Anti-acetylcholine antibodies and first immunocytochemical application in insect brain

Neuroscience Letters, 57 (1985) 1-6

1

Elsevier Scientific Publishers Ireland Ltd. NSL 03224

A N T I - A C E T Y L C H O L I N E A N T I B O D I E S A N D FIRST I M M U N O C Y T O C H E M I C A L A P P L I C A T I O N IN INSECT B R A I N

MICHEL GEFFARD 1'*, JEAN VIEILLEMARINGE2, ANNE-MARIE HEINRICH-ROCK 1 and PASCAL DUR1S3 ~Institut de Biochimie Cellulaire et Neurochimie du C.N.R.S. et U-259 INSERM, 1 rue Camille SaintSa~ns, F-33077 Bordeaux Cedex; 2Laboratoire de Neuroendocrinologie, UA C.N.R.S. 683, Universit~ de Bordeaux L Avenue des Facult~s, F-33405 Talence Cedex; and ~Laboratoire de Biologie Appliqu~e ?t I'EPS, Universit6 de Bordeaux I1, F-33076 Bordeaux Cedex (France)

(Received June 28th, 1984; Revised version received November 8th, 1984; Accepted January 1lth, 1985)

Key words: acetylcholine - choline-glutaryl-protein - enzyme-linked immunosorbent assay - ELISA -

immunocytochemistry - choline acetyltransferase - locust brain

A specific immunological approach was developed to enable acetylcholine (ACh) to be visualized in biological tissues. A variety of ACh-like immunogens were synthesized, and injected into rabbits. Antibody specificity was tested using an enzyme-linked immunosorbent assay (ELISA) method. The most immunoreactive ACh derivative was found to be choline-glutaryl-lysine.A mixture of allyl alcohol and formaldehyde was found to be the best fixative of ACh in tissues. The specificity of this antibody recognition was tested in vitro and in immunochemistry. There was excellent agreement between the in vitro results and the ACh staining. Moreover, visualization using these anti-ACh antibodies appeared identical to the results using anti-choline acetyltransferase antibodies. I n spite o f the fact that acetylcholine (ACh) was o n e o f the first n e u r o t r a n s m i t t e r s to be characterized, histological i n f o r m a t i o n o n the a n a t o m i c a l o r g a n i z a t i o n o f A C h p a t h w a y s is relatively scanty [3, 4, 15, 16]. O n e o f the m a i n reasons in the lack o f u n e q u i v o c a l a n a t o m i c a l m e t h o d s for the i d e n t i f i c a t i o n o f these n e u r o n s a n d their p r o j e c t i o n s . Over the last few years, more detailed i n f o r m a t i o n has come f r o m studies which have visualized the specific cholinergic enzymes [l l, 12, 17]. However, the n e u r o t r a n s m i t t e r itself has never been directly or specifically localized. This p r o b l e m has b e e n o v e r c o m e for other n e u r o t r a n s m i t t e r s by the p r o d u c t i o n o f antibodies directed against the t r a n s m i t t e r molecule itself [6-9, 13, 14]. Such antibodies have also been e m p l o y e d for i m m u n o c y t o c h e m i c a l studies [1, 19, 20]. A l t h o u g h a n t i - A C h a n t i b o d i e s have been o b t a i n e d for a n i m m u n o a s s a y o f A C h [18], their use in i m m u n o c y t o c h e m i s t r y has n o t yet been reported. Here, we report a m e t h o d for the p r o d u c t i o n o f specific a n t i - A C h a n t i b o d i e s a n d for the direct v i s u a l i z a t i o n o f A C h p a t h w a y s in the locust b r a i n . I n view o f i m m u n o c y t o c h e m i c a l detection o f A C h , a specific strategy was developed. The synthesized h a p t e n m u s t be ACh-like. Several likely i m m u n o g e n s *Author for correspondence and reprint requests. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.

were done, using a cross-linking method that enabled many haptens to be attached to the same proteins. The first step consisted in a 'glutarylation' of immunogenic proteins, such as bovine serum albumin (BSA; Sigma), human serum albumin (HSA; Centre de Transfusion Sanguine) and hemoglobin (Hb; Sigma). This was carried out by mixing 100 mg BSA, HSA or Hb in alkaline medium with 100 mg of glutaric anhydride (GA; Sigma), dissolved in 500 #1 of dimethylformamide (DMF; SDS). After dialysis against distilled water at 4°C over two days, each glutarylated protein was lyophilized. The second step coupled this glutarylated protein to choline. For example, 30 mg of glutarylated BSA (GA-BSA) was dissolved in 2 ml of DMF containing 40 #1 of triethylamine (TEA; Merck) and activated by 150 ~1 of a 1:16 dilution of ethylchloroformate (ECF; Fluka) in DMF. All these experiments were performed at 4°C. After 5 min, the mixture was added to 1 ml of an aqueous solution containing 20 mg of choline (Fluka) plus 2 #1 of [3H]choline (spec. act. 80 Ci/mmol; NEN) and 40/~1 of TEA. The mixture was then dialyzed at 4°C over two days, and then clarified by centrifugation. Beta-counting was carried out on a 100-~d aliquot, both before and after dialysis, in order to calculate the choline-GA concentration required to mimic ACh. After weighing the lyophilized immunogen from 1 ml of the solution, the molar coupling ratio was calculated to be around 90. The synthesized immunogens were injected s.c. into two rabbits. Every 3 weeks for 6 months, the rabbits received in rotation: choline-GA-BSA, choline-GA-HSA and choline-GA-Hb. The antisera was collected 10 days after the last immunization injection and tested for anti-ACh antibody specificity, both in vitro and in immunochemistry. The antibody affinity and specificity were initially evaluated with an enzymelinked immunosorbent assay (ELISA) method using a choline-GA-protein bearing the antigenic determinant fully recognized by the anti-ACh antibodies. The antibody titer was measured by a colored peroxidase reaction. Before the experiments, the antisera were adsorbed on glutarylated proteins: GA-HSA, GA-BSA, GA-Hb used during immunization. T he polystyrene well plates (Nunc) were coated with 200/~1 of choline-GA-BSA solution (1 #g/ml) or a GA-BSA solution (1 /~g/ml) in 0.05 M carbonate buffer, pH 9.6, for 16 h at 4°C. At the end of this period they were filled up with phosphate-buffered saline (PBS) containing 0.05o7o Tween 100 and 1 g/l BSA, and incubated at 37°C for 30 min. They were then rinsed 3 times with PBS-Tween. The anti-ACh antibodies were incubated at 37°C for 90 min, either alone, or with added compound. The final dilution of anti-ACh antibodies was 1:2000 in PBS-Tween-BSA. The wells were washed again 3 times with PBS-Tween and then filled up with 200/~1 of 1:1000 goat anti-rabbit serum labelled with horseradish peroxidase (Miles) in PBS-Tween-BSA. After 45 min incubation at 37°C the wells were re-washed 3 times with PBS-Tween. Peroxidase was assayed by a 10-min incubation with O-phenylenediamine hypochloride (I0 mg/ml) in 0.1 M citrate phosphate buffer, pH 5.0, containing 200 /~1 of 30O7o hydrogen peroxide. The reaction was stopped by the addition of 50 ~tl per well of 4 M H2SO4, and the color read at 492 nm in a Multiskan Titertek apparatus. Experimental values were corrected by subtracting values of blanks from wells coated with just the

gtutarylated protein (GA-BSA). The specific colored reaction seen for the choline-GA-protein enabled us to establish the competition between the conjugate (choline-GA-BSA) coated and either choline (Fig. 1, curve 1), phosphatidyl choline (Fig. 1, curve 1), ACh (Fig. 1, curve 2) or choline-GA-poly-lysine (Fig. 1, curve 3) incubated with the ACh antiserum dilution. The most immunoreactive compound was choline-GA-poly-L-lysine. Self-displacement was found at between 10-s and 10-6 M, indicating fairly high antibody affinity. The other compounds only exhibited slight immunoreactivity and were poorly recognized. These results are in agreement with an antibody recognition site based on the three amino acid residues, choline-GA-lysine. Use of this ACh antibody as an immunocytochemical reagent required a suitable coupling agent to: (i) fix the ACh molecule in the tissues; (ii) reproduce the most immunoreactive structure as clearly as possible; (iii) preserve the structural integrity of the tissues. Cerebroid ganglia of the adult locust were quickly removed and immersed in 0.1 M cacodylate buffer, pH 11.0, containing 1 M allyl alcohol-12o70 formalin-lo70 sodium metabisulfite, for 2 min at 4°C in the dark. They were then post-fixed in 'Bouin-Holland' without acetic acid, containing 12°70 formalin-l% sodium metabisulfite and a saturated solution of mercuric chloride, at 4°C for 36 h in the dark. After washing overnight at the same temperature, the cerebroid ganglia were rapidly dehydrated, embedded in paraffin and cut into 7-/zm sections. Q5

0,4

•

•

>-

~- 0.3 z 0.2

0.10

, -8

-7

.6 log

.5

i .4

_3

C

Fig. 1. Displacement curves established with ELISA system by competition experiments between choline-GA-BSA coated and (1) choline or phosphatidyl-choline; (2) ACh; (3) choline-GA-poly-L-lysine incubated with anti-ACh antibodies 2 h at 37°C. Before competition experiments, antibodies were adsorbed on glutaryled proteins for one night at 4°C. From these displacement curves obtained by competition experiments, a cross-reactivity ratio was calculated as follows: choline-GA-lysine concentration upon each compound concentration at half displacement. The cross-reactivity ratio values were for choline and phosphatidyl-choline, 1:> 105; for ACh, 1:104; and choline-GA-poly-L-lysine, 1.

The sections were then de-paraffinized, re-hydrated relatively quickly and immersed in veronal buffer (diluted 1:5) for 20 min. The anti-ACh antibodies diluted to 1:100 in the same buffer were put in contact with the sections overnight at 4°C. After a rapid rinse, a fluorescein-isothiocyanate conjugated sheep anti-rabbit lgG (Institut Pasteur) diluted to 1:50, detected the primary immunoreaction. Finally, after another wash and 20-min immersion in veronal buffer, they were counter-stained with Evans Blue (diluted to 1:1000) for 3 min. The stained sections were mounted in buffered glycerin and observed under a fluorescence microscope. As far as possible, all steps should be carried out in the dark. The transmitter was visualized in neuropils and some cell bodies of the locust brain. Fig. 2a shows some perikarya located in the pars intercerebralis, and the beginning of their neuronal processes can be seen in Fig. 2b. ACh neurons have been demonstrated histologically in insect brains [5, 10], and some measurements of ACh, choline acetyltransferase (CHAT) and ACh-esterase (ACHE) in cerebroid ganglia of Locusta migratoria have been reported in the literature [2]. In order to check the specificity of the ACh staining, brains from adult locusts were either fixed in 'Bouin-Holland' or in a mixture of 4°70 paraformaldehyde-1 °70 glutaraldehyde. Tissues were cut and incubated in a solution of anti-ChAT antibodies (Immunonuclear) at a 1:200 dilution. The cells and neuronal processes that were stained (Fig. 3a, b), reproduced the structures stained with the anti-ACh an-

Fig. 2. lmmunohistochemical detection of ACh in the brain of mature locusts (Locusta migratoria migratorioides) using a specific anti-ACh serum. Some ACh cell bodies are observed in the pars intercerebralis (a). Their processes are labelled by the antiserum (b), especially the collaterals. Bars = 2(I t~m. Fig. 3. lmmunohistochemical detection of C h A T in the brain of mature locusts with a C h A T antiserum (Immunonuclear). The neurons stained in the pars intercerebralis (perikarya a, and processes b) are of the same type as those previously visualized with the anti-ACh antibodies. Bars = 20 Imp.

5

TABLE 1 CROSS-REACTIVITY RATIO ESTABLISHED BETWEEN CHOLINE-GA-LYSINE AND CONJUGATES SYNTHESIZED WITH ALLYL ALCOHOL OR/AND ALDEHYDE Compounds a Choline-GA-poly-L-lysine ACh-allyl alcohol-protein ACh-allyl alcohol-formalin-protein ACh-allyl alcohol-glutaraldehyde-protein ACh-formalin-protein ACH-glutaraldehyde-protein Choline-allyl alcohol-protein Choline-allyl alcohol-formalin-protein Choline-allyl alcohol-glutaraldehyde-protein Choline-formalin-protein Choline-glutaraldehyde-protein

Cross-reactivity ratio b 1

1:6.4 1:3 1:2

1:>1.4×10 4

aEach compound was synthesized in vitro. The coupling reactions were identical to those used during histological fixation. The addition of [3H]ACh or [3Hlcholine allowed us to calculate the molar coupling ratio of ACh or choline to protein after dialysis against water. bAfter drawing the displacement curves, the cross-reactivity ratio of choline-GA-poly-L-lysine concentration:conjugate concentration was determined at half displacement.

tibodies (Fig. 2). In addition, the specificity of the immunocytochemical fixation of ACh was demonstrated by: (i) absence of the specific anti-ACh antibodies where no specific staining was observed; (ii) absence of staining, when the anti-ACh antibodies were pre-incubated with l0 -2 M ACh or 4 x 10-5 M choline-GA-lysine before immunocytochemistry; and (iii) recognition of the anti-ACh antibodies after histological fixation with allyl alcohol and formalin. Using the ELISA method, competition studies were carried out between choline-GA-BSA and the ACh conjugates synthesized with allyl alcohol and aldehyde. These conjugates were fully recognized by the anti-ACh antibodies in the presence of 1 M allyl alcohol-12% formalin, or 5o7o glutaraldehyde (see Table I). The other conjugates; ACh-formaldehyde-protein, ACh-glutaraldehyde-protein, choline-allyl alcohol-protein, choline-formaldehyde-allyl alcohol-protein, choline-glutaraldehyde-allyl alcoholprotein, showed no immunoreactivity at all. The ACh-molecule appears preferentially conjugated using this procedure of fixation. These results show that ACh can be detected directly in biological tissues using an allyl alcohol-aldehyde fixative. This mixture produces a haptenic structure which is fully recognized by antibodies, and mimics the synthetic immunogen choline-GA-lysine. This technique may be of value in furthering our understanding of cholinergic pathways and their role in such conditions as senile dementia and Alzheimer's disease. The authors wish to thank Josette Dulluc for her technical assistance. This work was supported by grants from M.R.I. and INSERM.

1 Buijs, R.M., Geffard, M., Pool, C.W. and Hoorneman, E.M.D., The dopaminergic innervation of supraoptic and paraventricular nucleus. A light and electron microscopical study, Brain Res., 323 (1984) 65-72. 2 Breer, H., Comparative studies of cholinergic activities in the central nervous system of Locusta migratoria, J. Comp. Physiol., 141 (1981) 271-275. 3 Dhainaut-Courtois, N., Engelhart, R.P. and Dhainaut, A., Etude cytophysiologique des syst~mes monoaminergiques et cholinergiques des Nereis (Ann61ides Polych~tes). I. Syst~me nerveux p6riph6riq u e e t fonctions neuromusculaires, Arch. Biol. (Bruxelles), 90 (1979) 225-244. 4 Dhainaut-Courtois, N., Engelhardt, R.P. and Dhainaut, A., Etude cytophysiologique des syst6mes monoaminergiques et cholinergiques des Nereis (Ann61ides Polych6tes). II. Syst~me nerveux central, Arch. Biol. (Bruxelles), 90 (1979) 273-288. 5 Frontali, N., Piazza, R. and Scopoletti, R., Localization of acetylcholinesterase in the brain of Periplaneta americana, J. Insect. Physiol., 17 (1971) 1833-1842. 6 Geffard, M., Buijs, R.M., Seguela, P., Pool, C.W. and Le Moal, M., First demonstration of highly specific and sensitive antibodies against dopamine, Brain Res., 294 (1984) 161-165. 7 Geffard, M., Kah, O., Chambolle, P., Le Moal, M. and Delaage, M.A., A novel immunocytochemical application of antibodies against dopamine in the central nervous system, C.R. Acad. Sci. (Paris), 295 (1982) 797-802. 8 Geffard, M., Kah, O., Onteniente, B., Seguela, P., Le Moal, M., and Delaage, M.A., Antibodies to dopamine: radioimmunological study of specificity in relation with immunocytochemistry, J. Neurochem., 42 (1984) 1593-1599. 9 Geffard, M., Seguela, P. and Heinrich-Rock, A.M., Antisera against catecholamines: specificity studies and physicochemical data for anti-dopamine and p-tyramine antibodies, Molec. Immunol., 21 (1984) 515-522. 10 Hess, A., Histochemical localization of cholinesterase in the brain of the cockroach (Periplaneta americana), Brain Res., 46 (1972) 287-295. 11 Kimura, H., McGeer, P.L., Peng, F. and McGeer, E.G., Choline acetyltransferase containing neurons in rodent brain demonstrated by immunohistochemistry, Science, 208 (1980) 1057-1059. 12 Levey, A.I., Armstrong, D.M., Atweh, S.F., Terry, R.D. and Wainer, B.M., Monoclonal antibodies to choline acetyltransferase: production specificity and immunohistochemistry, J. Neurosci., 3 (1983) 1-9. 13 Ranadive, N.S. and Sehon, A.H., Antibodies to serotonin, Canad. J. Biochem., 45 (1967) 1701-1710. 14 Seguela, P., Geffard, M., Buijs, R.M. and Le Moal, M., Antibodies against GABA: specificity studies and immunocytochemical results, Proc. Natl. Acad. Sci. USA, 81 (1984) 3888-3892. 15 Shute, C.C.D. and Lewis, P.R., Cholinesterase-containing pathways of the hindbrain: afferent cerebellar and centrifugal cochlear fibers, Nature (Lond.), 205 (1965) 242-246. 16 Shute, C.C.D. and Lewis, P.R., The ascending cholinergic reticular system: neocortical, olfactory and subcortical projections, Brain, 90 (1967) 497-522. 17 Sofroniew, M.V., Eckenstein, F., Thoenen, H. and Cuello, A.C., Topography of choline acetyltransferase-containing neurons in the forebrain of the rat, Neurosci. Lett., 33 (1982) 7-12. 18 Spector, S., Felix, A., Semenuk, G. and Finberg, J.P.M., Development of a specific radioimmunoassay for acetylcholine, J. Neurochem., 30 (1979) 685-689. 19 Steinbusch, H.W.M., Verhofstad, A.A.J. and Joosten, H.W.F., Localization of serotonin in the central nervous system by immunochemistry: description of a specific and sensitive technique and some applications, Neuroscience, 3 (1980) 811-819. 20 Vieillemaringe, J., Duris, P., Geffard, M., Le Moal, M., Delaage, M., Bensch, C. and Girardie, J., Immunohistochemical localization of dopamine in the brain of the insect Locusta in comparison With the catecholamine distribution determined by histofluorescence technique, Cell Tiss. Res., 237 (1984) 391-394.