Cholinotoxicity of the ethylcholine aziridinium ion in primary cultures from rat central nervous system

298

Brain Research, 454 (1988) 298-307 Elsevier

BRE 13733

Cholinotoxicity of the ethylcholine aziridinium ion in primary cultures from rat central nervous system Adina Amir, Zipora Pittel, Abraham Shahar, Abraham Fisher and Eliahu Heldman Israel Institute for Biological Research, Ness-Ziona (Israel)

(Accepted 28 January 1988) Key words: Ethylcholine aziridinium ion; Cholinergic neuron; Cytotoxicity; Dopaminergic neuron; Primary rat brain culture

The cytotoxic effects of ethylcholine aziridinium ion (AF64A) were studied in primary cultures prepared from either whole brain, septum, or midbrain of fetal rats. AF64A, at concentrations up to 22.5 ktM, significantly reduced the number of acetylcholinesterasestained cells without affecting the number of dopaminergic neurons or their ability to take up and release [3H]dopamine. Many of the survived acetylcholinesterase-stained cells appeared with intact somata but damaged processes, indicating a retrograde degeneration starting at the nerve terminal. Higher concentrations of AF64A (>22.5 ktM), caused general toxicity which was expressed by degeneration of various neuronal and glial cells. Choline (500/zM), significantly protected the cells from AF64A induced cytotoxicity. The resuits are consistent with a previously described kinetic model, that predicted a dual action of AF64A: selective cholinotoxicity at low concentrations and non-selective cytotoxicity at higher concentrations. INTRODUCTION Ethylcholine aziridinium ion (AF64A) was shown to be a potent inhibitor of the high-affinity choline transport ( H A C h T ) in synaptosomes 23. This inhibition could be attenuated either by hemicholinium-3 or by choline 2°. In addition, enzymes that use choline as a substrate, such as choline dehydrogenase 2 and choline acetyltransferase 22, were inhibited by AF64A. Administration of A F 6 4 A into the central nervous system causes reduction in hippocampal choline acetyltransferase (CHAT) activity 17 as well as in acetylcholine (ACh) levels 7,26. These in vitro and in vivo studies indicate that A F 6 4 A damages cholinergic neurons. Therefore, it has been suggested to use A F 6 4 A in vivo as a specific cholinotoxin in order to produce cholinergic hypofunction 8. For such a purpose, selectivity of the toxin towards cholinergic neurons is required. Indeed, selective cholinergic lesion was obtained in the chicken retina following intravitreal injection of A F 6 4 A 19. Damages which were confined specifically to the cholinergic system were

also observed after intrastriata124 or intracerebroventricular 26 administration of AF64A. However, the specificity of A F 6 4 A in affecting solely the cholinergic system has been questioned. For example, Levy et a1.15 demonstrated that A F 6 4 A induces dopaminergic hypofunction after injection into the substantia nigra. However, Levy et al. did not eliminate the possibility that the dopaminergic damage could have been secondary to a cholinergic lesion. Nevertheless, there are additional studies which point out that A F 6 4 A may cause non-selective damages expressed as cavitary lesion in the hippocampus 4'18, reduction in serotonin 4,7, and long-lasting decrease in hippocampal norepinephrine 7. The discrepancies between studies showing cholinergic specificity of A F 6 4 A toxicity and those showing nonselective damages may be accounted for by differences in rates of the toxin injection and by differences in the local concentrations of the toxin produced at the site of the injection. To overcome such difficulties we used primary brain culture for studying A F 6 4 A cytotoxicity.

Correspondence: E. Heldman, Department of Organic Chemistry, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona 70450, Israel.

0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

299 Primary brain cultures represent a system in which the concentration of the tested drug can be controlled. In addition, the presence of mixed populations of neurons (i.e. cholinergic and non-cholinergic) in these cultures, makes them most appropriate for studying toxin selectivity. In the present study, we used morphological and biochemical parameters to determine the type of cells that are most sensitive to AF64A. Cultures from 3 different brain regions were used: septal cultures which are rich in cholinergic neurons were used for studying the cholinotoxic selectivity of AF64A; midbrain cultures, rich in dopaminergic neurons which have been well characterized 3'21, were used to test whether the neurotoxic effects of AF64A are also apparent in dopaminergic neurons; whole brain cultures, which represent a system of wide cellular heterogeneity were used to assess the general nonspecific toxicity levels. An important advantage of whole brain cultures is that the ontogenesis of choline transport had been characterized in this system 28. Therefore, we performed our studies of the effect of AF64A on choline uptake with this type of culture. The results of the present study help to estimate the concentrations of the toxin that are needed to produce selective lesion of cholinergic neurons. MATERIALS AND METHODS

Preparation of neuronal cultures Cultures were prepared from fetal rat brain - - 15, 16 and 17 day old fetuses were used for midbrain, whole brain, or septal cultures, respectively. The dissected brain regions were collected and washed in cold Gey's balanced salt solution and then dissociated mechanically in growth medium (1:1 Eagle's minimal essential medium (MEM): Ham F12, containing 10% heat inactivated fetal calf serum, 6 g/1 glucose, 292 mg/1 glutamine and 1.5 mg/1 gentamycin) by repeated passages through a series of gradually narrowing Pasteur pipettes. Cells were plated on culture dishes or on glass coverslips that were precoated (24 h) with an aqueous solution of 2/~g/ml poly-L-ornithine.HBr (mol. wt. 44,000). Seeding density was 1.6 × 105 cells/cm 2. Cultures were kept in a 5% CO 2 atmosphere. Fig. 1 shows the morphological appearance of the 3 types of cell cultures that were used in the present study. In all cases, various

Fig. 1. Phase contrast micrographs of cultures prepared from three different brain regions of fetal rat brain. Cultures are in their 2nd week in vitro, a: whole brain culture, b: septal culture. c: midbrain culture. Bar = 50ktm.

types of neurons with a single neurite or with bundles of neurites are visible on top of a confluent monolayer of glial cells.

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. The pH of the solution was adjusted to 11.5 by addition of NaOH and maintained for 20 min. The pH was then adjusted to 7.3 with HC1, and the solution (containing the active aziridinium species)

300 was kept on ice and used within 4 h. Under these conditions the concentration of the active aziridinium species (AF64A) was 4.5 _+ 0.2 mM as determined by thiosulfate titration 9 and by 1H-NMR analysis (Waysbort, Pittel, Fisher and Heldman, manuscript in preparation).

centage of the total [3H]DA incorporated for each plate.

Morphological evaluation

The cultures were rinsed in Earle's balanced salt solution (EBSS) containing 6 g/1 glucose and incubated at 37 °C for 2 h with various concentrations of AF64A dissolved in EBSS. Then, the toxin-containing medium was removed and replaced with fresh growth medium.

For [3H]DA autoradiography, cultures were treated as was described for the uptake measurement and then fixed for 30-40 min in 2.5% glutaraldehyde at 4 °C. The fixed cells were rinsed, dehydrated with increasing concentrations of ethanol, air dried, and mounted (face up) with permount. Coating was done using 3-fold diluted Ilford L4 emulsion. Exposure time was two weeks. Cells were stained for acetylcholinesterase (ACHE) according to Karnovsky and Roots 12.

Biochemical procedures

RESULTS

Treatment with AF64A

Choline transport was measured according to Yavin28. Briefly, plated cells were incubated with 0.5 ~M [3H]choline ([3H-methyl]choline chloride 1/~Ci/ plate) for 10 min at 37 °C in a CO 2 incubator. Parallel incubations were carried out on ice for an equivalent period (for blanks). Following the incubation period, the plates were placed on ice, rinsed three times with EBSS containing 1 mM choline and precipitated in 1 ml 5% trichloroacetic acid. Aliquots of the supernatant were then taken for radioactivity measurement. Protein content was determined 16 after solubilizing the pellet in 1 M NaOH. Under these conditions choline uptake was inhibited by 50% with 25/~M hemicholinium-3. For 3,4-[8-3H(N)]dopamine ([3H]DA) uptake 21 and [3H]DA release 5, cultures were preincubated with EBSS containing 44 nM [3H]DA (1 ~Ci/ml) at 37 °C for 45 min. For the uptake measurements, nonspecific uptake was determined in the presence of 3 x 10-5 M dopamine. For release studies, the cultures were reincubated (after 3 washes) with EBSS for 30 min, washed and stimulated with 50 mM K ÷ (substituted for an equivalent amount of Na ÷ in EBSS) for an additional 15 min. The released radioactivity in the medium was counted. The cells were then precipitated in 5% trichloroacetic acid and radioactivity was counted in the supernatant to determine the cellular content of [3H]DA. The precipitate was dissolved in 1 M NaOH and protein content was measured. Under these conditions at least 75% of the tritium released by potassium was associated with [3H]DAS. [3H]DA release was calculated as a per-

Previous studies 11'2° indicated that AF64A exerts its cholinotoxic action by interacting with the choline transport system. Therefore, it was necessary to ascertain that this parameter was fully expressed in the tested cultures. Since whole brain cultures include cells from regions such as midbrain and septum, we thought that the ontogenesis of the choline transport system in whole brain culture may closely represent the situation in cultures from more discrete regions such as those mentioned above. Fig. 2 shows the ontogenesis of the choline transport in primary whole brain cultures starting at the time of cell plating. As can be seen, marked elevation in choline transport

7

5 4

0

2

4

6

8 tO t2 t4 t6 t8 20 Days in culture Fig. 2. Ontogenesis of choline transport in primary whole brain culture. Choline transport was determined at various times after plating by incubating the cells with [3H]cholineas described in the method section. Each point represents the mean of 3 plates + S.E.M. (horizontal bars).

301 occurred at day 8 after plating and peaked at day 11. These results are consistent with the findings that neuronal cells from whole hemispheres 28'29, midbrain 3 and septum 1°, differentiate during the 2nd week in culture. Therefore, we exposed the cells to A F 6 4 A between days 11 and 14, a time period at which the toxin might interact with the maximum number of choline transport sites. In order to study morphological aspects of A F 6 4 A induced cholinotoxicity, we exposed septal cultures to A F 6 4 A and used A C h E staining to detect cholinergic neurons. In this type of culture it was shown that all cells which are stained for A C h E also show C h A T immunoreactivity 1° and therefore can be considered as cholinergic neurons. Fig. 3 shows ACHEstained neurons in septal cultures treated with 2.25 p M AF64A. Stained neurons, varying in size (10-40 #m), were visible in untreated (Fig. 3a) as well as in treated cultures (Fig. 3b). However, the number of intact AChE-stained neurons in the treated cultures was markedly reduced (Table I). As can be seen the reduction in the number of AChE-stained cells was dose-dependent. Careful examination of the damaged stained neurons showed that while many somata were intact, their processes were degenerated, particularly at the distal part of their axons (Fig. 3b). Thus it seems that the primary target of the toxin at the cholinergic neuron is the terminals of its processes. It was important to determine if this type of damage is unique to cholinergic neurons or can also be inflicted to non-cholinergic cells. In order to study possible non-cholinergic neurotoxicity of AF64A, we tested its cytotoxic effect on dopaminergic neurons. For this purpose, we used midbrain cultures in which dopaminergic neurons are well differentiated and are clearly visualized by [3H]DA autoradiography 1'3. Autoradiography of cells that accumulated [3H]DA is shown in Fig. 4. Cells that took up and accumulated [3H]DA were specifically labeled in the perikarya (Figs. 4a,b) and in the processes (Fig. 4c). At concentrations below 22.5 ~ M A F 6 4 A , no significant changes in the number of dopaminergic neurons as well as in [3H]DA uptake and release were observed (Table II). However, at a higher concentration (45 /~M AF64A), there were significant reductions in the number of dopaminergic neurons and in [3H]DA uptake and release (Table II). These reductions were

Fig. 3. AChE-stained cells in septal cultures treated with AF64A. Cultures, 13 days in vitro, were exposed to AF64A for 2 h, washed, and then returned to normal growth medium. Fixation and staining was 24 h after exposure of the cultures to the toxin, a: control, b: 2.25 #M AF64A. c: 2.25 ~tM AF64A in presence of 500 ~tM choline. Bar = 50 ~tm.

correlated with a decrease in the protein content. Nevertheless, intact [3H]DA-labeled cells were still present even in cultures exposed to 45 ktM A F 6 4 A (Figs. 4d,e) without apparent changes in the morphology of their processes (Fig. 4f).

302 TABLE I Quantitative analys& o f cholinergic parameters in whole brain and septal cultures treated with AF64A

Cultures were exposed to increasing concentrations of AF64A for 2 h, washed, and returned to growth medium. Determinations of protein content and choline uptake, and staining for AChE were performed 24 h after the toxin treatment. The number of ACHEstained cells was determined by counting stained cells with intact somata and processes on an area covering one third of each plate. Concentration o f

Whole brain cultures

Septal cultures

AF64A (¢tM)

Protein content" (mg/plate)

Choline uptake a (cpm/plate)

Protein contentc (rag~plate)

Number o f A ChE-stained neurons b (cells~field)

0 2.25 4.50 9.00 22.50 45.00

0.82 ± 0.01 0.75 + 0.04 0.22 ± 0.08*

9538 ± 741 8269 ± 512 3077 ± 731"

0.63 + 0.05 0.43 + 0.06 0.17 ± 0.03*

20.3±4.1 10.7±2.6 10.3±4.4 5.0±1.2" 0.0±0.0" 0.3±0.3*

45.00 (in presence of 500pM choline)

0.67 ± 0.02

9008 + 298

0.45 ± 0.03*

10.3±1.4

Values represent means + S.E.M. from a2; b3; and c4 replicates. * Values found to be significantly different (P < 0.05) from control (t-test).

Comparison between the cholinergic and the dopaminergic toxicity of A F 6 4 A shows that at low concentrations of the toxin, only the cholinergic toxicity is expressed. Thus, at a concentration of 9 ktM, the number of AChE-stained cells was reduced significantly (Table I) while the number of dopaminergic neurons was not altered (Table II). Only at a concentration of 22.5 ~M A F 6 4 A some reduction in the number of the dopaminergic neurons started to appear (Table II). When the changes in [3H]choline and [3H]DA uptake were compared, it also appeared that the inhibition in choline uptake exceeded the inhibition in [3H]DA uptake. Thus, at 22.5/~M A F 6 4 A , we observed 13% inhibition in [3H]choline uptake and no inhibition in [3H]DA uptake, and at 45 ,uM A F 6 4 A we observed 68% inhibition in choline uptake and only 39% inhibition in [3H]DA uptake. The reduction in the dopaminergic indices may indicate general toxicity. Such reductions started to appear at 22.5/~M AF64A. Fig. 5 demonstrates the general appearance of whole brain and septal cultures before and after treatment with AF64A. As can be seen, the monolayer was not severely damaged even at a concentration of 22.5 ktM A F 6 4 A (Fig. 5b,f) and only at 45 ktM A F 6 4 A , significant damage to the monolayer is apparent (Fig. 5c,g). Thus, significant general toxicity is inflicted at 45 ~ M AF64A. A quantitative measure to the general cytotoxicity was the protein content of the cultures. Significant reductions

in the protein content of whole brain and septal cultures (Table I) as well as of midbrain cultures (Table II) appeared only at 45 p M AF64A. It appears that not only the cholinotoxicity but also the general toxicity is associated with the choline-like structure of AF64A. Thus, 500 p M choline significantly reduced all damages, cholinergic and noncholinergic, which are caused by A F 6 4 A (Figs. 3c and 5d,h and Tables I and II). DISCUSSION In previous studies 11,2° we observed that A F 6 4 A is capable to induce reversible and irreversible inhibition of the H A C h T in isolated synaptosomes and suggested that each of these inhibitions may be related to a selective or a non-selective cytotoxicity induced by this toxin in vivo 2°. In the present work we attempted to determine whether A F 6 4 A can indeed induce selective cholinotoxicity at low toxin concentrations and general toxicity at higher toxin concentrations and to find a mechanism explaining this dual action of AF64A. For this purpose, we used primary cell cultures that are characterized by their cellular heterogeneity and exposed them to various concentrations of AF64A. Our results showed that after treatment with concentrations up to 9 ~ M A F 6 4 A , cholinergic neurons were damaged while general toxicity was not observed. Thus, it seems that within

303 an a p p r o p r i a t e c o n c e n t r a t i o n r a n g e , A F 6 4 A acts as a

viving A C h E - s t a i n e d n e u r o n s a p p e a r e d with intact

selective c h o l i n o t o x i n . T h e c h o l i n o t o x i c i t y was ex-

s o m a t a but d a m a g e d processes. This suggests that

pressed by a specific r e d u c t i o n in the n u m b e r of

the p r i m a r y site of A F 6 4 A c h o l i n o t o x i c i t y was at the

A C h E - s t a i n e d cells, which in septal cultures h a v e

n e r v e t e r m i n a l s and that r e t r o g r a d e d e g e n e r a t i o n ,

b e e n d e m o n s t r a t e d as c h o l i n e r g i c 1°. M a n y of the sur-

which results in a p r o g r e s s i v e lesion of the c h o l i n e r g i c

Fig. 4. [3H]DA autoradiography of primary midbrain cultures treated with AF64A. Cultures, 14 days in vitro, were exposed to the toxin for 2 h and returned to normal growth medium. Twenty-four h after treatment with the toxin, [3H]DA was added to the cells for 45 min. The cultures were washed, and autoradiography was performed as described in the method section, a-c: control neurons and processes, d-f: cultures exposed to 45¢tM AF64A. Bar = 25Bm.

304 TABLE II

Quantitative analysis of dopaminergic parameters in midbrain cultures treated with AF64A Cultures were treated with AF64A as described in Table I. Twenty-four h after the exposure, the cells were incubated with [3H]DA for uptake and release measurements and for [3H]DA autoradiography. The number of labeled cells was counted on an area covering 1/10 of each plate.

Concentration of AF64A (/~M)

Protein content (mg/plate)

Number of labeled neurons (cells~field)

[3H]DA uptake (cpm/plate)

K+-induced [3H]DA release (% released)

0 4.50 11.25 22.50 45.00

0.43 --- 0.02e 0.43 + 0.02c 0.39 + 0.03e 0.25 + 0.01c'*

24.4 + 3 . 5 d 21.0 + 2.0 16.5 + 3.2c 5.2 + 3.0c'*

10,299 + 11,616 + 10,758 + 6,304 +

36.4 --- 0 . 4 b 38.3 + 1.9b 32.1 + 0.7b 18.9 + 0.6a'*

45,00 (in presence of 500/xM choline)

0.37 + 0.02c

-

389e 434c 1902e 752c'*

9,157 _+1119c

37.9 -I- 0 . 8 b

Values represent means + S.E.M. from a2; b 3 ; c4; d5 and e6 replicates. * Values found to be significantly different (P < 0.05) from control (t-test).

neurons, follows afterward. A t higher concentrations of A F 6 4 A (>22.5 p M ) , we observed general toxicity which was expressed by a decrease in the n u m b e r of dopaminergic n e u r o n s and in their ability to take up and release [3H]DA. In addition, the general toxicity was also expressed by damage to the monolayer paralleled by a decrease in the protein content. The irreversible inhibition of the choline uptake, as detected 24 h after the removal of the toxin from the cells, indicates a reduction in the n u m b e r of cells which are engaged in choline transport or alternatively in the n u m b e r of choline transport sites per cell. The inhibition in choline transport was small when the toxin concentrations were below those that caused general cytotoxicity (regardless whether the m e a s u r e m e n t was performed with whole brain cultures or with septal cultures). However, after treatment with these low toxin concentrations (9 p M ) about 5 0 - 7 5 % of the A C h E - s t a i n e d cells disappeared. Therefore, we concluded that not all the cells that transport choline are stained for ACHE. Hence, choline transport does not seem to be a reliable marker for cholinergic neurons, and instead, A C h E histochemistry seems to be a better parameter for the detection of cholinotoxicity in primary cultures. The reduction in the n u m b e r of A C h E - s t a i n e d cells may originate either from inhibition of A C h E activity or from a destruction of these specific cells by the cholinotoxin. The fact that the reduction in the n u m b e r of A C h E - s t a i n e d cells was persistent up to 72

h post exposure (data are not shown), conditions under which de novo synthesis of the enzyme should have occurred 27, suggests that the reduction in the n u m b e r of stained cells was due to cell destruction rather than inhibition of A C h E activity. This interpretation is also in accordance with the expectation that A F 6 4 A (which is a choline analog) will not interact with A C h E that uses acetylcholine rather than choline as a substrate. Indeed, enzymes which use choline as a substrate were shown to be more sensitive to A F 6 4 A than A C h E 25, which uses A C h as the substrate. The dual action of A F 6 4 A , namely, the induction of selective toxicity at low concentrations and general toxicity at high concentrations, is consistent with a model which was previously described by us z°. According to this model, we expect that selective cholinotoxicity would occur at toxin concentrations that produce reversible inhibition, since at these concentrations the toxin is transported via the H A C h T , which is confined mainly to the terminals of cholinergic neurons a4. O n the other hand, at higher concentrations, A F 6 4 A inhibits irreversibly the H A C h T and directs further transport via the low-affinity choline carrier (present in many types of cells) thus causing general toxicity. Our results are also consistent with studies performed with whole animals. For example, Millar et al. 19 showed that A F 6 4 A caused selective cholinotoxicity when injected intravitreally into the chick eye. Millar et al. ~9 estimated the extra-

305

Fig. 5. Phase contrast micrographs of septal and whole brain cultures 24 h after treatment with AF64A. Cultures, 12 days in vitro, were exposed to the toxin for 2 h, washed and returned to normal growth medium. Septal cultures before (a) and after treatment with 22.5 ~tM AF64A (b), 45 pM AF64A (c), and 45 BM AF64A in presence of 500 pM choline (d). Whole brain cultures before (e) and after treatment with 22.5 BM AF64A (f), 45/zM AF64A (g) and 45 pM AF64A in presence of 500 #M choline (h). Bar = 100 pro.

306 cellular concentration of the intravitreally injected toxin to be 12 ~tM, which is within the same concentration range that caused selective cholinotoxicity in our study. M o r e o v e r , when A F 6 4 A was injected into the nucleus basalis of M y n e r t 13 it caused selective cholinergic damage at concentrations (calculated) of 10-20 ktM and general toxicity at concentrations of 50-100/~M 13'18. O u r calculation of the toxin concentration in the experiments by Kozlowski et a1.13 did not take into consideration diffusion rate and volume of distribution but we rather considered the concentration which was f o r m e d i m m e d i a t e l y at the site of the injection. Thus, the estimated concentration should be r e g a r d e d as maximal. The correlation between these maximal concentrations and those used in the present study is striking. O u r results are also in agreement with the work of Davies et al. 6, who studied the cholinergic neurotoxicity induced by A F 6 4 A in chick e m b r y o neuronal cultures. They showed that selective cholinergic damage was found at 10 ~ M

A F 6 4 A , while non-selective damage a p p e a r e d at 100 ktM. T a k e n together these studies and ours, it m a y be concluded that p r i m a r y cultures and n o r m a l tissues in situ show similar sensitivities to A F 6 4 A . Similar sensitivities of primary brain culture and brain tissue in situ were shown by us recently also for a n o t h e r selective toxin, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) 1. In contrast to p r i m a r y cell culture, the cholinergic cell line NG108-15 showed relatively low sensitivity to A F 6 4 A . In this cell line, cellular damage started to show up at a toxin concentration around 100 ktM25. Thus, p r i m a r y cell cultures seem to represent better than cell lines the in vivo situation.

REFERENCES

(1987) 181-186. 8 Fisher, A. and Hanin, I., Potential animal models for senile dementia of Alzheimer's type with emphasis on AF64A-induced cholinotoxicity, Annu. Rev. Pharmacol. Toxicol., 26 (1986) 161-181. 9 Golumbic, C., Fruton, J.S. and Bergmann, M., Chemical reactions of the nitrogen mustard gases. 1. The transformation of methyl-bis (fl-chloroethylamine) in water, J. Org. Chem., 11 (1946) 518-535. 10 Hefti, E., Hartikka, J., Eckenstein, F., Gnahn, H., Heumann, R. and Schwab, M., Nerve growth factor increases choline acetyltransferase but not survival or fiber growth of cultured septal cholinergic neurons, Neuroscience, 14 (1985) 55-68. 11 Heldman, E., Amir, A., Pittel, Z., Shahar, A. and Fisher, A., AF64A-induced cholinotoxicity in rat brain synaptosomes and primary cell cultures. In M.Y. Dowdall (Ed.), Cellular and Molecular Basis of Cholinergie Functions, Ellis, Horwood, Chichester, 1987, pp. 618-626. 12 Karnovsky, M.J. and Roots, L., A direct coloring thiocholine method for cholinesterase, J. Histochem. Cytochem., 12 (1964) 219-22l. 13 Kozlowski, M.R. and Arbogast, R.E., Specific toxic effects of ethylcholine nitrogen mustard on cholinergic neurons of the nucleus basalis of Meynert, Brain Research, 372 (1986) 45-54. 14 Kuhar, M.J., Sethy, V.H., Roth, R.H. and Aghajanian, G.K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1975) 581-593. 15 Levy, A., Kant, J.G., Meyerhoff, J.L. and Jarrard, L.E., Non-cholinergic neurotoxic effects of AF64A in the substantia nigra, Brain Research, 305 (1984) 169-172. 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.

1 Amir, A., Shahar, A., Pittel, Z., Korczyn, A.D., Fisher, A. and Heldman, E., Selective lesion of neurons in primary cell cultures by MPTP and ethylcholine aziridinium ion (AF64A). In A. Shahar and A. Goldberg (Eds.), Model Systems in Neurotoxicology: Alternative Approaches to Animal Testing, Progress in Clinical and Biological Research,

Liss, New York, 1987, pp. 239-253. 2 Barlow, P. and Marchbanks, R.M., Effect of ethylcholine mustard on choline dehydrogenase alJd other enzymes of choline metabolism, J. Neurochem., 43 (1984) 1568-1573. 3 Berger, B., Di Porzio, U., Daguet, M.C., Gay, M., Vigny, A., Glowinsky, J. and Prochiantz, A., Long term development of mesencephalic dopaminergic neurons of mouse embryos in dissociated primary cultures: morphological and histochemical characteristics, Neuroscience, 7 (1982) 193-205. 4 Colhoun, E.H., Myles, L.A. and Rylett, R.J., An attempt to produce cholinergic hypofunction in rat brain using choline mustard aziridinium ion: neurochemical and histological parameters, Can. J. Neurol. Sci., 13 (1986) 517-520. 5 Daguet, M.C., DiPorzio, U., Prochiantz, A., Kato, A. and Glowinski, J., Release of dopamine from dissociated mesencephalic dopaminergic neurons in primary culture in absence or presence of striatal target cells, Brain Research, 191 (1980) 564-568. 6 Davies, D.L., Sakellarides, N., Valcana, T. and Vernadakis, A., Cholinergic neurotoxicity induced by ethylcholine aziridinium (AF64A) in neuron-enriched cultures, Brain Research, 378 (1986) 251-261. 7 Eva, C., Fabrazzo, M. and Costa, E., Changes of cholinergic, noradrenergic and serotonergic synaptic transmission indices elicited by ethylcholine aziridinium ion (AF64A) infused intraventricularly, J. Pharmacol. Exp. Ther., 241

ACKNOWLEDGEMENTS W e wish to thank Mr. M. Peled for his help in taking the p h o t o m i c r o g r a p h s , and Mrs. D. Wallach for the secretarial work.

307 17 Mantione, C.R., Zigmond, M.J., Fisher, A. and Hanin, I., Selective presynaptic cholinergic neurotoxicity following intrahippocampal AF64A injection in rats, J. Neurochem., 41 (1983) 251-255. 18 McGurck, S.R. and Butcher, L.R., Cholinergic neuropathology following intracerebral infusion of ethylcholine mustard aziridinium (AF64A), Abstr. Am. Soc. Neurosci., 11 (1985) 1240. 19 Millar, T.J., Ishimoto, I., Boelen, M., Epstein, M.L., Johnson, C.D. and Morgan, I.G., The toxic effects of ethylcholine mustard aziridinium ion on cholinergic cells in the chicken retina, J. Neurosci., 7 (1987) 343-356. 20 Pittel, Z., Fisher, A. and Heldman, E., Reversible and irreversible inhibition of high-affinity choline transport caused by ethylcholine aziridinium ion, J. Neurochem., 49 (1987) 468-474. 21 Prochiantz, A., DiPorzio, V., Kato, A., Berger, B. and Glowinski, J., In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5387-5391. 22 Rylett, B.J. and Colhoun, E.H., The interactions of choline mustard aziridinium ion with choline acetyltransferase (EC 2.3.1.6), J. Neurochem., 32 (1979) 553-558. 23 Rylett, B.J. and Colhoun, E.H., Kinetic data on the inhibition of high-affinity choline transport into rat forebrain syn-

24

25

26

27

28

29

aptosomes by choline like compounds and nitrogen mustard analogues, J. Neurochem., 34 (1980) 713-719. Sandberg, K., Hanin, I., Fisher, A. and Coyle, J.T., Selective cholinergic neurotoxins: AF64A effects in rat striatum, Brain Research, 293 (1984) 49-55. Sandberg, K., Schnaar, R.L., McKinney, M., Fisher, A. and Coyle, J.T., AF64A: an active site directed irreversible inhibitor of choline acetyltransferase, J. Neurochem., 44 (1985) 439-445. Walsh, T.J., Tilson, H.A., DeHaven, D.L., Mailman, R.B., Fisher, A. and Hanin, I., AF64A, a cholinergic neurotoxin, selectively depletes acetylcholine in hippocampus and cortex, and produces long-term passive avoidance and radial-arm maze deficits in the rat, Brain Research, 321 (1984) 91-102. Walker, K.B. and Wilson, B.W., Regulations of acetylcholinesterase in cultured chick embryo spinal cord neurons, FEBS Lett., 93 (1978) 81-85. Yavin, E., Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Ontogenesis of carrier-specific transport of choline and N-methyl-substituted choline analogs, J. Neurochem., 34 (1980) 178-183. Yavin, Z. and Yavin, E., Synaptogenesis and myelogenesis in dissociated cerebral cells from rat embryo on polylysine coated surfaces, Exp. Brain Res., 29 (1977) 137-147.