ANG II modulates both slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rabbit rostral ventrolateral medulla.

Am J Physiol Regul Integr Comp Physiol 306: R538–R551, 2014. First published February 12, 2014; doi:10.1152/ajpregu.00285.2013.

ANG II modulates both slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rabbit rostral ventrolateral medulla Takeshi Saigusa and Jun Arita Department of Physiology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan Submitted 10 June 2013; accepted in final form 7 February 2014

Saigusa T, Arita J. ANG II modulates both slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rabbit rostral ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol 306: R538 –R551, 2014. First published February 12, 2014; doi:10.1152/ajpregu.00285.2013.—This study investigated the effects of ANG II on slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rostral ventrolateral medulla (RVLM) in urethane-anesthetized rabbits to determine whether the sympathetic baroreflex modulation induced by application of ANG II into the RVLM can be explained by the total action of ANG II on individual RVLM neurons. In response to pharmacologically induced slow ramp changes in mean arterial pressure (MAP), individual RVLM neurons exhibited a unit activity-MAP relationship that was fitted by a straight line with upper and lower plateaus. Iontophoretically applied ANG II raised the upper plateau without changing the slope, and, thereby, increased the working range of the baroreflex response. An asymmetric sigmoid curve that was determined by averaging individual unit activity-MAP relationship lines became more symmetric with ANG II application. The characteristics of the average curves, both before and during ANG II application, were consistent with the renal sympathetic nerve activity-MAP relationship curves obtained under the same experimental conditions. ANG II also affected rapid baroreflex responses of RVLM neurons that were induced by cardiac beats, as application of ANG II predominantly raised the average unit activities in the downstroke phase of arterial pulse waves. The present study provides a possible explanation for the ANG II-induced sympathetic baroreflex modulation based on the action of ANG II on barosensitive bulbospinal RVLM neurons. Our results also suggest that ANG II changes both static and dynamic characteristics of baroreflex responses of RVLM neurons. angiotensin II; baroreflex; sympathetic nervous system; single-unit recording; medulla oblongata THE ROSTRAL VENTROLATERAL medulla (RVLM) contains a group of neurons that project directly to the sympathetic preganglionic neurons in the spinal cord (8, 16, 20, 38) and that provide an essential source of the sympathetic nerve activity (SNA) to maintain mean arterial pressure (MAP) within normal levels (14, 21, 43, 53). A substantial portion of the bulbospinal neurons in the RVLM receive inhibitory inputs driven by arterial baroreceptor inputs, and, thus, display phasic activities that are synchronized to the cardiac cycle (8, 28, 55). Since blockade of inhibitory neurotransmission in the RVLM largely interferes with sympathetic baroreflex responses (7), the barosensitive bulbospinal neurons in the RVLM are believed to be a key component of the sympathetic baroreflex mechanism.

Address for reprint requests and other correspondence: T. Saigusa, Dept. of Physiology, Interdisciplinary Graduate School of Medicine and Engineering, Univ. of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan (e-mail: [email protected]). R538

ANG II is one of the peptides that have been identified in nerve terminals within the RVLM (13). Although the origins of the ANG II-containing axon tracts have not been fully identified, several supramedullary regions, such as the lateral hypothalamic area and the lateral parabrachial nucleus, are known to contain ANG II-immunopositive neurons that project to the RVLM (40). Therefore, it is possible that neurogenic ANG II may be released in the RVLM when these supramedullary regions are activated. ANG II receptors are also localized in the RVLM (1, 2, 4, 5, 25). Blockade of ANG II receptors in the RVLM decreases the baseline levels of SNA (44, 45) and attenuates sympathetic responses evoked by stimulation of the hypothalamic paraventricular nucleus (PVN) (54), which suggests that endogenous ANG II or related peptides are involved in controlling the sympathetic outflow. When ANG II is exogenously applied to the RVLM, a pressor response is induced that is, in part, mediated by an increase in SNA (2, 39, 47). Because the RVLM is a key component of the sympathetic baroreflex mechanism, it is possible that ANG II modulates not only the baseline SNA, but also sympathetic baroreflex functions. When we previously tested this hypothesis, we found that application of ANG II to the RVLM in anesthetized rabbits modulates the renal sympathetic baroreflex induced by slow ramp changes in MAP (44, 45). ANG II resets the renal sympathetic nerve activity (RSNA)-MAP relationship curve to a higher-pressure area that parallels an increase in baseline MAP. ANG II increases the upper plateau of the curve (the maximum RSNA that can be evoked by lowering blood pressure), while the lower plateau of the curve (the minimum RSNA that can be evoked by increasing blood pressure) and the average slope of the curve are not affected. Therefore, ANG II expands the working range of the renal sympathetic baroreflex, which enables a sensitive baroreflex control over a wider range of MAP. The upper plateau of the sympathetic baroreflex curve is thought to reflect the excitatory capacity of the sympathetic motoneuron pool when baroreceptor inputs are unloaded (18). Thus, the previously mentioned results suggest that ANG II activates the barosensitive bulbospinal neurons in the RVLM, and consequently, increases excitatory inputs to sympathetic preganglionic neurons. This idea is further supported by results showing that iontophoretically applied ANG II increases the baseline activity of a subpopulation of barosensitive bulbospinal RVLM neurons in rats (9). However, the overall effects of ANG II on the baroreflex responses in these neurons have not been fully characterized. On the other hand, when ANG II is applied intravenously, it attenuates barosensitivity of splanchnic sympathetic nerve activity, which is accompanied with reduced barosensitivity of approximately half of the barosensitive bulbospinal RVLM neurons (36). This observation sug-

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ANG II-INDUCED BAROREFLEX MODULATION

gests that the attenuation of sympathetic baroreflex induced by circulating ANG II is a result of altered barosensitivity of cardiovascular premotor neurons in the RVLM. The first purpose of the present study was to determine whether the sympathetic baroreflex modulation induced by local application of ANG II into the RVLM in urethaneanesthetized rabbits can be explained by the total action of iontophoretically applied ANG II on individual RVLM neurons. For this purpose, we derived the average baroreflex curves both before and during ANG II application from those of individual RVLM neurons and compared them with the renal sympathetic baroreflex curves. The second purpose of the present study was to determine whether ANG II modulates not only the static (time-independent) baroreflex property but also dynamic (temporal) baroreflex property of both the RVLM neuronal activities and the sympathetic nerve activities. For this purpose, we analyzed the rapid baroreflex responses of RVLM neuronal activities and RSNA that were induced by pulsatile changes in arterial pressure (AP). Since manipulation of blood pressure when assessing the slow ramp baroreflex induces considerable displacement of the medulla, it is difficult to maintain stable unit recordings with conventional glass electrodes. Therefore, we developed a carbon-fiber floating electrode that was able to move as the brain moved and was used for single unit recordings in the present study. MATERIALS AND METHODS

Experiments were performed on 41 male Japanese-White rabbits that weighed 2.4 –3.8 kg. The rabbits were housed in individual cages (room temperature: 23–25°C) with free access to laboratory chow and tap water. All protocols were approved by the Ethical Committee of Animal Experiments of the University of Yamanashi. General Procedures The rabbits were pretreated with scopolamine methylbromide (50 ␮g/kg iv; Sigma, St. Louis, MO) to reduce bronchial secretion and to inhibit vagal reflexes during surgical procedures. The animals were anesthetized with a slow intravenous infusion of a 10% urethane solution (1.5 g/kg, 0.05 g·kg⫺1·min⫺1; Sigma) (22, 45). The adequacy of anesthesia was verified by the absence of a withdrawal reflex to hindpaw pinching, and, if required, a supplementary dose of urethane (0.1– 0.2 g/kg) was given. Rectal temperatures were monitored with a thermistor probe and were kept at 38 –39°C with an electric heating pad. The rabbit was tracheotomized and allowed to breathe spontaneously with oxygen-enriched room air. End-tidal CO2, which was monitored with a CO2 analyzer (CapStar-100; CWE, Ardmore, PA), ranged between 3 and 4%. The left central ear artery was cannulated with a plastic catheter to measure pulsatile AP and MAP. ECG was recorded to calculate heart rate (HR) and, in unit-recording experiments, to construct cardiac phase-synchronized spike histograms. The rabbit was placed in a stereotaxic apparatus, and the neck was flexed in the ventral direction, so that the lambda-bregma line inclined at an angle of 75° with respect to the horizontal plane. This alignment placed the medulla at an angle of 30° with the rostral side down. The dura mater was cut through a midline incision between the atlas and the occipital crest to expose the dorsal surface of the medulla for drug applications and/or single-unit recordings. Slow-ramp baroreflex. Reflex changes in RSNA and unit activities in response to both slow ramp increases and decreases in MAP were recorded to construct RSNA-MAP and unit activity-MAP relationship curves, respectively. The slow ramp changes in MAP were induced by infusions of L-phenylephrine hydrochloride (PHE; 250 ␮g/ml, Sigma) and nitroglycerin (NG; 500 ␮g/ml; Millisrol, Nippon Kayaku, Tokyo,

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Japan) into the right marginal ear vein via a triple-lumen vinyl catheter (45). These PHE and NG solutions were alternately infused for 30 – 60 s with a minimum of 5 min between each infusion. Each infusion began at a rate of 25 ␮l·kg⫺1·min⫺1, and when MAP started to change, the rate was gradually increased up to 250 ␮l·kg⫺1·min⫺1 so that MAP changed in a linear fashion at a rate of 1–2 mmHg/s. Baseline levels of MAP, RSNA, and unit activities were calculated from the average values in the 30 s just before the NG and PHE infusions. Single-Unit Recordings Manufacturing of the electrode. For each electrode that was used, the main body was manufactured with seven-barreled borosilicateglass capillaries (OD: 1.00 mm, ID: 0.56 mm, with a glass filament in each capillary except for the central one, Hilgenberg, Germany). A single-strand carbon fiber (7 ␮m) was inserted into the central capillary as a conductor to reduce the resistance of the recording electrode. This improved not only the signal-to-noise ratio of the unit recordings (especially when the recordings were combined with iontophoretic application of drugs), but also the stability of the unit recordings against brain movements (by increasing the resistance-coupling components of the signal and relatively reducing capacitance-coupling components) (37). The capillaries were pulled using a vertical puller (PE-2; Narishige, Tokyo, Japan) with a two-stage pulling process. A Cashew resin-coated tungsten wire (0.1 mm␾; Unique Medical, Tokyo, Japan) was connected to the carbon fiber inside the central capillary with electrically conductive glue (Silver Epoxy; World Precision Instruments, Sarasota, FL). This tungsten wire protruded 15 mm out of the open end of the capillary to serve as a flexible shaft for the electrode and as a cable for signal output. The carbon fiber that protruded from the tip of the electrode was cut so that only 10 ␮m protruded and sharpened with a spark-etching method (37). Using a microscope, a tungsten electrode with a tip diameter of 5 ␮m was placed close to the carbon fiber that protruded from the tip of the glass electrode. High-voltage (100 –200 V) pulses were then applied between the tungsten electrode and the carbon fiber to generate small sparks. This gradually trimmed and cut the carbon fiber, so that it was as sharp as a fine metal electrode. Recordings. For each recording, the carbon-fiber electrode was vertically hung from an electrode holder, and its flexible shaft was temporarily fixed to a platinum heating coil installed on the holder with a drop of cetyl alcohol (melting point 49 –53°C). The electrode was then inserted into the dorsal surface of the medulla at the rostrocaudal level of the obex (AP0) ⫾ 0.5 mm and 3.2 ⫾ 0.2 mm lateral to the midline. The electrode was then advanced 5.5 ⫾ 1.0 mm at an angle of 30° rostroventral to the coronal planes. When a unit recording was established, the heating coil on the electrode holder was warmed with a DC current (1.5 A) to melt the cetyl alcohol that fixed the shaft of the electrode to the heating coil. This released the electrode from the holder and allowed it to move with the brain. The electrode could subsequently be released from and fixed to the holder repeatedly, by warming and cooling the heating coil, respectively. The signal from the electrode was amplified and filtered (bandwidth 300 Hz–3 kHz), and a single-unit activity was selected with a voltage-gated window discriminator. The firing rates of the single unit were stored on a hard disk with MAP and HR for later analyses. The neural signal, pulsatile AP, and ECG were sampled at a rate of 25 kHz with another computer system to construct perievent spike histograms and to identify spinal projection of the neuron, as described in Single-Unit Recording Experiments. At the conclusion of each experiment, the last recording site was marked with application of a DC current (0.1 mA, 10 s) using the carbon-fiber electrode as a cathode to allow histological identification. Sympathetic Nerve Recording Experiments A renal nerve electrode was implanted as previously described (45). The signal from the electrode was amplified, filtered (bandwidth

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00285.2013 • www.ajpregu.org

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100 Hz–1 kHz), full-wave rectified, and then integrated with a time constant of 0.3 s. The integrated RSNA was stored on a hard disk with MAP for later analyses. In rapid baroreflex experiments, the filtered RSNA, pulsatile AP, and ECG were sampled at a rate of 25 kHz with another computer system for later analysis. L-glutamate monosodium salt (Glu) and ANG II were administered into the RVLM with a glass micropipette connected to a hydraulic microinjection system, as previously described (44, 45). The micropipette was inserted into the dorsal surface of the medulla at AP0 and 3.2 mm lateral to the midline at an angle of 30° rostroventral to the coronal planes and was then advanced up to a depth of 6 mm. Glu (10 mM; Sigma) dissolved in normal saline was microinjected with a volume of 100 nl, and ANG II (0.2 mM; Sigma) dissolved in artificial cerebrospinal fluid (31) was microinfused at a rate of 20 nl/min. At the beginning of each experiment, Glu (1 nmol) was microinjected into the RVLM to determine the position of maximum renal sympathetic activation. ANG II (4 pmol/min) or vehicle solutions were then microinfused into this site for 20 min in a random order with a minimum of 1 h between infusions. This dose of ANG II was used to give a pressor response of 10 –15 mmHg based on the results of previous experiments (44, 45). In slow ramp baroreflex experiments, RSNA-MAP relationship curves were derived from NG/PHEinduced slow ramp changes in MAP 10 –20 min before and after the initiation of the ANG II or vehicle microinfusion. In rapid baroreflex experiments, RSNA-AP relationship curves were derived from the phasic responses of RSNA and AP caused by cardiac beats 0 –1 before and 5– 6 min after the initiation of ANG II or vehicle microinfusion. At the conclusion of each experiment, the ganglionic blocker hexamethonium (10 mg/kg; Sigma) was intravenously injected to measure the baseline noise level in the RSNA recording. This was later subtracted from the original integrated RSNA values. The microinfusion site was marked with a 2% pontamine sky blue solution (50 nl) to allow histological identification. Single-Unit Recording Experiments Identification of barosensitive bulbospinal neurons. Single-unit activities were recorded in the RVLM with carbon-fiber floating electrodes. For each recording, it was confirmed that iontophoretically applied Glu promptly increased the unit activity. This ensured that the iontophoretic capillaries that surrounded the recording electrode were located close enough to the somato-dendritic portion of the neuron that was being recorded. A perievent spike histogram triggered by R-waves on the ECG (time span: 0.5 s; bin width: 5 ms) and simultaneously sampled and averaged AP and ECG curves were constructed to identify the barosensitivity of the neuron. If the spike histogram showed an inhibitory pattern in response to the peaks of the averaged AP wave, the neuron was determined to be “barosensitive”. A collision test was then conducted to identify a direct projection of the neuron to the spinal cord. According to the method established by Terui et al. (55) in rabbits, electrical stimuli were applied to the dorsolateral funiculus of the spinal cord. The dorsal surface of the upper thoracic vertebrae was exposed with a midline incision and was then held with a vertebral clamp. The second thoracic vertebra was partially laminectomized, and a small hole was made in the dura. A Parylene-coated tungsten unipolar electrode (0.1 mm of the tip was exposed, 0.1 M⍀ at 1 kHz; World Precision Instruments) was inserted just lateral to the dorsal root entry of the spinal cord (1.2–1.5 mm from the midline) and ipsilateral to the site of the unit recording. The depth of the electrode from the spinal surface was adjusted 0.5–1.0 mm to obtain the maximum pressor response to electrical stimulation (negative 0.2 mA, duration 0.5 ms, interval 20 ms, 50 trains). The hole in the dura was covered with agar, and then the electrode was fixed to the adjacent vertebrae with a self-curing silicone elastomer (Kwik-Sil, World Precision Instruments). The RVLM neuron that was being recorded was examined to determine whether it could be activated antidromically with a constant latency by a single electrical pulse

applied to the spinal cord (negative 0.6 mA, duration 0.5 ms). If the neuron was antidromically activated, it was then determined whether this activation disappeared if the spinal stimulation were applied within a critical period following a spontaneous firing of the neuron. Only the neurons with confirmed barosensitivities and spinal projections were used in the following experiments. Microiontophoresis. The capillaries that surrounded the carbonfiber electrode were filled with ANG II (0.01–5 mM, pH 4.0), Glu (0.5 M, pH 8.0), and normal saline (for current balance). A carbon fiber that was used for current supply was inserted into each capillary and, to ensure electrical insulation from other capillaries, the upper ends of the capillaries were sealed with a self-curing silicone elastomer. ANG II and Glu were iontophoretically ejected with currents of ⫹50 nA and ⫺50 nA, respectively, and the retaining currents were set to ⫺10 nA and ⫹10 nA, respectively. Baseline experiments. After each stable unit recording, cumulatively increasing doses of ANG II (0.01, 1, and 5 mM or 0.1, 1, and 5 mM) were iontophoretically applied for 3 min each, and the baseline unit activity at each dose was evaluated. Since the neuronal response to ANG II achieved a steady state in 2 min, the average firing rate between 2 and 3 min was adopted as the baseline unit activity for each dose. Slow ramp baroreflex experiments. Unit activity-MAP relationship curves were derived from NG- and PHE-induced slow ramp changes in MAP both before and during iontophoretic application of 1 mM ANG II. This dose was chosen from the above baseline experiments because, on average, it induced submaximum activation of the neurons. Similar to the RSNA baroreflex experiments, the unit activityMAP relationship curves were evaluated 10 –20 min before and after the initiation of the ANG II applications. Rapid baroreflex experiments. The effects of ANG II on the phasic responses of unit activities caused by arterial pulse waves were assessed. Spike histograms triggered by consecutive R-waves on the ECGs and simultaneously sampled and averaged AP curves were constructed both 0 –1 min before and 2–3 min after the initiations of iontophoretic applications of ANG II (1 mM). Phasic unit activitypulsatile AP relationship curves were then derived from these data. Histology At the completion of each experiment, the animal was euthanized with pentobarbital sodium (50 mg/kg iv). The medulla was removed and immersed in 10% neutral buffered formalin for 1 wk and then immersed in a 30% sucrose solution overnight. Frozen coronal sections (40 ␮m) were cut through the medulla, and alternate sections were then counterstained with cresyl violet. Both the stained and unstained sections were scanned into a computer, and the microinjection sites and sites of unit recordings were identified with a standard rabbit-brain atlas (50). Data Analysis RSNA-MAP relationship. The RSNA-MAP paired data derived from slow ramp changes in MAP were averaged over 2-s intervals and fitted into five-parameter asymmetric sigmoid curves using Igor Pro (WaveMetrics, Lake Oswego, OR) data analysis software. The fiveparameter sigmoid model, originally introduced for evaluation of asymmetry of baroreflex curves by Ricketts and Head (42), is an extended model of the more well-known four-parameter symmetric sigmoid model that uses two curvature parameters instead of just one. The equation of this model is RSNA ⫽ P1 ⫹

P2 1 ⫹ w · e⫺P3(MAP⫺P4) ⫹ (1 ⫺ w) · e⫺P5(MAP⫺P4)

, (1)

where P1 is the lower plateau; P2 (⬎ 0) is the range between the upper and lower plateaus; P3 and P5 are the curvature parameters (or the range-independent gains) that operate predominantly at lower and upper MAPs, respectively, and that have negative values when RSNA



is negatively correlated with MAP; P4 is the MAP at half the baroreflex response, or MAP50. Finally, w is a sigmoid function that is given by the following equation, which determines the weights of the two exponential terms in Eq. 1 depending on MAP: w⫽

1

1⫹e

Cw(MAP⫺P4) ,

(2)

where Cw is the curvature of w, defined as the absolute value of the harmonic mean of the two curvature parameters (P3 and P5). As P3 approaches P5, the curve becomes symmetric, and when P3 equals P5, the curve is identical to a four-parameter symmetric sigmoid curve. To evaluate the asymmetry of the curve, the following index was calculated (42): Asymmetry index ⫽

P3 ⫺ P5 ¯ C

,

(3)

where C is the mean of the two curvature parameters. When P3 and P5 are of the same sign, the asymmetry index has a value between ⫺2 and ⫹2. When this index has a positive or negative value, the curve skews to the left (lower MAPs) or to the right (higher MAPs), respectively. The slope at MAP50 is given by P2·C/4. Because there was considerable variation between rabbits in the amplitudes of the raw nerve activities (in ␮V·s), the RSNA-MAP curves were normalized for each rabbit by expressing RSNA in terms of the upper plateau of the control curve, which was equal to 100 normalized units (n.u.). Unit activity-MAP relationships. Similar to the RSNA-baroreflex analyses, the data points of unit activities (in terms of firing rates) and MAP during slow ramp changes in MAP were averaged over 2-s intervals and fitted into the function defined below. A straight-line model was used rather than a curvilinear model, such as a sigmoid curve, because the unit activity-MAP relationships obtained from individual neurons demonstrated good linearity almost throughout the range where unit activities were dependent on MAP. The model consisted of three segments: a horizontal line for the subthreshold portion (the upper plateau), a tilted line for the MAP-dependent portion, and another horizontal line for the saturated portion (the lower plateau). Since all of the barosensitive neurons that were recorded in the present study completely ceased to fire when MAP exceeded a certain level, the value of the lower plateau was considered to be zero. Therefore, the fitting model was defined by the following equations that include three fitting parameters (P1–P3): Unit activity P1

冦

if MAP ⬍ MAPthreshold ,

⫽ P1[P2(MAP ⫺ P3) ⫹ 1 ⁄ 2] if MAPthreshold ⱕ MAP ⱕ MAPsaturation , (4) 0 if MAP ⬎ MAPsaturation ,

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ECGs and simultaneously sampled and averaged AP curves. The data in the spike histograms were smoothed with a 10th-order binomial filter (⫺3 dB at ⬃17 Hz) and spline-interpolated at an interval of 1 ms to obtain unit-activity curves. The unit-activity curves were then normalized in terms of the average level of the control curve in each unit, which was equal to 100 n.u. Because the neural conduction and transmission from arterial baroreceptors to RVLM neurons are timeconsuming processes, a pure delay, or dead time, consistently existed in the relationship between the pulsatile APs and the phasic unit activities. Therefore, unit-activity curves were phase-adjusted to offset these pure delays, which were derived from the time difference between the peak of the APs and the nadir of unit activities in the control curves. After that, unit activity-AP relationship curves before and during ANG II were derived. Finally, to compare the effects of ANG II on unit activities between the upstroke (anacrotic) and downstroke (catacrotic) phases of arterial pulse waves, the average unit activities in each phase were calculated. RSNA-pulsatile AP relationships. The RSNA signals were fullwave rectified and integrated every 5 ms, the same interval as the bin width of spike histograms, and then RSNA, AP, and ECG curves that were synchronized to consecutive R-waves were averaged. Using the same procedure to derive a unit-activity curve, as described above, the averaged RSNA curve was smoothed, interpolated, normalized, and then phase-adjusted. From the phase-adjusted data, RSNA-AP relationship curves before and during ANG II or vehicle application were derived. To compare the effects of ANG II on RSNA between the upstroke and downstroke phases of arterial pulse waves, the average of RSNA in each phase was calculated. Statistical Analysis Values are expressed as means ⫾ SE. All of the statistical tests were performed with SPSS 15.0J (SPSS/IBM), and P ⬍ 0.05 was considered to be statistically significant. In the RSNA-recording experiments, the relationships between the depths of Glu injection sites and the AP and RSNA responses were analyzed using the one-way ANOVA with repeated measures and polynomial contrasts. The effects of ANG II on baroreflex and baseline parameters were assessed using the two-way ANOVA with repeated measures that includes two factors, drug (vehicle and ANG II) and time (before and during drug application). If drug ⫻ time interaction was significant, the data were divided into subsets, including a single level of either factor, and then the effect of another factor was assessed using the paired t-test with Bonferroni adjustment. In the single-unit recording experiments, the effects of different doses of ANG II on baseline unit activities were assessed using the one-way ANOVA and polynomial contrasts, and the baroreflex and baseline parameters before and during ANG II applications were compared using the paired t-test. RESULTS

where MAPthreshold ⫽ P3 ⫹ MAPsaturation ⫽ P3 ⫺

1 2P2 1 2P2

; ;

(5) (6)

P1 (⬎0) is the range between upper and lower plateaus; P2 is the range-independent gain between the threshold and saturation MAPs, which has a negative value when the unit activity is negatively correlated with MAP and has an actual slope given by P1·P2; and P3 is the MAP at half the response, or MAP50. Similar to the RSNAMAP baroreflex curves, the unit activity-MAP relationship lines were normalized for each unit by expressing firing rates in terms of the upper plateau of the control baroreflex, which was equal to 100 n.u. Unit activity-pulsatile AP relationships. Relationships between pulsatile AP and phasic unit activities were analyzed on the basis of perievent spike histograms triggered by consecutive R-waves on the

Slow-Ramp RSNA Baroreflex Experiments We previously reported that when ANG II is microinfused into the RVLM, the working range of the RSNA baroreflex that is induced by slow ramp changes in MAP expands. Therefore, in the present study, we performed experiments to confirm these findings using a more refined analytical model and also to directly correlate these findings with the effects of ANG II on the unit activities of barosensitive bulbospinal RVLM neurons. At the beginning of each experiment, Glu (1 nmol) was microinjected into the RVLM to determine the position of maximum renal sympathetic activation (Fig. 1A). Glu induced inhibitory, excitatory, or biphasic RSNA responses depending on the sites of injections (Fig. 1B, bottom). As depths increased from 4.5 to 6.0 mm (D4.5 to D6.0), the average excitatory and inhibitory components obtained from six rabbits increased and


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Fig. 1. Effects of glutamate (Glu) microinjections into the rostral ventrolateral medulla (RVLM) on mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). A: schematic drawings of a sagittal section 3.2 mm lateral to the midline (L3.2) (left) and a coronal section 3 mm rostral to the obex (A3) of the rabbit medulla oblongata (right), respectively. A micropipette for drug injections was inserted through the dorsal surface of the medulla at the rostrocaudal level of the obex (AP0) and 3.2 mm lateral to the midline at an angle of 30° with respect to coronal planes, as shown by the solid line, and was then advanced up to 6 mm in depth (D6) to reach the rostral ventrolateral medulla in the A3 coronal plane. FN, facial nucleus; RFN, retrofacial nucleus; NTS, nucleus of the solitary tract; IO, inferior olivary nucleus. B: typical responses of MAPs and RSNAs induced by microinjections of Glu (1 nmol) at D4.5-D6.0. C: average changes in MAPs and RSNAs induced by microinjections of Glu at various depths (n ⫽ 6). In the RSNA responses, excitatory and inhibitory components are shown as filled and open bars, respectively. Error bars represent means ⫾ SE.

8 4 0

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0

decreased, respectively (P ⬍ 0.01 for the linear trends over D4.5–D6.0, Fig. 1C, bottom). The maximum excitatory responses were induced at D6.0, the most rostroventral site tested, which corresponded to a site close to the ventral surface of the medulla and just caudal to the facial nucleus. On the other hand, Glu consistently induced pressor responses in the range of D4.5 to D6.0 (Fig. 1B, top). The average pressor response revealed a peak at D5.0 (P ⬍ 0.01 for the quadratic trend over D4.5–D6.0, Fig. 1C, top). Namely, the site of maximum renal sympathoexcitation was located more rostroventrally to that of maximum pressor response. This finding was in good agreement with the topographical organization of the RVLM in cats that the region preferentially driving renal sympathetic outflow locates more rostroventrally to that driving visceral and muscle vasoconstrictors, which mainly determine the total peripheral vascular resistance (35). ANG II (4 pmol/min) was infused into the sites where Glu induced the maximum sympathoexcitatory responses, and RSNA baroreflexes were assessed both before and during the ANG II infusions. A typical change of the RSNA baroreflex that was recorded from a rabbit is shown in Fig. 2A, left. When the data were subjected to the five-parameter asymmetric model, well-fitted RSNA-MAP relationship curves were ob-

2

4 6 time (min)

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tained both before and during ANG II infusion (Fig. 2A, right). The RSNA-MAP relationship curves that were produced by averaging the five parameters over the individual curves obtained from the six rabbits revealed that ANG II markedly increased the upper plateau (Fig. 2B, right). Statistical analyses of the averaged parameters showed that the increase in the upper plateau induced by ANG II was statistically significant (P ⬍ 0.05, Table 1) and that the lower plateau was not changed. This resulted in a significant increase in the reflex range (P ⬍ 0.05). The slope at MAP50 was not significantly changed. Therefore, ANG II expanded the working range of the renal sympathetic baroreflex, which enabled sensitive baroreflex control over a wider range of MAP. In addition, ANG II significantly increased MAP50 (P ⬍ 0.01) and shifted the reflex curve to the right. The asymmetry index of the pre-ANG II baroreflex curve significantly deviated from zero (⫹0.52 ⫾ 0.16, P ⬍ 0.05), which indicated that the baroreflex curve was asymmetric with a greater curvature at lower MAPs. ANG II reduced the asymmetry index to ⫹0.14 ⫾ 0.19, which was not significantly different than zero. ANG II significantly raised the baseline MAP (⌬MAP: ⫹10.7 ⫾ 2.0 mmHg, P ⬍ 0.01) but did not significantly change the baseline RSNA (⌬RSNA: ⫹9.7 ⫾ 5.3 n.u., Fig. 2B,


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Fig. 2. Effects of ANG II microinfusions into the RVLM on RSNA baroreflexes induced by slow ramp changes in MAP. A: typical example of RSNA baroreflexes before (dashed line and open symbols) and during (solid line and filled symbols) microinfusion of ANG II (4 pmol/min) into the RVLM. Left: ramp changes in MAP were induced by intravenous infusions of nitroglycerin (NG; circles) and L-phenylephrine hydrochloride (PHE; triangles). Right: RSNA-MAP baroreflex curves were constructed by fitting a five-parameter asymmetric sigmoid function to the RSNAMAP paired data. The square on each curve represents the baseline point. B: RSNA-MAP baroreflex curves before (dashed line) and during (solid line) infusions of either vehicle (left) or ANG II (right) in six rabbits. The curves were reconstructed with the average values of baroreflex parameters based on the five-parameter asymmetric model. RSNA was normalized in terms of the upper plateau of the control (pre-ANG II or pre-vehicle) baroreflex in each rabbit, which was equal to 100 normalized units (n.u.). The square and circle on each curve represent the baseline and midrange points, respectively. Error bars represent means ⫾ SE.

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right). Infusion of an equal volume of vehicle solution did not significantly alter any of the baroreflex or baseline parameters (Fig. 2B, left, Table 1). Single-Unit Recording Experiments Baseline experiments. Baseline single-unit activities were recorded before and during iontophoretic applications of three doses of ANG II in a total of 21 barosensitive bulbospinal RVLM neurons that were obtained from 14 rabbits. Fig. 3 shows an example of a single unit recording from a barosensitive bulbospinal RVLM neuron (A and B) and the response to both Glu and ANG II (C). The baseline firing rates before ANG II application ranged from 0.5 to 19.6 spikes/s (8.7 ⫾ 1.3 spikes/s). Although the effects of ANG II on baseline activities varied among neurons, on average, ANG II increased the baseline activities of neurons in a dose-dependent manner (P ⬍

140

0.01, Fig. 4A). The baseline MAP before ANG II application was 74.3 ⫾ 3.0 mmHg, and it was not significantly altered by iontophoretic application of any ANG II dose (⌬MAP: ⫺0.4 ⫾ 0.9 at 0.01 mM; ⫹0.2 ⫾ 1.0 at 0.1 mM; ⫹0.3 ⫾ 1.1 at 1 mM; and ⫺1.2 ⫾ 1.1 mmHg at 5 mM). Although it has been reported that ANG II predominantly activates RVLM neurons with relatively low firing rates in rat slice preparations (30), the percentage changes in unit activities observed with ANG II application (1 mM) in the present experiments did not significantly correlate with the baseline firing rates (Fig. 4B). The conduction velocities determined by antidromic activations of the neurons varied with a range of 4.4 –17.7 (10.9 ⫾ 0.9 m/s). There were no correlations between the effects of ANG II and the conduction velocities (Fig. 4C). Slow ramp baroreflex experiments. The effects of iontophoretically applied ANG II (1 mM) on the neuronal responses to

Table 1. Effects of ANG II infusion into the rostral ventrolateral medulla on the renal sympathetic nerve activity–mean arterial pressure relationship: five-parameter asymmetric model analysis Lower plateau (P1), n.u. Range (P2), n.u. Range-independent gains at lower MAPs (P3), 1/mmHg at higher MAPs (P5), 1/mmHg MAP50 (P4), mmHg Upper plateau (P1 ⫹ P2), n.u. Slope at MAP50 (P2 ⫻ C/4), n.u./mmHg

Pre-Vehicle

Vehicle

Pre-ANG II

ANG II

0.4 ⫾ 2.1 99.6 ⫾ 2.1

1.1 ⫾ 2.0 96.2 ⫾ 4.3

0.3 ⫾ 2.2 99.7 ⫾ 2.2

0.4 ⫾ 1.1 127.7 ⫾ 6.8*†

⫺0.18 ⫾ 0.03 ⫺0.11 ⫾ 0.01 83.9 ⫾ 2.7 100 ⫺3.6 ⫾ 0.4

⫺0.20 ⫾ 0.04 ⫺0.11 ⫾ 0.01 82.4 ⫾ 3.3 97.3 ⫾ 2.8 ⫺3.7 ⫾ 0.5

⫺0.18 ⫾ 0.02 ⫺0.14 ⫾ 0.02 ⫺0.11 ⫾ 0.02 ⫺0.12 ⫾ 0.01 83.6 ⫾ 3.3 92.5 ⫾ 4.2*† 100 128.1 ⫾ 6.6*† ⫺3.6 ⫾ 0.3 ⫺4.2 ⫾ 0.4 Values are expressed as means ⫾ SE; n ⫽ 6. MAP, mean arterial pressure; MAP50, MAP at half-maximum activation; C ⫽ (P3 ⫹ P5)/2; n.u., normalized units (100 n.u. ⫽ the upper plateau in each preinfusion baroreflex). *P ⬍ 0.05 vs. pre-ANG II. †P ⬍ 0.05 vs. vehicle. AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00285.2013 • www.ajpregu.org

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slow ramp changes in MAP were investigated in a total of 14 barosensitive bulbospinal RVLM neurons that were obtained from 11 rabbits. Typical changes in unit activities induced by ANG II infusion and the unit activity-MAP relationships are shown in Fig. 5A. Although the unit activities included spontaneous fluctuations, the overall profile of the unit activityMAP relationships was well fitted with a straight line, rather

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than a sigmoid curve, with upper (threshold) and lower (saturation) plateaus determined by three fitting parameters (Fig. 5A, right). Fig. 5B, left shows the individual fitted lines that were obtained from the 14 neurons both before and during ANG II application and were normalized in terms of the upper plateaus of their control lines. Statistical analyses of the averaged parameters of individual straight lines obtained from the

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Fig. 3. An example of a single-unit recording from a barosensitive bulbospinal neuron in the RVLM. A: perievent spike histogram triggered by R-waves on the ECG at intervals of ⬎0.5 s and simultaneously sampled and averaged arterial pressure (AP) and ECG curves (time span: 0.5 s; bin width: 5 ms; accumulated 282 times in this example). B: an example of the collision test to confirm monosynaptic projection(s) of the recorded neuron to the spinal cord. We examined whether the recorded neuron was activated antidromically () with a constant latency by a single electrical stimulation applied to the spinal electrode (constant current: 0.6 mA; duration: 0.5 ms; ). If the neuron was activated, we determined whether this activation disappeared if spinal stimulation was applied within a critical period (ⱕ6 ms, in this example) after a spontaneous firing of the neuron (Œ). Five traces are overlaid in each graph. C: typical response of a recorded neuron to iontophoretic application of Glu (0.5 M, ⫺50 nA) and cumulative doses of ANG II (0.1, 1, and 5 mM, ⫹50 nA). HR, heart rate;, UNIT, single-unit activity.

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Fig. 4. The effects of ANG II on the baseline activities of 21 barosensitive bulbospinal RVLM neurons. A: individual (Œ) and average (䊐) changes in baseline unit activities induced by cumulatively applied ANG II at doses of 0.01, 1, and 5 mM, or 0.1, 1, and 5 mM. Error bars represent means ⫾ SE. The dose-response curve was derived from the individual data points by fitting a semilogarithmic sigmoid curve. The numbers of individual data points for each dose are shown in parenthesis. B: correlation between the percentage changes in unit activities induced by ANG II (1 mM) relative to the baseline firing rates. The result of a linear regression is shown as a dashed line. r2: the coefficient of determination based on the linear regression. C: correlation between the percentage changes in unit activities induced by ANG II (1 mM) relative to the conduction velocities. The result of a linear regression is shown as a dashed line. AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00285.2013 • www.ajpregu.org


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Fig. 5. Effects of ANG II on the slow ramp MAP changeinduced baroreflexes of barosensitive bulbospinal RVLM neurons. A: typical example of the unit activity-MAP relationship of a barosensitive bulbospinal neuron in the RVLM before (dashed line and open symbols) and during (solid line and solid symbols) iontophoretic application of ANG II (1 mM, ⫹50 nA). Left: slow ramp changes in MAP were induced by intravenous infusions of NG (circles) and PHE (triangles). Right: unit activity–MAP-paired data were fitted with a straight-line model with threshold (upper plateau) and saturation (lower plateau) lines. The square on each line represents the baseline point. B: individual (left) and average (right) unit activity-MAP relationship curves before (dashed line) and during (solid line) iontophoretic application of ANG II (1 mM, ⫹50 nA, n ⫽ 14). The unit activities were normalized in terms of the upper plateau of each pre-ANG II curve, which was equal to 100 n.u. Each average curve was derived from the average of the individual curves. The square and circle on each curve represent the baseline and mid-range points, respectively. Error bars represent means ⫾ SE.

0 20

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14 neurons showed that ANG II significantly raised the upperplateau level (P ⬍ 0.01), and, therefore, increased the range of baroreflexes (Table 2). ANG II did not significantly change either the slope or MAP50. Consequently, the saturation MAP was significantly increased (P ⬍ 0.01), leading to an expanded working range of MAP. Fig. 5B, right shows average response curves of 14 neurons both before and during ANG II applications that were derived from the individual fitting lines by calculating the average unit activities at each MAP. The average curves were well approximated by the five-parameter asymmetric model and were quite similar to those observed in the RSNA-recording experiments. The curve before ANG II application was a sigmoid curve that was slightly asymmetric and had a greater curvature at lower MAPs. The range-independent gains at the lower and higher MAPs (P3 and P5) were ⫺0.15 and ⫺0.08/mmHg, respectively, which resulted in an asymmetry index of ⫹0.60. On the other hand, the average curve with ANG II application was Table 2. Effects of iontophoretic application of ANG II on the unit activity–MAP relationship in barosensitive bulbospinal RVLM neurons: three-parameter straight-line model analysis Pre-ANG II

ANG II

100 125.4 ⫾ 6.6* Upper plateau, range (P1), n.u. Range-independent gain (P2), 1/mmHg ⫺0.034 ⫾ 0.004 ⫺0.025 ⫾ 0.003* MAP50 (P3), mmHg 93.3 ⫾ 2.8 96.0 ⫾ 3.2 Slope (P1⫻P2), n.u./mmHg ⫺3.4 ⫾ 0.4 ⫺3.0 ⫾ 0.3 MAPthreshold [P3 ⫹1/(2P2)], mmHg 74.9 ⫾ 3.6 71.9 ⫾ 3.8 MAPsaturation [P3 ⫺1/(2P2)], mmHg 111.7 ⫾ 3.8 120.1 ⫾ 4.7* Values are expressed as means ⫾ SE; n ⫽ 14. *P ⬍ 0.05 vs. pre-ANG II.

more symmetric. The estimated values of P3 and P5 were ⫺0.10 and ⫺0.07/mmHg, respectively, which resulted in an asymmetry index of ⫹0.28. Thus, application of ANG II reduced the asymmetry of the average curve by reducing the curvature at the lower MAPs. Rapid baroreflex experiments. The effects of ANG II application on the phasic responses of unit activities caused by arterial pulse waves were assessed in 24 barosensitive bulbospinal RVLM neurons that were obtained from 10 rabbits (including six rabbits that were used in the baseline experiments). Fig. 6 shows a typical example of the phasic responses recorded from a single neuron both before and during ANG II application. The unit activities were decreased and increased in response to the rise and fall of APs, respectively, with a delay of tens of milliseconds (Fig. 6A). The phase-adjusted unitactivity curve revealed that unit activities decreased in an almost linear fashion when correlated to APs in the upstroke phase of the pulse wave. By contrast, the unit activities increased in a monomodal, bimodal, or polymodal pattern in the downstroke phase (Fig. 6, B and C). In most of the neurons that were recorded, the unit activities reached maximum peaks at the end of the rapid fall in APs and reached other peaks at the end of the downstroke phase. On average, ANG II raised the maximum unit activity by 42.0 ⫾ 13.3 n.u. and raised the minimum by only 13.8 ⫾ 5.4 n.u. (Table 3). This change in the maximum was significantly greater than the change in the minimum (P ⬍ 0.05, Fig. 7, left), which led to increases in the amplitudes of the phasic unit activities. ANG II also significantly increased the overall unit activities (P ⬍ 0.05, Table 3). When the effects of ANG II on average unit activities were compared between the upstroke and downstroke phases of the pulse wave, it became


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apparent that ANG II significantly increased the average unit activities with respect to time in both phases (P ⬍ 0.05). However, the increase in the downstroke phase was significantly greater than that in the upstroke phase (P ⬍ 0.01, Fig. 7, middle). The average levels of APs with respect to time were significantly different between phases (upstroke vs. downTable 3. Effects of iontophoretic application of ANG II on arterial pressure-related phasic unit activities of barosensitive bulbospinal RVLM neurons Peak unit activities Maximum, n.u. Minimum, n.u. Average unit activities with respect to time Overall, n.u. Upstroke, n.u. Downstroke, n.u. Average unit activities with respect to arterial pressure Overall, n.u. Upstroke, n.u. Downstroke, n.u.

ANG II

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(mmHg)

Fig. 6. Effects of ANG II on arterial pulse-induced phasic responses of a barosensitive bulbospinal RVLM neuron. A: spike histograms (bin width: 5 ms, accumulated for 1 min) triggered by consecutive R-waves on the ECG and simultaneously sampled and averaged ECG and AP curves before (left) and during (right) iontophoretic application of ANG II (1 mM, ⫹50 nA). The original spike-histogram data were smoothed and interpolated to obtain smoothed unit-activity curves. u0 and u0= indicate the start of the upstroke phases of the arterial pulse wave. B: AP and unit-activity curves before (dashed line) and during (solid line) ANG II application. Both unit-activity curves were phase-adjusted to align the nadir of the pre-ANG II unit-activity curve with the peak of AP curve (the start of the downstroke phase of the arterial pulse wave, d0). The unit activities were normalized in terms of the average level of the pre-ANG II unit-activity curve over one cardiac cycle (from u0 to u0=), which was equal to 100 n.u. C: phase-adjusted unit activity-AP relationship curves before (dashed line) and during (solid line) ANG II application. Arrows indicate the direction of the trajectory. The circle and triangle on each trajectory indicate u0 and d0, respectively.

pre-ANG II u0 u0'

Pre-ANG II

ANG II

166.9 ⫾ 6.5 28.5 ⫾ 3.3

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104.9 ⫾ 6.7* 84.3 ⫾ 6.8 125.5 ⫾ 7.8*

Values are expressed as means ⫾ SE; n ⫽ 24. Upstroke and downstroke, the rising and falling phases of the arterial pulse wave, respectively. n.u., normalized unit (100 n.u. ⫽ the overall average unit activity with respect to time before ANG II application). *P ⬍ 0.05 vs. pre-ANG II.

0.1 0.2 time (sec)

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stroke of the control AP curve, 80.8 ⫾ 1.8 mmHg vs. 75.4 ⫾ 1.7, respectively, P ⬍ 0.01), so the average unit activity with respect to AP was derived from the unit activity-AP curve and compared between the upstroke and downstroke phases. ANG II significantly increased the average unit activity with respect to AP in the downstroke phase (P ⬍ 0.01) but not in the upstroke phase, indicating a preferential effect of ANG II on unit activities in the downstroke phase (Table 3, Fig. 7, right). Rapid RSNA Baroreflex Experiments To clarify whether the ANG II-induced rapid baroreflex modulation in RVLM neurons is reflected in the peripheral sympathetic baroreflex, the effects of microinfusion of ANG II into the RVLM on rapid renal sympathetic baroreflex were assessed in six rabbits. Fig. 8 shows a typical example of the phasic RSNA responses both before and during ANG II application. RSNA was decreased in response to the rise of APs with a delay of more than 100 ms (Fig. 8A). The delay was much larger than that observed in unit activities due to the extended neural pathway from the RVLM to peripheral nerves. Similarly to the unit activity curve, the phase-adjusted RSNA curve revealed that RSNA was decreased in an almost linear fashion when correlated to AP in the upstroke phase of the pulse wave, while the relationship between RSNA and AP showed nonlinear pattern in the downstroke phase (Fig. 8, B and C). On average, ANG II increased the baseline MAP from



60

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*

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* 40

*

20

0 min

max

uAvg dAvg

(

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uAvg dAvg with ) (averaged respect to AP )

Fig. 7. The average changes in the peak and average unit activities induced by iontophoretic application of ANG II (1 mM, ⫹50 nA) in 24 barosensitive bulbospinal RVLM neurons. min and max, minimum and maximum unit activities observed in response to arterial pulses, respectively. uAvg and dAvg, the average unit activities in the upstroke and downstroke phases of the arterial pulse wave, respectively. These averages were calculated with respect to time (middle) and with respect to AP (right). The overall average with respect to time before ANG II application was equal to 100 n.u. Error bars represent means ⫾ SE. *P ⬍ 0.05.

78.3 ⫾ 3.6 mmHg to 94.3 ⫾ 3.6 mmHg (P ⬍ 0.01). ANG II raised the maximum RSNA by 43.5 ⫾ 7.2 n.u., whereas it did not change the minimum (⫺0.3 ⫾ 1.9 n.u., Table 4). As a result, the amplitudes of the phasic RSNA were significantly increased (P ⬍ 0.01, Fig. 9, left). ANG II also significantly increased the overall RSNA, and the average RSNA with respect to time in both upstroke and downstroke phases (P ⬍ 0.01, Table 4). However, the increase in the downstroke phase was significantly greater than that in the upstroke phase (P ⬍ 0.01, Fig. 9, middle). The preferential effect of ANG II on RSNA in the downstroke phase was also confirmed, when the effects of ANG II on average RSNA with respect to AP were compared between these phases (P ⬍ 0.05, Fig. 9, right). Infusion of an equal volume of vehicle solution did not significantly alter any of the baseline and phasic responses of RSNA (Table 4). DISCUSSION

In the present study, we have confirmed our previous results that ANG II microinfused into the RVLM modulates the renal sympathetic baroreflex, which is induced by slow ramp changes in MAP (44, 45). Using a newly developed floating electrode, we showed that iontophoretically applied ANG II increased the baseline activities of barosensitive bulbospinal RVLM neurons, expanded the baroreflex working range of the unit activity-MAP relationship curve, and modified the asymmetry of the curve. The present study also demonstrated that ANG II augments the rapid baroreflex responses of unit activities and RSNA to pulsatile AP predominantly in the downstroke phase of arterial pulse waves. The present study showed that ANG II increased the upper plateau of the RSNA-MAP relationship curve that was derived from slow ramp changes in MAP, while the lower plateau and the average slope of the curve were not affected. As a result, ANG II expanded the working range of the renal sympathetic

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baroreflex, which enabled sensitive baroreflex control over a wider range of MAPs. ANG II also reset the baroreflex curve to a higher-pressure area in parallel with increased baseline MAPs. The results are in agreement with those found in our previous study (44, 45). The four-parameter symmetric sigmoid model has been widely used for analyses of the sympathetic baroreflex (18, 44, 46). This model is useful when it is acceptable to assume that the reflex curve is symmetrical with respect to the midpoint; in other words, when the slope of the curve is symmetrical with respect to MAP50. However, when we attempted to fit actual baroreflex data with this model, we frequently found that the upper and/or lower parts of the fitting curve systematically deviated from the data points because of their asymmetric distribution. This deviation results in inappropriate estimates of the upper and/or lower plateaus. Therefore, in the present study, we utilized the five-parameter asymmetric model to assess the renal sympathetic baroreflex without the assumption that the curve would be symmetrical. This model was originally introduced by Ricketts and Head (42) to evaluate the asymmetry of baroreflex curves. With the five-parameter model, we found that the control renal sympathetic baroreflex curve was slightly, but significantly, asymmetric, with a larger curvature at lower MAPs. This finding is contradictory to the findings observed in a previous study where the asymmetry index of the renal sympathetic baroreflex curve was not significantly different than zero in conscious rabbits (42). This discrepancy may be caused by the presence or absence of anesthesia. In the present study, the baseline level of RSNA was elevated beyond the midpoint of the reflex curve, presumably by the peripheral and central actions of anesthesia (18, 24), while in conscious rabbits, the baseline RSNA was relatively low (mostly less than the midpoint). Therefore, it is possible that in anesthetized animals, the shift of baseline RSNA toward the upper plateau may compress the reflexive RSNA activation, and, consequently, increase the curvature at lower MAPs. For the assessment of neuronal responses induced by slow ramp changes in MAP, a line with two bending points (threshold and saturation points), rather than a curve, was fitted to the data to depict a profile of individual neuronal responses. This model was used because the unit activity-MAP relationship showed good linearity almost throughout the range where the unit activities were sensitive to MAPs. Others have reported similar results (8, 49). Because the threshold and saturation points varied among neurons, the average unit activity-MAP relationship curve, which was derived from the individual fitting lines by calculating average unit activities at each MAP, was a curved line. The present study revealed that the average unit activity-MAP relationship curve before ANG II applications was quite similar to that observed in the RSNA-recording experiments. Application of ANG II resulted in a more symmetric curve with a higher upper plateau and an unaffected slope, which led to an expanded range of the unit-activity baroreflex. Importantly, these ANG II-induced changes observed in unit activity were consistent with those observed in RSNA, which provides a possible explanation for the ANG II-mediated sympathetic baroreflex modulation in the RVLM. However, it is difficult to quantitatively compare the effects of ANG II on RVLM neuronal activities with the effects on the RSNA because the doses are not comparable between the


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Fig. 8. Effects of ANG II on arterial pulse-induced phasic responses of RSNA. A: ECG, AP, and RSNA curves, which were triggered by consecutive R-waves on the ECG and averaged over 1 min, before (left) and during (right) microinfusion of ANG II (4 pmol/min) into the RVLM. The original RSNA, which was integrated every 5 ms, was smoothed and interpolated to obtain smoothed RSNA curves. B: AP and RSNA curves before (dashed line) and during (solid line) ANG II application. Both RSNA curves were phase-adjusted to align the nadir of the pre-ANG II RSNA curve with the peak of AP curve (d0). The RSNA was normalized in terms of the average level of the preANG II RSNA curve over one cardiac cycle (from u0 to u0=), which was equal to 100 n.u. C: phase-adjusted RSNA-AP relationship curves before (dashed line) and during (solid line) ANG II application. Arrows indicate the direction of the trajectory. The circle and triangle on each trajectory indicate u0 and d0, respectively.

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iontophoretically applied and microinfused ANG II, and also because the sympathetic preganglionic neurons may not integrate the inputs from individual RVLM neurons in a linear fashion. The RSNA-baroreflex curve was shifted to the right with ANG II application, as indicated by a significant increase in MAP50. A similar shift was not observed in the unit-activity baroreflex. This horizontal shift was probably due to a change in baseline MAPs induced by ANG II microinfusion, because

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arterial baroreceptors can be rapidly reset (within 15 min) in parallel with baseline MAP levels (17). By contrast, ANG II that was applied by microiontophoresis did not change the baseline MAP, and, therefore, no baroreceptor resetting occurred in unit-recording experiments. Alternatively, it is possible that ANG II microinfused into the RVLM activates barosensitive bulbospinal neurons that were silent at baseline MAP before ANG II application. These neurons were not included in either the individual or averaged unit-activity

Table 4. Effects of microinfusion of ANG II into the rostral ventrolateral medulla on arterial pressure-related phasic renal sympathetic activities Peak activities Maximum, n.u. Minimum, n.u. Average renal sympathetic activities with respect to time Overall, n.u. Upstroke, n.u. Downstroke, n.u. Average renal sympathetic activities with respect to arterial pressure Overall, n.u. Upstroke, n.u. Downstroke, n.u.

Prevehicle

Vehicle

Pre-ANG II

ANG II

138.4 ⫾ 2.6 58.0 ⫾ 3.9

136.7 ⫾ 6.5 57.1 ⫾ 3.9

138.6 ⫾ 3.7 53.3 ⫾ 5.3

182.1 ⫾ 8.9*† 53.0 ⫾ 5.7

100 78.4 ⫾ 3.4 106.2 ⫾ 0.7

99.2 ⫾ 3.0 76.2 ⫾ 4.8 105.9 ⫾ 3.2

100 81.7 ⫾ 3.2 105.9 ⫾ 0.7

115.6 ⫾ 1.9*† 88.7 ⫾ 2.4* 124.8 ⫾ 2.2*†

83.4 ⫾ 3.1 80.3 ⫾ 3.1 86.4 ⫾ 3.3

83.4 ⫾ 4.2 77.9 ⫾ 4.5 88.8 ⫾ 4.0

85.7 ⫾ 3.4 84.2 ⫾ 4.0 87.2 ⫾ 3.8

95.7 ⫾ 2.6*† 92.4 ⫾ 3.9* 98.9 ⫾ 3.0*†

Values are expressed as means ⫾ SE; n ⫽ 6. Upstroke and downstroke denote the rising and falling phases of the arterial pulse wave, respectively. n.u., normalized unit (100 n.u. ⫽ the overall average renal sympathetic activity with respect to time before each drug application). *P ⬍ 0.05 vs. pre-ANG II; †P ⬍ 0.05 vs. vehicle. AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00285.2013 • www.ajpregu.org

ANG II-INDUCED BAROREFLEX MODULATION (n.u.)

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averaged with respect to time

uAvg dAvg with ) (averaged respect to AP )

Fig. 9. The average changes in the peak and average RSNA induced by microinfusion of ANG II (4 pmol/min) into the RVLM in six rabbits. These averages were calculated with respect to time (middle) and with respect to AP (right). The overall average with respect to time before ANG II application was equal to 100 n.u. Error bars represent means ⫾ SE. *P ⬍ 0.05.

baroreflex curves because of no spontaneous activity at baseline MAP but could contribute to the horizontal shift of the RSNA-baroreflex curve. The present results demonstrated that ANG II shifted the individual reflex curves upward without a significant change in MAP50. This change seemed to expose a portion of the slope that had previously been hidden below the cut-off level, which led to an increase in the saturation (cut-off) MAP. The observed vertical shift of the curve suggests that ANG II excites bulbospinal neurons independently of the synaptic inputs driven by baroreceptor inputs. Therefore, it is likely that ANG II excites these neurons directly. This is supported by the observations that ANG II increases baseline activities of a subpopulation of the RVLM neurons not only in vivo, as shown in the previous (9) and present study, but also in vitro, where synaptic inputs are substantially attenuated (30). In addition, it was demonstrated in rat slice experiments under total removal of synaptic inputs in low Ca2⫹ and high Mg2⫹ environment that ANG II decreases resting K⫹-membrane conductance of RVLM neurons, which promotes depolarization (29, 52). Using brain-spinal cord preparations from neonatal mice, Matsuura et al. (33) demonstrated that ANG II depolarizes bulbospinal RVLM neurons via ANG II type I (AT1) receptors. However, potential contributions of other mechanisms cannot be ruled out. It is possible that ANG II also presynaptically facilitates excitatory inputs that are independent of baroreceptor signals. Consistent with this, microinjection of ANG II into the RVLM increases baseline Glu release in this area (56). At the same time, it is also possible that ANG II inhibits GABAergic transmission presynaptically to activate bulbospinal neurons. Such a mechanism has been known in the PVN (12), although ANG II rather facilitates GABAergic transmission in the nucleus of the solitary tract (41). Alternatively, ANG II may also modulate neurotransmission postsynaptically. A majority of the ANG II-responsive bulbospinal neurons can be inhibited by a selective agonist of ␣2-adrenergic receptors (␣2-ARs) in rats (29, 30). If ANG II decreases the affinities of ␣2-ARs on RVLM neurons, as has been demonstrated in the nucleus of the solitary tract (19), the RVLM

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neurons would then be activated by ANG II. Therefore, multiple mechanisms can produce activation of bulbospinal RVLM neurons. The relative importance of these actions remains to be clarified. In the present study, no significant correlation was observed between ANG II responses and conduction velocities of barosensitive bulbospinal RVLM neurons. However, the recorded neurons in this study did not include slow-conducting (⬍1 m/s) cells, which were reported to account for 14% of the barosensitive bulbospinal neurons in rabbits (55). The lack of slowconducting cells in the present study may be due to a technical difficulty of the collision test in the rabbit, in which the distance between the stimulating and recording electrodes is much longer than in the rat, resulting in the failure to sample the slow-conducting cells. Nevertheless it seems likely that the majority of slow-conducting neurons respond to ANG II because it has been demonstrated that a majority of the ANG II-responsive bulbospinal neurons in rats are sensitive to ␣2-AR agonists, as mentioned above, and that most of the ␣2-AR agonist-sensitive neurons are slow-conducting cells (3). On the basis of the findings of Schreihofer and Guyenet (48) that the rat barosensitive bulbospinal RVLM neurons with fast conduction velocities included both C1 and non-C1 cells, while slow conducting cells were exclusively C1 cells, the neurons recorded in the present study may include both C1 and non-C1 cells. Because we did not distinguish between adrenergic C1 and non-C1 cells, it remains to be determined whether the ANG II-responsive RVLM neurons identified in our study are C1 or non-C1 cells. However, it seems likely that the ANG II-responsive neurons in our study are predominantly C1 cells, because a strong association between ANG II responsiveness and C1 neurons has been demonstrated. ANG II excited 91% of bulbospinal C1 cells, but 23% of bulbospinal non-C1 cells in neonatal rat slice preparations (29). Furthermore, recent studies using mice demonstrated that the majority of C1 neurons express AT1A receptors and that AT1A receptor expression by C1 neurons was required for the sympathoexcitatory and pressor responses induced by microinjection of ANG II into this region (10, 11). The rapid baroreflex experiments of the RVLM neurons demonstrated that ANG II increased the amplitude of phasic variations in unit activities that were evoked by pulsatile changes in AP. This suggests that ANG II potentiates the baroreflex responses of RVLM neurons that are induced by rapid changes in AP, such as arterial pulse waves. The results also demonstrated that ANG II increased the average unitactivity level predominantly in the downstroke phase of arterial pulse waves. Because the average level of AP in the downstroke phase was significantly lower than the level in the upstroke phase, this phase-dependent effect of ANG II may have been caused by a change in the static (steady-state) characteristics of baroreflex functions that increased the unit activities predominantly at lower APs. However, when the average unit activities with respect to AP were compared with balance of the static baroreflex effects of ANG II between the upstroke and downstroke phases, the effects of ANG II remained greater in the downstroke phase than in the upstroke phase. This result suggests that the phase-dependent effect was produced, in part, by a change in dynamic baroreflex characteristics, such as derivative and integral elements.


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To clarify whether the ANG II-induced modulation of rapid baroreflex at the level of RVLM neurons is reflected in the peripheral sympathetic outflow, we examined the effects of ANG II on rapid renal sympathetic baroreflex. ANG II increased the maximum activation of RSNA induced by pulsatile changes in AP, which is consistent with the observation in the unit-activity baroreflex. On the other hand, the minimum level of RSNA was not changed on average by ANG II. This is probably due to a static baroreflex effect caused by upward shift of baseline MAP that was induced by microinfusion of ANG II, but not by iontophoretic application. Nevertheless, the amplitude of phasic variation of RSNA was significantly increased by ANG II, which is consistent with the result of the unit-activity baroreflex. Furthermore, ANG II increased the average level of RSNA predominantly in the downstroke phase of arterial pulse waves. Again, this is consistent with the result of unit-activity experiments. Therefore, the primary effects of ANG II on rapid RSNA baroreflex can be explained by the action of this peptide on the phasic responses of barosensitive bulbospinal RVLM neurons. In the present study, we analyzed rapid baroreflex responses that were induced by arterial pulse waves caused by spontaneous cardiac beats. Because the main frequency components of the arterial pulse waves were HR-frequency waves (4 –5 Hz in urethane-anesthetized rabbits) and their harmonics, the ANG II-induced changes in phasic unit activities and RSNA that were observed in the present experiments only reflect the action of ANG II on dynamic baroreflex characteristics at these frequencies. On the other hand, system-analysis studies on sympathetic baroreflex control in rabbits (26, 27) demonstrate that baroreceptor signals in a frequency range of ⬃0.8 Hz are most effectively transferred to peripheral sympathetic outflow. Therefore, the action of ANG II on baroreflex responses of RVLM neurons in this frequency range would be more significant in the phasic control of sympathetic outflow, which remains to be determined in future studies. In conclusion, the present study revealed that the ANG II-induced modulation of the average baroreflex curves of bulbospinal RVLM neurons was characterized by increases in the upper plateau, the reflex range, and the working rage, and a decrease in the asymmetry index, without a significant change in the maximum slope. Importantly, these effects successfully explain the ANG II-induced sympathetic baroreflex modulation in the RVLM. The present study also clarified that ANG II augments the phasic response of these neurons to arterial pulse waves predominantly in the downstroke phase, which suggests that ANG II modulates dynamic property of baroreflex response. Perspectives and Significance Recent studies have elucidated that, contrary to the neuroexcitatory and sympathoexcitatory actions, the endogenous and exogenous ANG II in the RVLM exerts neuroinhibitory (6) and sympathoinhibitory actions (51), or exerts little effect (32, 34). Bertram and Coote (6) have demonstrated that iontophoretic application of ANG II inhibits barosensitive RVLM neurons in rats. ANG II applied to the RVLM in anesthetized rabbits potentiates the RSNA baroreflex, as shown in the previous (44, 45) and present studies, whereas it does not change the RSNA baroreflex in conscious rabbits (34). One of the interpretations

of these inconsistent observations is that ANG II mediates both neuroexcitatory and neuroinhibitory action to the sympathetic premotor neurons in the RVLM, and the balance of these actions can be altered depending on tonic synaptic inputs arising from peripheral receptors, such as chemoreceptors and baroreceptors and other central nuclei, including the PVN (15, 23). Further work is needed to evaluate the actions of ANG II on sympathetic premotor neurons under various physiological and pathological conditions that alter the tonic synaptic inputs to these neurons. GRANTS This work was supported by Grant-in-Aids for Scientific Research 07770045 and 13670059, which were provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: T.S. and J.A. conception and design of research; T.S. and J.A. performed experiments; T.S. and J.A. analyzed data; T.S. and J.A. interpreted results of experiments; T.S. and J.A. prepared figures; T.S. and J.A. drafted manuscript; T.S. and J.A. edited and revised manuscript; T.S. and J.A. approved final version of manuscript. REFERENCES 1. Aldred GP, Chai SY, Song K, Zhuo J, MacGregor DP, Mendelsohn FAO. Distribution of angiotensin II receptor subtypes in the rabbit brain. Regul Pept 44: 119 –130, 1993. 2. Allen AM, Dampney RAL, Mendelsohn FAO. Angiotensin receptor binding and pressor effects in cat subretrofacial nucleus. Am J Physiol Heart Circ Physiol 255: H1011–H1017, 1988. 3. Allen AM, Guyenet PG. ␣2-Adrenoceptor-mediated inhibition of bulbospinal barosensitive cells of rat rostral medulla. Am J Physiol Regul Integr Comp Physiol 265: R1065–R1075, 1993. 4. Allen AM, MacGregor DP, McKinley MJ, Mendelsohn FAO. Angiotensin II receptors in the human brain. Regul Pept 79: 1–7, 1999. 5. Benarroch EE, Schmeichel AM. Immunohistochemical localization of the angiotensin II type 1 receptor in human hypothalamus and brainstem. Brain Res 812: 292–296, 1998. 6. Bertram D, Coote JH. Inhibitory effects of angiotensin II on barosensitive rostral ventrolateral medulla neurons of the rat. Clin Exp Pharmacol Physiol 28: 1112–1114, 2001. 7. Blessing WW. Depressor neurons in rabbit caudal medulla act via GABA receptors in rostral medulla. Am J Physiol Heart Circ Physiol 254: H686 –H692, 1988. 8. Brown DL, Guyenet PG. Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ Res 56: 359 –369, 1985. 9. Chan RKW, Chan YS, Wong TM. Responses of cardiovascular neurons in the rostral ventrolateral medulla of the normotensive Wistar Kyoto and spontaneously hypertensive rats to iontophoretic application of angiotensin II. Brain Res 556: 145–150, 1991. 10. Chen D, Bassi JK, Walther T, Thomas WG, Allen AM. Expression of angiotensin type 1A receptors in C1 neurons restores the sympathoexcitation to angiotensin in the rostral ventrolateral medulla of angiotensin type 1A knockout mice. Hypertension 56: 143–150, 2010. 11. Chen D, Jancovski N, Bassi JK, Nguyen-Huu TP, Choong YT, PalmaRigo K, Davern PJ, Gurley SB, Thomas WG, Head GA, Allen AM. Angiotensin type 1A receptors in C1 neurons of the rostral ventrolateral medulla modulate the pressor response to aversive stress. J Neurosci 32: 2051–2061, 2012. 12. Chen Q, Pan HL. Signaling mechanisms of angiotensin II-induced attenuation of GABAergic input to hypothalamic presympathetic neurons. J Neurophysiol 97: 3279 –3287, 2007. 13. Coveñas R, Fuxe K, Cintra A, Aguirre JA, Goldstein M, Ganten D. Evidence for the existence of angiotensin II like immunoreactivity in subpopulations of tyrosine hydroxylase immunoreactive neurons in the A1



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Intracellular analysis in vivo of different barosensitive bulbospinal neurons in the rat rostral ventrolateral medulla.

Characteristics of caudal ventrolateral medullary neurons antidromically activated from rostral ventrolateral medulla in the rabbit.

Angiotensin II excites vasomotor neurons but not respiratory neurons in the rostral and caudal ventrolateral medulla.

Quantitative analysis of bulbospinal projections from the rostral ventrolateral medulla: contribution of C1-adrenergic and nonadrenergic neurons.

Expression and functions of β1- and β2-adrenergic receptors on the bulbospinal neurons in the rostral ventrolateral medulla.

Responses of single units in the midline medulla to stimulation of the rostral ventrolateral medulla.

Selective sensitization by L-glutamate of baroreflex-mediated bradycardia following microinjection into the rostral ventrolateral medulla.

Electrophysiological properties of neurons in the rostral ventrolateral medulla of normotensive and spontaneously hypertensive rats.

Swimming Training Modulates Nitric Oxide-Glutamate Interaction in the Rostral Ventrolateral Medulla in Normotensive Conscious Rats.

Electrophysiological properties of rostral ventrolateral medulla presympathetic neurons modulated by the respiratory network in rats.

Projections from the rostral ventrolateral medulla to brainstem monoamine neurons in the rat.

Effects of clonidine on sympathoexcitatory neurons in the rostral ventrolateral medulla.

Responses of cardiovascular neurons in the rostral ventrolateral medulla of the normotensive Wistar Kyoto and spontaneously hypertensive rats to iontophoretic application of angiotensin II.

Rostral ventrolateral medulla and sympathorespiratory integration in rats.

Sex differences in NMDA GluN1 plasticity in rostral ventrolateral medulla neurons containing corticotropin-releasing factor type 1 receptor following slow-pressor angiotensin II hypertension.

Neurons in the caudal ventrolateral medulla mediate the somato-sympathetic inhibitory reflex response via GABA receptors in the rostral ventrolateral medulla.

Spinal cord substance P mediates carbachol-induced cardiovascular responses from the rostral ventrolateral medulla.

Localization of vasodepressor neurons in the caudal ventrolateral medulla in the rabbit.

Chronic infusion of lisinopril into hypothalamic paraventricular nucleus modulates cytokines and attenuates oxidative stress in rostral ventrolateral medulla in hypertension.

Immunohistochemical evidence of tissue hypoxia and astrogliosis in the rostral ventrolateral medulla of spontaneously hypertensive rats.

Neuroinflammation contributes to autophagy flux blockage in the neurons of rostral ventrolateral medulla in stress-induced hypertension rats.

Lesions of rostral ventrolateral medulla abolish some cardio- and cerebrovascular components of the cerebellar fastigial pressor and depressor responses.

Involvement of endogenous hydrogen sulfide (H2S) in the rostral ventrolateral medulla (RVLM) in hypoxia-induced hypothermia.

Role of the rostral ventrolateral medulla in the generation of synchronized sympathetic rhythmicities in the rat.