Proc. Natl. Acad. Sci. USA Vol. 75, No. 2, pp. 1016-1020, February 1978

Neurobiology

Nonequivalence of a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons (acetylcholine sensitivity/ganglionic transmission/glutaraldehyde crosslinking)

S. T. CARBONETTO, D. M. FAMBROUGH, AND K. J. MULLER Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, Maryland 21210

Communicated by Donald D. Brown, November 28, 1977

ABSTRACT a-Bungarotoxin binds selectively to chick sympathetic neurons that are responsive to iontophoretically applied acetylcholine. a-Bungarotoxin (125 nM) does not affect the response of cultured neurons to acetylcholine, nor does it affect a cholinergic synaptic potential recorded from sympathetic ganglia. d-Tubocurarine (100 ,uM) inhibits a-bungarotoxin binding and blocks acetylcholine receptor function in both preparations, but a-bungarotoxin does not protect acetylcholine receptors against d-tubocurarine blockade of acetylcholine responses. The receptor for a-bungarotoxin can be extracted from neuronal membranes with nonionic detergents and, when assayed by velocity sedimentation in sucrose gradients, sediments at a rate faster than that of skeletal muscle acetylcholine receptors. Treatment of a-bungarotoxin-receptor complexes with glutaraldehyde (0.1%, wt/vol) increases their stability from a half-time for dissociation of 3.5 hr to greater than 6 days at 230. This permits a quantitative assay of a-bungarotoxin-receptor complexes after relatively long periods of velocity sedimentation. It is concluded that a-bungarotoxin does not bind to the acetylcholine-binding site of neuronal acetylcholine receptors. These results compel a reevaluation of studies that assume that a-bungarotoxin is a specific ligand for neuronal acetylcholine receptors.

a method for stabilizing them so that they can be characterized conveniently. Our efforts in this regard as well as our failure to establish an equivalence between the neuronal AcCh receptor and the a-BuTX receptor are documented here.

METHODS Cell Culture. Primary cultures of chick sympathetic neurons were made from 11- to 15-day embryos after the methods of Varon and Raiborn (14). Cells were grown in Eagle's minimal essential medium plus 2% embryo extract, 10% horse serum or 5% fetal calf serum, 2 units of nerve growth factor (Burroughs-Wellcome) per ml, and 50 mg of gentamicin (Schering) per ml at pH 7.2. Autoradiography. Light microscope autoradiographs were prepared by incubating cultures with 125I-labeled a-BuTX (125I-a-BuTX) for 60 min at 370, washing cultures thoroughly with Hanks' solution, and then fixing the cultures for 60 min at 50 in 2% (wt/vol) paraformaldehyde/1% (wt/vol) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2. They were then dehydrated in alcohols and coated with Kodak NTB-2 emulsion diluted 1:1 with water. Autoradiographs were exposed for 7 days and developed in Kodak D-19 developer for 2.5 min at 18°, fixed, and washed. Physiology. For intracellular recording, the culture dish was mounted on the stage of an inverted phase microscope. A warming collar around the dish maintained its temperature at 370, and this sat inside a larger chamber around which 95% air/5% CO2 flowed, equilibrating with the minimal essential medium in the bath to maintain a constant pH. Recording electrodes were about 100 MQ) and were filled with 2 M potassium acetate. AcCh electrodes were filled with 2 M acetylcholine chloride (Merck) and had resistances of 80-100 MQ. A small holding current was applied to the electrode to prevent leakage of AcCh from the tip. For extracellular recording, a chain of lumbosacral sympathetic ganglia was dissected from 18- to 20-day embryos. This was pinned in a Sylgard (Dow) coated chamber, bathed in a known volume of chick Ringer's solution (15), and gassed with 95% 02/5% CO2. One suction electrode was placed on the interganglionic connective (preganglionic) and another suction electrode on the ventral root leading from one of the ganglia (postganglionic). Conventional electronic apparatus was used for extracellular and intracellular recording, stimulation, and acetylcholine iontophoresis. Measurement of Dissociation Rate. Neuronal cultures were incubated with 3 nM 125I-a-BuTX for 60 min at 370. They were washed four times for 2 min each at 230 to remove unbound a-BuTX and equilibrated at 370. At time zero, 1 ml of medium (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acidbuffered Hanks' solution/0. 1% bovine serum albumin, pH 7.0)

a-Bungarotoxin (a-BuTX) satisfies three criteria that certify it as a ligand for the acetylcholine (AcCh) receptor in muscle: (i) its binding to muscle is saturable and agonists and antagonists of AcCh compete for this binding; (ii) it binds to cells and regions of cells that respond physiologically to AcCh; (iii) it blocks the physiological response of muscle to AcCh. a-BuTX binds with a high affinity to neuronal as well as to muscle membranes, and this binding is competitively inhibited by cholinergic agonists and antagonists (1-12). On the basis of the binding kinetics of a-BuTX, its subcellular distribution, and the hydrodynamic properties of its receptor, it has been inferred that a-BuTX binds to neuronal AcCh receptors (1, 3-11). There is, however, little physiological evidence to support this inference. The strongest case for a-BuTX as a ligand for neuronal AcCh receptors is in the chick sympathetic nervous system, where it is clear that a-BuTX binds selectively to neurons (1) that are responsive to AcCh (13). One of the goals of the present study was to test the hypothesis that a-BuTX receptors are, indeed, neuronal AcCh receptors. The a-BuTX receptor, regardless of its function, is a specific component of the plasma membrane of chick sympathetic neurons. We began this study to determine where and how membrane proteins are metabolized during the period of rapid outgrowth of neuronal processes. In keeping with this, our initial goal was to identify a-BuTX-receptor complexes and devise The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: a-BuTX, a-bungarotoxin; AcCh, acetylcholine; d-TC, d-tubocurarine.

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FIG. 1. Autoradiographs of cultured chick sympathetic neurons incubated with 125I-a-BuTX. When viewed with phase optics the neuron (N) and a single non-neuronal cell (F) are visible (Upper Left). With bright-field illumination only the exposed silver grains are visible and these cover the neuron (Lower Left). Preincubation of neurons with nonradioactive a-BuTX at 125 nM blocks subsequent labeling with radioactive a-BuTX (Right). Calibration bar is 30 Am.

plus or minus 100 ,uM d-tubocurarine (d-TC) was pipetted onto each plate. The medium was drawn off at each time point and replaced with fresh medium. Samples were centrifuged at 10,000 X g for 5 min to sediment cell debris and an aliquot of the supernatant was assayed for radioactivity in scintillation vials containing 3 ml of Aquasol (New England Nuclear) in a Packard Tri-Carb scintillation spectrometer. At the end of the sampling period the cells were dissolved in 1 M NaOH and assayed for radioactivity. Nonspecific binding of a-BuTX was estimated by preincubating parallel sets of cultures with 100 gM d-TC for 20 min and then incubating with a-BuTX (3 nM) in the presence of d-TC (100 AtM). Nonspecific binding was less than 10% of specific binding of a-BuTX and was subtracted from specific binding. Preparation of a-BuTX-Receptor Complexes. Iodinated derivatives of a-BuTX were prepared as previously described (16). Neuronal a-BuTX-receptor complexes were prepared by dissecting lumbosacral chains from 13- to 20-day embryos, which were then homogenized in 10 mM Tris-HCI/1 mM phenylmethylsulfonyl fluoride/i mM EDTA, pH 7.8, and the homogenate was centrifuged at 27,000 X g for 60 min. The resulting membrane pellet was extracted in 1% (vol/vol) Triton X-100/1 mM phenylmethylsulfonyl fluoride/i mM EDTA (Sigma). The extract was centrifuged at 27,000 X g for 60 min and the supernatant was divided into equal aliquots for incubation with 1.3 nM radioactive a-BuTX for 25-60 min at 37'. Free a-BuTX was separated from a-BuTX-receptor complexes by chromatography on a Bio-Gel P-60 column. Crosslinked a-BuTX-receptor complexes were prepared by incubating ganglionic chains with radioactive a-BuTX, washing away the

unbound a-BuTX, and extracting receptors directly from ganglia with Triton X-100. The extract was centrifuged for 45 min at 27,000 X g at 50 and the supernatant was treated with 0.1% glutaraldehyde (Electron Microscopy Sciences) for 20 min at 23'. Muscle a-BuTX-receptor complexes were prepared by similar methods, as described previously (16), and subsequently were incubated with unlabeled a-BuTX to ensure that all receptors were saturated. Sucrose Gradient Centrifugation. Velocity sedimentation was carried out by layering a-BuTX-receptor complexes in a thin band over a 5-20% linear sucrose gradient (5 ml) or 25-40% linear sucrose gradients in deuterium oxide (11 ml) both containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA. Five to 20% sucrose gradients were centrifuged in a Beckman SW 50.1 rotor at 48,000 rpm for 5-6 hr at 80. Twenty-five to 40% sucrose/deuterium oxide gradients were centrifuged in a Beckman SW 41 rotor at 40,000 rpm for 48 hr at 100. Thirty to 40 fractions were collected and counted in a Packard -y scintillation spectrometer. Data were corrected for crossover between the 1311 and 125I channels. RESULTS The presence of receptors on the plasma membrane can be inferred from autoradiographs of neurons incubated with radioactive a-BuTX (Fig. 1). 125I-Labeled a-BuTX binds selectively to neurons and not to the other cells in the culture. When cultures are pretreated with unlabeled a-BuTX (125 nM) or d-TC (10MuM), binding of labeled a-BuTX is blocked (Fig. 1). This suggests that the receptor is: (i) saturable at less than 125 nM a-BuTX [in fact, we have confirmed previous observations

Proc. Nati. Acad. Sci. USA 75 (1978)

Neurobiology: Carbonetto et al.

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C FIG. 2. The effect of d-TC and a-BuTX on the AcCh response in neurons. (A) Responses of two neurons to short pulses of iontophoretically applied AcCh in the absence (control) and presence of 125 nM a-BuTX. Upper trace: 100 nA, 10 msec; lower trace: 10 mV, 10 msec. (B) Response of two neurons to release of the AcCh holding current in the absence (control) and presence of 125 nM a-BuTX. Upper trace: onset and offset of the release of the holding current; lower trace: 10 mV, 5 sec. (C) Response of one neuron to short pulses of AcCh applied before (Left) and 3.5 min after (Right) the addition of d-TC (100 AM) in the presence of 125 nM a-BuTX. Upper trace: 50 nA, 10 msec; lower trace: 10 mV, 2 msec.

(1) that a-BuTX binding to sympathetic neurons, incubated for 1 hr at 370, is saturable at about 5 nM]; (ii) located on the surface membrane, because d-TC is a water-soluble molecule and probably does not penetrate into the cell; and (iii) an AcCh receptor, because d-TC is a well-known antagonist of AcCh receptors. The presence of AcCh receptors in the plasma membranes of chick sympathetic neurons can be independently demonstrated by physiological methods. Neurons in culture are easily identified by their spherical cell bodies that refract light (Fig. 1). Penetration of these cells with a microelectrode reveals that they have membrane potentials of -40 to -70 mV and generate overshooting action potentials in response to intracellular stimulation. As early as 3 days in culture, pulses of AcCh applied iontophoretically onto neurons produce graded depolarizations that can give rise to action potentials (Fig. 2A). With prolonged pulses of AcCh (Fig. 2B) the response desensitizes and, after the pulse, the membrane hyperpolarizes. Both the long and the short duration depolarizations are blocked by d-TC (100 ,uM) (Fig. 2C). The short latency of the AcCh response, its similarity to synaptic potentials, and its antagonism by d-TC are generally regarded as demonstrative of AcCh receptors in the plasma membrane. a-BuTX acts as antagonist of the AcCh response in muscle, where it binds to AcCh receptors. Because it also appears to bind to neuronal AcCh receptors, it should also antagonize AcCh responses in neurons. However, neurons incubated with 125 nM a-BuTX for more than 60 min (a dose that saturates receptors; see Fig. 1 and also ref. 1) still respond to iontophoresis of AcCh (Fig. 2 A and B). Measurement of AcCh sensitivity in a number of neurons showed that there is no significant difference be-

60 Time, min

FIG. 3. Effect of d-TC on the dissociation of 1251-a-BuTX from chick sympathetic neurons. 0, Hanks' solution; 0, Hanks' solution plus 100MLM d-TC.

tween neurons treated with a-BuTX (137 32 mV/nC, n = 12) and untreated neurons (109 ± 20 mV/nC, n = 12). Furthermore, a-BuTX is unable to protect receptors from d-TC, which blocked responses even in neurons pretreated and coincubated with a-BuTX (Fig. 2C). It is not that d-TC appreciably displaces a-BuTX from receptors in the 3-5 min d-TC takes to act, because it only decreases the half-time for dissociation of a-BuTX from 104 to 58 min at 370 (Fig. 3). Because a-BuTX also binds specifically to chick sympathetic ganglia (2), the effect of a-BuTX was tested on an extracellular "ganglionic potential" that can be recorded from the postganglionic root of excised lumbosacral ganglia. This potential is bimodal and similar to extracellular potentials recorded from chick ciliary ganglia (15). The second component of this response is thought to be mediated by chemical synapses because antidromic stimulation of the postganglionic root produces only a unimodal response in the connective with the same latency as the first component of the orthodromic response, and d-TC (100 /AM) reversibly blocks the second component of the response (Fig. 4A). The first component of this response may be due to axonal through-fibers or, as in the ciliary ganglion, electrical synapses; the second component seems to be mediated by cholinergic synapses. High doses of a-BuTX (12 ,uM) have no effect on the ganglionic potential (Fig. 4B), although the toxin penetrates into the ganglionic neuropil, as seen by electron microscope autoradiography (unpublished observations; see also ref. 18). d-TC evidently acts at the synapse and does not simply block axonal conduction, because the compound action potential recorded from a length of postganglionic nerve is unaffected by d-TC (100 ,uM), but is abolished by lidocaine (100 -

/AM).

The present study and previous observations (1, 2) have shown that a-BuTX binds to a membrane component specifically located in sympathetic neurons. We have extracted this toxin-bound macromolecule with nonionic detergents from membranes of sympathetic neurons, and it can be readily identified on sucrose gradients with a peak that is completely

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Proc. Natl. Acad. Sci. USA 75 (1978)

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d-TC

A 104

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I40 AV 40 msec FIG. 4. Effect of d-TC and a-BuTX on the ganglionic response of chick sympathetic ganglia. (A) Ganglionic responses before and 5 min after application of 100 kiM d-TC. (B) Ganglionic responses before and 1 hr after application of 12 ,uM a-BuTX.

absent when membrane preparations are treated with 100 JAM d-TC (Fig. 5A). This concentration of d-TC also blocks a-BuTX binding to neurons in culture. The second peak in these gradients is only slightly affected by d-TC and represents unbound a-BuTX as well as "nonspecific" binding of a-BuTX to low molecular weight proteins. 125I-a-BuTX can be made to label its receptor covalently by crosslinking at-BuTX-receptor complexes with glutaraldehyde (0.1%). Crosslinked a-BuTXreceptor complexes sediment at the same rate as untreated a-BuTX-receptor complexes (Fig. 5B). Before crosslinking, a-BuTX-receptor complexes dissociate with a half-time of 3.5 hr at 230; afterwards they dissociate with a half-time of greater than 6 days. This is very helpful in assaying molecules having similar sedimentation rates that are distinguished by velocity sedimentation for long periods of time (Fig. 5C). DISCUSSION One component of synaptic transmission in chick sympathetic ganglia is reversibly blocked by d-TC, indicating that sympathetic ganglion cells may contain nicotinic AcCh receptors. In culture, chick sympathetic neurons will, within 3 days of plating, respond to focally applied AcCh (see also ref. 13). This response is reversibly blocked by d-TC (100 AM) as expected for nicotinic receptors. The present experiments also confirm that a-BuTX, which binds to nicotinic receptors in muscle, will bind selectively to chick sympathetic neurons rather than fibroblasts in culture. Moreover, this binding to a single site is saturable and is subject to competition with physiologically effective concentrations of d-TC. In contrast to muscle AcCh receptors, the a-BuTX-receptor complex in neurons dissociates with a half-time of about 2 hr, rather than days or weeks. aBuTX receptors can be extracted from the membranes of sympathetic neurons and routinely assayed by velocity sedimentation in sucrose gradients. As in vivo, the stability of the solubilized complex is low, but it can be increased by glutaraldehyde fixation. By analogy with muscle AcCh receptors, a-BuTX should block the response of chick sympathetic neurons to AcCh. In intact ganglia or in neuronal cultures it does not, even at concentrations in excess of those that saturate toxin binding sites. a-BuTX also does not block the response of rat superior cervical ganglia to bath-applied carbachol, an AcCh agonist, nor does

Fraction

FIG. 5. (A) Velocity gradient sedimentation of neuronal and muscle a-BuTX receptors in 5-20% sucrose gradients. 0-0, Neuronal receptors labeled with 1311-a-BuTX; --- -0, muscle receptors labeled with 1251-BuTX; 0-0, neuronal receptors labeled in the presence of 100 ,M d-TC. (B) Velocity gradient sedimentation of glutaraldehyde crosslinked a-BuTX receptors in 5-20% sucrose gradients. *-*, Neuronal receptors labeled with 1251-a-BuTX; O--- -0, muscle receptors labeled with 131I-BuTX. (C) Velocity gradient sedimentation of muscle and glutaraldehyde crosslinked neuronal a-BuTX receptors in 25-40% sucrose deuterium oxide gradients. 0-0, Neuronal receptors labeled with 1251-a-BuTX; --- -0, muscle receptors labeled with 13I-a-BuTX.

it affect other ganglionic AcCh receptors (17, 18). More significant, however, is our finding that a-BuTX does not interfere with the d-TC block of the response to applied AcCh, which is unexpected because d-TC and a-BuTX compete for a common site. d-TC cannot rapidly displace a-BuTX from that site; d-TC reduces the half-time for a-BuTX dissociation by a factor of only 2. Yet, d-TC rapidly acts to block the AcCh response by binding to the AcCh receptor, and this occurs whether a-BuTX is there or not. A simple model in which d-TC and a-BuTX compete for a single, physiologically active site is not consistent with the present data. One possibility is that d-TC binds to two sites on the AcCh receptor, one of which is not important to the action of AcCh but does bind a-BuTX. Multiple a-BuTXbinding sites have been demonstrated for AcCh receptors from Torpedo californica, but these have different affinities (19), whereas a-BuTX binds to neurons with a single affinity. Furthermore, there is no evidence that either of the a-BuTX sites on Torpedo AcCh receptors is physiologically inactive. Another possibility is that d-TC binds to two separate molecules, the AcCh receptor and another molecule that binds a-BuTX. This possibility seems quite likely now, on the basis of recent experiments by Patrick and Stallcup (20) showing that antibodies to AcCh receptors block carbachol-induced uptake of 22Na by cell line PC12 but do not crossreact with a-BuTX-binding sites. The role of the a-BuTX receptor is puzzling, given its similarities to muscle AcCh receptors and its localization at synaptic regions in chick retina (21) and rat brain (22). There remains an exciting possibility that it is involved in modulating synaptic transmission or in long term nerve-nerve interactions. On the basis of the present data, we conclude that a-BuTX does not bind to the ligand-binding site of neuronal AcCh receptors. This is surprising, because the ability of AcCh receptor agonists and antagonists to block a-BuTX binding to neurons indicates that it should bind to this site if it binds to AcCh receptors at all. These results compel a reconsideration of a number of studies in which a-BuTX has been used to characterize and purify neuronal AcCh receptors (5-11, 23, 24). Also

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open to question are studies in which a-BuTX has been used to study the subcellular and regional distribution of AcCh receptors in the nervous system (3,4, 6, 10; 25-27). Although in chick sympathetic cultures a-BuTX binds to neurons that have AcCh receptors, this might not be true elsewhere. Clearly the relationship between the molecule that binds a-BuTX and the AcCh receptor ought to be determined, and this might be done partly by immunological and further by physicochemical characterization. Ultimately, some physiological test would be required to show that a-BuTX binding sites occur on the physiologically important AcCh receptors. We thank Dr. Richard Rotundo for his helpful suggestions throughout this work. S.C. is supported by a National Institutes of Health Postdoctoral Fellowship. Research in the authors' laboratories is supported in part by a grant from the Whitehall Foundation. 1. Greene, L. A., Sytkowski, A. J., Vogel, Z. & Nirenberg, M. W.

(1973) Nature 243, 163-166. 2. Greene, L. A. (1976) Brain Res. 111, 135-145. 3. Kouvelas, E. D. & Greene, L. A. (1976) Brain Res. 113, 111126. 4. Salvaterra, P. M. & Moore, W. J. (1973) Biochem. Biophys. Res.

Commun. 55, 1311-1318. 5. Bosmann, H. B. (1972) J. Biol. Chem. 247, 130-145. 6. Salvaterra, P. M., Mahler, H. R. & Moore, W. J. (1975) J. Biol.

Chem. 250, 6469-6475. 7. Salvaterra, P. M. & Mahler, H. R. (1976) J. Biol. Chem. 251,

6327-6334. 8. McQuarrie, C., Salvaterra, P. M., DeBlas, A., Routes, J. & Mahler, H. R. (1976) J. Biol. Chem. 251, 6335-6339.

9. Seto, A., Arimatsu, Y. & Amano, T. (1977) Neurosci. Lett. 4, 115-119. 10. Eterovic, V. A. & Bennett, E. L. (1974) Biochim. Biophys. Acta

362,346-355. 11. Moore, W. M. & Brady, R. N. (1977) Biochim. Biophys. Acta 498, 331-340. 12. Lowy, J., McGregor, J., Rosenstone, J. & Schmidt, J. (1976) Biochemistry 15, 1522-1527. 13. Chalazonitis, A., Greene, L. A. & Nirenberg, M. (1974) Brain Res. 68,235-252. 14. Varon, S. & Raiborn, C. (1972) J. Neurocytol. 1, 211-221. 15. Martin, A. R. & Pilar, G. (1963) J. Physiol. (London) 168, 443-463. 16. Devreotes, P. N. & Fambrough, D. M. (1975) J. Cell Biol. 65,

5-358. 17. Brown, D.A. & Fumagalli, L. (1977) Brain Res. 129, 165-168. 18. Bursztajn, S. & Gershon, M. D. (1977) J. Physiol. (London) 269,

17-31. 19. Raftery, M. A., Vandlen, R. L., Reed, K. L. & Lee, T. (1975) Symp. Quant. Biol. 40, 193-202. 20. Patrick, J. & Stallcup, W. B. (1977) Proc. Natl. Acad. Sci. USA

74,4989-4692. 21. Vogel, Z., Maloney, G. J., Long, A. & Daniels, M. P. (1977) Proc. Natl. Acad. Sci. USA 74,3268-3272. 22. Lentz, T. L. & Chester, J. (1977) J. Cell Biol. 75, 258-267. 23. Romine, W. O., Goodall, M. C., Peterson, J. & Bradley, R. J. (1974) Biochim. Biophys. Acta 367,316-325. 24. Goodall, M. C., Bradley, R. J., Saccomani, G. & Romine, W. 0. (1974) Nature 250,68-69. 25. Wang, G. K. & Schmidt, J. (1976) Brain Res. 114,524-529. 26. Vogel, Z. & Nirenberg, M. (1976) Proc. Natl. Acad. Scd. USA 73, 1806-1810. 27. Polz-Tejera, G., Schmidt, J. & Karten, H. J. (1975) Nature 258, 349-351.

Nonequivalence of alpha-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons.

Proc. Natl. Acad. Sci. USA Vol. 75, No. 2, pp. 1016-1020, February 1978 Neurobiology Nonequivalence of a-bungarotoxin receptors and acetylcholine re...
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