Functional characterization of the α1-adrenoceptor in adult male rat locus coeruleus neurons ex vivo

Abstract

Introduction: The α₁-adrenoceptor (α₁AR) is involved in the physiopathology of the central nervous system (CNS), but its function in the adult male rat locus coeruleus (LC) has not been fully studied. We aimed to characterize the role of the α₁AR in the regulation of the firing rate (FR) of LC neurons and to describe the signaling pathways involved.

Methods: We measured, through single-unit extracellular recordings of LC neurons from adult male rats were used to measure the effect of adrenergic agonists in the presence and absence of adrenergic antagonists or inhibitors of several signalling pathways.

Results: Noradrenaline (NA) (100 µM) and phenylephrine (PE) (100 µM) induced a stimulatory effect in the presence of α₂-adrenoceptor (α₂AR) antagonist RS 79948 (0.1 µM). The α₁AR agonist cirazoline (1-100 µM) also stimulated the FR of LC neurons. The stimulatory effects of NA (100 µM), PE (100 µM), and cirazoline (1 μM and 10 µM) were blocked by α₁AR antagonist WB 4101 (0.5 µM). NA (100 µM)-induced stimulation was reduced in the presence of G_i/o protein inactivator pertussis toxin (PTX) (500 ng ml^-1) and the transient receptor potential (TRP) channel blocker 2-APB (30 µM), but not by protein kinase C (PKC) inhibitor Go 6976 (1 µM), G protein-activated inward rectifier potassium (GIRK) channel blocker BaCl₂ (300 µM), or protein kinase A (PKA) inhibitor H-89 (10 µM). The stimulatory effect of cirazoline was not reduced by any of the tested inhibitors.

Conclusions: From α₁AR activation stimulates the FR of adult rat LC neurons through a signaling pathway that involves G_i/o proteins and TRP channels.

Keywords: cirazoline; firing; locus coeruleus; noradrenaline; phenylephrine; rat; slice; α1-adrenoceptor.

FIGURE 1

Effect of nonselective adrenergic agonist NA before and after the administration of α₂AR antagonist RS 79948 or RS 79948 and α₁AR antagonist WB 4101 on the spontaneous FR of LC neurons. (A,B) Representative examples of recordings of single LC neurons showing the inhibitory effect of NA (100 μM, 1 min) or its stimulatory effect in the presence of α₂AR antagonist RS 79948 (0.1 μM, 10 min) on the basal FR (A) and the blockade of NA-induced effects in the presence of both RS 79948 (0.1 μM, 10 min) and α₁AR antagonist WB 4101 (0.5 μM, 10 min) (B). Vertical lines represent the integrated firing rates (spikes per 10 s). Drugs were bath-applied at the concentrations and for the durations indicated by horizontal bars. (C) Bar histograms showing the mean ± SEM of LC neurons FR before and after the application of NA (100 μM, 1 min, n = 5), NA (100 μM, 1 min) + RS 79948 (0.1 μM, 10 min, n = 5), NA (100 μM, 1 min) + WB 4101 (0.5 μM, 10 min, n = 6), or NA (100 μM, 1 min) + RS 79948 (0.1 μM, 10 min) + WB 4101 (0.5 μM, 10 min, n = 5). *P < 0.05, compared with the FR before the application of NA (100 μM, 1 min) using a paired Student’s t-test. †P < 0.05, compared with the effect (normalized as the percentage change from the baseline FR) induced by NA (100 μM, 1 min) in the absence of WB 4101 (0.5 μM, 10 min) during RS 79948 (0.1 μM, 10 min) perfusion using an unpaired Student’s t-test.

FIGURE 2

Effect of α₁AR agonists PE and cirazoline before and after the administration of α₂AR antagonist RS 79948, α₁AR antagonist WB 4101 or RS 79948 and WB 4101 on the spontaneous FR of LC neurons. (A–D) Representative examples of recordings of single LC neurons showing the inhibitory effect of PE (100 μM, 1 min) on the basal FR (A), the stimulatory effect of cirazoline (1, 10, and 100 μM; 5–10 min) (B), the stimulatory effect of PE (100 μM, 1 min) in the presence of α₂AR antagonist RS 79948 (0.1 μM, 10 min) (C), and the blockade of the stimulatory effect of cirazoline (1, 10 μM, 5–10 min), but not cirazoline (100 μM, 5–10 min), in the presence of α₁AR antagonist WB 4101 (0.5 μM, 10 min) (D). Vertical lines represent the integrated firing rates (spikes per 10 s). Drugs were bath-applied at the concentrations and for the durations indicated by horizontal bars. (E) Bar histograms showing the mean ± SEM of LC neurons FR before and after the application of PE (100 μM, 1 min, n = 5), PE (100 μM, 1 min) + RS 79948 (0.1 μM, 10 min, n = 5), PE (100 μM, 1 min) + WB 4101 (0.5 μM, 10 min, n = 7), or PE (100 μM, 1 min) + RS 79948 (0.1 μM, 10 min) + WB 4101 (0.5 μM, 10 min, n = 5). (F) Symbols representing the mean ± SEM of LC neurons FR before and after the application of cirazoline (1, 10, and 100 μM; 5–10 min; n = 12) or cirazoline (1, 10, and 100 μM; 5–10 min) + WB 4101 (0.5 μM, 10 min, n = 5). *P < 0.05, compared with the FR before the application of PE (100 μM, 1 min) using a paired Student’s t-test or with the FR before the application of cirazoline (1, 10, and 100 μM, 5–10 min) using a repeated-measures ANOVA, followed by Bonferroni’s multiple-comparison post hoc test. †P < 0.05, compared with the effect (normalized as the percentage change from the baseline FR) induced by PE (100 μM, 1 min) in the absence of WB 4101 (0.5 μM, 10 min) during RS 79948 (0.1 μM, 10 min) perfusion using an unpaired Student’s t-test. ‡P < 0.05, compared with the effect of cirazoline in the presence of WB 4101 (0.5 μM, 10 min) using an unpaired Student’s t-test.

FIGURE 3

Effect of NA in the presence of RS 79948 before and after the application of PKC inhibitor Go 6976, GIRK blocker BaCl₂, or PKA inhibitor H-89. (A–C) Representative examples of recordings of single LC neurons showing the stimulatory effect of NA (100 μM, 1 min) in the presence of RS79948 (0.1 μM, 10 min) before and after the application of Go 6976 (1 μM, 30 min) (A), BaCl₂ (B), and H89 (C). Vertical lines represent the integrated firing rates (spikes per 10 s). Drugs were bath-applied at the concentrations and for the durations indicated by the horizontal bars. (D) Bar histograms showing the mean ± SEM of the stimulatory effect (normalized as the percentage change from the baseline FR) of NA (100 μM, 1 min) in the presence of RS 79948 (0.1 μM, 10 min), before and after the application of Go 6976 (1 μM, 30 min, n = 5), BaCl₂ (300 μM, 15 min, n = 5), or H-89 (10 μM, 20 min, n = 5).

FIGURE 4

Effect of NA in the presence of RS 79948 before and after the application of the catalyst of ADP-ribosylation of G_i/o proteins PTX or the TRPC5/M7 channel blocker 2-APB. (A,B) Representative examples of the recordings of single LC neurons showing inhibitory effects of ME (0.8 μM, 1 min) and NA (100 μM, 1 min), and effect of NA (100 μM, 1 min) in the presence of RS 79948 (0.1 μM, 10 min), in a slice treated with PTX (500 ng ml^-1, 18 h) (A), or the effect of NA (100 μM, 1 min) in the presence of RS 79948 (0.1 μM, 10 min) before and after the application of 2-APB (3, 10, 30 μM, 10 min) (B). Vertical lines represent the integrated firing rates (spikes per 10 s). Drugs (except PTX) were bath-applied at the concentrations and for the durations indicated by horizontal bars. (C) Bar histograms showing the mean ± SEM of the stimulatory effect of NA (100 μM, 1 min) in the presence of RS 79948 (0.1 μM, 10 min) in slices treated with PTX (500 ng ml^-1, 18 h, n = 5) or its vehicle (n = 5). (D) Symbols representing the mean ± SEM of the stimulatory effect (normalized as the percentage change from the baseline FR) of NA (100 μM, 1 min) in the presence of RS 79948 (0.1 μM, 10 min), before and after the application of 2-APB (3, 10, and 30 μM; 10 min). *P < 0.05, compared with the stimulatory effect of NA (100 μM, 1 min) during RS 79948 (0.1 μM, 10 min) perfusion in slices that were not treated with PTX using an unpaired Student’s t-test. *P < 0.05, compared with the stimulatory effect of NA (100 μM, 1 min) during RS 79948 (0.1 μM, 10 min) perfusion before the application of 2-APB (3, 10, and 30 μM; 10 min) using a repeated-measures ANOVA, followed by Bonferroni’s multiple-comparison post hoc test.

FIGURE 5

Effect of cirazoline in the absence or presence of PTX, 2-APB, Go 6976, chelerythrine, H-89, and U73122. (A,B) Representative examples of the recordings of single LC neurons showing the stimulatory effect of cirazoline (10 μM, 5–10 min) in a slice treated with PTX (500 ng ml^-1, 18 h) (A) or in the presence of 2-APB (30 μM, 10 min) (B), Go 6976 (1 μM, 30 min) (C), chelerythrine (10 μM, 30 min) (D), H-89 (10 μM, 20 min) (E), or U73122 (10 μM, 30 min) (F). Vertical lines represent the integrated firing rates (spikes per 10 s). Drugs (except PTX) were bath-applied at the concentrations and for the durations indicated by horizontal bars. (G) Bar histograms showing the mean ± SEM of the stimulatory effect of cirazoline (normalized as the percentage change from the baseline FR) in the absence (control) and presence of PTX (500 ng ml^-1, 18 h), 2-APB (30 μM, 10 min), Go 6976 (1 μM, 30 min), chelerythrine (10 μM, 30 min), H-89 (10 μM, 20 min), and U73122 (10 μM, 30 min). *P < 0.05, compared with the stimulatory effect of cirazoline (10 μM, 5–10 min) in the presence of the vehicle using one-way ANOVA, followed by the Dunnett post hoc test.