Nicotinic Acetylcholine Receptor α9 and α10 Subunits Are Expressed in the Brain of Mice

Abstract

The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are likely to be the evolutionary precursors to the entire cys-loop superfamily of ligand-gated ion channels, which includes acetylcholine, GABA, glycine and serotonin ionotropic receptors. nAChRs containing α9 and α10 subunits are found in the inner ear, dorsal root ganglia and many non-excitable tissues, but their expression in the central nervous system has not been definitely demonstrated. Here we show the presence of both α9 and α10 nAChR subunits in the mouse brain by RT-PCR and immunochemical approaches with a range of nAChR subunit-selective antibodies, which selectivity was demonstrated in the brain preparations of α7-/-, α9-/- and α10-/- mice. The α9 and α10 RNA transcripts were found in medulla oblongata (MO), cerebellum, midbrain (MB), thalamus and putamen (TP), somatosensory cortex (SC), frontal cortex (FC) and hippocampus. High α9-selective signal in ELISA was observed in the FC, SC, MO, TP and hippocampus and α10-selective signal was the highest in MO and FC. The α9 and α10 proteins were found in the brain mitochondria, while their presence on the plasma membrane has not been definitely confirmed The α7-, α9- and α10-selective antibodies stained mainly neurons and hypertrophied astrocytes, but not microglia. The α9- and α10-positive cells formed ordered structures or zones in cerebellum and superior olive (SO) and were randomly distributed among α7-positive cells in the FC; they were found in CA1, CA3 and CA4, but not in CA2 region of the hippocampus. The α9 and α10 subunits were up-regulated in α7-/- mice and both α7 and α9 subunits were down-regulated in α10-/- mice. We conclude that α9 and α10 nAChR subunits are expressed in distinct neurons of the mouse brain and in the brain mitochondria and are compensatory up-regulated in the absence of α7 subunits.

Keywords: RT-PCR; brain; immunohistochemistry; sandwich ELISA; α10 nicotinic acetylcholine receptors; α7; α9.

Figure 1

Sandwich ELISA assays with the brain preparations of the wild type (WT), α7−/−, α9−/− and α10−/− mice demonstrating subunit selectivity of α7(179–190)-, α9(11–23)- and α10(404–417)-specific antibodies. The brain samples were captured with α7(1–208)-specific antibody and revealed with α7(179–190)-, α9(190–200)- or α10(404–417)-specific antibodies. The columns correspond to M ± SE; *p < 0.05, ***p < 0.0005 compared to corresponding OD values of WT mice, n = 5.

Figure 2

Characterization of mitochondria (Mch) and mitochondria-depleted brain fraction (Brain (-Mch)) (A) and subunit composition of nicotinic acetylcholine receptors (nAChRs) in the whole brain, mitochondria or mitochondria-depleted brain fraction of WT and α9−/− mice (B). (A) The samples were studied by Sandwich ELISA using the antibodies against voltage-dependent anion channel (VDAC), lamin B1 or inositol-requiring enzyme-1α (IRE-1α). (B) The samples were captured with α7(1–208)-specific antibody and revealed with α3(181–192)-, α4(181–192), α5(180–191), α7(179–190)-, α9(190–200)-, α10(404–417)-, β2(190–200) or β4(190–200)-specific antibodies. The columns correspond to M ± SE; **p < 0.005, ***p < 0.0005 compared to corresponding OD values of WT mice, n = 5.

Figure 3

Sandwich ELISA (A) and RT-PCR (B) of various brain regions of the WT mice. In (A), the brain samples were captured with α7(1–208)-specific antibody and revealed with α7(179–190)-, α9(190–200)- or α10(404–417)-specific antibodies. Each column corresponds to M ± SE of data obtained from five mice per brain region. MO, medulla oblongata; C, cerebellum; MB, midbrain; TP, thalamus and putamen; SC, somatosensory cortex; FC, frontal cortex; Hip, hippocampus; -RT negative control where no enzyme was used during cDNA synthesis, H₂O, negative control where water was used instead of cDNA; M, marker; bp, base pair.

Figure 4

α9-specific staining in the hippocampal CA3 region of the WT (A,B), α9−/− (C,D) and α7−/− (E,F) mice with either α9-selective antibody (A,C,E) or α-conotoxin PeIA (B,D,F), bar is 100 μm.

Figure 5

Immunochemical staining of the CA2-CA3 regions of hippocampus of the WT mouse. Green—α7-specific, red—α9- or α10-specific staining, blue—DAPI (cell nuclei) with α9- or α10-selective antibody and DAPI (A,B) and with α7- and α9-selective (C,C1,C2) or α7- and α10-selective antibodies (D,D1,D2). (A,B) Bar is 200 μm; (C,D) bar is 100 μm.

Figure 6

α7- (green) and α9- or α10-specific (red) staining in the dentate gyrus (DG, A,B) and superior olive (SO, C,D) of the WT mice, blue—DAPI (cell nuclei). MHB, medial habenula; LSO, lateral superior olive; SPO, superior; VPO, ventral periolivary nuclei; TZ, corpus trapezoid. In (A,B) bar is 100 μm, in (C,D) 200 μm.

Figure 7

The presence of α7 (green), α9 and α10 (red) nAChR subunits in the II/III and V/VI layers of SC (A,B, insert); cortical layers specification according to Ahissar and Staiger, 2010) and cerebellum (C,D). Pcl, Purkinje and granule cell layers; Ml, molecular layer. Yellow—merge of the red and green staining. In (A,C,D) bar is 200 μm, in (B) 100 μm.

Figure 8

The double staining for α7, α9 or α10 nAChR subunits (red) and markers of microglia (Iba1, green, A,C,E) or astrocytes glial fibrillary acidic protein (GFAP, green, B,D,F) in the cerebellum (Crb), cortex (Crtx) or putramen (Put). Abbreviations: WM, white matter; GL, granular layer of cerebellum; V, internal pyramidal layer; VI, multiform layer of cortex. In (A) bar is 20 μm, in (B–F) 50 μm.