Transgenic mice whose axons and Schwann cells express fluorescent chromophores enable new imaging techniques and augment concepts in developmental neurobiology. The utility of these tools in the study of traumatic nerve injury depends on employing nerve models that are amenable to microsurgical manipulation and gauging functional recovery. Motor recovery from sciatic nerve crush injury is studied here by evaluating motor endplates of the tibialis anterior muscle, which is innervated by the deep peroneal branch of the sciatic nerve. Following sciatic nerve crush, the deep surface of the tibialis anterior muscle is examined using whole mount confocal microscopy, and reinnervation is characterized by imaging fluorescent axons or Schwann cells (SCs). One week following sciatic crush injury, 100% of motor endplates are denervated with partial reinnervation at two weeks, hyperinnervation at three and four weeks, and restoration of a 1:1 axon to motor endplate relationship six weeks after injury. Walking track analysis reveals progressive recovery of sciatic nerve function by six weeks. SCs reveal reduced S100 expression within two weeks of denervation, correlating with regression to a more immature phenotype. Reinnervation of SCs restores S100 expression and a fully differentiated phenotype. Following denervation, there is altered morphology of circumscribed terminal Schwann cells demonstrating extensive process formation between adjacent motor endplates. The thin, uniformly innervated tibialis anterior muscle is well suited for studying motor reinnervation following sciatic nerve injury. Confocal microscopy may be performed coincident with other techniques of assessing nerve regeneration and functional recovery.

End-to-side (ETS) nerve repair remains an area of intense scrutiny for peripheral nerve surgeonscientists. In this technique, the transected end of an injured nerve, representing the "recipient" is sutured to the side of an uninjured "donor" nerve. Some work suggests that the recipient limb is repopulated with regenerating collateral axonal sprouts from the donor nerve that go on to form functional synapses. Significant, unresolved questions include whether the donor nerve needs to be injured to facilitate regeneration, and whether a single donor neuron is capable of projecting additional axons capable of differentially innervating disparate targets. We serially imaged living transgenic mice (n=66) expressing spectral variants of GFP in various neuronal subsets after undergoing previously described atraumatic, compressive, or epineurotomy forms of ETS repair (n=22 per group). To evaluate the source, and target innervation of these regenerating axons, nerve morphometry and retrograde labeling was further supplemented by confocal microscopy as well as Western blot analysis. Either compression or epineurotomy with inevitable axotomy were required to facilitate axonal regeneration into the recipient limb. Progressively more injurious models were associated with improved recipient nerve reinnervation (epineurotomy: 184±57.6 myelinated axons; compression: 78.9±13.8; atraumatic: 0), increased Schwann cell proliferation (epineurotomy: 72.2% increase; compression: 39% increase) and cAMP response-element binding protein expression at the expense of a net deficit in donor axon counts distal to the repair. These differences were manifest by 150 days, at which point quantitative evidence for pruning was obtained. We conclude that ETS repair relies upon injury to the donor nerve.

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Glial cell line-derived neurotrophic factor (GDNF) is known for its potent effect on neuronal survival, but its role in the development and function of synapses is not well studied. Using Xenopus nerve-muscle co-cultures, we show that GDNF and its family member neurturin (NRTN) facilitate the development of the neuromuscular junction (NMJ). Long-term application of GDNF significantly increased the total length of neurites in the motoneurons. GDNF also caused an increase in the number and the size of synaptic vesicle clustering, as demonstrated by synaptobrevin-GFP fluorescent imaging, and FM dye staining. Electrophysiological experiments revealed two effects of GDNF on synaptic transmission at NMJ. First, GDNF markedly increased the frequency of spontaneous transmission and decreased the variability of evoked transmission, suggesting an enhancement of transmitter secretion. Second, GDNF elicited a small increase in the quantal size, without affecting the average rise and decay times of synaptic currents. Imaging analysis showed that the size of acetylcholine receptor clusters at synapses increased in muscle cells overexpressing GDNF. Neurturin had very similar effects as GDNF. These results suggest that GDNF and NRTN are new neuromodulators that regulate the development of the neuromuscular synapse through both pre-and postsynaptic mechanisms.Studies in the last few years suggest that neurotrophins, originally defined as a family of trophic factors essential for neuronal survival, also regulate synaptic transmission and plasticity (for reviews, see Refs. 1-3). The first evidence for such a new role was the demonstration that brain-derived neurotrophin (BDNF) 1 and neurotrophin-3 (NT3) acutely potentiate synaptic transmission at the Xenopus neuromuscular synapse in culture (4). Subsequent experiments from many laboratories have demonstrated regulatory effects of neurotrophins on synapses in a variety of model systems. For example, changes in the level of BDNF in the visual cortex alter the development of ocular dominance synapses (5, 6). Consistent with this, neurotrophins seem to have profound effects on the growth of dendrites of cortical neurons and afferent axons of thalamic neurons (7,8). In the hippocampus, BDNF acutely facilitates long-term potentiation (9 -12). Neurotrophins have also been shown to rapidly regulate synaptic transmission in various cultured neurons (13-16). Mechanistic studies of the role of neurotrophins in synaptic transmission have largely been carried out in the Xenopus nerve-muscle co-cultures. Two major effects of neurotrophins have been described on the neuromuscular synapse: acute enhancement of neurotransmitter release (4, 17-22), and long-term regulation of synapse maturation (23-26). Despite of the rapid progress, a number of important issues still await to be addressed. For example, while the acute effects of neurotrophic factors on synaptic transmission have attracted a great deal of interest, much less is known about cellular and molecular mechanisms underlying the long-term ...

Retrograde labeling has become an important method of evaluation for peripheral nerve regeneration after injury. We review the features of the commonly used retrograde tracers Fast Blue, Fluoro-Gold, and Fluoro Ruby in addition to the various application methods (conduit reservoir, intramuscular injection, and crystal powder application) and the techniques used to count stained neurons. Upon application of the staining techniques and dyes in a rat and mouse nerve injury model, Fluoro-Gold was found to stain the greatest number of neurons with all application methods. However, due to variability of staining intensity, neuron size, and background staining, it is difficult to count the stained neurons accurately. Fast Blue stains consistently using intramuscular injection in the mouse but fails to provide adequate staining using the muscle injection method in the rat model and shows high failure rates using the conduit reservoir technique. However, crystal dye application with Fast Blue to the cut nerve end provides excellent results. We believe that it is imperative to use the various tracers and application methods prior to their experimental use to develop a consistent standardized approach to retrograde labeling.