Functional characterization of alpha9-containing cholinergic nicotinic receptors in the rat adrenal medulla: implication in stress-induced functional plasticity

Abstract

An increase in circulating adrenal catecholamine levels constitutes one of the mechanisms whereby organisms cope with stress. Accordingly, stimulus-secretion coupling within the stressed adrenal medullary tissue undergoes persistent remodeling. In particular, cholinergic synaptic neurotransmission between splanchnic nerve terminals and chromaffin cells is upregulated in stressed rats. Since synaptic transmission is mainly supported by activation of postsynaptic neuronal acetylcholine nicotinic receptors (nAChRs), we focused our study on the role of alpha9-containing nAChRs, which have been recently described in chromaffin cells. Taking advantage of their specific blockade by the alpha-conotoxin RgIA (alpha-RgIA), we unveil novel functional roles for these receptors in the stimulus-secretion coupling of the medulla. First, we show that in rat acute adrenal slices, alpha9-containing nAChRs codistribute with synaptophysin and significantly contribute to EPSCs. Second, we show that these receptors are involved in the tonic inhibitory control exerted by cholinergic activity on gap junctional coupling between chromaffin cells, as evidenced by an increased Lucifer yellow diffusion within the medulla in alpha-RgIA-treated slices. Third, we unexpectedly found that alpha9-containing nAChRs dominantly (>70%) contribute to acetylcholine-induced current in cold-stressed rats, whereas alpha3 nAChRs are the main contributing channels in unstressed animals. Consistently, expression levels of alpha9 nAChR transcript and protein are overexpressed in cold-stressed rats. As a functional relevance, we propose that upregulation of alpha9-containing nAChR channels and ensuing dominant contribution in cholinergic signaling may be one of the mechanisms whereby adrenal medullary tissue appropriately adapts to increased splanchnic nerve electrical discharges occurring in stressful situations.

Figure 1.

Involvement of α9-containing nAChRs in excitatory synaptic neurotransmission between splanchnic nerve endings and chromaffin cells. A, Representative chart recordings of spontaneous excitatory synaptic events recorded in a chromaffin cell voltage-clamped at −80 mV, before (left panel) and 4 min after bath-applied α-RgIA (right panel). α-RgIA (200 nm) induces a decrease in both sEPSC frequency and amplitude. B, Double immunostaining for α9 nAChRs and the presynaptic vesicle protein synaptophysin. As illustrated in the merge picture, labelings codistribute, indicative of a close localization between the two proteins.

Figure 2.

Effect of α9-containing nAChR blockade on sEPSC frequency. A, Histogram illustrating the blocking effect of the toxin α-RgIA on the mean sEPSC frequency calculated in 17 cells. *p < 0.05 (paired t test), compared with control frequency calculated before toxin application. B, Distribution of mean sEPSC frequency in the nine cells in which synaptic events were recorded before, during, and after α-RgIA application. Note that the effect of the toxin is reversible.

Figure 3.

Effect of α9-containing nAChR blockade on sEPSC amplitude. A, Histogram illustrating the blocking effect of the toxin α-RgIA on the mean sEPSC amplitude. A 4–10 min washout is sufficient for a complete recovery. *p < 0.05 (paired t test), compared with control sEPSC amplitude calculated before toxin application. B, Illustration of the sEPSC amplitude variance distribution for each recorded cell. Lines represent the four cells in which the number of sEPSCs recorded before, during, and after toxin application was large enough to calculate the amplitude variance (from 10 to 150 synaptic events). α-RgIA reduced the amplitude variance value. C, Distribution of the cumulative probability plotted from sEPSC amplitudes recorded in one representative cell. Note that the two-modal distribution observed under control condition disappeared in the presence of α-RgIA, indicating a preferential action of the toxin on high amplitude synaptic events.

Figure 4.

Effect of α9-containing nAChR blockade on sEPSC quantal size. Aa, Ab, Representative histograms (bin size of 2 pA) of the distribution of sEPSC amplitudes in a chromaffin cell, recorded in control (a) and in α-RgIA-containing saline (b). Quantal size was estimated from the mean value of the first Gaussian fitted to the amplitude histogram. α-RgIA induced both a switch from a bimodal to a unimodal amplitude distribution and a decrease in sEPSC quantal size (arrows). B, Pooled data from the five cells in which the quantal analysis was performed. As symbolized by the dashed lines, toxin application decreased the mean quantal size by ∼27% (*p < 0.05, paired t test).

Figure 5.

Increased LY diffusion after α9-containing nAChR blockade. A, Time-lapse recordings (one frame per 5 min for 30 min) of LY diffusion in adrenal slices under different experimental conditions. 1* represents the LY-injected cell. Whereas the number of LY-labeled cells does not change in the control slice (top row), it gradually increases after 15–20 min in the α-RgIA-exposed slice (middle row). The increased LY diffusion occurs through a gap junctional pathway, as evidenced by its blockade in the α-RgIA+carbenoxolone-treated slice (bottom row). B, Pooled data. Each histogram bar represents the pooled data obtained from 4 to 31 cells.

Figure 6.

Upregulation of α9 nAChR protein and transcript expression levels in cold-stressed rats. A, Representative immunoblots showing specific detection of α9 nAChR in the adrenal medulla of control and cold-stressed rats (top panel). Actin was used as an internal loading control. Histogram summarizing the densitometric analysis of immunoblots (normalized by actin) from six control and six stressed rats (bottom panel). Quantitative analysis shows that α9 nAChR expression level was significantly increased (1.8-fold) in stressed rats. *p < 0.05 compared with unstressed rats. B, Expression levels of α9-, α3-, and α7 nAChR subunits were determined by quantitative PCR. Total RNA was extracted from macrodissected adrenal medulla from 13 control and 12 cold-stressed rats. Expression of nAChR mRNA was normalized to the geometric mean of the expression levels of Hprt, GAPDH, and Gus mRNA. A significant increase in relative expression level was observed for α9 nAChR but not for α3 nAChR and α7 nAChR transcripts. *p < 0.05, compared with unstressed rats.

Figure 7.

Dominant contribution of α9-containing nAChRs in acetylcholine-evoked current in cold-stressed rats. A, Individual chromaffin cells from control and cold-stressed rats were voltage-clamped at −40 mV and stimulated by a 1 s application of ACh (100 μm). A, Representative ACh-triggered current recorded in the absence of toxin and 4 min after α-RgIA (200 nm), hexamethonium (200 μm), or α-RgIA+hexamethonium applications. Note that the stronger blockade of ACh-evoked current is observed for hexamethonium in control rats and for α-RgIA in cold-stressed rats. B, Histograms illustrating the blockade efficiency of α-RgIA, hexamethonium, and α-RgIA+hexamethonium on ACh current density in control and cold-stressed rats. Note that in each condition, toxins' effects are fully reversible. C, Histogram illustrating the percentage of inhibition of the ACh-evoked inward current by α-RgIA, hexamethonium, and α-RgIA+hexamethonium in control and cold-stressed rats.

Figure 8.

Time course of the blocking effect of α-RgIA and hexamethonium (hexameth) on acetylcholine-evoked current in control and cold-stressed rats. A, B, ACh current density was calculated before and 1, 4, and 5 min after blocker application and after 1 and 5 min washout (wash) in control (A) and cold-stressed (B) rats. ACh-evoked current did not rundown over the 10 min recording (insets). In control rats, the blocking effect of α-RgIA was complete after 1 min, whereas it required at least 4 min in cold-stressed rats. Regarding hexamethonium, the blocking effect was complete within 4 min in control animals and was still under process after 5 min in stressed rats.

Figure 9.

Preferential codistribution of α9-containing nAChRs with synaptophysin in cold-stressed rats. A, B, Synaptophysin and α9-containing nAChRs were detected in control (A) and cold-exposed (B) rats by immunofluorescence. The pictures (pseudocolored in green and red for α9 nAChRs and synaptophysin, respectively) represent the thresholded images to which a binary transformation was applied (see Material and Methods). Original pictures for each staining are shown in insets.