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Anesth Analg 2000;91:1537-1541
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

The Involvement of Adenosine Neuromodulation in Pentobarbital-Induced Field Excitatory Postsynaptic Potentials Depression in Rat Hippocampal Slices

Yurie Tohdoh, MD, Eichi Narimatsu, MD, PhD, Mikito Kawamata, MD, PhD, and Akiyoshi Namiki, MD, PhD

Department of Anesthesiology, Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo, Hokkaido 060-0061, Japan

Address correspondence and reprint requests to Yurie Tohdoh, MD, Department of Anesthesiology, Sapporo Medical University, School of Medicine, South 1, West 16, Sapporo, Hokkaido 060-0061, Japan. Address e-mail to yurie-toudou{at}ghs.hospital.hokkaido.east.ntt.co.jp


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We investigated the contribution of adenosine neuromodulation to mechanisms of pentobarbital-induced depression of excitatory synaptic transmission in vitro. Transverse hippocampal slices were prepared from brains removed from isoflurane-anesthetized male Wistar rats. Field excitatory postsynaptic potentials (fEPSPs), elicited by orthodromic electrical stimulation of Schaffer collateral at 0.05 Hz, were recorded from the CA1 region in oxygenated artificial cerebrospinal fluid. Amplitude of fEPSP was analyzed for assessing drug effects. Pentobarbital (100 µM) transiently depressed fEPSPs (P < 0.01); i.e., fEPSP was initially depressed to approximately 60% of control and then recovered to approximately 80% of control. The fEPSP depression was partially suppressed by pretreatment with 50 µM aminophylline, a nonselective adenosine receptor antagonist, and 0.2 µM 3, 7-Dimethyl-1-propagylxanthine, an adenosine A1 receptor antagonist (P < 0.01 each). However, the fEPSP depression was not affected by pretreatment with 10 µM 8-cyclopentyl-1, 3-dipropylxanthine, an A2 receptor antagonist, or 10 µM bicuculline, a {gamma}-aminobutyric acid (GABA) A receptor antagonist. The results indicate that adenosine neuromodulation through A1 receptors and other undefined mechanisms, which are independent from GABAergic mechanisms, are involved in pentobarbital-induced depression of excitatory synaptic transmission.

Implications: Adenosine neuromodulation contributes to mechanisms of pentobarbital-induced excitatory postsynaptic potentials depression.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Barbiturates act mainly to depress the function of the central nervous system, primarily by enhancing {gamma}-aminobutyric acid (GABA)-ergic synaptic transmission (1). Inhibitory postsynaptic potentials, mediated by GABAA receptors, are facilitated by pentobarbital (2) and other barbiturates. In addition to this mechanism, pentobarbital depresses excitatory postsynaptic potentials (EPSPs) (3), but the mechanism of EPSP depression is not clear.

However, it has been reported that pentobarbital inhibits adenosine uptake (4). Adenosine uptake inhibition (5) results in accumulation of extracellular endogenously released adenosine, which stimulates subtypes of adenosine receptors. Stimulation of the adenosine A1 receptor induces adenylate cyclase inhibition (6) and phospholipase C activation (7). Conversely, A2 receptor stimulation elicits adenylate cyclase activation (6). The balance of A1 and A2 receptor stimulation regulates adenylate cyclase activity in the adenosine neuromodulatory system (6), which modulates synaptic transmission in the central nervous system. Adenosine accumulation reduces adenylate cyclase activity through A1 receptors, thereby reducing transmitter release (6,8–10) and depressing postsynaptic excitability (8,10,11). We have reported that dipyridamole, a standard adenosine uptake inhibitor, and midazolam, a benzodiazepine that may also depress adenosine uptake, depressed field EPSPs (fEPSPs) in the CA1 region of rat hippocampal slices (12,13). From these findings, we suspected that the pentobarbital-induced adenosine uptake inhibition might also activate the adenosine neuromodulatory system and then depress excitatory synaptic transmission.

We evaluated the contribution of adenosine neuromodulation to the pentobarbital-induced depression of excitatory synaptic transmission. We investigated the effects of adenosine receptor antagonists on pentobarbital-induced fEPSP depression in rat hippocampal slices.


   Methods
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 Abstract
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 Methods
Results
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All of the experimental protocols were approved by the Animal Use Committee of Sapporo Medical University.

Wistar rats, 5 wk old and weighing 100–140g, were anesthetized with isoflurane and decapitated. The brain was rapidly removed from each rat, and from each hemisphere four or five transverse hippocampal slices (400-µm thick) were prepared with a vibratome (Leica VT 1000S; Leica, Nusslcoch, Germany) in artificial cerebrospinal fluid (ACSF) oxygenated with 95%O2-5%CO2 gas at 2–3°C. Slices were then incubated for at least one hour in oxygenated ACSF at room temperature. The slices were placed in a submerged recording chamber (volume of 2 mL) and constantly superfused (3 mL/min) with oxygenated ACSF at 28.0 ± 0.3°C. The composition of the ACSF was as follows (in mM): NaCl, 123.4; KCl, 4.5; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25.0; NaH2PO4, 1.2; and glucose, 10. During oxygenation of the ACSF, the pH value was 7.40 ± 0.05.

The fEPSPs, elicited by orthodromic electrical stimulation on CA1-Schaffer collateral (inframaximal square pulse: duration, 0.1 ms; intensity, 0.02–0.10 mA; frequency, 0.05 Hz) with bipolar tungsten microelectrodes (Microprobe; Protomoc, MD), were recorded by using extracellular tungsten microelectrodes (Microprobe; resistance, 0.9–1.0 M{Omega}) placed in the CA1-stratum radiatum. The intensity of the stimulation was regulated so as not to elicit action potential-induced upward waves on the fEPSPs. Signals from the microelectrodes were amplified (1000 times) and filtered (bandpass, 0.01–3 kHz) with a DAM80 AC differential amplifier (WPI, Sarasota, FL). As an index of synaptic strength, the fEPSP amplitudes (Figure 1a), averaged in groups of three, were analyzed online by using a computer program (MacLab; AD Instruments, Castle Hill, NSW, Australia).



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  		Figure 1. A: A representative waveform of a field excitatory postsynaptic potential (EPSP) recorded from the CA1 region of a rat hippocampal slice. The amplitude of the field EPSP was measured as indicated. B: An example of the effect of 100 µM pentobarbital on field EPSPs in normal artificial cerebrospinal fluid. C: An example of the effect of 100 µM pentobarbital on field EPSPs in a hippocampal slice pretreated with 0.2 µM 3, 7-dimethyl-1-propagylxanthine (DPCPX).

 
Pentobarbital, a barbiturate; bicuculline methiodide, a GABAA receptor antagonist; aminophylline, a nonselective adenosine receptor antagonist; and dimethyl sulfoxide (DMSO) were obtained from Sigma (St. Louis, MO). 3, 7-dimethyl-1-propagylxanthine (DPCPX), an A1 receptor antagonist; and 8-cyclopentyl-1, 3-dipropylxanthine (DMPX), an A2 receptor antagonist, were obtained from Research Biochemicals International (Natick, MA). All drugs were applied to the slices extracellularly and continuously through the superfusing ACSF. We evaluated the effects of pentobarbital (50, 100, 200, and, 500 µM) on fEPSPs in normal ACSF and the effect of 100 µM pentobarbital on fEPSPs pretreated for 30 min with 10 µM bicuculline, 50 µM aminophylline, 0.2 µM DPCPX or 10 µM DMPX. Pentobarbital and DPCPX were first dissolved in DMSO and then in ACSF. The final concentration of DMSO in ACSF was <0.1%.

The amplitude of the fEPSP was expressed as mean ± SE (n = 6–7). Statistical analysis was performed by using the paired Student’s t-tests, one- or two-way repeated measures analysis of variance (ANOVA), or one-way factorial measures ANOVA when appropriate. Data were considered to be statistically significant when P < 0.05.


   Results
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 Abstract
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 Methods
 Results
Discussion
 References
 
Inframaximal stimulation of Schaffer collaterals elicited CA1-fEPSPs (range 0.12–0.52 mV) without a significant influence of action potentials (Figure 1). The drug effects on the fEPSPs were investigated after stabilization of the fEPSP amplitude, which usually required a period of >=30 min from the start of stimulation at 0.05 Hz. The stabilization of the fEPSP amplitude was confirmed when the variability of the amplitude was within the range of 7% of the mean value for a period of >=15 min.

Continuous application of 100 µM pentobarbital transiently depressed the fEPSPs (P < 0.01); i.e., pentobarbital initially reduced the fEPSP amplitude to 58.9 ± 3.1% of control at 8 min after its application (P < 0.01), then the reduced amplitude gradually recovered to 77.3 ± 4.3% of control within 16 min. The partially recovered amplitude remained stable at that level (n = 7, Figure 1B and 2). At more than 100 µM, pentobarbital further transiently and dose-dependently decreased the fEPSP amplitude. Pentobarbital (200 and 500 µM) decreased the amplitude to 40.1 ± 2.5% and 18.7 ± 2.1% of control at 8 min (P < 0.01 each), that recovered to 56.4 ± 3.9% and 29.3 ± 3.3% of control within 16 min, respectively, and thereafter remained stable (n = 6 each). However, at 50 µM, pentobarbital conversely increased the fEPSP amplitude to 142.5 ± 7.9% of control within 12 min (n = 6, P < 0.01), and the increased amplitude thereafter remained stable.

The influence of GABAA receptor-mediated inhibitory synaptic transmission on pentobarbital-induced fEPSP depression was investigated. Application of 10 µM bicuculline had no significant effect on the fEPSP amplitude (98.6 ± 4.5% of control, n = 7). Pentobarbital (100 µM) transiently depressed (P < 0.01) fEPSPs in slices pretreated with 10 µM bicuculline in a manner similar to that with 100 µM pentobarbital alone (P > 0.05); i.e., pentobarbital initially reduced the fEPSP amplitude to 53.0 ± 9.8% of control at 8 min (P < 0.01), that recovered to 77.0 ± 5.9% of control within 16 min and thereafter remained stable (n = 6, Figure 2).



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  		Figure 2. The time courses of the effects of 100 µM pentobarbital (Pento) on field excitatory postsynaptic potential (EPSP) amplitude in normal artificial cerebrospinal fluid (n = 7) and in slices pretreated with 10 µM bicuculline (Bic) for 30 min (n = 6). Mean field EPSP amplitude (averaged for 3 min) immediately before the application of pentobarbital is defined as the control value (100%) in each experimental group. Mean ± SE, *: P < 0.05 and **: P < 0.01 vs control.

 
The influences of adenosine antagonists were investigated. Within 15 min, fEPSP amplitude was increased (P < 0.01 each) by 50 µM aminophylline, 0.2 µM DPCPX, and 10 µM DMPX to 119.8 ± 4.7% (n = 6), 135.3 ± 5.1% (n = 6), and 111.6 ± 4.9% (n = 6) of control, respectively, and the increased amplitude thereafter remained stable in each case. Next, the effects of pentobarbital on slices pretreated with each of these adenosine antagonists were investigated. The fEPSP depression was partially inhibited by aminophylline and DPCPX. Pentobarbital (100 µM) continuously depressed fEPSPs in slices pretreated with 50 µM aminophylline or 0.2 µM DPCPX (P < 0.01 each); i.e., the fEPSP amplitude was gradually decreased by pentobarbital to 75.6 ± 6.5% (n = 6) or 82.6 ± 3.6% of control (n = 7), respectively (P < 0.01 each), within 12 min and thereafter remained stable (Figure 1C and 3). The effects of 100 µM pentobarbital on slices pretreated with aminophylline or DPCPX were different (P < 0.01) from the effects of 100 µM pentobarbital alone. However, 100 µM pentobarbital transiently depressed (P < 0.01) fEPSPs in slices pretreated with 10 µM DMPX in a manner similar (P > 0.05) to that with 100 µM pentobarbital alone; i.e., pentobarbital reduced the fEPSP amplitude to 66.0 ± 5.3% of control at 8 min (P < 0.01), recovered to 80.5 ± 4.6% of control within 16 min, and thereafter remained stable (n = 6, Figure 3).



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  		Figure 3. The time courses of the effects of 100 µM pentobarbital (Pento) on field excitatory postsynaptic potential (EPSP) amplitude in slices pretreated for 30 min with 50 µM aminophylline (Ami, n = 6), 0.2 µM 3, 7-dimethyl-1-propagylxanthine (DPCPX) (n = 7) or 10 µM 8-cyclopentyl-1, 3-dipropylxanthine (DMPX) (n = 6). Mean field EPSP amplitude (averaged for 3 min) immediately before the application of pentobarbital is defined as the control value (100%) in each experimental group. Mean ± SE, *P < 0.05 and **P < 0.01 vs control.

 
The pentobarbital-induced maximal decrease in fEPSP amplitude at 8 min in slices that had not been pretreated was more intense than that in slices pretreated with aminophylline or DPCPX (P < 0.01 each) but was not significantly different from that in slices pretreated with bicuculline or DMPX (P > 0.05 each). There were no significant differences in the 100 µM pentobarbital-induced decrease in fEPSP amplitude at 16 or 20 min among the five experimental groups (P > 0.05).

There were no significant differences in the absolute values of the fEPSP amplitudes before drug application among the above-described five experimental groups: 0.29 ± 0.03mV (pentobarbital alone, n = 7), 0.25 ± 0.04mV (bicuculline and pentobarbital, n = 6), 0.32 ± 0.04mV (aminophylline and pentobarbital, n = 6), 0.27 ± 0.04 mV (DPCPX and pentobarbital, n = 7), and 0.30 ± 0.5mV (DMPX and pentobarbital, n = 6).


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The present study revealed the existence of an adenosine-mediated mechanism in pentobarbital-induced depression of excitatory synaptic transmission. We used CA1-fEPSPs, which mainly reflect glutamatergic excitatory synaptic transmission (12,13) and are preferred because of their stability during long-term recording.

Continuous application of 100 µM pentobarbital, which was the smaller limit of the range of concentrations that could induce fEPSP depression in the hippocampus (3), transiently, but not continuously, depressed fEPSPs. This result demonstrated that pentobarbital transiently depresses glutamatergic EPSPs, which are generated on dendrites of CA1-pyramidal cells. Pretreatment with bicuculline did not significantly influence pentobarbita l-induced fEPSP depression. Furthermore, bicuculline did not affect fEPSPs, as was reported previously (12,13). These results indicate that pentobarbital has a mechanism to transiently depress glutamatergic excitatory synaptic transmission, which is independent from its conventional GABAergic action. However, the details of the nonGABAergic mechanism of pentobarbital are not known.

Because pentobarbital inhibits adenosine uptake (4) and because transient fEPSP depression mediated by adenosine receptors and subsequent adenosine neuromodulatory system has been observed when adenosine uptake inhibitors have been used (12,13), we presumed that pentobarbital-induced adenosine uptake inhibition may be a possible mechanism of the pentobarbital-induced fEPSP depression.

Pentobarbital-induced fEPSP depression was partially antagonized by pretreatment with aminophylline and DPCPX; these suppressed the initial and transient parts of fEPSP depression, which peaked at 8 min and lasted until 16 min. However, pentobarbital-induced fEPSP depression was not influenced by pretreatment with DMPX. These results indicate that the initial transient part of fEPSP depression is mediated by A1 receptors, not by A2 receptors.

Furthermore, there were no significant differences in pentobarbital-induced fEPSP depressions at 16 min and at 20 min between the experimental groups. These results indicate that the effect of pentobarbital is because of at least two individual mechanisms; i.e., 1) an A1 receptor-dependent mechanism that elicits initial and transient fEPSP depression, and 2) adenosine receptor-independent mechanisms that produce subsequent continuous fEPSP depression. The initial part of the pentobarbital-induced fEPSP depression may be a result of the addition of these mechanisms.

The details of the adenosine-independent mechanism are unknown. Other effects of barbiturates, such as modulation of sodium channels (14), blockade of {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (15), depression of membrane depolarization (16), and modulation of other second messenger systems (17), are possible causes of the induction of adenosine-independent EPSP depression.

We have reported that adenosine uptake inhibition-induced transient fEPSP depression was mediated mainly by presynaptic adenosine neuromodulation through A1 receptors but was not mediated by postsynaptic adenosine neuromodulation (13). These findings suggest that the initial transient part of fEPSP depression induced by pentobarbital may also be mediated mainly by the presynaptic mechanism. Presynaptic A1 receptors are linked to deactivation of adenylate cyclase (8) and subsequent decrease in glutamate release (8–10) and spontaneous recovery of adenosine uptake inhibition-induced fEPSP depression requires protein kinase C activation (13). It is possible that the initial transient part of the pentobarbital-induced fEPSP depression may also be a result of such adenosine receptor-mediated presynaptic mechanisms.

The contribution of the adenosine receptor-mediated mechanism of pentobarbital to its clinical effect is unknown. However, it is possible that this mechanism influences the strength of the clinical effect of pentobarbital in the period immediately after its administration.

In summary, this study showed that both adenosine neuromodulation through A1 receptors and other unrevealed mechanisms, both of which are independent from GABAergic mechanisms, are involved in the pentobarbital-induced depression of excitatory synaptic transmission.


   References
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 Abstract
 Introduction
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 Discussion
 References
 

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Accepted for publication August 8, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2000 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press