Article Figures & Data
Figures
- Figure 1.
Tactile allodynia, S1 excitability, and long-term imaging. A, Development and maintenance of tactile allodynia following PSL (n = 12, pooled of 6 wild-type and 6 M-line mice) as shown by reduced mechanical threshold. Sham, Sham-operated mice (n = 12). Left and right, Left and right hindpaws. B, Top, Representative traces of somatosensory-evoked potentials in the S1 showing the typical primary response, positive-negative (P1–N1) wave. Bottom, Amplitude of P1–N1 wave increased in the early phase (PSL + 3 d, n = 3) and to a greater extent in the late phase (PSL + 9 d, n = 3) (*p < 0.04 vs ShamCont: pooled of 3 sham-operated and 2 normal control mice). C, Timeline of long-term imaging experiments. Di, Schematic of in vivo imaging of the S1 (blue circle); Dii, intrinsic optical signal map of the S1 hindpaw area (scale bar, 500 μm); Diii, low-magnification z-projection image of a layer 5 pyramidal cell (scale bar, 50 μm) taken in the region indicated in Dii (yellow square); Div, schematic of PSL injury. E, High-magnification repeated imaging of the same dendritic segment shown in Diii (red box). All types of dendritic protrusions are included in analysis, except the spines near dendritic tips. Arrowheads indicate spines generated (red) or eliminated (blue) when compared with the previous imaging session. Scale bar, 5 μm.
- Figure 2.
Change in spine dynamics following PSL injury and its prevention by immediate local nerve blockade. A, Spine turnover rate (*p < 0.02, **p < 0.003). B, Spine gain rate (*p < 0.03, **p < 0.007). C, Spine loss rate (*p < 0.05). D, Normalized spine density (*p < 0.04). Control, 356 spines from 9 dendrites in 3 mice; Sham, 386 spines from 11 dendrites in 4 mice; PSL, 522 spines from 12 dendrites in 5 mice. E, Representative images of the same dendrite taken before and after PSL with local nerve blockade using Elvax-TTX implantation. Arrowheads indicate spines generated (red) or eliminated (blue) when compared with the previous session. Scale bar, 2 μm. F, G, Local nerve blockade throughout the early phase of neuropathic pain inhibited the increase of spine gain and loss (255 spines from 6 dendrites in 3 mice, F) and development of allodynia (each group: n = 6, G).
- Figure 3.
Phase-specific survival/growth of new spines. A, Schematic of transient, NP, and total new (TN) spines. B, Density of preinjury and postinjury NP spines (*p < 0.04). C, Ratio of NP spines to TN spines. ShamCont, 17 dendrites from 7 mice; PSL, 12 dendrites from 5 mice. D, Preinjury NP spines (n = 9) increase in normalized brightness before PSL, but gradually decrease after injury [*p < 0.02 vs ShamCont (n = 14)]. E, Postinjury NP spines (n = 10) show a marked increase in brightness during the late phase of neuropathic pain [*p < 0.05 vs ShamCont (n = 15)]. Yellow arrowheads indicate NP spines. Scale bars, 2 μm.
- Figure 4.
Size-dependent elimination of preexisting stable spines and LTP-like long-lasting enlargement of always-present spines following neuropathic injury. A, Schematic of LP, AP, and preexisting stable spines. B, Survival rate of preexisting stable spines fitted with one exponential (S = Te−d/τ + P, where S is the survival rate, T is an estimate of the percentage of spines that will ultimately disappear with a time constant τ, and P is an estimate of the percentage of spines that persist throughout life). C, Comparison of integrated brightness of AP (PSL: n = 32; ShamCont: n = 40) and LP spines (PSL: n = 28; ShamCont: n = 30) just before injury (day 0; *p < 0.02, **p < 0.002). Red circles (n = 8) indicate individual AP spines showing long-lasting enlargement (criteria: >30% increase in brightness at PSL + 3 d and >10% increase at all subsequent time points, based on the graph in D). D, Top, Representative images of AP spines before and after PSL. Scale bars, 2 μm. Note that a large AP spine (yellow arrowhead, corresponding to “L3” in C, E) shows long-lasting enlargement over weeks following injury. Bottom, LTP-like increase in normalized brightness of AP spines (n = 32) after PSL injury [*p < 0.03, vs ShamCont (n = 40)]. Note that Elvax-TTX implantation (blue square) immediately after injury blocked the increase in AP spine brightness at PSL + 3 d. E, Long-term traces of normalized brightness in three large (L1–L3) and small (S1–S3) AP spines indicated in C.
