WAVE1 in Neurons Expressing the D1 Dopamine Receptor Regulates Cellular and Behavioral Actions of Cocaine

WAVE1 in neurons expressing the D1 dopamine receptor regulates cellular and behavioral actions of cocaine

Ilaria Cegliaa,1, Ko-Woon Leea,1, Michael E. Cahillb,1, Steven M. Gravesc, David Dietzd, Dalton J. Surmeierc, Eric J. Nestlerb, Angus C. Nairna,e, Paul Greengarda,2, and Yong Kima,2

aLaboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10065; bFishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; cDepartment of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; dDepartment of Pharmacology and Toxicology, Research Institute on Addictions, Program in Neuroscience, State University of New York at Buffalo, Buffalo, NY 14214; and eDepartment of Psychiatry, Yale University School of Medicine, New Haven, CT 06508

Contributed by Paul Greengard, December 23, 2016 (sent for review October 26, 2016; reviewed by Huda Akil and Solomon H. Snyder) Wiskott-Aldrich syndrome protein (WASP) family verprolin homolo- upon stimulation of D1 dopamine and of NMDA glutamate re- + gous protein 1 (WAVE1) regulates actin-related protein 2/3 (Arp2/3) ceptors, acting through cAMP- and Ca2 -mediated activation of complex-mediated actin polymerization. Our previous studies have protein phosphatase 2A and 2B, respectively (4, 7). These results found WAVE1 to be inhibited by Cdk5-mediated phosphorylation indicate that WAVE1 is a neuronal activity-regulated phospho- in brain and to play a role in the regulation of dendritic spine mor- protein in the striatum. phology. Here we report that mice in which WAVE1 was knocked out We previously identified a pivotal role for Cdk5/p35 in cocaine- (KO) in neurons expressing the D1 dopamine receptor (D1-KO), but induced adaptive changes in brain (8). Cocaine is a highly addictive not mice where WAVE1 was knocked out in neurons expressing the psychostimulant that exerts its effects on brain reward regions such D2 dopamine receptor (D2-KO), exhibited a significant decrease in as the nucleus accumbens (NAc), a ventral part of the striatum (9– place preference associated with cocaine. In contrast to wild-type 11). The NAc receives dopaminergic input from the ventral teg- (WT) and WAVE1 D2-KO mice, cocaine-induced sensitized locomotor mental area (VTA) and glutamatergic inputs from the medial behavior was not maintained in WAVE1 D1-KO mice. After chronic prefrontal cortex (mPFC), hippocampus, and amygdala, among cocaine administration and following withdrawal, an acute cocaine other regions (12). Cocaine elevates synaptic levels of dopamine by challenge induced WAVE1 activation in striatum, which was assessed blocking reuptake of dopamine into presynaptic terminals (13, 14). by dephosphorylation. The cocaine-induced WAVE1 dephosphoryla- However, chronic exposure to cocaine dampens dopamine signaling tion was attenuated by coadministration of either a D1 dopamine through increases in ΔFosB and Cdk5/p35 (8). Cdk5/p35 phos- receptor or NMDA glutamate receptor antagonist. Upon an acute phorylates Thr75 of DARPP-32, a master regulator of dopamine challenge of cocaine following chronic cocaine exposure and with- signaling in striatal medium spiny projection neurons (MSNs), and drawal, we also observed in WT, but not in WAVE1 D1-KO mice, a thereby negatively regulates DARPP-32 function (15). Chronic decrease in dendritic spine density and a decrease in the frequency of exposure to cocaine also changes glutamatergic synapses in the excitatory postsynaptic AMPA receptor currents in medium spiny projection neurons expressing the D1 dopamine receptor (D1-MSNs) Significance in the nucleus accumbens. These results suggest that WAVE1 is involved selectively in D1-MSNs in cocaine-evoked neuronal activity- Cocaine-induced synaptic plasticity in the nucleus accumbens is mediated feedback regulation of glutamatergic synapses. implicated in neural adaptations that underlie addiction. Here we demonstrate that Wiskott-Aldrich syndrome protein (WASP) WAVE1 | cocaine | striatum | medium spiny projection neurons | Cdk5 family verprolin homologous protein 1 (WAVE1) plays a selective role in medium spiny projection neurons that express D1 dopa- iskott-Aldrich syndrome protein (WASP) family verprolin mine receptors (D1-MSNs) in the cellular and behavioral actions Whomologous protein (WAVE) initiates de novo actin poly- of cocaine. Our results suggest that chronic exposure to cocaine, merization through its ability to activate the actin-related protein 2/3 followed by withdrawal, potentiates the signaling capacity of (Arp2/3) complex (1). Three members of the family (WAVE1–3) D1 dopamine and NMDA glutamate receptors, allowing WAVE1 exist, with WAVE1 being highly expressed in brain (2). WAVE1 activation in response to an acute cocaine challenge. WAVE1 was purified from bovine or rat brain as part of a heteropentameric activation is associated with morphological and functional de- protein complex together with PIR121 [also called cytoplasmic creases in glutamatergic synapses on D1-MSNs. Thus, we pro- FMR1-interacting protein 2, (Cyfip2)], Nck-associated protein 1 pose that WAVE1 is involved in a negative feedback mechanism (Nckap1), Abl-interactor 2 (Abi2), and HSPC300 (3, 4). Whereas of regulation of glutamatergic synapses as a part of the process WAVE1 plays a key role in Arp2/3 complex binding and actin of cocaine-induced synaptic plasticity. polymerization, other protein components in the complex are important for its subcellular localization and interaction with upstream Author contributions: I.C., K.-W.L., M.E.C., S.M.G., D.D., D.J.S., E.J.N., A.C.N., P.G., and Y.K. ligands or activators (5, 6). designed research; I.C., K.-W.L., M.E.C., S.M.G., D.D., and Y.K. performed research; Y.K. contributed new reagents/analytic tools; I.C., K.-W.L., M.E.C., S.M.G., and Y.K. analyzed In our initial study in striatum, the WAVE1 complex was found

data; and I.C., M.E.C., S.M.G., D.J.S., E.J.N., A.C.N., P.G., and Y.K. wrote the paper. NEUROSCIENCE to interact with p35, the regulatory subunit of cyclin-dependent Reviewers: H.A., University of Michigan; and S.H.S., Johns Hopkins University School kinase 5 (Cdk5) (4). Phosphorylation of WAVE1 at Ser310, Ser397, of Medicine. and Ser441 by Cdk5/p35, in the complex purified from rat brain, or The authors declare no conflict of interest. with recombinant WAVE1, resulted in inhibition of actin poly- Freely available online through the PNAS open access option. merization in vitro (4). We also observed that the stoichiometry of 1I.C., K.-W.L., and M.E.C. contributed equally to this work. WAVE1 phosphorylation by Cdk5 is high in brain, suggestive of the 2To whom correspondence may be addressed. Email: [email protected] or protein being largely inactive under basal conditions (4). However, [email protected]. studies using mouse striatal slices indicated that WAVE1 can be This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dephosphorylated at all three Cdk5 sites and thereby activated 1073/pnas.1621185114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1621185114 PNAS | February 7, 2017 | vol. 114 | no. 6 | 1395–1400 Downloaded by guest on September 25, 2021 NAc, including increasing dendritic spine density (9, 10, 16, 17). We showed that a Cdk5 inhibitor attenuated baseline dendritic spine density as well as cocaine-induced dendritic spine outgrowth in NAc core and shell, suggesting an involvement of Cdk5 in the regulation of dendritic spine density (18). WAVE1 is also known to regulate dendritic spine morphology (4). In the present study, we investigated the function of WAVE1 in the cellular and behavioral responses to cocaine. We found a critical role of WAVE1 expressed in a subpopulation of MSNs in cocaine-induced feedback regulation of dendritic spine density, maintenance of cocaine-induced locomotor sensitization, and cocaine reward. Results Expression of WAVE1 in D1- and D2-MSNs and Generation of Cell- Type–Specific WAVE1 KO Mice. The WAVE1 complex is highly expressed in the forebrain, including cerebral cortex, hippocampus, striatum, and thalamus, and this expression pattern is well correlated with that of p35 (Fig. S1A) (2). MSNs, the major neuronal type in the Fig. 1. Cocaine-induced CPP and locomotor sensitization in WAVE1 D1- or D2- KO mice. (A and B) Cocaine-associated place preference in D1-KO mice (A)or striatum, are largely segregated into two distinct subpopulations D2-KO mice (B) and WT mice was assessed using a “regular” CPP test with co- expressing high levels of either dopamine D1 receptor (D1) or D2 caine (7.5 mg/kg, i.p.): WT versus D1-KO mice [t = 2.663, *P < 0.05, n = 17 (WT), – 31 receptor (D2) (19 21). We used bacterial artificial chromosome 16 (D1-KO)] and WT versus D2-KO mice [n = 10 (WT), 10 (D2-KO)]. (C and D) (BAC) transgenic mice expressing EGFP under the control of D1 “Sensitization” CPP was assessed in D1-KO (C)orD2-KO(D)andWTmicein (Drd1) or D2 (Drd2) promoters and examined the expression of which cocaine was administered (15 mg/kg) for 7 consecutive days followed by a WAVE1 and EGFP in D1- and D2-MSNs. Similar levels of WAVE1 5-d withdrawal (WD). Preference for a low dose of cocaine (5 mg/kg) was then = < = immunostaining were observed in both D1- and D2-MSNs (Fig. S1 B assessed: WT versus D1-KO mice [t15 2.271, *P 0.05, n 7 (WT), 10 (D1-KO)] and C). Anatomically, D1-MSNs and D2-MSNs in dorsal striatum and WT versus D2-KO mice [n = 8 (WT), 8 (D2-KO)]. (E and F)WTmiceand project to distinct brain areas (22) but projections of D1-MSNs and WAVE1 D1-KO mice (E)orD2-KOmice(F) were treated daily with saline for 2 d. Cocaine (10 mg/kg, i.p.) was administered for 10 consecutive days. After chronic D2-MSNs from the NAc are more overlapping and remain to be cocaine administration, WT and D1-KO mice were divided into two groups (n = further defined (11, 23). Importantly, stimulation of MSNs by do- 8 per group) and subjected to 2-wk or 3-wk WD (E), whereas WT and D2-KO pamine results in opposite regulation of cAMP and PKA via stimu- mice were subjected to only a 2-wk WD (n = 11 per group) (F). Acute cocaine latory G protein (Gs/Golf)-mediated signaling in D1-MSNs compared challenge (10 mg/kg) was administered following the 2-wk (day 25) or 3-wk WD – with inhibitory G protein (Gi/Go)-mediated signaling in D2-MSNs (day 32). Locomotion was measured daily for 1 h after saline, cocaine (days 1 5), (24). Furthermore, molecular manipulations of D1-MSNs or D2- or acute cocaine challenge. Data represent means ± SEM. WT versus D1-KO MSNs results in distinct behavioral effects of drugs of abuse (24). mice were as follows: saline (days 1–2) and chronic cocaine (days 1–5) (n = 16 per = < = Thus, we decided to generate cell-type–selective KO lines to more group), cocaine challenge day 25 (t14 2.331, *P 0.05, n 8 per group) and = < = specifically investigate WAVE1 function. cocaine challenge day 32 (t14 4.745, **P 0.01, n 8 per group). WT versus D2-KOmicewereasfollows:salineday1(t20 = 3.619, **P < 0.01, n = 11 per Previously we generated mice in which exon 4 of the WAVE1 group) and saline day 2 (t = 3.611, **P < 0.01, n = 11 per group). t test was used. gene was flanked by loxP sites (25). We bred floxed WAVE1 with 20 either Drd1 or Drd2 promoter-driven Cre-recombinase lines (26) to delete WAVE1 in neurons expressing D1 (D1-KO) or D2 (D2- cocaine (Fig. 1 E and F). However, after 2 or 3 wk of drug with- KO) receptors (Fig. S2A). WAVE1 protein in striatal lysates was drawal, D1-KO mice showed a lower response to subsequent acute reduced in both D1- and D2-KO mice, with a greater reduction cocaine administration (Fig. 1E). D2-KO mice responded the same ∼ ∼ being found in D1-KO mice ( 40% versus 17% of reduction) as WT mice to a cocaine challenge following withdrawal (Fig. 1F). B (Fig. S2 ). Previous studies of Drd1-EGFP or Drd2-EGFP mice These results suggest further that WAVE1 in neurons expressing the have indicated a comparable number of D1-MSNs and D2-MSNs + D1 receptor is required for the enduring effects of cocaine as mea- (∼50% of neurons as Drd1-EGFP versus ∼40% of neurons as + sured by CPP and locomotion sensitization tests. Drd2-EGFP ) (27). The reason for the greater reduction in total striatal WAVE1 in the D1-KO mice is therefore unclear, but could Regulation of WAVE1 Phosphorylation by Cocaine. Previously, our reflect higher expression of WAVE1 in D1-MSNs compared with studies showed that stimulation of striatal slices with a D1 agonist – that in D2-MSNs. We also used a Cre-recombinase dependent or NMDA led to dephosphorylation of WAVE1 at Ser310, Ser397, tdTomato reporter line and confirmed that WAVE1 deletion was and Ser441, indicating neuronal activity-mediated stimulation of selectively dependent on Drd1-promoter–driven Cre-recombinase C WAVE1 (4, 7). We anticipated that WAVE1 would be dephos- activity (Fig. S2 ). phorylated in response to cocaine administration in vivo, because cocaine increases synaptic levels of dopamine, and activation of D1 Role for WAVE1 in D1-MSNs in Cocaine-Induced Behavior. We used – noncontingent cocaine administration (cocaine administration by is known to increase NMDA-receptor mediated signaling (13, 28, experimenter) and assessed cocaine reward in a conditioned place 29). However, there was no significant change in phosphorylation, preference (CPP) test. WAVE1 D1-KO mice showed a signifi- or of the total protein level, of WAVE1 in the striatum in response cantly reduced CPP score in both a standard CPP test (Fig. 1A)and to acute injection of cocaine (45 min after 15 mg/kg) or chronic asensitizationCPPtest(Fig.1C) compared with WT mice. In injection of cocaine (15 mg/kg daily for 14 d; 45 min after the last contrast, WAVE1 D2-KO mice did not show any significant dif- injection). Instead, we found that WAVE1 was significantly ference compared with WT mice in either of the CPP tests (Fig. 1 dephosphorylated at all three sites in response to an acute chal- B and D), suggesting a selective role for WAVE1 in D1-neurons in lenge with cocaine following chronic cocaine administration and a cocaine reward. subsequent 14-d withdrawal period (Fig. 2). We did not see any We next investigated the effect of cocaine on locomotor sensi- significant change in total protein levels of WAVE1, p35, or Cdk5 tization using WAVE1 D1-KO or D2-KO mice and their corre- (Fig. S3 A–C). Coinjection of a D1 antagonist (SCH23390) or an sponding WT controls. WAVE1 D1-KO mice and D2-KO mice both NMDA-receptor antagonist (MK801) with the acute cocaine developed locomotor sensitization during chronic administration of challenge largely blocked the dephosphorylation of WAVE1 (Fig. 3

1396 | www.pnas.org/cgi/doi/10.1073/pnas.1621185114 Ceglia et al. Downloaded by guest on September 25, 2021 WT mice (Fig. 6). This effect of cocaine on AMPA current frequency was abolished in WAVE1 D1-KO mice, supporting a critical role for WAVE1 in the decrease of functional glutamatergic synapses on D1-MSNs under these conditions (Fig. 6). Discussion In this study, we identified a role for WAVE1 in cocaine reward and maintenance of locomotor sensitization. Using selective KO of WAVE1, we found decreased cocaine CPP and maintenance of locomotor sensitization in mice lacking WAVE1 in D1-expressing neurons. In contrast, mice lacking WAVE1 in D2-expressing neu- Fig. 2. Dephosphorylation of WAVE1 by acute cocaine challenge following rons showed normal behavioral responses to cocaine. We also found chronic administration of cocaine and withdrawal. (A) Cocaine treatment a role for WAVE1 in the regulation of dendritic spine density in D1- regimens. Saline (S) or cocaine (C, 15 mg/kg, i.p.) was administered for 14 consecutive days followed by 14-d withdrawal (WD). After acute cocaine MSNs in NAc in response to an acute challenge of cocaine following (15 mg/kg) or saline administration, mice were killed 45 min later using focused chronic cocaine exposure and 2 wk of withdrawal. This finding was microwave irradiation. (B–D) Phosphorylation of WAVE1 at S310 (P-S310) (B), associated with a decrease in the frequency of excitatory post- S397 (P-S397) (C), and S441 (P-S441) (D) and total WAVE1 were measured in total synaptic AMPA receptor currents in D1-MSNs. In further support striatum by immunoblotting. The levels were quantified and normalized to the of a selective role of WAVE1 in D1-mediated signaling, we found levels of saline/saline (S/S). Data represent means ± SEM, n = 6micepergroup. that following the acute challenge of cocaine in conjuction with < ’ *P 0.05, C/S versus C/C, one-way ANOVA, Tukey s post hoc test. chronic cocaine administration and withdrawal, cocaine-induced WAVE1 dephosphorylation was attenuated by coadministration and Fig. S3 D and E), suggesting critical roles of D1- and NMDA- of a D1 or an NMDA glutamate receptor antagonist. receptor signaling in WAVE1 dephosphorylation. Our previous studies indicated that WAVE1 is phosphorylated to a high level by Cdk5/p35 and as a result is largely inactive under Role for WAVE1 in Cocaine-Induced Changes in Dendritic Spine basal conditions (4). Chronic cocaine exposure increases the levels Morphology. Plasticity of glutamatergic transmission in the NAc of Cdk5 and p35 in the striatum (8). Increased levels of Cdk5/p35 has emerged as a major contributing factor for addictive behavior (9, phosphorylate DARPP-32 at Thr75 and reduce the efficacy of 10, 17). Previous studies showed that chronic administration of co- dopamine/cAMP/PKA/DARPP-32/protein phosphatase-1 signal- caine increases dendritic spine density in D1-MSNs (30–32). Den- ing (8, 15). Cdk5/p35 also regulates the NR2B subunit of NMDA dritic spines are the postsynaptic part of glutamatergic synapses and receptors by direct interaction and phosphorylation at Ser1116, actin polymerization is highly implicated in spine morphogenesis resulting in facilitation of degradation of NR2B by calpain and and stability (33, 34). Because WAVE1 plays a role in the regulation inhibition of surface expression, respectively (37, 38). Thus, chronic of dendritic spine morphology (4) and cocaine-induced behavior is cocaine-induced Cdk5/p35 expression would be expected to altered in WAVE1 D1-KO mice (Fig. 1), we examined a possible maintain WAVE1 in an inactive state through direct inhibitory role for WAVE1 in chronic cocaine-induced increase of spine phosphorylation as well as through inhibition of D1 and NMDA density in D1-MSNs. We injected WT and WAVE1 D1-KO mice receptor signaling, which are the major pathways for WAVE1 ac- daily with saline or cocaine (15 mg/kg) for 14 d. Two days after the tivation (7). Consistent with this interpretation, WAVE1 was not last injection, dendritic spine morphology of D1-MSNs was ana- required for the development of locomotor sensitization or for the lyzed. We quantified total dendritic protrusions and also four classes of dendritic spines based on their length (35, 36). Class 1 represents stubby spines without a discernible neck, classes 2 and 3 represent short and long spines, respectively, and class 4 represents filopodial protrusions. In response to cocaine, we observed a comparable increase in the density of total spines and class 2 spines in the NAc core of WT or WAVE1 D1-KO mice (Fig. 4). The effect of chronic cocaine on dendritic spines in the NAc shell was relatively minimal in this experiment (Fig. S4). Our results suggest that WAVE1 is not required for the chronic cocaine-induced increase in dendritic spine density in D1-MSNs observed under these conditions. We next investigated possible changes in dendritic spines after an acute challenge with cocaine following withdrawal because WAVE1 dephosphorylation was observed under this condition (Fig. 2). Notably, an acute challenge with cocaine reduced total spine density in D1-MSNs in the WT NAc core but no decrease was found in D1-MSNs in WAVE1 KO NAc core (Fig. 5). The decrease in total spine density in WT mice was due to a decrease in spine density in classes 1–3 spines, whereas an opposing direction of

spine changes in classes 1 and 2 was observed in WAVE1 D1-KO Fig. 3. Effect of D1 or NMDA receptor antagonists on cocaine-induced WAVE1 NEUROSCIENCE mice (Fig. 5). We also observed an acute cocaine-challenge– dephosphorylation. After 14-d of withdrawal following 14-d of daily injection of induceddecreaseintotalandclass 3 spine density in D1-MSNs in cocaine (15 mg/kg), mice were coinjected with saline (C/S) or cocaine (15 mg/kg) the NAc shell but the decrease was not observed in WAVE1 KO (C/C) plus vehicle, a D1 antagonist, SCH23390 (0.25 mg/kg) (A–C), or an NMDA – D1-MSNs in the NAc shell (Fig. S5). receptor antagonist, MK801 (0.25 mg/kg) (D F). Mice were killed 45 min later To examine the potential physiological consequences of a de- using focused microwave irradiation. The levels of phospho- and total WAVE1 were measured by immunoblotting. Quantified data were normalized to C/S crease in spine density in the NAc core, we examined AMPA vehicle. Data represent means ± SEM, n = 7 mice per group. *P < 0.05, **P < † ††† glutamate receptor currents in this region. Consistently, acute 0.01, and ***P < 0.001 (C/S versus C/C). P < 0.05 and P < 0.001 (C/C vehicle cocaine challenge following withdrawal caused a significant re- versus C/C antagonist as indicated). One-way ANOVA and Tukey’s post hoc test duction in frequency but not in amplitude of AMPA currents in were used.

Ceglia et al. PNAS | February 7, 2017 | vol. 114 | no. 6 | 1397 Downloaded by guest on September 25, 2021 D1-MSNs in response to a cocaine challenge following withdrawal from chronic cocaine. We also observed that cocaine challenge decreased the frequency in AMPA currents in D1-MSNs in a WAVE1-dependent manner, supporting a role for WAVE1 in the pruning of glutamatergic synapses. Thus, our previous and current results together implicate WAVE1 in mediating distinct aspects of morphological plasticity that depend on the stage of spine development and the strength of input signals. The dopamine system is intricately connected with the glutamatergic system in responses to acute and chronic exposure to cocaine. Dopaminergic neurons in the VTA provide dopaminergic input to NAc and mPFC and receive glutamatergic input from several corticolimbic structures such as mPFC, amygdala, and hippocampus (48–50). Glutamatergic input to the VTA increases the activity of dopaminergic neurons and enhances dopamine release in NAc (51–53). Dopamine also affects glutamatergic transmission by increasing the firing probability in pyramidal neurons in Fig. 4. Chronic cocaine-induced changes in dendritic spine density. WT mice mPFC (54). The interactions of these two neurotransmitter systems (A) and WAVE1 D1-KO mice (B) were treated daily (i.p.) with saline (Sal) or in the VTA and mPFC might be involved in the increased gluta- cocaine (Coc) (15 mg/kg) for 14 d. Two days after the last injection, mice were perfused and dendritic spines were analyzed in DiI-labeled D1-MSNs (identified mate release in the NAc observed after repeated cocaine admin- as enkephalin− cells) in the NAc core. (Upper panels) Representative images of istration (55, 56). Glutamate transmission in the NAc plays a key dendritic segments. (Scale bars, 5 μm.) (Lower graphs) Quantification of total role in cocaine addiction, which is predominantly mediated by and four classes of dendritic protrusion density. Class 1 represents stubby spines AMPA receptors in MSNs (17, 41, 57, 58). The synaptic strength of without a discernible neck, classes 2 and 3 represent short and long spines, AMPA receptors in MSNs is also modulated by intracellular do- respectively, and class 4 represents filopodial protrusions. Data represent pamine signaling. Cocaine-activated D1/cAMP/PKA signaling means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Kolmogorov–Smirnov = – leads to phosphorylation of AMPA receptors and primes them for test. n 14 16 mice per group. The total numbers of dendrites analyzed were synaptic insertion (59, 60). The WAVE1-regulated decrease of 36 (WT-Sal), 30 (WT-Coc), 45 (KO-Sal), and 30 (KO-Coc). glutamatergic synapses on D1-MSNs in NAc in response to cocaine challenge following withdrawal might be part of a homeostatic increase of dendritic spine density observed following chronic ex- feedback mechanism to avoid excessive increases in excitatory input posure to cocaine. Instead, WAVE1 was dephosphorylated after an and to thereby maintain network stability (61, 62). It is also possible acute challenge of cocaine following chronic cocaine plus with- that WAVE1-mediated synaptic pruning is input specific. Recent drawal, in a D1 and NMDA receptor signaling-dependent manner. studies indicate that cocaine-evoked plasticity in NAc specifically in Our previous studies showed that D1/cAMP/PKA signaling or glutamatergic afferents from mPFC onto D1-MSNs mediates cor- NMDA receptor/calcium signaling leads to dephosphorylation and rect action-outcome selection during cocaine seeking, whereas co- activation of WAVE1 (4, 7). Stimulation of D1 increases but caine-evoked plasticity specifically in glutamatergic afferents from stimulation of D2 decreases cAMP/PKA signaling (24). Further- ventral hippocampus onto D1-MSNs mediates recognition of the more, stimulation of D1 facilitates, whereas stimulation of D2 at- correct context where cocaine can be obtained (41). It will be tenuates NMDA-mediated calcium responses in MSNs (39, 40). Thus, WAVE1 in D1-MSNs but not in D2-MSNs would be activated in response to cocaine. This biochemical activation mechanism likely underlies the selective role for WAVE1 in D1-MSNs in cellular and behavioral responses to cocaine observed in this study. In this study, we used a noncontingent cocaine administration paradigm involving chronic cocaine administration, withdrawal, and acute challenge of cocaine. In this paradigm, we were able to ob- serve preserved sensitization of cocaine reward (measured by CPP) and sensitization of locomotor activity. Following withdrawal from chronic cocaine exposure, morphological and functional plasticity of glutamatergic synapses in the NAc has been suggested as a critical mechanism underlying cocaine craving and relapse (10, 11). Strengthening of excitatory synapses in MSNs in the NAc involves the initial formation of silent synapses lacking AMPA receptors during chronic exposure to cocaine, followed by insertion of AMPA receptors into spines during withdrawal (9–11). Accumulating evidence indicates that adaptive cocaine action involves potentiation of glutamatergic afferents arising from mPFC, amygdala, and ventral hippocampus preferentially onto D1-MSNs (41–44), al- Fig. 5. Effect of acute cocaine challenge after withdrawal on dendritic spine though D2-MSNs still contribute to certain behavioral features of density. WT mice (A) and WAVE1 D1-KO mice (B) were treated daily (i.p.) with cocaine action (45, 46). Previous studies showed that WAVE1 is cocaine (15 mg/kg) for 14 d. The mice were subsequently subjected to 14 d of highly localized to dendritic spines (47). WAVE1 KO neurons had withdrawal (WD) and then acutely challenged with saline or cocaine (15 mg/kg). decreased dendritic spine density, suggesting a role for WAVE1 in One hour after the last injection, mice were perfused and dendritic spines were dendritic spine formation (4, 47). In the current study, we also analyzed in DiI-labeled D1-MSNs in the NAc core as in Fig. 4. (Upper panels) Representative images of dendritic segments. (Scale bars, 5 μm.) (Lower graphs) observed lower baseline dendritic spine density in WAVE1 D1-KO Quantification of total and four classes of dendritic protrusion density. Data MSNs compared with WT MSNs. However, WAVE1 was dis- represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Kolmogorov– pensable for chronic cocaine-induced spine formation. Instead, Smirnov test. n = 15–16 mice per group. The total numbers of dendrites ana- WAVE1 was required for a selective decrease in spine density in lyzed were 29 (WT-Sal), 30 (WT-Coc), 31 (KO-Sal), and 30 (KO-Coc).

1398 | www.pnas.org/cgi/doi/10.1073/pnas.1621185114 Ceglia et al. Downloaded by guest on September 25, 2021 maintenance of sensitized locomotor activity. In the case of WAVE1 D2-KO mice and their WT mice, following a 2-wk withdrawal, mice received a challenge of cocaine (i.p. 10 mg/kg). Locomotor activity was measured daily in an open field chamber (42 × 42 × 30.5 cm) for 60 min after saline treatments, chronic cocaine treatments (first 5 d), and acute challenge with cocaine. Automated Accuscan software (Gerber Technology) was used to calculate total distance traveled.

Conditioned Place Preference. CPP was carried out in a three-chambered box and a pretest, which preceded all training sessions, was performed as described previously (69). During 30-min training sessions in which partitions between end chambers were lowered, mice received two saline and two cocaine injections on 2 consecutive days (saline in the morning and cocaine in the afternoon) in opposite end chambers. One day after training, CPP was assessed during a Fig. 6. Effect of acute cocaine challenge after withdrawal on AMPA glu- training session in which all partitions between chambers were raised, mice tamate receptor-mediated synaptic currents in D1-MSNs in the NAc core. were placed in the middle chamber, and time spent in individual chambers was After 14 d of withdrawal (WD) from chronic exposure to cocaine (i.p., 15 mg/kg), monitored during a 20-min session. The testing session preference score was WT (A–C)andWAVE1D1-KOmice(D–F) were acutely challenged with saline or calculated as time spent in the cocaine-paired end chamber minus time spent in cocaine (5 mg/kg). Sample traces of mEPSC from WT (A)orD1-KO(D)D1-MSNsin the saline-paired end chamber. For normal CPP tests (nonsensitization) a 7.5 mg/kg the NAc core are shown with vertical and horizontal scale bars. mEPSC frequency cocaine dose (i.p.) was used during the training session. For sensitization CPP (B) was reduced by an acute cocaine challenge without altering mEPSC experiments, mice were injected i.p. with a 15-mg/kg cocaine dose one amplitude (C) in WT mice (Sal challenge: n = 9 cells, 6 mice; Coc challenge: time per day for 7 consecutive days in their home cage. This dosing was n = 6 cells, 3 mice). mEPSC frequency (E) and amplitude (F) were unchanged followed by a 5-d withdrawal period in which no cocaine was given. Mice by an acute cocaine challenge after withdrawal in D1-KO mice (Sal chal- were then injected with a low cocaine dose (5 mg/kg) for the sensitization lenge: n = 10 cells, 7 mice; Coc challenge: n = 8 cells, 6 mice). Data were CPP training sessions. analyzed by Mann–Whitney nonparametric analysis (*P < 0.05) and pre- sented as box-and-whisker plots indicating the median, quartiles, and range. Phosphorylation Analysis and Immunoblotting. Saline- or cocaine-injected mice were killed by focused microwave irradiation of the brain (model: TMW-6402C, Muromachi Kikai). The killing of rodents by microwave irradiation results in rapid protein denaturation, effectively protecting phosphorylated proteins important to identify specific afferents regulated by WAVE1 and from postmortem activity of protein phosphatases (70). Preparation of brain behavioral consequence of WAVE1-mediated synaptic plasticity. lysates, BCA protein assay, and immunoblotting using the Odyssey infrared Cyfip2 (PIR121) is a component of the WAVE1 complex, which imaging system (Licor) were done as described (7). Antibodies for total WAVE1, we isolated as a Cdk5/p35 binding protein from the striatum (4). phosphorylation site-specific WAVE1, PIR121, Nckap1, Abi2, p35, and Cdk5 Recently, genetic screening of C57BL/6 substrains identified the were used as described (4). Cyfip2 gene mutation S968F as a causative variant for different acute and sensitized locomotor responses to cocaine (63). Another Dendritic Spine Analysis. Perfusion of mouse brain, preparation of brain slices, and ′ ′ ′ genetic study of BXD mouse strains also identified Cyfip2 as a labeling brain slices with the lipophilic fluorescence dye 1,1 -diotadecyl-3,3,3 ,3 - tetramethylindocarbocyanine perchlorate (DiI) were performed as described (30, candidate gene underlying a different intake response in i.v. cocaine Cyfip2 36). Fluorescent dendritic images of MSNs were taken using a confocal microscope self-administration (64). As in the case of the mouse S968F (Zeiss LSM 710) with an oil immersion lens (EC Plan-Neofluar 100×, 1.4 N.A.). DiI variant providing a differential cocaine response in mouse sub- was excited using an Argon 514-nm laser line. A filterless scanhead was used for strains (63), certain polymorphisms in WAVE1 complex proteins in emission collection. Images of distal dendrites (second to fourth order dendrites) humans might contribute to vulnerability in functional plasticity of were examined in this study. For dendritic spines, a stack of images was acquired glutamatergic synapses underlying addiction behavior such as co- in the z dimension with an optical slice thickness of 0.4 μm (with oversampling). caine craving and relapse. Characterization of WAVE1 mouse The pixel sizes along x and y dimensions were both 0.06 μm (with oversampling). To identify D1-MSNs, enkephalin was immnunostained and dendritic spine im- models using drug self-administration to measure clinically relevant − drug craving and compulsive drug taking behavior (10, 65, 66), ages were acquired from enkephalin cells (D1-MSNs). All measurements were made manually with Metamorph image analysis software (Universal Imaging together with further understanding WAVE1-mediated synaptic Corporation). Protrusions from dendrites were classified into four types based on plasticity in D1-MSNs, will be important topics for future research. their length as described (35, 36). Class 1 protrusions (also called stubby) were less than 0.5 μm, lacked a large spine head, and did not appear to have a neck; class 2, Methods or shorter spines, were between 0.5 and 1.25 μm long; class 3, or longer spines, Animals and Treatments. All procedures involving animals were approved by ranged between 1.25 and 3.0 μm; and class 4, or filopodial extensions, were fil- The Rockefeller University Institutional Animal Care and Use Committee and amentous protrusions longer than 3.0 μm. were performed in accordance with National Institutes of Health guidelines. BAC transgenic Drd1a-EGFP, Drd2-EGFP, Drd1a-Cre, and Drd2-Cre were from Electrophysiological Analysis. WT and WAVE1 D1-KO mice expressing Drd1- GENSAT (The Rockefeller University) (26, 67). A floxed WAVE1 line (25) was promoter–driven tdTomato were injected i.p. with a 15 mg/kg cocaine dose per crossed with Drd1a-Cre (EY262) or Drd2-Cre (ER44) mouse lines. A double day for 14 consecutive days. This dose was followed by a 14-d withdrawal and transgenic line harboring floxed WAVE1 and Drd1a-Cre alleles was crossed then an acute challenge of either saline or cocaine (5 mg/kg). Coronal slices with a tdTomato reporter line (The Jackson Laboratory, 007909). Progeny were were prepared from WT and WAVE1 D1-KO mice following procedures ap- produced using in vitro fertilization and embryo-transfer techniques to gen- proved by the Northwestern University Animal Care and Use Committee, as erate the animals needed for the biochemical and behavioral tests. Cocaine described (62). Patch pipettes (3–4MΩ) were filled with internal recording so- (Sigma-Aldrich) was administered by i.p. injection. lution (in millimolar concentration): 120.0 CsMeSO3,5.0NaCl,0.16CaCl2,10.0 TEA-Cl, 10.0 Hepes, 5.0 QX-314, 4.0 Mg-ATP, 0.3 Na-GTP. Recordings were Immunohistochemistry. Mouse perfusion, preparation of brain slices, and performed on a submersion-style recording chamber with a fixed-stage Olym- NEUROSCIENCE

immunohistochemistry were performed as described (30, 36). Anti-GFP pus BX51 upright microscope with continual perfusion of 95% O2/5% CO2 (rabbit polyclonal, 1:1,000; Abcam), anti-WAVE1 (rabbit polyclonal, 1:1,000; bubbled artificial cerebrospinal fluid (in millimolar concentration): 124 NaCl, 3

V0101) (68) and anti-enkephalin (clone: NOC1, 1:250; Millipore) were used. KCl, 2 CaCl2, 1 MgCl2,26NaHCO3,1NaH2PO4, and 16.66 glucose with (in mi- cromolar concentration) 100.0 4-AP, 1.0 TTX, 10.0 gabazine, and 1.0 CGP 35348. Locomotor Behavior. WT, WAVE1 D1-KO, and D2-KO mice (12–16 wk of age) tdTomato expression was used to verify D1-MSNs. Whole-cell patch clamp re- were habituated to saline injection (i.p.) for 2 consecutive days and then they cordings were made using a Multiclamp 700B amplifier using custom-written received injections of cocaine (i.p. 10 mg/kg) for 10 consecutive days. WT and freeware package WinFluor (John Dempster, University of Strathclyde, Glas- WAVE1 D1-KO mice were then divided into two groups: one group for 2-wk gow, UK) as described (62, 71). Miniature excitatory postsynaptic currents withdrawal and the other group for 3-wk withdrawal. After 2-wk or 3-wk (mEPSCs) were recorded in voltage clamp held at −70 mV. Data were analyzed withdrawal, mice received a challenge of cocaine (i.p. 10 mg/kg) to test the using the MiniAnalysis program (Synaptosoft).

Ceglia et al. PNAS | February 7, 2017 | vol. 114 | no. 6 | 1399 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. We thank Xiaozhu Zhang, Grant Martin, Hersh Trivedi, by Department of Defense US Army Medical Research Acquisition Activity Caroline Holmes, Ryan Malpass, Cassandra Kooiker, Junghee Jin, and Jodi Gresack Grants W81XWH-09-1-0392 (to Y.K.) and W81XWH-09-1-0402 (to P.G.); Na- for technical assistance and discussion; Rada Norinsky and The Rockefeller tional Institutes of Health Grants DA010044 and MH090963 (to P.G.), University Transgenic Services Laboratory for in vitro fertilization and embro R01DA014133 (to E.J.N.), and NS34696 (to D.J.S.); and grants from the JPB transfer services; and Elisabeth Griggs for graphics. This work was supported Foundation (to P.G. and D.J.S.).

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