Article Figures & Data
Figures
- Figure 1.
Alcohol activates Rac1 specifically in the DMS of male mice. A, Intermittent access to 20% alcohol in two-bottle choice (IA20%2BC) experimental timeline. B–D, The NAc (B), the DLS (C), and the DMS (D) were harvested 4 h into the last drinking session (binge, B) or after 24 h of abstinence (withdrawal, WD). Rac1-GTP pulldown assay was conducted on cell lysates. Levels of GTP-bound Rac1 were normalized to total Rac1 and presented as percentage of the average of the water values. E, After 4 weeks of IA20%2BC, the DMS was harvested, and Rac1-GTP pulldown assay was conducted on cell lysates. Levels of GTP-bound Rac1 were normalized to total Rac1 and presented as the percentage of the average of the water values. F, Female mice underwent IA20%2BC before the DMS was harvested and the percentage of Rac1-GTP was calculated. RhoA-GTP (G) or Cdc42-GTP (H) pulldown assay was conducted on cell lysates after IA20%2BC and quantified using the Western blot. Levels of GTP-bound RhoA or Cdc42 were normalized to total respective protein and presented as the percentage of average of the water values. I, Mice underwent 21 sessions of 10%CA, and the DMS was harvested. Rac1-GTP pulldown assay was conducted on cell lysates and quantified using the Western blot. Rac1-GTP was normalized to total Rac1 and presented as the percentage of average of water values. J, Mice underwent intermittent access to 1% sucrose for 2 weeks, and the DMS was harvested. Rac1-GTP pulldown assay was performed on cell lysates, and Rac1-GTP was normalized to total Rac1 and presented as average of water values. K, Mice underwent 7 weeks of IA20%2BC, and LIMK and cofilin phosphorylation in the DMS were examined using Western blot analysis. The levels of phospho-LIMK (pLIMK) and phospho-cofilin (pCofilin) were normalized to total respective protein and quantified as a percentage of the average of the water values. Data are represented as mean ± SEM and analyzed using one-way ANOVA (C, D, F, K) with post hoc Šidák's multiple-comparison test, Kruskal–Wallis test (B, E, G), or unpaired two-tailed t test (H, I, J). *p < 0.05, **p < 0.01, ***p < 0.001; ns, nonsignificant. n = 4–7 per group.
- Figure 2.
Alcohol activation of the LIMK/cofilin signaling pathway and subsequent F-actin formation in the DMS depends on Rac1. A, Rac1-DN mechanism of action. Rac1-DN has a mutation in its P-loop and does not bind GTP. GEFs bind to Rac1-DN but are unable to exchange GDP for GTP. The GEFs remain bound to Rac1-DN and ignore endogenous Rac1. B, HEK293 cells infected with AAV-Rac1-DN imaged in both bright-field and GFP to detect viral infection (2× magnification). Western blot analysis of Rac1 protein, phospho-LIMK, and phosphorylation of ERK1/2 in noninfected cells and cells infected with AAV-Rac1-DN after stimulation with 10% FBS. C, Confirmation of viral overexpression during dissection on the DMS of a mouse infected with AAV-Rac1-DN. A 2× image from EVOS FL tabletop fluorescent microscope. D, E, Left images (4× magnification) depict the specificity of the infection site for AAV-GFP (D) and AAV-Rac1-DN (E); scale bar, 500 μm. Right images (20× magnification) depict solely neurons infected in the DMS by both AAV-GFP (D) and AAV-Rac1-DN (E); scale bar, 100 μm. Each slice is stained with anti-GFP (green) and anti-NeuN (red) antibodies, along with DAPI (magenta). F, Experimental timeline. Mice received bilateral infusion of AAV-Rac1-DN or AAV-GFP in the DMS and were allowed 3 weeks for overexpression. After IA20%2BC or water only for 7 weeks, the DMS was harvested. G, Mice were infected with AAV-GFP or AAV-Rac1-DN before undergoing IA20%2BC. The DMS was harvested, and the total Rac1 protein level was examined using a Western blot and normalized to GAPDH. Data are presented as mean ± SEM and analyzed using two-way ANOVA with post hoc Šidák's multiple-comparison test. ***p < 0.001 ****p < 0.0001; ns, nonsignificant. n = 6 per group. H–J, Phosphorylation of LIMK and cofilin were examined using Western blot analysis. Levels of pLIMK and pCofilin were normalized to total respective protein and quantified as a percentage of AAV-GFP-infected, water-only animals. Data are represented as mean ± SEM and analyzed by two-way ANOVA with post hoc Šidák's multiple-comparison test. *p < 0.05; ****p < 0.0001; ns, nonsignificant. n = 6 per group. K–M, G-Actin/F-actin assay was conducted on the DMS of mice after overexpression surgery and IA20%2BC or water-only drinking. The filamentous (F) or globular (G) actin contents were examined using Western blot analysis and normalized to total actin and quantified as a percentage of AAV-GFP-infected, water-only animals. Data are represented as mean ± SEM and analyzed using two-way ANOVA with Šidák's multiple comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, nonsignificant. n = 6 per group.
- Figure 3.
Rac1 promotes remodeling of dendritic arbors in the DMS. Low titer of AAV-GFP or AAV-Rac1-DN (7 × 109–3.5 × 1010 vg/ml) was infused bilaterally into the DMS. After 1 week of recovery, mice underwent 7 weeks of IA20%2BC. Mice consuming water only were used as the control. Twenty-four hours after the last drinking session, mice were perfused, and brains were sliced into 100 μm sections before MSN morphology was analyzed. A, A sample image (40× magnification) of a GFP-positive DMS MSN. Scale bar, 50 μm. B, An example of the reconstruction of the GFP-positive neuron. C, Scheme of the morphological parameters measured in each neuron. Dendritic order increased after each branching point, defined as a dendritic intersection. D, E, Analysis of neuronal dendritic arborization. Sholl analysis was performed on reconstructed neurons. The number of intersections was analyzed for each point (D) and the AUC (E) was calculated. F, Measurement of total dendritic length. G, The number of branching points. H, The number of ending points. I, The number of branches per dendritic order. Data are represented as mean ± SEM and analyzed by two-way ANOVA. Main effect of virus is represented graphically. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. Water + AAV-GFP, 19 neurons from 6 mice; alcohol + AAV-GFP, 23 neurons from seven mice; water + AAV-Rac1-DN, 15 neurons from six mice; alcohol + AAV-Rac1-DN, 10 neurons from three mice.
- Figure 4.
Rac1 in the DMS is required for the alcohol-mediated morphological changes in dendritic spines. Low titer of AAV-GFP or AAV-Rac1-DN (7 × 109–3.5 × 1010 vg/ml) was infused bilaterally into the DMS. After 1 week of recovery, mice underwent 7 weeks of IA20%-2BC. Mice consuming water only were used as the control. Twenty-four hours after the last drinking session, mice were perfused, and MSN dendritic spine morphology was analyzed. A, A sample image of a GFP-positive DMS MSN (100× magnification) from each group. Scale bar, 10 μm. B, Average dendritic spine density (number of spines/10 µm). C, Average dendritic spine area (µm2). D, Average dendritic spine volume (µm3). E, Average dendritic spine length (µm). F, Average diameter of dendritic spine heads (µm). G, Dendritic spine length-to-width ratio. H, The percentage of filopodia, thin, stubby, and mushroom dendritic spines for water- and alcohol-drinking mice infected with either AAV-GFP or AAV-Rac1-DN. Data are represented as mean ± SEM and analyzed using two-way ANOVA (B–G) or three-way ANOVA (H) and Šidák's post hoc multiple-comparison test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns: non-significant. Water + AAV-GFP: 14 neurons from five mice; alcohol + AAV-GFP, 13 neurons from five mice; water + AAV-Rac1-DN: 14 neurons from five mice; alcohol + AAV-Rac1-DN, 11 neurons from four mice.
- Figure 5.
Rac1 does not play a role in the development and maintenance of voluntary alcohol intake. A, Development: surgery and IA20%2BC timeline. Mice received a bilateral, dual infusion of AAV-Rac1-DN or AAV-GFP as a control. After 3 weeks of recovery, IA20%2BC began and continued for 7 weeks. B, Weekly average alcohol consumption (g/kg/24 h) of development drinking mice. C, Water consumption averages for the IA20%2BC development drinking groups. D, Preference for alcohol during the development IA20%2BC drinking, with an index above 50 indicating a preference for alcohol. E, Maintenance: surgery and IA20%2BC timeline. Mice first underwent IA20%2BC for 7 weeks. Groups were balanced based on average daily alcohol consumption in the last six drinking sessions. Mice received a bilateral, dual infusion of AAV-Rac1-DN or AAV-GFP as a control and were allowed 1 full week of recovery. IA20%2BC resumed for 4 weeks. F, Weekly average consumption (g/kg/24 h) for maintenance-drinking mice. G, Water consumption average for maintenance-drinking mice. H, Preference for alcohol for maintenance-drinking mice. All data are represented as mean ± SEM and analyzed with two-way ANOVA with repeated measures. n = 9–10.
- Figure 6.
Rac1 in the DMS is required for alcohol-associated goal–directed learning. A, OSA of 20% alcohol paradigm. Mice underwent surgery to overexpress AAV-Rac1-DN or AAV-GFP in the DMS. After 3 weeks of recovery, mice underwent 7 weeks of IA20%2BC to develop a preference for alcohol before transitioning to 20% alcohol OSA for 1 week of training and 4 experimental weeks. B, C, The group average of active (B) and inactive (C) lever presses during the 2 h sessions. D, Discrimination index for the active lever. Calculated as proportion of active lever presses compared with total lever presses. E, Proportion of rewarded lever presses. Calculated as rewarded lever presses compared with total lever presses. F, Alcohol consumption during each session. G, Proportion of inter-response interval between lever presses during the final session. Data are presented as mean ± SEM and analyzed using RM two-way ANOVA and Šidák's post hoc multiple-comparison test. *p < 0.05; **p < 0.01; ***p < 0.001. n = 6, 7. H, Individual mouse behavioral profiles from OSA Session 9. Each line corresponds with an active (green) or inactive (red) lever press, and triangles represent reward (purple). I, Total distance moved in an open field. Mice were placed in an open field, and locomotion was recorded for 20 min. Data are represented as mean ± SEM and analyzed using an unpaired t test. n = 9 per group.
- Figure 7.
Rac1 in the DMS is required for alcohol action–outcome associations. A, Contingency degradation paradigm. Mice received a dual infusion of AAV-GFP or AAV-Rac1-DN into the DMS. After a recovery period, mice underwent IA20%2BC for 7 weeks before being trained to operant self-administer alcohol. Mice were first trained on an FR1 schedule before progressing to a RR schedule of reinforcement. Under RR, mice received a reward after a random number of active lever presses within a range (e.g., RR2 1–3 presses, RR3 2–4 presses, RR4 3–5 presses). Mice were trained for 5 sessions at each reinforcement step and spent 10 sessions at RR4 before testing. During the contingency degradation test, there were two types of sessions: ND and D. During nondegraded sessions, rewards were delivered on an RR4 schedule. During degraded sessions, rewards were delivered at a rate equal to the average of the last week of training, but active lever presses had no effect. Testing consisted of a ND session followed by a D session, repeated three times. B, Average active lever presses during ND and D testing sessions for AAV-GFP and AAV-Rac1-DN mice. C, Average port entries during ND and D testing sessions for AAV-GFP and AAV-Rac1-DN mice. Data are represented as mean ± SEM and analyzed using two-way ANOVA with Šidák's post hoc multiple-comparison test. ***p < 0.001, ns: non-significant. n = 7 per group.
- Figure 8.
Rac1 in the DMS is not required for sucrose goal-directed learning. A, OSA of 1% sucrose paradigm. Mice underwent surgery to overexpress AAV-Rac1-DN or AAV-GFP in the DMS. After 3 weeks of recovery, mice underwent IA20%2BC for 7 weeks. One percent sucrose OSA began with 1 week of training and 4 weeks of experimental tracking. Variables tracked during the session include (B) active lever presses and (C) inactive lever presses. D, Discrimination and (E) proportion of rewarded lever presses were calculated. Data are represented as mean ± SEM and analyzed using RM two-way ANOVA. n = 7, 6.
Tables
Figure Average consumption (g/kg/24 h) SEM (±g/kg/24 h) 1B NAc 13.38 1.025 1C DLS 13.38 1.060 1D DMS 14.53 0.982 1F Female DMS 19.59 0.979 1G RhoA 12.31 0.4461 1H Cdc42 13.67 0.7354 1I 10%CA 8.787 0.2611 1E 4 week DMS 12.560 1.678 1K pLIMK pCofilin 15.34 0.5169 2H–M AAV-GFP 14.00 0.7264 2H–M AAV-Rac1-DN 13.68 0.4589 3, 4 AAV-GFP 10.63 0.5058 3, 4 AAV-Rac1-DN 10.88 1.223 6 AAV-GFP 12.10 0.7463 6 AAV-Rac1-DN 12.36 1.046 1J Sucrose 174.81 (ml/kg) 17.87 (ml/kg)
