The membrane cytoskeletal protein adducin is phosphorylated by protein kinase C in D1 neurons of the nucleus accumbens and dorsal striatum following cocaine administration

Abstract

Repeated cocaine administration results in persistent changes in synaptic function in the mesolimbic dopamine system that are thought to be critical for the transition to addiction. Cytoskeletal rearrangement and actin dynamics are essential for this drug-dependent plasticity. Cocaine administration increases levels of F-actin in the nucleus accumbens and is associated with changes in the phosphorylation state of actin-binding proteins. The adducins constitute a family of proteins that interact with actin and spectrin to maintain cellular architecture. The interaction of adducin with these cytoskeletal proteins is regulated by phosphorylation, and it is therefore expected that phosphorylation of adducin may be involved in morphological changes underlying synaptic responses to drugs of abuse including cocaine. In the current study, we characterized the regulation of adducin phosphorylation in the nucleus accumbens and dorsal striatum in response to various regimen of cocaine. Our results demonstrate that adducin is phosphorylated by protein kinase C in medium spiny neurons that express the dopamine D1 receptor. These data indicate that adducin phosphorylation is a signaling event regulated by cocaine administration and further suggest that adducin may be involved in remodeling of the neuronal cytoskeleton in response to cocaine administration.

Figure 1. Time course of adducin phosphorylation in the striatum after acute cocaine injection

Detection of phospho-adducin in striatal and NAc homogenates using an antiserum raised against phospho-serine 724 in adducin 5 min (Coc 5), 10 min (Coc 10), 20 min (Coc 20) and 30 min (Coc 30) after acute cocaine injection (20 mg/kg, i.p.). 1A, Western blot showing the time course of phospho-adducin induction after cocaine treatment (data are representative of 3-4 independent animals in each group).Quantitation of phospho-adducin compared to total adducin in each sample. 1B Immunoblots showing phospho-adducin levels in homogenates of striatum and NAc, from WT and beta-adducin KO mice after acute injection of cocaine (10 min; 20 mg/kg ip). Note the absence of phospho-adducin signal in beta-adducin KO mice compared to WT (data are representative of 3-4 independent animals in each group).

Figure 2. Cocaine-induced adducin-phosphorylation measured by western blotting is blocked by dopamine receptor antagonists

SCH 23390 (sch; 0.25 mg/kg, i.p.) or raclopride (raclo; 0.25 mg/kg, i.p.) were injected 30 min before saline (sal) or cocaine administration (coc). A, Immunoblots showing phospho-adducin levels in homogenates of the dorsal striatum from cocaine-injected mice (20 mg/kg; ip), in the presence or absence of dopamine receptor antagonists. B, Quantitation of phospho-adducin measured by Western blotting in the dorsal striatum (6-8 independent mice/group).**p<0.001 when comparing sch+coc group to sal+coc group and ***p<0.0001 when comparing raclo+coc group to sal+coc group C, Immunoblot showing phospho-adducin in homogenates of the NAc from cocaine-injected mice (20 mg/kg, i.p.), in the presence or absence of dopamine receptor antagonists. D, Quantitation of phospho-adducin measured by Western blotting in the NAc (6-8 independent mice/group). *p<0.05 when compare sch+coc group to sal+coc group.

Figure 3. Cocaine-induced adducin-phosphorylation measured by immunocytochemistry in striatal slices depends on D1 receptor activity

A, Immunostaining for phospho-adducin in NAc induced by saline (sal) or cocaine (coc; 10 min, 20 mg/kg, i.p.) in striatal slices from WT and β-adducin KO mice. Note the absence of phospho-adducin staining in slices from β-adducin KO mice. B, Quantitation of phospho-adducin immunopositive neurons in medial striatum. ***p<0.0001 when comparing sch+coc group to sal+coc group and ***p<0.0001 when comparing raclo+coc group to sal+coc group C, Quantitation of phospho-adducin immunopositive neurons in NAc core***p<0.001 when comparing sch+coc group to sal+coc group D, Quantitation of phospho-adducin immunopositive neurons in NAc shell. ***p<0.001 when comparing sch+coc group to sal+coc group. SCH 23390 (sch; 0.25 mg/kg, i.p.) or raclopride (raclo; 0.25 mg/kg, i.p.) were injected 30 min before cocaine administration. The number of neurons immunopositive for phospho-adducin were counted in medial striatum, NAc shell and NAc core following cocaine-injection in mice (20 mg/kg; ip) in the presence or absence of DA receptor antagonists.

Figure 4. Acute or chronic cocaine administration induces adducin phosphorylation only in D1 receptor-expressing striatal neurons

A, B, C, Immunostaining for phospho-adducin (red) and EGFP fluorescence (green) in striatal subregions from drd1aEGFP mice (A, dorsal striatum; B, NAc core; C, NAc shell) 10 min after saline (sal) or cocaine administration (coc; 20 mg/kg; ip) or saline (sal). Arrowheads show phospho-adducin immunoreactivity in EGFP-positive (D1 receptor expressing) neurons. Quantitation of phospho-adducin immunoreactive/EGFP-positive (D1+) neurons in striatal subregions of drd1a-EGFP mice. *p < 0.05 when comparing sal+sal group to sal+coc and coc+coc groups in both striatal subregions.

Figure 5. Chelerythrine inhibits cocaine-induced adducin phosphorylation in the nucleus accumbens and the striatum

A,C, Phospho-adducin staining in the dorsal striatum (A) or the NAc (C), in the absence (sal) or presence of the general PKC inhibitor chelerytrine (chel; 1, 3 or 10 mg/kg). B, D, Quantitation of the number of neurons immunopositive for phospho-adducin in dorsal striatum (B) or NAc (D) in the absence or presence of chelerythrine. Data are representative of 4-5 independent mice for each group. ***p < 0.0001 when comparing sal+coc group to chel (3 mg)+coc group or chel (10 mg)+coc group in dorsal striatum and NAcc.