Propofol Modulates γ-Aminobutyric Acid–mediated Inhibitory Neurotransmission to Cardiac Vagal Neurons in the Nucleus Ambiguus

Experiments were performed in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association and the National Institutes of Health publication Guide for the Care and Use of Laboratory Animals  15and are in compliance with the institutional guidelines at George Washington University (Washington, D.C.).

In an initial surgery, rats (2–6 days old) were anesthetized with ketamine and xylazine and hypothermia. The heart was then exposed through a right thoracotomy, and the retrograde fluorescent tracer rhodamine (XRITC; Molecular Probes, Eugene, OR; 2% solution) was injected into the pericardial sac and applied to the site of the terminals of preganglionic cardiac vagal neurons that are located in the fat pads at the base of the heart. 16One to 3 days later, the animals were anesthetized with halo-thane and humanely killed by cervical dislocation. The brain was emerged in cold (4°C) buffer of the following composition: 140 mm NaCl, 5 mm KCl, 2 mm CaCl², 5 mm glucose, and 10 mm HEPES, oxygenated continually with 100% O². Under a dissection microscope, the cerebellum was removed, and the hindbrain was isolated. Slices of 400-μm thickness were obtained using a vibratome and were transferred to the recording chamber and continuously perfused at 2 ml/min with a solution of the following composition: 125 mm NaCl, 3 mm KCl, 2 mm CaCl², 26 mm NaHCO³, 5 mm dextrose, and 5 mm HEPES, aerated with 95% O²–5% CO²at a pH of 7.4. In experiments that examined mIPSCs, tetrodotoxin (1 μm) was included in the perfusion to block action potential–dependent events. Individual cardiac vagal neurons in the nucleus ambiguus were identified by the presence of the fluorescent tracer, imaged with differential interference contrast optics, and studied using patch clamp electrophysiologic methodologies.

Patch pipettes were pulled from glass capillaries (World Precision Instruments, Inc., Sarasota, FL) with an OD of 1.5 mm on a two-stage puller (PP-830; Narishige, Tokyo, Japan) and filled with solution of the following composition: 150 mm KCl, 2 mm MgCl², 2 mm EGTA, 10 mm HEPES, and 2 mm Mg-ATP at a pH of 7.4. The membrane potential was held at −80 mV to examine spontaneous GABAergic and glycinergic IPCS and mIP-SCs. Electrophysiologic recordings were sampled at 200 μs with a low-pass Bessel filter at 1 kHz.

To isolate GABAergic synaptic events, slices were perfused with D-2-amino-5-phosphonovalerate (AP5; 50 μm), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μm) and strychnine (1 μm) to block postsynaptic N -methyl-d-aspartate and non–N -methyl-d-aspartate glutamatergic and glycinergic receptors, respectively. Glycinergic events were isolated by perfusion with AP5 (50 μm), CNQX (50 μm), and gabazine (50 μm) to block postsynaptic N -methyl-d-aspartate and non–N -methyl-d-aspartate glutamatergic and GABAergic receptors, respectively. Propofol was applied by inclusion in the bath perfusion at concentrations of 0.1, 1, 5, 10, 50, and 100 μm. Each dose of propofol was administered for 2 min. At the end of each experiment that examined GABAergic and glycinergic neurotransmission, gabazine (50 μm) and strychnine (1 μm) were applied to block the remaining GABAergic and glycinergic activity, respectively. GABAergic mIPSCs were isolated in the same solution as described above for isolating GABAergic neurotransmission, except tetrodo-toxin (1 μm) was included in the perfusate to block action potential–dependent events.

Propofol (Diprivan) was obtained from Zeneca Limited (Macclesfield, Cheshire, United Kingdom). AP5, CNQX, strychnine, gabazine, and tetrodotoxin were purchased from Sigma Aldrich (St. Louis, MO).

Data were acquired using pCLAMP 8.0 software (Axon Instruments Inc., Union City, CA). Analysis of spontaneous synaptic currents was performed using MiniAnalysis (Synaptosoft, Decatur, GA; version 4.3.1) with amplitude thresholds of 20 pA for GABAergic IPSCs, 30–35 pA for glycinergic IPSCs, and 8 pA for mIPSCs. Values are expressed as mean ± SEM. Statistic analysis was performed using analysis of variance or paired t  test to detect differences between control and propofol-modulated GABAergic or glycinergic neurotransmission. A P  value less than 0.05 was accepted as significantly different.

Propofol at a concentration of 0.1 μm had no significant effect on GABAergic IPSC decay time or frequency. However, at concentrations from 1 to 100 μm, propofol evoked significant increases in the decay time of spontaneous GABAergic IPSCs (fig. 1). The average IPSC decay time increased from 78.1 ± 7.5 to 101.7 ± 8.8 ms in the presence of propofol at a concentration of 1.0 μm. The decay time was prolonged to 102.0 ± 9.5, 110.2 ± 11.0, 111.7 ± 7.2, and 110.1 ± 9.8 ms when propofol was applied at concentrations of 5, 10, 50, and 100 μm, respectively.

To assess the reversibility of the effects of propofol on GABAergic IPSC decay time, changes in decay time were measured during application and on washout of 10 μm propofol. Propofol increased the decay time of GABAergic IPSCs, and this increase in decay time persisted for 5 min after propofol application (fig. 2). The effects of-propofol were no longer significant 10 min after propofol application, and after 30 min, the decay time was indistinguishable from control values.

To examine whether this increase in GABAergic IPSC decay time could be caused by changes in the action potential in the preceding GABAergic neuron or whether the increase in decay time was action potential independent and likely due to postsynaptic changes, GABAergic mIPSCs were isolated in the presence of tetrodotoxin. Propofol at concentrations from 1 to 100 μm but not 0.1 μm significantly augmented the decay time of GABAergic mIPSCs (fig. 3). At concentrations of 1, 5, 10, 50, and 100 μm, propofol increased the mIPSC decay time from 77.6 ± 7.4 to 93.8 ± 8.0, 95.1 ± 8.7, 98.8 ± 11, 98.1 ± 11, and 92.9 ± 4.6 ms, respectively (fig. 3).

Propofol also seems to have presynaptic effects indicated by reductions in both GABAergic IPSC and mIPSC frequency, as shown in figure 4. GABAergic IPSC frequency was inhibited by 30% and 50%, and GABAergic mIPSCs were diminished by 50% and 70% with propofol concentrations of 50 and 100 μm, respectively (fig. 4). However, these presynaptic actions occurred at only propofol concentrations greater than 50 μm. Propofol did not evoke any significant changes in the amplitude of either GABAergic IPSC or mIPSCs.

To test whether propofol acts at another important inhibitory pathway to cardiac vagal neurons, the effect of propofol on glycinergic neurotransmission to cardiac vagal neurons was examined. Even at concentrations up to 50 μm, propofol did not elicit any significant alteration of glycinergic IPSCs frequency, amplitude, or decay time (fig. 5).

Propofol has been reported to act on various receptors and ion channels in neurons and other neuronal preparations, including hippocampus CA1 pyramidal neurons, 17,18cortical neurons, 19cerebellar synaptosomes, 20,Xenopus  oocytes 12,21,22and in spinal cord. 23However, the effect of propofol on neurons in the brainstem involved in cardiovascular homeostasis is particularly clinically relevant and has received little attention. This study examined the effects of propofol on parasympathetic cardiac vagal neurons in the nucleus ambiguus that dominate the control of heart rate.

Propofol at clinically relevant concentrations evoked changes in the GABAergic neurotransmission to cardiac vagal neurons. Propofol elicited a prolongation of both GABAergic IPSC and mIPSC decay times at concentrations of 1 μm and greater. This alteration of decay time is consistent with what has been observed in other neurons within the central nervous system. Propofol has been shown to increase the duration of spontaneous and endogenous inhibitory synaptic responses by reducing desensitization of postsynaptic GABA^Areceptors in cultured hippocampal neurons 17,18and dissociated spinal dorsal horn neurons. 24In primary cultured rat cortical neurons, propofol prolongs the decay phase of GABAergic mIPSCs and IPSCs at concentrations from 1 to 10 μm. 19It has been proposed that this prolongation of decay time is due to a reduction in postsynaptic GABA^Areceptor desensitization.

The results of this study show that in addition to an increase in decay time, at higher concentrations, propofol inhibits the frequency of GABAergic IPSCs and mIPSCs in cardiac vagal neurons. However, this inhibition of GABAergic frequency only occurred at higher concentrations of propofol (≥50 μm). Because propofol decreased the frequency of action potential–independent GABAergic mIPSCs, the site of action of propofol is likely on the presynaptic GABAergic terminal. Other work has also shown that propofol can alter GABAergic frequency. Propofol at a concentration of 10 μm increased the frequency of GABAergic mIPSCs in granule cells of the dentate gyrus. 25 

Studies on the effect of propofol on glycinergic receptors are controversial. Propofol (10 μm) prolonged the glycinergic mIPSCs in spinal cord by slowing desensitization and deactivation of glycinergic receptors, 26and propofol has also been shown to inhibit glycinergic currents at high doses (100 μm). 24Lower doses of propofol (1–10 μm) have been shown to enhance glycine-evoked responses in the optic nerve, 27but in another study using human homomeric α¹glycine receptors 28in cultured hippocampal neurons, glycine-evoked currents were not altered by propofol. 27In our study, propofol did not significantly alter glycinergic IPSCs in cardiac vagal neurons, suggesting propofol acts selectively on inhibitory GABAergic but not glycinergic neurotransmission to cardiac vagal neurons.

Clinically, propofol has been shown to increase heart rate when it is constantly infused intravenously at low doses (0.2–0.4 mg · kg⁻¹· min⁻¹) to induce light sedation. 2However, this situation does not occur commonly in clinical propofol anesthesia. A plasma propofol concentration of 1 μm only produces sedation, and propofol concentrations of greater than 10 μm are typically necessary to achieve anesthesia when propofol is applied as the sole anesthetic agent. 29Because propofol has a narrow therapeutic index and causes respiratory arrest when administered in excessive doses, it is usually administered with opioids or other volatile anesthetic agents in surgical anesthesia. Propofol is highly protein bound (as much as 98%) and plasma free propofol concentrations are typically maintained between 2 and 6 μm when coadministered with other anesthetic agents in surgical anesthesia. 29,30At comparable concentrations in this study (1 and 5 μm), propofol evoked an increase in GABAergic IPSC decay time, which would inhibit cardioinhibitory cardiac vagal neurons and elicit an increase in heart rate. At higher concentrations (≥50 μm), propofol evoked a decrease in the inhibitory GABAergic frequency to cardiac vagal neurons, which would be expected to increase the activity of cardioinhibitory parasympathetic neurons, thereby decreasing heart rate. This is consistent with the clinical situations in which propofol anesthesia induces decreases in blood pressure, heart rate, and inhibition of respiratory rate if it is applied at high concentrations or if propofol is injected rapidly. 6Experiments in animal studies also show that high doses of propofol induce bradycardia. 5 

In conclusion, this study showed that propofol at clinically relevant concentrations augments GABAergic activity to cardiac vagal neurons by increasing GABAergic IPSC and mIPSC decay time. At higher concentrations (>50 μm), propofol also reduced GABAergic IPSC and mIPSC frequency. These propofol-induced alterations in the inhibitory GABAergic pathway to cardiac vagal neurons are likely involved in the cardiovascular changes that occur with propofol anesthesia.