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Active avoidance learning in old rats chronically treated with levocarnitine acetyl
Physiology & Behavior, Vol. 52, pp. 185-187, 1992
0031-9384/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA.
BRIEF COMMUNICATION
Active Avoidance Learning in Old Rats Chronically Treated With Levocarnitine Acetyl ORLANDO
G H I R A R D I , *l A N T O N I O C A P R I O L I , * S T E F A N O M I L A N O , * A L E S S A N D R O G I U L I A N I , * M A R I A T E R E S A R A M A C C I * A N D L U C I A N O ANGELUCCI~"
*Institute for Research on Senescence, Sigma Tau S.p.A., 00040 Pomezia, Rome, and ~-Institute of Pharmacology II, School of Medicine, "La Sapienza'" University of Rome, 00187 Rome, Italy Received 3 July 1991 GHIRARDI, O., A. CAPRIOLI, S. MILANO, A. GIULIANI, M. T. RAMACCI AND L. ANGELUCCI. Active avoidance learning in old rats chronically treated with levocarnitine acetyl. PHYSIOL BEHAV 52( 1) 185-187, 1992.--The aging laboratory
animal is recognized as a suitable experimental model for the investigation on drugs potentially able to retard the age-dependent decline in cognitive functions. There is robust evidence that levocarnitine acetyl (ALCAR), the acetyl derivative of carnitine, when administered chronically, prevents some age-related deficits of the central nervous system, mainly at the hippocampal level. On the basis of this evidence and because learning of active avoidance was demonstrated to become impaired with age, we decided to investigate the effect of ALCAR in rats. For statistical evaluation of results, the Cluster Analysis technique was chosen. This procedure pointed out the great heterogeneity of the old population and allowed the classification of the animals into homogeneous groups according to their response pattern. The effect of ALCAR was evident in the higher number of treated old animals yielding escape responses, indicating that ALCAR can preserve, at least partially, learning and memory from the natural decay occurring with age. Levocarnitine acetyl
Active avoidance
Old rats
Aging
IN behavioral studies, the aging animal has been assessed as a suitable experimental model and it is widely used for the investigation on the possible effect of drugs on the decline of the cognitive function naturally accompanying old age. With some differences according to the strain used, age-related deficits in active avoidance learning have been demonstrated reliably in tests carried out over repeated (3) or single sessions (8). Levocarnitine acetyl (ALCAR) is the acetyl derivative ofcarnitine; both are biological substances that in eukaryotics play an important role in energy and lipid metabolism (6,7). Levocarnitine acetyl's structural similarity to acetylcholine accounts for a very mild cholinergic activity (6). As widely reported, in aging rats chronic treatment with ALCAR retards and/or reduces some age-related deficits. This activity is particularly evident for some morphological, endocrine (2), and behavioral (4,9) parameters linked to the hippocampal function (1,11,12). Based on these findings, we decided to further investigate the effect of ALCAR on learning deficits as exhibited by aged Sprague-Dawley rats in the active avoidance test.
The recurrent observation of heterogeneity in the learning impairment due to age prompted the choice of a statistical approach, namely Cluster Analysis, which allowed taking into account explicitly the discontinuities of the data (natural classes of performance). METHOD
Animals and Treatment Male Sprague-Dawley (Charles River Italia) rats, 15 months old at the beginning of treatment, were used. Treated animals (n = 24) were given daily 75 mg/kg ALCAR added to drinking water for 7 months; control animals (n = 24) received drinking water alone.
Apparatus and Procedure The apparatus used was a conventional shuttle-box (U. Basile, Italia) divided into two identical compartments by a partition provided with an opening to allow communication
Requests for reprints should be addressed to Dr. Orlando Ghirardi, Institute for Research on Senescence, Psychobiology Dept., Sigma Tau, S.p.A., Via Pontina Km 30,400, 00040 Pomezia, Rome, Italy.
185
186
G H I R A R I ) I E l AI..
~6
( m a x i m u m intergroup variance) (5). This enabled us to find out by means o f a Fisher's exact test (generalization to the r c tables) (10) to what extent our a priori division into treatment classes would be reflected in our data field as a natural structure (mathematical classes). This procedure takes profitably and simultaneously into account all the aspects of the p h e n o m e n o n under study.
~14
c 12 < ~
8
RESULTS
0
I
r
1
2
i 3
/
4
i
i
5 6 Sessions
i
i
7
8
FIG. l. Percent (+SEM) of avoidance responses in the shuttle-box for each 20-trial session. Empty circles, control animals; filled circles, animals treated with ALCAR.
between them. Each c o m p a r t m e n t was illuminated by a 10watt bulb hanging above the Plexiglas cage lid. Foot shock was delivered through the stainless steel grid floor. Trial sessions were programmed on a schedule of conditioned stimulus (CS = light on), followed 3 s afterwards by an unconditioned stimulus (US = a 4-s, 0.2-mA electric shock). Eight consecutive daily sessions of 20 trials at l - m i n intervals were performed: the rat was placed in one c o m p a r t m e n t and the start of the trial was signalled by the CS followed by delivery of the US. The rat was given a few seconds to avoid shock and escape to the adjacent compartment. Responses during CS were considered as avoidances, responses during US as escapes, and failures to avoid shock as failures.
The results indicated an extremely reduced learning capability in old rats. They could reach, at most, 7.1% avoidances at the 6th trial, whereas ALCAR-treated animals exhibited constantly higher performances than control animals, reaching up to 15.6% avoidances at the 7th trial (Fig. 1). The high dispersion of the data within the old animal population was reminiscent of the discrete character of the distribution, which was evidenced by Cluster Analysis showing 78.8% of explained variability with a three-cluster solution (Table 1). The rats scoring a high number of avoidances (43.8%) and escapes (47.9%), i.e., optimal performances, were grouped under cluster G (n = 7). Those scoring a high number of escapes (59.4%), i.e., mean performances yielding an escape response only when the US was activated, were grouped under cluster M (n = 13). The rats scoring a high number of failures (78.3%), i.e., animals that totally failed to learn the test, were grouped under cluster P (n = 28). Seventy-live percent of control animals were included in cluster P, whereas only 41.7% of treated animals were in cluster P and another 41.7% in cluster M. This distribution into performance classes was significantly different for treated versus control rats (Fisher's exact test: p = 0.04}. DISCUSSION
The use of a multivariate Cluster Analysis procedure allowed us to point out the heterogeneity of the old population and to classify the rats into homogeneous groups as per their performances in active avoidance learning. Such a classification made it possible to discriminate the effect of treatment that modified the animals' distribution in the three classes. The effect of chronic treatment with A L C A R was substantially manifest in the increase of the number of rats capable of yielding escapes responses. This process is reminescent of the progression of learning observed in young animals, going from failures to avoidances while passing through an intermediate phase of a high rate of escape responses. Levocarnitine acetyl
Statistical Methodology We adopted a two-step data analysis strategy: first, Cluster Analysis outlined the structure of a multivariate matrix having rats as rows and n u m b e r of avoidances, escapes, and failures as columns; and second, the structure generated by Cluster Analysis (internal structure) was compared with the control/ treated subdivision of the animals (external structure). Cluster Analysis evidenced natural groups, pointing to the creation of classes as compact as possible inside ( m i n i m u m within-group variance) and as separate as possible from the other groups
TABLE 1 CLUSTER ANALYSISON PERCENT RESPONSESTHROUGHOUT THE EIGHT SESSIONSAND DISTRIBUTION IN THE THREE PERFORMANCECLASSES Number
Avoidances* Entire set Cluster G Cluster M Cluster P
8.8 _+ 2.2 43.8 ± 5.6 4.8 ± 0.9 1.9 _+ 0.4
Escapes* 34.6 ± 47.9 ± 59.4 ± 19.8 ±
3.1 6. l 3.7 1.9
Failures* 56.6 8.2 35.8 78.3
The percent of the animals in each cluster is shown in parentheses. * Values are mean _+ SE. t Fisher's exact test: p = 0.04.
+_ 4.3 ± 3.6 _+ 4.0 ± 2.1
of Animals
Total
Control
ALCAR
48 7 13 28
24 3 (12.5)t 3 (12.5)t 18 (75.0)t
24 4 (16.7)t 10 (41.7)¢ 10 (41.7)t
A L C A R A N D A C T I V E A V O I D A N C E IN O L D R A T S
can once more be regarded as capable of preserving old animals from the gradual decay of some aspects of learning and m e m o r y occurring with age.
187 ACKNOWLEDGEMENT The authors gratefully acknowledge the assistance of Mrs. L. Mattace for English translation and editing.
REFERENCES 1. Amenta, F.; Ferrante, F.; Lucreziotti, R.; Ricci, A.; Ramacci, M. T. Reduced lipofuscin accumulation in senescent rat brain by longterm acetyl-L-carnitine treatment. Arch. Gerontol. Geriatr. 9:147153; 1989. 2. Angelucci, L.; Patacchioli, F. R.; Taglialatela, G.; Maccari, S.; Ramacci, M. T.; Ghirardi, O. Brain glucocorticoid receptor and adrenocortical activity are sensitive markers of senescence retarding treatments in the rat. In: Biggio, G.; Spano, P. F.; Toffano, G.; Gessa, G. L., eds. Modulation of central peripheral transmitter function. Padova: Liviana Press; 1986:337-343. 3. Barrett, R. J.; Ray, O. S. Behavior in the open field, Lashley III maze, shuttle-box, and Sidman avoidance as a function of strain, sex, and age. Dev. Psychol. 3:73-77; 1970. 4. Caprioli, A.; Ghirardi, O.; Ramacci, M. T.; Angelucci, L. Age-dependent deficits in radial maze performance in the rat: Effect of chronic treatment with acetyI-L-carnitine. Prog. Neuropsychopharmacol. Biol. Psychiatry 14:359-369; 1990. 5. Everitt, B. Cluster analysis. New York: Halsted; 1980. 6. Falchetto, S.; Kato, G.; Provini, L. The action ofcarnitines on cortical neurons. Can. J. Physiol. Pharmacol. 49:1-7; 1971.
7. Fritz, I. B. Carnitine and its role in fatty acid metabolism. Adv. Lipid Res. 1:285-334; 1963. 8. Fuchs, A.; Martin, J. R.; Bender, R.; Harting, J. Avoidance acquisition in adult and senescent rats. Gerontology 32:91-97; 1986. 9. Ghirardi, O.; Milano, S.; Ramacci, M. T.; Angelucci, L. Long term acetyl-L-carnitine preserves spatial learning in the senescent rat. Prog. Neuropsychopharmacol. Biol. Psychiatry 13:237-245; 1989. 10. Mehta, C. R.; Patel, N. R. A network algorithm for performing Fisher's exact test in rxc contingency tables. J. Am. Stat. Assoc. 78: 427-434; 1983. 11. Napoleone, P,; Ferrante, F.; Ghirardi, O.; Ramacci, M. T.; Amenta, F. Age-dependent nerve cell loss in the brain of Sprague Dawley rats: Effect of long-term acetyl-L-carnitine treatment. Arch. Gerontol. Geriatr. 10:173-185; 1990. 12. Ricci, A.; Ramacci, M. T.; Ghirardi, O.; Amenta, F. Age-related changes of the mossy fibre system in rat hippocampus: Effect of long term acetyl-L-carnitine treatment. Arch. Gerontol. Geriatr. 8:6371; 1989.
0031-9384/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA.
BRIEF COMMUNICATION
Active Avoidance Learning in Old Rats Chronically Treated With Levocarnitine Acetyl ORLANDO
G H I R A R D I , *l A N T O N I O C A P R I O L I , * S T E F A N O M I L A N O , * A L E S S A N D R O G I U L I A N I , * M A R I A T E R E S A R A M A C C I * A N D L U C I A N O ANGELUCCI~"
*Institute for Research on Senescence, Sigma Tau S.p.A., 00040 Pomezia, Rome, and ~-Institute of Pharmacology II, School of Medicine, "La Sapienza'" University of Rome, 00187 Rome, Italy Received 3 July 1991 GHIRARDI, O., A. CAPRIOLI, S. MILANO, A. GIULIANI, M. T. RAMACCI AND L. ANGELUCCI. Active avoidance learning in old rats chronically treated with levocarnitine acetyl. PHYSIOL BEHAV 52( 1) 185-187, 1992.--The aging laboratory
animal is recognized as a suitable experimental model for the investigation on drugs potentially able to retard the age-dependent decline in cognitive functions. There is robust evidence that levocarnitine acetyl (ALCAR), the acetyl derivative of carnitine, when administered chronically, prevents some age-related deficits of the central nervous system, mainly at the hippocampal level. On the basis of this evidence and because learning of active avoidance was demonstrated to become impaired with age, we decided to investigate the effect of ALCAR in rats. For statistical evaluation of results, the Cluster Analysis technique was chosen. This procedure pointed out the great heterogeneity of the old population and allowed the classification of the animals into homogeneous groups according to their response pattern. The effect of ALCAR was evident in the higher number of treated old animals yielding escape responses, indicating that ALCAR can preserve, at least partially, learning and memory from the natural decay occurring with age. Levocarnitine acetyl
Active avoidance
Old rats
Aging
IN behavioral studies, the aging animal has been assessed as a suitable experimental model and it is widely used for the investigation on the possible effect of drugs on the decline of the cognitive function naturally accompanying old age. With some differences according to the strain used, age-related deficits in active avoidance learning have been demonstrated reliably in tests carried out over repeated (3) or single sessions (8). Levocarnitine acetyl (ALCAR) is the acetyl derivative ofcarnitine; both are biological substances that in eukaryotics play an important role in energy and lipid metabolism (6,7). Levocarnitine acetyl's structural similarity to acetylcholine accounts for a very mild cholinergic activity (6). As widely reported, in aging rats chronic treatment with ALCAR retards and/or reduces some age-related deficits. This activity is particularly evident for some morphological, endocrine (2), and behavioral (4,9) parameters linked to the hippocampal function (1,11,12). Based on these findings, we decided to further investigate the effect of ALCAR on learning deficits as exhibited by aged Sprague-Dawley rats in the active avoidance test.
The recurrent observation of heterogeneity in the learning impairment due to age prompted the choice of a statistical approach, namely Cluster Analysis, which allowed taking into account explicitly the discontinuities of the data (natural classes of performance). METHOD
Animals and Treatment Male Sprague-Dawley (Charles River Italia) rats, 15 months old at the beginning of treatment, were used. Treated animals (n = 24) were given daily 75 mg/kg ALCAR added to drinking water for 7 months; control animals (n = 24) received drinking water alone.
Apparatus and Procedure The apparatus used was a conventional shuttle-box (U. Basile, Italia) divided into two identical compartments by a partition provided with an opening to allow communication
Requests for reprints should be addressed to Dr. Orlando Ghirardi, Institute for Research on Senescence, Psychobiology Dept., Sigma Tau, S.p.A., Via Pontina Km 30,400, 00040 Pomezia, Rome, Italy.
185
186
G H I R A R I ) I E l AI..
~6
( m a x i m u m intergroup variance) (5). This enabled us to find out by means o f a Fisher's exact test (generalization to the r c tables) (10) to what extent our a priori division into treatment classes would be reflected in our data field as a natural structure (mathematical classes). This procedure takes profitably and simultaneously into account all the aspects of the p h e n o m e n o n under study.
~14
c 12 < ~
8
RESULTS
0
I
r
1
2
i 3
/
4
i
i
5 6 Sessions
i
i
7
8
FIG. l. Percent (+SEM) of avoidance responses in the shuttle-box for each 20-trial session. Empty circles, control animals; filled circles, animals treated with ALCAR.
between them. Each c o m p a r t m e n t was illuminated by a 10watt bulb hanging above the Plexiglas cage lid. Foot shock was delivered through the stainless steel grid floor. Trial sessions were programmed on a schedule of conditioned stimulus (CS = light on), followed 3 s afterwards by an unconditioned stimulus (US = a 4-s, 0.2-mA electric shock). Eight consecutive daily sessions of 20 trials at l - m i n intervals were performed: the rat was placed in one c o m p a r t m e n t and the start of the trial was signalled by the CS followed by delivery of the US. The rat was given a few seconds to avoid shock and escape to the adjacent compartment. Responses during CS were considered as avoidances, responses during US as escapes, and failures to avoid shock as failures.
The results indicated an extremely reduced learning capability in old rats. They could reach, at most, 7.1% avoidances at the 6th trial, whereas ALCAR-treated animals exhibited constantly higher performances than control animals, reaching up to 15.6% avoidances at the 7th trial (Fig. 1). The high dispersion of the data within the old animal population was reminiscent of the discrete character of the distribution, which was evidenced by Cluster Analysis showing 78.8% of explained variability with a three-cluster solution (Table 1). The rats scoring a high number of avoidances (43.8%) and escapes (47.9%), i.e., optimal performances, were grouped under cluster G (n = 7). Those scoring a high number of escapes (59.4%), i.e., mean performances yielding an escape response only when the US was activated, were grouped under cluster M (n = 13). The rats scoring a high number of failures (78.3%), i.e., animals that totally failed to learn the test, were grouped under cluster P (n = 28). Seventy-live percent of control animals were included in cluster P, whereas only 41.7% of treated animals were in cluster P and another 41.7% in cluster M. This distribution into performance classes was significantly different for treated versus control rats (Fisher's exact test: p = 0.04}. DISCUSSION
The use of a multivariate Cluster Analysis procedure allowed us to point out the heterogeneity of the old population and to classify the rats into homogeneous groups as per their performances in active avoidance learning. Such a classification made it possible to discriminate the effect of treatment that modified the animals' distribution in the three classes. The effect of chronic treatment with A L C A R was substantially manifest in the increase of the number of rats capable of yielding escapes responses. This process is reminescent of the progression of learning observed in young animals, going from failures to avoidances while passing through an intermediate phase of a high rate of escape responses. Levocarnitine acetyl
Statistical Methodology We adopted a two-step data analysis strategy: first, Cluster Analysis outlined the structure of a multivariate matrix having rats as rows and n u m b e r of avoidances, escapes, and failures as columns; and second, the structure generated by Cluster Analysis (internal structure) was compared with the control/ treated subdivision of the animals (external structure). Cluster Analysis evidenced natural groups, pointing to the creation of classes as compact as possible inside ( m i n i m u m within-group variance) and as separate as possible from the other groups
TABLE 1 CLUSTER ANALYSISON PERCENT RESPONSESTHROUGHOUT THE EIGHT SESSIONSAND DISTRIBUTION IN THE THREE PERFORMANCECLASSES Number
Avoidances* Entire set Cluster G Cluster M Cluster P
8.8 _+ 2.2 43.8 ± 5.6 4.8 ± 0.9 1.9 _+ 0.4
Escapes* 34.6 ± 47.9 ± 59.4 ± 19.8 ±
3.1 6. l 3.7 1.9
Failures* 56.6 8.2 35.8 78.3
The percent of the animals in each cluster is shown in parentheses. * Values are mean _+ SE. t Fisher's exact test: p = 0.04.
+_ 4.3 ± 3.6 _+ 4.0 ± 2.1
of Animals
Total
Control
ALCAR
48 7 13 28
24 3 (12.5)t 3 (12.5)t 18 (75.0)t
24 4 (16.7)t 10 (41.7)¢ 10 (41.7)t
A L C A R A N D A C T I V E A V O I D A N C E IN O L D R A T S
can once more be regarded as capable of preserving old animals from the gradual decay of some aspects of learning and m e m o r y occurring with age.
187 ACKNOWLEDGEMENT The authors gratefully acknowledge the assistance of Mrs. L. Mattace for English translation and editing.
REFERENCES 1. Amenta, F.; Ferrante, F.; Lucreziotti, R.; Ricci, A.; Ramacci, M. T. Reduced lipofuscin accumulation in senescent rat brain by longterm acetyl-L-carnitine treatment. Arch. Gerontol. Geriatr. 9:147153; 1989. 2. Angelucci, L.; Patacchioli, F. R.; Taglialatela, G.; Maccari, S.; Ramacci, M. T.; Ghirardi, O. Brain glucocorticoid receptor and adrenocortical activity are sensitive markers of senescence retarding treatments in the rat. In: Biggio, G.; Spano, P. F.; Toffano, G.; Gessa, G. L., eds. Modulation of central peripheral transmitter function. Padova: Liviana Press; 1986:337-343. 3. Barrett, R. J.; Ray, O. S. Behavior in the open field, Lashley III maze, shuttle-box, and Sidman avoidance as a function of strain, sex, and age. Dev. Psychol. 3:73-77; 1970. 4. Caprioli, A.; Ghirardi, O.; Ramacci, M. T.; Angelucci, L. Age-dependent deficits in radial maze performance in the rat: Effect of chronic treatment with acetyI-L-carnitine. Prog. Neuropsychopharmacol. Biol. Psychiatry 14:359-369; 1990. 5. Everitt, B. Cluster analysis. New York: Halsted; 1980. 6. Falchetto, S.; Kato, G.; Provini, L. The action ofcarnitines on cortical neurons. Can. J. Physiol. Pharmacol. 49:1-7; 1971.
7. Fritz, I. B. Carnitine and its role in fatty acid metabolism. Adv. Lipid Res. 1:285-334; 1963. 8. Fuchs, A.; Martin, J. R.; Bender, R.; Harting, J. Avoidance acquisition in adult and senescent rats. Gerontology 32:91-97; 1986. 9. Ghirardi, O.; Milano, S.; Ramacci, M. T.; Angelucci, L. Long term acetyl-L-carnitine preserves spatial learning in the senescent rat. Prog. Neuropsychopharmacol. Biol. Psychiatry 13:237-245; 1989. 10. Mehta, C. R.; Patel, N. R. A network algorithm for performing Fisher's exact test in rxc contingency tables. J. Am. Stat. Assoc. 78: 427-434; 1983. 11. Napoleone, P,; Ferrante, F.; Ghirardi, O.; Ramacci, M. T.; Amenta, F. Age-dependent nerve cell loss in the brain of Sprague Dawley rats: Effect of long-term acetyl-L-carnitine treatment. Arch. Gerontol. Geriatr. 10:173-185; 1990. 12. Ricci, A.; Ramacci, M. T.; Ghirardi, O.; Amenta, F. Age-related changes of the mossy fibre system in rat hippocampus: Effect of long term acetyl-L-carnitine treatment. Arch. Gerontol. Geriatr. 8:6371; 1989.