Chronic diazepam treatment produces regionally specific changes in GABA-stimulated chloride influx

European Journal of Pharmacology, 159 (1989) 217-223 Elsevier

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Chronic diazepam treatment produces regionally specific changes in GABA-stimulated chloride influx R o d n e y J. Marley * and D o r o t h y W. Gallager Yale Unwerstty School of Medtclne, Department of Psychtatry, Abraham Rtblcoff Research Facthttes and Connecttcut Mental Health Center, 34 Park Street, New Haven, CT 06508. U S A

Received 27 September 1988. accepted 18 October 1988

GABA-stlmulated 36C1- reflux was used to investigate regional differences in response to chronic dmzepam treatment by c o m p a n n g cortical and cerebellar tissue from rats chronically treated w~th dlazepam for 3 weeks Using a treatment protocol which has prewously been shown to produce behavioral tolerance and physical dependence, cortical membrane preparations from chromc dmzepam-treated rats were found to exhibit a decreased responsiveness to the stimulation of 36C1- reflux by G A B A and a corrresponding decrease in the ablhty of flunltrazepam to enhance GABA-stimulated 36C1- influx This decrease in sensmvlty to flumtrazepam, however, appears to reflect the underlying decrease m sensmvity to G A B A In contrast, in membrane vesicles prepared from cerebella of chromc diazepam-treated rats, there was no measurable effect on GABA-st~mulated 36C1--lnflux or on the enhancement of GABA-sUmulated 36C1- influx by flumtrazepam These results support the suggestxon that there is a regaonally specific reduction m G A B A / b e n z o d i a z e p i n e receptor function following chronic benzodiazepme treatment

GABA (y-armnobutync acid), Benzodmzeplnes, Dlazepam (chronic), Chloride influx, Tolerance

1. I n t r o d u c t i o n

Benzodmzeplnes exert their pharmacological effects by binding to recognition sites on the 3'armnobutyrlc aod (GABA) receptor complex (Haefely et al, 1983, Tallman and Gallager, 1985) The binding of benzodlazepme agomsts results m an increase m the frequency of opemng of GABA-dependent chloride channels, generally facilitating the mhihitory actions of GABA (Barker and Owen, 1986) Chnlcal and behavioral studies have shown that tolerance develops to many of the therapeutic actions of benzodlazepmes following chromc exposure Following prolonged exposure

* To whom all correspondence should be addressed Yale School of Medicine, Department of Psyctuatry, 34 Park St, New Haven, CT 06508, U S A

to benzodmzeplnes, decreases m sensitivity to the sedative (Lister et al, 1983, Rosenberg and Chlu, 1985), muscle relaxant (Rosenberg and Chiu, 1981, Ryan and Bolsse, 1983), antlconvulsant (Garratt et al, 1988, Gonsalves and Gallager, 1987) and anxaolytlc (Davis and Gallager, 1988, File, 1985, Stephens and Schneider, 1985) properties of benzodlazepines have been observed While the exact mechanism responsible for the development of tolerance to benzodiazepmes ~s unknown, It does not appear that tolerance to benzodiazepmes can be accounted for solely by changes m pharmacokinetic parameters (Gonsalves and Gallager, 1987, Greenblatt and Shader, 1986, Halgh et al., 1986) Rather, the evidence suggests that the development of tolerance to benzodlazeplnes is assoctated with alterations in the GABA/benzodlazepme receptor complex Early studies from our laboratory suggested that tolerance to benzodlazepmes could

0014-2999/89/$03 50 © 1989 Elsevier Science Pubhshers B V (Biomedical Dlvaslon)

218 be correlated with a functional subsensltlvlty to GABA rather than a change in benzodiazeplne binding site parameters (Gallager et al, 1984) Rats exposed to the benzodaazeplne, dlazepam, for 2 weeks show a decreased responsiveness of dorsal raphe neurons to iontophoretically applied G A B A and a loss of the ability of benzodiazeplnes to enhance sensitivity to GABA in this region Furthermore, tolerance to the antlconvulsant effects of diazepam against blcuculhne-induced seizures is temporally related to these electrophyslologlcal changes (Gonsalves and Gallager, 1987) More recent studies (Wilson and Gallager, 1987), however, have found major differences between brain regions in the effects of chronic diazepam treatment In the substantia nlgra, chronic dlazepam treatment failed to alter the sensitivity of retlculata neurons to G A B A and the enhancement of GABA sensitivity observed following acute dlazepam exposure, was not altered by chronic benzodiazeplne exposure in this region (Gallager et al, in press) Thus, the data from these two brain areas indicate that there are regional differences in response to chronic benzodlazeplne exposure. To investigate regional differences in response to chronic dlazepam exposure further, we have measured GABA-stlmulated 36C1 influx into cortical and cerebellar membrane vesicles prepared from rats chronically treated with diazepam or vehicle Benzodiazepine hgands have been shown to modulate GABA-stlmulated 36C1- influx (Lehoulller and Tlcku, 1987, Morrow and Paul, 1988, Obata and Yamamura, 1986) In our preparations from chronically treated rats, we also evaluated the modulation of GABA-stimulated ~6C1 influx by the benzodlazeplne, flunltrazepam

2. Materials and methods

Adult male Sprague-Dawley rats (Charles River. Kingston. NY) were housed in groups of three to five in a temperature-controlled environment with food and water available ad hbitum and maintained on a 12 12 hght-dark cycle Chronic dlazepam exposure, using the sllastic implant method (Gallager et al, 1985). began 4 days after the animals (150-200 g) arrived Rats were anesthe-

tlzed with halothane and two 60 mm sllastlc capsules filled with 90 mg of crystalline dlazepam-HC1 (recrystalhzed in ethanol, a gift from Dr Peter Sorter. Hoffman-LaRoche. Nutley, N J) were implanted s c through a small incision in the back Rats received one additional capsule 10 days later This treatment protocol maintains essentially constant dlazepam levels of 250 n g / g brain tissue over the entire 3-week treatment period Control animals received onljy empty capsules Levels of residual dlazepam in the membrane vesicle preparations from chronically treated rats were evaluated using radloreceptor assay techniques (Gallager et al, 1985) The procedures employed for the preparation of membrane vesicles (mlcrosacs) and for the measurement of GABA-stlmulated 36C1- influx were modifications of those outlined previously (Harris and Allan, 1985) Following decapitation, the brains were removed, placed on ice and the cerebral cortex and cerebellum dissected out Each assay required the use of one cortical hemisphere and two cerebella (pooled from two identically treated rats) The tissue was homogenized by hand (10-12 strokes) in 70 volumes of ice-cold assay buffer (mM 145 NaC1, 5 KC1, 1 MgCI 2, 10 D-glucose, 1 CaCl 2 and 10 HEPES, pH 7 5) using a glass-Teflon homogenizer and centrifuged at 1000 × g for 15 mln at 4 ° C The resulting pellet was resuspended in 70 volumes of ice-cold buffer and again centrifuged at 1000 × g for 15 mln The final pellet was resuspended in assay buffer to a final concentration of 6-8 mg protein per ml Initial characterization of GABA-stimulated ~6C1influx found influx to be linear from 1-8 m g / m l protein However, the enhancement of GABAstimulated ~6C1- influx by benzodlazeplnes, was found to be optimal at 6-8 m g / m l protein Aliquots (200/xl) of the membrane vesicle suspensions were prelncubated in a shaking water bath for 10 mln at 3 0 ° C 36C1 uptake was then initiated by the addition and immediate vortexlng of 200 ~tl of a solution containing ~6C1- (1 6/~C1/ml of assay buffer) and increasing concentrations of G A B A (0-300/~M) Evaluation of the modulation of 3 /~M GABA-stlmulated ~6C1 influx by flunltrazepam, was accomplished in a similar manner, except that the membrane vesicle ahquots

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were premcubated in the presence of 10-1°-10 -5 M flumtrazepam. GABA, 3/~M, was chosen based on p r e h m m a r y observations that GABA-stimulated 36C1- influx was optimally enhanced at tlus concentration A previous report (Morrow and Paul, 1988) has noted that benzodiazepmes enhance GABA-stimulated 36C1- influx in filtered membrane vesicle preparations in a concentration dependent manner only at low (1-10 /~M) concentratlons of G A B A Three seconds after the addition of the 36C1- solution, influx was terminated by the addition of ice-cold buffer (4 ml × 2) containing 1 m M plcrotoxln, followed by immediate and rapid filtration under vacuum (10 inches Hg) onto a 2 4 cm W h a t m a n G F / C glass rmcrofibre filter, using a Hoefer manifold (Hoefer Scientific, San Franslsco, CA) The filters were further washed with 8 ml of cold assay buffer contaxmng 1 m M plcrotoxm with the manifold towers removed. The addition of plcrotoxan to the wash solutions was found to greatly reduce replicate variability Following filtration and washmg, the filters were dried in a 30 ° C oven, placed in 10 ml scintillation fluid, and the amount of 36C1taken up by the vesicles deternuned using l i q u i d scintillation s p e c t r o p h o t o m e t r l c t e c h n i q u e s GABA-stlmulated 36C1- uptake (nmol chloride/

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mg protein per 3 s) was calculated as the difference between basal uptake in the absence of G A B A and total uptake in the presence of G A B A Protein concentrations were deternuned by the method of Lowry et al. (1951). Analysis of vanance techniques were used to evaluate differences between the two chromcally treated groups in the two brain regions Where significant differences were observed between the dose-response curves, the two treatment groups were further compared at each concentration of drug using post-hoc Student's t-tests

3. Results The mean results + S E. from four to six assays of GABA-stlmulated 36C1- uptake in cortical and cerebellar tissue preparations from chromc dlazepam- and chronic vehicle-treated rats are illustrated in fig 1 Baseline 36C1- influx did not differ between the two groups In tissue from either region In the absence of added GABA, the mean v a l u e s + S E . were 1 5 4 _ + 0 5 and 161 + 0 3 n m o l / m g protein per 3 s for cortical tissue from the chronic dlazepam and chronic vehicle ammals, respectively (t (8) = 0.10, n s ) In cerebellar tissue,

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Fzg l GABA-stlmulated 36C1 influx in cortical (left panel) and cerebellar (right panel) membrane preparations from rats chromcally treated for 3 weeks with sdasUc implants contatmng either crystalline dlazepam or vehicle Influx is expressed as total chloride accumulation (36C1- plus unlabeled C1- ) per mg protein/3 s Each point represents the mean+ S E from 4-6 assays * P < 0 05 vs velucle

220 crosacs p r e p a r e d f r o m c e r e b e l l a r tissue were virtually i d e n t i c a l (F(1,74) = 1.2, n s ). F i g u r e 2 illustrates the m e a n results + S E from five to six assays of f l u n l t r a z e p a m e n h a n c e m e n t of 3 /~M G A B A s t i m u l a t e d 36C1- influx In cortical a n d c e r e b e l l a r m e m b r a n e p r e p a r a t i o n s In b o t h regions, f l u n l t r a z e p a m e n h a n c e d G A B A - s t i m u l a t e d 36C1- influx in a d o s e - d e p e n d e n t m a n n e r (F(6,65) = 27 7, P < 0 0001 a n d F(6,66) = 19 6, P < 0 0001, for c o r t e x a n d cerebellum, respectively). I n cortical m e m b r a n e p r e p a r a t i o n s , c h r o n i c dia z e p a m t r e a t m e n t r e s u l t e d in a d e c r e a s e d sensitivity to the m o d u l a t o r y effects of f l u m t r a z e p a m u p o n G A B A - s t l m u l a t e d 36C1- influx ( F ( 1 , 6 5 ) = 8 0, P < 0 01) H o w e v e r , as d e s c r i b e d above, in the a b s e n c e of f l u m t r a z e p a m , 3/~M G A B A s t i m u l a t e d 36C1- Influx to a lesser degree in cortical tissue f r o m c h r o n l c - d i a z e p a m t r e a t e d rats ( t ( 9 ) = 1 85, P < 0 05). I n fact, the slopes of the d o s e r e s p o n s e curves f r o m vehicle- a n d d l a z e p a m - t r e a t e d tissue in this region were i d e n t i c a l as i n d i c a t e d b y the l a c k of a significant i n t e r a c t i o n b e t w e e n the m a i n effects of c h r o n i c d l a z e p a m t r e a t m e n t a n d the c o n c e n t r a t i o n of f l u m t r a z e p a m ( F ( 6 6 5 ) = 0 . 1 6 , n.s.) This w o u l d suggest that the o b s e r v e d diff e r e n c e s in f l u n l t r a z e p a m e n h a n c e m e n t of GABA-stlmulated 36C1- influx are the result of

the c o r r e s p o n d i n g b a s e h n e values were 20.0 ___1 0 a n d 20.1 + 0.6 n m o l / m g p r o t e i n p e r 3 s ( t ( 8 ) = 0 09, n s ) P r e l n c u b a t l o n of cortical a n d cerebellar macrosacs from c h r o n i c d l a z e p a m a n d c h r o m c vehicle t r e a t m e n t g r o u p s with the G A B A a n t a g o nist, S R 95531 ( H e a u l m e et a l , 1987), fmled to decrease b a s e l i n e 36C1- influx. T h e s e d a t a suggest that the differences in 36C1 influx o b s e r v e d in our s t u d y were n o t d u e to differences in the levels of e n d o g e n o u s G A B A in the m e m b r a n e p r e p a r a tions. In a d d i t i o n , n o d e t e c t a b l e a m o u n t s ( < 3 p m o l ) of residual d l a z e p a m c o u l d b e m e a s u r e d in mlcrosac preparations from chromc dlazepamtreated ammals In b o t h cortex a n d cerebellum, G A B A stimul a t e d a6C1- influx In a d o s e - d e p e n d e n t m a n n e r (F(8,72) = 321 6, P < 0 0001, F(8,72) = 41 6, P < 0.0001 for cortex a n d cerebellum, respectively) I n s p e c t i o n of fig 1 indicates that in rmcrosacs p r e p a r e d f r o m cortex, c h r o m c d i a z e p a m t r e a t m e n t resulted in a d e c r e a s e d sensitivity to G A B A as m e a s u r e d b y G A B A - s t l m u l a t e d 36C1- Influx S l g m f i c a n t differences in G A B A - s t l m u l a t e d 36Elinflux b e t w e e n c h r o m c d l a z e p a m a n d c o n t r o l t r e a t m e n t groups were f o u n d in cortical tissue (F(1,72) = 28.2, P < 0.0001) T h e d o s e - r e s p o n s e curves for G A B A - s t l m u l a t e d 36C1- Influx in ml-

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Fig 2 Enhancement of 3 FM GABA-stlmulated 36C1 influx by increasing concentrations of flumtrazepam Data are shown for cortical (left panel) and cerebellar (nght panel) membrane preparations from rats chromcally treated for 3 weeks with sdasuc implants contalmng either crystalhne dtazepam or vehxcle Influx is expressed as total chloride accumulation (36C!- plus unlabeled C1- ) per mg protein/3 s Each point represents the mean + S E from 5-6 assays * P < 0 05 vs vehicle

221 baseline differences in the ablhty of G A B A to stimulate 36C1- reflux in cortex from the chromc dlazepam treatment group. In cerebellar preparations, chronic dlazepam treatment had no effect on rather 3 /~M GABA-stimulated 36C1- Influx (t(8) = 0.1, n s.) or on the modulation of G A B A stimulated 36C1- Influx by flumtrazepam (F(1,66) =03, ns)

4. Discussion

The demonstration of tolerance to benzodiazepines using the rat as a model is complicated by the rapid metabolism of diazepam in this species. The resulting inablhty to maintain constant receptor occupancy has made it difficult to demonstrate tolerance to m a n y of the effects of benzodiazeplnes in this species. However, in our studies of tolerance to benzodlazeplnes, we have employed a sustained release preparation conSlSting of sealed silastlc tubing contalmng crystalline diazepam, implanted s.c in the back of the rats. This method allows for the slow, but continuous, release of diazepam and the maintenance of therapeutically relevant (250 n g / g ) tissue concentratlons of dlazepam in brain. Using this method, long-term exposure to benzodlazeplnes has been shown to result in the development of tolerance to the anticonvulsant, sedative and antifear properties of diazepam (Davis and Gallager, 1988). Although some laboratories have reported changes in benzodlazepme binding parameters following various chromc benzodlazepme regimens (Crawley et al., 1982; Miller et al., 1988, Rosenberg and Ctuu, 1981; Szczawlnska et al., 1988), previous biochemical characterizations from our laboratory have indicated that chromc diazepam treatment, using a sustained-release method, does not directly affect either the number of affinity of benzodlazeplne receptors in the cerebral cortex (Gallager et al, 1985) Using [3H]blcuculhne/ thiocyanate binding to evaluate low-affinity G A B A binding sites in cerebral cortex from chromc dlazepam-treated rats, an increase in the apparent affimty of G A B A for its low-affimty (~M) binding site was observed (Gallager et al.,

1985) The low affinity state of the G A B A receptor is believed to represent the active form of the chloride channel-linked G A B A receptor (Olsen et al., 1984) This would suggest that chronic dlazepam treatment results in a shift of the G A B A receptor to a high-affinity, perhaps desensmzed, state In the present study, the ablhty of G A B A to stimulate the influx of 36C1- was reduced in cortical m e m b r a n e preparations from rats treated chromcally with dlazepam. These results are m agreement with those of Miller et al. (1988) who found that chromc lorazepam treatment, using a sustained release preparation, also results in a decrease in muscxmol-stlmulated chloride uptake in cortical synaptoneurosome preparations. Thus the subsensltlVlty to GABA-stlmulated 36C1- Influx observed in cortical preparations from chromc dlazepam-treated animals and to musclmolstimulated 36C1- influx in cortical preparations from chromc lorazepam-treated ammals would seem to reflect a decrease in the functional responSlvlty of G A B A receptors. In contrast to the results from our studies and from those of Miller et al. (1988), Yu et al. (1988) have reported that in the rat, the long-term adrmmstratlon of flurazep a m in drinking water results in an increase in GABA-stlmulated 36C1- uptake and a reduction in the modulation of GABA-sumulated 36C1- uptake by benzodlazeplnes There are several possible reasons for these discrepancies, including differences in the pharmacoklnetlc characteristics of flurazepam (Miller et a l , 1988), lorazepam (Greenblatt et al., 1983) and dlazepam (Friedman et al, 1986), differences in the methods of chromc drug administration, and differences in the methodologms employed to evaluate GABA-stlmulated

36C1- influx. Chronic dlazepam treatment has prevmusly been shown to decrease the ablhty of G A B A to enhance benzodiazepine agomst binding (Gallager et a l , 1984) and to decrease inverse agomst bindlng in cortical membranes (Wilson et al., 1987). It was originally suggested that chromc dlazepam treatment resulted in an alteration in the nature of allostenc couphng between G A B A and benzodlazeplne binding sites The apparent decrease in the degree of allosterlc modulation of benzodlazepine receptor agomst and inverse agomst bind-

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ing, however, could also be reflective of a decrease in the sensitivity of G A B A receptors The modulation of benzodlazepine binding by G A B A is also associated with the low affinity state of the G A B A receptor (Browner et al, 1981). Since chronic diazepam treatment appears to produce a decrease m the number of low affinity, functional G A B A receptors (Gallager et al, 1985), it is possible that the apparent decrease in allosterlc coupling is due to decreased G A B A receptor sensitivity In our studies using cortical membrane preparauons, chronic dmzepam treatment also resulted in a decreased sensitivity to the modulatory effects of flunItrazepam upon GABA-stimulated 36C1- Inf l u x However, the parallel slopes of the dose response curves in fins region provide evidence that this difference may reflect underlying differences in the ability of GABA to stimulate 36C1 influx Our findings that chronic diazepam treatment has no effect on GABA-stlmulated 36C1- influx In cerebellum and no effect on the enhancement of GABA-stimulated 36C1- Influx by flunitrazepam would predict that the changes in the G A B A / benzodiazepIne receptor complex observed m cortical preparations may not occur in cerebellum The binding characteristics of cerebellar G A B A receptors have not yet been specifically evaluated m chronic diazepam-treated rats However, thts data is conslstant with a lack of binding stte differences in cerebellum following chronic lorazepam exposure (Miller et al, 1988) Further evidence for a lack of changes in G A B A / benzodiazepme receptor complex m cerebellum ts provided by the observation that in cerebellar Purkmje cells, sensitivity to iontophoretically applied G A B A is not decreased by chronic benzo&azeplne exposure (Waterhouse et al., 1984). Such data are consistant with electrophyslolog~cal evtdence for regional differences in G A B A sensitivity following chronic diazepam exposure (Gallager and Wtlson, 1988) Thus, the results from the studies presented here indicate that chronic dlazepam treatment produces a functional subsensltiVlty to G A B A in cortical, but not cerebellar tissue when evaluated using established protocols for measuring the modulation of GABA-stimulated 36C1- influx by

benzodlazepmes (Obata and Yamamura, 1986, Morrow and Paul, 1988) These data provide further evidence for regionally spectftc changes occurnng at the G A B A receptor complex following chronic benzodlazeplne exposure

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