A role for calmodulin-stimulated adenylyl cyclases in cocaine sensitization

Abstract

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca(2+)/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that, whereas AC1 and AC8 single knock-out mice (AC1(-/-) and AC8(-/-)) exhibit Ca(2+)-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knock-out (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization after chronic cocaine treatment. Because of the known role for the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated ERK (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase-positive interneurons in DKO mice relative to wild-type (WT) controls. After acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581 and cAMP response element-binding protein (pCREB) at Ser133 after acute cocaine treatment. Our results demonstrate that the Ca(2+)-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs.

Figure 1.

Ca²⁺-stimulated adenylyl cyclase activity as a percentage of basal adenylyl cyclase activity in the striatum depends on AC1 and AC8. A, Striatal adenylyl cyclase activity in naive WT mice from Charles River Laboratories and AC1^+/+ littermate controls under baseline conditions and in the presence of 4 μm free calcium. B, Hippocampal cyclase activity under same conditions as in A. C, Striatal adenylyl cyclase activity in AC1^−/−, AC8^−/−, and DKO mice under baseline and calcium⁺ conditions. All values are means ± SEM of three separate experiments. Individual experiments were performed on tissue pooled from three to five animals with samples analyzed in triplicate. Two-way ANOVA, **p < 0.001, ***p < 0.001 (Bonferroni's test).

Figure 2.

Enhanced locomotor responses to acute cocaine in DKO mice. A–C, Mice were habituated to the testing chamber for 60 min before injection with 10 mg/kg cocaine injected intraperitoneally at time 0. Ambulatory distance traveled is shown for AC1^−/−, AC8^−/−, DKO, and WT (AC1^+/+) mice. D, Ambulatory distance traveled over the first 30 min after injection for saline and three doses of cocaine in WT and DKO mice. n = 9–15 mice per group. Two-way ANOVA, **p < 0.01 (Tukey's post hoc test).

Figure 3.

Absence of locomotor sensitization in DKO mice after chronic cocaine treatment. A–D, Mice were tested on day 1 with a 10 mg/kg acute dose of cocaine. On days 2–6, they received 20 mg/kg intraperitoneal cocaine injections in their home cage. On day 8, they received a 10 mg/kg challenge dose of cocaine. Ambulatory distance traveled on days 1 and 8 is shown for WT(AC1^+/+), AC1^−/−, AC8^−/−, and DKO mice. E, Summary data. WT, AC1^−/−, and AC8^−/− mice sensitize significantly to chronic cocaine, whereas DKO mice do not. n = 10–13 mice per group analyzed by two-way ANOVA, **p < 0.01 (Tukey's post hoc test).

Figure 4.

Altered PKA activation after acute cocaine treatment in DKO. A, B, Full image of immunoblot probed with pPKA-s antibody. Individual bands from pPKA-s blot and actin shown in B. Bands labeled 1–3 were analyzed. C, Data obtained by recording the integrated density of cocaine-treated WT (Charles River Laboratories) and DKO pPKA-s bands normalized to the integrated density of corresponding actin band, reported as average percentage change over saline-treated controls. Saline control groups for WT and DKO were set to 100%. n = 4 per condition. One-way ANOVA, *p < 0.05 (Tukey's post hoc test).

Figure 5.

Elevated basal striatal pERK levels and impaired activation in response to acute cocaine in DKO mice. A, Immunohistochemistry for pERK in the dorsal striatum from sections representative of WT (AC1^+/+) and DKO mice 30 min after injection with saline or 10 mg/kg cocaine. Scale bar, 50 μm. B–D, pERK-positive cell count summary data for the dorsal striatum, nucleus accumbens core, and nucleus accumbens shell. Values are means ± SEM for five to seven animals, whose means were determined from three to six sections per subregion. Data analyzed by two-way ANOVA, *p < 0.05, **p < 0.01 (Tukey's post hoc test).

Figure 6.

Basal pERK levels are elevated in non-MSNs in DKO striatum. Images are three-dimensional reconstructions of optical z-stacks taken from the rostral, dorsomedial caudate–putamen. A, Immunodetection of pERK and STEP in naive DKO mice. Arrowheads indicate pERK⁺ neurons that lacked STEP expression. Bar graph summarizes the percentage distribution of pERK⁺ cells relative to the expression of STEP in control WT and DKO mice (n = 5 per group). A total of 1547 cells were analyzed from DKO mice, and 898 cells were analyzed from WT mice. Scale bar, 40 μm. B, Immunodetection of pERK and ChAT in naive WT (Charles River Laboratories) and DKO mice. Arrowheads indicate neurons in which pERK⁺ neurons colocalized with ChAT⁺ neurons. Bar graph displays the percentage of pERK⁺, cholinergic cells in WT and DKO mice; p = 0.009 between WT and DKO. Scale bar, 80 μm. C, Immunodetection of pERK and DARPP32. Arrowheads indicate some pERK⁺ cells. Note that, although pERK was segregated from DARPP-32 in control DKO mice, they strictly colocalized after cocaine injection in WT (Charles River Laboratories) mice. Scale bar, 80 μm. n = 4–5 mice per group. Data analyzed by two-tailed t test (p = 0.006 between DARPP32⁻ cells in saline-treated groups; p = 0.017 between DARPP32⁺ cells in cocaine-treated groups).

Figure 7.

Increases in ERK, MSK1, and CREB phosphorylation after cocaine injection are suppressed in DKO mice. Immunodetection of pERK^{Thr183/Tyr185}, pMSK1^Ser376, pMSK1^Thr581, and pCREB^Ser133 in WT (Charles River Laboratories) and DKO mice. Images are three-dimensional reconstructions of optical z-stacks taken from the rostral, dorsomedial caudate–putamen. Arrows indicate neurons with nuclear pMSK1^Ser376 immunolabeling. Scale bar, 80 μm. A significant interaction between treatment and genotype was found for all four phosphorylation events (one-way ANOVA, p < 0.0001; pairwise comparisons with Tukey's post hoc test: p < 0.05 between saline pERK groups, as well as between all cocaine groups; n = 4–5 mice per group). Dynph, Dynorphin.

Figure 8.

Cocaine treatment coactivates ERK, MSK1, and CREB in the same neurons. Images are three-dimensional reconstructions of optical z-stacks taken from the rostral, dorsomedial caudate–putamen of WT (Charles River Laboratories) mice. Representative examples of neurons showing nuclear colocalization of pERK and pMSK1^Ser376 immunosignals (first row), pMSK1^Ser376 and pMSK1^Thr581 immunosignals (second row; arrows illustrate low pMSK1^Thr581 levels in neurons lacking nuclear pMSK1^Ser376), and pMSK1^Ser376 and pCREB immunosignals (third row; arrows illustrate low pCREB levels in neurons lacking nuclear pMSK1). Bar graphs summarize the observed (Ob) percentage of colocalization versus colocalization predicted by randomness (Rd) for each pair of antibodies (the latter estimated by multiplying the individual percentages of immunopositive MSNs after cocaine). Between 150 and 200 neurons from four mice were sampled per staining group. Two-tailed t test: p < 0.000001 between random and observed values for all groups. Scale bar, 40 μm.