Dr. Tsien presented new and unpublished work that provides new insights into signaling at neuronal synapses. One area of research focuses on the existence and functional meaning of multiple modes of synaptic vesicle fusion triggered by Ca2+. The opening of the Ca2+ channels drives at least two distinct forms of fusion. In the classical mode, known as “full-collapse fusion,” the vesicle membrane fully merges with and flattens into the presynaptic membrane. In a newly characterized mode, termed “kiss-and-run” (K&R), the connection between the vesicle interior and the external medium lasts long enough to allow passage of neurotransmitter, but the connection is severed before the identity of the vesicle is lost. Dr. Tsien studies the dynamic properties and functional implications of both fusion modes by loading single synaptic vesicles with single photoluminescent reporter particles—quantum dots. Sharp distinctions between full-collapse fusion (FCF) and K&R are now in hand. The same vesicle could undergo K&R and then FCF, as soon as the next stimulus, ten seconds later. Rapid imaging of the decay of fluorescence associated with a typical K&R event indicated that the fusion pore was open for well below 1 s and that the reacidification proceeded rapidly, with a time constant of ~1 s. The prevalence of K&R and FCF depends on the stimulus pattern during the processes of Qdot loading and unloading. Imaging of Qdots not only provides a new perspective on intriguing and controversial issues of vesicle dynamics, but also offers the possibility of tracking presynaptic activity within neuronal circuits over extended periods.
Another topic concerns the fundamental unit of cell-cell communication between brain neurons: quantal synaptic transmission. Presynaptic release of a packet of neurotransmitter—for example, glutamate—causes activation of postsynaptic receptors and a brief flow of current that promotes firing of the postsynaptic cell. Dr. Tsien’s group works on neuronal mechanisms that allow synapses to adapt to a sudden or long-lasting change in their level of activity. For example, blocking impulses or postsynaptic glutamate receptors causes a cascade of biochemical events that eventually leads to readjustment of critical molecular players on both sides of the synapse. The group uses state-of-the-art methods to pin down the cell biology of changes in synaptic strength in cultures of isolated neurons and brain slices. Their experiments demonstrate that adaptation may take place both post- and presynaptically, with patterns of modification that differ sharply from one kind of synapse to another in hippocampal circuits.
One of the more profound effects of synaptic transmission and cellular depolarization is to cause changes in neuronal gene expression. Despite its importance, signaling from synapse or surface membrane to nucleus is only partly understood. One example of such signaling involves a local increase in Ca2+ concentration near class of Ca2+ channels (L-type) different from those that trigger presynaptic release, subsequently leading to activation of exemplar transcription factor, CREB, a transcription regulator of many important neuronal genes.
The group’s approach is to combine physiological approaches (how fast, how steeply voltage-dependent, how signal is transduced) and biochemical experiments using cDNA microarrays (which genes, in what context, what relationship to learning and memory). Recently, they have shown that excitation-transcription is every bit as steeply voltage-dependent as excitation-dependent as excitation-contraction or excitation-secretion coupling, but shows its own unique biological features.
