Enhancing VTA Cav1.3 L-type Ca2+ channel activity promotes cocaine and mood-related behaviors via overlapping AMPA receptor mechanisms in the nucleus accumbens

Abstract

Genetic factors significantly influence susceptibility for substance abuse and mood disorders. Rodent studies have begun to elucidate a role of Ca_v1.3 L-type Ca²⁺ channels in neuropsychiatric-related behaviors, such as addictive and depressive-like behaviors. Human studies have also linked the CACNA1D gene, which codes for the Ca_v1.3 protein, with bipolar disorder. However, the neurocircuitry and the molecular mechanisms underlying the role of Ca_v1.3 in neuropsychiatric phenotypes are not well established. In the present study, we directly manipulated Ca_v1.3 channels in Ca_v1.2 dihydropyridine insensitive mutant mice and found that ventral tegmental area (VTA) Ca_v1.3 channels mediate cocaine-related and depressive-like behavior through a common nucleus accumbens (NAc) shell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) mechanism that requires GluA1 phosphorylation at S831. Selective activation of VTA Ca_v1.3 with (±)-BayK-8644 (BayK) enhanced cocaine conditioned place preference and cocaine psychomotor activity while inducing depressive-like behavior, an effect not observed in S831A phospho-mutant mice. Infusion of the CP-AMPAR-specific blocker Naspm into the NAc shell reversed the cocaine and depressive-like phenotypes. In addition, activation of VTA Ca_v1.3 channels resulted in social behavioral deficits. In contrast to the cocaine- and depression-related phenotypes, GluA1/A2 AMPARs in the NAc core mediated social deficits, independent of S831-GluA1 phosphorylation. Using a candidate gene analysis approach, we also identified single-nucleotide polymorphisms in the CACNA1D gene associated with cocaine dependence in human subjects. Together, our findings reveal novel, overlapping mechanisms through which VTA Ca_v1.3 mediates cocaine-related, depressive-like and social phenotypes, suggesting that Ca_v1.3 may serve as a target for the treatment of neuropsychiatric symptoms.

Figure 1

Pharmacological inhibition and activation of VTA Ca_v1.3 channels oppositely regulates cocaine behaviors. (a) Western blot showing the presence of Ca_v1.3 protein in VTA PSD fractions from wildtype (WT) and Ca_v1.3 knockout (KO) mice. (b) Schematic of Ca_v1.3 showing its ITTL in the C-terminal cytoplasmic domain and its interaction with PSD-95 and Shank. (c) Western blots showing immunoprecipitated PSD95 and Shank with Ca_v1.3 antibody in VTA protein lysates. (d) Disruption of Ca_v1.3 PDZ domain ITTL with an inhibitory peptide blocks KCl- (Two-way ANOVA, [Peptide x KCl, F_{(1, 20)} = 4.831, P=0.0399]; Bonferroni post hoc, Control Peptide: Vehicle vs KCl *P<0.05, KCl: Control Peptide vs ITTL Peptide ^†P<0.05) and BayK- (Two-way ANOVA, [Peptide x BayK, F_{(1, 20)} = 7.593, P=0.0122]; Bonferroni post hoc test: Control Peptide: Vehicle vs BayK *P<0.05, BayK: Control Peptide vs ITTL Peptide ^††P<0.01) induced Ser 133 P-CREB phosphorylation in VTA slices from Ca_v1.2 DHP^−/− mice. For all groups, n = 6. (e, f, i, l) Schematic timeline of behavioral protocol and VTA infusion of either Veh or Nif, Veh or BayK in Ca_v1.2DHP^−/− mice, or Ca_v1.3 shRNA in C57BL/6 mice. (g-h) Intra-VTA microinjection of Nif in Ca_v1.2 DHP^−/− mice administered prior to each cocaine conditioning session attenuated (g) expression of CPP on Day 5 (WD1) and Day 34 (WD30) (Two-way ANOVA, [VTA infusion x day, F_(2,60) = 7.595, P=0.0011]; Bonferroni post hoc test: Veh: Day 1 vs Day 5 ***P<0.001, Veh: Day 1 vs Day 34 ***P<0.001, Day 5: Veh vs Nif ^††P<0.01, Day 34: Veh vs Nif ^†††P<0.001. Veh n = 12, Nif n = 10), and (h) the cocaine-induced locomotor activity measured on day 36 (t₍₁₇₎ = 2.86, *P=0.0108. Veh n = 10, Nif n = 9). (i) Inset, image shows green fluorescent protein (GFP-green), tyrosine hydroxylase (TH-red) and dual-labeled (yellow) cells. (j, k) Intra-VTA stereotaxic delivery of Ca_v1.3 shRNA (21 days before the start of CPP) attenuated (j) the expression of CPP tested on day 5 and 34 (Two-Way ANOVA, [VTA injection x day, F_(2,51) = 14.09, P< 0.0011]; Bonferroni post hoc test: Ctrl shRNA: Day 1 vs Day 5 ***P<0.001, Ctrl shRNA Day 1 vs Day 34 ***P<0.001, Day 5: Ctrl shRNA vs Ca_v1.3 shRNA ^††P<0.01, Day 34: Ctrl shRNA vs Ca_v1.3 shRNA ^†††P<0.001. Ctrl shRNA n= 10, Ca_v1.3 shRNA n = 9), and (k) cocaine-induced locomotor activity measured on day 36 (t-test, t₍₁₇₎ = 3.066, **P=0.0070. Ctrl shRNA n = 10, Ca_v1.3 shRNA n = 9). (m, n) Intra-VTA infusion of BayK prior to each cocaine conditioning session enhanced (m) expression of cocaine CPP on day 5 and 34 (Two-way ANOVA, [VTA infusion x day F_(2,48) = 3.206, P=0.0493]; Bonferroni post hoc test: Veh: Day 1 vs Day 5 ***P<0.001, Veh: Day 1 vs Day 34 **P<0.01, Day 5: Veh vs BayK ^†P<0.05, Day 34: Veh vs BayK ^†P<0.05. Veh n = 9, BayK n = 9), and (n) enhanced cocaine-induced locomotor response on day 36 (t₍₁₆₎ = 3.955, **P=0.0011. Veh n = 9, BayK n = 9). Error bars represent ± s.e.m.

Figure 2

VTA Ca_v1.3 channels mediate long-term increase in CP-AMPARs at the NAc PSD of cocaine-exposed mice. (a) Schematic representation of VTA projection to the NAc and PFC. (b) Cocaine administration increased GluA1 but not GluA2 protein levels at the NAc PSD at WD30 that was blocked by Nif pretreatment (GluA1: Two-way ANOVA, [pre-treatment x post-treatment, F_(1,22) = 18.82, P< 0.0003]. Bonferroni post hoc test: Veh-Sal vs Veh-coc ***P<0.001, Veh-coc vs Nif-coc ^†††P<0.001; Veh-Sal n = 7, Nif-Sal n = 5, Veh-Coc n = 7, Nif-Coc n = 8). (c) No difference in GluA1 or GluA2 levels was seen in the PFC PSD (Veh-Sal n = 7, Nif-Sal, n = 5, Veh-Coc, n = 7, Nif-Coc, n = 8). ***P <0.0001, †††P < 0.001. (d) Experimental timeline of Naspm microinjection. (e) Schematic of Naspm infusion in the NAc shell. (f-g) Naspm infusion in the NAc shell prior to (f) cocaine CPP test on day 34 attenuated expression of cocaine CPP (Day 1 and 5, Two-way ANOVA, [day, F_(1,36) = 51.05, P<0.001]; Bonferroni post hoc test: Veh and Naspm groups: Day 5 vs Day 1 ***P<0.001; Day 34, t-test, t₍₁₈₎=2.881, ^††P=0.0099. Veh n = 8, Naspm n = 12) and (g) cocaine-induced locomotor activity test on day 34 attenuated expression of psychomotor sensitization (Day 1 and 5, Two-way ANOVA, [day, F_(1,28) = 23.76, P<0.001]; Bonferroni post hoc test: Veh group: Day 5 vs Day 1 **P<0.01, Naspm group: Day 1 vs. Day 5 *P<0.05; Day 34, t-test, t₍₁₄₎= 2.767, ^†P=0.0151. Veh n = 8, Naspm n = 12). (h) VTA Nif pretreatment in Ca_v1.2DHP^−/− mice blocked cocaine-induced increase in CaMKIIα, P-T286 CaMKIIα and P-S831 GluA1 in the NAc of cocaine exposed mice examined 30 days later (Two-way ANOVA, [CaMKIIα: pretreatment x posttreatment, F_(1,23) = 9.091, P=0.0062]; Bonferroni post hoc test: Veh-Sal vs Veh-Coc ***P<0.001, Veh-Coc vs Nif-Coc ^†††P<0.001; [P-T286 CaMKIIα: F_(1,23) = 13.11, P=0.0014]; Veh-Sal vs Veh-Coc ***P<0.001, Veh-Coc vs Nif-Coc ^†††P<0.001; [P-S831 GluA1: F_(1,23) = 4.247, P=0.05]; Veh-Sal vs Veh-Coc ***P<0.001, Veh-Coc vs Nif-Coc ^††P<0.01. Veh-Sal n = 7, Nif-Sal n = 6, Veh-Coc n = 7, Nif-Coc n = 7). (i) S831A GluA1 phospho-mutant mice presented a blunted cocaine CPP response (Two-Way ANOVA, [day x genotype, F_(2,50) = 3.211, P=0.0488]; Bonferroni post hoc test: WT and S831A: Day1 vs Day 5 ***P<0.001, WT: Day1 vs Day 34 ***P<0.001, Day 34: WT vs. S831A ^†P<0.05. WT n = 10, S831A n = 9), and (j) blunted cocaine-induced locomotor activity on Day 34 (Two-Way ANOVA, [day x genotype, F_(2,48) = 8.863, P=0.0005]; Bonferroni post hoc test: WT: Day1 vs Day 5 **P<0.01, S831A: Day1 vs Day 5 ***P<0.001, WT: Day 1 vs Day 34 ***P<0.001, Day 34: WT vs S831A ^†P<0.05. WT n = 8, S831A n = 10). Error bars represent ± s.e.m.

Figure 3

Repeated VTA BayK 8644 treatment results in depressive-like behavior, social interaction deficits and enhanced cocaine psychomotor activity. (a) Experimental timeline of VTA-BayK infusion and behavioral testing. (b–d) Repeated administration of BayK in the VTA decreased sucrose preference (b, t₍₁₈₎ = 3.486, **P=0.0026. Veh n = 10, BayK n = 10), increased immobility time in FST (c, t₍₁₂₎ = 2.725, *P=0.0184. Veh n = 7, BayK n = 7), and impaired social approach behavior (d, Two-Way ANOVA, [genotype x contact zone, F_(1,36) = 13.56, P=0.0008]; Veh: stranger vs empty cup **P<0.01. Veh n = 10, BayK n = 10). (e) BayK administration in the VTA increased GluA1 and GluA2 protein in the NAc PSD when tested 30 days later (GluA1: t₍₁₇₎ = 3.247, **P=0.0047. Veh n = 9, BayK: n = 10; GluA2: t₍₁₇₎ = 2.366, *P=0.0301. Veh n = 9, BayK n = 10). (f) Experimental timeline. (g–i) Naspm infusion in the NAc shell prior to (g) SPT (Two-Way ANOVA, [pre-treatment (Veh or BayK): F_(1,34) = 6.351, P=0.016]; Bonferroni post hoc test: Veh-Veh vs. BayK-Veh **P=0.0079. Veh-Veh n = 8, Veh-Naspm, n = 9, BayK-Veh n = 10, BayK-Naspm n = 11) or (h) FST (Two-Way ANOVA, [pretreatment x posttreatment, F_(1,34) = 4.903, P=0.0336]; Bonferroni post hoc test: Veh-Veh vs BayK-Veh *P<0.05, BayK-Veh vs BayK-Naspm ^†P<0.05. Veh-Veh n = 8, Veh-Naspm n = 9, BayK-Veh n = 10, BayK-Naspm n = 11), rescued depressive-like behavior and (i) decreased the enhanced cocaine-induced locomotor response (Two-Way ANOVA, [pre-treatment (Veh or BayK): F_(1,34) = 19.96, P<0.0001]; post-treatment (Veh or Naspm): F_(1,34) = 6.117, P=0.0185]; Bonferroni post hoc test: Veh-Veh vs BayK-Veh ***P<0.001, BayK-Veh vs BayK-Naspm ^†P<0.05. Veh-Veh n = 8, Veh-Naspm n = 9, BayK-Veh n = 10, BayK-Naspm n = 11), observed in BayK treated mice. (j–m) Naspm infusion in the (j) NAc shell (Two-way ANOVA, [pre-treatment (Veh or BayK): F_(1,34) = 16.83, P< 0.0002]; Bonferroni post hoc test: Veh-Veh vs BayK-Veh **P<0.01, Veh-Naspm vs BayK-Naspm *P<0.05. Veh-Veh n = 9; Veh-Naspm n = 9, BayK-Veh n = 10, BayK-Naspm n = 10), or (k) in the NAc core (Two-way ANOVA, [pre-treatment (Veh or BayK): F_(1,32) = 11.67, P< 0.0017]; Bonferroni post hoc test: Veh-Veh vs BayK-Veh *P<0.05, Veh-Naspm vs BayK-Naspm P=0.0526. Veh-Veh n = 8, Veh-Naspm n = 8, BayK-Veh n = 10, BayK-Naspm n = 10) had no effect on BayK-induced social approach deficit, whereas NBQX infusion in (l) the NAc core (Two-Way ANOVA, [pretreatment x posttreatment, F _(1,35) = 5.403, P=0.0260]; Bonferroni post hoc test: Veh-Veh vs BayK-Veh *P<0.01, BayK-Veh vs BayK-NBQX ^†P<0.05. Veh-Veh n = 9, Veh-NBQX n = 9, BayK-Veh n = 10, BayK-NBQX n = 11), but not in (m) the NAc shell [pre-treatment (BayK or Veh), F_(1,28) = 15.74, P=0.0005]; Post-treatment (NBQX or Veh), F_(1,28) = 0.1436, P = 0.7076]; Bonferroni post hoc test: Veh-Veh vs BayK-Veh *P<0.05. Veh-NBQX vs BayK-NBQX P=0.0829. Veh-Veh n = 8, Veh-NBQX n = 8, BayK-Veh n = 8, BayK-NBQX n = 8), rescued the BayK- induced social approach deficit. Error bars represent ± s.e.m.

Figure 4

Repeated BayK-induced depressive-like and cocaine behavior is dependent on phosphorylation of GluA1 at S831. (a) Repeated BayK infusion in the VTA increased CaMKIIα and P-S831 GluA1 phosphorylation in the NAc examined 30 days later (CaMKIIα: t₍₁₂₎ = 2.589, *P=0.0237; P-T286 CaMKIIα: t₍₁₂₎ = 2.132, P=0.0543; P-831 GluA1: t₍₁₂₎ = 2.378, *P=0.0349. VTA-Veh n = 7, VTA-BayK n = 7). (b, c) Repeated BayK infusion in the VTA of WT mice but not S831A mice resulted in depressive-like behavior as revealed in (b) the SPT (Two-Way ANOVA, [genotype x pretreatment, F_(1,32) = 4.983, P=0.0327]; Bonferroni post hoc test: WT: Veh vs BayK *P<0.05, BayK: WT vs S831A ^†P<0.05. WT-Veh n = 9, WT-BayK n = 10, S831A-Veh n = 8, S831A-BayK n = 9) and (c) FST (Two-Way ANOVA, [genotype x pretreatment, F_(1,32) = 6.145, P=0.0186]; Bonferroni post hoc test: WT:Veh vs BayK *P<0.05, BayK:WT vs S831A Mut ^†P<0.05. WT-Veh n = 9, WT-BayK n = 10, S831A-Veh n = 8, S831A-BayK n = 9). (d) Repeated BayK infusion in the VTA of WT and S831A mutant mice induced social approach deficits in both genotypes (Two way ANOVA, [pretreatment, F_(1,32) = 14.10, P=0.0007]; Bonferroni post hoc test: WT: Veh vs BayK *P<0.05, S831A: Veh vs BayK *P<0.05. WT-Veh n = 9, WT-BayK n = 10, S831A-Veh: n = 8, S831A-BayK n = 9). (e) Repeated BayK treatment in the VTA resulted in higher cocaine-induced locomotor activity in WT but not S831A mutant mice (Two-Way ANOVA, [genotype F_(1,32) = 13.58, P=0.0008], Bonferroni post hoc test: WT: Veh vs BayK **P<0.01. WT-Veh n = 9, WT-BayK n = 10, S831A-Veh n = 8, S831A-BayK n = 9). Error bars represent ± s.e.m.

Figure 5

Regional association plot of SNPs in and around CACNA1D. (a) SNPs are plotted with their −log10 (p-value) on the y-axis along with their physical position (NCBI build 36) on the x-axis. The SNPs are color coded according to their correlations (r²) with the most significant SNP rs4687735 shown in purple. The light blue line and right y-axis indicates the observed recombination rates in the HapMap CEU samples.