Neurotoxicologyand Teratology,Voi. 14, pp. 259-264, 1992

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Effects of Postnatal Aluminum Exposure on Choline Acetyltransferase Activity and Learning Abilities in the Rat G I L B E R T C H E R R O R E T , * V I V I A N E B E R N U Z Z I , * D I D I E R DESOR,t" M A R I E - F R A N C E H U T I N , ~ D A N I E L B U R N E L ~ A N D P A U L R. L E H R *1

*Centre des Sciences de l'Environnement, Neurotoxicologie, Universitd de Metz, Metz "fLaboratoire de Biologie du Comportement, Facultd des Sciences, Universitd de Nancy 1, Nancy ~Service de Chimie gdndrale appliqude ~ ia Mddecine, Facultd de Mddecine, Universitd de Nancy 1, Nancy, France Received 9 M a y 1991 CHERRORET, G., V. BERNUZZI, D. DESOR, M.-F. HUTIN, D. BURNEL AND P. R. LEHR. Effectsof postnatal aluminum exposure on choline acetyltransferaseactivityand learningabilitiesin the rat. NEUROTOXICOL TERATOL 14(4) 259-264, 1992.-Young rats were treated by gastric intubation with aluminum lactate (0, 100, and 200 mg Al/kg/day) from postnatal days 5 to 14 to determine the treatment's influence on brain choline acetyltransferase activity and learning abilities, The results indicated that aluminum concentrations in the cerebral areas increased in parallel to plasma aluminum at the dose of 200 mg. In the same case, choline acetyltransferase activity was reduced. At postnatal days 50 and 100, the treated rats did not show alterations in their learning abilities in the 2 tests which are based on different motivations (avoidance of an aversive light or alimentary motivation) and different ways of achievement (pressing on a lever or running in a maze). A low reduction in the general activity, particularly in the radial maze test, was only observed in rats treated with 200 mg Al/ kg/day. Rat intoxication

Aluminum lactate

Acetylcholinetransferase (CAT) activity

IN healthy human subjects, aluminum is mainly absorbed by the gastrointestinal tract (7,31) and readily excreted in the urine (34). In patients with chronic renal failure this excretion is impaired. Uremic patients undergoing dialysis or given oral preparations o f aluminum-containing phosphate binding gels exhibit rapid accumulation o f this metal in various tissues, often leading to toxicity (8). Aluminum is considered to be the causal factor for a high incidence of dialysis encephalopathy (1). Microcytic anemia and osteomalacia usually appear before the neurologic symptoms (27,35). These conditions seem to be especially prevalent in young children with uremia who received large doses o f aluminum to control their serum phosphorus levels (2,17,24). In infants with chronic renal failure, the increased incidence of encephalopathic and osteomalacia symptoms may also be due to an immaturity of the gastrointestinal absorption mechanism and metabolism, which increases the vulnerability of the child to tissue accumulation of aluminum (2). However, in the experimental field, there is relatively little information regarding the behavioral effects in young animals

Learning abilities

directly intoxicated by this metal. After injection of aluminum tartrate (3 /zM) into the right lateral ventricle of 2-day-old rabbits, retention of an active avoidance task is disrupted and a high mortality rate is observed 17 days after infusion (29). Immature rabbits injected in the cisterna magna with 1~70 aluminum chloride on the 15th day of life show a statistically significant learning deficit in a water maze (30). The subcutaneous injection of aluminum lactate in pregnant rabbits on day 2 of gestation produces aluminum dose dependent effects on the rate of acquisition o f a classically conditioned response by the offspring; the learning was facilitated by lower and impaired by higher aluminum exposure (38). In young rats, the direct toxic effects of aluminum compounds have received little attention. Nevertheless, several reports indicate that maternal dietary exposure to aluminum during gestation or gestation and lactation can result in developmental alterations in young rats (4,23) and in young mice (12). A transitory delay of neuromotor development was observed in pups treated by gastric intubation with aluminum lactate from postnatal day 5 to 14 (5).

Requests for reprints should be addressed to Paul R. Lehr, Centre des Sciences de l'Environnement, Neurotoxicologie, Universit~ de Metz, 1, rue des R6collets, B.P. 4025, 57040 Metz Cedex I, France. 259

260

CHERRORET ET AL.

Rats are termed nonprecocial animals because they are very poorly developed at birth; visual perception appears only after eye opening (postnatal days 13-15) and coordinated motor activity, which is very rudimentary at birth, progressively improves with age. In the brain in vivo, the period between day 10 and 15 of postnatal growth is called the criticalperiod (15). It corresponds to the development of the aerobic glycolytic metabolism enzymes (6) and to the appearance of the rapid incorporation of glucose carbon into amino acids associated with the compartmentation of the glutamate metabolism, which are characteristic of the adult mmnmalian brain (15,28). The activity of choline acetyltransferase (CAT), which synthesizes acetylcholine also increases rapidly in the basal forebrain of the rat between day 10 and 20 after birth (Cherroret, unpublished results). Aluminum salt may affect the proteins of cholinergic transmission, in particular the CAT (18,37, Cherroret, unpublished results). In the present work, we examined the effects of aluminum lactate on brain CAT activity and learning abilities in rats, treated by gastric intubation from postnatal day 5 to 14, before and during the critical period when the brain is vulnerable (11).

For the evaluation of CAT activity, the incubation mixture contained (final concn.): 0.2 mM acetylCoA, 50 mM sodium phosphate buffer (pH 7.4), 300 mM NaCI, 8 mM choline chloride, 20 mM EDTA (pH 7.4), and 0.1 mM physostigmine. The incubation solution (5/~1) and 2/~1 of the labeled acetylCoA solution (acetyl 1-14C C o A - N e w England N u c l e a r 48.1-59.3 mCi/mol) were placed in a microtube (23 m m x 2 ram) and the homogenate (2/d) was added. The solution was mixed and incubated for 15 rain at 37°C. The microtube was placed in a scintillation vial containing 5 ml of 10 mM sodium phosphate buffer (pH 7.4). Then, 2 ml acetonitrile containing 10 mg of sodium tetraphenylborate (Kalignost) and 10 ml of toluene scintillation mixture (0.05°7o PPO, 0.02o70 POPOP, to|uene q.s.p.) were added to the vial which was sightly shaken. The radioactivity of acetylcholine (ACh) which was extracted in the toluene phase was measured with a liquid scintillation spectrometer (Minibeta 1211, LKB). The enzyme activity was calculated from the specific activity of a given batch of 14Cacetyl CoA. The specific activity of CAT was expressed as nmol ACh synthetized/h/mg protein.

METHOD

The protein content of the homogenates was determined using the Lowry et al. method (20) modified by Markwell et ai. (21) with bovine serum albumine (fraction V) as a reference.

Subjects Pregnant female rats of the Wistar strain (Iffa Credo, L'Arbresle, France) weighing 220 =l: 4 g (mean + SD) were used. The animals were housed in individual plastic cages, allowed free access to food (Extra-Labo, Provins, France) and water, and maintained under constant environmental conditions (22-23°C and alternative cycle of 12 L:I2 D. The litter sizes were reduced to about 12 pups for homogeneity. Each litter was randomly divided into three groups of four pups, a control group and two groups which received 100 and 200 mg Al/kg body weight/day, respectively. Each pup was tattooed with spots of alizarine blue. Aluminum lactate, dissolved in distilled water (0 to 0.5 ml) was administered by gastric intubation to newborn rats; the control pups received an equal volume of distilled water. All pups were treated from postnatal day 5 to 14. First, we studied the effect of aluminum lactate on CAT activity in homogenates from the basal forebrain and neostriaturn in 15-day-old rats (one day after the end of treatment). Second, we explored the behavioral consequences of aluminum administration using operant conditioning and radial arm maze tests in 50- and 100-day-old rats, respectively.

Assay of CA T Activity CAT activity was measured by the micromethod of Fonnum (14). The rats were sacrificed by decapitation. The basal forebrain and the neostriatum were quickly isolated in a cold room by dissection (16) then weighed. In each litter, the brain areas of the four pups from each experimental group were pooled. They were homogenized at 0°C in a Potter Elvejhem apparatus with a Teflon pestle (A. H. Thomas Co., Philadelphia, Size A) in a 10 mM EDTA solution (pH 7.4). Ten minutes after adjunction of an equal volume of a 10 mM EDTA solution (pH 7.4) containing 0.5°70 Triton X100 (v/v), a 10 mM EDTA solution (pH 7.4) was used to complete the treatment. The final concentration of the homogenate was 5 % (w/ v). This homogenate was then divided into three aliquots to evaluate CAT activity, proteins, and aluminum concentrations, respectively.

Protein Evaluation

Aluminum Determination In each experimental group, the blood from the four rats was collected in heparinized plastic tubes and centrifuged at 900 g for 10 min. Aluminum concentration in plasma and cerebral homogenate was determined using an atomic emission spectrometer (Spectra Span V, Beckman). To avoid aluminum contamination from environmental sources, the treatment of the aiiquots from the three experimental groups was similar and the number of decantings was limited. The contamination sources, such as the distilled water used for the homogenate and the vials from which aliquots were taken for aluminum evaluation were negligible. Indeed, aluminum concentration of the distilled water evaluated in such a type of vial was very low (4/zg/l).

Rat's Learning Abilities Operant conditioning. Male and female rats were tested on postnatal day 50. The number of rats were 22, 15, and 18 in the control group, group 1, (100 mg Al/kg) and group 2 (200 mg/Al/kg), respectively. The method used was an operant conditioning test using light as an aversive stimulus in order to avoid any experimental change related to the rats weight. Details of procedure are given elsewhere (9,23). Briefly, a single 1.5 h session was carried out during the active phase of the animals. The rat was placed in a metal cage (standard 42 × 28 x 24 cm) where it could press 2 rectangular plastic pedals (4 x 7 cm). Locomotor activity was evaluated through the intersections of 2 infrared beams. Forty cm above the cage, 4 20W fluorescent tubes delivered 400 lux illumination to the floor of the cage. The rat could press either the inactive pedal, which had no effect on the luminous environment, or the active pedal which turned out the light for 30 s. If the rat pressed the active pedal during the 30 s of darkness, it had no effect, but if it was still pressing at the moment when the light was due to come back on, a new 30 s period of darkness was added (cumulative strategy).

A L U M I N U M A N D L E A R N I N G ABILITIES IN RAT

261

Previous studies (9,23) indicated that according to their behavior in this test, rats can be classified into three categories corresponding to different levels of performance. These categories had to be compared separately in the various experimental groups, in order to gain accuracy in the analysis. 1. Nondiscriminant type rats which did not discriminate between the active and active pedals (tested rat × rat × X 2 test). These rats also exhibited the lowest amounts o f locomotor activity, presses, and reinforcements. 2. Simple discrimination type r a t s - i n these rats, X 2 test indicated a significant level o f discrimination between the active and inactive pedals. These rats exhibited average amounts of locomotor activity, presses, and reinforcements. 3. Cumulative type r a t s - o n at least one occasion, these rats obtained several aggregated dark periods by presses of more than 30 s in duration. Furthermore, this cumulative type behavior was correlated in these rats to high levels of locomotor activity and early discrimination between the pedals during the session. This category of rats was considered to exhibit the best style of learning.

Radial Arm Maze Test In order to avoid variation in locomotor activity linked to the oestral cycle, only male rats were tested on postnatal day 100. The numbers o f rats were 10, 10, and 9 in the control group, group 1 (100 mg Al), and group 2 (200 mg AI), respectively. Apparatus. The apparatus was a closed wooden eight-arm radial maze painted in gray according to the Olton procedure (25). The center was an octagonal area, 40 cm in diameter. All arms were identical, 80 cm large, 10 cm wide, and 15 cm high. A circular food well (4 cm in diameter, 1 cm deep) was positioned at the end o f each arm. The ceiling of the central platform and the arms were made of transparent plastic. Procedure. Prior to experimentation, body weight was reduced by food deprivation and maintained at 80% of the free-feeding value. The rats were tested individually over 3 days. Rats were placed at the center o f the platform and left in the apparatus until all 8 food pellets, placed at the end o f each arm, had been consumed. A n arm entrance was recorded when the four paws were placed in an arm. Number of arms entered and times o f entrance were recorded.

TABLE 1 ALUMINUM CONCENTRATION IN PLASMA AND CEREBRAL HOMOGENATES Plasma0~g/l) Controlgroup

27.2 + 8.5

CerebralHomogenate ~g/g wet weight) 1.72 + 0.21

(4)

(4)

Group 1

31.0 + 6.5

1.74 + 0.40

(4)

(4)

Group2

66.0 + 10.0"

3.47 + 1.18t

(4)

(4)

Values are means + SD. Numbers in parentheses indicate the number of experiments. Significantly different from control: *p < 0.001. tP < 0.05.

versus group 2 (p < 0.001). No difference was detected between the control group and group 1. A N O V A applied to aluminum data in the cerebral areas indicated an overall significant effect of aluminum treatment, F(2, 6) = 6.15, p < 0.035. Multiple comparisons by Fischer's PLSD test indicated significant differences in the comparisons: control group versus group 2 (p < 0.05) and group 1 versus group 2 (p < 0.05). No difference was detected between the control group and group 1. In conclusion, aluminum concentrations in the cerebral areas and in plasma increased in group 2 (200 mg Al) versus control group: 142.6 and 101.7%, respectively.

Effect o f Aluminum on CA T Activity A N O V A applied to CAT activity data (Table 2) indicated an overall significant effect of the aluminum treatment, F(2, 6) = 6.445, p < 0.032. Multiple comparisons by Fischer's PLSD test indicated significant differences in the comparisons: control group versus group 2 (p < 0.05) and group 1 versus group 2 (/7 < 0.05). In conclusion, the administration of 200 mg A l / k g body weight by gastric intubation to newborn rats inhibited CAT activity in the cerebral areas under study (7.4%). At 100 mg A l / k g body weight, aluminum lactate had no significant effect on the enzyme activity as compared with the control group.

Operant Conditioning Statistical Analysis For CAT activity and aluminum concentrations, the data were analyzed using a single factor analysis o f variance (ANOVA) procedure. Multiple comparisons were performed using Fischer's PLSD test (36). For behavioral data, nonparametric statistics were used: X2, Kruskal-Wallis, Mann Whitney tests (32). RESULTS

Aluminum Levels Aluminum concentration in plasma and cerebral homogenate was determined in the three experimental groups (Table 1). A N O V A applied to plasma aluminum data indicated an overall significant effect o f aluminum treatment, F(2, 6) = 285.8, p < 0.0001. Multiple comparisons using Fischer's PLSD test indicated significant differences in the comparisons: control group versus group 2 (/7 < 0.001) and group 1

The rats from the three experimental groups were tested on postnatal day 50. The rats distributions into different operant conditioning behavioral types (Table 3) showed a tendency toward heterogeneity between the 3 groups 0c2 = 8.89, 4 df, p < 0.10). The number o f nondiscriminant type rats did not

TABLE 2 EFFECTOF ALUMINUMLACTATE ON CAT ACTIVITY (nmol/mgprotein/h) Controlgroup Group 1 Group 2

13.61 + 3.18 13.95 + 2.88 12.60 + 2.74*

(4) (4) (4)

Values are means + SD. Numbers in parentheses indicate number of experiments. Significantly different from control: *p < 0.05.

262

CHERRORET ET AL. TABLE 5 OPERANT CONDITIONING:DISCRIMINATIONLEVEL OF RATS OF SIMPLE DISCRIMINANTTYPE*

TABLE 3 REPARTITION INTO DIFFERENTBEHAVIORALTYPES IN OPERANT CONDITIONINGTEST (50-DAY-OLDRATS) TreatedGroups Types

ControlGroup

Nondiscriminant Simple discriminant Cumulative

7 4 11

1 8 3 4

2 8 8 2

significantly differ between control and treated groups. A significantly greater number of cumulative type rats appeared into the control group (73.30/0) as compared with group 2 (20%). Further analyses were performed only upon discriminant and cumulative rat variables. For these two types of rat, the following parameters were evaluated: locomotor activity (number of intersections of IR beams), total number of presses (active + inactive presses), active presses, discrimination level (active presses/total number of presses in %). This assessment was carried out at different moments of the session: • Period 1: first half hour; • Period 2: first + second half hour; • Period 3: first + second + third half hour. For the rats of simple discriminant type, locomotor activity measured by the number of crossed infrared beams was generally lower in the treated groups versus control group (Table 4) but there was no significant difference between the three groups (Kruskal-Wallis tes0. The active presses variable showed no significant difference at any period of the test between the three groups (Kruskal-Wallis test). On the contrary, the discrimination level was significantly higher in group 2 versus control group and group 1 (Table 5). For period three: comparisons control group versus group 1, U = 6, ns; control group versus group 2, U = 2, p = 0.008; group 1 versus group 2, U = 2, p = 0.012 (Mann Whitney test). For the rats of cumulative type, the different parameters (locomotor activity, active presses, discrimination level) showed no significant difference at any period of the test between the three groups.

Spatial Learning: Radial Arm Maze Test Only male rats from the three experimental groups were tested on postnatal day 100. Six variables were recorded on

Period 1

2 3

Group 1

I

495 (374.5 - 585)

424 (269 - 481)

2

717 (515 - 821) 821.5 (575.5 - 899)

557 (342 - 694) 569 (352 - 1207)

(63 ~ 66) 66 (65 - 73) 66 (65 - 68)

72

(68.5 - 83.5) 77.5 (73 - 84.5) 79.5 (73 - 84.5)

DISCUSSION In a previous study (5), we observed that the mortality of rats treated by gastric intubation with 300 mg Al/kg/day from postnatal day 5 to 14 was very high: 63%0 of the initial population at postnatal day 20. Because the number of surviving rats was too low to carry out behavioral tests, the present study was limited to the administration of 200 mg Al/kg body weight/day. Although a study on immature rabbits (39) has shown that aluminum is poorly absorbed following oral ingestion, we observed that the plasma aluminum concentration increased significantly in group 2 (200 mg Al) after intoxication by gastric intubation. The aluminum level also increased

TABLE 6 RADIAL ARM MAZE TEST: TOTAL TIME FOR THE TEST AT THE THREE TRIALS* ControlGroup

Group1

Group2

191 (129 - 332) 205 (193 - 236) 196 (104 - 213)

271 (163 - 324) 259 (195 - 323) 191 (160 - 274)

392 (169 - 558) 275 (211 - 409) 290 (251 - 342)

Group2 1

3

64

(60.5 - 72) 67.5 (65 - 69.5) 67 (63.5 - 69.5)

Group2

each radial arm maze test trial: (a) total number of arms visited before reaching the 8 pellets, (b) total time of the test, (c) time before occurrence of first error, (d) latency of entrance in the first arm, (e) number of arms visited before occurrence of first error, (t) number of different arms visited in the 8 first entrances. Results of the three experimental groups indicated that generally they did not significantly differ, except for variable n°2 (total time taken for the test) (Table 6). On the first day, Mann-Whitney test indicated a significantly longer time in group 2 (U = 14, p < 0.025; U = 19, p < 0.05 in the comparisons: control group versus group 2 and group 1 versus group 2, respectively). These differences were significant, again, on the third day of testing (U = 15, p < 0.05; U = 16, p < 0.05 in the same comparisons). Thus, results indicate a general slowing down of group 2 rats in the course of the test but do not reveal any impairment in their learning abilities.

Day ControlGroup

65.5

GroupI

*Median values; in parentheses: limits of interquartile ranges.

TABLE 4 OPERANT CONDITIONING:LOCOMOTORACTIVITY OF RATSOF SIMPLEDISCRIMINANTTYPE* Period

ControlGroup

372.5 (305 - 422.5)

531.5 (387.5 - 752.5) 552 (389 - 799)

*Median values; in parentheses: limits of interquartile ranges.

2 3

*Median values; in parentheses: limits of interquartile ranges.

ALUMINUM AND LEARNING ABILITIES IN RAT significantly in the cerebral areas studied in parallel to plasma aluminum. This elevation of aluminum concentration observed in the cerebral homogenate couM be attributed to an increase in permeability of the blood brain barrier, particularly in young rats. Indeed, aluminum significantly increases transmembrane diffusion across the blood brain barrier (BBB); it also selectively affects membrane transport systems (3). Thus, permeability of the BBB to transferrin may be increased, inducing a greater access for aluminum to the brain. Indeed, transferrin may be transported across the BBB because binding sites for it have been found on brain endothelial cells and brain tissues (13,19,26). As aluminum binds to transferrin and circulates primarily in a bound form (33), transferrin may be the pathway by which aluminum gains entry to the CNS (3). The rate of aluminum penetration into the brain could also be associated with the development of the rat brain. A relationship between transferrin accumulation in developing neurons and oxidative metabolism ontogeny had been demonstrated in the rat's brain (10); the peak of intraneural transferrin and Fe 2+ preceded the onset of a number of oxidative enzymes of the mitochondrial chain (22). The significant decrease in CAT activity in some areas of the brain in the rat from ~group 2 could be due to the presence of aluminum at 4.6.10-°M. Cherroret (unpublished results) observed in vitro that aluminum at 10-3M inhibited CAT activity of homogenates from the same areas of the rat's brain at postnatal day 15; no effects on CAT were observed at 10-4M AI. Thus, the effects of aluminum on CAT differ in vitro and in vivo according to concentrations. The consequences of an early aluminic intoxication upon the learning abilities of rats were studied using 2 tests, very different in the motivations involved and in the methods employed in their carrying out. The Olton radial maze is based on alimentary motivation, spatial memory, and requires walking

263 over a distance of at least 6.4 m in order to be achieved. The operant .conditioning used in this study is based on the possibility of avoiding an aversive light by discriminating the correct lever and pressing it. The complete set of results does not indicate any effect of the treatment upon the learning abilities per se but rather suggests a lower level of activity in the most intoxicated rats: group 2 rats need a longer time to finish the radial maze trial. In the same way, the greater proportion of cumniative-type rats in control group versus group 2 attests the differences in the levels of activity in the rats: only the most active animals which get a lot of information about the roles of the two levers can reach this level of performance. The higher level of discrimination in group 2 rats is another aspect of their reduced activity: when, in rats which have learned the differential roles of the two levers, the total number of presses decreases, the reduction is more m a r k e d for the inactive lever, and the discrimination ration (number of presses on the active lever/number of presses on the inactive one) increases. Finally, the few differences detected in the comparisons, control group/group 2 rats, seems to indicate a general "slowing down" of the latter rather than a deficiency in their learning abilities. At least three hypotheses (which are not mutually exclusive) can be raised to explain these observations in the treated rats: (a) an impairment of their sensory inputs and/or processing, Co) an impairment in their motor abilities, and (c) a general drop in their basal metabofism. At this point in the study, it is impossible to decide between these possibifities and further studies are needed to clarify this point. ACKNOWLEDGEMENTS This research was supported by grants from the Fondation pour la Recherche M6dicale (Comit6 Lorraine). We are also thankful for the helpful comments of the referees.

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