Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats

Abstract

The central nervous system plays an important role in regulating sympathetic outflow and arterial pressure in response to ethanol exposure. However, the underlying neural mechanisms have not been fully understood. In the present study, we tested the hypothesis that injection of ethanol in the central nucleus of the amygdala (CeA) increases sympathetic outflow, which may require the activation of local ionotropic excitatory amino acid receptors. In anesthetized rats, CeA injection of ethanol (0, 0.17, and 1.7 μmol) increased splanchnic sympathetic nerve activity (SSNA), lumbar sympathetic nerve activity (LSNA), and mean arterial pressure (MAP) in a dose-dependent manner. A cocktail containing ethanol (1.7 μmol) and kynurenate (KYN), an ionotropic excitatory amino acid receptor blocker, showed significantly blunted sympathoexcitatory and pressor responses compared with those elicited by CeA-injected ethanol alone (P < 0.01). A cocktail containing ethanol and d-2-amino-5-phosphonovalerate, an N-methyl-d-aspartate (NMDA) receptor antagonist, elicited attenuated sympathoexcitatory and pressor responses that were significantly less than ethanol alone (P < 0.01). In addition, CeA injection of acetate (0.20 μmol, n = 7), an ethanol metabolite, consistently elicited sympathoexcitatory and pressor responses, which were effectively blocked by d-2-amino-5-phosphonovalerate (n = 9, P < 0.05). Inhibition of neuronal activity of the rostral ventrolateral medulla (RVLM) with KYN significantly (P < 0.01) attenuated sympathoexcitatory responses elicited by CeA-injected ethanol. Double labeling of immune fluorescence showed NMDA NR1 receptor expression in CeA neurons projecting to the RVLM. We conclude that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in CeA neurons projecting to the RVLM.

Keywords: N-methyl-d-aspartate; acetate; amygdala; ethanol; glutamate receptors; sympathetic nerve activity.

Fig. 1.

Schematic drawings of the rat amygdala in coronal section. A: the shaded area indicates regions of the central nucleus of the amygdala (CeA) exposed to injected dye (100 nl). B: representative image of a single injection (100 nl) within the CeA. The inset shows a representative immune fluorescent image of retrograde-labeled CeA neurons with axons projecting to the rostral ventrolateral medulla (CeA-RVLM) identified by cholera toxin subunit B (CTB; red) in a separate animal. Scale bar = 1 mm. C: N-methyl-d-aspartate (NMDA) NR1 receptors (green; left) expressed in CeA-RVLM neurons identified by CTB (red; right) as indicated by the arrows (yellow; middle). LaD, lateral nucleus of amygdala; BLA, basolateral nucleus of amygdala; BMA, basomedial nucleus of amygdala; MeA, medial nucleus of amygdala; OPT, optic tract.

Fig. 2.

A: representative traces showing splanchnic sympathetic nerve activity (SSNA), lumber sympathetic nerve activity (LSNA), and arterial blood pressure (ABP) responses to unilateral microinjection of ethanol (0.17 μmol) into the CeA. A 100-nl injection of ethanol (arrow) was completed over a period of ∼1 min. All tracings were recorded in the same animal. Int, integrated. B, left: 2.5-s specimen traces of SSNA (top) and LSNA (bottom) before the injection of ethanol. Right, 2.5-s specimen traces of SSNA (top) and LSNA (bottom) after the microinjection of ethanol.

Fig. 3.

A: representative traces showing SSNA, LSNA, and ABP responses to unilateral microinjection of ethanol (1.7 μmol) into the CeA. A 100-nl injection of ethanol (arrow) was completed over a period of ∼1 min. All tracings were recorded in the same animal. B, left: 2.5-s specimen traces of SSNA (top) and LSNA (bottom) before the injection of ethanol. Right, 2.5-s specimen traces of SSNA (top) and LSNA (bottom) after the microinjection of ethanol.

Fig. 4.

Summary data showing the changes (Δ) in SSNA, LSNA, mean arterial pressure (MAP), and heart rate [HR; in beats/min (bpm)] in response to unilateral microinjections of varying doses of ethanol [0 μmol (saline), 0.17 μmol, and 1.7 μmol] into the CeA. Note that graded concentrations of ethanol elicited a dose-dependent increase in these recorded variables. *P < 0.05 vs. saline; #P < 0.05 vs. 0.17 μmol ethanol.

Fig. 5.

Representative traces showing SSNA, LSNA, and ABP responses to unilateral microinjection of a cocktail containing ethanol (1.7 μmol) and the nonselective ionotropic excitatory amino acid receptor antagonist kynurenate (KYN; 7.2 nmol) into the CeA. A 100-nl injection of the cocktail (arrow) was completed over a period of ∼1 min. All tracings were recorded in the same animal.

Fig. 6.

Representative traces showing SSNA, LSNA, and ABP responses to unilateral microinjection of a cocktail containing ethanol (1.7 μmol) and the NMDA receptor antagonist d-2-amino-5-phosphonovalerate (AP5; 3.0 nmol) into the CeA. A 100-nl injection of cocktail (arrow) was completed over a period of ∼1 min. All tracings were recorded in the same animal.

Fig. 7.

Group data showing SSNA, LSNA, MAP, and HR responses to CeA injections of ethanol (1.7 μmol) alone (n = 8), a cocktail containing ethanol (1.7 μmol) and KYN (n = 7), a cocktail containing ethanol (1.7 μmol) and AP5 (n = 7), and a cocktail containing ethanol (1.7 μmol) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX; n = 9), respectively. Note that sympathoexcitatory and pressor responses to either a cocktail containing ethanol and KYN or a cocktail containing ethanol and AP5 or a cocktail containing ethanol and NBQX were significantly blunted compared with responses to CeA injection with ethanol (1.7 μmol) alone. *P < 0.05 vs. the ethanol-alone group.