Optic nerve regeneration. (A and B) Ten weeks after optic nerve injury, CTB⁺ axons (red) extend the full length of the optic nerve in Group I mice (A) but only part-way in Group II (B). (C–E) Region in the white rectangle of A double-labeled for CTB and GAP-43. (F) Time-course of axon regeneration in Group I mice as visualized by CTB-labeling. Bars show numbers of CTB⁺ axons counted 2.5 mm (gray) and 5 mm (black) from the injury site at 1, 6, and 10 wk after nerve injury. Note progressive increase in lengthy axons over time. (G–I) High-power EM images through optic nerves of Group I mice show axons that appear to be undergoing remyelination (G, arrow), unmyelinated axons (yellow arrows), and axons with thick myelin (blue arrow, H) that are seen at higher magnification to contain multiple lamellae of tightly-packed myelin (I). (J–M) RGC survival. (J) Average number of viable RGCs counted in the retinas of mice in Groups I and II. (K–M) RGC survival visualized by immunostaining for βIII-tubulin in uninjured (K), Group I (l), and Group II (M) mice. (N) CTB⁺ axons in the optic chiasm. Note axons coursing in the optic tract on the right side (contralateral to the regenerating optic nerve) and a smaller number on the left side (white arrowheads). Some CTB⁺ profiles are seen within the SCN bilaterally (blue arrowheads) and some are outside this area (yellow arrowheads). Error bars in F and J show SEM. **P < 0.01; ***P < 0.001. (Scale bars: A and B, 200 μm; C–E, 50 μm; K–M, 50 μm. All others as indicated.)

Reinnervation of other target areas. (A–D, F, and G) Superior colliculus. CTB⁺ axons are seen at the rostral (A), middle (B), and caudal (C) levels of the SC of a Group I mouse. (D) Counterstaining with an antibody to NeuN shows that CTB⁺ axons are within the neuropil of the SC. Dotted line indicates approximate ventral extent of the SC. Absence of CTB⁺ axons in the SC across from the side shown in A–D (E) and in a Group II mouse (F). G–L Reinnervation of the OPT and MTN. CTB⁺ fibers are abundant on the side contralateral to the regenerating optic nerve (G and J) but are absent from the ipsilateral side (H and K) and from either side in Group II mice I and L. (Scale bars: 100 μm. Bar in C is for panels A–C, E, and F; bar in I is for G–I; bar in L is for J–L).

Reinnervation of the lateral geniculate nucleus. CTB⁺ fibers at the rostral (A), middle (B), and caudal (C) levels of the DLG contralateral to the regenerating optic nerve in a Group I mouse. Counterstaining for the neuronal protein NeuN shows CTB⁺ fibers to be confined to the neuropil of the DLG (dotted line). (E) CTB⁺ axons are absent in the DLG ipsilateral to the regenerating nerve in the same mouse shown in A–D. (F) No CTB⁺ fibers are seen in the DLG of any Group II mice. (G–I) Reinnervation of the VLG contralateral to the regenerating optic nerve. CTB⁺ axons (G) show intense labeling for the RGC-specific marker ERRβ (H and I), and colocalize with the presynaptic marker Vglut2 (J). (K and L) Distribution of the postsynaptic marker PSD-95 at low (K) and high magnification (L). L is a single confocal plane showing apposition of CTB⁺ terminals and PSD-95⁺ structures. Side panels show z-stacks of images in the orthogonal planes. (Scale bars: A–I, 100 μm; all others, 20 μm.)

Partial recovery of visually guided behaviors. (A) Top-down view of visual cliff. (B) Average latency to step off shallow end (Left) and total time spent on shallow end (Right). ***P < 0.01. (C) Population frequency histogram for time spent on shallow end. (D) Top-down view of apparatus used to evaluate OMR. (E) Average OMR (response threshold, cycles/degree) as a function of time. (F) Frequency distribution of the OMR. Note that y-axes in E and F are discontinuous. (G) Circadian photoentrainment: percentage of overall activity in 1-h bins for individual mice (Left) and group averages (Right). Mice were maintained on a continuous cycle of lights on at 7:00 AM and off at 7:00 PM before testing and for the first 2.5 d in the activity monitor. The light cycle was set back 6 h on day 3. Error bars represent SEM.