Table 1.
Primers used to generate in situ hybridization probes.
Fig 1.
In situ hybridization of Chrnb2 digoxigenin labeled anti-sense RNA probe in FF and CV taste bud cells.
(FF) Shows images of a WT mouse FF taste papilla section labelled with Alexa Fluor® 488 (FITC), DAPI, the merged image of DAPI and FITC, and the DIC image. The anti-sense (AS) Chrnb2 riboprobe labeled a subset of FF taste bud cells. No significant fluorescence was observed when the sense probe (S) was used. (CV) Shows low magnification images of a WT mouse CV taste papilla section labelled with Alexa Fluor® 488 (FITC), DIC image, and the merged image of FITC and the DIC image. The anti-sense (AS) Chrnb2 riboprobe labeled a subset of WT mouse CV taste bud cells. No significant fluorescence was observed when the sense (S) probe was used. Bar = 10 μm.
Fig 2.
In situ hybridization of Chrnb4 digoxigenin labeled anti-sense RNA probe in WT mouse CV taste bud cells.
Shows merged images of a WT mouse FF taste papilla section labelled with Alexa Fluor® 488 (FITC) and the DIC image. The anti-sense (AS) riboprobe Chrnb4 labeled a subset of CV taste bud cells (A) and FF taste bud cells (B). No significant fluorescence was observed when the sense probe (S) was used in a FF taste papilla section (C).
Fig 3.
In situ hybridization of Chrna7 digoxigenin labeled anti-sense RNA probe in rat CV taste papillae sections.
Shows images of rat CV papilla sections labelled with Alexa Fluor® 488 (FITC), DAPI, the merged images of FITC and DAPI, and the DIC image. The anti-sense (AS) Chrna7 riboprobe labeled a subset of rat CV taste bud cells (AS). No significant fluorescence was observed when the sense (S) probe was used. Bar = 10 μm.
Fig 4.
In situ hybridization of Trpm5 digoxigenin labeled anti-sense RNA probe in rat CV taste papillae sections.
Shows low magnification images of rat CV papilla sections labelled with Alexa Fluor® 488 (FITC), DAPI, the merged images of FITC and DAPI, and the DIC image. The anti-sense (AS) Trpm5 riboprobe labeled a subset of rat CV taste bud cells (AS). No significant fluorescence was observed when the sense (S) probe was used. Bar = 10 μm.
Fig 5.
Immunostaining of β2 nAChR in WT mouse CV taste bud cells.
The images are merged images of DIC, DAPI, and secondary antibody fluorescence (Alexa Fluor® 488). (A) A low magnification image of mouse CV taste papilla section that shows preferential binding of the nAChR β2 antibody to the apical pole of CV taste bud cells (red arrows). (B) and (C) High resolution images of the CV taste bud cells showing preferential binding of the antibody to the apical pole of the TRCs. Some binding was also observed in the intracellular/basal compartment of TRCs. (D) No antibody binding was observed when the primary antibody step was omitted (NC). Horizontal bars = 10 μm.
Fig 6.
Immunostaining of α4 nAChR in WT mouse CV and FF taste bud cells.
(A) Secondary antibody fluorescence (Alexa Fluor® 488; FITC), (B) DIC image, (C) DAPI, and (D) Merged images of DIC, DAPI and FITC. Panel (P1) shows a low magnification image of a WT mouse CV section. Only a subset of CV taste bud cells demonstrated specific binding to nAChR α4 antibody. Panels (P2) and (P3) show high magnification images of a WT mouse FF taste papillae section. Again, only a subset of FF taste bud cells showed binding to nAChR α4 antibody mainly in the basolateral/intracellular region of TRCs. Panels (P4) and (P5) show that no antibody binding was observed when the primary antibody step was omitted (NC) or in tissue sections from α4 KO mouse (KO), respectively. Horizontal bars = 10 μm.
Table 2.
Fraction of CV TRCs labelled with nAChR antibodies.
Fig 7.
Immunostaining of α3 nAChR in WT mouse CV taste bud cells.
The images are overlay of DIC image and secondary antibody fluorescence (Alexa Fluor® 488; FITC). Only a subset of CV taste bud cells showed the binding of nAChR α3 antibody (A and B). No antibody binding was observed when the primary antibody step was omitted (C; NC).
Fig 8.
Immunostaining of α7 nAChR in WT mouse FF taste bud cells.
(A) DIC image (B) DAPI; (C) secondary antibody fluorescence (Alexa Fluor® 488; FITC) and (D) Merged image of DAPI and FITC. In panels (P1) and (P2) only a subset of TRCs within the FF taste buds showed binding to the nAChR α7 antibody. In panel (P2) nAChR α7 antibody binding was also observed in the apical pole of TRCs. In panels (P3) and (P4) no antibody binding was observed when the primary antibody step was omitted (NC) or in tissue sections from α7 KO mouse (KO), respectively. Horizontal bars = 10 μm.
Fig 9.
Immunostaining of α3 nAChR and β4 nAChR in CV taste bud cells from Trpm5-GFP transgenic mice.
Panel (P1) shows images of Trpm5-GFP cells (green); nAChR α3 binding (using Cyamine 5 Amplification Reagent and secondary donkey//rabbit antibody), and the merged images of Trpm5-GFP and the 590 red-fluorescent dye. A subset of Trpm5-GFP cells demonstrate the binding of nAChR α3 antibody. Panel (P2) shows images of Trpm5-GFP cells (green); nAChR β4 binding (using Cyamine 5 Amplification Reagent and secondary donkey/goat antibody), and the merged images of Trpm5-GFP and the 590 red-fluorescent dye. A subset of Trpm5-GFP cells demonstrate the binding of nAChR β4 antibody. Bar = 10 μm.
Fig 10.
Immunostaining of α7 nAChR in CV taste bud cells from Trpm5-GFP transgenic mice.
Shows merged images of Trpm5-GFP cells (green); nAChR α7 binding (using Cyamine 5 Amplification Reagent and secondary donkey//rabbit antibody) and DAPI (blue). A subset of Trpm5-GFP cells demonstrate the binding of nAChR α7 antibody.
Fig 11.
Immunostaining of α4 nAChR in CV taste bud cells from Trpm5-GFP transgenic mice.
Shows merged images of Trpm5-GFP cells (green); nAChR α4 binding (using Cyamine 5 Amplification Reagent and secondary donkey/goat antibody), DAPI (blue), and the DIC image. A subset of Trpm5-GFP cells demonstrate the binding of nAChR α4 antibody.
Fig 12.
Relative distribution of nAChRs in TRPM5-GFP CV taste bud cells.
Shows the percent distribution of nAChRs in TRPM5-GFP cells in various CV taste papillae sections as a Venn diagram.
Fig 13.
Immunostaining of α3 nAChR in enteroendocrine cells in the Trpm5-GFP mouse gut.
Shows DIC image, DAPI, Trpm5-GFP, α3 nAChR antibody binding (secondary Donkey/Rabbit-590 antibody), and the merged images of DAPI, GFP and 590 red-fluorescent dye. In each of the 8 slides from 3 different gut sections examined 1 to 4 enteroendocrine cells were positive for α3 nAChR in the crypts (P1, P2, and P3; red). The α3 nAChR-positive cells were also positive for Trpm5-GFP (green). Negative control (NC) without primary antibody (P4).
Fig 14.
Effect of nicotine (Nic) and ethanol (ETOH) exposure on the nAChR mRNA expression levels in CV taste bud cells in WT mice.
(A) Relative to control WT mice (H2O), in WT mice exposed to Nic (100 μg/ml) for 3 weeks, the p values for fold change in chrna3, chrna4, chrna5, chrna6, chrnb2 and chrnb4 mRNAs were 0.0292, 0.0018, 0.0028, 0.0026, 0.0049, and 0.0016, respectively. (B) Relative to control WT mice (H2O) mice, in WT mice exposed to ETOH (5%) for 3 weeks p values for chrna3, chrna4, chrna5, chrna6, chrnb2 and chrnb4 were 0.0001, 0.0001, 0.0003, 0.0194, 0.0002, and 0.0253, respectively. The values are mean ± SEM of triplicate runs.
Fig 15.
Detection of qRT-PCR α7 product by gel electrophoresis.
Following qRT-PCR, the final qRT-PCR product was loaded onto 2% agarose gel to detect the presence of the amplified product.