The focus of my laboratory's work is to understand the mechanisms by which neurons form synaptic connections, how synapses transmit information, and how synapses change during learning and memory. We combine molecular biology, protein biochemistry, electrophysiology, and imaging approaches with Drosophila genetics to address these questions. Neurotransmitter release from synaptic vesicle fusion is the fundamental mechanism for neuronal communication at synapses. Since the work of Bernard Katz, it has been known that synaptic vesicle fusion is triggered by Ca2+ influx into the presynaptic terminal. Vesicular trafficking pathways are ubiquitous among eukaryotes, with fusion driven by the SNARE complex. Neurotransmitters can be released during evoked fusion following an action potential, or through spontaneous fusion of vesicles (termed “minis”) in the absence of nerve stimulation. One of the major goals of my research program has been to define how vesicular trafficking mediates synaptic transmission, and how this process can be altered to change neuronal communication. At synapses, SNARE-mediated fusion is uniquely regulated to allow rapid and Ca2+-triggered synaptic vesicle fusion. Several key adaptations to the core fusion machinery require synapse specific SNARE-binding proteins, including Synaptotagmin 1 (Syt 1) and Complexin (Cpx). We have extensively studied how these proteins control synaptic output. Syt 1 is a synaptic vesicle protein that binds to SNARE complexes and membrane phospholipids in a Ca2+-dependent manner. My lab and others have show that Syt 1 functions as the Ca2+ sensor for fast synchronous neurotransmitter release. Cpx is a small cytosolic α-helical protein that binds assembled SNARE complexes. We have shown that association of Cpx with SNAREs allows it to function as a facilitator for synaptic vesicle fusion and as a fusion clamp to prevent premature exocytosis in the absence of Ca2+. A key avenue of our ongoing research program is to understand how these proteins, together with other key fusion regulators, mediate cycles of SNARE assembly and disassembly in a precisely coordinated fashion to support synaptic transmission. Similarly, we also seek to understand how the core fusion machinery can be rapidly modified, for example through phosphorylation, to alter presynaptic function and synaptic plasticity.
We use the Drosophila NMJ as a model synapse to uncover general principles of synapse biology. Recent work in the lab is also seeking to understand how synaptic vesicle release probability and release mode are determined at individual release sites. Evoked release following an action potential has been well characterized for its function in activating the postsynaptic cell, but the significance of spontaneous release is less clear. Whether receptors on the postsynaptic cell have the ability to distinguish between neurotransmitters released through the two independent fusion pathways is still under debate. If both vesicle release mechanisms activate the same set of receptors, crosstalk would occur between the two modes of fusion. However, if spontaneous release occurs at distinct release sites, the postsynaptic cell may be capable of differentiating between the two modes of release, suggesting spontaneous fusion may represent a separate information channel independent of the traditional Ca2+-activated evoked release pathway. The major confound to this question has been the inability to examine vesicle fusion at individual active zones. Classical electrophysiological studies of synaptic transmission measure the postsynaptic effect of neurotransmitter release over a population of release sites, precluding an analysis of how individual active zones participate in and regulate these two modes of vesicle fusion. We have developed transgenically expressed Ca2+ sensors that allow imaging of postsynaptic glutamate receptor activation following vesicle fusion, providing a mechanism to visualize all exocytotic events occurring through both spontaneous and evoked release pathways at Drosophila glutamatergic neuromuscular junctions. This toolkit allows us to generate release probability maps for both spontaneous and evoked fusion for all active zones at a synapse. By imaging a defined neuromuscular connection with ~ 300 active zones between the synaptic partners, we have begun to analyze the relationship between spontaneous and evoked release and determine how release mode and release probability are regulated at individual active zones at this model glutamatergic synapse. Unexpectedly, we have discovered that a subset of active zones are dedicated to spontaneous release, indicating a population of postsynaptic receptors is uniquely activated by this mode of vesicle fusion. We have also found that release probability can vary more than 100-fold between neighboring active zones. This is an exciting finding and provides us a mechanism to understand how single active zone release probability is regulated. The ability to measure individual active zones for their release probability also allows us to determine how active zone release properties can change during synaptic plasticity. It is likely that evoked and spontaneous active zone release probability is not a static property of synaptic connections, and can be differentially regulated during distinct modes of synaptic plasticity. Synaptic plasticity mechanisms can be manifested through changes in N (the number of release sites), P (probability of release) or Q (quantal size). Many factors have been suggested to regulate these properties. However, the lack of a tool to examine single active zone release probability has made it difficult to dissect how these changes play out over a population of release sites. We are now in a position to examine how plastic changes in release properties at the NMJ map onto alterations in individual active zone release probability. By mapping single active zone release probability during both acute and homeostatic synaptic plasticity and following the properties, we can gain new insights into presynaptic mechanisms underlying synaptic communication and plasticity at a model glutamatergic synapse.
Troy Littleton received his MD and Ph.D. from Baylor College of Medicine in Houston, TX. He completed his postdoctoral training at the University of Wisconsin. In 2000, he joined the faculty of the Department of Biology and the Picower Institute for Learning and Memory at MIT.
Jorquera, R.A., Huntwork-Rodriguez, S., Akbergenova, Y., Cho, R.W. & Littleton, J.T. (2012) Complexin controls spontaneous and evoked neurotransmitter release by regulating the timing and properties of Synaptotagmin activity. J. Neuroscience 32: 18234-18245.
Stevens, R., Akbergenova, Y., Jorquera, R.A. & Littleton, J.T. (2012) Abnormal synaptic vesicle biogenesis in Drosophila synaptogyrin mutants. J. Neuroscience 32: 18054-18067.
Buhl, L.K., Jorquera, R.A., Akbergenova, Y., Huntwork-Rodriguez, S., Volfson, D. & Littleton, J.T. (2013) Differential regulation of evoked and spontaneous neurotransmitter release by C-terminal modifications of Complexin. Mol. Cell. Neuroscience 52: 161-172.
Lee, J., Guan, Z., Akbergenova, Y. & Littleton, J.T. (2013) Genetic analysis of Synaptotagmin C2 domain specificity in regulating spontaneous and evoked neurotransmitter release. J. Neuroscience 33: 187-200.
Melom, J.E. & Littleton, J.T. (2013) Mutation of a NCKX eliminates glial microdomain calcium oscillations and enhances seizure susceptibility. J. Neuroscience 33: 1169-1178.
Bykhovskaia, M., Jagota, A., Gonzalez, A., Vasin, A. & Littleton, J.T. (2013) Interaction of the Complexin accessory helix with the C-terminus of the SNARE complex: Molecular dynamics model of the fusion clamp. Biophysical Journal 105: 679-690.
Melom, J.E., Akbergenova, Y., Gavornik, J.P. & Littleton, J.T. (2013) Spontaneous and evoked release are independently regulated at individual active zones. J. Neuroscience 33: 17253-17263.
Piccioli, Z.D. & Littleton, J.T. (2014) Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM Kinase regulation of Cofilin. J. Neuroscience 34: 4371-4381.
Cho, R.W., Kummel, D., Li, F., Baguley, S., Coleman, J., Rothman, J.E. & Littleton, J.T. (2014) Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo. PNAS 111:10317-10322.
Vasin, A., Zueva, L., Torrez, C., Volfson, D., Littleton, J.T. & Bykhovskaia, M. (2014) Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction. J. Neuroscience, In Press.
Poitras Scholar Award in Neuroscience
Alfred P. Sloan Research Fellow
Human Frontier Science Program Junior Faculty Fellowship
Searle Scholar Award
Surdna Research Foundation Award
Wade Fund Award
David and Lucile Packard Foundation Fellowship for Science and Engineering
Fred and Carole Middleton Career Development Professorship
Member, Faculty of 1000
Distinguished Alumni Award, Baylor College of Medicine