7 SMITHSONIAK SCI NCE INFORMATION EXCHA GE r U.S. DEPARTMENT OF PROJECT NUMBER PROJECT NUMBER 00 NOT use thia space HEALTH EDUCATION AND WELFARE PtBLl#44T$SERVICE INTRAYURAL RESEARCJi PROJECT ZOl HI, 00017-04 LBG PERIOD COVERED October 1, 1978 - September 30, 1979 TITLE OF PROJECT (80 characters or leas) Acetylcholine Receptors NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES CF PRINCIPAL INVESTIGATORS AND AU OTHER PROFESSIONAL PERSONNEL ENGAGED ON TtiE PROJECT PI: Mathew P. Daniels Research Biologist LBG NHLBI OTRRRS: P. Nelson Chief, Laboratory of LDN ??ICHD Developmental Neuro- biology C. Christian Senior Staff Fellow LDN NICHD Z. Vogel Assistant Professor Weizmann Institute Marshall Nirenberg Chief, LBG LBG NRLBI Hans Bauer Visiting Scientist LDN NICHD Joan, Prives Guest Worker LDN NICHD Anne Schaffner Guest Worker LBG NHLBI I COOPERATING UNITS (if any) Laboratory of Developmental Neurobiology, NICRD Neurobiology Unit, Weizmann Institute of Science LAB/ BRANCH Laboratory of Biochemical Genetics SECT I ON Section on Molecular Biology INSTITUTE AND LOCATION NRLBI, NIH, Bethesda, MD 20205 TOTAL MANYEARS: PROFESSICBAL: OTHER: 6.5 4.5 2.0 CHECK APPROPRIATE BOX(ES) 0 (a) HUMAN SUBJECTS c (b) HUMAN TISSUES s (c) NEITHER 0 (at) MINORS 0 (a2) INTERVIEWS SUMMARY OF WORK (200 words or less - underline keyworda) 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. Tc this purpose histochemical local- ization of a-bungarotoxin bound to the-receptors is used in conjunction with light and electron microscopy. In the past year we have continued our study of the form- ation of cholinergic synapses in developing chick retina, using an a-bungartoxin- we have extended our studies on the control of aggregation on cultured skeletal muscle cells by cromolecular factors secreted by`neuroblastoma-glioma hybrid cells and embryonic and we have initiated work on the structural interaction between the cyto receptors in cultured skeletal muscle cells. T Pi S-6040 (Rev. IO-j6) 201 HI, 00017-04 LBG Project Description: Methods Employed: We have used fluorescence staining of monolayer cultured muscle cells with rhodamine-labeled a-bungarotoxin (oBT) and peroxidase stain- ing of tissues incubated in vitro with peroxidase-labeled aBT. These materials -- are subsequently examined by light or electron microscopy to visualize and quan- titate uicotinic acetylcholine receptor sites (AChR). Ion exchange chromatography, ultrafiltration, and isoelectric focusing have been used to characterize and purify the AChR aggregation factor. Primary cul- tures of dissociated embryonic neurons and serial cultures of clonal cell lines have been grown as sources of AChR aggregating factor. 125 I-aBT binding, detergent treatment and light and electron microscopy have been used to study AChR-cytoskeleton interactions. Major Findings: An aBT-horseradish peroxidase conjugate was used to study the distribution of AChR (aBT binding sites) in developing chick retina. Incuba- tion of the retina in vitro with the conjugate allowed quantitative comparison -- of developmental stages. aBT-binding synapses were found at the early stages of synapse formation and comprised between 5 and 11% of the inner plexiform layer synapse population during in ovo development. -- The AChR aggregation factor from neuroblastoma x glioma hybrid cells was par- tially purified by ion exchange chromatography, gel filtration, and preparative isoelectric focusing. Factors with similar activity were detected in embryonfc brain and cultures of sympathetic ganglion neurons and spinal cord neurons, but not in liver, adult brain or embryonic glial cell cultures. c Detergent treatment under appropriate conditions removed most lipid and soluble protein from cultured skeletal muscle cells, but left the cytoskeleton and bound components intact. This extraction was used to distinguish tightly bound and loosely bound populations of AChR, which may be correlated with the degree of receptor aggregation. Significance to Biomedical Research: Knowledge of the ultrastructural distrib- ution of acetylcholine receptors is of clear importance in any attempt to under- stand the role of neurotransmitters and their receptors in the function and de- velopment of the nervous system. The results obtained with developing chick retina represent the beginning of an understanding of the role of neurotransmitter receptors in the formation and maturation of chemical synapses, as seen on the ultrastructural level. The cultured muscle studies may lead to a better understanding of the mechanism whereby neurons control the distribution of receptors on muscle cells and on other neurons. Proposed Course of Research: (1) We have developed a monolayer culture system for physiological and histo- chemical observation of rabbit retina neurons, which we hope to exploit to learn more about the relationships between aBT binding sites and AChR in central neurons. 2 1079 ZOl HL 00017-04 LBG (2) We will continue the biochemical characterization of the AChR aggregation factor, adding immunochemical techniques to the array. We will also con- tinue to probe the cellular specificity of factor formation and target receptor specificity of the factor. (3) We will pursue the study of AChR-cytoskeletal interactions with biochem- ical and morphological techniques. Publications: 1) Christian, C.N., Daniels, M.P., Sugiyama, H., Vogel, Z., Jacques, L., and Nelson, G.: A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells. Proc. Natl. Acad. Sci. USA 75: 4011-4015 (1978) - 2) Vogel, Z., Towbin, M., and Daniels, M.P.: a-Bungarotoxin-horseradish per- oxidase conjugate: Preparation, properties and utilization for the histo- chemical detection of receptors to acetylcholine. J. Histochem. Cytochem.. 27: 846-851, 1979. 3