Age-related changes of the effects of a group of nootropic drugs on the content of rat brain biogenic monoamines

Gen. Pharmac. Vol. 22, No. 5, pp. 873-877, 1991 Printed in Great Britain. All rights reserved

0306-3623/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pie

AGE-RELATED CHANGES OF THE EFFECTS OF A GROUP OF NOOTROPIC DRUGS ON THE CONTENT OF RAT BRAIN BIOGENIC MONOAMINES STEFANKA L. STANCHEVA,* VESSELIN D. PETKOV, CHRISTINA I. HADJIIVANOVA and VESSELINV. PETKOV Department of Experimental Pharmacology, Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 23, 1113 Sofia, Bulgaria (Received I1 December 1990) Abstract--l. The changes in the levels of brain biogenic monoamines (BMAs) after chronic (7 days) treatment with piracetam, aniracetam and structural analogues of aniracetam (p-H, p-F, p-Cl, p-P and m-D) were studied in young and old rats. 2. An age-related significant decrease in the BMA content was established in old rats. 3. Most of the investigated compounds increased the level of one or other BMA in one or other of the brain structures studied. This elevation was predominantly established in old rats. 4. The present results and those from previous behaviour studies show that elevation of one or more of the BMA levels in one or more brain regions plays a beneficial role in the realization of their effects on the processes of learning and memory.

INTRODUCTION The studies o f many authors (Bartus, 1981; Bartus et al., 1982; Zornetzer, 1984; La Poncin Lafitte and Rapin, 1986; Soumireu-Mourat, 1986; Fuchs et al., 1987), as well as some studies of ours (Petkov, 1985) showed that difficulties in learning and deterioration of m e m o r y processes often accompany ageing. The decrease in functional capacity of brain neurotransmission can be assumed as one of the neurochemical correlates of the impaired cognitive processes accompanying ageing, and a sign of this decrease is usually the reduction of the content and turnover rate of biogenic monoamines (BMAs) in most brain structures and the decrease in the density of their receptors (Petkov, 1985; Petkov et al., 1988). One of the basic properties o f the nootropic drugs studied in experiments on rats is their ability to improve the process of learning and to facilitate retention of memory traces. Our studies showed that nootropic drugs provoked changes in the content and turnover rate of B M A s - - a n important element of the mechanism of their action (Petkov, 1985; Petkov et aL, 1984, 1989b; Stancheva et al., 1988). Bearing in mind the basic role of neurotransmission for acquisition, storage and recall of information, it is natural to suppose that pharmacological influence on the cerebral neurotransmission would restore to a greater or smaller extent the age-related m e m o r y deficiencies that are c o m m o n l y observed in advanced age. The aim of the present study was to investigate the effect o f piracetam and aniracetam, and o f a group of newly synthesized pyrrolidinone derivatives with known effect on retention processes (Petkov et al., 1989a), on the B M A levels in various brain regions of young and old rats. Simultaneously, we tried to *To whom all correspondence should be addressed. 873

evaluate how substitution in the benzene ring of piracetam influences the pharmacological activity o f the derivatives. MATERIALS AND METHODS

The substances used were piracetam (Pharmachim, Sofia), aniracetam and the structural analogues of aniracetam with code names p-H, p-F, p-CI, p-P and m-D (synthesized in the Organic Chemistry Institute of Bulgarian Academy of Sciences by B. Milenkov) (Fig. 1). White male Wistar rats 3-5 months old (young) and 24 months old (old), kept on a standard diet and water ad libitum (6-15 rats in each group), received for 7 days piracetam, aniracetam and its analogues p-H, p-F, p-Cl, p-P and m-D. The drugs, tested at a dose of 50 mg/kg body weight, were administered orally twice daily (daily dose I00 mg/kg), dissolved in water with I-2 drops of Tween-80. The control groups of young and old rats received orally the same volume of water with Tween-80 added to it, according to the same schedule. On the day 7 the entire daily dose (100 mg/kg weight) was administered at once and 1 hr later the animals were decapitated. Frontal cortex, striatum, bypothalamus and hippocampus were rapidly removed on ice and were kept frozen at -40°C. Analogous experiments were carried out in another season of the year to observe the influence of the aniracetam analogues p-F and p-P on the BMA content in the four previously studied brain structures. The drugs were administered at a daily dose of 100 mg/kg dissolved in water with 1-2 drops of Tween-80 added to it, once a day for 7 days. This experiment again involved two age groups--4-monthold (young) and 21-month-old (old) rats. During the same period of time the control young and old rats were administered daily the same quantity of water with Tween-80 added. The content of BMAs--dopamine (DA), noradrenaline (NA) and serotonin (5-HT)--was determined by the fluorescent method of Jacobowitz and Richardson (1978) and the BMA levels were expressed in micrograms BMA per gram fresh brain tissue. The data obtained from both series of experiments were statistically analyzed by Student's t-test at P ~<0.05.

STEFANKA L. STANCHEVA et al.

874

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The results obtained are presented in Tables 1-4. It can be seen that the content of BMAs in the brain regions tested shows substantial age-determined differences which manifest themselves in decreased content in old rats. In a number of the brain regions tested, the drugs studied considerably influenced, but most often to a different extent, the levels of one or more of the BMA. Thus, piracetam did not affect the DA and NA levels in any of the tested brain structures of young rats whereas in old animals it increased significantly the DA levels in all four brain regions; also, the NA levels were increased in the brain cortex and hypothalamus as

compared with the levels in the same brain regions of the control old rats. The 5-HT level, under the influence of piracetam, was increased in the frontal cortex and hypothalamus of young rats whereas in old rats it was increased in all four brain regions tested. Aniracetam increased significantly the DA content in the frontal cortex and striatum of young rats, whereas in old rats it increased the DA content in the striatum and hippocampus. This nootropic drug increased NA content in young animals in all the brain regions tested except the hypothalamus whereas in old rats it increased NA level only in the hippocampus. Aniracetam decreased 5-HT content in the hypothalamus of young rats and in old rats it

Table 1. Effects of piracetam, aniracetam, p-H, p-C1 and m-D on dopamine content (gg DA/g fresh tissue + SD) in cerebral cortex, striatum, hypothalamus and hippocampus of young and old rats Drugs Brain structure

Controls

Piracetam

Aniracetam

p-H

p-CI

m-D

Cortex

Young Old

0.53 ± 0.17 0.29 ± 0.06?

0.62 _+0.20 1.55 + 0.19"

0.87 ± 0.21" 0.50 ± 0.28

-1.05 ± 0.55*

2.17 _+0.93* 0.53 ± 0.30

0.81 ± 0.32 --

Striatum

Young Old

1.64 ± 0.75 0.83 ± 0.18"t

1.85 _+0.18 3.42 ± 2.61 *

2.91 ± 1.15* 2.27 ± 1.08"

-5.09 :t: 2.89*

5.20 + 2.14" --

2.24 ± 0.99 --

Hypothalamus

Young Old

5.26 _+2.28 2.07 ± 0.80?

3.40 + 2.25 9.62 + 1.89'

4.23 ± 2.00 2.83 ± 0.84

-4.33 + 2.60

4.17 ± 2.05 --

5.02 ± 1.77 --

Young Old

1.58 + 0.97 0.47 ± 0.28?

1.98 _+ 1.04 6.94 ± 1.11"

1.59 ± 0.70 1.01 + 0.35*

-2.32 ± 2.27

1.56 ± 0.26 --

1.52 ± 0.75 --

Hippocampus

*Significant difference between drug treatment and the controls at P ~<0.05. "['Significant difference between the control young and old rats at P ~<0.05. Table 2. Effects of piracetam, aniracetam, p-H, p-Cl and m-D on noradrenaline content ( ~ g N A / g f r c s h tissue±SD) in cerebral cortex, striatum, hypothalamus and hippocampus of young and old rats Drugs Brain structure

Controls

Piracetam

Aniracetam

p -H

p -CI

m -D

Young Old

0.36 ± 0.15 0.32 ± 0.13

0.32 + 0.09 0.69 ± 0.23*

1.14 ± 0.58* 0.29 ± 0.09

-0.78 _+0.21"

I. I I ± 0.53* 0.24 + 0.09

0.52 _+0.32 --

Striatum

Young Old

1.01 + 0.36 0.86 + 0.28

0.81 ± 0.41 0.76 + 0.28

2.07 ± 0.95* 1.20 ± 0.65

-2.24 + 0.76*

2.73 + 1.62" 0.68 _+0.13

0.75 ± 0.46 --

Hypothalamus

Young Old

3.14 _+ 1.55 2.93 __ 1.69

3.26 _+ 1.40 6.79 _+ 2.77*

3.66 ± 1.57 3.93 ± 2.27

-12.83 ± 5.53*

2.53 ± 1.21 1.84 _+ 1.38

3.98 _+ 1.79 --

Young Old

1.30 + 1.05 0.39 _+0.23?

1.18 + 0.78 0.59 + 0.31

2.69 ± 1.60" 0.88 ± 0.34*

-3.56 + 1.89"

1.24 _+0.45 0.93 + 0.46*

1.47 ± 0.66 --

Cortex

Hippocampus

*Significant difference between drug treatment and the controls at P ~ 0.05. ?Significant difference between the control young and old rats at P ~<0.05.

Nootropics and brain biogenic monoamines in aging

875

Table 3. Effects of piracetam, aniracetam, p-H, p-CI and m-D on serotonin content (p g 5-HT/g fresh tissue + SD) in cerebral cortex, stria?urn, hypothalamus and hippocampus of young and old rats Drugs Brain structure

Controls

Piracetam

Aniracetam

p -CI

m -D

Cortex

Young Old

1.65 + 0.71 0.74 -+ 0.36?

2.40 _+0.52* 2.51 -+ 0.44*

1.34 + 0.29 0.97 _+0.31

p-H -1.42 _+0.32*

2.64 _+0.87 0.33 -+ 0. I 1

1.68 -+ 0.55 --

Striatum Hypothalamus

Young Old Young Old

3.08 _ 1.18 1.36 _+0.65? 6.11 _+3.81 2.07 _+0.88?

1.27 _+0.43 4.10 _+0.90* 2.02 _+ 1.62* 4.86 _+ 1.48"

1.65 -+ 0.48 2.96 _ 0.54* 3.30 _+0.88* 1.44 _+0.76

-0.76 -+ 0.28 -4.94 _+2.40

3.97 _+0.78 0.75 _+0.41 7.75 _+2.18 2.04 _+0.34

1.99 + 0.57 -1.87 _+0.60* --

Hippocampus

Young Old

3.92 _+0.93 1.40 _+0.48?

2.92 _+0.84 3.05 _+0.79*

2.33 _+ 1.03 0.29 + 0.19"

-1.32 _+0.75

4.07 _+ 1.47 0.23 _+0.08*

2.57 -t- 0.80 --

*Significant difference between drug treatment and the controls at P ~<0.05. tSignificant difference between the young and old rats at P ~<0.05. i n c r e a s e d t h e 5 - H T c o n t e n t in the s t r i a t u m a n d d e c r e a s e d 5 - H T level in t h e h i p p o c a m p u s . T h e c h a n g e s o f b r a i n B M A s c a u s e d by the s t r u c t u r a l a n a l o g u e s o f a n i r a c e t a m ( p - H , p - F , p-C1 and p-P) demonstrate both similarities a n d differences as c o m p a r e d w i t h the c h a n g e s c a u s e d by piracetam and aniracetam. The p-H, p-F, p-Cl and p - P i n d u c e d different effects o n t h e B M A levels depending both on the brain structure studied and on t h e age o f t h e rats. T h e c o m p o u n d p - H , a d m i n i s t e r e d only to o l d rats, similarly to p i r a c e t a m h a d p r o n o u n c e d effects o n B M A levels o f this age g r o u p . A s c a n be seen f r o m T a b l e s 1-3, t h e effect o f p - H o n this age g r o u p was to i n c r e a s e the D A c o n t e n t in t h e f r o n t a l c o r t e x a n d s t r i a t u m , a n d to increase N A in all t h e b r a i n r e g i o n s tested a n d 5 - H T in t h e f r o n t a l cortex. T h e a n i r a c e t a m a n a l o g u e p - C l i n c r e a s e d t h e c o n t e n t o f D A a n d N A in t h e f r o n t a l c o r t e x a n d s t r i a t u m a n d t h e c o n t e n t o f 5 - H T in the f r o n t a l c o r t e x o f y o u n g rats. In old rats p - C l i n f l u e n c e d t h e N A a n d 5 - H T c o n t e n t only in t h e h i p p o c a m p u s . T h e a n a l o g u e m - D , a d m i n i s t e r e d to

y o u n g rats only, d e c r e a s e d only the 5 - H T level in hypothalamus. I n t h e s e c o n d series o f e x p e r i m e n t s , in all t h e s t r u c t u r e s tested, the B M A c o n t e n t in o l d ( 2 1 - m o n t h old) rats s h o w e d a t e n d e n c y to d e c r e a s e in c o m p a r i s o n w i t h the c o n t e n t in y o u n g rats. H o w e v e r , this d e c r e a s e was n o t significant for D A in a n y o f t h e b r a i n s t r u c t u r e s tested; for N A it w a s significant only in t h e h i p p o c a m p u s ; a n d f o r 5 - H T in t h e f r o n t a l c o r t e x , s t r i a t u m a n d h i p p o c a m p u s (Table 4). I n y o u n g rats, p - F a n d p - P i n c r e a s e d significantly o n l y the level o f N A in the f r o n t a l cortex. T h e influence o f p - P a n d p - F o n old a n i m a l s w a s also slight. T h u s , p - F i n c r e a s e d the level o f d o p a m i n e in t h e h i p p o c a m p u s , a n d p - P d e c r e a s e d t h e level o f 5 - H T in t h e f r o n t a l cortex. DISCUSSION T h e results o f the p r e s e n t investigation, s o m e previous studies o f o u r s ( P e t k o v et al., 1987, 1988) a n d i n v e s t i g a t i o n s o f o t h e r a u t h o r s (Bartus, 1981;

Table 4. Effects ofp-F and p-P on the content of dopamine, noradrenaline and serotonin (expressed as #g BMA/g fresh tissue _+SD) in cerebral cortex, striatum, hypothalamus and hippocampus of young and old rats Brain structure Drugs Young

Cortex Striatum Hypothalamus Hippocampus Dopamine (,ag/g fresh tissue) 0.15 5:0.02 0.49 _+0.20 0.99 _+0.48 0.51 _ 0.18

Old

0. I0 +_ 0.05

--

0.50 _+ 0.28

0.47 _+ 0.13

Young

0.38+0.02

0.34_+0.11

I.II _+0.34

0.58_+0.26

Old

0.09 _+0.06

--

0.64 _+0.32

1.87 _+ 1.04'

P-P

Young Old

0.49 _+0.19 0.48 _+0.16

Controls

Young Old Young Old

0.13 _+0.08 0.26 _+0.12 0.79 _+0.35 0.08 _+0.02 --Noradrenaline (~g/g fresh tissue) 0.15 _+0.07 0.66 _+0.23 1.77 _+0.90 0.18 -+ 0.08 0,28 -+ 0,14 0.72+0.35 0.27 -+ 0.10* 0,65 _+0,23 1.76 _+0.30 0.21 _+0.06 0,14_+0,07 0.77_+0.38

p.p

Young Old

0.28 -+ 0.10* 0.21 _+0.05

0.30 _+0.14 0.16 _+0.03

Controls

Young Old

Serotonin (/zg/g fresh tissue) 0.59 + 0.10 1.31 -+ 0.41 4.01 _+ 1.92 0.44 + 0.02t 0.69+ 0.23? 1.42 + 0.68

1.11 _+0.20 0.81 + 0.23?

p-F

Young Old Young Old

0.52 + 0.09 0.39 + 0.05 0.63 + 0.08 0.38 + 0.04*

1.22 + 0.29 0.64 + O.12 0.95 + 0.17 0.62 + 0.06

Controls p-F

p-F

p.p

0.35 -+ 0.14 0.23 _+0.12

1.17 + 0.34 0.63 + 0.06 1.17 + 0.36 0.63 + 0.16

1.79 -+ 0.45 0.71 _+0.56

3.89 _+ 1.52 1.28 + 0.40 4.23 + 1.60 1.82 + 0.92

*Significant difference between drug treatment and the controls at P ~< 0.05. tSignificant difference between the young and old control rats at P ~<0.05.

0.42 _+0.17 0.15 -+ 0.06? 0.51 _+0.39 0.28 -+ 0.13

876

STEFANKAL. STANCHEVAel al.

Carlsson, 1987; Racagni et al., 1989) establish the development of age-related deficiencies of one or other of the BMAs which accompany ageing. In both series of experiments it was observed again (although to a different extent, probably because of the different age of the old animals--24 and 21 months) that the content of BMAs decreases in most of the brain structures tested. Our previous studies and those of other authors show the essential importance of these neurochemical correlates for the cognitive and memory deficiencies that develop with ageing (Mosharrof et al., 1987; Zornetzer, 1985). The observed decreases of the content of the various BMAs in different brain structures in advanced age undoubtedly cause disturbances in the correlations established in maturity between the various BMAs both in particular brain structures and in the brain as a whole. It may be suggested that those disturbances of the already balanced content of BMAs in the brain or in some brain regions are of greater importance for age-related memory disturbances than the decrease of only one BMA type. Correction of the disturbances in learning and memory processes in old rats with apparent deficiency in cognitive functions is widely recognized as one of the experimental models for studying the activity of nootropic drugs (Voronina, 1989). That is why the effect of the tested substances, whose nootropic action is known or implied, was tested not only on young rats but also on old ones. Our finding that BMA is affected by piracetam mainly in old rats further confirms the opinion that restoration of the balance of BMAs in deficiency states is an important element of the mechanism of action of nootropic drugs. In our previous investigations it was established that aniracetam and its analogues improved the acquisition traces and retention of memory when different behavioural methods are applied (Petkov et al., 1989a). According to our results, the effect of aniracetam analogues on the BMA content in different brain structures was found to depend both on the type of substitution in the benzene ring of aniracetam and on the age of the test animals. Thus, for example, the compounds p - F and p-Cl have a considerably slight correcting effect on the established age-related deficiency of BMA in the tested brain structures of old animals than the compound without a substitute--p-H. It should also be pointed out that p-C1 is more active than p - F but its effects are more pronounced on young animals. On the other hand, when the methoxy group is taken out of the aniracetam ring, as is the case for p-H, a considerable resemblance to piracetam activities is evident--p-H increased significantly the level of BMAs in brain regions of old rats. Thus p-H, in spite of the presence of a benzene ring attached to the pyrrolidinone ring, resembles the action of piracetam more than that of aniracetam. Our results prove that the strongest influence on the level of the brain BMAs in old rats is exerted by the aniracetam derivative without a substitute in the benzene ring, i.e. p - H (which has the lowest molecular weight among the aniracetam analogues tested), and the least active are the compounds of comparatively high molecular weight such as p-P and m-D.

The analogues with average molecular weight (p-F and p-C1) have a moderate effect on the BMA level. The present study on the effect of the series of aniracetam analogues on BMA level does not make it possible to find a simple explanation for the mechanism of action of these nootropic drugs. It is possible, of course, that the reduction of the effect of p - F and p-Cl on the BMA levels might be due not to changes in "nootropic" properties but to different pharmacokinetics of these substances in which the charged C1 and F atoms reduced their lipophility. The aniracetam derivatives with higher molecular weight, p - P and m-D, affect the BMA level insignificantly. However, the established improvement of learning and memory after treatment with p - P in some behaviour tests, without influencing the BMA level to any great extent, suggest the co-participation of some other mechanisms in the action of the nootropic drugs. In conclusion, it can be pointed out that the beneficial effects of nootropic drugs on learning and memory in old rats suggest that the restoration of the impaired balance of BMAs may take place by the "nootropic" action of piracetam-like compounds. REFERENCES

Bartus R. T. (1981) Age-related memory loss and cholinergic dysfunction: possible directions based on animal models. In Strategies for the Development of an Effective Treatment for Senile Dementia (Edited by Crook T. and Gersham K. S.), pp. 71-92, Mark Powley, New Canaan. Bartus R. T., Dean R. L., Beer B. and Lippa A. S. (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 217, 408-417. Carlsson A. (1987) Brain neurotransmitters in aging and dementia: similar changes across diagnostic dementia groups. Gerontology 33, 159-167. Fuchs A., Martin J. R., Bender R. and Harting J. (1987) Avoidance acquisition in adult and senescent rats. Gerontology 32, 91-97. Jacobowitz D. M. and Richardson J. S. (1978) Method for the rapid determination of norepinephrine, dopamine, and serotonin in the same brain region. Pharmac. Biochem. Behav. 8, 515-519. La Poncin Lafitte M. and Rapin J. R. (1986) Age and learning: experimental and clinical aspects. Gerontology 32, (Suppl. 1), 53-59. Mosharof A. H., Petkov V. D. and Petkov V. V. (1987) Effects of meclofenoxate and citicholine on learning and memory in aged rats. Acta Physiol. Pharmac. Bulg. 13(4), 17-24. Petkov V. D. (1985) Psychotropic drugs and aging. In Proc. 4th Congr. Hung. Pharmac. Soc., Budapest, Vol. 3, Section 5. Dopamine, Ageing and Diseases (Edited by

Borsy J., Kerecsen L. and Gyorgy L.), pp. 103-108. Petkov V. D., Petkov V. V. and Stancheva S. L. (1988) Age-related changes in brain neurotransmission. Gerontology 34(1,2), 14-21. Petkov V. D., Mosharrof A. H., Milenkov B. and Enev V. (1989a) Learning and memory effects of four newly synthesized aniracetam analogues. Acta Physiol. Pharmac. Bulg. 15(2), 18-32. Petkov V. D., Stancheva S. L., Petkov V. V. and Alova L. G. (1987) Age-related changes in brain biogenic monoamines and monoamine oxidase. Gen. Pharmac. 18, 397-401. Petkov V. D., Stancheva S. L., Tokuschieva L. and Petkov V. V. (1989b) Changes in brain biogenic monoamines induced by the nootropic drugs adafenoxate and

Nootropics and brain biogenic monoamines in aging meclophenoxate and by citicholine (experiments on rats). Gen. Pharmac. 21(0, 71-75. Petkov V. D., Grahovska T., Petkov V. V., Konstantinova E. and Stancheva S. (1984) Changes in the brain biogenie monoamines of rats, induced by piracetam and aniracetam. Acta Physiol. Pharmac. Bulg. 10(4), 6--15. Racagni G., Rovescalli A. C., Galimberti R. and Brunello N. (1989) Ncurotransmitter systems and receptor plasticity in brain aging. In Diagnosis and Treatment of OM Dementias (Edited by Ban E. and Lehmann H. E.) Mod. Probl. Pharmacopsychiatry 23, 21-27. Karger, Basel. Soumireu-Mourat B. (1986) Experimental studies of ageing: behavioural and physiological correlates. Gerontology 32, (Suppl. 1), 24-29.

877

Stancheva S. L., Petkov V. D. and Petkov V. V. (1988) Effects of the nootropic agents adafenoxate and meclofenoxate on brain biogenic monoamines in aged rats. Acta Physiol. Pharmac. 8ulg. 140), 14-21. Voronina T. A. (1989) Experimental psychopharmacology of nootropes. In Pharmacology of Nootropes (Edited by Valdman A. V. and Voronina T. A.), pp. 8-19. Moscow (in Russian). Zornetzer S. F. (1984) Brain Substrates of senescent memory decline. In The Neurophysiology of Memory (Edited by Squire L. R. and Butters N.), pp. 588-600. Guilford Press, New York. Zornetzer S. F. (1985) Catecholamine system involvement in age related memory disfunction. Ann. N. Y. Acad. Sci. 444, 242-254.