Aging Clin. Exp. Res. 7: 17-22, 1995
Effects of aging upon recent memory in Microcebus
murinus
J-L. Picq Laboratoire de Conservation des Especes Animales , Museum National d'Histoire Naturelle, Parc Zoologique de Paris, Paris, France ABSTRACT. Four young (2 to 4-year-old) and four aged (9 to 10-year-old) grey mouse lemurs (Microcebus murinus) were gi ven a simple visual discrim inat ion task, and a delayed response visual dis crim ination task with variable retention delay. Th e response to the test is a motor one, that consists of choosing one out of four corridors of an apparatus based upon the degree of illumination . The aged animals did not show any learning deficiency, and were capable of memorizing the task for several months. However, they were more sensitive to the length of the delay in the delayed response task than the young animals. It is, therefore, possible to argue that the memory for recent stimulus events is affected during aging. These results corroborate those obtained in other primates, and demonstrate the usefulness of the grey mouse lemur as a new animal model for analyzing aging. (Aging Clin. Exp. Res. 7: 17-22, 1995)
INTRODUCTION Cognitive deterioration and memory impairment in particular are among the most severe symptoms identified in the elderly. Choosing reliable animal models is a necessary requirement for understanding human aging . Animal models of declining memory in the aged would provide an invaluable tool for studying relationships between brain alterations and behavioral disorders. Moreover, they could be employed in testing drugs for use in geriatric dysfunction. Until now, studies on animal aging have based essentially on rats and mice (rodents) (1-4) and on macaques (primates) (5-8). However, these two groups have certain disadvantages: rodents are phylogenetically remote from humans, and have a short life span with a brief aging phase; macaques, like most monkeys or apes, are rare , expensive , difficult to breed and handle , of-
ten of uncertain age , and possess too long a life span for convenient longitudinal studies. Results from various recent studies (9, 10) have revealed the value of a new animal model, the grey mouse lemur (Microcebus murinus), a nocturnal primate from Madagascar belonging to the prosimian group. Its small size (length: 12 em, weight: 100 g), and relative ease of breeding are invaluable qualities. The grey mouse lemur has a maximum life span in captivity (12 years), and the length of its senescent stage (about 4 years) makes it ideal for combining cross-sectional and longitudinal studies. Furthermore, morphological and histopathologic alterations resembling those of aged humans have been observed in the brains of aged grey mouse lemurs. We recently began an analysis of the effects of aging on various behavioral aspects of Microcebus murinus, that included spontaneous social behavior and radial maze performance (11 , 12). The aim of this preliminary report is to present a comparative study of the performances of young and aged grey mouse lemurs in a delayed response task. This type of test has several advantages: a) It demands cognitive faculties of a "high order" (13). The discrimination of stimuli, not physically present at the time of the response , indicates a representative treatment. This test, therefore , is am ongst the most sens itive to phylogenetic differences. And, clearly, the aging process in humans affects mainly superior cognitive faculties. b) It involves a working memory parameter that seems to be especially altered during aging (14 , 15). c) It is sensitive to prefrontal lesions in monkeys , and various studies suggest that the prefrontal cortex is affected during aging (16 , 17). d) Studies involvinq this type of experiment in primates have shown how sensitive it is to the age of the subjects. The memory of recent stimulus events may be especially affected in aged monkeys (18 , 19).
Key words : Aging, memory , primate. Correspo nde nce : J-L. Picq , Pare Zoologique de Paris, Avenue de Saint-Maurice 53 , 7501 2 Paris, France . Received January 31 ,1994; accepted in revised form Sept ember 6 . 1994 .
Aging Clin. Exp. Res., Vol. 7, No.1
17
J-L. Picq
Therefore, it is of particular value to use delayed response tasks to assess the possible cognitive dysfunctions of the aged grey mouse lemur. MATERIAL AND METHODS Animals Eight grey mouse lemurs were divided into two groups: four aged between 2 to 4 years were the young group, and four 9 to 10-year olds were the aged group. Each group consisted of two males and two females. All grey mouse lemurs were derived from A. and J-J. Petter's breeding unit that was initiated in Paris in 1953. Prior to the test, all animals were subjected to the same behavioral testing history. All grey mouse lemurs used in this experiment had previously participated in studies concerned with social behavior, exploratory activityand motor abilities; in addition, they had been tested on radial maze task. Husbandry The animals were reared in individual wire mesh cages (0.5xO.5x1m high), containing a wooden nest box (12x12x12 em) and one plastic trough. The walls of the cages could easily be climbed by the grey mouse lemurs. The cages were located in a single closed room in which the light cycle was inverted in relation to natural daylight, so that the tests were performed durinq their activity phase. During the tests, the room was dimly lit by red light to which the colourblind grey mouse lemur shows negligible responses (20). The animals were fed daily at the beginning of each dark period. Food was varied and substantially based on fruits (bananas, apples, oranges, figs), carrots, mealworms and milk pap; water was freely available in the home cage. No food deprivation procedure was used for these tests. During testing, food and water were not available in the apparatus. Apparatus for testing The entire apparatus is made of plywood, and covered with a plexiglas roof to prevent the grey mouse lemur from escaping and to allow easy observation of their behavior. The apparatus, especially designed for grey mouse lemurs, is shown in Figure 1. It is composed of a vertical cylinder (height: 40 em) into which each animal is inserted when the test is begun. The lower part of the cylinder leads to a corridor giving access to the work chamber via a sliding door opened by the experimentalist. The base of the wall opposite the access corridor has four circularopenings (diameter: 35 mm); these openings are similar to the natural cavities found in trees in which the grey mouse lemur seeks refuge during the day, and also re-
18 Aging Clin. Exp. Res., Vol. 7, No.1
semble those of their nest-boxes and thus are especiallyappealing to these animals. Behind each opening is a corridor (length: 25 em) leading to the reinforcement chamber housing the grey mouse lemurs' usual nest-box. The corridors can be shut off at each end by sliding doors. They can also be lit by a 15 W bulb situated 4 cm above a plexiglas window located in the ceiling. Opaque partitions surrounding each bulb ensure that light does not radiate laterally towards the other corridors. Procedures The grey mouse lemurs, like other prosimians, are highly strung and easily distracted. In view of this fact, the following general rules were applied: 1) As change in environmental stimuli tends to be disruptive, the animals were tested in their home room; 2) A prolonged habituation was necessary to accommodate the grey mouse lemur to the test situation (see below); and 3) To avoid to subjecting the grey mouse lemurs to handling stress, their own nest-box was used to transfer them from their home cage into the apparatus (and conversely). Simple visual discrimination task The test starts by opening the door allowing a grey mouse lemur access to the work chamber. The task consists in selecting the only corridor out of four
reinforcement chamber
response corridors
*
*
sliding opaque sreen ~~~~~~~_~~~iV sliding transparent screen . , circular openings
t
30cmi
I •••• 1 switcns
starting
corridor
t _
vertical cylinder
Figure 1 - Apparatus used in Grey Mouse Lemur studies for discrimination tasks (* only for the delayed response visual discrimination task).
Memory in aged lemurian primates
training stage , a clear plexiglas screen is placed in front of the corridor openings, thus preventing access. After the grey mouse lemur has watched the illuminated corridor for 4 seconds , a second opaque screen is lowered behind the clear one, masking all corridors. The two screens are immediately lifted, and the corridor stays lit for another 2 seconds, so that the animal has to go through it unlit to reach its nest-box. Each grey mouse lemur is given a session of 20 trials everyday, until the criterion of 19 out of 20 correct responses is reached for two consecutive sessions. Training schedule-phase 2 : The grey mouse lemurs are trained to memorize the position of the illuminated corridor for a minimal delay ("O-sec" delay) before the availability of the response. A new series of trials is started where the lighting stimulus is stopped when the opaque screen is lowered. The grey mouse lemurs are given daily sessions of 20 trials, until the criterion of 16 out of 20 correct answers for two consecutive sessions is reached. Testing phase: Each grey mouse lemur does 10 sessions of 30 trials at the rate of a session every two days . Each session starts by 5 trials with the light kept on for 2 seconds after the lifting of the screens. These five initial trials were considered warm-up trials, and were not used in the data analysis. For the remaining 25 trials , the lighting stimulus is stopped when the opaque screen is lowered . The screens
which gives access to the nest-box situated in the reinforcement chamber; the stimulus guiding the choice is light. If a grey mouse lemur enters the illuminated corridor, the sliding exit door is lifted allowing free passage to the nest box, where it may rest for 2 minutes. If it enters one of the three unlit corridors, the exit door remains shut and the sliding entrance door is lowered behind the grey mouse lemur, thus blocking it inside the corridor, whose two exits are closed. The corridor is removable and allows the grey mouse lemur to be put again in the starting cylinder for another trial. The grey mouse lemur's urge to find its nest box is the only motivation; no food reward is necessary. Each grey mouse lemur is given a session of 20 consecutive trials every other day, until the criterion of 19 out of 20 correct responses is reached for three consecutive sessions. The illuminated corridor is chosen at random for every trial, but is the same for all the grey mouse lemurs. Three months after the criterion was reached, each grey mouse lemur was given one session of 20 consecutive trials . Delayed response visual discrimination task Training schedule-phase 1 : The grey mouse lemurs are trained to defer their response because the entry into a corridor was temporarily impossible , and the visibility of the stimulus was interrupted. During this
immovable response corridors
/-
----.JA
r--
-
,.....
position 1
-
-
-
-
rotation
-
" -
,.....
-
"
,.....
position 2
Figure 2 - 180 0 rotation of the work chamber during retention interval.
Aging Clin. Exp. Res. , Vol. 7 , No.1 19
J-L. Picq
stay down for a variable period: 0", 5", 15",30", 1'. Each session has 5 trials for each of the 5 delay periods. The presentation order of the 5 delay intervals was randomized with the restriction that the same delay interval not be presented on two successive trials. When the delays are between 5" and 1', half the trials involve a 180 0 rotation of the experimental enclosure again before making its choice (Fig. 2). Selection of the correct corridor (the one illuminated before the opaque screen came down) cannot be done by simply keeping position during the delay period, or by an eventual scent mark on the floor in front of the correct opening.
Statistical analysis In order to assess the influence of age and delay interval, a two-way analysis of variance technique was used. RESULTS
Simple visual discrimination tests The learning stage was rapid. All grey mouse lemurs had at least a 70 % success rate from the second session. Three grey mouse lemurs (two aged and one young) reached a 95% success rate at the third session, and the remaining grey mouse lemurs at the fourth session (Table 1). After the three-month break, the aged animals performed comparably to the young. All animals reached or exceeded an 85% success rate (Table 1).
Delayed response visual discrimination tests Rgure 3 shows the mean percentage of successful trials for each delay period. Statistical analysis reveals that all grey mouse lemurs had a much lower success
rate as the delay period increased (F (4.30) = 28.53, p
The delayed response task (DR) is one of the most widely used tasks for examining cognitive aging in the non human primate. A number of laboratories have .shown, in a variety of species, the high sensitivity of this procedure for revealing age-related deficits. Indeed, the vast majority of aged animals exhibit deficits on DR task. However, the principal question addressed by these studies is whether these deficits are attributable to a memory deficiency (21-23), or whether they may be related, for instance, to attention deterioration in the aged animals (24, 25). To answer this question, it is necessary to examine whether the diminished accuracy of an aged animal on DR tasks is dependent on delay intervals. Aged grey mouse lemurs were apparently sensitive to the length of the retention delay. In fact, they did not show any learning deficit for the simple visual discrimination test (non memory control condition), nor for the delayed response test with the zero-second delay (minimal memory control condition). The poor results of the aged grey mouse lemurs cannot be explained by a lack of motivation, psychomotor coordination, sensory processing or global comprehension of the task. The length of the delay was, therefore, the key factor ac-
Table 1 - Simple visual discrimination task. Scores are number of correct responses in 20 trials for each session (*indicates the criterion was reached), session 1
session 2
session 3
session 4
session 5
A
10
14
20'
19'
20'
B
11
15
15
19'
20'
20'
18
C
12
15
16
20'
19'
19'
20'
0
10
12
15
20'
20'
20'
19'
E
9
18
20'
20'
20'
F
10
15
19'
19'
19'
G
12
13
17
19'
19'
20'
20'
H
11
14
17
19'
20'
20'
18
session 6
session after 3-month break
young animals 20'
old animals
20 Aging Clin. Exp. Res., Vol. 7, No.1
19' 17
Mem ory in aged lemurian primates
REFERENCES
100
1. Elias P .K. : Effects of age on learning ability: contribution from the animal literature . Ex p. Ag. Res. 2 : 16 5-1 86, 1976 . 2 . Dean R.L., Scozzafava J ., Goas J A, Reagan B., Beer B., Bartus R.T. : Age-relat ed differ ences in behavior accross th e life span of the C57 B/6 J mous e . Exp. A g. Res. 7 : 427-451 , 1981. 3. Kubanis P ., Zornetzer S .F.: Age related beh avioral and neurob iological changes: a review with an emphasis on memory. Beha u. Ne ur. Bio I. 3 1: 115-1 72 , 19 81. 4 . Wallace J .E., Krauter E.E., Campbell B.A.: Animal mode ls of declining memory in th e aged : sho rt-te rm and spatial mem ory in th e age d rat. J. Gerontal. 35: 355-363 ,1980.
t; 80 UJ
""""
0
u
~
z
UJ
u
cc
UJ
0..
60
Z
-c
UJ
::;:
-G-
......
40
-
20 L...-..L..-_..L..-_ _- ' -
o
5
15
YOUNG AGED
chance performance
-...L
_
60
30
RETENTION INTERVAL (Sec)
Figure 3 - Effect of age o n delayed - response perform ance.
counting for the different performance level in the two age groups . Furthermore, for each delay period , the resultswere the same whether or not the work chamber was rotated. The problem , therefore, was not solved by maintaining a fixed position towards the positive stimulus during the retention delay. It is thus possible to interpret these results as the consequence of a short -term memory alteration during aging in grey mouse lemurs. This memory of recent stimulus events (working memory type) seems to be selectively affected by age , because the memory needed to achieve rapid learning of a visual discrimination task appears to be unaffected . Also, after a three-month break, the aged grey mouse lemurs managed to do the task again, showing that their long-term memory was unaffected . These experiments need to be repeated with a larger group of grey mouse lemurs. It would also be beneficial to follow the same subjects over several years to determine how their performance changes. Noneth eless, it Is interesting to note that the results presented in this study are close to those obtained by Bartus et al. (18) with aged monkeys . As such, these preliminary results seem to confirm that cognitive disorders are gene ral in aged primates, and that the grey mouse lemur represents a spe cies of interest as an animal aging model.
ACKNOWLEDGEMENTS I would like to thank A and J-J . Petter who allowed me to work in their precious lemurian breed ing unit.
5 . Dean R.L. , Bartus R.T .: Animal mode ls of geriatric cog nitive dysfunction : evide nce for an impo rtant cho line rgic involmen t. In: T raber J ., Gispen W.H . (Eds .), Sen ile dem entia of the Alzheim er type. Springer-Verlag, Berlin , 19 8 5 , pp . 269282 . 6. Dean R.L., Dean L.K., Bartus R.T.: Modeles comportementaux et ph armacologiques du vieillissement chez les primates. In: Le uieillissement cerebral. P .U.F. , Paris, 19 90, pp. 159188. 7 . Rapp P .R.: Visual discrim inat ion and rever sal learni ng in the ag ed monke y (Maca co mu latt a). Behau. Ne u rosci. 104: 876-8 84, 19 90. 8 . Bachevalier J ., Landis L.S ., Walker L.e.. Brickson M., Mishkin M., Price D.L., Co rk L.e. : Aged mon keys exhibit behavioral deficits indicative of widespread cerebral dysfunction. Neurobiol. Aging 12 : 99-1 11 , 199 1. 9 . Bon s N., Mestr e N., Petter A : Se nile plaques and neu rofibrillary changes in th e brain of an ag ed lemurian primat e . Neurob iol. Aging 13: 9 9-105,1991. 10. Bons N. : Le microcebe, un modele animal pou r l'e tude de la malad ie d'Alzh eimer. Al zh eim er Actualites 8 2 : 8- 11 , 1993 . 11. Picq J-L. : Aging and socia l behavi our in captivity in Microcebu s murinu s. Folia Primatol . 59: 2 17-220 , 19 92. 12 . Picq J-L. : Rad ial maz e pe rforma nces in young an d age d grey m ou se lemurs (Microceb us murinus). Prima tes 34 : 2 2 32 26,1 9 9 3 . 13 . Flet ch er H .J .: Th e delayed res po nse problem . In: Schrie r A.M., Harlow H.F., Sto llnitz F. (Eds.), Behavior of non-hum an primates. Academ ic Press, New-York , 19 65 , pp. 129-166 . 14. Olt on D .S .: Memory function and th e h ip p ocampus. In: Seifert W. (Ed.), Neurobiology of the hippocampus. Academ ic Press, New-York, 1983. 15. Badd eley AD., Logie R. , Bressi S. , Della Salla S.S , Spinnler H. : Dem e nt ia and working memory. Q. J. Exp. Psychol. 38: 6 03-6 08, 1986. 16. Goldm an P .S ., Rosvold H .E.: Localization of funct ion within th e dor solateral prefrontal cortex of th e Rhesus monkey. Exp. Neur al. 27 : 291-304, 1970. 17 . Forett e F., O rgogozo J .M., Bouchacourt P ., Hervy M.P .: Aspects cUniques et therapeutiq ues du vieillissement cerebral. In: Le vieillissement cerebral, P.U .F., Paris, 1990, pp. 26 5-2 8 6 . 18 . Bartus R.T .: Effects of aging on visual mem ory , sensory processing and discrimination learning in the non-h uman primate. In: Ordy J .M., Brizzer K. (Eds.), Aging. Raven Press, NewYork, 1979, pp. 85-114. 19. Rapp P .R., Amaral D.G .: Individual differences in the cognitive and neurobiolog ical consequences o f n ormal agin g . Trends Neu rosci. 15: 34 0 -345 , 1992.
Aging Clin. Exp. Res., Vol. 7, No.1 21
J-L. Picq
20 . Pariente G.: App roche eco-etho loqique de la vision chez les prosimiens malgaches . J. Physio!. 1: 141 -164, 1977. 21. Bartus R.T., Remming D.L., Jo hnson H.R: Aging in the Rhesus mon key: Debilitating effects on short-term memory. J . Geron tol. 33 : 858-87 1, 197 8 . 22 . Bartus RT. , Dean RL. , Beer B.: Memory deficits in aged cebus monkeys and facilitation with central cholinomimetics. Neurobio!. Aging 1: 145-152, 1980 .
22
Aging Clin . Exp. Res. , Vo l. 7 , No.1
23 . Medin D.L.: Form percept ion and pattern reproduction by monkeys. J. Camp. Physio!. Psycho!. 68 : 4 12-4 19. 1969 . 24 . Riopelle A.J ., Rogers C.M.: Age changes in chimpanzees . In: Schrier A.M., Harlow H.F., Stollnitz F. (Eels.), Behav ior of non -human primat es. Academic Press, New-York, 196 5. pp. 449-462 . 25. King J.E., Michels RR : Erroranalysis of delayed response in aged squirrel monkeys. An im. Learn. Behav. 17: 157 -162. 1989.
