrow). B: A Type II cell containing two large “atypical” mitochondria (arrows) that are in contact with a P2X2-LIR nerve process (N). m: regular mitochondrion. Scale bars = 0.5 μm.

Figure 6

Immunogold micrographs showing that 6 nm gold particles are found only at the junctions between Type II cells and nerve processes. The colloidal gold particles are marked by transparent red dots. A and B: Gold particles are concentrated near the membrane of nerve processes (N) in close apposition with Type II cells (Nu). m: mitochondria. Scale bars = 0.2 μm.