Am J Physiol Heart Circ Physiol 307: H701–H709, 2014. First published July 3, 2014; doi:10.1152/ajpheart.00005.2014.

Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats Andrew D. Chapp,1* Le Gui,1,2* Michael J. Huber,1 Jinling Liu,2 Robert A. Larson,1 Jianhua Zhu,2 Jason R. Carter,1 and Qing-Hui Chen1 1

Department of Kinesiology and Integrative Physiology, Michigan Technological University, Houghton, Michigan; and 2Department of Cardiology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China Submitted 3 January 2014; accepted in final form 1 July 2014

Chapp AD, Gui L, Huber MJ, Liu J, Larson RA, Zhu J, Carter JR, Chen QH. Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats. Am J Physiol Heart Circ Physiol 307: H701–H709, 2014. First published July 3, 2014; doi:10.1152/ajpheart.00005.2014.—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 ␮mol) 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 ␮mol) 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 ␮mol, 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. amygdala; acetate; ethanol; glutamate receptors; sympathetic nerve activity; N-methyl-D-aspartate

is able to cause an increase in arterial pressure (AP) and/or sympathetic outflow in both humans (8, 19, 21, 26, 37, 59) and animals (9, 38, 39, 42, 57). Increased secretion of arginine vasopressin (38) and corticotrophin-releasing hormone (37), as well as enhanced vascular reactivity to the vasoconstrictor agent phenylephrine (39, 57), may underlie the mechanisms responsible for the alcohol-induced increased in AP. Although these studies have suggested that sympathetic activation could contribute to the elevated AP in

ALCOHOL CONSUMPTION

* A. D. Chapp and L. Gui contributed equally to this work. Address for reprint requests and other correspondence: Q.-H. Chen, Dept. of Kinesiology and Integrative Physiology, Michigan Technological Univ., SDC, 1400 Townsend Dr., Houghton, MI 49931 (e-mail: [email protected]). http://www.ajpheart.org

response to alcohol consumption, the central mechanisms responsible for alcohol-induced sympathetic activation have yet to be explored. The amygdala is composed of several groups of neurons that integrate sensory and cognitive information to initiate behavioral and physiological responses. Among these groups of neurons, the central nucleus of the amygdala (CeA) plays a pivotal role in regulating cardiovascular function in response to many stressful experiences. Although ethanol can cross the blood-brain barrier and elicit effects globally within the brain, the CeA could be one of the key areas involved in cardiovascular responses to ethanol exposure. First, studies (2, 29, 35) have demonstrated that ethanol administration increases c-Fos expression in the CeA nucleus in both rats and mice, indicating that CeA neurons are activated with ethanol consumption. Second, other studies have reported that chemical lesions of the CeA in spontaneous hypertensive rats delay the development of hypertension (48) and attenuate cardiovascular responses to acute stress in rats with borderline hypertension (47). Most importantly, anatomic and electrophysiological studies (6, 17, 45, 46) have identified CeA neurons that have axons projecting to presympathetic neurons in the rostral ventrolateral medulla (RVLM). The RVLM provides a key source of excitatory drive to sympathetic preganglionic neurons in the spinal cord regulating the sympathetic nervous system; therefore, it is reasonable to speculate that CeA is one of the key nuclei that plays an important role in the control of sympathetic outflow and AP in response to ethanol challenge. A significant population of CeA neurons with axons projecting to the RVLM (CeA-RVLM neurons) are glutamatergic (53). Because chronic and acute alcohol intake increase sympathetic outflow and alcohol is able to freely cross the bloodbrain barrier (48a) to access the CeA, we tested the hypothesis that acute injection of ethanol into the CeA causes sympathoexcitation and increases AP via CeA-RVLM projections. Moreover, the CeA is innervated by several excitatory transmitters (22, 50), but among the most prominent is L-glutamate. Not only does the CeA contain an abundance of glutamatergic nerve terminals (41), evidence also indicates that neurons in the CeA express ionotropic excitatory receptors: the ␣-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the N-methyl-D-aspartate (NMDA) receptor (5, 43, 49, 51). Given the evidence that local CeA administration of glutamate increases AP (27) and alcohol consumption could activate NMDA receptors expressed in the CeA nucleus in vivo (40, 49), we further hypothesized that sympathoexcitatory and pressor responses evoked by CeA injection of ethanol may involve the activation of local NMDA receptors. A portion of these results has been previously reported in abstract form (13).

0363-6135/14 Copyright © 2014 the American Physiological Society

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METHODS

Experiments were performed in 90 male Sprague-Dawley rats (350 – 450 g) anesthetized with an intraperitoneal injection containing a mixture of ␣-chloralose (80 mg/kg) and urethane (800 mg/kg). An adequate depth of anesthesia was assessed before surgery by the absence of pedal and corneal reflexes and by failure to withdraw the hindlimb in response to a paw pinch. Animals were instrumented with an arterial catheter inserted into the aorta through a femoral artery. The catheter was connected to a pressure transducer to measure AP. Heart rate (HR) was obtained from the R-wave of the electrocardiogram (lead I). A catheter was also placed in the left femoral vein to administer drugs. After tracheal cannulation, rats were paralyzed with gallamine triethiodide (25 mg·kg⫺1·h⫺1 iv) and artificially ventilated with oxygen-enriched room air. After paralysis, anesthesia was monitored by the stability of AP and HR, and supplements equal to 10% of the initial dose were given when needed. End-tidal PCO2 was continuously monitored and maintained within normal limits (35– 40 mmHg) by adjusting ventilation rate (80 –100 breaths/min) and/or tidal volume (2.0 –3.0 ml). Body temperature was held at 37°C with a water-circulating pad. All experimental and surgical procedures were approved by the Institutional Animal Care and Use Committee of Michigan Technological University. Recording of sympathetic nerve activity. Using a flank incision, a left lumbar and a postganglionic splanchnic sympathetic nerve were isolated from the surrounding tissue (52), mounted on a stainless steel wire electrode (0.127-mm outer diameter, A-M Systems), and covered with silicon-based impression material (Coltene, Light Body) to insulate the recording from body fluids. The recorded signal was directed to an alternating current amplifier (P511, Grass Technologies) equipped with half-amplitude filters (band pass, 100 –1,000 Hz) and a 60-Hz notch filter. The processed signal was rectified, integrated (10-ms time constant), and digitized at a frequency of 5,000 Hz using a 1401 Micro3 analog-to-digital converter and Spike 2 software (7.04 version, Cambridge Electronic Design, Cambridge, UK). Background noise was determined by a bolus injection of hexamethonium (30 mg/kg iv), a ganglionic blocker, at the end of the experiment and was subtracted from all integrated values of sympathetic nerve activity (SNA). Microinjection of drugs. Animals were placed in a stereotaxic head frame, and the skull was leveled between the bregma and lambda. A section of the skull was removed so that a single-barreled glass microinjector pipette could be lowered vertically into the CeA and RVLM. The following stereotaxic coordinates were used: CeA, caudal to the bregma, ⫺2.4 ⬃ ⫺2.5 mm; lateral to the midline, 4.8⬃5.0 mm; and ventral to the dura, 7.7⬃8.0 mm; RVLM, caudal to the bregma, ⫺12.5 ⬃ ⫺12.9 mm; lateral to the midline, 1.7⬃1.9 mm; and ventral to the dura, 9.0⬃9.2 mm. It should be noted that we have previously reported that L-glutamate injected into the RVLM using the above stereotaxic coordinates evokes a maximal pressor and sympathoexciatory response (12). Injected compounds were purchased from Sigma, including ethanol, acetate, the nonselective ionotropic excitatory amino acid (EAA) receptor antagonist kynurenate (KYN), non-NMDA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX), and the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (AP5). Experimental protocols. Animals were allowed to stabilize for at least 2 h after surgery. Different dose responses to CeA injection of ethanol [0 ␮mol (saline), 0.17 ␮mol, and 1.7 ␮mol] were determined in separate animals. Changes in mean AP (MAP), HR, splanchnic SNA (SSNA), and lumbar SNA (LSNA) were recorded in response to each injection. To determine the effects of KYN, AP5, and NBQX on sympathoexcitatory and pressor responses elicited by CeA injection of ethanol (1.7 ␮mol), a cocktail containing ethanol (1.7 ␮mol) and KYN (7.2 nmol), ethanol (1.7 ␮mol) and AP5 (3.0 nmol), or ethanol (1.7 ␮mol) and NBQX (1.3 nmol) was injected into the CeA, and responses were tested in separate animals. To test whether the ethanol

metabolite acetate elicited a sympathoexcitatory response and whether activation of NMDA receptors was involved in these responses, the effects of CeA injection of acetate (0.20 ␮mol) and a cocktail containing acetate (0.20 ␮mol) and AP5 (3.0 nmol) on SSNA, LSNA, and MAP were determined in separate groups of animals. It should be noted that the reasons for using microinjection of a cocktail in the present study as opposed to multiple microinjections to test the effect of glutamate receptor antagonist on the sympathoexcitatory response evoked by ethanol were as follows. First, the cocktail containing ethanol-acetate and the glutamate receptor antagonist was a homogenous mixture and, upon injection, would diffuse over the same area. Two injections, the first ethanol injection followed by another injection, or pretreatment of the glutamate receptor antagonist could not guarantee the exact same overlap. Second, multiple microinjections would increase the mechanical damage to the CeA and surrounding brain tissue. In addition, the effects of KYN, AP5, and NBQX on baseline MAP and SNA were examined in separate animals. The doses of KYN, AP5, and NBQX used in the present study have been previously confirmed to effectively block EAA, NMDA, and nonNMDA receptors, respectively, by our previous study (14). Compounds were microinjected into the CeA unilaterally. Ethanol (1.7 ␮mol) was injected intravenously (100 nl) from the femoral vein to assess the extent to which evoked responses were influenced by peripheral actions. Furthermore, experiments were performed to ensure that the effects of ethanol were due to an action within the CeA by testing effects of ethanol microinjected outside the CeA (⬃6.5 lateral to the midline, anatomic control) on MAP, HR, and SNA responses. Finally, to determine whether inhibition of RVLM neurons is able to effectively block sympathoexcitatory responses elicited by CeA-injected ethanol, the effect of CeA-injected ethanol (1.7 ␮mol) on SNA and AP was determined ⬃10 min after ipsilateral RVLM administration of KYN (7.2 nmol). All compounds were microinjected in a volume of 100 nl with a pneumatic pump (WPI). The volume of each injection was determined by measuring the movement of the fluid meniscus within the microinjector pipette using a dissecting microscope equipped with an eyepiece reticle. Histology. At the end of each experiment, Chicago blue dye (100 nl) solution (2% in saline) was injected into the CeA to mark the site of each injection. Rats were then perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in PBS. Brains were removed and postfixed for 4 h at room temperature in 4% paraformaldehyde. Brains were then transferred to 30% sucrose-PBS for 24 h. The CeA was then cut into 40-␮m-thick coronal sections, and microinjection sites were identified under bright-field microscopy. Retrograde labeling of CeA-RVLM neurons. CeA neurons were retrograde labeled from the RVLM as previously described (11, 12). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and placed in a stereotaxic frame, and a small hole was made to expose the cerebellum. A glass micropipette was lowered into the pressor region of the RVLM (coordinates: ⫺12.9 mm caudal to the bregma, 1.9 mm lateral to the midline, and 9.2 mm below the skull) as previously described (11, 12), and a 0.5% solution of cholera toxin B subunit (CTB; List Biologicals, Campbell, CA) dissolved in PBS was microinjected in a volume of 50 nl. The micropipette was left undisturbed for 10 min to avoid diffusion of the tracer along the pipette track. Each animal received a daily injection of the antibiotic penicillin G (300,000 U/kg sc) and the analgesic Metacam (1 mg/kg sc) for 3 days to prevent postoperative infection and reduce pain, respectively. Immunohistochemistry. After 5–7 days after RVLM injection of retrograde tracers, rats were deeply anesthetized with pentobarbital sodium (50 mg/kg ip) and perfused transcardially with 20 ml heparinized saline followed by 150 –200 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were removed and cryoprotected overnight in 30% sucrose in PBS at 4°C. Brain blocks were frozen on dry ice, and serial sections at 30 ␮m thicknesses were cut on a cryostat and collected in 0.01 M PBS. Sections were washed (3 ⫻ 10 min) in 0.01

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M PBS followed by an incubation in 1% BSA (containing 0.3% Triton X-100 in PBS) for 30 min to prevent nonspecific conjugate binding. Sections were incubated with monoclonal mouse anti-NMDA NR1 selective antibody (1:800, BD Bioscience) for 2 h at 37°C and then for 14 h at 4°C. Sections were then washed in PBS for 30 min and incubated with secondary green fluorescent protein-conjugated goat anti-mouse IgG (Invitrogen) for 2 h at room temperature. All sections were then observed and analyzed with the fluorescence microscope. Data analysis. SSNA and LSNA were determined as an average of the rectified, integrated signal. Baseline values of all recorded variables were obtained by averaging a 10-min segment of data recorded immediately before each treatment. MAP, HR, and SNA responses to CeA-injected ethanol, acetate, a cocktail containing ethanol and KYN, a cocktail containing ethanol and AP5, a cocktail containing ethanol and NBQX, a cocktail containing acetate and AP5, anatomy control, vehicle control (saline), and intravenously injected ethanol were measured by averaging a 2-min period centered on the plateau responses. The 2-min period of responses to injected compounds was recorded ⬃60 min after the start of injection. Responses to each compound injected were compared by repeatedmeasures ANOVA. For analyses that yielded a significant difference, pairwise comparisons were made using Bonferroni multiple-comparison tests. All values in the text and figures are expressed as means ⫾ SE. Differences were considered statistically significant at a critical value of P ⬍ 0.05. RESULTS

Histological analysis. Histological examination of brain sections showed that tracings of the outermost distribution of dye were made by overlying areas from similar rostral-caudal sections taken from different brains (Fig. 1A). Areas shown are larger than the dye distribution observed in any single brain but represent the widest possible distribution of injected dye for the entire group of ethanol-treated animals. Figure 1B shows a representative of a single injection tracing within the CeA (100 nl of 2% Chicago blue dye). The inset in Fig. 1B shows a representative immunofluorescent image of CeA-RVLM retrograde-labeled neurons identified by CTB in a separate animal.

Anatomic control injections were located lateral (⬃6.5 mm) to the midline (data not shown) and did not significantly invade lateral portions of the CeA. Effects of CeA-injected ethanol on SSNA, LSNA, MAP, and HR. Unilateral CeA microinjection of ethanol (100 nl) consistently increased SSNA, RSNA, and MAP. Figure 2 shows an example of the responses to the lower dose of ethanol (0.17 ␮mol/100 nl) injected into the CeA. Figure 3 shows an example of the response to the higher dose of ethanol (1.7 ␮mol/100 nl) injected into the CeA. Note that this larger dose of ethanol caused sympathoexcitatory and pressor responses. Sympathoexcitatory responses to CeA injection of 1.7 ␮mol ethanol (Fig. 3) began within 4 – 6 min, reached a plateau within 30 – 60 min, and remained at an elevated steady state throughout the 90-min experimental period. Figure 4 shows summary data. In these experiments, graded increases in the doses of CeA-injected ethanol (0.0 ␮mol, n ⫽ 5; 0.17 ␮mol, n ⫽ 6; and 1.7 ␮mol, n ⫽ 8) produced corresponding and significant (P ⬍ 0.05⬃0.01) increases in MAP, SSNA, and LSNA, respectively. Maximal effects occurred in response to CeA injection of 1.7 ␮mol ethanol. No significant change in HR was observed at any dose of ethanol. Ethanol effects appeared to be site-specific since microinjections of ethanol (1.7 ␮mol/100 nl, n ⫽ 5) placed outside of the CeA (⬃6.5 mm lateral to the midline) failed to significantly change SSNA (⫹3.5 ⫾ 4.3%, P ⬎ 0.05), LSNA (⫹1.2 ⫾ 1.6%, P ⬎ 0.05), MAP (⫹0.2 ⫾ 0.6 mmHg, P ⬎ 0.05), and HR (⫺4 ⫾ 4 beats/min, P ⬎ 0.05; Table 1). To exclude the possibility that responses evoked by ethanol injection into the CeA were influenced by peripheral actions, ethanol was injected intravenously from the femoral vein. Peripheral administration of this small dose of ethanol (1.7 ␮mol/100 nl, n ⫽ 7) failed to alter resting SSNA, LSNA, MAP, and HR (Table 1). Effects of KYN on responses to CeA-injected ethanol. To test whether the ethanol-induced sympathoexcitatory response involved local actions of nonselective ionotropic EAA receptors,

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: Nmethyl-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.

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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 ␮mol) 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.

SSNA, LSNA, and MAP responses to a CeA-injected cocktail containing ethanol (1.7 ␮mol) and KYN (7.2 nmol, n ⫽ 7) were determined. Figure 5 shows an example of the response to a cocktail injected into the CeA. Note that sympathoexcitatory and pressor responses elicited by the cocktail containing ethanol and KYN were obviously attenuated compared with responses evoked by ethanol (1.7 ␮mol) alone. In a separate group of animals, the effects of KYN (7.2 nmol, n ⫽ 5) on baseline recorded parameters were determined. KYN had no significant effect on resting SSNA, LSNA, MAP, and HR (Table 1). Effects of AP5 on responses to CeA-injected ethanol. To test whether the ethanol-induced sympathoexcitatory response involved local actions of NMDA receptors, SSNA, LSNA, and MAP responses to a CeA-injected cocktail containing ethanol (1.7 ␮mol) and AP5 (3.0 nmol, n ⫽ 7) were determined. Figure 6 shows an example of the response to a cocktail injected into the CeA. Note that sympathoexcitatory and pressor responses elicited by the cocktail containing ethanol and AP5 were clearly attenuated compared with responses evoked by ethanol (1.7 ␮mol) alone. Fig. 7 shows summary data for the results shown in Figs. 5 and 6. In a separate group of animals, the effects of AP5 (3.0 nmol, n ⫽ 5) on baseline recorded parameters were determined. AP5 had no significant effect on resting SSNA, LSNA, MAP, and HR (Table 1). Effects of NBQX on responses to CeA-injected ethanol. To test whether the ethanol-induced sympathoexcitatory response involved local actions of non-NMDA receptors, SSNA, LSNA,

Fig. 3. A: representative traces showing SSNA, LSNA, and ABP responses to unilateral microinjection of ethanol (1.7 ␮mol) 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.

and MAP responses to a CeA-injected cocktail containing ethanol (1.7 ␮mol) and NBQX (1.3 nmol, n ⫽ 9) were determined. The summary data shown in Fig. 7 demonstrate the changes in SSNA, LSNA, MAP, and HR responses to a CeA-injected

Fig. 4. Summary data showing the changes (⌬) 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 ␮mol (saline), 0.17 ␮mol, and 1.7 ␮mol] 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 ␮mol ethanol.

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Table 1. Effect of injected compounds on resting MAP, HR, SSNA, and LSNA MAP, mmHg Injected Compounds

Saline (into the CeA) Anatomy control (ethanol out of the CeA) Ethanol (intravenous) KYN (into the CeA) AP5 (into the CeA) NBQX (into the CeA) KYN (into the RVLM)

HR, beats/min

SSNA, ␮V

LSNA, ␮V

No. of Rats/Group

Before injection

After injection

Before injection

After injection

Before injection

After injection

Before injection

After injection

5

110 ⫾ 2

110 ⫾ 3

428 ⫾ 13

432 ⫾ 14

0.02 ⫾ 0.005

0.02 ⫾ 0.004

0.04 ⫾ 0.003

0.04 ⫾ 0.005

5 7 5 5 5 5

109 ⫾ 4 108 ⫾ 4 110 ⫾ 3 107 ⫾ 4 107 ⫾ 4 105 ⫾ 3

110 ⫾ 3 110 ⫾ 4 107 ⫾ 2 108 ⫾ 5 108 ⫾ 4 106 ⫾ 3

422 ⫾ 19 399 ⫾ 12 396 ⫾ 14 409 ⫾ 12 401 ⫾ 14 400 ⫾ 8

415 ⫾ 15 397 ⫾ 16 401 ⫾ 15 411 ⫾ 13 407 ⫾ 17 402 ⫾ 12

0.02 ⫾ 0.002 0.03 ⫾ 0.005 0.03 ⫾ 0.006 0.03 ⫾ 0.006 0.03 ⫾ 0.004 0.05 ⫾ 0.005

0.02 ⫾ 0.005 0.03 ⫾ 0.005 0.03 ⫾ 0.007 0.03 ⫾ 0.005 0.03 ⫾ 0.004 0.05 ⫾ 0.004

0.04 ⫾ 0.004 0.06 ⫾ 0.002 0.06 ⫾ 0.003 0.04 ⫾ 0.006 0.06 ⫾ 0.002 0.06 ⫾ 0.005

0.04 ⫾ 0.008 0.06 ⫾ 0.002 0.06 ⫾ 0.004 0.04 ⫾ 0.006 0.06 ⫾ 0.002 0.07 ⫾ 0.008

Values are mean ⫾ SE. MAP, mean arterial pressure; HR, heart rate; SSNA, splanchnic sympathetic nerve activity; LSNA, lumber sympathetic nerve activity; CeA, central nucleus of the amygdala; KYN, kynurenate; AP5, D-2-amino-5-phosphonovalerate; NBQX, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium; RVLM, rostral ventrolateral medulla.

cocktail containing ethanol and NBQX. Note that the increases in LSNA and MAP elicited by the cocktail containing ethanol and NBQX were significantly attenuated compared with those evoked by CeA injection of ethanol (1.7 ␮mol) alone. There was no statistical difference in the increase of SSNA between groups injected with ethanol alone and the cocktail containing ethanol and NBQX. In a separate group of animals, the effects of NBQX (1.3 nmol, n ⫽ 5) on baseline recorded parameters were determined. NBQX had no significant effect on resting SSNA, LSNA, MAP, and HR (Table 1). Effects of AP5 on responses to CeA-injected acetate. Unilateral CeA microinjection of acetate (0.20 ␮mol/100 nl) consistently increased SSNA, RSNA, and MAP (n ⫽ 7). To test whether acetate-induced sympathoexcitatory and pressor responses involved activation of local NMDA receptors, SSNA, LSNA, and MAP responses to a CeA-injected cocktail containing acetate (0.20 ␮mol) and AP5 (3.0 nmol) were determined (n ⫽ 9). The increases in SNA and MAP elicited by CeA-injected acetate alone (SSNA: ⫹72 ⫾ 13%, LSNA: ⫹62 ⫾ 12%, and MAP: ⫹5 ⫾ 2 mmHg) were significantly blunted

Fig. 5. Representative traces showing SSNA, LSNA, and ABP responses to unilateral microinjection of a cocktail containing ethanol (1.7 ␮mol) 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.

compared with those evoked by CeA injection of a cocktail containing acetate and AP5 (SSNA: ⫹20 ⫾ 10%, P ⬍ 0.05 vs. CeA-injected acetate alone; LSNA: ⫹22 ⫾ 11%, P ⬍ 0.05 vs. CeA-injected acetate alone; and MAP: ⫺6 ⫾ 3 mmHg, P ⬍ 0.05 vs. CeA-injected acetate alone). Effects of RVLM preadministration of KYN on responses to CeA-injected ethanol. To test whether sympathoexcitatory responses to CeA-injected ethanol involve activation of the CeA-RVLM neuronal circuit/pathway, the effect of CeA-injected ethanol (1.7 ␮mol) on SNA and MAP was determined after ipsilateral RVLM administration of KYN (7.2 nmol, n ⫽ 5). The increases in SNA elicited by CeA-injected ethanol (SSNA: ⫹115 ⫾ 24% and LSNA: ⫹70 ⫾ 11%) were significantly attenuated by RVLM injection of KYN (SSNA: ⫹23 ⫾ 3%, P ⬍ 0.01 vs. CeA-injected ethanol; LSNA: ⫹18 ⫾ 9%, P ⬍ 0.05 vs. CeA-injected ethanol). The pressor response elicited by CeA-injected ethanol (⫹11 ⫾ 3 mmHg) was obviously blunted by RVLM preadministration of KYN, although it did not reach a significant statistical difference (⫹6 ⫾ 1 mmHg, P ⬎ 0.05). RVLM-injected KYN had no significant effect on resting SSNA, LSNA, MAP, and HR (Table 1).

Fig. 6. Representative traces showing SSNA, LSNA, and ABP responses to unilateral microinjection of a cocktail containing ethanol (1.7 ␮mol) 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.

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Fig. 7. Group data showing SSNA, LSNA, MAP, and HR responses to CeA injections of ethanol (1.7 ␮mol) alone (n ⫽ 8), a cocktail containing ethanol (1.7 ␮mol) and KYN (n ⫽ 7), a cocktail containing ethanol (1.7 ␮mol) and AP5 (n ⫽ 7), and a cocktail containing ethanol (1.7 ␮mol) and 2,3-dioxo-6nitro-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 ␮mol) alone. *P ⬍ 0.05 vs. the ethanol-alone group.

NMDA NR1 receptors are expressed in CeA-RVLM neurons. Five to seven days after injections of CTB conjugated with red fluorophores into the RVLM, clusters of neuronal cell bodies in the CeA were retrograde labeled with CTB (Fig. 1B, inset). These data suggest that CeA neurons have axons projecting to the RVLM (CeA-RVLM). To determine whether expression of NMDA receptors exists within CeA-RVLM neurons, NMDA NR1 antibody was used to perform immune fluorescence staining in brain slices. The data show that NMDA NR1 receptors were present extensively in CeA-RVLM neurons. Figure 1C shows a representative section with double-labeled CeA neurons (CTB ⫹ NMDA NR1). Note that NMDA NR1 receptors (green; Fig. 1C, left) were expressed in CeA-RVLM neurons identified by the CTB retrograde label (red; Fig. 1C, right), as indicated by the arrows (yellow; Fig. 1C, middle). DISCUSSION

This study is the first to investigate the in vivo effects of ethanol and its metabolite microinjected into the CeA in regulating SNA and AP as well as the role of local EAA receptors in mediating these responses. Here, we report five novel findings. First, CeA-injected ethanol elicited a consistent dose-dependent increase in SSNA, LSNA, and MAP. Second, blockade of NMDA receptors in the CeA markedly attenuated sympathoexcitatory and pressor responses elicited by CeAinjected ethanol. Third, CeA injection of the ethanol metabolite acetate consistently elicited sympathoexcitatory and pressor responses, which were effectively blocked by NMDA receptor antagonist. Fourth, inhibition of RVLM neurons effectively attenuated sympathoexcitatory and pressor responses elicited by CeA injection of ethanol. Finally, we observed that NMDA

NR1 receptors are abundantly expressed in CeA-RVLM neurons. Taken together, these findings indicate that sympathoexcitatory and pressor responses elicited by CeA-injected ethanol and the ethanol metabolite acetate are mediated by activation of NMDA receptors expressed in CeA-RVLM neurons. Acute ethanol administration in the CeA elicited significant increases in SNA and AP in anesthetized rats. The method to activate CeA neurons was performed by local microinjection of a small volume of ethanol (100 nl). The distribution of injected dye limited in the area of the CeA (Fig. 1, A and B) indicates that the effect of ethanol appears to originate from within the CeA. The fact that ethanol injected outside the CeA failed to evoke significant changes in the recorded variables supports the view that responses were due to an action within the CeA where CeA-RVLM neurons are located (Fig. 1B, inset). Because acute intravenous administration of ethanol (0.5 g/kg over a period of 45 min) appears capable of increasing sympathetic outflow and AP (37), peripheral mechanisms could contribute to sympathoexcitatory and pressor responses elicited by CeA injection of ethanol. However, this possibility in the present study is unlikely due to the observation that the small dose of ethanol (1.7 ␮mol/100 nl) injected into the CeA was also administrated intravenously and failed to change the recorded variables (Table 1). Although in vitro studies (41, 49) have reported that ethanol inhibits glutamatergic activity of CeA neurons in brain slice preparations, alcohol consumption has been reported to increase the expression of NMDA receptors in the CeA in rats (40). This evidence suggests that increased glutamatergic activity in the CeA might underlie the central neural mechanisms of increased SNA and AP in response to ethanol administration. Combined with the fact that ethanol is able to cross the blood-brain barrier (48a), local CeA administration of glutamate could increase AP (27), and there is an abundant expression of NMDA receptors in the CeA (5, 44, 50, 53), we hypothesized that the manifestation of cardiovascular and sympathetic responses to CeA injection of ethanol requires the activation of NMDA receptors expressed in CeA neurons in vivo. Consistent with this hypothesis, the present in vivo study found that sympathoexcitatory responses to CeA administration of ethanol were significantly attenuated by blockade of local NMDA receptors (Fig. 6). The data in the present study show that NMDA NR1 receptors were located in CeA-RVLM neurons (Fig. 1C) and that sympathoexcitatory responses elicited by CeA-injected ethanol were effectively blunted by inhibiting RVLM neurons, further supporting our hypothesis. It should be mentioned that present study also found that either KYN or AP5 injected into the CeA failed to alter resting AP and SNA. This is consistent with our previous study (14) showing that the same dose of KYN or AP5 injected into the paraventricular nucleus (PVN) failed to affect resting AP and SNA. These data indicate that tonic actions of glutamate in the CeA may not support resting AP and ongoing SNA. However, previous studies have also reported that pressor responses to GABA-A receptor blockade in either the RVLM (25) or PVN (14) are mediated by glutamate receptors, although no effect was seen when KYN or AP5 was given before the GABA-A receptor blockade. These data suggest that there is tonic glutamatergic activity in either the PVN or RVLM and that tonic activity could be dominantly inhibited by GABAergic inputs. Whether GABAergic inputs play a role in inhibiting tonic

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glutamatergic activity in the CeA region remains unclear. Interestingly, it has been previously reported that under conditions such as hypertension, actions of glutamate in the PVN (30) and RVLM (23, 24, 31) appear to contribute to tonic neuronal activity. Similarly, it could be speculated that glutamate may contribute to tonic drive to CeA neurons to underlie the potential mechanisms of increased sympathetic outflow to contribute to the hypertension elicited by chronic alcohol consumption. Whether chronic alcohol consumption increases glutamatergic activity in the CeA and contributes to the elevated sympathetic outflow remains to be studied in the future. It is at present unknown what cellular mechanisms are responsible for ethanol-induced sympathoexcitatory and pressor responses. The fact that there was a delayed onset of action (⬃4 – 6 min) and that ethanol increased both SNA and AP, ethanol metabolism by cytochrome P-450 and catalase to acetaldehyde and acetate metabolites may be the contributing factor to the increase in SNA and AP through the following two mechanisms (60 – 62). First, ethanol metabolism in brain tissue occurs via two oxidative pathways: cytochrome P-450, a major enzyme in drug metabolism that uses NADPH to convert ethanol to acetaldehyde (62), and catalase, which uses hydrogen peroxide in the oxidative pathway of ethanol to acetaldehyde (60). Zimatkin and colleagues (60, 61) have demonstrated that ethanol oxidation and elimination (⬃98%) in brain tissue occurs rapidly, with acetaldehyde production in as little as ⬃5 min in brain hemispheres. Acetaldehyde is then rapidly metabolized to acetate in the brain (1, 34). Increases in the acetate (structurally similar at the COOH-terminal of glutamate and NMDA) concentration can mimic the excitatory neurotransmitter glutamate or NMDA. Therefore, it is speculated that binding of acetate in the active site of NMDA receptors might cause depolarization of the neuron to increase the neuronal excitability and sympathetic outflow. Consistent with this speculation, the present study found that CeA injection of the ethanol metabolite acetate consistently elicited sympathoexcitatory and pressor responses, which were effectively blocked by the NMDA receptor antagonist. To further support this speculation, our preliminary data revealed that bath application of acetate increased the in vitro excitability of CeA-RVLM neurons identified by retrograde labeling in brain slice preparations and that the increase in neural excitability elicited by acetate was effectively blocked by pretreatment with AP5, the NMDA receptor antagonist (10). Acetaldehyde has been identified as a generator of free radicals (7), which can subsequently activate external G protein receptors (20, 28, 56, 58), leading to an intracellular downstream response (i.e., channel internalization, release of Ca2⫹ from intracellular stores, etc.) (15, 16), which may lead to an increases in neuronal activity. It is noteworthy that in vitro studies of brain slice preparations have reported that acute administration of ethanol in bath solution significantly inhibits glutamatergic transmission of CeA neurons. Specifically, ethanol inhibits NMDA receptors, as seen through a decrease in excitatory postsynaptic potential in CeA neurons (41). It has been previously reported that a significant population of CeA-RVLM neurons are glutamatergic (53) and that CeA neurons innervating the nucleus tractus solitarii nucleus (NTS) are GABAergic (4, 44); therefore, coupling this inhibition of NMDA receptors with CeA projections to the RVLM and NTS, ethanol itself would be expected to reduce SNA and AP. However, this is not the case in our in

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vivo study, which demonstrates that acute ethanol injected in the CeA evoked a significant increase in AP and SNA in rats. Due to the lack of enzymes to convert ethanol to the acetate metabolite in vitro, bath application of ethanol could inhibit NMDA receptors instead of activation of NMDA receptors through the acetate metabolite. This possibility may explain the discrepancy between previous in vitro (41) and present in vivo studies. It should be noted that besides activation of NMDA receptors by CeA injection of ethanol, AMPA receptors also seem to be involved in sympathoexcitatory responses in vivo (Fig. 7). As speculated previously and supported by our data, the ethanol metabolite acetate activates NMDA receptors, which causes an influx of Ca2⫹ into the cell. One of the subsequent intracellular signaling mechanism responses to a rise in intracellular Ca2⫹ is activation of Ca2⫹/calmodulin-dependent kinase II (CaMKII) (55). The translocation of CaMKII to synaptic sites and facilitation of increasing apical surface expression of AMPA receptors have been previously identified in long-term potentiation (32, 33). Additionally, phosphorylation of AMPA receptors itself by CaMKII increases ion conductance through AMPA receptors (3). Whether the above intracellular signaling cascade mechanism is able to explain the activation of AMPA receptors induced by CeA injection of ethanol remains to be determined in the future. Anatomic and electrophysiological studies have identified several groups of autonomic CeA neurons. One group of CeA neurons innervates the presympathetic neurons in the RVLM (6, 17, 45, 46). Another group of CeA neurons has axons innervating the NTS in the dorsal brain stem (6, 36, 45). The third group of CeA neurons has axons projecting to the hypothalamic PVN (6, 31, 54). Therefore, it is likely that different anatomic CeA neuronal pathways could innervate different sympathetic targets, supporting the orchestration of sympathetic responses. Here, we sought to determine whether CeA injection of ethanol or acetate contributes to differential patterning between SSNA and LSNA. The present study demonstrates that both SSNA and LSNA increased in response to either ethanol or acetate injected into the CeA, with each expressing a similar pattern of excitation. An important question that arises from the present findings is whether the dose of ethanol used in the present study is within the physiological range, i.e., the solution of 1.7 ␮mol ethanol in 100 nl microinjected in the central nervous system. The concentration at which blood alcohol starts to cause intoxication is ⬃0.05%. It should be emphasized that the present study used a small volume of solution, 100 nl of 200 proof ethanol (100%), microinjected into the CeA. As described in METHODS, Chicago blue dye solution (2% in saline, 100 nl) was injected into the CeA at the end of each experiment to mark the site of each injection and thus draw the estimated diffusion area of visualized dye. We checked the visualized diffusion area of Chicago blue dye solution, which was estimated ⬃1 mm3, i.e., ⬃1 ml (Fig. 1). Therefore, the microinjected ethanol can be interpreted to also diffuse in the area of CeA, and the final concentration of 100% ethanol (100 nl injected) diffusing over the 1-ml area was estimated at or below 0.01%. In fact, the visualized diffusion area of dye did not reflect the exact diffusion area of the injected compound since rats were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in PBS and part of the dye

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could have been affected by washout, reducing the visualized volume area of Chicago blue dye. Although we did not directly measure the concentration of ethanol in the CeA after microinjection, we believe that the final concentration of ethanol in the area of CeA is within the physiological range, i,e, at least ⬍0.05%. This is an important point of emphasis, as any final CeA-injected ethanol concentration outside of the normal physiological range obtained by drinking would have little physiological relevance. It is also noteworthy that the present study used an anesthetized preparation, a potential limitation in considering the effect of ethanol on SNA since anesthesia may inhibit the activity of CeA neurons to alter cardiovascular responses. A study (27) that measured AP in response to either electrical or chemical (L-glutamate) stimulation of the CeA has shown differential responses in conscious versus anesthetized rats. Therefore, anesthesia could potentially underestimate the role of ethanol in the CeA in regulating SNA and AP. Nevertheless, it is clear that ethanol increases the sympathetic outflow and AP, which may involve the activation of NMDA receptors in CeA-RVLM neurons. Perspective. The CeA has been recognized as an important contributor to cardiovascular function both under normal physiological conditions and in disease states. This study provides a novel finding that acute ethanol injection into the CeA in animals causes an increase in SNA and AP. Activation of NMDA receptors among CeA-RVLM neurons may contribute to the neural mechanisms of sympathoexcitatory and pressor responses. Whether another group of CeA neurons that have axons innervating either the NTS or hypothalamic PVN are involved in the elevated sympathetic outflow in response to local ethanol administration remains to be determined in the future. Further studies are needed to test the hypothesis that increased glutamatergic activity among CeA-RVLM neurons may underlie the neural mechanisms of increased sympathetic outflow and arterial blood pressure in response to chronic alcohol consumption. Additionally, identifying a neural mechanism that contributes to increased sympathetic outflow with regard to ethanol stimulation in the CeA may result in further investigation in ethanol research with regard to learning/memory, addiction, motor function, and other areas. ACKNOWLEDGMENTS The authors gratefully acknowledge Mingjun Gu for excellent technical assistance. GRANTS This work was funded by the following grants: NSFC81370344, JST2013WSN073, and AHA10SDG2640130. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: A.D.C., L.G., M.J.H., J.L., and R.A.L. performed experiments; A.D.C., J.Z., J.R.C., and Q.-H.C. interpreted results of experiments; A.D.C., L.G., M.J.H., and Q.-H.C. prepared figures; A.D.C., L.G., and Q.-H.C. drafted manuscript; A.D.C., J.R.C., and Q.-H.C. edited and revised manuscript; A.D.C., L.G., M.J.H., R.A.L., J.Z., J.R.C., and Q.-H.C. approved final version of manuscript; L.G., M.J.H., R.A.L., and Q.-H.C. analyzed data; J.R.C. and Q.-H.C. conception and design of research.

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