Period Covered: October 1, 1978 - September 30, 1979
Title of Project: Call Recognition and Synapse Formation.
Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged
on the Project:
PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI
OTHER: Andrej Rotter, Visiting Fellow, LBG NHLBI
Radharaman Ray, Staff Fellow, LBG NHLBI
Michael Adler, Staff Fellow, LBG NHLBI
George Eisenbarth, Research Associate, LBG NHLBI
Frank Walsh, Guest Worker, LBG NHLBI
Jeffrey Thompson, Staff Fellow, LBG NHLBI
Cooperating Units (if any): G. Cantoni and P. Chiang, Laboratory of General and Comparative Biochem., NIMH
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section of Molecular Biology
Institute and Location: NIH, NHLBI, Bethesda, Maryland 20205
Total Man Years: 7.5
Summary of Work: Regulatory reactions were identified that turn synapses on or off.
Major Findings: The formation of synapses between clonal cells of neural origin, such as NBr10A or NG108-15 hybrid cells,
and rat striated muscle cells was found to be regulated. Exposure of hybrid cells for 3-7 days to PGE1, which results in
activation of adenylate cyclase, or exposure to various cyclic nucleotide phosphodiesterase inhibitors, markedly increases
the number of synapses formed. The effects of putative neurotransmitters or hormones on intra-cellular cyclic AMP or cyclic
GMP levels, voltage-sensitive Ca2+ channel activity, and acetylcholine secretion were determined. Receptor-mediated increases
in intracellular cyclic AMP or cyclic GMP levels had no immediate effect on K+-dependent 45Ca2+ uptake by cells or on acetylcholine
secretion from cells. However, prolonged exposure of hybrid cells to PGE1 results both in an increase in cellular cyclic
AMP and the gradual acquisition by cells of functional voltage-sensitive Ca2+ channels. Concomitantly cells acquire the ability
to secrete acetylcholine in response to a depolarizing stimulus and can then form functional synapses with muscle cells.
D600 inhibits 45Ca2+ uptake dependent on 80 mM K+ (IC50 = 2 X 10^(-7) M), but has little or no effect on 45Ca2+ uptake in
the presence of 5 mM K+. 45Ca2+ uptake also is inhibited to 10 mM La3+, Co2+, Ni2+, Mn2+, Sr2+, or Ba2+, but not by 10 mu-M
tetrodotoxin, 20 mM tetraethylammonium, or 1 mM 3,4-diaminopyridine
Other cell lines were found that synthesize acetylcholine but do not form synapses with striated muscle cells. Various types
of synapse defects were detected; including defects in voltage-sensitive 45Ca2+ channels, vesicles, and an additional unidentified
reaction that is required for acetylcholine secretion. These results show that cell lines with or without defects in synapse
formation can be generated and that voltage-sensitive Ca2+ channel activity can be regulated by a receptor-mediated reaction
which is coupled to activation of adenylate cyclase, or by inhibition of adenlyate clyclase, or by inhibition of cyclic nucleotide
phosphodies-terase. Voltage-sensitive Ca2+ channel activity increases slowly over a period of days and this reaction is required
for stimulus-dependent secretion of transmitter and the formation of functional synapses.
To identify molecules required for synaptogenesis or communication across the synapse, hybrid cell lines which synthesize
mono-specific antibodies were obtained by fusion of clonal myeloma cells with spleen cells immunized against cells from the
nervous system. Some of the hybridoma cell lines that were obtained synthesize mono-specific antibodies of high titre directed
against membrane antigens found on some cells from the nervous system that were not detected with cells from other tissues.
One of these cell lines, A2B5, synthesizes antibody directed against an antigen that was shown by indirect immunofluorescence
to be associated with plasma membranes of most, or all, neuron cell bodies in chick retina; however, the antigen was not detected
on axons or dendrites of neurons, on retina Muller cells, or pigment cells, or on cells from non-neuraltissues.
Antigen A2B5 activity is relatively stable at l00C, is insensitive to trypsin, exhibits the solubility properties of a ganglioside,
and is destroyed by neuraminidase. Antibody A2B5 cytotoxicity against retina cells is inhibited by a tetrasialo GQ ganglioside
fraction from bovine brain (estimated half-maximal inhibition, 0.2 uM), or N-acetylneuraminic acid (half-maximal inhibition,
5,000 uM), but not by other purified gangliosides tested. These results suggest that the antigen is a GQ ganglioside in plasma
membranes of retina neuron cell bodies but not membranes of axons or dendrites.
A solid-phase 125I-Protein A radioassay for anti-cell surface antibodies was devised which employs target cell monolayers
cultured on fenestrated poly-vinyl chloride, 96-well plates ("transfer plates"). The calibrated aperture in the bottom
of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to
enter the wells when transfer plates are lowered in receptacles containing washing buffer or test sera. To assay for antibodies
directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with
corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of
hybrid cell colonies for production of antibodies with desired specificity.
Methyltransferases can be inhibited by S-adenosyl homocysteine or by analogs which either increase S-adenosyl homocysteine
levels or inhibit methyl-transferases directly such as 3-deazaadenosine (DZA), adenosine-2',3'-deazido- 5'-carboxamide
(744-99), 5'-deoxy-5'-isobutylthioadenosine (SIBA), and 5'-deoxy- 5'-isobutylthio-3-deazaadenosine (DZ-SIBA).
In collaboration with P. Chiang and G. Cantoni the effects of these and other compounds on synapses between dissociated chick
embryo retina neurons and cultured rat striated muscle cells were investigated to determine whether inhibition of transmethylation
affects synapse formation, acetylcholine release, or muscle responses to acetylcholine mediated by nicotinic acetylcholfne
receptors. The frequency of spontaneous synaptic responses of muscle cells was markedly reduced by these compounds; half-maximal
inhibition was obtained with 1.5 x 10^-6 M DZ-SIBA, 1.5 x 10^(-5) M DZA, 3 x 10^(-5) M SIBA, or 1 x 10^(-4) M 744-99. DZ-SIBA
reduced the frequency of muscle synaptic responses by 50 percent in 3.5 minutes via a reaction which exhibits first-order
kinetics. Homocysteine thiolactone, 5-deoxy-adenosine, or tubercidin, which do not increase levels of S-adenosine homoeysteine
or inhibit methyltransferase activity, do not affect the frequency of spontaneous synaptac responses of muscle cells. However,
homocysteine thiolactone potentiates the inhibition of muscle synaptic responses by DZA by 6-fold. These results suggest
that a transmethylation reaction may be required for acetylcholine secretion or vesicle cycling in synaptic terminals of neurons.
Significance to Biomedical Research: Cultured cell systems have been established and used as model systems for biochemical
and electrophysiological studies on synapses. A reaction was found that regulates synapse plasticity.
Proposed Course: Current studies focus on determining the reactions which are required for synapse formation and termination
and factors regulating these reactions.
1. Nirenberg, M., Wilson, S., Higashida, H., Thompson, J., Eisenbarth, G ., Walsh, F., Rotter, A., Kenimer, J., and Sabol,
S. Synapse Plasticity. In Pontificiae Academiae Scientarvm Scripta Varia, In Press.
2. Eisenbarth, G. S., Walsh, F. S., and Nirenberg, M. Monoclonal Anti-body To A Plasma Membrane Antigen of Neurons, Proc.
Natl. Acad. Sci., In Press.
3. Schneider, M. D., and Eisenbarth, G. S. Transfer Plate Radioassay Using Cell Monolayers To Detect Anti-Cell Surface Antibodies
Synthesized By Lymphocyte Hybridomas, 2. Immunol. Methods, In Press.
4. Eisenbarth, G. S., Ruffalo, R. R., Walsh, F. S., and Nirenberg, M. Lactose Sensitive Lectin Of Chick Retina And Spinal
Cord, Biochem. and Biophys. Res. Comm. 83, 1246-1252 (1978).
5. McGee, R., Smith, C., Christian, C., Mata, M., Nelson, P., and Nirenberg, M. A New Method For Measurement Of The Uptake
And Release Of Materials From Cultured Cells. Anal. Biochem, In Press.
6. DeMello, F. G., The Ontogeny Of Dopamine-dependent Increase Of Adenosine 3', 5'-cyclic Monophosphate In The Chick
Retina, J. Neurochem. 2, 1049-1053 (1978).