Hyperactivity and instrumental learning deficits in methylazoxymethanol-treated rat offspring

Neurotoxicologyand Teratology, Vol. 10, pp. 341-347. ©PergamonPress plc, 1988. Printedin the U.S.A.

0892°0362/88$3.00 + .00

Hyperactivity and Instrumental Learning Deficits in Methylazoxymethanol-Treated Rat Offspring T R E V O R A R C H E R , * A R T O J. H I L T U N E N , t T O R B J O R N U. C. J A R B E , t M. R E Z A K A M K A R , t J O H A N L U T H M A N , ~ E R I K S U N D S T R O M $ AND ANNA TEILING§

*Department of Psychobiology, University o f Gothenburg, Box 14158, S-400 20 Gothenburg, Sweden tDepartment of Psychology, Uppsala University, S-751 04 Uppsala SDepartment of Histology, Karolinska Institute, S-104 09 Stockholm §AB Astra, Safety Assessment, S-151 85 Sfdertiilje R e c e i v e d 26 J u n e 1987 ARCHER, T., A. J. HILTUNEN, T. U. C. JARBE, M. R. KAMKAR, J. LUTHMAN, E. SUNDSTROM AND A. TEILING. Hyperactivity and instrumental learning deficits in methylazoxymethanol-treated rat offspring. NEUROTOXICOL TERATOL 10(4)341-347, 1988.--Several changes of spontaneous motor and learned behaviours were obtained in the male offspring of pregnant rats that were treated on gestation day 15 with the antimitotic agent methylazoxymethanol (MAM, 25 mg/kg). MAM-treated offspring, when tested at adult ages, showed notable increases in motor activity parameters as measured by direct observation or in automated photocell test cages. This hyperactive state was accompanied by clear impairments by MAM offspring in the acquisition of instrumental learning in a radial arm maze and in a circular swim maze. In Skinner boxes, MAM offspring made fewer responses during the Fixed Ratio (FR) 1 schedule but did not differ from the saline offspring in the acquisition of the difficult differential-reinforcement-of-low-rates (DRL) 72 sec task. Neurochemical assays indicated that the MAM rats had elevated noradrenaline and dopamine levels in several brain regions. These fin.dingsare discussed with regard to possible alterations of habituation processes in MAM rats. Methylazoxymethanol Forebrain microencephaly Operant schedules Noradrenaline Dopamine

Hyperactivity Habituation

M E T H L A Z O X Y M E T H A N O L (MAM) is an antimitotic agent derived from cycasin. It has a cytotoxic effect, presumably caused by alkylation of nucleic acids. Since MAM has a short half-life in vivo it is widely used as an experimental tool to kill discreet populations of neurons during development. The administration of MAM to pregnant rats on gestation day 14 or 15 results in offspring with a severe telencephalic hypoplasia from the MAM mothers [8,27], with the cerebral cortex and hippocampus most markedly affected [7,10]. The importance of this developmental retardation, induced during fetal life, has been discussed in the context of animal models of certain human disorders in which significant cortical hypoplasia is obtained [30]. A relative hyperinnervation of monoamine nerve terminals has been clearly indicated from the neurochemical analyses [11-14]. The neurochemical and morphological changes have been most consistently correlated with impairments in the performance of instrumental learning tasks [5,9], a marked hyperactivity, as measured by several tech-

Instrumental learning tasks Male rats

niques [31,35], and severe alterations in the conditioning of context-dependent phenomena [3, 20, 21]. Thus, Haddad et al. [9] reported that MAM-treated rats showed learning impairment in a Hebb-Williams maze and others have reported deficits in the acquisition of passive avoidance responses [6,35]. MAM-treated rats have also been found to demonstrate marked and long-lasting hyperactivity over several parameters of activity as well as with a variety of test procedures (cf. [29]). Prenatal MAM treatment has also been reported to cause an array of physical effects such as general growth retardation [8], reduced organ weights [10] and retinal dysplasia [18]; in no case do these studies suggest undernutrition on the part of the pregnant rats. The purpose of the present investigation was to study MAM- and salinetreated offspring in the performance of several types of learned and spontaneous behaviours. Thus, two instrumental maze learning tasks were selected, in a radial arm maze and a circular swim maze, that require very different behaviour patterns, the former requiting appetitive, food-

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342 seeking behaviour and the latter swimming to escape onto a platform in the middle of a circular bath. Spontaneous motor behaviour was measured both by direct observation (in the modified radial arm maze) and by the use of automated test cages. Operant behaviour was measured over a long period (I0 weeks) using a simple Fixed Ratio 1 schedule and a difficult differential-reinforcement-of-low-rates (DRL) schedule to further characterize the behavioural changes induced by the prenatal MAM treatment [20]. The purpose of employing the DRL schedule was that some deficit by the MAM rats was predicted in view of the well documented hyperactivity and "irritability" noted in the animals. METHOD

Animals and Drug Treatment Five pregnant Sprague-Dawley rats (ALAB, Sollentuna, Sweden) were injected intravenously (IV) with 25 mg/kg MAM acetate under light anaesthesia on gestation day 15 (day of mating=day 1). MAM acetate was diluted in physiological saline (0.9% NaC1) and injected in a concentration of 10 mg/ml. Control dams were also anaesthetized and received saline alone in an equal mode of administration. Litter size was limited to 4-6 pups per dam. The offspring were weaned at 25 days of age and 20 male pups, 2 from each litter, were retained for behavioural testing and neurochemical analysis. Of these, 16 rats (8 MAM and 8 Control) were randomly chosen for the experiment and the remaining 4 were kept as reserve. The animals were housed in groups of 2 or 3 rats under laboratory conditions involving a 12 hour on/12 hour off lighting schedule (lights on at 07.00) in an air-conditioned room thermostatically maintained at 21-+1°C. The animals were maintained on ad lib food (Lab Chow R3 Ewos, S6dert/ilje, Sweden) and water, except where indicated. No cross-fostering procedure was employed in keeping with the general outline used in MAM studies (e.g., [25, 30, 35]). However, the effects of crossfostering have been investigated in a later study (manuscript in preparation).

Apparatus Radial arm maze. The radial arm maze task, a procedure sensitive to deficits in learning performance [23,24], was adapted both to evaluate spontaneous motor activity and cognitive function [1]. Each of the 8 arms (each 54 cm long and l0 cm wide) of the Olton maze was " m a r k e d " off (divided) into three units (each 18 cm long and l0 cm wide) which gave a total of 25 units (counting the central hub, 25 by 25 cm) within walls that were 25 cm high. The radial arm maze was always placed on the floor, and a video-camera positioned in the ceiling filmed each rat. Behavior was monitored on a TV screen placed in an adjoining room. The food cups, on the floor of the maze at the extremity of each arm, were not accessible during motor activity testing due to the presence of an extra, removable back wall. For the learning test the 'false' back walls were removed and each food cup was exposed. Standard food pellets (45 g) were obtained from Campden Instruments Ltd. (London, England). Swim maze (Morris-type). The swim maze was similar to that described by Morris [22], although a different procedure to that of Morris was used here and thus the term swim maze is applied. The circular pool, diameter 140 cm, was invariably filled with water to a depth of 30 cm and was equipped with both a heating element and a thermostat at the bottom of the pool. At testing the temperature of the water was

maintained at a constant 25°C. A Plexiglas platform was placed at a specific and constant position in the pool, remaining at a depth 1 cm below the surface of the water. Drying cages equipped with blow-dryers were always provided to aid each rat to dry itself properly following testing. Spontaneous motor activity. Locomotion, rearing and motility were measured over 30-min periods using Motron Mark 24 Fc/R instruments, automated devices utilising photocell beams to register the counts from each parameter of activity [21]. The test cages were identical to the rat home cages (40x25x15 cm). High level beams (13 cm from floor level) measured rearing and photocells under the floor of the cages measured locomotion when at least 4 were activated. Motility was measured as soon as any beams were activated. Spontaneous motor activity was measured over a 7-day period on days 71 to 77 after birth. Differential-reinforcement-of-low-rates (DRL) schedule. Rate of reinforcement and rate of responding under Fixed Ratio (FR) and DRL schedules were measured using standard rodent Skinner boxes (Model No. 410, Campden Instruments Ltd., England) with two retractable levers (bars). During the DRL experiment only one of the bars was available, the left one in two boxes, and the right one in the other two boxes. Thus assignments of bar position were such that half the number of rats had to press a lever placed to the left of the drinking cup whereas the other half had to press a right hand lever to produce the reward, a 4-sec access to sweetened water (0.1% saccharin). The four chambers were sound attenuated and ventilated by exhaust fans. Masking sound was provided by a white noise generator. Schedule programming and the event recording employed electromechanical equipment located away from the experimental chambers.

Procedure All behavioural testing was performed during daytime from 0900 to 1500 hours. Locomotor activity. Activity in the radial arm maze was measured on days 61 to 64 after birth. Each rat's behaviour was monitored with a video TV apparatus placed in a separate room. Ambulation and rearing were measured over 10-min periods of each test occasion. Ambulation is defined as the passage of a rat's body from one unit to another, and rearing was scored each time a rat raised itself onto its hind legs. After each rat was tested the maze was wiped carefully with water containing detergent so that the maze was clean and any odors left by each rat removed. Following motor activity measurements all the rats were placed on a total food deprivation for 48 hours. Acquisition performance. Acquisition in the radial arm maze was tested at ages 66 and 67 days (Tests 1 and 2). For this, the 'false' back walls were removed, exposing the 8 food cups, into each of which a 45 mg food pellet was placed. At the start of each test, each rat was placed in the central hub and then monitored for its performance in taking all the pellets. Body weights at the onset of testing were: MAM=190_+15 g, saline=230_+10 g. This is in agreement with reports of generalized growth retardation in MAM offspring [8]. Two measures of performance were taken: latency to take all the 8 food pellets and the number of arms visited in acquiring all 8 pellets. Immediately after the second test each rat was given free access to food. Spontaneous motor activity. Spontaneous motor activity was measured in the Motron automated test cages on days 71

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FIG. 1. Number of ambulations (top panel) and number of rearings (bottom panel) by MAM-treated rats and control (saline-treated) rats in a radial arm maze. Motor activity was measured on two occasions (days 1 and 2) over a 10-rainperiod in each case. Access to the food cups was blocked off by the placement of the 'false' back walls. The results are expressed as means±s.e.m.

and 77 of age. Although it is not recommended that activity be tested so soon after a deprivation schedule, in this case it was deemed necessary in order to compare activity levels with those obtained in other nonconfounded designs. On the day after the last activity test (78 days of age), the measure of acquisition performance in the Morris swim maze was started. Swim maze performance. Five trials were presented to each rat, after the platform had been placed at a particular position within the circular pool. For each trial, each rat was placed at the same point in the pool and allowed to swim around to find the platform and escape from the water onto a platform which was located 1 cm below the water level. Five trials were presented to each animal on each of 4 consecutive days (78 to 81 days after birth). On reaching the platform each rat was allowed to remain upon it for 30 sec before being placed in the water again for the next trial. If a rat failed to locate the platform within 65 sec it was removed from the water and placed on the platform for 30 sec. After the fifth trial each animal was placed in the drying cage, a cage confronted by a blow-dryer which blew warm air towards it. Thus, care was taken that each rat was allowed to dry itself completely before being replaced in its home cage. In addition to a drawing of the swim path of each rat on each trial, two measures of performance were made: (1) the latency to reach and climb onto the platform and (2) the number of trials on which the animal failed to reach the platform during a 65-sec swimming period. Operant conditioning to a D R L 72 schedule. One week after the swim maze test all the rats were placed on a water deprivation schedule by which water intake was restricted to

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FIG. 2. Latency to take all eight pellets (top panel, A) and the number of arms visited in taking all eight pellets (bottom panel, B) by MAM-treated rats and control (saline-treated) rats in the radial arm maze. Maze learning performance was measured on two occasions (Test 1 and 2) with a twenty-four hour interval between each test. Access to the eight food cups (each loaded with one pellet) was open by removal of the 'false' back wall. Results are expressed as means+s.e.m.

a 30-rain period each day. During this period the animals were shaped to press a bar in the box, each bar press producing access to the reinforcer, a 4-sec presentation of sweetened water. During this week the rats remained on an effective FR l schedule. Thereafter, on the 9 .succeeding weeks the rats were placed on DRL 6, 12, 18, 24, 36, 48, 60, 72 and 72-sec schedules, respectively, whereby during each week the rats were subjected to each incremented DRL schedule for 5 days and then a 2-day rest period before the onset of the next DRL schedule. For the performance of the DRL schedule it was necessary for the rats to withhold a bar-pressing response in order to be reinforced, i.e., they were introduced to the DRL schedule in which only responses that had been withheld for a given length of time were rewarded. The time that bar pressing had to be withheld was successively increased from 6 sec with increments of first 6, until DRL 24 sec, and then 12 sec until DRL 72 sec was in effect whereby at least 72 sec must have elapsed since the last bar-press in order to give access to the reinforcer, a 4-sec presentation of 0.1% saccharin in water. Premature responses reset the clocks so that the delay between the last lever press and the reinforced lever press was always >~72 sec (in the final stage of training). This requirement was, of course, in effect throughout the various stages of DRL training even though the length of the delay period varied with the DRL requirement in effect. The DRL 72 schedule was continued for two weeks, i.e., two 5-day periods.

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TABLE 1 MEAN (-s.e.m.) LOCOMOTION, REARING AND MOTILITY COUNTS BY MAM- AND SALINE-TREATED OFFSPRING IN AUTOMATED ACTIVITY TEST BOXES. Test Days (age)

Locomotion Rearing Motility

MAM saline MAM saline MAM saline

71

72

73

74

75

76

77

68-- 4* 37_+ 2 138 ± 9 136 ± 7 3963* -+355 2668 ± 107

58-±- 6* 28--_ 2 131 ± 8 131 ± 6 2872* ±342 2106 ±97

61 ± 3* 26 ± 2 148 ± 10 128 ± 7 3017" ±322 1663 ±88

5 6 ± 3* 26± 3 139 ± 11 129 ± 11 3108" ±317 1738 ±86

59 ± 4* 24 ± 4 142 ± 7 127 ± 6 2466* ±286 1669 ±91

61 ± 7* 23 ± 3 173 ± 12" 126 ___5 2787* ±273 1603 ±92

94 ± 2* 26± 4 248 ± 16" 129 ± 9 4026* ±411 1528 -+79

The rats were tested at 71 to 77 days of age during 30 rain testing periods. *p<0.01, Tukey's HSD test.

A

Neurochemical analysis. Following the D R L testing the animals were sacrificed and forebrain regions were analysed for N A and D A concentrations, as described by Jonsson and Hallman [14] and Keller et al. [16]. E n d o g e n o u s catecholamine c o n c e n t r a t i o n s were d e t e r m i n e d using high pressure liquid c h r o m a t o g r a p h y with e l e c t r o c h e m i c a l detection. Statistics The s p o n t a n e o u s m o t o r activity data, the acquisition data from the radial arm m a z e and the Morris swim m a z e , as well as the data from the operant conditioning tasks w e r e all submitted to t w o - w a y analysis o f variance ( A N O V A ) according to S n e d e c o r and C o c h r a n [33]. Pairwise testing was p e r f o r m e d using T u k e y H S D tests. Data on c a t e c h o l a m i n e levels w e r e tested by the S t u d e n t ' s t-test. The 1% level of significance was maintained throughout unless o t h e r w i s e stated. RESULTS

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Radial Arm Maze Activity The M A M - t r e a t e d rats d e m o n s t r a t e d a clear hyperactivity for b o t h the ambulation and rearing measures. Figure 1 presents the n u m b e r of ambulations and rearings by M A M and saline-treated rats on each day of testing. T w o - w a y ANOVA indicated a significant Interaction effect, F(1,28)=4.9, for the ambulation data and a significant Interaction effect, F(1,28)=4.3, for the rearing data. Testing with T u k e y ' s H S D tests indicated that the M A M - t r e a t e d rats exhibited significantly m o r e ambulation and rearing than the saline rats during Test 2.

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FIG. 3. Latency to reach the platform (top panel, A) and the number of failures to locate the platform (bottom panel, B) by MAM-treated rats and control (saline-treated) rats in the swim maze test. Results are expressed as means_s.e.m.

Radial Arm Maze Learning Performance The M A M - t r e a t e d rats disclosed an i m p a i r m e n t of acquisition p e r f o r m a n c e in the m a z e learning task. Figure 2 presents the latencies to take all 8 pellets and the n u m b e r of arms visited in taking all 8 pellets, by M A M - t r e a t e d and salinetreated rats. T w o - w a y A N O V A indicated significant Groups effects both for the latency data, F(1,28)=19.1, and the n u m b e r o f arms visited data, F(1,28)=11.3. T u k e y ' s H S D tests indicated longer latencies and a greater n u m b e r of arms visited to obtain all eight pellets by the M A M - t r e a t e d rats in c o m p a r i s o n with the saline-treated rats. M A M and saline

rats were tested for food intake during a 4-hour period bet w e e n the tests but no differences were obtained. The intakes were as follows: MAM=7.9--+0.6 g, saline=8.3-+0.4 g (means_+s.e.m.).

Spontaneous Motor Activity M A M - t r e a t e d rats d e m o n s t r a t e d notable hyperactivity in the a u t o m a t e d (photocell) test cages during recordings o v e r a 7-day period. Table 1 presents the m e a n l o c o m o t i o n , rearing

MAM E F F E C T S ON HYPERACTIVITY AND L E A R N I N G A

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TABLE 2 MEAN BODYWEIGHTSOF MAM AND CONTROLRATS DURING THE PERIODOF THE DRL SCHEDULEAND WATERDEPRIVATION ( A G E 88-156 DAYS AFTERBIRTH). Day

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FIG. 4. Number of reinforcements received (top panel, A) and number of responses made (bottom panel, B) by MAM-treated and control (saline-treated) rats during the FR 1 and DRL schedules. Results are expressed as means±s.e.m.

and motility counts over daily 30-min periods by the MAMand saline-treated rats. Two-way ANOVA indicated significant Groups effects for all three behaviour parameters [Locomotion, F(1,98)=21.4; Rearing, F(1,98)=5.1; and Motility, F(1,98)= 19.8]. Pairwise testing with Tukey's HSD test indicated significantly more locomotion and motility counts by the MAM rats on all the test occasions and significantly more rearing counts when tested at 76 and 77 days of age.

Swim Maze Learning Performance The MAM-treated rats disclosed a drastic impairment in the acquisition of the swim maze task. Figure 3 presents the mean latencies to reach the platform and the mean number of failures to locate the platform by the MAM-treated and saline groups. ANOVA indicated significant groups effect, F(1,56)=50.4, for the latency data as well as for the failures to locate the platform data, F(1,56)=41.0. Tukey's HSD tests indicated significantly longer latencies to reach the platform and more failures to locate the platform by MAM rats as compared to the saline animals.

The MAM-treated rats made fewer responses on the FR 1 schedule and received fewer reinforcements. On the DRL schedule leading up to DRL 72, no differences between the MAM and saline groups were obtained for either the number of reinforcements or the number of responses, except that the MAM-treated rats made fewer responses than the control rats during the DRL 6 schedule. Figure 4 presents the mean number of reinforcements and responses for MAM- and saline-treated rats in the performance of the FR and DRL schedules. Two-way ANOVA indicated a significant Groups x Schedule interaction both for the number of reinforcements, F(9,180)=28.1, and for the number of responses, F(9,169)= 14.9. Tukey's HSD tests indicated that the MAMtreated rats made fewer responses on the FR 1 schedule and received fewer reinforcements. No significant effects of the prenatal treatment upon the performance of responses resulting in reinforcement (i.e., the number of reinforcements) in the various DRL schedules (i.e., DRL 6 sec-DRL 72 sec) were obtained. For the total responses parameter on the DRL 6 schedule the MAM rats made significantly fewer responses, but received a similar number of reinforcements. Table 2 presents the body weights of the MAM and control rats during the period of the DRL schedule (9 weeks), i.e., when the animals were placed under the water deprivation schedule. From the table it appears evident that both control and MAM rats remained at essentially unaltered body weights during DRL training.

Neurochemical Analysis Prenatal MAM treatment caused marked increases in catecholamine concentration in the occipital cortex and hippocampus but no alterations were obtained in the hypothalamus and cerebellum. In the mesencephalon, NA concentrations were significantly increased but not DA concentrations. DISCUSSION Offspring of pregnant rats that had been administered MAM (25 mg/kg) or saline on gestation day 15 were tested for spontaneous motor activity and instrumental learning performance from 61 days of age till just over 22 weeks of age, after which they were sacrificed for neurochemical analysis. The results of the various behavioural tests may be summarized as follows: (1) The prenatally MAM-treated rats

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TABLE 3 THE EFFECT OF PRENATALADMINISTRATIONON GESTATION DAY 15 ON CATECHOLAMINECONCENTRATIONSIN VARIOUS BRAIN REGIONS. Noradrenaline (ng/g)

Occipital Cortex Hippocampus Hypothalamus Cerebellum Mesencephalon

Dopamine (ng/g)

MAM

Saline

MAM

Saline

4582* _+473 1472* ± 164 2418 _+210 156 ±5 571t ±17

1909 ± 152 561 _+29 2008 ± 129 158 _+8 511 ±9

492t ± 115 41* +5 378 ± 15 7 ±5 165 ±11

270 _+71 10 ±1 399 _+17 8 ±1 145 ±14

Catecholamine concentrations were assayed as described previously [14]. Values are expressed as means--s.e.m. (n=7). *p<0.001, tp<0.01, Student's t-test.

demonstrated marked hyperactivity, whether measured by direct observation of the rats in an adapted radial arm maze or by automated photocell devices in test cages. (2) MAMtreated rats were impaired in the acquisition of the radial arm maze task, for both the latency and number of arms visited parameters, and they were also impaired in the acquisition of the swim maze task, for both the latency and the number of failures to reach the platform parameters. (3) MAM-treated rats showed a clear decrease in responding on the FR 1 schedule and as a result received fewer reinforcements. On the DRL schedules MAM-treated rats did not differ significantly from the saline animals on either the number of reinforcements or responses. However, the MAM rats did make significantly fewer responses on the DRL 6 schedule but received the same number of reinforcements as the saline rats. (4) The neurochemical analysis indicated that both NA and DA concentrations were increased in several of the regions analysed. These results are consistent with the findings of previous neurochemical assays [14,20]. Earlier investigations have indicated marked forebrain microencephaly with weight reductions of the cerebral cortex, hippocampus and striatum [11,30], and the volume reduction of the forebrain structures has been associated with a relative hyperinnervation of catecholamine terminals [34]. The alterations in the performance of radial arm maze and swim maze learning and operant responding on an FR 1 schedule suggest a reliable disruption in the ability of MAMtreated rats to acquire some, but not all, instrumental responses. This debility is accompanied consistently by marked hyperactivity of MAM rats over several parameters of motor activity. In the present instance, it is interesting to note that the locomotion and ambulation measures indicated the MAM hyperactivity effect on the first test occasions whereas the rearing measure developed with repeated testing, as has been suggested from the previous observations [3, 21, 35]. It has been argued that MAM treatment results in a disruption of habituation [21,35] and this seems to be a

reasonable explanation with some degree of exploratory behaviour and it is therefore to be expected that both MAM and control animals show rearing behaviour to a comparable extent during the initial exposures to the test apparatus. However, whereas control animals demonstrate less rearing or exploratory behaviour over repeated presentations, the MAM animals show, if anything, an increase, i.e., there appears to be some alteration of habituation processes in the MAM rats [3]. It is interesting to note that the MAM-treated rats tended to make more responses during the DRL 12, 18, 24 and 36 schedules but at the same time there was a consistent tendency for these rats to receive more reinforcements, although not significantly so. As a general rule, a greater number of responses by a rat leads to the presentation of fewer rewards in the DRL schedule of reinforcement. Therefore, it ought to be considered that through some circumstance, as yet inexplicable, the responding of MAM animals in the DRL schedule is unusually effective, a conclusion underlined by the observation that at DRL 6 the MAM groups performed fewer responses but received as many reinforcements as the control group (see Fig. 4). This result was surprising since we had expected the hyperactive MAM rats to show an impairment in acquiring the DRL schedule. Note, however, that Rabe and Haddad [27] found MAM rats to perform as well as controls on a DRL 20 sec schedule. It is important to note that the body weights of MAM and control rats appear to remain at a constant level during the period of the DRL schedule (see Table 2). The hyperkinetic effect of prenatal MAM treatment is the most consistent effect seen with these animals [3, 6, 15, 17, 25, 26, 28, 29, 31, 32, 35]. Seo et al. [32] found no change in rearing behaviour but significant increases in ambulation around the open-field perimeter, just as Cannon-Spoor and Freed [6] obtained locomotion increases during 60-min tests in photocell cages. Sanberg et al. [30] make the important observation that the MAM-induced increases in activity could reflect an increased exploration and/or a failure to habituate to the test situation. There seems to be three lines of evidence in support of this: (1) During extended periods of activity (> 12 hours) testing of MAM rats there appears not to be any increase in the activity [17,31]. (2) Measures of exploratory behaviour indicated increases by MAM rats [26,35]. (3) When MAM and control rats are tested over both daytime and nighttime periods the major increase in activity seems to occur during the nighttime period [17,28], during which the hyperactivity effect appears to develop earlier in MAM rats [15]. Thus, Sanberg et al. [30] associate nighttime activity in rats with greater levels of arousal and exploration and subsequently predict the most marked activity increases of MAM rats to be during the nocturnal hours. In the present study all testing was performed during daytime hours and over short periods (10 min in the radial arm maze, 30 min in photocell cages) but it may be relevant to note that the hyperactivity effect was noted both before and after the food deprivation schedule, which was incorporated for the purpose of testing radial arm maze acquisition. In this context it should be noted that the impaired acquisition seen here in the radial arm maze confirms the results seen earlier by Pevsner et al. [25]. In addition to the instrumental learning deficits indicated above, MAM-treated offspring also show marked alterations of some classical conditioning phenomena studied in conditioned taste-aversion learning. Thus, the contextdependent latent inhibition effect [2], whereby the conditioning deficits induced by prior repeated preexposure to the

MAM EFFECTS ON HYPERACTIVITY AND LEARNING conditioned stimulus (latent inhibition [19] effect) are altered by manipulation o f c o n t e x t u a l stimuli, was abolished in M A M offspring. Similarly, context-dependent extinction effects may be manipulated by varying the contextual stimuli present during the conditioning and extinction phases [4]. M A M treated rats failed completely to show the context-dependent extinction effects in the taste-aversion conditioning and ex-

347 tinction o f a saccharin a v e r s i o n [3]. T a k e n together, the various results indicating considerable functional changes of M A M - t r e a t e d rats may be a c o n s e q u e n c e of alterations in the attentional p r o c e s s e s underlying the rat's p e r f o r m a n c e of the behavioural tests e m p l o y e d [20], and certainly the considerable n e u r o c h e m i c a l changes would suggest that there exists a strong correlate for the behavioural impairments.

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