Printed in Sweden Copyright 0 1978 by Academic Press, inc. All rights of reproduction in any form reserved 0014-4827/78/11 I1-0l85$02.00/0

Experimental

ACETYLCHOLINE

RECEPTORS

SYSTEM

IN

OF DROSOPHILA EBERHARD

Institut

111 (1978) 185-190

Cell Research

ftir Biologie

Ill,

D-7800

THE

CENTRAL

NERVOUS

MELANOGASTER

RUDLOFF Freiburg

i. Br.,

Germany

SUMMARY Using [125J]-a-bungarotoxin (lzsJ-@t) as a marker, evidence was obtained for the presence of acetylcholine receptors in the central nervous system of Drosophila. Autoradiographic analyses show that bound toxin is associated onlv with the neuronile of larvae. nuoae and adults. both within the brain and the thoracic ganglion.-In the first optic ganglion, the l&&a gan ‘onaris,‘much less binding was found than in other Darts of the brain. Studies on the binding of i?F5J-aBat to the homogenate of flies’ heads exhibited a receptor site with a binding constant of 1.8f0.2 ;M. The pharmacological properties of the receptor were investigated with competitive binding experiments using various cholinergic ligands known from vertebrate studies. These properties were found to be very similar to those of the nicotinic acetylcholine receptors of vertebrates.

Acetylcholine (ACh) seems to play a major role in excitatory chemical transmission within the insect central nervous system (CNS). This view is supported by the finding that the insect CNS is rich in ACh, choline acetyltransferase and acetylcholinesterase, as well as by the strong electrophysiological effects of applied ACh (for reviews see [ 1, 21). The purpose of the experiments described here was to investigate the postsynaptic receptor, which must be assumed to be involved in the excitatory action of ACh during chemical transmission. [lzsJ]aBungarotoxin (125J-arBgt), a specific ligand of the nicotinic ACh receptor in vertebrates, was used to detect the receptor in cryotome sections of developing and adult Drosophila and to show its distribution within the CNS. Hall & Teng have de-

scribed essentially the same autoradiographic results for the brain of adult Drosophila [3].

Experiments on the binding of 125J-c> to homogenates of flies’ heads were performed in order to investigate the chemical and pharmacological properties of the receptor and compare them with those of vertebrate ACh receptors. A first approach in this direction has apparently been undertaken by Dudai [4]. MATERIALS

AND

METHODS

Reagents Carrier-free Na’=J was obtained from NEN Chem., Boston, Mass.; ol-bungarotoxin from Miami Serpentarium Lab., Miami, Fla; acetylcholine chloride and eserine from E. Merck, Darmstadt; n-tubocurarine chloride, hexamethonium chloride and atropine from Fluka AG, Buchs; bovine serum albumin from Serva, Heidelberg; or=muscarine and nicotine from Sigma Chem. Co., St Louis, MO. Exp Cell

Res I I1 (1978)

Fig. 1. (a) Horizontal section through a fly’s head, treated with le5J-oBgt; very few silver grains can be seen on the cell body layers (* ) and the optical chiasmata (x). The lamina ganglionaris (la) shows less labeling than other uarts of the brain. mbr. Midbrain: toe, compound eye; og, optical ganglia; me, medulla; lo, lobula and lobula mate: (b) same nreoaration as in (a) but with preincubation‘of the s’ect’on with unlabeled oBgt; no silver grains can be seen. mu, head muscles; (c) longitudinal section through the rostral

part of a third instar larva. The brain (br) is labeled more weakly than the compound ventral ganglion (cvg), and only a few silver grains can be seen on the enveloping cell body layers ( * ). mp, mouth parts; (d) longitudinal section through the rostra1 half of a 3-day-old pupa. Labeling of brain (br) and thoracic ganglion (thg) can be seen clearly. No grains can be detected on the wing muscles (wmu). All figures are bright field micrographs of autoradiographs. Bars, 0.1 mm.

Preparation of 125J-o13gtaccording to Vogel et al. [5] with 5 mCi of carrier-free Na**“J yielded diiodo-abungarotoxin exclusively. The initial specific activity was 340 Ci/mmole. The labeled toxin was stored and handled in the presence of 2 mg/ml bovine serum albumin unless otherwise stated.

carried out with 2% glutaraldehyde in 0.2 M sodium cacodylate at pH 7.2. After fixation the slides were rinsed three times in distilled water, air-dried, and coated with Ilford L4 emulsion, using a dipping technique. After 5 days of exposure the autoradiographs were developed in Kodak D 19, dehydrated by increasing ethanol concentrations and embedded in Rhenohistola.

Autoradiography

of cryotome sections

Flies’ heads, whole larvae, or pupae (wild-type strain “Oregon R”) were embedded in small pieces of pig liver and frozen. From this block, 16 pm sections were cut with a cryotome, fixed to slides bv ranid meltinn and incubated in ‘“J-&&t (0.2 nM) -in DrosophilaRinger with 2 mg/ml bovine serum albumin for 10 min at room temnerature. Excess unbound toxin was extracted with bovine serum albumin containing Drosophila-Ringer (1 min), and Drosophila-Ringer (2 x 1 min at 0°C). The subsequent fixation (30 min at O’C) was Exp Cell Res 111 (1978)

Preparation homogenate

and fractionation

of

The heads of about 10000 flies (Oregon R) were harvested according to the technique described by Harris et al. [6], and homogenized in 0.32 M ice-cold sucrose solution in two steps; (1) Chopping with electrically moved razor blades in 5 ml; and (2) final homogenization with a Potter-Elvehjem homogeni-

ACh receptors

loI L 01

,'

-10

-9

-0

-7

I I

-6

Fig. 2. Abscissa: log molarity of aBgt; ordinate: % ‘?I-+$ bound. Binding of 125J-oBgt to membrane particles of “pellet 2” in the presence of increasing amounts of unlabeled &at. Each assay tube contained 1.2 mn membrane protein in 1 ml homogenate solution 00 pM 125J-crBat). In the Scatchard-Plot debicted in the inset the tota amount of aBgt bound specifically to particles (i.e., concentration of toxin-receptor site complex) is represented on the abscissa, while the ordinate shows the nercent of sneciticallv bound toxin. Specific binding >s defined as binding- minus binding value at 1 +M cd3gt (unspecific binding). RS is the receptor site concentration. The mean of 7 experiments yielded a KD of 1.8f0.2 nM and a specific receptor site concentration of 0.19+0.07 pmoleslmg protein.

zator in 20 ml. The whole preparation and the following binding assays were done on ice. The crude homogenate was centrifuged for 10 min at 1000 g, after which the pellet was extracted with another 20 ml of 0.32 M sucrose and centrifuged as before. The resulting pellet will be referred to as “pellet 1”. The combined supernatants were then centrifuged for 20 min at 20000 g; the supematant of this centrifugation was centrifuged for 20 min at 30000 g (“pellet 3”). The pellet of the 20000 g centrifugation was then exposed to osmotic shock by resuspending and stirring it in 30 ml of distilled water for 30 min. This solution was centrifuged for 20 min at 95OO’g in order to remove soluble proteins, and the pellet (“pellet 2”) used for most of the binding assays because it exhibited the highest degree of binding (see Results). Protein measurements done according to Lowry et al. [7] yielded approx. 10 pg protein/mg heads in “pellet l”, 40 pg proteinlmg heads in “pellet 2”, and 10 pg proteinlmg heads in “pellet 3”.

Binding assay In order to determine binding of lwJ-cuBgt, the abovementioned pellets wereC=suspended in 50 mM Tris/ citrate pH 7.1 to a final concentration of 0.8-1.2 mg protein/ml. Test tubes (Eppendorf 1 ml plastic centrifuge tubes) contained 1 ml of membrane suspension; ‘05J-aBgt (final concentration 50 pM, unless otherwise stated), and increasing amounts of unlabeled crBgt,

in Drosophila

187

or inhibitors of aBgt binding were added simultaneously. After 30 min of incubation. the tubes were cent-rifuged at 30000 g, and ahquots of the supematants and the pellets resuspended in 0.1 ml of distilled water were counted in 10 ml of Bray’s solution (10% naphthalene, 0.5 % diphenyloxazole-in dioxene) in a Beckmann liquid scintillation counter with a counting efficiency of 63 %. Binding is expressed as: l”J-aBgt bound (total cpm of pellet) x loo free ‘=J-> (total cpm of supematant) (=% lZ5J-aBgt bound).

RESULTS

AND

DISCUSSION

Autoradiography

Autoradiography of flies’ heads, which were prepared for histological examination after in toto incubation in lz5J-aBgt was unsuccessful. Therefore the heads were first freeze-sectioned and then treated with 125J-CyBgt. This procedure results in signiticant binding of toxin in the brain (fig. 1 a). Fig. 1 b shows the result of preincubation of the section in 0.1 PM unlabeled arBgt before treatment with lz5J-CuBgt. No radioactivity can be seen, indicating that the binding is saturable by relatively low amounts of unlabeled toxin, and thus is not due to unspecific adsorption to the brain tissue. lz5J-aBgt binds exclusively in the neuropile, as can be seen in fig. 1 a ; cell bodies and optical chiasmata show considerably fewer silver grains than the neuropile. Neurotransmitter receptors are not to be expected on either the surfaces of cell bodies, which are neurologically inactive in insects, or on the synapse-free processes of the chiasmata. Thus the selective labeling pattern found can be interpreted as binding of the toxin to post-synaptic ACh receptors. The whole brain, except for one ganglion, appears to be labeled fairly homogeneously. This ganglion, the first optic ganglion or lamina ganglionaris, shows significantly less label. The compound eye exhibits no Exp Cell

Res 111 (1978)

188

E. Rudloff

In 3-day-old pupae the main parts of the CNS can be visualized autoradiographically (fig. 1 d). In particular the three-fold segmentation of the thoracic ganglion can be seen very well. Moreover, the figure shows that muscles are not labeled, indicating that the receptor at the neuromuscular junction is different from that found in the CNS. In fact, glutamate is widely accepted to be the transmitter at the neuromuscular junction of insects.

255

0’ -9

-8

-7

-6

-5

4

-3

Fig. 3, Abscissa: log molarity of ligand; ordinate: % ‘25J-oBgt bound. n , > 0, o-tubocuratine; 0, nicotine; 0, ACh plus 5 PM eserine; A, hexamethonium; A, atropine; @, oL-muscarine. Binding of rz5J-aBgt to membrane particles of “pellet 2” in the presence of increasing amounts of various agonists and antagonists of vertebrate ACh receptor. Each assay tube contained 0.9 mg of membrane protein in 1 ml of homogenate solution, which was also 0.1 nM of 125J-oBgt.

more silver grains than the background. This supports the assumption that binding of 125J-cd3gt occurs to synaptic receptors, since this layer is known to be free of synapses. Already in larval neuronal tissue 125J-(uBgt binding is found. Fig. 1c shows an unequal distribution of label between brain hemisphere and the compound ventral ganglion. This ’ perhaps reflects that the compound ventral ganglion is of higher functional significance for the larva [8].

Binding assays

The aim of the in vitro binding assays described here was to investigate the properties of the c> binding receptor for purpose of comparison with the ACh receptors of vertebrates. Fig. 2 shows the binding of 125J-> in the presence of increasing amounts of unlabeled crBgt to material of “pellet 2” (see Materials and Methods), which was found to exhibit the highest degree of binding. Only very little unspecific binding can be seen in the presence of saturating amounts (1 PM) of unlabeled cuBgt. The Scatchard-Plot in the inset shows a uniform slope over the whole range of binding; this suggests a binding reaction to only one site at the receptor without any cooperative effect (Hill-coefficient= 1.2). The equilibrium constant of binding of

Table 1. Pharmacological properties of ACh receptors from various systems K, refers to the equilibrium constant of aBgt binding to the receptor, and I, to the concentration required for 50% inhibition of maximum 125J-aBgt binding KD (nW

System Chick sympath. ganglia neurons Chick retina Chick muscle cells Drosophila brain

Exp Ceil

Res 111 (1978)

of drug

f&M) o-Tuboc.

Nicotine

ACh

Hexameth. Atropine

Ref.

2

0.25

1

0.6 0.3

10

500

r91

1.3

1 1

1.8

0.3

I0 70

r100 20

E:;’

Present results

ACh receptors

125J-CuBgtto the receptor was found to be 1.8kO.2 nM and the specific receptor site concentration was calculated to be 0.19+ 0.07 pmoleslmg protein; both values are the means f standard deviation of 7 separate experiments. Specific receptor site concentration in “pellet 1” was 0.014 pmoles/ mg protein, and 0.056 pmoleslmg protein in “pellet 3”. In order to prove that 125J-czBgt actually binds to membrane particles, binding was measured in the presence of various amounts of “pellet 2” material. A linear increase of binding with increasing amounts of material was found between 0.2 and 1.0 mg of membrane protein per 1 ml homogenate solution (data not shown). In vertebrates various drugs are known to be either agonists or antagonists of the neurophysiological effect of ACh at various types of ACh receptor. Fig. 3 shows the inhibitory effects of some of these drugs on the binding of 125J-(uBgt. The substances used can be classified into two distinct groups according to their inhibitory effect. D-Tubocurarine and nicotine, forming one group, exhibit very effective inhibition of binding, which is best explained by a high affinity competitive binding of these substances. Inhibition of 50% (ZsO)is achieved with 0.3 Z.LM D-tubocurarine and 0.5 /.LM nicotine. The other group consists of atropine (Z,,=20 PM), hexamethonium (Z,,=70 PM) and muscarine (Z5,,=1 mM, but the t&form was used). ACh itself, used in the presence of 5 ,uM eserine to avoid destruction by acetylcholinesterase, exhibits a medium affinity (Z,,=3 PM) and the slope of its binding curve is weaker, which suggests negative cooperativity (Hill-coefftcient= 0.67) in ACh binding. These data suggest that the aBgt binding receptor in the Drosophila CNS is a nicotinic ACh receptor, similar to those found in cultured chick muscle

in Drosophila

cells [5], cultured chick sympathetic rons [9], and chick retina [lo].

189

neu-

CONCLUSIONS The cwBgt binding receptor site in Drosophila shown in this study has been found to be very similar to the nicotinic cholinergic receptor within the CNS and the neuromuscular junction of vertebrates. This can be seen from table 1, which shows that the binding constants and the inhibitory effects of some cholinergic ligands measured in various systems are in the same range as the values found in Drosophila. Only atropine, known to interact with the muscarinic receptor, shows a more prominent effect in Drosophila than in chick retina and muscle. However, the difference between the inhibitory effects of nicotine and atropine is great enough to allow the classification of the Drosophila receptor as a nicotinic cholinergic receptor. Hexamethonium is normally used to distinguish between nicotinic receptors from the neuromuscular junction and from autonomic ganglia of mammals. The results from Drosophila thus suggest the presence of a receptor similar to the muscular type receptor of vertebrates. In fact, this conclusion must be regarded with caution, since findings of Greene et al. with hexamethonium in chick sympathetic ganglia neurons show that the results from mammals cannot be extrapolated to all vertebrates [9]. From the receptor concentrations in all pellets (see Results and Discussion) a total amount of 6.8X lo-l6 moles of receptor sites/single fly’s head was calculated. The brain of Drosophila is assumed to contain approx. 80000 neurons. This very rough estimation is based on values given by Strausfeld for Musca [ 1I], and corrected for the fewer number of ommatidia and the Exp Cell Res

1I I (1978)

190

E. Rudloff

smaller brain volume in Drosophila. Thus the surface of a single neuron may contain at least 5 000 receptor sites. The results presented here do not prove that the aBgt binding receptor is a constituent of the post-synaptic membrane. This would be required of a receptor taking part in chemical transmission with ACh. Further studies by electronmicroscope autoradiography of brain sections and/or sections of pellets from binding experiments treated with 125J-> are presently being performed in order to clarify this point. Note added in proof After this work was submitted, similar results were published by Y. Dudai, FEBS lett 76 (1977) 211, Y. Dudai & A. Amsterdam, Brain res 130 (1977) 551 and L. Hall, J neurochem. In press (1977). The results presented in these papers are in very good agreement with those presented here. I am indebted to Miss Regine Leutenecker for her skilful technical assistance and to Drs P. Nevers, J.-A. Campos-Ortega and R. Hertel for helpful criticism during preparation of the manuscript. I thank Dr S. Benzer for the kind permission to cite unpublished

EX/J Cell

Res 111 (1978)

work from his laboratory and for calling my attention to the publication of Hall & Teng 131. This work was supported by the Deutsche For~chungsgemeinschaft SFB 46.

REFERENCES 1. Gerschenfeld, H M, Physiol rev 53 (1973) 1. 2. Pitman, R M, Comp gen pharmacol2 (1971) 347. 3. Hall, L M & Teng, N N H, Developmental biology-pattern formation-gene regulation. ICN-UCLA symposia on molecular and cellular biology (ed D McMahon & C F Fox) vol. 2, p. 282. W A Benjamin, Menlo Park, Calif. (1975). 4. Dudai, Y, Caltech 1975 biology annual report, no. 101, p. 72 (1975). 5. Vogel, Z, Sytkowski, A J & Nirenberg, M W, Proc natl acad sci US 69 (1972) 3180. 6. Harris, W A, Stark, W S &Walker, J A, J physiol 256 (1976) 415. 7. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 8. Hertweck, H, Z wiss Zoo1 139 (1931) 559. 9. Greene, L A, Sytkowski, A J, Vogel, Z & Nirenberg, M W, Nature 243 (1973) 163. 10. Vogel, Z & Nirenberg, M, Proc natl acad sci US 73 (1976) 1806. 11. Strausfeld, N J, Atlas of an insect brain. Springer-Verlag, Heidelberg, New York (1976). Received May 18, 1977 Revised version received August 5, 1977 Accepted August 12, 1977