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(Circulation Research. 1996;78:274-282.)
© 1996 American Heart Association, Inc.

Articles

Angiotensin II Decreases a Resting K⁺ Conductance in Rat Bulbospinal Neurons of the C1 Area

Yu-Wen Li, Patrice G. Guyenet

From the University of Virginia, Department of Pharmacology, Charlottesville, Va.

Correspondence to P.G. Guyenet, PhD, University of Virginia, Department of Pharmacology, Box 448, Health Sciences Center, Charlottesville, VA 22908. E-mail pgg@virginia.edu.

	Abstract

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Abstract In the rostral ventrolateral medulla (RVLM), angiotensin II (Ang II) receptors are concentrated in the region that contains neurons innervating sympathetic preganglionic neurons. We sought to determine whether these bulbospinal cells are sensitive to Ang II. Retrogradely labeled bulbospinal RVLM neurons (N=125) were recorded in thin slices from neonatal rats. Most (33 of 46) histologically recovered bulbospinal neurons were C1 cells (immunoreactive for tyrosine hydroxylase [TH-ir] or phenylethanolamine N-methyltransferase [PNMT-ir]). Bulbospinal RVLM neurons were spontaneously active (2.7±0.2 spikes per second, n=69) with `resting' potential of -54±0.4 mV (n=77) and input resistance of 879±53 M (n=47). Ang II (0.3 to 1 µmol/L) increased the spontaneous firing rate of most bulbospinal neurons (+250%, 28 of 39). In current-clamp mode, Ang II (1 µmol/L) produced depolarization (+6.8±0.6 mV, n=59 neurons) and increased input resistance (+21±2%, n=36 neurons). In voltage-clamp mode, Ang II elicited an inward current (9.7±0.9 pA; holding potential, -40 to -55 mV; n=25 neurons) that reversed polarity at the K⁺ equilibrium potential (n=8 neurons) and was barium sensitive (n=4 neurons). Ang II–evoked conductance change was voltage independent (-40 to -140 mV, n=8 neurons). The effects of Ang II were blocked by losartan (9 of 9 neurons) but persisted in low Ca²⁺/high Mg²⁺ (7 of 7 neurons). Ang II–sensitive cells were inhibited by ₂-adrenergic receptor agonists (12 of 15 neurons). Ang II excited 91% (30 of 33) of TH-ir or PNMT-ir cells but 23% (3 of 13) of non–TH-ir neurons. In conclusion, RVLM bulbospinal cells express Ang II type-1 receptors whose activation leads to a reduction in resting K⁺ conductance.

Key Words: rostral ventrolateral medulla • C1 neurons • sympathetic vasomotor tone • angiotensin II • resting K⁺ conductance

	Introduction

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The RVLM is crucial for sympathetic tone generation and cardiovascular reflexes.^{1 2} The integrative role of the RVLM seems to be regulated by Ang II, since pressor and depressor effects are produced by microinjection of Ang II receptor agonists and antagonists, respectively, at this level.^{3 4 5 6 7 8} The depressor effect of the antagonist [Sar¹,Thr⁸]Ang II is enhanced in spontaneously hypertensive rats,⁹ suggesting that abnormally high levels of Ang II within the RVLM may contribute to hypertension in this model. The increase in blood pressure produced by the injection of Ang II into the RVLM is mediated by the sympathetic system and is probably due to direct effects on RVLM neurons rather than to indirect effects via vasoconstriction, since the RVLM contains neurons that are excited "in vitro" by Ang II via AT₁ receptors.¹⁰

The RVLM contains Ang II–immunoreactive nerve terminals and a moderately high density of Ang II receptors, predominantly of the AT₁ variety.^{11 12 13 14} In several species, the distribution of Ang II receptors in the RVLM is strikingly similar to the location of C1 neurons, a group of phenotypically adrenergic neurons with monosynaptic projection to sympathetic preganglionic neurons and a presumed role in controlling sympathetic tone.^{6 12 14 15} These results suggest that C1 neurons of the RVLM may express high levels of AT₁ receptors and that these cells could mediate the increase in arterial pressure caused by microinjection of Ang II into the RVLM "in vivo."^{3 4 5 6 7 8} Accordingly, the main objectives of the present study are to determine whether bulbospinal C1 neurons of the RVLM have functional Ang II receptors and to analyze what type of electrophysiological response Ang II produces in these cells. To address this issue, whole-cell recordings were performed in thin slices from neonatal rats, in which RVLM bulbospinal cells can be visualized by retrograde labeling with a fluorescent tracer.^{16 17} Since up to 80% of the RVLM neurons retrogradely labeled by this procedure in the neonate are C1 cells,¹⁷ this approach provides a unique opportunity to focus on this sparse population of reticular formation cells.

	Materials and Methods

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Whole-cell recordings of bulbospinal RVLM neurons were obtained using modifications of the original method of Edwards et al.¹⁸ The procedures have been detailed in a previous article¹⁷ and will be described only briefly.

Retrograde Labeling of Bulbospinal Neurons and Slice Preparation
Sprague-Dawley rat pups (1 to 5 days old) were anesthetized by hypothermia. The upper thoracic spinal cord was exposed, and a suspension of either FITC- or rhodamine-labeled microspheres (total volume, 0.3 to 0.5 µL; Molecular Probes) was injected bilaterally into the spinal cord. Two to 5 days after the spinal injection, the pups (3 to 10 days old) were deeply anesthetized by hypothermia and decapitated. The brain stem was immersed in ice-cold Cl^-- and Ca²⁺-poor aCSF (sucrose aCSF¹⁹ ) equilibrated with 95% O₂/5% CO₂ (pH 7.3 to 7.4) of the following composition (mmol/L): NaHCO₃ 26, KH₂PO₄ 1.3, KCl 2.5, MgSO₄ 5, CaCl₂ 0.5, glucose 10, and sucrose 248. Coronal slices (120 µm thick) were cut at 4°C to 8°C with a Microslicer (EM Dosaka Co, Ltd). The slices were preincubated for 1 to 6 hours at room temperature (18°C to 22°C) in lactic acid aCSF equilibrated with 95% O₂/5% CO₂ (pH 7.3 to 7.4) of the following composition (mmol/L): NaCl 124, NaHCO₃ 26, KH₂PO₄ 1.3, KCl 2.5, MgSO₄ 2, CaCl₂ 2, glucose 10, and lactic acid 4.5. For recording, a single medullary slice was selected under a dissection scope using the following criteria: location caudal to the last slice in which the facial motor nucleus was visible and characteristic shape of the rostral tip of the inferior olive. The slice was placed in a custom-designed recording chamber on an upright epifluorescence microscope (Olympus BH-2). The characteristic pattern of the retrograde labeling in the RVLM region was used as the final means to ensure that the correct slice had been selected (for details, see Reference 17). In the chamber, the slice was continuously superfused at a rate of 2 to 3 mL/min with normal aCSF, equilibrated with 95% O₂/5% CO₂ (pH 7.3 to 7.4; composition was identical to lactic acid aCSF without the lactate). All experiments were performed at room temperature.

Recordings and Data Analysis
Individual retrogradely labeled neurons were visualized with a water-immersion x40 objective via epifluorescence and Hoffman modulation optics. Gentle cleaning with a stream of normal aCSF was done to access cells embedded below the surface of the slice. Patch pipettes were pulled from thin-walled borosilicate glass capillaries (outer diameter, 1.5 mm; Clark) on a horizontal pipette puller (Sutter P87) and were filled with a solution of the following composition (mmol/L) potassium gluconate 114, KCl 17.5, NaCl 4, MgCl₂ 4, HEPES 10, EGTA 0.2, Mg²⁺-ATP 3, and Na⁺-GTP 0.3, along with 0.02% Lucifer yellow (Molecular Probes). The pH was adjusted to 7.3, and the osmolarity was adjusted to 270 mOsm. Electrode resistance was 5 to 9 M. Three types of recordings were made, all with an Axoclamp-2A amplifier: extracellular unit recordings using the cell-attached configuration described by Alreja and Aghajanian,²⁰ whole-cell current clamp, and whole-cell voltage clamp. For extracellular single-unit recording, a patch pipette was placed close to the cell membrane, and a gigaseal (>2 G) was formed by very gentle suction. The extracellular spikes were amplified, filtered (0.3 to 3 kHz), and displayed on-line on a Gould chart recorder. An integrated rate histogram of the unit discharges was generated by passing the signal serially through a window discriminator and counter. Whole-cell current-clamp and voltage-clamp recordings were made with an Axoclamp-2A amplifier. In current-clamp mode, input resistance was measured by determining the voltage drop elicited by hyperpolarizing current pulses (10 to 20 pA, 1 second) after membrane capacitance charge. For voltage clamp, neurons were first recorded in current-clamp bridge mode and then transferred to continuous single-electrode voltage-clamp mode. Liquid junction potential was measured, and all reported voltage measurements have been corrected for these potentials. Because of the very small amplitude of the currents recorded in voltage-clamp mode, voltage errors due to series resistance were small, and no series resistance compensation was performed. Current and voltage signals were collected through a DigiData-1200 Interface using pCLAMP software (version 6.0, Axon Instruments) and stored on videotape for off-line analysis.

Histology
After recording, the slices were fixed by immersion in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.3) for 1 to 3 days at 4°C. Immunostaining for TH was done using an avidin-biotin–based reaction as previously described¹⁷ (mouse anti-TH monoclonal antiserum from Chemicon [dilution, 1:500], biotinylated goat anti-mouse antiserum from Vector [dilution, 1:150], and avidin-conjugated Texas red from Molecular Probes [dilution, 1:200]). In five cases, the sections were stained for PNMT using a similar protocol (antibody from Eugene Tech International Inc [dilution, 1:500]). The slices were mounted, dried, and coverslipped with Krystalon. TH-ir was generally preferred because it gave a stronger signal than PNMT-ir. Others have previously demonstrated that virtually all bulbospinal TH-ir cells in the RVLM also express PNMT.²¹ C1 neurons are defined by the presence of TH and PNMT.^{21 22}

The tissue was examined by epifluorescence microscopy using incident wavelength and filters appropriate for Texas red and rhodamine (simultaneous detection of TH-ir or PNMT-ir and rhodamine-labeled microspheres) or for fluorescein (simultaneous detection of Lucifer yellow and fluorescein-conjugated microbeads; for full details on spectral characteristics of the filters used, see Reference 23, and for details on triple-label procedure in thin slice, see Reference 17). The punctate nature of the rhodamine-labeled microspheres made them clearly visible even in cells immunostained for TH with rhodamine-tagged antibodies. Similarly, the presence of Lucifer yellow did not impair the detection of fluorescein-tagged microspheres as shown in Fig 1.

View larger version (60K): [in this window] [in a new window]   Figure 1. A, Photograph taken under UV illumination using fluorescein filter. Lucifer yellow–filled bulbospinal neuron of the RVLM (thick arrow) is located among other nonrecorded bulbospinal cells (small arrows). Bulbospinal cells are identified by the presence of fluorescein-tagged microbeads (small dots within cells). B, Higher power view of recorded cell under same illumination (both Lucifer yellow and the microbeads are visible). C, Same field photographed under green incident light with rhodamine filter revealing presence of TH-ir in the recorded cell. Note that microbeads are now invisible. Bar=25 µm (A) and 10 µm (B and C).

The location of retrogradely labeled TH-ir (or PNMT-ir) and/or Lucifer yellow–stained neurons was plotted by computer using a Ludl motor-driven stage and Neurolucida software (MicroBrightfield) as described previously.^{17 23} The atlas of Paxinos and Watson²⁴ was used for reference and nomenclature. Photographs of fluorescent cells were taken with 400ASA Ektachrome film, and the color slides were scanned with a Nikon scanner using Adobe Photoshop software.¹⁷

Drugs, Chemicals, and Statistics
Drugs and solutions of different ionic content were applied to the slice by switching the perfusion solution via a three-way electronic valve system. The following drugs were used: tetrodotoxin (fast Na⁺ channel blocker, 1 µmol/L, Sigma Chemical Co), Ang II (human, 0.1 to 3 µmol/L, Behring Diagnostic), MN (₂-adrenergic receptor agonist, 30 µmol/L, Research Biochemicals Inc), losartan (selective AT₁ receptor antagonist, 3 µmol/L, a gift from Dupont), and UK-14,304 (selective ₂-adrenergic receptor agonist, 1 µmol/L, Research Biochemicals Inc). All drugs were dissolved in normal aCSF. The effects of Ang II on firing rate, membrane potential, input resistance, and membrane current were tested by a paired-sample Student's t test. The effects of low Ca²⁺/high Mg²⁺ or losartan on Ang II–evoked responses were analyzed by performing one-way ANOVA with Tukey post hoc tests. Significance was set at P<.05. All data were expressed as mean±SEM.

	Results

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The present study is based on results from 125 recorded RVLM neurons, all of which were retrogradely labeled by either FITC- or rhodamine-conjugated microspheres. Therefore, they were all bulbospinal neurons projecting to the thoracic spinal cord. However, not all recorded neurons were recovered after histology, and not all recovered neurons were TH-ir. To avoid confusion, the term "RVLM bulbospinal neurons" will be used when describing the properties of histologically identified and unidentified neurons together as a single group. The terms "C1 neurons" and "non-C1 neurons" will be applied only to histologically recovered TH-ir (or PNMT-ir) and non–TH-ir bulbospinal RVLM neurons, respectively.

General Characteristics of RVLM Bulbospinal Neurons Recorded
Of the 125 neurons sampled, 24 were recorded only extracellularly in the cell-attached mode. The remaining 101 cells were recorded in whole-cell configuration (current clamp, voltage clamp, or both) and therefore were stained with Lucifer yellow. Forty-six of the 101 bulbospinal cells were recovered after histology, and 33 (72%) of them were either TH-ir (n=30) or PNMT-ir (n=3). Although the cell recovery rate after histology was only 46% (46 of 101 stained cells) for a variety of technical reasons, the total number of recovered cells was large enough to suggest that the figure of 72% was representative of the percentage of C1 cells in the total population of cells recorded (N=125). Fig 1 shows an example of a recorded bulbospinal RVLM neuron that was TH-ir (C1 cell). Note the presence of FITC-labeled microbeads in the Lucifer yellow–labeled neuron (panel B), indicating that it projects to the spinal cord. Fig 2 shows the location of the 33 recorded cells that were subsequently identified as TH-ir or PNMT-ir. These cells were found within 0.25 mm of the ventral surface, 1.0 to 1.4 mm lateral to the midline, and extending rostrocaudally for 0.2 to 0.4 mm at the level of the rostral tip of the inferior olive (Fig 2). Histologically recovered bulbospinal neurons not identified as TH-ir were located in the same area and were interspersed with spinally projecting TH-ir neurons.

View larger version (16K): [in this window] [in a new window]   Figure 2. Location of 33 RVLM bulbospinal neurons histologically identified as TH-ir (n=30) or PNMT-ir (n=3) (computer-assisted plot). Coronal section corresponds to the interaural level (-2.8-mm level of the atlas of Paxinos and Watson).24 IO indicates inferior olive; NA, compact portion of nucleus ambiguus; NTS, nucleus of the solitary tract; P, pyramidal tract; and Vsp, spinal trigeminal nucleus, interpolar. Bar=1 mm.

The vast majority of RVLM bulbospinal cells were spontaneously active (69 of 77 cells recorded in extracellular or whole-cell current-clamp mode) with an average firing rate of 2.7±0.2 spikes per second (ranging from 0.3 to 7 spikes per second, n=69). Thirty of the 33 cells identified as TH-ir or PNMT-ir also exhibited spontaneous discharges (2.8±0.3 spikes per second). The mean resting membrane potential (average interspike membrane potential) of RVLM bulbospinal neurons was -54±0.4 mV (n=77), and their input resistance was 879±53 M (n=47). No difference in membrane potential, input resistance, or resting discharge rate was detected between the 33 neurons identified as TH-ir (PNMT-ir) and the 13 identified as non–TH-ir.

Effect of Ang II on Firing Rate of RVLM Bulbospinal Neurons: Extracellular Recordings
Cell-attached extracellular single-unit recordings were made to examine the response of RVLM bulbospinal cells to bath applications of Ang II under conditions in which the intracellular environment of the neurons was intact (n=39). Whole-cell recordings were later obtained in 15 of these 39 cases after completion of the extracellular experiment. Slice superfusion with 0.3 to 1 µmol/L Ang II for 1 to 2 minutes excited 28 (72%) of the cells. The excitatory response usually started within 1 minute after the beginning of Ang II application and peaked within 3 minutes (Fig 3A). Baseline discharge rates were usually recovered within 20 minutes after washout. The effect of Ang II was repeatable at least twice without apparent reduction in magnitude or duration when the peptide was reapplied after full recovery of baseline firing rate (n=12, example in Fig 3B). The minimum effective concentration of Ang II was 0.1 to 0.3 µmol/L. In the presence of 1 µmol/L Ang II, the mean firing rate of the 28 Ang II–sensitive neurons increased to 250% of control from 1.9±0.2 to 4.8±0.4 spikes per second (P<.01, Fig 3C). The remaining 11 cells were not significantly affected by 1 µmol/L Ang II (baseline, 3.7±0.6 spikes per second; Ang II, 3.9±0.7 spikes per second; P>.05).

View larger version (45K): [in this window] [in a new window]   Figure 3. A, Response of one bulbospinal cell to bath application of 1 µmol/L Ang II (at bar). Unit activity (recorded extracellularly in cell-attached mode) is displayed as an integrated rate histogram. B, Different single unit showing reproducibility of excitation by 1 µmol/L Ang II (applied at bar). The interval between the two excerpts was 10 minutes. C, Spontaneous discharge rate of Ang II–sensitive bulbospinal neurons of the RVLM recorded extracellularly in cell-attached mode before and during application of Ang II (1 µmol/L).

Effect of Ang II on Membrane Potential and Input Resistance of RVLM Bulbospinal Neurons
In current-clamp mode, Ang II produced a slow depolarization and an increase in firing rate in 77% (59 of 77) of RVLM bulbospinal cells recorded in this mode (Fig 4). On average, 1 µmol/L Ang II depolarized the cells by 6.8±0.6 mV (3 to 20 mV, from -54±0.6 mV, n=59). In the remaining 18 cells, Ang II produced no observable depolarization.

View larger version (51K): [in this window] [in a new window]   Figure 4. A, Typical depolarizing effect of 1 µmol/L Ang II on bulbospinal C1 cell (whole-cell recording). Membrane trajectory at times indicated by arrows a and b is illustrated in insets (action potentials clipped). B, Effect of 1 µmol/L Ang II in a different cell. A constant amount of hyperpolarizing current (5 pA) was injected throughout to maintain the membrane potential at -60 mV (resting membrane potential, -51 mV) before Ang II application. Hyperpolarizing current pulses (20 pA, 1 second, 0.1 Hz) were given to measure input resistance. A small amount of additional constant hyperpolarizing current (4 pA) was injected during the period identified by the letter b (at arrow) to restore membrane potential to the pre–Ang II level. Insert illustrates the increase in input resistance produced by Ang II (a, before; b, during Ang II application after restoration of predrug membrane potential).

An increase in input resistance was generally associated with the depolarization. This increase in input resistance was clear when, after application of Ang II, the membrane potential was restored to the predrug level by injection of an appropriate amount of hyperpolarizing current (Fig 4B) or when tetrodotoxin was present to avoid action potentials altogether. The input resistance was increased 21±2% by 1 µmol/L Ang II (baseline, 870±54 M; Ang II, 1047±64 M; P<.05; n=36).

Effect of Low Ca²⁺/High Mg²⁺ Solution on Ang II–Evoked Excitation of RVLM Bulbospinal Neurons
We tested whether the effect of Ang II on RVLM bulbospinal neurons was dependent on extracellular Ca²⁺ in 7 Ang II–sensitive cells recorded in current-clamp mode. The membrane potential was set at -60 to -70 mV in each case by injecting a small amount of hyperpolarizing current before 1 µmol/L Ang II was applied (1 to 2 minutes). After recovery from a first application of Ang II, the slices were superfused with low Ca²⁺ (0.1 mmol/L)/high Mg²⁺ (5 mmol/L) solution for at least 10 minutes, and then 1 µmol/L Ang II was reapplied for the same amount of time. As shown in Fig 5, the low Ca²⁺/high Mg²⁺ solution did not substantially affect the excitatory effect of Ang II. On average, 1 µmol/L Ang II depolarized the cells by 8±0.6 mV (n=7) in the presence of the low Ca²⁺/high Mg²⁺ solution, which was not significantly different from that (8.2±0.5 mV) evoked by 1 µmol/L Ang II in the absence of the solution.

View larger version (34K): [in this window] [in a new window]   Figure 5. Left, 1 µmol/L Ang II (at bar) depolarized one bulbospinal cell superfused with normal aCSF (resting membrane potential, -54 mV). Right, In the same cell, Ang II still evoked depolarization in the presence of low Ca2+/high Mg2+ solution. In both cases, a constant amount of hyperpolarizing current (<10 pA) was injected to maintain the membrane potential at -65 mV and stop the cell from discharging before Ang II application. Hyperpolarizing current pulses (10 pA, 1 second, 0.1 Hz) were given to measure input resistance. The interval between the first and second applications of Ang II was 20 minutes.

Effect of Losartan on Ang II–Evoked Excitation of RVLM Bulbospinal Neurons
We tested whether the effect of Ang II was mediated by AT₁ receptors in 9 Ang II–responsive RVLM bulbospinal neurons (4 recorded in extracellular mode and 5 in whole-cell current-clamp mode). These cells were exposed to a first application of 1 µmol/L Ang II and allowed to recover their original discharge rate and/or membrane potential. They were then superfused with 3 µmol/L losartan, and a second application of 1 µmol/L Ang II was made (at least 20 minutes after the first and 2 to 4 minutes after the beginning of the application of losartan). As shown in the Table and Fig 6, losartan did not change the basal discharge rate and/or membrane potential but prevented the increase in discharge rate and/or depolarization evoked by Ang II. Recovery from losartan was very slow and was observed in only 2 cells. Generally, recovery could not be observed because the cells could rarely be held for >60 minutes.

View this table: [in this window] [in a new window]   Table 1. Effects of Losartan on Ang II–Evoked Responses

View larger version (44K): [in this window] [in a new window]   Figure 6. A, One bulbospinal cell (extracellular recording; integrated rate histogram of discharge rate is shown) exposed to 1 µmol/L Ang II in the absence (left) and, 20 minutes later, in the presence (right) of 3 µmol/L losartan (dotted line). B, Whole-cell current-clamp recording. At the left, 1 µmol/L Ang II (solid bar) evoked a slow depolarization and an increase in firing rate in one bulbospinal cell. At the right, in the presence of 3 µmol/L losartan (dotted line) the second application of 1 µmol/L Ang II (solid bar) was ineffective. Hyperpolarizing current pulses (10 pA, 1 second, 0.1 Hz) were given to measure input resistance. The interval between the first and second applications of Ang II was 25 minutes. Action potentials were clipped.

Effect of Ang II on Bulbospinal RVLM Neurons in Voltage-Clamp Mode
In voltage-clamp mode (1 µmol/L tetrodotoxin present; holding potential, -40 to -55 mV), Ang II (0.1 to 3 µmol/L) elicited an inward current with the same kinetics as the depolarization observed in current-clamp mode (9 pA at peak in example shown in Fig 7A) and a reduction in input conductance (Fig 7B). Ang II–evoked inward current was observed in 25 of 29 RVLM bulbospinal cells tested. In the 25 responsive cells, the peak current induced by 1 µmol/L Ang II averaged 9.7±0.9 pA (range, 3 to 18 pA) at holding potentials of -40 to -55 mV.

View larger version (18K): [in this window] [in a new window]   Figure 7. A, Inward current produced by 1 µmol/L Ang II in one cell recorded under voltage-clamp mode (holding potential, -55 mV). B, Different cell clamped at -40 mV and exposed to three concentrations of Ang II. Constant voltage steps (-20 mV, 1 second, 0.2 Hz) were imposed to measure input conductance. Note reduction of input conductance in the presence of 1 and 3 µmol/L Ang II. Asterisks indicate the level at which potential ramps were imposed (-40 to -140 mV; trace was clipped). Dashed lines indicate zero current levels.

Voltage ramps (from -40 to -140 mV in 2 seconds) were imposed in voltage-clamp mode to analyze the voltage dependence of the current induced by 1 µmol/L Ang II (n=8 cells; typical case is shown in Fig 8A). The current evoked by Ang II was determined by subtracting the ramp current generated in the absence of Ang II from that recorded in the presence of the peptide. In the example shown in Fig 8A, Ang II–evoked current was linearly related to voltage (ie, ohmic) from -40 to -140 mV (range of voltage examined) and reversed polarity at -90 mV. For all 8 cells, E_rev averaged -86±1 mV. E_rev was very close to the predicted value of E_K calculated from the Nernst equation (-89 mV, with [K⁺]_o=3.8 mmol/L and [K⁺]_i=130 mmol/L). Ang II–induced currents were recorded in 4 other neurons in the presence of a higher [K⁺]_o (10 mmol/L). E_rev of the current evoked by Ang II shifted to more depolarized values (-64±1.8 mV, n=4, Fig 8B). This shift is according to the Nernst equation for a current predominantly carried by K⁺ (predicted value of E_K, -65 mV with [K⁺]_o=10 mV and [K⁺]_i=130 mmol/L). The conductance change evoked by Ang II was calculated by dividing the evoked current by the driving force (conductance=I/[V-E_rev], where I is current and V is voltage). As indicated in Fig 8C, the averaged conductance change (n=8) recorded in the presence of 3.8 mmol/L [K⁺]_o was independent of voltage in the range examined.

View larger version (28K): [in this window] [in a new window]   Figure 8. A, On the left, ramp currents in the absence (control) and in the presence of 1 µmol/L Ang II; on the right, Ang II–elicited current (difference between control and Ang II curves shown on the left). Experiment was performed with [K+]o=3.8 mmol/L. B, Similar experiment performed with [K+]o=10 mmol/L in a different cell (ramp currents on left and Ang II–elicited current on right). C, Average conductance change elicited by 1 µmol/L Ang II in 8 cells ([K+]o=3.8 mmol/L). Data points were obtained by measuring Ang II–induced conductance [I/(V-Erev), where I is current and V is voltage] at 10-mV intervals from -40 to -140 mV from tracings such as shown in panel B. D, Ang II–evoked current in one cell in the absence and presence of 0.5 mmol/L BaCl2.

We tested whether the nonselective K⁺ channel blocker BaCl₂ affected Ang II–evoked current in 4 RVLM bulbospinal Ang II–sensitive neurons. After the cells had recovered from a first application of Ang II, the slices were superfused with 0.5 to 1 mmol/L BaCl₂ for 2 to 5 minutes before a second Ang II application was performed. In all cells, BaCl₂ itself induced an inward current of 4 to 10 pA and reduced input conductance by 5% to 10%. In the presence of BaCl₂, Ang II–evoked inward current was reduced by 70% (pre-BaCl₂, 12±1.8 pA; post-BaCl₂, 3.5±1.2 pA; n=4) at a holding potential level of -40 mV. Fig 8D shows an example of the effect of 0.5 mmol/L BaCl₂ on Ang II–evoked current. The inward current elicited by Ang II (18 pA) at -50 mV was reduced to 3 pA in the presence of BaCl₂. However, E_rev of the Ang II–induced current remained very similar in the presence of BaCl₂ (close to the predicted value of E_K, with [K⁺]_o=3.8 mV and [K⁺]_i=130 mmol/L).

Phenotype of Ang II–Sensitive RVLM Bulbospinal Cells
All but 3 of the 33 bulbospinal neurons histologically identified as C1 cells (either TH-ir [n=30] or PNMT-ir [n=3]) showed some form of excitatory response to Ang II, as detailed below. All 10 C1 cells tested extracellularly in cell-attached mode had an increased firing rate in response to 1 µmol/L Ang II application (baseline, 2±0.7 spikes per second; Ang II, 5.2±0.8 spikes per second). Of 15 C1 neurons (including 12 TH-ir and 3 PNMT-ir) tested in the current-clamp mode, 13 exhibited membrane depolarization (6.2±0.7 mV, from -53±0.8 mV). Of 8 C1 neurons tested in the voltage-clamp configuration, 7 exhibited an inward current (9.4±1.4 pA) at holding potentials of -40 to -55 mV.

In contrast, most (10 of 13) of the non-C1 bulbospinal cells recovered histologically were unaffected by 1 µmol/L Ang II, whereas only 3 were excited. Thus, the vast majority (30 of 33) of the C1 cells were excited by Ang II, whereas the majority (10 of 13) of the non-C1 cells were insensitive to the peptide.

Ang II–Sensitive RVLM Bulbospinal Neurons Are Inhibited by ₂-Adrenergic Agonists
In a previous study, we found that the predominant catecholaminergic receptors of bulbospinal C1 adrenergic cells are ₂-adrenergic receptors that couple to inwardly rectifying K⁺ channels.¹⁷ In the present experiment, we examined the responses of 15 Ang II–sensitive bulbospinal RVLM neurons (8 cells in extracellular recording mode and 7 in whole-cell current or voltage-clamp mode) to the ₂-adrenergic agonists MN (30 µmol/L) or UK-14,304 (1 µmol/L). Of the 8 Ang II–sensitive RVLM bulbospinal cells tested in extracellular mode, 6 were inhibited and 2 were not responsive. The discharges of the 6 responsive cells (basal firing rate, 1.9±0.4 spikes per second) stopped for 2 to 7 minutes after ₂-adrenergic agonist application (Fig 9A). Four of 5 Ang II–sensitive RVLM bulbospinal cells recorded in the current-clamp mode were hyperpolarized (-7.8±0.9 mV from baseline potential of -52 to -57 mV), which was associated with cessation of action potentials, in the presence of MN. In 2 of 2 Ang II–sensitive RVLM bulbospinal neurons clamped at -40 and -55 mV, respectively, MN elicited an outward current (Fig 9B).

View larger version (15K): [in this window] [in a new window]   Figure 9. A, Extracellular unit recording of a bulbospinal neuron inhibited by 30 µmol/L MN and excited by 1 µmol/L Ang II. B, Voltage-clamp recording of different bulbospinal neuron (holding potential, -55 mV). Outward current was elicited by 30 µmol/L MN; inward current, by 1 µmol/L Ang II. Dashed line indicates zero current level.

	Discussion

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The present study confirms that the vast majority of bulbospinal neurons in the RVLM of neonatal rats are autoactive in vitro.^{16 17} This includes neurons identified as C1 cells on the basis of spinal projections and the presence of immunoreactivity for TH or PNMT (30 active cells out of 33). Similar rates of discharge were observed when the cells were recorded extracellularly or in whole-cell configuration. Therefore, the autoactivity of the cells is not an artifact of the whole-cell recording method. The present study also confirms the existence of neurons that are excited by Ang II via AT₁ receptors in the RVLM of the rat.¹⁰ The new finding of the present study is the identification of some of these Ang II–sensitive cells as C1 bulbospinal neurons. Furthermore, an analysis of the ionic mechanism involved in the excitatory action of Ang II is provided.

Three Quarters of RVLM Bulbospinal Neurons Express Functional AT₁ Receptors
The majority of RVLM bulbospinal neurons were excited by Ang II (72% of cells recorded extracellularly, 79% of cells recorded intracellularly in current- or voltage-clamp mode). The effect of Ang II that we observed was largely postsynaptic, since Ang II was effective in slices superfused with solution containing tetrodotoxin or low Ca²⁺/high Mg²⁺, ie, conditions in which synaptic transmission is greatly reduced. Ang II concentrations in excess of 0.1 µmol/L were required to produce effects on RVLM cells. This low sensitivity is not unusual for an in vitro preparation of brain^{25 26} and could be due to the lability of the peptide. Alternately, angiotensinergic synaptic transmission in the brain may require high concentrations of the peptide. All RVLM cells that responded to Ang II had the same type of response, and when tested, the response was blocked by losartan used in a concentration that is specific for the AT₁ receptor.¹⁴ The above evidence demonstrates that 75% of RVLM bulbospinal neurons are activated by Ang II via AT₁ receptors. The high percentage of Ang II–sensitive cells in the bulbospinal projection of the RVLM is in contrast to the small proportion of sensitive cells found when units are randomly sampled from the same region in thick slices of the adult rat brain.^{10 27} Despite differences in age and type of recording, this discrepancy suggests that Ang II–sensitive cells are a minority in the RVLM.

Ninety-one Percent of Bulbospinal C1 Cells Are Excited by Ang II
The vast majority (30 of 33) of the C1 neurons (bulbospinal cells of the RVLM identified histologically as TH-ir or PNMT-ir) were excited by Ang II. This evidence indicates that C1 cells express functional AT₁ receptors, a hypothesis based on the striking overlap in the location of catecholaminergic cell bodies and autoradiographic binding of labeled angiotensin-related ligands in the RVLM of several species.^{6 12 14 15}

Only 3 of the 13 non-C1 cells (histologically recovered cells that were found devoid of TH-ir) were excited by Ang II. Since the histological technique presumably generates some false-negative results (TH-ir may not be distinctly above background in some cells that may be mistaken as noncatecholaminergic), it is conceivable that the 3 responders in this series of 13 cells could have been C1 cells that remained undetected. In any case, the result suggests that the noncatecholaminergic component of the projection from the RVLM to the spinal cord may not have functional Ang II receptors.

The proportion of Ang II–sensitive cells among the entire population of RVLM bulbospinal neurons (72% with extracellular recording, 79% with whole-cell recordings) closely approximates the previously reported proportion of C1 cells among the retrogradely labeled RVLM cells (78% to 80%).¹⁷ This and the observation that >90% of C1 cells are sensitive to Ang II suggest that Ang II receptors may be selectively located in C1 cells as opposed to other types of bulbospinal cells in the RVLM.

Mechanism Underlying the Depolarizing Effect of Ang II on RVLM Bulbospinal Neurons
Ang II elicited a relatively small inward current (9.7 pA on average in response to 1 µmol/L peptide). However, because of the high input resistance of the cells (0.8 to 0.9 G), this current is sufficient to produce the 7- to 8-mV depolarization consistently observed in current-clamp experiments. Because the cells are slowly active at rest, a depolarization of this magnitude leads to a substantial increase in discharge rate (250% of control).

The depolarization (current clamp) or inward current (voltage clamp at -40 to -55 mV) was associated with a decrease in membrane conductance. This reduction of conductance is likely due to closure of K⁺ channels for the following reasons: First, in the presence of 3.8 mmol/L [K⁺]_o, the current elicited by Ang II reversed polarity very close to E_K (E_rev, -89 mV; Fig 8A), suggesting that it was carried selectively by K⁺. Second, raising [K⁺]_o to 10 mmol/L shifted the E_rev of the Ang II–induced current to between -60 and -68 mV, a shift that is according to the Nernst equation for a current predominantly carried by K⁺. Moreover, barium, a nonselective K⁺ channel inhibitor,²⁸ attenuated the effect of Ang II (Fig 8D).

The Ang II–induced conductance change was not voltage dependent in the range of -40 to -140 mV (Fig 8C). This is in contrast to the conductance induced by ₂-adrenergic agonists, which exhibits marked inward rectification in the same cells.¹⁷ From this finding, we suggest that in the RVLM, Ang II does not close inwardly rectifying K⁺ channels as it does in some brain endothelial cells,²⁹ although this type of channel is clearly present in C1 cells.¹⁷ In addition, the inward current elicited by Ang II in C1 cells was not altered by lowering [Ca²⁺]_o and raising [Mg²⁺]_o. Therefore, the K⁺ conductance reduced by Ang II does not appear to be Ca²⁺ dependent, as may be the case in vascular muscle cells of the rabbit aorta.³⁰ The lack of voltage dependence of the current in the range of -40 to -140 mV also excludes the possibility that Ang II inhibits an M current, such as in sympathetic ganglionic cells.³¹ Therefore, we are led to propose that in RVLM cells, Ang II decreases a resting K⁺ conductance akin to a "leak." The leak K⁺ conductance (also known as resting or persistent K⁺ current) is a relatively voltage-independent K⁺ conductance that is active at resting membrane potential and contributes to setting the resting membrane potential.³² This conductance is the target of several G protein–coupled receptors found commonly on neurons, such as receptors to thyrotropin-releasing hormone,^{28 33} substance P,³⁴ glutamate (metabotropic),³⁵ , acetylcholine (muscarinic),^{35 36} histamine,³⁷ noradrenaline (₁-receptors),³⁸ and serotonin (5HT₂ receptors).³⁹ To the best of our knowledge, the coupling of AT₁ receptors to leak K⁺ channels has not been described before. In hypoglossal motor neurons, the inward current evoked by thyrotropin-releasing hormone is due to a combination of leak K⁺ current reduction and activation of a small Ba²⁺-resistant, presumably cationic, conductance.²⁸ As a result, E_rev of the thyrotropin-releasing hormone–evoked current is more negative than E_K by 12 mV. The possibility that Ang II may also be coupled to a similar type of cationic conductance in RVLM bulbospinal cells seems unlikely for two reasons. First, E_rev of the Ang II–evoked current did not deviate significantly from E_K in the presence of either 3.8 or 10 mmol/L [K⁺]_o. Second, in the presence of Ba²⁺, the small residual current still reversed close to E_K.

Elsewhere, AT₁ receptors are coupled to numerous other types of ion channels, including Ca²⁺, Cl^-, and nonselective cationic ion channels.^{25 31 40 41 42} Coupling to Cl^- channels seems unlikely in C1 cells, since the Cl^- equilibrium potential was -45 mV under the present experimental conditions. However, a modulation of voltage-dependent Ca²⁺ channels is not excluded by the present experiments.

Functional Implications
Although the present results were obtained in neonatal tissue, the following clues indicate that the effects of Ang II on RVLM cells persist in the adult. First, in the C1 region of the RVLM of adult rats, there is a distinct population of slowly firing neurons that are excited to 200% to 300% of the control firing rate by Ang II via AT₁ receptors.¹⁰ Since this cell population in the adult is also selectively inhibited by ₂-adrenergic receptor agonists, it is likely to include the C1 neurons. Second, microinjection of Ang II excites some RVLM bulbospinal neurons in vivo.⁴³

Since C1 cells have a vasomotor sympathoexcitatory function,^{22 44 45 46} a depolarizing effect of Ang II would tend to increase sympathetic outflow and arterial pressure, which is indeed observed when the peptide is injected into the RVLM in vivo.^{3 4 5 6 7 8} The increased input resistance caused by Ang II should also have the effect of potentiating the synaptic inputs onto these cells (excitatory as well as inhibitory), and this has been found also in vivo. In anesthetized rabbits, Ang II facilitates both the baroreflex (an inhibitory reflex mediated by a GABA input onto bulbospinal cells of RVLM⁷ ) and the somatosympathetic reflex (an excitatory reflex mediated by excitatory amino acids in the RVLM⁸ ).

In summary, C1 cells have AT₁ receptors whose activation leads to the reduction of a resting potassium conductance. This effect increases the discharge rate and excitability of these cells, which is likely to contribute to the sympathoexcitation observed when Ang II is injected into the RVLM in vivo.

	Selected Abbreviations and Acronyms

 

MN = -methylnoradrenaline aCSF = artificial cerebrospinal fluid Ang II = angiotensin II AT1 receptor = type-1 angiotensin receptor EK = K+ equilibrium potential Erev = reversal potential FITC = fluorescein isothiocyanate PNMT = phenylethanolamine N-methyltransferase PNMT-ir = PNMT immunoreactivity RVLM = rostral ventrolateral medulla TH = tyrosine hydroxylase TH-ir = TH immunoreactivity

	Acknowledgments

 
This study was supported by a grant from the National Institutes of Health (HL-28785) to Dr Guyenet. Dr Li is an International Research Fellow of the American Heart Association. We thank Dr Douglas Bayliss for comments on the manuscript.

Received June 12, 1995; accepted November 2, 1995.

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