Effects of ventrolateral medullary NMDA-receptor antagonism on biogenic amines and pressor response to muscle contraction

Neuroscience Research 32 (1998) 47 – 56

Effects of ventrolateral medullary NMDA-receptor antagonism on biogenic amines and pressor response to muscle contraction Gudbjorn Asmundsson, David J. Mokler, Ahmmed Ally * Departments of Pharmacology and Biochemistry, Uni6ersity of New England, College of Osteopathic Medicine, 11 Hills Beach Road, Biddeford, ME 04005, USA Received 1 January 1998; accepted 14 June 1998

Abstract Effects of D(− )2-amino-7-phosphonohepatanoic acid (AP-7), an N-methyl-D-aspartic acid (NMDA) receptor antagonist, administered into rostral ventrolateral medulla (RVLM) on changes in mean arterial pressure (MAP), heart rate (HR), extracellular levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) during static muscle contraction were investigated in anesthetized rats. Tibial nerve stimulation-evoked muscle contraction increased MAP and HR by 25 9 3 mmHg and 29 9 4 bpm, respectively. Microdialysis of AP-7 (1 mM) into the RVLM for 30 min attenuated the contraction-evoked cardiovascular responses with similar developed muscle tensions, without baseline HR or blood pressure changes. Administration of AP-7 into the caudal ventrolateral medulla had no effect on MAP or HR responses during contraction. Muscle contraction increased extracellular 5-HT in the RVLM by 144 9 35%, DA by 104 9 15% and NE by 62 912%. Perfusion of AP-7 for 30 min into the RVLM attenuated contraction-evoked increases in monoamines, concomitant to attenuating cardiovascular responses. Results demonstrate that NMDA-receptor blockade within the RVLM, but not the CVLM, inhibits cardiovascular responses during muscle contraction. Furthermore, NMDA receptor antagonism within the RVLM results in a decrease of biogenic amine release during muscle contraction, suggesting that extracellular biogenic amine concentrations are modulated by NMDA receptors. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rostral ventrolateral medulla; Caudal ventrolateral medulla; Microdialysis; Norepinephrine; Dopamine; Serotonin

1. Introduction The ventrolateral medulla (VLM) is a functionally identified area of the reticular formation of the medulla oblongata that has been implicated in autonomic regulatory processes (Ross et al., 1984; Ciriello et al., 1986; Ally, 1998). Furthermore, the rostral portion of the VLM, i.e. the RVLM, has been shown to be involved in the regulation of increases in mean arterial pressure (MAP) and heart rate (HR) during static muscle contraction in anesthetized animals, commonly known as * Corresponding author. Tel.: + 1-207-283-0171, Ext. 2285; Fax: +1-207-286-9493.; E-mail: [email protected]

the exercise pressor reflex (McCloskey and Mitchell, 1972; Bauer et al., 1989, 1992; Ally et al., 1997; Asmundsson et al., 1997; Ally, 1998). Lesioning the RVLM (Bauer et al., 1992) abolishes the pressor response during muscle contraction. In addition, radioactive glucose (Iwamoto et al., 1982) and c-Fos expression (Li et al., 1997) studies have identified the RVLM as an important area active during the exercise pressor reflex. Thus, the RVLM is an important integration brainstem area in the integration of cardiovascular responses during static exercise (Ally, 1998). Blockade of excitatory amino acid (EAA) receptors within the VLM eliminates the increase in arterial blood pressure in response to sciatic nerve stimulation

0168-0102/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 9 8 ) 0 0 0 6 7 - 4

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G. Asmundsson et al. / Neuroscience Research 32 (1998) 47–56

(Kiely and Gordon, 1993). Furthermore, EAA receptors in the RVLM appear to play a role in mediating the exercise pressor reflex, since microinjection of kynurenic acid into the RVLM diminishes the pressor response to static muscle contraction (Bauer et al., 1989). Kynurenic acid is an antagonist of all ionotropic EAA receptors, including NMDA, kainic acid, and AMPA receptors (Collingridge and Lester, 1989). Recently, it has been demonstrated that antagonism of AMPA receptors in the RVLM attenuates cardiovascular responses during static muscle contraction while similar administration into the CVLM augments the responses (Kobayashi et al., 1997). It would be of interest to determine the effects of selective NMDA receptor-antagonism on pressor and HR responses during static exercise. We hypothesize that bilateral administration of a competitive NMDA antagonist, D(− )2-amino-7-phosphonohepatanoic acid (AP-7), into the RVLM would attenuate increases in arterial blood pressure and HR evoked by static muscle contraction, whereas administration of AP-7 into the CVLM would produce an augmentation of such responses. Studies have identified that bulbospinal neurons arising from RVLM contain several neurotransmitters, including serotonin, catecholamines and EAAs that contribute to the regulation of cardiovascular functions (Ross et al., 1984; Chalmers et al., 1987; Phillippu, 1988; Collingridge and Lester, 1989; Singewald and Phillippu, 1996). It has been shown that serotonin (5-HT) in the RVLM is involved in control of autonomic functions and sympathetic outflow via descending medullary neuronal projections to sympathetic or somatomotor regulatory regions in the spinal cord (McCall and Clement, 1994). A recent study has demonstrated an increase in extracellular 5-HT concentration within the RVLM during static muscle contraction in the rat (Asmundsson et al., 1997). Furthermore, activation of 5-HT1A receptors in the RVLM inhibits increases in MAP and HR during muscle contraction via an attenuation of the exercise-induced increases in extracellular 5-HT (Asmundsson et al., 1997). Likewise, several studies have also demonstrated that changes in blood pressure modify the release of catecholamines in the RVLM (Dev et al., 1992; Rhee et al., 1992). For example, pharmacologically induced rises and falls in blood pressure are associated with increases and decreases, respectively, in the release of norepinephrine (Dev et al., 1992). Recently it was shown that extracellular glutamate concentrations are increased in the RVLM during the exercise pressor reflex (Caringi et al., 1998). In vivo microdialysis of L-glutamate or NMDA results in increased simultaneous release of dopamine (DA), norepinephrine (NE), and 5-HT in the striatum (Ohta et al., 1994). Since exercise pressor reflex activates RVLM neurons (Bauer et al., 1992) that are

mediated, at least in part, via an increase in glutamate concentrations (Caringi et al., 1998), we propose that the exercise pressor reflex increases extracellular concentrations of NE, DA and 5-HT, similar to the mechanism demonstrated by Ohta et al. (1994). Furthermore, since blockade of NMDA receptors in the striatum suppressed NMDA-evoked monoamine release (Ohta et al., 1994), antagonism of NMDA receptors in the RVLM would result in attenuated increases in the biogenic amines following an exercise pressor reflex and thereby, inhibiting cardiovascular responses. Taken together, we hypothesize that NMDA-receptors exist on monoaminergic terminals located within the RVLM (similar to that in the striatum as demonstrated by Ohta et al. (1994)) and that these receptors contribute to modulation of the exercise pressor reflex via an alteration in monamine release.

2. Methods

2.1. Surgery and microdialysis Experiments were performed using procedures described in previous papers (Ally et al., 1997; Asmundsson et al., 1997; Kobayashi et al., 1997). Male Sprague-Dawley rats (300–350 g) were initially anesthetized with 25 mg kg − 1 sodium pentobarbital (Sigma, St. Louis, MO, USA) and 75 mg kg − 1 chloral hydrate (Sigma) given i.p. The rats were maintained at 37–38°C by a heating pad and an IR heat lamp. Maintenance of anesthesia was done by additional doses of chloral hydrate given upon appearance of a corneal reflex and/or changes in blood pressure during surgical manipulation. A common carotid artery was catheterized and connected to a Model P231D Statham pressure transducer to measure arterial pressure (AP) using a Grass Model 79D (Quincy, MA, USA) physiological chart recorder. Mean arterial pressure (MAP) and HR were obtained by integrating the AP signal (time constant= 2 s). The trachea was intubated and the rat was allowed to breath spontaneously. However, during experiments involving neuromuscular blockade with an intravenous administration of pancuronium bromide through a cannula inserted into the jugular vein, a Harvard Apparatus respirator (Model 681, Natick, MA, USA) was used for artificial ventilation (room air, 60 strokes min − 1, 1 ml per 100 g body weight). The left tibial nerve was isolated and placed on a bipolar platinum hook electrode for electrical stimulation in order to evoke a static muscle contraction using a Model S88 Grass stimulator (Quincy) connected with a Model SIU5C Grass stimulus isolation unit (Quincy). The hip and left knee joint were secured to prevent movement during muscle contractions. The triceps surae muscle was exposed and consistently moistened

G. Asmundsson et al. / Neuroscience Research 32 (1998) 47–56

with mineral oil and covered with wet gauze. Developed muscle tensions evoked by the tibial nerve stimulation were measured using a Model FT03 Grass force transducer (Quincy) attached to the left Achilles tendon. After fixing the head of the rat in a stereotaxic frame (Kopf, Tujunga, CA, USA), a static muscle contraction was elicited by stimulating the tibial nerve (3× motor threshold, 40 Hz, 0.1 ms) while monitoring AP, MAP, HR and developed tension. Model CMA-11 microdialysis probes (CMA, Acton, MA, USA) with a 1 mm membrane and 0.24 mm outer diameter were inserted bilaterally into the RVLM (2.0 mm rostral to the caudal tip of the area postrema, 1.9 mm lateral to midline, and 2.4 mm ventral to the floor of the fourth ventricle) or the CVLM (0.5 mm rostral to the caudal tip of area postrema), serving as control outside the RVLM, based on the rat atlas of Paxinos and Watson (1982). Using a CMA/100 microdialysis pump (CMA), the probes were continuously perfused at 1 ml min − 1 with artificial cerebrospinal fluid (CSF: 125 mM NaCl, 1.26 mM CaCl2, 2.5 mM KCl, 1.18 mM MgCl2), at a pH 7.4 and osmolality of : 309 mOs kg − 1. This artificial fluid served as the delivery system for the drug, D(− )2-amino-7-phosphonohepatanoic acid (AP-7; RBI, Natick, MA) used in the experiments. Verification of proper placement of microdialysis probes was performed prior to each experiment by perfusing 1 nM L-glutamate (RBI) into either the RVLM or the CVLM. If the probes were inserted into the RVLM, an increase in MAP was noted within a minute subsequent to L-glutamate administration. Conversely, L-glutamate dialysis into the CVLM resulted in a decrease in MAP. After functionally assessing correct placement of probes, a static muscle contraction was evoked by stimulating the tibial nerve at similar parameters described above. Arterial pressure, MAP, HR, and developed tension were recorded and compared with those prior to insertion of probes. This was performed to determine if insertion of probes disrupted the functional integrity of the RVLM or the CVLM.

2.2. Protocols 2.2.1. Effects of microdialysis of AP-7 into the RVLM on MAP and HR during muscle contraction (Protocol 1) Following placement of dialysis probes in the RVLM or CVLM, the preparation was allowed to stabilize for 2 h, during which artificial CSF was continuously microdialyzed. Then, a tibial nerve stimulation-evoked static contraction of the left triceps surae muscle was performed for 30 s (3 ×MT, 40 Hz, 0.1 ms), and AP, MAP, HR, and muscle tension were measured. Then, a dose-dependent effect of microdialyzing AP-7 into the RVLM on cardiovascular responses during contraction was determined (n =6). This was done by perfusing the

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drug at incremental log doses (0.1 mM and 1.0 mM) sequentially for 30 min and repeating the muscle contractions. Five animals were used to determine a dosedependent effect following administration into the CVLM, thus illustrating drug effects outside of the RVLM. Thereafter, a set of experiments (n= 8) was performed with only the most effective dose of 1.0 mM AP-7 perfused into the RVLM. In all experiments, artificial CSF solution was microdialyzed for 90 min after ceasing the perfusion of AP-7 and a muscle contraction was evoked to determine if the cardiovascular responses returned to control levels (recovery). At the conclusion of each experiment, the animal was paralyzed by an i.v. administration of pancuronium bromide (200 mg kg − 1) and artificially ventilated. The tibial nerve was then stimulated for 30 s using the prior stimulation parameters. If stimulation of the nerve after paralysis evoked no cardiovascular responses, this demonstrated that there had been no direct activation of muscle afferents and changes in MAP and HR were due to contraction-evoked activation of muscle afferents.

2.2.2. Extracellular concentration of serotonin (5 -HT), norepinephrine (NE) and dopamine (DA) in the RVLM during muscle contraction before and after administration of AP-7 (Protocol 2) A separate set of eight animals were used in this protocol to determine extracellular levels of 5-HT, NE and DA in the RVLM during a 2-min static muscle contraction. Following surgical set-up and insertion of microdialysis probes into the RVLM, perfusion of artificial CSF continued at 1 ml min − 1 and nine 10-min collections were performed to establish baseline (control) for 5-HT, NE and DA release. A 10-min collection was necessary for 20 ml of dialysate to be collected due to the sensitivity of the assay (Asmundsson et al., 1997). Then, a 2-min static muscle contraction was evoked along with a 10-min collection period and measurement of cardiovascular changes. The animal was allowed to recover for 60 min, during which there were six 10-min collections. Thereafter, AP-7 was microdialyzed into the RVLM for 30 min during three 10-min collections. A muscle contraction was then repeated and the dialysate was collected for 10 min along with monitoring changes in MAP and HR. Artificial CSF was dialyzed for an additional 60 min (six 10-min collections) to determine if 5-HT, NE and DA levels returned to pre-contraction values. Following this, experiments were conducted after the animal was paralyzed with pancuronium bromide. The tibial nerve was stimulated for 2 min using prior stimulation parameters with 10min dialysate collections and MAP and HR measurements. A similar protocol with microdialysis of AP-7 into the CVLM (:1.5 mm caudal to the RVLM) to study its effect on biogenic amines was performed, thus

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in effect serving as controls. Dialysate samples were immediately stored at −80°C. On a separate day, 5-HT, NE and DA concentrations of the perfusates were measured using high pressure liquid chromatography with electrochemical detection (HPLC-EC; see below).

2.3. High pressure liquid chromatography analysis of 5 -HT, NE and DA Analyses of 5-HT, NE and DA were done using HPLC-EC. A reverse phase C18 3 mm was used with a mobile phase consisting of 75 mM monobasic sodium phosphate, 1.4 mM sodium octanyl sulphonate, 100 mM EDTA, and 10% acetonitrile brought to pH 5.6 with NaOH. Analysis of the eluent was by electrochemical detection (ESA Coulochem II, Bedford, MA, USA). Area under the curve was compared with standards of the biogenic amines injected onto the column at the beginning of each run. Standards were prepared daily from stock standards stored at − 80°C. The Justice Innovations ChromPerfect (Palo Alto, CA, USA) software allowed for determination of regression for the standard. A minimum correlation coefficient of 0.95 was used for all standard curves.

2.4. Histology At the completion of each experiment, the animal was perfused transcardially with 0.9% saline and then with 10% phosphate-buffered formalin. The brain was removed, fixed in 10% phosphate-buffered formalin, and then stored at −4°C. The location of microdialysis probes was determined by mounting the medullary region containing the VLM on the stage of a vibratome, taking 50 mm transverse sections, and examining for probe location under a microscope.

2.5. Statistical analyses All data are expressed as mean 9S.E.M. Normality of all data was tested so that appropriate parametric or nonparametric statistics could be performed. Baseline and peak values of MAP, HR, tension and % biogenic amine release elicited by muscle contractions were analyzed using a one-way analysis of variance with repeated measures (RM-ANOVA). Baseline values are the average of a 2-min period prior to a manipulation. Peak changes in MAP, HR and developed tension are defined as the maximum values obtained during the contraction periods. Dose – response data with AP-7 was analyzed using the one-way RM-ANOVA. A twoway RM-ANOVA was used to compare the hemodynamic, tension and biogenic amine data before and after AP-7, and following recovery. Post-hoc analyses for the ANOVAs were performed by Student –Neu-

man–Keul’s tests and for all statistical evaluations PB 0.05 was considered significant.

3. Results

3.1. Confirmations of proper microdialysis probe placement Proper probe placements were confirmed by bilateral administration of L-glutamate (1 nM) into either the RVLM or CVLM and the elicitation of either a pressor (4595 mmHg) or a depressor (44 9 6 mmHg) response, respectively. Additionally, bilateral insertion of the microdialysis probes into the RVLM or CVLM had no effect on changes in MAP, HR, and developed tension generated by triceps surae muscle contraction in response to tibial nerve stimulation. Prior to probe placement MAP, HR and tension in all rats (n=27) increased by 3093 mmHg, 349 4 bpm and 555 932 g, respectively. Following probe insertion, changes in cardiovascular parameters and tension were not significantly different from prior values, i.e. MAP, HR and tension increased by 2893 mmHg, 329 4 bpm and 5659 34 g, respectively. Histological analysis (see below) confirmed proper implantation of probes in the targeted areas.

3.2. Effects of AP-7 microdialyzed into the RVLM After probe placement and 2 h of stabilization, tibial nerve stimulation-evoked static muscle contractions were performed. Experiments with the RVLM (n=6) resulted in a developed muscle tension of 6209 35 g, an increase in MAP from 1119 6 to 142 9 4 mmHg (D= 3194 mmHg), and a rise in HR from 40198 to 42994 bpm (D =289 4 bpm). Microdialysis of AP-7 (0.1 mM and 1.0 mM) bilaterally into the RVLM for 30 min produced a dose-dependant decrease in MAP and HR responses during muscle contraction compared with controls (0.1 mM: MAP = 2095 mmHg, HR = 1894 bpm, tension= 630930 g; 1.0 mM: MAP= 109 3 mmHg, HR = 892 bpm, tension = 625937 g). Neither dose of AP-7 affected baseline MAP and HR. Administration of 1.0 mM AP-7 into the RVLM (n= 8) for 30 min resulted in a significant attenuation (PB 0.05) of MAP and HR responses during muscle contraction with equivalent change in developed tension (Fig. 1; Table 1). In addition, during 60 min perfusion of the drug, the cardiovascular responses remained attenuated. Following discontinuation of AP7 perfusion, artificial CSF was microdialyzed for approximately 90 min. Thereafter, a subsequent muscle contraction resulted in cardiovascular responses similar to pre-drug levels (recovery), suggesting that the observed attenuations were the effects of NMDA receptor

G. Asmundsson et al. / Neuroscience Research 32 (1998) 47–56

blockade in the RVLM (Fig. 1). In all experiments, tibial nerve stimulation following muscle paralysis elicited no changes in MAP or HR, further suggesting that the cardiovascular responses were mediated by contraction-evoked activation of muscle afferents. In experiments involving the CVLM serving as controls (n=5), bilateral microdialysis of AP-7 (0.1 or 1.0 mM) had no effect on MAP or HR responses during muscle contraction following 30 and 60 min of perfusion of the drug (Table 1). These data suggest that blockade of NMDA receptors within the CVLM by either doses of AP-7 do not appear to modulate cardiovascular responses during muscle contraction.

3.3. Changes in extracellular concentrations of 5 -HT, DA and NE in the RVLM during muscle contraction Extracellular 5-HT concentrations were elevated in the RVLM following static muscle contraction supporting results from a recent study (Asmundsson et al., 1997). After insertion of microdialysis probes within the

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RVLM, 5-HT, NE and DA levels were 199 3, 34 94, and 30 95 fmol per 20 ml, respectively, measured in the first 10 min of sampling. The levels gradually declined in the next 20 min and were stable during the remaining 60 min (Table 2). Extracellular fluid concentrations of 5-HT in the RVLM significantly increased by 1449 35% (n=8; PB0.05; Fig. 2; Table 2) following muscle contraction and paralleling a pressor response of 2794 mmHg, an increase in HR of 309 4 bpm, and a developed tension of 590930 g. Dopamine concentrations also increased by 1049 15% (Fig. 2; PB 0.05) in response to static muscle contraction, concomitant to increased MAP and HR. Lastly, extracellular concentrations of norepinephrine (NE) increased following the exercise pressor reflex by 629 12% (Fig. 2; P B0.05). In experiments involving the CVLM serving as controls (n= 5), bilateral microdialysis of AP-7 (1.0 mM) had no effect on these biogenic amines during muscle contraction (data not shown). This corresponds to a previous study where levels of 5-HT in the CVLM did not increase following a static muscle contraction (Asmundsson et al., 1997).

3.4. Extracellular concentrations of biogenic amines in the RVLM during muscle contraction following microdialysis of AP-7

Fig. 1. Average peak changes in mean arterial pressure (MAP), heart rate (HR), and developed tension during a 30-s tibial nerve stimulation-evoked muscle contraction before (open bars, control), 30-min (hatched bars) and 60-min (closed bars) after microdialysis of 1.0 mM AP-7, and 90 min following perfusion of artificial cerebrospinal fluid after discontinuing AP-7 (cross-hatched bars, recovery) into the rostral ventrolateral medulla in anesthetized rats. Values are means 9 S.E.M. (n =8). * P B0.05 compared with control and recovery.

Extracellular levels of 5-HT in the RVLM returned to pre-contraction baseline values within 20 min following a 2-min muscle contraction. During administration of AP-7 into the RVLM there was no change in baseline 5-HT, MAP, and HR levels. However, extracellular concentrations of 5-HT decreased to 479 7% of baseline values following a static muscle contraction after 30 min perfusion of AP-7 (Fig. 2; Table 2). Attenuation of MAP and HR were also observed after AP-7 with developed tension similar to that generated prior to the drug. Extracellular concentrations of DA and NE were significantly elevated (PB 0.05) following the initial muscle contraction and also returned to pre-contraction baseline levels within 20 min. Administration of AP-7 for 30 min increased baseline DA and NE concentrations without a change in resting cardiovascular variables. However, following AP-7 perfusion, the increase in the extracellular concentration of DA in the RVLM during a muscle contraction was attenuated when compared with that before the drug (209 11%; PB0.05; Fig. 2; Table 2). Likewise, significant attenuation (PB 0.05) of the extracellular increases of NE occurred during static muscle contraction after perfusion of AP-7 when compared with those before the drug (Fig. 2). Following discontinuation of AP-7, 5-HT, NE and DA levels returned to baseline values in 10–15 min. A muscle contraction after 60 min dialysis of artificial CSF increased 5-HT, NE and DA release similar to pre-AP-7 levels (Fig. 2; Table 2).

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Table 1 Hemodynamic and tension data before, after 30 min and after 60 min of microdialyzing AP-7 (1.0 mM) into RVLM and CVLMa Before AP-7

30 min after AP-7

60 min after AP-7

Recovery

Control

Peakb

Control

Peak

Control

Peak

Control

Peak

RVLM c MAP (mmHg) HR (bpm) Tension (g)

114 9 5 410 9 6 10 9 2

1399 4* 4399 4* 555 9 31*

110 95 403 95 11 9 3

119 94** 413 9 5** 560 935*

108 9 6 414 9 5 12 94

121 9 4** 4239 5** 570 9 30*

106 9 4 412 9 6 10 9 2

13295* 4439 5* 590940*

CVLM d MAP (mmHg) HR (bpm) Tension (g)

110 9 4 402 9 5 10 92

1359 3* 42694* 6049 28*

114 93 4109 5 12 92

138 9 4* 432 94* 616 927*

112 9 4 408 94 10 93

1349 4* 429 9 4* 600 9 31*

Values are means 9 S.E.M. Peak tension is the difference between the absolute maximum and control. c RVLM, rostral ventrolateral medulla; ; MAP, mean arterial pressure; HR, heart rate. d CVLM, caudal ventrolateral medulla. * Significantly different vs. corresponding control (PB0.05). ** Significantly different vs. corresponding control and peak values before AP-7 (PB0.05). a

b

At the end of all experiments and following muscle paralysis, a subsequent tibial nerve stimulation for 2 min using the prior parameters evoked no changes in MAP, HR, 5-HT, DA and NE, suggesting that these responses were elicited by contraction-evoked activation of muscle afferents (Table 2).

3.5. Histology Histological sections of the medullary region showed that the membranes of the probes were within the RVLM or the CVLM when compared with the rat brain atlas of Paxinos and Watson (1982). The placements of probes correspond to recent studies using similar techniques (Ally et al., 1997; Asmundsson et al., 1997; Kobayashi et al., 1997) and is shown in Fig. 3.

4. Discussion Several novel findings are reported in this study. First, microdialysis of AP-7 (1.0 mM), an NMDA receptor antagonist, into the RVLM, but not into the CVLM, attenuated MAP and HR increases associated with static muscle contraction in anesthetized rats. Therefore, it appears that static muscle contractionevoked cardiovascular changes are modulated by an NMDA receptor-mediated mechanism in the RVLM. Secondly, this research demonstrates changes in extracellular concentrations of biogenic amines in the RVLM during contraction-evoked increases in MAP and HR, i.e. a significant increase in all three biogenic amines, DA, NE and 5-HT, during a pressor response to muscle contraction. Lastly, we report that blockade of NMDA receptors in the RVLM attenuates MAP and HR responses during muscle contraction associated

with a reduction in extracellular NE, DA and 5-HT levels. The VLM is a principle brainstem locus regulating static muscle contraction-induced pressor responses (Iwamoto et al., 1982; Li et al., 1997; Ally, 1998). Kobayashi et al. (1997) demonstrated that microdialysis of CNQX (1.0 mM) into the RVLM at a concentration that is selective for AMPA receptors attenuates contraction-evoked increases in MAP and HR in anesthetized rats. Alternatively, they showed that CNQX administration into the CVLM potentiated the cardiovascular responses during static muscle contraction, thus demonstrating a possible tonic role of AMPA receptors in the CVLM on neurons mediating the exercise pressor reflex (Kobayashi et al., 1997). In the present study, we determined that blockade of NMDA receptors in the RVLM by AP-7 attenuates increases in MAP and HR during tibial nerve stimulation-evoked static muscle contraction. Therefore, it appears that in addition to AMPA receptors (Kobayashi et al., 1997), NMDA receptors in the RVLM are also involved in glutaminergic regulation of cardiovascular responses during static muscle contraction. Also of interest in the present study, 1.0 mM AP-7 perfused into the RVLM (final concentration : 200 nM), significantly attenuated the increases in MAP and HR during contraction without a change in baseline cardiovascular parameters. These results correspond to those of Kao et al. (1991), who reported that localized pressure microejection of AP-7 (300 nM) into the RVLM produced no change in resting blood pressure, though it antagonized NMDA receptor stimulation-evoked hypertension. In our study, since microdialysis of 5.0 mM AP-7 into the RVLM resulted in a decreased baseline blood pressure, the 1.0 mM dose was chosen for further experiments.

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Table 2 Hemodynamic, tension, norepinephrine (NE), dopamine (DE), and serotonin (5-HT) data in response to a 2-min tibial nerve stimulation-evoked muscle contraction before and after administration of AP-7 (1.0 mM) into the RVLM, after recovery, and after neuromuscular blockade with an intravenous injection of pancuronium bromide (n= 8)a Control

MAPb HR Tension NE DA 5-HT

After AP-7

Recovery

After paralysis

Baseline

Peak

Baseline

Peak

Baseline

Peak

Baseline

Peak

110 94 390 95 10 92 15 93 690.7 2.4 90.3

1379 4* 4259 6* 5749 30* 249 2* 129 1.1* 5.992.0*

1139 5 3859 6 119 3 169 2 99 1.0 4.09 1.0

127 93** 400 95** 582 928* 19 92** 11 91.1** 2.1 90.6**

108 9 5 380 9 4 13 9 4 14 9 2 5 9 1.0 2.2 9 0.4

138 94* 420 950* 578 9 22* 24 92* 12 91.1* 5.4 9 0.6

110 9 4 380 9 9 10 9 3 14 9 2 4.8 9 0.8 2.7 9 0.4

10995 37098 1292 1392 4.7 90.8 2.2 90.6

Values are mean 9 S.E.M. MAP, mean arterial pressure (mmHg); HR, heart rate (bpm); Tension (g); NE, DA and 5-HT (fmol per 20 ml). * Significantly different vs. corresponding control (PB0.05). ** Significantly different vs. corresponding control and peak values before AP-7 (PB0.05).

a

b

Furthermore, microdialysis of 1.0 mM AP-7 into the CVLM had no effect on changes in HR and blood pressure in response to static muscular contraction performed at 30 or 60 min following the drug perfusion. This result corresponds with that of Solomon et al. (1994), where NMDA receptor antagonism by (9 )2-amino-5-phosphonovaleric acid (AP-5) infusion into the CVLM had no effect on cardiovascular responses during hindlimb skeletal muscle contraction in anesthetized dogs. Therefore, NMDA receptors in the CVLM that are blocked by the 1.0 mM dose of AP-7, as opposed to AMPA receptors described previously (Kobayashi et al., 1997), do not have a direct role in regulation of the pressor reflex. A higher dose of AP-7,

Fig. 2. A bar graph showing percent extracellular fluid serotonin (5-HT, open bars), dopamine (DA, hatched bars), and norepinephrine (NE, closed bars) concentrations sampled from the rostral ventrolateral medulla during 2-min tibial nerve stimulation-evoked static muscle contractions before and after 30-min microdialysis of AP-7 (1.0 mM), and following recovery after discontinuing AP-7. Values are means 9 S.E.M. (n= 8). * PB 0.05 compared with respective controls (values prior to each contraction).

administered into the CVLM, could have produced an effect on cardiovascular responses during muscle contraction. In our study (data not shown), since a dose of 5.0 mM AP-7, microdialyzed into the CVLM, increased baseline blood pressure and heart rate, 1.0 mM AP-7 was chosen as the experimental dose. Several neurotransmitters in the VLM, including catecholamines and neuropeptides, contribute to the regulation of cardiovascular activity (Ross et al., 1984; Phillippu, 1988; Guyenet, 1990; Singewald and Phillippu, 1996; Ally, 1998). Bulbospinal neurons arising from the RVLM have been shown to contain multiple neurotransmitters, e.g. biogenic amines colocalized within the RVLM in such cellular groups as B3 and C1 (Chalmers et al., 1987). Serotonin neurons descending to the spinal cord from B1 and B3 areas in the RVLM have been described to be involved in elevating and maintaining blood pressure (Chalmers et al., 1987). A recent study demonstrated significant (PB 0.05) increases in 5-HT in the RVLM, but not in the CVLM during the exercise pressor reflex (Asmundsson et al., 1997). Furthermore, Caringi et al. (1998) showed that extracellular glutamate increases significantly (PB 0.05) in the RVLM during static muscle contraction. These results indicate the involvement of 5-HT and EAA within the RVLM during the exercise pressor reflex. The present study utilizing microdialysis techniques reveals several findings regarding extracellular concentrations of biogenic amines within the RVLM associated with increases in MAP and HR following static muscle contraction. Our results documented a significant increase in three biogenic amines, namely DA, NE and 5-HT, in the RVLM following a pressor reflex. This furthers the findings of Ross et al. (1984), which in addition to increased plasma levels of catecholamines (NE, DA and epinephrine) following electrical stimulation of RVLM neurons, we observed localized in-

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Fig. 3. A transverse histological section of the medulla at the level +2.5 mm rostral to the calamus scriptorius (the caudal—most part of obex) that corresponds to the right rostral ventrolateral medullary region. The microdialysis probe tract (tip membrane being 1 mm) is indicated by the arrow.

creased concentrations of NE and DA in the RVLM during the pressor reflex. In contrast, the exercise pressor reflex did not increase nor decrease DA, NE or 5-HT in the CVLM, suggesting that neurochemical changes during the exercise pressor reflex is limited to the RVLM site and not the caudal region of the VLM with respects to 5-HT, NE, or DA. Bazil and Gordon (1991) suggest that the RVLM neurons partially maintains and regulates sympathetic drive to the cardiovascular system not only via spinal NMDA receptors, but also possibly through other neurotransmitters, such as monoamines, that are involved in maintaining sympathetic outflow. It has been known that monoamines act as excitatory and/or inhibitory neurotransmitters depending on specific brain regions (see Singewald and Phillippu, 1996 for a review). It may be possible that glutamate modulates monoamine release in the RVLM which, in turn, regulates descending sympathoexcitatory neurons. In vivo microdialysis of L-glutamate, AMPA, kainate or NMDA in the rat

striatum has been shown to increase simultaneous release of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) in the striatum (Ohta et al., 1994). Further, given a role of 5-HT (Asmundsson et al., 1997) and glutamate (Caringi et al., 1998) in the exercise pressor reflex, these neurotransmitters may interact in the RVLM to alter MAP and HR during static exercise. The increases in MAP and HR during the exercise pressor reflex may be the result of increases in monoamine concentrations within the RVLM, thereby exciting descending sympathoexcitatory neurons. It is possible that glutamate receptors exist on both catecholaminergic and indoleaminergic terminals in the RVLM in a manner similar to that in the striatum (Ohta et al., 1994), thus facilitating the release of monoamines in response to the exercise pressor reflex. In the present study, antagonism of the NMDA receptor resulted in attenuated increases in the neurotransmitters following an exercise pressor reflex. This lends further support to our hypothesis that glutamate stimulates monoamine release in the RVLM. Following the initial muscle contraction, 5-HT, DA and NE levels returned to pre-contraction baseline values after 10 min. Microdialysis of AP-7 for 30 min into the RVLM did not alter baseline MAP, HR, 5-HT or NE levels. In contrast, AP-7 resulted in an increase of baseline DA extracellular concentrations in the absence of changes in resting MAP and HR. It may be possible that blockade of NMDA receptors in the RVLM caused a re-uptake inhibition of DA. Similar reuptake inhibition of DA by MK-801, a noncompetitive NMDA receptor antagonist, mediated via NMDA receptor blockade has been suggested in a previous study (Venero et al., 1996). However, increases in DA during muscle contraction were abolished during stimulation of the tibial nerve after AP-7 administration into the RVLM. In addition, 5-HT levels following AP-7 microdialysis into the RVLM resulted in a level significantly lower than baseline in response to a muscle contraction. Serotonin was reduced by 549 4% during a second contraction in the presence of AP-7. These results appear to correspond to a previous study where a 5-HT1A receptor agonist, 8-OH-DPAT, microdialyzed into the RVLM, evoked extracellular concentrations of 5-HT lower than baseline during a muscle contraction (Asmundsson et al., 1997). However, further studies are necessary to elucidate potential mechanisms by which AP-7 decreases 5-HT lower than control during muscle contraction but not during rest. In our experiments, microdialysis of AP-7 into the RVLM or the CVLM, and sampling of 5-HT, DA and NE were presumed to be localized. Confirmation can be made by: (1) administration of L-glutamate into the RVLM and the CVLM evoked pressor and depressor responses, respectively; (2) diffusion of AP-7 more than 1.0 mm from the CVLM into the RVLM would have

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resulted in an attenuation of the cardiovascular responses during muscle contraction; (3) lastly, similar techniques have been used in previous studies where diffusion of drugs or microdialysis sampling of neurotransmitters have been demonstrated to be within and from the extracellular space adjacent to the microdialysis probes (Taylor and Basbaum, 1995; Wilson, 1996; Ally et al., 1997; Asmundsson et al., 1997; Kobayashi et al., 1997; Caringi et al., 1998). In conclusion, our results demonstrate that the exercise pressor reflex is modulated by blockade of NMDA receptors within the RVLM. In addition, this study demonstrates that increases in extracellular fluid concentrations of NE, DA and 5-HT in the RVLM are associated with cardiovascular responses during static muscle contraction, suggesting that these monoamines influence neural pathways descending from the RVLM to the spinal cord to regulate cardiovascular activity. Furthermore, it appears that an NMDA receptor-mediated mechanism in the RVLM is involved in regulating noradrenergic, dopaminergic and serotonergic systems within the RVLM during exercise-induced pressor responses. This was demonstrated by the fact that cardiovascular responses during muscle contraction were attenuated after blockade of NMDA receptors along with decreased elevations in extracellular concentrations of NE, DA and 5-HT. However, the physiological significance of these biogenic amines and their correlation to cardiovascular responses during static exercise needs to be further clarified. Studies after depletion of 5-HT, NE and DA in the RVLM followed by muscle contraction will add further insight into the role of these neurotransmitters in the neural control of circulation.

Acknowledgements The authors thank Professor Allen Bell of the Department of Anatomy for kindly performing photographs of the histology sections. G.A. was supported by a fellowship from the American Heart Association (ME/NH/VT Affiliate) in 1997 and the 1996 University of New England Dean’s summer research grant. A part of this study has been previously presented in abstract form (Soc. Neurosci Abstract 23(1), 724, 1997).

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