Our goal is to elucidate the mechanisms of synapse elimination at the mammalian neuromuscular junction. There are three related Specific Aims. In preliminary studies, we have characterized fluorescent dyes which selectively stain adult and neonatal motor nerve terminals in rat fourth deep lumbrical (4DL) muscle. The terminals are stained in an activity-dependent fashion. The dyes are non-toxic. We will study neonatal end plates during the period of synapse elimination, when muscle fibers receive synaptic inputs from more than one motoneuron (polyneuronal innervation). We will stain and identify nerve terminal boutons belonging to single motoneurons. We will also measure transmitter release characteristics of the same terminals using electrophysiological techniques (voltage clamp of post-synaptic muscle fibers). These experiments will be performed on acutely dissected preparations. In addition, we will perform similar experiments on an organ culture preparation, consisting of neonatal spinal cord fragment, peripheral nerve, and muscle, all dissected and cultured in continuity. This preparation offers the possibility of continuous in vitro observation of identified end plates. In other preliminary work, we have described an inhibitory interaction between nerve terminals in neonatal muscle. Stimulation of one input produces an inhibition of the response to stimulation of a different input to the same muscle. This heterosynaptic inhibition lasts several tens of milliseconds. We will continue and extend our studies of the phenomenon. We will look for signs of the inhibition in adult muscle undergoing reinnervation. We will also test the hypothesis that ATP, ACh, or similar substances released by one nerve produce inhibition of transmitter release by the other nerve (presynaptic inhibition). In a third project, we have measured the spatial arrangements of muscle fibers in the 4DL muscle. We found that muscle fibers belonging to a single adult motor unit are scattered throughout the muscle in a random pattern. Preliminary observations of neonatal muscles suggest, however, that the initial pattern is nonrandom. We will test this hypothesis quantitatively.
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