Cocaine activates Rac1 to control structural and behavioral plasticity in caudate putamen.

Neurobiology of Disease 75 (2015) 159–176

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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

Cocaine activates Rac1 to control structural and behavioral plasticity in caudate putamen Juan Li a,b, Lei Zhang b, Zhenzhong Chen a, Minjuan Xie a, Lu Huang a, Jinhua Xue a, Yutong Liu a, Nuyun Liu c, Fukun Guo d, Yi Zheng d, Jiming Kong e, Lin Zhang b,⁎, Lu Zhang a,c,e,⁎⁎ a

Key Laboratory of Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, China Elderly Health Services Research Center, Southern Medical University, Guangzhou 510515, China d Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, Cincinnati, OH, USA e Southern Medical University-University of Manitoba Geriatric Medicine Joint Laboratory, Guangzhou 510515, China b c

a r t i c l e

i n f o

Article history: Received 28 March 2014 Revised 19 November 2014 Accepted 18 December 2014 Available online 13 January 2015 Keywords: Dendritic plasticity Cocaine Behavioral plasticity Caudate putamen (CPu) Rac1

a b s t r a c t Repeated exposure to cocaine was previously found to cause sensitized behavioral responses and structural remodeling on medium spiny neurons of the nucleus accumbens (NAc) and caudate putamen (CPu). Rac1 has emerged as a key integrator of environmental cues that regulates dendritic cytoskeletons. In this study, we investigated the role of Rac1 in cocaine-induced dendritic and behavioral plasticity in the CPu. We found that Rac1 activation was reduced in the NAc but increased in the CPu following repeated cocaine treatment. Inhibition of Rac1 activity by a Rac1-specific inhibitor NSC23766, overexpression of a dominant negative mutant of Rac1 (T17N-Rac1) or local knockout of Rac1 attenuated the cocaine-induced increase in dendrites and spine density in the CPu, whereas overexpression of a constitutively active Rac1 exert the opposite effect. Moreover, NSC23766 reversed the increased number of asymmetric spine synapses in the CPu following chronic cocaine exposure. Downregulation of Rac1 activity likewise attenuates behavioral reward responses to cocaine exposure, with activation of Rac1 producing the opposite effect. Thus, Rac1 signaling is differentially regulated in the NAc and CPu after repeated cocaine treatment, and induction of Rac1 activation in the CPu is important for cocaine exposure-induced dendritic remodeling and behavioral plasticity. © 2015 Elsevier Inc. All rights reserved.

Introduction Drug addiction is a long-lasting brain disease. Long-term exposure to cocaine produces enduring neuronal alterations in intracellular signaling pathways, structural changes in dendritic morphology, and behavioral plasticity (Di Ciano and Everitt, 2004; Hyman and Malenka, 2001; Nestler, 2001; Taylor et al., 2007; Vanderschuren and Kalivas, 2000; White and Kalivas, 1998). One of the long-lasting neural adaptations observed in several animal models of addiction is an increase in dendritic spine density on dopaminoceptive medium spiny neurons (MSNs) in striatum (Li et al., 2012; Nestler, 2004; Pulipparacharuvil et al., 2008; Robinson et al., 2001; Robinson and Kolb, 1999, 2004; Zhang et al., 2012). Such neuroadaptations may contribute to the changes in synaptic plasticity underlying cocaine addiction. Although several

⁎ Correspondence to: L. Zhang, Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, China. ⁎⁎ Correspondence to: L. Zhang, Department of Pathophysiology, Elderly Health Services Research Center, Southern Medical University, Guangzhou 510515, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (L. Zhang). Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2014.12.031 0969-9961/© 2015 Elsevier Inc. All rights reserved.

groups have documented that repeated cocaine exposure increases spine density in striatum (Lee et al., 2006; Norrholm et al., 2003; Pulipparacharuvil et al., 2008; Robinson et al., 2001; Robinson and Kolb, 1999), the precise molecular mechanisms leading to these morphological changes have yet to be fully investigated. Dendritic remodeling requires architectural changes in dendrites and spines through modification of the actin cytoskeleton (Lin et al., 2005). Previous studies indicated that changes in spine morphology are controlled by modification of the actin cytoskeleton (Luo et al., 1996; Nakayama et al., 2000; Tashiro and Yuste, 2004). The Rho family of small GTPases, including Rac1, RhoA, and Cdc42, are key regulators of the actin cytoskeleton rearrangement and play important roles in dendritic morphogenesis. It is generally believed that RhoA and Rac1/ Cdc42 have antagonistic effects on dendritic spine morphology, in that Rac1/Cdc42 promotes the development of new spines, while RhoA inhibits their formation and maintenance (Nakayama et al., 2000; Tashiro and Yuste, 2004). Using Rac1 knockout mice, recent findings show that Rac1 plays critical roles in neural progenitor regulation during brain development (Chen et al., 2007, 2009). Rac1 contributes to the development and structural remodeling of dendrites and spines (Luo et al., 1996; Nakayama et al., 2000; Tashiro and Yuste, 2004).

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J. Li et al. / Neurobiology of Disease 75 (2015) 159–176

Furthermore, Rac1 signaling has been implicated in cognitive disorders (Chen et al., 2010) and the Fragile X syndrome (Bongmba et al., 2011), which are all characterized by abnormal dendritic structure. The striatum is comprised of the CPu and NAc (Berke and Hyman, 2000; Hyman, 1996; Koob et al., 1998). The NAc and CPu differ in many aspects of their anatomy and physiology, and may respond in different ways to cocaine administration. Previous results have shown that there may be different dopamine mechanism in the NAc and CPu for the mediation of stereotypy and hyperactivity (Costall et al., 1977). The CPu and NAc mediate distinct facets of the reinforcing properties of cocaine, related to its rewarding and motivational aspects (Veeneman et al., 2012). In addition, there is evidence that the CPu and NAc display different gene expression patterns after cocaine exposure (Pozzi et al., 2011; Zhang et al., 2007). On the basis of connectivity and function, the striatum is comprised of three distinct parts: the dorsolateral CPu (dlCPu), the dorsomedial CPu (dmCPu) and ventral striatum (also known as NAc) (Chen et al., 2011). The dmCPu presents high similarities with the core of NAc and could react similarly, while the dlCPu has different physiological properties and functions (Voorn et al., 2004). A recent study by Dietz et al. has documented the role of Rac1 in cocaine-induced structural and behavioral plasticity in the NAc, particularly the NAc shell (Dietz et al., 2012). However, the role of Rac1 signaling in the CPu, especially in the dlCPu, following chronic cocaine treatment remains unknown. Given that Rac1 plays a crucial role in the regulation of dendritic morphology (Etienne-Manneville and Hall, 2002; Jaffe and Hall, 2005; Mack et al., 2011; Tolias et al., 2005; Zhang et al., 2005), we hypothesized that Rac1 is involved in chronic cocaine-induced dendritic remodeling in the CPu and, thereby, contributes to cocaine-induced behavioral responses. We have used a complex method, including viral-mediated gene transfer, gene knockout mice and specific chemical inhibitors to address this issue. Here, we present evidence that Rac1 activity is differentially regulated in CPu (increase) and NAc (decrease) after repeated cocaine exposure. The change of Rac1 activity in the CPu is important for cocaine to alter the dendritic plasticity and the rewarding behavior. Our findings reveal an important role of Rac1 GTPase in regulating many aspects of cocaine-induced dendritic and behavioral plasticity in the CPu, and highlight the importance of understanding regional differences in Rac1 signaling. Materials and methods Animals The Rac1 floxed mice were generated as described previously (Cancelas et al., 2005). Seven-to-ten-week-old Kunming strain male mice (mean age was 8 weeks) were obtained from the Southern Medical University Animals Center (Guangzhou, China). Kunming strain mice were derived from Swiss mice that were from the Indian Haffkine Institute in 1944 (Shang et al., 2009). All mice had unrestricted access to food and water and were maintained in a temperature-controlled colony room on a 12:12-h light/dark cycle. All experimental procedures were in compliance with the National Institutes of Health guidelines and were approved by the local Animal Care and Use Committee of the Southern Medical University. Drugs and treatments Cocaine hydrochloride (Qinghai Pharmaceutical Factory, China) and the Rac1 inhibitor NSC23766 (Tocris, England) were dissolved in normal saline, which will hereinafter be referred to as saline (Jiao et al., 2007; Zhang et al., 2004, 2005). Saline was used as the vehicle control (dose 0). All injections were administered intraperitoneally (i.p.) in volumes of 10 ml/kg. Injections were performed during the light phase of the light/dark cycle.

Three cocaine treatment regimens were used. For 5 or 7 day regimen, mice were injected with 20 mg/kg of cocaine or saline each day for 5 or 7 days, and were sacrificed 15 min, 45 min, 2, 4, 8, or 24 h following the last injection for virus/structural plasticity analyses (5 days) or biochemistry studies (7 days). For 28 day regimen, the mice were treated with 20 mg/kg of cocaine or saline 5 days/week for 4 weeks and then analyzed 15 min, 45 min, 2, 4, 8, or 24 h following the last injection for virus/structural plasticity and biochemistry studies. The Rac1 inhibitor NSC23766 (2.5 mg/kg) was injected 30 min prior to cocaine administration. Plasmid constructions and preparation of viral stocks For lentivirus expression, cDNAs encoding the dominant-negative mutant Rac1 (T17N-Rac1) and constitutive active mutant of Rac1 (Q61L-Rac1) were ligated into the BamHI and XhoI site of the lentiviral vector plenti6/V5-topo, which expresses enhanced fluorescent protein (EGFP) bicistronically (Gao et al., 2004). Recombinant lentiviruses were produced using the ViralPower Lentiviral Expression System (Invitrogen, K4970-00). The virus was concentrated 10 to 15 times by centrifugation in a Centricon plus-20 filter (Millipore) following the manufacturer's instructions. Aliquots were stored at − 80 °C. All virus preparations were titered according to the Virapower protocol and contained 4 × 108 TU/ml. The adenovirus pAV.Des1d-Cre-EGFP and pAV.Des1d-EGFP were constructed by Cyagen Bioscience Inc. (China), the former of which expressed Cre recombinase (4 × 1010 PFU/ml) with GFP as a tag, and the latter only expressed GFP (4 × 1011 PFU/ml) as a control. Surgical procedures and injection of the lentiviral vector For virus infusion of CPu, mice were anesthetized deeply with a ketamine/dormitor cocktail (100 mg/kg/10 mg/kg, i.p.). After 10 min, the surgical area was shaved and cleaned using 75% ethanol. The animals were placed into a stereotaxic instrument. Stereotaxic coordinates for the mice were determined empirically and based on the Mouse Brain in Stereotaxic Coordinates. Coordinates of the dorsolateral CPu were 1.2 mm anterior and 1.8 mm lateral to bregma and 3.0 mm below bregma. The head was positioned in the stereotaxic frame so that the skull was leveled between lambda and bregma. Unilateral infusions (for morphology analysis) or bilateral infusions (for behavior testing) of the plenti-EGFP, plenti-EGFP-T17N-Rac1, plenti-EGFP-Q61L-Rac1 lentivirus, or pAV.Des1d-Cre-EGFP, pAV.Des1d-EGFP adenovirus were delivered over a 10-min period through a Hamilton syringe with a 33-gauge tip needle. A total of 2 μl/side lentivirus or 0.5 μl/side adenovirus was infused at a rate of 0.1 μl/min as previously reported (Chhatwal et al., 2007; Kim et al., 2011). The injection needle was kept in place for an additional 5 min following infusions to allow for diffusion of the virus and then withdrawn very slowly to prevent backflow of solution. The wound site was closed with braided silk sutures. For the lentivirus, all mice were returned to their home cages and allowed to recover for up to 14 days before receiving cocaine injections or other detection. For the adenovirus, all mice were allowed to recover for 7 days before receiving cocaine injections or other detection. Biocytin staining and GFP immunohistochemistry Twenty-four hours following the last injection of five days or twenty-eight days of cocaine treatment, the mice were deeply anesthetized with ethylether and decapitated. The appropriate part of the brain was then removed rapidly and placed into ice-cold artificial cerebrospinal fluid (containing (in mM): 223 sucrose, 25 NaHCO3, 1.2 NaH2PO4, 3.6 KCl, 2 CaCl2, 1 MgCl2, 0.4 ascorbic acid, 2 pyruvate, and 11 D-glucose) and incubated for 2 min. Four to six coronal sections (300-μm thickness) containing the CPu area were cut with a vibrating slicer (LEICA, VT1200, Germany). Then the brain slices were incubated in ACSF equilibrated


with 95%/5% O2/CO2 at room temperature for at least 1 h. The slices were transferred to a recording chamber (Nikon, FN-S2N, Nikon Corporation, Japan). The biocytin-filled electrodes were implanted into living neurons expressing EGFP. The neurons focused into the middle section of the slice were chosen to connect the biocytin-filled electrodes to insure integrity of the neuronal dendritic branching. After a minimum of 20 min in the whole-cell, tight-seal patch-clamp configuration, the biocytin-filled electrodes were withdrawn from the targeted neuron in CPu. The brain slices from the patch pipette were then fixed overnight in cold 4% paraformaldehyde, rinsed in phosphate-buffered saline (PBS), blocked in 1% bovine serum albumin (BSA) diluted in 2.5% TritonX-100 in 0.01 M PBS at RT for 4 h, and incubated with rabbit polyclonal anti-GFP antibody (abcam; 1:200) and Streptavidin, Alexa Fluor 546 Conjugate (Invitrogen; 1:200) at 4 °C for 48 h. Secondary donkey anti-rabbit Alexa 488 antibody (Invitrogen; 1:200) was applied overnight. For mice pretreated with Q61L-Rac1, pAV.Des1d-Cre-EGFP or their control virus, after five days (for adenovirus) or twenty-eight days (for lentivirus) of cocaine injection regimen, the coronal sections (40 μm) containing CPu of the mice were cut using a freezing microtome. The stained cells were observed and photographed using an LCS confocal microscope (LEICA, DMIRE2, Germany) with a ×100 objective. The dendrites or dendritic spines were expressed as mean ± SEM. Initial analyses of spines and dendrites data were performed using ANOVA with a 2 × 4 factorial design, including day (5 day treatment and 28 day treatment), treatment (plenti-EGFP + saline, plenti-T17NRac1 + saline, plenti-EGFP + cocaine, and plenti-T17N-Rac1 + cocaine) as factors, or 2 × 2 factorial design, including drugs (saline and cocaine treatment), and treatment (plenti-EGFP and plentiRac1L17 or pAV.Des1d-Cre-EGFP and pAV.Des1d-EGFP) as factors. When a significant day × treatment interaction or drug × treatment interaction was observed, simple main effects analyses were conducted separately by each factor. When no interaction effect was observed, the main effects of each factor were analyzed. p b 0.05 was considered statistically significant.

PAK-PBD binding assay and Western blotting The GTPase pull-down assay was performed according to the manufacturer's protocol (Rac/cdc42 Assay Reagent, no. 14-325, Millipore). 15 min, 45 min, 2, 4, 8, or 24 h following the injections, the mice (n = 9 mice per group) were anesthetized deeply with ethylether and decapitated. The NAc and CPu tissues were isolated by gross dissection, and extracts were prepared as described (Zhang et al., 2004). The samples were lysed with Mg2 + containing buffer (MLB; 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% TritonX-100, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin) (300 μl for CPu, 200 μl for NAc). Protein concentrations were determined by the Bradford method as before (Zhang et al., 2004). The cell lysates were divided into two parts, one part was used for detecting total Rac1, Tiam1 (Santa Cruz; 1:200; sc-872), RacGAP1 (abcam; 1:500; ab61192), p-cofilin (Cell Signaling; 1:1000; #3311), cofilin (Cell Signaling; 1:1000; #3312) and other signaling molecules. Another part was proceeded to PAK-PBD binding assay as follows: 10 μg/ml final concentration of GST-PAK-PBD agarose was added to the cell lysates, and were then incubated at 4 °C for 60 min while being constantly rotated, the bound proteins were collected by centrifugation and the pellets were washed three times in MLB and finally suspended in 2 × Laemmli sample buffer (40 μl). Proteins were subjected to SDS-PAGE (10%), transferred to PVDF membranes, and blocked with Tris-buffered saline-Tween 20 containing 5% (v/v) non-fat powdered milk for 1 h. Primary antibody against Rac1 (BD; R5622S1P) was diluted 1:1000 in blocking agent. The secondary antibody was diluted 1:1000 in TBST. The bands were visualized using the enhanced chemiluminescence (ECL)

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detection system. Loading controls were performed using antibodies against mouse β-actin (Santa Cruz; 1:500; sc-1616). Golgi-Cox impregnation and data analysis Twenty-four hours, one week or two weeks after the final injection, the brains (n = 5 mice per group) were removed and processed for Golgi-Cox impregnation. The brains were cut into 150 μm sections, and MSNs in CPu were analyzed. Dendritic morphological analysis was carried out in both hemispheres of the CPu using three methods (Li et al., 2004; Norrholm et al., 2003; Robinson and Kolb, 1997; Zhang et al., 2006). First, the dendritic complexity was calculated using a Sholl analysis of ring intersections (Robinson and Kolb, 1997; Zhang et al., 2006). Secondly, the total number of dendritic branches was counted at each branch point from the cell body. Thirdly, the spine density was quantified by counting spines on the third order (or greater) dendritic terminal tip of each MSN. The spines were counted from the last branch point to the terminal tip of the dendrite. The dendritic branches were counted on 8–12 neurons from each mouse in the CPu. The dendritic spine density was counted at 1000 × magnification (at least 20 μm in length) from different neurons in the CPu, of each mouse (Robinson and Kolb, 1997; Zhang et al., 2006). All neurons were reconstructed using Image Pro Plus version 5.1 (Media Cybernetics, Silver Spring, Md., USA), which allows for the three-dimensional analysis of dendritic trees. All measurements were made by a person blinded to the experimental conditions. The number of dendrites and spines was expressed as mean ± SEM and was compared using oneway ANOVA followed by Bonferroni post-hoc test. p b 0.05 was considered statistically significant. Electron microscopy and data analysis Twenty-four hours after the last injection, the mice (n = 4 mice per group) were anesthetized with methoxyflurane and cardiac-perfused with ice-cold saline. Mice were then perfused with freshly prepared 4% paraformaldehyde with 0.2% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. The brains were rapidly removed and stored in the same fixative containing 2.5% glutaraldehyde overnight at 4 °C. Then the coronal sections (30 μm) containing CPu were cut using a freezing microtome (Christensen, 1971; Liu and Schumann, 2014). One-square-millimeter blocks were excised from the CPu at the level of the anterior commissure. The blocks were treated with 1% osmium for 1 h on ice, counterstained with 2% aqueous uranyl acetate for 1 h, dehydrated through an ascending series of ethanols, rinsed in propylene oxide, and flat-embedded in Epon. The flat-embedded specimens were sectioned with an ultramicrotome at the same set and mounted on 150-mesh formvar-coated slot grids serially. The grids were stained with uranyl acetate followed by lead citrate and then examined with a transmission electron microscope (Hitachi H-7500, Japan). We analyzed 25–30 dissectors for each animal per treatment group. The area of dissector is 18 μm2 (4.55 μm × 3.96 μm). The synapses were identified by the presence of pre- and postsynaptic membrane specializations, a visible synaptic cleft, and the accumulation of synaptic vesicles in the presynaptic profile (Antonopoulos et al., 2002; Meng et al., 2002). The number of asymmetric synapses located within the box was counted. The results were expressed as mean ± SEM and were analyzed using one-way ANOVA followed by the Bonferroni post-hoc test. Significance was set at p b 0.05. Calculation of the numerical density of synapses per unit volume, Nv, was calculated for each animal with the formula: Nv = Q −/v (dis). In this formula, Q is the mean number of synapses counted per dissector and v (dis) the mean dissector volume. The ultrathin section thickness was determined according to Small's method of minimal folds (Small, 1968): 4–6 serial ultrathin sections were cut and collected on pioloform-coated slot grids, at least 3-folds from each of the sections used for analysis were photographed at 25,000 × magnifications.

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Section thickness (h), the height of the dissector, which is the distance (μm) between dissector planes, was estimated as half the mean width of the measured folds (Morshedi et al., 2009). Cocaine-induced locomotor sensitization Procedures for sensitization experiments were adapted from published methods (Pulipparacharuvil et al., 2008). To minimize stress, establish baseline activity, and habituate them to the novel environment, mice (n = 12 mice per group) received saline injections on days 1–3. On days 4–10, mice received cocaine injections (10 mg/kg, 20 mg/kg or 30 mg/kg). Challenge doses of cocaine (10 mg/kg, 20 mg/kg, 30 mg/kg) occurred on day 17 (1 week of withdrawal) of the experiment. Day 1 of the experiment was 21 days after lentiviral delivery or 7 days after adenoviral delivery, a time point at which high levels of expression were verified. For each day, the sum of locomotor activity at 1 h after injection is displayed (Fig. 6). Conditioned place preference Methods for conditioned place preference were adapted from published procedures (Pulipparacharuvil et al., 2008). Eighteen to 21 days for lentivirus (T17N-Rac1, Q61L-Rac1 or their control lentivirus) or seven days for adenovirus (pAV.Des1d-Cre-EGFP or pAV.Des1d-EGFP) following stereotactic delivery of virus into the CPu, mice (n = 8 mice per group) were conditioned to cocaine in a standard three-chamber conditioned place preference box (gray side, middle, and striped side). Using an unbiased 6 day paradigm, mice were pretested on day 1 to balance pre-existing side bias. On days 2 and 4, mice received cocaine injection (10 mg/kg) and were confined to the appropriate chamber. On days 3 and 5, mice received a saline injection and were confined to the opposite chamber. On the final day, mice were placed in the middle chamber 24 h after the final injection with both doors open and allowed to explore freely for 20 min. The time spent on each side was quantified. Data are expressed as time spent on the cocaine-paired side minus the time spent on the saline-paired side (CPP score). Statistics Statistical analyses were performed using SPSS 13.0 software. Behavioral analyses were performed using two-way repeated measures ANOVA or Student's t-tests as appropriate. When two-way ANOVA showed a significant group × day interaction, simple main effects analyses were conducted separately by group or day. Quantification of Western blots was performed using one-way ANOVA followed by Bonferroni post-hoc test. Data were expressed as means ± SEMs. Significance was set at p b 0.05. Results Repeated cocaine exposure induces opposite Rac1 signaling in NAc versus CPu Given that Rac1 is critical for neuronal morphogenesis and neural transmitter release (Govek et al., 2005; Le et al., 2005; Linseman et al., 2001), we first measured Rac1 activity in the NAc and CPu following cocaine exposure. We found that Rac1 activity was significantly decreased in the NAc after 7 and 28 days of repeated cocaine injections, which is consistent with the recent observation by Nestler's group (Dietz et al., 2012). Surprisingly, Rac1 activity was increased in the CPu at 15 min after the last cocaine administration in both the 7 and 28 day groups of cocaine treatment (Figs. 1A and B). This increased activation lasted for 2 h and returned to baseline 4 h after the last cocaine injection in the 7 day cocaine group (Fig. 1). The increased activation of Rac1 after 28 days of cocaine exposure lasted longer than that observed following 7 days of cocaine injections. That being said, the increased Rac1 activity

in the 28 day cocaine treatment group lasted to 4 h and returned to baseline 8 h after the last cocaine administration (Figs. 1A and B). Meanwhile, we measured the acute effects of cocaine on Rac1 activation in CPu, and found that Rac1 was activated 2 h following single cocaine (30 mg/kg) injection (Fig. 1C). These data suggest that Rac1 activity is differentially regulated in NAc and CPu following repeated cocaine treatments. Next, we attempted to identify the mechanisms by which cocaine downregulates Rac1 activity in the NAc but upregulates it in the CPu. To this end, we examined the guanine nucleotide exchange factor (GEF) Tiam1, an upstream positive regulator of Rac1, and the GTPase activating protein (GAP) RacGAP1, an upstream negative regulator of Rac1. In the NAc, 7 and 28 days of cocaine administration significantly downregulated Tiam1 expression (Figs. 2 and 3), correlating with increased RacGAP1 expression (Figs. 2 and 3), which is consistent with the findings by Dietz et al. (2012). On the contrary, 7 and 28 days of cocaine administration induced significantly increased Tiam1 expression (Figs. 2 and 3), correlating with reduced RacGAP1 expression in the CPu (Figs. 2 and 3). These data indicate that repeated cocaine exposure regulates Rac1 activity in the NAc and CPu through differentially controlling Tiam1 as well as RacGAP1 expression. Cofilin, a downstream target of Rac1 signaling, was recently reported to be activated after repeated cocaine treatment, as evidenced by the decrease in the phosphorylated form of cofilin (Dietz et al., 2012). We thus determined the levels of phosphorylated cofilin in the NAc and CPu after repeated cocaine exposures. We found that in line with the opposite activation of Rac1 activity after repeated cocaine exposure in the NAc and CPu, the amount of phosphorylated cofilin was decreased in the NAc (Figs. 2 and 3), but increased in the CPu (Figs. 2 and 3) after 7 or 28 days of cocaine treatment (Figs. 2 and 3). These results suggest that cocaine differentially regulates Rac1-cofilin signaling circuitry in the NAc and CPu. NSC23766 attenuates chronic cocaine-induced structural remodeling of dendrites and spines in CPu Next, we determined whether increased Rac1 activity is important for cocaine-induced structural and behavioral plasticity in the CPu. To this end, we injected i.p. NSC23766, a Rac1 activation-specific inhibitor (Gao et al., 2004), 30 min prior to each cocaine administration by using the 28 day regimen, and investigated the structural remodeling of dendrites and spines in the CPu at 24 h following the last injection of cocaine. As shown in Fig. 4, repeated exposure to cocaine led to an increase in dendritic branching (22.80% increase: 30.86 ± 1.240 versus 25.13 ± 0.640 branches, p = 0.001) (Figs. 4A and C) and spine density (29.30% increase: 12.40 ± 0.140 versus 9.59 ± 0.270 spines/10 μm, p b 0.001) (Figs. 4B and D) of the MSNs. Pretreatment of mice with 2.5 mg/kg of NSC23766 attenuated the cocaine-induced increase in the number of dendritic branching (14.42% fewer: 26.41 ± 0.570 versus 30.86 ± 1.240 branches, p = 0.002) (Figs. 4A and C) and spine density (13.95% fewer: 10.67 ± 0.350 versus 12.40 ± 0.140 spines/10 μm, p = 0.001) (Figs. 4B and D), respectively, when compared to the CPu cocaine group. We also determined dendritic branch numbers (dendritic complexity) by a standard Sholl analysis as previously described (Zhang et al., 2012). As shown in Fig. 4E, there was an increase in CPu dendritic complexity following repeated cocaine treatment compared to saline-treated mice, as reflected by an increased number of dendritic intersections from cocaine-treated mice. This increase was abolished by pretreatment of mice with NSC23766 (Fig. 4E). The numbers of dendrites on MSNs in the CPu were similar between the NSC23766-alonetreated mice and saline-treated mice (p N 0.05). Together, these results suggest that increased Rac1 activity is essential for cocaine-induced elevations in dendrites and spine density in the CPu. In addition, in order to explore the long-term effect of Rac1 inhibition, we investigated the dendritic changes 1 week and 2 weeks after treatment stoppage. We injected i.p. NSC23766 30 min prior to each


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Fig. 1. Regulation of Rac1 activity by cocaine in NAc and CPu. (A) Mice (n = 9 mice per group) were treated with saline or cocaine for 7 days (20 mg/kg each day) or 28 days (20 mg/kg, 5 days/week for 4 weeks), and sacrificed 15 min, 45 min, 2, 4, 8, or 24 h following the last injection. The protein extracts from the NAc and CPu of these mice were subjected to GTPase pulldown assay with GST-PBD. Rac1 proteins were detected by immunoblotting with monoclonal anti-Rac1 antibodies. (B) Data are expressed as mean ± SEM. The saline treatment group was set as 1 for quantifications. *p b 0.05, compared with saline treatment. (C) Mice (n = 9 mice per group) were treated with saline or cocaine for one injection (30 mg/kg), and sacrificed 2 h following the injection. The protein extracts from the CPu of these mice were subjected to GTPase pull-down assay with GST-PBD. Rac1 proteins were detected by immunoblotting with monoclonal anti-Rac1 antibodies. Data are expressed as mean ± SEM. The saline treatment group was set as 1 for quantifications. *p b 0.05, compared with saline treatment.

cocaine administration by using the 14 day regimen, and investigated the structural remodeling of dendrites and spines in the CPu 7 or 14 days after the last cocaine treatment. As shown in Fig. 5, repeated

exposure to cocaine led to an increase in dendritic branching (one week after withdrawal of cocaine: F(3,15) = 143.488, p b 0.001; 14 days after last cocaine treatment: F(3,15) = 77.295, p b 0.001)

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Fig. 2. Regulation of Tiam1, racGAP1 and p-cofilin by cocaine in NAc and CPu following cocaine treatment for 7 days. (A) Mice (n = 9 mice per group) were treated with saline or cocaine for 7 days (20 mg/kg each day), and sacrificed 15 min, 45 min, 2, 4, 8, or 24 h following the last injection. The protein extracts from the NAc and CPu of these mice were subjected to detect Tiam1, racGAP1, p-cofilin and cofilin. (B) Data are expressed as mean ± SEM. The saline treatment group was set as 1 for quantifications. *p b 0.05, compared with saline treatment.


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Fig. 3. Regulation of Tiam1, racGAP1 and p-cofilin by cocaine in NAc and CPu following cocaine treatment for 28 days. (A) Mice (n = 9 mice per group) were treated with saline or cocaine for 28 days (20 mg/kg, 5 days/week for 4 weeks), and sacrificed 15 min, 45 min, 2, 4, 8, or 24 h following the last injection. The protein extracts from the NAc and CPu of these mice were subjected to detect Tiam1, racGAP1, p-cofilin and cofilin. (B) Data are expressed as mean ± SEM. The saline treatment group was set as 1 for quantifications. *p b 0.05, compared with saline treatment.

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Fig. 4. Rac1 inhibition by NSC23766 attenuates cocaine-induced increase in dendritic branching and spine density in CPu. Mice (n = 9 mice per group) were treated with saline or cocaine for 28 days (20 mg/kg, 5 days/week for 4 weeks), and sacrificed 24 h following the last injection. (A) Representative dendritic branching images of MSNs from the CPu by Golgi-Cox analysis. Scale bars, 10 μm. (B) Representative dendritic spine images of MSNs from the CPu by Golgi-Cox analysis. Scale bars, 10 μm. (C and D) Quantification of dendritic branching and dendritic spine density in the indicated groups of mice. For dendritic branching, F(3,16) = 11.092. For spine density, F(3,16) = 28.24. Data are expressed as mean ± SEM. (E) The changes in dendrite complexity in the CPu revealed by Sholl analysis of the intersection number per 20 μm radial unit distance from soma of MSNs. *p b 0.05 compared with saline-treated mice, ○p b 0.05 compared with cocaine-treated mice.

(Figs. 5A and C) and spine density (7 after last cocaine treatment: F(3,15) = 14.088, p b 0.001; 14 days after last cocaine treatment: F(3,15) = 14.750, p b 0.001) (Figs. 5B and C) of the MSNs 7 or 14 days after last cocaine treatment. Pretreatment of mice with 2.5 mg/kg of NSC23766 still attenuated the cocaine-induced increase in the number of dendritic branching (p b 0.001) (Figs. 5A and C) and spine density (p b 0.001) (Figs. 5B and C) 7 or 14 days after the last cocaine treatment, respectively, when compared to the cocaine group. Meanwhile, as shown in Fig. 5D, Sholl analysis reveals an increase in CPu dendritic complexity 7 or 14 days after last cocaine treatment compared to saline-treated mice, as reflected by an increased number of dendritic intersections from cocaine-treated mice. This increase was inhibited by pretreatment of mice with NSC23766 (Fig. 4E). The numbers of dendrites on MSNs in the CPu were similar between the NSC23766-alone-treated mice and saline-treated mice (p N 0.05). Together, these results suggest that Rac1 inhibition has long-term effect on cocaine-induced elevations in dendritic remodeling in the CPu.

NSC23766 reduces the number of asymmetric spine synapses induced by chronic cocaine exposure Previous studies have demonstrated that exposure to drugs of abuse increases the number of synapses in cortex and NAc of rats (Alcantara et al., 2011; Morrow et al., 2007; Morshedi et al., 2009; Robinson and Kolb, 2004; Russo et al., 2010). In line with this, we recently reported that cocaine treatment increased the number of excitatory asymmetric synapses in the NAc and CPu (Zhang et al., 2012). As changes in synapse number require a dynamic actin cytoskeleton (Bonhoeffer and Yuste, 2002; Calabrese et al., 2006; Dillon and Goda, 2005; Toda et al., 2010) and since the Rac1-GTPase signaling pathway is one of the best characterized pathways functioning to regulate actin dynamics (Hall, 1998; Nobes and Hall, 1995; Tapon and Hall, 1997; Toda et al., 2010), here we determined whether the cocaine-induced increase in asymmetric spine synapse numbers depends on the Rac1 signaling pathway. To this end, mice were treated with NSC23766 prior to cocaine by using the 28 day regimen. Consistent with our previous observation, the


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Fig. 5. Rac1 inhibition by NSC23766 exerts long-term effects in attenuating cocaine-induced increase in dendritic branching and spine density after one or two week's withdrawal from cocaine treatment for 14 days. (A) Representative dendritic branching images of MSNs from the CPu by Golgi-Cox analysis. Scale bars, 10 μm. (B) Representative dendritic spine images of MSNs from the CPu by Golgi-Cox analysis. Scale bars, 10 μm. (C) Quantification of dendritic branching and dendritic spine density in the indicated groups of mice. (D) The changes in dendrite complexity in the CPu revealed by Sholl analysis of the intersection number per 20 μm radial unit distance from soma of MSNs. *p b 0.05 compared with saline-treated mice, ○p b 0.05 compared with cocaine-treated mice. Data are expressed as mean ± SEM.

estimated number of excitatory asymmetric synapses was significantly increased in the CPu following repeated exposure to cocaine (cocaine versus saline: 1.263 ± 0.027 versus 1.036 ± 0.022 (109 mm− 3), F(3,12) = 28.995, p b 0.001, n = 4 mice per group) (Fig. 6). As expected,

this increase was partially abolished in the CPu of NSC23766-pretreated mice (NSC23766 + cocaine versus cocaine: 1.123 ± 0.019 versus 1.263 ± 0.027 (109 mm−3), p = 0.004), whereas NSC23766 had no effect on the number of excitatory asymmetric synapses at steady-state

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(saline versus NSC23766, p N 0.05). These results indicate that the Rac1 signaling contributes to chronic cocaine exposure-induced increase in the number of asymmetric spine synapses in the CPu. Rac1 activity in the CPu regulates the number of dendrites and spine density induced by repeated cocaine treatment

Fig. 6. Rac1 inhibition by NSC23766 attenuates the increase in the number of asymmetric spine synapses induced by cocaine treatment for 28 days in CPu. (A) A pair of electron micrographs, taken from two consecutive ultrathin sections in the CPu area demonstrating the application of the dissector method for synapse counting. Profiles of asymmetric synapses (as) and mitochondria (m) are shown. The dissector is formed by a reference section (X) and a look-up section (Y). The unbiased counting frame indicated by white lines is randomly placed over the reference section micrograph. Synapses are counted with the dissector if their PSD is not seen in the look-up section, but observed in the reference section being positioned within the counting frame or intersected by its inclusion edges (broken lines) and not by the exclusion edges or their extensions (solid lines). Asterisks mark the bottoms of synapses. Analysis of the micrographs presented here shows that only three synapses among those having their PSD within the counting frame can be counted according to the dissector counting rules. × 25,000. Scale bars, 500 μm. (B) Quantification of synaptic numerical density in the indicated groups of mice. Data are expressed as mean ± SEM. p b 0.05 compared with cocaine-treated mice.

Noting that NSC23766 may not act specifically in CPu, we injected the dominant-negative Rac1 mutant T17N-Rac1, or the plenti-GFP control vector, directly into the CPu. Using two cocaine-injection protocols to induce dendrites and spines in the CPu (Dietz et al., 2012; Norrholm et al., 2003), mice were treated with either a 5 day regimen (cocaine or saline once a day for 5 days) or a 28 day regimen (cocaine or saline once a day for 4 weeks) before CPu MSN dendrites and spine density were analyzed. As T17N-Rac1 was coexpressed with GFP, GFP-positive MSNs were analyzed for the neural dendrites and dendritic spine density using serial optical sections (z stacks) gathered by laser-scanning confocal microscopy. In our previous in vitro study, the properties of lentivirus used were reported (Li et al., 2014). In this study, we used immunohistochemistry, Tunnel staining, and western-blotting techniques to further determine the expression levels, distribution and toxicity of the used virus in vivo. After 7 days (for adenoviruses) or 14 days (for lentiviruses) of recovery from virus injection, the brain slices containing virus expression region were analyzed. As shown in Supplementary Fig. 1, GFP immunostaining indicated that transgene expressions were at high efficiency in the CPu 1 week (adenovirus) or two weeks (lentivirus) after stereotaxic injection (Supplementary Fig. 1A). Meanwhile, we found the complete loss of Rac1 from Cre + MSNs in CPu of the floxed Rac1 mice transfected with Cre adenovirus (Supplementary Fig. 1B). Next, Tunnel staining was used to detect the apoptosis in dlCPu of mice injected with viruses. The regions around the injected area were captured. As shown in Supplementary Fig. 1C, D, and E, there is no significant differences between mice injected with different viruses and their control PBS groups 1 week (adenovirus) or two weeks (lentivirus) after injection. These data suggest that the viruses applied in the study do not cause toxicity in vivo. Then we analyzed the morphological changes of neurons by using our viruses' tools. As shown in Fig. 7, dendrite analysis revealed a significant main effect of days (5 and 28 days; F = 54.584, p b 0.001) as well as treatment (plenti-EGFP + saline, plenti-T17N-Rac1 + saline, plenti-EGFP + cocaine, and plenti-T17N-Rac1 + cocaine; F = 74.085, p b 0.001), while no significant interactions between day and treatment were found (days × treatment, p N 0.05). The main effect of days may be associated with mouse age, because the 5 and 28 day group mice ended up at different age, although they had the same beginning age. Both 5 and 28 days of cocaine injections induced a significant increase in CPu dendrites compared to chronically saline-treated mice infected with the control EGFP lentivirus (Figs. 7A and B). Expression of dominant negative Rac1 (T17N-Rac1) significantly attenuated the cocaineinduced increase in dendrites of the MSNs (Figs. 7A and B), whereas T17N-Rac1 did not affect basal CPu dendritic branching in salinetreated mice (Figs. 7A and B). We then carried out spine type analysis according to a published method (Lee et al., 2006). Protrusions from dendrites were classified into three types: Stubby protuberances are b0.5 μm in length, lack a large spine head, and do not appear to have a neck; mushroomshaped spines are between 0.5 and 1.25 μm long and characterized by a short neck and large spine head; thin spines, range from 1.25 to 3.0 μm and have elongated spine necks with small heads. There was a significant main effect of treatment (plenti-EGFP + saline, plentiT17N-Rac1 + saline, plenti-EGFP + cocaine, and plenti-T17N-Rac1 + cocaine; F = 54.848, p b 0.001), but no significant main effect of days (5 and 28 days; F = 0.519, p N 0.05) and interactions were found (day × treatment, p N 0.05). The 5 day cocaine injections induced a significant increase in total spine density compared to saline-treated


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Fig. 7. Rac1 inhibition by dominant negative mutant attenuates cocaine-induced increase in dendritic branching in CPu. (A) Two weeks after stereotactic delivery of control lentivirus or plenti-EGFP-T17N-Rac1 into the CPu, mice were injected with saline or cocaine for 5 days (20 mg/kg each day) or 28 days (20 mg/kg, 5 days/week for 4 weeks). Representative dendritic branching images of MSNs were visualized by GFP and biocytin immunostaining colocalization. Scale bars, 50 μm. (B) Expression of plenti-T17N-Rac1 in the CPu significantly attenuated cocaine-induced increases in dendritic branching but did not affect basal dendritic branching in saline-treated mice. Data are expressed as mean ± SEM. n = 6 mice per condition. *p b 0.001 compared with saline-treated mice, ○p b 0.001 compared with cocaine-treated mice. Coc = cocaine, Sal = saline, EGFP = plenti-EGFP, T17N-Rac1 = plenti-T17N-Rac1.

mice infected with the control EGFP lentivirus (Figs. 8A and B), and this increase was mainly driven by an increase in thin spines, not of stubby and mushroom spines. Expression of T17N-Rac1 significantly blocked the cocaine-induced increase in total spine density and thin spine density of the MSNs (Figs. 8A and B). The 28 days of cocaine injections also induced a significant increase in total spine density compared to salinetreated mice infected with the control EGFP lentivirus (Figs. 8A and B). However, unlike 5 days of cocaine injections, the 28 day cocaine injection-induced increase in spine density was driven by increases in all three types of spine, namely thin spines, stubby and mushroom spines. Expression of T17N-Rac1 significantly blocked the cocaineinduced increase in spine density of all three types of spines (Figs. 8A and B). T17N-Rac1 itself reduced basal levels of total and mushroom spine density (Figs. 8A and B), but to a lesser extent compared to its inhibitory effect on cocaine-induced increase in the

total and mushroom spine density, particularly for 28 days of cocaine injections. These findings demonstrate that upregulation of Rac1 activity in the CPu contributes to repeated cocaine-induced dendritic remodeling. Rac1 dominant-negative mutant (e.g. T17N-Rac1) works by sequestering the upstream GEF family that includes over 80 members in humans and mice, many of which are known as capable of serving multiple Rho GTPases (Zheng, 2001). Therefore, the Rac1 dominantnegative mutant could impact on the function of other Rho GTPases (Debreceni et al., 2004). To determine the specific and physiological role of Rac1 in dendritic remodeling, we carried out genetic deletion of Rac1 in the CPu of Rac1 conditional knockout mice and then performed morphological analysis. To delete Rac1, we injected the pAV.Des1d-Cre-EGFP and pAV.Des1d-EGFP adenovirus into the dorsolateral CPu of the Rac1 floxed mice. Five day cocaine treatment-

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Fig. 8. Rac1 inhibition by dominant negative mutant attenuates cocaine-induced increase in dendritic spine density in CPu. (A) Two weeks after stereotactic delivery of control lentivirus or plenti-T17N-Rac1 into the CPu, mice were injected with saline or cocaine for 5 days (20 mg/kg each day) or 28 days (20 mg/kg, 5 days/week for 4 weeks). Representative dendritic spines images of MSNs were visualized by GFP and biocytin immunostaining colocalization. Scale bars, 10 μm. (B) Expression of plenti-T17N-Rac1 in the CPu significantly blocked cocaine-induced increases in dendritic spine density, including thin, mushroom and stubby spines, and attenuated the basal dendritic spine density in saline-treated mice in the CPu. Data are expressed as mean ± SEM. n = 6 mice per condition. *p b 0.05 compared with saline-treated mice, ○p b 0.05 compared with cocaine-treated mice. Coc = cocaine, Sal = saline, EGFP = plenti-EGFP, T17N-Rac1 = plenti-T17N-Rac1.

induced changes of the dendritic spine density were then detected in Rac1-proficient and -deficient CPu. Consistent with the T17N-Rac1 results, depletion of Rac1 in CPu by expression of Cre significantly blocked the cocaine-induced increase in total spine density and thin spine density of the MSNs (Figs. 9A and C), whereas Rac1 deletion only modestly reduced basal levels of total and thin spine density (Figs. 9A and C). These findings demonstrate that upregulation of Rac1 activity is physiologically important for repeated cocaine-induced dendritic remodeling in the CPu.

To investigate if Rac1 is sufficient for cocaine-induced dendritic remodeling in the CPu, we overexpressed Rac1 by injection of a constitutively active Rac1 mutant construct, plenti-Q61L-Rac1, into the dorsolateral CPu and then detected the changes of the dendritic spine density with or without exposure to cocaine for 28 days. Our data showed that there were significant main effects of drug (saline and cocaine; F = 11.080, p b 0.01) and treatment (Q61L-Rac1 and EGFP lentivirus; F = 9.070, p b 0.01), but no interactions were found (drug × treatment, p N 0.05). Cocaine for 28 days induced increase in total, thin, and


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Fig. 9. Rac1 activity is necessary and sufficient for cocaine-induced increase in dendritic spine density in CPu. (A) One day after stereotactic delivery of pAV.Des1d-Cre-EGFP and pAV.Des1d-EGFP into the dorsolateral CPu of the floxed Rac1 mice, the mice were injected with saline or cocaine for 5 days (20 mg/kg each day). Representative dendritic spine images of MSNs were visualized by GFP immunostaining. Scale bars, 10 μm. (C) Expression of Cre in the dorsolateral CPu of the floxed Rac1 mice significantly inhibited cocaine-induced increases in total spine density and thin spine density, and attenuated the total and thin dendritic spine density in saline-treated mice in the CPu. Data are expressed as mean ± SEM. n = 6 mice per condition. *p b 0.05 compared with saline-treated mice, ○p b 0.05 compared with GFP group. Cre = pAV.Des1d-Cre-EGFP, EGFP = pAV.Des1d-EGFP. (B) Two weeks after stereotactic delivery of control lentivirus or plenti-Q61L-Rac1 into the CPu, mice were injected with saline or cocaine for 28 days (20 mg/kg, 5 days/week for 4 weeks). Representative dendritic spines images of MSNs were visualized by GFP immunostaining. Scale bars, 10 μm. (D) Expression of plenti-Q61L-Rac1 in the CPu significantly promoted cocaine-induced increases in dendritic spine density, including thin and mushroom spines, and increased the basal dendritic spine density in saline-treated mice in the CPu. Data are expressed as mean ± SEM. n = 6 mice per condition. *p b 0.05 compared with saline-treated mice, ○p b 0.05 compared with GFP group. EGFP = plenti-EGFP, Q61L-Rac1 = plenti-Q61L-Rac1.

mushroom spine density that was further potentiated by overexpression of the constitutively active Rac1 mutant (Figs. 9B and D). However, overexpression of the constitutively active Rac1 mutant also caused a significant increase in basal levels of total, thin, and mushroom spine densities in the CPu without cocaine treatment and the extent of this increase was comparable to that induced by cocaine, particularly for thin spine density (Figs. 9B and D). Together, these data suggest that the increased Rac1 activity is sufficient for cocaine-induced dendritic remodeling. Rac1 activity in the CPu modulates behavioral responses to cocaine Given that regulation of Rac1 activity by cocaine has a potent effect on CPu neuronal morphological plasticity, we sought to examine what outcome this might have on mice's behavioral responses to cocaine. To this end, we bilaterally injected the plenti-T17N-Rac1, plenti-Q61LRac1 Lentivirus into the dorsolateral CPu of mice or pAV.Des1d-CreEGFP adenovirus into the dorsolateral CPu of the Rac1 floxed mice, and checked the cocaine reward response by testing conditioned place preference. Lentivirus or adenovirus expressing GFP was used as

control. Plenti-T17N-Rac1 in the CPu reduced the rewarding effects of cocaine. That is, mice expressing plenti-T17N-Rac1 in their CPu spent significantly less time in a cocaine-paired environment compared to its control group (t = 2.530, *p = 0.019, n = 12 mice per group, Student's t test) (Fig. 10A). Consistent with the effect of T17N-Rac1, deletion of Rac1 by expression of Cre adenovirus showed a significant decrease in cocaine preference compared to its control group (t = 2.344, *p = 0.031, n = 10 mice per group, Student's t test) (Fig. 10B). Conversely, plenti-Q61L-Rac1 expression in the CPu induced the rewarding effects of low dosage (5 mg/kg) cocaine treatment (t = 2.677, *p = 0.020, n = 12 mice per group, Student's t test) (Fig. 10C). These data indicate that Rac1 activation is necessary and sufficient for cocaine-induced reward learning. To further examine the role of Rac1 in mice's behavioral responses to cocaine, we carried out locomotor sensitization tests. We found that there was no significant interaction between the effects of group (plenti-EGFP pretreated group and plenti-T17N-Rac1 pretreated group) and day on locomotor activity (10 mg/kg cocaine: F = 0.375, p = 0.694; 20 mg/kg cocaine: F = 0.432, p N 0.05; 30 mg/kg cocaine: F = 0.377, p = 0.892). Although the main effect of treatment (saline

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Fig. 10. Rac1 activation is necessary and sufficient for cocaine-induced behavioral responses to cocaine. (A) Mice expressing plenti-T17N-Rac1 in the CPu spend less time in a cocainepaired (10 mg/kg) environment as measured by conditioned place preference. EGFP = plenti-EGFP, T17N-Rac1 = plenti-T17N-Rac1. (B) Mice expressing Cre virus in the dorsolateral CPu of the floxed Rac1 mice spend less time in a cocaine-paired (10 mg/kg) environment as measured by conditioned place preference. (C) Mice expressing plenti-Q61L-Rac1 in the CPu spend more time in a cocaine-paired (5 mg/kg) environment as measured by conditioned place preference. EGFP = plenti-EGFP, Q61L-Rac1 = plenti-Q61L-Rac1. (D, E, F) Expression of plenti-T17N-Rac1 in the CPu has no effect on the sensitivity to repeated cocaine administration at 10 mg/kg (D), 20 mg/kg (E), and 30 mg/kg (F) dosage (n = 8 mice per group, two-way repeated ANOVA). (G) Mice expressing Cre virus in the dorsolateral CPu of the floxed Rac1 mice have no effect on the sensitivity to repeated 20 mg/kg cocaine administration. (H, I, J) Mice expressing plenti-EGFP-T17N-Rac1 in their CPu have no effect on the sensitivity to a challenge dose of cocaine [10 mg/kg (H), 20 mg/kg (I), 30 mg/kg (J)] after 1 week of withdrawal. (K) Mice expressing Cre virus in the dorsolateral CPu of the floxed Rac1 mice have no effect on the sensitivity to a challenge dose of cocaine (20 mg/kg) after 1 week of withdrawal.

and cocaine) was significant (10 mg/kg cocaine: F = 5.628, p = 0.008; 20 mg/kg cocaine: F = 30.00, p b 0.001; 30 mg/kg cocaine: F = 6.632, p b 0.001), there was no significant difference in the main effect of group (plenti-EGFP pretreated group and plenti-T17N-Rac1 pretreated group) (all ps N 0.05). In other words, mice expressing plenti-EGFP and plenti-T17N-Rac1 in the CPu exhibited similar increase in their locomotor activity after cocaine treatment (Figs. 10D, E, and F). Meanwhile, floxed-rac1 mice expressing GFP and Cre also exhibited similar increase in their locomotor activity after cocaine treatment (Fig. 10G). In addition, after one week of withdrawal of cocaine, the plenti-EGFP and plenti-T17N-Rac1-expressing mice or GFP and Cre-expressing floxed rac1 mice remained similar sensitivity to a cocaine challenge dose (Fig. 10E).

Finally, we determined whether the effects of rac1 on spines after cocaine treatment are mediated by cofilin. To this end, we investigated the effect of manipulation of Rac1 activity on cofilin phosphorylation after cocaine treatment. As shown in Supplementary Fig. 2, T17N-Rac1 inhibited the basal Rac1-GTPase activity, as well as the Rac1-GTPase activity induced by repeated cocaine. On the other hand, Q61L-Rac1 had opposite effect on basal and cocaine-induced Rac1-GTPase activity. As a result, T17N-Rac1 was able to inhibit the basal and cocaine-induced cofilin phosphorylation levels, and Q61L-Rac1 exerted opposite effects. Although at this point we don't have a definitive answer for whether cofilin is required for Rac1 effects on spines, the changes in cofilin activity upon Rac1 inhibition by T17N-Rac1 or Rac1 activation by Rac1L61 are well correlated with the changes in dendritic branching and


behavioral responses induced by the same perturbation of Rac1 activity, suggesting that Rac1 might exert its effect on spines by cofilin after cocaine treatment. Discussion It has been hypothesized that dendritic remodeling contributes to the long-lasting behavioral sensitization following chronic cocaine administration (Ridley and Hall, 1992; Robinson and Kolb, 1999). Likewise, it has recently become clear that the structural plasticity of dendritic spines is associated with synaptic plasticity (Kopec et al., 2006; Xie et al., 2007). Previous studies have demonstrated that neuronal plasticity requires proteins that are involved in cytoskeleton remodeling (Hall, 1994; Lai et al., 2012; Ng et al., 2002; Rex et al., 2007; Sebeo et al., 2009). Rac1, one of Rho GTPases, plays an important role in activitydependent spine enlargement during synapse maturation (Tashiro et al., 2000; Tashiro and Yuste, 2004; Wiens et al., 2005). In the current study, we show that cocaine promotes Rac1 activation in the CPu. Importantly, Rac1 activation by cocaine is required for cocaine-induced structural and behavioral plasticity in the CPu. Interestingly, we found that Rac1 activity is differentially regulated in the CPu (increase) and NAc (decrease) after repeated cocaine exposure. Our observation on the inhibition of Rac1 activity in NAc by cocaine is consistent with that reported by Dietz et al. (2012). They show that Rac1 inhibition by cocaine is essential for cocaine-induced structural and behavioral plasticity in the NAc, while we show that Rac1 activation by cocaine is important for cocaine-induced structural and behavioral plasticity in the CPu. These seemingly contradictory findings underscore a complex and site-specific role of Rac1 in the NAc and CPu in cocaine-seeking behavior, highlighting the importance of understanding regional differences in Rac1 signaling. The differential effects of cocaine on Rac1 activity in CPu and NAc and the opposing effects of Rac1 on cocaine-induced structural and behavioral plasticity in the CPu and NAc could be attributable to two folds. First, although the NAc and CPu are structures in continuity, they have different afferent inputs and efferent projections (Kourrich and Thomas, 2009). As such, the NAc is influenced by both dopamine (DA) release from the ventral tegmentum and glutamatergic afferents from the PFC, amygdala, and hippocampus (Kauer and Malenka, 2007), whereas the CPu mainly receives innervation by dopamine (DA) afferents and important glutamate innervations from motor cortex and thalamus (Belin and Everitt, 2008; Fallon and Moore, 1978). Along this line, growing evidence indicates regional specificity of drug actions on the striatum. In this aspect, the dlCPu plays key role in the development of habitual drug use, while the NAc appears to have important roles in environmental control of alcohol drinking and relapse (Chen et al., 2011). Furthermore, the NAc and CPu are thought to serve different functions in drug reward behavior (Kourrich and Thomas, 2009). It is thus not surprising that the CPu and NAc may respond in different ways to cocaine administration (Costall et al., 1977; Veeneman et al., 2012). This is supported by the report that cocaine can produce larger increases in extracellular DA concentrations in the ventral as compared with dorsal striatum of rodents (Carboni et al., 1989; Cass et al., 1992; Wu et al., 2002), monkeys (Bradberry, 2000; Bradberry et al., 2000), or humans (Drevets et al., 2001; Martinez et al., 2003). And CPu and NAc mediate distinct facets of the rewarding effects of cocaine (Veeneman et al., 2012). Thus, it is plausible that Rac1 is differentially regulated in NAc and CPu by cocaine treatment, through different cascading serial circuits, and that the differentially regulated Rac1 activity regulates different aspects of dendrite structural plasticity and of cocaine rewarding responses in CPu compared to NAc. Our findings are echoed by a previous report that while decreased Rac1 activity suppresses Collapsin-1induced growth cone collapse, increased Rac1 activity suppresses Myelin-induced growth cone collapse, in a neuronal culture system (Kuhn et al., 1999). Together, these data suggest the complexity of Rac1 signaling in neuronal cell biology.

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Second, the striatum is mainly composed of two subpopulations of MSNs: direct-pathway MSNs expressing the D1 receptor (Drd1) and indirect-pathway MSNs expressing the D2 receptor (Drd2) (Civelli et al., 1993; Lee et al., 2006; Lu et al., 1998; Russo et al., 2009; Thompson et al., 2010; Zhang et al., 2004). These two populations of neurons are thought to participate in distinct circuits with opposing functional properties. Recent evidences suggest that Drd1 and Drd2 MSNs exert a cell-type specific regulation of gene function and behavioral responses after cocaine exposure (Bertran-Gonzalez et al., 2008; Maze et al., 2014). In support, TrkB deletion leads to opposite behavioral effects on cocaine reward in Drd1 and Drd2 MSNs (Lobo et al., 2010). Interestingly, it was reported that the percentage of Drd1 MSNs for ERK signaling differed from CPu to NAc after cocaine treatment. Together, these data indicate that the direct and indirect pathway in the ventral and dorsal striatal regions may be out-of-step in cocaine addiction. Therefore, the Rac1 activation in Cpu and inhibition in NAc after cocaine treatment may occur in different populations of MSNs (e.g. Drd2 in Cpu but Drd1 in NAc), reminiscent of the variable expression of BDNF in cortical and sub-cortical structures on cocaine-seeking (McGinty et al., 2010). Under normal circumstances, spine shapes, including thin, mushroom and stubby, differ categorically. Spines tend to stabilize over time and by maturation. The thin spines have been shown to be highly mobile and plastic, whereas mushroom spines remain stable (Dumitriu et al., 2012; Kasai et al., 2010; Shen et al., 2009). Here, we demonstrate that the 5 day cocaine injection mode mainly induces the thin spine formation in the CPu, while the 28 day cocaine injection mode increases all forms of spine formation. The different outcome following 28 days of cocaine injection might result from the greater total cocaine intake as compared to only 5 days of cocaine injection. Importantly, we have found that inhibition of Rac1 activity inhibits the cocaine-induced increase of spine formation at both the 5 and 28 day modes of cocaine injection. Thus, Rac1 is involved in cocaine-induced dendritic remodeling in the CPu. In the striatum, synapses to dendritic spines are primarily asymmetric synapses formed by excitatory afferents from the cortex and thalamus (Ingham et al., 1998). A recent study has demonstrated that prenatal exposure to cocaine is associated with increased numbers of asymmetric spine synapses (Morrow et al., 2007). In most cases, the changes in dendritic structures assessed by Golgi staining are accompanied by changes in the number of synapses analyzed with electron microscopy (Kolb and Whishaw, 1998). We have recently found that chronic cocaine exposure leads to increased asymmetric spine synapses in the NAc and CPu (Zhang et al., 2012). In the present study, we show that the cocaine-induced increase in the number of asymmetric synapses is associated with increased Rac1 activation, thus indicating an important role of Rac1 in the regulation of the number of asymmetric spine synapses in the CPu. Rac1 is critical for long-term plasticity. Altered Rac1 impairs LTP and LTD induction (Bongmba et al., 2011). The MSNs in the CPu may therefore rely on Rac1-dependent signaling pathways to control homeostatic synaptic plasticity after repeated cocaine exposure. Interestingly, several recent studies show that exposure to cocaine generates silent synapses in striatum (Brown et al., 2011; Huang et al., 2009). Thus, the increased asymmetric spine synapses observed here might represent an increased pool of silent synapses. Nonetheless, the role of Rac1 signaling on silent synapses formation needs further investigation. In animal models of drug addiction, changes in locomotor sensitization and conditioned place preference have been shown to be related to structural remodeling and have long been used to evaluate the effect of factors that contribute to addiction (Benavides et al., 2007; Kiraly et al., 2010; Norrholm et al., 2003; Pulipparacharuvil et al., 2008; Taylor et al., 2007; Zhang et al., 2006). It has been hypothesized that cocaine induction of dendritic branching and spine formation in the MSNs of NAc mediate behavioral responses to the drug after repeated exposure (Robinson and Kolb, 1997, 2004; Russo et al., 2009). Specifically, in c-

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Fos-deficient mice, decreased behavioral sensitization and increased conditioned place preference to cocaine are accompanied by decreased spine density (Zhang et al., 2006). Additionally, in Kalirin7 knock-out mice, decreased spine density is accompanied by increased behavioral sensitization and decreased conditioned place preference to cocaine (Kiraly et al., 2010). In myocyte enhancer factor 2 (MEF2) overexpressing mice, both behavioral sensitization and conditioned place preference to cocaine were increased, accompanied by decreased spine density (Pulipparacharuvil et al., 2008). Several groups have reported that the CPu is heavily involved in habitual behaviors, while motivated behaviors are attributable to the NAc (Bachtell et al., 2005; Bari and Pierce, 2005; Everitt and Robbins, 2005; Packard and Knowlton, 2002; Pierce and Vanderschuren, 2010; Suto et al., 2009; Yin and Knowlton, 2006). However, accumulating evidences have shown that CPu is also involved in brain motivation circuits that contribute to the compulsive pursuit of cocaine reinforcement (Liu et al., 2013; Minogianis et al., 2013; Veeneman et al., 2012), while NAc is a necessary contributor to cocaine-mediated behavioral sensitization (Crombag et al., 2002; Girault et al., 2007; Valjent et al., 2005). In the current study, we found that inhibition of Rac1 in the CPu has different effects on locomotor sensitization and conditioned place preference after cocaine treatment. For locomotor sensitization, our results show that inhibition of Rac1 in the CPu has no obvious effect on cocaine-induced behavioral sensitization. In contrast, inhibition of Rac1 inhibits the rewarding effects of cocaine as measured by conditioned place preference. This suggests that activation of Rac1 in the CPu, a condition that induces cocaine-induced dendritic remodeling, is responsible for the rewarding effects of cocaine, providing a novel mechanistic insights into the regulation of motivated behaviors in the CPu. In addition, the lack of effect of Rac1 inhibition on cocaine-induced behavioral sensitization suggests that cocaine induces behavioral sensitization independent of Rac1 activity in the CPu.

Conclusion The present study helps to shed light on the role of Rac1 in dendritic remodeling of MSNs in the CPu and behavioral plasticity following chronic cocaine exposure. Cocaine upregulates Rac1 activity in the CPu, which is important for controlling the dendritic and behavioral plasticity processes after repeated cocaine treatment. Rac1 signaling may, therefore, play a critical role in mediating cocaine-induced neuroadaptation in the CPu. These data provide further insights into the signaling pathways that control the cocaine-induced structural and behavioral plasticity that have been implicated in the persistence of drug addiction. On the basis of previous report by Dietz et al. that cocaine treatment reduces Rac1 activity, leading to drug addiction in NAc (Dietz et al., 2012), restoring Rac1 activity seems to be beneficial in treating cocaine addiction. However, our study cautions this potential strategy because it may aggravate cocaine addiction in CPu. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.nbd.2014.12.031. Author contributions Lu Zhang and Lin Zhang were responsible for the overall study design and wrote the paper with the help of other authors. Juan Li, Lei Zhang, Zhenzhong Chen, Minjuan Xie, Lu Huang, Jinhua Xue, Yutong Liu and Nuyun Liu conducted the experiments. Fukun Guo, and Jiming Kong provided help in writing the manuscript. Yi Zheng provided the Rac1 mutant and the Rac1 floxed mice. Conflict of interest The authors declare no conflict of interest.

Acknowledgments This work is supported by National Natural Science Foundation of China (81371509, 81071120, 81371719, 81472166 and 81430045), Foundation for High-level Talents in Higher Education of Guangdong (C1031118, C2050205), the Key Project of the Chinese Ministry of Education (211132), Major Breakthroughs in Key Areas and Projects of Guangdong and Hongkong (2011A011304001), Science and Technology Program of Guangzhou (12C32121608, 201300000183), Major cooperation project of the Science and Technology of Guangdong Province (2011A090100025) and Research Fund for the Doctoral Program of Higher Education of China (201334433110017), and the Key Program of the Natural Science Foundation of Guangdong Province (92510515000008). References Alcantara, A.A., et al., 2011. Cocaine- and morphine-induced synaptic plasticity in the nucleus accumbens. Synapse 65, 309–320. Antonopoulos, J., et al., 2002. Postnatal development of the dopaminergic system of the striatum in the rat. 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