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Can nootropic drugs be effective against the impact of ethanol teratogenicity on cognitive performance?
European Neuropsychopharmacology 11 (2001) 33–40 www.elsevier.com / locate / euroneuro
Can nootropic drugs be effective against the impact of ethanol teratogenicity on cognitive performance? Julia Vaglenova*, Vesselin Vesselinov Petkov Laboratory of Experimental Psychopharmacology, Institute of Physiology, Bulgarian Academy of Science, 1113 Sofia, Bulgaria Received 28 September 1999; accepted 19 October 2000
Abstract Rats exposed pre- (PA) and postnatally (PNA) to ethanol at a dose of 1 g / kg for 24 h developed fetal alcohol effects (FAE). This was measured using a condition-reflex method for active avoidance with punishment reinforcement (shuttle-box) in which pronounced learning and memory deficits in 3-month-old rats were found after ethanol exposure (Vaglenova and Petkov, 1998. Fetal alcohol effects in rats exposed pre- and postnatally to a low dose of ethanol. Alcohol. Clin. Exp. Res. 22(3), 697–703). In the present study the effects of piracetam (Pyramem) at a dose of 600 mg / kg body weight, aniracetam at 50 mg / kg, and meclophenoxate (Centrophenoxine) at 100 mg / kg were studied. The drugs were administered orally during 10 days to separate groups of naive and pre- and postnatally exposed to ethanol rats. All the investigated nootropic drugs showed a significant possibility to alleviate learning and memory disability of rats with FAE. Aniracetam was administered to 1-month-old rats, demonstrating a prolonged (2 months) therapeutic effect, observed in rats aged 3 months. As previously reported (Vaglenova and Petkov, 1998), between male rats with FAE and controls, 66 and 33% were ‘poor learners’, respectively. In all nootropic treatment groups the percentage of ‘poor learners’ dropped to 28%. The positive effects of piracetam, aniracetam and meclophenoxate suggest that these drugs could be used for both treatment and prophylactic of FAE-connected disturbances of cognition. 2001 Elsevier Science B.V. All rights reserved. Keywords: Prenatal ethanol exposure; Fetal alcohol effects; Learning; Memory; Nootropic drugs
1. Introduction CNS dysfunctions in children and in experimental animals with fetal alcohol effects (FAE) are a result of ethanol teratogenicity. Deviations of the normal mental, cognitive and behavior development that can occur in the presence and absence of associated gross morphological defects manifest them. It was established that pre- (PA) and postnatal (PNA) exposure to high, moderate and low doses of ethanol cause behavior teratogenicity. The neurotoxic effects of ethanol on the developing CNS are not uniform, but are carried out by different mechanisms at different periods of development and in different brain areas. Clinical and experimental practice, however, showed *Corresponding author. Present address: Department of Pharmacal Sciences, 401 Pharmacy Bldg., Auburn University, Auburn, AL 36849, USA. Tel.: 11-334-844-8333; fax 11-334-844-8331. E-mail address: [email protected] (J. Vaglenova).
identical key components in the behavior of some victims of PA ethanol exposure, such as hyperactivity, attention deficits, learning and memory disabilities and other cognitive impairments (Streissguth and Martin, 1983). Brain, and other organs may undergo very subtle changes that can only be recognized by their belonging functional disturbances. All damages caused by teratogens, however, are stable and permanent; therefore, the fight against them using pharmacological agents is difficult. Today there are no specific pharmacological treatments recommended for children to address the syndrome problems following fetal alcohol exposure (Hannigan and Randal, 1996). Some nootropic drugs are effective against other brain disorders, caused by age, injury, ischaemic stroke, hypoxia, chemical agents, etc. It is logical to suppose that damage to the CNS, and behavior of individuals with FAE can be treated with such psychotropic drugs. The first attempts to influence some behavior reactions resulting from PA ethanol exposure were with psychothera-
0924-977X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0924-977X( 00 )00129-2
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peutic CNS stimulants (Means et al., 1984; Ulug and Reley, 1983). Clinical efficiency of methylphenidate (Ritalin), d-amphetamine (Dextrine), premoline (Cylert) and, in rarer cases caffeine, was studied with regards to attention disorders and hyperactivity in children with FAS (Elia et al., 1991; Rapport et al., 1994). No clinical data was found for the pharmacological improvement of more complicated behavior deviations such as learning and memory deficits. Pharmacological experimental data are scanty (Petkov et al., 1991, 1993; Vaglenova, 1992; Kovalev et al., 1993, Trofimov et al., 1992; Chepkova et al., 1995). These data suggest that some nootropic drugs might control learning and memory deficits in experimental rats PA exposed to ethanol. The nootropic agents in our experiments were chosen considering their pharmacological characteristics. Twentyeight years have passed since the discovery of piracetamlike nootropics. Giurgea (1972) coined the term nootropic, from Greek noos (mind) and tropos (turn) to describe the properties of 2-pyrrolidinone derivatives. These properties included enhancement of learning and memory; facilitation of the flow of the information between the cerebral hemispheres; enhancement of the resistance towards chemical and physical injuries; lack of the usual psychological and general cardiovascular pharmacological activity of psychopharmaca (Giurgea, 1972, 1980, 1982; Gouliaev and Senning, 1994). Aniracetam and rolziracetam have, among other compounds, been categorized as piracetam analogues (Gouliaev and Senning, 1994). Several compounds with nootropic action like citicholine, meclophenoxate and others have been investigated in our lab (Petkov et al., 1991, 1993; Vaglenova, 1992). It is known that nootropic drugs are harmless in clinical practice and have no side effects (Coper and Herrman, 1988). They are usually prescribed to treat brain disturbances and intellectual disorders caused by alcohol, tranquilizers, neuroleptics, depressants, barbiturates and agents impairing the brain circulation. They increase resistance of the brain to enumerating harmful actions. So-called cognition enhancers are also widely used in pediatrics and geriatrics for the treatment of cerebroischemic and encephalopathic disturbances (Platt et al., 1993). Nootropic drugs also showed a possibility for eliminating the intrauterine hypoxia in prematurely born infants and other illnesses (Foltyn et al., 1983; Gamzu et al., 1989; Voronina, 1989; Senin et al., 1991; Canonico et al., 1991; Levinson, 1991; Herrman and Stephan, 1991; Domna, 1998). Recently, piracetam has been investigated as a cerebroprotective agent in ischaemic stroke (De Deyn et al., 1997; Hitzenberger et al., 1998). The present study has been proposed to screen classic nootropic drugs — piracetam, aniracetam and meclophenoxate — for the ability to alleviate learning and memory deficits, using our model for behavior teratogenicity of ethanol in rats (Vaglenova and Petkov, 1998). Another aim was to detect if there is any prolonged effect of their action.
2. Material and methods
2.1. Animals and drugs The alcohol groups were formed by 3-month-old offspring of Wistar rats, PA and PNA exposed to ethanol and cross-fostered using the procedure reported in Vaglenova and Petkov (1998). Ethanol, 1 g / kg / 24 h (12.5% v / v in distilled water), was administered to rat mothers per oral intubation beginning on day 1 of pregnancy and continued throughout this period (22 days) and lactation (23 days). Sucrose solution (equicaloric to ethanol) was administered in the same manner to the control dams. A second group of control offspring was treated PA and PNA with water. Each dam delivered different numbers of pups. For identical rearing conditions – food (equal quantities of milk) and care — equal numbers of pups (11) were put in one nest. Each control group in our pharmacological experiments consisted of 33 3-month-old rats (offspring of sucrose and water treated dams). Piracetam (Pyramem) was used at a dose of 600 mg / kg body weight, aniracetam at a dose of 50 mg / kg, and meclophenoxate (Centrophenoxine) at 100 mg / kg. Pharmachim (Sofia, Bulgaria) provided all drugs. Piracetam and meclophenoxate were administered orally during 10 days to separate groups of 17 naive and 17 PA and PNA exposed 3-month-old rats. The doses and the chronic type of administration were chosen according to nootropics specific ‘therapeutic window’ detected by experiments in our laboratory over the past 20 years and supported by other authors. In all previous investigations, nootropic compounds were effective in models, measuring impaired cognition caused by different pharmacological agents. Nootropic drugs display a bell-shaped dose / response curve and have been effective in doses mentioned above (Gobert, 1972; Nickolson and Wolthuis, 1976; Petkov et al., 1983, 1988, 1990, 1991; Poschel et al., 1985; Mosharrof, 1987; Genkova and Lazarova, 1988; Gamzu et al., 1989; Voronina, 1989; Genkova-Papazova et al., 1993). Piracetam and meclophenoxate groups were treated 5 days before training, and the treatment was continued throughout 5 days of the learning session. Aniracetam was administered for 10 days to 17 1-month-old rats, practically 2 months before the training session started. The nootropic drugs were dissolved in distilled water. Controls were treated in the same manner as experimental animals but with distilled water in the same adequate volume (1 ml / kg). The animals were kept under standard laboratory conditions. The light / dark cycle was 12 h, starting at 06:00 h; the temperature was 20–228C and the humidity was 69–70%. Animals were given laboratory chow (SLH granules) and tap water ad libitum.
2.2. Two-way active avoidance (shuttle-box) test A repeated training procedure using 3-month-old rats with the shuttle box active avoidance test was performed
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as previously reported (Vaglenova and Petkov, 1998). The shuttle box apparatus (47.5321321 cm), divided in two equal compartments, was placed in a sound-proof room, connected by a round opening at the center. The combined presentation of a quiet buzzer (30 dB, mounted in the middle of the two compartments) and light (15 W, switched on alternately in the compartments) was used as the conditioned stimulus (CS). The unconditioned stimulus (US) was an electric shock (0.2 mA, AC) applied to the grid floor. The CS (sound and light) continued for 4 s and during this time the animals could avoid the subsequent US by passing from one compartment to the other. The US continued for 11 s, after which another cycle was started for a period of 15 s. An avoidance response was recorded when the animal avoided the US within 4 s after the onset of the CS. Each rat was trained for five consecutive days with 30 trials in each training session. A retention test was given 12 days after the last training day, the results of which show the level of intermediate memory of experimental animals (Yonkov et al., 1983; Heise, 1984).
2.3. Data and statistical analysis The data were statistically analyzed using a repeated measure analysis of variance (ANOVA). The factors of ANOVA were two: factor one — treatment with four levels (four types of treatment); and factor two — training days with six levels (five training days and one retention day). ANOVA was followed by individual post-hoc comparisons using Scheffe-test and x 2 analysis for ‘poor’ and ‘good’ learners in all groups. Offspring in each treatment group was divided into ‘poor’ and ‘good’ learners. ‘Good’ learners were defined as displaying a permanently increasing number of avoidances on each successive day of learning (2, 5, 12, 16, and 18) according to the procedure previously reported (Vaglenova and Petkov, 1998). Their number or this of ‘poor learners’ is represented versus the number of all experimental animals in each group. The number of avoidances of ‘poor learners’ were much lower with an average of 1, 2, 4, 4, 6 for each experimental day.
3. Results As previously reported ethanol exposure, 1 g / kg / 24 h significantly increased the mortality rate (23–32 vs.7% in controls) in offspring exposed to ethanol during pregnancy, continued PNA exposure had no additional effect on first month of the development. No dead pups were found after PND 28 in any treatment group. Thus, a high percentage of the week, and probably damaged, animals died before PND 28: and, in all the groups after this period, there were only healthy and strong rat pups (Vaglenova and Petkov, 1998).
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The results of our first experiment with piracetam are presented in Fig. 1. PA and PNA exposed to ethanol 3-month-old rats (E) produced a significantly lower number of avoidances from the first to the last training day and on day 12, when compared to the control group. (Figs. 1–3). Rats treated with both ethanol and piracetam (EP) showed an increasing number of avoidances from the third to the last day of the investigation as compared to the group with FAE but performed a worse learning avoidance task as compared to controls during all time points of the training. The differences in numbers of avoidances was significant during the whole investigation [F(5,190 ) 5124.26; P#0.001] for all treatment groups [F( 3,38 ) 520.06; P# 0.001]. Factor interactions were also significant [F(15,190 ) 5 20.24; P#0.001]. Learning and memory abilities of naive animals treated only with piracetam, meclophenoxate or only with aniracetam in the shuttle-box avoidance task did not differ at any of the time points of the investigation when compared to each of their control groups (Figs. 1–3). Aniracetam treatment experiments showed learning and memory improvement effects of the nootropic agent (Fig. 2) on animals with FAE. We found significant main effects of the treatment factor [F( 3, 35 ) 516.52; P#0.001], the time factor [F(5, 176) 5263.62; P#0.001] and also the factor interactions [F( 15, 176) 598.49; P#0.001]. The post-hoc analysis also showed a full normalization of learning (significantly increasing number of avoidances when compared to only ethanol treated rats). Ethanol–aniracetam (EA) group gave significantly less condition responses only on day 5 of the investigation as compared to controls. Under our experimental conditions, the rats treated only with meclophenoxate, did not show significantly better results in acquiring the two-way active avoidance tasks. Meclophenoxate applied to rats exposed PA and PNA to ethanol (EM group) normalized their conditioned-reflex activity, i.e. the number of avoidances was not significantly different from that of the controls, but was higher (in all training days) than the number of rats with FAE [F( 3,38 ) 521.05; P#0.001] (Fig. 3). When we performed retention test on day 12, we observed that the nootropic drugs aniracetam, piracetam and meclophenoxate administered to animals with FAE, significantly increased not only the acquisition, but also completely restored intermediate memory (Figs. 1–3). In the present study, two-thirds of the offspring of the control groups were ‘good learners’. This number dropped to 40% in PA- and PNA exposed (E) groups (Table 1). These last groups were significantly different from the controls: P#0.05. The percentage of ‘good learners’ among the ethanol– nootropic groups — EP, EA and meclophenoxate (EM) — were significantly increased when compared to the E group. On the contrary, the percentage of ‘poor learners’ dropped significantly.
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Fig. 1. Effect of piracetam (600 mg / kg body weight, administered 10 days per os) on learning and memory of rats with FAE (E). Left ordinate: number of avoidances. Right ordinate: the type of administration (E) rats pre- and postnatally exposed to ethanol. P, piracetam treatment rats; EP, piracetam treatment ethanol exposed pre- and postnatally rats; C, controls rats exposed pre and postnatally to sucrose. Abscissa: training days. * Statistical significance vs. controls (P#0.001). 8 Statistical significance vs. piracetam group (P#0.001).
4. Discussion As previously reported (Vaglenova and Petkov, 1998), PA and PNA ethanol exposure results in long-lasting impairment of performance in learning and memory tasks, indicating that ethanol administered even at a very low dose during gestation acts as a behavioral teratogen in the offspring. The behavioral teratogenicity of ethanol has been evidenced by deficits in performance of adolescent, mature and old rats in a task that measured learning and memory deficits. In our present investigation we used mature rats (3-month-old) with evident FAE. Nootropic drugs piracetam, aniracetam and meclophenoxate completely abolished their learning and memory. This finding supports our previous data for retention-improving effects of piracetam on rats exposed PA and PNA to a much higher dose of ethanol (9 g / kg body weight) (Petkov et al., 1991). Our present results showed a common capability of nootropic drugs belonging to different chemical families to improve learning and memory of rats with FAE. Cognitive disturbances appear after PA- and PNA-exposure and those caused by low doses of ethanol can be observed using a more complicated learning task (see Vaglenova and Petkov, 1998; Kerr and Hindmarch, 1998). Piracetam, aniracetam and meclophenoxate were found to be promising for
controlling the cognitive deficits induced by PA and PNA ethanol exposure and it was also evident that the type of nootropic drug was not of essential importance. Interestingly, using nootropic agents did not change learning and memory when they have been applied on naive experimental animals. These observations are supported by other authors (Gamzu et al., 1989). A 5-day training two-way avoidance task allowed us to establish that three studied nootropic drugs can improve not only learning and memory, but are also able to increase significantly the percentage of ‘good learners’ in a group of FAE rats. The percentage of ‘good learners’ in a group of FAE rats, treated with nootropic drugs is the same as that of controls and nootropic groups (P, A, M). It has also been shown that aniracetam can efficiently prevent learning and memory deficits in rats PA and PNA exposed to ethanol before their appearance, during adolescence and maturity. Aniracetam was administered to young, 1-month-old rats and showed a prolonged (2 months) prophylactic effect, observed in rats aged 3 months. To explain the identical beneficial effects of the three different nootropic agents, their pharmacological characteristics and the mechanism of action of ethanol on the developing brain should be taken into account. According to a general opinion, multiple mechanisms are involved in the teratogenic action of ethanol. We found that fetal
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Fig. 2. Effect of aniracetam (50 mg / kg body weight, administered 10 days per os on 1-month-old rats) on learning and memory of 3-month-old FAE rats (E). Left ordinate: number of avoidances. Right ordinate: the type of administration (E) rats pre- and postnatally exposed to ethanol. A, piracetam treatment rats; EA, piracetam treatment ethanol exposed pre- and postnatally rats; C, controls rats exposed pre and postnatally to sucrose. Abscissa: training days. * Statistical significance vs. controls (P#0.001). 8 Statistic significance vs. aniracetam group (P#0.001).
alcohol exposure increased levels of lipid peroxidation in all brain areas measured in rats, including neocortex, hippocampus and cerebellum (Petkov et al., 1992). Nootropic agents alter brain biomembranes by accumulating into them, and might change the membrane fluidity by partitioning into the phospholipid bilayer (Ostrowski et al., 1975; Ostrowski and Keil, 1978). Piracetam changes the membrane physical properties because it interacts with phosphate head groups of artificial phospholipid bilayers (Pevout et al., 1995). The metabolites of meclophenoxate also accumulate in the nerve cell membranes and remain there for a long time. Their strong OH ? radical scavenger ability was discovered by Nagy (1994). Meclophenoxate increases the synthesis of the alcalin ethanolamine phospholipids, lecitin and holin. All these events lead to the stabilization of brain biomembranes and are a basis for the fixation of traces of the neuronal circle. Precisely, these traces are the biological structure of memory (Vaglenova, 1992). When favorable effects of nootropic drugs on the CNS dysfunction in animals and humans are discussed, their alteration at neurotransmitter systems should be considered too. It has been concluded that in utero and early PNA exposure to ethanol markedly impairs several neurotransmitter systems. Using our model of behavior teratogenicity of ethanol
(Vaglenova and Petkov, 1998) alterations in muscarin cholinoceptors and b-adrenoceptors in the hippocampus with the peak of the blood ethanol concentration (BAC) of 35 mg% (Hadjiivanova et al., 1991; Vaglenova, 1992) were demonstrated. Changes in the synapses and activity of enzymes associated with the synthesis and degeneration of many CNS transmitters in amygdala and hippocampus were also found (Lolova et al., 1987, 1989; Vaglenova, 1992) with a higher ethanol dose. Piracetam increases the density of muscarinic cholinergic receptors in the frontal ¨ cortex of aged rats (Pilch and Muller, 1988) which pointed to the involvement of cholinergic effects in learning and memory. On the other hand, there are data that piracetam, aniracetam and meclophenoxate induced alterations on the level and turnover of the biogenic monoamines in the main brain structures: cortex, striatum, hippothalmus, and pons (Petkov et al., 1984, 1985). Therefore, the level of activity of different brain neurotransmitter systems and the relations between them determine the character of the cognitive processes in each type of training task. In recent publications, a new research line has shown the interactions between long-term potentiation (LTP)) that is a cell model of cognition and the glutamate transmission. Lynch (1998) discussed that LTP is expressed by changes in NMDA and AMPA receptors operations. A main
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Fig. 3. Effect of meclophenoxate (100 mg / kg body weight, administered 10 days per os) on learning and memory of rats with FAE (E). Left ordinate: number of avoidances. Right ordinate: the type of administration (E) rats pre- and postnatally exposed to ethanol. M, meclophenoxate treatment rats; EM, meclophenoxate treatment ethanol exposed pre- and postnatally rats; C, controls rats exposed pre and postnatally to sucrose. Abscissa: training days. * Statistical significance vs. controls (P#0.001). 8 Statistical significance vs. meclophenoxate group (P#0.001).
hypothesis emerges that enhancement of the NMDA receptor tone increases cognitive performance. Low ethanol concentration (BACs as low as 30 mg%) leads to significant reduction of NMDA receptors in hippocampus and cerebellum during their formation (Savage et al., 1991; Valles et al., 1995). Hamelin and Lehmann (1995) showed that piracetam and aniracetam could act like cognition Table 1 Stratification to ‘good’ and ‘poor’ learners of rats with FAE, treated with piracetam (600 mg / kg), aniracetam (50 mg / kg), and meclophenoxate (100 mg / kg) during the performance shuttle-box avoidance task a Groups
‘Good learners’ (%)
‘Poor learners’ (%)
Sucrose (n533) Water (n533) FAE (n525) Meclophenoxate (n517) FAE1Mecloph. (n517) Piracetam (n517) FAE1Piracetam (n517) Aniracetam (n517) FAE1Aniracetam (n517)
66.6 (n522) 66.6 (n522) 40.0* (n510) 71.2 † (n512)
33.3 (n511) 33.3 (n511) 60.0* (n515) 28.8 † (n55)
71.2 † (n512) 71.2 † (n512)
28.8 † (n55) 28.8 † (n55)
71.2 † (n512)
28.8 † (n55)
71.2 † (n512)
28.8 † (n55)
71.2 † (n512)
28.8 † (n55)
a n5number of animals; * P#0.05 compared to control (sucrose or water); † P#0.05 compared to animals with FAE.
enhancers via at least some subtypes of NMDA receptors. High levels of aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors (Fallarino et al., 1995). Aniracetam reduced AMPA-receptor desensitisation (Isaacson and Nicol, 1991; Tang et al., 1991) by allosteric potentiation of quisqualate receptors (Ito et al., 1990), thus amplified excitatory synaptic transmission. Piracetam also interacts with the brain glutamate transmission (see Gouliaev and Senning, 1994). Finally, we can conclude that nootropic drugs have favorable effects on cognitive disruptions of rats pre- and postnatally exposed to ethanol. These agents are effective a long time after stopping the alteration of the teratogen ethanol. We accept that the recovery of memory traces in rats with FAE are probably carried into effect by a normalization of the protein synthesis, disrupted in an earlier stage of ontogenesis and also by the activation and recovery of the equilibrium of monoaminergic, cholinergic and glutamatergic transmitter systems in different brain structures. Also, nootropic drugs could act as scavengers of lipid peroxidation, provoked by perinatal exposure to ethanol. By all affections mentioned above, nootropic drugs support neuronal plasticity in adult rat brain, disturbed during development by ethanol exposure. Therefore, we have completed a positive answer to the question: can nootropic drugs be effective against the impact of the
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behavioral teratogenicity of ethanol or its main characteristics — learning and memory disability?
Acknowledgements The authors gratefully acknowledge Dr Mauro Santos ´ from Departmento di Genetica y Microbiologia, Universitat Autonoma de Barcelona, Spain for the statistical analysis.
References Canonico, P.L., Forgione, L., Paoletti, C., Casini, A., Collona, C.V., Bertini, M., Acito, R., Rengo, F., 1991. Efficacy and tolerance of aniracetam in elderly patients with primary or secondary mental deterioration. Riv. Neurol. 61, 92–96. Chepkova, A.N., Doreulee, N.V., Trofimov, S.S., Gudasheva, T.A., Ostrovskay, R.U., Skrebitsky, V.G., 1995. Nootropic compound Lpyroglutamyl-D-alanine-amide restores hippocampal long-term potentiation impaired by exposure to ethanol in rats. Neurosci. Lett. 188, 163–166. Coper, H., Herrman, W.M., 1988. Psychostimulants, analeptics, nootropics: an attempt to differentiate and assess drugs designed for the treatment of impaired brain functions. Pharmacopsychiatry 21, 211– 217. Domna, M.M., 1998. Clinical efficacy of piracetam in treatment of breath-holding spells. Pediatr. Neurol. 18 (1), 41–45. De Deyn, P.P., De Reuck, J., Deberdt, W., 1997. Treatment of acute ischemic stroke with piracetam. Stroke 28, 2347–2352. Elia, J., Borcherdinging, B.G., Raport, J.L., Keysor, C.S., 1991. Methylphenidate and dextroamphetamine treatments of hyperactivity: Are there true nonresponders? Psychiatry Res. 36, 141. Fallarino, F., Genazzani, A.A., Silla, S., L’Episcopo, M.R., Camici, O., Corazzi, L., Nicoletti, F., Fioretti, M.C., 1995. [ 3 H]aniracetam binds to specific recognition sites in brain membranes. J. Neurochem. 65, 912–918. Foltyn, P., Lucker, P.W., Schnitker, J., Wetzelsberger, N., 1983. A test model for cerebrally active drugs as demonstrated by the example of the new substance aniracetam. Arzneim.-Forsch. Drug Res. 33, 865– 867. Gamzu, E.R., Hoover, T.M., Gracon, S.I., Ninteman, M.V., 1989. Recent developments in 2-pyrolidine-containing nootropic. Drug Dev. Res. 18, 177–189. Genkova, M.G., Lazarova, M.B., 1988. Influence of fipexide on the learning and memory impairing effects of diethyldithiocarbamate, clonidine and electroconvulsive shock in albino rats. C.R. Acad. Bulg. Sci. 41 (3), 73–76. Genkova-Papazova, M.G., Stancheva, S., Alova, L.G., LazarovaBaracova, M.B., 1993. Adafenoxate abolishes the amnesia induced by neonatal 6-hydroxydopamine treatment in rats. Methods Find. Exp. Clin. Pharmacol. 15 (5), 267–271. ´ Giurgea, C., 1972. Vers une pharmacologie de l’activite´ integrative du cerveau. Tentative du concept nootrope en psychopharmacologie. Actual Pharmacol. 25, 115–156. Giurgea, C., 1980. A drug for the mind. Chem. Technol. 10, 360–365. Giurgea, C., 1982. The nootropic concept and its prospective implications. Drug Dev. Res. 2, 441–446. Gobert, J.G., 1972. Genese d un medicaments: Le piracetam. Metabolisation et recheche biochemique. J. Pharm. Belg. 27, 281–304. Gouliaev, A.H., Senning, A., 1994. Piracetam and other structurally related nootropics (review). Brain Res. Dev. 19, 180–222. Hadjiivanova, C., Petkov, V.D., Alova, L., Petkov, V.V., Vaglenova, J.,
39
1991. Effects of chronic maternal ethanol consumption on offspring brain muscarinic and beta-adrenoceptors. Acta Physiol. Pharmacol. Bulg. 17 (1), 44–51. Hamelin, S.M., Lehmann, J.C., 1995. Effects of putative cognition enhancers on the NMDA receptor by [ 3 H]MK801 binding. Eur. J. Pharmacol. 281, R11–R13. Hannigan, J.H., Randal, S., 1996. Behavioral pharmacology in animals exposed prenatally to alcohol. In: Abel, E.L. (Ed.), Fetal Alcohol Syndrome from Mechanism to Prevention. CRC Press, Boca Raton, FL, pp. 191–213. Heise, G.A., 1984. Behavioral methods for measuring effects of drugs on learning and memory in animals. Med. Res. Rev. 4, 535–546. Herrman, W.M., Stephan, K., 1991. Efficacy and clinical relevance of cognition enhancers. Alzheimer Dis. Assoc. Disord. 5, 7–12. Hitzenberger, G., Rameis, H., Manigley, C., 1998. Pharmacological properties of piracetam. Rationale for use in stroke patients. CNS Drugs 9 (1), 19–27. Isaacson, J., Nicol, R.A., 1991. Aniracetam reduced glutamate receptor desensitization and slows a decay of fast excitatory synaptic currents in the hippocampus. Proc. Natl. Acad. Sci. USA 88, 10936–10940. Ito, I., Tanabe, S., Kohda, A., Sugiyama, H., 1990. Allosteric potentiation of quisqualate receptors by a nootropic drug aniracetam. J. Physiol. (Lond.) 424, 533–543. Kerr, J.S., Hindmarch, I., 1998. The effects of alcohol alone or in combination with other drugs on information processing, task performance and subjective responses. Hum. Psychopharmacol. Clin. Exp. 13 (1), 1–9. Kovalev, G.I., Budigin, E.A., Gainetdinov, R.R., Kudrin, V.S., Trofimov, S.S., Ostrovskay, R.U., 1993. The sodium oxybutyrate and nooglutil correction of dopamine release in the striatum of prenatally alcoholized rat pups. Biull. Eksp. Biol. Med. 116 (7), 56–58. Levinson, H.N., 1991. Dramatic favorable responses of children with learning disabilities or dyslexia and attention deficit disorder to antimotion sickness medications: four case reports. Percept. Mot. Skills 73, 723–738. Lolova, I., Petkov, V.V., Vaglenova, J., 1987. Enzyme histochemical studies on rat amygdala in fetal alcohol syndrome. Folia Histochim. Citobiol. 25 (2), 119–128. Lolova, I., Lolov, V., Petkov, V.V., Vaglenova, J., 1989. Changes in the synapses of the rat hippocampus following pre- and postnatal alcohol exposure. Anat. Anz. 169, 285–294. Lynch, G., 1998. Memory and the brain: unexpected chemistries and a new pharmacology. Neurobiol. Learn. Memory 70, 82–90. Means, L.W., Medlin, C.W., Hughes, V.D., Gray, S.L., 1984. Rats exposed in utero to ethanol are hyperresponsive to methylphenidate when tested as neonates or adults. Neurobehav. Toxicol. Teratol. 6, 187. Mosharrof, A.H.M., 1987. Investigation of effects of synthetic and plants psychopharmacological agents on learning and memory. PhD thesis, Sofia, Bulgaria, 204 p. Nagy, Z.S.I., 1994. A survey of the available data on a new nootropic drug, BCE-001. Ann. NY Acad. Sci. 10 (717), 102–114. Nickolson, V.J., Wolthuis, O.L., 1976. Effect of the acquisition-enhancing drug piracetam on rat cerebral energy metabolism. Comparison with naftidrofuryl and methamphetamine. Biochem. Pharmacol. 25, 2241– 2244. Ostrowski, J., Keil, M., 1978. Autoradiographische untersuchungen zur verteilung von piracetam 14 C im affengehirm. Arzneim.-Forsch. Drug Res. 25 (1), 29–35. Ostrowski, J., Keil, M., Schraven, E., 1975. Autoradiographische untersuchungen zur verteilung von piracetam 14 C bei ratte un hund. Arzneim.-Forsch. Drug Res. 25 (4), 589–596. Petkov, V.V., Grahovska, T., Petkov, V.D., Konstantinova, E., 1985. Effects of meclophenoxate on the level and turnover of biogenic monoamines in the rat brain. Arzneim.-Forsch. Drug Res. 35, 1778–1781. Petkov, V.D., Grahovska, T., Petkov, V.V., Konstantinova, E., Stancheva, S., 1984. Changes in the brain biogenic monoamines of rats infused by piracetam and aniracetam. Acta Physiol. Pharmacol. Bulg. 10 (4), 6–15.
40
J. Vaglenova, V. Vesselinov Petkov / European Neuropsychopharmacology 11 (2001) 33 – 40
Petkov, V.D., Kehayov, R.A., Mosharoff, A.H., Petkov, V.V., Getova, D., Lazarova, M.B., Vaglenova, J., 1993. Effects of cytidine diphosphate choline on rats with memory deficits. Arzneim.-Forsch. Drug Res. 43 (8), 822–828. Petkov, V.D., Konstantinova, E., Petkov, V.V., Vaglenova, J., 1991. Learning and memory in rats exposed pre- and postnatally to alcohol. An attempt at pharmacological control. Methods Find. Exp. Clin. Pharmacol. 13, 43–50. Petkov, V.D., Mosharoff, A., Petkov, V.V., 1988. Comparative studies on the effects of adenofenoxate, meclophenoxate and piracetam and citicholine on scopolamine — impaired memory, exploratory behavior and physical capabilities (Experiments on rats and mice). Acta Physiol. Pharmacol. Bulg. 1, 3–13. Petkov, V.D., Mosharoff, A., Petkov, V.V., Kehayov, P., 1990. Age-related differences in memory and in the memory effects of nootropic drugs. Acta Physiol. Pharmacol. Bulg. 16, 28–35. Petkov, V.V., Rousseva, S., Dimov, S., 1983. Behavioral effects of piracetam in rats with isolation syndrome. Acta Physiol. Pharmacol. Bulg. 10, 3–9. Petkov, V.V., Stoianovski, D., Petkov, V.D., Vaglenova, J., 1992. Lipid peroxidation changes in the brain in fetal alcohol syndrome. Biull. Eksp. Biol. Med. 113 (5), 500–502. Pevout, J., Schank, A., Deleers, M., Brasseur, R., 1995. Piracetam induced changes to membrane physical properties. Biochem. Pharmacol. 50, 1129–1134. ¨ Pilch, H., Muller, W.E., 1988. Piracetam elevates muscarinic cholinergic receptor density in the frontal cortex of age but not in young mice. Psychopharmacology 94, 74–78. Platt, D., Horn, J., Summa, J.-D., Achmitt-Ruth, R., Kauntz, J., Kronert, E., 1993. On the efficacy of piracetam in geriatric patients with acute cerebral ischemia: a clinically controlled double-blind study. Arch. Gerontol. Geriatr. 16, 149. Poschel, B.P.H., Ho, P.M., Ninteman, E.W., Callahan, M.J., 1985. Pharmacologic therapeutic window of piracetam demonstrated in behavior. EEG and single neuron firing rates. Experientia 41, 1153– 1156. Rapport, M.D., Denny, C., DupPaul, G.J., Garder, M.J., 1994. Attentional deficit disorder and methylphenidate: Normalization rates, clinical effectiveness, and response in 76 children. J. Am. Acad. Child. Adolesc. Psychol. 33, 882.
Savage, D.D., Montano, C.Y., Otero, M.A., Paxton, L.L., 1991. Prenatal ethanol exposure decreases hippocampal NMDA-sensitive 3 H-glutamate binding site density in 45-day-old rats. Alcohol 8, 193–201. Senin, U., Abate, G., Fieschi, C., Gori, G., Guala, A., Marini, G., Villardita, C., Parnettti, L., 1991. Aniracetam (Ro 13-5057) in the treatment of senile dementia of Alzheimer’s type (SDAT): results of a placebo controlled multicentre clinical study. Eur. Neuropsychopharmacol. 1, 511–517. Streissguth, A.P., Martin, J.C., 1983. Prenatal effects of alcohol abuse in humans and laboratory animals. In: Kissin, P., Begleiter, H. (Eds.), The Pathogenesis of Alcoholism. York, Plenum Press, New, pp. 539–589. Tang, C.-M., Shi, Q.-Y., Katchman, A., Lynch, G., 1991. Modulation of the time course of fast EPSCs and glutamate channel kinetics by aniracetam. Science 254, 288–290. Trofimov, S.S., Ostrovskay, R.U., Smolnikova, N.M., Khromova, I.V., Krapivin, S.V., Krilova, A.M., Kovalev, G.I., Gainetdinov, R.R., Liubimov, B.I., Voronina, T.A., 1992. The nooglutil correction of functional disorders of central nervous system caused by prenatal alcoholization in rats. Eksp. Klin. Farmakol. 55 (1), 18–21. Ulug, S., Reley, E.P., 1983. The effect of methylphenidate on overactivity in rats prenatally exposed to alcohol. Neurobehav. Toxicol. Teratol. 5, 35–39. Vaglenova, J., Petkov, V.V., 1998. Fetal Alcohol Effects in rats exposed pre- and postnatally to a low dose of ethanol. Alcohol. Clin. Exp. Res. 22 (3), 697–703. Vaglenova, J., 1992. Fetal alcohol syndrome in rats and correction with nootropic drugs. PhD thesis, Sofia, Bulgaria, 171 p. Valles, S., Felipo, V., Montoliu, C., Guerri, C., 1995. Alcohol exposure during brain development reduces 3 H-MK-801 binding and enhances metabotropic-glutamate receptor-stimulated phosphoinositide hydrolysis in rat hippocampus. Life Sci. 56, 1373–1383. Yonkov, D.I., Petkov, V.V., Roussinov, K., Vaglenova, J., 1983. A new model of apparatus for two-way avoidance (shuttle-box) for pharmacological memory studies of small laboratory animals. C.R. Acad. Bulg. Sci. 36 (8), 1123–1126. Voronina, T.A., 1989. In: Valdman, A.V., Voronina, T.A. (Eds.), Pharmacology of Nootropics. Experimental and Clinical Studies. Nauka, Moscow, p. 105.
Can nootropic drugs be effective against the impact of ethanol teratogenicity on cognitive performance? Julia Vaglenova*, Vesselin Vesselinov Petkov Laboratory of Experimental Psychopharmacology, Institute of Physiology, Bulgarian Academy of Science, 1113 Sofia, Bulgaria Received 28 September 1999; accepted 19 October 2000
Abstract Rats exposed pre- (PA) and postnatally (PNA) to ethanol at a dose of 1 g / kg for 24 h developed fetal alcohol effects (FAE). This was measured using a condition-reflex method for active avoidance with punishment reinforcement (shuttle-box) in which pronounced learning and memory deficits in 3-month-old rats were found after ethanol exposure (Vaglenova and Petkov, 1998. Fetal alcohol effects in rats exposed pre- and postnatally to a low dose of ethanol. Alcohol. Clin. Exp. Res. 22(3), 697–703). In the present study the effects of piracetam (Pyramem) at a dose of 600 mg / kg body weight, aniracetam at 50 mg / kg, and meclophenoxate (Centrophenoxine) at 100 mg / kg were studied. The drugs were administered orally during 10 days to separate groups of naive and pre- and postnatally exposed to ethanol rats. All the investigated nootropic drugs showed a significant possibility to alleviate learning and memory disability of rats with FAE. Aniracetam was administered to 1-month-old rats, demonstrating a prolonged (2 months) therapeutic effect, observed in rats aged 3 months. As previously reported (Vaglenova and Petkov, 1998), between male rats with FAE and controls, 66 and 33% were ‘poor learners’, respectively. In all nootropic treatment groups the percentage of ‘poor learners’ dropped to 28%. The positive effects of piracetam, aniracetam and meclophenoxate suggest that these drugs could be used for both treatment and prophylactic of FAE-connected disturbances of cognition. 2001 Elsevier Science B.V. All rights reserved. Keywords: Prenatal ethanol exposure; Fetal alcohol effects; Learning; Memory; Nootropic drugs
1. Introduction CNS dysfunctions in children and in experimental animals with fetal alcohol effects (FAE) are a result of ethanol teratogenicity. Deviations of the normal mental, cognitive and behavior development that can occur in the presence and absence of associated gross morphological defects manifest them. It was established that pre- (PA) and postnatal (PNA) exposure to high, moderate and low doses of ethanol cause behavior teratogenicity. The neurotoxic effects of ethanol on the developing CNS are not uniform, but are carried out by different mechanisms at different periods of development and in different brain areas. Clinical and experimental practice, however, showed *Corresponding author. Present address: Department of Pharmacal Sciences, 401 Pharmacy Bldg., Auburn University, Auburn, AL 36849, USA. Tel.: 11-334-844-8333; fax 11-334-844-8331. E-mail address: [email protected] (J. Vaglenova).
identical key components in the behavior of some victims of PA ethanol exposure, such as hyperactivity, attention deficits, learning and memory disabilities and other cognitive impairments (Streissguth and Martin, 1983). Brain, and other organs may undergo very subtle changes that can only be recognized by their belonging functional disturbances. All damages caused by teratogens, however, are stable and permanent; therefore, the fight against them using pharmacological agents is difficult. Today there are no specific pharmacological treatments recommended for children to address the syndrome problems following fetal alcohol exposure (Hannigan and Randal, 1996). Some nootropic drugs are effective against other brain disorders, caused by age, injury, ischaemic stroke, hypoxia, chemical agents, etc. It is logical to suppose that damage to the CNS, and behavior of individuals with FAE can be treated with such psychotropic drugs. The first attempts to influence some behavior reactions resulting from PA ethanol exposure were with psychothera-
0924-977X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0924-977X( 00 )00129-2
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peutic CNS stimulants (Means et al., 1984; Ulug and Reley, 1983). Clinical efficiency of methylphenidate (Ritalin), d-amphetamine (Dextrine), premoline (Cylert) and, in rarer cases caffeine, was studied with regards to attention disorders and hyperactivity in children with FAS (Elia et al., 1991; Rapport et al., 1994). No clinical data was found for the pharmacological improvement of more complicated behavior deviations such as learning and memory deficits. Pharmacological experimental data are scanty (Petkov et al., 1991, 1993; Vaglenova, 1992; Kovalev et al., 1993, Trofimov et al., 1992; Chepkova et al., 1995). These data suggest that some nootropic drugs might control learning and memory deficits in experimental rats PA exposed to ethanol. The nootropic agents in our experiments were chosen considering their pharmacological characteristics. Twentyeight years have passed since the discovery of piracetamlike nootropics. Giurgea (1972) coined the term nootropic, from Greek noos (mind) and tropos (turn) to describe the properties of 2-pyrrolidinone derivatives. These properties included enhancement of learning and memory; facilitation of the flow of the information between the cerebral hemispheres; enhancement of the resistance towards chemical and physical injuries; lack of the usual psychological and general cardiovascular pharmacological activity of psychopharmaca (Giurgea, 1972, 1980, 1982; Gouliaev and Senning, 1994). Aniracetam and rolziracetam have, among other compounds, been categorized as piracetam analogues (Gouliaev and Senning, 1994). Several compounds with nootropic action like citicholine, meclophenoxate and others have been investigated in our lab (Petkov et al., 1991, 1993; Vaglenova, 1992). It is known that nootropic drugs are harmless in clinical practice and have no side effects (Coper and Herrman, 1988). They are usually prescribed to treat brain disturbances and intellectual disorders caused by alcohol, tranquilizers, neuroleptics, depressants, barbiturates and agents impairing the brain circulation. They increase resistance of the brain to enumerating harmful actions. So-called cognition enhancers are also widely used in pediatrics and geriatrics for the treatment of cerebroischemic and encephalopathic disturbances (Platt et al., 1993). Nootropic drugs also showed a possibility for eliminating the intrauterine hypoxia in prematurely born infants and other illnesses (Foltyn et al., 1983; Gamzu et al., 1989; Voronina, 1989; Senin et al., 1991; Canonico et al., 1991; Levinson, 1991; Herrman and Stephan, 1991; Domna, 1998). Recently, piracetam has been investigated as a cerebroprotective agent in ischaemic stroke (De Deyn et al., 1997; Hitzenberger et al., 1998). The present study has been proposed to screen classic nootropic drugs — piracetam, aniracetam and meclophenoxate — for the ability to alleviate learning and memory deficits, using our model for behavior teratogenicity of ethanol in rats (Vaglenova and Petkov, 1998). Another aim was to detect if there is any prolonged effect of their action.
2. Material and methods
2.1. Animals and drugs The alcohol groups were formed by 3-month-old offspring of Wistar rats, PA and PNA exposed to ethanol and cross-fostered using the procedure reported in Vaglenova and Petkov (1998). Ethanol, 1 g / kg / 24 h (12.5% v / v in distilled water), was administered to rat mothers per oral intubation beginning on day 1 of pregnancy and continued throughout this period (22 days) and lactation (23 days). Sucrose solution (equicaloric to ethanol) was administered in the same manner to the control dams. A second group of control offspring was treated PA and PNA with water. Each dam delivered different numbers of pups. For identical rearing conditions – food (equal quantities of milk) and care — equal numbers of pups (11) were put in one nest. Each control group in our pharmacological experiments consisted of 33 3-month-old rats (offspring of sucrose and water treated dams). Piracetam (Pyramem) was used at a dose of 600 mg / kg body weight, aniracetam at a dose of 50 mg / kg, and meclophenoxate (Centrophenoxine) at 100 mg / kg. Pharmachim (Sofia, Bulgaria) provided all drugs. Piracetam and meclophenoxate were administered orally during 10 days to separate groups of 17 naive and 17 PA and PNA exposed 3-month-old rats. The doses and the chronic type of administration were chosen according to nootropics specific ‘therapeutic window’ detected by experiments in our laboratory over the past 20 years and supported by other authors. In all previous investigations, nootropic compounds were effective in models, measuring impaired cognition caused by different pharmacological agents. Nootropic drugs display a bell-shaped dose / response curve and have been effective in doses mentioned above (Gobert, 1972; Nickolson and Wolthuis, 1976; Petkov et al., 1983, 1988, 1990, 1991; Poschel et al., 1985; Mosharrof, 1987; Genkova and Lazarova, 1988; Gamzu et al., 1989; Voronina, 1989; Genkova-Papazova et al., 1993). Piracetam and meclophenoxate groups were treated 5 days before training, and the treatment was continued throughout 5 days of the learning session. Aniracetam was administered for 10 days to 17 1-month-old rats, practically 2 months before the training session started. The nootropic drugs were dissolved in distilled water. Controls were treated in the same manner as experimental animals but with distilled water in the same adequate volume (1 ml / kg). The animals were kept under standard laboratory conditions. The light / dark cycle was 12 h, starting at 06:00 h; the temperature was 20–228C and the humidity was 69–70%. Animals were given laboratory chow (SLH granules) and tap water ad libitum.
2.2. Two-way active avoidance (shuttle-box) test A repeated training procedure using 3-month-old rats with the shuttle box active avoidance test was performed
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as previously reported (Vaglenova and Petkov, 1998). The shuttle box apparatus (47.5321321 cm), divided in two equal compartments, was placed in a sound-proof room, connected by a round opening at the center. The combined presentation of a quiet buzzer (30 dB, mounted in the middle of the two compartments) and light (15 W, switched on alternately in the compartments) was used as the conditioned stimulus (CS). The unconditioned stimulus (US) was an electric shock (0.2 mA, AC) applied to the grid floor. The CS (sound and light) continued for 4 s and during this time the animals could avoid the subsequent US by passing from one compartment to the other. The US continued for 11 s, after which another cycle was started for a period of 15 s. An avoidance response was recorded when the animal avoided the US within 4 s after the onset of the CS. Each rat was trained for five consecutive days with 30 trials in each training session. A retention test was given 12 days after the last training day, the results of which show the level of intermediate memory of experimental animals (Yonkov et al., 1983; Heise, 1984).
2.3. Data and statistical analysis The data were statistically analyzed using a repeated measure analysis of variance (ANOVA). The factors of ANOVA were two: factor one — treatment with four levels (four types of treatment); and factor two — training days with six levels (five training days and one retention day). ANOVA was followed by individual post-hoc comparisons using Scheffe-test and x 2 analysis for ‘poor’ and ‘good’ learners in all groups. Offspring in each treatment group was divided into ‘poor’ and ‘good’ learners. ‘Good’ learners were defined as displaying a permanently increasing number of avoidances on each successive day of learning (2, 5, 12, 16, and 18) according to the procedure previously reported (Vaglenova and Petkov, 1998). Their number or this of ‘poor learners’ is represented versus the number of all experimental animals in each group. The number of avoidances of ‘poor learners’ were much lower with an average of 1, 2, 4, 4, 6 for each experimental day.
3. Results As previously reported ethanol exposure, 1 g / kg / 24 h significantly increased the mortality rate (23–32 vs.7% in controls) in offspring exposed to ethanol during pregnancy, continued PNA exposure had no additional effect on first month of the development. No dead pups were found after PND 28 in any treatment group. Thus, a high percentage of the week, and probably damaged, animals died before PND 28: and, in all the groups after this period, there were only healthy and strong rat pups (Vaglenova and Petkov, 1998).
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The results of our first experiment with piracetam are presented in Fig. 1. PA and PNA exposed to ethanol 3-month-old rats (E) produced a significantly lower number of avoidances from the first to the last training day and on day 12, when compared to the control group. (Figs. 1–3). Rats treated with both ethanol and piracetam (EP) showed an increasing number of avoidances from the third to the last day of the investigation as compared to the group with FAE but performed a worse learning avoidance task as compared to controls during all time points of the training. The differences in numbers of avoidances was significant during the whole investigation [F(5,190 ) 5124.26; P#0.001] for all treatment groups [F( 3,38 ) 520.06; P# 0.001]. Factor interactions were also significant [F(15,190 ) 5 20.24; P#0.001]. Learning and memory abilities of naive animals treated only with piracetam, meclophenoxate or only with aniracetam in the shuttle-box avoidance task did not differ at any of the time points of the investigation when compared to each of their control groups (Figs. 1–3). Aniracetam treatment experiments showed learning and memory improvement effects of the nootropic agent (Fig. 2) on animals with FAE. We found significant main effects of the treatment factor [F( 3, 35 ) 516.52; P#0.001], the time factor [F(5, 176) 5263.62; P#0.001] and also the factor interactions [F( 15, 176) 598.49; P#0.001]. The post-hoc analysis also showed a full normalization of learning (significantly increasing number of avoidances when compared to only ethanol treated rats). Ethanol–aniracetam (EA) group gave significantly less condition responses only on day 5 of the investigation as compared to controls. Under our experimental conditions, the rats treated only with meclophenoxate, did not show significantly better results in acquiring the two-way active avoidance tasks. Meclophenoxate applied to rats exposed PA and PNA to ethanol (EM group) normalized their conditioned-reflex activity, i.e. the number of avoidances was not significantly different from that of the controls, but was higher (in all training days) than the number of rats with FAE [F( 3,38 ) 521.05; P#0.001] (Fig. 3). When we performed retention test on day 12, we observed that the nootropic drugs aniracetam, piracetam and meclophenoxate administered to animals with FAE, significantly increased not only the acquisition, but also completely restored intermediate memory (Figs. 1–3). In the present study, two-thirds of the offspring of the control groups were ‘good learners’. This number dropped to 40% in PA- and PNA exposed (E) groups (Table 1). These last groups were significantly different from the controls: P#0.05. The percentage of ‘good learners’ among the ethanol– nootropic groups — EP, EA and meclophenoxate (EM) — were significantly increased when compared to the E group. On the contrary, the percentage of ‘poor learners’ dropped significantly.
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Fig. 1. Effect of piracetam (600 mg / kg body weight, administered 10 days per os) on learning and memory of rats with FAE (E). Left ordinate: number of avoidances. Right ordinate: the type of administration (E) rats pre- and postnatally exposed to ethanol. P, piracetam treatment rats; EP, piracetam treatment ethanol exposed pre- and postnatally rats; C, controls rats exposed pre and postnatally to sucrose. Abscissa: training days. * Statistical significance vs. controls (P#0.001). 8 Statistical significance vs. piracetam group (P#0.001).
4. Discussion As previously reported (Vaglenova and Petkov, 1998), PA and PNA ethanol exposure results in long-lasting impairment of performance in learning and memory tasks, indicating that ethanol administered even at a very low dose during gestation acts as a behavioral teratogen in the offspring. The behavioral teratogenicity of ethanol has been evidenced by deficits in performance of adolescent, mature and old rats in a task that measured learning and memory deficits. In our present investigation we used mature rats (3-month-old) with evident FAE. Nootropic drugs piracetam, aniracetam and meclophenoxate completely abolished their learning and memory. This finding supports our previous data for retention-improving effects of piracetam on rats exposed PA and PNA to a much higher dose of ethanol (9 g / kg body weight) (Petkov et al., 1991). Our present results showed a common capability of nootropic drugs belonging to different chemical families to improve learning and memory of rats with FAE. Cognitive disturbances appear after PA- and PNA-exposure and those caused by low doses of ethanol can be observed using a more complicated learning task (see Vaglenova and Petkov, 1998; Kerr and Hindmarch, 1998). Piracetam, aniracetam and meclophenoxate were found to be promising for
controlling the cognitive deficits induced by PA and PNA ethanol exposure and it was also evident that the type of nootropic drug was not of essential importance. Interestingly, using nootropic agents did not change learning and memory when they have been applied on naive experimental animals. These observations are supported by other authors (Gamzu et al., 1989). A 5-day training two-way avoidance task allowed us to establish that three studied nootropic drugs can improve not only learning and memory, but are also able to increase significantly the percentage of ‘good learners’ in a group of FAE rats. The percentage of ‘good learners’ in a group of FAE rats, treated with nootropic drugs is the same as that of controls and nootropic groups (P, A, M). It has also been shown that aniracetam can efficiently prevent learning and memory deficits in rats PA and PNA exposed to ethanol before their appearance, during adolescence and maturity. Aniracetam was administered to young, 1-month-old rats and showed a prolonged (2 months) prophylactic effect, observed in rats aged 3 months. To explain the identical beneficial effects of the three different nootropic agents, their pharmacological characteristics and the mechanism of action of ethanol on the developing brain should be taken into account. According to a general opinion, multiple mechanisms are involved in the teratogenic action of ethanol. We found that fetal
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Fig. 2. Effect of aniracetam (50 mg / kg body weight, administered 10 days per os on 1-month-old rats) on learning and memory of 3-month-old FAE rats (E). Left ordinate: number of avoidances. Right ordinate: the type of administration (E) rats pre- and postnatally exposed to ethanol. A, piracetam treatment rats; EA, piracetam treatment ethanol exposed pre- and postnatally rats; C, controls rats exposed pre and postnatally to sucrose. Abscissa: training days. * Statistical significance vs. controls (P#0.001). 8 Statistic significance vs. aniracetam group (P#0.001).
alcohol exposure increased levels of lipid peroxidation in all brain areas measured in rats, including neocortex, hippocampus and cerebellum (Petkov et al., 1992). Nootropic agents alter brain biomembranes by accumulating into them, and might change the membrane fluidity by partitioning into the phospholipid bilayer (Ostrowski et al., 1975; Ostrowski and Keil, 1978). Piracetam changes the membrane physical properties because it interacts with phosphate head groups of artificial phospholipid bilayers (Pevout et al., 1995). The metabolites of meclophenoxate also accumulate in the nerve cell membranes and remain there for a long time. Their strong OH ? radical scavenger ability was discovered by Nagy (1994). Meclophenoxate increases the synthesis of the alcalin ethanolamine phospholipids, lecitin and holin. All these events lead to the stabilization of brain biomembranes and are a basis for the fixation of traces of the neuronal circle. Precisely, these traces are the biological structure of memory (Vaglenova, 1992). When favorable effects of nootropic drugs on the CNS dysfunction in animals and humans are discussed, their alteration at neurotransmitter systems should be considered too. It has been concluded that in utero and early PNA exposure to ethanol markedly impairs several neurotransmitter systems. Using our model of behavior teratogenicity of ethanol
(Vaglenova and Petkov, 1998) alterations in muscarin cholinoceptors and b-adrenoceptors in the hippocampus with the peak of the blood ethanol concentration (BAC) of 35 mg% (Hadjiivanova et al., 1991; Vaglenova, 1992) were demonstrated. Changes in the synapses and activity of enzymes associated with the synthesis and degeneration of many CNS transmitters in amygdala and hippocampus were also found (Lolova et al., 1987, 1989; Vaglenova, 1992) with a higher ethanol dose. Piracetam increases the density of muscarinic cholinergic receptors in the frontal ¨ cortex of aged rats (Pilch and Muller, 1988) which pointed to the involvement of cholinergic effects in learning and memory. On the other hand, there are data that piracetam, aniracetam and meclophenoxate induced alterations on the level and turnover of the biogenic monoamines in the main brain structures: cortex, striatum, hippothalmus, and pons (Petkov et al., 1984, 1985). Therefore, the level of activity of different brain neurotransmitter systems and the relations between them determine the character of the cognitive processes in each type of training task. In recent publications, a new research line has shown the interactions between long-term potentiation (LTP)) that is a cell model of cognition and the glutamate transmission. Lynch (1998) discussed that LTP is expressed by changes in NMDA and AMPA receptors operations. A main
38
J. Vaglenova, V. Vesselinov Petkov / European Neuropsychopharmacology 11 (2001) 33 – 40
Fig. 3. Effect of meclophenoxate (100 mg / kg body weight, administered 10 days per os) on learning and memory of rats with FAE (E). Left ordinate: number of avoidances. Right ordinate: the type of administration (E) rats pre- and postnatally exposed to ethanol. M, meclophenoxate treatment rats; EM, meclophenoxate treatment ethanol exposed pre- and postnatally rats; C, controls rats exposed pre and postnatally to sucrose. Abscissa: training days. * Statistical significance vs. controls (P#0.001). 8 Statistical significance vs. meclophenoxate group (P#0.001).
hypothesis emerges that enhancement of the NMDA receptor tone increases cognitive performance. Low ethanol concentration (BACs as low as 30 mg%) leads to significant reduction of NMDA receptors in hippocampus and cerebellum during their formation (Savage et al., 1991; Valles et al., 1995). Hamelin and Lehmann (1995) showed that piracetam and aniracetam could act like cognition Table 1 Stratification to ‘good’ and ‘poor’ learners of rats with FAE, treated with piracetam (600 mg / kg), aniracetam (50 mg / kg), and meclophenoxate (100 mg / kg) during the performance shuttle-box avoidance task a Groups
‘Good learners’ (%)
‘Poor learners’ (%)
Sucrose (n533) Water (n533) FAE (n525) Meclophenoxate (n517) FAE1Mecloph. (n517) Piracetam (n517) FAE1Piracetam (n517) Aniracetam (n517) FAE1Aniracetam (n517)
66.6 (n522) 66.6 (n522) 40.0* (n510) 71.2 † (n512)
33.3 (n511) 33.3 (n511) 60.0* (n515) 28.8 † (n55)
71.2 † (n512) 71.2 † (n512)
28.8 † (n55) 28.8 † (n55)
71.2 † (n512)
28.8 † (n55)
71.2 † (n512)
28.8 † (n55)
71.2 † (n512)
28.8 † (n55)
a n5number of animals; * P#0.05 compared to control (sucrose or water); † P#0.05 compared to animals with FAE.
enhancers via at least some subtypes of NMDA receptors. High levels of aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors (Fallarino et al., 1995). Aniracetam reduced AMPA-receptor desensitisation (Isaacson and Nicol, 1991; Tang et al., 1991) by allosteric potentiation of quisqualate receptors (Ito et al., 1990), thus amplified excitatory synaptic transmission. Piracetam also interacts with the brain glutamate transmission (see Gouliaev and Senning, 1994). Finally, we can conclude that nootropic drugs have favorable effects on cognitive disruptions of rats pre- and postnatally exposed to ethanol. These agents are effective a long time after stopping the alteration of the teratogen ethanol. We accept that the recovery of memory traces in rats with FAE are probably carried into effect by a normalization of the protein synthesis, disrupted in an earlier stage of ontogenesis and also by the activation and recovery of the equilibrium of monoaminergic, cholinergic and glutamatergic transmitter systems in different brain structures. Also, nootropic drugs could act as scavengers of lipid peroxidation, provoked by perinatal exposure to ethanol. By all affections mentioned above, nootropic drugs support neuronal plasticity in adult rat brain, disturbed during development by ethanol exposure. Therefore, we have completed a positive answer to the question: can nootropic drugs be effective against the impact of the
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behavioral teratogenicity of ethanol or its main characteristics — learning and memory disability?
Acknowledgements The authors gratefully acknowledge Dr Mauro Santos ´ from Departmento di Genetica y Microbiologia, Universitat Autonoma de Barcelona, Spain for the statistical analysis.
References Canonico, P.L., Forgione, L., Paoletti, C., Casini, A., Collona, C.V., Bertini, M., Acito, R., Rengo, F., 1991. Efficacy and tolerance of aniracetam in elderly patients with primary or secondary mental deterioration. Riv. Neurol. 61, 92–96. Chepkova, A.N., Doreulee, N.V., Trofimov, S.S., Gudasheva, T.A., Ostrovskay, R.U., Skrebitsky, V.G., 1995. Nootropic compound Lpyroglutamyl-D-alanine-amide restores hippocampal long-term potentiation impaired by exposure to ethanol in rats. Neurosci. Lett. 188, 163–166. Coper, H., Herrman, W.M., 1988. Psychostimulants, analeptics, nootropics: an attempt to differentiate and assess drugs designed for the treatment of impaired brain functions. Pharmacopsychiatry 21, 211– 217. Domna, M.M., 1998. Clinical efficacy of piracetam in treatment of breath-holding spells. Pediatr. Neurol. 18 (1), 41–45. De Deyn, P.P., De Reuck, J., Deberdt, W., 1997. Treatment of acute ischemic stroke with piracetam. Stroke 28, 2347–2352. Elia, J., Borcherdinging, B.G., Raport, J.L., Keysor, C.S., 1991. Methylphenidate and dextroamphetamine treatments of hyperactivity: Are there true nonresponders? Psychiatry Res. 36, 141. Fallarino, F., Genazzani, A.A., Silla, S., L’Episcopo, M.R., Camici, O., Corazzi, L., Nicoletti, F., Fioretti, M.C., 1995. [ 3 H]aniracetam binds to specific recognition sites in brain membranes. J. Neurochem. 65, 912–918. Foltyn, P., Lucker, P.W., Schnitker, J., Wetzelsberger, N., 1983. A test model for cerebrally active drugs as demonstrated by the example of the new substance aniracetam. Arzneim.-Forsch. Drug Res. 33, 865– 867. Gamzu, E.R., Hoover, T.M., Gracon, S.I., Ninteman, M.V., 1989. Recent developments in 2-pyrolidine-containing nootropic. Drug Dev. Res. 18, 177–189. Genkova, M.G., Lazarova, M.B., 1988. Influence of fipexide on the learning and memory impairing effects of diethyldithiocarbamate, clonidine and electroconvulsive shock in albino rats. C.R. Acad. Bulg. Sci. 41 (3), 73–76. Genkova-Papazova, M.G., Stancheva, S., Alova, L.G., LazarovaBaracova, M.B., 1993. Adafenoxate abolishes the amnesia induced by neonatal 6-hydroxydopamine treatment in rats. Methods Find. Exp. Clin. Pharmacol. 15 (5), 267–271. ´ Giurgea, C., 1972. Vers une pharmacologie de l’activite´ integrative du cerveau. Tentative du concept nootrope en psychopharmacologie. Actual Pharmacol. 25, 115–156. Giurgea, C., 1980. A drug for the mind. Chem. Technol. 10, 360–365. Giurgea, C., 1982. The nootropic concept and its prospective implications. Drug Dev. Res. 2, 441–446. Gobert, J.G., 1972. Genese d un medicaments: Le piracetam. Metabolisation et recheche biochemique. J. Pharm. Belg. 27, 281–304. Gouliaev, A.H., Senning, A., 1994. Piracetam and other structurally related nootropics (review). Brain Res. Dev. 19, 180–222. Hadjiivanova, C., Petkov, V.D., Alova, L., Petkov, V.V., Vaglenova, J.,
39
1991. Effects of chronic maternal ethanol consumption on offspring brain muscarinic and beta-adrenoceptors. Acta Physiol. Pharmacol. Bulg. 17 (1), 44–51. Hamelin, S.M., Lehmann, J.C., 1995. Effects of putative cognition enhancers on the NMDA receptor by [ 3 H]MK801 binding. Eur. J. Pharmacol. 281, R11–R13. Hannigan, J.H., Randal, S., 1996. Behavioral pharmacology in animals exposed prenatally to alcohol. In: Abel, E.L. (Ed.), Fetal Alcohol Syndrome from Mechanism to Prevention. CRC Press, Boca Raton, FL, pp. 191–213. Heise, G.A., 1984. Behavioral methods for measuring effects of drugs on learning and memory in animals. Med. Res. Rev. 4, 535–546. Herrman, W.M., Stephan, K., 1991. Efficacy and clinical relevance of cognition enhancers. Alzheimer Dis. Assoc. Disord. 5, 7–12. Hitzenberger, G., Rameis, H., Manigley, C., 1998. Pharmacological properties of piracetam. Rationale for use in stroke patients. CNS Drugs 9 (1), 19–27. Isaacson, J., Nicol, R.A., 1991. Aniracetam reduced glutamate receptor desensitization and slows a decay of fast excitatory synaptic currents in the hippocampus. Proc. Natl. Acad. Sci. USA 88, 10936–10940. Ito, I., Tanabe, S., Kohda, A., Sugiyama, H., 1990. Allosteric potentiation of quisqualate receptors by a nootropic drug aniracetam. J. Physiol. (Lond.) 424, 533–543. Kerr, J.S., Hindmarch, I., 1998. The effects of alcohol alone or in combination with other drugs on information processing, task performance and subjective responses. Hum. Psychopharmacol. Clin. Exp. 13 (1), 1–9. Kovalev, G.I., Budigin, E.A., Gainetdinov, R.R., Kudrin, V.S., Trofimov, S.S., Ostrovskay, R.U., 1993. The sodium oxybutyrate and nooglutil correction of dopamine release in the striatum of prenatally alcoholized rat pups. Biull. Eksp. Biol. Med. 116 (7), 56–58. Levinson, H.N., 1991. Dramatic favorable responses of children with learning disabilities or dyslexia and attention deficit disorder to antimotion sickness medications: four case reports. Percept. Mot. Skills 73, 723–738. Lolova, I., Petkov, V.V., Vaglenova, J., 1987. Enzyme histochemical studies on rat amygdala in fetal alcohol syndrome. Folia Histochim. Citobiol. 25 (2), 119–128. Lolova, I., Lolov, V., Petkov, V.V., Vaglenova, J., 1989. Changes in the synapses of the rat hippocampus following pre- and postnatal alcohol exposure. Anat. Anz. 169, 285–294. Lynch, G., 1998. Memory and the brain: unexpected chemistries and a new pharmacology. Neurobiol. Learn. Memory 70, 82–90. Means, L.W., Medlin, C.W., Hughes, V.D., Gray, S.L., 1984. Rats exposed in utero to ethanol are hyperresponsive to methylphenidate when tested as neonates or adults. Neurobehav. Toxicol. Teratol. 6, 187. Mosharrof, A.H.M., 1987. Investigation of effects of synthetic and plants psychopharmacological agents on learning and memory. PhD thesis, Sofia, Bulgaria, 204 p. Nagy, Z.S.I., 1994. A survey of the available data on a new nootropic drug, BCE-001. Ann. NY Acad. Sci. 10 (717), 102–114. Nickolson, V.J., Wolthuis, O.L., 1976. Effect of the acquisition-enhancing drug piracetam on rat cerebral energy metabolism. Comparison with naftidrofuryl and methamphetamine. Biochem. Pharmacol. 25, 2241– 2244. Ostrowski, J., Keil, M., 1978. Autoradiographische untersuchungen zur verteilung von piracetam 14 C im affengehirm. Arzneim.-Forsch. Drug Res. 25 (1), 29–35. Ostrowski, J., Keil, M., Schraven, E., 1975. Autoradiographische untersuchungen zur verteilung von piracetam 14 C bei ratte un hund. Arzneim.-Forsch. Drug Res. 25 (4), 589–596. Petkov, V.V., Grahovska, T., Petkov, V.D., Konstantinova, E., 1985. Effects of meclophenoxate on the level and turnover of biogenic monoamines in the rat brain. Arzneim.-Forsch. Drug Res. 35, 1778–1781. Petkov, V.D., Grahovska, T., Petkov, V.V., Konstantinova, E., Stancheva, S., 1984. Changes in the brain biogenic monoamines of rats infused by piracetam and aniracetam. Acta Physiol. Pharmacol. Bulg. 10 (4), 6–15.
40
J. Vaglenova, V. Vesselinov Petkov / European Neuropsychopharmacology 11 (2001) 33 – 40
Petkov, V.D., Kehayov, R.A., Mosharoff, A.H., Petkov, V.V., Getova, D., Lazarova, M.B., Vaglenova, J., 1993. Effects of cytidine diphosphate choline on rats with memory deficits. Arzneim.-Forsch. Drug Res. 43 (8), 822–828. Petkov, V.D., Konstantinova, E., Petkov, V.V., Vaglenova, J., 1991. Learning and memory in rats exposed pre- and postnatally to alcohol. An attempt at pharmacological control. Methods Find. Exp. Clin. Pharmacol. 13, 43–50. Petkov, V.D., Mosharoff, A., Petkov, V.V., 1988. Comparative studies on the effects of adenofenoxate, meclophenoxate and piracetam and citicholine on scopolamine — impaired memory, exploratory behavior and physical capabilities (Experiments on rats and mice). Acta Physiol. Pharmacol. Bulg. 1, 3–13. Petkov, V.D., Mosharoff, A., Petkov, V.V., Kehayov, P., 1990. Age-related differences in memory and in the memory effects of nootropic drugs. Acta Physiol. Pharmacol. Bulg. 16, 28–35. Petkov, V.V., Rousseva, S., Dimov, S., 1983. Behavioral effects of piracetam in rats with isolation syndrome. Acta Physiol. Pharmacol. Bulg. 10, 3–9. Petkov, V.V., Stoianovski, D., Petkov, V.D., Vaglenova, J., 1992. Lipid peroxidation changes in the brain in fetal alcohol syndrome. Biull. Eksp. Biol. Med. 113 (5), 500–502. Pevout, J., Schank, A., Deleers, M., Brasseur, R., 1995. Piracetam induced changes to membrane physical properties. Biochem. Pharmacol. 50, 1129–1134. ¨ Pilch, H., Muller, W.E., 1988. Piracetam elevates muscarinic cholinergic receptor density in the frontal cortex of age but not in young mice. Psychopharmacology 94, 74–78. Platt, D., Horn, J., Summa, J.-D., Achmitt-Ruth, R., Kauntz, J., Kronert, E., 1993. On the efficacy of piracetam in geriatric patients with acute cerebral ischemia: a clinically controlled double-blind study. Arch. Gerontol. Geriatr. 16, 149. Poschel, B.P.H., Ho, P.M., Ninteman, E.W., Callahan, M.J., 1985. Pharmacologic therapeutic window of piracetam demonstrated in behavior. EEG and single neuron firing rates. Experientia 41, 1153– 1156. Rapport, M.D., Denny, C., DupPaul, G.J., Garder, M.J., 1994. Attentional deficit disorder and methylphenidate: Normalization rates, clinical effectiveness, and response in 76 children. J. Am. Acad. Child. Adolesc. Psychol. 33, 882.
Savage, D.D., Montano, C.Y., Otero, M.A., Paxton, L.L., 1991. Prenatal ethanol exposure decreases hippocampal NMDA-sensitive 3 H-glutamate binding site density in 45-day-old rats. Alcohol 8, 193–201. Senin, U., Abate, G., Fieschi, C., Gori, G., Guala, A., Marini, G., Villardita, C., Parnettti, L., 1991. Aniracetam (Ro 13-5057) in the treatment of senile dementia of Alzheimer’s type (SDAT): results of a placebo controlled multicentre clinical study. Eur. Neuropsychopharmacol. 1, 511–517. Streissguth, A.P., Martin, J.C., 1983. Prenatal effects of alcohol abuse in humans and laboratory animals. In: Kissin, P., Begleiter, H. (Eds.), The Pathogenesis of Alcoholism. York, Plenum Press, New, pp. 539–589. Tang, C.-M., Shi, Q.-Y., Katchman, A., Lynch, G., 1991. Modulation of the time course of fast EPSCs and glutamate channel kinetics by aniracetam. Science 254, 288–290. Trofimov, S.S., Ostrovskay, R.U., Smolnikova, N.M., Khromova, I.V., Krapivin, S.V., Krilova, A.M., Kovalev, G.I., Gainetdinov, R.R., Liubimov, B.I., Voronina, T.A., 1992. The nooglutil correction of functional disorders of central nervous system caused by prenatal alcoholization in rats. Eksp. Klin. Farmakol. 55 (1), 18–21. Ulug, S., Reley, E.P., 1983. The effect of methylphenidate on overactivity in rats prenatally exposed to alcohol. Neurobehav. Toxicol. Teratol. 5, 35–39. Vaglenova, J., Petkov, V.V., 1998. Fetal Alcohol Effects in rats exposed pre- and postnatally to a low dose of ethanol. Alcohol. Clin. Exp. Res. 22 (3), 697–703. Vaglenova, J., 1992. Fetal alcohol syndrome in rats and correction with nootropic drugs. PhD thesis, Sofia, Bulgaria, 171 p. Valles, S., Felipo, V., Montoliu, C., Guerri, C., 1995. Alcohol exposure during brain development reduces 3 H-MK-801 binding and enhances metabotropic-glutamate receptor-stimulated phosphoinositide hydrolysis in rat hippocampus. Life Sci. 56, 1373–1383. Yonkov, D.I., Petkov, V.V., Roussinov, K., Vaglenova, J., 1983. A new model of apparatus for two-way avoidance (shuttle-box) for pharmacological memory studies of small laboratory animals. C.R. Acad. Bulg. Sci. 36 (8), 1123–1126. Voronina, T.A., 1989. In: Valdman, A.V., Voronina, T.A. (Eds.), Pharmacology of Nootropics. Experimental and Clinical Studies. Nauka, Moscow, p. 105.