For example, might some of the excess innervation originate from axons that were sending long collateral branches to multiple muscles in the embryonic period, or, alternatively, might some of the branches originate from motor neurons that are at the tail end of the period of naturally

occurring motor neuron cell death and are destined to die? The idea of cell death was ruled out by finding that there were no activated caspase-3 or TUNEL-positive ventral horn cholinergic cells in the spinal cord at birth, even though CHIR-99021 purchase we could induce caspase-3 or TUNEL labeling in the sternomastoid muscle motor neurons by axotomy in the spinal accessory nerve of pups at P0 (Figure 6C). We also found no evidence of axons branching to more than one muscle at birth by examining both retrograde labeling of motor neurons projecting to different muscles and lipophilic axon tracing from different muscles (Figure 6B). This study shows extensive connectivity in the developing neuromuscular system that resolves over the first few postnatal days into the much simpler pattern that has been well described in previous studies. Motor axons innervate

roughly an order of magnitude more target cells, and target cells each receive input from an order of magnitude more axons at birth than 2 weeks later. The loss occurs precipitously because even by postnatal day 6, many of these muscle fibers are singly innervated (Keller-Peck et al., 2001), meaning that the postsynaptic cells must be losing innervation from more than buy MLN0128 an axon per day during the first postnatal week. This data also shows that the peak of the “exuberance” is just before birth, suggesting perhaps that postnatal life may be a critical impetus for this synapse elimination.

Although there are many possible reasons for a die off of axonal branches, the studies presented here indicate that neither late apoptosis of a subset of neurons (Landmesser Phosphatidylinositol diacylglycerol-lyase and Pilar, 1974), nor the pruning of long intermuscular axon collaterals that projected erroneously to multiple targets (Bunt and Lund, 1981, Innocenti, 1981 and Stanfield et al., 1982), nor the pruning of large intramuscular branches with many synaptic terminals explains the result. Rather, the results show that pruning of terminal synaptic branches explains the large reduction in axonal complexity beginning in the perinatal period. We have studied the excessive branching using light and electron microscopical anatomical methods. Light and electron microscopy were necessary because of technical limitations of electrophysiological and more traditional light microscopic assays when used in developing systems. We measured the size of neonatal motor units anatomically because the several physiological methods previously used are insensitive to subthreshold innervation. One approach measures the muscle tension elicited by individual motor axons and compares it with the total tension a muscle is capable of generating (Brown et al., 1976).