Effect of thiopental on hippocampal GABA receptors in rat brain

Neurochem. Int. Vol. 8, No. 3, pp. 417--421, 1986 Printed in Great Britain.All rights reserved

0197-0186/86 $3.00+ 0.00 Pergamon Journals Ltd

EFFECT OF THIOPENTAL ON HIPPOCAMPAL GABA RECEPTORS IN RAT BRAIN RYOICHI TAKADA*, KIHACHI SAITOt, HI1)EO MATSUURA* and RExzo INOKIt Departments of *Anesthesiology and "t'Pharmacology, Osaka University, Faculty of Dentistry, Yamadaoka I-8, Suita 565, Japan (Received 1 August 1985; accepted 14 October 1985)

Abstract--The effect of thiopental on rat brain hippocampal GABA receptors was studied in slice preparations and membrane fractions. In slice preparations, thiopental at a concentration of 10-5 M enhanced the GABA (1-5 x 10-4 M) inhibition of the field potentials evoked in pyramidal neurons by stratum radiatum stimulation. In hippocampal slices obtained from chronically barbital-administered (100 mg/kg, b.i.d., 10 days) rats, less enhancement of thiopental on GABA inhibitionof the field potentials was observed. In binding experiments, two components of specific [3H]GABA binding to membrane fractions were observed. While thiopentat was without effect on high-affinitysites, [3H]GABAbindingwith low affinity was enhanced by 80% in the presence of 10-s M thiopental. The results are discussed in relation to the phenomena underlying chronic barbiturate administration.

INTRODUCTION Despite the long use of barbiturates as a hypnotics, sedatives and anticonvulsants, the mechanism of their action has been only partially clarified. Nicoll et al. (1975) demonstrated that pentobarbital prolonged the time-course of inhibitory postsynaptic potentials (IPSP) recorded in hippocampal pyramidal cells wherein GABA is supposed to elicit inhibitory action. There are many reports demonstrating the prolongation of IPSP by barbiturates (Larson and Major, 1970; Scholfield, 1977). Thus, it is now generally accepted that barbiturates enhance the action of GABA. Recently, it has been shown that barbiturates enhance the binding of [3H]GABA to its receptors (Asano and Ogasawara, 1981; Olsen and Snowman, 1982; Willow and Johnston, 1981). Hippocampal in vitro slice preparations are useful materials in physiological and biochemical CNS experiments, since the stability of the slices make it possible to record responses for a period of several hours or more and to conduct biochemical analyses. Particular important findings concerning the mechanisms of action of morphine and opioid peptides have been made using hippocampal slice preparations (Siggins and Zieglg/insberger, 1981; Valentino and Dingledine, 1982). In the present study, using hippocampal slice preparations, the effect of thiopental on GABA inhibition of the field potentials evoked in pyramidal cells by stratum radiatum stimulation was

studied in conjunction with chronic barbital administration. The effect of thiopental on [3H]GABA binding to hippocampal membranes was also examined. EXPERIMENTAL PROCEDURES

Preparation of brain slices Sprague--Dawley rats were killed by decapitation and their brains were quickly removed. Parasagittal slices containing hippocampns were cut to a thickness of 300/~m (Saito et al., 1985) and preincubated in modified oxygenated Krebs-Ringer solution (composition: NaCI 124mM, KCI 5 mM, KH2PO4 1.24mM, MgSO4 1.3 raM, CaCI2 2.6 raM, NaHCO3 26 mM, glucose 10 mM) for about I h at 35°C. Electrophysiological experiments (Nakahiro et al., 1985) Slices were transferred to a recording chamber and perfused with Krebs-Ringer solution (35°C) at a rate of 1.3 ml/ min. Stimuli were applied through a bipolar stainless-steel electrode to the stratum radiatum with a frequency and duration of 0.5 Hz and 50 tt s, respectively. Field potentials were recorded in cornu ammonis 1 (CA1) with glass micropipettes (3-5M~, filled with Krebs--Ringer solution) positioned on the pyramidal. Ten responses were averaged with a data processor (ATAC-350, Nihon Kohden) and displayed on an X - Y recorder for analysis. Preparation of hippocampal membrane fractions Rats were decapitated and hippocampi were homogenized with 10 vol of 0.32 M sucrose. The homogenate was centrifuged at 900 g for 10 min. The supernatant was centrifuged at ll,500g for 30rain and the pellet was stored at - 8 0 ° C overnight. The frozen membranes were thawed and suspended in 10 vol of 50 mM Tris--maleat¢buffer (pH 7.4) and centrifuged at ll,500g for 30min. This was repeated 417

418

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four times and the resultant pellet was suspended in 50 mM Tris maleate buffer.

[~H]GABA binding For assay of [3H]GABA binding, the membrane suspension (300/tg/tube) was incubated at 0 C for 20 rain in 1 ml of 50raM Tris maleate buffer (pH 7.41 containing 1 200 nM [3H]GABA and 100 mM KC1 in the presence and absence of 10 3 M GABA. The reaction was terminated by filtering the incubation medium through Whatman glass filters (GF/C). The filters were then washed three times with 5 ml of ice-cold incubation medium and the radioactivity was counted in a liquid scintillation counter. The differences in the amount of [3H]GABA bound in the presence and absence of 10-3M GABA were designated as specific [3H]GABA binding.

Administration of barbital Barbital was i.p. administered to rats at a dose of 100 mg/kg twice a day for 10 days. Thirty-six hours following the last administration of barbital, the rats were killed and slices were prepared.

Materials [3H]GABA was purchased from New England Nuclear (Boston, Mass., U.S.A.). Thiopental (Tanabe Pharmaceuticals Co. Ltd, Osaka, Japan) and sodium barbital (Wako Pure Chemical Co. Ltd, Osaka, Japan) were also used.

RESULTS

Effect of thiopental and GABA on the.fieM potentials evoked in pyramidal neurons Stimulation of stratum radiatum evoked responses in C A I pyramidal neurons consisting of two or three

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negative population spikes (0.5-I .5 mV, F i g !. inset) Thiopental and G A B A at the various concentrations were perfused and their effects on the field potentials were examined by measuring the first peak height (Tielen et al., 1981). Thiopental at a concentration of 5 × 10 6M slightly enhanced the amplitude of the field potentials (Fig. 1). Increasing the concentration of thiopental gradually reduced the amplitude of the field potentials over a range of 2 orders of magnitude. As shown in Fig. 2, while 10 4 M G A B A was without effect, complete reduction of the field potentials was obtained with 10- ~ M G A B A . Thus, the inhibition of the field potentials was observed within a narrow range of G A B A concentration.

Effect of thiopental on GABA inhibition qt the field potentials To examine the effect of thiopental on G A B A inhibition of the field potentials, various concentrations of G A B A were perfused in the presence of 10 5M thiopental. At this concentration of thiopental, no effect on the field potentials was observed. Concomitant perfusion of thiopental with G A B A significantly shifted the concentration-inhibition curve to the left (Fig. 3a). ICs0-values for G A B A in inhibiting the field potentials in the absence and presence ofthiopental were 4 × 10 `4 and 2 × 10 4 M, respectively. There was a reduced thiopental effect in the chronically barbital-treated rats; a significant effect being observed with G A B A concentrations of higher than 5 x 1 0 - a M (Fig. 3b).

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Fig. 1. Effect of thiopental on field potentials evoked in hippocampal CA 1 pyramidal neurons. Inset: field potentials recorded in CA1 pyramidal neurons. Bars represent the means of the number of experiments (indicated in parentheses + SD).

Thiopental and GABA receptors

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Fig. 2. Effect of GABA on field potentials evoked in hippocampal CA1 pyramidal neurons. Bars represent the means of the number of experiments (indicated in parentheses ± SD).

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DISCUSSION

A number of studies have reported the reduction of excitatory postsynaptic potentials (EPSP) without alteration of resting membrane potentials by barbiturates (Barker and Gainer, 1973; Eccles, 1946). The inhibition of the release of excitatory synaptic transmitters would account for the reduction of EPSP. However, in most studies, it has been shown

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Effect of thiopental on [3H]GABA binding to hippocampal membranes Two components of the specific [3H]GABA binding to hippocampal membranes were observed after extensive washing of the samples (Fig. 4; Table 1). The high-affinity site maximally b o u n d 1 3 . 9 + 0 . 6 fmol/mg protein and had a KD-value of 6.8 + 0.2 nM. The low-affinity sites were observed to have a KDvalue of 308.4 ___11.4 n M and a Bm~ of 301.2 _ 8.6 fmol/mg protein. When [3H]GABA binding was measured in the presence of 10-SM thiopental, no alteration in the KD-value and Bm~ of high-affinity sites was observed. However, in the presence of 10-SM thiopental the a m o u n t of low-affinity sites increased significantly by 80% without a change in the KD-value.

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Fig. 3. Effect of thiopental (10 -5 M) on GABA inhibition of the field potentials evoked in hippocarnpal CAI pyramidal neurons in slices prepared from (a) saline- and Co) barbitaladministered rats. Bars represent the means of the number of experiments (indicated in parentheses + SD). *Significantly different from that observed in the absence of thiopental (P < 0.01).

that higher concentration of barbiturates were required to inhibit the release of transmitters (Richter and Waller, 1977). Stabilization of axonal or somal membranes leading to the impaired conduction may alternatively account for the action of barbiturates. However, this is also unlikely since the majority of papers have reported no change of conduction by barbiturates. Finally, the augumentation of GABA

Table 1. Effect of thiopental on [3H]GABA binding High-affinitysite Low-affinity site KD (riM) Bm~ (fmol/mg) Ko (nM) B=,, (fmol/mg) 6.8+_0.2 13.9+_0.6 308.4+11.4 301.2+-8.6

Thiopental (10-5 M) 6.9+-0.1 13.4+-0.9 310.2+7.6 546.1 +- 11.8" *Significantlydifferent from the [3H]GABA binding observed in the absence of thiopental (P < 0.01, n = 3).

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Fig. 4. Scatchard analysis of [3H]GABA binding to hippocampal membranes in the absence (O) and presence (C)) of thiopental. action by barbiturates should be discussed. Nicoll et al. (1975) reported that pentobarbitat prolonged

the time-course of IPSP recorded in hippocampal pyramidal cells. There are many reports demonstrating the prolongation of IPSP by barbiturates in the CNS where G A B A exerts an inhibitory action (Eccles, 1946; Scholfield, 1977). Thus, the action of barbiturates is most likely due to the enhancement of G A B A inhibition. In the present in vitro slice experiments, the inhibition of the field potentials by GABA was enhanced with thiopental at concentrations as low as 10 -5 M. Furthermore, at this concentration of thiopental, the enhancement of the specific [3H]GABA binding to membrane fractions was also observed. Our results are compatible with the above notion. It also appeared that in vitro slice preparations are feasible for studying the mechanisms of the action of barbiturates. Original studies to demonstrate enhancement of [3H]GABA binding to its receptors by barbiturates were unsuccesful. However, freezing-thawing and extensive washing of membrane fractions removed endogenous inhibitors for [3H]GABA binding and enabled the effect of barbiturates on the binding to be detected. After the removal of endogenous inhibitors, two components of [3H]GABA binding to its receptors and the effects of barbiturates on both low- and high-affinity sites were observed (Olsen and Snowman, 1982). Furthermore, it has been shown that the potency of barbiturates enhancing [3H]GABA binding correlates with their anesthetic activity (Asano and Ogasawara, 1982). In the present study, however, thiopental enhanced the low-affinity [3H]GABA binding sites only. The major difference is the concentration of barbiturates employed. In our study, I0 5 M thiopental, based on slice experiments, was

uscd whereas barbiturates at conccntrauons ~I iHolc 0~an 10 4M were employed in published reports obtaining detectable enhancemcnt of [~H]GABA binding. The somewhat lower value of [q-I]GABA binding observed in the present study may bc explained by the use of hippocampal membranes (Young et al., 1976). In the present study, long-acting barbital was administered to produce a chronic effect in rats, while short-acting thiopental was used for assessing m vitro action. Development of cross-tolerance is a wellknown phenomenon for a number of barbiturates. Furthermore, enhancement of [3H]GABA binding has been observed for barbiturate congeners (Asano and Ogasawara, 1981, 1982; Olsen and Snowman, 1982; Willow and Johnson, 1981 ). Inhibition of ['H]phenobarbitone binding by those barbiturates has also been reported (Willow et al., 1981). Thus, it seems likely that all of these barbiturates act on common sites. The use of barbital for the administration and thiopental for in vitro experiments is thus permissible. It is well-known that tolerance development for barbiturates is attributable to the enhancement of liver drug metabolizing enzyme. However, involvement of central components including the GABA system in tolerance is also suggested. Thus, the decrease of GABA binding to the membrane fraction in barbiturate-treated rats has been reported previously (M6hler et al., 1978). The present study demonstrated reduced effectiveness of thiopental on GABA inhibition for the held potentials, which strongly indicates a decrease in either barbiturate or GABA receptors. Such an experiment is now in progress at our laboratory. REFERENCES

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