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Preservation of a remote fear memory requires new myelin formation

Abstract

Experience-dependent myelination is hypothesized to shape neural circuit function and subsequent behavioral output. Using a contextual fear memory task in mice, we demonstrate that fear learning induces oligodendrocyte precursor cells to proliferate and differentiate into myelinating oligodendrocytes in the medial prefrontal cortex. Transgenic animals that cannot form new myelin exhibit deficient remote, but not recent, fear memory recall. Recording population calcium dynamics by fiber photometry, we observe that the neuronal response to conditioned context cues evolves over time in the medial prefrontal cortex, but not in animals that cannot form new myelin. Finally, we demonstrate that pharmacological induction of new myelin formation with clemastine fumarate improves remote memory recall and promotes fear generalization. Thus, bidirectional manipulation of myelin plasticity functionally affects behavior and neurophysiology, which suggests that neural activity during fear learning instructs the formation of new myelin, which in turn supports the consolidation and/or retrieval of remote fear memories.

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Fig. 1: Fear learning induces OPC proliferation in the mPFC.
Fig. 2: Fear learning experience induces OPC maturation into myelinating OLs in the mPFC.
Fig. 3: OPC maturation into myelinating OLs occurs over several weeks in adult gray matter.
Fig. 4: Inhibition of new myelin formation impairs remote fear memory recall.
Fig. 5: Immediate early gene expression following remote recall is impaired in the absence of new myelin formation.
Fig. 6: Prefrontal population calcium dynamics in the mPFC are altered in the absence of new myelin formation.
Fig. 7: Induction of new myelin formation preserves remote fear memory recall.
Fig. 8: Induction of new myelin formation increases immediate early gene expression following remote fear memory recall.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

Custom Matlab software written for extraction, analyses and visualization of photometry signals is available as Supplementary Software 13 and online at https://github.com/sp808/Fiber-photometry.

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Acknowledgements

We are grateful to K. Hoi for providing reagents, insightful discussion, technical assistance and comments on the manuscript. We also thank members of the Chan and Kheirbek laboratories for technical assistance and scientific discussion, and V. Sohal, S. Pleasure, K. Poskanzer and A. Molofsky for insightful discussions. This work was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS062796, R01NS097428 and R01NS095889), the Adelson Medical Research Foundation (ANDP grant A130141) and the Rachleff Family Endowment to J.R.C., and the NIMH (R01 MH108623, R01 MH111754 and R01 MH117961), a Weill Scholar Award, the Pew Charitable Trusts, the Esther A. and Joseph Klingenstein Fund, and an IMHRO/One Mind Rising Star Award to M.A.K.

Author information

Affiliations

Authors

Contributions

S.P. conceptualized the project. S.P. designed and performed all the experiments. M.A.K. and J.R.C. provided technical expertise, funding and advice. S.P., M.A.K. and J.R.C. drafted and edited the manuscript. S.P. wrote all of the code for photometry signal processing and analyses, performed the quantification and data analyses and prepared the figures. S.R.M. prepared the electron microscopy samples and performed image quantification. H.S.C. assisted with histological preparations and quantification.

Corresponding authors

Correspondence to Jonah R. Chan or Mazen A. Kheirbek.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Ekaterina Likhtik, Stephen Maren, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Proliferation, differentiation, and maturation of OPCs following fear learning.

a, EdU+ cell density of home cage (HC), fear conditioned (CFC), no shock (NS), and immediate shock (IS) animals 24 hours post-conditioning; unpaired two-tailed t-tests comparing HC vs. CFC for PL (difference: 2.060 ± 0.8565, 95% CI: 0.1942 to 3.927, t12 = 2.406, p = 0.0332), IL (difference: 2.239 ± 0.8566, 95% CI: 0.3731 to 4.106, t12 = 2.614, p = 0.0226), ACC (difference: 1.176 ± 0.5811, 95% CI: -0.09067 to 2.442, t12 = 2.406, p = 0.0760), BLA (difference: 3.865 ± 1.395, 95% CI: 0.8506 to 6.880, t12 = 2.77, p = 0.0159), dHPC (difference: 2.060 ± 0.8565, 95% CI: 0.1942 to 3.927, t12 = 2.406, p = 0.6694), vHPC (difference: 0.2300 ± 0.5265, 95% CI: -0.9075 to 1.368, t12 = 0.4369, p = 0.3128), paired two-tailed t-test comparing PL vs. IL for CFC animals (difference: -0.1025 ± 0.7635, 95% CI: -1.971 to 1.766, t6 = 0.1343, p = 0.8975). b, EdU+/Olig2+ cell density at 24 hours post-conditioning; unpaired two-tailed t-tests comparing HC vs. CFC for PL (difference: 1.801 ± 0.7408, 95% CI: 0.1867 to 3.415, t12 = 2.431, p = 0.0317), IL (difference: 1.547 ± 0.6297, 95% CI: 0.1750 to 2.919, t12 = 2.457, p = 0.0302), ACC (difference: 0.2161 ± 0.5518, 95% CI: -0.9863 to 1.418, t12 = 0.3916, p = 0.7022), BLA (difference: 3.619 ± 1.338, 95% CI: 0.7295 to 6.509, t12 = 2.706, p = 0.018), dHPC (difference: 0.2882 ± 0.4868, 95% CI: -0.7634 to 1.340, t12 = 0.592, p = 0.564), vHPC (difference: -0.008014 ± 0.5297, 95% CI: -1.162 to 1.146, t12 = 0.01513, p = 0.9882), paired two-tailed t-test comparing PL vs. IL for CFC animals (difference: -0.2322 ± 0.4552, 95% CI: -1.346 to 0.8817, t12 = 0.5102, p = 0.6289). For (a-b), n = 7 mice (HC), 8 mice (CFC), 7 mice (NS), 8 mice (IS). c, EdU+/ASPA+ density post-conditioning; unpaired two-tailed t-tests HC vs. CFC for PL (difference: 3.576 ± 1.006, 95% CI: 1.404 to 5.749, t13 = 3.556, p = 0.035), IL (difference: 2.472 ± 0.7097, 95% CI: 0.9503 to 3.995, t13 = 0.5102, p = 0.037), ACC (difference 0.9596 ± 0.5516, 95% CI: -0.2234 to 2.143, t13 = 0.5102, p = 0.1038), BLA (difference: 1.520 ± 2.071, 95% CI: -3.165 to 6.204, t13 = 0.7339, p = 0.4817), dHPC (difference: 0.3704 ± 0.5950, 95% CI: -0.9150 to 1.656, t13 = 0.6225, p = 0.5444), vHPC (difference: 0.0536 ± 0.6227, 95% CI: -1.303 to 1.410, t13 = 0.086, p = 0.9328), paired two-tailed t-test comparing PL vs. IL for CFC animals (difference: -0.9171 ± 0.7719, 95% CI: -2.742 to 0.9081, t13 = 1.188, p = 0.5466). d, EdU+/Olig2+ density 30 days post-conditioning; unpaired two-tailed t-tests HC vs. CFC for PL (difference: 4.512 ± 5.558, 95% CI: -7.599 to 16.62, t13 = 0.8117, p = 0.4328), IL (difference: 4.514 ± 6.705, 95% CI: -10.10 to 19.12, t13 = 0.6731, p = 0.5136), ACC (difference: 3.086 ± 6.324, 95% CI: -10.69 to 16.86, t13 = 0.488, p = 0.6343), BLA (difference: 19.00 ± 11.32, 95% CI: -5.452 to 43.45, t13 = 1.679, p = 0.1171), dHPC (difference: 4.325 ± 2.933, 95% CI: -2.011 to 10.66, t13 = 1.475, p = 0.1641), vHPC (difference: -0.8542 ± 3.854, 95% CI: -9.251 to 7.543, t13 = 0.2216, p = 0.8283), paired two-tailed t-test comparing PL vs. IL for CFC animals (difference: -4.630 ± 6.858, 95% CI: -21.41 to 12.15, t13 = 0.6751, p = 0.5248). For (c-d), n = 7 mice (HC) and 8 mice (CFC). Quantification of GFP+/MBP+ (e), GFP+/MBP (f), and EdU+/ASPA+ (g) cell density in the dHPC and BLA; n = 6 mice (7 days), 9 mice (14 days), and 7 mice (30 days). e-g One-way ANOVA with Sidak’s post hoc tests comparing cell densities across days; (e) BLA: F2,19 = 4.369, p = 0.0275, 7 vs. 14 day, (difference: -3.760 ± 1.672, 95% CI: -7.818 to 0.2981, p = 0.0718), 7 vs. 30 day (difference: -5.041 ± 1.765, 95% CI: -9.324 to -0.7572, p = 0.0201), dHPC: F2,22 = 5.397, p = 0.0124, 7 vs. 14 day, (difference: -3.108 ± 1.241, 95% CI: -6.086 to -0.1300, p = 0.0399), 7 vs. 30 day (difference: -4.124 ± 1.303, 95% CI: -7.251 to -0.9968, p = 0.009); (f) BLA: F2,19 = 0.07572, p = 0.9274, 7 vs. 14 day, (difference: 2.354 ± 6.908, 95% CI: -14.41 to 19.12, p = 0.9308), 7 vs. 30 day (difference: 2.540 ± 7.292, 95% CI: -15.16 to 20.24, p = 0.9279), dHPC: F2,20 = 0.4852, p = 0.6226, 7 vs. 14 day, (difference: 1.629 ± 3.759, 95% CI: -7.456 to 10.71, p = 0.8907), 7 vs. 30 day (difference: -1.900 ± 4.049, 95% CI: -11.69 to 7.888, p = 0.8733); (g) BLA: F2,18 = 47.49, p < 0.0001, 7 vs. 14 day, (difference: -0.6155 ± 0.6763, 95% CI: -2.265 to 1.034, p = 0.6092), 7 vs. 30 day (difference: -5.632 ± 0.6565, 95% CI: -7.233 to -4.031, p < 0.0001), dHPC: F2,16 = 46.74, p < 0.0001, 7 vs. 14 day, (difference: -0.03272 ± 0.7020, 95% CI: -1.764 to 1.699, p = 0.9987), 7 vs. 30 day (difference: -6.038 ± 0.7285, 95% CI: -7.835 to -4.242, p < 0.0001). h, Proportion of GFP+/MBP+ cells that are ASPA+ (left bars) and the proportion of ASPA+/GFP+ cells that are MBP+ (right bars) at 30 days post-conditioning; n = 7 mice. Representative DAPI (gray) images the sub-regional dHPC (i) and amygdalar (j) cytoarchitecture, scale bars: 250 μm (k) Schematic of the sampled area within the mPFC (green rectangle); dashed lines demarcate the approximate region cut for electron microscopy. For box-and-whisker plots, the center, boxes, and whiskers represent the median, interquartile range, and the 10th and 90th percentiles, with asterisks indicating the following p-value ranges: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001.

Extended Data Fig. 2 Overall myelination status and microglial density are unchanged in the absence of new myelin formation.

a, Representative images of ASPA (green) staining in the PFC of cre-negative and Myrf icKO mice 30 days post-conditioning, quantified in (b); unpaired two-tailed t-test, difference: -26.50 ± 26.20, 95% CI: -81.15 to 28.15, t20 = 1.012, p = 0.3238, n = 11 mice per genotype. c, Representative images of MBP (gray) staining in the PFC of cre-negative and Myrf icKO mice 30 days post-conditioning, quantified in (d); unpaired two-tailed t-test, difference: 0.1927 ± 12.13, 95% CI: -25.12 to 25.50, t20 = 2.406, p = 0.9875, n = 11 mice per genotype. e, Representative images of Iba1 (magenta) staining in the PFC of cre-negative and Myrf icKO mice 30 days post-conditioning, quantified in (f); unpaired two-tailed t-test, difference: -3.698 ± 29.55, 95% CI: -69.53 to 62.14, t10 = 0.1251, p = 0.9029, n = 6 mice per genotype. Scale bars: 100 μm (a,c,e). Data are presented as mean ± SEM.

Extended Data Fig. 3 Inhibition of new myelin formation impairs remote fear memory recall.

a, Extended experimental timeline for cohort represented in Fig. 4a,e, quantified in (e); two-way ANOVA (F4,80 = 3.437, p = 0.0121) with Sidak’s post hoc tests comparing cre-negative vs. Myrf icKO during conditioning (difference: -2.416 ± 5.790, 95% CI: -17.58 to 12.74, p = 0.9965), 24 hour (difference: -5.169 ± 5.790, 95% CI: -20.33 to 9.991, p = 0.9040), 7 day (difference: -9.901 ± 5.790, 95% CI: -25.06 to 5.260, p = 0.9040), 21 day (difference: -12.35 ± 5.790, 95% CI: -27.51 to 2.811, p = 0.3772), and 30 day (difference: -24.37 ± 5.790, 95% CI: -39.53 to -9.209, p = 0.0003), and comparing 7 day (difference: 5.685 ± 5.685, 95% CI: -6.004 to 17.37, p = 0.6277), 21 day (difference: 17.05 ± 17.05, 95% CI: 5.361 to 28.74, p = 0.0015), and 30 day (difference: 26.18 ± 26.18, 95% CI: 14.49 to 37.87, p < 0.0001) to 24 hour freezing within Myrf icKO animals, n = 11 animals per genotype. b, Extended experimental timeline for cohort represented in Fig. 4b,f,g, quantified in (f); two-way ANOVA (F4,80 = 1.435, p = 0.2291) with Sidak’s post hoc tests comparing cre-negative vs. Myrf icKO during conditioning (difference 1.467 ± 7.398, 95% CI: -13.19 to 16.13, p = 0.8431), 24 hour (difference: 0.9199 ± 7.398, 95% CI: -13.74 to 15.58, p = 0.9013), 7 day (difference: -1.946 ± 7.398, 95% CI: -16.61 to 12.71, p = 0.7930), 21 day (difference: 6.165 ± 7.398, 95% CI: -8.496 to 20.83, p = 0.4064), and 30 day (difference: 18.43 ± 7.398, 95% CI: 3.767 to 33.09, p = 0.0142), n = 14 cre-negative and 10 Myrf icKO mice. c, Experimental timeline for cohort in which tamoxifen was administered after 24 hour recall, quantified in (h); two-way ANOVA (F4,72 = 1.724, p = 0.1541), n = 10 animals per genotype. d, Experimental timeline for cohort in which tamoxifen was administered seven weeks prior to conditioning, quantified in (h); two-way ANOVA (genotype, F1,26 = 0.03642, p = 0.8501) with Sidak’s post hoc tests comparing cre-negative vs. Myrf icKO during conditioning (difference: 5.241 ± 6.245, 95% CI: -9.579 to 20.06, p = 0.6507) and 24 hour (difference: 3.556 ± 6.245, 95% CI: -11.26 to 18.37, p = 0.8185), n = 8 cre-negative and 7 Myrf icKO mice. i, Expanded quantification of Fos+ cell density across brain regions following 30-day retrieval sessions; unpaired two-tailed t-tests, PL (difference: 62.14 ± 24.56, 95% CI: 10.54 to 113.7, t21 = 72.53, p = 0.0209), IL (difference: 65.70 ± 28.64, 95% CI: 5.532 to 125.9, t21 = 2.294, p = 0.034), ACC (difference: 99.84 ± 21.22, 95% CI: 55.27 to 144.4, t21 = 4.706, p = 0.0002), BA (difference: 80.55 ± 24.87, 95% CI: 28.66 to 132.4, t21 = 3.238, p = 0.0041), LA (difference: 6.494 ± 9.138, 95% CI: -12.70 to 25.69, t21 = 0.7107, p = 0.4864), DG (difference: 163.5 ± 55.29, 95% CI: 48.50 to 278.5, t21 = 2.957, p = 0.0075), CA3 (difference: 175.4 ± 63.83, 95% CI: 42.70 to 308.2, t21 = 2.749, p = 0.012), NR (difference: 84.08 ± 22.50, 95% CI: 35.83 to 132.3, t21 = 3.737, p = 0.0022), vDG (difference: 88.20 ± 32.23, 95% CI: 18.58 to 157.8, t21 = 2.737, p = 0.017), vCA3 (difference: 55.39 ± 24.33, 95% CI: 2.831 to 108.0, t21 = 7.361, p = 0.0404), vPAG (difference: 105.9 ± 18.71, 95% CI: 66.04 to 145.8, t21 = 5.662, p < 0.0001), SCC (difference: 21.86 ± 53.48, 95% CI: -89.35 to 133.1, t21 = 0.4088, p = 0.6868), n = 12 cre-negative and 11 Myrf icKO mice. j, Within-genotype comparisons of Fos+ cell density between the PL and IL; paired two-tailed t-tests, cre-negative (difference: 35.68 ± 33.66, 95% CI: -40.47 to 111.8, t9 = 0.4088, p = 0.3168), Myrf icKO (difference: 3.320 ± 21.65, 95% CI: -45.65 to 52.29, t9 = 0.4088, p = 0.8815), n = 10 mice per genotype. Within-genotype comparisons of freezing responses between males and females for animals represented in a-e for cre-negative (k) and Myrf icKO animals (l); two-way ANOVA for cre-negative (F4,44 = 0.6815, p = 0.6085) and Myrf icKO animals (F4,44 = 1.649, p = 0.1833), n = 8 males and 5 females for cre-negative mice, n = 5 males and 6 females for Myrf icKO mice. Data are presented as mean ± SEM, with asterisks indicating the following p-value ranges: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001.

Extended Data Fig. 4 Innate anxiety-like and locomotor behaviors are unchanged in the absence of new myelin formation.

Quantification of time spent in the closed arms of the elevated plus maze (EPM) (a) and the periphery of the open field test (OFT) (b); unpaired two-tailed t-test, EPM (difference: -16.38 ± 12.29, 95% CI: -42.59 to 9.818, t15 = 1.333, p = 0.2025), OFT (difference: 20.94 ± 22.43, 95% CI: -27.17 to 69.05, t15 = 0.9334, p = 0.3664). (c) Quantification of total distance traveled in the open field test; unpaired two-tailed t-test (difference: 788.3 ± 780.0, 95% CI: -884.6 to 2461, t15 = 1.011, p = 0.3293). Heatmaps representing relative frequency (red - frequent, blue - infrequent) of cre-negative and Myrf icKO animals in the elevated plus maze (d) (dashed lines demarcate the closed arms), and open field test (e). Data are presented as mean ± SEM. For a-c, n = 5 cre-negative and 11 Myrf icKO mice.

Extended Data Fig. 5 Supplementary data for fiber photometry experiments.

a, Schematic of fiber photometry recording setup. b, Representative trace of a recorded retrieval session depicting the raw GCaMP (blue) and isobestic (black) signal, with no detectable motion artifact. Quantification of the z-scored mean ΔF/F during the two seconds pre- and post-bout transition at 24 hours and 30 days for control (c) and Myrf icKO (d) animals; n = 10 bouts across 7 mice (controls) and 7 mice (Myrf icKO); paired two-tailed t-tests comparing z-scored signal intensity pre- and post-bout for control 24 hour (difference: -0.2016 ± 0.1564, 95% CI: -0.5095 to 0.1064, t138 = 1.289, p = 0.0202), control 30 day (difference 0.1051 ± 0.1118, 95% CI: -0.1150 to 0.3251, t138 = 0.9396, p = 0.055), Myrf icKO 24 hour (difference: -0.2216 ± 0.1650, 95% CI: -0.5494 to 0.1063, t138 = 1.343, p < 0.0001), and Myrf icKO 30 day (difference: -0.1837 ± 0.1502, 95% CI: -0.4810 to 0.1136, t138 = 1.223, p = 0.0032). e, Quantification of the mean ΔF/F over a five-minute home cage recording taken just prior to the 24-hour and 30-day retrieval sessions; n = 7 (controls) and 7 mice (Myrf icKO); unpaired two-tailed t-tests, 24 hour (difference: -0.0001284 ± 0.006472, 95% CI: -0.01423 to 0.01397, t13 = 0.01984, p = 0.6534), 30 day (difference: 0.004054 ± 0.01353, 95% CI: -0.03350 to 0.04161, t13 = 0.9334, p = 0.8892). f, Representative image of Iba1 staining under the implant site, quantified in (g); paired two-tailed t-test (difference: -32.40 ± 26.10, 95% CI: -92.58 to 27.78, t16 = 0.908, p = 0.2496), n = 9 mice. Scale bar: 100 μm. For box-and-whisker plots, the center, boxes, and whiskers represent the median, interquartile range, and the 10th and 90th percentiles, with asterisks indicating the following p-value ranges: * ≤ 0.05, ** ≤ 0.01, **** ≤ 0.0001.

Extended Data Fig. 6 Induction of new myelin formation preserves remote fear memory recall.

a, Extended experimental timeline for cohort represented in Fig. 7a,e. b, Freezing responses in the conditioning context; two-way ANOVA (F4,108 = 5.884, p = 0.0003) with Sidak’s post hoc tests for conditioning (difference: 0.8941 ± 4.116, 95% CI: -9.860 to 11.65, p = 0.9928), 24 hour (difference: -0.8004 ± 4.116, 95% CI: -11.55 to 9.954, p = 0.9948), 7 day (difference: -11.91 ± 4.116, 95% CI: -22.67 to -1.160, p = 0.0221), 21 day (difference: -16.77 ± 4.116, 95% CI: -27.52 to -6.013, p = 0.004), and 30 day (difference: -17.49 ± 4.116, 95% CI: -16.00 to -0.3832, p = 0.0002), n = 15 vehicle and 14 clemastine mice. c, Freezing responses in the similar context; two-way ANOVA (F1,27 = 8.277, p = 0.0077) with Sidak’s post hoc tests for 7 day (difference: 0.4158 ± 3.894, 95% CI: -7.391 to 8.222, p = 0.9154) and 21 day (difference: -8.190 ± 3.894, 95% CI: -28.24 to -6.733, p = 0.401), or across days within vehicle- (difference: -5.845 ± 2.078, 95% CI: -10.77 to -0.9248, p = 0.018) and clemastine-treated (difference: -14.45 ± 2.151, 95% CI: -19.54 to -9.357, p < 0.0001) animals, n = 15 vehicle and 14 clemastine mice. d, Experiment assessing the effects of continuous vehicle injections/handling, compared against home cage animals; two-way ANOVA (F4,36 = 9.926, p < 0.0001) with Sidak’s post hoc tests for conditioning (difference: -0.6013 ± 5.615, 95% CI: -15.66 to 14.46, p > 0.9999), 24 hour (difference: 0.2373 ± 5.615, 95% CI: -14.82 to 15.30, p > 0.9999), 7 day (difference: 22.20 ± 5.615, 95% CI: 7.137 to 37.25, p = 0.0014), 21 day (difference: 31.96 ± 5.615, 95% CI: 16.91 to 47.02, p < 0.0001), and 30 day (difference: 26.78 ± 5.615, 95% CI: 11.72 to 41.83, p < 0.0001), n = 5 mice per treatment group. e, Expanded quantification of Fos+ cell density following 30-day retrieval sessions; unpaired two-tailed t-tests, PL (difference: 279.0 ± 43.19, 95% CI: 188.6 to 369.4, t19 = 6.459, p < 0.0001), IL (difference: 198.6 ± 68.81, 95% CI: 49.90 to 347.2, t19 = 2.886, p = 0.274), ACC (difference: 127.4 ± 51.52, 95% CI: 18.66 to 236.1, t19 = 2.472, p = 0.0069), BA (difference: 86.21 ± 19.27, 95% CI: 45.87 to 126.5, t19 = 4.473, p = 0.0044), LA (difference: 128.6 ± 44.43, 95% CI: 35.89 to 221.2, t19 = 2.894, p = 0.009), DG (difference: 268.7 ± 56.69, 95% CI: 150.0 to 387.3, t19 = 4.739, (p < 0.0001), CA3 (difference: 270.5 ± 77.34, 95% CI: 109.2 to 431.8, t19 = 3.497, p =0.0023), NR (difference: 110.7 ± 34.65, 95% CI: 37.59 to 183.8, t19 = 3.195, p = 0.0026), vDG (difference: 153.9 ± 34.77, 95% CI: 80.51 to 227.2, t19 = 4.426, p = 0.0013), vCA3 (difference: 139.2 ± 36.88, 95% CI: 61.42 to 217.0, t19 = 3.775, p = 0.0019), vPAG (difference: 193.0 ± 48.67, 95% CI: 90.28 to 295.6, t19 = 3.965, p = 0.0017), SSC (difference: 79.25 ± 55.73, 95% CI: -38.33 to 196.8, t19 = 1.422, p = 0.1731), n = 13 vehicle and 8 clemastine mice. f, Within-treatment group comparisons of Fos+ cell density between the PL and IL; unpaired two-tailed t-tests, vehicle (difference: 80.43 ± 48.18, 95% CI: -20.78 to 181.6, t18 = 1.67, p = 0.3657), n = 13 PL- and 7 IL-sampled mice, clemastine (difference: -24.05 ± 65.62, 95% CI: -162.5 to 114.4, t17 = 0.3666, p = 0.7185), n = 8 PL- and 11 IL-sampled mice. Freezing responses for experimental timeline described in (a) for home cage (g), no shock (h), and immediate shock (i) animals; (g) two-way ANOVA (F3,24 = 2.217, p = 0.1122) (h) two-way ANOVA (F4,32 = 2.674, p = 0.0496) (i) two-way ANOVA (F4,32 = 0.7811, p = 0.5496); for g-I, n = 5 animals per condition. j, Extended experimental timeline for clemastine injections in Myrf icKO animals. k, Individual fear expression for vehicle- and clemastine-treated Myrf icKO animals represented in (j); two-way ANOVA (F4,48 =1.357, p = 0.2629), n = 6 vehicle and 8 clemastine mice. l, Quantification of the z-scored mean ΔF/F during pre- and post-bout transitions at 30 days for vehicle- (left) and clemastine-treated (right) animals; paired two-tailed t-tests comparing pre- and post-transition for vehicle (difference: 1.075 ± 0.1560, 95% CI: 0.7685 to 1.382, t78 = 6.894, p < 0.0001) and clemastine (difference: 0.8792 ± 0.1548, 95% CI: 0.5745 to 1.184, t78 = 5.678, p < 0.0001). m, Quantification of the z-scored mean ΔF/F of the pre- and post-bout transition for vehicle- and clemastine-treated animals; unpaired two-tailed t-test (difference: -0.1963 ± 0.2099, 95% CI: -0.6094 to 0.2168, t78 = 0.9352, p = 0.3504). For l-m, n = 10 bouts per animal, 4 animals per treatment group. For box-and-whisker plots, the center, boxes, and whiskers represent the median, interquartile range, and the 10th and 90th percentiles. For dot plots, data are presented as mean ± SEM, with asterisks indicating the following p-value ranges: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001.

Supplementary information

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Supplementary Software 1

Subject management, used in conjunction with photometry_f_cond.m and photometry_f_v2.m.

Supplementary Software 2

Master function for conditioning session analyses.

Supplementary Software 3

Master function for retrieval session analyses.

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Pan, S., Mayoral, S.R., Choi, H.S. et al. Preservation of a remote fear memory requires new myelin formation. Nat Neurosci 23, 487–499 (2020). https://doi.org/10.1038/s41593-019-0582-1

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