Indian Journal of Expcrimcnt al Biology Vol. 4 1, January 2003, pp. 47-52

Involvement of GABA-A receptor chloride channel complex in isolation stress-induced free choice ethanol consumption in rats

Pramod K Mediratla*, Prabha Mahajan , Krishna K Sharma, Ri shabh Bhandari & Shilla P Dubey

Dcpartmcnt of Pharmacology, Univcrsity Collcgc of Mcdical Sc icnccs and GTI3 Hosp ital, Dclhi 110095 , India

Tcl: +9 1 II 25H297 I , Fax: +91 II 2590495, E-mail : dbmi @ucms.crnct.in

Received 28 May 2002; revised 6 September 2002

Thc prcscnt study rcvcal cd thc cfTcct of diazcpam, a hcnzodiazcpine, and progcstcronc, a pregnanc prccursor of IlCUI'OS­tcroids, which ac t via modul ating GABA-A chloridc channcl complcx on thc isolation strcss- induccd frcc choice ethanol consumption in adult rats. Isolation strcss for 24 hI' ovcr a period of 6 days produecd a signifi cant inereasc in eth anol eon­sUlllption , which pcrs istcd during thc 6-day recovcry peri od. Pretreating thc animals w ith diazepam (5 mg/kg, ip), or proges­teronc (5 mg/kg, ip), blocked the isolati on stress- induced increasc in ethanol consumption. l3icuculline (2 mg/kg, ip), a GAI3A-A reccptor antagonist significantl y allenuatcdthc effect of both diazcpam and progcstcrone on strcss- inducedmodu­lation of cthanol consumption. Isolation stress also caused an increasc in total nuid consumpti on, which was antagoni scd by both diazepam and progestcrone. Li ke ethanol consu mption, thi s effect of diazepam and progcsterone on isolation strcss­induced incrcasc in total fluid consumption was all cnuated by bicucu llinc. Ncither diazcpam nor progesteronc produced an incrcase in ethanol consumption in non-strcssed rat s. Ilowcver, unlike diazepam, progestcronc admin istl'ation to non­strcsscd rats causcd a significant incrcase in tota l fluid consumption. Results of the prcsent study thus show that GABAcrgic mcchan isms may be playing an important role in isolation stress- induced incrcase in cthanol consumption.

Several ep idemiologica l studi es have reported an as­soc iation between stress ful life events and high con­sumption of alcohol l

.3

. Research on stress and ethanol consumption shows a mixed but often positive rela-. h' 45 V . d . tl ons Ip ·. anous stressors are repone to IIlcrease

ethanol consumpt ion in ex peri mental an i mals6.x.

Iso lation stress has been shown to be one of the important factors, which determines the inclinati on to vo luntary alcohol intake. It was reported that level o f alcohol moti vati on directly corre lated with the degree of stress, and isolated rats drank significantl y more alcohol than con trol animalslJ

.1o

. In fact soc ial isola­

li on and loneliness may contri bute to alcoho l con­sumption in human be ings. Elevated anxiety levels might pred ispose to eth anol consumption while alco­hol consumption may reli eve fee lings of tension and anxiety in stressed sUbjects". In previous reports a relationship between voluntary ethanol intake and escape from the consequences of stress has been shown to ex ists.C!. This reduction o f tension by alcohol should reinforce drinking and contribute to the devel­opment of alcohol dependence ' 2.

*Address for correspondence: Dr. P K M ediralla, OOXII II , Kirti Apartmcnt s, M ayur Vihar, Phasc I Ex t. , Dclhii 110 09 I , India. Tc l: +91 112710070, E-mail: drpramod_k@yahoo.com

Benzod iazep ine (BZD) - GA BA-A receptor COI11-

pl ex has been implicated in the ph ys iolog ica l regu la­ti on of many stress respon ses. BZDs which act by modulating GAB A-A receptor-med iat ed chloride ion conductance have been shown to exert antianxiety and anti stress effects13

-15

• Progesterone which per se is devoid of binding to GABA-A receptor l 6

, is metabo­li sed to a\lopregnano lone and tetrahydrodeoxycorti­cos terone in the neuronal glia of the brain l7 and both these compounds are among the most potent of the known neurosteroids acti ve at GABA-A receptors

. I f'f"·· I BZD IX IY S . I WIt 1 a ' Inltles greater t nil s ' . ome 01 t lC

important effects of progesterone, such as anr istress, anx io lytic, ana lges ic, neuroprotecti ve, locomotor sup­pressive activity, sex ual recepti vity, neural control of circulation, etc are reported to be mediated via these

. d ?(l-?6 I h 1 I neurosterol s- - . n t e present stue y an attempt las been made to in ves ti gate the effect of diazepam, a BZD and progesterone, a pregnane precursor or nell­rosteroids, on the iso lation stress-induced free choice ethanol consumption in rats in order to delineate the role of GABA-A receptor chloride channel complex in this response.

Materials and Methods Allil/lals-The study was ca rried ou t in male W is­

tar rats 8-10 months old, we ighing between 375 and

48 INDI AN J EXP 810 1.., JANUA RY 2003

400 g. The animal s were housed 6/cage in polyvinyl cages ( 17x II x6 inches) in temperature (22°±rC) and humidity controlled conditions with 12 hI' li ght - 12 hI' dark cycle. The rats were allowed to accl i matize for one week before commencing the ex periment , so as to nulli fy any effect of stress du e to ex perimental group housing. The food (Pellet diet, Go lden Feeds, Delhi ) and drin ki ng solutions were availabl e ad Iibi/1I11 1.

Drinking s(JIII/ ions- Two so lutions were pre­sented to animals fo r drinking. One consisted of 0.2% saccharin solution in tap water and the other of 10% ethanol (v/v) in 0.2% saccharin solution. The solu­ti ons were fres hly prepared every day.

t..".\perilllen/ol grollps- Si xty rats were randoml y divided into 2 main groups, Group I and II. Each group was furth er di vided into 5 subgroups consisting of 6 ani ma ls each. The subgroups of group] were subjected to the foll owing treatment : group V­vehicle, Group D- diazepam (5 mg/kg, :p), group S­iso lation stressed rats inj ected with vehic le, Group SD - isolati on stressed rats inj ected with diazepam (5 mg/kg, ip ), Group SDB - isolati on stressed rats in­jected with di azepam (5 mg/k g, ip) + bicuculline me­thi od ide (2 mg/kg, ip). Similarl y, animals of Group II were treated with : Group V - vehi cle, Group P­proges terone (5 mg/kg, ip), Group S - iso lation stressed rats injected with vehicle, Group SP­isolat ion stressed rats inj ected with proges terone (5 mg/kg, i p), Gro up S PB - stressed rats inj ected with progesterone (5 mg/kg, ip) + bi cuculline methiodide (2 mg/kg, ip). Since the data of control (vehicl e) and st ressed rats were similar in the two groups, the va l­ues were poo led together for compari son with the drug-treated groups.

SII'ess procedllre- Fo ll owin g a 6-day base line pe­riod of group housing (6 rats/eage), the animal s in the stressed groups were subjected to 6 days of iso lation stress (tes t peri od). The isolation stress was induced by pl ac ing the animals alone I rat/cage ( 17x II x6 inc hes) in a novel environment of separate room. Stress pe ri od ranged fro m I to 7 hI' on any parti cular day follow ing a random unpredi ctable schedul e. The total durati on of exposure of iso lati on stress to each an imal was of 24 hI' over a peri od of 6 days . The non­stressed rats were exposed to continuous group hous­ing conditi on of the animal roo m during this period. The iso lation stress peri od was fo ll owed by a recov­ery peri od of 6 days. Consumption of both 10% etha­nol in saccharin so lution and saccharinated water were measured separately for baseline period , test period and recovery peri od.

Measurelllen/ of alcohol cOl1sumpliol1 - Sacchari­nated water sJ lution was presented in two bottles for one week of acc limati za tion peri od. Following thi s peri od one bottle of saccharinated water solution was replaced by the ethanol + saccharin solution for the entire duration of the study. The volume of solution remaining in each bottl e was recorded da ily between 9.00 and 10.00 AM. The bottles were then refill ed and the positi ons (I eft- ri ght) alternated to prevent the de­velopment of pos iti onal preference. Daily fluid con­sumption (both 10% ethanol and sacchari nated water) and daily ethanol intake (in terms of absolute alcohol ) in ml /kg/day were ca lculated6

.

Drugs- Diazepam (Ranbaxy , lndia), proges terone and bicuculline methi odid \:: (Sigma Chemical Co., US A) were used for the study. Diazepam injections containing benzy l alcohol USP NF 1.5% (w/v) were used. Progesterone was di spersed in 1 % Tween RO and diluted with saline. Drugs/vehicle were injected daily at 10.00 A M in a volume of 5 ITil /kg just before the initia tion of tes t (stress) period.

S/a/is/ical analysis- Mean 24 hr total fluid and eth anol consumption for eaeh group of animals was ca lculated in ml/kg body weight. Data were analysed by using Hi erarchical ANOV A and mult ipl e compari­sons were made by Tukey's test at 5% level of sig­nifi cance.

The ex perimental protocol was approved by the In­stituti onal Animal Ethi cs and Research Rev iew Committee. The care of the animals was as per the "Guidelines for the Care and Use of Animals in Sc ientifi c Research" prepared by the Ind ian National Science Academy, New Delhi .

Results Adult rats ex posed to isolation stress showed sig­

nifi cantl y increased ethanol consumpt ion as compared to non-stressed animals [3.88 ±0.49 (mean± SD) in vehicle- treated control Vs 7.3 1± 0.99 ml/kg/day in stressed animals or in terms of 10% ethanol solution 38.8 ±4.9 Vs 73.1 ± 9.9 mllkg/day l (P < O.OOI ; F=37.992). Further, stressed animal s exhibited a greater ethanol consumption during the test (stress) peri od (7 .3 1 ± 0.99 or 73 .1±9.9 ml/kg/day of 10% ethanol solution) when compared to base line (non­stressed) period (3.89 ± 0.49 or 38.9 ± 4.9 ml/kg/day of [0% ethanol solution) (P < O.OOI ; F=I I 1.603). This increase in ethanol consumpti on was observed to per­sist during the pos t- stress recovery peri od (Table I). Pretreati ng the animal s with diazepam (5 mg/k g, ip) or progesterone (5 mg/k g, ip) significantly attenuated

"

MEDIRATTA el af.: ROLE OF GABA-A RECEPTOR CO MPL EX IN ETHANOL CONSUMPTION 49

the effect of isolation stress on ethanol consumption. These effects of both di azepam and progesterone were blocked by bicuculline (2 mg/kg, ip ; Fig. I). Neither diazepam nor progesterone when administered to non­stressed rats were observed to modulate the free choice ethanol consumption (Table 1).

Total fluid consumption was significant ly in­creased in isolat ion stressed rats ( 102.68 ±8.3 1 in ve­hicle control V s 131.28 ± 6.5 mg/kg/day in stressed animals) (P < 0.00 I ; F=30.S06) (Table I ). This stress­induced increase in tota l fluid consumption was sig­nificantly blocked by diazepam as wel l as by proges­terone treatment. Further, like in case of ethanol con­sumption, effects of both diazepam and progestcrone were antagonized by bi cucullinc pretreatment (Fig. 2). However, unlike diazepam, progesterone admini stra­tion increased total fluid consumption cven in non­stressed rats (Table I ).

Discussion The results of the present study show that iso lation

stress produced a signifi cant increase in free eth anol consumption in adult rats. These results corroborate the findings of other workers who also observed an increase ethanol consumption in young and adult rats

h d d'ff f' I ' . 6-X?7 TI w en ex pose ' to I erent streSS'Ll SltLIatlons '- . l e increase in ethanol consumption was effecti ve ly blocked by diazepam, a BZD and progesterone which is metabol ized to neurosteroids, Ii kc allopregnanolone and tetrahydrodeox ycorti cos terone in thc neuronal glia of the brain 17. Both BZDs and neurostcroids have bcen shown to ex hibit antistress activitics and to at-

tenuate various effects of stress, including rI SC In I . I I 11- 1 S ?O ?X -,0 Th d I P asma corticosterone eve s · --_.- -. e a rena

corticosterone hypersec reti on produced by ACTH was reported to modulate stress-induced increase in etha­nol consumptionJ I

. For example, adrenalectomized rats fail ed to show an increase in ethanol consu lllrtion as compared to sham operated rats duri ng stress i n­duced by food restriction. Further, treatment of rats subjec ted to food restriction stress with cyanoketone, an inhi bitor of enzyme in volved in the stress-induced hypersecretion of adrenal corti costerone blocked the effect of stress on ethanol consumptionJ2 Hence, di­azepam and progesterone in the present study may be inhibiting iso lat ion stress-induced increase in eth anol consumption by modul ating cort icosterone seCl·crion. The observed increase in ethanol consumption seen during the stress (test) period was maintained during the post-stress (recovery) peri od when compared to non-stressed control animals. This could be due to a decrease in negati ve feed back sensiti v ity of hypotha­lamic-pituitary-ad renocortical (HPA) axis to gluco­corticoids due to persistentl y hi gh corti costerone lev­els during the stress period. Alternatively maintenance o f increased ethanol consumption during recovery period could be due to development of dependence to alcohol intake. Both diazepam and progesterone, when administered to non-stressed rats failed to modul ate eth anol consumption. Thi s observat ion is in agreement with the findings of Wolfe ef 0 / J3. who also reported that progesterone when administered to Myers High Ethanol Preferrin g rats did not ch ange ethanol consumption.

Tabl e 1- Erreci o r progeslerone. d iazepam and isolati on stress on ethano l and lola I f1uici consumption in rat s

I Values arc mean (±SD) 24 hI' co nsum pti on o r 10% e thanol so luti on (expressed in te rm s or absolute al cohol)/to tal fl uid by 6 rats over a 6-day period represented in Illllkg/day 1

Ethanol consumpt ion Treat Illent (mg/kg) Baseline period

V Vehicle (Control ) 3 .79 ± 0.44 P Progesterone (5) 3 .66 ± 0.49 D Diazepam (5) 4 .21 ± 0.50 S Stress 3.89 ± 0.49

Total flui d consumpti on

V Vehicle (Contro l) 100 59±7.86 P Proges terone (5) 10 1.27±7 .7 1 D Di azepam (5) 98.77± 10.16 S Stress 102. 7 1±8.44

jJ value .;. < 0 .00 I (Hierarchical ANOVA with Tukey's test). a. Compared to control (vehi cle) g roup b. Compared to progesterone/d iazepam trea ted group B. Compared to basel ine data or the respec tive g ruup.

Test pe ri od Recovery pe ri od

3.88 ± 0.49 3.88 ± 0.5 I 4 .05 ± 0.75 3.66 ± 0. 39 4 .42 + 0.56 4.48 + 0. 77 7.3 1 ± 0.99': 11,1) 7. 11 ± O.72· :o hll

F = 37.992

102.68+8.3 1 102.35+6 .39 123.16±4.05· :o1l 120.08±6.0 1' :01)

101.65+5.5 1 102.87+6.74 13 1.28±6.55· :Oh8 130.11± 930' abB

F=30.506

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I:ig. I - Effect of progesterone and diaze pam on et hano l consumpti on in stressed rat s (Va lucs arc mea n (±S D) 24 hnur consu mpti on of 10% et hanol sol ution by 6 rats over a 6 day period represe nt ed in mllkg/day) S = St ress: SP = Strcss + Progcsterone (5 mg/kg): S D = St ress + Dia/.epam (5 mg/kg): SPB = Stress + Progesterone + l3icucull i nc (2 mg/"g): SDB = St rcss + Diaze pam + l3 icuculline I) value * < 0.00 I (ll ierarchical ANOVA wit h Tukey's test) c. Compa rcd to stress group. d. Compared to st ress + progesterone/stress + diazepam grou p. 13. Compared to baselilll: data or thc n.:spec-tive group

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(V,liues arc mean (±S D) 24 hou r tot al fluid consumpti on by 6 rat s over a 6 day period represented in mllkg/day) S = Stress: SP = St ress + Progesterone.: (5 mg/kg) : SD = Stress + Diaze pam (5 mg/kg) : SPB = St ress + Progestcrone + Blcucull ine (2 mg/kg): SDB = Stress + Diazepam + Bicuc ulline fJ va lue * dl.OO I (Hi era rchi cal ANOV A with Tukey's test) c. CO l1lpared to stress group. d. CO l1l pared to stress + progestcroneh,tress + diazepam group. B. Co mparcd to baseli ne data of tile respec­ti ve group

MED IR ATTA 1'1 01.: ROLE OF GABA-A RECEPTOR COMPLEX IN ETHANOL CONSUMPTION 5 I

BZOs arc know n to produce their effec ts via modu­lati on of GABA-A chloridc channel co mpl ex' -l . Fur­ther, neurosteroids, like all opregnanolone and tetra­hydrodeoxycorticosteronc are among the most potent of the known ncuroste roids acti ve at GA BA-A recep­tors and produce a pos itive allosteri c modulati on of GABA evoked chl oride currents l 9

. Both BZOs and neurosteroids have been shown to attenuatc stress­induced increase in corti costerone levels 1.1.2X . Bes ides, exposure to corticosterone is reported to modul ate rat hippocampal GABA system's. Thus, diazepam, a BZO and progesterone, which is metaboli sed to allo-

I d . 17 pregnanolone and tetra lydro eoxycortlcosterone may be producin g thcir effect by modulatin g ce ntral BZO-GABA-A receptor chloride channel complex whi ch may in turn regul ate the release of neuroendo­cri ne hormones. This suggesti on gains furth er cre­dellCe from the observa ti ons of present study th at the inhi bitory effect of di azepam and progesterone on iso lati on stress-induced increase in ethanol co nsump­tion was signifi cantl y antagoni zed by bi cuculline, a GABA-A receptor antagoni st, im pli cating thereby the in vo lvement of GABA-A receptor in thi s stress re­sponse.

Besides eth anol, total fluid consumpti on was al so increased in strcssed animal s. Thi s change appears to be due to an associated increase in eth anol consum p­tion sincc, li ke ethanol intake, thi s increase in fluid consumption was attenu ated by both diazepam and progesterone. Further, like ethanol consumpti on, these effects of di azepam and progesterone were al so bloc ked by bicuculline. However, unlike di azepam, progesterone per se has increased total fluid co nsump­ti on in non-stressed animals. Although at thi s poin t it is diffi cult to exactl y ex plain thi s increase in total fluid consumption produced by progesterone in non­stressed rats, it could be related to its reported an­timineralocorti coid acti vity resulting in natriuresis'6 leading to activation of renin-angiotensin system which in turn would modulate thirst response'7.

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