Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons

Abstract

Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.

Figure 1. Cocaine regulation of Rac1 signaling in NAc

Repeated, but not acute, cocaine administration: (a) transiently decreased Rac1 activity in NAc 15 min after the final drug injection (n=7–8 mice per group) (F2,21=4.50, *p<0.05), (b) but this decrease was no longer present at 45 min (n=7–8 mice) (p>0.05). At both the 15 and 45 min time points, there were no differences in total Rac1 levels (p >0.05). (c) The inactive (phosphorylated) form of cofilin (p-cofilin) was significantly decreased 15 min after repeated, cocaine (n=9 mice per group) (F1,17=6.29, *p<0.05), and remained decreased at 4 hours (n=5–6 mice per group) (F1,10=22.5, ***p<0.001). Total cofilin was unaltered by cocaine at both time points (p>0.05). (d) 24 hours after the final cocaine treatment, Tiam1 protein levels, normalized to tubulin (which was not altered), were significantly reduced by repeated cocaine (n=7–8 mice per group) (F2,22=3.54, *p<0.05). (e) RacGap1 protein levels, normalized to tubulin, were significantly increased at this same 24 hour time point (n=8–9 mice per group) (F2,14=6.73, **p<0.01). Full-length blots are presented in Supplementary Fig. 2). Data were analyzed by ANOVA and represented as mean ± s.e.m.

Figure 2. Rac1 signaling regulates behavioral responses to cocaine

(a) HSV-mediated overexpression of Rac1-ca (n=10 mice) blocked cocaine conditioned place preference at 7.5 mg/kg compared to dsRed controls (n=7) (F2,23=4.00, *p<0.05), while HSV-Rac1-dn (n=9 mice) did not further enhance cocaine’s rewarding effects at this higher dose. (b) Using a lower dose of cocaine (4 mg/kg), which did not induce a conditioned place preference in control mice (n=13 mice), HSV-Rac1-dn (n=8) induced a robust preference to the drug (F3,37=3.33, *p<0.05 compared to control ). Overexpression of a constitutively active form of cofilin (HSV-cofilin-ca; n=10 mice) also increased the reward sensitivity to cocaine compared to controls (*p<0.05). (c) Rac1-ca (n=10 mice) attenuated locomotor activation following an acute injection of cocaine (7.5 mg/kg) (F3,41=3.06, p<0.05 compared to control), (d) but had no effect on locomotor responses to a saline challenge (p>0.05). (e) Anatomical placement of viral infection in NAc after HSV injection. Cartoon shows the location of the injection site at 1.77 mm from Bregma. (f) A representative HSV-infected NAc medium spiny neuron imaged at 40X. Rectangular boxes highlight areas used for dendritic spine analysis in Fig. 4. (g–i) High powered immunohistochemical images of Cre (green) and Rac1 (red) after HSV-Cre injection into the NAc of a floxed Rac1 mouse. Blue arrows highlight a Cre+ neuron in which Rac1 staining is completely absent, while white arrows indicate an adjacent non-Cre expressing neuron where Rac1 protein is strongly expressed. Consistent with previous reports of Rac1 cellular localization, in NAc neurons, the Rac1 immunostaining concentrates at soma subplasma membrane. This result is representative of the analysis of numerous sections from 4 animals. Data were analyzed by ANOVA and represented as mean ± s.e.m.

Figure 3. Temporal regulation of Rac1 signaling regulates behavioral responses to cocaine

(a) Optical stimulation of Rac1 activity (by use of Rac1-pa) increased the phosphorylation state of cofilin (p-cofilin) (F2,13=8.72, ***p<0.005; n=4 mice) while light stimulation of the light insensitive mutant did not alter p-cofilin levels (p>0.05) compared to HSV-GFP controls (n=5 mice per group). Full-length blots are presented in Supplementary Fig. 3. (b) Such optical stimulation of Rac1 during cocaine pairing [Rac1-pa (S)] also prevented the formation of a conditioned place preference (n=8 mice) (F5,43=2.66, **p<0.01). This blockade required light activation of Rac1, since expression and stimulation of the light insensitive C450A mutant (n=7 mice) or HSV-GFP (n=7 mice) led to the formation of equivalent cocaine preferences as unstimulated (US) control mice (n=8 mice). (c) Light activation of the Rac1-pa construct blocked cocaine-induced increases in locomotion (n=7–8 mice per group) (F4,45=2.43, **p<0.01), (d) but had no effect on locomotor responses to saline (n=7–8 mice per group). (e) A representative infected NAc medium spiny neuron, imaged at 40X, infected with HSV-Rac1-pa. (f) Anatomical placement of viral infection in NAc after HSV injection. (g) A representative infected NAc medium spiny neuron, imaged at 40X, infected with HSV-Rac1-pa-C450A mutant. Rectangular boxes in f and g highlight areas used for dendritic spine analysis. Cartoon shows the location of the injection site at 1.77 mm from Bregma. Data were analyzed by ANOVA and represented as mean ± s.e.m.

Figure 4. Decreased Rac1 signaling mediates induction of NAc spines by cocaine

Representative confocal images of viral-mediated dsRed expression after treatment with (a) saline or (b) cocaine; viral overexpression of Rac1-ca with (c) saline or (d) cocaine; viral overexpression of Rac1-dn with (e) saline or (f) cocaine; and viral overexpression of cofilin-ca with (g) saline or (h) cocaine. To correspond with the transient timescale of HSV expression, spine analyses were performed 4 hours after the last of 5 cocaine injections. (i) Cocaine significantly increased spine density of NAc medium spiny neurons (F1,16=6.50, p<0.001; main effect of cocaine effect by two-way ANOVA). Subsequent Tukey’s multiple comparison post-hoc test revealed that mice injected with dsRed and treated with cocaine (n=4 mice) had a significant increase in dendritic spine density compared with saline dsRed controls (n=4; **p<0.01). HSV-Rac1-ca (n=4 mice) blocked the cocaine-induced increase in spine density (#p<0.01 compared to dsRed cocaine), while HSV-Rac1-dn (n=3 mice) alone mimicked cocaine’s effects (**p<0.01) and this action was not further potentiated by cocaine (n=4 mice). There was a trend of HSV-cofilin-ca to increase spine number in the saline condition (n=5 mice), with the effect significant in the HSV-cofilin-ca cocaine group (n=5 mice; **p<0.01). (j) NeuronStudio spine type analysis of the same images (same n per group) revealed a main effect demonstrating that repeated cocaine alters the number of thin spines (F1,16=3.63, p<0.05). Further multiple comparisons post-hoc analysis revealed that, when compared to dsRed saline controls, dsRed cocaine treated mice had a significant increase in thin spines (**p<0.01) and this effect was mimicked by HSV-Rac1-dn (**p<0.01) as well as HSV-cofiliin-ca (*p<0.05), with no further effect of cocaine (***p<0.001). (k) There were no significant changes in the density of stubby spines in any of the treatment conditions. However, there was a trend for an increase in stubby spine density in the Rac1-dn saline group compared to dsRed saline. (l) Neither cocaine Rac1-dn nor cofilin-ca had any effect on mushroom spines, although in both the saline and cocaine conditions Rac1-ca overexpression reduced the density of mushroom spines (F2,20=2.83, *p<0.05). Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test and represented as mean ± s.e.m. (m) Total spine density in floxed Rac1 mice after HSV-Cre (n=3 mice) or HSV-GFP injection into NAc (n=4 mice). Data analysis by two-tailed t-test demonstrated that such local knockdown of Rac1 leads to an increase in spine density (t=3.71, df=5, *p<0.05), and (n) this increase is selective for thin spines (t=4.69, df=5, **p<0.01) with no change in the density of stubby spines or mushroom spines. Data are represented as mean ± s.e.m.