, SbllTHSONIAN SCI E NCE INFORMATIO:~ EXCHA U.S. DEPARTMENT OF PROJECT ?ROJECT NUMBER Do NOT use this space HEALTH EDUCAT I C:l AND WELFARE `"""`~O~~~~T~~SERV'CE ZOl INTRAYURAL RESEARCH PROJECT NUMBER HL 00001-05 LBG PER IO0 COVERED July 1, 1975 through June 30, 1976 TITLE OF PROJECT (80 characters or less) Acetylcholine Receptors NAMES, LABORATORY AND 1NSTlTUTE AFFILIATIONS, AND TITLES OF PRlNClPAL INVESTIGATORS AND ALL OTHER PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT PI: Mathew P. Daniels OTHER: Marshall W. Nirenberg P. Nelson C. Christian G. Maloney Zvi Vogel Staff Fellow LBG NHLI Chief, Lab. of Biochem. Genetics LBG NHLI Chief, Behavioral Biology Branch LBG NHLI Special Fellow BB NICHD NIH Postdoctoral Fellow LBG NHLI Assistant Professor Weizmann Inst& tute COOPERATING UNITS (if any) Behavioral Biology Branch, NICHD Neurobiology Unit, Weizmann Institute of Science LAB/BRANCH Laboratory of Biochemical Genetics SECT tON Section on Molecular Biology iNSTlTUTE AND LOCATION NHLI, NIH, Bethesda, Maryland 20014 TOTAL MANYEARS: 1 PROFESSI ONAL: IOTHER: 2.0 1.5 I 0.5 SUMMARY OF WORK (200 words or less - underline keywords) Our aim is to study the distribution of nicotinic acetylcholine receptors in intact and cultured tissues of the peripheral and central nervous system in relationship to the development and function of synapses. To this purpose histochemical localization of a-bungarotoxin bound to the receptors is used in conjunction with light and electron microscopy. In the past year we have studied the ultrastructural distribution of receptors on cultured skeletal muscle fibers and have initiated the following investigations: 1) location and characterization of synapses formed by neuroblastoma hybrid cells in culture 2) modification of histochemical methods in order to permit ultrastructural analysis of receptor distribution in the central nervous system and 3) analysis of the distribution of receptors in the visual system of the goldfish with re- lationship to optic nerve damage and regeneration. PHS-6040 (12-75) ZOl HL 00001-05 LBG Project Description: 125 Objectives: Investigators in this laboratory and others have utilized I labelled a-bungarotoxin (aBT) as a label for nicotinic acetylcholine receptors in intact and cultured skeletal muscle, and in embryonic and mature retina. The objectives of this study were to devise a histochemical technique of greater sensitivity and resolution for localizing bound aBT and to apply this technique to studying the ultrastructural distribution of acetylcholine receptors in the peripheral and central nervous system during development, in culture, and in the mature state. Methods Employed: We have employed indirect immunoperoxidase staining of cryostat sectioned, teased, or monolayered cultured materials to which aBT has been bound. These materials are subsequently examined by light and elec- tron microscopy. Major Findings: Acetylcholine receptor-rich regions on the surface of muscle fibers grown in cultu previously been observed by light micro- scope autoradiography with [ fs5had I]-labelled aBT. The appearance of these regions could be explained either by (1) the localized presence of complex folds in the plasma membrane or (2) a high local concentration of receptors iri the plasma membrane, unrelated to membrane folding. Using the aBT-iumrunoper- oxidase technique with light- and electronmicroscopy we have shown that hy- pothesis 2 is correct; the plasma membranes of these regions contain at least 7 times the concentration of receptors found in other regions, with no distinc- tions in cell surface topography. Significance to Biomedical Research: Knowledge of ultrastructural distri- bution of acetylcholine receptor is of clear importance in any attempt to understand the role of neurotransmitters and their receptors in the function and development of the nervous system. The a-bungarotoxin-immunoperoxidase technique already has shown promise for the diagnosis and analysis of mecha- nisms in human neuromuscular disorders. Proposed Course: (1) We are using the aBT-immunoperoxidase technique. to help locate and characterize the ultrastructure of synapses which have been detected electrophysiologically in cultures of neuroblastoma hybrid cells with skeletal muscle fibers. (2) We are developing new reagents to adapt the histochemical technique to ultrastrucutral visualization of acetylcholine receptor sites in mature and developing central nervous system tissues. (3) We plan to study the distribution of acetylcholine receptors in the visual system of the goldfish with relationship to the destruction and reformation of synapses during optic nerve degeneration and regeneration. Publications: 1. Ringel, S. P., Bender, A. N., Festoff, B. W., Engel, W. K., Vogel, Z. and Daniels, M. P.: Ultrastructural demonstration and analytical application of extrajunctional receptors of denervated human and rat skeletal muscle fibres. Nature 255: 730-731, 1975. 2 ZOl HL 00001-05 LBG 2. Vogel, Z. and Daniels, M. P.: The ultrastructure of acetylcholine recep- tor clusters on cultured muscle fibers. J. Cell Biol. 69: in press. 3. Vogel, Z. and Daniels, M. P.: The acetylcholine receptor of intact and cultured chicken retina cells. Proc. VI Int. Cong. Pharmacol. Vol. 1 Receptors and Cellular Pharmacology. Pergamon Press, New York, 1976, pp. 59-66. 3