Brain Research, 591 (1992) 327-331

327

© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

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Uncoupling of GABA-benzodiazepine receptors in chick cerebral cortical neurons requires co-activation of both receptor sites A. Prasad a n d J.N. Reynolds Faculty of Medicine, Memorial Universityof Newfoundland, St. John's, Nfld. (Canada) (Accepted 16 June 1992)

Key words: Benzodiazepine; GABA; Cerebral cortical neuron; Ncuromodulation; Tolerance; Voltage clamp

Primary cultures of chick cerebral cortical neurons were exposed tc i p.M flurazepam in vitro, and the effect of flurazepam on GABA-activated membrane current (IoAnA) was examined using whole-cell voltage-clamp recording. Exposure of chick cerebral cortical neurons to flurazepam alone for 3-10 days resulted in a significant decrease in the degree of potentiation of IGAnA elicited by 0.5 p.M flurazepam. When GABA or nipecotic acid (a GABA uptake blocker) were added with flurazepam during chronic drug exposure, neuronal responses to GABA were significantly less sensitive to modulation by 0.5 p,M flurazepam compared to flurazepam treatment alone. Furthermore, this effect was significantly reduced by co-administration of the GABA A receptor antagonist bicuculline. These results suggest that tolerance to benzodiazepines in vitro requires activation of both the GABA and the benzodiazepine binding sites on the GABA A receptor-channel complex.

Benzodiazepines are widely used in clinical medicine as anxiolytics, sedative-hypnotics, and antieonvulsants. There is ample evidence from behavioral, electrophysiological and neurochemical studies to suggest that benzodiazepines exert their effects on the central nervous system by potentiating inhibitory synaptic transmission mediated by y.aminobutyric acid (GABA) acting at the GABAA receptor subtype. However, chronic administration of benzodiazepines can produce tolerance to the effects of these drugs a, which can limit their clinical efficacy. The mechanism of tolerance to benzodiazepines remains uncertain. Numerous studies have appeared in the literature concerning the neurochemical effects of chronic exposure to benzodiazepine agonists, but results are often conflicting. Benzodiazepine binding sites in neuronal membranes are reported to be either unchanged H)'tT'l~'2~ or decreased ~'~6'2° after chronic exposure to benzodiazepines. GABA A receptor function is also reported to be either unchanged ~3'2m or decreased 7,9.11 after chronic exposure to a benzodiazepine agonist. However, virtually all studies agree that chronic exposure to a benzodiazepine agonist results in a significant decrease in the functional coupling

of the benzodiazepine and GABA binding sites (for review see ref. 8). Benzodiazepine agonists are positive modulators of GABAA receptor function. Benzodiazepines do not themselves activate the receptor-channel complex. Rather, they produce an allosteric modulation of the complex which results in an increase in the frequency of bursting activity in the presence of GABA2a. The purpose of this study was to test the hypothesis that tolerance to benzodiazepines is influenced by the degree of activation of the GABA A receptor. Primary cultures of chick cerebral cortical neurons were exposed to flurazepam for varying lengths of time in the presence or absence of GABA or nipecotic acid, a GABA uptake blocker. Whole-cell voltage clamp recordings were used to directly assess neuronal responses to GABA and the ability of flurazepam to potentiate GABA A receptor function. Primary cultures of chick cerebral cortical neurons were prepared as previously described ~6. In brief, cerebral cortices were dissected from 7-8 day-old chick embryos (stages 31-34 as described by Hamburger and Hamilton3) into ice-cold Ca2+/Mg2+-free Tyrode's buffer. Tissue was prepared by enzymatic dissociation

Correspondence: J.N. Reynolds, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Nfld., Canada AIB 3V6.

328 with papain (30 U/ml, Boehringer Mannheim), or mechanically dissociated followed by filtration through a Nitex membrane (pore size 45/~m). Cells were resuspended in the growth medium and plated at a density of 0.5 x 106 cells/dish into poly-D-lysine-coated 35 mm tissue culture dishes. The growth medium consisted of Minimum Essential Medium (MEM, Gibco) supplemented with 1 mM glutamine, 4.5 g/! D-glucose, 50 U/ml penicillan, 50 /J,g/ml streptomycin, 10% calf serum (Seru-Max 3, Sigma Chemical Co.), and 2% Nu-serum (Collaborative Research Inc.). After 3-4 days the growth medium was replaced by serum-free N3 medium supplemented as described by Romijn et al ms. Thereafter the medium was changed every 5 days. Cells were maintained in a humidified 37°C incubator with 5% CO,. After 5 days in vitro drug treatments were initiated. All drugs were dissolved in sterile water and added directly to the culture dishes in a volume of 10-20 ~!. Control cultures received sterile water alone. The final concentrations of drugs added to the culture dishes were: Flurazepam HCi, 1 /~M; GABA, 1-5 p.M; Nipecotic Acid, 10/~M; Bicuculline, 10 ~M. Neuronal cultures were exposed to various combinations of these drugs for 1-10 days. Fresh drug was added each day, except for bicuculline which was added every second day. Individual neurons were visualized on the stage of an inverted microscope (Zeiss IM35). For electrophysi. ological recording the cells were continuously perfused (I-2 ml/min) with a physiological saline containing (in raM): 140 NaCI, 5 KCI, 2 CaCI 2, 1 MgCI,, 10 (N-[2-hy. droxycthyl]piperazine-N'-[2-ethanesulf onic acid]) (HEPES), l0 D-glucose, adjusted to pH 7.3 with NaOH. Flurazepam.HCI was dissolved in the extracellular saline and applied by bath perfusion. GABA (25/zM) was dissolved in the extracellular saline and applied by brief (20-100 ms) pressure pulses (Picospritzer ll) directly to the soma of the cell under study. Following chronic exposure to flurazepam, cells were washed with drug-free solution for 1-3 hours prior to record. ing. in some cases cells were washed with drug-free culture media for 24 h prior to r¢',ording. Whole-cell voltage clamp recordings were obtained using recording electrodes (3-5 Mr2) filled with (in raM): 140 KCi, 5 MgCI2, 10 HEPES, 4 Na-ATP, pH 7.3. All recordings were obtained using an Axoclamp.2A amplifier in discontinuous single-electrode voltage-clamp mode (switching frequency 10 kHz). Data acquisition and analysis were performed using pClamp software (Axon Instruments). All recordings were obtained at a holding potential of - 6 0 inV. In each cell, 2-3 control responses to GABA were obtained prior to starting

perfusion with flurazepam. Care was taken to ensure that the control responses to GABA were submaximal. Drug effects were evaluated by calculating the potentiation (expressed as a percentage of the control response) of the GABA response in the presence of flurazepam. Data are expressed as the mean ± S.E.M. Brief pulses (20-100 ms)of 25 p,M GABA produced membrane currents in control cells (709 + 90 pA~ n - 5) which were submaximal. Long (10 s) applications of 50 /z M GABA from a large bore pipette could elicit much larger currents (3.18 + 0.14 nA, n - 3). Chronic exposure of chick cerebral cortical neurons to flurazepam, or to combinations of flurazepam with other drugs for 1-10 days had no apparent effect on the initial response to brief pulses of 25 /zM GABA. After 5-10 days exposure to 1/zM flurazepam plus nipecotic acid or GABA, the initial response to 25 ~M GABA was similar to control cells (697 ± 123 pA, n = 6). In control dishes, bath perfusion of 0.5/zM flurazepam produced a pronounced, reversible potentiation of IOAaA (Fig. 1). LOW concentrations (100 nM) of flurazepam produced relatively small and inconsistent changes in IOABA, but higher concentrations of flurazepam (1-10 ~M) did not produce substantially larger increases in IOABA compared to 0.5/zM (data not shown). Thus, a concentration of 0.5/zM flurazepam was chosen to test the functional integrity of GABA A receptors in drugtreated cultures. Exposure of chick cerebral cortical neurons to GABA (1-5 ~M) 4. 10/~M nipecotic acid (a blocker of high affinity GABA uptake), or to nipecotic acid alone, for 1-10 days had no effect on the response to GABA or the degree of potentiation of loan^ by 0.5 /zM flurazepam (Fig. 2). Chronic exposure of chick cerebral cortical neurons to 1 /~M flurazepam alone for 3-10 days resulted in a small but statistically signifcant

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Fig, 1. GABAreceptor.activatedcurrents in cultured chick cerebral cortical neurons are reversiblypotentiated by 0.5 ~.M flurazepam. Brief (20 ms) pressure pulses of GABA (25 ~M) were delivered every 60 s to a cell voltage-clampedat -60 mV. Downwarddeflections are inwardcurrent responsesto applicationof GABA.Current responses are shown before (G), during (G+ F), and after (Wash) bath applicationof 0.5 p.M flurazepam.

329 degree of potentiation of IGABAby 0.5/zM flurazepam was significantly greater than the potentiation obtained in cells chronically exposed to flurazepam + GABA or nipecotic acid without bicuculline (Fig. 2). Bicuculline treatment alone had no effect on the degree of potentiation of IGABA by flurazepam (data not shown). The mechanism(s) involved in the development or maintenance of tolerance are unknown. Previous studies which have examined the binding of benzodiazepines and GABA A receptor-activated CI- flux using animal models of tolerance have produced a somewhat contradictory array of results, (for review see ref. 8) showing no change, decreases or increases in benzodiazepine binding and receptor-activated CI- flux. Primary neuronal cell cultures offer several advantages for examining the cellular mechanisms of drug tolerance. In particular, the problems of drug metabolism and distribution are largely avoided, and the concentration of drug at the active site can be more closely controlled. However, even in these simpler in vitro model systems contradictory results have been obtained. Binding of benzodiazepine agonists following chronic exposure to benzodiazepines has been reported to be unchanged~7 or decreased9'2°. Several studies however have shown that chronic benzodiazepine exposure in vitro results in allosteric uncoupling of benzodiazepine-GABA responses without any change in benzodiazepine receptor number 17'1~'21. In the present study, chronic exposure of chick cerebral cortical neurons to a combination of low doses

reduction in the degree of potentiation of IGABAby 0.5 /~M flurazepam (86 + 9.6% in control, 49 + 7.6% after flurazepam treatment, P < 0.05) (Fig. 2). There was no apparent time-dependence to this effect of flurazepam, so these data have been pooled. Exposure of chick cerebral cortical neurons to 1/zM flurazepam+either GABA (1-5 /zM) or 10 /.tM nipecotic acid produced a time-dependent reduction in the degree of potentiation of IGABA by 0.5 /~M flurazepam (Fig. 2). Co-administration of flurazepam with either GABA or nipecotic acid produced similar results, and therefore these data have been pooled. After 1-3 days exposure to either of these two drug treatments, the sensitivity of 1GABAto flurazepam was reduced in comparison to control cultures, and was similar to cells exposed to flurazepam alone for 3-10 days. However, longer periods of exposure (5-10 days) to flurazepam + GABA or nipecotic acid resulted in neuronal responses to GABA which were significantly less sensitive to modulation by 0.5 ~M flurazepam compared to flurazepam treatment alone (Fig. 2). Furthermore, this effect persisted even after several hours of washing the cells with drug-free extracellular solution. The results of these experiments suggested that the presence of both GABA and flurazepam was necessary to induce long-lasting changes in GABAA receptor function. Therefore, cultures of chick cerebral cortical neurons were exposed to 1 ttM flurazepam + 5 /.tM GABA or 10 ttM nipecotic acid + 10 p,M bicuculline for periods ranging from 5-10 days. In these cells, the

E f f e c t s of Chronic Flurazepam on IGABA in Chick Cerebral Cortical Neurons ~. 150] m

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Control GABA + Nipecotic Actd Flurazepam F + NA (1-3 d) F + NA (5-10 d) F + NA + B (5-10 d)

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Fig. 2. Effects of various drug treatments on the modulation of/GABA by 0.5/zM flurazepam. Data represent the degree of potentiation of the GABA response in the presence of 0.5/~M flurazepam. In control cells (A), 0.5 ~M flurazepam increased IGABAby 86.6+9.6%. Exposure to GABA (1-5/~M) plus 10 ~M nipecotic acid (B) had no significant effects on the neuronal response to GABA or modulation by flurazepam. Exposure to 1/zM flurazepam for 3-10 days (C) significantly reduced the degree of potentiation of/GABA by 0.5/zM flurazepam to 49.1 + 7.6% (P < 0.05). Adding GABA (1-5/~M) or nipecotic acid (10/~M) together with 1 p,M flurazepam reduced the subsequent modulation of 1GABAby 0.5/~M flurazepam to 39.4+ 12% (1-3 days, D) and 23.6+6.8% (5-10 days, E). Treatment E is significantly less than treatments A and C (P < 0.05). Co-administration of bicuculline (10/zM) with flurazepam and GABA or nipecotic acid (F) attenuated this effect on modulation of 1OABAby 0.5/~M flurazepam (53.4 + 10.8%). The number of cells tested is shown in parentheses above each drug treatment.

330

of flurazepam and GABA markedly reduced the benzodiazepine potentiation of /GABA" Control studies showed that the effects of flurazepam on IGAB^ were easily reversed over a period of several minutes (Fig. 1), making it very unlikely that the lack of potentiation after chronic exposure ;s simply due to the occupation of benzodiazepine binding sites. This suggestion is supported by the results of Sher et al 2°, who reported that spinal cord cultures chronically exposed to benzodiazepene exhibited decreased potentiation of GABAergic responses although there was no detectable benzodiazepine in the supernatant. In the present study, control responses to GABA were apparently unchanged by any of the drug treatments. GABA receptor activation appears to be crucial in the development of tolerance to benzodiazepines. The effect was most consistently produced in cultures with increased levels of GABA with flurazepam. The importance of GABA receptor activation was further emphasized by results showing that tolerance was reduced or eliminated when the GABA A receptor antagonist bicuculline was included with the GABA and flurazepam treatment. This contrasts with the findings of Roca et al. '7, who reported an allosteric uncoupling of the GABA and benzodiazepine receptors even in the presence of the GABA antagonist picrotoxin. However the dosage and duration of drug exposure was less than that used in our studies, and there is evidence to suggest that picrotoxin is a use-dependent blocker of the GABA^ channel, being most effective during pro. longed activation of the channel s. The GABA^ receptor-chloride channel complex is a heteromeric structure composed of a, ~, ?, and subunits. Multiple subtypes of these subunits have been detected, and there is wide variation in the expression of different subtypes between different neuronal populations and in different regions of the central nervous system r''~4''4. The results of several studies have confirmed that the pharmacology of the GABA^ receptorchannel comp!ex is determined by the subunit composition, in parti~.ular, the ),2 subunit has been shown to be required for the coupling of benzodiazepinc binding with potentiation of ic;^u^ 2,1"~,22. Long-term exposure of mice to Iorazepam has been shown to cause a decrease in specific subunits of the GABA^ receptor in the cerebral cortex, specifically the otl and ),2 subunits 4. in addition, chronic exposure to ethanol, which is also believed to potentiate GAB^^ receptor function, alters the expression: of sere,el GABA^ receptor subunits in mouse brain ~a2. Further experiments will be needed to determine whether chronic exposure of cultured cerebral cortical neurons to benzodiazepine agonists in vitro causes a change in the

expression of GABA A receptor subunits in parallel with the loss of functional coupling between benzodiazepine binding and chloride channel activation. This work was supported by the Medical Research Council of Canada. Flurazepam-HCI was kindly supplied by Hoffmann-La Roche Ltd.

Buck, K.J., Hahner, L., Sikela, J. and Harris, R.A., Chronic ethanol treatment alters brain levels of gamma-aminobutyric acid^ receptor subunit mRNAs: relationship to genetic differences in ethanol withdrawal seizure severity, J. Neurochem., 57 (1991) 1452-1455. Burt, D.R. and Kamatchi, G.L., GABAA receptor subtypes: from pharmacology to molecular biology, FASEB J., 5 (1991) 29162923. Hamburger, V. and Hamilton, H.L., A series of normal stages in the development of the chick embryo, J. Morphol., 88 (1951) 49-92. Kang, I. and Miller, L.G., Decreased receptor subunit mRNA concentrations following chronic Iorazepam administration, Br. J. Pharmacol., 103 ( 199 ! ) ! 285-1287. Knoflach, F., Backus, K.H., Giller, T., Malherbe, P., Pflimlin, P., Mohler, H. and Trube, G., Pharmacological and electrophysiological properties of recombinant GABA A receptors comprising the a3,/31 and y2 subunits, Eur. J. Neurosci., 4 (1992) I-9. MacLennan,. A.J., Brecha, N., Khrestchatisky, M., Sternini, C., Tillakaratnc, N.J. K, Chiang, M.-Y., Anderson, K., Lai, M. and 'robin, AJ., Independent cellular and ontogenetic expression of mRNAs encoding three a polypeptides of the rat GABA^ receptor, Neuroscience, 43 (1991) 369-380. Miller, L.G., Greenblatt, DJ., Barnhill, J.G. and Shader, R.I., Chronic benzodiazepine administration, l. Tolerance is associ. ated with bcnzodiazcpine receptor downregulalion and decreased gamma-aminobutyric acid^ receptor function, I. Pharmacol. 'Exp. 77wr., 246 (1988) 170-176. Miller, L.G., Grcenblatt, DJ., Lopez, F., Schatzi, J.14., Lumpkin, M, and Shadcr, R.I., Chronic henzodiazepine administration: Effects in viw~ lind in vitro, in G, Biggio and E. Costa (Eds.), GABA and Benzodia=epine Receptor Subtypt,.~', Raven, N~w York,

199{), pp. 167-175. Miller, L.G., Roy, R.B. and Weill, C.L., Chronic clonazepam administration decreases GABA^ receptor function in cultured cortical neurons, Afol. Pharmacol., 3fi (19~0) 796-802. 10 Miller, L.G., Roy, R.B,, WeiU, C.L. and Lopez, F., Persistent alterations in GABA^ receptor binding and function after prenatal Iorazepam administration in the chick, Brain Res.Bull., 23 (1989) 171-174. Miller, L.G., WeiU, C.L., Roy, R.B. and Gaver, A., Lxlrazepam administration during embryonic development alters GABA^ receptor binding and function, Der,. Brain Re.v.,44 (1988) 241-246. Montpied, P., Morrow, A.L., Karanian, J.W., Ginns, E.I., Martin, B.M. and Paul, S.M., Prolonged ethanol inhalation decreases gamma-aminobutryic acid^ receptor alpha subunit mRNAs in the rat cerebral cortex, Mol. Pharmacol., 39 (1991) 157-163. Ngur, D.O., Rosenberg, H,C., and Chiu, T.H., Modulation of GABA-stimulated CI- flux by a benzo0iazepine agonist and an 'inverse agonist' after chronic flurazepam treatment, Eur. J. Pharmacol., 176 (1990) 351-356. 14 Persohn, E., Malherbe, P. and Richards, J,G., In situ hybridization histochemistry reveals a diversity of GABA^ receptor subunit mRNAs in neurons of the rat spinal cord and dorsal root ganglia, Neuroscience, 42 (1991) 497-507. 15 Puia, G., Vicini, S., Seeburg, P.H. and Costa, E., Influence of recombinant 7-aminobutyric acid-^ receptor subunit composition on the action of allosteric modulators of 7-aminobutyric acidgated CI- currents, Mol. Pharmacoi., 39 (1991) 691-696. 16 Reynolds, J.N. and Prasad, A., Ethanol enhances GABA^ receptor-activated chloride currents in chick cerebral cortical neurons, Brain Res., 564 (1991) 138-142.

331 17 Roca, D.J., Schiller, G., D, Friedman, L., Rozenberg, !., Gibbs, T.T. and Farb, D.H., GABA A receptor regulation in culture: altered allosteric interactions following prolonged exposure to henzodiazepines, barbiturates, and methylxanthines, Mol. Pharmacol., 37 (1990) 710-719. 18 Romijn, H.J., Van Huizen, F. and Wolters, P.S., Towards an improved serum-free chemically defined medium for long-term culturing of cerebral cortex tissues, Neurosci. Biobehav. Rev., 8 (1984) 301-334. 19 Schiller, G., and Farb, D.H., Enhancement of Benzodiazepine Binding by GABA is reduced rapidly during chronic exposure to Flurazepam, Ann. NYAcad. Sci., 435 (1986) 221-223. 20 Sher, P.K., Study, R.E., Mazzetta, J., Barker, J.L. and Nelson, P.G., Depression of benzodiazepine binding and diazepam potentiation of GABA-mediated inhibition after chronic exposure of spinal cord cultures to diazepam, Brain Res., 268 (1983) 171-176.

21 Shibla, D.B., Gardeil, M.A. and Neale, J.H,~ The insensitivity of developing benzodiazepine receptors to chronic treatment with diazepam, GABA and muscimol in brain cell cultures, Brab~ Res., 210 (1981) 471-474. 22 Sigel, E., Baur, R., Trube, G., Mohler, H. and Malherbe, P., The effect of subunit composition of rat brain GABA A receptors on channel function, Neuron, 5 (1990) 703-711. 23 Twyman, R.E., Rogers, C.J. and Macdonald, R.L., Differential regulation of ~,-aminobutyric acid receptor channels by diazepam and pentobarbital, Ann. Neuroi., 25 (1989) 213-220. 24 Zimprich, F., Zezula, J., Sieghart, W. and Lassmann, H., Immunohistochemicai localization of the al, a2 and or3 subunits of the GABA A receptor in the rat brain, Neurosci. Lett., 127 (1991) 125-128.

Uncoupling of GABA-benzodiazepine receptors in chick cerebral cortical neurons requires co-activation of both receptor sites.

Primary cultures of chick cerebral cortical neurons were exposed to 1 microM flurazepam in vitro, and the effect of flurazepam on GABA-activated membr...
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