Piracetam Prevents Pentylenetetrazol Kindling-Induced Neuronal Loss and Learning Deficits

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Seizure 1997; 6: 467474

Piracetam prevents pentylenetetrazol kindling-induced neuronal loss and learning deficits

W. POHLE, A. BECKER, G. GRECKSCH, A. JUHRE & A. WILLENBERG

Department of Pharmacology and Toxicology, Faculty of Medicine, Otto-von-Guericke-University-Magdeburg, Leipziger Str. 44, Germany

Correspondence to: Axe1 Becker, Department of Pharmacology and Toxicology, Med. Fat. Otto-von-Guericke-University-Magdeburg, Leipziger Str. 44, D-391 20 Magdeburg, Germany

The effect of the nootropic drug piracetam (100 mgkg) on kindled seizures, kindling-induced learning deficits, and histological alterations due to changes in central excitability was investigated in Wistar rats. The animals were kindled by repeated i.p. injections of an initially subconvulsive dose of pentylenetetrazol (PTZ). As a control, piracetam or physiological saline was given 60 minutes before PTZ. Twenty-four hours after completion of kindling the rats were tested in a shuttle-box paradigm. Seven days after the final kindling injection, the animals received a challenge dose of PTZ. Finally, the brains of the rats were processed for histological investigation. Pentylenetetrazol-kindled animals showed increasing seizure scores, and a learning deficit in the shuttle-box. Piracetam had no effect either on kindling development or on the reaction to a challenge dose of PTZ, but it protected the animals against the kindling-induced reduction of learning performance. The substance had no effect on learning performance in control animals. In distinct hippocampal structures, a neuronal cell loss was found in kindled rats. Interestingly, piracetam counteracted this damage efficaciously. The effects of piracetam are discussed in terms of its cytoprotective action. It is suggested that a coadministration of piracetam with clinically used antiepileptic drugs might be useful in antiepileptic therapy.

Key words: kindling; pentylenetetrazol; hippocampus neuronal loss; learning deficit; piracetam.

INTRODUCTION impairment9 . Interestingly enough, the opiate receptor antagonist naloxona which had no effect on Epilepsy causes neuronal damage which is to be seizure development, ameliorated the learning deficit seen preferentially in the hippocampal formation14. in the kindled groupg. These results indicate that mo- Another morphological alteration related to specific tor seizures are unrelated to the learning deficit. forms of epilepsy is sprouting of mossy fibres3w5s6. As regards morphological alterations in epileptic These morphological changes may contribute to patients, equivalent results were found in kindled different behavioural and cognitive impairments in animals. After kindling by electrical stimulation of epileptic patients. different brain structures and after sustained perforant Kindling represents a useful experimental tool for path stimulation respectively, several authors found the study of epilepsy-related alterations in animals. neuronal damage and loss of cells’* lo-‘* or synapses’ The term ‘kindling’ refers to a process in which as well as sprouting of mossy fibres”, 11,18,20. periodic application of an initially subeffective dose Changes in neuropeptide Y, somatostatin and en- of a compound or an electrical stimulus induces cephalin were also reported2’y2*. Morphological progressive intensification of evoked electroen- changes were found after pilocarpine23, kainic cephalographic and behavioural seizures’. As a acid2426 and flurothyl-induced seizures2’. result of kindling, several behavioural alterations In contrast to other epilepsy models morphologi- occur. Recently, we reported on learning impairments cal findings after pentyleneterazol kindling are rel- in kindled rats depending on both the mode ofkin- atively rare2t* 28*2g. Thus, it is questionable to what dling induction and the learning paradigms used*. extent morphological alterations contribute to leam- It was shown that doses of diazepam, effective in ing deficits in rats kindled by repeated injections of seizure suppression, exerted no effect on learning pentylenetetrazol (PTZ). To investigate this, wead-

1059-l 31 l/97/060467 + 08 $12.00/O @ 1997 British Epilepsy Association 468 W. Pohle et a/

ministered piracetam prior to each kindling stimu- stage 3: myoclonic jerks, upright position with bi- lation. Previously, it was shown that this substance lateral forelimb clonus; injected in a dose of 100 mg/kg did not interfere with stage 4: clonic-tonic seizures; PTZ kindled seizures, but the substance was effective stage 5: generalized clonic-tonic seizures, loss of in counteracting kindling-induced learning deficits3’. postural control. According to Leonard3’ the main feature of Piracetam (pir) (100 mg/kg) or saline (Sal) was nootropic drugs, as exemplified by the prototype given i.p. in a volume of 10 ml/kg body weight piracetam are: (1) ability to enhance memory and 60 minutes prior to each PTZ or saline applica- learning; (2) facilitation of flow of information be- tion. The following experimental groups were used: tween the cerebral hemispheres; (3) enhancement of (1) sal + sal(n = 13); (2) sal + PTZ(n = 15); (3) the resistance of the brain to physical and chemical pir + sal(n = 15); (4) pir + PTZ(n = 20). In total, assaults; and (4) lack of effect on peripheral system the rats received 13 kindling injections. and lack of sedative, analgesic or neuroleptic activity. Twenty-four hours after the last injection, the rats The mode of action of this heterogeneous group of were tested in a shuttle-box. At this time the drugs agents is unknown. Despite learning and memory en- would be expected to have cleared. hancing effects in rodents, there is little evidence that The automatic shuttle-box was divided into two nootropics are therapeutically effective in dementia compartments (25 x 25 x 60 cm) separated by a 5- in humans. The bell-shaped dose-effect relationship, cm hurdle. The conditioned stimuli were light (40-W the existence of responder and non-responder, and bulbs located on the central ceiling of each compart- the possibility that these drugs might only increase ment) and sound produced by a buzzer.The uncondi- alertness, have been suggested as the reasons for the tioned stimulus was an electrical foot shock (max- limited clinical results3*. In the present study we imum 0.8 mA according to individual susceptibil- tested the effect of piracetam on the development ity of the animal) delivered through stainless-steel of kindling seizures, the kindling-induced learning rods covering the floor. The conditioned stimulus- deficit in the shuttle-box, and histological alterations unconditioned stimulus interval was 4 seconds. Each in the hippocampal formation. trial was limited to 20 seconds if the animal failed to react. Intertrial intervals were randomized (mean 30 seconds). Each session consisted of 20 trials and METHODS was repeated on three consecutive days. Sessions were performed during the light part of the 12: 12 hour Animals cycles at the same time f 1 hour. Prior to the first session, the rats were allowed to explore the box for The experiments were carried out with male Wis- 5 minutes, and on the following days 1 minute was tar rats [Mol:Wist(Shoe), Mollegaard Breeding Cen- allowed. tre Deutschland GmbH] with an age of 8 weeks at The number of escapes (reaction time >4 seconds) the beginning of the experiments. The animals were and conditioned reactions (reaction time c4 seconds) kept under controlled laboratory conditions (light was recorded for further evaluation. After comple- regime of 12 hours light/l2 hours dark, light on at tion of the learning experiments, i.e. 7 days after the 0600 hours), temperature 20 f 2 “C, air humidity 55- final kindling injection all animals received a chal- 60%. They had free access to commercial rat pellets lenge dose of 30 mg/kg PTZ or saline, respectively. (Altromin 1326) and tap water. The rats were housed Resultant seizures were scored as mentioned above. in groups of five per cage.

Histology Treatment and learning test Twenty-four hours after the last challenge dose or For PTZ-kindling an initially subeffective dose of saline injection the animals were processed for his- 45 mg/kg body weight pentylenetetrazol was injected tological examination. Under deep barbiturateanaes- intraperitoneally once every 48 hours. After each in- thesia, the rats were decapitated. The brains were re- jection, the convulsive behaviour was observed for at moved and placed into fixative solution which had least 20 minutes. The resultant seizures were classi- been saturated with H2S gas. After dehydration the fied according to a modified Racine scale33 as fol- brains were imbedded in paraffin. For histological lows: investigation horizontal paraffin sections, 10 pm in stage 0: no response; thickness, were cut in the plane of the nucleus habe- stage 1: ear and facial twitching; nulae. The sections were alternatively stained with stage 2: myoclonic jerks without upright position; toluidine blue or were used for the TIMM’s sulphide- Piracetam prevents neuronal loss and learning deficit 469

silver reaction3” respectively, to show the morphol- Table 1: Reaction to a challenge dose of PTZ (30 mgkg i.p.) shown in means of convulsive ogy of neuronesas well as that of the zinc-containing staaesfSEM mossy fibres. All brains were processedin an identi- Group Convulsive stage f SEM cal manner. To investigate mossy fibre sprouting af- Saline + saline 1.77 f0.34 ter PTZ kindling, five animals were investigated also Saline + PTZ 3.93 +0.32“ 4 weeks after the last kindling injection. Piracetam + saline I .47 + 0.22 Piracetam + PlZ 3.55 +0.29b For comparison with other results, we focused the ’ = P c 0.05 saline+saline vs. saline+PTZ. histological investigations on the hippocampal for- b = P < 0.05 piracetam + saline vs. mation. Dentate area granular cells, hilar neurones piracetam + PTZ. as well as pyramidal cells of the hippocampal sec- saline + PTZ vs. piracetam + PTZ exhibits no significant difference. tors CA3, CA1 and of the subiculum were counted in squares of 500 x 500 Km by using a counting of conditioned reactions in the piracetam treated net. Ten fields per animal and structure were aver- group (pir + sal) (Ft .29 = 0.442 vs. sal+ Sal). How- aged in 10 different sections stained with toluidine ever, the learning performance in the piracetam- blue. In TIMM-stained sections 24 hours, as well as treated kindling group (pir+ PTZ) was signifi- 4 weeks, after the last kindling injection, the sprout- cantly higher compared with the rats kindled with- ing of mossy fibres was examined. The histological out piracetam pretreatment (Sal+ PIZ) (Ft.31 = analyses were performed in a blind manner. 5 1.49, P c 0.0001). The difference between sal+ sal and pir + PTZ is insignificant (F1,3i = 3.09, P = 0.088). Statistics

The data obtained were analysed using ANOVA. To Histology evaluate the seizure development and the learning performance, the repeated-measuresmodel was ap- As shown in Figs 3 and 4, the number of neurons plied. The basis of statistical decision was a signif- (over all structures under investigation) in control ani- icance level of 0.05. The calculations were carried mals (sal + sal) and rats which had received piracetam out by means of SPSS/PC+software (ANOVA and (pir+sal) was identical. In the granular layer of the MANOVA). dentate area, in the hilus as well as in the pyramidal layer of the CA3 sector (Fig. 3) a striking loss of neurons was caused by PTZ-kindling group (sal+ PTZ) RESULTS as compared to both saline controls (sal+ sal) and piracetam controls (pir + Sal). In these structures, the Kindling reduction of the number of neurons was between 20 and 25% (P < 0.01, ANOVA). In the CA1 sector The results of the kindling experiments are shown in the loss of cells was not so strong, but also statisti- Fig. 1. The repeated application of PTZ resulted in cally significant (P = 0.016, ANOVA), whereas in an increasing seizure activity. The pretreatment with the subiculum no significant neuronal loss was found either saline or 100 mg/kg piracetam had no effect on (F3.25 = 2.977, P = 0.52). seizure stages(Ft.59 = 15.52, P < 0.0001, ANOVA). In rats treated with piracetam during kindling The differences between the control group (sal + sal) (pir +PTZ), the number of neurons was not signif- vs. the kindled group (sal + PTZ) and the piracetam- icantly reduced in comparison with saline controls treated rats (pir + sal) vs. the piracetam-treated kin- (sal + sal) and piracetam controls (pir + Sal). Obvi- dled animals (pir+PTZ) were significant (P < 0.05, ously, piracetam prevents the PTZ kindling-induced Newman-Keuls test), but there was no significant neuronal loss (Fig. 4). difference between kindled animals (sal+PTZ and After TIMM-staining, it was investigated whether pir + PTZ) . kindling induces mossy fibre sprouting or not. In PTZ-kindled rats (sal+ PTZ), no sprouting of mossy fibres was found, neither in the CA3 sector nor in the Learning experiments molecular layer of the dentate area (not shown).

As shown in Fig. 2, PTZ-kindled animals (Sal+ PTZ) exhibited a significantly lower learning perfor- DISCUSSION AND CONCLUSION mance compared to control rats (Sal+ saI) (Ft.26 = 31.2, P < 0.0001). Piracetam administered in a After PTZ-kindling, a significant neuronal cell loss dose of 100 mg/kg did not increase the number was found in all hippocampal structures except the 470 W. Pohle et el

” I 2 3 4 5 6 7 8 9 10 II 12 13 Kindling sessions

Fig. 1: Effect of piracetam pretreatment (100 mg/kg i.p.) on PTZ kindling development (repeated injections of 45 mg/kg pentylenetetrazol i.p.), mean seizures scores f SEM.

16 A sd+Pn 2 0 pir + sal 2 I4 t- &pir + FTZ t; ; 12 B 10 8 .- 8 % 5 6 u 4

Day 1 Day 2 Day 3

Fig. 2: Influence of piracetam (100 mg/kg i.p.) on the kindling-induced learning deficit in shuttle-box learning consecutive on three training days. sal = saline; PTZ = pentylenetetrazol; pir = piracetam; * = P c 0.05 sal + sal versus sal + PTZ; + = P -C 0.05 sal + PTZ versus pir + PTZ, mean f SEM.

subiculum, but there was no sprouting of mossy fi- ing neither in CA3 nor in the dentate area in PTZ- bres. PTZ-kindled rats exhibited a diminishedperfor- kindled animals under our experimental conditions. mance in the shuttle-box. Piracetam (100 mg/kg i.p.) Ebert36 found differences in mossy fibre sprouting did not influence the development of kindling (Fig. 1) between conventional and rapid kindling, and sug- but it prevented neuronal cell loss (Figs 3 and 4) as gested that sprouting dependson the mode of stimu- well as the learning deficit (Fig. 2). lation. Golarai2* found mossy fibre sprouting in the Human epilepsy is characterized by convulsions subgranularlayer only when PTZ-kindling was pro- and a reduced mental capability. From literature, it longed until 10-l 5 generalized tonic-clonic seizures is known that specific types of epilepsy resulted in had appeared; in usual PTZ-kindling he did not find morphological alterations. The seizure-induced neu- sprouting. Also several convulsive doses(55 mg/kg) ronal damage is time-dependent4’. Histological ex- causedsprouting without kindling. Therefore, sprout- aminations of epileptic brains revealed a neuronal ing seemsnot to be dependent on kindling, but on cell loss which is concentrated on the hippocampal the number of generalized seizures. In unpublished formation’a sprouting of mossy fibres3*5*6,and an investigations, using the same method, mossy fibre increasein density of kainate binding sites2’. sprouting in the CA3 sector was found to be induced In different animal models of epilepsy, simi- by ‘amygdala kindling’ and tetanic stimulation; there- lar morphological alterations have been found, i.e. fore, an insufficiency of the method can be excluded. loss of neurones’vlo* 12-15, ‘7*36,37 and mossy fibre To exclude a time-dependentfactor, five rats were ex- sprouting6*lo* “9 18*20*23*24*36.In contrast to amygdala amined 4 weeks after the last kindling injection, but kindling6*20we did not find any mossy fibre sprout- again no sprouting was found. In behavioural investi- Piracetam prevents neuronal loss and learning deficit 471

pir + FTZ

Fig. 3: Hippocampal CAB-pyramidal cells stained with toluidine blue CA3 sector of (a), a control animal; (b), control animals pretreated with the piracetam; (c), a PTZ kindled animal; (d), an animal which was pretreated with piracetam prior to each kindling injection. Bar = 50 Wm.

0 = saline + saline m = piracetam + saline

a = saline + FTZ I = piracetam + FIZ

Dent.area Hilus CA3 CA1 Subiculum

Fig. 4: Influence of piracetam (100 mg/kg on i.p.) on neuronal cell loss induced by PTZ kindling. Number of neurons f SEM counted in a field of 500 x 500 pm in control animals (saline-saline), pirazetam controls (pirazetam-saline), kindled rats (saline-PTZ) and animals kindled under piracetam (Pir-PTZ). * = P < 0.05, ** = P -C 0.01.

gations, learning deficits were described9*39*40.Neu- were reduced46and the release of somatostatin was rochemical investigations have shown an enhanced enhanced4’. Moreover, neuropeptide Y and nerve activity of the glutamatergic system including an in- growth factor were found to be changed2”29,48. creased sensitivity of glutamatergic receptors14, an The glutamatergic neurotransrnissionsystem plays increase in glutamate binding4t and a decreasein an important role for mechanismsof learning and the number of GABA-related parameters42*43.Fur- memory as well as in convulsions. Furthermore, an thermore, the number of axospinal synapses was excessive stimulation of this system resulted in cell changed# and the number of muscarinergic recep- damageand cell death. The neuronal cell loss found tors was reduced45.The enckephalin immunoreac- in brains of kindled animals might partly be a rea- tivity was elevated whereas dynorphine and CCK son for learning deficits. The mechanismof neuronal 472 W. Pohle et a/

death is discussed as follows. The enhanced activ- These actions may explain the beneficial effects of ity of the glutamatergic system plays a crucial role piracetam on the kindling-induced learning deficit. in epilepsy. Seizures and seizure-related hypoxic is- Results by Schmidt5658 demonstrate an anticonvul- chaemia induce an elevation of free radicals, which sant effect of high doses of piracetam (300 mg/kg) in can increase more basal release of excitatory amino rats, and Morgenstem er aPg found a slight increase acids4g. An increased liberation of glutamate leads in seizure threshold in mice (maximum electroshock to an increase of intracellular calcium50v51 which at seizure). These findings and our results support the least results in cell death. The importance of excessive idea, that the co-administration of piracetam in the calcium liberation is underlined by the fact that the antiepileptic therapy could be a useful attempt aim- calcium antagonist flunarizine counteracts the leam- ing at both seizure suppression and the prevention of ing deficit in kindled animals3’. The loss of calbindin epilepsy-related secondary disturbances in the field of in the dentate area may be another indicator for the cognition. importance of calcium in these processes. As in human epilepsy, we could demonstrate a significant neuronal loss in all hippocampal structures ACKNOWLEDGEMENT except the subiculum after completion of PTZ kindling. The question arises whether neuronal loss can This study was supported by a grant from DFG. be considered a cause for the learning deficit, since in- tact neurones can take over the function of damaged ones. 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