Fig 1.
A. The magnitude of the direct muscle (M) EMG responses evoked optically. The M response amplitudes always reached 100% prior to maximizing the laser output. Optical treatments were performed at a supramaximal intensity (similar to electrical stimulation paradigms) B. The magnitude of the direct EMG (M) response evoked electrically (top) or optically (bottom). A subset of axons within the sciatic nerve are ChR2+. Thus, the maximum electrically-evoked M responses were always larger than the maximum optically-evoked M responses. C. EMG responses to a continuous train of optical activations. Muscles will follow up to 20 Hz trains of blue light pulses during an hour of optical treatment.
Table 1.
Summary of Experiments.
Fig 2.
Anatomical expression ChR2/YFP.
Image of a whole mount of the L4 dorsal root ganglion, dorsal root, and ventral root from a Thy1-ChR2/YFP mouse. Note that a subset of axons in the ventral root (VR) and dorsal root (DR) are ChR2+ as indicated by the presence of YFP in the axons. The insets are a higher magnification of the image marked with a white box. Inset 1: Strong YFP expression was observed in the cell bodies of primary sensory neurons. Inset 2: Small, punctate regions were observed along the motor axons.
Fig 3.
Retrograde labeling to assess axon regeneration.
Sciatic nerves were cut and repaired by end-to-end anastomosis. The mean (+SEM) number of motoneurons whose axons had regenerated at least four mm into the distal stump and were labeled by retrograde tracer applied via nerve soak three weeks after transection and repair is shown in panel A. The mean (+SEM) number of motoneurons whose axons had regenerated successfully and had re-innervated the lateral and medial gastrocnemius muscles after tracer injection into muscles four weeks after sciatic nerve transection and repair is shown in panel B. Results from optically treated and untreated mice were compared. C. The ratio of the number of motoneurons in optically treated to untreated mice is shown as mean (+SEM) values. The results from both retrograde labeling methods indicate that more than twice as many motoneurons regenerated following optical treatment.
Fig 4.
Synaptic Vesicle Protein 2 (SV2) staining to assess muscle reinnervation.
A. Using the binding of fluorescent alpha bungarotoxin (α-Btx), two motor endplates are shown (top and bottom left panels). At one endplate, immunoreactivity to SV2 is found (top right, SV2+), but no similar immunoreactivity is found at the second one (bottom right, SV2-). B. Mean (+SEM) percentage of all endplates that were re-innervated (SV2+) at three and four weeks after transection and repair is shown for each group. As expected, significantly more neuromuscular junctions were SV2+ over time when untreated. Optical treatment markedly enhances the number of SV2+ neuromuscular junctions by four weeks after transection and repair * p ≤ 0.001 vs all groups.
Fig 5.
Functional assessment of the gastrocnemius innervation following nerve injury.
M responses elicited by optical and electrical nerve stimulation were measured three and four weeks after transection and repair of the sciatic nerve. A. Examples of electrically evoked (solid traces) and optically evoked (dashed traces) M responses recorded from the gastrocnemius muscle of a thy-1-ChR2/YFP mouse. Recordings were made prior to (Intact) and four weeks after transection and surgical repair of the sciatic nerve, followed by one hour of optical stimulation. B. Mean (+SEM) M response amplitudes, evoked optically from the gastrocnemius muscles at three and four weeks after sciatic nerve transection and repair are shown for both: optically treated and untreated animals. * p < 0.05 vs all other groups. C. The mean (+SEM) ratio of optically evoked to electrically evoked M responses recorded from gastrocnemius muscles of optically treated and untreated mice is shown. Measurements presented were made in Intact animals and three or four weeks after transection and repair of the sciatic nerve. The horizontal dashed line is placed at the mean ratio found in intact mice. * p < 0.05 vs all other groups.