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Silent synapses dictate cocaine memory destabilization and reconsolidation

Abstract

Cocaine-associated memories are persistent, but, on retrieval, become temporarily destabilized and vulnerable to disruptions, followed by reconsolidation. To explore the synaptic underpinnings for these memory dynamics, we studied AMPA receptor (AMPAR)-silent excitatory synapses, which are generated in the nucleus accumbens by cocaine self-administration, and subsequently mature after prolonged withdrawal by recruiting AMPARs, echoing acquisition and consolidation of cocaine memories. We show that, on memory retrieval after prolonged withdrawal, the matured silent synapses become AMPAR-silent again, followed by re-maturation ~6 h later, defining the onset and termination of a destabilization window of cocaine memories. These synaptic dynamics are timed by Rac1, with decreased and increased Rac1 activities opening and closing, respectively, the silent synapse-mediated destabilization window. Preventing silent synapse re-maturation within the destabilization window decreases cue-induced cocaine seeking. Thus, cocaine-generated silent synapses constitute a discrete synaptic ensemble dictating the dynamics of cocaine-associated memories and can be targeted for memory disruption.

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Fig. 1: Memory retrieval re-silences cocaine-generated synapses.
Fig. 2: Spine morphology correlate with memory destabilization and reconsolidation.
Fig. 3: Synapse re-silencing destabilizes cocaine memories.
Fig. 4: Decreased Rac1 activity primes cue-induced synaptic re-silencing.
Fig. 5: Increasing Rac1 activity prevents cue-induced synaptic re-silencing.
Fig. 6: Active Rac1 stabilizes synaptic states to regulate cocaine memory.

Data availability

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

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Acknowledgements

We thank K. Tang and K. Churn for excellent technical support, as well as M. Varkey, M. Mulloth, S. Beriwal, S. Maddukkuri, Y. Jung, O. Ikwuegbu, R. Moazzam and A. Kang for assistance with behavioral training. This work was supported by NIH grant nos. NS007433 (W.J.W.), DA043940 (W.J.W.), DA023206 (Y.D.), DA044538 (Y.D.), DA040620 (Y.D., E.J.N.), DA047861 (Y.D.), DA035805 (Y.H.H.), MH101147 (Y.H.H.), DA008227 (E.J.N.) and DA014133 (E.J.N.); the NIDA Intramural Research Program (Y.S.); and a Mellon Fellowship (W.J.W.).

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Contributions

W.J.W., N.M.G., Y.H.H., Y.S., O.M.S., E.J.N. and Y.D. designed the experiments and analyses and wrote the manuscript. W.J.W., N.M.G. and P.A.N. performed electrophysiology experiments. W.J.W., N.M.G., L.F., A.S., N.M.-B. and K.I. performed behavioral training and testing. N.M.G. performed ELISAs. W.J.W. performed spine analysis, immunohistochemistry and confocal microscopy. P.J.H. and H.M.C. made the HSVs.

Corresponding author

Correspondence to Yan Dong.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Rita A. Fuchs, Raphael Lamprecht and Manuel Mameli for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Fig. 1 Training results and example traces for silent synapse experiments in Fig. 1.

(a) Diagram showing the timeline of self-administration training with two withdrawal time points at which the behavioral and electrophysiological experiments were performed. (b-e) Self-administration training results from rats whose electrophysiology results are presented in Fig. 1f–h (saline 1w/d: infusions - d1 = 18.63 ± 3.73, d2 = 10.88 ± 2.82, d3 = 11.14 ± 2.31, d4 = 9.86 ± 5.62, d5 = 12.21 ± 3.01, inactive – d1 = 12.88 ± 2.93, d2 = 9.63 ± 3.29, d3 = 12.00 ± 3.98, d4 = 9.00 ± 5.46, d5 = 8.89 ± 2.37, n = 5 animals; cocaine 1w/d: infusions – d1 = 34.60 ± 3.71, d2 = 29.40 ± 5.84, d3 = 31.20 ± 3.85, d4 = 33.00 ± 2.88, d5 = 35.00 ± 4.00, inactive – d1 = 25.80 ± 10.52, d2 = 16.67 ± 9.27, d3 = 14.60 ± 5.73, d4 = 10.00 ± 2.59, d5 = 12.60 ± 3.44, n = 6 animals; saline 45w/d: infusion – d1=13.70 ± 2.60, d2 = 11.90 ± 2.60, d3= 8.40 ± 2.30, d4 = 8.90 ± 1.90, d5 = 9.30 ± 2.30, inactive – d1= 12.20 ± 3.0, d2 = 9.30 ± 2.00, d3 = 6.30 ± 1.60, d4 = 7.00 ± 1.20, d5 = 5.80 ± 1.10, n = 5 animals; cocaine 45w/d: infusions – d1 = 19.70 ± 3.03, d2 = 21.45 ± 3.26, d3 = 20.64 ± 3.37, d4 = 22.36 ± 4.00, d5 = 21.36 ± 1.38, inactive – d1 = 7.10 ± 3.49, d2 = 7.45 ± 2.53, d3 = 4.64 ± 1.43, d4 = 6.00 ± 1.22, d5 = 11.36 ± 2.62, n = 12 animals). (f-h) Example EPSCs over 100 trials from the minimal stimulation assay for the summarized results presented in Fig. 1h. (i) Diagram showing different time points after cue re-exposure at which silent synapses were assessed. (j-o) Self-administration training results of rats whose electrophysiology results are presented in Fig. 1j–m, s–v (saline re-exp: infusions – d1 = 13.80 ± 1.72, d2 = 12.40 ± 3.44, d3 = 7.20 ± 1.32, d4 = 8.60 ± 2.14, d5 = 10.40 ± 3.66, inactive – d1 = 10.20 ± 0.735, d2 = 11.80 ± 3.07, d3 = 7.60 ± 0.678, d4 = 6.60 ± 1.29, d5 = 6.60 ± 2.21, n = 5 animals; cocaine 45w/d: infusions – d1 = 27.73 ± 3.34, d2 = 23.60 ± 2.53, d3 = 24.73 ± 2.54, d4 = 22.00 ± 1.77, d5 = 23.53 = 2.03, inactive – d1= 5.71 ± 1.48, d2 = 8.13 ± 3.07, d3 = 7.14 ± 2.38, d4 = 5.86 ± 2.29, d5 = 4.33 ± 1.41, n = 16 animals; cocaine re-exp: infusions – d1 = 32.91 ± 5.17, d2 = 30.05 ± 3.78, d3 = 26.09 ± 2.06, d4 = 26.46 ± 2.33, d5 = 32.67 ± 4.34, inactive – d1 = 12.95 ± 4.10, d2 = 16.18 ± 5.16, d3 = 8.14 ± 2.79, d4 = 8.14 ± 2.80, d5 = 9.38 ± 3.15, n = 22 animals; cocaine 2hr: infusions – d1 = 40.20 ± 3.56, d2 = 36.40 ± 2.62, d3 = 30.20 ± 2.80, d4 = 34.80 ± 2.44, d5 = 36.20 ± 4.21, inactive – d1 = 7.00 ± 3.56, d2 = 8.00 ± 3.27, d3 = 8.00 ± 2.85, d4 = 8.00 ± 3.35, d5 = 6.60 ± 2.60, n = 5 animals; cocaine 4hr: infusions – d1 = 28.86 ± 1.49, d2 = 30.14 ± 4.26, d3 = 22.43 ± 1.07, d4 = 22.57 ± 2.26, d5 = 23.57 ± 2.99, inactive – d1= 13.71 ± 4.85, d2 = 11.86 ± 4.04, d3 = 6.57 ± 1.82, d4 = 9.34 ± 2.51, d5 = 8.14 ± 2.12, n = 7 animals; cocaine 6hr: infusions – d1 = 27.15 ± 3.52, d2 = 26.92 ± 2.18, d3 = 31.23 ± 6.20, d4 = 30.46 ± 4.26, d5 = 25.69 ± 2.36, inactive – d1 = 17.62 ± 11.29, d2 = 7.54 ± 4.49, d3 = 2.75 ± 1.18, d4 = 3.39 ± 2.03, d5 = 1.60 ± 0.743, n = 13 animals). (p) Summary showing no difference in nose poke responding during the 10-min cue re-exposure session in rats in which NAcSh silent synapses were assessed 10 min, 2 hr, 4 hr, and 6 hr after cue re-exposure (Fig. 1v) (10min = 29.45 ± 3.44, n = 22 animals; 2hr = 37.40 ± 3.98, n = 5 animals; 4hr = 21.86 ± 3.12, n = 7 animals; 6hr = 31.92 ± 2.68, n = 13 animals, F3,43=1.55, p=0.22, one-way ANOVA, n.s. > 0.05). (q-s) Example EPSCs over 100 trials from the minimal stimulation assay for the summarized results presented in Fig. 1m, v. Data presented as mean±SEM.

Supplementary Fig. 2 Training results for NMDAR and I-V experiments in Fig. 1.

(a-d) Self-administration training results of rats whose electrophysiology results are presented in Fig. 1n, o (saline 1w/d: infusions – d1 = 17.00 ± 3.62, d2 = 9.40 ± 2.54, d3 = 2.40 ± 3.12, d4 = 11.00 ± 3.08, d5 = 15.50 ± 4.29, inactive – d1 = 12.40 ± 2.50, d2 = 7.80 ± 1.32, d3 = 9.20 ± 2.04, d4 = 8.40 ± 3.44, d5 = 10.75 ± 3.95, n = 5 animals; cocaine 1w/d: infusions – d1 = 42.20 ± 11.43, d2= 32.00 ± 8.06, d3= 39.40 ± 9.88, d4= 40.40 ± 7.94, d5= 41.60 ± 7.71, inactive – d1= 20.40 ± 7.81, d2= 30.60 ± 9.94, d3= 13.00 ± 4.37, d4= 10.60 ± 2.93, d5 = 12.00 ± 3.65, n = 5 animals; saline 45w/d: infusions – d1= 17.00 ± 3.46, d2= 9.33 ± 6.33, d3= 5.33 ± 2.85, d4= 9.67 ± 5.67, d5= 10.67 ± 1.33, inactive – d1= 19.00 ± 8.02, d2= 8.33 ± 4.49, d3= 4.00 ± 3.06, d4= 7.00 ± 1.53, d5= 7.67 ± 2.33, n = 3 animals; cocaine 45w/d: infusions – d1= 33.00 ± 6.78, d2= 26.00 ± 1.92, d3= 26.00 ± 2.30, d4 = 28.40 ± 4.20, d5= 23.40 ± 1.83, inactive – d1 = 14.80 ± 3.76, d2= 15.00 ± 5.06, d3= 11.00 ± 1.30, d4= 11.20 ± 2.29, d5= 11.00 ± 4.55, n = 5 animals; cocaine re-exp: infusions – d1= 30.60 ± 4.76, d2= 28.80 ± 3.06, d3= 26.00 ± 2.67, d4= 30.20 ± 2.76, d5 = 31.20 ± 3.29, inactive – d1= 21.60 ± 14.94, d2= 7.20 ± 3.84, d3= 6.40 ± 3.36, d4 = 9.20 ± 2.65, d5= 8.00 ± 1.41, n = 5 animals). (d, right). Nose poke responding during the cue re-exposure session of rats whose electrophysiology results are presented in Fig. 1o (37.80 ± 7.70, n = 5 animals). (e,f) Self-administration training results of rats whose electrophysiology results are presented in Fig. 1p, q (saline: infusions – d1= 15.50 ± 2.39, d2= 10.67 ± 2.97, d3= 7.83 ± 2.97, d4= 9.50 ± 2.66, d5=8.00 ± 1.34, inactive – d1 = 15.00 ± 4.26, d2= 9.67 ± 2.28, d3= 7.00 ± 2.56, d4= 8.17 ± 1.17, d5= 6.17 ± 1.62, n = 6 animals; cocaine 45w/d: infusions – d1= 35.00 ± 11.24, d2= 26.00 ± 2.76, d3= 26.80 ± 2.81, d4= 28.20 ± 4.66, d5= 28.20 ± 2.46, inactive – d1= 15.80 ± 4.58, d2= 10.40 ± 2.29, d3= 9.20 ± 1.56, d4= 8.20 ± 2.56, d5= 12.00 ± 4.99, n = 5 animals; cocaine re-exp: infusions – d1= 45.75 ± 18.54, d2= 27.50 ± 2.40, d3= 23.25 ± 3.59, d4= 24.40 ± 5.39, d5= 25.25 ± 5.14, inactive – d1= 21.00 ± 11.23, d2= 13.75 ± 7.42, d3= 5.75 ± 2.59, d4= 5.25 ± 3.25, d5= 3.25 ± 2.29, n = 4 animals). (f, right). Nose poke responding during the cue re-exposure session of rats whose electrophysiology results are presented in Fig. 1p, q (20.50 ± 3.57, n = 4 animals). Data presented as mean±SEM.

Supplementary Fig. 3 Cocaine-induced changes in spine morphology and training results for Fig. 2.

(a) Example NAcSh dendrites from saline-trained rats (upper) and cocaine-trained rats (lower) after one day from the 5-day self-administration procedure. Scale bars, 2.5 µm. (b) Summary showing that cocaine-trained rats exhibited an increase in the density of total dendritic spines on withdrawal day 1 after cocaine self-administration compared to saline-trained rats (saline = 11.29 ± 0.40, n = 4 animals; cocaine = 16.46 ± 0.298, n = 4 animals, t6=10.45, p<0.0001, two-tail, unpaired t-test). (c) Summary showing no difference in the density of mushroom-like spines between cocaine- and saline-trained rats on withdrawal day 1, there was (saline = 3.89 ± 0.044, n = 4 animals; cocaine = 4.12 ± 0.150, n = 4 animals, t6=1.41, p=0.21, two-tail, unpaired t-test). (d) Summary showing an increase in the density of thin spines in cocaine-trained rats compared to saline-trained rats on withdrawal day 1 (saline = 5.97 ± 0.450, n = 4 animals; cocaine = 10.19 ± 0.292, n = 4 animals, t6=7.87, p=0.0002, two-tail, unpaired t-test). (e) Summary showing an increased in the density of stubby spines in cocaine-trained rats compared to saline-trained rats on withdrawal day 1 (saline = 1.43 ± 0.092, n = 4 animals; cocaine = 2.15 ± 0.092, n = 4 animals, t6=5.55, p=0.0014, two-tail, unpaired t-test). (f,g) Self-administration training results of rats used for spine analysis on withdrawal day 1 (Supplementary Fig. 3a–e) (saline 1w/d: infusions – d1= 22.25 ± 1.38, d2= 17.00 ± 2.65, d3= 18.25 ± 3.90, d4= 14.00± 3.58, d5= 15.00 ± 2.16, inactive – d1= 32.25 ± 5.59, d2= 23.50± 6.96, d3= 15.75 ± 3.75, d4= 13.50 ± 2.18, d5= 15.00 ± 4.97, n = 4 animals; cocaine 1w/d: infusions – d1= 42.75 ± 5.41, d2= 42.25±3.52, d3= 41.00 ± 4.06, d4= 42.00 ± 6.44, d5= 37.25 ± 1.84, inactive – d1=17.50 ± 5.98, d2= 14.50 ± 6.51, d3= 12.50 ± 5.35, d4= 13.75 ± 3.97, d5= 8.50 ± 3.78, n = 4 animals). (h-k) Self-administration training results of rats whose morphological results are presented in Fig. 2d–h (saline re-exp: infusions – d1= 9.50 ± 3.01, d2= 10.00 ± 2.35, d3= 14.25 ± 5.14, d4= 8.50 ± 2.60, d5= 13.00 ± 4.66, inactive – d1= 7.25 ± 1.11, d2= 5.75 ± 1.38, d3= 8.25 ± 0.854, d4= 6.75 ± 1.65, d5= 6.75 ± 1.93, n = 4 animals; cocaine 45w/d: infusions – d1= 48.43 ± 7.46, d2= 34.29 + 2.45, d3= 38.71 ± 1.89, d4= 38.57 ± 1.60, d5= 36.14 ± 2.25, inactive – d1= 8.71 ± 2.75, d2= 4.57 ± 0.812, d3= 4.86 ± 1.58, d4= 2.75 ± 1.07, d5= 1.86 ± 0.884, n = 7 animals; cocaine re-exp: infusions – d1= 45.57 ± 5.05, d2= 41.86 ± 3.67, d3= 43.14 ± 2.65, d4= 41.71 ± 4.01, d5= 43.29 ± 4.28, inactive – d1= 30.86 ± 9.12, d2= 15.57 ± 6.29, d3= 7.86 ± 1.64, d4= 3.71 ± 2.06, d5= 4.86± 3.21, n = 7 animals; cocaine 6hr: infusions- d1= 35.50 ± 4.03, d2= 32.75 ± 2.72, d3= 32.00 ± 2.55, d4= 28.25 ± 2.75, d5= 34.75 ± 4.01, inactive – d1= 18.50 ± 15.86, d2= 8.50 ± 4.48, d3= 3.25 ± 2.63, d4= 2.75 ± 2.14, d5= 3.50 ± 3.50, n = 4 animals). (l) Nose poke responding during the cue re-exposure session of rats whose morphological results are presented in Fig. 2d–h. Cocaine-trained rats exhibited higher levels of nose pokes compared to saline-trained rats. There was no difference in cocaine-trained rats examined 10 min or 6hr after cue re-exposure (saline = 12.25 ± 1.81, n = 4 animals; cue re-exp = 31.71 ± 2.07, n = 7 animals; 6hr post = 33.50 ± 2.99, n = 4 animals, F2,12=23.65, p<0.0001, one-way ANOVA; **p<0.01, Bonferroni posttest). See Supplementary Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Supplementary Fig. 4 Peptide validation and training results of rats presented in Fig. 3.

(a) (left and bottom) Diagram showing experimental setup for the induction of LTP at Schaffer collateral to CA1 synapses, with example field EPSPs before and after LTP induction. (right) Summarized results showing that this form of LTP, which was sensitive to the NMDAR-selective antagonist APV, was abolished in sliced pre-incubated with TGL, but not AGL (at time 60min: control = 1.48 ± 0.10, n = 8 cells; APV = 0.969 ± 0.05, n = 6 cells; AGL = 1.45 ± 0.092, n = 7 cells; TGL = 1.174 ± 0.113, n = 8 cells, F207, 1725=3.07, p<0.0001, two-way RM ANOVA; *p<0.05, Bonferroni posttest, cell-based statistics). (b,c) Self-administration training results of rats whose electrophysiology are presented in Fig. 3c–f (saline AGL: infusions – d1= 11.20 ± 3.60, d2= 7.20 ± 2.52, d3= 7.40 ± 1.91, d4= 11.00 ± 2.97, d5= 11.00 ± 2.35, inactive – d1= 12.40 ± 4.20, d2= 5.20 ± 1.69, d3= 3.80 ± 0.97, d4= 7.00 + 1.41, d5= 5.00 ± 1.79, n = 5 animals; saline TGL: infusions – d1= 10.00 ± 1.95, d2= 6.80± 1.07, d3= 7.40 ± 1.44, d4= 6.40 ± 1.72, d5= 5.60 ± 1.17, inactive – d1= 10.60 ± 3.47, d2= 5.80 ± 2.01, d3= 5.80 ± 1.69, d4= 4.40 ± 0.68, d5= 5.60 ± 1.89, n =5 animals; cocaine AGL: infusions – d1= 44.73 ± 3.96, d2= 40.27 ± 6.37, d3= 41.82 ± 5.47, d4= 35.36 ± 4.60, d5= 37.00 ± 7.60, inactive – d1= 34.45 ± 18.39, d2= 14.45 ± 5.17, d3= 18.45 ± 9.14, d4= 8.46 ± 2.40, d5= 9.18 ± 2.24, n = 11 animals; cocaine TGL: infusions – d1= 41.00 ± 7.18, d2= 35.29 ± 5.07, d3= 31.71 ± 3.78, d4= 28.71 ± 3.16, d5= 30.00 ± 4.71, inactive – d1= 12.43 ± 4.07, d2= 13.86 ± 4.38, d3= 15.83 ± 4.73, d4= 9.71 ± 1.49, d5= 11.57 ± 3.56, n = 7 animals). (d) Summary showing no difference in nose poking responding during the 10-min cue re-exposure session in rats presented in Fig. 3c–f (saline AGL = 7.00 ± 0.775, n =5 animals; saline TGL = 13.40 ± 3.30, n = 5 animals, t8=1.89, p=0.09, saline-AGL vs saline-TGL; cocaine AGL = 24.00 ± 2.34, n = 11 animals; cocaine TGL = 22.57 ± 2.94, t16=0.38, p=0.71, cocaine-AGL vs cocaine-TGL, two-tail, unpaired t-test, n.s. > 0.05). (e-g) Example EPSCs over 100 trials from the minimal stimulation assay for the summarized results presented in Fig. 3f. (h-i) Self-administration training results of rats whose morphology results are presented in Fig. 3g–l (saline AGL: infusions – d1= 9.67 ± 1.86, d2= 15.33 ± 2.91, d3= 11.67 ± 2.03, d4= 7.67 ± 0.882, d5= 8.33 ± 1.86, inactive – d1= 16.33 ± 4.63, d2= 7.00 ± 2.52, d3= 6.67 ± 1.76, d4= 4.67 ± 1.67, d5= 7.33 ± 1.86, n = 3 animals; saline TGL: infusions – d1= 7.00 ± 2.00, d2= 14.00 ± 1.53, d3= 20.00 ± 9.50, d4= 11.33 ± 2.03, d5= 12.67 ± 4.18, inactive – d1= 4.67 ± 2.33, d2= 11.33 ± 4.26, d3= 8.33 ± 4.84, d4= 7.67 ± 1.33, d5= 8.00 ± 1.53, n = 3 animals; cocaine AGL: infusions – d1= 43.00 ± 4.22, d2= 43.50 ± 7.26, d3= 41.00 ± 7.63, d4= 46.75 ± 5.76, d5= 43.75 ± 6.08, inactive – d1= 29.50 ± 24.24, d2= 4.00 ± 0.816, d3= 3.50 ± 1.66, d4= 4.25 ± 1.84, d5= 1.25 ± 0.946, n = 4 animals; cocaine TGL: infusions – d1= 44.00 ± 2.86, d2= 46.75 ± 4.11, d3= 54.50 ± 8.91, d4= 41.75 ± 4.91, d5= 38.75 ± 4.11, inactive – d1= 6.25 ± 3.97, d2= 3.00 ± 2.35, d3= 7.75 ± 2.50, d4= 5.50 ± 1.26, d5= 2.75 ± 1.32, n = 4 animals). (j) Summary showing no difference in nose poking responding during the 10-min cue re-exposure session in rats presented in Fig. 3g–l (saline AGL = 12.33 ± 5.04, n =3 animals; saline TGL = 13.00 ± 2.52, n = 3 animals, t4=0.118, p=0.9116, saline-AGL vs saline-TGL; cocaine AGL = 29.50 ± 1.56, n = 4 animals; cocaine TGL = 30.00 ± 5.12, t6=0.094, p=0.9285, cocaine-AGL vs cocaine-TGL, two-tail, unpaired t-test, n.s. > 0.05). See Supplementary Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Supplementary Fig. 5 Additional behavioral controls for experiments in Fig. 3.

(a,b) Summary showing that nose poke responding (seeking) in saline (a)- and cocaine (b)-trained rats was not altered when tested 6 hr after cue re-exposure (saline active = 24.18 ± 2.69, n = 11 animals; saline inactive = 10.82 ± 1.16, n =11 animals; cocaine active = 85.20 ± 4.38, n = 10 animals; cocaine inactive = 22.30 ± 3.86, n =11 animals). (c) Summary showing that cocaine-trained rats exhibited higher levels of nose poke responding during the 10-min cue re-exposure session compared to saline-trained rats on withdrawal day 45 (saline = 14.33 ± 2.10, n = 11 animals; coaine = 37.40 ± 2.66, n = 10 animals, t17=6.69, p<0.0001, two-tail, unpaired t-test). (d) Summary showing that intra-NAcSh infusions of AGL or TGL 2 hr after cue re-exposure did not affect cue-induced nose poke responding in saline-trained rats, measured 6 hr after re-exposure (AGL active = 17.87 ± 4.14, n = 8 animals; TGL active = 12.25 ± 2.07, n =8 animals; AGL inactive = 7.00 ± 1.65, n = 8 animals, TGL in active = 7.00 ± 1.71, n = 8 animals, F1,7=4.95, p=0.0614, RM two-way ANOVA, withdrawal day 45 peptide x hole interaction). Nose poke responding during the 10-min cue re-exposure session (right) was also not different between AGL and TGL rats (AGL = 13.00 ± 2.73, n = 8 animals; TGL = 13.00 ± 2.27, n =8 animals, t14=0.000, p=1.00, two-tail unpaired t-test). (e) Summary showing that intra-NAcSh infusions of AGL or TGL 6 hr after cue re-exposure did not affect cue-induced nose poke responding, tested 6.5 hr after cue re-exposure, in saline-trained rats (AGL active = 22.13 ± 4.88, n = 8 animals; TGL active = 20.88 ± 5.92, n = 8 animals; AGL inactive = 6.75 ± 1.37, n = 8 animals; TGL inactive = 7.13 ± 1.46, n = 8 animals, F1,7=0.0, p=0.7986, RM two-way ANOVA, withdrawal day 45 peptide x hole interaction). Nose poke responding during the 10-min cue re-exposure session (right) was also not different between AGL and TGL rats (AGL = 16.25 ± 2.72, n = 8 animals; TGL = 12.13 ± 2.91, n = 8 animals, t14=1.036, p=0.3179, two-tail, unpaired t-test). (f) Summary of nose poke responding during the 10-min cue re-exposure session in cocaine-trained rats whose behavioral results are presented in Fig. 3m, n. Nose poke responding was similar between rats with intra-NAcSh AGL vs rats with intra-NAcSh TGL when the peptides were injected either 2 hr (left) or 6 hr (right) after cue re-exposure (2 hr: AGL = 29.08 ± 3.96, n = 12 animals; TGL = 27.58 ± 3.58, n = 12 animals, t22=0.2811, p=0.7812; 6 hr: AGL = 32.10 ± 1.20, n = 10 animals; TGL = 29.30 ± 2.01, n = 10 animals, t18=1.196, p=0.2471, two-tail, unpaired t-test). (g-j) Diagrams showing the localization of the infusion needle tips for all rats included in the data analysis in Fig. 3m, n and Supplementary Fig. 5d, e. Data presented as mean±SEM.

Supplementary Fig. 6 Training and re-exposure results and viral localization for Fig. 4.

(a-b) Self-administration training results of rats whose electrophysiology results are presented in Fig. 4e–h (saline: infusions – d1= 9.17 ± 1.42, d2= 10.50 ± 2.45, d3= 8.33 ± 1.15, d4= 5.17 ± 1.20, d5= 6.50 ± 1.46, inactive – d1= 9.50 ± 3.54, d2= 13.83 ± 3.04, d3= 7.00 ± 1.77, d4= 3.33 ± 1.50, d5= 5.33 ± 1.50, n= 6 animals; cocaine: infusions – d1= 39.53 ± 5.03, d2= 36.07 ± 4.18, d3= 34.60 ± 3.18, d4= 36.47 ± 3.33, d5= 37.00 ± 4.75, inactive – d1= 14.80 ± 4.37, d2= 12.73 ± 3.92, d3= 8.47 ± 2.36, d4= 4.60 ± 1.10, d5= 7.40 ± 2.49, n = 15 animals). (c,d) Example EPSCs (inset) over trials in the minimal stimulation assay from rats whose electrophysiology results are presented in Fig. 4h. (e,f) Self-administration training results of rats whose electrophysiology results are presented in Fig. 4i–o (saline: infusions – d1= 7.00 ± 1.58, d2= 7.40 ± 1.50, d3= 6.80 ± 1.88, d4= 6.20 ± 1.11, d5= 7.20 ± 1.83, inactive – d1= 4.80 ± 0.800, d2= 7.20 ± 1.74, d3= 6.40 ± 1.03, d4= 3.60 ± 1.21, d5= 5.40 ± 2.29, n = 5 animals; cocaine: infusions – d1= 34.11 ± 4.57, d2= 39.67 ± 6.49, d3= 43.44 ± 6.58, d4= 42.56 ± 5.65, d5= 40.11 ± 5.16, inactive – d1= 32.33 ± 19.25, d2= 8.44 ± 3.35, d3= 4.78 ± 2.76, d4= 4.67 ± 1.80, d5= 6.11 ± 2.69, n = 9 animals). (i,j) Nose poke responding during the cue re-exposure session of rats whose behavioral results are presented in Fig. 4p, q (2hr post = 28.00 ± 3.20, n = 7 animals; pa-dnRac1 re-exp = 26.00 ± 1.84, n = 7 animals). (k-m) Example image showing HSV-mediated expression of pa-dnRac1 in the NAcSh and location of the optical fiber that delivered laser stimulation (k), and a magnified portion of the image showing viral expression in individual neurons (l) from a cocaine-trained rats used in behavioral experiments presented in Fig. 4q. Viral expression and fiber tip localization for all rats that were included in data analysis are presented in (m). (n-p) Example image showing HSV-mediated expression of pa-dnRac1 in the NAcSh and location of the optical fiber that delivered laser stimulation (n), and a magnified portion of the image showing viral expression in individual neurons (o) from a cocaine-trained rat used in behavioral experiments presented in Fig. 4q. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (p). Data presented as mean±SEM.

Supplementary Fig. 7 HSV-mediated expression of C450M, an inactive control for caRac1, does not affect silent synapse levels.

(a,b) Self-administration training results of rats whose electrophysiology results are presented in Supplementary Fig 7d–h (saline: infusions – d1= 15.40 ± 3.01, d2= 8.60 ± 1.44, d3= 9.60 ± 1.99, d4= 13.00 ± 2.24, d5= 9.00 ± 0.894, inactive – d1= 10.20 ± 1.02, d2= 6.40 ± 1.12, d3= 7.20 ± 0.80, d4= 8.80 ± 2.38, d5= 6.20 ± 1.72, n = 5 animals; cocaine: infusions – d1= 39.50 ± 2.74, d2= 34.75 ± 2.16, d3= 31.58 ± 1.82, d4= 32.50 ± 2.16, d5 = 36.25 ± 2.85, inactive – d1= 36.92 ± 10.46, d2= 13.42 ± 2.92, d3= 10.58 ± 3.26, d4= 6.58 ± 1.75, d5= 8.25 ± 2.15, n = 12 animals). (c) Diagram showing the experimental timeline for self-administration, withdrawal, HSV-mediated expression of C450M, and electrophysiology. (d-f) Example EPSCs (inset) over trials in the minimal stimulation assay of C450M-expressing NAcSh MSNs in saline (d)- and cocaine-trained rats (e), and the effects of CP-AMPAR inhibition (f). (h) Summary showing that C450M expression did not affect the % silent synapses in saline- or cocaine-trained rats, and the low % silent synapses in cocaine-trained rats was restored to high levels by naspm inhibition of CP-AMPARs (saline = 6.64 ± 4.15, n = 5 animals; cocaine = 6.04 ± 2.27, n = 6 animals; cocaine naspm = 36.76 ± 3.48, n = 6 animals, F2,14=29.20, p<0.0001, one-way ANOVA; **p<0.01, Bonferroni posttest). See Supplementary Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Supplementary Fig. 8 Self-administration training results for rats used in Fig. 5.

(a,b) Self-administration training results of rats whose electrophysiology results are presented in Fig. 5b–e (saline: infusions – d1= 14.14 ± 2.70, d2= 12.14 ± 2.44, d3= 9.43 ± 2.33, d4= 11.14 ± 3.23, d5= 8.71 ± 2.12, inactive – d1= 12.29 ± 1.43, d2= 8.29 ± 1.41, d3= 6.71 ± 1.38, d4= 10.71 ± 3.26, d5= 10.14 ± 2.06, n = 7 animals; cocaine: infusions – d1= 37.72 ± 2.26, d2= 32.22 ± 1.14, d3= 34.56 ± 2.24, d4= 40.33 ± 3.64, d5= 40.33 ± 3.02, inactive – d1= 19.56 ± 3.74, d2= 8.67 ± 1.67, d3= 7.17 ± 1.64, d4= 10.00 ± 3.59, d5= 7.89 ± 2.07, n = 18 animals). (c) Summary of nose poke responding during the 10-min cue re-exposure session from rats whose electrophysiology results are presented in Fig. 5b–e. Cocaine-trained rats exhibited higher levels of cue-induced nose poke responding compared to saline-trained rats (saline = 12.29 ± 2.39, n = 7 animals; cocaine = 28.67 ± 1.69, n = 18 animals, t23=5.29, p<0.0001, two-tail, unpaired t-test). (d,e) Example EPSCs (inset) over trials in the minimal stimulation assay from rats whose electrophysiology results are presented in Fig. 4e. (f,g) Self-administration training results of rats whose electrophysiology results are presented in Fig. 5i–l (saline: infusions – d1= 9.40 ± 1.50, d2= 10.00 ± 3.52, d3= 6.60 ± 2.09, d4= 7.80 ± 2.59, d5= 6.60 ± 1.57, inactive – d1= 7.20 ± 1.88, d2= 8.00 + 2.57, d3= 10.20 ± 3.20, d4= 7.20 ± 1.49, d5= 8.00 ± 1.14, n = 5 animals; cocaine: infusions – d1= 44.42 ± 4.14, d2= 35.75 ± 3.39, d3= 37.83 ± 3.19, d4= 39.33 ± 2.59, d5= 36.00 ± 2.31, inactive – d1= 21.25 ± 5.05, d2= 11.17 ± 3.78, d3= 8.75 ± 2.32, d4= 6.33 ± 2.02, d5= 8.58 ± 2.74, n = 12 animals). (h) Summary of nose poke responding during the 10-min cue re-exposure session from rats whose electrophysiology results are presented in Fig. 5i–l. The nose poke responding was higher in cocaine-trained rats compared to saline-trained rats (saline = 4.60 ± 0.51, n= 5 animals; cocaine = 21.08 ± 1.62, n = 12 animals, t15=6.41, p<0.0001, two-tail, unpaired t-test). (i,j) Self-administration training results of rats whose electrophysiology results are presented in Fig. 5t–v (vehicle: infusions – d1= 33.25 ± 9.76, d2= 40.25 ± 6.65, d3= 30.25 ± 1.91, d4= 34.75 ± 3.20, d5= 38.75 ± 2.78, inactive – d1= 35.75 ± 28.13, d2= 21.00 ± 13.02, d3= 9.75 ± 6.22, d4= 6.75 ± 3.82, d5= 6.00 ± 4.38, n = 4 animals; LIMKi: infusions – d1= 44.75 ± 1.89, d2= 31.00 ± 2.12, d3= 37.75 ± 6.36, d4= 43.00 ± 5.82, d5= 41.25 ± 6.38, inactive – d1= 11.50 ± 2.78, d2= 14.00 ±6.38, d3= 5.75 ± 5.11, d4= 7.50 ± 3.79, d5= 5.50 ± 5.17, n = 4 animals) . (k) Summary of nose poke responding during the 10-min cue re-exposure session from rats whose electrophysiology results are presented in Fig. 5i–l. The nose poke responding was not different in animals treated with vehicle or LIMKi (vehicle = 23.50 ± 3.80, n = 4 animals; LIMKi = 19.25 ± 1.03, n = 4 animals, t6=1.08, p=0.3215, two-tail,unpaired t-test). (l,m) Self-administration training results of rats whose morphology results are presented in Fig. 5m–r (saline: infusions – d1= 9.50 ± 0.645, d2= 8.50 ± 1.56, d3= 5.50 ± 2.18, d4= 10.50 ± 1.94, d5= 6.00 ± 2.48, inactive – d1= 6.25 ± 0.629, d2= 4.75 ± 2.18, d3= 3.50 ± 0.866, d4= 8.25 ± 0.250, d5= 3.00 ± 0.707, n = 4 animals; cocaine: infusions – d1= 40.25 ± 1.93, d2= 30.00 ± 5.31, d3= 31.75 ± 4.13, d4= 40.50 ± 1.04, d5 = 40.00 ± 4.88, inactive – d1= 20.50 ± 10.91, d2= 10.25 ± 7.92, d3= 9.50 ± 7.86, d4= 1.25 ± 0.629, d5= 1.00 ± 0.707, n = 4 animals). (n) Summary of nose poke responding during the 10-min cue re-exposure session from rats whose morphology results are presented in Fig. 5m–r. The nose poke responding was higher in cocaine-trained rats compared to saline-trained rats (saline = 5.50 ± 1.19, n = 4 animals; cocaine = 22.00 ± 3.24, n = 4 animals, t6=4.78, p=0.0031, two-tail, unpaired t-test). Data presented as mean±SEM.

Supplementary Fig. 9 Self-administration training results for rats used in Fig. 6.

(a,b) Self-administration training results of rats whose electrophysiology results are presented in Fig. 6c–f (saline: infusions – d1= 14.00 ± 2.12, d2= 3.50 ± 2.96, d3= 11.75 ± 0.854, d4= 13.50 ± 2.90, d5= 15.25 ± 2.56, inactive – d1= 10.25 ± 3.19, d2= 10.50 ± 1.94, d3= 7.75 ± 2.14, d4= 12.75 ± 4.88, d5 = 5.25 ± 1.84, n = 4 animals; cocaine: infusions – d1= 40.00 ± 4.03, d2= 44.75 ± 4.07, d3= 41.58 ± 2.81, d4= 42.42 ± 2.90, d5= 43.67 ± 3.45, inactive – d1= 26.00 ± 5.81, d2= 12.92 ± 4.63, d3= 9.83 ± 3.79, d4= 4.75 ± 1.71, d5= 3.67 ± 1.12, n = 12 animals). (c) Summary of nose poke responding during the 10-min cue re-exposure session from rats whose electrophysiology results are presented in Fig. 6c–f. The nose poke responding was higher in cocaine-trained rats compared to saline-trained rats (saline = 8.50 ± 1.26, n = 4 animals; cocaine = 26.50 ± 2.13, n = 12 animals, t14=4.69, p=0.0003, two-tail, unpaired t-test). (d,e) Self-administration training results of rats whose electrophysiology results are presented in Fig. 6n–p (vehicle: infusions – d1= 32.00 ± 9.34, d2= 27.75 ± 5.19, d3= 33.50 ± 7.53, d4= 33.24 ± 7.98, d5= 30.50 ± 4.84, inactive – d1= 22.50 ± 10.66, d2= 5.25 ± 3.97, d3= 7.00 ± 2.67, d4= 7.00 ± 2.86, d5= 8.25 ± 4.39, n = 4 animals; LIMKi: infusions – d1= 50.75 ± 5.47, d2= 45.25 ± 3.90, d3= 41.50 ± 5.90, d4= 46.00 ± 11.57, d5= 39.50 ± 2.53, inactive – d1= 53.50 ± 8.97, d2= 20.00 ± 10.75, d3= 13.75 ± 4.73, d4= 6.25 ± 3.28, d5= 4.00 ± 2.12, n = 4 animals). (f) Summary showing nose poke responding during the 10-min cue re-exposure session from rats whose electrophysiology results are presented in Fig. 6n–p. The nose poke responding was not different in animals treated with vehicle or LIMKi (vehicle = 24.00 ± 4.18, n = 4 animals; LIMKi = 26.00 ± 1.73, n = 4 animals, t6=0.44, p=0.6742, two-tail, unpaired t-test). (g,h) Self-administration training results of rats whose morphology results are presented in Fig. 6g–l (saline: infusions – d1= 7.25 ± 0.629, d2= 7.00 ± 3.72, d3= 14.50 ± 3.12, d4= 3.50 ± 0.645, d5= 12.25 + 2.18, inactive – d1= 7.00 ± 2.61, d2= 5.50 ± 3.18, d3= 17.25, ± 5.49, d4= 11.00 ± 7.08, d5= 10.25 ± 2.18, n = 4 animals; cocaine: infusions – d1= 28.00 ± 5.86, d2= 35.00 ± 9.85, d3= 35.33 ± 8.95, d4= 35.33 ± 9.49, d5= 38.67 ± 6.17, inactive – d1= 8.67 ± 5.18, d2= 5.33 ± 2.60, d3= 7.67 ± 3.76, d4= 3.67 ± 1.86, d5= 4.00 ± 2.08, n = 3 animals). (i) Summary showing nose poke responding during the 10-min cue re-exposure session from rats whose morphology results are presented in Fig. 6g–l. The nose poke responding was higher in cocaine-trained rats compared to saline-trained rats (saline = 7.00 ± 2.27, n = 4 animals; cocaine = 27.00 ± 2.65, n = 3 animals, t5=5.74, p =0.0022, two-tail, unpaired t-test). Data presented as mean±SEM.

Supplementary Fig. 10 Effects of paRac1 stimulation on cue-induced nose poke responding in saline controls.

(a) Diagram showing the experimental timeline and setup. (b) Summary showing that saline-trained rats with photostimulation of intra-NAcSh paRac1 and photostimulation of intra-NAcSh C450M (control) during cue re-exposure exhibited similar cue-induced seeking, measured 6 hr after cue re-exposure (C450 active = 17.88 ± 2.95, n = 8 animals; paRac1 active = 21.83 ± 4.31, n = 6 animals; C450 inactive = 5.13 ± 0.915, n = 8 animals; paRac1 inactive = 9.17 ± 2.60, n = 6 animals, F1,12=0.0002, p=0.9878, two-way RM ANOVA, withdrawal day 45 hole x virus interaction, n.s. > 0.05). (c) Nose poke responding during the 10-min cue re-exposure session in saline-trained rats whose behavioral results presented in Supplementary Fig. 10b. Rats with intra-NAcSh stimulation of paRac1 and intra-NAcSh stimulation of C450M exhibited similar nose poke responding during the cue re-exposure session (C450M = 6.00 ± 0.707, n = 8 animals; paRac1 = 5.67 ± 1.48, n = 6 animals, t12=0.22, p=0.8286, two-tail, unpaired t-test, n.s. > 0.05). (d) Nose poke responding during the 10-min cue re-exposure session in cocaine-trained rats whose behavioral results are presented in Fig. 6q. Rats with intra-NAcSh stimulation of paRac1 and intra-NAcSh stimulation of C450M exhibited similar nose poke responding during the cue re-exposure session (C450M = 18.78 ± 2.52, n = 9 animals; paRac1 = 18.38 ± 1.97, n = 8 animals, t15=0.12, p=0.9033, two-tail, unpaired t-test, n.s. > 0.05). (e) Diagram showing the experimental timeline and setup. (f) Summary showing that rats with NAcSh paRac1 and rats with NAcSh C450M (control) receiving intra-NAcSh photostimulation 2 hr after cue re-exposure exhibited similar cue-induced nose poke responding, measured 6 hr after cue re-exposure (C450 active = 23.43 ± 7.57, n = 7 animals; paRac1 active = 17.43 ± 3.72, n = 7 animals; C450 inactive = 7.57 ± 2.31, n = 7 animals; paRac1 inactive = 5.71 ± 1.96, n = 7 animals, F1,12=0.23, p=0.6370, two-way RM ANOVA, withdrawal day 45 hole x virus interaction, n.s. > 0.05). (g) Nose poke responding during the 10-min cue re-exposure session in saline-trained rats used in experiments in Supplementary Fig. 10f. Rats with intra-NAcSh paRac1 and intra-NAcSh C450M exhibited similar nose poke responding over the cue re-exposure session (C450M = 11.29 ± 1.92, n = 7 animals; paRac1 = 10.57 ± 1.96, n =7 animals, t12=0.26, p=0.7993, two-tail, unpaired t-test, n.s. > 0.05). (h) Nose poke responding during the 10-min cue re-exposure session in cocaine-trained rats whose behavioral results are presented in Fig. 6r. Rats with intra-NAcSh paRac1 and intra-NAcSh C450M exhibited similar nose poke responding during the cue re-exposure session (C450M = 28.63 ± 2.16, n = 8 animals; paRac1 = 25.80 ± 1.79, n = 10 animals, t16=1.02, p=0.3246, two-tail, unpaired t-test, n.s. > 0.05). (i) Nose poke responding during the 10-min cue re-exposure session in cocaine-trained rats whose behavioral results are presented in Fig. 6s. Rats with intra-NAcSh paRac1 and intra-NAcSh C450M exhibited similar nose poke responding during the cue re-exposure session (C450M = 27.50 ± 2.31, n = 8 animals; paRac1 = 26.00 ± 2.00, n = 8 animals, t14=0.42, p=0.6308, two-tail, unpaired t-test, n.s. > 0.05). Data presented as mean±SEM.

Supplementary Fig. 11 Viral localization and expression for cue re-exposure photostimulation in behavioral experiments.

(a-c) Example image showing HSV-mediated expression of C450M in the NAcSh and location of the optical fiber that delivered laser stimulation (a), and a magnified portion of the image showing viral expression in individual neurons (b) from a saline-trained rat used in behavioral experiments presented in Supplementary Fig. 10b. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (c). (d-f) Example image showing HSV-mediated expression of C450M in the NAcSh and location of the optical fiber that delivered laser stimulation (d), and a magnified portion of image showing viral expression in individual neurons (e) from a cocaine-trained rat used in behavioral experiments presented in Fig. 6q. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (f). (g-i) Example image showing HSV-mediated expression of paRac1 in the NAcSh and location of the optical fiber that delivered laser stimulation (g), and a magnified portion of image showing viral expression in individual neurons (h) from a saline-trained rat used in the behavioral experiments presented in Supplementary Fig. 10b. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (i). (j-l) Example image showing HSV-mediated expression of paRac1 in the NAcSh and location of the optical fiber that delivered later stimulation (j), and a magnified portion of the image showing viral expression in individual neurons (k) from a cocaine-trained rat used in behavioral experiments presented in Fig. 6q. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (l).

Supplementary Fig. 12 Viral localization and expression for 2 hr post photostimulation in behavioral experiments.

(a-c) Example image showing HSV-mediated expression of C450M in the NAcSh and location of the optical fiber that delivered laser stimulation (a), and a magnified portion of the image showing viral expression in individual neurons (b) from a saline-trained rats used in behavioral experiments presented in Supplementary Fig. 10f. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (c). (d-f) Example image showing HSV-mediated expression of C450M in the NAcSh and location of the optical fiber that delivered laser stimulation (d), and a magnified portion of the image showing viral expression in individual neurons (e) from a cocaine-trained rats used in behavioral experiments presented in Fig. 6r. Viral expression and fiber tip localization for all rats that were included in data analysis are presented in (f). (g-i) Example image showing HSV-mediated expression of paRac1 in the NAcSh and location of the optical fiber that delivered laser stimulation (g), and a magnified portion of image showing viral expression in individual neurons (h) from a saline-trained rat used in behavioral experiments presented in Supplementary Fig. 10f. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (i). (j-l) Example image showing HSV-mediated expression of paRac1 in the NAcSh and location of the optical fiber that delivered laser stimulation (j), and a magnified portion of the image showing viral expression in individual neurons (k) from a cocaine-trained rat used in behavioral experiments presented in Fig. 6r. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (l).

Supplementary Fig. 13 Viral localization and expression for photostimulation in long-term memory experiments.

(a-c) Example image showing HSV-mediated expression of C450M in the NAcSh and location of the optical fiber that delivered laser stimulation (a), and a magnified portion of the image showing viral expression in individual neurons (b) from a cocaine-trained rats used in behavioral experiments presented in Fig. 6s. Viral expression and fiber tip localization for all rats that were included in data analysis are presented in (c). (d-f) Example image showing HSV-mediated expression of paRac1 in the NAcSh and location of the optical fiber that delivered laser stimulation (d), and a magnified portion of the image showing viral expression in individual neurons (e) from a cocaine-trained rat used in behavioral experiments presented in Fig. 6s. Viral expression and fiber tip localizations for all rats that were included in data analysis are presented in (f).

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Wright, W.J., Graziane, N.M., Neumann, P.A. et al. Silent synapses dictate cocaine memory destabilization and reconsolidation. Nat Neurosci 23, 32–46 (2020). https://doi.org/10.1038/s41593-019-0537-6

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