Stress-induced cocaine sensitization: changes in the actin cytoskeleton.

BOLLATI F *EQUAL CONTRIBUTION; ESPARZA MA *EQUAL CONTRIBUTION; GARCIA KELLER C; VIRGOLINI MB; KALIVAS PW; CANCELA LM

Reno

Encuentro; NIDA International Forum Commonalities Among the Diseases of Addiction.; 2009

Drug addiction is associated with long-term changes in the synaptic function, including the actin cytoskeleton. There is evidence about the proactive influence of stress on drug addiction, a process that is exerted on excitatory synapses by the activation of common mechanisms between drugs and stress. The present study sought to investigate whether the neurobiological mechanisms that modulate repeated cocaine administration also occur in a stress-induced cocaine sensitization model. These experiments were designed to evaluate whether repeated stress induces alterations in actin rearrangement in the striatum and prefrontal cortex. Male Wistar rats (250-350 g) were restrained daily for 2 hours for 7 days. Control rats were left undisturbed in their home cages. Three weeks after the last restraint stress, the animals were decapitated 45 minutes after an injection of saline or cocaine (30 mg/kg i.p.), and the prefrontal cortex and the striatum were dissected. After subcellular fractionation by differential centrifugation to separate the F-actin from G-actin, the immunoreactivity of the F-actin (1:500, Santa Cruz), homer 1 b/c (1:100, Santa Cruz), Arp2 (1:100, Santa Cruz), and p-cofilin (1:100, a gift from Dr. J. Bamburg) was detected by Western blot, using tubulin (1:2,000, Sigma) as a loading control.

Our experiments revealed that a chronic repeated restraint stress which induces sensitization to cocaine also was able to induce an increase in F-actin levels in the prefrontal cortex, whereas any change was observed in the striatum. The increase in F-actin observed in the prefrontal cortex after chronic restraint stress was slightly attenuated following a cocaine injection, although the values did not return to baseline levels after drug injection. Since the p-cofilin levels did not change in the different groups, it is unlikely to attribute the increase in F-actin levels following stress to changes in the p-cofilin activity. Cofilin is an actin- depolymerizing protein and a direct regulator of actin reorganization and dynamics. Its activity is regulated through the phosphorylation at a single site (ser 3) by LIMKs, an event that inhibits the binding to actin monomers and its actin-depolymerizing activity. Given that the same proteins that participate in the modulation of actin cytoskeleton are altered by chronic stress, our experiments aimed to study proteins that regulate actin turnover in the synapses. Since the stress and cocaine-induced changes in Arp2 followed the same pattern to that observed with F-actin, it is likely to relate the increase in F-actin with the influence of these treatments on the polymerizing protein Arp2. We also will evaluate the changes in LIM K1, as well as other actin-binding proteins implicated in the actin cytoskeleton reorganization. Behavioral experiments will determine the relevance of these changes for stress-induced sensitization to cocaine.