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.

Figure 1.. Memory retrieval re-silences cocaine-generated synapses

(a) Diagram showing experimental timeline. (b and c) Summary showing that after cocaine (b), but not saline (c), self-administration, cue-induced seeking was higher on withdrawal day 45 than withdrawal day 1 (withdrawal day 1 active = 3.08 ± 5.787, n = 12; withdrawal day 45 active = 87.13 ± 20.367, n = 11, F_1,38=12.27, p=0.0012, two-way ANOVA; **p<0.01, Bonferroni posttest). (d) Diagram showing the recording area. (e) Example EPSCs in the minimal stimulation assay, in which failed and successful responses were readily discernable at both −70 mV and +50 mV, and the small vs. large failure rate differences between these two holding potentials connotes low % (upper) vs. high % silent synapses. (f and g) EPSCs evoked at −70 mV or +50 mV (insets) over 100 trials from example recordings 1 day after saline (f) or cocaine (g) self-administration. (h) Summary showing that the % silent synapses was increased on withdrawal day 1 after cocaine self-administration (saline = 5.93 ± 1.44, n = 5 animals; cocaine = 24.95 ± 7.13, n = 6 animals, t₉=2.38, p=0.04, two-tail unpaired t-test). On withdrawal day 45, the % silent synapses returned to basal levels, while CP-AMPAR inhibition restored the high % silent synapses (saline = 11.71 ± 5.35, n =5 animals; cocaine = 10.53 ± 1.49, n = 12 animals; cocaine naspm = 28.37 ± 4.68, n = 6 animals, F_2,19=8.61, p=0.0022, one-way ANOVA; *p<0.05, **p<0.01, Bonferroni posttest). (i) Diagram showing experimental timeline. (j-l) EPSCs evoked at −70 mV or +50 mV by minimal stimulation (insets) over 100 trials from example recordings after cue re-exposure from saline- (j) and cocaine-trained rats (k) in the absence or presence (l) of naspm. (m) Summary showing that cue re-exposure increased the % silent synapses in cocaine-trained, but not saline-trained, rats on withdrawal day 45, and the effects of naspm (saline re-exp = 7.74 ± 1.89, n = 5 animals; cocaine 45w/d = 8.01 ± 1.89, n = 16 animals; cocaine 45w/d naspm = 31.31 ± 5.22, n = 8 animals; cocaine re-exp = 32.89 ± 5.41, n = 12 animals; cocaine re-exp naspm = 29.74 ± 3.85, n = 10, F_4,47=10.11, p<0.0001, one-way ANOVA; n.s.>0.05 *p<0.05, **p<0.01, Bonferroni posttest). (n) Summary showing increased sensitivity to Ro256981 of NMDAR EPSCs in rats 1 day after cocaine self-administration (saline = 0.91 ± 0.03 at 24min, n = 5 animals; cocaine = 0.74 ± 0.03 at 24min, n = 5 animals, F_26,104=7.66, p<0.0001, two-way ANOVA repeated measure; **p<0.01, Bonferroni posttest). Subsequent application of APV (50 μM) confirmed that currents were mediated by NMDARs. Inset showing example NMDAR EPSCs before and during Ro256981 application. (o) Summary showing cue re-exposure did not affect the Ro256981 sensitivity of NMDAR EPSCs in NAcSh MSNs (saline = 0.93 ± 0.03 at 24min, n =3 animals; cocaine = 0.92 ± 0.06 at 24min, n = 5 animals; cocaine re-exp = 0.93 ± 0.05 at 24min, n = 5, F_52,260=0.50, p=0.9984, two-way ANOVA repeated measures). (p) Example AMPAR EPSCs evoked from holding potentials of −70 mV to +50 mV with 10 mV increments. (q) (Left) I-V curves of AMPAR EPSCs showing rectification in cocaine-trained rats on withdrawal day 45, and the rectification was abolished by cue re-exposure (saline = 52.51 ± 5.68, n = 6 animals; cocaine = 29.98 ± 3.17, n = 5 animals; cocaine re-exp = 43.93 ± 6.01, n = 4 animals, F_4,33=4.15, p=0.0078, two-way ANOVA; *p< 0.05, Bonferroni posttest). EPSC amplitudes at −70 mV were used to normalize EPSCs at other membrane potentials. (Right) Summary showing that on withdrawal day 45, the decreased rectification index in cocaine-trained rats was abolished by cue re-exposure (saline = 0.79 ± 0.051, n = 6 animals; cocaine = 0.54 ± 0.041, n = 5 animals; cocaine re-exp = 0.79 ± 0.057, n = 4 animals, F_2,12=8.06, p=0.006, one-way ANOVA; *p < 0.05, Bonferroni posttest). (r) Diagram showing the timepoints at which the effects of cue re-exposure on silent synapses were assessed. (s-u) EPSCs evoked at −70 mV or +50 mV by minimal stimulation (insets) over 100 trials from example recordings 2 (o) and 6 hr (p) after cue re-exposure in cocaine-trained rats, and 6 hr after re-exposure in the presence of naspm (q). (v) Summary showing that after cue re-exposure, % silent synapse was immediately increased, remained at high levels for a few hr, and declined to basal levels by ~6 hr, and the declined % silent synapses were restored to high levels by naspm (10-min = 32.89 ± 5.404, n = 12 animals; 2hr = 37.23 ± 4.86, n = 5 animals; 4hr = 23.62 ± 7.78, n = 8 animals; 6hr = 10.91 ± 3.07, n = 12 animals; 6hr naspm = 39.47 ± 5.77, n = 7, F_4,39=5.02, p=0.0023, one-way ANOVA; *p<0.05, **p<0.01, Bonferroni posttest). The 10-min data taken from m. See Supplemental Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Figure 2.. Spine morphology correlate with memory destabilization and reconsolidation

(a) Example image showing a NAcSh MSN filled with Alexa 594 dye, and a magnified secondary dendrite (inset). Replicates for each group are presented in d-h with n values. (b) Example spiny dendrites (upper) and three subtypes of spines (lower) whose dendritic locations are indicated by arrows; mushroom (red), thin (blue), and stubby (yellow). Replicates for each group are presented in d-h with n values. (c) Example NAcSh dendrites from saline-trained rats with cue re-exposure (upper left), cocaine-trained rats without (upper right), 10 min after (lower left), and 6hr after (lower right) cue re-exposure on withdrawal day 45. Replicates for each group are presented in d-h with n values. (d) Summary showing that the density of total spines was increased after 45 days of withdrawal from cocaine compared to saline controls, and this increase was not altered by cue re-exposure and reconsolidation (saline = 11.66 ± 0.49, n = 4 animal; cocaine 45w/d = 16.86 ± 1.12, n = 7 animals; cocaine re-exp = 16.53 ± 0.716, n = 7 animals; cocaine 6hr post = 16.46 ± 0.665, n = 4 animals, F_3,18=5.98, p=0.0051, one-way ANOVA; *p< 0.05, **p<0.01, Bonferroni posttest). (e) Summary showing that the density of mushroom-like spines was increased after 45 days of withdrawal from cocaine compared to saline controls. Cue re-exposure decreased mushroom-like spine density to saline levels, while this cue re-exposure-induced effect disappeared 6 hr later (saline = 4.68 ± 0.129, n =4; cocaine 45w/d = 7.57 ± 0.531, n =7; cocaine re-exp = 4.84 ± 0.215, n =7; cocaine 6hr post = 8.50 ± 0.493, n =4, F_3,18=19.86, p<0.0001; one-way ANOVA; **p<0.01, Bonferroni posttest). (f) Summary showing that the density of thin spines was increased by cue re-exposure in cocaine-trained rats compared to saline-trained rats or cocaine-rats without re-exposure. Density of thin spines normalized 6 hr after re-exposure (saline = 5.43 ± 0.598, n =4 animals; cocaine 45w/d = 6.86 ± 0.568, n =7 animals; cocaine re-exp = 9.21 ± 0.643, n =7 animals; cocaine 6hr post = 6.12 ± 0.107, n = 4 animals, F_3,18=7.89, p=0.0014, one-way ANOVA; *p<0.05, **p<0.01, Bonferroni posttest). (g) Summary showing that the density of stubby spines was increased in cocaine-trained rats with or without cue re-exp, compared to saline-trained rats (saline = 1.56 ± 0.142, n = 4 animals; cocaine 45w/d = 2.41 ± 0.115, n = 7 animals; cocaine re-exposure = 2.47 ± 0.09, n = 7 animals; cocaine 6hr post = 1.85 ± 0.295, n = 4 animals, F _3,18=8.28, p=0.001, one-way ANOVA; **p<0.01, Bonferroni posttest). (h) Summary of cumulative frequency (left) and mean values (right) showing an increase in the overall spine head diameter in cocaine-trained rats compared to saline controls. Spine head diameter in cocaine-trained rats decreased to saline levels after cue re-exposure, which normalized 6 hr after re-exposure (saline = 0.429 ± 0.019, n = 4 animals; cocaine 45w/d = 0.488 ± 0.011, n = 7 animals; cocaine re-exp = 0.401 ± 0.011, n = 7 animals; cocaine 6hr post = 0.503 ± 0.005, n = 4 animals, F _3,18=14.67, p<0.0001, one-way ANOVA; *p<0.05, **p<0.01, Bonferroni posttest). (i) Schematic illustration depicting the hypothesized dynamics of cocaine-generated silent synapses during acquisition, consolidation, destabilization, and reconsolidation of cocaine-associated memories. See Supplemental Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Figure 3.. Synapse re-silencing destabilizes cocaine memories

(a) Diagram illustrating the hypothesis that preventing CP-AMPAR re-insertion during the reconsolidation window locks cocaine-generated synapses in the silent state and compromises cue-induced cocaine seeking. (b) Diagram showing experimental timeline. (d-e) Example EPSCs evoked at −70 mV and +50 mV by minimal stimulation (insets) over 100 trials from saline- or cocaine-trained rats with NAcSh TGL infusion and the effects of naspm. (f) Summary showing that while intra-NAcSh TGL or AGL did not affect the % silent synapses in saline-trained rats, intra-NAcSh TGL, but not AGL, maintained the cue re-exposure-induced high % silent synapses in cocaine-trained rats beyond the presumable 6-hr destabilization window, and CP-AMPAR inhibition by naspm did not further affect the % silent synapses (saline AGL = 6.40 ± 3.68, n = 5 animals; saline TGL = 7.06 ± 3.07, n = 5 animals; cocaine AGL = 9.70 ± 1.05, n = 5 animals; cocaine AGL naspm = 33.76 ± 3.71, n = 6 animals; cocaine TGL = 37.10 ± 3.71, n = 4 animals; cocaine TGL naspm = 29.70 ± 3.08, n = 3 animals, F_5,22=14.85, p<0.0001, one-way ANOVA; n.s. > 0.05, *p<0.05, **p<0.01, Bonferroni posttest). (g) Example NAcSh dendrites from saline-trained rats and cocaine-trained rats with AGL and TGL infusions. Scale bar, 2.5 μm. (h) Summary showing the density of total spines was increased in cocaine-trained rats with either AGL or TGL compared to saline-trained rats (saline AGL = 11.66 ± 0.171, n = 3 animals; saline TGL = 11.20 ± 0.146, n =3 animals; cocaine AGL = 16.52 ± 0.69, n =4 animals; cocaine TGL = 16.89 ± 0.488, n = 4 animals, F_3,10,=36.01, p<0.0001, one-way ANOVA; n.s.>0.05 **p<0.01, Bonferroni posttest). (i) Summary showing the density of mushroom-like spines was increased in cocaine-trained rats with AGL compared to saline-trained rats, while TGL treatment normalized mushroom-like spine density to saline levels (saline AGL = 4.46 ± 0.19, n = 3 animals; saline TGL = 4.23 ± 0.163, n = 3 animals; cocaine AGL = 8.18 ± 0.383, n = 4 animals; cocaine TGL = 5.04 ± 0.302, n = 4 animals, F_3,10=38.59, p<0.0001, one-way ANOVA; **p<0.01, Bonferroni posttest). (j) Summary showing the density of thin spines was significantly increased in cocaine-trained TGL rats compared to cocaine-trained AGL rats or saline-trained rats (saline AGL = 5.60 ± 0.148, n = 3 animals; saline TGL = 5.54 ± 0.132, n = 3 animals; cocaine AGL = 6.33 ± 0.309, n = 4 animals; cocaine TGL = 10.16 ± 0.363, n = 4 animals, F_3,10=60.80, p<0.0001, one-way ANOVA; **p<0.01, Bonferroni posttest). (k) Summary showing densities of stubby spines in saline- and cocaine-trained rats with AGL or TGL treatment (saline AGL = 1.60 ± 0.076, n = 3 animals; saline TGL = 1.45 ± 0.117, n = 3 animals; cocaine AGL = 2.01 ± 0.051 n = 4 animals; cocaine TGL = 1.69 ± 0.118, n = 4 animals, F_3,10=6.32, p=0.0112, one-way ANOVA; *p<0.05, Bonferroni posttest). (l) Summary showing the mean spine head diameter was increased in cocaine-trained AGL rats compared to saline-trained rats, while TGL treatment normalized this increase to saline control levels (saline AGL = 0.417 ± 0.013, n = 3 animals; saline TGL = 0.415 ± 0.005, n = 3 animals; cocaine AGL = 0.492 ± 0.015, n = 4 animals; cocaine TGL = 0.400 ± 0.004, n = 4 animals, F_3,10=16.17, p=0.0004, one-way ANOVA; **p<0.01, Bonferroni posttest). (m) Summary showing that intra-NAcSh infusion of TGL, but not AGL, at 2hr after cue re-exposure decreased cue-induced cocaine seeking in cocaine-trained rats, measured 6 hr after cue re-exposure (AGL active = 72.25 ± 6.68, n = 12 animals; TGL active = 43.25 ± 2.07, n = 12 animals; AGL inactive = 17.58 ± 2.90, n =12 animals; TGL inactive = 7.92 ± 1.34, n =12 animals, F_1,22=12.26, p=0.002, RM two-way ANOVA, withdrawal day 45 peptide x lever interaction; **p<0.01, Bonferroni posttest). (n) Summary showing that rats with intra-NAcSh infusion of TGL 6 hr after cue re-exposure exhibited similar cue-induced cocaine seeking as AGL rats measured 6.5 hr after cue re-exposure (AGL active = 68.20 ± 2.87, n = 10 animals; TGL active = 68.20 ± 3.74, n =10 animals; AGL inactive = 13.30 ± 1.69, n = 10 animals; TGL inactive = 15.70 ± 2.94, n = 10 animals, F_1,18=0.23, p=0.64, RM two-way ANOVA, withdrawal day 45 peptide x lever interaction). See Supplemental Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Figure 4.. Decreased Rac1 activity primes cue-induced synaptic re-silencing

(a) Summarized ELISA results showing transiently decreased levels of active Rac1 in NAcSh upon cue re-exposure in cocaine-trained but not saline-trained rats (saline withdrawal = 1.24 ± 0.122, n = 6 animals; saline re-exp = 1.19 ± 0.136, n = 6 animals; cocaine withdrawal = 1.23 ± 0.071, n = 7 animals; cocaine 10min = 0.708 ± 0.033, n = 7 animals; cocaine 75min = 0.658 ± 0.041, n = 9 animals; cocaine 6hr = 1.36 ± 0.125, n = 8 animals; cocaine 24hr = 1.23 ± 0.136, n = 6 animals, F_6,42=, p<0.0001, one-way ANOVA; * p<0.05, Bonferroni posttest). (b) Diagram illustrating the hypothesis that the transient downregulation of active Rac1 triggers re-silencing of the already matured silent synapses in cocaine-trained rats. (c) Diagram showing the timeline for experiments involving intra-NAcSh expression of dnRac1. (d) Example images showing dnRac1-expressing NAcSh MSNs during recordings. (e-g) EPSCs evoked at −70 mV and +50 mV during the minimal stimulation assay (insets) over 100 trials from example dnRac1-expressing MSNs in saline- (e) and cocaine-trained rats (f), and the effects of naspm (g). (h) Summary showing that expressing dnRac1 in NAcSh MSNs increased the % silent synapses selectively in cocaine-trained rats on withdrawal day 45, and CP-AMPAR inhibition did not further increase this percentage (saline dnRac1 = 7.32 ± 2.73, n = 6 animals; cocaine non-trans = 6.32 ± 2.59, n = 5 animals; cocaine non-trans naspm = 33.97 ± 4.14, n = 6 animals; cocaine dnRac1 = 36.47 ± 3.46, n = 9 animals; cocaine dnRac1 naspm = 31.67 ± 4.83, n = 7 animals, F_2,28=14.46, p<0.0001, one-way ANOVA; **p<0.01, Bonferroni posttest). (i) Diagrams showing the experimental design, in which NAcSh pa-dnRac1 was photoactivated for 10 min while rats remained in their home cage. (j) Example images of a NAcSh slice (left) and MSNs (right) showing HSV-mediated expression of pa-dnRac1. All animals used in Fig 4o and Fig 4q (n = 26 animals) had pa-dnRac1 expression localized within the NAcSh. (k) Diagram illustrating the design concept of pa-dnRac1. (l-n) EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example pa-dnRac1-expressing MSNs in saline- (l) and cocaine-trained rats (m), and the effects of naspm (n). (o) Summary showing that stimulating pa-dnRac1 on withdrawal day 45 did not affect the % silent synapses in saline-trained rats, but increased the % silent synapses in cocaine-trained rats, and this increase was not affected by naspm (saline = 9.08 ± 1.70, n = 5 animals; cocaine = 35.91 ± 4.89, n = 5 animals; cocaine naspm = 33.07 ± 4.21, n = 4 animals, F_2,11=15.56, p =0.0006, one-way ANOVA; **p<0.01, Bonferroni posttest). (p) Summary showing that the nose poke responding remained at high levels in cocaine-trained rats when tested 2 hr after cue re-exposure (cocaine 2hr post = 84.86 ± 8.08, n = 7 animals). (q) (left) Diagrams showing the experimental timeline for cocaine-trained rats that received stimulation of pa-dnRac1 without (upper) and with (lower) prior cue re-exposure. (right) Summary showing that rats that received pa-dnRac1 stimulation without prior cue re-exposure exhibited decreased cue-induced cocaine seeking compared to rats with prior (2 hr before) cue re-exposure (pa-dnRac1 active = 52.00 ± 4.54, n = 10 animals; pa-dnRac1 re-exp active = 85.71 ± 11.23, n = 7 animals; pa-dnRac1 inactive = 10.50 ± 1.17, n = 10 animals; pa-dnRac1 re-exp inactive = 12.14 ± 2.01, n = 7 animals, F_1,15=9.17, p=0.0085, RM two-way ANOVA, withdrawal day 45 lever x group interaction; **p<0.01, Bonferroni posttest). (r) Hypothetical diagrams illustrating the dissociation between the functional state of cocaine-generated synapses and the behavioral expression (seeking) following reactivation of cocaine memories. Upper: after cue re-exposure-induced memory reactivation, cocaine-generated synapses are re-silenced, while cocaine seeking remains at high levels for a few hr. Lower: when cocaine-generated synapses are re-silenced and weakened beforehand, cue re-exposure does not induce high-level cocaine seeking. Thus, cocaine-generated silent synapses involve in the storage or reactivation of cocaine memories, while cocaine seeking expressed during the memory destabilization window is driven by an independent mechanism. See Supplemental Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Figure 5.. Increasing Rac1 activity prevents cue-induced synaptic re-silencing

(a) Diagram showing the experimental timeline. (b-d) Example EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example caRac1-expressing MSNs in saline- (b), and cocaine-trained rats (c), and the effects of naspm (d). (e) Summary showing that caRac1 expression prevented the cue re-exposure-induced increase in the % silent synapses in cocaine-trained rats after 45 days of withdrawal from self-administration, while perfusion of naspm restored this % to high levels (saline caRac1 = 10.34 ± 2.90, n = 7 animals; cocaine non-trans = 37.22 ± 5.40, n = 7 animals; cocaine non-trans naspm = 27.94 ± 4.94, n = 6 animals; cocaine caRac1 = 8.49 ± 2.10, n =6 animals; cocaine caRac1 naspm = 30.76 ± 3.17, n = 5 animals, F_4,26=10.44, p<0.0001, one-way ANOVA; *p<0.05, **p<0.01, Bonferroni posttest). (f) Diagrams showing the experimental design, in which NAcSh pa-Rac1 was photoactivated during the 10 min cue re-exposure in rats on withdrawal day 45. (g) Example images of a NAcSh slice (left) and MSNs (right) showing HSV-mediated expression of paRac1. All animals used in Fig 5l, Fig 5n-r, Fig 5v, and Fig 6q (n = 41 animals) had pa-dnRac1 expression localized within the NAcSh. (h) Diagram illustrating the design concept of paRac1. (i-k) EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example paRac1-expressing MSNs in saline- (i) and cocaine-trianed rats (j), and the effects of naspm (k). (l) Summary showing that stimulating paRac1 during cue re-exposure prevented cue re-exposure-induced re-silencing of the matured silent synapses in cocaine-trained rats, which was revealed by perfusion of naspm; paRac1 stimulation did not affect the % silent synapses in saline-trained rats (saline = 12.32 ± 2.74, n = 5 animals; cocaine = 9.39 ± 2.81, n = 6 animals; cocaine naspm = 34.83 ± 5.09, n = 6 animals, F_2,14=13.74, p=0.0005, one-way ANOVA; **p<0.01, Bonferroni posttest). (m) Example NAcSh dendrites of MSNs without and without paRac1 expression from saline-trained rats and cocaine-trained rats with photostimulation during cue re-exposure. Scale bar, 2.5 μm. (n) Summary showing that the total spine density was increased in cocaine-trained rats after cue re-exposure for both non-transduced and transduced MSNs compared to saline-trained rats (saline non-trans = 11.62 ± 0.138, n = 4 animals; saline trans = 11.23 ± 0.363, n = 4 animals; cocaine non-trans = 17.83 ± 0.631, n = 4 animals; cocaine trans = 17.94 ± 1.11, n =4 animals, F_1,6=55.47, p=0.0003, RM two-way ANOVA, drug main effect; **p<0.01, Bonferroni posttest). (o) Summary showing the increased density of mushroom-like spines was preserved in pcRac1-expressing MSNs from cocaine-trained rats after cue re-exposure, while the density in non-transduced MSNs decreased. paRac1 stimulation also led to a small, but significant increase in mushroom-like spine density in saline-trained rats (saline non-trans = 3.46 ± 0.216, n = 4 animals; saline trans = 4.28 ± 0.140, n = 4 animals; cocaine non-trans = 4.77 ± 0.175, n = 4 animals; cocaine trans = 8.04 ± 0.295, n =4 animals, F_1,6=57.03, p=0.0003, RM two-way ANOVA, drug x transduced interaction; *p<0.05, **p<0.01, Bonferroni posttest). (p) Summary showing the decreased density of thin spines was preserved in paRac1-expressing MSNs from cocaine-trained rats after cue re-exposure, while the density in non-transduced MSNs increased. paRac1 stimulation also led to a small, but significant decrease in thin spine density in saline-trained rats (saline non-trans = 6.80 ± 0.230, n = 4 animals; saline trans = 5.76 ± 0.253, n = 4 animals; cocaine non-trans = 11.12 ± 0.439, n = 4 animals; cocaine trans = 7.70 ± 0.618, n =4 animals, F_1,6=30.88, p=0.0014, RM two-way ANOVA, drug x transduced interaction; *p<0.05, **p<0.01, Bonferroni posttest). (q) Summary showing the density of stubby spines is increased in cocaine-trained rats after cue re-exposure for both non-transduced and transduced MSNs compared to saline-trained rats (saline non-trans = 1.37 ± 0.060, n = 4 animals; saline trans = 1.22 ± 0.050, n = 4 animals; cocaine non-trans = 1.95 ± 0.070, n = 4 animals; cocaine trans = 2.20 ± 0.225, n =4 animals, F_1,6=35.71, p=0.0010, RM two-way ANOVA, drug main effect; *p<0.05, **p<0.01, Bonferroni posttest). (r) Summary showing the increased mean spine head diameter was preserved in paRac1-expressing MSNs from cocaine-trained rats after cue re-exposure, while the density in non-transduced MSNs normalized back to saline control levels. paRac1 stimulation also led to a small, but significant increase in spine head diameter in saline-trained rats (saline non-trans = 0.375 ± 0.005, n = 4 animals; saline trans = 0.405 ± 0.006, n = 4 animals; cocaine non-trans = 0.377 ± 0.004, n = 4 animals; cocaine trans = 0.488 ± 0.006, n =4 animals, F_1,6=37.43, p=0.0009, RM two-way ANOVA, drug x transduced interaction; *p<0.05, **p<0.01, Bonferroni posttest). (s) Diagram showing the experimental timeline for LIMKi experiments. (t,u) EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example paRac1-expressing MSNs from cocaine-trained rats with photostimulation during cue re-exposure with pretreatment of vehicle (t) or LIMKi (u). (v) Summary showing that pretreatment of LIMKi prevented the effect of paRac1 stimulation on preserving cocaine-generated synapses against re-silencing, such that the % silent synapses were increased compared to vehicle-treated rats (cocaine vehicle = 8.55 ± 1.59, n = 4 animals; cocaine LIMKi = 41.73 ± 6.34, n = 4 animals, t₆=5.08, p=0.0023, two-sided unpaired t-test). See Supplemental Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.

Figure 6.. Active Rac1 stabilizes synaptic states to regulate cocaine memory

(a) Diagrams showing the experimental design in which paRac1 is photoactivated 2 hr after cue re-exposure in cocaine-trained rats on withdrawal day 45. (b) Example images of NAcSh slices (upper) and MSNs (lower) showing HSV-mediated expression of paRac1. All animals used in Fig 6f, Fig 6h-l, Fig 6p, and Fig 6r,s (n = 49 animals) had pa-dnRac1 expression localized within the NAcSh. (c-e) EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example NAcSh MSNs with paRac1 photoactivation after cue re-exposure in saline- (c) and cocaine-trained rats (d), and the effects of naspm (e). (f) Summary showing that stimulating paRac1 after cue re-exposure did not affect the % silent synapses in saline-trained rats, but locked cocaine-generated silent synapses within their silent state in cocaine-trained rats beyond the presumable 6-hr destabilization window, and CPAMPAR inhibition by naspm did not further increase the % silent synapses (saline = 8.30 ± 2.46, n = 4 animals; cocaine = 32.82 ± 3.48, n = 7 animals; cocaine naspm = 36.32 ± 7.65, n =5 animals, F_2,13=7.64, p=0.0064, one-way ANOVA; *p<0.05, **p<0.01, Bonferroni posttest). (g) Example NAcSh dendrites of MSNs non-transduced and transduced with paRac1 from saline- and cocaine-trained rats that received photostimulation 2 hr after cue re-exposure. Scale bar, 2.5 μm. (h) Summary showing the total spine density was increased in cocaine-trained rats 6 hr after cue re-exposure for both non-transduced and transduced MSNs compared to saline-trained rats (saline non-trans = 11.22 ± 0.484, n = 4 animals; saline trans = 11.03 ± 0.199, n = 4 animals; coaine non-trans = 17.88 ± 0.827, n = 3 animals; cocaine trans = 17.67 ± 0.298, n =3 animals, F_1,5=148.0, p<0.0001, RM two-way ANOVA, drug main effect; **p<0.01, Bonferroni posttest). (i) Summary showing that the density of mushroom-like spines was decreased in transduced MSNs from cocaine-trained rats 6 hr after cue re-exposure, while the density in non-transduced MSNs remained high (saline non-trans = 3.69 ± 0.130, n = 4 animlas; saline trans = 3.71 ± 0.175, n = 4 animals; cocaine non-trans = 7.96 ± 0.451, n = 3 animals; cocaine trans = 4.69 ± 0.091, n = 3 animals, F_1,5=58.92, p=0.0006, RM two-way ANOVA, drug x transduction interaction; **p<0.01, Bonferroni posttest). (j) Summary showing that the density of thin spines was increased in transduced MSNs from cocaine-trained rats 6 hr after cue re-exposure, while the density in non-transduced MSNs returned to the saline control level (saline non-trans = 6.20 ± 0.280, n =4 animals; saline trans = 5.83 ± 0.128, n = 4 animals; cocaine non-trans = 7.45 ± 0.498, n = 3 animals; cocaine trans = 10.53 ± 0.461, n = 3 animals, F_1,5=55.71, p=0.0007, RM two-way ANOVA, drug x transduction interaction; **p<0.01, Bonferroni posttest). (k) Summary showing that the density of stubby spines was increased in cocaine-trained rats 6 hr after cue re-exposure for both non-transduced and transduced MSNs compared to saline controls (saline non-trans = 1.32 ± 0.080, n = 4 animals; saline trans = 1.49 ± 0.135, n = 4 animals; cocaine non-trans = 2.47 ± 0.159, n =3 animals; cocaine trans = 2.45 ± 0.120, n = 3 animals, F_1,5=79.42, p=0.0003, RM two-way ANOVA, drug main effect; **p<0.01, Bonferroni posttest). (l) Summary showing that the mean spine head diameter was decreased in transduced MSNs from cocaine-trained rats 6 hr after cue re-exposure, while the spine head diameter in non-transduced MSNs remained high (saline non-trans = 0.387 ± 0.005, n = 4 animals; saline trans = 0.402 ± 0.002, n =4 animals; cocaine non-trans = 0.482 ± 0.003, n = 3 animals; cocaine trans = 0.375 ± 0.017, n = 3 animals, F_1,5=65.19, p=0.0005, RM two-way ANOVA, drug x transduction interaction; **p<0.01, Bonferroni posttest). (m) Diagram showing the experimental timeline for LIMKi experiments. (n-o) EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example paRac1-expressing MSNs from cocaine-trained rats receiving photostimulation 2 hr after cue re-exposure with pretreatment of vehicle (n) or LIMKi (o). (p) Summary showing that pretreatment with LIMKi prevented the effect of paRac1 stimulation on keeping cocaine-generated synapses in a silent state, such that the % silent synapses were decreased compared to vehicle-treated rats (cocaine vehicle = 32.52 ± 1.45, n = 4 animals; cocaine LIMKi = 7.02 ± 2.11, n = 4 animals, t₆=9.95, p<0.0001, two-sided, unpaired t-test). (q) Summary showing that cocaine-trained rats with photostimulation of paRac1 during cue re-exposure exhibited comparable levels of cue-induced cocaine seeking as in C450M control rats, measured 6 hr after cue re-exposure (C450 active = 63.89 ± 4.15, n = 9 animals; pa-Rac1 active = 70.38 ± 6.00, n = 8 animals; C450 inactive = 9.33 ± 1.53, n = 9 animals; pa-Rac1 inactive 13.75 ± 2.20, n = 8 animals,F_1,15=0.07, p=0.79, RM two-way ANOVA, withdrawal day 45 lever x virus interaction, n.s. >0.05). (r) Summary showing that cocaine-trained rats with photostimulation of paRac1 2 hr after cue re-exposure exhibited decreased cue-induced cocaine seeking compared to C450M control rats when measured 6 hr after re-exposure (C450 active = 69.88 ± 4.69, n = 8 animals; pa-Rac1 active = 36.20 ± 2.59, n = 10 animals; C450 inactive = 15.13 ± 2.66, n = 8 animals; pa-Rac1 inactive = 10.10 ± 2.15, n = 10 animals, F_1,16=31.89, p<0.0001, RM two-way ANOVA, withdrawal day 45 lever x virus interaction; **p<0.01, Bonferroni posttest). (s) Summary showing that cocaine-trained rats with photostimulation of paRac1 2 hr after cue re-exposure exhibited decreased cue-induced cocaine seeking compared to C450M control rats when measured 24 hr after re-exposure (C450 active = 86.13 ± 3.82, n = 8 animals; pa-Rac1 active = 55.88 ± 4.40, n = 8 animals; C450 inactive = 13.00 ± 2.47, n = 8 animals; pa-Rac1 = 9.63 ± 2.61, n = 8 animals, F_1,14=15.55, p=0.0015, RM two-way ANOVA, withdrawal day 45 lever x virus interaction; **p<0.01, Bonferroni posttest). See Supplemental Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.