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Physiology& Behavior, Vol. 47, pp. 755-760. ©Pergamon Press plc, 1990. Prin~d in the U.S.A.
0031-9384/90 $3.00 + .00
Chronic Administration of Phosphatidylserine During Ontogeny Enhances Subject-Environment Interactions and Radial Maze Performance in C57BL/6 Mice M A R T I N E A M M A S S A R I - T E U L E , *t S A N D R O F A G I O L I , t M A R C E L L A M A R I T A T I , : ~ ROBERTA POPULIN* AND FLAMINIA PAVONE*
*Istituto di Psicobiologia e Psicofarmacologia, CNR, Roma, Italy f'Dipartimento di Genetica e Biologia Molecolare, Universitd di Roma "La Sapienza," Italy 4:lstituto di Farmacologia Medica, III Cattedra, Universit?t di Roma "La Sapienza," Italy R e c e i v e d 31 July 1989
AMMASSARI-TEULE, M., S. FAGIOLI, M. MARITATI, R. POPULIN AND F. PAVONE. Chronic administration of phosphatidylserine during ontogeny enhances subject-environment interactions and radial maze performance in C57BL/6 mice. PHYSIOL BEHAV 47(4) 755-760, 1990.--A longitudinal behavioral study was performed in mice exposed to the bovine brain phospholipid phosphatidylserine (BC-PS) from birth until sixty days. Examination of treated and control pups revealed no effect of the treatment on body weight nor on sensorimotor reflexes. At one and two months of age, when placed in an open field and, particularly, in the presence of a novel object, treated mice were found more interactive with their environment than control mice. Finally, when submitted to a radial eight-arm maze problem, choice accuracy was higher and maze-running strategies more adaptive in treated than in control adult mice. These results suggest a stimulating effect of the treatment on subject-environment interactions during ontogeny underlying improved cognitive abilities in adulthood. Phosphatidylserine
Development
Open field-emotionality
A number of investigations have demonstrated the effects of chronic administration of bovine brain phosphatidylserine (BCPS) in counteracting age-related as well as pharmacologically induced electrophysiological and behavioral alterations in rats. On the one hand, facilitative effects of BC-PS on performance of aged animals have been reported in a number of memory and learning tasks (6, 9, 23), the improvement of behavioral responses being associated with a reduction of abnormal EEG discharges which characterize senescent EEG patterns (3). On the other hand, BC-PS has been found to antagonize the well-documented amnestic effect of scopolamine on passive avoidance conditioning (24). Investigations on the mechanisms involved in these effects have shown that BC-PS can affect the functional state of neuronal membrane (6) and modify several parameters of brain metabolism. Since BC-PS is the precursor of phosphatidylcholine, a primary brain source of choline for acetylcholine (ACh) synthesis, several works have focused on the modifications induced by that phos-
Spatial memory
pholipid on the cholinergic function. In particular, it has been observed that BC-PS increases ACh output from cerebral cortex in rats (7), and restores the age-related reduction of acetylcholinestimulated release from cortical slices (19). However, other classes of neurotransmitters are also affected by exposure to BC-PS which has been found to enhance the turnover of dopamine and noradrenaline, this last effect being particularly evident at the hypothalamic level (20). Moreover, at the structural level, recent data have shown that BC-PS can prevent the age-related loss of dendritic spines in rat hippocampus (14). This set of observations has led to consider BC-PS as an activator of brain metabolism, able to affect a large range of physiological systems and, therefore, to enhance neural and behavioral plasticity. For obvious clinical implications, the researches until now carried out on the effect of BC-PS have focused on the alleviation of neural and behavioral dysfunctions associated
~Requests for reprints should be addressed to M. Ammassari-Teule, Istituto di Psicobiologia e Psicofarmacologia, CNR, Via Reno 1,00198 Roma, Italy.
755
AMMASSARI-TEULE ET AL.
756
with aging. However, in order to define to which extent BC-PS can enhance neural and behavioral functions, it seemed to us interesting to examine its effects when administered during development, in mice of the C57BL/6 strain characterized by a precocious maturation (16), rigid behavioral patterns (13) and a reduced response to cholinergic agents during ontogeny (8,18). This study investigates the effect of postnatal administration of BC-PS on various physiological and behavioral parameters measured in developing C57BL/6 mice. Subsequently, learning performance was examined in adult mice submitted to the radial maze task. METHOD
Subjects Pregnant mice ( N = 2 0 ) belonging to the C57BL/6 strain (Charles River, Italy) were housed one per cage with water and food ad lib until delivery. On the day of parturition, the litters were cut to 6 pups and assigned to different experimental conditions. Half of the lactating mothers received an aqueous suspension of BC-PS (Fidia Research Laboratories, Italy) in order to early expose the pups to this compound through milk intake, while the other half received only water. The concentration of BC-PS was adjusted on the basis of the mother's weight and its daily liquid intake to supply a daily dose of BC-PS of 50 mg/kg. At one month of age, the pups were weaned and housed with four animals per cage. Treated animals directly received an aqueous solution of BC-PS from the bottle while control animals received water. Body weights and daily liquid intake were also checked each day in each treated litter in order to supply a mean daily dose of 50 mg/kg per pup until two months of age.
groups of two-month-old treated (8 males and 8 females) and control (8 males and 8 females) mice were estimated in a radial eight-ann maze. The apparatus was made of grey plastic material and consisted in eight arms (7 z 35 cm) radiating from an octogonal platform (perimeter 7 x 8 cm). The maze was elevated 60 cm above the floor and surrounded by cardboard walls forming a cubic enclosure. Three days before the beginning of pretraining, the animals were slightly food deprived in order to bring their body weight at 85% of the initial level. For three consecutive days, each mouse was placed on the central platform and allowed to explore the maze in which a large amount of food pellets had been placed. On the fourth day, the actual testing procedure started. Each mouse was placed on the central starting platform, all the arms being previously baited with a 0.25 g piece of Purina chow, and allowed to make 8 runs. The correct responses consisted in running all the 8 arms, once only, in order to consume the eight pieces of food. The animals went through one learning trial per day during ten consecutive days. On each trial, three dependent variables were recorded: the number of unrepeated arm choice differences, the rank of occurrence of the first error, and the degree of divergence of the sequence of runs. The calculation of this parameter has been described elsewhere (2). Statistics. The data concerning the reflexes were statistically evaluated by a Mann-Whitney U-test. The behavioral measures recorded in the open field were compared by a two-factor (treatment x sex) analysis of variance. Finally, between group differences in radial maze performance were estimated by a three factor (treatment x sex × repeated measures) analysis of variance. RESULTS
Body Weight and Reflexes Experimental Design The following physiological and behavioral measures were performed. Body weights and reflexes. Pups from control (N = 42) and treated (N = 48) litters were tested daily from postnatal day 2 to postnatal day 20. The following items were recorded: body weights, rooting, cliff aversion, hair growth, righting, forelimb placing, forelimb grasping, opening of ears and eyes, and auditory startle response. Reactivity to open field and novelty. Eight males and eight females from each treatment were randomly chosen from 4 litters. The animals were examined, both at one month of age and at two months of age, in an open field situation and successively tested for their reactivity to novelty by introducing a novel object in the same apparatus. The open field was a grey Plexiglas box consisting of a squared floor ( 3 0 x 3 0 cm) surrounded by 15-cm high walls. The floor was painted white and divided into 36 squares by drawing black lines. Each mouse was introduced in the center of the open field and retained for 15 sec in a transparent Plexiglas cylinder. After the removal of the cylinder, behavioral recordings were made for five minutes. At the end of this period, a transparent Plexiglas box (8.5 x 8.5 x 11 cm), with holes irregularly distributed on each side, was placed on the four central squares. After a free observation period of 15 sec, behavioral recordings were performed for another three minutes. The mouse was then removed from the apparatus, the defecation boluses were counted and both the open field and the object were cleaned with alcohol. The following behavioral items were recorded by the mean of an Apple IIE P.C.: number of crossed squares, number of rearing responses (against the walls or not), time spent in freezing, in grooming then in sniffing and rearing against the novel object. Radial maze performance. The learning performance of two
Measurements performed during development indicate no difference in body weights according to the treatment and a similar weight gain during ontogeny. As shown by Fig. 1, no significant effect of the treatment nor of the sex factor was found in the onset of reflexes between treated and control pups.
Reactivity to Open Field and Novelty The scores recorded in each group with the values of the statistical comparisons concerning the main effect of the treatment factor are reported on Table 1 and 2. At one month of age, no main effect of the treatment factor nor of the sex factor is detected when the animals are placed in the open field situation. A marginal significant sex x treatment interaction, F(1,28) = 2.83, p = 0.1, is evident for freezing behavior only. However, in the presence of the novel object, treated mice of both sexes display more grooming behavior and higher sniffing object scores than control mice (Table 1). At two months of age, treated animals still display more grooming but also less freezing as soon as they are placed in the open field situation. Moreover, in the presence of the novel object, treated mice are more interactive with their environment: their rearing behavior and sniffing object scores are significantly higher than those recorded in control mice. In treated mice, this sustained interest towards the novel object is accompanied by a significant reduction of the number of crossed squares (Table 2). No main effect of the sex factor is evident throughout the entire set of comparisons: only a marginal significant effect is observed in the case of freezing behavior, which is lower in males than in females when placed in the presence of the novel object, F(41,28) = 3.32, p<0.07. Conversely, significant treatment × sex interactions are observed for three behavioral items in the novelty situation. The
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A M M A S S A R I - T E U L E ET AL,
758
TABLE 1 OPEN FIELD AND NOVELTY SCORES IN ONE-MONTH-OLDOFFSPRING Prenatal Treatment Saline
BC-PS
F(1,28)
p
Open Field (5 min) Males Crossed squares Freezing Grooming Rearing Boluses
147.37 11.50 14.62 31.00 1.62
Females
_-_ 6.4 --- 8.1 ___ 3.4 _ 2.1 --- 0.5
153.00 0.25 8.75 32.37 0.87
± --+ __. ----+
Males
14.2 0.2 2.4 3.9 0.3
159.00 0.62 12.50 31.75 1.25
Females
__- 56.3 ± 0.4 __. 3.0 --+ 1.4 ± 0.4
151.87 3.12 14.12 33.62 1.37
• ----+ -+ ---
10.3 0.8 2.1 1.9 0.1
0.26 0.95 0.33 0.15 0.02
57.50 12.50 9.87 13.62 16.00 6.25
--± -+ --___ ---
7.2 1.3 1.9 4.1 2.6 2.5
0.50 0.17 13.94 0.87 3.73 0.07
ns ns ns ns ns
Novelty (3 min) Crossed squares Rearing Grooming Freezing Sniffing object Leaning object
54.75 11.50 3.37 26.50 11.00 6.00
--- 7.4 --- 1.6 --- 0.7 --_ 7.8 __+ 2.3 _+ 2.2
67.37 11.50 2.25 23.62 9.87 6.75
_+ _+ _+ ---+ ---
5.4 1.4 0.9 8.9 2.0 2.9
74.75 9.25 7.50 23.00 14.25 5.12
__. _--+ '~ __. •
8.0 1.5 2.2 7.0 2.5 2.1
ns ns 0.001 ns ns ns
Mean ___ S.E. scores from eight subjects. Freezing, grooming, sniffing object and rearing object are expressed in seconds.
n u m b e r o f crossed squares diminishes more in treated male than in treated female mice, F(1,28) = 6.63, p < 0 . 0 0 2 . Freezing does not vary in treated males but is drastically reduced in treated females, F ( 1 , 2 8 ) = 3 . 9 5 , p < 0 . 0 5 . Finally, the treatment has an opposite effect on leaning object behavior according to the sex, F ( 1 , 2 8 ) = 3.98, p < 0 . 0 5 .
Radial Maze Performance For each parameter, a significant effect of the repeated measures factor indicates that, in all groups, the n u m b e r of errors
decreases, F ( 9 , 2 5 2 ) = 3.40, p < 0 . 0 0 1 , the rank o f occurrence of the first error within each trial increases, F ( 9 , 2 5 2 ) = 3 . 5 0 , p < 0 . 0 0 1 , and the degree o f divergence of the patterns of choice diminishes, F ( 9 , 2 5 2 ) = 6 . 9 0 , p < 0 . 0 0 1 , with practice. A significant effect of the treatment factor, found in analyzing the n u m b e r of unrepeated path choice and the degree o f divergence o f the patterns of choice, s h o w s that treated mice of both sexes 1) make less errors, F ( 1 , 2 8 ) = 5.35, p < 0 . 0 3 , and 2) optimize their exploratory schemas in collecting food by choosing more adjacent paths, F(1,28) = 14.66, p < 0 . 0 0 1 , than control mice. For this last param-
TABLE 2 OPEN FIELD AND NOVELTY SCORES IN TWO-MONTH-OLDOFFSPRING Prenatal Treatment Saline
BC-PS
F( 1,28)
p
0.52 5.3 9.06 0.46 5.23
ns <0.05 0.005 ns <0.05
14.1 19.4 0.1 5.9 6.3 0.12
0.001 0.001 ns <0.05 0.01 ns
Open Field (5 min) Males Crossed squares Freezing Grooming Rearing Boluses
162.25 1.31 3.54 27.25 1.37
Females
--- 15.0 --- 0.3 • 1.2 --- 4.0 ___ 0.3
182.25 2.06 1.29 20.25 0.75
-+ 12.0 _+ 0.1 --- 0.6 ___ 3.4 --- 0.3
Males 149.37 0.27 6.66 24.7 1.75
Females
± 6.7 --- 0.1 --- 1.4 ___ 3.6 __. 0.4
176.75 0.52 5.47 28.10 2.37
___ 4.0 ___ 1.0 --- 2.0 --- 0.3 __. 1.0 --_ 2.0
86.25 12.87 3.05 1.12 7.00 11.93
--- 16.0 __- 0.2 ~ 1.3 _*_- 4.5 ± 0.5
Novelty (3 min) Crossed squares Rearing Groming Freezing Sniffing object Leaning object
125.25 6.37 5.31 1.57 5.51 12.56
___ --+ ------±
9.0 1.1 2.3 0.5 1.4 4.9
95.87 6.87 5.43 4.33 3.22 3.56
--- 6.0 • 1.4 ± 1.9 ± 0.9 ± 1.4 --- 0.7
73.75 11.12 8.94 1.24 7.69 6.74
--__--____. ---
Mean ___ S.E. scores from eight subjects. Freezing, grooming, sniffing object and rearing object are expressed in seconds.
7.0 1.8 1.1 0.3 1.1 3.6
BEHAVIORAL EFFECTS OF CHRONIC PHOSPHATIDYLSERINE
eter, a significant main effect of the sex factor, F(1,28)=4.18, p<0.05, and a marginal significant sex x treatment interaction, F(1,28) = 3.70, p<0.08, reveal that the degree of divergence of the patterns of choice is lower in males but more influenced by the treatment in female than in male mice. Finally, the rank of occurrence of the first error within each trial is not significantly higher [treatment effect, F(1,28)=2.94, p = 0 . 0 9 , n.s.] but increases more rapidly with practice in treated than in control mice [significant treatment x repeated measure interaction, F(9,252) =
1.97, p<0.04]. DISCUSSION The present results indicate no effect of perinatal BC-PS administration on developmental measures such as body weight gain and sensorimotor reflexes. This observation points out that BC-PS does not affect gross maturational processes and does not modify the reactivity of mice to sensory stimulation during ontogeny. At one month of age, slight behavioral changes related to the treatment are observed after the novel object has been placed in the open field: higher grooming and sniffing object scores are recorded in mice postnatally exposed to BC-PS. At two months of age, treated mice first display more grooming but less freezing behaviors in the open field situation, then, when placed in the presence of the novel object, more rearings and higher sniffing object scores. It is interesting to note that both freezing and grooming behaviors can be considered as indexes of emotionality since they generally occur in novel and stressful contexts (21). It seems, therefore, that exposure to BC-PS does not actually modify the level of emotionality of treated animals but the way (passive versus active behaviors) in which emotionality is expressed. Moreover, as previously reported (4,5) grooming generally occurs after other kinds of directed behavior such as drinking, eating, and walking. These observations indicate that, regardless of emotionality, a high rate of grooming behavior may also reflect the occurrence of previous interactions with the environment. It can, therefore, be assumed that, at two months of
759
age, mice exposed to BC-PS interact more intensively with thek environment than control mice. When successively tested in the radial eight-arm maze, treated mice perform significantly better than controls. In the treated group, the number of errors is lower and the rank of occurrence of the first error increases more rapidly throughout learning. Moreover, a marked effect of the treatment on the organization of maze-running strategies is observed. As shown by the lower degree of divergence of their patterns of choice, mice exposed to BC-PS tend to optimize their exploratory schemas in running the maze by choosing more adjacent arms than controls. On the one hand, these observations show a positive effect of early exposure to BC-PS on learning and memory processes of adult C57BL/6 mice. On the other hand, a possible interference of the treatment with the development of cognitive processes is supported by the sustained interactions with the environment displayed by treated mice during ontogeny and the adaptive strategies they develop in collecting food in the maze. It is interesting to note that radial maze performance is highly dependent on the cholinergic system. Damages to cholinergic pathways (2,17) or structures (11,15), as well as administration of cholinergic antagonists (21,22), produce important spatial memory dysfunctions. Cholinergic stimulation, induced by cholinomimetics, has also been found to improve spatial learning in aged animals (10) or to correct learning deficits in animals beating lesions of cholinergic structures (15) or pathways (12). Since it has been shown that chronic exposure to BC-PS enhances brain cholinergic metabolism (7,19), the improvement of spatial memory performance is therefore consistent with an effect of the treatment on this neurotransmitter system. Injections of various classes of cholinergic agents at different stages of development in perinatally BC-PS exposed animals are necessary to define which modifications of the cholinergic system outline the enhanced learning abilities observed in the present experiment. ACKNOWLEDGEMENT Dr. S. Fagioli was supported by grant No. 7020022/CR/SS from Fidia s.p.a., Abano Terme, Italy.
REFERENCES 1. Ammassari-Teule, M.; Caprioli, A. Spatial learning and memory, maze running strategies and cholinergic mechanisms in two inbred strains of mice. Behav. Brain Res. 17:9-16; 1985. 2, Amrnassari-Teule, M.; Maho, C. Properties of mapping induced by fornix damages: Learning and memorizing the radial maze task. Physiol. Psychol. 13:230-233; 1985. 3. Aporti, F.; Borsato, R.; Calderini, G.; Rubini, R.; Toffano, G.; Zanotti, A.; Valzelli, L.; Goldstein, L. Age dependent spontaneous EEG bursts in rats: Effects of brain phosphatidylserine. Neurobiol. Aging 7:115-120; 1986. 4. Bindra, D.; Spinner, N. Response to different degrees of novelty: the incidence of various activity. J. Exp. Anal. Behav. 1:341-350; 1958. 5. Bolles, R. C. Grooming behavior in the rat. J. Comp. Physiol. Psychol. 53:306-310; 1960. 6. Calderini, G.; Aporti, F.; Bellini, F.; Bonetti, A. C.; Rubini, R.; Teolato, S.; Xu, C.; Zanotti, A.; Toffano, G. Phospholipids as pharmacological tools in the aging brain. In: Horrocks, L. A., ed. Phospholipids in the nervous system. New York: Raven Press; 1985:11-19. 7. Casamenti, F.; Mantovani, P.; Amaducci, L.; Pepeu, G. Effect of phosphatidylserine on acetylcholine output from the cerebral cortex of the rat. J. Neurochem. 32:529-533; 1979. 8. Castellano, C.; Oliverio, A.; Schwab, C.; Brueckner, G.; Biesold, D. Age-dependent differences in cholinergic drug response in two strains of mice. Neurosci. Lett. 84:335-338; 1988. 9. Corwin, J.; Dean, R. L.; Bartus, R. T.; Rotrosen, J.; Watkins, D. L. Behavioural effects of phosphatidylserine in the aged Fisher 344 rat:
10. 11. 12.
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15. 16. 17. 18.
Amelioration of passive avoidance deficits without changes in psychomotor task performance. Neurobiol. Aging 6:11-15; 1985. Eppich, C.; Barnes, C. A.; Baldwin, J. 3,4-Diaminopyridine improves spatial working memory but not spatial reference memory in aged rats. Soc. Neurosci. Abstr. 11:724; 1985. Jarrard, L. E. Selective hippocampal lesions and behavior. Physiol. Psychol. 8:198-206; 1980. Maho, C.; Dutrieux, G.; Ammassari-Teule, M. Parallel modifications of spatial memory performance, maze-running strategies and hippocampal theta frequencies in fornix-damaged rats: reversal by oxotremorine. Behav. Neurosci. 102:601--604; 1988. Malomi, W.; Oliverio, A. Imprinting to light-dark cycles or rearing in constant light affects circadian locomotor rhythm of mice. Neurosci. Lett. 9:93-96; 1978. Milan, F.; Nnnzi, M. G.; Guidolin, D.; Calderini, G.; Toffano, G. Age-related loss of dendritic spines in rat hippocampus: effect of phosphatidylserine administration. In: B~s, A., ed. Senile dementias: Early detection. London: John Libbey Eurotext; 1986:408-414. Murray, C. L.; Fibiger, H. C. Pilocarpine and physostigmine attenuate spatial memory impairments produced by lesions of the nucleus basalis magnocellularis. Behav. Neurosci. 100:23-32; 1986. Oliverio, A.; Castellano, C. Postnatal brain maturation and learning in the mouse. In: Oliverio, A., ed. Genetics, environment and intelligence. Amsterdam: Elsevier/North Holland; 1977:117-131. Olton, D. S.; Walker, J. A.; Gage, F. H. Hippocampal connections and spatial discrimination. Brain Res. 139:295-308; 1978. Pavone, F.; Consorti, D.; Fagioli, S. Developmental differences of
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antinociceptive effects of oxotremorine in two inbred strains of mice. Dev. Brain Res 49:156-160; 1989. 19. Pedata, F. G.; Giovannelli, G.; Spignoli, G.; Giovannini, M. G.; Pepeu, G. Phosphatidylscrine increases acetylcholine release from cortical slices in aged rats. Neurobiol. Aging 6:337-339; 1985. 20. Toffano, G.; Leon, A.; Mazzari, S.; Savoini, G.; Teolato, S.; Orlando, P. Modification of noradrenergic hypothalamic system in rat injected with phosphatidylserlne liposomes. Life Sci. 23:1093-1102; 1978. 21. Walsh, R. N.; Cummins, R. A. The open field test: A critical review. Psychol. Bull. 83:482-504; 1976.
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22. Watts, J.; Stevens, R.; Robinson, C. Effects of scopolamine on radial maze performance in rats. Physiol. Behav. 26:845-851; 1981. 23. Zanotti, A.; Aporti, F.; Rubini, R.; Toffano, G. Learning and memory deficits in rats during aging: effect of phosphatidylserine treatments. In: Biggio, G.; Spano, P. F.; Toffano, G.; Gessa, G. L., eds. Symposia in Neuroscience III. Padova: Liviana Press; 1986: 323-330. 24. Zanotti, A.; Valzelli, L.; Toffano, G. Reversal of scopolamineinduced amnesia by phosphatidylserine in rats. Psychopharrnacology (Berlin) 90:274-275; 1986.
0031-9384/90 $3.00 + .00
Chronic Administration of Phosphatidylserine During Ontogeny Enhances Subject-Environment Interactions and Radial Maze Performance in C57BL/6 Mice M A R T I N E A M M A S S A R I - T E U L E , *t S A N D R O F A G I O L I , t M A R C E L L A M A R I T A T I , : ~ ROBERTA POPULIN* AND FLAMINIA PAVONE*
*Istituto di Psicobiologia e Psicofarmacologia, CNR, Roma, Italy f'Dipartimento di Genetica e Biologia Molecolare, Universitd di Roma "La Sapienza," Italy 4:lstituto di Farmacologia Medica, III Cattedra, Universit?t di Roma "La Sapienza," Italy R e c e i v e d 31 July 1989
AMMASSARI-TEULE, M., S. FAGIOLI, M. MARITATI, R. POPULIN AND F. PAVONE. Chronic administration of phosphatidylserine during ontogeny enhances subject-environment interactions and radial maze performance in C57BL/6 mice. PHYSIOL BEHAV 47(4) 755-760, 1990.--A longitudinal behavioral study was performed in mice exposed to the bovine brain phospholipid phosphatidylserine (BC-PS) from birth until sixty days. Examination of treated and control pups revealed no effect of the treatment on body weight nor on sensorimotor reflexes. At one and two months of age, when placed in an open field and, particularly, in the presence of a novel object, treated mice were found more interactive with their environment than control mice. Finally, when submitted to a radial eight-arm maze problem, choice accuracy was higher and maze-running strategies more adaptive in treated than in control adult mice. These results suggest a stimulating effect of the treatment on subject-environment interactions during ontogeny underlying improved cognitive abilities in adulthood. Phosphatidylserine
Development
Open field-emotionality
A number of investigations have demonstrated the effects of chronic administration of bovine brain phosphatidylserine (BCPS) in counteracting age-related as well as pharmacologically induced electrophysiological and behavioral alterations in rats. On the one hand, facilitative effects of BC-PS on performance of aged animals have been reported in a number of memory and learning tasks (6, 9, 23), the improvement of behavioral responses being associated with a reduction of abnormal EEG discharges which characterize senescent EEG patterns (3). On the other hand, BC-PS has been found to antagonize the well-documented amnestic effect of scopolamine on passive avoidance conditioning (24). Investigations on the mechanisms involved in these effects have shown that BC-PS can affect the functional state of neuronal membrane (6) and modify several parameters of brain metabolism. Since BC-PS is the precursor of phosphatidylcholine, a primary brain source of choline for acetylcholine (ACh) synthesis, several works have focused on the modifications induced by that phos-
Spatial memory
pholipid on the cholinergic function. In particular, it has been observed that BC-PS increases ACh output from cerebral cortex in rats (7), and restores the age-related reduction of acetylcholinestimulated release from cortical slices (19). However, other classes of neurotransmitters are also affected by exposure to BC-PS which has been found to enhance the turnover of dopamine and noradrenaline, this last effect being particularly evident at the hypothalamic level (20). Moreover, at the structural level, recent data have shown that BC-PS can prevent the age-related loss of dendritic spines in rat hippocampus (14). This set of observations has led to consider BC-PS as an activator of brain metabolism, able to affect a large range of physiological systems and, therefore, to enhance neural and behavioral plasticity. For obvious clinical implications, the researches until now carried out on the effect of BC-PS have focused on the alleviation of neural and behavioral dysfunctions associated
~Requests for reprints should be addressed to M. Ammassari-Teule, Istituto di Psicobiologia e Psicofarmacologia, CNR, Via Reno 1,00198 Roma, Italy.
755
AMMASSARI-TEULE ET AL.
756
with aging. However, in order to define to which extent BC-PS can enhance neural and behavioral functions, it seemed to us interesting to examine its effects when administered during development, in mice of the C57BL/6 strain characterized by a precocious maturation (16), rigid behavioral patterns (13) and a reduced response to cholinergic agents during ontogeny (8,18). This study investigates the effect of postnatal administration of BC-PS on various physiological and behavioral parameters measured in developing C57BL/6 mice. Subsequently, learning performance was examined in adult mice submitted to the radial maze task. METHOD
Subjects Pregnant mice ( N = 2 0 ) belonging to the C57BL/6 strain (Charles River, Italy) were housed one per cage with water and food ad lib until delivery. On the day of parturition, the litters were cut to 6 pups and assigned to different experimental conditions. Half of the lactating mothers received an aqueous suspension of BC-PS (Fidia Research Laboratories, Italy) in order to early expose the pups to this compound through milk intake, while the other half received only water. The concentration of BC-PS was adjusted on the basis of the mother's weight and its daily liquid intake to supply a daily dose of BC-PS of 50 mg/kg. At one month of age, the pups were weaned and housed with four animals per cage. Treated animals directly received an aqueous solution of BC-PS from the bottle while control animals received water. Body weights and daily liquid intake were also checked each day in each treated litter in order to supply a mean daily dose of 50 mg/kg per pup until two months of age.
groups of two-month-old treated (8 males and 8 females) and control (8 males and 8 females) mice were estimated in a radial eight-ann maze. The apparatus was made of grey plastic material and consisted in eight arms (7 z 35 cm) radiating from an octogonal platform (perimeter 7 x 8 cm). The maze was elevated 60 cm above the floor and surrounded by cardboard walls forming a cubic enclosure. Three days before the beginning of pretraining, the animals were slightly food deprived in order to bring their body weight at 85% of the initial level. For three consecutive days, each mouse was placed on the central platform and allowed to explore the maze in which a large amount of food pellets had been placed. On the fourth day, the actual testing procedure started. Each mouse was placed on the central starting platform, all the arms being previously baited with a 0.25 g piece of Purina chow, and allowed to make 8 runs. The correct responses consisted in running all the 8 arms, once only, in order to consume the eight pieces of food. The animals went through one learning trial per day during ten consecutive days. On each trial, three dependent variables were recorded: the number of unrepeated arm choice differences, the rank of occurrence of the first error, and the degree of divergence of the sequence of runs. The calculation of this parameter has been described elsewhere (2). Statistics. The data concerning the reflexes were statistically evaluated by a Mann-Whitney U-test. The behavioral measures recorded in the open field were compared by a two-factor (treatment x sex) analysis of variance. Finally, between group differences in radial maze performance were estimated by a three factor (treatment x sex × repeated measures) analysis of variance. RESULTS
Body Weight and Reflexes Experimental Design The following physiological and behavioral measures were performed. Body weights and reflexes. Pups from control (N = 42) and treated (N = 48) litters were tested daily from postnatal day 2 to postnatal day 20. The following items were recorded: body weights, rooting, cliff aversion, hair growth, righting, forelimb placing, forelimb grasping, opening of ears and eyes, and auditory startle response. Reactivity to open field and novelty. Eight males and eight females from each treatment were randomly chosen from 4 litters. The animals were examined, both at one month of age and at two months of age, in an open field situation and successively tested for their reactivity to novelty by introducing a novel object in the same apparatus. The open field was a grey Plexiglas box consisting of a squared floor ( 3 0 x 3 0 cm) surrounded by 15-cm high walls. The floor was painted white and divided into 36 squares by drawing black lines. Each mouse was introduced in the center of the open field and retained for 15 sec in a transparent Plexiglas cylinder. After the removal of the cylinder, behavioral recordings were made for five minutes. At the end of this period, a transparent Plexiglas box (8.5 x 8.5 x 11 cm), with holes irregularly distributed on each side, was placed on the four central squares. After a free observation period of 15 sec, behavioral recordings were performed for another three minutes. The mouse was then removed from the apparatus, the defecation boluses were counted and both the open field and the object were cleaned with alcohol. The following behavioral items were recorded by the mean of an Apple IIE P.C.: number of crossed squares, number of rearing responses (against the walls or not), time spent in freezing, in grooming then in sniffing and rearing against the novel object. Radial maze performance. The learning performance of two
Measurements performed during development indicate no difference in body weights according to the treatment and a similar weight gain during ontogeny. As shown by Fig. 1, no significant effect of the treatment nor of the sex factor was found in the onset of reflexes between treated and control pups.
Reactivity to Open Field and Novelty The scores recorded in each group with the values of the statistical comparisons concerning the main effect of the treatment factor are reported on Table 1 and 2. At one month of age, no main effect of the treatment factor nor of the sex factor is detected when the animals are placed in the open field situation. A marginal significant sex x treatment interaction, F(1,28) = 2.83, p = 0.1, is evident for freezing behavior only. However, in the presence of the novel object, treated mice of both sexes display more grooming behavior and higher sniffing object scores than control mice (Table 1). At two months of age, treated animals still display more grooming but also less freezing as soon as they are placed in the open field situation. Moreover, in the presence of the novel object, treated mice are more interactive with their environment: their rearing behavior and sniffing object scores are significantly higher than those recorded in control mice. In treated mice, this sustained interest towards the novel object is accompanied by a significant reduction of the number of crossed squares (Table 2). No main effect of the sex factor is evident throughout the entire set of comparisons: only a marginal significant effect is observed in the case of freezing behavior, which is lower in males than in females when placed in the presence of the novel object, F(41,28) = 3.32, p<0.07. Conversely, significant treatment × sex interactions are observed for three behavioral items in the novelty situation. The
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FIG. 2. Radial eight-arm maze performances in sixty-day-old mice neonatally exposed to BC-PS. Each point corresponds to the mean number of unrepeated arm choices (A) and to the mean degree of divergence of the sequence of choices (B) recorded in two consecutive learning trials.
A M M A S S A R I - T E U L E ET AL,
758
TABLE 1 OPEN FIELD AND NOVELTY SCORES IN ONE-MONTH-OLDOFFSPRING Prenatal Treatment Saline
BC-PS
F(1,28)
p
Open Field (5 min) Males Crossed squares Freezing Grooming Rearing Boluses
147.37 11.50 14.62 31.00 1.62
Females
_-_ 6.4 --- 8.1 ___ 3.4 _ 2.1 --- 0.5
153.00 0.25 8.75 32.37 0.87
± --+ __. ----+
Males
14.2 0.2 2.4 3.9 0.3
159.00 0.62 12.50 31.75 1.25
Females
__- 56.3 ± 0.4 __. 3.0 --+ 1.4 ± 0.4
151.87 3.12 14.12 33.62 1.37
• ----+ -+ ---
10.3 0.8 2.1 1.9 0.1
0.26 0.95 0.33 0.15 0.02
57.50 12.50 9.87 13.62 16.00 6.25
--± -+ --___ ---
7.2 1.3 1.9 4.1 2.6 2.5
0.50 0.17 13.94 0.87 3.73 0.07
ns ns ns ns ns
Novelty (3 min) Crossed squares Rearing Grooming Freezing Sniffing object Leaning object
54.75 11.50 3.37 26.50 11.00 6.00
--- 7.4 --- 1.6 --- 0.7 --_ 7.8 __+ 2.3 _+ 2.2
67.37 11.50 2.25 23.62 9.87 6.75
_+ _+ _+ ---+ ---
5.4 1.4 0.9 8.9 2.0 2.9
74.75 9.25 7.50 23.00 14.25 5.12
__. _--+ '~ __. •
8.0 1.5 2.2 7.0 2.5 2.1
ns ns 0.001 ns ns ns
Mean ___ S.E. scores from eight subjects. Freezing, grooming, sniffing object and rearing object are expressed in seconds.
n u m b e r o f crossed squares diminishes more in treated male than in treated female mice, F(1,28) = 6.63, p < 0 . 0 0 2 . Freezing does not vary in treated males but is drastically reduced in treated females, F ( 1 , 2 8 ) = 3 . 9 5 , p < 0 . 0 5 . Finally, the treatment has an opposite effect on leaning object behavior according to the sex, F ( 1 , 2 8 ) = 3.98, p < 0 . 0 5 .
Radial Maze Performance For each parameter, a significant effect of the repeated measures factor indicates that, in all groups, the n u m b e r of errors
decreases, F ( 9 , 2 5 2 ) = 3.40, p < 0 . 0 0 1 , the rank o f occurrence of the first error within each trial increases, F ( 9 , 2 5 2 ) = 3 . 5 0 , p < 0 . 0 0 1 , and the degree o f divergence of the patterns of choice diminishes, F ( 9 , 2 5 2 ) = 6 . 9 0 , p < 0 . 0 0 1 , with practice. A significant effect of the treatment factor, found in analyzing the n u m b e r of unrepeated path choice and the degree o f divergence o f the patterns of choice, s h o w s that treated mice of both sexes 1) make less errors, F ( 1 , 2 8 ) = 5.35, p < 0 . 0 3 , and 2) optimize their exploratory schemas in collecting food by choosing more adjacent paths, F(1,28) = 14.66, p < 0 . 0 0 1 , than control mice. For this last param-
TABLE 2 OPEN FIELD AND NOVELTY SCORES IN TWO-MONTH-OLDOFFSPRING Prenatal Treatment Saline
BC-PS
F( 1,28)
p
0.52 5.3 9.06 0.46 5.23
ns <0.05 0.005 ns <0.05
14.1 19.4 0.1 5.9 6.3 0.12
0.001 0.001 ns <0.05 0.01 ns
Open Field (5 min) Males Crossed squares Freezing Grooming Rearing Boluses
162.25 1.31 3.54 27.25 1.37
Females
--- 15.0 --- 0.3 • 1.2 --- 4.0 ___ 0.3
182.25 2.06 1.29 20.25 0.75
-+ 12.0 _+ 0.1 --- 0.6 ___ 3.4 --- 0.3
Males 149.37 0.27 6.66 24.7 1.75
Females
± 6.7 --- 0.1 --- 1.4 ___ 3.6 __. 0.4
176.75 0.52 5.47 28.10 2.37
___ 4.0 ___ 1.0 --- 2.0 --- 0.3 __. 1.0 --_ 2.0
86.25 12.87 3.05 1.12 7.00 11.93
--- 16.0 __- 0.2 ~ 1.3 _*_- 4.5 ± 0.5
Novelty (3 min) Crossed squares Rearing Groming Freezing Sniffing object Leaning object
125.25 6.37 5.31 1.57 5.51 12.56
___ --+ ------±
9.0 1.1 2.3 0.5 1.4 4.9
95.87 6.87 5.43 4.33 3.22 3.56
--- 6.0 • 1.4 ± 1.9 ± 0.9 ± 1.4 --- 0.7
73.75 11.12 8.94 1.24 7.69 6.74
--__--____. ---
Mean ___ S.E. scores from eight subjects. Freezing, grooming, sniffing object and rearing object are expressed in seconds.
7.0 1.8 1.1 0.3 1.1 3.6
BEHAVIORAL EFFECTS OF CHRONIC PHOSPHATIDYLSERINE
eter, a significant main effect of the sex factor, F(1,28)=4.18, p<0.05, and a marginal significant sex x treatment interaction, F(1,28) = 3.70, p<0.08, reveal that the degree of divergence of the patterns of choice is lower in males but more influenced by the treatment in female than in male mice. Finally, the rank of occurrence of the first error within each trial is not significantly higher [treatment effect, F(1,28)=2.94, p = 0 . 0 9 , n.s.] but increases more rapidly with practice in treated than in control mice [significant treatment x repeated measure interaction, F(9,252) =
1.97, p<0.04]. DISCUSSION The present results indicate no effect of perinatal BC-PS administration on developmental measures such as body weight gain and sensorimotor reflexes. This observation points out that BC-PS does not affect gross maturational processes and does not modify the reactivity of mice to sensory stimulation during ontogeny. At one month of age, slight behavioral changes related to the treatment are observed after the novel object has been placed in the open field: higher grooming and sniffing object scores are recorded in mice postnatally exposed to BC-PS. At two months of age, treated mice first display more grooming but less freezing behaviors in the open field situation, then, when placed in the presence of the novel object, more rearings and higher sniffing object scores. It is interesting to note that both freezing and grooming behaviors can be considered as indexes of emotionality since they generally occur in novel and stressful contexts (21). It seems, therefore, that exposure to BC-PS does not actually modify the level of emotionality of treated animals but the way (passive versus active behaviors) in which emotionality is expressed. Moreover, as previously reported (4,5) grooming generally occurs after other kinds of directed behavior such as drinking, eating, and walking. These observations indicate that, regardless of emotionality, a high rate of grooming behavior may also reflect the occurrence of previous interactions with the environment. It can, therefore, be assumed that, at two months of
759
age, mice exposed to BC-PS interact more intensively with thek environment than control mice. When successively tested in the radial eight-arm maze, treated mice perform significantly better than controls. In the treated group, the number of errors is lower and the rank of occurrence of the first error increases more rapidly throughout learning. Moreover, a marked effect of the treatment on the organization of maze-running strategies is observed. As shown by the lower degree of divergence of their patterns of choice, mice exposed to BC-PS tend to optimize their exploratory schemas in running the maze by choosing more adjacent arms than controls. On the one hand, these observations show a positive effect of early exposure to BC-PS on learning and memory processes of adult C57BL/6 mice. On the other hand, a possible interference of the treatment with the development of cognitive processes is supported by the sustained interactions with the environment displayed by treated mice during ontogeny and the adaptive strategies they develop in collecting food in the maze. It is interesting to note that radial maze performance is highly dependent on the cholinergic system. Damages to cholinergic pathways (2,17) or structures (11,15), as well as administration of cholinergic antagonists (21,22), produce important spatial memory dysfunctions. Cholinergic stimulation, induced by cholinomimetics, has also been found to improve spatial learning in aged animals (10) or to correct learning deficits in animals beating lesions of cholinergic structures (15) or pathways (12). Since it has been shown that chronic exposure to BC-PS enhances brain cholinergic metabolism (7,19), the improvement of spatial memory performance is therefore consistent with an effect of the treatment on this neurotransmitter system. Injections of various classes of cholinergic agents at different stages of development in perinatally BC-PS exposed animals are necessary to define which modifications of the cholinergic system outline the enhanced learning abilities observed in the present experiment. ACKNOWLEDGEMENT Dr. S. Fagioli was supported by grant No. 7020022/CR/SS from Fidia s.p.a., Abano Terme, Italy.
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