Possible Mechanism of Anticonvulsant Effect of Ketamine in Mice
Indian Jo urnal of Experimental Biology Vol. 39, October 200 I, pp. I 002-1008
Possible mechanism of anticonvulsant effect of ketamine in mice
Anshu Manocha*, Krishna Kumar Sharma & Pramod Kumari Mediratta Department of Pharmacology, University Coll ege of Medical Sciences & GTB Hospital, Shahdara, Delhi 110095 Received 8 September 2000; re vised 25 May 2001
The study was designed to investi gate the effect of ketamine on convulsive behaviour using maximal electroshock (MES) test. An attempt was also made to study the possible receptor mechanisms involved. MES seizures were induced in mice via transauricular electrodes (60mA, 0.2sec). Seizure severity was assessed by the duration of tonic hindlimb extensor phase and mortality due to convulsions. fntraperitoneal administration of ketamine produced a dose-dependent (5-50mg/kg) protection against hindlimb extensor phase. The anticonvulsant effect of ketamine was antagoni zed neither by naloxone (low as wel l as high doses) nor sulpiride, but was attenuated by haloperidol, a dopamine (D 2)/sigma receptor antagoni st. Co administration of y-aminobutyric acid (GABA)-ergic drugs (GABA, muscimo l, di?.zepam and baclofen) and N-meth yi-D aspartate (NMDA) receptor antagonist, dizocilpine (MK801) with ketamine facilitated the anticonvulsant action of the latter drug. In contrast, flumazenil, a benzodiazepine (BZD)-GABAA receptor antagonist, reversed the facilitatory effect of diaze pam on the anti-MES effect of ketamine. Similarly, 8-amin ovaleric acid (DA VA), antagonized the facilitatory effect of ba clofen on anti-MES action of ketamine. These BZD-GABAergic antagonists, flumazenil or DA VA per se al so attenuated th e anti-MES effect of ketamine given alone. The results suggest that besides its known antagoni stic effect on NMDA channel, other neurotransmitter systems i.e. sigma, GABAA-BZD-chloride chann el complex and GABA8 receptors may also be in vo lved in the anti-MES action of ketamine.
15 18 Ketamine, a dissociative anaesthetic and a known nergic and serotonergic neurotransmission - . These sigma receptor preferring ligand is reported to act as effects could be primarily due to its actions on the 15 18 an antagonist of N-methyl-0-aspartate (NMDA) NMDA receptors - • Additionally, y-aminobutyric mediated transmission by blocking the phencyclidine acid (GABA)ergic transmission can also be affected 1 11 (PCP) site at the NMDA receptor complex • In some by ketamine either directly or indirectli' ; however, studies, ketamine (5-40mg/kg) was found to be anti this contention needs further exploration. In light of convul sant against generalized tonic-clonic seizures these developments focussing the role of ketamine in (GTCS) induced by mercaptopropionic acid, pentyle seizures and to reconcile the rel ated divergent reports, 2 netet:-azole (PTZ), bicuculline and picrotoxin in rats · the present study has been designed to investigate its 5 . Ketamine also di splayed anticonvulsant action on pro- or anticonvulsant action using maximal electro seizures due to cortical application of penicil lin in shock (MES) seizure test, an animal analogue model rabbits and abolished the tonic phase of the seizures for grandma! epilepsy. An attempt has also been made evoked by sudden cooling of toad isolated spinal to determine the possible receptor mechanisms in 6 7 cords · . Further, ketamine suppressed lithium volved. pilocarpine- and ethacrynic acid-induced status epi 8 9 lepticus in rodents · . On the other hand, in some Materials and Methods studies ketamine (2.5-30mg/kg) has been found to be Animals-Albino Swiss mice of either sex (20- proconvulsant, since it activated epileptiform dis 25g) (procured from Central Animal Breeding House, charges in the neocortical cobalt generated focus or AIIMS, Delhi) were used. The animals were housed . . I 'l . 10 II B h . d cort1coret1cu ar e p1 epsy 111 cats · . ot ant1- an in plastic cages at an ambient temperature (25±2°C) proconvulsant effects of ketamine (1-13 mg/kg) have and 45-55% RH and maintained on a 12: 12hr light also been reported in mentally retarded human epi- . 12 dark (0700-1900 hrs) cycle. Food and water were 1eptiCS . provided ad libitum and animals were acclimatised to 'd . d' . NMDA I 13 14 B est es 1ts 1rect act1on on receptor · · , their environment for atleast one week before experi ketamine affects dopaminergic, n01·adrenergic, choli- mentation. The animals were randomly divided into different groups. *E- 99, Preet Vihar Delhi 110092, India. Each experimental group consisted of a minimum Phone 22 14073(R) of 10 animals. Each animal was housed separately MANOCHA et al.: ANTICONVULSANT EFFECT OF KETAMINE 1003 after recording its body weight and was randomized elicited complete tonic hindlimb extension (THE) in to receive the treatments according to a random num control mice. For recording various parameters, mice ber table. They had identification marks identifying were placed in a clear rectangular plastic cage with an the dose level group and individual number. All of the open top, permitting full view of the animals' motor experimental protocols were approved by the institu responses to seizure. In the pilot study, various phases tional review committee and experiments were con of convulsions, viz. tonic flexion, extension, clonus, ducted in accordance with the guidelines for the care stupor and mortality due to convulsions were timed. and use of laboratory animals as adopted by the Na To evaluate the drug effect on the seizure severity, the tional Institutes of Health (NIH). All the experiments duration of THE and mortality due to convulsions. were performed between 1000 and 1800 hrs. were selected as the parameters. Each animal was in Drugs-Ketamine hydrochloride (Sigma, USA), dividually observed for 2hr after MES seizures, and, naloxone hydrochloride (Sigma, USA), haloperidol at 24hr for mortality. A compound is known to pos (Sigma, USA), sulpiride (Sigma, USA), diazepam sess anticonvulsant property if it reduces or abolishes (Ranbaxy, Delhi, India), y-aminobutyric acid (GABA) the extensor phase of MES. (BDH, Poole, Dorset, UK), muscimol (Sigma, USA), Treatment schedule of ketamine, naloxone, halo baclofen (Ciba-Geigy, Switzerland), flumazenil peridol and sulpiride in MES test-Data were ob (F. Hoffmann La Roche, Basel, Switzerland), 8-amino tained on dose-response relationship of ketamine on valerie acid (DAVA) (Sigma, USA) and (+)-5- MES seizures. To this end, different groups of mice methyl-1 0, 11-dihydro-5H-dibenzo[a,d] cyclohepten- received a single ip injection of the compound (5 - 5,10-imine (dizocilpine) (Merck Sharp and Dohme, 50mg/kg) and after 30 min, animals were subjected to West Point, PA) were used. The drugs were used as MES seizure and duration of THE phase and mortal their respective salts. Doses of the drug are reported ity due to convulsions were recorded. Effect on gen as the actual amount of drug administered after cor eral behaviour of mice was also recorded. For nalox 20 rection for the salt content. Ketamine hydrochloride, one studies , after pretreating the mice with 50mg/kg naloxone hydrochloride, GABA, muscimol, baclofen, of ketamine in separate groups, at 20 min, a dose of DAVA and dizocilpine (MK801) were dissolved in naloxone (0.1 and 1mg/kg) was administered; the distilled water. Haloperidol was dissolved in a few animals' were then subjected to MES seizure 10 min 21 drops of 0.1 N lactic acid and the volume was ad later. For the haloperidol and sulpiride studies , mice justed with distilled water. Sulpiride was dissolved in were pretreated sc with haloperidol (0.5-2mg/kg), a distilled water with a few drops of 0.1 N hydrochloric dopamine (D2) and sigma receptor antagonist or sul acid. Diazepam injection (Calmpose®) was diluted to piride (20mg/kg), a D2 selective antagonist in separate the required volume with distilled water before use. groups and at 30 min, a dose of ketamine (50mg/kg) Flumazenil was uniformly suspended in distilled wa was administered. The animals were then subjected to ter with a few drops of tween 80. Ketamine, naloxone MES seizure 30 min later. Haloperidol and sulpiride and DAVA were administered intraperitoneally (ip) were injected 1hr prior to MES as preliminary data 2 1 22 while haloperidol, sulpiride, diazepam, GABA, mus and literature reports ' indicated that this time pe cimol, baclofen, flumazenil and MK801 were injected riod was sufficient to allow for maximum or near subcutaneously (sc) in the scruff of the animals. In maximum drug effect. The per se effects of naloxone, jection volume (1 Omllkg) was kept constant. The se haloperidol and sulpiride were also studied. lection of doses, route of administration and time Drug -interaction studies schedule of different compounds was based on pilot Interaction between GABAA, and benzodiazepine experiments and pharmacokinetic considerations. Ap receptor ligands and ketamine-GABAA receptor propriate vehicle controls were employed in all ex agonists, GABA (100 and 200 mg/kg, sc) and musci periments. For each series of experiments, separate mol (0.5 and 1mg/kg, sc) were studied alone and/or in controls were used in order to avoid variation in the combination with ketamine (5mg/kg, ip). These data from day to day. agents were administered 30 min prior to exposure to MES-induced convulsions-MES seizures 19 were MES. In combination study they were administered induced by an electroconvulsometer (Techno Instru concurrently. Benzodiazepine agonists, diazepam (2.5 ments, Lucknow). A 60mA current was delivered mg/kg sc, 30 min), and antagonist flumazenil (0.5 transauricularly for 0.2 sec in mice via small alligator mg/kg sc, 5 min) were administered alone and/or in clips attached to each pinna. This current intensity combination with ketamine. 1004 INDIAN J EXP BIOL, OCTOBER 2001
Interaction between GABA8 receptor ligands and tered with ketamine, these GABAergic drugs in their ketamine-GABA8 receptor agonist, baclofen (2.5 respective doses significantly enhanced (?<0.001) the and 5 mg/kg, sc) and antagonist DAY A (50mg/kg, ip) protective effect of ketamine against MES seizures. were studied alone and/or in combination with keta Flumazenil (0.5 mg/kg) when given along with keta mine (5mg/kg, ip). These agents were administered mine, significantly (P < 0.001) attenuated the anti 25-30 min prior toMES. MES effect of the latter drug (Table 1). In animals Interaction between glutamatergic ligands and ke previously treated with diazepam (2.5 mg/kg) and tamine-The effect of NMDA receptor antagoni st ketamine, when treated with flumazenil, there was a MK 801 (0.1 mg/kg, sc) was studied alone and/or in reversal of faci litatory effect of diazepam on the pro combin ation with ketamine (5mg/kg, ip). Drugs were tective effect of ketamine. However, the mortality was administered 30 min prior to MES. In case of combi not significantly altered in all the above groups (Table 1). nation study they were administered concurrently. Interaction between GABA 8 receptor ligands and Statistical analysis-The duration of THE phase ketamine-Baclofen (2.5 and 5 mg/kg) per se sig of MES convulsions expressed as the arithmetic mean nificantly (P<0.001) decreased the duration of THE ± SE was analysed by one-way analysis of variance phase. Although it also reduced the mortality inci 23 24 (ANOVA) followed by Dunnett's 't' test • . A dences (2/10,0/10) when compared to vehicle-treated grouped Chi-square test was initially used to deter control (4/1 0), the effect was not statistically signifi mine the overall differences in the mortality due to cant. DA VA, a GABA8 receptor blocker (50 mg/kg) convulsions. If a sign ificant effect was found, indi did not produce any effect per se, but it significantly vidual differences were determined by using a single (P<0.001) reversed the anti-MES effect of baclofen Chi-square test. P<0.05 was taken as a statistically and increased the mortality incidence (1110) [Table 2]. significant difference. When baclofen (2.5 and 5 mg/kg) was administered with ketamine (5mg/kg), it enhanced (P < 0.001) the Results protective effect of ketamine (Table 2) while DA VA Effect of ketamine-The lower doses of ketamine (50 mg/kg) attenuated the anticonvulsant effect of (5-lOmg/kg) produced mild sedation without affecting ketamine (?<0.001). In animals pretreated with both their gait /cage activity when compared to vehicle baclofen (5 mg/kg) and ketamine, DA VA reversed the treated control animals. However, ketamine in higher facilitatory action of baclofen on the anti-MES effect doses (25-50 mg/kg) produced motor incoordination of ketamine. However, the mortality was not signifi as observed with staggered gait whenever the animals cantly altered in all these groups (Table 2). moved. Interaction between glutamatergic ligands and ke MES induced tonic convul sions in all the animals. tamine-MK 801 (0.1 mg/kg) per se offered a signifi The end-point i.e. THE in vehicle treated control was cant protection (P Table 1-Effect of benzodiazepine-GABAA receptor acting drugs per se and on the anticonvulsant activity of ketamine (KT) in max imal electroshock (MES)-induced convulsions in mice Group Treatment Duration of hindlimb extensor phase( sec) Mortality## (mg/kg,ip or sc) (Mean± SE) # I. Vehicle 16.00 ± 0.2 1 3/10 2. DZP (2.5)* 3.60 ± 0.43** 0110 3. FLM (0.5)* 15.80 ± 0.25 3/10 4. DZP (2.5)* + FLM (0.5)* 12.40 ± 0.31 ** 1110 5. GABA (100)* 12.50 ± 0.27** 0/10 6. GABA (200)* 8.10±0.18** 0/10 7. MUS (0.5)* 10.90 ± 0.31 ** OliO 8. MUS (1)* 5.00 ± 0.21 ** 0/10 9. KT (5) 10.90 ± 0.55** 2/10 10. KT(5) + DZP (2.5)* 2.60 ± 0.60 ** OliO 11. KT(5) + FLM (0.5)* 15.70 ± 0.15* * 3/10 12. KT(5) + DZP (2.5)*+FLM (0.5)* 10.30 ± 0.75** 1110 13. KT(5) + GABA (100)* 7.80 ± 0.30 ** 0/10 14. KT(5) + GABA (200)* 6.70 ± 0.26 ** OliO 15. KT(5) +MUS (0.5)* OliO 4.50 ± 0.17** 16 KT(5) +MUS (I)* OliO 2.30 ± 0.33** 2 F (15,144) = 167.094 X 1sd r = 6.03 (?<0.01) *Drugs administered by sc route. **?<0.001 as compared with control( vehicle) or per se effect of ketamine or diazepam or its combination. Group 2,3,5,6,7,8,9,10,13,14,15,16 vs 1; Group 4 vs 2; Group 10,11,13,14,15,16 vs 9; Group 12 vs 10; n=10. #One-way ANOVA followed by Dunnett's 't' test;## Grouped Chi-square test with Yates correction. DZP, diazepam; FLM, flumazenil; GABA, y-aminobutyric acid; MUS, muscimol. Table 2 -Effect of GABA8 ergic drugs and NMDA receptor antagonist, MK80 I per se and on the anticonvulsant activity of ketamine (KT) in maximal electroshock (MES)-induced convulsions in mice Group Treatment Duration of hindlimb extensor phase( sec) Mortality## (mg!kg,ip or sc) (Mean± SE) # 1. Vehicle 16.10 ± 0.75 4/10 2. BAC (2.5)* 10.00 ± 0.33** 2/10 3. BAC(5)* 5.10 ± 0.28** OliO 4. DAVA (50) 15.30 ± 0.37 3/10 5. BAC (5)* + DA VA (50) 14.30 ± 0.15** l/10 6. MK80 1 (0.1 )* 2.00 ± 0.56** 0/10 7. KT (5) 11.20 ± 0.42** 2/10 8. KT(5) + BAC (2.5)* 6.50 ± 0.43** 0/10 9. KT(5) + BAC (5)* 3.30 ± 0. 15** 0/10 10. KT(5) + DA VA (50) 16.40 ± 0.22** 3/10 11. KT(5) + BAC (5)* + DA VA (50) 11.80 ± 0.90 ** 1/10 12. KT(5) + MK801 (0.1)* 1.90 ± 0.46** 0/10 2 F(11,108) = 137.29 X 11ctr = 9.54 (?<0.01) *Drugs administered by sc route. ** P < 0.001 as compared with control (vehicle) or per se effect of ketamine or baclofen or its combination. Group 2,3,4,6,7, 8, 9, 12 vs 1; Group 5 vs 3; Group 8,9,10,12 vs 7; Group 11 vs 9; n=IO. # One-way ANOVA followed by Dunnett's 't' test; ## Grouped Chi-square test with Yates correction. BAC, baclofen; DA VA, &-amino valerie acid. 1006 INDIAN J EXP BIOL, OCTOBER 2001 Table 3- Effect of naloxone (NLX), haloperidol (HP) and sulpiride (SP) per se and on the anticonvulsant activity of ketamine (KT) in maximal electroshock (MES)-induced convulsions in mice Group Treatment Duration of hindlimb extensor phase (sec) Mortality## (mg/kg, ip or sc) (Mean± SE) # 1. Vehicle 15.60 ± 0.62 4110 2. NLX (0.1) 15.20 ± 3.36 3/10 3. NLX (I) 16.00 ± 1.14 4/10 4. HP (0.5)* 15.50 ± 2.39 1/10 5. HP (I)* 16.10 ± 2.26 2110 6. HP (2)* 2/10 16.90± 1.81 7. SP (20)* 1/10 14.90 ± 1.25 8. KT(50) 0/10 0.90 ± 0.38** 9. KT(50)+NLX(O.I) 0/10 0.50 ± 0.40 10. KT(50)+NLX( I) 0/10 11. KT(50)+HP(0.5)* 1.30 ± 0.73 0110 12. KT(50)+HP(I)* 3.90 ± 0.53 0/10 13. KT(50)+HP(2)* 6.30 ± 0.30* 1/10 14. KT(50)+SP(20)* 9.30 ± 0.70** 0/10 0.20 ± 0.13 F(l3,126) = 22.22 X213dr= 13.52 (P < 0.01) *Drugs administered by s.c. route. *P<0.05; ** P<0.001 as compared with control (vehicle) or per se effect of ketamine. Group 2,3,4,5, 6, 7, 8 vs l; Group 9,10, 11,12,13,14 vs 8; n=IO. #One-way AN OVA followed by Dunnett's ' t' test; ##Grouped Chi-square test with Yates correction. vulsions as compared to vehicle-treated control (Table suggest that GABAergic system may be involved in 2). When MK 801 ( O.lmg/kg ) was combined with the action of ketamine; since PTZ interferes with ketamine (5 mg/kg) in separate groups of mice, the GABA transmission and interacts with picrotoxinin protective effect of ketamine on MES seizures was binding site of GABA-benzodiazepine receptor com potentiated by MK 801, i.e. duration of THE was sig- plex and inhibits e5S]-t-butylbicyclophosphoro • nificantly reduced (?<0.001) and there was no mor 5 34 35 thionate (e S]-TBPS) binding ' . In the present tality as compared to mortality incidence (2110) in the study, the facilitation of protective effect of ketamine ketamine (Smg/kg) treated group (Table 2). on MES seizures by GABAergic agents, like diaze Interaction of ketamine with opioid and dopa 36 37 pam, GABA and muscimol suggests that GABAA mine/sigma receptor ligands-Naloxone (0.1 mg/kg BZO mechanisms may be participating in atleast and 1 mg/kg) failed to reverse the protective effect of some ofketamine's anti-MES effect. Additionally, the ketamine against MES-induced convulsions. While reversal of facilitatory effect of diazepam on ketamine haloperidol (0.5-2mg/kg) a 0 dopamine and so called 2 protection of MES convulsions by flumazenil, a short sigma receptor antagonist reversed the effects of ke acting pure antagonist of the benzocliazepine recogni tamine, sulpiride (20 mg/kg) a selective 0 2 receptor 38 antagonist failed to show any significant reversal of tion site of GABAA receptor indicates that BZO site ketamine effect. None of these agents had any effect on BZO-GABAA receptor complex could be possibly per se against MES-convulsions (Table 3). involved in action of ketamine in the brain. This sug gestion gets further credence from observations, Discussion where flumazenil per se also attenuated the anti-MES GABA plays a critical role in the etiopathology of effect of ketamine when given alone. Further, the fa 5 epileps/ . GABAergic mechanisms have been impli cilitation of anticonvulsant action of ketamine by ba 9 cated in protection from a variety of chemo- and clofen, a GABA8 receptor agonise and attenuation of e I ectros h oc k -.In d uce d setzures. 26.?-8 . B enzo d'tazepme . anti-MES effect of ketamine alone as well as when (BZD) agonists, like diazepam are positive allosteric given in combination with baclofen by OA VA , a 40 modul ators qf GABA-mediated neurotransmission in GABA8 antagonist , show that besides GABAA, 28 30 CNS - . Ketamine has been found to be effective GABA8 receptors also in some way play a role in the against PTZ-induced convulsions2.4,J I-JJ_ These reports an ti-MES effect of ketamine. MANOCHA et al.: ANTICONVULSANT EFFECT OF KET AMINE 1007 There is also growing evidence that NMDA sub References type of excitatory amino acid (EAA) receptors play an I Anis N A, Berry S C, BUJ1on N R & Lodge D, The dissocia 41 tive anesthetics, ketamine and phencyclidine, selectively re important role in epilepsy • In fact, competitive and duce excitation of central mammali an neurons by N-methyl- non-competitive antagonists of NMDA receptor have 0-aspartate, Br 1 Pharmacal, 79 ( 1983) 565. shown potent anticonvulsant activity in a wide range 2 Taberner P V, The anticonvulsant activity of ketamine against 42 of seizure models . Ketamine is a well known non seizures induced by pentylenetetrazo l and mercaptopropionic competitive NMDA receptor antagonist and exerts its acid, Eur 1 Pharmacal, 39 (1976) 305. 3 Myslobodsky M S, Golovchinsky V & Mintz M, Ketamine: effects by entering and blocking the open c<~;tionic 36 Convulsant or anti-convul sant?, Pharmacal Biochem Behav, channel linked to NMDA receptors . MK801 is also 14 (198 1) 27. a non-competitive NMDA receptor antagonist which 4 Velfsek L, Mikolasova R, Blankova-Vankova S & Mares P, acts at a site within the ion channel of NMDA recep Effects of ketamine on metrazol-induced seizures during on 43 44 tor complex . Sharma et al. demonstrated the pro togenesis in rats, Pharmacal Biochem Behav, 32 ( 1989) 405. 5 Velfskova J, Velfsek L, Mares P & Rokyta R, Ketamine sup tective profile of MK801 and ketarnine in both rats presses both bicuculline- and picrotoxin-induced generalized and mice against NMDA-induced epileptogenic ex tonic-clonic seizures during ontogenesis, Pharmacal Biochem citotoxicity. In this study, MK801 potentiated the an Behav, 37 (1990) 667. ticonvulsant effect of ketamine on MES seizures 6 Sagratella S, Ni gli o T & Scotti de Carol is A, An in vesti gati on which indicates th at the anti-MES effect of ketamine on the mechani sm of anticonvulsant action of ketamine and phencyclidine on convulsions due to cortical applicati on of is also mediated at NMDA receptors. peni cillin in rabbits, Phannacol Res Commwz, 17 ( 1985) 773 . 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