Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 1806-1810, June 1976 Biochemistry Localization of acetylcholine receptors during synaptogenesis in retina (a-bungarotoxin/neuron development/cultured neurons) ZVI VOGEL AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshull Nirenberg, January 9,1976 ABSTRACT Nicotinic acetylcholine receptors are synthe- sized in chick embryo retina before synapses appear. Most of the receptors are found associated with neurites in the inner synaptic la er of the retina; later in development the receptors anpear in t e outer svnaptic laver. K Cells dissociated from retina and cultured in vitro form ag- gregates and also sort out into regions comprised either of neurites or cell bodies. Most of the nicotinic acetylcholine re- ceptors of aggregates are found associated with neurite regions. The receptor distribution of cultured retina cells thus resembles that of intact retina. 1251-Labeled cu-bungarotoxin, a specific ligand of the nicotinic acetylcholine ( AcCh) receptor, was used to detect AcCh re- ceptors in the developing chick embryo retina. This species of A&h receptor is a synaptic marker, since the AcCh receptors of striated muscle cells (1, 2) and certain parasympathetic neurons (3) are found in abundance only at the site of the sy- napse. After denervation, the receptors are found over the entire surface of the cell. Most of the recent information regarding the nicotinic AcCh receptor has been obtained with the receptors of the electric organ of fish, and striated muscle (for reviews see refs. 4-6). a-Bungarotoxin has been used to detect AcCh receptors in sympathetic ganglia (7) and brain (g-lo), but the nicotinic AcCh receptors of retina have not been studied in this manner. The vertebrate retina is a model system for studies on synaptic communication, since relatively few types of neurons are present and cells dissociated from retina form synapses in vttro (11, 12). The retina contains acetylcholine and enzymes that catalyze the synthesis and the hydrolysis of this compound (13-17). In addition, electrophysiologic studies show that some neurons in retina respond to acetylcholine and that both nico- tinic and muscarinic AcCh receptors are present in the retina of certain organisms (18-22). In this report, the concentration of nicotinic AcCh receptors and their location in chick embryo retina are described as a function of the developmental age of the retina. MATERIALS AND METHODS Assay of Homogenates for `%I-Labeled cr-Bungarotoxin Binding. Neural retinas from white leghorn chick embryos were dissected in cold Dulbecco's phosphate-buffered saline (Gibco) and then homogenized in 0.05 M Tris-HCl, pH 7.4, in a ground glass Dual1 homogenizer (Kontes) using 0.25-1.25 ml of buffer per retina so that the final concentration of protein was 3-6 mg/ml. Homogenates were frozen immediately and stored at - 191' ; binding sites for a-bungarotoxin (aBT) were stable for at least 1 year. a-Bungarotoxin labeled with 2 atoms of iarI per molecule of toxin (`251-(rBT) was prepared as de- Abbreviations: aBT, cu-bungarotoxin; 1251.aBT, cz-bungarotoxin labeled with 2 atoms of `%I per toxin molecule; AcCh, acetylcholine. scribed previously (23). Stock solutions of 1251-aBT contained 0.25-0.5 PM 1251-cuBT; 3 mM sodium phosphate buffer, pH 7.5; 150 mM NaCl; and 2 mg of crystalline bovine serum albumin per ml. Each binding reaction mixture contained the following components in a final volume of 0.1 ml: 10 nM `%I-aBT (2-4 ~1 of a stock solution); 0.05 M Tris-HCl, pH 7.4; 0.2 mg of serum albumin; and O-180 pg of retina homogenate protein. Reaction mixtures were incubated for 30 min at 37", diluted with 3 ml of solution C (0.05 M Tris-HCl, pH 7.4, and 2 mg of serum al- bumin per ml) at 3", and immediately filtered (vacuum) through a wet cellulose acetate filter (Millipore, EGWP 25 mm in diameter, 0.2 pm pore size). The filter was washed four times with 3 ml portions of solution C, the filter was dissolved in 10 ml of Instabray (Yorktown Research), and radioactivity was determined. Zero time values (100-200 cpm, approximately 0.25 fmol of `s-&BT) were subtracted from the values shown. The amount of `251-(uBT bound was proportional to protein concentration in the range studied (O-180 pg of protein). Pro- tein was determined by modification of the method of Lowry et al. (24). Autoradiography of 1251-crBT Bound to Intact Retina. Neural retina was dissected in cold solution A [the Dulbecco- Vogt modification of Eagle's medium (Gibco Catalog no. H-21) with 20 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-eth- anesulfonic acid), pH 7.4, instead of NaHC03, and 2 mg of serum albumin per ml of medium]. Pieces of retina were in- cubated for 60 min at 37" in 0.5-1.0 ml of solution A with 10 nM 1251-crBT and then were washed five times with 5 ml of cold solution A and twice with solution A without serum albumin. The tissue was fixed with 10% (vol/vol) neutral formalin or 2.5% (wt/vol) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and embedded in paraffin. Serial sections, 8 I.trn thick, were mounted on glass slides, and the paraffin was removed; the slides were coated with Kodak NTB-2 emulsion diluted 1:l with water. Slides were cooled to gel the emulsion and stored at 4" in the dark. Autoradiographs were developed for 4 min at 20" with Kodak D-19 developer diluted 1:l with water and fixed for 5 min with Kodak F5. The slides were rinsed with water; some were stained with 0.05% toluidine blue in 0.66 M borax. Retina Cell Cultures. Cells were dissociated from retinas of 8-day-old chick embryos and aggregated in vitro by a modi- fication of the method described by Sheffield and Moscona (11). Single cells, dissociated from retina with 0.25% trypsin (three times crystallized, Worthington) and 50 pg/ml of DNase I (Worthington), were cultured in rotating flasks (80 rpm) in a 37" incubator in a humidified atmosphere of 5% CO*--95% air. Each flask contained 9 X 106 cells and 1.5 ml of medium (80% Eagle's Basal Medium and 20% fetal bovine serum). The me- dium was changed every other day. After 7 days 10 nM lz51- aBT was added to the aggregates in the rotating flasks. One hour later the aggregates were collected and washed four times 1806 Biochemistry: Vogel and Nirenberg Proc. Natl. Acad. Sci. USA 73 (1976) 1807 MINUTES FIG. 1. The rate of iz51-~BT binding. Portions of a P-week-old chick retina homogenate (62.2 rg of protein per reaction mixture) were incubated with 1 nM iz51-~BT with or without 2 MM unlabeled aBT at the temperatures and times specified. with growth medium by centrifugation, and three times with growth medium without serum. The aggregates were fixed with glutaraldehyde, embedded in paraffin, sectioned, and subjected to autoradiography as described above. RESULTS AND DISCUSSION L251-aBT Binding. The rate of binding of `251-(uBT to re- ceptors in a homogenate of chick retina is shown in Fig. 1. Maximum `251-aBT binding was obtained after 15 min of in- cubation at 37"; rates of binding were lower at 19" and 0". Nonspecific binding of `=I-aBT in the presence of a large ex- cess of unlabeled olBT (2 PM) was relatively low. The relation behveen `%I-arBT concentration and the amount of labeled toxin bound is shown in Fig. 2. The amount of 1251-cuBT bound increased sharply as the concentration of `~&YBT was elevated from 1 to 7.5 nM. Higher concentrations of 1251-~BT (IO-40 nM) increased binding of labeled aBT only slightly. Little nonspecific binding of `251-~BT was observed in the presence of 2 KM unlabeled aBT. The number of specific (rBT binding sites in chick retina, 2 weeks after hatching, esti- FIG. 2. Relation between lz51-diiodo-aBT concentration and the amount of labeled toxin bound. Portions of a 2-week-old chick retina homogenate (62.2 fig of protein per reaction mixture) were incubated for 5 min at 20o, in the absence (O), or presence (01, of 2 /.IM unlabeled aBT. `251-Labeled a-bungarotoxin then was added at the concen- trations specified and the reaction mixtures were incubated and as- sayed for bound radioactivity as described in Materials and Methods. A Scatchard plot of specific `*~I-LuBT binding; i.e., binding obtained after subtracting the radioactivity bound in the presence of 2 FM unlabeled toxin, is shown in the insert; the ordinate refers to the bound/free `251-aBT ratio; the abscissa corresponds to nM toxin bound per 62.2 rg of protein. t &, I -8 -7 -6 -5 L I -4 -3 LOG MOLARITY FIG. 3. Inhibitors of 1251-aBT binding. Portions of a P-week-old chick retina homogenate (50 ug of protein per 80 JII of reaction mix- ture) were incubated for 5 min at 20' in the presence of the com- pounds indicated. 1251-~BT (10 nM) then was added (the final volume was 100 bl) and tubes were incubated for an additional 10 min at 20". The final concentration of each compound is indicated on the abscissa. Tubes with acetylcholine also contained 3 PM eserine sulfate. One hundred percent on the ordinate corresponds to 6.25 fmol of `251-~BT specifically bound in the absence of inhibitor. This value was 35% of that obtained after 30 min of incubation at 37O. Abbreviations rep- resent the following: (u-BT, unlabeled a-bungarotoxin; NIC, nicotine; d-TC, d-tubocurarine; ACH, acetylcholme plus 3 FM eserine sulfate; CARB, carbamylcholine; ATR, atropine; ESE, eaerine sulfate; PILO, pilocarpine. mated from a Scatchard plot (Fig. 2, insert), is 525 fmol/mg of protein. The ( 1251-cyBT.receptor) complex dissociates in a bi- phasic manner in the presence of 2 FM unlabeled (YBT (50% dissociation in 30 min at 37"). Slower rates of dissociation were observed at 20" and 0" (data not shown). Receptor Specificity. Nicotinic and muscarinic AcCh re- ceptors can be distinguished by differences in affinity for li- gands. In Fig. 3, the effects of various ligands upon lssI-crBT binding are shown. Binding was inhibited 50% by 1 nM unla- beled (uBT, 0.3 MM nicotine, and 1 nM d-tubocurarine, each compound known to have a high affinity for nicotinic AcCh receptors. Fifty percent inhibition of toxin binding was obtained with 6 WM acetylcholine (3 FM eserine sulfate also was present, a concentration without effect on aBT binding), and with 100 nM carbamylcholine. Compounds that interact preferentially with muscarinic AcCh receptors, such as pilocarpine and at- ropine, either did not affect lzI-oBT binding or inhibited binding only at relatively high concentrations. Putative neu- rotransmitters such as L-glutamic acid, glycine, y-aminobutyric acid, dopamine, and I-norepinephrine did not affect olBT binding at 10-1000 FM; however, serotonin stimulated (YBT binding 15%. Choline (100 PM) inhibited 1251-aBT binding (data not shown). AcCh Receptors During Embryonic Development. The amount of bound lSI-oBT is shown in Fig. 4 as a function of the developmental age of chick retina. Under the conditions used (10 nM `251-(uBT, 30 min incubation at 37") approximately 80% of the receptors bind aBT. Specific binding sites for aBT are present in low concentration (7.5 fmol/mg of protein) in chick retina on the 6th embryonic day. The concentration of receptors increases IO-fold between the 6th and the 13th em- bryonic day; however, >80% of the receptors are synthesized between the 13th and last embryonic day during the period of retina synapse formation. These results show that the receptors 1808 Biochemistry: Vogel and Nirenberg Proc. Natl. Acad. Sci. USA 73 (1976) IN OVO DAYS POST HATCHING FIG. 4. Amount of ~-cuBT bound as a function of the develop- mental age of chick retina. Filled symbols (0) represent pmol of toxin bound specifically per mg of protein; open symbols (0) represent pmol of toxin bound specifically per retina. Each point is the mean of 18 to 60 values (3 to 10 retina homogenates; six concentrations of protein were assayed from each homogenate). Specific toxin binding is shown; nonspecific binding has been subtracted. are synthesized before synapses appear and suggest that ces- sation of both receptor accumulation and synapse formation may be coupled. The maximum receptor concentration is at- tained at the time of hatching (400 fmol of izr'I-~BT bound per mg of protein). Almost the same receptor concentration is found in adult retina; however, the number of specific binding sites for aBT per adult retina (2700 fmol per retina) is twice that of the newly hatched chick (1350 fmol per retina). Thus, adult retina either has more synapses, more receptors per synapse, or more extrajunctional receptors than the retina of the newly hatched chick. Adult rabbit retina bound 107 fmol of n?(rBT per mg of protein under the same conditions, Receptor Distribution in Retina. The distribution of nico- tinic AcCh receptors in retina was determined by autoradi- ography. Adult retina was incubated with rzsI-cuBT, fixed, sectioned, and either stained with toluidine blue or subjected to autoradiography as shown in Fig. 5A and B, respectively. Cell bodies and neurites rich in synaptic connections are found in separate layers (Fig. 5A). The upper layer consists of photore- ceptor cells; next is the outer plexiform layer which consists of neurites and synapses; next is a layer of cell bodies, the inner nuclear layer; and then there is a wide layer of synaptic con- nections and neurites, the inner plexiform layer. The lowest layer of cell bodies is composed of ganglion neurons; the last layer consists of ganglion neuron axons. The autoradiographs show that most of the binding sites for 1251-(rBT are located in the inner plexiform layer, which consists primarily of synaptic connections between processes of bipolar, amacrine, and ganglion neurons. Four horizontal bands with relatively high concentrations of silver grains can be distin- guished within the inner plexiform layer. Silver grains also are found in the outer plexiform layer, which contains synapses between photoreceptor, horizontal, and bipolar neurons. Rel- atively few grains are associated with cell body layers, or with FIG. 5. Section of an a-month-old chicken retina. (A) Tolui- dine-blue-stained section, not subjected to autoradiography. (B) The corresponding field in an unstained serial section, subjected to au- toradiography for 110 days. The initial specific activity lz51-aBT was 335 Ci/mmol of toxin. Bright field views are shown in both panels; the bar represents 25 pm. Abbreviations of retina layers are as follows: NF, nerve fiber layer, GC, ganglion cell layer, IP, inner plexiform layer (i.e., inner synaptic layer); IN, inner nuclear layer; OP, outer plexiform layer (i.e., outer synaptic layer); IS and OS, inner and outer segments of photoreceptor cell layer, respectively. axons of ganglion neurons. lzr'I-a-Bungarotoxin does not bind to pigment cells, choroid, pecten, or sclera. Autoradiographs of sections of rat and rabbit retina are shown in Fig. 6. Again, most of the silver grains are associated with the inner plexiform layer. Some putative ganglion and amacrine neurons are labeled in rat retina (Fig. 6A); however, the outer plexiform layer of the rat is essentially unlabeled. In rabbit retina (Fig. 6B and C) silver grains are found in both the inner and outer plexiform layer. Approximately lO-20% of the rabbit ganglion cell bodies and some cell bodies in the lower portion of the inner nuclear layer, presumably amacrine neurons, are FIG. 6. Autoradiography of sections of adult rat and rabbit retina labeled with 1251-aBT. (A) Bright field view of a stained section of Fisher rat retina subjected to autoradiography for 23 days. The initial specific activity of 1251-cuBT was 260 Ci/mmol of toxin. (B) and (C) Phase contrast views of stained sections of New Zealand white rabbit retina subjected to autoradiography for 27 days; the initial specific activity of 1251-aBT was 220 Ci/mmol of toxin. The bar represents 25 pm. Biochemistry: Vogel and Nirenberg Proc. Natl. Acad. Set. USA 73 (1976) 1809 FIG. 7. Autoradiography of sections of 6- to 17-day-old chick embryo retina, labeled with `251-aBT. Retina sections were subjected te autoradiography for 29 days. The initial specific activity of 1251-aBT was 485 Ci/mmol of toxin. The same magnification was used for each photomicrograph; the bars represent 25 pm. (A) Phase contrast view iIf a stained autoradiograph of 6-day-old embryo retina. (B) Bright t ield view of the area shown in panel A before staining. (C) Phase contrast view of a stained autoradiograph of g-day-old embryo retina. (D) Bright field view of the same area shown in panel C before stain- ing. (E) Bright field view of a stained section of LB-day-old embryo retina not subjected to autoradiography. (F) Bright field view of the corresponding area of an unstained autoradiograph of an adjacent section. (G) and (H) 17-Day-old embryo retina sections, treated as in panels E and F, respectively. labeled, Some cells in the outer synaptic layer, thought to be horizontal neurons, also are labeled. The concentration of ganglion neurons is considerably higher in the center of the eye compared to the periphery; however, the concentration of silver grains in the inner plexiform layer does not change markedly (