Cholinotoxicity induced by ethylcholine aziridinium ion after intracarotid and intracerebroventricular administration

Life Sciences, Vol. 44, pp. 1437-1448 Printed in the U.S.A

Pergamon Press

C H O L I N O T O X I C I T Y I N D U C E D BY E T H Y L C H O L I N E A Z I R I D I N I U M ION AFTER INTRACAROTID AND INTRACEREBROVENTRICULAR ADMINISTRATION Zipora Israel

Pittel,

Abraham

Fisher

and E l i a h u

I n s t i t u t e for B i o l o g i c a l Research, N e s s - Z i o n a , 70450, Israel

Heldman*. P~O.Box

19

(Received in final form March I0, 1989) Summary The effect of ethylcholine a z i r i d i n i u m ion (AF64A) after an intracerebroventricular (icv) i n j e c t i o n was compared to that obtained after an intravascular administration. R e d u c t i o n s in c h o l i n e a c e t y l t r a n s f e r a s e (CHAT) and acetylcholinesterase activities in the hippocampus but not in the cerebral cortex or the corpus striatum were o b s e r v e d 10 days a f t e r b i l a t e r a l injection of AF64A into the rat c e r e b r o v e n t r i c l e s (3 nmol/side). However, when A F 6 4 A was i n j e c t e d into the carotid artery (1 p m o l / k g ) following a unilateral opening of the blood-brain barrier by a hypertonic treatment, a s i g n i f i c a n t d e c r e a s e in C h A T a c t i v i t y was observed in the i p s i l a t e r a l side of the c e r e b r a l c o r t e x but not in h i p p o c a m p u s , corpus striatum, or cerebellum. High-affinity choline transport was reduced significantly 11 days a f t e r an icv i n j e c t i o n of A F 6 4 A in all the above m e n t i o n e d b r a i n regions, and r e c o v e r e d 60 days post injection in the c e r e b r a l c o r t e x and in the corpus striatum but not in the h i p p o c a m p u s . Our results suggest that in v a r i o u s b r a i n regions, A F 6 4 A causes various degrees of damage to cholinergic neurons, depending on the q u a n t i t y of the t o x i n that reaches the t a r g e t tissue. Ethylcholine aziridinium ion (AF64A) causes cholinergic hypofunction when injected into the brain (I). It has been reported that after an i n t r a c e r e b r o v e n t r i c u l a r (icv) injection, the damage i n f l i c t e d by A F 6 4 A is not u n i f o r m l y spread t h r o u g h o u t the brain (2,3). To explain this uneven d i s t r i b u t i o n of the effect of AF64A, it was suggested that v a r i o u s b r a i n r e g i o n s possess different sensitivities to the toxin (1,4). However, injection of the toxin directly into d i s c r e t e b r a i n regions, which are generally not a f f e c t e d a f t e r an icv injection, c a u s e d cholinotoxicity (5,6) or e v e n n o n s e l e c t i v e lesions (7,8) of these otherwise "resistent" regions. One p o s s i b l e e x p l a n a t i o n of the nonspecific c e l l u l a r d a m a g e is that a h i g h local c o n c e n t r a t i o n of AF64A, which is f o r m e d a f t e r the i n j e c t i o n of the toxin d i r e c t l y into the tissue, causes lesions that o t h e r w i s e w o u l d not occur.

*To w h o m

correspondence

should

be mailed.

0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc

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Cholinotoxiclty in Various Brain Regions

Vol. 44, No. 20, 1989

We assumed that if AF64A were delivered via the vascular system, its distribution would be more extensive and homogeneous than that obtained after icv injection or after direct application of the toxin into the tissue. Therefore, we predicted that after intravascular administration, cholinotoxicity would be observed in regions that usually are not affected when AF64A is injected directly into the brain. However, when the toxin is injected intravascularly, the blood-brain barrier (BBB) might prevent the quaternary compound, AF64A, crossing from the blood to the neuronal tissue. To overcome the problem of permeability, we injected the toxin into the carotid artery after a unilateral opening of the BBB by pretreatment with hypertonic glycerol. In this article we demonstrate that the regional distribution of the cholinotoxicity obtained after an intracarotid injection is different from that obtained after icy injection. In addition, we demonstrate that AF64A causes long-lasting hypofunction of the cholinergic cells and that this hypofunction can be partially reversed.

Methods

Preparation of AF64A The precursor acetylethylcholine mustard hydrochloride (prepared in our laboratory, >99% pure) was dissolved in distilled water at a final concentration of 10 mM. Hydrolysis of the acetoxy group and cyclization of the aziridinium moiety were initiated by raising the pH of the solution to 11.5 with 4 M NaOH. The reaction was terminated after 20 min by lowering the pH to 7.3 with 6 M HCI, and the solution was kept on ice and used within 4 h. This solution, now containing AF64A (9), was diluted to the desired concentration (assuming 100% conversion of the precursor to the active aziridinium species) by adding artificial cerebrospinal fluid (CSF) or saline, for icy or intracarotid injection, respectively. Preparation of homo~enates and crude synaptosomal fraction (P2) Rat brain was quickly removed after decapitation. Cerebral cortex, hippocampus, striatum and cerebellum were freehand dissected, weighed and diluted as follows: 10% wt/vol in 0.32 M sucrose for high-affinity choline transport (HAChT) determination (assayed with P2 fraction) and 5% wt/vol in 75 mM phosphate buffer pH 7.4 (cortex, hippocampus and cerebellum) or 2% wt/vol (striatum) for ChAT and AChE determinations (assayed with crude homogenates). Homogenates for P2 fractions (10) were prepared in a teflon-glass homogenizer (0.6 mg protein/ml) and crude homogenates were prepared in a Branson 130 sonicator.

Determination

of enzyme activities

Choline acetyltransferase modified method described homogenates (10 ~i) were

and choline

transport

(CHAT) activity was determined by a by Fonnum (11). Aliquots of brain incubated for 20 min at 37°C with a

Vol. 44, No. 20, 1 9 8 9

Cholinotoxicity in Various Brain Regions

1439

reaction mixture (10 ~i) containing 0.6 M NaCl, 40 mM MgCI~, 2 mM eserine, 0.05% w t ~ o l bovine serum albumin, 10 mM choline iodide, and 0.9 mM [" C]acetyl-CoA (0.22 mCi/mmol). Parallel tubes, containing phosphate buffer instead of homogenates, were used as blanks. The reaction was terminated by transferring the tubes to ice and adding 150 ~i solution containing 15 mg/ml tetraphenyl boron in 3-heptanoneThe tubes were mixed thoroughly and the aqueous phase was separated from the organic phase by centrifugation. Aliquots of the upper organic phase were taken for measurements of radioactivity by scintillation spectrometry. Acetylcholinesterase (ACHE) activity was measured by a modified method described by Fonnum (11). 14Allquots of brain homogenate (5 ~i) were added to 20 ~i of I mM [^ C]acetylcholine (0.5 ~Ci/ml) and incubated for 3D min at 30~C. Parallel tubes, containing phosphate buffer instead of brain homogenate, were used as blanks. The reaction was terminated by adding cold distilled water (20 ~i) and 150 pl solution containing 15 mg/ml tetraphenyl boron in 3-heptanone. The tubes were thoroughly mixed, centrifuged, and immersed in acetone/dry-ice to freeze the lower aqueous phase. After removing the upper phase, aliquots of the thawed lower phase were taken for scintillation spectrometry. High-affinity choline transport (HAChT) was measured as described by Pittel et al. (12~. In brief, aliquots of P2 fraction (50 ~I) were incubated at 30vC for 6 min with 450 ~i reaction mixture (140 mM NaCl, 4.75 mM KCI, 1.2 mM CaCI2, 20mM~Tris pH 7.4, 1.4 mM MgCIg, 2 mg/ml dextrose, and 0.8 ~M [~H]choline, 5 Ci/mmol). The'reaction was terminated by placing the tubes on ice. Parallel tubes, containing homogenates, were incubated for an equivalent period on ice for determination of nonspecific counts. Aliquots of the synaptosomal suspension were then placed over 0.5 M sucrose, centrifuged at 10,000 X g for 30 min and radioactivity was determined in the pellet. Administration

of AF64A

Intracerebroventricular administration: Sprague-Dawley rats were anesthetized with 2.5-3.5 ml/kg (i.p.) equithesin (27.6% propylene glycerol, I mM MgS04, 4.1% chloral hydrate, 5.5% alcohol, and 0.9% nembutal) and -injected bilaterally, with 28 G cannula, using stereotactic technique (A-P = 0.8 nun, L = 1.5 nun from bregma, D-V = 4.7 mm from skull surface). AF64A, dissolved in artificial CSF (147 mM NaCI, 2.9 mM KCI, 1.6 mM MgCl2, 2.2 mM dextrose, and 1.7 mM CaCl2) was injected at a dose of 3.0 nmol/side in a volume of 0.5 or 2 ~i, and in a rate of 0.25 ~I/min. Control rats were injected under the same conditions with ~rtificial CSF. Intracarotid administration: Sprauge-Dawley rats (300-400 g) were anesthetized as described above. Unilateral opening of the BBB (13) was achieved by injection of 0.2 ml/kg of 40% glycerol (dissolved in saline) into the left carotid artery. AF64A dissolved in saline (0.1 or 1.0 ~mol/kg), or saline alone (both in volume of 0.1 ml) was injected Via the same artery, either with or without opening of the BBB. The right side remained intact and served as a control for each animal.

1440

Cholinotoxicity in Various Brain Regions

Vol. 44, No. 20, 1989

1 5 C -140

(b)

.

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.......

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.

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"--" 3 nmoles/0.5~l/side "--- 3nmoles/ 2 jul/side (a) p~< 0.05 (b) p~< o.o i (c) p.< O.OOi

1 I I I 1 ' ~.-.l I0 20 30 40 50 60 70

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Days after injection FIG.

I

ChAT activity at various times after bilateral icv a d m i n i s t r a t i o n of A F 6 4 A (3 n m o l / s i d e ) . V a l u e s are percent of c o n t r o l a c t i v i t y . E a c h p o i n t r e p r e s e n t s an a v e r a g e from 3-8 rats. Individual percentages were calculated by d i v i d i n g the a c t i v i t y f o u n d for e a c h e x p e r i m e n t a l a n i m a l by the a v e r a g e a c t i v i t y of the c o n t r o l group. The v a l u e s for ChAT a c t i v i t i e s (nmoles A C h / m g p r o t e i n / m i n ) in the c o n t r o l g r o u p s w e r e as follows: h i p p o c a m p u s - 0.40Z0.14; s t r i a t u m 1.38+0.58; f r o n t a l c o r t e x - 0.38+0.03. M e a n s +SEM are from 5 dTfferent experiments (3-8 a n i m a l s / e a c h --experiment). V e r t i c a l bars r e p r e s e n t S E M values. The s i g n i f i c a n c e level for v a l u e s w h i c h w e r e found to be statistically (t-test) d i f f e r e n t from the c o n t r o l a r e shown in p a r e n t h e s e s .

Results C h A T a c t i v i t y a f t e r icy i n j e c t i o n of A F 6 4 A We followed the time c o u r s e of the e f f e c t of AF64A on ChAT activity in t h r e e d i f f e r e n t b r a i n r e g i o n s s t a r t i n g at day 10, a time p e r i o d at w h i c h the r e d u c t i o n s in several c h o l i n e r g i c m a r k e r s w e r e s h o w n to be m a x i m a l (14).

Vol. 44, No. 20, 1989

"-'

f....

Cholinotoxicity in Various Brain Regions

1441

120

co

"6

100 80

I ~"

60

-t-4 • r"1

~ ,,,

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20

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!

I

I

I

10

15

20

25

30

35

Days afteP injectt0n FIG.

2

AChE activity at v a r i o u s times after bilateral icv a d m i n i s t r a t i o n of A F 6 4 A (3 n m o l / s i d e ) . For e x p e r i m e n t a l details and c a l c u l a t i o n s see Fig. I. The v a l u e s for AChE a c t i v i t i e s (nmoles p r o d u c t f o r m e d / m g protein/min) in the c o n t r o l g r o u p s w e r e as follows: hippocampus 39.1; s t r i a t u m - 170.8; f r o n t a l c o r t e x - 58.5 .

Since we p r e v i o u s l y s h o w e d that the m i n i m a l d o s e of A F 6 4 A which is r e q u i r e d to c a u s e s i g n i f i c a n t r e d u c t i o n in C h A T a c t i v i t y is 3 n m o l / s i d e (15), we s e l e c t e d this d o s e for o u r c u r r e n t e x p e r i m e n t s . As c a n be s e e n f r o m Fig. 1, ten d a y s a f t e r icv a d m i n i s t r a t i o n of the toxin, C h A T a c t i v i t y w a s r e d u c e d in the h i p p o c a m p u s to 36% of control. This r e d u c t i o n w a s t h e n f o l l o w e d by a g r a d u a l recovery of the e n z y m e a c t i v i t y , r e a c h i n g 80% of control, 52 days a f t e r the injection of the toxin. The e n z y m e a c t i v i t y w a s t h e n s t a b i l i z e d and s u s t a i n e d at a r e d u c e d level (Ca. 80% of control) u p to 283 days after the injection of the toxin (our last point of measurement). However, unlike its e f f e c t in the hippocampus, A F 6 4 A d i d not c a u s e a s i g n i f i c a n t d e c r e a s e in C h A T a c t i v i t y in the c e r e b r a l c o r t e x (Fig. I) and c a u s e d a t r a n s i e n t increase, f o l l o w e d by up a n d d o w n f l u c t u a t i o n in the c o r p u s striatum. To a s c e r t a i n t h a t a f t e r an icy a d m i n i s t r a t i o n A F 6 4 A is rapidly diluted into the c e r e b r o s p i n a l fluid, we i n j e c t e d the t o x i n at a c o n s t a n t d o s e but in t w o d i f f e r e n t volumes. As s h o w n in Fig. I, a s i m i l a r r e d u c t i o n in C h A T a c t i v i t y w a s o b t a i n e d w h e n AF64A, at a

1442

Cholinotoxlcity in Various Brain Regions

Vol. 44, No. 20, 1989

dose of 3 nmoles, was injected in a volume of either 0.5 or 2 ul. These results suggest that in both cases, the toxin was mixed rapidly with the CSF and diluted to a similar concentration before reaching the target tissue. AChE activity after icv injection of AF64A AF64A caused significant reductions in AChE activity in the hippocampus (Fig. 2). The peak reduction was observed between days 10 and 15 after the injection of the toxin. Recovery of enzyme activity was then observed, reaching 80% of control, 28 days after the injection of the toxin. In the cerebral cortex or in the corpus striatum, the enzyme activity remained at the basal level throughout the entire period of the experiment, except for one time point in which a small but significant reduction in striatal AChE was observed (Fig. 2). Thus, it appears that AF64A-induced changes in AChE are similar to those described for CHAT. HAChT activity after icy injection of AF64A AS the damage induced by AF64A begins at the cholinergic nerve terminals (16,17,18), we followed changes in HAChT, a cholinergic marker which is confined to the nerve terminals (19), as an index of early damages. Table I summarizes the activities of the HAChT system at days 11 and 60 after an icv injection of AF64A. Significant reductions in HAChT were observed in the hippocampus, corpus striatum and cerebral cortex 11 days after the toxin injection. However, at 60 days post injection the activity of HAChT in the corpus striatum and the cerebral cortex returned to control levels, while the activity in the hippocampus remained significantly reduced.

TABLE I Reduction in HAChT Activity in Various Brain Regions Following an icv Injection of AF64A (3 nmol/side)

Brain Region

Percent reduction in HAChT activity 11 days 60 days post treatment post treatment

hippocampus

49210

(5)*

striatum

42+ 8 (4)**

cortex

30+ 5 (4)

44±2 (4)* 5+2 (4) -19+4

(4)*

Each value represents an average ~ SEM from 4 or 5 treated rats (in parentheses). Individual percentages were calculated by dividing the activity found for each experimental animal by the average activity of the control group (4-5 rats which were injected with artificial CSF). Values which are significantly different from control (t-test) are indicated as follows:

*p
Vol. 44, No. 20, 1989

Chollnotoxlelty in Various Brain Regions

STRIATUM

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The e f f e c t of A F 6 4 A on s y n a p t o s o m a l c h o l i n e transport: A Michaelis-Menten a n a l y s i s is d e s c r i b e d on the left hand side and L i n e w e a v e r - B u r k a n a l y s i s on the right. R a t s w e r e i n j e c t e d icv w i t h A F 6 4 A (3 n m o l / s i d e ) , 60 days later the animals were s a c r i f i c e d and P2 f r a c t i o n s w e r e prepared from hippocampus, corpus s t r i a t u m and c e r e b r a l cortex. Control rats r e c e i v e d a r t i f i c i a l CSF i n s t e a d of AF64A. The P2 f r a c t i o n s (filled c i r c l e s for c o n t r o l and empty squares for A F 6 4 A - t r e a t e d 3 rats) were incubated with v a r i o u s c o n c e n t r a t i o n s of H - c h o l i n e and the i n c o r p o r a t e d radioactivity was d e t e r m i n e d as d e s c r i b e d in the method section. Km and V m a x v a l u e s for c o n t r o l (CSF) a n d t r e a t e d rats (AF64A) are shown. V a l u e s w h i c h w e r e found to be s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (paired t-test) are indicated: +p<0.05.

1443

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Chollnotoxlclty in Various Brain Regions

Vol. 44, No. 20, 1989

These data s u g g e s t that d e g e n e r a t i o n of cholinergic neurons o c c u r r e d o n l y in the h i p p o c a m p u s but not in the corpus s t r i a t u m or in the c e r e b r a l cortex. C o n s i s t e n t w i t h this notion, L i n e w e a v e r B u r k a n a l y s i s (Fig. 3) showed that the d e c r e a s e in the h i p p o c a m p a l HAChT was mainly due to r e d u c t i o n in Vmax. These reductions indicate that the n u m b e r of sites w h i c h t r a n s p o r t choline were reduced, and that the a f f i n i t y of the r e m a i n i n g c h o l i n e carriers to the s u b s t r a t e was not s i g n i f i c a n t l y altered. ChAT a c t i v i t y a f t e r i n j e c t i o n of A F 6 4 A into the c a r o t i d a r t e r y A w i d e and h o m o g e n e o u s d i s t r i b u t i o n of A F 6 4 A m i g h t be achieved by conveying the t o x i n via the v a s c u l a r system. The carotid artery could be an a p p r o p r i a t e i n j e c t i o n site since its blood supply is d i r e c t e d t o w a r d the brain. However, b e c a u s e of its q u a t e r n a r y nature, A F 6 4 A m i g h t not p e n e t r a t e well into the brain. To overcome this d i f f i c u l t y we o p e n e d the BBB t e m p o r a r i l y by a hypertonic treatment (20) g i v e n prior to the injection of the toxin. As a c r i t e r i o n for the e f f e c t of the toxin, we f o l l o w e d the reduction in ChAT activity, an e n z y m e w h i c h was significantly a f f e c t e d by A F 6 4 A after the icv injection. This r e d u c t i o n in ChAT activity was maximal at 10-13 days after icv administration.

TABLE R e d u c t i o n in ChAT A c t i v i t y Following Administration

II

in V a r i o u s B r a i n R e g i o n s of A F 6 4 A Via the C a r o t i d

13 Days Artery

Dose of AF64A ~mol/kg

Hypertonic glycerol

Number of animals

0

-

5

3+10

-9+ 7

3+ 5

11+ 9

0

+

5

5+ 3

-2+10

3+ 4

O+ 8

1.0

-

3

5+ 5

14+

4+ 5

9+ 4

0.1

+

4

-2+ I

2+11

6+ 6

-3+18

1.0

+

3

7+ 8

-15+28

Percent Hippocampus

reduction Corpus striatum

4

in ChAT a c t i v i t y Frontal Cerecortex bellum

30+10"

The hypertonic treatment comprised of a unilateral i n j e c t i o n of 40% g l y c e r o l via the c a r o t i d artery, and the toxin was i n j e c t e d into the same a r t e r y i m m e d i a t l y after the glycerol. Each value r e p r e s e n t s an a v e r a g e (~ SEM) from 3-5 rats (as indicated). I n d i v i d u a l p e r c e n t a g e s were c a l c u l a t e d by d i v i d i n g the a c t i v i t y of the left h e m i s p h e r e (treated side) by the a c t i v i t y of the right hemisphere (untreated side), s e p a r a t e l y for each animal. V a l u e s w h i c h are s t a t i s t i c a l l y (t-test) d i f f e r e n t from control (p<0.05) are i n d i c a t e d by asterisks.

4+ 4

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Cholinotoxicity in Various Brain Regions

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Therefore, for comparison purposes, we s e l e c t e d the same time point for the i n t r a v a s c u l a r administration. From the results presented in Table 2, it a p p e a r s that an o p e n i n g of the BBB is indeed required for o b t a i n i n g the c h o l i n o t o x i c effect when the toxin is i n j e c t e d into the c a r o t i d artery. Thus, I umol/kg of AF64A without p r e t r e a t m e n t w i t h 40% g l y c e r o l did not cause any s i g n i f i c a n t change in ChAT activity, w h e r e a s s i g n i f i c a n t r e d u c t i o n in C h A T a c t i v i t y in the i p s i l a t e r a l side of the cerebral cortex was observed if a u n i l a t e r a l h y p e r t o n i c t r e a t m e n t p r e c e e d e d the injection of the toxin. However, the c o n t r a l a t e r a l side of the AF64A-treated a n i m a l s showed similar a c t i v i t i e s to those of the control group, indicating that there was no contralateral c o m p e n s a t i o n to the i p s i l a t e r a l reduction. As can be d e d u c e d by c o m p a r i n g the r e s u l t s p r e s e n t e d in Fig. I w i t h those p r e s e n t e d in Table 2, b o t h m e t h o d s of a d m i n i s t r a t i o n of the toxin r e s u l t e d in a r e d u c t i o n of ChAT activity. However, in each case a d i f f e r e n t b r a i n r e g i o n was affected, the h i p p o c a m p u s after an icv injection, and the cerebral cortex after an intracarotid administration.

Discussion In the present work we introduce a new route for the a d m i n i s t r a t i o n of AF64A, w h i c h e l i c i t s c h o l i n o t o x i c i t y in a brain r e g i o n that is g e n e r a l l y not a f f e c t e d by direct injections into the cerebroventricles. We c o m p a r e d this new t e c h n i q u e w i t h the frequently used icv i n j e c t i o n and found that w h e n the toxin was conveyed via the carotid artery subsequent to a hypertonic g l y c e r o l treatment, cholinotoxicity was observed only in the c e r e b r a l c o r t e x but not in the hippocampus, the striatum, or the cerebellum. In contrast, when the toxin was i n j e c t e d into the lateral ventricles, it caused a long-lasting cholinotoxicity in the h i p p o c a m p u s but not in the corpus s t r i a t u m or in the cerebral c o r t e x (although the latter two regions are also known to c o n t a i n c h o l i n e r g i c neurons). In a recent paper, Leventer et al. (14) focused on the p r o b l e m of why after an icv administration, the hippocampus is more susceptible to the neurotoxic effect of AF64A than other brain regions. Two alternative explanations were given: I) The hippocampus is proximal to the injection site or 2) The neurons in the h i p p o c a m p u s m a y be m o r e s u s c e p t i b l e to A F 6 4 A than n e u r o n s in other brain regions. Our results suggest that the m a j o r factor which determines where c h o l i n o t o x i c i t y occurs, is the a v a i l a b i l i t y of the toxin at the target site and not the differential sensitivities of various brain regions to the toxin as was s u g g e s t e d by V i c k r o y et al. (4). Our results are c o n s i s t e n t w i t h other in v i t r o and in v i v o studies showing that AF64A-induced toxicity is not n e c e s s a r i l y r e s t r i c t e d to the hippocampus. For example, we have r e c e n t l y d e m o n s t r a t e d that t r a n s p o r t of choline by s y n a p t o s o m e s p r e p a r e d from h i p p o c a m p u s , striatum, or c e r e b r a l cortex exhibited similar sensitivities to the toxin (12). Moreover, direct i n j e c t i o n of A F 6 4 A into d i s t i n c t b r a i n regions (5,21,22), w h i c h are g e n e r a l l y not a f f e c t e d by the t o x i n a f t e r icv injection, caused d a m a g e at the i n j e c t e d area or at r e g i o n s that receive direct projections from the injected site (23). We propose that when A F 6 4 A is i n j e c t e d into the ventricle, the hippocampus, w h i c h is e x t e n d e d along the v e n t r i c u l a r surfaces, is

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exposed to a higher concentration of the toxin as c o m p a r e d to remote regions such as the c e r e b r a l cortex. Therefore, a f t e r icv injection the h i p p o c a m p u s is the m a j o r d a m a g e d site. In contrast, when the toxin is i n j e c t e d into the c a r o t i d artery, it arrives first at the s u p e r f i c i a l v a s c u l a r i z a t i o n of the cortex, e x p o s i n g the cerebral cortex to the highest toxin concentration and thereby causes most of the damage in this b r a i n region. This hypothesis could be v e r i f i e d by a d i r e c t m e a s u r e m e n t of the toxin concentrations at various brain regions shortly after the injection. The synthesis of a r a d i o l a b e l e d A F 6 4 A will p r o v i d e a useful tool for such m e a s u r e m e n t s . We previously proposed that specific d a m a g e to c h o l i n e r g i c neurons would be expected at a c o n c e n t r a t i o n range of 2-20 uM AF64A (18,24). When the cells are exposed to higher concentrations, the toxin may cause n o n s e l e c t i v e cytotoxicity. Direct injections into the tissue or even into the c e r e b r a l ventricles (where the toxin m u s t be i n j e c t e d in small v o l u m e s and therefore at high concentrations), may cause nonselective cytotoxicity (7,8,25). In contrast, during intravascular injection, the toxin is first d i l u t e d by the large volume of the blood before entering into the brain, and thus e x c e s s i v e l y h i g h concentrations of the toxin are not formed and n o n s e l e c t i v e damages to the b r a i n tissue are avoided. As b l o o d cells, c o m p a r e d to neurons, are l e s s s e n s i t i v e to the toxin (26), r e l a t i v e l y h i g h amounts of the toxin m a y be i n j e c t e d into the blood w i t h o u t the appearance of undesirable effects, a l l o w i n g the toxin to reach the neuronal tissue at concentrations capable of causing selective cholinotoxicity. However, it still remains to be determined whether the cytotoxicity induced by AF64A after intracarotid injection is indeed confined merely to the cholinergic system. Studies d e s i g n e d to r e s o l v e this q u e s t i o n by determining the e f f e c t of A F 6 4 A on n o n c h o l i n e r g i c p a r a m e t e r s are n o w in progress. The l o n g - l a s t i n g r e d u c t i o n s in CHAT, ACHE, and H A C h T a c t i v i t i e s suggest that cholinergic cells were damaged. However, the recovery of these c h o l i n e r g i c m a r k e r s suggests that not all the damaged cholinergic n e u r o n s died. A n a l y s i s of the changes of the above m e n t i o n e d p a r a m e t e r s in v a r i o u s b r a i n regions suggests that only partial cholinergic damage occurs in the cortex and the corpus striatum a f t e r icy injection. This was c o n c l u d e d from the fact that full r e v e r s i b i l i t y of the HAChT o c c u r r e d in these brain regions with no c h a n g e s in the n u m b e r of c h o l i n e c a r r i e r s or in the affinity of choline to the c a r r i e r (as i n d i c a t e d by the Km and Vmax values). However, the hippocampus was more s e v e r e l y damaged as indicated by the reduction in the number of the choline transport sites (as indicated by the r e d u c t i o n in the Vmax), suggesting a loss of c h o l i n e r g i c neurons in this brain region. The r e c o v e r y of ChAT a c t i v i t y in the h i p p o c a m p u s may be a result of an increase in the e n z y m e a c t i v i t y in the r e m a i n i n g neurons, or alternatively, a result of n e u r o n a l s p r o u t i n g and a formation of new nerve e n d i n g s in the d a m a g e d area. Other studies already pointed to the p o s s i b i l i t y that some r e c o v e r y of d a m a g e d neurons may occur (14,17). However, detailed k i n e t i c s of the r e c o v e r y p r o c e s s is p u b l i s h e d now for the first time.

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As the most sensitive component of the cholinergic system to AF64A is the HAChT (14), it is possible that the primary site which is damaged by AF64A is indeed the HAChT system, and only secondary damage is then extended to ChAT and AChE activities. Consistent with this hypothesis, long-lasting reductions in ChAT and AChE activities were observed only in the hippocampus, where the HAChT was reduced irreversibly, while in regions such as the cortex and striatum, where the HAChT was recovered, only transient changes in ChAT and AChE activities were observed (at time points in which the HAChT was reduced). Such transient changes may also be related to a compensatory response of one brain region to a reduction of the enzyme activity in another brain region. Thus, whenever a decrease in ChAT activity occurred in the hippocampus (after icy injection) or in the cortex (after intra-carotid injection), an increase in this enzyme activity was observed in the striatum. Our experiments also show that AF64A does not penetrate well through the BBB. Only after hypertonic treatment that temporarily opens the BBB (20) could AF64A injected via the carotid artery, cause cholinotoxicity in the brain. The BBB is poorly permeable to the toxin despite its structural similarity to choline, which penetrates well via the BBB. One explanation of this difference in permeability could be related to the alkylation potency of AF64A as compared to choline. According to this explanation, AF64A alkylates the choline carrier, which is responsible for choline transport from the blood to the brain, and thus prevents its own transport. We presume that the changes in the volume of endothelial cells, which occur subsequent to the hyperosmotic treatment, opens pores through which AF64A penetrates the BBB. If so, then different sensitivities of the various endothelial cells at various brain r e g i o n s to the hypertonic treatment, may also play a role in determining the amount of the toxin which reaches each of the brain regions. This interesting possibility is now under investigation. Acknowledgments We wish to thank Professor S. Cohen for his useful suggestions and Dr. R. Brandeis for her help with the stereotactic technique. We also express our gratitude to Mr. L. Gefen for his technical assistance and to Mr. D. Alkalai for the graphical work. References I. 2. 3. 4.

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