Pharmacology Biochemistry & Behavior, Vol. 3, pp. 1003-1009. Copyright © 1975 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.

Effect of Aluminum upon Conditioned Avoidance Response Acquisition in the Absence of Neurofibrillary Degeneration G. A. KING 1, U. D E BONI 1 A N D D. R. C R A P P E R 2'3 University o f Toronto, Toronto, Ontario, Canada ( R e c e i v e d 27 May 1975) KING, G. A., U. DE BONI AND D. R. CRAPPER. Effect o f aluminum upon conditioned avoidance response acquisition in the absence ofneurofibrillary degeneration. PHARMAC. BIOCHEM. BEHAV. 3(6) 1003-1009, 1975. - A l u m i n u m induces neurofibrillary degeneration in cats but not rats. Cats develop a progressive encephalopathy in which an early manifestation is impaired learning-memory performance. At brain aluminum concentrations of 5 to 6 times that found in cat, rats demonstrate an initial transient weight loss and acquisition deficit immediately following intracranial injection. However, rats do not develop a progressive encephalopathy or a chronic learning deficit. Aluminum

Acquisition

Conditioned avoidance response

S O L U T I O N S o f a l u m i n u m salts, i n j e c t e d i n t r a c r a n i a l l y in the cat, p r o d u c e a progressive e n c e p h a l o p a t h y associated w i t h an increased n u m b e r o f n e u r o f i l a m e n t s a n d loss o f n e u r o t u b u l e s in s u s c e p t i b l e n e u r o n s [5, 11]. Deficits in r e t e n t i o n a n d a c q u i s i t i o n have b e e n o b s e r v e d in t h e early stages o f t h e e n c e p h a l o p a t h y [ 4 ] , and are c o r r e l a t e d b o t h w i t h t h e e x t e n t o f n e u r o f i b r i l l a r y d e g e n e r a t i o n ( N F D ) in t h e neo- a n d e n t o r h i n a l c o r t e x [ 5 ] , a n d w i t h t h e b r a i n a l u m i n u m c o n c e n t r a t i o n (B. A. C.) [ 6 ] . This l a b o r a t o r y previously p o s t u l a t e d t h a t N F D m a y be r e s p o n s i b l e for t h e i m p a i r m e n t o f learning a n d m e m o r y in t h e cat [4, 5 ] . To f u r t h e r test this h y p o t h e s i s we e x a m i n e d the effects o f i n t r a c r a n i a l i n j e c t i o n o f a l u m i n u m chloride s o l u t i o n s on t h e a c q u i s i t i o n o f a o n e - w a y a v o i d a n c e r e s p o n s e in 2 d i f f e r e n t strains o f rat, in 3 e x p e r i m e n t s . T h e rat was c h o s e n because previous u n p u b l i s h e d i n v e s t i g a t i o n s (H. Wigniewski, D. R. C r a p p e r ) have s h o w n t h a t this species does n o t develop light m i c r o s c o p i c evidence o f N F D in r e s p o n s e to i n t r a c r a n i a l i n j e c t i o n s of a l u m i n u m . METHOD

Neurofibrillary degeneration

Experiment 2. Twelve male wistar rats weighing b e t w e e n 280-324 g were o b t a i n e d from C a n a d i a n Biological L a b o r a t o r i e s . These animals were h o u s e d a n d t r e a t e d in t h e same way as t h o s e in the first e x p e r i m e n t . Experiment 3. T h i r t y - t w o male h o o d e d rats weighing b e t w e e n 216 248 g were o b t a i n e d from Bio-Breeding L a b o r a t o r i e s . These animals were h a n d l e d in a m a n n e r i d e n t i c a l to those in t h e first e x p e r i m e n t e x c e p t t h a t t h e y were n o t f o o d deprived prior to surgery. Apparatus In all 3 e x p e r i m e n t s the test c h a m b e r was a Lehigh Valley s h u t t l e b o x 20 x 45 x 19 cm, w i t h a tilt floor c o n s t r u c t e d o f 1/16 in. steel rod spaced 11 m m center-tocenter. This b o x was divided i n t o 2 equal c o m p a r t m e n t s by a m e t a l p a r t i t i o n , w h i c h h a d a 7.5 x 12.5 cm guillotine door. A p a r t from t h e clear Plexiglas p a n e l facing the e x p e r i m e n t e r one c o m p a r t m e n t was black and the o t h e r white. White noise at 78 dB, m e a s u r e d inside the c h a m b e r , was p r e s e n t d u r i n g h a b i t u a t i o n a n d testing.

Animals

Solutions for In/ection

Experiment 1. T h i r t y male h o o d e d rats, weighing b e t w e e n 2 2 9 - 3 0 8 g, were o b t a i n e d f r o m C a n a d i a n Biological L a b o r a t o r i e s , a n d were h o u s e d in individual cages w i t h f o o d a n d w a t e r available ad lib u n t i l 24 hr p r i o r to surgery. T h e y were k e p t o n an a l t e r n a t i n g 12 h o u r l i g h t - 1 2 h o u r dark cycle, a n d were weighed and h a n d l e d each day.

Experiment 1. A l u m i n u m , as A 1 C 1 3 , was i n j e c t e d in 2 c o n c e n t r a t i o n s : 0.25 M a n d 0 . 1 2 5 M at pH 3.0 in artificial C. S. F. ( A m e s Medium). The c o n t r o l s o l u t i o n was artificial C. S. F. a d j u s t e d to pH 3.0 by the a d d i t i o n of HC1. A t o t a l o f 6 ul o f o n e o f these s o l u t i o n s was i n j e c t e d bilaterally i n t o t h e v e n t r a l h i p p o c a m p u s of e a c h a n i m a l ( s t e r e o t a x i c

1Department of Physiology, Faculty of Medicine, University of Toronto. 2Departments of Physiology & Medicine, Faculty of Medicine, University of Toronto. 3Send reprint requests to D. R. Crapper, M. D., Department of Physiology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. 1003

t004 coordinates: P 2.8 mm from Bregma, L 4.8 mm, and 8.5 mm below the surface of the brain) [ 14], under pentobarbital anesthesia (50 mg/kg IP). A Hamilton microliter syringe No. 701 was used for intracranial injection. Ten animals each were injected with 1 of 3 solutions: Group 1 - C. S. F. Control, Group 2 low [ A 1 ] , Group I I I - high [A1]. Testing began on the ninth day postinjection for all animals. This delay between injection and testing is the same as that e m p l o y e d in the cat e x p e r i m e n t (4). Experiment 2. Six rats each were injected, as in Experiment 1, with 6 ul of an 0.25 M solution of A1C13 (group A lw) or C. S. F. adjusted to pH 3 (group Cw). All animals were tested 9 days postoperatively. Experiment 3. Only the 0.25 M A1C13, and C. S. F. control solutions were e m p l o y e d . Utilizing the same procedure, volume, and locus of injection as in the first experiment, 16 animals were injected with the aluminum solution, and 16 with the C. S. F. Group A1 ~, consisting of 8 a l u m i n u m injected rats, and group C1 consisting of 8 controls, began acquisition training on the first day postinjection. The remaining 8 aluminum injected and 8 control rats, group A19 and group C9 respectively, commenced testing on the ninth day postinjection.

KING, DE BONI A N D C R A P P E R

Histological Methods Histological examination was undertaken for animals in E x p e r i m e n t 1. Portions of neocortex, hippocampus, and midbrain were fixed in a 4 percent glutaraldehyde and processed for electron microscopy. The remainder of the brain was fixed in 10 percent Formalin, e m b e d d e d in paraffin, and sectioned at 5 u. Sections near the locus of injection were stained with haematoxilin and eosin (H. and E.). The Bielchowsky silver stain was e m p l o y e d to detect NFD.

Aluminum Assay B. A. C. in the hemisphere anterior to the colliculi was measured by a modification of an atomic absorption m e t h o d [111 e m p l o y i n g a carbon furnace (Perkin Elmer Model 305A). The hemispheres were divided into 3 portions. For animals in Experiments 1 and 2, the concentration of aluminum reported was taken as a weighted average of 6 assays, 2 each from the 3 portions of the cerebral hemisphere. In E x p e r i m e n t 3 one assay was taken from an homogenate of each portion, and the total brain concentration obtained as an unweighted average of 3 assays. EXPERIMENT 1

Test Procedure On the first day of testing the animals were placed in the shuttle box with the guillotine door removed and allowed to explore. After 15 min the door was closed, the animal was placed in the black c o m p a r t m e n t , and m o c k trials c o m m e n c e d . Each mock trial was initiated by raising the guillotine door, which turned on the conditioned stimulus (C.S.), a white noice chopped at 10/sec. If the animal did not cross to the opposite side after 30 sec, the C. S. was terminated and the door closed. If the animal did cross to the other side the trial was automatically terminated and the d o o r was closed. After 30 sec, the rat was returned to the black c o m p a r t m e n t . These trials were continued until the animals had made no crossings for 5 trials in succession. Twenty-five training trials were given on the first day and 30 each on the second and third days. Each trial was initiated by the experimenter, who raised the guillotine door. If the rat did not cross to the opposite side within 10 sec, a 0.8 mA electric current was delivered through the floor rods, from a Grayson Stadler Model 700 shock scrambler, until the animal crossed and terminated b o t h the C. S. and the shock. The door was lowered and the rat was allowed 30 sec in the safe c o m p a r t m e n t . The intertrialinterval was 15 sec. Each trial was registered as either an escape or an avoidance. Trials to criteria of I, 5, and l0 avoidances in succession, and the n u m b e r of escapes made each day were used as measures of performance. After the last trial on the third day of testing each animal was anesthetized with pentobarbital, the skull was opened and the brain was bisected in the saggital plane. One-half was taken for histology and one-half for aluminum assay. The above procedure was followed for all experiments with this exception: animals in Experiment 2 were tested for only 1 day (Day 9 postinjection) at the end of testing on that day they were sacrificed and B. A. C. was determined for 3 of the animals in group A 1 w. The brains of the other 3 animals were taken for e n z y m e analysis (results not reported here).

RESULTS Unlike several higher mammalian species, rats do not develop signs o f a progressive e n c e p h a l o p a t h y following brain aluminum application. Doses greater than those e m p l o y e d in the present study also did not produce the chronic neurological signs usually seen in cat, rabbit or guinea pig. F u r t h e r m o r e , animals observed for 12 months postinjection failed to develop m o t o r signs of an encephalopathy.

Brain Aluminum Content The average normal brain aluminum concentration in 3 saline injected rats was 3.5 -+ .61 ug/g dry weight. A l u m i n u m assay in 13 injected animals revealed concentrations ranging from 2.5 to 40.8 ug/g dry weight, (Table 1). The average concentration, in Group 3, 11 days after injection was 22.3 -+ 14.3 ug/g. For Group 2 the average concentration was 10.1 -+ 6.9 ug/g dry weight. Three aluminum injected rats, 1 from Group 2 and 2 from Group 3, and 1 C. S. F. injected control, all of w h o m were rejected from the acquisition study (see below), were allowed to survive for 12 m o n t h s postinjection. These animals were then sacrificed along with 3 other noninjected rats of the same age, and all brains were analysed for B. A. C. The 3 A1 injected animals had an average B. A. C. of 2.03 ug/g brain (S. D. 1.31), and the mean for the 3 normal and 1 injected controls was 0.96 ug/g (S. D. 0.46). It should be n o t e d that the mean concentrations for the 3 aluminum injected animals sacrificed 12 months postinjection is much less than that of either of the aluminum injected groups, sacrificed on the eleventh day postinjection.

Histology Detailed examination of 27 Bielchowsky and H. and E. stained slides of 7 A1 injected rats and 4 C. S. F. controls failed to reveal evidence of NFD. Also, no evidence of NFD was found in tissue e x a m i n e d in the electron microscope.

E F F E C T O F A L U M I N U M IN T H E R A T

1005 Specifically, tissue f r o m A n i m a l 7, w h i c h had 40.8 ug A 1 / g dry weight in t h e c o n t r a l a t e r a l h e m i s p h e r e , was e x a m i n e d in detail a n d e x h i b i t e d n o a l t e r a t i o n in u l t r a s t r u c t u r e .

TABLE 1 PERFORMANCE SCORES AND BRAIN ALUMINUM CONCENTRATION

A v o i d a n ce A cq u i s i t i o n

Number of Trials to: (A1) #g/g Dry 5 10 Weight Avoidances Avoidances

Number of Escapes Day 1

NFD

7#

40.81

26

38

17

none

23?

38.70

30

72

20

none

45 t

24.18

37

37

19

none

6*

19.29

19

31

17

29*

19.21

19

41

10

none

9?

17.36

16

16

11

none

43?

13.97

4

4

8

none

26*

12.07

9

9

8

22*

7.25

16

45

14

36*

5.17

17

35

11

42*

5.15

20

26

9

32t

4.28

9

9

8

35*

2.48

7

27

7

none

r = 0.65

r = 0.49

r = 0.76

(p0.05)

(0 0 . 2 5 . The m e a n s a n d s t a n d a r d deviations o f the n u m b e r of escapes m a d e b y each g r o u p o n each day are given in T a b l e 2 ( l o w e r part). Again, analysis o f variance s h o w e d the i n t e r g r o u p differences n o t to be significant, F ( 2 , 1 9 ) = 2.12, p > 0 . 2 5 . An e x a m i n a t i o n o f Table 1 i n d i c a t e s t h a t the i n d e p e n d e n t variable, B. A. C., was n o t c o m p l e t e l y c o n t r o l l e d . T h e r e is a wide range o f values, a n d 5 animals had c o n c e n t r a t i o n s less t h a n 10 ug/g dry brain weight. To test for a c o r r e l a t i o n b e t w e e n p e r f o r m a n c e a n d B. A. C. a rank o r d e r c o r r e l a t i o n was p e r f o r m e d b e t w e e n B. A. C. and trials to 5 a n d 10 a v o i d a n c e s in a row, and the n u m b e r o f escapes on Days 1, 2 a n d 3 [ 1 0 ] . T h e r e is a significant positive c o r r e l a t i o n b e t w e e n B. A. C. a n d trials to 5 a v o i d a n c e s in a row, a n d t h e n u m b e r o f escapes on Day 1. Individual scores a n d c o r r e l a t i o n coefficients are given in T a b l e 1. Correlat i o n s w i t h t h e n u m b e r o f escapes on Days 2 and 3 were n o t significant (r = 0.52, p > 0 . 0 5 ; a n d r = - 0 . 2 2 , p > 0 . 1 respectively).

DISCUSSION

*Group 2

-~Group 3

T h e significant c o r r e l a t i o n b e t w e e n B. A. C. a n d t h e n u m b e r o f trials to 5 avoidances in a row, and t h e n u m b e r o f escapes o n Day 1, i n d i c a t e s t h a t a l u m i n u m m a y have a d e l e t e r i o u s effect u p o n a c q u i s i t i o n of a c o n d i t i o n e d avoid-

N = animal number

TABLE 2 MEAN TRIALS TO CRITERIA AND ESCAPES

Trials

Days

N

1

5

10

Group 1

6

6.00 (2.83)

8.13 ( 3.31)

13.14 (11.29)

7.86 (2.91)

3.00 (1.91)

3.85 (2.12)

Group 2

9

4.89 (1.54)

13.44 ( 5.53)

25.56 (14.34)

10.33 (3.24)

5.78 (5.91)

4.55 (6.48)

Group 3

7

7.83 (4.07)

20.33 (12.87)

29.33 (25.25)

13.83 (5.49)

4.83 (3.76)

4.17 (3.19)

Standard deviation in parentheses

2

1006

KING, DE BONI AND C R A P P E R TABLE 3 MEANS AND SD's OF VARIABLES MEASURED FOR GROUPS IN EXPERIMENT 3

Group Variable

Al I

(n = 8)

B.A.C. (ug/g)

47.5

+ 18.9

Body weight loss: Day l postinjection (g)

18.38 ± 7.01

4.75 -+ 5.50

17.25 ± 8.29

3.86 + 6.08

1 avoidance

5.88 + 2.03

3.63 ~ 0.92

3.86 + 1.25

2.50 + 0.84

5 avoidances

17.75 ± 11.89

4.75 ~: 1.75

5.50 + 2.00

4.17 + 1.72

10 avoidances

27.00 ± 9.61

11.63 ± 13.46

6.75 ± 3.88

5.83 ± 4.79

1.60

4.88 ± 1.36

4.17 ± 1.72

No. of trials

C~ (n = 8)

AI~ (n = 8)

4.4

34.1

+ 2.0

÷ 9.3

C 9

(n = 6)

4.1

+ 1.4

to:

No. of escapes on: Day 1

12.50 ± 4.18

Day 2

2.75 ± 1.83

1.63 ± 0.74

1.38 -~ 0.52

1.50 ± 0.55

Day 3

1.75 +

1.25 -~ 0.46

0.88 _+ 0.64

1.00 + 0.63

1.83

a n t e response. Since the massive a m o u n t s o f a l u m i n u m injected intracranially in these animals did n o t p r o d u c e N F D the cause of any deficit resulting from a l u m i n u m injection must be sought elsewhere. The second e x p e r i m e n t in this series was conceived as an investigation o f the possible effects of a l u m i n u m on a n u m b e r of brain e n z y m e s . This follow-up s t u d y , however, b r o u g h t into q u e s t i o n c o n c l u s i o n s drawn from the first e x p e r i m e n t . EXPERIMENT 2 The only d i s c e m a b l e effect o f the a l u m i n u m injection in the first e x p e r i m e n t had been the correlations b e t w e e n B. A. C. and measures o f p o o r p e r f o r m a n c e on Day 1 o f acquisition (only 3 o f 22 animals failed to achieve 5 avoidances in a row on Day 1 ). T h e r e f o r e , it was d e c i d e d t o test the animals in this e x p e r i m e n t on Day 1 only, using total n u m b e r o f escapes as the measure o f p e r f o r m a n c e . RESULTS One a l u m i n u m injected animal was discarded f r o m the s t u d y because o f haematuria. All o t h e r A 1 injected animals appeared healthy and displayed no gross neurological s y m p t o m s , G r o u p Cw, with a mean o f 15 escapes (S. D. = 5.22), displa? ed p o o r e r p e r f o r m a n c e t h a n group A1 w (X= 10, S. D. = 3.24). These differences are n o t statistically significant, F( 1,9) = 3.62. Three animals in group A1 w had B. A. C.'s of 71.5 ~g/g, 50.4 ~g/g, and 9.7 ~g/g. The mean B. A. C. for 6 rats in group C w was 2.72 ug/g -+ 0.86.

4.63 ±

The results o f this e x p e r i m e n t do n o t s u p p o r t the h y p o t h e s i s that intracranial injection o f a l u m i n u m interferes with acquisition o f a C. A. R. EXPERIMENT 3 To replicate and e x t e n d the findings o f E x p e r i m e n t 1, h o o d e d rats were tested for their ability to acquire a C. A. R. the day a f t e r intracranial injection as well as 9 days p o s t i n j e c t i o n . Animals were tested 1 day after injection in o r d e r to ascertain w h e t h e r deficits w h i c h do exist may n o t o c c u r i m m e d i a t e l y and be ameliorated with time. Also, more subtle e f f e c t s o f A1 injections, wRich might also be correlated with p e r f o r m a n c e deficits, were l o o k e d for: specifically daily weight changes were r e c o r d e d and closely e x a m i n e d for all animals. RESULTS As in E x p e r i m e n t 1, no gross neurological i m p a i r m e n t s were seen in any animal injected with A1C13. However, an effect on weight regulation which was not n o t i c e d in E x p e r i m e n t 1 became evident in this e x p e r i m e n t . B.A.C.

The B. A. C. of animals in groups A l i and C1 were m e a s u r e d after 3 days p o s t i n j e c t i o n while the B. A. C. o f animals in groups AI9 and C9 were d e t e r m i n e d 11 days following injection. The average c o n c e n t r a t i o n for each group is given in Table 3. A l t h o u g h there is a large within group variation, the a l u m i n u m injected rats have a m u c h

F;FFECT OF ALUMINUM IN THE RAT higher average concentration than the C. S. F. injected controls.

Weight Changes On the average both the C. S. F. controls and the aluminum injected rats lost weight on the day after injection. The mean body weight loss for each group is recorded in Table 3. The difference in the amount of weight lost on the Day 1 postinjection between the aluminum injected animals and the controls was shown to be significant by an analysis of variance, F(1,26) = 30.5, p