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Am J Physiol Regul Integr Comp Physiol 274: R903-R911, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 4, R903-R911, April 1998

Neurosteroid modulation of arterial baroreflexsensitive neurons in rat rostral ventrolateral medulla

J. D. Laiprasert, R. C. Rogers, and C. M. Heesch

Department of Physiology, The Ohio State University, Columbus, Ohio 43210-1218

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The major metabolite of progesterone, 3alpha -OH-dihydroprogesterone (3alpha -OH-DHP), is the most potent endogenous positive modulator of central nervous system GABAA receptors. Acute intravenous administration of 3alpha -OH-DHP to virgin female rats potentiates arterial baroreflex sympathoinhibitory responses. The current experiments tested the possibility that circulating 3alpha -OH-DHP potentiates central GABAergic influences in the rostral ventrolateral medulla (RVLM). The unit activity of spontaneously active, spinally projecting, and arterial pressure-sensitive neurons was recorded in the RVLM of urethan-anesthetized rats. Arterial pressure sensitivity of RVLM neurons was tested before (control) and 10 min after bolus injection (44 µl iv) of 3alpha -OH-DHP (1.12 µg/kg, n = 19) or vehicle (40% beta -cyclodextrin, n = 8). Both threshold pressure and saturation pressure for inhibition of RVLM neurons were decreased after acute administration of a physiological dose of 3alpha -OH-DHP (1.12 µg/kg iv), which produces plasma concentrations similar to those seen during pregnancy (20-30 ng/ml), suggesting potentiated responsiveness to endogenously released GABA. Following suppression by 3alpha -OH-DHP, high doses of the inactive stereoisomer 3beta -OH-DHP (112-224 µg/kg iv; n = 8) restored unit activity, presumably by displacing 3alpha -OH-DHP from the neurosteroid binding site on GABAA receptors.

gamma -aminobutyric acid; baroreflex; progesterone

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ROSTRAL VENTROLATERAL MEDULLA (RVLM) is an essential component in the central nervous system (CNS) pathway for regulation of sympathetic outflow. Commonly accepted as the final site for tonic sympathoexcitatory drive to preganglionic sympathetic neurons in the intermediolateral cell column (IML) of the spinal cord, neurons in the RVLM receive and integrate cardiovascular input from multiple CNS sites (8). In particular, as the final step in the medullary baroreflex pathway, the RVLM receives direct GABAergic inhibitory input from the caudal ventrolateral medulla (CVLM) (7, 24). GABAergic influence on RVLM neurons represents the primary mechanism for arterial baroreflex inhibition of tonic drive to sympathetic preganglionic neurons in the IML.

Previous studies in our laboratory have demonstrated that pregnancy is associated with decreased mean arterial pressure (MAP), potentiated baroreflex sympathoinhibition, and attenuated baroreflex sympathoexcitation (6, 23). These results are consistent with an increased GABAergic influence in the RVLM of pregnant animals. Although the mediator for altered control of sympathetic outflow during pregnancy is not known, the ovarian hormones and/or their metabolites, particularly the primary metabolite of progesterone, 3alpha -OH-dihydroprogesterone (3alpha -OH-DHP), are likely candidates.

The progesterone metabolite 3alpha -OH-DHP is the most potent endogenous positive modulator of CNS GABAA receptor function (27, 28). Plasma levels of 3alpha -OH-DHP are elevated during pregnancy to concentrations that have been demonstrated to potentiate GABA-mediated inhibition in vitro (22). The mechanism of action of 3alpha -OH-DHP is thought to be nongenomic and due to immediate membrane effects produced by the binding of 3alpha -OH-DHP to a unique neurosteroid binding site on the GABAA receptor complex (22, 28).

Previously our laboratory reported that acute administration of 3alpha -OH-DHP to virgin rats produced an attenuated baroreflex sympathoexcitation and potentiated baroreflex sympathoinhibition (14, 23), an effect that is qualitatively similar to the effects of pregnancy (6, 23). These results suggest a CNS mechanism. Thus the purpose of the current study was to determine if circulating 3alpha -OH-DHP, in concentrations similar to those found in pregnancy, acutely altered the arterial pressure sensitivity of spinally projecting neurons in the RVLM to endogenously released GABA.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Surgical preparation. Experiments were performed in 21 virgin female Sprague-Dawley rats (3-5 mo old; Harlan Sprague Dawley, Indianapolis, IN) weighing 225-270 g. Rats were anesthetized with intraperitoneal urethan (1.5 g/kg) and supplemented (0.15 g/kg iv) as needed. A subcutaneous injection of dexamethasone (1.2 mg/kg) was also given to limit nervous tissue swelling. The trachea was cannulated, and the rat was artificially ventilated (CWE SAR830 ventilator) with O2-enriched room air. Body temperature was monitored and maintained at 37°C. An arterial catheter was placed into either the right carotid or right femoral artery to monitor arterial blood pressure, and three jugular catheters were implanted for subsequent systemic drug administration. The rat was then placed in a stereotaxic apparatus, and an occipital craniotomy was performed. The occipital parietal membrane and dura were cut and folded laterally to expose the brain stem.

A laminectomy was performed to expose the spinal cord between C2 and T2, and the spinal cord was stabilized on the same plane as the brain stem by means of a rigid clamp on the dorsal vertebral process of T2. The head was tilted forward until the calamus scriptorius was located 2.4 mm posterior to interaural zero (17). Tubocurarine (0.1 mg/kg iv) was administered to paralyze the rat, and a tungsten monopolar stimulating electrode (tip diameter 0.1 mm) was advanced into the dorsolateral funiculus on the left side of the spinal cord at the level of C2 (immediately medial to dorsolateral sulcus and 0.3 mm ventral to dorsal surface). This region contains descending axonal projections from the RVLM to spinal preganglionic sympathetic neurons in the IML (32). A pressor response (20-40 mmHg) during brief electrical stimulation of the spinal cord (5 mA, <1 ms, 5 Hz) verified the location of the electrode tip in the dorsolateral funiculus.

Drugs and solution. Urethan ethyl carbamate (99%) and phenylephrine (PE) were purchased from Sigma (St. Louis, MO). Tubocurarine chloride was obtained from Bristol Myers Squibb (Princeton, NJ). The urethan, tubocurarine, and PE were each diluted in isotonic saline. Dexamethasone sodium phosphate was purchased from Steris Laboratories (Phoenix, AZ). 2-Hydroxypropyl-beta -cyclodextrin was purchased from Research Biochemicals International (Natick, MA) and dissolved in a 50:50 solution of distilled water and isotonic saline to make 40% beta -cyclodextrin. The progesterone metabolites 5alpha -pregnan-3alpha -ol-20-one (3alpha -OH-DHP) and 5alpha -pregnan-3beta -ol-20-one (3beta -OH-DHP), also obtained from Sigma, were dissolved in 40% beta -cyclodextrin. Chicago sky blue 6B 80% was obtained from Aldrich (Milwaukee, WI), and neutral red was purchased from National Diagnostics (Highland Park, NJ).

Single-unit recordings. Extracellular unit recording from cells in the RVLM was performed using glass microelectrodes (outer tip diameter approx 1 µm, resistance 1-2 MOmega ) filled with 1% Chicago sky blue dye dissolved in 1 M NaCl. The electrode was advanced into the left side of the brain stem using a hydraulic microdrive (David Kopf). Spontaneously active neurons within anterioposterior coordinates of 0.5-0.8 mm rostral to calamus scriptorius, 1.7-2.2 mm lateral to midline, and 2.7-3.8 mm ventral to the dorsal surface were identified. The signal was amplified 10,000 times using two Grass (Quincy, MA) P15 preamplifiers and monitored on a loudspeaker as well as on a dual beam storage oscilloscope (R5031; Tektronics, Beaverton, OR). A rate meter/window discriminator (RAD-IIA, Winston Electronics) was used to determine the firing frequency of the unit. Unit activity (UA), heart rate, and MAP were monitored on a polygraph (79D, Grass Instruments) and stored on videotape (DR-886, Neuro Data Instrument) for later analysis using data acquisition software (RC Electronics, Computerscope).

Identification of spontaneously firing units as spinally projecting neurons involved in the regulation of cardiovascular function was determined by means of two different tests. First, spontaneous units were tested for antidromic spike production in response to electrical stimulation of the dorsolateral funiculus in the spinal cord (0.5 Hz, 5 mA, <1 ms). Neurons demonstrating a constant latency from the stimulus to the evoked spike and observation of collision with a spontaneous action potential suggested that the neuron projected to the spinal cord (Fig. 1). During antidromic activation of spontaneously active neurons, slight variability in latencies may occur due to the effect of different levels of membrane polarization on the delay between antidromic invasion of the initial segment and the soma-dendritic region. Therefore, neurons with latency variation of <0.2 ms during repeated spinal cord stimuli were considered antidromically activated (20, 33). Second, the baroreflex sensitivity of the cell was tested. Slow ramp increases in MAP were elicited by graded intravenous infusions (Razel infusion pump) of PE (9-300 µg/min) during the monitoring of the UA of the neuron. Arterial baroreceptors are rate sensitive and thus, for a given cell, care was taken to ensure that the rate of ramp increases in MAP was similar each time pressure sensitivity was tested. A progressive decrease in UA as MAP was elevated indicated arterial baroreflex sensitivity of the cell. Neurons that were both antidromically activated by dorsolateral funiculus stimulation and greatly inhibited by elevations in MAP were presumed to be presympathetic cardiovascular neurons of the RVLM (10) and were included in this study.


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  		Fig. 1.   Identification of a spinally projecting rostral ventrolateral medulla (RVLM) neuron; antidromic-evoked potentials in an RVLM neuron due to electrical stimulation of ipsilateral spinal dorsolateral column. Stimulus artifact is seen on far left. A-C demonstrate a constant latency for evoked potential, which is indicative of direct connection between RVLM neuron and spinal cord. Latency for this neuron was 4.92 ms. Collision, shown in D, occurred when a spontaneous action potential (star ) rendered the neuron refractory.

Experimental protocol. The relationship between MAP and UA was determined in RVLM neurons that met the above criteria during a gradual increase in MAP (control response). After MAP and UA returned to baseline values, a bolus intravenous injection of 3alpha -OH-DHP (1.12 µg/kg, n = 19) was administered. This dose was estimated to produce a maximum plasma concentration (approx 22 ng/ml) within the reported range of plasma concentrations seen during pregnancy (20-30 ng/ml) (28). Ten minutes after drug administration, the pressure sensitivity of the neuron was retested. The effect of vehicle (40% beta -cyclodextrin iv) was evaluated in eight experiments using a similar protocol, except that an equivalent volume of 40% beta -cyclodextrin (44 µl) instead of 3alpha -OH-DHP was administered.

Preliminary experiments evaluating the effects of a higher dose of 3alpha -OH-DHP were also performed. In seven cells that had received 1.12 µg/kg 3alpha -OH-DHP (iv), the effect of subsequent administration of a higher dose of 3alpha -OH-DHP (11.2 µg/kg iv) was also tested.

The response of identified RVLM neurons to elevation of MAP was quantified by four parameters: MAP and UA values recorded at threshold and at saturation. After the experiments, taped data of MAP and instantaneous unit discharge were digitized (RC Electronics computerscope) and 5-ms averages were obtained (Microsoft Excel, Seattle, WA). Maximum and minimum UA were identified during a 1-min period immediately preceding the PE-induced pressure ramp. During a slow pressure ramp (approx 5 mmHg/s), the pressure at which action potential firing frequency decreased below minimum baseline firing rate and continued to decrease as pressure increased, was defined in the current study as the threshold pressure for inhibition of the unit. This method of threshold determination served to standardize measurements both within and between animals. However, it should be noted that this experimentally defined threshold is most likely higher than the physiological threshold of RVLM neurons. The pressure at which the neuron ceased firing or reached a minimum firing rate was defined as the saturation pressure (Fig. 3). The rate of recovery for MAP and UA was also evaluated in this study by measuring four parameters. The half time for MAP recovery (t1/2 MAP) and the half time for recovery of UA (t1/2 UA) were both determined. Additionally, values for UA at t1/2 MAP and the MAP at t1/2 UA were obtained (Fig. 3).

The relatively long half-life of 3alpha -OH-DHP (~1 h in humans) (5) prohibited reliable assessment of recovery from the drug effect. However, high concentrations of 3beta -OH-DHP have been shown to compete with 3alpha -OH-DHP for binding sites on the GABAA receptor complex in vitro (30). In some experiments (n = 8) it was noted that UA did not completely return to pre-3alpha -OH-DHP baseline levels after the last pressure ramp. In these experiments (1.12 µg/kg 3alpha -OH-DHP, n = 5; 11.2 µg/kg 3alpha -OH-DHP, n = 3), a high concentration of the inactive stereoisomer, 3beta -OH-DHP (112-224 µg/kg iv), was administered to test for reversal of the effects of 3alpha -OH-DHP. MAP and UA before and after administration of 3beta -OH-DHP were compared.

Once a neuron was accepted for inclusion in the study, the experimental protocol lasted ~30-60 min. A neuronal recording was considered stable only if shape and height of the spike were consistent during the entire recording period. At the end of the experiment, the location of the tip of the recording electrode in the brain stem was marked by ejecting Chicago sky blue dye from the pipette by iontophoresis (-25 µA for 20 min). Standard histological techniques were used to fix and section the brain stem (50-µm sections, neutral red stain). The recording site was estimated by comparison with a rat brain atlas (29).

Statistical analysis. A paired t-test was used to compare control and treatment values. Data obtained in cells that received more than one dose of 3alpha -OH-DHP were analyzed using a one-way ANOVA for repeated measures followed by Student-Newman-Keuls post hoc test. P < 0.05 was considered significant. Values are means ± SE.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Identification and characterization of presympathetic RVLM pressure-sensitive neurons. For inclusion in the study, spontaneously firing neurons recorded in the RVLM were tested for antidromic activation that would indicate that these neurons were spinally projecting neurons. Figure 1 shows sequential oscilloscope traces of an RVLM unit that was antidromically activated by electrical stimulation of the dorsolateral funiculus in the spinal cord (0.5 Hz, 5 mA, 0.5 ms). A constant latency between the stimulus artifact and the evoked action potential (Fig. 1, A-C) and observation of collision of the stimulus with a spontaneous action potential (Fig. 1D) suggest that the neuron projected directly to the spinal cord. Baroreflex sensitivity of each neuron was also tested. Substantial inhibition of a neuron in response to increased MAP suggested that the neuron was part of the central baroreflex pathway.

Although many spontaneously firing neurons were recorded in the area of the RVLM, protocols were performed only on those neurons meeting the criteria described above. A total of 22 spontaneously active neurons were both antidromically activated from the dorsolateral funiculus in the spinal cord and inhibited by elevations in MAP. Elevated arterial pressure resulted in complete cessation of unit discharge in 15 neurons, and discharge was inhibited to 35.9 ± 9.0% of baseline in the remaining 7 neurons. These neurons were assumed likely to be presympathetic RVLM neurons involved in the baroreflex regulation of sympathetic outflow. Antidromic latencies ranging from 2 to 13 ms (mean 6.15 ± 0.8 ms) were observed in these neurons. Calculated conduction velocities, assuming a linear conduction pathway and an estimated distance of 2.5 cm between stimulus and recording site, ranged from 1.92 to 12.5 m/s (mean 5.9 ± 0.80 m/s). Baseline firing frequencies of identified neurons ranged from 3.2 to 32.4 pulses per second (pps). Of the 22 cells studied, pulse-synchronous activity was evident in 11 cells (Fig. 2). Although not stringently evaluated, several of the neurons appeared to also demonstrate a respiratory-like rhythm (n = 9).


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  		Fig. 2.   Pulse-synchronous discharge of RVLM neuron; ~60 superimposed traces are shown. Action potentials are correlated with early diastolic phase of arterial pressure pulse. UA, unit activity; AP, arterial pressure.

Figure 3 shows a typical response of an RVLM neuron to a ramp increase in MAP. Parameters used to characterize responses are indicated on the figure.


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  		Fig. 3.   Identification of response and recovery parameters; ramp increase in mean arterial pressure (MAP) produced inhibition of UA of a spinally projecting RVLM neuron. MAP and UA at threshold (T), point at which firing frequency decreased below baseline, and at saturation (S), point at which cell discharge reached minimum frequency, were measured. Recovery parameters were obtained after removal of phenylephrine stimulus. Point at which MAP and UA were half recovered (*) was determined, and half time to recovery for MAP (t1/2 MAP) and UA (t1/2 UA) were calculated. pps, Pulses per second.

Effect of 3alpha -OH-DHP on baseline, threshold, and saturation parameters. The effects of vehicle and 3alpha -OH-DHP on presumed presympathetic, baroreflex-sensitive RVLM neurons were evaluated, and the results are summarized in Tables 1 and 2. Baseline values of MAP and UA measured immediately before the pressure ramps were not affected by either vehicle or 3alpha -OH-DHP (1.12 µg/kg iv, Table 1). Vehicle alone did not have an effect on threshold or saturation values in the eight neurons tested (Table 2 and Fig. 4). Responses to intravenous administration of 3alpha -OH-DHP (1.12 µg/kg iv) were determined in a total of 19 neurons. The maximum plasma concentration that could be achieved with this dose (approx 22 ng/ml) was calculated to be within the range of concentrations seen in pregnancy (20-30 ng/ml) (28). Both the threshold MAP for inhibition of the unit and the saturation MAP were decreased by 3alpha -OH-DHP (1.12 µg/kg iv), indicating an increased sensitivity of RVLM neurons to increases in arterial blood pressure. In five of these neurons, responses to intravenous administration of vehicle (44 µl 40% beta -cyclodextrin) had been tested before 3alpha -OH-DHP. Statistical analysis of the data with and without these five neurons revealed the same significant differences, and therefore data from all 19 neurons are shown (Table 2 and Fig. 5).

                              
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  		Table 1.   Effects of treatments on baseline MAP and UA

                              
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  		Table 2.   Effects of treatments on threshold and saturation parameters


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  		Fig. 4.   Effects of vehicle. Administration of vehicle (40% beta -cyclodextrin) had no effect on baseline MAP, threshold pressure, or saturation pressure of RVLM neurons (n = 8). Bars are values ± SE.


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  		Fig. 5.   Effects of 3alpha -OH-dihydroprogesterone (3alpha -OH-DHP). There was no significant effect of 3alpha -OH-DHP (1.12 µg/kg iv, n = 19) on baseline MAP. However, 3alpha -OH-DHP significantly decreased threshold pressure and saturation pressure of RVLM neurons. This indicates that 3alpha -OH-DHP had a potentiating effect on baroreflex-mediated sympathoinhibition. Bars are values ± SE; * P <=  0.05.

Preliminary data evaluating the effect of subsequent administration of a higher dose of 3alpha -OH-DHP (11.2 µg/kg) were obtained in 7 of these 19 cells. Repeated-measures ANOVA on the subset of seven neurons exposed to both 1.12 µg/kg and 11.2 µg/kg 3alpha -OH-DHP revealed that subsequent administration of the higher dose of 3alpha -OH-DHP did not produce any further decrease in either threshold (117 ± 6.1 mmHg) or saturation MAP (152 ± 4.9 mmHg).

Effect of 3alpha -OH-DHP on recovery parameters. Recovery parameters for MAP and UA after the ramp increase in arterial pressure are summarized in Tables 3 and 4. Because of technical limitations or a prolonged time for recovery (>10 min), it was not possible to quantitate recovery parameters in all neurons. Recovery after PE-induced elevations in pressure was evaluated in 15 of 19 neurons exposed to 3alpha -OH-DHP (1.12 µg/kg). The t1/2 MAP (Table 3) and t1/2 UA (Table 4) were unaffected by vehicle or 3alpha -OH-DHP (1.12 µg/kg), indicating similar rates of recovery for both blood pressure and UA between control and treatment responses in these groups.

                              
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  		Table 3.   Recovery parameters for MAP

                              
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  		Table 4.   Recovery parameters for UA

Recovery parameters were obtained for five of seven neurons exposed to the highest dose of 3alpha -OH-DHP (11.2 µg/kg iv) after administration of the physiological dose of 3alpha -OH-DHP (1.12 µg/kg iv). Repeated-measures ANOVA on control, 1.12, and 11.2 µg/kg 3alpha -OH-DHP values in these five cells revealed that t1/2 UA was increased [control, 84.2 ± 41.2; after 3alpha -OH-DHP (11.2 µg/kg), 136.5 ± 55.5 s] and MAP at t1/2 UA was less [control, 117.8 ± 7.8; after 3alpha -OH-DHP (11.2 µg/kg), 105.3 ± 4.0 mmHg] at the highest dose of 3alpha -OH-DHP. Therefore, in the presence of 11.2 µg/kg 3alpha -OH-DHP, recovery of UA was prolonged, and thus MAP was lower at t1/2 UA.

Effect of 3beta -OH-DHP. Baseline values of MAP and UA obtained before (control) and 10 min after administration of 1.12 µg/kg 3alpha -OH-DHP (iv) were not different (Table 1). During the experiment, final MAP and UA values were also noted after the last PE-induced pressure ramp in the presence of 3alpha -OH-DHP.

The effect of administration of a high concentration of the inactive stereoisomer 3beta -OH-DHP was evaluated in eight neurons that, at the time of the experiment, did not appear to fully recover after the final pressure ramp in the presence of 3alpha -OH-DHP (1.12 µg/kg, n = 5; 11.2 µg/kg, n = 3). MAP and UA for these eight neurons were compared immediately before and 1 min after bolus administration of high concentrations of 3beta -OH-DHP (112-224 µg/kg). Despite a slight but significant increase in baseline MAP after administration of 3beta -OH-DHP, a significant increase in UA was observed (Table 5). This is consistent with reversal of the effects of 3alpha -OH-DHP due to competition at the binding site by the inactive stereoisomer 3beta -OH-DHP.

                              
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  		Table 5.   Effect of 3beta -OH-DHP on MAP and UA

Histology. Post hoc histological examination of the recording sites verified that the neurons were distributed within an area previously described as the RVLM (Fig. 6) (3, 9, 10, 35).


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  		Fig. 6.   Post hoc histological analysis of recording sites. Extracellular recording sites were marked with 1% Chicago sky blue dye in 1 M NaCl at end of each experiment by passing a 25-µA cathodal current through the electrode for 20 min. Serial sections through brain stem at level of RVLM show that neurons from which recordings were taken were indeed located in area of RVLM. bullet , Recording sites (21 neurons). Sol, nucleus of the solitary tract; Cu, cuneate nucleus; ECu, external cuneate nucleus; IO, inferior olive; Amb, ambiguus nucleus; Sp, spinal trigeminal tract.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The RVLM is an integral component in the central pathway for control of cardiovascular function and is considered to be the final site for tonic sympathoexcitatory drive to preganglionic sympathetic neurons in the spinal cord (10). Neurons in the RVLM receive and integrate cardiovascular input from both supramedullary and medullary nuclei (8). When arterial pressure increases, the medullary baroreflex pathway is activated. Increased discharge of afferent fibers from arterial baroreceptors results in excitation of neurons in the nucleus of the solitary tract (NTS), followed by excitation of neurons in the CVLM (8, 10). A monosynaptic projection from the CVLM to the RVLM (7, 24) inhibits tonically active neurons in the RVLM, which results in decreased discharge of preganglionic sympathetic neurons in the IML of the spinal cord (15). Inhibition of neurons in the RVLM by the CVLM is mediated by release of the amino acid neurotransmitter GABA (34). GABAergic influences represent the primary mechanism for arterial baroreflex inhibition of tonic sympathetic drive.

Earlier studies in our laboratory evaluating the effects of pregnancy on baroreflex function showed that pregnancy potentiated sympathoinhibitory and attenuated sympathoexcitatory baroreflex responses (6, 23). These effects are consistent with an increased GABAergic inhibition in the RVLM of pregnant animals. The mediator for these pregnancy-associated adaptations in control of sympathetic outflow is not known, but likely candidates are the ovarian hormones and/or their metabolites, which are elevated during pregnancy. It has been recently demonstrated that the primary metabolite of progesterone, 3alpha -OH-DHP, is the most potent endogenous positive modulator of CNS GABAA receptor function (27, 28). Plasma concentrations of 3alpha -OH-DHP are elevated during pregnancy to levels that have been demonstrated to potentiate GABA-mediated inhibition (22, 28).

3alpha -OH-DHP belongs to a class of compounds known as neurosteroids whose primary action appears to be positive modulation of GABAA receptor function. The mechanism of action is not thought to be genomic, because the effects are rapid (seconds to minutes) (22) and inhibition of protein synthesis does not alter the effect of neurosteroids (28). The neurosteroids bind to a unique and stereospecific site on the GABAA receptor complex. Administered in 10- to 30-nM concentrations, neurosteroids are potent modulators of GABAA receptor function, prolonging the duration of chloride channel opening (22). Neurosteroids have been shown to produce an increased duration of inhibitory postsynaptic currents in hippocampal neurons (11). At higher concentrations (micromolar range), neurosteroids have been shown to directly open the chloride channel (22).

Previous studies in our laboratory evaluated the effect of acute administration of 3alpha -OH-DHP on baroreflex function in both anesthetized (14) and conscious rats (23). Acute administration of 3alpha -OH-DHP to virgin rats resulted in attenuated sympathoexcitatory responses and potentiated sympathoinhibitory responses. In other words, the acute response to exogenously administered 3alpha -OH-DHP in virgin rats was qualitatively similar to the effects of pregnancy. Although a CNS mechanism was implied by these previous studies, direct evidence was not provided. The purpose of this study was to determine if circulating levels of 3alpha -OH-DHP, administered in concentrations similar to those found in pregnancy, altered the sensitivity of sympathoexcitatory neurons in the RVLM to endogenously released GABA.

Characterization of presympathetic RVLM cardiovascular neurons. In the current study, spinally projecting spontaneously firing neurons in the RVLM that were inhibited by elevations in arterial pressure were presumed to be presympathetic neurons. Baseline UA of RVLM neurons included in this study also exhibited a wide range of firing frequencies, varying between 3.2 and 32.4 pps, which is consistent with the baseline firing rates reported by others (0.5-40 pps) (1, 2, 4, 9). Antidromic latencies between 2 and 13 ms (mean 6.15 ± 0.8 ms) and conduction velocities between 1.9 and 12.5 m/s were observed in this study. Conduction velocities ranging between 0.4 and 11 m/s have been reported (2, 4, 9, 16, 21, 26). The wide distribution of conduction velocities reported in the literature suggests involvement of both myelinated (>1 m/s) and unmyelinated (<1 m/s) fiber types. None of the neurons included in this study had conduction velocities <1 m/s, indicating that the axons of neurons in this study were probably myelinated. The mean conduction velocity of neurons included in this study (5.9 ± 0.8 m/s) is similar to that reported for presympathetic RVLM neurons by Granata and Kitai (5.5 ± 2.6 m/s) (9), Kanjhan et al. (4.9 ± 2.7 m/s) (16), and Lipski et al. (5.2 ± 2.3 m/s) (21). Mean conduction velocities reported by Morrison and Reis (26) are somewhat lower (3.1 ± 0.1 m/s). However, almost one-half the RVLM neurons characterized in that study were silent (49%) (26). Compared with conduction velocities of silent RVLM neurons, the conduction velocities of spontaneously active neurons tend to be higher (26). In our experiments, only spontaneously active RVLM neurons were studied, and thus we may have selected for neurons with myelinated axons and higher conduction velocities. Additionally, the use of relatively low-resistance electrodes (1-2 MOmega ) in the current experiments would bias the recording toward larger cells with myelinated axons.

Although not used as a criterion in the positive identification of RVLM neurons, a correlation between neuronal firing pattern and cardiac cycle was observed in many units included in the study. As has been reported by others (4, 16), correlation between the cardiac cycle and unit discharge was evident primarily at elevated MAP, especially in those units exhibiting high baseline firing frequencies. Pulse synchrony was defined as a pattern of discharge that correlated with no more than one-half the cardiac cycle. With use of this criterion, evidence of pulse-synchronous discharge was observed in 11 of 22 neurons. The number of pulse-synchronous neurons may have been underestimated in this study. Because of the nature of the protocol, arterial pressure was not elevated for a prolonged period of time, which would have been necessary for definitive characterization of a neuron as pulse synchronous. It is likely that had enough data at high pressures been obtained, more neurons would have been characterized as pulse synchronous.

Although a direct correlation between UA and respiratory activity was not possible in this study because phrenic nerve activity was not recorded, a respiratory-like rhythm was noted in nine identified RVLM baroreflex-sensitive neurons. Of the nine neurons, five were additionally found to exhibit a pulse-synchronous pattern that was unmasked at high MAP. Recent reports have demonstrated an effect of central respiratory drive on the baroreflex at the level of the RVLM. Brown and Guyenet (4) demonstrated that spinally projecting barosensitive RVLM cells showing acute sensitivity to plasma CO2 levels have a prominent respiratory-related rhythm. A direct correlation between phrenic nerve discharge and UA of baroreflex-sensitive RVLM neurons has been shown by Miyawaki et al. (25). However, Granata and Kitai (9) reported that antidromically activated RVLM neurons with respiratory-related activity demonstrated no baroreceptor-modulated activity. This apparent inconsistency may be due to the differences in recording sites between the studies. Compared with the study by Granata and Kitai (9), baroreflex-sensitive RVLM neurons with respiratory-related rhythm in the study by Miyawaki et al. (25) were located more dorsally in the RVLM. Although a respiratory modulation was evident in approximately one-third of the neurons in the current experiments, these neurons did not exhibit the characteristic on-off firing pattern of respiratory neurons and were greatly inhibited by elevated arterial pressure, suggesting that they were primarily involved in the arterial baroreflex (4, 16).

Post hoc histological verification of the recording sites for 21 neurons recorded in this study was obtained and revealed that the neurons were within the area described in the literature as the RVLM (10, 16, 25). In the current study, only 3 of the 21 neurons were located within 400 µm of the ventral surface. The majority of recorded neurons were located more dorsally (Fig. 6), and this may account for the relatively large number of neurons exhibiting respiratory-like rhythm. This distribution is consistent with reports by Brown and Guyenet (4) and Miyawaki et al. (25), demonstrating that presympathetic neurons 600-700 µm dorsal to the ventral surface of the medulla and 300-400 µm caudal to the caudal tip of the facial nucleus show a prominent respiratory rhythm.

The large number of dorsally located neurons may be a peculiarity of the experimental protocol of the current study. The recording electrode was advanced from the dorsal surface of the brain stem, and as soon as a spinally projecting pressure-sensitive neuron was isolated, the experiment was begun. Thus more ventral sites were frequently not explored.

Effect of 3alpha -OH-DHP on identified RVLM neurons. The majority of results in this study were obtained at a physiological dose of 3alpha -OH-DHP (1.12 µg/kg iv). This dose of 3alpha -OH-DHP was chosen to produce circulating levels of 3alpha -OH-DHP comparable to those seen during pregnancy. Maximal plasma concentrations achieved at this dose were calculated to be approx 22 ng/ml, and normal plasma concentrations of 3alpha -OH-DHP during pregnancy are 20-30 ng/ml (28). At this dose, acute administration of 3alpha -OH-DHP produced a significant decrease in both threshold MAP and saturation MAP. This decrease in threshold and saturation pressures is consistent with an increased sensitivity of identified RVLM neurons to endogenously released GABA. The effects of 3alpha -OH-DHP were subtle (~10% change), as might be expected with a substance that modulates the response to an endogenous transmitter.

Preliminary studies in which a higher dose of 3alpha -OH-DHP (11.2 µg/kg, n = 7) was administered revealed no further effect on threshold or saturation, indicating that near-maximal effects of 3alpha -OH-DHP are seen at physiologically relevant circulating levels.

Effect of 3alpha -OH-DHP on recovery parameters of RVLM neurons. 3alpha -OH-DHP was also found to have an effect on recovery after inhibition in response to elevations in pressure. Half time to recovery for MAP was not different between control and treatment for any of the groups (vehicle or 3alpha -OH-DHP). This indicates that once the PE stimulus was removed, MAP recovered at the same rate, thus eliminating the possibly confounding factor of different MAP recovery rates, which could affect recovery of the neuron. An effect of 3alpha -OH-DHP on recovery of UA was observed only in the presence of the highest dose of 3alpha -OH-DHP (11.2 µg/kg iv, n = 5) used in this study. At this dose, the t1/2 UA was significantly prolonged [control, 84.2 ± 41.2; after 3alpha -OH-DHP (11.2 µg/kg), 136.5 ± 55.5 s]. Also, as expected with a longer time period over which to recover, MAP at t1/2 UA was significantly lower [control, 117.8 ± 7.8; after 3alpha -OH-DHP (11.2 µg/kg), 105.3 ± 4.0 mmHg]. This effect on recovery is consistent with positive modulation of GABAA receptors by 3alpha -OH-DHP. For a given level of GABA present, inhibition of neurons would be greater in the presence of 3alpha -OH-DHP compared with control. In the presence of 3alpha -OH-DHP, actual levels of endogenously released GABA would have to decrease further before UA could recover, and thus time to recovery would be prolonged.

Although plasma concentrations achieved after administration of the highest dose of 3alpha -OH-DHP (11.2 µg/kg iv) likely exceed plasma levels during pregnancy, the results are still potentially significant. The enzymes responsible for converting progesterone to neuroactive metabolites are located both peripherally and within the CNS. Both circulating and centrally synthesized progesterone are converted to 3alpha -OH-DHP in the brain, and CNS concentrations of 3alpha -OH-DHP may exceed plasma concentrations by 100-fold (31). Thus acute intravenous administration of the higher dose of 3alpha -OH-DHP in the current experiments would result in acute exposure of the brain to concentrations that might well be within the physiologically relevant range for the CNS.

Effect of 3beta -OH-DHP on RVLM neurons. Currently, specific antagonists for the neurosteroid binding site are not available. However, high concentrations of the inactive stereoisomer 3beta -OH-DHP have been shown to compete with 3alpha -OH-DHP for binding sites and thereby reverse the positive modulation of GABAA receptors by 3alpha -OH-DHP (30). In the current experiments, any modulatory effect of 3alpha -OH-DHP on baseline firing rate would be most evident after a manipulation whereby endogenous GABA is elevated (i.e., after increased MAP). In 8 of 19 RVLM neurons, incomplete recovery of both MAP and UA was observed after the final pressure ramp in the presence of 3alpha -OH-DHP (11.2 µg/kg, n = 3; 1.12 µg/kg, n = 5), suggesting an inhibitory effect. In these neurons, the effect of the inactive stereoisomer 3beta -OH-DHP was evaluated. The relatively high dose of 3beta -OH-DHP (112-224 µg/kg) used in this study was chosen in an effort to produce maximal competition at the neurosteroid binding site on GABAA receptors. Baseline levels of MAP increased slightly with administration of 3beta -OH-DHP. However, despite the slight increase in pressure, 3beta -OH-DHP produced a significant increase in UA within 1 min of administration, indicating reversal of the inhibitory effect of 3alpha -OH-DHP. Additionally, the rapid effect of 3beta -OH-DHP further suggests that the mechanism of action of the neuroactive metabolite of progesterone, 3alpha -OH-DHP, was through a stereospecific nongenomic action at a unique binding site on the GABAA receptor complex.

Although the results of this study show that 3alpha -OH-DHP, administered to achieve plasma concentrations similar to those seen in pregnancy, has an effect on neurons in the RVLM, it should be recognized that the actual site of action of 3alpha -OH-DHP remains uncertain. 3alpha -OH-DHP is a highly lipid-soluble molecule and therefore has access to all CNS sites after intravenous administration. GABAergic influences have been demonstrated in other medullary nuclei in the baroreflex pathway, including the NTS and the CVLM (8). However, potentiation of GABAergic responses in the NTS or the CVLM might be expected to produce sympathoexcitation and attenuation of baroreflex sympathoinhibition (10), an opposite effect from that observed in both this and previous baroreflex studies (14, 17, 23). As the final site for sympathoinhibitory influences, the RVLM is the most likely site in the medullary baroreflex pathway where an increase in GABAergic influences would produce a potentiation of sympathoinhibition.

One potential mechanism for the preferential effect of 3alpha -OH-DHP in the RVLM would be a greater affinity for 3alpha -OH-DHP by RVLM neurons compared with other regions involved in the central baroreflex pathway. Affinity of 3alpha -OH-DHP for the GABAA receptor is dependent on the subunit composition of the receptor. Studies have demonstrated that although the beta -subunit of the GABAA receptor complex has no effect on modulation of GABA-induced chloride current, different alpha - and gamma -subunit isoforms (12, 19) may significantly affect the efficacy of 3alpha -OH-DHP to modulate GABAA receptor binding and function (28). Although it has not been determined in the medulla, heterogeneity of GABAA receptors in other areas of the CNS has been proposed to account for regional differences in neurosteroid responsiveness (28). Thus it is possible that GABAA receptors are distributed such that more receptors in the RVLM contain the appropriate subunits to maximize modulation of the GABAA receptor complex by 3alpha -OH-DHP.

Lastly, although this study demonstrated an effect of intravenous 3alpha -OH-DHP on arterial pressure sensitivity of RVLM neurons, the CNS site of action for the effects of 3alpha -OH-DHP on control of sympathetic outflow may not necessarily be restricted to the RVLM. The RVLM receives tonic excitatory drive from several supramedullary structures (8, 10), and it is possible that potentiation of GABAergic inhibition at one of these sites could have contributed to the results of the current experiments.

Perspectives

The current study demonstrated that acute increases in circulating levels of the neuroactive metabolite of progesterone, 3alpha -OH-DHP, result in potentiation of baroreflex inhibition of brain stem RVLM neurons. The fact that near-maximal effects were observed after a dose calculated to produce plasma concentrations within the range seen during pregnancy was administered suggests that these results may be physiologically relevant. Thus variations in levels of ovarian hormones and their metabolites, as occur during the estrus cycle and during pregnancy, may affect CNS regulation of sympathetic outflow and cardiovascular function. Although acute administration of the progesterone metabolite to virgin female animals produced effects qualitatively similar to the effects of pregnancy, the effects of long-term exposure to elevated levels of 3alpha -OH-DHP, as would occur in pregnancy, remain to be evaluated. In addition, preliminary experiments in which baroreflex control of efferent sympathetic nerve activity has been evaluated in male (13) and ovariectomized female rats (18) suggest that prior exposure to ovarian hormones is necessary for the acute effects of 3alpha -OH-DHP to be fully evident. Thus it is likely that modulation of sympathetic outflow by ovarian hormones and their metabolites is the result of an interaction between genomic and nongenomic actions within the CNS.

    ACKNOWLEDGEMENTS

The authors thank Dr. Gerlinda Hermann and Sarbani Ghosh for expert advice and technical assistance in performing these experiments.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant RO1-36245.

Address for reprint requests: J. D. Laiprasert, Dept. of Physiology, The Ohio State Univ., 302 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210-1218.

Received 1 August 1997; accepted in final form 12 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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