Electroacupuncture facilitates remote fear memory erasure via promoting perineuronal net degradation

Abstract

Post-retrieval extinction has been effective in erasing recent pathological memories but has been shown failed in erasing remote memories. Pathological memories are often formed over the long term, therefore how to erase the remote pathological memory remains a long-lasting question. Here, we identified the increased perineuronal nets (PNNs) surrounding engram cells with memory being remote in the prelimbic cortex (PrL) which rigidify the remote memory from erasure. Microinjection of ChABC into PrL could facilitate remote memory erasure by degrading PNNs in post-retrieval extinction paradigm. In addition, electroacupuncture (EA) at Baihui (GV20) and Neiguan (PC 6) could degrade the PNNs in PrL by increasing the neuronal MMP-9 expression via BDNF-TrkB signaling, leading to the remote memory erasure under post-retrieval extinction condition. Finally, we found EA stimulation facilitated remote memory erasure led to increased overlap between fear and extinction cells in the PrL. Our data clarified the essential role of PNNs surrounding engram cell in preventing remote memory from erasure, and provided evidences of EA facilitates remote pathological memory erasure as a non-invasive therapeutic method.

Fig. 1. The PNNs surrounding engram cells was increased in the PrL at a remote timepoint.

A Labeling strategy of the inducible double transgenic TetTag mouse and the experimental design. B Representative images of H2B-GFP⁺ (green) and c-Fos⁺ (red) immunofluorescence in PrL, BLA and CeA.Scale bar, 200 μm (top), 20 μm (bottom). C Quantification of H2B-GFP+ cell number in PrL, BLA and CeA. D The levels of PNN expression in PrL, BLA and CeA (two-way ANOVA with Sidak’s multiple comparisons test, ***p < 0.001; n = 5 animals per group including 3 females and 2 males). E Quantification of the number of c-Fos surrounded by PNNs in PrL, BLA and CeA. F Quantification of theratio of c-Fos surrounded by PNNs in PrL, BLA and CeA (two-way ANOVA with Sidak’s multiple comparisons test, ***p < 0.001). Data are presented as mean ± SEM.

Fig. 2. Retrieval-extinction paradigm following 7 days of EA stimulation effectively erased remote fear memory.

A Schematic of EA treatment, WFA staining following chABC microinjection into PrL and behavioral paradigm. B The freezing levels during conditioning, retrieval, extinction, EM test and SR test (RM two-way ANOVA with Sidak’s multiple comparisons test, ****p < 0.0001; n = 8 animals per group including 4 females and 4 males). C Representative images of H2B-GFP (green) and WFA (Red) expression in the PrL. Scale bar, 200 μm (top), 20 μm (bottom). D The quantification of H2B-GFP⁺ cells. E The levels of PNN in the PrL (unpaired t-test, ****p <0.0001). F The cell number surrounded by PNNs expression of H2B-GFP⁺ cells or H2B-GFP^- cells in PrL (two-way ANOVA with Sidak’s multiple comparisons test, *p <0.05, ***p <0.0001). Data are presented as mean ± SEM.

Fig. 3. EA induces increased MMP-9 expression in PrL neurons.

A Schematic of EA treatments, WFA staining and behavioral paradigm. B The expression of c-fos, arc, bdnf, mmp9, adamts4 and adamts5 were verified by RT-PCR (two-way ANOVA with Sidak’s multiple comparisons test, ***p < 0.001, **p < 0.005, *p < 0.05; n = 7 animals in Sham group, n = 9 animals in EA group). C Top: Representative images of MMP-9 (red) and NeuN (green) expression in the PrL. Scale bar, 200 μm (Top) and 20 μm (bottom). Bottom: Representative images of MMP-9 (red) and Iba1 (green) expression in the PrL. Scale bar, 200 μm (Top) and 20 μm (bottom). D Quantification of MMP-9 positive cell number in PrL (unpaired t-test, **p < 0.005; n = 4 animals per group). E Quantification of MMP-9⁺NeuN⁺ cell number in PrL (unpaired t-test, ***p < 0.001; n = 4 animals per group). F Quantification of MMP-9+Iba1+ cell number in PrL. Data are presented as mean ± SEM.

Fig. 4. EA treatment could erase remote fear memory under R + E paradigm via promoting PNN degradation in a MMP9 dependent pathway.

A Schematic of EA treatments and behavioral paradigm. B The freezing levels during conditioning, retrieval, extinction, EM test and SR test (RM two-way ANOVA with Sidak’s multiple comparisons test, ***p < 0.001; n = 10 animals per group including 5 females and 5 males). C Representative expression of BDNF in PrL by western blots. D Quantification of BDNF protein levels for western blot experiment (one way ANOVA with Turkey’s post-hoc comparison, ***p <0.001). E Representative expression of MMP-9 in PrL by western blots. F Quantification of MMP-9 protein levels for western blot experiment (one way ANOVA with Turkey’s post-hoc comparison, ***p <0.001). G Representative images of WFA (green) expression in the PrL. Scale bar, 200 μm. H Quantification of PNN levels in PrL for immunostaining experiment. (I-K) Quantification of MMP-9 enzyme activity during RE(I), EM (J) or SR (K) (one way ANOVA with Turkey’s post-hoc comparison, **p <0.005, ***p <0.001). Data are presented as mean ± SEM.

Fig. 5. EA induced remote memory erasure depends on BDNF-TrkB signaling.

A Schematic of EA treatments and behavioral paradigm. B The freezing levels during conditioning, retrieval, extinction, EM test and SR test (RM two-way ANOVA with Sidak’s multiple comparisons test, ***p < 0.001; n = 10 animals per group including 5 females and 5 males). C Representative expression of BDNF in PrL by western blots. D Quantification of BDNF protein levels for western blot experiment (one way ANOVA with Turkey’s post-hoc comparison, ***p <0.001). E Representative expression of MMP-9 in PrL by western blots. F Quantification of MMP-9 protein levels for western blot experiment (one way ANOVA with Turkey’s post-hoc comparison, ***p <0.001). G Representative images of WFA (green) expression in the PrL. Scale bar, 200 μm. H Quantification of PNN levels in PrL for immunostaining experiment. (I-K) Quantification of MMP-9 enzyme activity during RE(I), EM (J) or SR (K) (one way ANOVA with Turkey’s post-hoc comparison, ***p <0.001). Data are presented as mean ± SEM.

Fig. 6. EA enhances overlap between fear and extinction cells in the PrL during remote fear memory erasure.

A Labeling strategy of fear engram cell by the inducible TetTag virus tool and the experimental design. B Representative images of mCherry⁺ (red) and c-Fos⁺ (green) immunofluorescence in PrL. The white arrow marked colabeled mCherry⁺c-Fos⁺ cells. Scale bar, 200 μm (top), 20 μm (bottom). C Quantification of mCherry⁺ cell density in PrL. D Quantification of c-Fos⁺ cell density in PrL. E Quantification of overlap levels in PrL (paired t test, ***P < 0.001; one-way ANOVA with Tukey’s multiple comparisons test, ^###P < 0.001). Data are presented as mean ± SEM.