About the Lab
For more than three decades this laboratory has been concerned with understanding how the nervous system’s synapses function, how they develop and how they regenerate after trauma. Such information is essential for a comprehensive understanding of the factors that bring about certain diseases of the nervous system and for developing ways of improving recovery after trauma.

We have done most experiments on vertebrate neuromuscular junctions, particularly those of frog, because they are the best understood of all synapses, but we have also studied neuron-to-neuron synapses in the brains of both vertebrates and invertebrates.

Over the years our experimental approaches have included in vivo microsurgery, tissue culture, light and electron microscopy, immunocytochemistry, protein purification, molecular genetics, in situ hydridization, and electrophysiology.

We are currently using the nascent technology of high-resolution electron microscope tomography to study at 2-3 nm spatial resolution the organization and behavior of macromolecules at synapses. The information that is obtained provides unique insights about the molecular mechanisms involved in synaptic impulse transmission and in synapse formation. To augment these studies we are exploring methods for localizing synaptic proteins characterized by biochemistry to specific macromolecules observed by electron microscope tomography.

We are also developing software, called EM3D, a unified application designed specifically for structural cell biologists that allows a user to proceed from an electron microscope tomography data set of a specimen to a collection of 3D surface models of structures within the specimen. EM3D also includes computational tools that quantify spatial characteristics on a vertex-by-vertex basis upon the surface models. These technologies can be used to examine how macromolecular organization regulates cell function in any tissue.

In addition to being equipped with the standard apparatus used for cellular and molecular neurobiological experiments, the lab operates the Stanford High Resolution Electron Microscope Facility for Biomedical Sciences, which features an FEI Polara electron microscope for tomography and single particle analysis.

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Some of the lab’s contributions are:

  • Showing that the position of individual axon terminals at synapses in autonomic ganglia and of neuromuscular junctions can be observed in living isolated preparations by Nomarski DIC optics. Although DIC optics had been used in a previous publication by others to examine the trafficking of large organelles, this was its first application to the study of cellular topography, in general, and synapses, in particular, in live preparations, and it led to the widespread use of DIC optics for these purposes today.

McMahan, U.J. and S.W. Kuffler. Visual Identification of synaptic boutons on living ganglion cells and of varicosities in post-ganglionic axons in the heart of the frog. Proc. Roy. Soc. Lond. B. 177:485-508, 1971.

McMahan, U.J. and Spitzer, N.C. and Peper, K. Visual identification of nerve terminals in living isolated skeletal muscle. Proc. Roy. Soc. Lond. B. l8l:42l-430, 1972.

  • Learning that the muscle fiber’s surface directly opposite the axon terminal at neuromuscular junctions (the postsynaptic membrane) has a far greater sensitivity to the direct iontophoretic application of the neurotransmitter acetylcholine than it does a few micrometers away. This study, which relied on the use of Nomarski DIC optics to visualize axon terminals on muscle fibers, together with a study published by others the same year , which relied based on α- bungarotoxin labeling, provided the first direct evidence that receptors for neurotransmitter are highly concentrated in the postsynaptic membrane.

Peper, K. and U.J.McMahan. Distribution of acetylcholine receptors in the vicinity of nerve terminals on skeletal muscle of the frog. Proc. Roy. Soc. Lond. B. l8l:43l-440, 1972.

  • Determining that acetylcholinesterase , which degrades acetylcholine after its interaction with the muscle fiber has terminated, is a component of the portion of the muscle fiber’s basal lamina sheath that occupies the synaptic cleft between the axon terminal and muscle fiber.

McMahan, U.J., Sanes, J.R. and Marshall , L.M. Cholinesterase is associated with the basal lamina at the neuromuscular junction. Nature 27l:l72-l74, 1977.

  • Finding that after damage to a motor nerve in the frog, regenerating axon terminals grow to precisely cover the portion of a muscle fiber’s surface that was formerly opposite the original axon terminals. It had been shown around the turn of the twentieth century that regenerating axon terminals reinnervate the general area of a muscle fiber that was the home of the original axon terminals, the endplate region. Knowledge about the precision of reinnervation of the narrow postsynaptic membrane within the endplate region was an essential step toward learning that the synaptic basal lamina contains synaptogenic proteins, which ultimately led to the discovery of agrin as described below.

Letinsky, M., Fischbeck, K.H., and U.J.McMahan. Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush. J. Neurocytol. 5:69l-7l8, 1976.

Rotshenker, S. and U.J.McMahan. Altered patterns of innervation in frog muscle after denervation. J. Neurocytol. 5:719-750, 1976.

  • Demonstrating that when a frog muscle is damaged in a way that causes the muscle fibers to degenerate but leaves their basal lamina sheaths intact, including that which lies between the axon terminal and the postsynaptic membrane, damaged axons regenerate to precisely cover the sites on the sheaths formerly occupied by the original axon terminals despite the absence of muscle fibers.

Marshall , L.M., Sanes, J.R. and U.J. McMahan. Reinnervation of original synaptic sites on muscle fiber basement membrane after disruption of the muscle cells. Proc. Natl. Acad. Sci. 74:3073-3077, 1977.

  • Showing that the portion of the muscle fiber’s basal lamina sheath that occupies the synaptic cleft at the frog’s neuromuscular junction contains synaptogenic proteins that induce the accumulation of synaptic vesicles and the formation of active zones in regenerating axon terminals, which are major constituents of the presynaptic apparatus of axon terminals at normal and regenerating neuromuscular junctions and are directly involved in the exocytosis of the neurotransmitter acetylcholine during synaptic transmission.

Sanes, J.R., Marshall , L.M. and U.J. McMahan. Reinnervation of muscle fiber basal lamina after removal of myofibers: Differentiation of regenerating axons at original synaptic sites. J. Cell Biol. 78:176-198, 1978.

  • Finding that the portion of the muscle fiber’s basal lamina sheath that occupies the synaptic cleft at the frog’s neuromuscular junction contains synaptogenic proteins that induce regenerating muscle fibers to form on their surface aggregates of acetylcholine receptors and acetylcholinesterase, which are major constituents of the postsynaptic apparatus at normal and regenerating neuromuscular junctions and are required for synaptic transmission.

Burden, S.J., Sargent, P.B. and U.J. McMahan. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J. Cell Biol. 82:4l2-425, 1979

McMahan, U.J. and Slater, C.R. The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscles. J. Cell Biol. 98:1453-1473, 1984.

Anglister, L. and U.J. McMahan. Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle. J. Cell Biol. 101:735-743, 1985.

  • Isolating, identifying and initially characterizing the protein agrin, and formulating the agrin hypothesis. The hypothesis holds that in developing vertebrates, agrin synthesized in the cell bodies of motor neurons is transported along their axons to be released from axon terminals into the synaptic cleft at neuromuscular junctions. There, it binds to an agrin receptor on the muscle fiber surface triggering a cascade of events leading to the formation of local aggregates of acetylcholine receptors, acetylcholinesterase and other molecular components of the postsynaptic apparatus including constituents of the synaptic basal lamina. Agrin becomes associated with this nascent basal lamina, and thus it is bound at the synaptic site. Release of agrin from axon terminals at adult neuromuscular junctions and its incorporation into synaptic basal lamina helps maintain the postsynaptic apparatus and induces the formation of postsynaptic apparatus on muscle fibers regenerating in the absence of axon terminals, while release of agrin by regenerating axons accounts for their ability to induce a new postsynaptic apparatus when experimentally positioned at ectopic sites on denervated muscle fibers.

Rubin, L.L. and U.J. McMahan. Regeneration of the neuromuscular junction: steps toward defining the molecular basis of the interaction between nerve and muscle. In: Disorders of the Motor Unit , Ed. by D.L. Schotland, John Wiley and Sons, New York , 187-196, 1982.

Godfrey, E.W., Nitkin, R.M., Wallace, B.G., Rubin, L.L., and McMahan, U.J. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. J. Cell Biol. 99:6l5-627, 1984.

Fallon, J.R., Nitkin, R.M., Reist, N.E., Wallace, B.G. and McMahan, U.J. Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions. Nature (Lond.). 3l 5:57 l-574, 1985.

Wallace, B.G., Nitkin, R.M., Reist, N.E., Fallon, J.R., Moayeri, N.N. and McMahan, U.J. Aggregates of acetylcholinesterase induced by acetylcholine receptor-aggregating factor. Nature (Lond.). 3l5 :574-577, 1985.

Nitkin, R.M., Smith, M.A., Magill, C., Fallon, J.R., Yao , Y-M.M., Wallace, B.G., and U.J. McMahan. Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J. Cell Biol. 105 :2471-2478, 1987.

Reist, N.E., Magill, C. and U.J. McMahan. Agrin-like molecules at synaptic sites in normal, damaged and denervated skeletal muscles. J. Cell Biol. 105:2457-2470, 1987.

McMahan, U.J. and B.G. Wallace. Molecules in basal lamina that direct the formation of synaptic specializations at neuromuscular junctions. Dev. Neurosci. 11:227-247, 1989.

McMahan, U.J. The agrin hypothesis. Cold Spring Harbor Symp. Quant. Biol. 50:407-418, 1990.

Smith, M.A., Magill-Solc, C., Rupp F., Tao, M. Y.-M., Schilling, J.W., Snow, P., and U.J. Mahan 1992 Isolation and Characterization of a cDNA that encodes an agrin homolog in the marine ray. Mol. Cell. Neurosci. 3: 406-417, 1992.

Tsim, K.W.K, Ruegg, M.A., Escher, G., Kröger, S. and U.J. McMahan. cDNA that encodes active agrin. Neuron 8:677-689, 1992.

Ruegg, M.A., Tsim, K.W.K., Horton, S.E., Kröger, S., Escher, G., Gensch, E.M. and U.J. McMahan. The agrin gene codes for a family of basal lamina proteins that differ in function and distribution. Neuron 8:691-699, 1992

Reist, N.E., Werle, M.J. and U.J. McMahan. Agrin released by motor neurons induces the aggregation of AChRs at neuromuscular junctions. Neuron 8: 865-868, 1992.

  • The agrin hypothesis has been repeatedly tested in this and other labs over the last 17 years and still stands. Indeed, there is now good evidence from this lab and others that agrin in addition to inducing the early events in postsynaptic apparatus formation also induces the late events, such as the local formation of infoldings in the muscle fiber’s plasma membrane, the local accumulation of muscle nuclei (in mammals), and the γ/ε acetylcholine receptor switch.

Cohen, I. , Rimer, M., Lømo, T. and U.J. McMahan. Agrin-induced postsynaptic-like apparatus in vivo . Mol. Cell. Neurosci. 4:237-253, 1997.

Rimer, M., Mathiesen, I. , Lømo, T. and U.J. McMahan. γ -AChR/ ε-AChR switch at agrin-induced postsynaptic-like apparatus in skeletal muscle. Mol. Cell. Neurosci. 4:254-263, 1997.

  • Using electron microscope tomography on sections from frog neuromuscular junctions to learn that the dense aggregates of proteins attached to the presynaptic membrane of typical synapses, known as active zone material, contain an organized network of elongate macromolecules. Our results indicate that the macromolecules help dock synaptic vesicles at the presynaptic membrane and anchor calcium channels in the membrane, maintaining them at a particular distance from each other. The macromolecules most likely contain proteins that mediate the calcium-induced exocytosis of acetylcholine from the docked vesicles into the synaptic cleft upon arrival of a nerve impulse.

CHarlow , M.L., Ress, D., Stoschek, A., Marshall , R.M. and McMahan, U.J. The architecture of active zone material at the frog’s neuromuscular junction. Nature 409: 479-484, 2001.

Ress, D.B., Harlow, M.L., Marshall, R.M. and McMahan, U.J. Methods for generating high-resolution structural models from electron microscope tomography data.
Structure : 12 (10):1763-1774, 2004.

Laboratory is located in the Interdisciplinary Life Sciences Building on the Texas A&M University main campus.