Differential Effects of GABAA Receptor Antagonists in the Control of Respiratory Neuronal Discharge Patterns

Z. Dogas1, 2, M. Krolo1, 2, E. A. Stuth1, 2, M. Tonkovic-Capin1, 2, F. A. Hopp1, 2, D. R. McCrimmon3, and E. J. Zuperku1, 2

1 Zablocki Veterans Affairs Medical Center; and 2 Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53295; and 3 Department of Physiology and Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Dogas, Z., M. Krolo, E. A. Stuth, M. Tonkovic-Capin, F. A. Hopp, D. R. McCrimmon, and E. J. Zuperku. Differential effects of GABAA receptor antagonists in the control of respiratory neuronal discharge patterns. J. Neurophysiol. 80: 2368-2377, 1998. To ascertain the role of the inhibitory neurotransmitter gamma -aminobutyric acid (GABA) in shaping and controlling the phasic discharge patterns of medullary respiratory premotor neurons, localized pressure applications of the competitive GABAA receptor antagonist bicuculline (BIC) and the noncompetitive GABAA receptor antagonist picrotoxin (PIC) were studied. Multibarrel micropipettes were used in halothane anesthetized, paralyzed, ventilated, vagotomized dogs to record single unit activity from inspiratory and expiratory neurons in the caudal ventral respiratory group and to picoeject GABAA receptor antagonists. The moving time average of phrenic nerve activity was used to determine respiratory phase durations and to synchronize cycle-triggered histograms of discharge patterns. Picoejection of BIC and PIC had qualitatively different effects on the discharge patterns of respiratory neurons. BIC caused an increase in the discharge rate during the neuron's active phase without inducing activity during the neuron's normally silent phase. The resulting discharge patterns were amplified replicas (×2-3) of the underlying preejection phasic patterns. In contrast, picoejection of PIC did not increase the peak discharge rate during the neuron's active phase but induced a tonic level of activity during the neuron's normally silent phase. The maximum effective BIC dose (15 ± 1.8 pmol/min) was considerably smaller than that for PIC (280 ± 53 pmol/min). These findings suggest that GABAA receptors with differential pharmacology mediate distinct functions within the same neuron, 1) gain modulation that is BIC sensitive but PIC insensitive and 2) silent-phase inhibition blocked by PIC. These studies also suggest that the choice of an antagonist is an important consideration in the determination of GABA receptor function within the respiratory motor control system.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The rhythmic discharge patterns of respiratory neurons are largely the result of periodic variations in excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) (Ballantyne and Richter 1984; Champagnat et al. 1982; Richter 1982) and intrinsic neuronal mechanisms (Ramirez and Richter 1996). In many of these neurons, postsynaptic inhibition produces the silent or inactive phase of the respiratory cycle of inspiratory (I) and expiratory (E) neurons (Feldman and Grillner 1982). In addition, phasic inhibition during the bursting phase appears to play a role in shaping the envelope of their discharge patterns (Ezure et al. 1989).

The inhibitory amino acid neurotransmitters gamma -aminobutyric acid (GABA) and glycine are generally believed to be the primary mediators of the fast synaptic inhibition in the control of breathing. The specific roles of GABA and glycine were studied by local application of their receptor antagonists while recording the activity of single respiratory neurons (Champagnat et al. 1982; Haji et al. 1990, 1992; Schmid et al. 1996; Toleikis et al. 1979). In a recent study by Schmid et al. (1996) the GABAA receptor antagonist bicuculline (BIC) and the glycine receptor antagonist strychnine altered the discharge pattern of respiratory neurons in restricted portions of the respiratory cycle. Activity was either selectively induced in restricted portions of their inactive phase or their discharge frequency was increased only in restricted parts of their active phase. Notably, BIC was most often effective throughout the active phase of each neuron but was without effect during the neuron's silent period (Schmid et al. 1996; Wang et al. 1982). We previously found that picoejection of BIC on I and E bulbospinal neurons resulted in an amplified replica of the underlying phasic discharge pattern with a proportionality constant of 2-3 (McCrimmon et al. 1997). This multiplicative relationship was maintained when the discharge patterns of these neurons were reflexly altered by different afferent inputs, such as those arising from pulmonary stretch receptors and central chemosensory inputs. These findings suggest the existence of a potent GABAergic gain modulation mechanism that controls the output patterns of these neurons. Picoejection of strychnine blocked glycine-induced inhibitions but had no effect on the spontaneous or reflexly induced activities of E neurons and had only a small effect on I neuronal activities (Krolo et al. 1997; Tonkovic-Capin et al. 1996).

In a preliminary study to ascertain the role of GABAergic inhibition in the absence of the BIC-induced amplification of discharge patterns, we investigated the effects of the GABAA receptor channel blocker picrotoxin (PIC) on these neurons. We noted that, unlike BIC, picoejection of PIC did not enhance the activity of respiratory neurons during their normally active phase but induced activity during their normally silent phase. These strikingly and qualitatively different responses produced by two different GABAA receptor antagonists are consistent with recent discoveries of multiple GABAA receptor subtypes with different subunit compositions and pharmacologies (Burt and Kamatchi 1991; MacDonald and Olsen 1994). For example, retinal GABA receptors containing rho -subunits and coupled to Cl- channels were found to be sensitive to PIC but not BIC (Matthews et al. 1994). The existence of differential pharmacology at the neuronal level in vivo and the related physiological and pharmacological implications motivated this study. The objective was to compare and contrast the effects of the competitive GABAA receptor antagonist BIC with those of the noncompetitive GABAA receptor antagonist PIC on the spontaneous discharge activities of single respiratory neurons. Specifically, the picoejection technique was used to locally apply BIC and PIC on I- and E-bulbospinal premotor neurons of the caudal ventral respiratory group (VRG) in dogs.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Experiments were performed on 16 mongrel dogs of either sex, weighing from 8 to 15 kg. Thiopental sodium was used for induction of anesthesia (15 mg/kg iv), and anesthesia was maintained with halothane (1.0-1.4% end-tidal concentration). The animal was monitored for signs of inadequate anesthesia, including salivation, lacrimation, and/or increases in blood pressure, and the anesthetic depth was increased immediately if such signs were present.

Surgical procedure

The animals were intubated with a cuffed endotracheal tube and mechanically ventilated with an air-O2 mixture. End-tidal CO2 concentration was continuously recorded with an infrared analyzer (POET II, Criticare Systems). Tracheal pressure (Pt) was measured from an airway side port with an air-filled catheter connected to a Gould-Statham P23 ID transducer. A triple-lumen catheter was placed in the femoral vein and used for continuous infusion of maintenance fluids (0.9% NaCl) as well as drug and anesthetic administration. The femoral arteries were cannulated for arterial blood sampling and continuous blood pressure monitoring (Gould-Statham P23 ID transducer). Blood gas samples were obtained periodically, and additional sodium bicarbonate was given to correct metabolic acidosis, if required. Esophageal temperature was monitored and maintained at 37.5-38.5°C with a servo-controlled heating pad.

The dogs were positioned in a Kopf (model 1530) stereotaxic apparatus with the head flexed ventrally by 30°. The vertebral column was maintained straight through caudal tension applied via a hip-pin clamp. The right C5 phrenic nerve rootlet was exposed via a dorsolateral neck dissection, cut distally, desheathed, and placed on bipolar platinum electrodes in a mineral oil pool formed from a neck pouch. The moving time average of phrenic nerve activity or the phrenic neurogram (PNG) was recorded and used to obtain I- and E-timing pulses.

An occipital craniotomy was performed, and the dura mater was opened along the midline and reflected laterally to expose the dorsal surface of the medulla oblongata. To minimize brain stem movements during neuronal unit recording and feedback from extravagal afferents, a bilateral pneumothorax was created, and the animal was paralyzed with a 0.1-mg/kg iv bolus of pancuronium bromide and supplemental doses of 0.05 mg/kg, as required. Bilateral cervical vagotomies were performed to eliminate afferent vagal input from pulmonary stretch receptors.

Multibarrel micropipettes (10- to 50-µm composite tip diameter), consisting of one recording barrel containing a carbon filament (7-µm diam) and three drug barrels, were used for extracellular neuronal recordings and picoejections. The ejected solutions consisted of the vehicle, an artificial cerebrospinal fluid (aCSF), the competitive GABAA receptor antagonist bicuculline (BIC; 50-250 µM; Research Biochemicals), the noncompetitive GABAA receptor antagonist, PIC (2-5 mM; Research Biochemicals), and GABA (1 mM; Research Biochemicals), all of which were dissolved in aCSF. The aCSF consisted of (in mM) 124 NaCl, 2 KCl, 2 MgCl, 1.3 KH2PO4, 0.9 CaCl2, 26 NaHCO3, and 11 glucose. The pH of each solution was adjusted to 7.2-7.4 by aeration with 5% CO2. The picoejection system was pressurized with compressed nitrogen, and the parameter ranges typically used were 1) ejection pressure, 10-100 psi; 2) duration of the pressure pulse, 10-100 ms; and 3) frequency of the ejection pressure pulses, 0.5-5 Hz. Ejected volume/time was measured via height changes of the meniscus in the pipette barrel with a ×50 magnification microscope equipped with a reticule (resolution approx 2 nL). To minimize vibration effects during pressure application, 3-ft long, soft catheter tubings were connected between the picoejector solenoid valves and the micropipette barrels. To obtain steady-state dose-response data, constant-rate picoejection was used, and the doses were increased via increases in ejection rate.

Single unit neuronal activities were recorded from I- and E-bulbospinal neurons in the caudal VRG, typically 1-5 mm caudal to the obex, 2.5-4.5 mm lateral to the midline, and 2.5-4.5 mm below the dorsal medullary surface. The amplified output of the microelectrode was monitored on a oscilloscope, and an amplitude-time window discriminator was used to generate a standard pulse for each neuronal spike. These pulses were counted during 100-ms intervals, and the resulting spike frequency was displayed on a polygraph (Grass model 7) via a D/A converter. The data were recorded on an eight-channel digital tape system (A. R. Vetter Digital PCM recording adaptor, model 3000A). The recorded signals consisted of neuronal unit activity, phrenic nerve activity, Pt, proximal airway CO2 concentration, and pressure picoejection marker.

Protocol

Once a stable extracellular recording of a VRG I- or E-neuronal unit was established, 1-2 min of preejection control data were recorded. Vehicle or drug ejection followed with step increases in dose (i.e., ejection rate). To permit calculation of effective doses, each ejection rate was maintained until a steady-state response was achieved. Before antagonist (BIC or PIC) applications, aCSF was ejected to verify that the vehicle and the ejected volume had little or no effect on neuronal discharge frequency.

Data analysis

Cycle-triggered histograms (CTHs; bin width, 50 ms), triggered from either the onset of the I or E phase and based on 5-15 respiratory cycles, were used to quantify the discharge frequency patterns at each dose level. Values of the peak discharge frequency during the normally active phase and the average discharge frequency during the normally silent phase of the neuron were calculated.

Because picoejection of BIC results in amplified replicas of the control discharge frequency pattern, the proportionality factors relating the control and test discharge patterns (CTHs) were obtained from the slopes of the best-fit lines through plots of Fn during BIC picoejection versus Fn of preejection control. The data for these plots were taken from the corresponding CTHs during the active phase. This method has the advantage of being insensitive to the geometric shape of the pattern. For quasilinear patterns, the proportionality factor gives the fractional increase, relative to control, in the augmenting or decrementing slopes.

Data obtained during picoejection of the antagonists and the aCSF vehicle were normalized to their preejection control values of the active phase, pooled, and expressed as means ± SE. These values obtained at the maximum effective doses were compared by a repeated measures one-way analysis of variance (ANOVA), with the type of agent as the main treatment factor. If the ANOVA revealed a significant difference among treatments, the treatment means were compared with the modified t-values and the Bonferroni procedure for multiple comparisons (Wallenstein et al. 1980). Differences were considered significant for P < 0.05.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

I Neurons

BIC EFFECTS. Picoejection of BIC produced dose-dependent increases in the phasic discharge frequency (Fn) of I neurons with no effect during the silent E phase (Fig. 1). The BIC-induced increases in phasic activity are due to gain-modulating effects (McCrimmon et al. 1997) on the discharge pattern as illustrated by the CTHs of Fig. 2, right, for three picoejection rates (BIC1, BIC2, and BIC3) that were applied to the same neuron as shown in Fig. 1. There were dose-dependent increases of 49, 175, and 352%, respectively, in the slope of the augmenting pattern. Representative records of the neuronal discharge activity (NA) are shown at the left of Fig. 2 for the preejection control period and during the BIC2 dose rate. Full recovery from the effects of BIC typically requires 20-30 min.


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FIG. 1. Picoejection of bicuculline (BIC) dose-dependently increases the phasic activity of I neurons. PNG, phrenic neurogram; Fn, neuronal discharge frequency. BIC (200 µM) was picoejected continuously at the dose rates indicated above the horizontal bars.


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FIG. 2. Picoejection of BIC results in discharge patterns that are amplified replicas of the underlying control pattern of I neurons. Data from same neuron as shown in Fig. 1. Left: time-expanded records of neuronal activity before (top) and during picoejection of BIC (bottom, BIC2). PNG, phrenic neurogram; NA, neuronal activity; Fn, neuronal discharge frequency. Right: cycle-triggered histograms (CTHs) of the neuronal discharge activity (6-8 cycles/histogram). Control, preejection control discharge pattern; dose-related increase in augmenting slope: BIC1, 49%; BIC2, 175%; BIC3, 352%. Dose rates (pmol/min): BIC1, 0.7; BIC2, 1.9; BIC3, 3.6.

PIC EFFECTS. In contrast to BIC, picoejection of PIC did not alter the peak Fn of I neurons but produced a dose-dependent increase in the silent phase activity (Fig. 3). This effect continued for >1 h after discontinuation of the PIC picoejection. The CTHs (Fig. 4B) also indicate no change in I-phase peak discharge frequency; however, the E-phase activity increased in a tonic fashion from zero to ~50% of the peak Fn of the I phase at the highest picoejection rate.


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FIG. 3. Continuous picoejection of picrotoxin (PIC, 2 mM) at increasing rates (indicated above the bar) dose dependently increases the normally silent E-phase activity of I neurons, without altering the peak discharge frequency.


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FIG. 4. PIC induces activity during the E phase of I neurons. A: time-expanded records for the neuron of Fig. 3 before (Control, top) and during picoejection of PIC (bottom). B: corresponding ensemble averages of PNG and CTHs (Fn) of unit discharge activity (10-12 cycles/histogram). Induced E-phase activity at the maximum effective dose rate of 112 pmol/min is ~50% of peak I-phase activity.

E Neurons

BIC EFFECTS. Similar to the effect on I neurons, picoejection of BIC on caudal VRG E neurons produced potent dose-dependent increases in discharge frequency during the normally active phase (E phase) without inducing activity during their normally silent I phase (Fig. 5). The effects of BIC persisted beyond the picoejection period, and the return to preejection activity levels required 20-30 min. Picoejection of the aCSF vehicle at rates that exceeded those used for BIC or PIC delivery had no effect (Fig. 6). The steady-state responses of neurons with time-dependent E-phase patterns demonstrate the amplification (gain modulation) of Fn by BIC picoejection (Fig. 7, right, CTHs). The discharge pattern during BIC is a proportional replica of the preejection control pattern. This is confirmed by the linear relationship of the plot of Fn during BIC versus Fn of the control period obtained from the CTH data (Fig. 7, bottom right). The slope of this relationship, obtained by linear regression, is the proportionality constant (2.04). The constancy of the gain factor is evident by the close correspondence of the BIC response CTH with a pattern that results when the control period CTH is multiplied by this proportionality constant or gain factor (triangular points, Fig. 7, top right).


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FIG. 5. Picoejection of BIC dose dependently increases the phasic activity of E neurons. Top: BIC (50 µM) was picoejected continuously at the dose rates indicated. At the dose-rate of 3.6 pmol/min the volume rate was 73 nL/min. Bottom: time-expanded records of the phrenic neurogram (PNG), neuronal activity (NA), and discharge frequency (Fn), taken during preejection period (A) and during the peak response (B).


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FIG. 6. Artificial cerebrospinal fluid (aCSF) vehicle has no effect on neuronal activity. Top: data from the same E neuron shown in Fig. 5 indicates no change in peak Fn at high vehicle ejection rates (103 nL/min) exceeding those used for maximal BIC delivery (73 nL/min). Bottom: time-expanded records of unit activity for the times indicated (A and B).


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FIG. 7. Picoejection of BIC indicates the presence of gain modulation of E neurons. Left: examples of E-neuronal activity with a decrementing discharge pattern before (top, Control) and during BIC (100 µM) picoejection at a dose rate of 5.8 pmol/min (bottom). Right, top: CTHs of unit activity for control and BIC application (11-12 cycles/histogram). Superimposed triangles indicate that the response to BIC is an amplified (×2.04 Control) replica of the underlying control pattern. The gain factor, 2.04, was obtained from the linear regression slope of data from a plot of Fn during BIC vs. Fn of CON (bottom right) taken from the histograms (top right) at corresponding times.

ANTAGONISM OF BIC EFFECTS BY GABA. The BIC-induced amplification of the underlying discharge patterns can be attenuated by picoejection of GABA immediately after termination of BIC picoejection when the effects of BIC still persist. For example, picoejection of BIC at a dose-rate of approx 5 pmol/min increased the peak Fn of an E neuron with a decrementing pattern from 125 to 240 Hz and resulted in a proportionality constant of 1.93 (BIC, Fig. 8, top left). Immediate, subsequent picoejection of GABA resulted in a reduction of both the peak Fn to approx 170 Hz and the proportionality constant to 1.22 (GABA, post-BIC, Fig. 8, top left).


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FIG. 8. gamma -Aminobutyric acid (GABA) antagonizes BIC-induced gain modulation. Left, top: CTHs of unit activity of an E neuron with a decrementing pattern shown for 3 conditions: preejection control, (Control), during BIC picoejection (250 µM at dose rate of 4.8 pmol/min), and during GABA picoejection (1 mM) immediately after BIC (GABA post-BIC). bullet : Control pattern multiplied by a gain factor (G) of 1.93; open circle : Control pattern × 1.22. Gain factors obtained by method shown in Fig. 7, bottom right. Top right: CTHs of unit activity from the same E neuron illustrating the effects of picoejection of a BIC (250 µM) and GABA (1 mM) mixture in the same pipette barrel. Mixture at a dose rate that was equal to that for BIC alone (top left) resulted in a gain factor of G = 1.30 vs. G = 1.93 for BIC, alone. black-down-triangle : Control histogram ×1.30. Bottom left: plots of peak Fn vs. ejection rate for the same E neuron. bullet : BIC (250 µM) alone; open circle : GABA (1 mM) immediately after BIC; black-square: GABA alone; black-down-triangle : BIC/GABA mixture. Bottom right: plots of the gain factor (G) vs. dose rate for the same 4 conditions as for peak Fn-ejection rate plots (bottom left). GABA Post-BIC, GABA in a mixture, and GABA alone all reduce the gain factor compared with BIC.

In addition, picoejection of a BIC/GABA mixture (250 µM BIC and 1 mM GABA from the same barrel) produced a much smaller response than 250 µM BIC alone at the same dose rate (approx 5 pmol/min). The mixture increased peak Fn from 130 Hz (Fig. 8, Control) to 175 Hz, and the corresponding proportionality constant was 1.30 (BIC/GABA Mix, Fig. 8, top right). The values of both parameters were much less than those produced by BIC alone (BIC, Fig. 8, top left). The dose-response data for peak Fn and slope of the plots of Fn during agent versus control Fn are shown in Fig. 8, bottom. The data of four picoejection protocols are shown: GABA alone, BIC alone, GABA (post-BIC), and the BIC/GABA mixture (Mix). Near maximal effects were observed at BIC doses of 7-8 pmol/min. Picoejection of GABA alone dose dependently reduced both peak Fn and the gain factor (G, regression slope). These data confirm that GABA antagonized the BIC-induced neuronal responses.

PIC EFFECT ON E NEURONS. In contrast to the BIC-induced responses, picoejection of PIC on E neurons produced little or no increase in the peak phasic activity but produced a dose-dependent increase in the normally silent I-phase activity of these neurons (Fig. 9). With large doses, the phasic I inhibition could be completely blocked in some E neurons. This produced a tonic discharge pattern similar to that seen during hypocapnic apnea (Fig. 10, top). Picoejection of the aCSF vehicle was able to restore the phasic pattern, most likely by dilution and/or washout of PIC (Fig. 10, bottom).


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FIG. 9. Picoejection of PIC produces a dose-dependent increase in the I-phase activity of an E neuron with no effect on peak Fn. Cumulative doses of PIC (5 mM) are shown. The highest dose-rate was 78 pmol/min. Top right: effects of PIC are long-lasting (>1 h). Bottom: time-expanded records of neuronal activity (NA) and discharge frequency (Fn) at times indicated by A and B. Shaded horizontal bars: I-phase indicator.


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FIG. 10. Example of the complete block of I-phase inhibition by PIC in an E neuron. Top: PIC application converted phasic activity to tonic activity. Bottom: continuation of upper record. Picoejection of aCSF gradually restored the phasic pattern through dilution and/or washout of PIC.

Summary data

The qualitative differences in neuronal responses to the two GABAA receptor antagonists are corroborated by the pooled data that were normalized to their respective preejection control values during the normally active phase for each neuron type. BIC, at maximal effective dose levels (15.0 ± 1.76 pmol/min), produced a 167 ± 15% increase in peak I-neuronal activity and a 162 ± 16% increase in peak E-neuronal activity with no significant increases in the normally silent phase activity of both neuron types (Fig. 11). PIC produced no change in the peak activity during the normally active phase of each neuron type but induced activity during the normally silent phase, which was 37.6 ± 4.9% of the peak Fn during the active phase for I neurons and 47.0 ± 11.7% of the peak Fn during the active phase for E neurons. The maximum effective dose (i.e., after which higher doses produced no further increases) for PIC was 280 ± 53.1 pmol/min. The aCSF vehicle produced no changes during either the normally active or inactive phases of both neuron types.


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FIG. 11. Pooled data that summarize and contrast the differential responses produced by BIC and PIC. Peak Fn is expressed as percent of preejection control values. Left: active respiratory phase, I phase for I neurons and E phase for E neurons. Number of neurons tested for each condition is shown within bars and also applies to data of the right panel. Picoejection of BIC produced significant increases in peak Fn of the active phase, whereas PIC and aCSF had no effect. Asterisks: P < 0.0001. Right: inactive phase, E phase for I neurons and I phase for E neurons. Picoejection of PIC produced significant increases in the inactive phase Fn relative to preejection control (of the inactive phase), whereas BIC and aCSF had no effect. *P < 0.0001.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The significant findings of this study are the striking, qualitatively different neuronal responses to local application of the two GABAA receptor antagonists BIC and PIC. The characteristics of the responses were similar for both I and E neurons. BIC amplified the underlying discharge frequency pattern of these neurons during their normally active phase without inducing activity during the normally silent phase. In contrast, PIC did not alter the activity during the normally active phase but dose dependently increased neuronal activity during the normally silent phase.

Respiratory neuron types in the caudal VRG

Although antidromic activation and collision studies were not performed in this study, our previous studies found that approx 88% (15/17) of the I neurons and approx 88% (50/57) of the E neurons in the same region of the caudal VRG yielded positive collision results with antidromic stimulation from the spinal C3 level (Bajic et al.1992; Stuth et al.1994). On the basis of these findings, the probability that >= 75% of the neurons we studied were bulbospinal neurons exceeds 98%.

The discharge patterns of the I neurons had ramp or step-ramp patterns (e.g., Figs. 2 and 4) similar to those seen in other species. In dogs, 75-80% of the E-bulbospinal neurons have a decrementing pattern, and the other 20-25% have an augmenting pattern that plateaus early in the E phase (Bajic et al. 1992). In contrast, the discharge patterns of the E neurons in the caudal VRG of the cat are either augmenting ramp (two-thirds) or step-ramp (one-third) patterns (Cohen et al. 1985). In rats, ~85% of the E neurons have decrementing patterns, and 15% have augmenting patterns (Hayashi et al. 1996). In this study of 21 E neurons, 18 had decrementing patterns and 3 had augmenting patterns. The neuronal responses to picoejection of BIC (e.g., Fig. 1 of McCrimmon et al. 1997) and PIC were similar for both E-neuron types.

Differential effects of GABAA receptors

It appears that BIC and PIC are affecting two different GABAergic mechanisms in the same neuron. BIC seems to antagonize GABAA receptors that are involved with gain modulation of the output of these respiratory premotor neurons. We have previously shown that this type of modulation is essentially multiplicative in nature (McCrimmon et al. 1997). The phasic discharge pattern during BIC application is an amplified, proportional replica of the preejection control pattern (e.g., Fig. 7). This tonic modulation appears to operate on the underlying discharge pattern even when it is altered by different reflex inputs and thus controls the final neuronal output pattern, possibly via a postsynaptic, shunting inhibition (McCrimmon et al. 1997). This mechanism is potent because its block by BIC results in 100-200% increases in peak discharge frequency (Fig. 11, left), indicating that neuronal output is normally attenuated to 33-50% of its potential output under our experimental conditions. The robust increases in phasic activity produced by picoejection of BIC are similar to those observed with microiontophoresis in cats (Schmid et al. 1996; Wang et al.1982). In contrast to the BIC-induced gain modulatory responses, local application of excitatory amino acid analogues produce a tonic or continuous increase in neuronal activity of an additive nature (Schmid et al. 1996).

The neuronal response to BIC is not mimicked by PIC (Figs. 1 vs. 3). PIC appears to antagonize a subset of GABAA receptors that mediates the phasic inhibition responsible for the normally silent phases but not the gain modulation mechanism. At maximum effective doses, PIC increased the average silent phase activity from 0 to 36.7% of active phase peak Fn of I neurons and from 0 to 47.0% of the peak Fn of E neurons. In one of eight neurons tested, it was even possible to obtain a complete block of the phasic I inhibition such that the E neuron discharged at a constant rate throughout the respiratory cycle. BIC at greater dose rates than those used in this study was also able to induce activity during the inactive phases, but this was overshadowed by the large increases in phasic activity because of antagonism of the gain modulatory mechanism (unpublished personal observations). This latter observation suggests that the GABA receptors that mediate the silent phase inhibition are GABAA receptors and not GABAC receptors, which are BIC insensitive (Matthews et al.1994).

Secondary role of glycinergic inhibition

A role for glycine in the production of the silent-phase inhibition was suggested by studies of the microiontophoretic application of the glycine receptor antagonist strychnine on medullary I neurons in cats (Champagnat et al. 1982; Schmid et al. 1996). Strychnine increased the discharge activity primarily in the last part of the I phase and early part of the E phase, suggesting that it may play an important role in the I-to-E phase transition. In contrast, GABA antagonists induced activity throughout the E phase (Champagnat et al. 1982), qualitatively similar to our results with PIC on canine I neurons.

In our previous studies, picoejection of strychnine on canine I neurons induced activity throughout the E phase (Krolo et al. 1997). However, the magnitude of this effect was much smaller than that produced by PIC (12 vs. 56% of peak Fn). In addition, strychnine has no effect on the spontaneous activity of E neurons (Tonkovic-Capin et al. 1996). Thus, in contrast to GABAA receptors, glycine receptors appear to play a minor role in the control of I- and E-bulbospinal neurons in dogs.

Effective antagonist doses

The average maximum effective BIC dose rate (15 ± 1.8 pmol/min) was considerably smaller than that for PIC (280 ± 53 pmol/min). Reported antagonist affinity constants for BIC obtained from in vitro preparations (0.5-1.5 µM) are very similar to IC50 values reported for PIC (0.4 to 1 µM) (Krishek et al. 1996). Because antagonist concentration decreases rapidly with distance from the point of ejection and because much higher dose rates for PIC were required to overcome the phasic inhibition than for BIC to interfere with gain modulation, it seems likely that the phase-dependent GABAergic inhibition of the I- and E-premotor neurons may be located at synaptic sites more distal to those mediating gain modulation. However, it is possible that other factors, such as differences in diffusion rates and/or accessibility to the receptors, may also account for the noted differences in the effective concentrations of the two antagonists.

Possible indirect effects of the picoejection method

It is also possible that part of the neuronal responses to the picoejected antagonists may be due to indirect effects via neurons other than the recorded cell. However, several lines of reasoning suggest that indirect effects are small if present. 1) The qualitative character of the responses was consistent for all neurons studied, including both I and E neurons. If indirect effects caused by the spread of antagonist to other surrounding antecedent neurons were important, more variability in the results would be expected to be caused by different combinations of altered presynaptic activities. 2) The response direction is consistent with the expected effect of the antagonist. Both BIC and PIC produced an increase in discharge frequency, which is expected when inhibitory inputs are antagonized. If, for example, a neighboring inhibitory interneuron was disinhibited by these antagonists, then a relative decrease in activity of the recorded premotor neuron would be expected. In this regard, the dose-dependent responses indicate no obvious change in the rate or direction of response as doses increase. 3) The qualitative character of the responses was consistent for all neurons studied, including both I and E neurons. If indirect effects caused by the spread of antagonist to other surrounding antecedent neurons were important, more variability in the results would be expected to be caused by different combinations of altered presynaptic activities. 4) The responses to the antagonists are consistent with the neurophysiology of these respiratory neurons. It is well established that the silent phase of medullary I and E neurons is due to active inhibition mediated by IPSPs, which can be reversed in polarity by Cl- injection (Richter et al. 1979). The increase in silent-phase activity produced by PIC is consistent with antagonism of this mechanism. Because neuronal activity during the active phase was not altered by PIC, presynaptic interneurons with tonic inputs or that discharge in phase with the recorded neurons do not appear to be affected. If the inhibitory presynaptic neurons responsible for the silent phase were disinhibited by PIC, then the presynaptically mediated inhibition would be increased and would offset some of the PIC-induced block of inhibition at the recorded neuron. Furthermore, the concentration of the picoejected antagonist is expected to be highest near the recorded neuron and to decrease rapidly with distance from the electrode tip. Thus concentrations are expected to be much less near any neighboring presynaptic neurons. Similar arguments can be made for BIC effects. Because the pipette concentration of BIC was less than one-tenth of that of PIC, the concentration of BIC at points somewhat distant to the electrode tip would also be expected to be much less than that of PIC, which had to be picoejected at much higher dose rates to show any effects.

Selectivity of BIC as a GABAA receptor antagonist

Although BIC is firmly established as a specific, competitive inhibitor of the GABAA receptor recognition site (MacDonald and Olsen 1994), the possibility that BIC mediates its gain-modulating effects via nonspecific or non-GABAergic-related mechanisms should be considered. It was reported that BIC may noncompetitively block the binding of acetylcholine to its receptor (IC50: 101 ± 9 µM for cultured embryonic rat skeletal muscle) (Liu et al. 1994). However, this is unlikely to be a factor in this study because our previous work with acetylcholine and its nicotinic antagonists dihydro-beta -erythroidine and hexamethonium and the muscarinic antagonist atropine show no effect on the spontaneous activity of the I- and E-bulbospinal neurons (Krolo et al. 1997; Tonkovic-Capin et al. 1996). BIC was also shown to exert nonsynaptic actions in cultured mouse spinal cord neurons (Heyer et al. 1981). In that preparation, BIC in low concentrations (IC50: approx 1 µM) antagonized GABA responses, but at higher concentrations (ED50: approx 30 µM) it induced a nonsynaptic membrane depolarization and reduction in membrane conductance, likely caused by a decrease in K+ conductance. In this study, however, the I- and E-neuronal responses to BIC did not exhibit evidence of a depolarizing baseline shift that would be expected when hyperpolarization caused by an equilibrium potential more negative than resting (EK: -80 to -90 mV) is antagonized. Even with pipette concentrations as small as 50 µM BIC, which would result in concentrations distal to the ejection source that are much lower, dose-dependent, graded responses were obtained throughout the neuronal discharge frequency range without any change in the gain-modulating character of the response (Fig. 5). In addition, GABA antagonized the gain modulation response produced by BIC (Fig. 8), which is consistent with the notion that BIC antagonizes GABAA receptors in our experimental paradigm. Thus, although a BIC mechanism that is independent of GABAA receptor antagonism cannot be excluded, it seems unlikely.

Antagonistic properties of PIC

PIC exerts a noncompetitive inhibition of the GABAA-Cl- channel complex via a receptor site distinct from that for GABA itself (MacDonald and Olsen 1994). Its binding either prevents the conformational change of the GABAA receptor protein that opens the Cl- channel on GABA binding and/or physically blocks the channel. Because PIC is a noncompetitive antagonist, GABA application will not reduce this type of block. We were unable to verify the selectivity of PIC as a GABAA receptor antagonist in our preparation. Although PIC induced activity during the neuron's normally silent phase, subsequent picoejection of GABA produced a dose-dependent tonic reduction in activity throughout the respiratory cycle that was not different from that observed before PIC. Normally, this would suggest that PIC was ineffective. However, coactivation of the GABAergic gain modulatory receptors by picoejection of GABA would lead to the observed dose-dependent decreases in Fn, which can be easily reduced to 0.

Differential effects in other neuronal systems

Differential responses to BIC and PIC were also observed in other in vivo neuronal systems. Shiekhattar and Aston-Jones (1992) reported that BIC application to noradrenergic neurons of the locus coeruleus potently enhanced their sensory responsiveness. This action of BIC was not mimicked by the antagonists PIC, penicillin, or the GABAB receptor antagonist 2-hydroxy-saclofen or by agents that directly depolarized locus coeruleus neurons. In addition, the response of the noradrenergic neurons was not modulated by benzodiazepine receptor agonists (Shiekhattar and Aston-Jones 1992). This novel pharmacology is consistent with the combination of GABA receptor subunit oligomers present in the locus coeruleus. Luque et al. (1994) identified a very low level of the gamma 1 and a lack of alpha 1 subunits that may account for the lack of PIC and benzodiazepine sensitivities.

Other examples of GABA-gated Cl- channels with differential sensitivities to both BIC and PIC were reported. Retinal GABAC receptors contain a rho  subunit and are sensitive to PIC but not BIC (Matthews et al. 1994). Zhang et al. (1995) found that homomers of insect (Drosophila) GABA receptor subunits were sensitive to PIC and insensitive to BIC. When these subunits were coexpressed with a homolog of the vertebrate GABAA receptor beta  subunit, the resulting receptors were sensitive to BIC and insensitive to PIC (Zhang et al. 1995). Point mutations in the second transmembrane domain of insect (French-Constant et al. 1993) and rat (Gurley et al. 1995) GABAA receptors can render GABA-activated membrane current insensitive to PIC. Thus, although the subunit composition of GABAA receptors on respiratory neurons is not known, the presence of GABAA receptors with different subunit compositions could explain the different responses to BIC and PIC in this study.

    SUMMARY

These studies demonstrate that the GABAA receptor antagonists BIC and PIC produce dramatically different responses in the neuronal activity of respiratory premotor neurons in dogs. BIC antagonizes a potent tonic gain modulatory mechanism, and PIC antagonizes the phasic inhibition responsible for producing the silent phases of these respiratory neurons. The ability to distinguish between the effects of these two mechanisms by differential pharmacology provides an effective tool for dissecting their respective influences and for choosing the appropriate GABAA receptor antagonist for a given investigational objective. Further studies on the modulatory effects of compounds (e.g., benzodiazepines, barbiturates, and steroids) acting at other GABAA receptor sites may help to resolve the subunit composition of these receptors.

    ACKNOWLEDGEMENTS

  The authors are indebted to J. Tomlinson for expert surgical assistance and to Criticare Systems for supplying a POET II anesthetic agent/CO2-O2 monitor.

  This work was support by the Department of Veterans Affairs Medical Research Funds and the Department of Anesthesiology of The Medical College of Wisconsin, Milwaukee.

    FOOTNOTES

  Address for reprint requests: E. J. Zuperku, Research Service/151, Zablocki VA Medical Center, Milwaukee, WI 53295.

  Received 2 April 1998; accepted in final form 16 July 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society