Brain Research, 556 (1991) 145-150 © 1991 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/91/$03.50 ADONIS 000689939124758C
145
BRES 24758
Responses of cardiovascular neurons in the rostral ventrolateral medulla of the normotensive Wistar Kyoto and spontaneously hypertensive rats to iontophoretic application of angiotensin II R.K.W. Chan 1, Y.S. Chan and T.M. Wong Department of Physiology, Facultyof Medicine, University of Hong Kong (Hong Kong) (Accepted 23 April 1991)
Key words: Spontaneous neuronal activity; Angiotensin II; Saralasin; Iontophoresis; Rostrai ventrolateral medulla; Wistar Kyoto rat; Spontaneously hypertensive rat
In female pentobarbital-anesthetized Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), changes in spontaneous discharges of cardiovascular neurons in the rostral ventrolateral medulla (RVL) in response to iontophoretic application of angiotensin II (Ang II) were studied and compared. It was found that iontophoretic application of Ang II to RVL increased the spontaneous neuronal activities of 30% of the cardiovascular neurons in both types of rats and that the increase was significantly greater in SHR than in WKY. In both types of rats, there was an increase in arterial blood pressure in response to iontophoretic release of Ang II to RVL. The pressor response was accompanied by tachycardia, which was significantly greater in SHR than in WKY. The present study provides evidence that Ang II acts directly on cardiovascular neurons in RVL, and in SHR, an enhanced sensitivity and responsiveness of the RVL cardiovascular neurons to Ang II may augment the sympathetic outflow from RVL and contribute to the genesis of hypertension.
The rostral ventrolateral medulla (RVL), a region known to play a pivotal role in providing the sympathetic drive and maintaining the arterial blood pressure (BP) 5' 22,29, has recently been suggested to be a new potential site of action of the brain renin-angiotensin system 2, 3,11,17,21,31. In rabbit 2°, cat 2, dog 32 and human 1, R V L has been shown to possess a high concentration of receptor binding sites to angiotensin II (Ang II), a neuropeptide implicated in the neurogenic control of BP 25'27 and the genesis of hypertension in spontaneously hypertensive rats (SHR) ~2,25,28. In normotensive rat, R V L neurons have been shown to lie in a matrix of angiotensin immunoreactive fibres and varicosities 16,17,18, suggesting that A n g II may be a neurotransmitter or neuromodulator at the R V L 2'18"21,31. Anesthetization of R V L with lidocaine has been shown to attenuate the pressor response to intracerebroventricular injection of Ang 1I26. Furthermore, microinjection of A n g II into R V L of anesthetized cat 2, rabbit 31 and normotensive rat 11 has been shown to produce a dose-dependent pressor effect and an increase in sympathetic nerve activity, which are blocked by the administration of A n g II antagonist, saralasin, whereas microinjection of saralasin into R V L produces a dose-dependent depressor response and a
decrease in sympathetic nerve activity 31. Taken together, these findings indicate that R V L may be responsible for mediating the tonic cardiovascular effects of brain A n g II. However, the ways by which brain A n g II and R V L neurons interact to produce the hypertensive effect remain elusive. In addition, the R V L of S H R , when compared with normotensive Wistar Kyoto rats (WKY), has also been demonstrated by our recent study to exhibit an enhanced cardiovascular responsiveness to electrical microstimulation 8 as well as to possess predominantly double discharge units with a fast conduction velocity and single discharge units with a higher firing rate than that of W K Y 9. In SHR, an altered interaction between R V L neurons and A n g II may possibly exist 25,28, thus contributing to a higher sympathetic outflow from R V L and ultimately an elevated arterial BP observed in SHR. The purposes of the present study are therefore, by employing iontophoretic method, (1) to determine whether Ang II acts directly on the spontaneously active cardiovascular neurons in R V L and (2) to compare the sensitivity and responsiveness of R V L cardiovascular neurons in W K Y and S H R to A n g II. Part of this study has been presented in an abstract 1°. Female W K Y (n = 12) and S H R (n = 11), weighing
1 Present address: The Salk Institute, 10010 North Torrey Pines Road, San Diego, CA 92186, U.S.A.
Correspondence: T.M. Wong, Department of Physiology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong. Fax: (852) 8559730.
146 210-230 g (19-20 weeks of age), were used in this study. The rat was anesthetized with pentobarbital sodium at a dose of 40 mg/kg intraperitoneally, and an adequate anesthesia was characterized by the absence of corneal reflex and no retraction of distal phalanges to nociceptive stimulation of the hindpaw 15. A supplementary dose of 20 mg/kg was given intravenously when the animal showed signs of wakefulness, such as (1) perturbations of BP and heart rate, and (2) increases in breathing rate. Experiments continued 20-30 min after intravenous administration of the supplementary dose of anesthetic. The femoral artery was cannulated with a polyethylene catheter which was connected to a Statham pressure transducer and a Gould recorder for the continuous measurement of BP throughout the experiment. The femoral vein was cannulated for the infusion of drugs. Electrocardiogram (ECG) signals were obtained from an E C G monitor via 4 platinum electrodes inserted into muscles of the limbs. The head of the animal was mounted onto a stereotaxic apparatus (Narishige). All pressure points were infiltrated with lidocaine. The rat was allowed to breathe spontaneously and the breathing rate was not significantly disturbed by any of the surgical procedures employed in this study. Body temperature was maintained at 37 °C with a heating pad. For iontophoresis, the occipital bone, the dura and pia mater overlying the cerebellum were removed to allow placement of the micropipette. Extracellular single unit activities of spontaneously active RVL cardiovascular neurons were recorded using a double-barrel glass micropipette, tip diameter 1.5/~m and DC resistance of 8-12 M£2, inserted vertically through the intact cerebellum in a dorsoventral fashion to the RVL on the right side according to the brain atlas of the rat 24. The glass micropipette was connected to an Electrometer (EEI, model 400B) for monitoring neuronal discharges and current injection. The barrel for recording RVL neuronal activities contained 10 -3 M saralasin dissolved in 2 M NaCl solution (pH 5.0) saturated with 7% Fast green. Saralasin was released by current injection to block the excitatory action of iontophoretic Ang II. The other barrel contained 10-3 M Ang II dissolved in distilled water (pH 3.5) for iontophoretic release of Ang II to the RVL cardiovascular units being studied. Each spontaneously active RVL cardiovascular unit was characterized by: (1) its barosensitivity in response to an intravenous bolus injection of 2-4/~g/kg phenylephrine, (2) synchronization of spontaneous discharge with the cardiac cycle, and (3) its spinal projection to the intermediolateral column (IML) using the antidromic collision test 15"22. To antidromically identify RVL neurons that project to IML of the thoracic spinal cord, the spinal cord was immobilized with a spinal clamp and laminectomy was performed
at T2-T 5 levels of the spinal cord to permit placement of an array of two stainless steel stimulating electrodes ipsilateral to the recording sites. The exposed nervous tissues were covered with warm paraffin oil. Monopolar cathodal pulses (0.5 ms, 0.5 Hz, single shock) were delivered to sites in the IML regions and the stimulus current (40-60/~A) was monitored on a storage oscilloscope. Those units with bursting activity linked to the respiratory cycle were not studied further. Prior to iontophoretic application of Ang II, the initial discharge rate of an RVL cardiovascular neuron at the respective basal BP level of WKY (mean arterial pressure, MAP = 101.6 + 2.3 mmHg) or SHR (MAP -- 161.1 _+ 1.8 mmHg) was first determined. Cationic current in the range of 10-60 nA was then applied to the barrel c o n t a i n i n g 10 -3 M Ang II for 1 min to release the Ang II to the RVL cardiovascular unit. It was demonstrated in our control study that iontophoretic application of saline at a current intensity of 10-50 nA did not alter the discharge rate of RVL cardiovascular units, and a current intensity of 60 nA would induce a 0.85% increase in the discharge rate, probably due to the direct depolarization effect of the injection current 15. In both types of rats, iontophoretic application of saline at a current intensity of 10-60 nA did not alter the cardiovascular parameters. Compensation anionic current (10-15 nA) was used to prevent any spontaneous leakage of drugs from the glass micropipette, and direct depolarization of the RVL neurons during current injection is. For each responsive RVL cardiovascular unit, the threshold current, which was defined arbitrarily as the current intensity eliciting a 10% change in discharge rate of an RVL neuron by iontophoretically released Ang II, was also determined. The interval between successive iontophoretic applications of Ang II to RVL cardiovascular units was at least 30 min to ensure complete recovery of BP and neuronal activities since the half-life for dissociation of Ang II from the Ang II receptors in WKY and S H R has been found to be around 15 min 28. To confirm the excitatory action of Ang II, the effect of Ang II blockade was studied by applying cationic current in the range of 10-60 nA simultaneously to both barrels of the glass micropipette for 1 min to enable concomitant iontophoretic release of Ang II and saralasin to the unit being studied. The amount of Ang II released was not determined in the present study since it has been demonstrated that the quantity released increases linearly with the intensity of ejecting current up to about 60 nA and the rate of release is approximately 41.5 + 4.8 fmol/nA/min 15. This guideline was employed in the present study. The position of the electrode in RVL was determined before electrophysiological recording with the aid of electrical microstimulation that would elicit the charac-
147 teristic pressor responses as described previously8. At the end of each experiment, Fast green was iontophoretically deposited at the most ventral site of pipette penetrations. Frozen coronal sections of the brain and spinal cord were cut at 60/zm with a sliding microtome. The recording and antidromic stimulation sites were reconstructed from Cresyl violet-stained serial frontal sections with reference to the dye deposits. Histological identification of RVL was based on anatomical landmarks described by Haselton and Guyenet 15 and Ruggiero et al. 3°. Only units within the confines of RVL were included in this study. Statistical comparisons between groups were made by using a one-way analysis of variance (ANOVA). A difference at the level of P < 0.05 was considered statistically significant. In agreement with our previous study, two kinds of discharge patterns, namely single and double discharges, were identified in the cardiovascular neurons of RVL in both types of rats; the RVL of SHR possesses predominantly double discharge units with a fast conduction velocity and single discharge units with a higher firing rate than that of WKY 11 (see Table I). In the present study, iontophoretic application of Ang II to the RVL increased the discharge rates of 30% of single discharge (WKY, n = 52 out of 150 sampled; SHR, n = 42 out of 118 sampled) and 30% of double discharge (WKY, n -21 out of 69 sampled; SHR, n = 61 out of 184 sampled) cardiovascular units dose-dependently in both WKY and SHR (Fig. 2). The increase in spontaneous discharges was abolished by co-administration of saralasin, indicating that the responses were mediated by specific Ang II receptors on the R V L cardiovascular neurons (Fig. 1A,B). The cardiovascular neurons excited by iontophoretic Ang II were found to distribute within RVL at coordinates 11.6-13.3 mm posterior to bregma, lateral
1.9-2.1 mm, and 6.0-6.7 mm deep from the brain surface (Fig. 1C). In both types of rats, the number of Ang II-responsive cells found in the medial and ventral portion of the RVL was 8-12% more than from regions closer to the subcompact division of the nucleus ambiguus. The number of responsive cells also became scarce at the lateral and medial border of the RVL. The remaining 70% of the cardiovascular neurons did not respond to iontophoretic application of Ang II even at a higher ejecting current of 100 nA, suggesting that the pressor response to microinjection of Ang II to RVL was mediated by some but not all of the R V L cardiovascular neurons. These unresponsive R V L cardiovascular neurons were scattered among the responsive cells. In both WKY and SHR, the increase in discharge rate elicited by iontophoretic Ang II was much greater in double discharge units than in single discharge units (Figs. 1 and 2). In comparison, both single and double discharge RVL units in SHR showed a statistically lower threshold current, shorter latency (Table I), greater increase in discharge rate and longer duration of response than those in WKY (Fig. 2), indicating a greater sensitivity and responsiveness of R V L cardiovascular neurons in SHR to Ang II. 80% of the Ang II-responsive units were also tested for their spinal projection and all of the tested units were found to project to the IML of the thoracolumbar spinal cord, a region from which the preganglionic neurons arise 22, suggesting that the responsive units may be important in determining the sympathetic vasomotor outflow to the peripheral vascular beds. In both types of rats, there was invariably an increase in mean arterial pressure following application of Ang II to RVL in the order of 2-5 m m H g in WKY and 4-7 m m H g in SHR; these values were too small for reliable statistical comparison. The pressor response was accompanied by a
TABLE I Response characteristics of RVL cardiovascular neurons to iontophoretically applied angiotensin H WKY
Responsive cells No. (total sampled) % Firing rate (Spikes/s) Conduction velocity (m/s) Slow-conducting group (%)
SHR
Single discharge
Double discharge
Singledischarge
Double discharge
52 (150) 34.7% 16.7 + 0.4
21 (69) 30.4% 21.2 + 1.4
42 (118) 35.6% 23.1 + 0.9***
61 (184) 33.2% 22.4 + 1.1
0.48 + 0.01 (35.6%) 2.67 + 0.06 (56.3%) 19.3 + 0.60 18.2 + 0.42
0.58 + 0.03 (1.1%) 3.2 + 0.02 (7.0%) 17.4 _+0.80 16.8 + 0.50
0.55 __+0.02
0.78 + 0.05 (3.8%) 4.23 + 0.11 (50.1%) 10.3 + 0.60*** 12.3 + 0.43*
(36.2%) Fast-conducting group (%) 2.75 + 0.03 (9.9%) Threshold current (nA) 12.5 + 0.30*** Latency(s) 15.9+0.11"** Values are mean + S . E . M . *,*** statistically different from corresponding values in WKY at levels P < 0.05 and ANOVA.
P
145
BRES 24758
Responses of cardiovascular neurons in the rostral ventrolateral medulla of the normotensive Wistar Kyoto and spontaneously hypertensive rats to iontophoretic application of angiotensin II R.K.W. Chan 1, Y.S. Chan and T.M. Wong Department of Physiology, Facultyof Medicine, University of Hong Kong (Hong Kong) (Accepted 23 April 1991)
Key words: Spontaneous neuronal activity; Angiotensin II; Saralasin; Iontophoresis; Rostrai ventrolateral medulla; Wistar Kyoto rat; Spontaneously hypertensive rat
In female pentobarbital-anesthetized Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), changes in spontaneous discharges of cardiovascular neurons in the rostral ventrolateral medulla (RVL) in response to iontophoretic application of angiotensin II (Ang II) were studied and compared. It was found that iontophoretic application of Ang II to RVL increased the spontaneous neuronal activities of 30% of the cardiovascular neurons in both types of rats and that the increase was significantly greater in SHR than in WKY. In both types of rats, there was an increase in arterial blood pressure in response to iontophoretic release of Ang II to RVL. The pressor response was accompanied by tachycardia, which was significantly greater in SHR than in WKY. The present study provides evidence that Ang II acts directly on cardiovascular neurons in RVL, and in SHR, an enhanced sensitivity and responsiveness of the RVL cardiovascular neurons to Ang II may augment the sympathetic outflow from RVL and contribute to the genesis of hypertension.
The rostral ventrolateral medulla (RVL), a region known to play a pivotal role in providing the sympathetic drive and maintaining the arterial blood pressure (BP) 5' 22,29, has recently been suggested to be a new potential site of action of the brain renin-angiotensin system 2, 3,11,17,21,31. In rabbit 2°, cat 2, dog 32 and human 1, R V L has been shown to possess a high concentration of receptor binding sites to angiotensin II (Ang II), a neuropeptide implicated in the neurogenic control of BP 25'27 and the genesis of hypertension in spontaneously hypertensive rats (SHR) ~2,25,28. In normotensive rat, R V L neurons have been shown to lie in a matrix of angiotensin immunoreactive fibres and varicosities 16,17,18, suggesting that A n g II may be a neurotransmitter or neuromodulator at the R V L 2'18"21,31. Anesthetization of R V L with lidocaine has been shown to attenuate the pressor response to intracerebroventricular injection of Ang 1I26. Furthermore, microinjection of A n g II into R V L of anesthetized cat 2, rabbit 31 and normotensive rat 11 has been shown to produce a dose-dependent pressor effect and an increase in sympathetic nerve activity, which are blocked by the administration of A n g II antagonist, saralasin, whereas microinjection of saralasin into R V L produces a dose-dependent depressor response and a
decrease in sympathetic nerve activity 31. Taken together, these findings indicate that R V L may be responsible for mediating the tonic cardiovascular effects of brain A n g II. However, the ways by which brain A n g II and R V L neurons interact to produce the hypertensive effect remain elusive. In addition, the R V L of S H R , when compared with normotensive Wistar Kyoto rats (WKY), has also been demonstrated by our recent study to exhibit an enhanced cardiovascular responsiveness to electrical microstimulation 8 as well as to possess predominantly double discharge units with a fast conduction velocity and single discharge units with a higher firing rate than that of W K Y 9. In SHR, an altered interaction between R V L neurons and A n g II may possibly exist 25,28, thus contributing to a higher sympathetic outflow from R V L and ultimately an elevated arterial BP observed in SHR. The purposes of the present study are therefore, by employing iontophoretic method, (1) to determine whether Ang II acts directly on the spontaneously active cardiovascular neurons in R V L and (2) to compare the sensitivity and responsiveness of R V L cardiovascular neurons in W K Y and S H R to A n g II. Part of this study has been presented in an abstract 1°. Female W K Y (n = 12) and S H R (n = 11), weighing
1 Present address: The Salk Institute, 10010 North Torrey Pines Road, San Diego, CA 92186, U.S.A.
Correspondence: T.M. Wong, Department of Physiology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong. Fax: (852) 8559730.
146 210-230 g (19-20 weeks of age), were used in this study. The rat was anesthetized with pentobarbital sodium at a dose of 40 mg/kg intraperitoneally, and an adequate anesthesia was characterized by the absence of corneal reflex and no retraction of distal phalanges to nociceptive stimulation of the hindpaw 15. A supplementary dose of 20 mg/kg was given intravenously when the animal showed signs of wakefulness, such as (1) perturbations of BP and heart rate, and (2) increases in breathing rate. Experiments continued 20-30 min after intravenous administration of the supplementary dose of anesthetic. The femoral artery was cannulated with a polyethylene catheter which was connected to a Statham pressure transducer and a Gould recorder for the continuous measurement of BP throughout the experiment. The femoral vein was cannulated for the infusion of drugs. Electrocardiogram (ECG) signals were obtained from an E C G monitor via 4 platinum electrodes inserted into muscles of the limbs. The head of the animal was mounted onto a stereotaxic apparatus (Narishige). All pressure points were infiltrated with lidocaine. The rat was allowed to breathe spontaneously and the breathing rate was not significantly disturbed by any of the surgical procedures employed in this study. Body temperature was maintained at 37 °C with a heating pad. For iontophoresis, the occipital bone, the dura and pia mater overlying the cerebellum were removed to allow placement of the micropipette. Extracellular single unit activities of spontaneously active RVL cardiovascular neurons were recorded using a double-barrel glass micropipette, tip diameter 1.5/~m and DC resistance of 8-12 M£2, inserted vertically through the intact cerebellum in a dorsoventral fashion to the RVL on the right side according to the brain atlas of the rat 24. The glass micropipette was connected to an Electrometer (EEI, model 400B) for monitoring neuronal discharges and current injection. The barrel for recording RVL neuronal activities contained 10 -3 M saralasin dissolved in 2 M NaCl solution (pH 5.0) saturated with 7% Fast green. Saralasin was released by current injection to block the excitatory action of iontophoretic Ang II. The other barrel contained 10-3 M Ang II dissolved in distilled water (pH 3.5) for iontophoretic release of Ang II to the RVL cardiovascular units being studied. Each spontaneously active RVL cardiovascular unit was characterized by: (1) its barosensitivity in response to an intravenous bolus injection of 2-4/~g/kg phenylephrine, (2) synchronization of spontaneous discharge with the cardiac cycle, and (3) its spinal projection to the intermediolateral column (IML) using the antidromic collision test 15"22. To antidromically identify RVL neurons that project to IML of the thoracic spinal cord, the spinal cord was immobilized with a spinal clamp and laminectomy was performed
at T2-T 5 levels of the spinal cord to permit placement of an array of two stainless steel stimulating electrodes ipsilateral to the recording sites. The exposed nervous tissues were covered with warm paraffin oil. Monopolar cathodal pulses (0.5 ms, 0.5 Hz, single shock) were delivered to sites in the IML regions and the stimulus current (40-60/~A) was monitored on a storage oscilloscope. Those units with bursting activity linked to the respiratory cycle were not studied further. Prior to iontophoretic application of Ang II, the initial discharge rate of an RVL cardiovascular neuron at the respective basal BP level of WKY (mean arterial pressure, MAP = 101.6 + 2.3 mmHg) or SHR (MAP -- 161.1 _+ 1.8 mmHg) was first determined. Cationic current in the range of 10-60 nA was then applied to the barrel c o n t a i n i n g 10 -3 M Ang II for 1 min to release the Ang II to the RVL cardiovascular unit. It was demonstrated in our control study that iontophoretic application of saline at a current intensity of 10-50 nA did not alter the discharge rate of RVL cardiovascular units, and a current intensity of 60 nA would induce a 0.85% increase in the discharge rate, probably due to the direct depolarization effect of the injection current 15. In both types of rats, iontophoretic application of saline at a current intensity of 10-60 nA did not alter the cardiovascular parameters. Compensation anionic current (10-15 nA) was used to prevent any spontaneous leakage of drugs from the glass micropipette, and direct depolarization of the RVL neurons during current injection is. For each responsive RVL cardiovascular unit, the threshold current, which was defined arbitrarily as the current intensity eliciting a 10% change in discharge rate of an RVL neuron by iontophoretically released Ang II, was also determined. The interval between successive iontophoretic applications of Ang II to RVL cardiovascular units was at least 30 min to ensure complete recovery of BP and neuronal activities since the half-life for dissociation of Ang II from the Ang II receptors in WKY and S H R has been found to be around 15 min 28. To confirm the excitatory action of Ang II, the effect of Ang II blockade was studied by applying cationic current in the range of 10-60 nA simultaneously to both barrels of the glass micropipette for 1 min to enable concomitant iontophoretic release of Ang II and saralasin to the unit being studied. The amount of Ang II released was not determined in the present study since it has been demonstrated that the quantity released increases linearly with the intensity of ejecting current up to about 60 nA and the rate of release is approximately 41.5 + 4.8 fmol/nA/min 15. This guideline was employed in the present study. The position of the electrode in RVL was determined before electrophysiological recording with the aid of electrical microstimulation that would elicit the charac-
147 teristic pressor responses as described previously8. At the end of each experiment, Fast green was iontophoretically deposited at the most ventral site of pipette penetrations. Frozen coronal sections of the brain and spinal cord were cut at 60/zm with a sliding microtome. The recording and antidromic stimulation sites were reconstructed from Cresyl violet-stained serial frontal sections with reference to the dye deposits. Histological identification of RVL was based on anatomical landmarks described by Haselton and Guyenet 15 and Ruggiero et al. 3°. Only units within the confines of RVL were included in this study. Statistical comparisons between groups were made by using a one-way analysis of variance (ANOVA). A difference at the level of P < 0.05 was considered statistically significant. In agreement with our previous study, two kinds of discharge patterns, namely single and double discharges, were identified in the cardiovascular neurons of RVL in both types of rats; the RVL of SHR possesses predominantly double discharge units with a fast conduction velocity and single discharge units with a higher firing rate than that of WKY 11 (see Table I). In the present study, iontophoretic application of Ang II to the RVL increased the discharge rates of 30% of single discharge (WKY, n = 52 out of 150 sampled; SHR, n = 42 out of 118 sampled) and 30% of double discharge (WKY, n -21 out of 69 sampled; SHR, n = 61 out of 184 sampled) cardiovascular units dose-dependently in both WKY and SHR (Fig. 2). The increase in spontaneous discharges was abolished by co-administration of saralasin, indicating that the responses were mediated by specific Ang II receptors on the R V L cardiovascular neurons (Fig. 1A,B). The cardiovascular neurons excited by iontophoretic Ang II were found to distribute within RVL at coordinates 11.6-13.3 mm posterior to bregma, lateral
1.9-2.1 mm, and 6.0-6.7 mm deep from the brain surface (Fig. 1C). In both types of rats, the number of Ang II-responsive cells found in the medial and ventral portion of the RVL was 8-12% more than from regions closer to the subcompact division of the nucleus ambiguus. The number of responsive cells also became scarce at the lateral and medial border of the RVL. The remaining 70% of the cardiovascular neurons did not respond to iontophoretic application of Ang II even at a higher ejecting current of 100 nA, suggesting that the pressor response to microinjection of Ang II to RVL was mediated by some but not all of the R V L cardiovascular neurons. These unresponsive R V L cardiovascular neurons were scattered among the responsive cells. In both WKY and SHR, the increase in discharge rate elicited by iontophoretic Ang II was much greater in double discharge units than in single discharge units (Figs. 1 and 2). In comparison, both single and double discharge RVL units in SHR showed a statistically lower threshold current, shorter latency (Table I), greater increase in discharge rate and longer duration of response than those in WKY (Fig. 2), indicating a greater sensitivity and responsiveness of R V L cardiovascular neurons in SHR to Ang II. 80% of the Ang II-responsive units were also tested for their spinal projection and all of the tested units were found to project to the IML of the thoracolumbar spinal cord, a region from which the preganglionic neurons arise 22, suggesting that the responsive units may be important in determining the sympathetic vasomotor outflow to the peripheral vascular beds. In both types of rats, there was invariably an increase in mean arterial pressure following application of Ang II to RVL in the order of 2-5 m m H g in WKY and 4-7 m m H g in SHR; these values were too small for reliable statistical comparison. The pressor response was accompanied by a
TABLE I Response characteristics of RVL cardiovascular neurons to iontophoretically applied angiotensin H WKY
Responsive cells No. (total sampled) % Firing rate (Spikes/s) Conduction velocity (m/s) Slow-conducting group (%)
SHR
Single discharge
Double discharge
Singledischarge
Double discharge
52 (150) 34.7% 16.7 + 0.4
21 (69) 30.4% 21.2 + 1.4
42 (118) 35.6% 23.1 + 0.9***
61 (184) 33.2% 22.4 + 1.1
0.48 + 0.01 (35.6%) 2.67 + 0.06 (56.3%) 19.3 + 0.60 18.2 + 0.42
0.58 + 0.03 (1.1%) 3.2 + 0.02 (7.0%) 17.4 _+0.80 16.8 + 0.50
0.55 __+0.02
0.78 + 0.05 (3.8%) 4.23 + 0.11 (50.1%) 10.3 + 0.60*** 12.3 + 0.43*
(36.2%) Fast-conducting group (%) 2.75 + 0.03 (9.9%) Threshold current (nA) 12.5 + 0.30*** Latency(s) 15.9+0.11"** Values are mean + S . E . M . *,*** statistically different from corresponding values in WKY at levels P < 0.05 and ANOVA.
P
