Summary of work on this project as indicated on the report: "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 alpha-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 formation of cholinergic
synapses in developing chick embryos, using an alpha-bugartoxin(sic)-horseradish peroxidase conjugate; we have extended our
studies on the control of nicotinic acetylcholine receptor aggregation on cultured skeletal muscle cells by macromolecular
factors secreted by the neuroblastoma-glioma hybrid cells and embryonic neurons; and we have initiated work on the structural
interaction between the cytoskeleton and nicotinic acetylcholine receptors in cultured skeletal muscle cells.
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1979-09 (September 1979)
Daniels, Mathew P.
Nirenberg, Marshall W.
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
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From Neuroblastoma to Homeobox Genes, 1976-1992
Annual Report of the Laboratory of Biochemical Genetics, October 1, 1978 - September 30, 1979 (September 1979)
Period Covered: October 1, 1978 - September 30, 1979
Title of Project: Acetylcholine Receptors
Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged
on the Project:
PI: Mathew P. Daniels, Research Biologist, LBG NHLBI
OTHERS: P. Nelson, Chief, Laboratory of Developmental Neurobiology, LDN NICHD
C. Christian, Senior Staff Fellow, LDN NICHD
Z. Vogel, Assistant Professor, Weizmann Institute
Marshall Nirenberg, Chief, LBG, LBG NHLBI
Hans Bauer, Visiting Scientist, LDN NICHD
Joav Prives, Guest Worker, LDN NICHD
Anne Schaffner, Guest Worker, LBG NHLBI
Cooperating Units (if any):
Laboratory of Developmental Neurobiology, NICHD
Neurobiology Units, Weizmann Institute of Science
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section on Molecular Biology
Institute and Location: NHLBI, NIH, Bethesda, MD 20205
Total Man Years: 6.5
Summary of Work:
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 alpha-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 formation of cholinergic synapses in developing chick retina, using an alpha-bungartoxin-horseradish
peroxidase conjugate; we have extended our studies on the control of nicotinic acetylcholine receptor aggregation on cultured
skeletal muscle cells by macromolecular factors secreted by neuroblastoma-glioma hybrid cells and embryonic neurons; and we
have initiated work on the structural interaction between the cytoskeleton and nicotinic acetylcholine receptors in cultured
skeletal muscle cells.
Methods Employed: We have used fluorescence staining of monolayer cultured muscle cells with rhodamine-labeled alpha-bungarotoxin
(alphaBT) and peroxidase staining of tissues incubated in vitro with peroxidase-labeled alphaBT. These materials are subsequently
examined by light or electron microscopy to visualize and quantitate nicotinic acetylcholine receptor sites (AChR).
Ion exchange chromatography, ultrafiltration, and isoelectric focusing have been used to characterize and purify the AChR
aggregation factor. Primary cultures of dissociated embryonic neurons and serial cultures of clonal cell lines have been grown
as sources of AChR aggregating factor.
125 I-alphaBT binding, detergent treatment and light and electron microscopy have been used to study AChR-cytoskeleton interactions.
Major Findings: An alphaBT-horseradish peroxidase conjugate was used to study the distribution of AChR (alphaBT binding sites)
in developing chick retina. Incubation of the retina in vitro with the conjugate allowed quantitative comparison of developmental
stages. alphaBT-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 partially purified by ion exchange chromatography,
gel filtration, and preparative isoelectric focusing. Factors with similar activity were detected in embryonic brain and
cultures of sympathetic ganglion neurons and spinal cord neurons, but not in liver, adult brain or embryonic glial cell cultures.
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 distribution of acetylcholine receptors 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 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 histochemical observation of rabbit retina neurons,
which we hope to exploit to learn more about the relationships between alphaBT binding sites and AChR in central neurons.
(2) We will continue the biochemical characterization of the AChR aggregation factor, adding immunochemical techniques to
the array. We will also continue to probe the cellular specificity of factor formation and target receptor specificity of
(3) We will pursue the study of AChR-cytoskeletal interactions with biochemical and morphological techniques.
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.: alpha-Bungarotoxin-horseradish peroxidase conjugate: Preparation, properties
and utilization for the histochemical detection of receptors to acetylcholine. J. Histochem. Cytochem. 27: 846-851, 1979.