Brain Research, 506 (1990) 153-158 Elsevier
153
BRES 23879
The rostral and caudal ventrolateral medulla in young spontaneously hypertensive rats J. K e v i n Smith 1 a n d Kirk W. B a r r o n 2 1Department of Internal Medicine, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL (U.S.A.) and 2Department of Physiology and Biophysics, Collegeof Medicine, Universityof Kentucky, Lexington, KY (U.S.A.) (Accepted 12 September 1989) Key words: Mean arterial pressure; Heart rate; Cardiovascular effect; Hypertension; Spontaneouslyhypertensive rat; Wistar-Kyoto rat; Ventrolateral medulla; L-Glutamate; Tetrodotoxin
This study was designed to compare the cardiovascular influences of the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla (CVLM) in young (5-6 weeks) spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats. 8H and WKY groups had similar pressor and depressor responses to microinjection of L-glutamate into the RVLM and the CVLM, respectively. In addition the results of this study indicate a reduced tonic sympathoinhibitory function of the CVLM in young SH rats, which may contribute to the development of hypertension in the spontaneously hypertensive rat.
There is evidence to suggest that sympathetic efferent activity is greater in spontaneously hypertensive (SH) rats compared to normotensive control rats 4A1A4'lS. Recent studies indicate that the level of tonic sympathetic outflow is closely associated with the neuronal function of two discrete sites in the ventrolateral medulla, the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla (CVLM) 2"17'24. Previous work in our laboratory found significant differences between the responses of adult SH and normotensive Wistar-Kyoto (WKY) rats to microinjections of tetrodotoxin (TTX) into the CVLM, indicating a decreased tonic depressor influence of the CVLM in adult SH rats 2~. The purpose of the present study was to examine the cardiovascular responses to activation and inhibition of the RVLM and CVLM in young (5-6 weeks of age) SH rats to determine if these areas exhibit differences in responsiveness or basal levels of activity when compared to age-matched normotensive control WKY rats. Young animals were employed in these experiments to circumvent secondary cardiovascular effects due to the prolonged elevation of arterial pressure found in mature SH rats and to determine if the differences in depressor function previously seen in adult SH rats were present during the developmental stages of hypertension. All experiments were performed acutely on 5- to 6-week-old (95-150 g) SH (n = 8) and age-matched WKY (n = 8) obtained from Taconic Farms. The animals
were anesthetized with urethane (1.25 g/kg b. wt., s.c.), and the femoral artery and vein were cannulated to record mean arterial blood pressure (MAP) and for intravenous injections, respectively. After the trachea was cannulated, the animals were artificially ventilated, and gallamine triethiodide was administered (10 mg/kg, i.v.) for muscular paralysis. Blood gases and pH were measured to verify adequacy of ventilation. Body temperature was monitored with a rectal temperature probe and maintained at 37 + 0.5 °C. Drugs used for CNS microinjection were dissolved in phosphate-buffered saline (pH 7.4) and injected in a volume of 30 nl. The solutions were delivered through glass micropipettes (15 /~m tip o.d.) using a pressurized air injection system TM. The ventral medulla was exposed as previously described 21, and injection sites for the RVLM and CVLM were located using published stereotaxic coordinates for the RVLM and CVLM as a first approximation 16'23. Adjustments of the coordinates were then made to obtain the maximum pressor and depressor response from the RVLM and CVLM, respectively, in response to test injections of L-glutamate (10 mM, 30 nl); these adjusted coordinates were used for subsequent injections. Mean arterial pressure and heart rate values are given as peak responses for microinjection of L-glutamate, while steady state values are given for ]~FX microinjections and i.v. phenylephrine and chlorisondamine. The
Correspondence: K.W. Barton, Department of Physiologyand Biophysics, Tobacco and Health Research Institute, University of Kentucky, Cooper and University Drives, Lexington, KY 40546-0236, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
154 751
60
45
30
r.)
15
0 5
I0 20 i.v. p h e n y l e p h r i n e ( P g / k g / m i n )
40
Fig. 1. Changes in mean arterial pressure (MAP) in mmHg due to intravenous infusion of phenylephrine (5, 10, 20, and 40/~g/kg/min) in 5- to 6-week-old spontaneously hypertensive (SH; n = 8) and normotensive control (WKY; n = 8) rats. Changes in mean arterial pressure were not significantly different between the two groups.
statistical analysis of this data employed an initial General Linear Models 2-way analysis of variance followed by multiple t-comparisons with Bonferroni's adjustment if main effects were significant. Data are expressed as group mean + S.E.M.; significance level was selected as P < 0.05. Following a 30-min stabilization period prior to the initiation of the experimental protocol, resting mean arterial pressure (MAP) and heart rate (HR) were significantly elevated in SH rats as compared to the normotensive control WKY rats (80.3 + 3.1 m m H g vs 71.6 + 2.0 m m H g MAP, P < 0.0001; 441 + 8 vs 408 + 9 bpm H R , P < 0.0001). Before the brainstem microinjection protocols, an assessment of pressor responsiveness was performed using intravenous infusions of phenylephrine (5, 10, 20, and 40 gg/kg/min). Intravenous phenylephrine infusions caused dose-dependent increases in mean arterial pressure and increases in heart rate in both SH and WKY rats. The changes in MAP in response to i.v. phenylephrine, shown in Fig. 1, were not significantly different between the SH and WKY groups. To assess responsiveness to excitatory stimulation, the R V L M and CVLM were then injected unilaterally in a random order with L-glutamate in three concentrations (1, 10, and 100 mM, 30 hi) and with a vehicle control. A period of 4-5 min was allowed to elapse between injections. Microinjections of L-glutamate into the R V L M increased MAP and H R in both SH and WKY rats, the changes in M A P and H R were not significantly different between SH and WKY groups (Fig. 2). Investigation of depressor area function by microinjection of 30 nl L-glutamate (1, 10, and 100 mM) unilaterally into the CVLM caused dose-dependent decreases in MAP and H R in both SH and WKY rats; however, the MAP
and the H R changes were not significantly different between the SH and WKY groups (Fig. 3). Following these injections the function of the CVLM was blocked with bilateral injection of T I ' X (10 ~M, 30 nl), which increased MAP in WKY rats but caused no significant change of MAP in the SH group (+ 17.8 -+ 3.0 m m H g for WKY vs +0.9 _+ 3.0 m m H g for SH, P -0.0015, Fig. 4). Changes in heart rate were somewhat variable and were not significantly different from zero (no change) in either group. Values of H R were no longer significantly different after TTX injection into the CVLM, while MAP values were significantly higher in the WKY group (P = 0.02, Fig. 4). Subsequent bilateral microinjection of TTX (10/~M, 30 nl) into the RVLM provided an estimation of tonic activity in this area after tonic input from the CVLM had been removed (Fig. 4). Under these conditions microinjection of T-FX into the RVLM caused significant
40
u 30
2O
ti
0
I Conc. L - g l u
10 (mM)
|00
16
T ..= m
12
m ~
g
~
4
SH
~ - ] WKY
0
1 Conc. L - g l u
10 (raM)
100
Fig. 2. Changes in mean arterial pressure (MAP; top panel) in mmHg and changes in heart rate (HR; bottom panel) in bpm due to microinjection of 30 nl L-glutamate (0 = phosphate-buffered saline, 1, 10, and 100 mM) into the rostral ventrotateral medulla (RVLM) in 5- to 6-week-old SH (n = 8) and WKY (n = 8) rats. Changes in HR and MAP were not significantly different between the two groups.
155 decreases in H R and MAP in both groups. Decreases in H R were significantly greater in the WKY group as compared to the SH group (-90 + 14 bpm for WKY vs -33 + 6 bpm for SH, P = 0.002); H R in the WKY rat dropped to a level significantly below that of the SH group (P --- 0.005). The decrease in MAP due to injection of T F X into the RVLM was also significantly greater in WKY rats compared to SH rats (-48 + 6 m m H g for WKY vs -29 + 4 m m H g for SH, P = 0.03), but, unlike HR, MAP dropped to similar levels in both the WKY and the SH groups. To appraise residual sympathetic outflow, H R and MAP were measured before and after administration of the nicotinic ganglionic antagonist chlorisondamine. After TTX, administration of chlorisondamine (10 mg/kg i.v.) caused further decreases in H R and small additional decreases in MAP in the SH rats but no significant changes in the WKY rats (HR: -52 + 17 bpm for SH vs
- 4 + 4 bpm for WKY, P = 0.003; MAP: -4.9 + 1.1 m m H g for SH vs -1.9 + 1.5 m m H g for WKY, P = 0.02). After chlorisondamine H R and MAP values were no longer significantly different between the SH and WKY groups. At the end of each experiment, injections (30 nl) of 1% Fast green were made into each area for later histological identification of sites. The brains were then fixed by intravascular perfusion of a buffered 10% formalin solution. Frozen sections (40 ~m) of appropriate medullary areas were cut, mounted on microscope slides, and stained with Cresyl violet. Histological localization of the injection sites are illustrated in Fig. 5. Maximum pressor sites were located ventral to the nucleus ambiguus and caudal to the seventh nerve motor nucleus, in
100
I SH E
8O
$
I wrv
-g E a_ <
153
BRES 23879
The rostral and caudal ventrolateral medulla in young spontaneously hypertensive rats J. K e v i n Smith 1 a n d Kirk W. B a r r o n 2 1Department of Internal Medicine, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL (U.S.A.) and 2Department of Physiology and Biophysics, Collegeof Medicine, Universityof Kentucky, Lexington, KY (U.S.A.) (Accepted 12 September 1989) Key words: Mean arterial pressure; Heart rate; Cardiovascular effect; Hypertension; Spontaneouslyhypertensive rat; Wistar-Kyoto rat; Ventrolateral medulla; L-Glutamate; Tetrodotoxin
This study was designed to compare the cardiovascular influences of the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla (CVLM) in young (5-6 weeks) spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats. 8H and WKY groups had similar pressor and depressor responses to microinjection of L-glutamate into the RVLM and the CVLM, respectively. In addition the results of this study indicate a reduced tonic sympathoinhibitory function of the CVLM in young SH rats, which may contribute to the development of hypertension in the spontaneously hypertensive rat.
There is evidence to suggest that sympathetic efferent activity is greater in spontaneously hypertensive (SH) rats compared to normotensive control rats 4A1A4'lS. Recent studies indicate that the level of tonic sympathetic outflow is closely associated with the neuronal function of two discrete sites in the ventrolateral medulla, the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla (CVLM) 2"17'24. Previous work in our laboratory found significant differences between the responses of adult SH and normotensive Wistar-Kyoto (WKY) rats to microinjections of tetrodotoxin (TTX) into the CVLM, indicating a decreased tonic depressor influence of the CVLM in adult SH rats 2~. The purpose of the present study was to examine the cardiovascular responses to activation and inhibition of the RVLM and CVLM in young (5-6 weeks of age) SH rats to determine if these areas exhibit differences in responsiveness or basal levels of activity when compared to age-matched normotensive control WKY rats. Young animals were employed in these experiments to circumvent secondary cardiovascular effects due to the prolonged elevation of arterial pressure found in mature SH rats and to determine if the differences in depressor function previously seen in adult SH rats were present during the developmental stages of hypertension. All experiments were performed acutely on 5- to 6-week-old (95-150 g) SH (n = 8) and age-matched WKY (n = 8) obtained from Taconic Farms. The animals
were anesthetized with urethane (1.25 g/kg b. wt., s.c.), and the femoral artery and vein were cannulated to record mean arterial blood pressure (MAP) and for intravenous injections, respectively. After the trachea was cannulated, the animals were artificially ventilated, and gallamine triethiodide was administered (10 mg/kg, i.v.) for muscular paralysis. Blood gases and pH were measured to verify adequacy of ventilation. Body temperature was monitored with a rectal temperature probe and maintained at 37 + 0.5 °C. Drugs used for CNS microinjection were dissolved in phosphate-buffered saline (pH 7.4) and injected in a volume of 30 nl. The solutions were delivered through glass micropipettes (15 /~m tip o.d.) using a pressurized air injection system TM. The ventral medulla was exposed as previously described 21, and injection sites for the RVLM and CVLM were located using published stereotaxic coordinates for the RVLM and CVLM as a first approximation 16'23. Adjustments of the coordinates were then made to obtain the maximum pressor and depressor response from the RVLM and CVLM, respectively, in response to test injections of L-glutamate (10 mM, 30 nl); these adjusted coordinates were used for subsequent injections. Mean arterial pressure and heart rate values are given as peak responses for microinjection of L-glutamate, while steady state values are given for ]~FX microinjections and i.v. phenylephrine and chlorisondamine. The
Correspondence: K.W. Barton, Department of Physiologyand Biophysics, Tobacco and Health Research Institute, University of Kentucky, Cooper and University Drives, Lexington, KY 40546-0236, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
154 751
60
45
30
r.)
15
0 5
I0 20 i.v. p h e n y l e p h r i n e ( P g / k g / m i n )
40
Fig. 1. Changes in mean arterial pressure (MAP) in mmHg due to intravenous infusion of phenylephrine (5, 10, 20, and 40/~g/kg/min) in 5- to 6-week-old spontaneously hypertensive (SH; n = 8) and normotensive control (WKY; n = 8) rats. Changes in mean arterial pressure were not significantly different between the two groups.
statistical analysis of this data employed an initial General Linear Models 2-way analysis of variance followed by multiple t-comparisons with Bonferroni's adjustment if main effects were significant. Data are expressed as group mean + S.E.M.; significance level was selected as P < 0.05. Following a 30-min stabilization period prior to the initiation of the experimental protocol, resting mean arterial pressure (MAP) and heart rate (HR) were significantly elevated in SH rats as compared to the normotensive control WKY rats (80.3 + 3.1 m m H g vs 71.6 + 2.0 m m H g MAP, P < 0.0001; 441 + 8 vs 408 + 9 bpm H R , P < 0.0001). Before the brainstem microinjection protocols, an assessment of pressor responsiveness was performed using intravenous infusions of phenylephrine (5, 10, 20, and 40 gg/kg/min). Intravenous phenylephrine infusions caused dose-dependent increases in mean arterial pressure and increases in heart rate in both SH and WKY rats. The changes in MAP in response to i.v. phenylephrine, shown in Fig. 1, were not significantly different between the SH and WKY groups. To assess responsiveness to excitatory stimulation, the R V L M and CVLM were then injected unilaterally in a random order with L-glutamate in three concentrations (1, 10, and 100 mM, 30 hi) and with a vehicle control. A period of 4-5 min was allowed to elapse between injections. Microinjections of L-glutamate into the R V L M increased MAP and H R in both SH and WKY rats, the changes in M A P and H R were not significantly different between SH and WKY groups (Fig. 2). Investigation of depressor area function by microinjection of 30 nl L-glutamate (1, 10, and 100 mM) unilaterally into the CVLM caused dose-dependent decreases in MAP and H R in both SH and WKY rats; however, the MAP
and the H R changes were not significantly different between the SH and WKY groups (Fig. 3). Following these injections the function of the CVLM was blocked with bilateral injection of T I ' X (10 ~M, 30 nl), which increased MAP in WKY rats but caused no significant change of MAP in the SH group (+ 17.8 -+ 3.0 m m H g for WKY vs +0.9 _+ 3.0 m m H g for SH, P -0.0015, Fig. 4). Changes in heart rate were somewhat variable and were not significantly different from zero (no change) in either group. Values of H R were no longer significantly different after TTX injection into the CVLM, while MAP values were significantly higher in the WKY group (P = 0.02, Fig. 4). Subsequent bilateral microinjection of TTX (10/~M, 30 nl) into the RVLM provided an estimation of tonic activity in this area after tonic input from the CVLM had been removed (Fig. 4). Under these conditions microinjection of T-FX into the RVLM caused significant
40
u 30
2O
ti
0
I Conc. L - g l u
10 (mM)
|00
16
T ..= m
12
m ~
g
~
4
SH
~ - ] WKY
0
1 Conc. L - g l u
10 (raM)
100
Fig. 2. Changes in mean arterial pressure (MAP; top panel) in mmHg and changes in heart rate (HR; bottom panel) in bpm due to microinjection of 30 nl L-glutamate (0 = phosphate-buffered saline, 1, 10, and 100 mM) into the rostral ventrotateral medulla (RVLM) in 5- to 6-week-old SH (n = 8) and WKY (n = 8) rats. Changes in HR and MAP were not significantly different between the two groups.
155 decreases in H R and MAP in both groups. Decreases in H R were significantly greater in the WKY group as compared to the SH group (-90 + 14 bpm for WKY vs -33 + 6 bpm for SH, P = 0.002); H R in the WKY rat dropped to a level significantly below that of the SH group (P --- 0.005). The decrease in MAP due to injection of T F X into the RVLM was also significantly greater in WKY rats compared to SH rats (-48 + 6 m m H g for WKY vs -29 + 4 m m H g for SH, P = 0.03), but, unlike HR, MAP dropped to similar levels in both the WKY and the SH groups. To appraise residual sympathetic outflow, H R and MAP were measured before and after administration of the nicotinic ganglionic antagonist chlorisondamine. After TTX, administration of chlorisondamine (10 mg/kg i.v.) caused further decreases in H R and small additional decreases in MAP in the SH rats but no significant changes in the WKY rats (HR: -52 + 17 bpm for SH vs
- 4 + 4 bpm for WKY, P = 0.003; MAP: -4.9 + 1.1 m m H g for SH vs -1.9 + 1.5 m m H g for WKY, P = 0.02). After chlorisondamine H R and MAP values were no longer significantly different between the SH and WKY groups. At the end of each experiment, injections (30 nl) of 1% Fast green were made into each area for later histological identification of sites. The brains were then fixed by intravascular perfusion of a buffered 10% formalin solution. Frozen sections (40 ~m) of appropriate medullary areas were cut, mounted on microscope slides, and stained with Cresyl violet. Histological localization of the injection sites are illustrated in Fig. 5. Maximum pressor sites were located ventral to the nucleus ambiguus and caudal to the seventh nerve motor nucleus, in
100
I SH E
8O
$
I wrv
-g E a_ <
