Restoration of Visual Function by Enhancing Conduction in Regenerated Axons

Abstract

Although a number of repair strategies have been shown to promote axon outgrowth following neuronal injury in the mammalian CNS, it remains unclear whether regenerated axons establish functional synapses and support behavior. Here, in both juvenile and adult mice, we show that either PTEN and SOCS3 co-deletion, or co-overexpression of osteopontin (OPN)/insulin-like growth factor 1 (IGF1)/ciliary neurotrophic factor (CNTF), induces regrowth of retinal axons and formation of functional synapses in the superior colliculus (SC) but not significant recovery of visual function. Further analyses suggest that regenerated axons fail to conduct action potentials from the eye to the SC due to lack of myelination. Consistent with this idea, administration of voltage-gated potassium channel blockers restores conduction and results in increased visual acuity. Thus, enhancing both regeneration and conduction effectively improves function after retinal axon injury.

Figure 1. Retinocollicular axon regeneration induced by co-deletion of PTEN and SOCS3 in a unilateral optic tract transection model at P6 mice

(A) Illustration of a mouse at postnatal day 6 showing a unilateral knife cut (red arrow) across the entire right superior colliculus (SC; outlined in green). (B) Illustration of a sagittal view showing a knife cut transecting the retinal axons (black arrowed lines) projecting to the superficial layers (green) of the SC. D, dorsal. V, ventral. R, rostral. C, caudal. (C) Schematic diagram showing the experimental timeline. Control group: BCL-2⁺/PTEN^f/f/SOCS3^f/f with AAV-PLAP and regeneration group: BCL-2⁺/PTEN^f/f/SOCS3^f/f with AAV-Cre (plus AAV-CNTF). (D–K) In control group (D–G), few CTB-labeled axons grew across the lesion site [arrows in (E)]. The images in (F) and (G) are the enlarged areas boxed in (D). (H–K) in regeneration group, many labeled axons cross the lesion. The images in (J) and (K) are enlarged areas boxed in (H). (L) Quantification of labeling intensities of regenerating axons at different distances caudal to the lesion sites (pre-lesion SC as 100%; Mean ± SEM, N = 6–7). There were significant differences between control and regeneration groups at every distance from 0 to 0.7 mm distal to the lesion (P < 0.05, ANOVA with Bonferroni post-test). (M) Representative images showing that little or extensive ChR2-mCherry-labeled regenerating axons across the lesion site (arrow and arrowheads) in control and regeneration groups, respectively. Scale bars in (D), (H) and (M): 300 μm. Scale bars in (F) and (J): 50 μm. See also Figures S1–2.

Figure 2. Optogenetic stimulation of regenerated axons at the SC triggers synaptic responses detected by local field potentials in vivo and whole-cell recording in vitro

(A) Schematic diagram showing the experimental timeline. Control group: BCL-2⁺/PTEN^f/f/SOCS3^f/f with AAV-PLAP and regeneration group: BCL-2⁺/PTEN^f/f/SOCS3^f/f with AAV-Cre (plus AAV-CNTF). (B) Illustration of in vivo optogenetic stimulation of post-lesion- (also referred to as terminal-evoked) local field potential (LFPs) in the SC. (C–E) Representative terminal-evoked LFPs from all three groups. No injury was performed in sham group. (F) Maximal terminal-evoked amplitudes in each mouse (each point represents one animal). (G, H) An example (G) of regeneration-group mice showing the terminal-evoked LFP reversibly reduced by local application of kynurenic acid (KA) and the quantification (H) of reduction of amplitude (N = 4). (I) Illustration of in vitro optogenetic whole-cell patch recording in the SC. (J) A representative example showing overlay of six repeated recordings of light evoked postsynaptic responses in a SC neuron at membrane holding potentials of both −70 and +55 mV (N =12). (K, L) AMPA currents recorded at −70 mV (K) were blocked with CNQX by 100% in amplitude (N = 4; in L); with the AMPA currents blocked, the NMDA currents recorded at +55 mV (K) were reduced by D-APV by 77% in amplitude (N=4, in L). Blue lines above traces indicate onset of light stimulation. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ANOVA with Bonferroni post-test except paired t test in L. See also Figure S3.

Figure 3. Regenerated axons fail to improve optomotor visual acuity and lack myelination

(A) Illustration of the optomotor task for quantifying the visual acuities of mice. (B) Optomotor acuities of intact or injured eyes in both control and regeneration groups. No significant differences for both intact and injured eyes in two groups. (C) Illustration of in vivo optogenetic recording of eye-evoked LFPs in the SC. (D–F) Eye-evoked LFPs recorded from the same sites in the SC after recordings of their respective terminal-evoked LFPs (Figure 2C–E). Blue lines above traces indicate onset of light stimulation. (G) Maximal eye-evoked amplitudes in each mouse (each point represents one animal). (H) Ratios of eye-evoked vs. paired terminal-evoked LFPs. (J) Representative images showing lack of co-localization with the myelin marker MAG for the ChR2-mCherry-positive regenerated axons. Scale bars: 10 μm and 1 μm (inset). (K) Quantification results showing that none of the regenerated axonal segments (0/404 in four mice) were co-localized with MAG while ~80% of axonal segments in the sham-operated SC were. N/A, not applicable. ns, not significant. *** P < 0.001, ANOVA with Bonferroni post-test.

Figure 4. 4-AP enhances conduction of regenerated axons and improves regenerated axons-mediated optomotor acuities

(A, B) Representative eye-evoked (A) and terminal-evoked LFPs (B) before and after local 4-AP treatment in a regeneration-group mouse. Blue lines above traces indicate onset of light stimulation. (C) Ratios of eye-evoked vs paired terminal-evoked LFPs showing the effect of 4-AP in regeneration-group mice. * P < 0.05, paired t-test. (D–F) Representative eye-evoked (D) and terminal-evoked LFPs (E) before and after local vehicle treatment in a regeneration-group mouse. (F) Ratios of eye-evoked vs paired terminal-evoked LFPs showing no significant effect of vehicle treatment on LFPs in regeneration-group mice. ns, not significant, paired t-test. (G–I) The effects of 4-AP (4 mg/kg, I.P.) on optomotor acuities of intact and injured eyes of different groups. 4-AP increased the optomotor acuities of injured eyes only in the regeneration group but not in control group (G). In the control group, 4-AP treatment failed to change the optomotor acuities of either injured or intact eyes (H). However, in the regeneration group, 4-AP increased the optomotor acuities of only injured eyes, but not intact ones (I). Data for 4-AP-treated, injured eyes of regeneration mice were re-plotted from those in G, showing the “before (no vehicle or 4-AP)-after” effect of 4-AP. * P < 0.05, *** P < 0.001, ANOVA with Bonferroni post-test. See also Figure S4 for all regeneration-group mice in (G) and (I).

Figure 5. Improved optomotor acuities achieved by combined treatment of 4-AP and axon regeneration in adult-injured mice

(A) Schematic diagram showing the experimental timeline. Control group: PTEN^f/f/SOCS3^f/f with AAV-PLAP and regeneration group: PTEN^f/f/SOCS3^f/f with AAVs-Cre/CNTF. Mice of 8 weeks or older are considered as adults. (B–D) Optomotor acuities in injured and intact eyes of both groups. 4-AP treatment increased the optomotor acuities of injured eyes only in the regeneration group but not in control group (B). In the control group, 4-AP did not change the acuities of either injured or intact eyes (C). However, in the regeneration group, 4-AP increased the optomotor acuities of only injured eyes, but not intact ones (D). N = 10 for control and 8 for regeneration. (E–H) Representative eye-evoked (E, G) or post-lesion terminal-evoked (F, H) LFPs in control (E, F) or regeneration (G, H) group. Post-behavioral in vivo recordings showed 4-AP, but not vehicle, significantly increased eye-evoked LFPs in regeneration-group mice. No LFPs were recorded in all control-injured mice with or without 4-AP. (I) Ratios of eye-evoked vs paired terminal-evoked LFPs showing the effect of 4-AP or vehicle in regeneration-group mice. N = 7 (out of 8; one failed recording due to technical reasons). No eye-evoked LFPs were recorded in two of them. * P < 0.05, ** P < 0.01 ANOVA with Bonferroni post-test. See also Figure S5.

Figure 6. Regenerated axons and their lack of myelination in adult-injured mice

(A–H) Representative images (A, B) and quantification (H) showing that ChR2-mCherry-labeled regenerated axons in regeneration-group (B) but not in control-group mice (A). The lesion sites (arrows) were identified with over-expression of the reactive astrocyte marker GFAP (C, D). The images in (E) and (F) are the enlarged areas boxed in (A) and (B), respectively. (G) The enlarged image of (B) showing only the ChR2 staining. There were significant differences between control and regeneration groups at every distance from 0 to 0.5 mm distal to the lesion (P < 0.05, ANOVA with Bonferroni post-test). Scale bars: 300 μm (A, G) and 50 μm (E). (I, J) ChR2-mCherry-positive regenerated axons are not co-localized with anti-MAG immunoreactivity signal in the adult-injured mice (I) and quantification (J) showing that ~80% of the ChR2-mCherry-labeled axonal segments in the sham-operated mice (272 out of 336 across three mice), but none of regenerated axonal segments (0 out of 330 across three mice) were co-localized with MAG. Scale bars: 10 μm and 1 μm (inset). N/A, not applicable. Data are represented as mean ± SEM. Histological analysis was performed in the mice that had previously undergone functional tests.

Figure 7. Improved behavioral performances mediated by regenerated axons induced by over-expression of OPN/IGF1/CNTF in the presence of 4-APs

(A) Timeline of the experiments. (B) Optomotor acuities in injured eyes of both PLAP and OPN/IGF1/CNTF treated mice. Mice with OPN/IGF1/CNTF treatment show increased optomotor acuities with 4-AP (4 mg/kg) or 4-AP-3-Me (1 mg/kg). N = 8 for each group. (C) Ratios of eye-evoked vs paired terminal-evoked LFPs showing the effect of locally applied 4-AP-3-Me (0.25 μM) or vehicle in regeneration-group mice. * P < 0.05, ** P < 0.01 ANOVA with Bonferroni post-test. (D)–(G) Images and quantification of ChR2-labeled regenerated axons. No axon regenerated was observed in the mice treatment with PLAP (D & G); many axons seem to retract from the injury site. In contrast, OPN/IGF1/CNTF treatment resulted in long-distance axon regeneration up to 4 mm away from the injury site (E & G). Some axons sprouted along the pathway (E) and some elaborated extensively within the SC (F). Axons in boxed area in E is enlarged in F. Scale bar in D and F: 300 μm and 50 μm respectively. (H) Images showing that ChR2-mCherry-positive regenerated axons are not co-stained with anti-MAG immunoreactivity signal (labeling myelin). Over 200 individual axonal segments were examined and no co-staining was observed. Scale bars: 10 μm. See also Figures S5–7.