Project Summary: IBN-9722785, Alan D. Grinnell, P.I. Mechanisms of Long-Term Transynaptic Regulation of Neurotransmitter Release" Competitive interactions between synapses innervating the same target cell are critical in the establishment and refinement of connectivity in the nervous system, and in the adjustment of synaptic strength with use or experience. One of the most intriguing forms is known as "Hebbian" plasticity, in which synaptic inputs that are active simultaneously with postsynaptic activity are retained or strengthened, while synapses that are not active at the same time as the postsynaptic cell are suppressed or lost. A particularly clear, accessible instance of such pre-synaptic suppression is that observed in synapses between Xenopus motoneurons and muscle cells in culture. Transmitter release at a synapse is suppressed by manipulations that elevate the calcium concentration in the muscle cell, simulating activation of another synaptic input. The synapse can be protected from suppression if it is active during the time of calcium-induced feedback. The mechanisms of suppression and protection are unknown. We have developed techniques for patch-clamping synaptic terminals and postsynaptic cells simultaneously in this system, allowing us to analyze rigorously the magnitude and time course of ionic currents in the terminal and the associated neurotransmitter release. With a third patch pipette it is possible to introduce reagents into the synaptic terminal through the neuronal cell body. Since calcium influx during a presynaptic action potential is critical to neurotransmitter release, we will first (1) determine whether long-term suppression of synaptic function is associated with a decrease in calcium- influx either by direct modulation of calcium channels or by changes in other ionic currents in the terminal, and what these changes are at the level of single channels. If suppression of release can be explained by changes in ionic currents, we will determine (2) whether the suppression effects are mediated by G-protein associated receptors, and whether this is by a direct ("membrane delimited") or indirect (second messenger mediated) pathway, and (3) whether cyclic nucleotides help mediate the effect. In addition, we will test (4) whether protection by simultaneous synaptic activity is due to prevention of the suppressive influence or to some compensatory change, and (5) what aspect of neuronal activity is responsible for this protection. The Xenopus motoneuron-muscle cell synapses are robust, characteristic in most respects of mature synapses. The Hebbian character of the modulation, the evidence for activity-dependent protection of synapses, the accessibility of the preparation, and above all the ability to monitor and manipulate pre-synaptic events make this an ideal preparation in which to obtain insights about mechanisms of long-term synaptic remodeling and plasticity.
