Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and autonomic nervous systems. The authors have previously shown that depressant and convulsant barbiturates both inhibit the ganglion-type nAchRs in PC12 cells. However, the central and gangliontype receptors have different subunit composition and pharmacologic properties. In this study, the authors investigated the effects of thiopental, depressant [R(-)] and convulsant [S(+)] stereoisomers of 1-methyl-5 phenyl-5-propyl barbituric acid (MPPB) on neuronal nAChRs in the rat central nervous system to explore significance of these effects in barbiturate anesthesia.
Whole-cell currents were measured in acutely dissociated rat medial habenula (MHb) neurons by applying 10 or 100 microM nicotine in the absence or presence of thiopental 3-100 microM. Effects of R(-)- and S(+)-MPPB on the nicotine-induced current were also studied.
Thiopental suppressed the nicotine-elicited inward current and accelerated the current decay dose-dependently at the clinical relevant concentrations. R(-)- and S(+)-MPPB both inhibited the nicotine-induced current dose-dependently without augmenting the current decay. There was no significant difference in the magnitudes of inhibition by R(-)- and S(+)-MPPB.
Although thiopental suppressed the current mediated through native nAchRs in rat MHb neurons at the clinically relevant concentrations, the depressant and convulsant stereoisomers of MPPB both inhibited the current in the same extent. These findings are consistent with the results previously obtained in the ganglion-type receptors of PC12 cells and suggest that inhibition of nAChRs in MHb neurons is not directly relevant to the hypnotic or anticonvulsive actions of barbiturates.
NEURONAL nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and autonomic nervous systems. 1,2They are thought to modulate synaptic transmission in the central nervous system (CNS) by regulating the release of neurotransmitters mainly through presynaptic mechanisms. 3–6Although roles of neuronal nAchRs in the CNS are poorly understood, these receptors are reported to be involved in cognitive performance, locomotor activities, nociception, and psychoneurologic disorders such as Alzheimer and Parkinson disease. 7–9It is known that both central and peripheral neuronal nAChRs are very sensitive to various types of anesthetics, including volatile anesthetics, ketamine, and barbiturates. 10–13Therefore, neuronal nAChRs are candidates for potential target sites of these anesthetics.
We reported that thiopental strongly suppresses the current mediated through neuronal nAChRs at clinically relevant concentrations in PC12 cells, a rat pheochromocytoma cell line, 11and that both stereoisomers of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) suppress neuronal nAChRs in PC12 cells. 14Since R (−)-isomer of MPPB induces loss of righting reflex, but S (+)-MPPB causes hyperactivity and convulsion in vivo , 15our results suggest that inhibition of neuronal nAchRs by barbiturates does not correlate with their hypnotic effects in vivo . However, central receptors may possibly show different sensitivity to thiopental and MPPB isomers compared with ganglionic receptors because of differences in subunit composition and pharmacologic properties. 3,16Thus, we intended to study the effects of these barbiturates on native nAchRs in the CNS.
A very recent study investigated the effects of thiopental stereoisomers on the recombinant receptors with subunit composition putatively expressed in the brain 17and showed that α7homomeric receptors and α4β2receptors are both sensitive to clinical concentrations of racemic thiopental. It also revealed that α7homomeric receptors do not discriminate the stereoisomers with different anesthetic potencies and α4β2receptors are more sensitive to the isomer less potent as an anesthetic. However, effects of barbiturates on native nAChRs in CNS neurons have not been clarified. Because it is known that native neuronal nAChRs show different electrophysiological behaviors from those observed in the recombinant receptors with putatively corresponding subunit composition, 1,18we cannot automatically extrapolate the findings obtained from the recombinant nAchRs to the native receptors.
The medial habenula nucleus (MHb), located in the medial part of the dorsal thalamus, receives cholinergic input from the basal forebrain and the brain-stem tegmentum. It is one of the regions in which nAChRs are strongly expressed, and electrophysiological properties of nAChRs in this region have been extensively studied using acutely dissociated neurons 19–21and slice preparations. 22,23In the current study, we evaluated whether thiopental inhibits native nAChRs in rat MHb neurons at clinically relevant concentrations. We also compared the effects of stereoisomers of MPPB on the nAChR-mediated current in these neurons to clarify if inhibition of these receptors correlates with anesthetic actions of barbiturates.
Material and Methods
Acute Isolation of Medial Habenula Neurons
This study was approved by the Animal Care and Use Committee at Yokohama City University School of Medicine (Yokohama, Japan). MHb neurons were isolated from the MHbs of rats using a modification of methods described elsewhere. 20,21Briefly, 10- to 25-day-old Sprague-Dawley rats were anesthetized with diethylether and decapitated, then the brains were rapidly placed in ice-cold and equilibrated with 100% O2PIPES saline containing 150 mm NaCl, 5 mm KCl, 5 mm MgCl2, 25 mm PIPES, and 25 mm d-glucose (pH was adjusted to 7.4 with NaOH). The transverse slices containing the MHb nuclei were made with Vibratome tissue slicer (DTK-1000; Dosaka, Kyoto, Japan) and maintained in well-oxygenated PIPES saline for 50–60 min at room temperature (22–26°C). Thereafter, the slices were incubated in PIPES saline containing papain (7.5 U/ml), bovine serum albumin (1 mg/ml), and l-cystein (0.2 mg/ml) for 25–35 min at 35°C. During incubation, the slices were constantly agitated using a gentle rocking motion. The slices were washed twice with PIPES saline containing 1 mg/ml each of bovine serum albumin and trypsin inhibitor. The MHbs were then dissected under a binocular microscope and triturated using fire-polished Pasteur pipette in Dulbecco modified Eagle medium containing 25 mm HEPES and supplemented with Ultroser G (2%). Dissociated neurons were plated onto polyornitine-coated coverslips in a 35-mm petri dish and maintained in a humid atmosphere of 95% air and 5% CO2at 37°C for more than 1 h and used up to 8 h after isolation.
PC12 cells were cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum and 5% horse serum, as previously described. 11For the experiment, cells were plated on collagen and poly-l-lysin–coated coverslips and used after additional 2–4 day culture.
Membrane currents were measured by conventional whole-cell voltage clamp method. 24Cells on the coverslips were placed in a recording bath, mounted on the stage of an inverted microscope (IX50, Olympus, Tokyo, Japan) with an approximate volume of 1.5 ml, and continuously perfused at the rate of 1–2 ml/min with external solution containing 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, and 11.1 mm d-glucose (pH was adjusted to 7.4 with NaOH). Recordings were made from small MHb neurons (< around 10 μm cell body diameter), which consistently responded to nicotinic agonists. Bicuculine methbromide (50 μm) was included in the external solution for the experiments using acutely dissociated neurons to prevent γ-aminobutyric acid receptor type A (GABAA)-mediated chloride current. Heat-polished patch pipettes had tip resistance of 2–10 MΩ when filed with an intracellular solution. An intracellular solution contained 140 mm CsF, 10 mm CsCl, 10 mm HEPES, 5 mm EGTA, 2 mm Mg-ATP (pH was adjusted to 7.3 with CsOH), which brought the reversal potential for Cl close to the holding potential. We compensated the junction potential between intracellular and external solutions but not the series resistance. The series resistance changed within approximately 20% from the initial value. In the experiments using PC12 cells, which lack GABAAreceptors, we used an intracellular solution containing 150 mm CsCl, 10 mm HEPES, 5 mm EGTA, and 2 mm Mg-ATP (pH was adjusted to 7.3 with CsOH). Cells were voltage clamped at −60 mV with a patch clamp amplifier (CEZ 2400, Nihon Koden, Tokyo, Japan and Axopatch 200A, Axon Instruments, Foster City, CA). Whole-cell currents were filtered at 0.5 kHz with Bessel filter and digitized at 2 kHz. The currents were stored on a computer using pClamp software (Axon Instrument) and analyzed using Axograph 3.5 software (Axon Instruments). All experiments were performed at room temperature (22–26°C).
A Y-tube method described elsewhere 11was used for delivery of the agonists and the barbiturates. The tip of the Y-tube was made by a glass micropipette (Microcaps, 2 μl, Drummond, Broomall, PA) with about a 100-μm opening and was positioned about 500 μm from the recorder cell. This method enabled the complete exchange of the external solution surrounding the cell around 100 ms, as estimated by recording the liquid junction current produced at an open patch pipette. To obtain concentration–response relation for nicotine, we recorded the whole-cell currents induced by 1–300 μm nicotine in MHb neurons. Nicotine was applied for 2.5 s, and each application was separated by 4 min. To study the effects of barbiturates on the nicotine-induce current, the agonist with or without the barbiturates was applied in the same manner. For preincubation with the barbiturates or the antagonists, the external solution containing the drugs was perfused at the rate of 5 ml/min for 4 min before rapid application. Cells were perfused with the plain external solution at the same rate for 4 min to wash out the drugs from the bath after the measurement. We applied both MPPB isomers to a given cell to compare the effects of the isomers on the nicotine-induced current in the same population of MHb neurons. One half of the recorded cells received R (−)-isomer first and the other half received S (+)-isomer first to eliminate time-dependent bias.
Drugs used in the current study included (−)-nicotine, hexamethonium (Wako, Osaka, Japan), Mg-ATP, acetylcholine bromide (Sigma), thiopental sodium (Tanabe, Osaka, Japan), Ultroser G (IVF, Villneuve-la-Garenne, France), cytisine, and bicuculine methbromide (Research Biochemicals Inc., Cambridge, MA). S (+)–R (−)-MPPB were provided by Professor J. Knabe (Saaland University, Saarbrücken, Germany). Acetylcholine bromide and cytisine were dissolved in distilled water to make 60 and 30 mm stock solution and stored at −80°C. Thiopental sodium was dissolved in 0.1N NaOH to make 100 mm stock solution and stored at −80°C. S (+)- and R (−)-MPPB were dissolved in 0.1N NaOH to make 30 mm stock solution just before experiments. They were diluted with the external solution to the designated concentration. The addition of the barbiturates to the external solution caused pH changes less than 0.05. 25
We measured the peak and the nondesensitized current, which was defined as the average amplitude from 2.45 to 2.5 s during agonist application. Because nicotine-induced currents declined slightly with each application of nicotine, the response in the presence of barbiturates was compared with the average amplitude of elicited currents before and after application of the barbiturates. This procedure was rationalized by the finding that the second response was almost the same as the average of the first and third responses when nicotine was applied successively three times at an interval of 4 min (data not shown). Concentration–inhibition curves were fitted to the following equation by a least-squares fit:
where I is the relative current normalized to the control currents, C is the concentration of barbiturates, n is the Hill coefficient, and IC50is the concentration for 50% inhibition. The decaying phases of the nicotine-induced current were fitted either to a single or a double exponential function of the following form by simplex method:
where I is the total peak current, Ifinalis the residual current at the steady state condition, Iiis the peak current amplitude of the each component, and tiis the time constant of the corresponding component. Goodness of fit was compared by chi-square test between single and double exponential models. This analysis was conducted for the experiments using barbiturates as well as the ones in which nicotine alone was applied successively. Time constant ratio was defined as the ratio of tiin the presence of the drugs relative to the average of precontrol and postcontrol. Desensitization was also evaluated by calculating the percent decay of the current during agonist application (% current decay) defined by the following equation 26:
The data were expressed as mean ± SEM. The significance of differences was analyzed using one-way analysis of variance followed by Dunnett test for comparison among different doses of thiopental. A paired t test was performed to analyze differences between R (−)- and S (+)-isomers. A P value < 0.05 was considered to indicate a significant difference.
Nicotine-induced Current in Acutely Dissociated Rat Medial Habenula Neurons
Nicotine elicited inward currents, which decayed rapidly during nicotine application because of desensitization, at the membrane potential of −60 mV in acutely dissociated rat MHb neurons. The dose–response relation for the peak current showed that EC50for nicotine was 12.9 μm and that the peak response was saturated at the doses equal to or more than 100 μm of nicotine (fig. 1A). For the nondesensitized current, the response was also saturated at 100 μm, and 10 μm nicotine produced near half maximal response. The nondesensitized current decreased at 300 μm because of augmented desensitization (fig. 1B). The currents elicited by 10 and 100 μm nicotine were suppressed by 10 μm hexamethonium, a competitive antagonist for neuronal nAChRs. When 10 μm hexamethonium was preincubated and coapplied, the peak and nondesensitized currents were 30.5 ± 7.9 and 27.7 ± 12.0 % of control at 10 μm nicotine (n = 5), and they were 62.7 ± 7.6 and 51.4 ± 16.0% of control at 100 μm nicotine (n = 5), respectively (fig. 2).
Effect of Thiopental on the Nicotine-induced Current
Thiopental alone produced no current response in the tested neurons at 100 μm. This finding confirmed that the direct activation of GABAAreceptors by thiopental was effectively blocked in the current condition. Three to 100 μm thiopental inhibited the peak and nondesensitized current in a dose-dependent manner. The inhibition was much stronger for the current elicited by nicotine 100 μm than that for the current elicited by nicotine 10 μm. Inhibitions of the nondesensitized current were much greater than that of the peak current (figs. 3A and 3B). The IC50 values for the peak and nondesensitized currents were 30.1 ± 7.5 and 19.0 ± 3.3 μm for 10 μm nicotine. Those were 21.9 ± 6.5 and 6.2 ± 1.1 μm for 100 μm nicotine, respectively (figs. 4A and 4B).
Percent current decay represents the fraction of the current that decays during nicotine application. Thiopental augmented the current decay in a dose-dependent manner. Increases in percent current decay caused by 100 μm thiopental were significantly larger than those caused by 3 μm thiopental. However, the difference between 10 and 100 μm thiopental did not reach statistical significance (table 1). The decaying phases of the elicited current were well fitted to single exponential functions for nicotine 10 μm and better fitted to double exponential functions for nicotine 100 μm. Thiopental at 100 μm significantly reduced time constant ratios of the currents induced by 10 μm nicotine. The fast decaying phases of the current induced by 100 μm nicotine were significantly accelerated by thiopental at 30 and 100 μm. Although thiopental tended to decrease time constant ratios of the slow phases, the changes did not reach statistical significance (table 2).
We compared the inhibitory effects of 30 μm thiopental on the nicotine-induced current in MHb neurons with those in PC12 cells (fig. 5). Thiopental at 30 μm suppressed the nondesensitized currents elicited by 100 μm nicotine more strongly than those elicited by 10 μm nicotine in MHb neurons. However, there was no significant difference in the magnitudes of inhibition of the current elicited by 100 μm nicotine between MHb neurons and PC12 cells.
Effect of MPPB Isomers on the Nicotine-induced Current
Both R (−)- and S (+)-MPPB reversibly inhibited the current induced by 10 μm nicotine in a dose-dependent manner (figs. 6A and 6B). There was no significant difference in the magnitudes of inhibition by these isomers at either doses (fig. 6C). Neither isomers of MPPB changed percent current decay at 30 or 100 μm (table 1). Both isomers tended to increase time constants of the decaying phases of the responses at 100 μm; however, the differences in time constant ratios did not reach statistical significance because of large variances in the values for 100 μm MPPB. There was no significant difference in time constant ratios between the isomers (table 3). These results showed that although the effects of MPPB isomers in vivo were completely opposite, both isomers showed non-stereoselective inhibition of native nAChRs expressed in rat CNS neurons.
Comparison of Pharmacologic Properties of Neuronal Nicotinic Acetylcholine Receptors in PC12 Cells and Rat Medial Habenula Neurons
We compared the responses to three different nAChRs agonists in PC12 cells and rat MHb neurons to characterize agonist pharmacology of nAChRs in these preparations. We used nicotine, acetylcholine, and cytisine at 30 μm. For the peak current, the rank order of the amplitudes of the responses was cytisine > nicotine > acetylcholine in both types of neuronal cells. For the nondesensitized current, it was nicotine = cytisine > acetylcholine in MHb neurons, and cytisine > nicotine > acetylcholine in PC12 cells. The normalized responses relative to the nicotine-induced response were not significantly different between these two types of neuronal cells (fig. 7). These results indicated that PC12 cells and rat MHb neurons share similar agonist pharmacology.
To our knowledge, the current study is the first to demonstrate that thiopental inhibits native nAChR-mediated current at clinically relevant concentrations (20–50 μm) 27in rat CNS neurons. Studies using native receptors have importance because recombinant nAchRs do not necessarily reflect characteristics of corresponding native receptors. 1,18The discrepancies may be raised by differences in subunit stoichiometry, presence of unidentified subunits, and distinct modulation by cellular elements of host cells. 18We also found that convulsant and depressant isomers of MPPB both inhibited native nAChRs in rat CNS neurons and that the inhibitory actions of MPPB isomers were almost the same in their potencies, despite their opposite behavioral effects. 15These results suggest that there is no correlation between inhibitory actions on central nAChRs and the hypnotic effects of the barbiturates in vivo . This finding suggests that inhibition of neuronal nAChRs in MHb neurons is not directly relevant to the hypnotic or anticonvulsive actions of barbiturates. Lack of correlation between inhibitory effects on neuronal nAchRs and anesthetic potencies of barbiturate stereoisomers is consistent with the results of the recent study investigating effects of thiopental stereoisomers on recombinant α4β2and α7homomeric receptors. 17
Comparison of Inhibitory Effects of the Barbiturates in PC12 cells and Medial Habenula Neurons
We previously reported that depressant [thiopental and R (−)-MPPB] and convulsant [S (+)-MPPB and -5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid] barbiturates both suppress neuronal nAchRs in PC12 cells. 14We have confirmed that thiopental and MPPB isomers also inhibit nAchR-mediated response in rat MHb neurons independent from their abilities to produce hypnosis. We have verified our assumption in the previous study that nAchRs in PC12 cells and the CNS have similar sensitivities to barbiturates. Thiopental at 30 μm reduced the current elicited by 100 μm nicotine to the same extent in PC12 cells and MHb neurons, and it accelerated the current decay in the similar manner. IC50values for thiopental are comparable in these two populations, even though agonist concentrations and application times were different. Although we studied effects of MPPB isomers using different nicotine concentrations, characteristics of inhibition by the isomers were slightly different between two preparations. Although MPPB isomers accelerated the current decay in PC12 cells, theydid not significantly increase percent current decay nor decrease the time constant of the decaying phases in MHb neurons. This might be a result of different agonist concentrations used and differences in characteristics of the receptors. It may be possible that the modes of inhibition by MPPB are different in these two populations. Taken together, it seems that thiopental showed the similar effects on two populations of nAchRs, but characteristics of inhibition by MPPB isomers were slightly different between these populations.
Nicotinic Acetylcholine Receptors in PC12 cells and Medial Habenula Neurons
We found that the rank order of agonist potencies was almost the same for PC12 cells and MHb neurons. mRNAs detected in MHb neurons are for α3, α4, α7, β2, β3, and β4subunits, 6,28and the predominant subunit composition of nAChRs on somas is considered to be α3β4. 23However, multiple subtypes of the receptors exist, and the β2subunit is thought to be additionally contained in variable subpopulation of these neurons. 23On the other hand, PC12 cells expressed mRNAs of α3, α5, β2, β3, and β4subunits, 29,30and predominant subunit composition is estimated to be α3β4or α3β4α5. 16,28,29Therefore, it is likely that α3β4-containing receptors are predominant in both populations. However, the exact stoichiometry and combination of the subunits are not identified in either populations.
Early biochemical studies indicated that a major subtype of nAChRs responsible for high affinity nicotine binding sites in the CNS contains α4β2subunits. 3However, consistent with the diversity in subunit gene expression, 31a broad spectrum of nAChRs subtypes are identified by the recent electrophysiological studies in several regions of the brain, such as hippocampus, 32MHb, 19,21,22interpedunclar nucleus (IPN), 33and midbrain nucleus (e.g. , Locus coeleus). 34And in light of recent studies showing more extensive β4expression in the CNS than initially thought, β4-containing nAChRs are also important in the CNS. 31,35
Roles of the Medial Habenula
We have chosen to study MHb neurons because they respond to external application of nicotinic agonists robustly, and characteristics of the responses are relatively homogenous. 23,33In addition, there are few models suitable for electrophysiological studies of native nAchRs with relatively homogenous properties in the brain. However, physiological roles of the MHb are poorly understood. MHb neurons give rise to a large cholinergic projection through the fasciculus retroflexus to the IPN and the median raphe. 33The MHb-IPN system is thought to be involved in rapid-eye-movement sleep, avoidance response, arousal, and attentive behavior. 36–39It has been reported that destruction of the MHb and its primary efferent pathway, the fasciculus retroflexus, impaired avoidance learning, 31,37and decreased rapid-eye-movement sleep and hippocampal theta rhythm of electroencephalogram. 39It is known that nAchRs with the same pharmacologic properties are present at the somas as well as the terminals of MHb axons connecting with the IPN. 33Therefore, barbiturates are likely to affect activity of the MHb-IPN system by inhibition of these receptors, which may result in modulation of rapid-eye-movement sleep and avoidance learning.
Our study shows that thiopental and depressant and convulsant isomers of MPPB all inhibit native α3β4-containing nAchRs in MHb neurons. This type of nAchR is not predominant in the brain, and MHb is not thought to be the primary responsible site governing consciousness. Therefore, these receptors are unlikely to represent the family of native central nAchRs. Taking account of the results of our experiment and the study by Downie et al. , 17however, anesthetic potencies of barbiturate stereoisomers do not correlate with inhibitory effects on brain α3β4-containing receptors, recombinant α4β2or α7homomeric receptors. On the contrary, it is known that potentiating effects on the activity of GABAAreceptors matched well anesthetic actions of enantiomers of thiopental and MPPB. 25,40Therefore, modulation of neuronal nAchRs is likely to be much less important for hypnotic action of barbiturates than that of GABAAreceptors. In conclusion, since thiopental inhibits native nAchRs in rat MHb neurons at the clinically relevant concentrations, this effect may contribute some aspects associated with barbiturate anesthesia. However, the finding that convulsant and depressant isomers of a barbiturate both inhibited the nAChR-mediated currents in the same degree suggests that the inhibition of nAChRs in MHb neurons is not directly relevant to hypnotic or anticonvulsive actions of barbiturates.
The authors thank Dr. J. Knabe (Saaland University, Saarbrücken, Germany) for providing us the stereoisomers of MPPB, and Takeaki Miyamae, M.D., Ph.D., and Ikuo Kawamoto, M.D., Ph.D. (Yokohama City University, Yokohama, Japan), for technical advice.