Cocaine HCL, CaM kinase inhibitor KN93, LTCC inhibitor nifedipine, and the MEK inhibitor, U0126 were obtained from Sigma (St. Louis, MO). Anti-rabbit Ser 831 and Ser 845 phospho-GluA1 antibodies were obtained from Abcam (Cambridge, MA). Anti-rabbit GluA1 and GluA2 antibodies was obtained from Millipore (Billerica, MA). Anti-rabbit Thr 286 P-CaMKII, CaMKII, Thr183/Tyr 185 P-ERK1/2 and ERK1/2 antibodies were obtained from Cell Signaling (Danvers, MA). Anti-mouse Ca_v1.3 antibody was obtained from UC Davis/NINDA/NIMH NeuroMab facility c/o Antibodies Incorporated (Davis, CA) and anti-mouse β-actin antibody was from Chemicon (Temecula, CA). For immunohistochemistry, donkey Cy3 anti-mouse and Cy2 anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). For immunoblot analyses, goat anti-rabbit and horse anti-mouse secondary antibodies were obtained from Vector Laboratories (Burlingame, CA).

ERK2 siRNA was identified using an identical approach as that described for the Ca_v1.3 siRNAs described above and is published in Xu et al., (2008). The siRNA sequence used for this study was 5’-GGAGCAGTATTATGACCCA-3’. The sequence was reversed to produce a control sequence, 5’-ACCCAGTATTATGACGAGG-3’.

NAc co-ordinates for 9–10 week old male C57BL/6 mice were +1.6 mm A/P, +/−1.52 mm M/L and −4.55 mm D/V at an angle of 10° relative to Bregma (Paxinos and Franklin, 2004). To confirm injection placement in the NAc 300µm sections were obtained from freshly dissected brain. GFP fluorescence was utilized to confirm accurate bilateral targeting of the NAc. Mice with inaccurate targeting on either side of the NAc were eliminated from the study.

For delivery of pharmacological drugs into the NAc, guide cannulae were implanted bilaterally in adult male mice under ketamine (100 mg/ml) and xylazine (20 mg/ml) anesthesia in a stereotaxic frame. Coordinates used were +1.6 mm A/P, +/−1.52 mm M/L and −4.55 mm D/V at an angle of 15° relative to Bregma (Paxinos and Franklin, 2004). The tip of the cannula was positioned 1.5 mm above the NAc. Stainless steel guide cannula (5 mm in length, CMA Microdialysis, North Chelmsford, MA, model CMA/7) was secured to the skull with dental acrylic resin and 5 mm stainless-steel stylets inserted until the time of experiments. Following surgery, mice were individually housed with ad libitum access to food and water and allowed a minimum of 7 days recovery prior to all experiments. Pharmacological agents were delivered into the NAc using an injection cannula connected by flexible polyethylene tubing to a microinjection system, mounted with a 5 ml Hamilton syringe. Accurate bilateral cannulae placement was confirmed in a 50µM paraformaldehyde perfused section by Cresyl violet Nissl staining. Mice with inaccurate targeting on either side of the NAc were eliminated from the study. The CaM Kinase inhibitor KN93 (3µg in 0.2µl) or MEK inhibitor U0126 (80 ng in 0.2µl) was microinjected into the NAc 20 min prior to challenge with cocaine. KN93 and U0126 doses were chosen following an initial dose response with 1, 3, and 5µg/0.2µl and 60, 80, and 100 ng/µl, respectively. Both drugs were resuspended and dissolved in 0.9% saline containing 1.5% DMSO and 1.5% Tween-80 used as vehicle.

Cocaine psychomotor sensitization was performed as previously described in Giordano et al., (2010). Briefly, mice were habituated to open-field locomotor activity chambers (Med Associates Inc., St. Albans, VT) for 30 min. Mice were then administered saline or cocaine (15 mg/kg, i.p.) once a day for five days (Day 1 to 5) and locomotor activity was measured for 30 min on each testing day. Following a 21-day drug-free period, mice were challenged with saline or 15 mg/kg i.p. cocaine and locomotor activity measured for 30 min. Expression of cocaine psychomotor sensitization was calculated as the difference in total distance traveled (cm) on challenge day (day 26) minus that traveled on Day 1 and is reported as a difference score. For pharmacological experiments, nifedipine, KN93 and U0126 were administered 30 mins prior to cocaine treatment. Cocaine and SKF82958 were dissolved in 0.9% saline. Nifedipine, KN93 and U0126 were dissolved in 0.9% saline containing 1.5% DMSO and 1.5% Tween 80.

Immunoblotting was performed as previously described in Giordano et al., 2010. Briefly, mice were decapitated 30 min following behavioral testing and brains rapidly dissected and frozen in isopentane at −40°C. To obtain NAc tissue, brains were sectioned in the coronal plane on a cryostat to the rostral end of the NAc and 0.5 mm bilateral tissue punches (containing NAc shell and core), spanning approximately +1.7 to +1.2 mm A/P relative to Bregma (Paxinos and Franklin, 2004), were isolated with a 17-gauge stainless steel stylet. To obtain VTA tissue, brains were sectioned to the rostral end of the VTA and an approximate 0.5 mm unilateral punch (spanning −3.16 to −3.64 A/P relative to Bregma, Paxinos and Franklin, 2004), was isolated with a 17-gauge stylet. Tissue was processed as previously described in Giordano et al., 2010. Twenty to forty µg of protein was loaded on 12% SDS-polyacrylamide gels and run at 200 volts constant voltage. Blots were probed with anti-rabbit (1:850 Ser 845 P-GluA1, Ser 831 P-GluA1, GluA2, 1:1000 GluA1, P-ERK1/2, ERK1/2, Thr 286 P-CaMKII and CaMKII) or anti-mouse (1:30,000 β-actin and 1:1000 Ca_v1.3) primary antibodies overnight at 4°C. Blots were then incubated with goat anti-rabbit (1:5000 for all anti-rabbit primary antibodies) or horse anti-mouse (1:40,000 for actin and 1:5000 for Ca_v1.3) horseradish peroxidase-linked IgG. Protein bands were visualized by chemiluminescence. Kaleidoscope Prestained standards (Bio-Rad, Hercules, CA) were used for protein size determination. For quantitation, β-actin was used as a loading control as we have found that repeated cocaine does not significantly alter total protein levels in the NAc, compared to repeated saline treated mice (sal, 100 ± 10% vs. coc, 97 ± 7%). Blots were scanned with an HP Scanjet 7400c scanner (Hewlett Packard, Palo Alto, CA). Intensity of the protein bands was measured as optical density, using the NIH Image J program (National Institutes of Health, Bethesda, MD).

Experiments were performed using a modified protocol of that published in Boudreau and Wolf (2005). A single NAc tissue punch (spanning +1.7 – +1.2 mm relative to Bregma, Paxinos and Franklin, 2004) was rapidly dissected using a stainless steel stylet (15 gauge) from 0.5 mm coronal section placed on an ice-cold surface, obtained from a mouse brain matrix. NAc tissue was pooled from three mice. Tissue was incubated in ice-cold artificial CSF (aCSF) containing 2mM BS³ (Pierce, Rockford, IL) and incubated for 30 min at 4°C on a rotator. Cross-linking reaction was quenched with 100 mM glycine in aCSF for 10 min at 4°C on a rotator. Samples were centrifuged for 2 min at 4°C. Supernatants were discarded and pellets washed once with aCSF. Samples were re-centrifuged, supernatants were discarded, and pellets were sonicated in ice-cold lysis buffer (0.1% NP40 buffer in Tris-EDTA, pH7.4 containing 1X protease inhibitor mixture (Sigma-Aldrich), 5 mM NaF, and 1X phosphatase inhibitor mixture (Sigma-Aldrich). Protein concentration was determined by BCA assay and 15µg protein was loaded on a 4–15% gradient Tris-HCl gel (BioRad, Hercules, CA) and run at 100 volts constant voltage. Gels were processed for GluA1 and GluA2 immunoblot analysis as described above and in Tropea et al., 2008. Blots were probed with anti-rabbit GluA1 (1:850) and GluA2 (1:1000) and goat anti-rabbit horseradish peroxidase-linked IgG (1:5000, Vector Laboratories).

The effect of systemic pretreatment of the LTCC antagonist, nifedipine, during the development of cocaine psychomotor sensitization on NAc S831 P-GluA1, NAc surface GluA1 and NAc S845 P-GluA1 21 days later was examined in Cav1.2DHP^−/− mutant mice. In this experiment (see Figs. 2B–D) a factorial design that included the between-subject factors of pretreatment (vehicle, nifedipine) and pre-exposure (saline, cocaine) was used to assess the effect of blocking Ca_v1.3 channels during development on cocaine-induced NAc S831 P-GluA1 (see Fig. 2B), S/I GluA1 (see Fig. 2 C), and S845 P-GluA1 (see Fig. 2 D). N = 6 mice were used for s-s, v-s and n-s groups, and n= 7 for the v-c and n-c groups.

The effect of systemic pretreatment of the LTCC antagonist nifedipine administered immediately prior to cocaine challenge 21 days following development of psychomotor sensitization on NAc S831 P-GluA1, NAc surface GluA1 and NAc S845 P-GluA1, was examined in Ca_v1.2DHP insensitive mutant mice. In this experiment (see Fig. 2F–H), a factorial design that included the between-subject factors of pretreatment (vehicle, nifedipine) and challenge (saline, cocaine) was used to assess the effect of blocking Ca_v1.3 channels at expression on cocaine-induced S831 P-GluA1 (see Fig. 2D), S/I GluA1 (see Fig. 2E), and S845 P-GluA1 (see Fig. 2F). N = 6 mice were used for s-s, v-s and n-s groups, and n= 7 for the v-c and n-c groups.

The effect of knockdown (KD) of Ca_v1.3 in the VTA on the development of psychomotor sensitization, on the subsequent expression of cocaine sensitization examined 21 days later and on NAc S831 P-GluA1 was assessed in control and Ca_v1.3 siRNA injected mice. Ca_v1.3 KD in the VTA was achieved with site-specific microinjection of rAAV expressing Ca_v1.3 siRNA prior to the start of the sensitization regimen. In this experiment (see Figs. 3E–F), between-subject factors of viral treatment (control siRNA, Ca_v1.3 siRNA) and day (1, 5 or 26) was used to assess the effect of VTA Ca_v1.3 KD on development and expression of sensitization (Fig. 3E) and a between-subject factor of viral treatment (control siRNA, Ca_v1.3 siRNA) was used to assess the effect of VTA Ca_v1.3 KD on NAc S831 P-GluA1 (Fig. 3F). N = 12 and 14 for ctrl and Ca_v1.3 siRNA, respectively in Fig. 3E and n = 10 for ctrl and Ca_v1.3 siRNA in Fig. 3F.

The effect of knockdown (KD) of Ca_v1.3 in the NAc on the development of psychomotor sensitization, on the subsequent expression of cocaine sensitization examined 21 days later, and on NAc S831 P-GluA1 was assessed in control and Ca_v1.3 siRNA injected mice. Ca_v1.3 KD in the NAc was achieved with site-specific microinjection of rAAV expressing Ca_v1.3 siRNA prior to the start of the sensitization regimen. In this experiment (see Figs. 3H–I), a between-subject factor of viral treatment (control siRNA, Ca_v1.3 siRNA) and day (1, 5 or 26) was used to assess the effect of NAc Ca_v1.3 KD on development and expression of sensitization (see Fig. 3H) and a between subject factor of viral treatment (control siRNA, Ca_v1.3 siRNA) was used to assess the effect of NAc Ca_v1.3 KD on NAc S831 P-GluA1 (see Fig. 3I). N = 10 mice for ctrl and Ca_v1.3 siRNA groups.

The effect of KD of ERK2 in the VTA during the development of psychomotor sensitization on the subsequent expression of cocaine sensitization 21 days later and on NAc S831 P-GluA1 was assessed in mice microinjected with rAAV expressing ERK2 siRNA. ERK2 KD in the VTA was achieved prior to the start of development. In this experiment (see Fig. 4C, D), a between-subject factor of viral treatment (control siRNA, ERK2 siRNA) was used to assess the effect of VTA ERK2 KD on development and expression of sensitization (see Fig. 4C) and on NAc S831 P-GluA1 (see Fig. 4D). N = 10 and 12 for ctrl and Ca_v1.3 siRNA, respectively in Fig. 4C and n = 10 for ctrl and Ca_v1.3 siRNA in Fig. 4D.

The effect of brain-specific neuronal genetic deletion of Ca_v1.2 on the development and expression of cocaine psychomotor sensitization and NAc S831 P-GluA1 was examined in CNS-specific Ca_v1.2 conditional knockout (Cav1.2^CNSKO) mice. In this experiment (see Fig. 5), a factorial design that included the between-subject factors of day (1, 5 and 26) and genotype (WT, KO) was used to assess the effect of loss of neuronal Ca_v1.2 on development and expression of cocaine sensitization (see Fig. 5A). A between-subject factor of genotype (Ca_v1.2^CNSWT, Ca_v1.2^CNSKO) was used to assess S831 P-GluA1 on Day 26 (see Fig. 5B). N = 8 and 12 mice for Ca_v1.2^CNSWT and Ca_v1.2^CNSKO, respectively in Fig. 5A and n = 8 for both genotypes in Fig. 5B.

The effect of knockdown (KD) of Ca_v1.2 in the NAc on cocaine-induced expression of sensitization and S831 P-GluA1 was assessed in Ca_v1.2 floxed mice microinjected in the NAc with AAV-Cre. Twenty four hours after the development of cocaine sensitization, AAV-Cre-GFP or control AAV-GFP was microinjected into the NAc. Fourteen days later, mice were tested for expression of cocaine sensitization and NAc S831 P-GluA1 levels. Fourteen days of withdrawal was chosen as maximal KD is achieved at this time point using AAV-Cre. In this experiment (see Figs. 5E–F), a between-subject factor of viral treatment (control AAV-GFP, AAV-Cre-GFP) was used to assess the effect of NAc Ca_v1.2 KD on expression of sensitization (see Fig. 5E) and on NAc S831 P-GluA1 (see Fig. 5F). N = 8 mice for ctrl and n = 10 mice for AAV-Cre-GFP groups.

The effects of intra-NAc administration of the CaMK inhibitor, KN93, and the MEK inhibitor, U0126, on cocaine-induced expression of sensitization and S831 P-GluA1 were examined in cocaine sensitized C57BL/6 mice. In this experiment (see Fig. 6F, G), the between-subject factor of pretreatment (veh, KN93 or veh, U0126) was used to assess the effect of blocking CaM kinases or ERK on expression of cocaine sensitization (see Fig. 6F) and on S831 P-GluA1 (see Fig. 6G). KN93 (3µg in 0.2µl) and U0126 (80ng in 0.2µl) were microinjected into the NAc 30 min prior to cocaine challenge. N = 12 and 14 mice were used for veh and KN93 treatment groups, respectively and n = 13 and 14 for veh and U0126 treatment groups, respectively.

The effect of systemic pretreatment of the MEK inhibitor, U0126, administered immediately prior to cocaine challenge on NAc surface GluA1 was examined in cocaine sensitized C57BL/6 mice. In this experiment (see Fig. 6H), a between-subject factor of pretreatment (veh, U0126) was used to assess the effect of blocking ERK on surface GluA1 levels. U0126 (80ng in 0.2µl) was microinjected into the NAc 30 min prior to cocaine challenge. N = 6 mice were used for veh and U0126 groups.

The effect of knockdown (KD) of VTA Ca_v1.3 and ERK2 during the development of cocaine sensitization, on NAc Ca_v1.2 mRNA 21 days later, was assessed in C57BL/6 mice microinjected with rAAV expressing the respective siRNA. Ca_v1.3 and ERK2 KD were achieved prior to the start of development of sensitization. In this experiment (see Fig. 7C,D), a between-subject factor of viral injection (ctrl siRNA, Ca_v1.3 siRNA or ctrl siRNA, ERK2 siRNA) was used to assess the effect of VTA Ca_v1.3 KD (see Fig. 7C) and VTA ERK2 KD (see Fig. 7D) on NAc Ca_v1.2 mRNA levels. N = 10 mice for ctrl and Ca_v1.3 siRNA in Fig. 7C and n = 10 and 12 for ctrl and ERK2 siRNA, respectively.

Next, to identify the neuroanatomical site of Ca_v1.3’s actions we targeted the VTA as this region has been implicated in initiating mechanisms during the development of sensitization that are essential for long-term expression of psychomotor sensitization following withdrawal (Vanderschuren and Kalivas, 2000). Additionally, Ca_v1.3 is highly expressed in VTA DA neurons (Rajadhyaksha et al., 2004) and activation of LTCCs in this region is sufficient to induce long-term expression of cocaine sensitization (Licata et al., 2000). As no Ca_v1.3-specific pharmacological agents currently exist, we generated neuron-specific recombinant adenoassociated virus (rAAV)-expressing Ca_v1.3 siRNA (described in the methods section). rAAV-Ca_v1.3 siRNA or rAAV-Ctrl siRNA was stereotaxically delivered bilaterally to the VTA three weeks prior to the start of the cocaine sensitization regimen (Fig. 3A,B). Ca_v1.3 siRNA generated a 50 (± 10)% knockdown (KD) of Ca_v1.3 mRNA (Figure 3C, F_1,14 = 12.757; p < 0.01) as demonstrated by quantitative real-time PCR (qPCR) and 55 (±8)% KD of Ca_v1.3 protein (Figure 3D, F_1,14 = 8.232; p < 0.01) using immunoblot analysis. We found that KD of VTA Ca_v1.3 channels attenuated development of cocaine sensitization and expression of the sensitized response when examined 21 days later (Fig. 3E, significant day×viral treatment interaction, F_{2, 72} = 32.68; p < 0.001). Locomotor activity was significantly lower in Ca_v1.3 siRNA injected mice compared to control siRNA injected at day 5 and 26 (Fig. 3E). KD of VTA Ca_v1.3 had no effect on acute cocaine-induced locomotor response (Fig. 3E, day 1). Examination of S831 P-GluA1 levels revealed significantly lower levels in Ca_v1.3 siRNA injected mice compared to control mice (Fig. 3F, F_{1, 18} = 8.373; p < 0.01). No change in phosphorylation was seen at S845 (data not shown). In contrast, KD of Ca_v1.3 in the NAc, which was also achieved prior to the start of the sensitization regimen, had no effect on development or expression of sensitization (Fig. 3G–I). There was no difference in locomotor activity between NAc Ca_v1.3 siRNA and control siRNA microinjected mice at day 5 or 26 or following acute cocaine treatment on day 1 (Fig. 3H), nor was there a difference in NAc S831 P-GluA1 levels examined at day 26 (Fig. 3I).

Next we examined the role of VTA ERK2, as we have shown previously that the LTCC-activated ERK pathway is involved in psychostimulant-mediated changes in the VTA (Rajadhyaksha et al., 2004). By utilizing Ca_v1.3 KO mice, we found that repeated cocaine treatment increased ERK2 phosphorylation in the VTA via Ca_v1.3 channels. Cocaine-induced P-ERK2 was significantly higher in Ca_v1.3 WT mice but not KO mice (Fig. 4A, significant treatment × genotype interaction, F_{1, 20} = 5.832; p < 0.05). To specifically test the causal role of ERK2 in the VTA on behavior and on S831 P-GluA1 in the NAc, we utilized a previously characterized rAAV-expressing ERK2 siRNA (Xu et al., 2008), as this is one of two existing strategies for targeting ERK2 in vivo. For this experiment, rAAV-ERK2 siRNA or rAAV-Ctrl siRNA was stereotaxically delivered into the VTA three weeks prior to the start of the cocaine sensitization regimen as shown in Fig. 3A. ERK2 siRNA generated a 55 (± 13)% KD of ERK2 protein in the VTA (Figure 4B, F_1,14 = 7.235; p < 0.01). Knockdown of VTA ERK2 attenuated both the development and cocaine-induced expression of sensitization (Fig. 4C, significant day × viral treatment interaction, F_2,60 = 20.50; p < 0.0001). Locomotor activity was significantly lower in ERK2 siRNA injected mice compared to control siRNA injected at day 5 and 26 (Fig. 4C). KD of VTA ERK2 had no effect on acute cocaine-induced locomotor response (Fig. 4C, day 1). KD of VTA ERK2 also blocked the increase in phosphorylation at S831 in the NAc (Fig. 4D, F_1,18 = 16.348; p < 0.0001), when examined 21 days later. VTA ERK2 KD did not affect levels of phosphorylated S845 (data not shown).

The above experiments that utilized rAAV Ca_v1.3 siRNA and Ca_v1.2DHP mutant mice established that Ca_v1.3 channels in the VTA mediate cocaine’s effects during the development of sensitization and that VTA Ca_v1.3 channels are necessary for the cocaine-induced increase in S831 P-GluA1 observed 21 days later. Utilizing Ca_v1.2 DHP insensitive mice we also showed that Ca_v1.3 channels do not play a role in mediating cocaine-induced increase in S831 P-GluA1 at expression. Thus, in order to elucidate the mechanism regulating the cocaine-induced increase in S831 GluA1 in the NAc, we explored the role of Ca_v1.2 channels as our recent data using Ca_v1.2DHP insensitive mice has suggested that Ca_v1.2 channels mediate expression (Giordano et al., 2010). In the present study we performed experiments in CNS-specific Ca_v1.2 conditional KO mice (Moosmang et al., 2005). Ca_v1.2 channels were found to mediate cocaine-induced expression of psychomotor sensitization with no role during development (Fig. 5A, significant day × genotype interaction, F_{1, 63} = 14.12; p < 0.0001). Locomotor activity was significantly lower in Ca_v1.2^CNSKO mice compared to Ca_v1.2^CNSWT mice on day 26 but not day 5 or day 1 (Fig. 5A). Examination of S831 P-GluA1 levels in the NAc following behavioral testing revealed significantly lower levels in Ca_v1.2 KO mice compared to WT mice (Fig. 5B, F_{1, 14} = 11.859; p < 0.01) with no difference in S845 P-GluA1 levels (data not shown). Next to directly test the role of Ca_v1.2 channels in the NAc we utilized AAV-Cre to knockdown Ca_v1.2 in the NAc of Ca_v1.2 floxed mice (Fig. 3C–F). For this experiment mice were sensitized to cocaine (once a day for 5 days; Fig. 5C). Twenty-four hours later (day 6) mice were bilaterally microinjected in the NAc with AAV-Cre-GFP or AAV-GFP (Fig. 5C). When examined fourteen days later, a time point that results in maximal knockdown, 55 (± 10)% KD of Ca_v1.2 mRNA was achieved in AAV-Cre-GFP micro-injected mice compared to AAV-GFP microinjected mice (Fig. 5D). Following cocaine challenge, locomotor activity was significantly attenuated in NAc AAV-Cre microinjected mice compared to AAV-GFP microinjected mice that displayed expression of cocaine sensitization (Fig. 5E, significant day × viral treatment interaction, F_{2, 48} = 26.57; p < 0.001). Cocaine-induced increase in NAc S831 P-GluA1 was also significantly lower in AAV-Cre microinjected mice (Fig. 5F, F_1,16 = 4.612; p < 0.05) demonstrating that NAc Ca_v1.2 channels mediate cocaine-induced increase in S831 P-GluA1 that underlies expression of cocaine sensitization. Additionally, we found that treating cocaine sensitized Ca_v1.2 WT but not KO mice with the dopamine D₁ agonist SKF82958 significantly increased NAc phosphorylation of S831 (Fig. 5G, skf vs. veh in WT but not KO mice, significant treatment × genotype interaction, F_{1, 28} = 26.51; p < 0.0001).

As LTCC-mediated activation of CaMKII and ERK in the NAc contributes to psychostimulant-induced behaviors (Pierce et al., 1998; Anderson et al., 2008; Giordano et al., 2010), we next tested the role of CaMKII and ERK2 in mediating the cocaine-induced increase in Ser 831 that underlies expression of cocaine psychomotor sensitization. Western blots revealed that Ca_v1.2 channels mediated cocaine-induced increase in Thr 286 P-CaMKII and Thr202/Tyr204 P-ERK2 in the NAc when examined 21 days following withdrawal. Cocaine significantly increased P-CaMKII (Fig. 6A, significant treatment × genotype interaction, F_1,18 = 4.839, p < 0.05) and P-ERK2 (Fig. 6B, significant treatment × genotype interaction, F_1,18 = 5.944, p < 0.05) in Ca_v1.2 WT but not KO mice. To directly test the role of NAc CaM kinases (CaMKs) and the ERK pathway on expression of cocaine sensitization and S831 P-GluA1 we next pharmacologically inhibited these two kinases in the NAc 30 min prior to the cocaine challenge on day 26 (Fig. 6C). Intra-NAc administration of KN93 was used to inhibit CaMKs and ERK was inhibited by U0126 (Fig. 6D,E). However, it should be noted that KN93 has additionally been found to inhibit LTCCs in vitro (Gao et al., 2006). Administration of KN93 attenuated both expression of sensitization (Fig. 6F, F_1,24 = 4.567; p < 0.05) and levels of phosphorylated S831 in the NAc (Fig. 6G, F_1,24 = 4.851; p < 0.05). Contrary to this, U0126, attenuated expression (Fig. 6F, F_1,25 = 5.166; p < 0.05) but not S831 P-GluA1 (Fig. 6G) or S845 P-GluA1 (data not shown). However, U0126 blocked the cocaine-induced increase in NAc cell surface GluA1 levels (Fig. 6H, F_1,12 = 4.159; p < 0.05), suggesting that cocaine-induced GluA1 trafficking to the cell surface requires both a CaMKII-dependent S831 phosphorylation event and an ERK2-dependent event. Taken together, the above experiments demonstrated that D1/Ca_v1.2-activated P-CaMKII and P-ERK2 mediate cocaine-induced increase in NAc S831 GluA1 phosphorylation following protracted withdrawal.