Expression of transient receptor potential channels and two-pore potassium channels in subtypes of vagal afferent neurons in rat

Abstract

Vagal afferent neurons relay important information regarding the control of the gastrointestinal system. However, the ionic mechanisms that underlie vagal activation induced by sensory inputs are not completely understood. We postulate that transient receptor potential (TRP) channels and/or two-pore potassium (K2p) channels are targets for activating vagal afferents. In this study we explored the distribution of these channels in vagal afferents by quantitative PCR after a capsaicin treatment to eliminate capsaicin-sensitive neurons, and by single-cell PCR measurements in vagal afferent neurons cultured after retrograde labeling from the stomach or duodenum. We found that TRPC1/3/5/6, TRPV1-4, TRPM8, TRPA1, TWIK2, TRAAK, TREK1, and TASK1/2 were all present in rat nodose ganglia. Both lesion results and single-cell PCR results suggested that TRPA1 and TRPC1 were preferentially expressed in neurons that were either capsaicin sensitive or TRPV1 positive. Expression of TRPM8 varied dynamically after various manipulations, which perhaps explains the disparate results obtained by different investigators. Last, we also examined ion channel distribution with the A-type CCK receptor (CCK-R(A)) and found there was a significant preference for neurons that express TRAAK to also express CCK-R(A), especially in gut-innervating neurons. These findings, combined with findings from prior studies, demonstrated that background conductances such as TRPC1, TRPA1, and TRAAK are indeed differentially distributed in the nodose ganglia, and not only do they segregate with specific markers, but the degree of overlap is also dependent on the innervation target.

Fig. 1.

Detection of transcripts of selected transient receptor potential (TRP) and two-pore potassium (K2p) channels in the nodose ganglia. Electrophoresis clearly revealed amplification products of the predicted size for TRPC1, TRPC3, TRPC5, TRPC6, TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, TRAAK, TREK1, TASK1, TASK2, TRPA1, and TWIK2 from whole nodose ganglia preparations (NG) but not controls (Neg; parallel prepared without added reverse transcriptase).

Fig. 2.

Changes in ion channel expression after systemic capsaicin treatments. TRPV1 (a), TRPA1 (e), TRPC6 (i), and TASK1 (k) exhibited the greatest reduction in relative mRNA abundance after capsaicin. The expression levels in experimental group (Cap) relative to control group (Con) were 30 ± 3.9, 29.9 ± 2.8, 29.2 ± 9.2, and 29.0 ± 4.2%, respectively. Genes that decreased but to a lesser extent included TRPC1 (f), TRPC3 (g), and TASK2 (l), which were reduced to 67.8 ± 0.9, 62 ± 2.8, and 72.6 ± 2.3% of control levels after capsaicin, respectively. In contrast, the relative expression levels of TRPV3 (c), TRPV4 (d), and TRPM8 (j) were increased to 300 ± 104, 262 ± 92, and 331 ± 56% of control levels after capsaicin, respectively. Finally, TRPV2 (b), TRPC5 (h), TREK1 (m), TRAAK (n), and TWIK2 (o) were not significantly altered by the treatment. Symbols over data indicate P values of comparisons (t-tests): *significantly different than control, P < 0.05; #significantly different than control, P < 0.01; †significantly less than control but significantly greater than TRPV1, P < 0.05. The decrease in TREK1 (m) was of borderline significance, P = 0.06.

Fig. 3.

Distribution of TRP channels in the nodose ganglia. a–d: Distribution of TRPA1 (a), TRPC1 (b), TRPC3 (c), and TRPM8 (d) in TRPV1-positive vs. TRPV1-negative vagal afferent neurons determined by single-cell PCR. Black, cells positive for the gene of interest; gray, cells negative for the gene of interest. a: 133 TRPV1-positive neurons tested, 88 TRPA1 positive and 45 negative; 53 TRPV1-negative neurons tested, 11 TRPA1 positive and 42 negative. b: 76 TRPV1-positive neurons tested, 32 TRPC1 positive and 44 negative; 34 TRPV1-negative neurons tested, 7 TRPC1 positive and 27 negative. c: 72 TRPV1-positive neurons tested, 33 TRPC3 positive and 39 negative; 30 TRPV1-negative neurons tested, 14 TRPC3 positive and 16 negative. d: 78 TRPV1-positive neurons tested (pooled data regardless of time in culture): 37 TRPM8 positive and 41 negative; 37 TRPV1-negative neurons tested, 20 TRPM8 positive and 17 negative. e: TRPM8 expression after 5 h and 24 h in culture is shown. At 5 h, 27 of 41 neurons expressed TRPM8; at 24 h, 36 of 82 neurons were TRPM8 positive. f: TRPC1 expression in selected populations of nodose neurons: Control, distribution in randomly selected nodose neurons (15 of 48 positive); Duodenum, distribution in neurons positive for tracer when DiI was injected into the duodenum (22 of 41 positive); Stomach, distribution in neurons positive for tracer when DiI was injected into the stomach (7 of 33 positive). g: Acute upregulation of TRPM8 message (dark gray) observed 3 days postcapsaicin treatment was not maintained at 60 days, whereas TRPV1 message (light gray) was still reduced to ∼30% of control. Symbols over data indicate P values of comparisons (χ² tests except for g, which is a t-test, values compared with control): *P < 0.05; #P < 0.01.

Fig. 4.

Distribution of K2p channels in the nodose ganglia. Single cell PCR did not reveal any preferred expression of TASK-1 (a) or TASK-2 (b) to either TRPV1-positive or TRPV1-negative neurons. Black, cells positive for the gene of interest; gray, cells negative for the gene of interest. a: 94 TRPV1-positive (VI+) neurons tested, 30 TASK1 positive and 64 negative; 33 TRPV1-negative (VI−) neurons tested, 8 TASK1 positive and 25 negative. b: 95 TRPV1-positive neurons tested, 17 TASK2 positive and 78 negative; 32 TRPV1-negative neurons tested, 6 TASK2 positive and 26 negative. c: 102 TRPV1-positive neurons tested, 48 TRAAK positive and 54 negative; 45 TRPV1-negative neurons tested, 8 TRAAK positive and 37 negative. d: A higher percentage of GI-innervating (GI) vagal afferent neurons expressed TRAAK mRNA (30 of 60) than the randomly selected (Ran) neurons (26 of 87). Symbols over data indicate P values of comparisons (χ² tests): *P < 0.05; #P < 0.01.

Fig. 5.

Distribution of CCK A-type receptor (CCK-R_A) in the nodose ganglia and its coexpression with TRAAK. a: CCK-R_A was preferentially expressed by the stomach-innervating vagal afferent neurons (Sto; 34 of 48 positive), compared with randomly selected neurons (Ran; 61 of 118 positive) or duodenal-innervating neurons (Duo; 20 of 36 positive). b: 141 TRPV1-positive neurons tested, 85 CCK-R_A positive and 56 negative; 60 TRPV1-negative neurons tested, 31 CCK-R_A positive and 29 negative. Black, cells positive for the gene of interest; gray, cells negative for the gene of interest. c: TRAAK coexpression with CCK-R_A; 44 of 94 neurons expressing CCK-R_A expressed TRAAK; whereas only 12 of 41 neurons that did not express CCK-R_A expressed TRAAK. d: Segregation of TRAAK with CCK-R_A appeared to be even greater when restricted to neurons projecting to the duodenum: 16 of 26 CCK-R_A-positive neurons coexpressed TRAAK but only 1 of 10 CCK-R_A negative neuron coexpressed TRAAK. Symbols over data indicate P values of comparisons (χ² tests): *P < 0.05; #P < 0.01.