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Sector-dependent neurotoxicity of ethylchoune aziridinium (AF64A) in the rat hippocampus
Neuropharmacology Vo1.29, N0.10, pp.961-964, 1990 Printed in Great Britain
SE~OR-DEPE~E~
0028-3908,'90 $3.00+0.00 Pergamon Press plc
NBUROTOXIC!ITY OF ETHYLCHOLINE? ATOM (AF64A) IN THE RAT HIPPOCAMPUS S. Laganiere, M. Marinko, Janice Corey, E. Wulfert* and I. Hanin
Loyola University of Chicago Stritch School of Medicine 2160 South First Avenue Maywood, IL 60153. U.S.A. and *UCB sa. Pharmaceutical Sector, Brussels ~Accepted 5 SUay 1990)
SUMMARY
The present study was aimed at measuring the distribution of ethylcho~ne ~i~dinium (AF64A)induced cholinoto~~~ within the hipp~pus 6 days after bilateral (icv) a~~tration of 1, 2 or 3 nmol, or vehicle. The dissected hippocampus was sectioned with a vibratome into 5 parallel sectors distributed along its long axis from its thalamic surface (medial) to its cortical surface (lateral). In vehicle-treated rats, the high affinity choline@ transport (HAChT), choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were distributed according to a gradient of increasing activity, extending from the lateral to the medial surface of the hippocampus. After treatment with AF64A, the normal gradient of enqme activity was profoundly disrupted at all doses of AF64A and the core sectors of the hip~c~pus were significantly more affected than the superficial sectors. The HAChT gradient was progressively abolished with increasing doses of toxin, and the effect was maximal at 2 mnol. Key words - AF64A, hippocampus, cholinotoxin, ChAT, AChE, HAChT
Ethylcho~ne aziridinium (AF64A) has recently received a great deal of attention as a selective cholinergic neurotoxin. Dose-related losses of hippocampal levels of acetylcholine, HAChT, ChAT and AChE activities, as well as behavioral deficits have been observed reproducibly after icv administration of AF64A (Hanin, Fisher, Hortnagl, Leventer, Potter and Walsh, 1987). Although it is well known that an aziridinium moiety is a potent alkylator, the mode of action of AF64A in vivo still remains to be determined. Some investigators have demonstrated that this choline analog binds irreversibly to the high affinity transporter of choline in vitro (Curti and Marchbanks, 1984; Pittel, Fisher and Heldman, 1987). It was therefore postulated that a similar mechanism might take place in the brain, since pre-treatment with inhibitors of HAChT was shown to blunt the effects of icv AF64A (Chrobak, Spates, Stackman and Walsh, 1989; Potter, Tedford, Kindel and Hanin, 1989). Pittel et al (1987) have shown that AF64A may actually diffuse into the cell cytoplasm, implying that the mechanism of cholinotoxicity induced by AF64A may also occur at intracellular, cytoplasmic loci, rather than at the nerve terminal membrane. However, Mantione, Zigmond, Fisher and Hanin (1983) reported no decrease in AChE after intra-hippocampal injection and Blaker and Goodwin 0987) clearly showed that after similar injection, the hippocampal choline-deficiency was only restricted to a narrow zone surrounding the needle track. This implies a very restricted diffusion of AF64A, indicating also that the aziridinium ring half-life may be very short in vivo. Accordingly, icv administration of the toxin should hypothetically show more potency towards the superficial sector of the hippocampus, which is surrounded by the ventricular fluid, rather than the core portion. Thus, our aim was to determine how the AF64A-induced cholinoto~~~ is distributed within the hippo~mpu~ after its icv administration.
961
962
Preliminary
Notes
METHODS -ration of AF64A AF64A was prepared freshly, according to the procedure described by Fisher, Mantione, Abraham, and Hanin (1982). The extent of formation of the aziridinium moiety was quantitated by the method of Gill and Rang (1966). Under these conditions, aziridinium cyclization was routinely found to be between 80 and 85%. Male Sprague-Dawley rats (275350 g, Zivic Miller Laboratories, Allison Park, PA) were anesthetized with pentobarbital and positioned in a Kopf stereotaxic apparatus. AF64A (1, 2, 3 nmol or vehicle, 3 ul/side), was infused bilaterally (icv) at a rate of 1 ul/min. Stereotaxic coordinates (Paxinos and Watson, 1986) were: anterior -0.8 mm, lateral +1..5 mm, ventral +3.6 mm from bregma.
* *
Dissection and sectroutug o f tissa. Following decapitation, the hippocampus of each hemisphere was dissected on ice. Tissues were immediately placed in oxygenated isotonic buffer at 4 “C (120 mM sucrose, 11.5 mM glucose, 4.7 mM KCl, 1.2 mM sodium phosphate, 1.25 mM CaCl 2, and 1.2 mM MgCl J. The dissected hippocampus presents two distinctive surfaces: the thalamic-facing surface (medial) is flat whereas the opposite, cortical-facing surface (lateral), is concave. The medial surface of the hippocampus was secured to the sectioning block of a vibratome (Campden Instruments Ltd.) with a cyanoacrylate-containing adhesive. Longitudinal sections of 225 urn thickness were obtained and immediately homogenized in appropriate buffer. Biochemical (1970). High (1973), on a was defined accumulated and Randall
marker u ChAT and AChE activity was determined as described by Fonnum affinity transport of choline was determined as described by Yamamura and Snyder synaptosomal fraction, in the presence of 1 uM choline. The Nat-dependent HAChT as the amount of choline transported in the presence of Na’, minus that in the absence of Na’. Protein was assayed according to Lowry, Rosebrough, Farr (1951).
. . &&&A
analvsis, Dose and sector differences were compared by multiple analysis of variance and post-hoc analysis using the Systat Statistical System (Evanston, 11, U.S.A.). RESULTS
In vehicle treated rats, the distribution of ChAT, AChE and HAChT was clearly organized into a linear gradient of activity (Fig. 1, 2, 3; p
-20 z
-
l
\._-
l\.
p 16-\ 0’12-I ?L D 8-Y ;
4--
6 4
0
0 1 nmol A 2 nmol 0 3 nmol
.\.
q :\
HQ
.8\*-. O\o/O-n
1 HI&XAMF’:L
4 SECTORS
5
FIGURE 1. Choline acetyltransferase (ChAT) activity in sectors distributed from the thalamic (1) to the cortical (5) surface of the hippocampus of vehicle or AF64A-infused rats. Multiple contrasts on the dose effects: veh vs 1 nmol,p
Preliminary
Notes
963
AF64A-induced loss of AChE activity completely abolished the normal gradient (Fig.2). The AChE activity rern~~g was decreased d~~e~nden~y between 1 to 3 mnol of AF64A, and varied from 40% to approximately 20%. In AF64A-treated rats, the gradient of HAChT activity was still perceptible after 1 mnol/lateral ventricle (p< 0.001) but was completely abolished following 2 and 3 nmol AF64A. The decrease in HAChT was dose-dependent at 1 and 2 nmol and varied from 20 to 45% throughout the hippocampus, being more severe at the thalamic surface of the hippocampus. Surprisingly, there was no further reduction in transport activity at 3 mnol/lateral ventricle. * 0 vehicle a1 500
-
0
0~
1
4
2 3 HlPPOCAMPAl
4
1 nmol
5
SECTORS
FIGURE 2 Acetylcholinesterase (AChE) activity in sectors distributed from the thalamic (1) to the cortical (5) surface of the hippocampus of vehicle or AF64A-infused rats. Multiple contrasts on the dose effects: veh vs 1 mnol, p
A 2 nmol Cl 3 nmol
i\i $6..
g_;:q
0” 12-q z
w a-z dI 4--
T
%r’ Q--e---
(j-q\ I
8 i’u
0
o-
1
1 5
di’OCAM&
SECT:RRS
FIGURE 3. High aftlnity choline transport (HAChT) activity in hipp~p~ sectors distributed from the thalamic (1) to the cortical (5) surface in vehicle or AF64A-infused rats. Multiple contrasts on the dose effect: veh vs 1 nmol, p
964
Preliminary
Notes
with AChE and I-IAChT. Most importantly, in AF64A-treated rats, ChAT, and to a lesser extent AChE activity, was significantly more affected within the core of the hippocampus than the outlying sectors. Despite the fact that CbAT and AChE were further decreased by the administration of 3 nmol of AF64A, the loss in HAChT was comparable at 2 and 3 nrnol/ventricle (Fig. 3). Thus, reduction in HAChT with: 2 nmol of AF64A represents the saturation concentration to be used in our conditions. Residual activity of cholinergic markers persists in hippocampus, even if high, nonspecific doses of AF64A are used (Hanin et al, 1987). However, having shown that AF64A acted in every sector of the hippocampus, but that each cholinergic marker reacted differently to a given dose of the toxin, it is conceivable that certain cholinergic terminals either do not transport AF64A, or are more resistant to its toxicity. Such a mecha~sm may also apply to striatal cholinergic neurons, which do not react to &-administered AF64A at either low or high doses (Hanin et al, 1987). Together, these findings indicate that the hippocampal cholinergic innervation is highly organized. Moreover, caution should be used to interpret neurochemical data obtained in the hippocampus, because of the regional nature of cholinergic orientation in this brain region. Acknowledgements: This work was supported by the UCB s.a. Pharmaceutical and by NIMH Grant #MH42572. REFERENCES Blaker, W.D. and Goodwin, SD. (1987) Biochemical and behavioral AF64A in rats. Pharmacoi. Biochem. Behav. 28:157-163.
Sector, Brussels
effects of ~n~ahip~mpal
Chrobak J.J., Spates M.J., Stackman R.W. and Walsh T.J. (1989) He~choii~~-3 prevents the working memory impairments and the cholinergic hypofunction induced by ethylchohne aziridinium ion (AF64A). Brain Res. 504~269-275. Curti D. and Marchbanks R.M. (1984) Kinetics of irreversible inhibition of choline transport synaptosomes by ethylcholine mustard aziridinium. J. Membr. Biol 82:259-268.
in
Fisher A., Mantione C.R., Abraham D.J. and Hanin I. (1982) Long-term central cholinergic hypofunction induced in mice by ethylcholine aziridinium ion (AF64A) in vivo. J. Pharmacol. Exp. Ther. 222:140-145. Fonnum F. (1970) Topographical and subcellular localization hippocampal regions. J. Neurochem. 17: 1029-1037,
of choline acetyltransferase
Gill E.W. and Rang H.P. (1966) An al~lating derivative of be~lcholine lasting par~~pathol~ic activity. Mol. Pharmacol. 2: 284-297.
in rat
with specific and long-
Hanin I., Fisher A., Hortnagl H., Leventer S.M., Potter P.E. and Walsh T.J. (1987) Ethylcholine aziridinium (AF64A, ECMA) and other potential cholinergic neuron-specific neurotoxins. In: Psychopharmacolw The Third Generation of Progress (Meltzer H.Y, Ed.), pp.341-349. Raven Press, New York. Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J. (1951) Protein measurement Folin phenol reagent. .I. Biol. Chem. 193:265-275. Mantione C., igmond M.J., Fisher A. and Hanin I. (1983) Selective presynaptic neurotoxicity E llowing intrahippocampal AF64A injection in rats. J. Neurochem Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Academic Press, Orlando, Fla.
Coordina@.
with the
cholinergic 41:251-255.
Second Edition.
Pittel Z., Fisher A. and Heldman E. (1987) Reversible and irreversible inhibition of high-af~ni~ choline transport caused by ethylcholine aziridinium ion. J. Neurochem. 49: 468-474. Potter P.E., Tedford C., Kindel G. and Hanin I. (1989) Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A). Brain Res. 487: 238-244. Yamamura H.I. and Snyder S.H. (1973) High affinity transport rat brain. J. Neurochem 21: 13551374.
of choline into synaptosomes
of
SE~OR-DEPE~E~
0028-3908,'90 $3.00+0.00 Pergamon Press plc
NBUROTOXIC!ITY OF ETHYLCHOLINE? ATOM (AF64A) IN THE RAT HIPPOCAMPUS S. Laganiere, M. Marinko, Janice Corey, E. Wulfert* and I. Hanin
Loyola University of Chicago Stritch School of Medicine 2160 South First Avenue Maywood, IL 60153. U.S.A. and *UCB sa. Pharmaceutical Sector, Brussels ~Accepted 5 SUay 1990)
SUMMARY
The present study was aimed at measuring the distribution of ethylcho~ne ~i~dinium (AF64A)induced cholinoto~~~ within the hipp~pus 6 days after bilateral (icv) a~~tration of 1, 2 or 3 nmol, or vehicle. The dissected hippocampus was sectioned with a vibratome into 5 parallel sectors distributed along its long axis from its thalamic surface (medial) to its cortical surface (lateral). In vehicle-treated rats, the high affinity choline@ transport (HAChT), choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were distributed according to a gradient of increasing activity, extending from the lateral to the medial surface of the hippocampus. After treatment with AF64A, the normal gradient of enqme activity was profoundly disrupted at all doses of AF64A and the core sectors of the hip~c~pus were significantly more affected than the superficial sectors. The HAChT gradient was progressively abolished with increasing doses of toxin, and the effect was maximal at 2 mnol. Key words - AF64A, hippocampus, cholinotoxin, ChAT, AChE, HAChT
Ethylcho~ne aziridinium (AF64A) has recently received a great deal of attention as a selective cholinergic neurotoxin. Dose-related losses of hippocampal levels of acetylcholine, HAChT, ChAT and AChE activities, as well as behavioral deficits have been observed reproducibly after icv administration of AF64A (Hanin, Fisher, Hortnagl, Leventer, Potter and Walsh, 1987). Although it is well known that an aziridinium moiety is a potent alkylator, the mode of action of AF64A in vivo still remains to be determined. Some investigators have demonstrated that this choline analog binds irreversibly to the high affinity transporter of choline in vitro (Curti and Marchbanks, 1984; Pittel, Fisher and Heldman, 1987). It was therefore postulated that a similar mechanism might take place in the brain, since pre-treatment with inhibitors of HAChT was shown to blunt the effects of icv AF64A (Chrobak, Spates, Stackman and Walsh, 1989; Potter, Tedford, Kindel and Hanin, 1989). Pittel et al (1987) have shown that AF64A may actually diffuse into the cell cytoplasm, implying that the mechanism of cholinotoxicity induced by AF64A may also occur at intracellular, cytoplasmic loci, rather than at the nerve terminal membrane. However, Mantione, Zigmond, Fisher and Hanin (1983) reported no decrease in AChE after intra-hippocampal injection and Blaker and Goodwin 0987) clearly showed that after similar injection, the hippocampal choline-deficiency was only restricted to a narrow zone surrounding the needle track. This implies a very restricted diffusion of AF64A, indicating also that the aziridinium ring half-life may be very short in vivo. Accordingly, icv administration of the toxin should hypothetically show more potency towards the superficial sector of the hippocampus, which is surrounded by the ventricular fluid, rather than the core portion. Thus, our aim was to determine how the AF64A-induced cholinoto~~~ is distributed within the hippo~mpu~ after its icv administration.
961
962
Preliminary
Notes
METHODS -ration of AF64A AF64A was prepared freshly, according to the procedure described by Fisher, Mantione, Abraham, and Hanin (1982). The extent of formation of the aziridinium moiety was quantitated by the method of Gill and Rang (1966). Under these conditions, aziridinium cyclization was routinely found to be between 80 and 85%. Male Sprague-Dawley rats (275350 g, Zivic Miller Laboratories, Allison Park, PA) were anesthetized with pentobarbital and positioned in a Kopf stereotaxic apparatus. AF64A (1, 2, 3 nmol or vehicle, 3 ul/side), was infused bilaterally (icv) at a rate of 1 ul/min. Stereotaxic coordinates (Paxinos and Watson, 1986) were: anterior -0.8 mm, lateral +1..5 mm, ventral +3.6 mm from bregma.
* *
Dissection and sectroutug o f tissa. Following decapitation, the hippocampus of each hemisphere was dissected on ice. Tissues were immediately placed in oxygenated isotonic buffer at 4 “C (120 mM sucrose, 11.5 mM glucose, 4.7 mM KCl, 1.2 mM sodium phosphate, 1.25 mM CaCl 2, and 1.2 mM MgCl J. The dissected hippocampus presents two distinctive surfaces: the thalamic-facing surface (medial) is flat whereas the opposite, cortical-facing surface (lateral), is concave. The medial surface of the hippocampus was secured to the sectioning block of a vibratome (Campden Instruments Ltd.) with a cyanoacrylate-containing adhesive. Longitudinal sections of 225 urn thickness were obtained and immediately homogenized in appropriate buffer. Biochemical (1970). High (1973), on a was defined accumulated and Randall
marker u ChAT and AChE activity was determined as described by Fonnum affinity transport of choline was determined as described by Yamamura and Snyder synaptosomal fraction, in the presence of 1 uM choline. The Nat-dependent HAChT as the amount of choline transported in the presence of Na’, minus that in the absence of Na’. Protein was assayed according to Lowry, Rosebrough, Farr (1951).
. . &&&A
analvsis, Dose and sector differences were compared by multiple analysis of variance and post-hoc analysis using the Systat Statistical System (Evanston, 11, U.S.A.). RESULTS
In vehicle treated rats, the distribution of ChAT, AChE and HAChT was clearly organized into a linear gradient of activity (Fig. 1, 2, 3; p
-20 z
-
l
\._-
l\.
p 16-\ 0’12-I ?L D 8-Y ;
4--
6 4
0
0 1 nmol A 2 nmol 0 3 nmol
.\.
q :\
HQ
.8\*-. O\o/O-n
1 HI&XAMF’:L
4 SECTORS
5
FIGURE 1. Choline acetyltransferase (ChAT) activity in sectors distributed from the thalamic (1) to the cortical (5) surface of the hippocampus of vehicle or AF64A-infused rats. Multiple contrasts on the dose effects: veh vs 1 nmol,p
Preliminary
Notes
963
AF64A-induced loss of AChE activity completely abolished the normal gradient (Fig.2). The AChE activity rern~~g was decreased d~~e~nden~y between 1 to 3 mnol of AF64A, and varied from 40% to approximately 20%. In AF64A-treated rats, the gradient of HAChT activity was still perceptible after 1 mnol/lateral ventricle (p< 0.001) but was completely abolished following 2 and 3 nmol AF64A. The decrease in HAChT was dose-dependent at 1 and 2 nmol and varied from 20 to 45% throughout the hippocampus, being more severe at the thalamic surface of the hippocampus. Surprisingly, there was no further reduction in transport activity at 3 mnol/lateral ventricle. * 0 vehicle a1 500
-
0
0~
1
4
2 3 HlPPOCAMPAl
4
1 nmol
5
SECTORS
FIGURE 2 Acetylcholinesterase (AChE) activity in sectors distributed from the thalamic (1) to the cortical (5) surface of the hippocampus of vehicle or AF64A-infused rats. Multiple contrasts on the dose effects: veh vs 1 mnol, p
A 2 nmol Cl 3 nmol
i\i $6..
g_;:q
0” 12-q z
w a-z dI 4--
T
%r’ Q--e---
(j-q\ I
8 i’u
0
o-
1
1 5
di’OCAM&
SECT:RRS
FIGURE 3. High aftlnity choline transport (HAChT) activity in hipp~p~ sectors distributed from the thalamic (1) to the cortical (5) surface in vehicle or AF64A-infused rats. Multiple contrasts on the dose effect: veh vs 1 nmol, p
964
Preliminary
Notes
with AChE and I-IAChT. Most importantly, in AF64A-treated rats, ChAT, and to a lesser extent AChE activity, was significantly more affected within the core of the hippocampus than the outlying sectors. Despite the fact that CbAT and AChE were further decreased by the administration of 3 nmol of AF64A, the loss in HAChT was comparable at 2 and 3 nrnol/ventricle (Fig. 3). Thus, reduction in HAChT with: 2 nmol of AF64A represents the saturation concentration to be used in our conditions. Residual activity of cholinergic markers persists in hippocampus, even if high, nonspecific doses of AF64A are used (Hanin et al, 1987). However, having shown that AF64A acted in every sector of the hippocampus, but that each cholinergic marker reacted differently to a given dose of the toxin, it is conceivable that certain cholinergic terminals either do not transport AF64A, or are more resistant to its toxicity. Such a mecha~sm may also apply to striatal cholinergic neurons, which do not react to &-administered AF64A at either low or high doses (Hanin et al, 1987). Together, these findings indicate that the hippocampal cholinergic innervation is highly organized. Moreover, caution should be used to interpret neurochemical data obtained in the hippocampus, because of the regional nature of cholinergic orientation in this brain region. Acknowledgements: This work was supported by the UCB s.a. Pharmaceutical and by NIMH Grant #MH42572. REFERENCES Blaker, W.D. and Goodwin, SD. (1987) Biochemical and behavioral AF64A in rats. Pharmacoi. Biochem. Behav. 28:157-163.
Sector, Brussels
effects of ~n~ahip~mpal
Chrobak J.J., Spates M.J., Stackman R.W. and Walsh T.J. (1989) He~choii~~-3 prevents the working memory impairments and the cholinergic hypofunction induced by ethylchohne aziridinium ion (AF64A). Brain Res. 504~269-275. Curti D. and Marchbanks R.M. (1984) Kinetics of irreversible inhibition of choline transport synaptosomes by ethylcholine mustard aziridinium. J. Membr. Biol 82:259-268.
in
Fisher A., Mantione C.R., Abraham D.J. and Hanin I. (1982) Long-term central cholinergic hypofunction induced in mice by ethylcholine aziridinium ion (AF64A) in vivo. J. Pharmacol. Exp. Ther. 222:140-145. Fonnum F. (1970) Topographical and subcellular localization hippocampal regions. J. Neurochem. 17: 1029-1037,
of choline acetyltransferase
Gill E.W. and Rang H.P. (1966) An al~lating derivative of be~lcholine lasting par~~pathol~ic activity. Mol. Pharmacol. 2: 284-297.
in rat
with specific and long-
Hanin I., Fisher A., Hortnagl H., Leventer S.M., Potter P.E. and Walsh T.J. (1987) Ethylcholine aziridinium (AF64A, ECMA) and other potential cholinergic neuron-specific neurotoxins. In: Psychopharmacolw The Third Generation of Progress (Meltzer H.Y, Ed.), pp.341-349. Raven Press, New York. Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J. (1951) Protein measurement Folin phenol reagent. .I. Biol. Chem. 193:265-275. Mantione C., igmond M.J., Fisher A. and Hanin I. (1983) Selective presynaptic neurotoxicity E llowing intrahippocampal AF64A injection in rats. J. Neurochem Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Academic Press, Orlando, Fla.
Coordina@.
with the
cholinergic 41:251-255.
Second Edition.
Pittel Z., Fisher A. and Heldman E. (1987) Reversible and irreversible inhibition of high-af~ni~ choline transport caused by ethylcholine aziridinium ion. J. Neurochem. 49: 468-474. Potter P.E., Tedford C., Kindel G. and Hanin I. (1989) Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A). Brain Res. 487: 238-244. Yamamura H.I. and Snyder S.H. (1973) High affinity transport rat brain. J. Neurochem 21: 13551374.
of choline into synaptosomes
of