Propofol, a newer diisopropylphenol compound, is the most widely used IV drug for induction of general anesthesia. The cellular mechanisms underlying its sedative effect are not completely defined. Accumulating evidence, however, indicates that the ventrolateral preoptic area (VLPO) of the hypothalamus in the endogenous sleep pathway plays a critical role.1 The VLPO contains 2 major types of neurons: the majority are γ-aminobutyric acid (GABA)-containing projecting neurons targeting the arousal-producing nuclei in the tuberomammillary nucleus (TMN).2 This type of neuron is inhibited by noradrenalin (NA(−) neuron). Conversely, the minority are excited by noradrenalin (NA(+) neurons),3 which are probably interneuron and may contain GABA as well.4 Importantly, NA(−) neurons may normally be under the inhibitory control of NA(+) neurons.3,4 Using c-Fos expression, a previous study demonstrates that GABAergic agents, including propofol, activate the VLPO neurons.1 These investigators interpret their data to indicate that by activating VLPO neurons, and the release of GABA into the TMN, propofol inhibits the activity of the arousal-producing nucleus, and consequently induces sedation.1 However, it remains unclear how propofol activates VLPO neurons.
General anesthetics have marked effects on synaptic transmission. Most previous studies found that propofol enhances the function of GABAA receptors (GABAARs)5–7 and may depress the release of glutamate, the major excitatory neurotransmitter.8
In the present study, by using a combination of pharmacological and electrophysiological approaches, we show that there are functional GABAARs on the NA(+) neurons in the VLPO. By activating these GABAARs, propofol suppresses NA(+) neurons and the release of GABA onto the NA(−) neurons, which results in the excitation of NA(−) neurons.
Brain Slice Preparation
All procedures were performed in accordance with National Institutes of Health guidelines and with the approval of the Animal Care and Utilization Committee of University of Medicine and Dentistry of New Jersey. All efforts were made to minimize the number of animals used and to minimize their suffering. We studied 40 Sprague-Dawley rats at postnatal days 20 to 30. Brain slices were prepared as described previously.4 Rats were anesthetized and then decapitated. Coronal slices (230–250 µm thick) containing the VLPO were cut with a Compresstome VF-200 slicer (Precisionary Instruments Inc., Greenville, NC), then immediately transferred to a holding chamber and incubated for at least 1 hour at room temperature (24°C–25°C) in oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) before the electrophysiological recording. The ACSF contained the following (in millimolar): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 NaHCO3, 1 L-ascorbate, and 11 glucose, and oxygenated with 95% O2/5% CO2 (carbogen).
Electrophysiological recordings were obtained with an Axon 200B amplifier, a Digidata 1440A A/D converter, and Clampfit 10.3 software (Molecular Devices Co., Union City, CA). Data were filtered at 2 kHz and sampled at 5 kHz. All electrophysiological recordings were obtained at 32.6°C. ACSF was perfused at a rate of 1.5 to 2.0 mL/min. Patch pipettes had a resistance of 6 to 8 MΩ when filled with internal solution containing (in millimolar)1 140 cesium methanesulfonate, 5 KCl, 2 MgCl2, 10 Hepes, 2 MgATP, 0.2 GTP for voltage-clamp recordings, or 140 potassium gluconate, 5 KCl, 2 MgCl2, 10 Hepes, 2 MgATP, and 0.2 GTP for current clamp recordings. The pH of both pipette solutions was adjusted to 7.2 with Tris-base and the osmolality to 310 mOsmol/L with sucrose. In voltage-clamp experiments, spontaneous inhibitory postsynaptic currents (sIPSCs) were examined in whole-cell configuration at a holding potential of 0 mV in the presence of AP5 (50 µM), 6, 7-dinitroquinoxaline-2, 3-dione (DNQX) (20 µM) and strychnine (1 µM) to block glutamate and glycine receptors. Spontaneous discharges of VLPO neurons were recorded by the loose-patch cell-attached technique or in whole-cell mode.
Chemicals and Application
We purchased DNQX, DL-2-amino-5-phosphono-valeric acid (DL-AP5), strychnine, gabazine, 2, 6-diisopropylphenol (propofol), and noradrenaline (NA) from Sigma (St Louis, MO). All chemicals were applied to the cell by bath perfusion.
Data Analysis and Statistics
Electrophysiological events were analyzed using Clampfit 10.3 (Molecular Devices Co.). Drug-induced changes in the frequency and amplitude of sIPSCs and the frequency of firings in a given cell were calculated by averaging the changes induced by drug administration (a 3-minute period at the peak of a drug response from each cell) and normalized to the predrug baseline. The data from the same treatment were pooled. All experimental values are presented as the mean ± SEM while n indicated the number of neurons tested. Changes in the firing/membrane potential/sIPSC within groups induced by propofol were analyzed by 1-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test using corrected P-values. A paired t test was used to analyze differences between control and drug groups (i.e., 10 µM gabazine on firing rate). Dose–response data were fitted to the logistic equation: y = 100xa/(xa + xoa), where y is the percentage change, x is the concentration of propofol, a the slope parameter, and x0 the propofol concentration which induces a half-maximal change. Two-tailed unpaired Student t tests were used to compare the change in firing rate/membrane potential induced by propofol in the absence and presence of gabazine. To compare the frequency or amplitude of sIPSCs between control and propofol conditions, cumulative interevent-interval/amplitude distributions were constructed, and the Kolmogorov–Smirnov (K-S test) statistic was applied to detect statistically important differences between interevent-interval/amplitude distributions. Significance for all analyses was determined at P < 0.05.
Identification of VLPO Neurons
Experiments were conducted in acute brain slices containing the VLPO. In keeping with previous reports,3,4,9 we found 2 major types of cells within the VLPO: the majority were triangular and multipolar in shape (Fig. 1A1), characterized by a potent low-threshold spike (LTS) (not shown) and inhibited by NA (100 μM, 15 seconds) (NA(−) neurons, Fig. 1B1). Conversely, the minority was fusiform and bipolar in shape (Fig. 1A2), had no LTS (not shown), and excited by NA (NA(+) neurons, Fig. 1B2). In the following experiments, we identified the cell type based on their pharmacological and morphological characteristics.
Propofol Stimulates NA(−) Neurons Involving GABAARs
We first examined the effects of propofol on the spontaneous firing of NA(−) neurons under current clamp (Fig. 2A). Initially, these neurons spontaneously fired at 3.6 ± 0.5 Hz under control conditions and had a resting potential of −59.2 ± 3.1 mV (range: −53.7 to − 64.3 mV, n = 32). The propofol-induced membrane depolarization depended on its concentration with a 50% effective concentration of 70 nM (F(5,32) = 16.5, P < 0.0001, 1-way ANOVA; Fig. 2B). Specifically, 1 nM, 100 nM, and 10 µM propofol induced depolarization by 0.4 ± 0.05 mV (n = 6, P = 1), 3.2 ± 0.5 mV (n = 9, P < 0.0001), and 5.6 ± 1.2 mV (n = 4, P < 0.0001) (Bonferroni posttest, Fig. 2, A1 and B), respectively. Likewise, propofol concentration dependently induced an acceleration of firing rate (F(5,32) = 65.3, P < 0.0001, 1-way ANOVA, Fig. 2C): between 1 and 100 nM, propofol-induced acceleration increases with its concentrations (1 nM: 3.3% ± 1.8%, n = 6, P = 1; 10 nM: 23.5% ± 3.6%, n = 7, P < 0.0001; 100 nM: 47.3% ± 4.5%, n = 9, P < 0.0001, Bonferroni posttest; Fig 2, A and C). The effects of propofol were reversible after washout of propofol. Conversely, at 1 µM, propofol had no significant effect (2.5% ± 1.6%, n= 6, P = 1), and at 10 µM, propofol significantly reduced the firing rate (−12.8% ± 2.6%, n = 4, P = 0.026, Bonferroni posttest; Fig. 2C).
General anesthetics including propofol have marked effects on synaptic transmissions. Most previous studies found that propofol enhances the function of GABAARs (10–13 from9). To determine whether propofol-induced acceleration of firing of NA(−) neurons involves GABAARs, we compared propofol’s action in the absence and presence of the specific GABAAR antagonist gabazine. The application of gabazine (10 µM) alone substantially accelerated the ongoing firing rate (by 35.7% ± 4.5%, n = 6, P < 0.0001) and induced membrane depolarization (by 3.1 ± 0.3 mV, n = 6, P < 0.0001, paired t test; Fig. 2D), indicating that NA(−) neurons are normally under strong tonic inhibition, mediated by GABAARs.4 After a new stable baseline was established in the continuous presence of gabazine, adding propofol (100 nM) only slightly changed the firing rate (5.2% ± 0.3%, n = 5), or the membrane potential (0.3 ± 0.1 mV, n = 5; Fig. 2E), which are much less than the increases produced by 100 nM propofol in the absence of gabazine (firing rate: P < 0.0001; membrane potential: P = 0.001; unpaired t test). When GABAARs are blocked, the stimulating effect of propofol may be underestimated due to nonlinear summation of excitation. That is, propofol’s effect may be less in neurons with a higher firing rate. To test this possibility, we compared the effect of propofol on NA(−) neurons of different basic firing rates. As illustrated in Figure 2F, propofol exerted similar acceleration on firing of NA(−) neurons regardless of their basic firing rates.
Propofol Decreases the Frequency of sIPSCs on NA(−) Neurons
We next recorded sIPSCs from NA(−) neurons in the presence of AP5 (50 μM), DNQX (20 μM), and strychnine (1 μM) and at a holding potential of 0 mV. Under these conditions, the spontaneous events were eliminated by 10 μM gabazine (data not shown), indicating that they were mediated by GABAARs. Propofol (100 nM) significantly decreased sIPSC frequency (by 57.5% ± 5.5%, from 2.6 ± 0.3 to 1.1 ± 0.2 Hz, n = 7, P < 0.0001, Fig. 3, A and B) and increased the mean amplitude (by 6.4% ± 0.8%, from 42.4 ± 3.8 pA in control to 45.3 ± 4.5 pA in propofol, n = 7, P < 0.0001; paired t test). These were further illustrated by cumulative plots of the incidence of various intervals (K-S = 59.2%, P < 0.0001) and amplitudes (K-S = 25.3%, P < 0.0001) between successive sIPSCs (K-S test, Fig. 3C).
Propofol Suppresses the Firing of NA(+) Neurons
A major GABAergic input to the NA(−) neuron is from the local GABAergic interneurons: the NA(+) neurons. To determine the role of NA(+) neurons in propofol-induced reduction of sIPSCs in NA(−) neurons, we examined the effects of propofol (1–10,000 nM) on the spontaneous firing of NA(+) neurons (Fig. 4A). As expected, propofol suppressed the ongoing firing. The concentration dependence was well fitted by a logistic equation (r2 = 0.99), giving a half-maximal inhibitory concentration of 120 nM (F(5,30) = 12.5, P < 0.0001, 1-way ANOVA; Fig. 4B), and the maximal inhibition was −78.2% ± 14.8% of baseline (n = 6, P < 0.0001; Bonferroni posttest). This effect of propofol was recovered to control levels (97.8% ± 3.1% for firing rate, n = 6 and 98.3% ± 3.3% for membrane potential, n = 6) after washout of propofol.
Finally, to determine whether propofol-induced suppression of firing of NA(+) neurons involves GABAARs, we compared propofol’s action in the absence and presence of gabazine. The application of gabazine (10 µM) alone substantially accelerated the ongoing firing rate (by 37.8% ± 3.9%, n = 5, P < 0.0001) and induced membrane depolarization (by 3.0 ± 0.4 mV, n = 5, P < 0.0001, paired t test; Fig. 4C), indicating that NA(+) neurons are normally under GABAAR-mediated tonic inhibition. After a new stable baseline was established in the continuous presence of gabazine, adding propofol (100 nM) slightly inhibited the firing rate (0.3% ± 0.1%, n = 5).
Our major finding is that propofol excites VLPO NA(−) neurons. This is probably mediated by GABAARs, and by suppressing presynaptic GABA release, which is at least partly due to the inhibition of local GABAergic NA(+) neurons. Since NA(−) neurons send inhibitory projections to TMN histaminergic neurons, NA(−) neuron excitation will suppress TMN neurons; less histamine will be released and sedation may occur. This may contribute to propofol-induced sedation.
Propofol, one of the most potent IV anesthetics, is effective at submicromolar concentrations,10 but the underlying cellular and molecular mechanisms have not been well explored. Accumulating evidence, however, indicates that the VLPO of the hypothalamus in the endogenous sleep pathway plays a critical role.1 The VLPO is a brain structure that plays a pivotal role in sleep regulation and rhythmicity.2 Neurons in the VLPO were identified as sleep-active and involved in promoting and maintaining natural sleep, which provides inhibitory control of many of the arousal nuclei, including the TMN.2
Intracellular recordings in slices revealed only 2 cell types within VLPO: the NA(−) neurons, also named as LTS, and the NA(+) neurons, also named as non-LTS neurons.3,11 Most NA(−) neurons release GABA, but some also release the small inhibitory peptide galanin.1,10,12 We have previously shown that VLPO NA(−) neurons may normally be under the inhibitory control of NA(+) neurons.2 In the current study, we showed that gabazine significantly stimulated NA(−) neurons, indicating that NA(−) cells are tonically inhibited by GABA, which might at least in part be released from NA(+) neurons. Gabazine eliminated propofol-induced stimulation of NA(−) neurons, suggesting that propofol’s effect is mediated by GABAARs. However, in the presence of gabazine, the inhibitory effect of the NA(+) cell on the NA(−) cell should be suppressed (Fig. 5), and the effect of propofol on the NA(+) cell is difficult to evaluate. To get around this, we examined the effect of propofol on sIPSCs in NA(−) neurons. Propofol decreased the frequency without significantly changing the mean amplitude of sIPSCs, indicating a reduction of presynaptic release of GABA. We further showed that propofol suppressed the firing of NA(+) neurons, indicating that propofol’s suppression of sIPSCs in NA(−) neurons may be due at least in part by suppressing NA(+) firing. Taken together, these results support the hypothesis that propofol accelerates the firing of NA(−) neurons by the reduction of GABA, which might be released from NA(+) neurons.
Interestingly, the dose-dependent relationship of propofol’s action on NA(−) neurons firing is a reversed U shape: while propofol at a lower concentration range facilitates the firing, at a higher concentration, propofol suppresses it. Although the mechanism underlying this observation warrants further investigation, we can speculate that although propofol may exert its effect through enhancing the function of inhibitory GABAARs, these receptors are present on both the NA(−) and NA(+) neurons. The GABAARs in the NA(+) neurons may be more sensitive to propofol than those in NA(−) neurons; probably due to variations in subunit compositions, density of the receptors and their distributions, or a combination of these. In support of this, previous studies using point mutation indicated the sites on the β-subunit of the GABAAR are relevant for the action of propofol,14,15 and propofol may selectively activate extrasynaptic GABAARs, which have subunits different from the synaptic counterpart.6 Furthermore, a previous study using positron emission tomography data from volunteers found that propofol’s action negatively correlated most significantly with the regional distribution of [3H]diazepam and [3H]flunitrazepam (benzodiazepine) binding site densities.16 Therefore, while propofol’s enhancement of GABAARs may inhibit both the NA(−) and NA(+) neurons, at lower concentrations, propofol-induced enhancement of the GABAARs in the NA(+) neurons or their terminals which make synapses on NA(−) neurons is dominated, which leads to the suppression of NA(+) neurons and the reduction in GABA release onto the NA(−) neurons. This will result in the excitation of NA(−) neurons by a mechanism of disinhibition. Conversely, at higher concentrations, the direct inhibitory effect of propofol on NA(−) neurons may become dominant and thus a suppression of NA(−) neurons occurs. Remarkably, in contrast to the reversed U shape dose-dependent relationship of propofol’s effect on firing, the dose dependence of propofol-induced depolarization of the NA(−) cells is sigmoid. One plausible mechanism for this difference is that although depolarization induced by lower concentrations of propofol could increase firing of NA(−) neurons, stronger depolarization induced by higher concentrations of propofol could lead to reduced firing by the activation of the M type K+ channels17 or by inactivation of the voltage-gated Na+ channels on the NA(−) neurons.9
Previously, we have shown that propofol can facilitate glutamatergic excitatory postsynaptic currents to VLPO neurons, probably to the NA(−) neurons.9 Together, our work demonstrates that propofol can work in at least 2 ways to stimulate VLPO NA(−) neurons: by increasing excitatory postsynaptic currents and reducing IPSCs. Since NA(−) neurons send their inhibitory GABAergic projections to TMN histaminergic neurons, the excitation of NA(−) neurons will lead to the inhibition of TMN histaminergic neurons, resulting in sedation.
In conclusion, the present study shows that propofol at nanomolar concentrations enhanced discharges of NA(−) neurons by reducing axonal GABA release; at least in part by inhibiting VLPO NA(+) neurons. This may be a critical mechanism underlying propofol-induced sedation.
Name: Yu-Wei Liu, PhD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Yu-Wei Liu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Wanhong Zuo, MD, MS.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Wanhong Zuo has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Jiang-Hong Ye, MS, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Jiang-Hong Ye has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
We thank Dr. Hong Nie for consulting on statistical data analysis.
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