Aberrant information transfer interferes with functional axon regeneration
- Yale University, United States
Figures
Figure 1 with 1 supplement
DA9 axon regeneration and degeneration.
(A) Diagram of DA9 morphology and the laser cutting site. (B) A control adult expressing GFP::RAB-3 and mCherry in DA9. mCherry expression is also observed in the posterior gut in these micrographs. (C) A control animal 12 hr after axotomy. The distal axon fragment remains and overlaps with the regenerating axon. The white lightning mark indicates the axotomy site. (D) A ric-7(n2657) animal 12 hr after axotomy. The distal fragment has degenerated. Scale bars = 10 µm.
Figure 1—figure supplement 1
ric-7(n2657) animals show normal DA9 development, better regeneration and increased degeneration of GABA motor neurons.
(A and B) GFP::RAB-3 and mCherry labeling in DA9 of control (A) and ric-7(n2657) (B) animals (2-day-old adults). Scale bars = 10 µm. (C) Length of the asynaptic region and the synaptic region in control and ric-7(n2657) animals (2-day-old adults). Mean ± SEM. ns, not significant. Unpaired t test. (D) Number of axonal RAB-3 puncta in control and ric-7(n2657) animals (2-day-old adults). Mean ± SEM. ns, not significant. Unpaired t test. (E and F) Regeneration and degeneration of GABA motor neurons in control (E) and ric-7(n2657) (F) animals 24 hr after axotomy. White arrowheads point to the remaining distal axon segments in control (E) which have degenerated in ric-7(n2657) (F). The red line in (F) indicates the length of the regenerating axon along the dorsal-ventral axis and the white line indicates the width of the animal. Scale bars = 10 µm. (G) Relative length of regenerating axons (the ratio of the length of red line and white line in (F)) in control and ric-7(n2657) animals 24 hr after axotomy. Black line is median. ***p<0.0001. χ2 test.
Figure 2 with 2 supplements
DA9 forms normal and dendritic synapses during regeneration.
(A) Diagram of a regenerated DA9 axon. (B–D) DA9 axonal and synaptic regeneration at different timepoints after axotomy. The white lightning mark indicates the axotomy site. Scale bars = 10 μm. (E) Number of axonal RAB-3 puncta at different timepoints with or without axotomy. Mean ± SEM. *p<0.05; **p<0.01; ****p<0.0001; ns, not significant. Unpaired t test for comparisons between two groups and one-way ANOVA for more than two groups. (F) Length of the asynaptic and the synaptic region in intact and axotomized animals 48 hr after axotomy. Mean ± SEM. ****p<0.0001; ns, not significant. Unpaired t test. (G and H) Total axon length and number of dendritic RAB-3 puncta at different timepoints with or without axotomy. Mean ± SEM. ***p<0.001; ****p<0.0001; ns, not significant. Unpaired t test for comparisons between two groups and one-way ANOVA for more than two groups.
Figure 2—figure supplement 1
Axotomy in the DNC enables accurate axon growth following the original path.
(A) DA9 regeneration 48 hr after axotomy in the DNC. The lightning mark indicates the cutting site. The scale bar = 10 μm. (B) DA9 regeneration 48 hr after axotomy in the dorsal-ventral midline of the commissure. The lightning mark indicates the cutting site. The arrowhead indicates the growth cone. The scale bar = 10 μm. (C) Quantification of regenerated DA9 axons following the original axon path or an alternative path. The number indicates number of animals.
Figure 2—figure supplement 2
Regeneration of other presynaptic components.
(A–A’’) UNC-10 localization in intact DA9. Scale bars = 10 μm. (B–B’’) UNC-10 localization in regenerated DA9. Scale bars = 10 μm. (C–C’’) CLA-1S localization in intact DA9. Scale bars = 10 μm. (D–D’’) CLA-1S localization in regeneration DA9. Scale bars = 10 μm. (E) Quantification of axonal UNC-10 puncta number. ****, p<0.0001. Unpaired t test. (F) Quantification of axonal CLA-1S puncta number. ****, p<0.0001. Unpaired t test.
Figure 3 with 1 supplement
Postsynaptic receptors to DA9 maintain their localization and are aligned with DA9 SV clusters after axotomy.
(A) Labeling of DA9 axon and ACR-12 in GABA motor neurons in intact animals (2d old adults). Scale bars = 10 μm. (B) Labeling of DA9 axon and ACR-12 in GABA motor neurons in axotomized animals. Scale bars = 10 μm. (C) ACR-12::GFP puncta number in the putative DA9 synaptic region (~85 μm anterior from the most posterior ACR-12 puncta in the dorsal nerve cord) in both intact and axotomized animals. ns, not significant. Unpaired t test. (D) Length of posterior DA9 axon without ACR-12 in both intact and axotomized animals. ns, not significant. Unpaired t test. (E) BFP::RAB-3 puncta in DA9 axon appose ACR-12::GFP puncta in the postsynaptic GABA motor neurons in intact animals. Scale bars = 10 μm. (F) BFP::RAB-3 puncta in DA9 axon appose ACR-12::GFP puncta in the postsynaptic GABA motor neurons 48 hr after axotomy. Scale bars = 10 μm. (G) Percentage of BFP::RAB-3 apposing ACR-12::GFP in intact and axotomized animals. ns, not significant. Unpaired t test.
Figure 3—figure supplement 1
No change of postsynaptic receptors in body wall muscles after axotomy.
(A–A’’) Muscle ACR-16 localization in intact animals. Note its localization in both the dorsal and ventral nerve cords. Scale bars = 10 μm. (B–B’’) Muscle ACR-16 maintains its localization 48 hr after DA9 axotomy. Scale bars = 10 μm. (C) ACR-16::GFP puncta number in the putative DA9 synaptic region (~85 μm anterior from the most posterior ACR-16 puncta in the dorsal nerve cord) in both intact and axotomized animals. ns, not significant. Unpaired t test. (D) Length of posterior DA9 axon without ACR-16 in both intact and axotomized animals. ns, not significant. Unpaired t test. (E) Number of ACR-16::GFP puncta overlapping with DA9 dendrite in both intact and axotomized animals. ns, not significant. Unpaired t test.
Figure 4 with 2 supplements
Aberrant recovery of a single neuron-driven behavior.
(A) Diagram of the optogenetically triggered tail-bending behavior. (B) Images from an example movie showing the dorsal tail-bending behavior. (C–H) Quantification of the tail-bending behavior of intact animals (C), axotomized animals 5 min after axotomy (D), 12 hr after axotomy (E), 24 hr after axotomy (F), 48 hr after axotomy (G) and 72 hr after axotomy (H). Both averaged data (upper, Mean ± SEM) and heat maps (lower) of individual responses are shown. Shaded areas represent the 5 s light stimulation. The color bar indicates the tail angle. Positive numbers indicate dorsal bending, and negative numbers indicate ventral bending. (I) Averaged tail angle during the 5 s stimulation at different timepoints after axotomy of animals in (C–H). Mean and SEM. **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant. Unpaired t test. (J and K) Recovery of dorsal bending is abolished 12 hr after the second cut of the regenerating axon (K) but stays the same in the absence of the second cut (J). ****p<0.0001; ns, not significant. Paired t test.
Figure 4—figure supplement 1
Optogenetically-triggered tail-bending behavior requires Chrimson and ATR and is DA9 specific.
(A) ric-7(n2657) animals expressing mCherry instead of Chrimson supplied with ATR show no dorsal tail bending. Mean ± SEM. The shaded area represents the 5 s stimulation. (B) ric-7(n2657) animals expressing Chrimson in the absence of ATR show no dorsal tail bending. Mean ± SEM. The shaded area represents the 5 s stimulation. (C) WT animals expressing Chrimson supplied with ATR but with their DA9 cell body ablated show no dorsal tail bending. Mean ± SEM. The shaded area represents the 5 s stimulation. (D) Averaged tail angle during stimulation of positive controls and animals in (A), (B) and (C). The positive controls are the same as in Figure 4C. Mean and SEM. ****p<0.0001. Unpaired t test.
Figure 4—figure supplement 2
Optogenetically-triggered tail-bending behavior depends on synaptic transmission.
(A) Tail-bending behavior of intact unc-13(e51) animals (2-day-old adults). (B) Tail-bending behavior of unc-13(e51) animals 48 hr after axotomy. (C) Averaged tail angle during stimulation of both intact and axotomized unc-13(e51) animals in (A) and (B). ns, not significant. Unpaired t test.
Figure 5 with 1 supplement
Simultaneous optogenetic stimulation and calcium imaging reveals rerouted information transfer in the regenerated circuit.
(A–D) Heat maps of calcium signal fold changes (df/f) in intact (A and C) and axotomized animals (B and D) 12 hr or 48 hr after axotomy. White dashed lines delineate the animals’ tails. Scale bars = 20 μm. (E–H) Calcium signal traces (E and F) and peak amplitudes during stimulation (G and H) of both dorsal and ventral BWMs in intact and axotomized animals 12 hr after axotomy (n = 29 for axotomized and 25 for intact animals). Shaded areas indicate the 5 s light stimulation. Mean ± SEM. *p<0.05; ****p<0.0001. Unpaired t test. (I–L) Calcium signal traces (I and J) and peak amplitudes during stimulation (K and L) of both dorsal and ventral BWMs in intact and axotomized animals 48 hr after axotomy (n = 30 for axotomized and 31 for intact animals). Shaded areas indicate the 5 s light stimulation. Mean ± SEM. *p<0.05; **p<0.01. Unpaired t test.
Figure 5—figure supplement 1
Optogenetically induced muscle calcium response requires all-trans retinal and depends on synaptic transmission.
(A) Calcium responses in intact adult animals cultured with all-trans retinal (ATR) (n = 17). (B) No calcium responses in intact adult animals cultured without ATR (n = 24). (C) No calcium responses in intact unc-13(e51) animals with ATR (n = 25). (D) No calcium responses in unc-13(e51) animals 48 hr after axotomy with ATR (n = 24).
Figure 6 with 1 supplement
Aberrant information transfer is independent of dlk-1 and suppresses behavioral recovery.
(A and B) GFP::RAB-3 and mCherry labeling of DA9 in intact and axotomized ric-7(n2657); dlk-1(ju476) animals 48 hr after axotomy. Scale bars = 10 μm. (C and D) Comparison of length of regenerating axons (C) and number of dendritic RAB-3 puncta (D) between ric-7(n2657) and ric-7(n2657); dlk-1(ju476) animals 48 hr after axotomy. Mean ± SEM. ****p<0.0001; ns, not significant. Unpaired t test. (E) Ventral tail-bending behavior of ric-7(n2657); dlk-1(ju476) animals 48 hr after axotomy. The shaded area indicates the 5 s light stimulation. Mean ± SEM. (F) Traces of tail-bending behavior of ric-7(n2657) intact (red) and axotomized animals (green) 48 hr after axotomy. Dendrites of axotomized (orange) and intact animals (blue) were then cut and behavior was measured 2 hr later. Mean ± SEM. The shaded area indicates the 5 s light stimulation. (G) Averaged tail angles during stimulation of the animals in (F). Mean and SEM. ****p<0.0001; ns, not significant. Paired t test between green and orange. Unpaired t test between orange, red and blue.
Figure 6—figure supplement 1
Behavior response of ric-7(n2657); dlk-1(ju476) animals with and without axotomy.
(A) ric-7(n2657); dlk-1(ju476) intact animals show robust dorsal tail bending. The shaded area represents the 5 s stimulation. Mean ± SEM. (B) ric-7(n2657); dlk-1(ju476) axotomized animals show robust ventral tail bending 12 hr after axotomy. Mean ± SEM. (C) Averaged tail angle during stimulation of animals in (A) and (B). Mean and SEM. ****p<0.0001. Unpaired t test. (D) Images from a movie showing the ventral-bending behavior of a ric-7(n2657); dlk-1(ju476) animal 12 hr after axotomy.
Figure 7 with 1 supplement
Dendritic microtubule polarity is maintained after axotomy.
(A) Diagram of regions of EBP-2::GFP imaging in DA9. (B–E) Kymographs of EBP-2 traces in both DA9 dendrite and axon in intact and axotomized animals 12 hr after axotomy. Scale bars are 5 μm and 5 s. (F) Direction of EBP-2 traces in DA9 dendrite and axon. Dendritic microtubule polarity is opposite to axonal microtubule polarity. Numbers represent the number of EBP-2 traces. (G) Total number of EBP-2 traces in DA9 dendrite and axon with and without axotomy. Numbers represent the number of animals. Mean and SEM. **p<0.01. Unpaired t test.
Figure 7—figure supplement 1
ACR-2, a subunit of the nicotinic acetylcholine receptor, maintains dendritic localization after axotomy.
(A) ACR-2::GFP localizes to the dendrite and soma of DA9 in intact ric-7(n2657) animals. The scale bar = 10 μm. (B) ACR-2::GFP still localizes to the dendrite and soma of DA9 in axotomized ric-7(n2657) animals 12 hr after axotomy. The scale bar = 10 μm.
Figure 8 with 1 supplement
JNK-1 and dynein-mediated transport mediate SV mislocalization to the dendrite and loss of JNK-1 improves behavioral recovery.
(A–D) GFP::RAB-3 localization 48 hr after axotomy in ric-7(n2657) (A), ric-7(n2657); dhc-1(js319) (B), ric-7(n2657); nud-2(ok949) (C) and ric-7(n2657); jnk-1(gk7) (D) animals. Asterisk indicates cell body and bracket indicates dendrite. Scale bars = 10 μm. (E) Quantification of the number of dendritic RAB-3 puncta in intact and axotomized animals 48 hr after axotomy. Mean ± SEM. **p<0.01; ****p<0.0001; ns, not significant. Unpaired t test. (F) Traces of the tail-bending behavior of ric-7(n2657) and ric-7(n2657); jnk-1(gk7) animals with and without axotomy 48 hr after axotomy. The shaded area indicates the 5 s stimulation. Mean ± SEM. (G) Averaged tail angle during stimulation of the animals in (F). Numbers represent the number of animals. Mean and SEM. ***p<0.001; ****p<0.0001. Unpaired t test.
Figure 8—figure supplement 1
DA9 in dhc-1(js319) animals develops normally and loss of JNK-1 rescues RAB-3 mis-localization in DA9 dendrite 12 hr after axotomy.
(A–B) RAB-3::GFP and mCherry labeling of DA9 in intact ric-7(n2657) and ric-7(n2657); dhc-1(js319) animals (2-day-old adults). Scale bars = 10 μm. (C–F) RAB-3 localization to the dendrite inArxiocn-7ti(pn2657) and ric-7(n2657); jnk-1(gk7) animals with and without axotomy 12 hr after axotomy. Magnified GFP channels are shown at the top right corner. Scale bars = 10 μm. (G) Quantification of dendritic RAB-3 puncta number in animals in (C–F). Mean ± SEM. *p<0.05; ****p<0.0001; ns, not significant. Unpaired t test.
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- https://doi.org/10.7554/eLife.38829.021
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Aberrant information transfer interferes with functional axon regeneration
eLife 7:e38829.
https://doi.org/10.7554/eLife.38829
