Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3542-3546, July 1979

Neurobiology

Localization of horseradish peroxidase-a-bungarotoxin binding in crustacean axonal membrane vesicles and intact axons (a-neurotoxin/peripheral nerve membrane/microsacs)

JANICE CHESTER*, THOMAS L. LENTZ*, JUDITH K. MARQUISt, AND HENRY G. MAUTNERt *Section of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510; and tDepartment of Biochemistry and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts 02111

Communicated by David Nachmansohn, April 19, 1979

ABSTRACT A conjugate of a-bungarotoxin with horseradish peroxidase was used to visualize a-bungarotoxin binding sites at the fine structural level in isolated axonal membrane vesicles from lobster walking leg nerve. These plasma membrane vesicles have previously been shown to exhibit saturable binding of [3H nicotine and [3H]acetylcholine. Binding of the toxin was identified in the axon plasma membrane and could be blocked by pretreatment with excess free a-bungarotoxin or d-tubocurarine. Binding sites for a-bungarotoxin were identified by the same technique in sections of intact nerve fibers from both lobster and spider crab and were found to be localized primarily in the axolemma rather than in the Schwann cell membrane. Several lines of evidence are suggestive of the presence of a cholinergic system in lobster axons. For example, acetylcholine (AcCho) is released from cut nerves (1). Acetylcholinesterase is localized near or on the surface of the axolemma (2). Working with lobster nerve fibres, Dettbarn (3, 4) showed that AcCho and d-tubocurarine (d-TC) could affect electrical conduction, even though the Schwann cell enveloping the axon is more permeable to tertiary than to quaternary amines (4, 5). The lobster axonal membrane contains a component that binds nicotine and other cholinergic ligands (6-9) and constitutes 1% of the membrane proteins (7). It has also proved possible to demonstrate direct binding of radioiodinated a-bungarotoxin (a-BuTx) with an apparent KD of 0.27 ,uM to lobster axon plasma membrane fragments and to solubilized membranes (10). This neurotoxin is widely believed to be capable of selective attachment to nicotinic AcCho receptors. The present studies were undertaken to obtain more detailed information regarding the binding of a-BuTx to axonal membrane using lobster and crab walking leg nerves and membrane fragments isolated from the lobster nerves. Binding sites were localized by using a conjugate of a-BuTx with horseradish peroxidase (HRP) (11, 12). This procedure permits the electron microscopic localization of toxin binding sites at high resolution. MATERIALS AND METHODS Nerve Plasma Membrane Fragment Preparation and In-

cubation. The preparation of plasma membrane fragments from lobster (Homarus americanus) walking leg nerve bundles has been described in detail by Denburg (7). The plasma membrane fraction with maximal nicotine binding activity resides in a microsomal (100,000 x g) pellet of a hypotonic (0.32 M sucrose) extract of the nerve tissue. The procedures for separation of a-BuTx from crude Bungarus multicinctus venom and conjugation with HRP have The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate

been described (11). For the localization of AcCho receptor, the pellets were resuspended in 1 ml of 10 mM Tris-HCl (pH 7.8), containing 0.1 ml of the HRP-a-BuTx conjugate, incubated for 0.5 hr with mixing at 4VC, and centrifuged in a Beckman Model L ultracentrifuge at 40,000 rpm for 1 hr. They were then resuspended in 3 times volume of 10 mM Tris-HCl (pH 7.8), washed for 0.5 hr with mixing at 4VC, and centrifuged for 1 hr at 40,000 rpm. Controls consisted of preincubation of suspended membranes for 1 hr in native a-BuTx (0.5 mg in 1 ml of 10 mM Tris1HCl) or d-TC (10 AiM in 10 mM Tris-HCl). The suspensions were centrifuged at 40,000 rpm for 0.5 hr and then resuspended and incubated in the conjugate and washed as described above. Some membranes were incubated in Tris.HCl alone. All the pellets were fixed for 1 hr in 3% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2). Tissue Incubation and Cytochemistry. Axon segments were dissected from the walking legs of the lobster and the spider crab (Libinia emarginata). Several procedures were employed in an effort to obtain both satisfactory morphology and localization of reaction product. These included variation in length of incubation time, brief prefixation in 2% glutaraldehyde or 2% paraformaldehyde or both, and teasing the lobster nerve fibers longitudinally into smaller bundles. Prolonged incubation of unfixed nerve tissue resulted in unsatisfactory preservation of morphology. Prefixation, while preserving structure, did not permit specific localization of reaction product. Adequate preparations were obtained with both crab and lobster nerves by teasing the nerve along the long axis of the bundle to facilitate penetration of the conjugate and by reducing the length of incubation to 0.5 hr. Crab nerves withstood longer periods of incubation (1.5 hr) and, when cut, separated into numerous small fascicles, thereby increasing exposure to the conjugate. Incubation in the conjugate (diluted 1:10 with 10 mM Tris-HCl, pH 7.8) was performed in-the cold (40C) with gentle mixing. After incubation, the nerves were rinsed in 10 mM Tris buffer for 15 min and fixed for 1 hr in 3% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2). The pellets and nerve fiber segments were washed overnight in 0.05 M cacodylate buffer (pH 7.2). Nerves were trimmed into small blocks, and the blocks and membrane pellets were assayed for peroxidase activity by incubation in 3,3'-diaminobenzidine (50 mg/100 ml of Tris buffer, pH 7.2) and H202 (0.01%) for 60-90 min. The tissues were rinsed for 1 hr in buffer and fixed in OS04 for 1 hr. Small pieces were trimmed from the outer regions of the reacted pellets. All of the blocks were dehydrated and infiltrated and embedded in Epon 812. Thin, unstained sections were viewed with a Hitachi HS8 or HUI 1-ES electron microscope. Abbreviations: AcCho, acetylcholine; HRP, horseradish peroxidase; d-TC, d-tubocurarine; a-BuTx, a-bungarotoxin.

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Proc. Natl. Acad. Sci. USA 76 (1979)

RESULTS Membrane fragments were found to form closed vesicles ranging from 0.1 to 0.4 pm in diameter (Figs. 1 and 2). On occasion, particulate material and dense granules resembling lysosomes were also seen. Fractions incubated in the presence of HRP-a-BuTx conjugate and then treated with 3,3'-diaminobenzidine contained many vesicles with dense reaction product lying within the membrane (Fig. 1). Activity occurred over the entire circumference of some profiles, whereas in others, reactive membrane regions were separated by unreac-

tive segments. Because of the variability of labeling in different preparations and different regions of the pellets, it was not possible to assign a ratio of reactive to unreactive profiles. Labeling was reduced or absent in the central core of the pellets, possibly because of insufficient penetration of the diaminobenzidine. In other regions, a few reactive vesicles were scattered among unreactive profiles (Fig. 1A), whereas in some areas the majority of the vesicles were labeled (Fig. 1B).

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FIG. 1. (A) Lobster walking leg nerve membrane fraction incubated in the HRP-a-BuTx conjugate and assayed for peroxidase activity. The fraction consists largely of membranous vesicles, vacuoles, and fragments of various sizes and some particulate material. Some of the profiles are reactive. Reaction product is localized to the membrane forming the vacuoles. (X67,500). (B) Membrane fraction after incubation in the conjugate and assayed for peroxidase. Most of the vesicles in this preparation are reactive. (X41,000.)

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Neurobiology: Chester et al.

3544

Proc. Natl. Acad. Sci. USA 76 (1979)

Reaction product did not occur in membranes incubated in buffer and assayed for peroxidase, indicating that the membrane labeling was not the result of endogenous peroxidase activity (Fig. 2A). Pretreatment with native a-BuTx (Fig. 2B) or with d-TC (Fig. 2C) rendered membrane fragments unreactive to labeling with the HRP-a-BuTx conjugate. In all preparations, some of the larger dense granules were reactive, presumably due to the presence of endogenous peroxidase activity. Dense dots were seen at the edge of some membranous profiles in all preparations and in intact tissues; presumably these are nonspecific products of the preparative or fixation procedures. The site of binding of the HRP-a-BuTx conjugate was also studied in intact lobster and spider crab nerves. Nerve fibers of various sizes usually were grouped in bundles, although some large fibers occurred singly. Schwann cells enveloped the bundles and larger fibers. A looser wrapping of thin connective tissue cell processes was situated outside the Schwann cells. Further details of the fine structural morphology of the lobster walking leg nerve have been described (13). After incubation in the conjugate, reaction product was found within the membranes of both crab and lobster nerve fibers (Fig. 3). In densely labeled specimens, accumulation of reaction product obscured the membrane and extended into the narrow intercellular space. In some cases, the entire circumference of the axon was reactive, whereas in others only portions of the membrane were reactive. Such differences could be due to variable penetration of the conjugate. It was often difficult to distinguish portions of Schwann cells from adjacent axons. Schwann cell cytoplasm, however, could be found enveloping bundles of nerve fibers. In addition, Schwann cells could be identified with certainty and distinguished from nerves in sections that included the entire cell and its nucleus wrapped around an axon (Fig. 4A). In these cases, reaction product outlined and was confined to the axonal pro-

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file. The outer Schwann cell membrane was largely unreactive. At high magnification of the axon-Schwann cell interface, activity was more intense over the axolemma (Fig. 4B). However, the density of the Schwann cell membrane was sometimes enhanced opposite to reactive axolemma and some reaction product occurred in the intercellular space. Endogenous peroxidase activity was found in membranebound structures and was seen frequently in connective tissue cells and Schwann cells and rarely in nerve fibers. These structures probably are the same as those encountered in the vesicle fractions and may represent lysosomes. At the edges of the blocks, activity occurred over collagenous and fibrous extracellular material, possibly the result of nonspecific binding of conjugate. For this reason, peripheral regions of blocks were not included in determining localization of conjugate binding. DISCUSSION These studies demonstrate the binding of HRP-a-BuTx conjugate to vesicles obtained from membrane fragments of lobster walking leg nerves. Binding of the conjugate can be virtually abolished by pretreatment with native BuTx or with d-TC. In a previous study, it was shown that lobster axon plasma membrane fragments are capable of binding AcCho with a KD Of 6.7 ,gM (10). These findings support the notion that we are localizing axonal AcCho binding material, although questions have arisen in recent years regarding the reliability of a-BuTx binding as an absolute indicator of the presence of nicotinic AcCho receptors in nervous tissue (14-18). The sidedness of the vesicular membrane fragments to which the HRP-a-BuTx conjugate is bound has not been established unequivocally. However, the earlier finding that tertiary amine local anesthetics and their less permeable quaternary ammonium analogues have equal abilities to displace nicotine from such membrane fragments (9) suggests that the anionic site to

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Proc. Natl. Acad. Sci. USA 76 (1979)

Neurobiology: Chester et al.

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FIG. 4. (A) Large lobster axon (Ax) enveloped by a Schwann cell (SC). The nerve was incubated in conjugate and assayed for HRP. The outer contour of the axon is outlined by dense reaction product. The outer membrane of the Schwann cell is devoid of activity. The axon contains

large numbers of microtubules. N, Schwann cell nucleus (X9000.) (B) Portion of a large crab axon (Ax) and adjacent Schwann cell (SC), identified by its nucleus (N), was incubated in conjugate and assayed for HRP. Note that the axolemma is more dense than the membranes of the Schwann cell and connective tissue (CT) cell. (X42,500.)

which binding occurs resides on the outside of the vesicles. The observation that HRP-a-BuTx conjugate binds to the axonal membranes of the walking leg nerve of the spider crab (Libinia emarginata) is of interest, because Lazdunski and his collaborators (8) reported the absence of nicotine binding to axonal membranes isolated from the spider crab Maia squinado. With the HRP-a-BuTx technique, a different localization of reaction product is observed in nerve tissue and at the postsynaptic surface of the neuromuscular junction. In the latter, activity occurs both within the membrane and in a thin overlying layer in a configuration that is consistent with a transmembrane location of receptors projecting slightly above the membrane (11). In motor axon terminals (11), presynaptic processes of central synapses (12), and the axonal membranes observed in the present study, reaction product is restricted to the membrane without an overlying layer. The two patterns of localization of activity after HRP-a-BuTx binding could be due to a different positioning or nature of receptors in the neuronal membranes and at the neuromuscular junction. Such differences in position or accessibility of the a-BuTx binding sites might account for the presence or absence of an effect of a-BuTx on receptor function. In isolated nerve membrane fragments, a number of membrane profiles bound the HRP-a-BuTx conjugate whereas others were unreactive. It is likely that these fractions contain both axonal and Schwann cell membranes, although it cannot be determined to which the reaction product is localized. Study of intact nerves, however, clearly reveals binding of the con-

jugate to the axolemma. Where Schwann cell membranes could

be identified and distinguished from axolemma, they were generally unreactive. In some instances, however, Schwann cell membranes appeared to contain reaction product where they were adjacent to reactive axolemma. This activity could be due to diffusion of reaction product from the axolemma. Rawlins and Villegas (19) recently demonstrated the binding of 125I-labeled a-BuTx at the axon-Schwann cell boundary in squid nerve fibers. The suggestion by Villegas (20) that AcCho receptors might play an essential role in the Schwann cell hyperpolarization that is seen after axonal excitation may be relevant to our findings. However, in contrast to their report that '25I-labeled a-BuTx binding is located primarily on the Schwann cell membrane (19, 20), our experiments show more binding of HRP-a-BuTx conjugate to the axonal membrane. The postulate of Nachmansohn (21) that AcCho plays an essential role in axonal conduction has been the subject of considerable controversy. The results reported here, in conjunction with earlier data (9, 10), are compatible with the presence of cholinergic binding material in axonal membranes. This work was supported in part by grants from the National Science Foundation: BNS 78-13729 (to T.L.L.) and BNS 77-22356 (to H.G.M.). 1. Dettbarn, W.-D. & Rosenberg, P. (1966) J. Gen. Physiol. 50, 447-460. 2. DeLorenzo, A. J. D., Dettbarn, W.-D. & Brzin, M. (1968) J. Ultrastruct. Res. 28, 27-33. 3. Dettbarn, W.-D. (1960) Biochim. Biophys. Acta 41,377-386.

3546

Neurobiology: Chester et al.

4. Dettbarn, W.-D. (1960) Nature (London) 186,891-892. 5. Rosenberg, P. & Mautner, H. G. (1967) Science 155, 15691571. 6. Denburg, J. L., Eldefrawi, M. E. & O'Brien, R. D. (1972) Proc. Natl. Acad. Sci. USA 69, 177-181. 7. Denburg, J. L. (1972) Biochim. Biophys. Acta 282, 453-458. 8. Balerna, M., Fosset, M., Chicheportiche, R., Romey, G. & Lazdunski, M. (1975) Biochemistry 14,5500-5511. 9. Marquis, J. K., Hilt, D. C., Papadeas, V. A. & Mautner, H..G. (1977), Proc. Natl. Acad. Sci. USA 74,2278-2282. 10. Marquis, J. K., Hilt, D. C. & Mautner, H. G. (1977) Biochem. Biophys. Res. Commun. 78,476-482. 11. Lentz, T. L., Mazurkiewicz, J. E. & Rosenthal, J. (1977) Brain Res. 132, 423-442. 12. Lentz, T. L. & Chester, J. (1977) J. Cell Biol. 75, 258-267.

Proc. Nati. Acad. Sci. USA 76 (1979) 13. DeLorenzo, A. J. D., Brzin, M. & Dettbarn, W.-D. (1968) J. Ultrastruct. Res. 24,367-384. 14. Hess, G. P. & Andrews, J. P. (1977) Proc. Natl. Acad. Sci. USA

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15. Carbonetto, S. T., Fambrough, D. M. & Muller, K. J. (1978) Proc. Natl. Acad. Sci. USA 75, 1016-1020. 16. Yazulla, S. & Schmidt, J. (1977) Brain Res. 138,45-57. 17. Hanley, M. R., Bennett, E. L. & Lukasiewicz, R. J. (1978) Soc. Neurosci. Abstr. 4,514. 18. Patrick, J. & Stallcup, W. B. (1977) Proc. Natl. Acad. Sci. USA

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19. Rawlins, F. A. & Villegas, J. (1978) J. Cell Biol. 77,371-376. 20. Villegas, J. (1977) in Cholinergic Mechanisms and Psychopharmacology, ed. Jenden, D. J. (Plenum, New York), pp. 387-399. 21. Nachmansohn, D. (1955) Harvey Lect. 49,57-99.