1. Volume 89, Issue 3, Pages 566-582 (February 2016)
Bidirectional Synaptic Structural Plasticity after Chronic Cocaine Administration Occurs through Rap1 Small GTPase Signaling 
Michael E. Cahill, Rosemary C. Bagot, Amy M. Gancarz, Deena M. Walker, HaoSheng Sun, Zi-Jun Wang, Elizabeth A. Heller, Jian Feng, Pamela J. Kennedy, Ja Wook Koo, Hannah M. Cates, Rachael L. Neve, Li Shen, David M. Dietz, Eric J. Nestler 
Neuron 
Volume 89, Issue 3, Pages (February 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1 Cocaine Alters RhoA Signaling in NAc
(A–C) Western blots showing PDZ-RhoGEF levels in NAc P1 (crude nuclear) (A), S2 (cytoplasm) (B), and P2 (crude synaptoneurosomal) (C) fractions from saline- or cocaine- (20 mg/kg i.p. × 7 days) treated mice studied 24 hr after last injection.
(D–F) PDZ-RhoGEF NAc protein levels were increased in P1 (D) (t29 = 2.861, ∗∗p < 0.01, n = 15 [s], 16 [c]) and decreased in S2 (E) (t29 = 2.558, ∗p < 0.01, n = 15 [s], 16 [c]) by cocaine, with no effect in P2 (F) (t29 = , p > 0.05, n = 15 [s], 16 [c]).
(G and H) Linear regression of NAc PDZ-RhoGEF protein levels in P1 versus S2 for individual saline- (G) and cocaine-treated (H) mice. Statistical correlation values are shown above each graph, n = 15 (s), 16 (c).
(I–L) Blot shows increased RhoA NAc levels in P1 by cocaine (I and K) (t52 = 2.268, ∗p < 0.05, n = 27 [s], 27 [c]), with no difference in S2 (J and L) (t30 = 0.305, p > 0.05), n = 16 [s], 16 [c]).
(M and N) Blots show increased active RhoA (RhoA-GTP) NAc levels in P1 by cocaine (t20 = 2.197, ∗p < 0.05, n = 11 [s], 11 [c]).
(O and P) Blots show levels of MAL in G-actin and F-actin subfractions of P1 in NAc of saline- and cocaine-treated mice, with elevated MAL F-actin to MAL G-actin ratios after cocaine (t19 = 2.342, ∗p < 0.05, n = 10 [s], 11 [c]).
(Q and R) MAL was immunoprecipitated (IP) from NAc P1 fractions of saline- and cocaine-treated mice. Blots show increased SRF protein in MAL NAc P1 immunoprecipitates after cocaine; SRF was normalized to the amount of MAL immunoprecipitated (t4 = 2.896, ∗p < 0.05, n = 3 [s], 3 [c]).
All summary data are the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2 Cocaine-Mediated Rap1b Expression Controls Cocaine Reward
(A) qPCR in mice treated with saline or cocaine (20 mg/kg i.p. × 7 days) revealed that Rap1b transcript levels (t15 = 3.724, ∗∗p < 0.01) but not those of other small GTPases were elevated 24 hr post-cocaine (p > 0.05). n = 9 (s), 8 (c).
(B–D) Blots show Rap1b levels in NAc P2 (B), P1 (C), or S2 (D) fractions in cocaine-treated mice.
(E) Cocaine treatment increases Rap1b protein levels in NAc P2 fractions (t42 = 2.074, ∗p < 0.01, n = 21 [s], 23 [c]).
(F) Cocaine does not alter Rap1b protein levels in NAc P1 fractions (t30 = , p > 0.05, n = 16 [s], 16 [c]).
(G) No alterations in Rap1b protein levels occurred in response to cocaine in NAc S2 fractions (t30 = , p > 0.05, n = 16 [s], 16 [c]).
(H) HSV-GFP or HSV-Cre-GFP was infused into NAc of floxed SRF mice and mice were given five cocaine injections over a 3-day period. One NAc hemisphere was infused with HSV-GFP and the other with HSV-Cre-GFP.
(I and J) Blots show the effects of SRF knockdown in NAc on P2 Rap1b levels in cocaine-treated mice. SRF levels in the P1 fraction show SRF knockdown (see Figure S3E for P1 quantification). HSV-Cre-GFP decreased Rap1b levels in NAc P2 fractions (paired t test, t7 = 2.696, ∗p < 0.05, n = 8 pairs).
(K) GGTI-298 or vehicle was infused into NAc. Twenty-four hours post-infusion, saline/cocaine pairing began (7.5 mg/kg cocaine); cocaine CPP behavior was assessed 3 days post-infusion.
(L) Cocaine CPP shows that GGTI-298 NAc infusion reduced cocaine preference (t31 = 2.157, ∗p < 0.05, n = 17 [v], 16 [g]).
(M) GGTI-298 or vehicle was infused into the NAc. Twenty-four hours post-infusion, mice were tested for 8 consecutive days in a cocaine (7.5 mg/kg) locomotor sensitization task.
(N) Reduced locomotor responses to cocaine occurred in GGTI-298 infused mice. There was a significant main effect for drug treatment across days 1–8 (two-way repeated-measures ANOVA, F1,14 = 5.800, ∗p < 0.05, n = 8 [v], 8 [g]).
(O) Images show the targeting of HSV-GFP and HSV-Rap1b-GFP to NAc. The anterior commissure landmark is labeled (a.c.). Scale bar, 400 μm.
(P) HSV-GFP or HSV-Rap1b-GFP was infused into NAc. Three days post-infusion, saline/cocaine pairing began (5 mg/kg cocaine).
(Q) Rap1b overexpression in NAc increases cocaine preference (t21 = 2.142, ∗p < 0.05, n = 12 [g], 11 [r]).
(R) Rap1b or vehicle was infused into NAc. Three days post-infusion, mice were tested over 7 days for cocaine (5 mg/kg)-induced locomotion.
(S) Increased locomotor responses to cocaine were seen in mice overexpressing Rap1b in NAc across all testing days (two-way repeated-measures ANOVA, main effect of Rap1b expression, F1,16 = 6.199, ∗p < 0.05, n = 9 [g], 9 [r]).
All summary data are the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
Cocaine-Mediated Regulation of Akt/mTOR Activity Controls Cocaine Reward
(A) HSV-GFP or HSV-Rap1b-GFP was infused into NAc of mice. For each mouse one NAc hemisphere was infused with HSV-GFP and the other with HSV-Rap1b-GFP. Five days post-infusion, NAc was dissected and P2 fractions were isolated.
(B and C) Blot shows the effects of Rap1b overexpression on NAc P2 protein levels. Pairwise analysis indicates that Rap1b NAc overexpression increased levels of p-Akt T308 (t6 = 2.755, ∗p < 0.05), p-mTOR S2448 (t6 = 2.744, ∗p < 0.05), and total mTOR (t6 = 3.645, ∗p < 0.05); the virus elevated Rap1b levels as well (t6 = 2.698, ∗p < 0.05). Levels of p-Akt S473 were not altered (t6 = , p > 0.05). n = 7 pairs.
(D) HSV-GFP or HSV-Cre-GFP was infused into NAc of floxed SRF mice. Two days post-surgery, mice were given five cocaine injections over a 3-day period. NAc was dissected and P2 fractions isolated. For each mouse one NAc hemisphere was infused with HSV-GFP and the other with HSV-Cre-GFP.
(E and F) Blots show the effects of SRF knockdown on NAc P2 protein expression profiles in cocaine-treated mice. Data are derived from the same experiment in Figures 2I and 2J. Pairwise analysis indicates that SRF knockdown reduced levels of p-Akt T308 (t7 = 2.657, ∗p < 0.05), p-mTOR (t7 = 2.008, ∗p < 0.05), and total mTOR (t7 = 2.410, ∗p < 0.05), with no effect on p-Akt S473 (t7 = , p > 0.05). n = 8 pairs.
(G–I) Blots show Akt-mTOR P2 protein levels from mice injected with saline or cocaine (i.p., 20 mg/kg cocaine) for 7 days analyzed 24 hr (G), 1 week (H), or 2 weeks (I) after the last injection.
(J) At 24 hr, cocaine increased P2 levels of p-Akt T308 (t28 = 2.063, ∗p < 0.05, n = 15 [s], 15 [c]) and p-mTOR S2448 (t29 = 2.149, ∗p < 0.05, n = 16 [s], 15 [c]) with no effects on total Akt (t29 = , p > 0.05, n = 16 [s], 15 [c]) or total mTOR (t29 = , p > 0.05, n = 16 [s], 15 [c]).
(K and L) Cocaine did not alter P2 levels of p-Akt T308, p-mTOR S2448, total Akt, or total mTOR at 1 (K) or 2 (L) weeks (p > 0.05; 1 week n = 12 [s], 13 [c]; 2 week n = 10 [s], 9 [c]).
(M) Images show the targeting of the indicated virus to NAc; the anterior commissure (a.c.) landmark is labeled. Scale bar, 400 μm.
(N) HSVs were infused into NAc and 3 days post-infusion saline/cocaine pairing began (5 mg/kg or 7.5 mg/kg cocaine).
(O) Akt, but not DN-Akt, increased cocaine preference to a low dose of cocaine (5 mg/kg) (one-way ANOVA, F2,35 = 3.656, p < 0.05; post hoc GFP versus Akt, ∗p < 0.05; post hoc GFP versus DN-Akt, p > 0.05, n = 15 [g], 13 [akt], 10 [dn-akt]).
(P) DN-Akt reduced cocaine preference to a higher dose of cocaine (7.5 mg/kg); Akt overexpression had no effect (one-way ANOVA, F2,64 = 4.020, p < 0.05, post hoc GFP versus Akt, p > 0.05; post hoc GFP versus DN-Akt, ∗p < 0.05, n = 26 [g], 23 [akt], 18 [dn-akt]).
(Q) Graphs depict locomotor responses during the second day of saline/cocaine pairing with a 5 mg/kg cocaine dose. No differences between groups were detected (one-way ANOVA, F2,36 = 1.893, p > 0.05, post hoc GFP versus Akt and DN-Akt, p > 0.05). One sample actual versus theoretical mean analyses revealed while both GFP (t = 2.198, ∗p < 0.05) and Akt (t = 2.206,∗p < 0.05) overexpression increased locomotor activity in response to cocaine as compared to saline (above a chance level of 0), DN-Akt overexpression locomotor activity did not differ from chance levels (t = , p > 0.05). n = 15(g), 14(akt), 10(dn-akt).
(R) Locomotor responses during the second day of saline/cocaine pairing with a 7.5 mg/kg cocaine dose. Akt, but not DN-Akt, increased cocaine activity relative to control mice (one-way ANOVA, F2,63 = 3.275, p < 0.05; post hoc GFP versus Akt, ∗p < 0.05; post hoc GFP versus DN-Akt, p > 0.05, n = 25 [g], 23 [akt], 18 [dn-akt]).
All summary data are the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
Rap1b Regulates NAc MSN Dendritic Spine Morphogenesis through Akt
(A) Low-magnification image of a NAc MSN with an adjacent high-magnification image of a dendritic segment illustrating different spine subtypes. This neuron overexpresses HSV-GFP, shown in black and white. Scale bar for left image, 25 μm; for right image, 5 μm.
(B) Experimental design. HSV-GFP, HSV-Rap1b-GFP, HSV-DN-Akt-GFP, and HSV Rap1b+HSV-DN-Akt-GFP were infused into NAc. Three days post-infusion, brains were perfused.
(C) High-magnification 3D reconstruction images of NAc MSN dendrites bearing dendritic spines for all viral conditions. Differences in brightness across a dendrite reflect different Z-plane depths. Images are cropped from larger dendrite segments. Scale bar, 5 μm.
(D) Only neurons overexpressing Rap1b show altered total spine density on NAc MSNs relative to those overexpressing GFP only (one-way ANOVA, F3,75 = 3.693, p < 0.05; post hoc GFP versus Rap1b, ∗p < 0.05; post hoc GFP versus DN-Akt and DN-Akt+Rap1b, p > 0.05). n = 21 cells from four brains (GFP), 22 cells from three brains (Rap1b), 12 cells from three brains (DN-Akt), and 24 cells from three brains (DN-Akt+Rap1b).
(E) Only Rap1b overexpression increased the density of thin spines on NAc MSN dendrites (one-way ANOVA, F3,75 = 5.920, p < 0.01; post hoc GFP versus Rap1b, ∗∗p < 0.01; post hoc GFP versus DN-Akt and DN-Akt+Rap1b, p > 0.05).
(F and G) No conditions altered stubby (F) or mushroom (G) spine density in NAc MSNs (stubby, one-way ANOVA, F3,75 = 0.465, p > 0.05; post hoc GFP versus all conditions, p > 0.05) (mushroom, one-way ANOVA, F3,75 = 1.350, p > 0.05; post hoc GFP versus all conditions, p > 0.05).
(H) Across all spine types only Rap1b overexpression alters mean spine head diameter relative to cells overexpressing GFP (one-way ANOVA, F3,3316 = 10.92, p < 0.0001; post hoc GFP versus Rap1b, ∗∗∗p < 0.001; post hoc GFP versus DN-Akt and DN-Akt+Rap1b, p > 0.05). n = 746 spines (GFP), 1,211 spines (Rap1b), 408 spines (DN-Akt), 955 spines (Rap1b+DN-Akt).
(I) Cumulative plot of thin spine head diameter reveals no significant differences between Rap1b overexpression and control neurons (spine log-rank test, χ2 = 1.596, p > 0.05).
(J and K) Rap1b overexpression in NAc MSNs results in a leftward shift of the cumulative stubby spine (J) and mushroom spine (K) head diameter curve relative to GFP overexpression (stubby log-rank test, χ2 = 7.164, ∗∗p < 0.01; mushroom log-rank test, χ2 = 4.362, ∗p < 0.05).
All summary data are the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 5
Rap1b and Akt Are Necessary for Cocaine-Induced Spine Remodeling in NAc MSNs
(A) HSV-GFP or HSV-Cre-GFP was infused into NAc of floxed Rap1 mice. Two days post-surgery, mice were administered five saline or cocaine injections (i.p., 20 mg/kg cocaine) over a 3-day period and brains were perfused 4 hr after the last injection.
(B) High-magnification 3D reconstruction images of NAc MSN dendrites bearing dendritic spines for all four experimental conditions. Differences in brightness across a dendrite reflect different Z-plane depths. Images are cropped from larger dendrite segments. Scale bar, 5 μm.
(C) Cocaine increased total spine density in GFP-expressing but not Cre-expressing neurons (2x2 ANOVA main effect of drug treatment F1,62 = 7.064, p = 0.01; post hoc GFP saline versus GFP cocaine, ∗p < 0.05; Cre saline versus Cre cocaine, p > 0.05). n = 19 cells from three brains (GFP saline), 16 cells from three brains (GFP cocaine), 12 cells from three brains (Cre saline), and 19 cells from three brains (Cre cocaine).
(D) Cocaine increased thin spine density in GFP-expressing but not Cre-expressing NAc MSNs (2x2 ANOVA main effect of drug treatment, F1,62 = 11.30, p = 0.01; post hoc GFP saline versus GFP cocaine, ∗∗∗p < 0.001; Cre saline versus Cre cocaine, p > 0.05).
(E and F) Cocaine did not alter the density of stubby (E) or mushroom (F) spines in either GFP- or Cre-expressing NAc MSNs (p > 0.05). There was a main effect of Rap1 level on mushroom spine density (2x2 ANOVA, F1,62 = 17.56, ∗∗∗p < ).
(G) Analysis of total spine head diameter across all spine types revealed main effects of drug treatment (2x2 ANOVA, F1,2571 = 37.80, ∗∗∗p < ) and Rap1 expression (2x2 ANOVA, F1,2571 = 82.65, ∗∗∗p < ). In both GFP- and Cre-expressing NAc MSNs, cocaine reduced mean spine head diameters (post hoc, ∗∗∗p < for both GFP and Cre saline versus cocaine). n = 670 spines (GFP saline), 644 spines (GFP cocaine), 490 spines (Cre saline), and 771 spines (Cre cocaine).
(H) Cumulative plot of thin spine head diameter indicates that only the GFP cocaine group differed from the GFP saline group, with a significant leftward shift in the curve (spine log-rank test, χ2 = 8.305, ∗p < 0.05).
(I) No difference in the cumulative frequency plot of stubby spine head diameter was detected between any groups and the GFP saline condition (p > 0.05).
(J) Cumulative plot of mushroom spine head diameter indicates a significant rightward shift in the curve in the Cre saline group compared to the GFP saline group (spine log-rank test, χ2 = 20.91, ∗∗∗p < ). No other groups differed from GFP saline (p > 0.05).
(K) GFP or DN-Akt were infused into the NAc. Two days post-surgery, mice were administered five saline or cocaine injections (i.p., 20 mg/kg cocaine) over a 3-day period and brains were perfused 4 hr after the last injection.
(L) High-magnification 3D reconstruction images of NAc MSN dendrites bearing dendritic spines for all experimental conditions. Differences in brightness across a dendrite reflect different Z-plane depths. Images are cropped from larger dendrite segments. Scale bar, 5 μm.
(M) Cocaine increased total spine density in GFP-expressing, but not DN-Akt-expressing, NAc MSNs (2x2 ANOVA interaction effect, F1,88 = 5.264, p < 0.05; post hoc GFP saline versus GFP cocaine, ∗p < 0.05; DN-Akt saline versus DN-Akt cocaine, p > 0.05). n = 19 cells from three brains (GFP saline), 22 cells from five brains (GFP cocaine), 25 cells from four brains (DN-Akt saline), and 26 cells from four brains (DN-Akt cocaine).
(N) Cocaine increased thin spine density in GFP-expressing but not DN-Akt-expressing NAc MSNs (2x2 ANOVA interaction effect, F1,88 = 10.28, p < 0.01; post hoc GFP saline versus GFP cocaine, ∗∗∗p < 0.001; DN-Akt saline versus DN-Akt cocaine, p > 0.05).
(O and P) Cocaine did not alter stubby (O) or mushroom (P) spine density in GFP- or DN-Akt-expressing NAc MSNs (p > 0.05). There was a main effect of DN-Akt expression on mushroom spine density (2x2 ANOVA, F1,88 = 12.83, ∗∗∗p < 0.001).
(Q) Analysis of spine head diameter across all spine types revealed main effects of drug treatment (2x2 ANOVA, F1,3148 = 9.630, ∗∗p < 0.01) and DN-Akt expression (2x2 ANOVA, F1,3148 = 68.15, ∗∗∗p < ). In GFP- but not DN-Akt-expressing NAc MSNs, cocaine reduced mean spine head diameters (post hoc, ∗∗p < 0.01 for GFP saline versus GFP cocaine; p > 0.05 for DN-Akt saline versus DN-Akt cocaine). n = 579 spines (GFP saline), 801 spines (GFP cocaine), 919 spines (DN-Akt saline), and 853 spines (DN-Akt cocaine).
(R) Cumulative frequency plot analysis of thin spine head diameters revealed no significant differences between groups (p > 0.05).
(S) Cumulative plot analysis of stubby spine head diameters revealed a rightward shift in the curve in the DN-Akt saline group as compared to the GFP saline group (spine log-rank test, χ2 = 9.978, ∗∗p < 0.01); no other groups differed from GFP saline.
(T) Cumulative frequency plot of mushroom spine head diameter revealed a rightward shift in the curve in both the DN-Akt saline and DN-Akt cocaine groups compared to the GFP saline group (DN-Akt saline spine log-rank test, χ2 = 16.47, ∗∗∗p < ; DN-Akt cocaine spine log-rank test, χ2 = 9.382, ∗∗p < 0.01).
All summary data are the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 6
Self-Administered Cocaine Bidirectionally Regulates P1 and P2 Protein Expression Profiles as a Function of Time after Last Cocaine Exposure
(A) Mice self-administered saline or cocaine during 6-hr sessions for 10 consecutive days. Twenty-four hours after the last session, NAc tissue was dissected and cell fractionated. Graphs show that mice volitionally self-administered cocaine at high rates (one-way ANOVA cocaine versus saline, ∗∗∗p < ).
(B and C) Decreased S2 levels of PDZ-RhoGEF 24 hr post-cocaine (t17 = 2.686, ∗p < 0.05, n = 8 [s], 11 [c]).
(D and E) Increased P1 PDZ-RhoGEF levels 24 hr post-cocaine (t17 = 2.461, ∗p < 0.05, n = 8 [s], 11 [c]).
(F and G) P2 levels of Rap1b (t16 = 3.359, ∗∗p < 0.01), p-Akt T308 (t16 = 4.848, ∗∗∗p < 0.001), p-mTOR (t16 = 3.807, ∗∗p < 0.01), and total mTOR (t16 = 4.135, ∗∗∗p < 0.001) were increased 24 hr post-cocaine. Levels of total Akt were not affected (t16 = , p > 0.05). n = 7 [s], 11 [c]).
(H) Mice self-administered saline or cocaine as above, but were analyzed 4 weeks after the last session. Graphs show that mice volitionally self-administered cocaine at high rates (one-way ANOVA cocaine versus saline, ∗∗∗p < ).
(I and J) Cocaine increased S2 PDZ-RhoGEF levels 4 weeks post-cocaine (t16 = 3.400, ∗∗p < 0.01, n = 8 [s], 10 [c]).
(K and L) Decreased P1 PDZ-RhoGEF levels 4 weeks post-cocaine (t28 = 2.068, ∗p < 0.05).
(M and N) P2 levels of Rap1b (t28 = 2.141, ∗p < 0.05), p-Akt T308 (t28 = 2.578, ∗p < 0.05), p-mTOR S2448 (t28 = 2.765, ∗∗p = 0.01), and total mTOR (t28 = 3.053, ∗∗p < 0.01) were decreased 4 weeks post-cocaine. Levels of total Akt were not affected (t28 = 1.171, p > 0.05). n = 14 (s), 16 (c).
All summary data are the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 7
Pathway-Specific Inputs to the NAc Differentially Regulate Nuclear and Synaptic Signaling
(A) AAV5-CaMKIIa/ChR2/EYFP was infused into ventral hippocampus (vHIPP), basolateral amygdala (BLA), or ventromedial prefrontal cortex (vmPFC; infralimbic targeting). NAc shell-targeting optical fibers were implanted, and mice underwent 7 days of 20-min burst stimulation sessions in which AAV5-expressing axon terminals in NAc shell were stimulated; mock-stimulation was used as a control. Twenty-four hours after the final stimulation day, NAc shell tissue was dissected and fractionated.
(B) Image shows the placement of AAV5 to the vmPFC (infralimbic targeting).
(C and D) Increased P1 PDZ-RhoGEF levels after vmPFC terminal stimulation (t17 = 2.246, ∗p < 0.05, n = 9 [mock], 10 [stim]).
(E and F) Increased P2 levels of Rap1b (t37 = 2.048, ∗p < 0.05) and p-mTOR S2448 (t37 = 2.298, ∗p < 0.05) after vmPFC terminal stimulation. n = 19 (mock), 20 (stim).
(G) Image shows the placement of AAV5 to the vHIPP.
(H–K) No alterations in P1 PDZ-RhoGEF levels (H and I) (t11 = 0.050, p > 0.05), or in P2 Rap1b or p-mTOR S448 levels (J and K) (Rap1b, t11 = 0.300 p > 0.05; p-mTOR, t11 = 1.301, p > 0.05), after vHIPP terminal stimulation. n = 8 (mock), 5 (stim).
(L) Image shows the placement of AAV5 to the BLA.
(M–P) No alterations in P1 PDZ-RhoGEF levels (M and N) (t11 = 0.368, p > 0.05), or in P2 Rap1b or p-mTOR S2448 levels (O and P) (Rap1b, t11 = 0.728, p > 0.05; p-mTOR, t11 = , p > 0.05), after BLA terminal stimulation. n = 7 (mock), 6 (stim).
(Q) AAV5-CaMKIIa/ChR2/EYFP was infused into vmPFC of floxed Rap1 mice. After >6 weeks post-AAV5 infusion, HSV-GFP or HSV-Cre-GFP (Rap1 knockdown) was infused into NAc, and NAc shell-targeting optical fibers were implanted. Mice were trained in a CPP paradigm in which one end-chamber was paired with vmPFC terminal NAc stimulation and the other with mock-stimulation, or in some mice both chambers were paired with mock stimulation.
(R) Stimulation increases CPP scores in mice expressing HSV-GFP, but not those expressing HSV-Cre-GFP in NAc (2x2 ANOVA stimulation effect, F1,33 = 6.55; post hoc GFP mock versus GFP stim, ∗p < 0.05; post hoc Cre mock versus Cre stim, p > n = 12 (gfp mock), 10 (gfp stim), 9 (cre mock), 6 (cre stim).
All summary data are the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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9. Figure 8 Summary Schematic
Levels of PDZ-RhoGEF, an activator of the RhoA small GTPase, decrease in the cytoplasm and increase in the nucleus of NAc MSNs 24 hr after the last chronic cocaine dose. This is associated with increased levels of active RhoA in the nucleus, which promotes the formation of F-actin. These actions, in turn, activate the serum response factor (SRF) transcription factor by controlling the localization of the SRF co-activator MAL. MAL is unable to interact with SRF when bound to G-actin, but 24 hr post-cocaine MAL shifts away from G-actin and toward F-actin due to higher RhoA activity, with a resulting increase in MAL-SRF-dependent transcription. This PDZ-RhoGEF-RhoA-actin-MAL-SRF signaling cascade drives Rap1b expression in synaptoneurosomes, where it activates the PI3K-Akt-mTOR pathway. Activation of this pathway drives the formation of thin, immature spines 24 hr post-cocaine. Conversely, the activity of this signaling pathway is reversed 3–4 weeks after cocaine administration and results in the formation of mature mushroom spines.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions