Specificity of piracetam's anti-amnesic activity in three models of amnesia in the mouse

PharrnacologJ, Blochemtstry&Behawor, Vol 29, pp 625-629 ~ Pergamon Press plc. 1988 Pnnted m the U S A

0091-3057/88 $3 00 + 00

Specificity of Piracetam's Anti-Amnesic Activity in Three Models of Amnesia in the Mouse A LENI~GRE,* R. CHERMAT,t I AVRIL,* L. STt~RU* AND R. D PORSOLT *I *I T E . M . - L a b o , 93 ave de F o n t a m e b l e a u , 94270 K r e m l i n - B t c k t r e , F r a n c e tFacultO de M O d e c m e Pitt~-SalpOtrtkre, 75013 Parts, F r a n c e

Received 28 May 1987 LENI~GRE, A , R CHERMAT, I AVRIL, L STI~RU AND R D PORSOLT Spectfictty oJ'ptracetam's antt-amnestc acttvlty m three models of amnesta tn the mouse PHARMACOL BIOCHEM BEHAV 29(3)625-629, 1988 ---The effects of p,racetam on the amnesias reduced by scopolamme, dmzepam and electroconvulslve shock (ECS) were stud,ed in a passive avoidance procedure in the mouse and compared with the interactions of p,racetam with the major behavioral effects of these treatments, namely scopolamine-reduced hyperacttvlty, dmzepam-mduced release of pumshed behavior (Four Plates Test) and ECS-mduced convulsions Amnesm was reduced by rejecting scopolamine or dlazepam (1 mg/kg, IP) 30 mmutes before or applying ECS immediately after the first session (S1) of the passive avoidance task P~racetam was studied at 3 doses (512, 1024 and 2048 mg/kg) administered PO 60 minutes before SI Retentmn was measured 24 hours later ($2) in the absence of any treatment lhracetam dose-dependently attenuated the memory deficits reduced by the three amnesic treatments but did not affect either scopolamine-induced hyperactw,ty, dmzepam-mduced release of pumshed behawor or ECS-lnduced convulsmns These results pomt to the spec,fic~ty of plracetam's anU-amnesic activity and, m parttcular, suggest that p~racetam can suppress the memory d~sturbances reduced by dmzepam without affecting dmzepam's anxlolyt~c activity The test battery employed would therefore seem highly statable for evaluating the potential nootroplc activity of novel compounds Piracetam Amnesm Passive avoidance Specificity of action Nootroplc drugs

Scopolamine

Dlazepam

Electroconvulslve shock

treatments, namely the hyperactivity induced by scopolamine, the release of punished b e h a v i o r by d l a z e p a m and the convulsions induced by E C S The rationale for choosing amnesias o f three different origins in the same test situation was that an antagonism by piracetam of all three amnesias would rule out interpretations of p i r a c e t a m ' s effects in terms of a specific antagonism of the intrinsic propertles of any one of the amnesic agents. Such a conclusion w o u l d be c o m f o r t e d by demonstrating the absence of antagonism by plracetam of the o t h e r behavioral c o n s e q u e n c e s of the amnesic treatments

E X P E R I M E N T A L a m n e s i a can be induced in animals by a variety o f treatments including e l e c t r o c o n v u l s i v e shock [7], antichohnerglc agents [5] and b e n z o d l a z e p l n e s [13] Antagonism of different e x p e r i m e n t a l amnesias has p r o v i d e d the m a j o r a p p r o a c h towards the d e v e l o p m e n t of drugs useful for t r e a t m e n t o f m e m o r y p r o b l e m s in old age [3,11]. The predictive value of such models is by no m e a n s well established but it seems reasonable to suppose that a c o m p o u n d which antagonizes amnesias of different origins is more likely to be generally effective F u r t h e r m o r e , antagonism by the same c o m p o u n d of dtfferent kinds of amnesias p r o v i d e s e v i d e n c e for the specificity of the anti-amnesic activity o f the compound as o p p o s e d to its effects on one o r m o r e systems [8] The term nootroplc was suggested by Giurgea [4] for classifying drugs which e n h a n c e m e m o r y and attenuate experimental amnesia, the p r o t o t y p e drug in this category is piracetam. The e x p e r i m e n t s described in the present paper investigated the antagonism by plracetam o f amnesias induced by scopolamine, d i a z e p a m and e l e c t r o c o n v u l s i v e shock (ECS) and then e x a m i n e d the interaction o f piracetam with the major behavioral effects o f these three amnesic

METHOD

Ammals The subjects were male N M R I mice, weighing 23-25 g, supplied by the Centre d ' E l e v a g e Roger Janvler (CERJ), 53940 Le G e n e s t Saant Isle, F r a n c e T h e y were housed In groups of 10 in transparent m a c r o l o n cages (25 5× 19 5 x 13 5 cm) containing sawdust with free access to food ( U A R 113) and tap w a t e r All animals w e r e delivered to the laboratory

1Requests for repnnts should be addressed to Dr R D Porsolt, SoentlfiC Director, ITEM-Labo, 93 ave de Fontmnebleau, 94270 Kremhn-BlcStre, France

625

LENI~GRE E T A L

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Drugs

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The following drugs were used dmzepam (Roche), piracetam (UCB) and scopolamine hydrobromlde (Sigma) They were either dissolved m distilled water (plracetam, scopolamine) or dispersed in an aqueous suspension of acacia gum (5%) (dlazepam) All drugs were injected in a volume of 0 25 ml/20 g body weight Doses are expressed in terms of the base or salt where approprmte

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Pro( edure Passtve avoidance tests Mice were placed individually into the smaller (10×10×29 cm), but brightly lit, compartment of a two compartment box When they crossed with all four paws into the larger darker compartment (19 5× 16 5×29 cm) they received a light foot-shock (0 35 mA) until they returned to the lighted compartment from which they were immedmtely removed (S1) Twenty-four hours later they were replaced in the lighted compartment without any pretreatment and their latency before crossing to the dark compartment was measured with a cut-off time of 180 seconds ($2) Twenty mice were used per group In the experaments where amnesia was induced by scopolamine mice were given an IP injection of scopolamine (1 mg/kg) 30 minutes before S1 In the experaments where amnesia was induced by dlazepam mice were given an IP reJection of diazepam (1 mg/kg) 30 minutes before S1 In the experaments where amnesm was induced by ECS mice were given ECS (rectangular current, 0 4 sec, 50 mA, 50 Hz, via temporal electrodes connected to a Ugo Baslle Shock Generator) on removal from the apparatus immediately after S1 In all amnesia experiments piracetam (512, 1024 and 2048 mg/kg) was given m a single PO administration 60 minutes before S1 In each experament two control groups were employed Normal controls, instead of receiving the amnesic agent, received IP rejections of distilled water (scopolamine) or 5% acacia gum (dlazepam) or simple placement of the electrodes without shock (ECS) Controls (normal and amnesic), instead of receiving plracetam, were given an oral administration of distilled water 60 minutes before S1 Spontaneous motor acttvtty For investigating the interaction of piracetam with the motor stimulatory effects of scopolamine a photo-cell activity meter similar to that described by Bolssler and Simon [2] was used The activity meter consisted of 6 covered Plexlglas enclosures ( 2 5 5 × 2 0 5 × 9 cm) each equipped with two crass-cross photo-cell assemblies contained within a darkened enclosure. The number of interruptions of the photo-electric beams by each ammal was counted during a 30 minute test Twelve animals were studied per group The experiment consisted of four groups control, plracetam (2048 mg/kg) alone, scopolamine (1 mg/kg) alone, plracetam (2048 mg/kg)+scopolamine (1 mg/kg) Piracetam was administered PO 60 minutes before the test and scopolamine was administered IP 30 minutes before the test All ammals received two injections, when a drug was not given they received the vehicle Four Plates Test For investigating the Interaction of p~racetam with the anxlolytlc effects of dlazepam the Four Plates Test [1] was used Mice were placed individually in a

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FIG 1 Scopolamine-reduced amnesm The effects of plracetam, admlmstered PO 60 minutes before the first session (S1) on the mean (-+s e m ) step-through latency at S1 (left columns) and $2 (right columns) of a passive avoidance task Twenty mice were used per group All ammals, except the non-amnesic control group, received an IP injection of scopolamine (1 mg/kg) 30 minutes before SI **=p<0 01, ***=p<0 001 (unpmred Student's t-test, two-tailed) white plastic enclosure (25 × 18 × 16 cm) with a floor consisting of four rectangular metal plates (8× 11 cm) The animal was left to explore freely for 15 sec and then for the next 60 sec it received a mild electnc shock (0 35 mA, 0 5 sec) every time it crossed from one plate to another The number of punished crossings during this period was counted Ten animals were studied per group The experiment consisted of four groups control, plracetam (2048 mg/kg) alone, diazepam (2 mg/kg) alone, plracetam (2048 mg/kg)+dlazepam (2 mg/kg) Plracetam was administered PO 60 minutes before the test and diazepam was admlmstered IP 30 minutes before the test All animals received two injections, when a drug was not gwen they received the vehicle Electroconvulslve shock (ECS) For investigating the interaction of piracetam with the effects of ECS, the number of animals showing full tonic-clonlc convulsions in the different experimental groups after administration of ECS on the first day (S1) of the passive avoidance task was counted There were thus 20 ammals per group

Stattsttcal Tests All quantitative results were analyzed for statistical significance using the Student's t-test for independent samples (two-tailed) RESULTS

Passtve Avotdance Tests Amnestas Induced by Scopolamme, Dtazepam and ECS The effects ofpiracetam on scopolamine-induced amnesia are shown in Fig 1 Control mice which had received only rejections of distilled water (normal controls) showed clear retention as Indicated by the close to maximal step-through latencles at $2 Control mice which had received an IP inJection of scopolamine 30 minutes before S 1 showed a clear and

ANTI-AMNESIC

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Doses (mg/kg) FIG 2 Dmzepam-mduced amnesm The effects of plracetam, administered PO 60 minutes before the first sessmn (SI) on the mean ( + s e m ) step-through latency at SI (left columns) and $2 (right columns) of a passive avoidance task Twenty mice were used per group All animals, except the non-amnesic control group, received an IP reJection of dmzepam (i mg/kg) 30 minutes before S1 * * = p < 0 01, ***=p<0 001 (unpaired Student's t-test, two-tailed) u

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FIG 4 Interaction of plracetam with scopolamine-reduced hyperactwlty The effects of scopolamine and piracetam either alone or admimstered together on the mean (_+s e m ) number of photocell beams interrupted dunng a 30 minute session l:hracetam (2048 mg/kg) was admimstered PO 60 minutes before testing and scopolamine (1 mg/kg) was administered IP 30 minutes before testmg Twelve mice were used per group ***=p<0 001 (unpmred Student's t-test, two-tailed) sngmficant d e c r e a s e in $2 l a t e n c l e s as c o m p a r e d w i t h the normal controls indicating scopolamine-reduced amnesia. P l r a c e t a m a d m i n i s t e r e d P O 30 m i n u t e s b e f o r e s c o p o l a m i n e at S1 h a d n o effect o n S1 l a t e n c l e s b u t c a u s e d a d o s e d e p e n d e n t i n c r e a s e in $2 l a t e n c l e s suggesting a n t a g o n i s m o f s c o p o l a m i n e - r e d u c e d a m n e s i a w h i c h w a s a l m o s t c o m p l e t e at t h e h i g h e s t d o s e t e s t e d (2048 mg/kg PO)

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FIG 3 ECS-mduced amnesm The effects of plracetam, administered PO 60 minutes before the first sessmn (S1) on the mean (+_s e m ) step-through latency at S1 (left columns) and $2 (right columns) of a passive avoidance task Twenty mice were used per group All ammais, except the non-amnesic control group, received ECS immediately after Si * = p < 0 05, ***=p<0 001 (unpmred Student's t-test, two-tailed)

T h e effects of p l r a c e t a m o n d l a z e p a m - l n d u c e d a m n e s i a are s h o w n in Fig 2 L i k e in the s c o p o l a m i n e e x p e r i m e n t d i a z e p a m , a d m i n i s t e r e d I P 30 m i n u t e s b e f o r e S1, c a u s e d a m a r k e d a n d slgnLficant d e c r e a s e m $2 l a t e n c l e s as c o m p a r e d w i t h the n o r m a l c o n t r o l g r o u p indicating d l a z e p a m - i n d u c e d a m n e s i a t h r a c e t a m , a d m i n i s t e r e d P O 30 m i n u t e s before d l a z e p a m , h a d n o effect o n S1 l a t e n c l e s b u t c a u s e d a c l e a r a n d a d o s e - d e p e n d e n t i n c r e a s e in $2 l a t e n c l e s suggesting, as in the s c o p o l a m i n e e x p e r i m e n t , a n t a g o n i s m o f d l a z e p a m i n d u c e d a m n e s i a w h i c h w a s vnrtually c o m p l e t e at the highest d o s e t e s t e d (2048 mg/kg PO) T h e effects o f p l r a c e t a m o n E C S - m d u c e d a m n e s i a are s h o w n in Fig 3 E C S , a d m i n i s t e r e d to vehicle t r e a t e d a n i m a l s i m m e d i a t e l y a f t e r S1, c a u s e d a m a r k e d d e c r e a s e in $2 l a t e n c i e s as c o m p a r e d to s h a m t r e a t e d c o n t r o l a n i m a l s i n d i c a t i n g E C S - l n d u c e d a m n e s i a . T h e d e c r e a s e in S2 latencles after E C S a p p e a r e d to b e r a t h e r m o r e m a r k e d t h a n t h a t o b s e r v e d in the s c o p o l a m i n e or d l a z e p a m e x p e r i m e n t s P l r a c e t a m , a d m i n i s t e r e d P O 60 m i n u t e s b e f o r e S1, c a u s e d a d o s e - d e p e n d e n t i n c r e a s e in $2 latencles, suggesting as In t h e p r e v i o u s t w o e x p e r i m e n t s , a n t a g o n i s m o f E C S - i n d u c e d amnesia. A l t h o u g h the s a m e d o s e s of p l r a c e t a m w e r e investigated, the a n t a g o n i s m b y p l r a c e t a m o f E C S - m d u c e d a m n e s i a a p p e a r e d to b e less c o m p l e t e t h a n t h a t o b s e r v e d in the scopolamine and diazepam experiments

Spontaneous Motor Actlvtty Interactton Between Ptracetam and Scopolamme T h e effects o f s c o p o l a m i n e a n d p i r a c e t a m a l o n e o r adm i n i s t e r e d t o g e t h e r o n s p o n t a n e o u s m o t o r activity are s h o w n in Fig 4 S c o p o l a m i n e (1 mg/kg IP) a l o n e c a u s e d a m a r k e d i n c r e a s e m t h e n u m b e r o f p h o t o - c e l l b e a m s interr u p t e d b y m i c e p l a c e d in the a c t i v i t y m e t e r P l r a c e t a m , at the dose which was active m antagonizing scopolamine-reduced a m n e s i a (2048 mg/kg PO), w a s itself w i t h o u t significant eff e c t s o n s p o n t a n e o u s m o t o r activity a n d did n o t sigmficantly modify scopolamine-induced hyperactivity

628

LENI~GRE ET AL °°~ -

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FIG 5 Interaction of plracetam with dlazepam-mduced release of pumshed responding The effects of dmzepam and plracetam either alone or administered together on the mean (_+s e m ) number of pumshed crossmgs dunng the Four Plates Test Piracetam (2048 mg/kg) was admlmstered PO 60 minutes before testing and dmzepam (2 mg/kg) was administered IP 30 minutes before testmg Ten mice were used per group ***=p<0 001 (unpmred Student's t-test, two-taded)

Four Plates Te~t Interaction Between Ptracetam and Dzazepam The effects of dmzepam and plracetam alone or administered together on the number of punished crossings are shown m Fig 5 Dlazepam (2 mg/kg IP) alone caused a slgmflcant increase m the number of pumshed crossings reflecting its anx~olytxc activity in th~s test Plracetam, at the dose which was actwe in antagonizing dlazepam-induced amnesia (2048 mg/kg PO), was itself without significant effect on the number of pumshed crossings and did not significantly modify the increase in pumshed crossings observed with dmzepam

Convulsions Induced by ECS Interaction With Ptracetam The effects of the three doses of ptracetam used m the amnesia experiment on the convulsions induced by ECS are shown m Fig 6 No convulsions were observed m sham treated ammals whereas all control ammals receiving ECS showed full tonlc-clomc convulsions Piracetam did not protect against electrically-induced convulsions at any of the doses tested DISCUSSION

The present experiments have shown that plracetam antagomzes the amnesias induced by three different amnesic treatments, scopolamine, dmzepam and ECS These results confirm the proposed nootroplc profile of this compound [4] and suggest that ~ts anti-amnesic effects are unrelated to the specific nature of these amnesia-reducing agents This concluslon is supported by the finding that plracetam in no way interacts with the major behaworal effects of these treatments namely the hyperactlwty induced by scopolamine, the ante-punishment effect of dmzepam or the convulsant effect of electroshock The anti-amnesic effects of plracetam occur

ECS Plrocetam p o

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FIG 6 Interaction of plracetam with convulsions reduced by electroshock The effects of three doses of plracetam on the number of ammals convulsing after apphcatlon of ECS through temporal electrodes Twenty mice were used per group at doses of this compound which have no observable effects on behavior either dunng the passive avoidance test itself or on any of the other behaviors investigated m the present experiments The anti-amnesic effects observed, moreover, occur m animals which are tested at $2 m the absence of the compound thereby ruling out possible interpretations m terms of the effects of plracetam on performance or even state-dependent effects [9] Although these experiments clearly suggest a speofic anU-amnesic action of plracetam, the absence of other effects provide no clue as to its mechanism of action The compound exerts no clear effects on any brain neurotransmitter system and, in particular, appears to be devoid of classical chohnerglc activity [6] although plracetam has been reported to act synergistically with the acetylchohne precursor choline in facilitating memory funcuon in rodents [10] Absence of direct chohnerg~c actlvlty ls also indicated in the present experiments by plracetam's failure to antagonize scopolamine-induced hyperactivity Furthermore, although structurally related to G A B A ~t did not interact with dlazepam's anxlolytlc activity nor possess convulsant or antlconvulsant activity as might be expected of substances mteractlng with GABAerglc neurotransmlsslon [12] It is possible that different thresholds exist for different behawors and that plracetam antagonizes the memory disrupting effects of different amnesic treatments w~thout blockmg their overt behavioral effects For example, plracetam could conceivably attenuate some of the disruption of CNS activity that presumably mediates amnesia but fad to block overt convulsions This seems an unlikely explanation for all the results obtained in the present experiments however, partlcularly where it was shown that diazepam, which is a potent antlconvulsant, itself reduced amnesm From a more pragmatic point of view. the present results suggest the usefulness of the test battery employed for evaluating the anti-amnesic activity of plracetam or other compounds potentmlly useful for treating memory disturbance Furthermore it may be of practical significance that plracetam could be shown to antagonize dlazepam-induced amnesia without apparently affectmg dmzepam's anxlolytlc activity

ANTI-AMNESIC ACTIVITY OF PIRACETAM

629

ACKNOWLEDGEMENTS These experiments were performed m collaboration w~th the Scientific Service of the Centre d'Elevage Roger Janvler We thank Ms Marianne Rotsztem for secretarial assistance

REFERENCES 1 Aron, C , P Simon, C Larousse and J R Bolssler Evaluation of a rap~d techmque for detecting minor tranqudhzers Neuropharmacology 10: 459-469, 1971 2 Bo~ssler, J R and P Simon Action de la caffeine sur ia mouht6 spontan6e de la souns Arch Int Pharmacodyn Ther 158: 212221, 1965 3 Cumin, M , E F Bandle, E Gamzu and W E Haefely Effects of the novel compound amracetam upon impaired learning and memory m rodents Psychopharrnacology (Berhn) 78:104-111, 1982 4 Glurgea, C and M Salama Nootroplc drugs Prog Neuropsychopharmacol Btol Psychtatry 1: 235-247, 1977 5 Ghck, S D and B Zlmmerberg Amnesic effects of scopolamine Behav Btol 7: 245-254, 1972 6 Gobert, J G G6n6se d'un m6dlcament le p~rac6tam, m6tabohsme et recherche blochlmlque J Pharma~ol Belg 27: 281-304, 1972 7 McGaugh, J L Time dependent processes in memory storage Sctence 153: 1351-1358, 1966

8 Martmez, J L , R A Jensen and J L McGaugh Attenuation of experamentally induced amnesia Prog Neuroblol 16: 155-186, 1981 9 Overton, D A Experimental methods for the study of statedependent learning Fed Proc 33: 1800-1813, 1974 10 Platel, A , M Jalfre, C Pawelec, S Roux and R D Porsolt Habttuat~on of exploratory activity m m~ce Effects of combinations of p~racetam and chohne on memory processes Pharmacol Btochem Behav 21: 209-212, 1984 11 Schmdler, U , D K Rush and S Fielding Nootruplc drugs animal models for studying effects on cognition Drug Dev Res 4: 567-576, 1984 12 Schmutz, M Benzodmzepmes, GABA and epdepsy--the ammal evidence In Benzodtazepmes DtvMed a Multtdlsctphnary Revtew, edited by M Tnmble Chlchester Wiley, 1983, pp 149-166 13 Thl6bot, M-H Some evidence for amnesic effects of benzodlazepmes in animals Neurosct Btobehav Rev 9: 95-100, 1985