Cholinergic but not GABAergic neuronal markers are decreased in primary neuronal cultures treated with choline mustard

Brain Research, 519 (1990) 209-216

209

Elsevier BRES 15580

Cholinergic but not GABAergic neuronal markers are decreased in primary neuronal cultures treated with choline mustard J.C. Baskey 1, E.H.

C o l h o u n 1 a n d R . J . R y l e t t 1'2

Departments of 1Pharmacology and 2physiology, University of Western Ontario, London, Ont. (Canada) (Accepted 5 December 1989)

Key words: Choline mustard; Neuronal culture; Acetylcholine; Choline transport; y-Aminobutyric acid; Choline acetyltransferase

Nitrogen mustard analogues of choline are potent irreversible inhibitors of high-affinity choline transport at the cholinergic presynaptic nerve terminal in vitro. Ethylcholine mustard aziridinium ion, and to a lesser extent choline mustard aziridinium ion, have been used as tools to chemically lesion cholinergic neurons in the central nervous system. The selectivity of these compounds as neurotoxins for cholinergic neurons in vivo has been questioned and the mechanism by which they mediate neuronal death has not been elucidated. The objective of the present study was to investigate the selectivity of choline mustard aziridinium ion on embryonic rat brain neurons maintained in primary culture, The effect of choline mustard aziridinium ion on levels of chohnergic neuronal markers was compared with markers for GABAergic neurons as a measure of neuronal specificity. Choline mustard aziridinium ion at 10 and 30 gM irreversibly inhibited hemicholinium-sensitive, high-affinity choline transport into the cultured neurons without altering sodium-dependent, high-affinity GABA transport. Similarly, incubation of the neurons for 30 min in the presence of 10 gM choline mustard aziridinium ion led to a decrease in choline acetyltransferase activity of the cultures which was maintained for 72 h; glutamic acid decarboxylase activity was not altered under the same experimental conditions. Protein and DNA content and DNA-to-protein ratios of the choline mustard aziridinium ion-treated cultures were monitored as indicators of generalized cellular damage. Neither protein nor DNA content of the neuronal cultures were changed up to 72 h after a 30 rain incubation in the presence of 10 gM choline mustard aziridinium ion indicating that cell numbers and the extent of neuronal processes were not altered. In summary, it appears that low concentrations of choline mustard aziridinium ion mediated selectivity of action for cholinergic neurons relative to GABAergic neurons maintained in vitro. INTRODUCTION Cholinergic neurons in forebrain play i m p o r t a n t roles in cognitive processes including learning and m e m o r y , and d e g e n e r a t i o n of these neurons is a consistent finding in a g e - r e l a t e d m e m o r y disorders such as A l z h e i m e r ' s disease 6. W h i l e the etiological basis of this n e u r o d e g e n erative process remains to be d e t e r m i n e d , attempts have been m a d e to p r o d u c e animal models which mimic some of the neurochemical changes occurring in A l z h e i m e r ' s disease. O n e potentially useful m o d e l which has received attention recently uses nitrogen mustard analogues of choline, ethylcholine m u s t a r d aziridinium ion 16 ( E C M A z or A F 6 4 A ) o r choline m u s t a r d aziridinium ion 12 ( C h M A z ) , as toxins to induce selective d e g e n e r a t i o n of cholinergic neurons in rat brain. C h M A z and E C M A z are structural analogues which a p p e a r to share c o m m o n sites and mechanisms of action 39. B o t h c o m p o u n d s irreversibly inhibit choline t r a n s p o r t into brain s y n a p t o s o m e s 13'35,39, and both comp o u n d s inhibit C h A T in rat brain h o m o g e n a t e s 4'38 or following partial purification 42,45. It was o b s e r v e d that

C h M A z d e c r e a s e d s y n a p t o s o m a l C h A T activity following incubation of nerve terminals with the analogue 4°, and m o r e recently it was d e m o n s t r a t e d that a m e m b r a n e b o u n d form of C h A T was most sensitive to inhibition 37. In synaptosomes, C h M A z a p p e a r s to selectively inhibit choline t r a n s p o r t without altering y-aminobutyric acid ( G A B A ) , serotonin or n o r a d r e n a l i n e u p t a k e 36, and E C M A z decreases choline u p t a k e without blocking serotonin transport 3°. Studies designed to evaluate w h e t h e r C h M A z serves as a t r a n s p o r t a b l e substrate for the high-affinity choline carrier thus being accumulated into cholinergic nerve terminals r e v e a l e d that [3H]ChM A z was taken up into rat brain s y n a p t o s o m e s at only a fraction of the rate of [3H]choline t r a n s p o r t and that it rapidly limited its own accumulation, p r e s u m a b l y through inactivation of the carriers 43. T h e actions of C h M A z and E C M A z when injected into rat brain in vivo are less well characterized. A n u m b e r of studies have concluded that E C M A z exerts selective actions on cholinergic neurons following injection into lateral ventricle 29 or directly into brain areas such as h i p p o c a m p u s 3° or striatum 44. H o w e v e r , several

Correspondence: R.J. Rylett, Department of Physiology, Medical Sciences Building, University of Western Ontario, London, Ont., Canada N6A 5C1. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

210 reports have demonstrated that injection of ECM Az into n u m e r o u s C N S l o c a t i o n s r e s u l t e d in n o n - s p e c i f i c , n o n c h o l i n e r g i c t i s s u e damage22'26"32"5°; s i m i l a r n o n - s p e c i f i c effects have been noted following intracerebral administration

of

ChM

A z 12. T h e

source

of

the

apparent

n o n - s e l e c t i v e e f f e c t s of t h e n i t r o g e n m u s t a r d a n a l o g u e s of c h o l i n e in v i v o a r e u n k n o w n

a n d t h e m e c h a n i s m s by

which these compounds mediate destruction of cholinergic n e u r o n s h a v e n o t b e e n d e t e r m i n e d . T h e o b j e c t i v e o f t h e p r e s e n t s t u d y w a s to d e t e r m i n e the selectivity of ChM Az for cholinergic neurons using rat b r a i n n e u r o n s m a i n t a i n e d in p r i m a r y c u l t u r e . S h o r t a n d l o n g - t e r m effects o f this a n a l o g u e w e r e e v a l u a t e d by measuring markers for cholinergic and GABAergic neur o n s in a d d i t i o n to t h e g e n e r a l c e l l u l a r m a r k e r s p r o t e i n and DNA.

It h a s b e e n

demonstrated

that ECM

Az

d e c r e a s e s m a r k e r s f o r c h o l i n e r g i c n e u r o n s in b r a i n cell c u l t u r e s 1'3A4"23"24 o r n e u r o b l a s t o m a 45, b u t little is k n o w n a b o u t t h e s e l e c t i v i t y o f t h e c h o l i n e a n a l o g u e s in this context. MATERIALS AND METHODS

Materials Butyl ethyl ketone (3-heptanone) was purchased from Aldrich Chemical Co., Milwaukee, WI; [N-methyl-3H]choline chloride (80 Ci/mmol) and [4-amino-(2,3-3H)]butyric acid (105 Ci/mmol) were obtained from Amersham Radiochemical Corp., Oakville, Ont.; acetylCoenzyme A, lithium salt was from Boehringer Mannheim, Dorval, Que.; Nitex mesh was obtained from B & SH Thompson Co., Scarborough, Ont.; Dulbecco's Modified Eagle medium, Puck's Saline A (PSA) solution, Hank's Balanced Salt solution, fetal bovine serum and horse serum were obtained from Gibco Laboratories, Burlington, Ont.; [acetyl-l-14C]Coenzyme A (49 mCi/mmol) was purchased from ICN Radiochemicals, Montreal, Que.; L[1-14C]glutamic acid (57.9 mCi/mmol) was obtained from New England Nuclear (Dupont Canada), Mississauga, Ont.; tissue culture dishes and flasks were from Nunclon Delta S1, Burlington, Ont.; crystalline bovine serum albumin, GABA, aminooxyacetic acid, choline iodide, deoxyribonucleic acid (type 1, calf thymus, sodium salt), eserine sulphate, fl-estradiol, hemicholinium-3, insulin from bovine pancreas, poly-D-lysine hydrobromide, progesterone, putrescine, pyridoxyl-5"-phosphate, sodium selenite, sodium tetraphenylboron and human transferrin were purchased from Sigma Chemical Co., St. Louis, MO. All other salts and reagents were obtained at the highest purity possible from standard sources. Acetylcholine mustard (AChM) was synthesized according to the procedure of Jackson and Hirst 2~ with a purity of greater than 97% measured by gas chromatography. The free base was dissolved in distilled water and stirred at room temperature for 50 min to generate the aziridinium ion (Az) species. ChM Az was obtained by alkaline hydrolysis of AChM Az (pH ll.0 for 20 rain). Concentrations of the Az ion in solution were determined by the iodinethiosulphate titration method of Golumbic et al. 18 and drug concentrations reported have been corrected to represent 100% Az. The ChM Az solution was prepared fresh daily then diluted to the desired concentration in Krebs-Ringer solution (mM: NaCl, 124; KCI, 5.0; Tris-PO a, 20; MgSO 4, 1.3; CaCI 2, 1.5; glucose, 10 at pH 7.4).

Cultures Astrocytes. Primary cultures of astrocytes were prepared from neonatal rat cerebral cortex by the method of Hamprecht and

Loffier 1'~.Cerebral hemispheres were removed from 10-15 newborn pups less than 24 h old and immersed in PSA. Meninges were dissected and tissue was washed twice in PSA then minced into 1-2 mm 3 pieces. Tissue was dissociated mechanically in l0 ml PSA using a Pasteur pipette, then filtered sequentially through beakers covered with Nitex mesh of pore size 233/tm and 132/~m. The cell suspension was concentrated by centrifugation (200 g for 10 min) and the pellet was resuspended in l0 ml Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 30 mM glucose, penicillin (100 U/ml), streptomycin (100 ~g/ml) and amphotericin B (0.25 gtg/ml). Cells were seeded into 175 cm 2 Nunc flasks at a density of 26 x 10 6 cells/flask (44 ml of 6 x 105 cell/ml) and maintained at 37 °C in a humidified atmosphere of 95% air/5% CO 2. The medium was renewed every 3-4 days for the duration of the culture, except during the time when serum-free astrocyte conditioned medium was being prepared. Preparation of astrocyte-conditioned medium. Astroglial cultures reached confluency by day 14 in culture and could then be used for the preparation of serum-free, astrocyte-conditioned medium (SFACM) 27. The medium was aspirated and cells were washed with 10 ml serum-free DMEM containing glucose and antibiotics as above. Culture was continued in this medium (45 ml/flask) for 2 days. This medium, the SFACM, was collected and fresh serum-free DMEM was added to the cells for an additional two day period. The resulting medium was collected, pooled with the first collection, and the cells were returned to serum-containing DMEM for 5 days. This process was repeated twice. The SFACM was pooled, sterilized by filtration through Nalgene 500 ml filter flasks (pore diameter 0.2 am), then aliquoted into 500 ml bottles and refrigerated until use, usually about two weeks. Prior to use for the neuronal cultures, N2 supplements plus fl-estradiol were added to the SFACM according to Bottenstein and Satog: fl-estradiol (l pM), insulin (5 ~g/ml), progesterone (20 nM), putrescine (100 ~tM), sodium selenite (30 nM) and transferrin (100 #g/ml). Neurons. Primary cultures of rat forebrain neurons were prepared using a combination of the methods of Yavin and Yavin 52, Yu and Hertz s3, Hertz et al. 2° and Loftier et al. 27. Embryos were removed aseptically from pregnant female Sprague-Dawley rats at midgestation, days 15-17. Forebrains were removed and immersed in PSA. The meninges were removed and tissue was washed twice in PSA then minced into 1-2 mm 2 pieces. The pieces were dissociated mechanically by trituration with a fire-polished pasteur pipette into 10 ml PSA, then filtered sequentially through Swinny adapters fitted with Nitex mesh of pore diameter 70 #m and 30/tm. The cell suspensions were concentrated by centrifugation (200 g for l0 min), then the pellet was washed and resuspended in DMEM containing 10% (v/v) horse serum, 30 mM glucose, penicillin (100 U/ml), streptomycin (100/~g/ml) and amphotericin B (0.25/~g/ml) at 8 × 105 viable cells per ml. Cells were plated at a density of 3.2 × 10 6 cells/plate in 60 mm Nunc culture dishes which had been precoated with poly-D-lysine according to the method of Sensenbrenner et al. 47. After 20 min incubation at 37 °C, unattached cells were removed with the medium by aspiration then fresh medium was added to the attached cells52. The cultures were incubated at 37 °C in a humidified atmosphere of 95% air/5% CO z. After 24 h, the medium was replaced with SFACM containing the N2 supplements. This medium was renewed at 3-4 day intervals.

Experimental protocol Cultures were washed twice with 2 ml Hank's Balanced Salt Solution (HBSS) at 37 °C, then incubated in the absence or presence of ChM Az for 30 min. Following incubation, the cells were washed twice with HBSS then processed immediately for assays as described below, or growth medium was added and plates were returned to the incubator. In the latter case, all procedures were performed under sterile conditions. Control cultures did not receive ChM Az, but an equal volume of vehicle was added and they were incubated in parallel. Plates used for the determination of CHAT, G A D or DNA

211 content were washed twice with 2 ml of Solution 1 (mM: NaCI, 137; KCI, 5.4; Na2HPO4, 0.17; KH2PO 4, 0.22; glucose, 5.5; sucrose, 59 and CaCI 2, 0.14 at pH 7.4), followed by 2 ml of Solution 2 (same as Solution 1 without CaCI2). Cells were scraped from the plate into 1 ml of Solution 2, then the plate was washed with 1 ml of Solution 2. The cell suspension was transferred to a conical centrifuge tube and centrifuged at 2000 g for 20 min. The supernatant was discarded and the cell pellet was frozen at -80 °C until assay. Plates used for membrane transport studies were equilibrated to 37 °C for 5 min in Krebs-Ringer solution. The incubation was continued for 5 min after the addition of [3H]GABA (0.5 pM, 2 Ci/mmol) or [3H]choline (0.5/~M, 4 Ci/mmol). Following incubation with the radiolabelled substrate, cells were washed twice with the preincubation solution. Cold 5% TCA (2 ml) was added to each plate, then cells were scraped from the culture surface and transferred to a conical centrifuge tube and centrifuged at 3200 rpm for 20 min. Aliquots of 200/A of the supernatants were added to 5 ml of scintillation cocktail (Beckman Ready Protein Plus) and tritium was quantitated by liquid scintillation spectrometry. The pellets were digested in 200 /tl of 0.5 M NaOH for protein quantitation according to the method of Lowry et al. 28 using bovine serum albumin as a standard. For G A B A transport, all solutions contained the G A B A transaminase inhibitor aminooxyacetic acid (10/~M) to block the degradation of [3H]GABA; some samples were incubated in a modified sodium-free Krebs-Ringer solution and high-affinity G A B A uptake was calculated by subtracting tritium accumulated in the absence of sodium from that taken up in the presence of sodium. For choline transport, some samples were incubated in the presence of the specific inhibitor hemicholinium-3 (10 pM) and high-affinity choline uptake was calculated by subtracting tritium accumulated in the presence of hemicholinium-3 from that taken up in the absence of this inhibitor.

and incubated at 60 °C for 15 min. Following centrifugation, DNA was extracted from the pellet at 75 °C in 1 M perchloric acid for 30 min. Samples were centrifuged and 100 #1 aliquots of the supernatant was added to 100 pl of 1.32 M diaminobenzoic acid hydrochloride and incubated at 60 °C for 30 min. This reaction mixture was diluted to a final volume of 2.5 ml with 0.6 M perchloric acid, then fluorescence intensity was monitored at excitation and emission wavelengths of 420 and 520 nm, respectively. DNA content of samples was determined from a standard curve of calf thymus DNA.

Data analysis Data is expressed as mean + S.E.M. Statistical analysis was by the Student's t-test or one-way analysis of variance (ANOVA) combined with Duncan's multiple range test using P < 0.05 as the criterion for statistical significance.

RESULTS

Cell culture model E m b r y o n i c r a t f o r e b r a i n n e u r o n s g r o w n f o r 11 d a y s in SFACM

vival 15 a r e s h o w n in Fig. 1. C u l t u r e s a p p e a r e d t o c o n s i s t predominantly

was measured according to the radioenzymatic method of Chalmers et al. 1°. Frozen culture pellets were homogenized in 125 mM potassium phosphate buffer, pH 6.8, containing 2.0 mM dithiothreitol and 1.0% Triton X-100. Aliquots of this homogenate were incubated with an equal volume of radiolabelled substrate L[1-14C]glutamic acid (6 mM, 1.33 pCi/pmol) at 37 °C for 30 min in a gas-tight system. The reaction was terminated by the addition of 200/~1 1 M H2SO 4 and samples were incubated for an additional 1.5 h to ensure maximum release of 14CO2. 14CO2 was trapped onto filter paper strips presoaked in Protosol (New England Nuclear); for quantitation~of radioactivity filter paper strips were placed in scintillation vials with 10 ml Aquasol containing 0.1% glacial acetic acid. Choline acetyltransferase. ChAT activity was quantitated using modifications of the method of Fonnum 17. The frozen cell pellets were homogenized in a solution containing 154 mM NaCI, 0.5 mM eserine sulphate and 0.2% Triton X-100. Aliquots of this homogenate were incubated in the presence of (final concentrations) 25 mM sodium phosphate buffer, pH 7.5, 3.4 mM L-cysteine, 0.5 mM disodium EDTA, 0.1 mM eserine sulphate, 300 mM NaCI, 10 mM choline iodide and 200/~M [14C]acetylcoenzyme A (14 mCi/mmol) for 1 h at 37 °C. [laC]AChproduced was extracted by liquid cation exchange into an organic phase comprised of 3-heptanone/acetonitrile (5:1, v/v) containing 20 mg/ml sodium tetraphenylboron. An aliquot of the organic phase was added to 5 ml scintillation cocktail for quantitation of radioactivity. Protein content of samples for ChAT and G A D activity determination was by the method of Markwell et al. 3~ because of the presence of detergent in the samples. Deoxyribonucleic acid. DNA content of the cultured neurons was quantitated using the fluorimetric method of Setaro and Morley as. Frozen cell pellets were washed twice with ice cold 0.6 M TCA, then resuspended in potassium acetate/absolute alcohol (1 g/100 ml) and allowed to stand at room temperature for 10 min. Following centrifugation, the pellet was resuspended in 1 ml absolute alcohol

of neurons displaying extensive neurite

outgrowth, with relatively few proliferating background n o n - n e u r o n a l cells. T h i s h i g h l y - e n r i c h e d n e u r o n a l c u l t u r e s y s t e m w a s d e v e l o p e d u s i n g a c o m b i n a t i o n o f (a) differential

Enzyme and DNA assays Glutamic acid decarboxylase. G A D activity in neuronal cultures

containing N2 supplements of Bottenstein and

S a t o 9 w i t h t h e a d d i t i o n o f f l - e s t r a d i o l t o e n h a n c e sur-

plating

which

allowed

for

relatively

selective

a t t a c h m e n t o f n e u r o n s b u t n o t n o n - n e u r o n a l cells t o t h e p o l y - l y s i n e c o a t e d c u l t u r e s u r f a c e 53, ( b ) s e r u m - f r e e culture conditions which discouraged cellular division and proliferation of non-neuronal

cells 8 w i t h o u t t h e u s e o f

m i t o t i c i n h i b i t o r s , a n d (c) d e f i n e d m e d i u m s u p p l e m e n t s 9 and growth factors released into conditioned medium by

Fig. 1. Phase contrast photomicrograph of embryonic rat brain neurons in primary culture. Rat forebrains at embryonic day 15-17 were dissociated then differentially plated 53 on poly-lysine coated plastic. They were maintained in a serum-free astrocyte conditioned medium 27 supplemented with fl-estradiol, insulin, progesterone, putrescine, transferrin and sodium selenite 9'~5 to promote survival and inhibit proliferation of nonneuronal cells. The culture is at day 11 in vitro.

212 iO0

glial cells27 which enhanced the survival and differentiation of the neurons.

80

Chofinergic neuronal markers

T

o r._

ChAT specific activity was measured in cultured neurons immediately following, or 24 and 72 h after incubation of day 12 cultures for 30 min in the presence of 10/~M ChM Az, and hemicholinium-sensitive highaffinity choline transport activity was quantitated after the 30 min incubation with 10 or 30/~M ChM Az. The development of ChAT activity was followed for 15 days in the cultured neurons. As shown in Fig. 2, there was a large (2.7-fold) increase in specific activity of this enzyme between days 6 and 12 in vitro, and although there was a trend towards increased specific activity of the enzyme between days 12 and 15 this was not found to be statistically significant (overall increase 3.2-fold). Incubation of day 12 cultures for 30 min with 10/~M ChM Az decreased neuronal ChAT activity to 55% of control (P < 0.01), and this decrease was maintained for up to 72 h after drug treatment as demonstrated in Fig. 3. There was no significant difference between the percentage change in ChAT activity immediately following or 24 or 72 h after incubation with ChM Az (P > 0.05, F2,13 = 0.027, one-way ANOVA). Hemicholinium-sensitive choline uptake represented only a small proportion, about 15%, of the total choline transported into the cultured cells. The hemicholiniuminsensitive portion of the choline uptake presumably represents transport on the low-affinity carrier present in all cells25 and in neurons in primary culture 7. A complete time course for the development of choline transport in the cultured neurons was not established, but it has been shown that high-affinity choline transport reaches near maximum levels during the second week in culture 1'24"51. As shown in Fig. 3, incubation of day 12 cultures with 10 and 30/~M ChM Az resulted in a concentration-related reduction in hemicholinium-sensitive choline uptake.

~

60

ii!i!ii!iiiiiiiii!i!iii iiiii!"il.-"i!i'fiii iiii iiiiiiii!!iiff!i

(3

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iiiiiiii!i!',ii!iii!iiii ',iiiii! i Choline Uptake

Choline Acetyltraneferase

Fig. 3. Effect of ChM Az on cholinergic neuronal markers in cultured rat brain neurons. ChM Az produced a dose-dependent inhibition of hemicholinium-sensitive [3H]choline uptake. Cells were incubated for 30 rain in the presence of 10/~M ([], n = 5) or 30/.tM ( ~ , n = 5) ChM Az, then washed and hemicholiniumsensitive choline transport was measured as described in Materials and Methods. ChAT activity was decreased following 30 rain incubation (l~, n = 9) in the presence of 10/~M ChM Az, and this inhibition was maintained at 24 h (Ira, n = 4) and 72 h (r~, n = 3) after the initial incubation. Control values were 14.4 + 5.0 pmol/mg protein/10 min for high-affinity choline transport and 0.90 + 0.14 nmol/mg protein/h for specific activity of CHAT. Data represent mean + S.E.M. Statistical significance was determined by Student's t-test: *P < 0.01; **P < 0.05.

Following 30 min incubation with 10 /~M ChM Az, high-affinity choline transport was irreversibly inhibited by 27% although this did not achieve statistical difference from control (P > 0.05; t = 1.673). The higher concentration of 30/~M ChM Az irreversibly decreased choline uptake to 55 + 15.5% of control (P < 0.05).

GABAergic neuronal markers To test the specificity of action of ChM Az in the cultured neurons, the GABAergic neuronal markers sodium-dependent, high-affinity G A B A transport and G A D activity were measured. As with the cholinergic neuronal markers, effects on the neurotransmitter syn-

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Fig. 2. Ontogenic development of choline acetyltransferase activity in cultured rat brain neurons as a function of time in culture. ChAT specific activity increased 2.7-fold between days 6 and 12, with a smaller change between days 12 and 15. Data represent the mean + S.E.M. of 6 (day 6), 6 (day 12) and 4 (day 15) separate cultures.

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Days in vitro Fig. 4. Ontogenic development o f glutamic acid decarboxylase

activity in cultured rat brain neurons as a function of time in culture. G A D activity increased 1.7-fold between days 6 and 15. Data represent mean + S.E.M. of 5 (day 6), 5 (day 12) and 6 (day 15) separate cultures.

213 activity was not changed during a 72 h incubation period following the 30 min exposure to the choline analogue.

i20 ,.~ lO0

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61uLamic Acid Decarboxylase

Fig. 5. Effect of ChM Az on GABAergic neuronal markers in cultured rat brain neurons. ChM Az did not alter activity of [3H]GABA uptake into cells. Cultures were incubated with 10/~M (D, n = 13) or 30/~M (l~, n = 7) ChM Az for 30 min, then washed and sodium-dependent G A B A transport was measured. G A D activity was not different from controls following 30 min incubation (t~, n = 5) in the presence of 10/~M ChM Az, and remained unchanged at 24 h (~', n = 4) and 72 h (1~, n = 2) after the initial incubation. Control values were 320 + 60 pmol/mg protein/10 min for high-affinity G A B A transport and 3.5 + 0.7 nmol/mg protein/h. Data represent mean + S.E.M. Analysis using the Student's t-test did not reveal any data to be statistically different from controls.

thesizing enzyme were monitored immediately following or 24 and 72 h after a 30 min incubation in the presence of 10/~M ChM Az, and effects on the transport system were measured after the 30 min incubation with 10 and 30 /~M ChM Az. Evaluation of GAD activity in the cultured cells between days 6 and 15 in vitro revealed little change in specific activity of the enzyme (1.7-fold increase) indicating a relatively stable population of GABAergic neurons, as shown in Fig. 4. Treatment of day 12 cultured cells with ChM Az did not significantly alter either of the GABAergic markers compared to controls, as illustrated in Fig. 5. In addition, GAD

TABLE I

Cellular markers DNA and protein in control and ChM Az-treated primary neuronal cultures Data represent the mean _+ S.E.M. with the number of cultures per group indicated in parentheses. Cultures were incubated for 30 min in the presence of 10/tM ChM Az, then returned to the incubator in growth medium for 24 or 72 h before the measurement of D N A and protein content as described in Materials and Methods. The units for D N A are/tg/plate and for protein are rag/plate.

24 h Control ChM Az-treated 72h Control ChM Az-treated

D NA

Protein

ttg D NA/mg protein

186.2 + 31.2 235.5 + 17.4

1.85 + 0.15 2.04 + 0.37

98.9 + 9.3 (4) 94.8 + 12.7 (4)

160.4 + 15.1 150.2 + 7.7

1.94 + 0.03 1.73 + 0.10

83.0 + 8.3 (3) 87.7 + 9.5 (3)

Cellular markers Cellular markers DNA and protein were quantitated 24 and 72 h after exposure of day 12 cultures to ChM Az to detect non-specific cellular changes or damage. Neither total protein and DNA content per culture dish, nor DNA content expressed per mg of protein, were significantly different from controls following ChM Az treatment as shown in Table I. This suggests that neither cell numbers nor the extent of neuronal processes were changed quantitatively at times up to 72 h after 30 min incubation with 10 /~M ChM Az. When observed by phase contrast microscopy, ChM Az-treated cultures did not appear to differ from control cultures at times immediately following or up to 72 h after drug treatment (data not shown). DISCUSSION

The objective of the present study was to test the specificity of action of the putative cholinergic neurotoxin ChM Az for cholinergic neurons in primary cultures of embryonic rat brain neurons. For this purpose, a model system enriched in neurons with few non-neuronal cells was developed to allow an unambiguous interpretation of results since some GABAergic neuronal markers such as high-affinity GABA transport are not restricted to neurons and appear also to be a characteristic of glial cells46. It was determined that under the conditions used in the present experiments ChM Az selectively decreased markers for cholinergic neurons without altering levels of GABAergic neuronal or general cellular markers. The actions of ChM Az on cholinergic neuronal markers were compared with those for GABAergic neurons since it has been shown in our laboratory34 and others 49'5° that the choline mustard analogues may alter levels of the GABAergic neuronal marker GAD following intracerebral administration. Injection of ChM Az at doses of 2 nmol or greater into medial septal nucleus, decreased G A D activity at the site of injection and in hippocampus. Similarly, injection of 0.1 nmol ECM Az into nucleus basalis of Meynert decreased GAD activity at the site of injection by more than 50% without altering ChAT activity49 and injection of 1 nmol ECM Az into interpeduncular nucleus led to significant decreases in both ChAT and G A D activity at the site of drug administration 5°. GABAergic neurons in striatum, however, appeared to be more resistant to damage by this neurotoxin with statistically significant decreases in GAD activity occurring at doses above 8 nmo132'44. In another investigation, it was reported that retinal G A B A levels

214 were not different from control one month after intraocular injection of 50 nmol ECM Az 33. It is of interest that even though G A D activity was significantly decreased following injection of low concentrations of ECM Az into interpeduncular nucleus in vivo, uptake of [3H]GABA into nerve terminals in the region was not altered relative to controls 5°. These investigators reasoned that since glial cells exhibit high-affinity G A B A uptake but not G A D activity as found in GABAergic neurons, hyperactivity of glial cells in response to tissue damage in vivo may compensate for and possibly mask damage to GABAergic neurons when G A B A uptake was used as a marker for this class of neurons 5°. In the present study, we used neuron-enriched cultures with minimal astroglial cell content in order to test this hypothesis directly and observed that sodium-dependent, highaffinity G A B A uptake in ChM Az-treated cultures was not changed relative to controls. This could indicate that in cultured neurons ChM Az, and presumably ECM Az, do not interact with the high-affinity G A B A carrier, in agreement with previous findings with synaptosomal preparations 36'5°. It was reported recently that ECM Az in concentrations in the range used in the present study did not inhibit dopamine uptake into cultured neurons 1. One possible route by which ChM Az could inhibit G A D after intracerebral administration would be by accumulation into GABAergic neurons by the ubiquitous low-affinity choline carrier. It was shown recently 43 that [3H]ChM Az was accumulated into rat brain synaptosomes by the high-affinity choline carrier at about 10% of the rate of choline transport, with much less than this being taken up by alternative mechanisms such as low-affinity transport at low micromolar concentrations such as those used in the present study and following intracerebral administration. The hemicholinium-insensitive, low-affinity choline carrier was active in the culture model used thus offering a possible route for accumulation of ChM Az, but we observed that G A D activity was not altered in the cultured neurons by ChM Az at a concentration which decreased ChAT activity by almost 50%. This suggests that either ChM Az was not accumulated into non-cholinergic neurons by the low-affinity choline carrier or an alternate mechanism, or that it did not interact with G A D if the neurotoxin were present in GABAergic neurons. ChM Az does not inhibit G A D activity in homogenates of the rat brain at concentrations up to 5 mM (unpublished observation). Actions of choline mustard in the neuronal cultures were qualitatively similar to observations made previously with brain synaptosomes, including irreversible inactivation of high-affinity choline transport and inhibition of choline acetyltransferase in situ by micromolar concentrations of the compound 37"39-41. It is apparent,

however, that the sensitivity of high-affinity choline transport in the cultured neurons to inhibition by ChM Az was less than that observed previously in synaptosomes 39. This same phenomenon was reported recently for ECM mz 23. One possible explanation for this is that the nitrogen mustard compounds were inactivated rapidly in the culture system by interaction with components other than cholinergic neurons thereby lowering the effective concentration of the inhibitor. Indeed, it was shown recently that the half-life of ECM Az in reaggregate neuronal cultures was only 15 min a. Similar loss of choline uptake inhibitory activity was observed when ChM Az was incubated with synaptosomes at higher protein-to-medium concentrations 11. Although the halflife of ChM Az in our culture system was not measured, the incubation time for the neurons with ChM Az was kept to 30 min since prolonged incubations as used in other studies probably did not increase exposure of the neurons to the drug. ChM Az caused a selective inhibition of cholinergic neuronal markers at low micromolar concentrations. ChM Az concentrations tested in the present study were chosen because it was shown that ECM Az at concentrations of 50/~M or above mediated massive release of L D H 3 and non-specific cellular damage an and 45 ,uM ECM Az caused non-selective neuronal effects by inhibiting dopamine uptake and decreasing dopamine release 1. The concentrations used were within the range of doses which might be present in brain tissue following intracerebral administration 49,5° as indicated by Amir et al. J. In agreement with the observations of Davies et a1.14, D N A or protein content of the cultures were not altered following incubation with 10 p M ChM Az and control and drug-treated cultures did not appear to differ on microscopic evaluation. Similarly, ChAT activity was decreased following incubation of neurons with 10 p M ChM Az as was observed with ECM AZ3'14; one study 24 reports no change in ChAT activity relative to controls after incubation of neuronal cultures with 30 #M ECM Az, but it is unclear why these results differ. Since the loss of ChAT activity did not correlate with decreased D N A content of the cultures, it would imply direct inhibition of the enzyme by ChM Az rather than a decrease in number of cholinergic neurons during the time frame studied. This could offer indirect evidence that some ChM Az was taken up into the cholinergic neurons by the high-affinity choline carrier. It should be noted, however, that ChAT may also be present on the extracellular surface of synaptosomes and some cholinergic neuroblastoma 5, although the physiological relevance of this form of ChAT remains to be demonstrated. Alternatively, if cholinergic neurons comprise only a very small proportion of the total neurons in culture, as seems

215 likely, then a small change in culture D N A content

to demonstrate that C h M Az can be a selective neuro-

resulting from ChM Az-mediated cholinergic n e u r o n a l

toxin for cholinergic n e u r o n s when compared to G A B A -

death may not be detectable within the range of experimental error. This latter point is supported by the observation that there was no recovery of ChAT activity by 72 h after t r e a t m e n t with ChM Az; if cholinergic

ergic neurons.

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