Stress-Induced Sensitization to Cocaine: Changes in the Actin Cytoskeleton from Nucleus Accumbens
Esparza, M.A.; Garcia Keller, C.; Virgolini, M.; Cancela L.M.
IFEC-CONICET, Universidad Nacional de Córdoba, Argentina
Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba. CP:5000
Introduction
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 Nucleus Accumbens.
Material and Methods
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 nucleus accumbens was dissected. After subcellular fractionation by differential centrifugation to separate the F-actin from G-actin ( Toda et al., 2006), the immunoreactivity of the actin (1:500, Santa Cruz), homer 1 b/c (1:100, Santa Cruz), Arp2 (1:100, Santa Cruz), p-cofilin (1:100, a gift from Dr. J. Bamburg), PSD 95 (1:250; Santa Cruz Biotechnology, Inc.) and p-cortactin (1: 200; Santa Cruz Biotechnology, Inc.) was detected by Western blot, using tubulin (1:2,000, Sigma) as a loading control. Locomotor activity was monitored in a photocell apparatus (actographs). Motor activity was quantified as total photocell counts. For these sessions, animals were allowed to habituate to the activity chambers for 60 min before latrunculin A or DMSO (1%) microinjections, which were followed by cocaine (15 mg/kg, i.p.) or saline, and behavior recorded was for 120 min over 10 min interval-bins.
Results
Our experiments revealed that repeated stress induces changes in protein levels involved in synaptic plasticity, such as actin and proteins that regulate actin cytoskeleton. Specifically, a decrease in NAc F-actin levels was observed twenty-one days after repeated stress exposure followed in the saline injection group, compared to all other groups. Thus, stress lowered F-actin levels and acute cocaine administration restored F-actin values to the levels observed in non-stress animals. The stress-induced decrease in F-actin levels may arise from a pronounced decrease in p-cofilin. Binding of the ADF/cofilin family of proteins to F-actin promotes filament disassembly, and actin binding by cofilin is terminated by phosphorylation (Ono, 2003). Thus, a decrease in p-cofilin may increase F-actin depolymerization, thereby reducing F-actin in the stressed animals. Along these lines, cortactin is known to activate the Arp2/3 complex which promotes the nucleation of the new actin filaments from F-actin, and this action of cortactin is inhibited by phosphorylation (Lua and Low, 2005. Thus, the cocaine-induced reduction of p-cortactin in stress-pretreated subjects may contribute to the restoration of F-actin. Latrunculin A binds to G-actin thereby preventing its polymerization into F-actin (Morton et al., 2000), and Interestingly the cross-sensitization between repeated stress pretreatment and cocaine was prevented by intra-accumbens lantrunculin A (0.5 µg/µl) administered 5 min before saline or cocaine.
Conclusions
This is the first evidence that increased actin cyling in the NAc is one of the shared underpinning molecular mechanisms of stress and drug-induced sensitization to cocaine.
References
Lua BL, Low BC (2005) Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control. FEBS Lett 579:577-585.
Morton WM, Ayscough KR, McLaughlin PJ (2000) Latrunculin alters the actin-monomer subunit interface to prevent polymerization. Nat Cell Biol 2:376-378.
Ono S (2003) Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin-interacting protein 1: new blades for twisted filaments. Biochemistry 42:13363-13370.
Toda S, Shen HW, Peters J, Cagle S, Kalivas PW (2006) Cocaine increases actin cycling: effects in the reinstatement model of drug seeking. J Neurosci 26:1579-1587.
