Plasticity of L-type Ca2+ channels after cocaine withdrawal

Abstract

Increased reactivity of certain frontal cortical brain regions to cocaine re-exposure or drug-associated cues in cocaine-abstinent human addicts is linked to drug craving. Similarly, in rats tested after withdrawal from repeated cocaine exposure, cocaine or other strong excitatory stimuli produce greater activation of pyramidal neurons in the medial prefrontal cortex (mPFC). Our recent findings indicate that the increased mPFC neuronal activation depends primarily upon enhanced voltage-sensitive Ca(2+) influx, most likely through high-voltage activated (HVA) L-type Ca(2+) channels, but the mechanism underlying the enhanced Ca(2+) currents is unknown. In this study, we used a protein crosslinking assay to show that repeated cocaine injections, resulting in behavioral sensitization, increased total protein levels and cell surface expression of HVA-Ca(v)1.2 L-type channels in pyramidal neurons in deep layers of the mPFC. These changes in Ca(v)1.2 L-channels were time dependent and subtype specific (i.e., differed from those observed for Ca(v)1.3 L-channels). Furthermore, we found enhanced PKA activity in the mPFC of cocaine-sensitized rats that persisted for 21 days after withdrawal. PKA phosphorylation of L-channels increases their activity, so Ca(2+) currents after cocaine withdrawal could be enhanced as a result of both increased activity and number of HVA-Ca(v)1.2 L-channels on the cell surface. By increasing the suprafiring threshold excitability of mPFC pyramidal neurons, excessive upregulation of HVA L-channel activity and number may contribute to the cortical hyper-responsiveness that enhances vulnerability to cocaine craving and relapse. More generally, our results are the first to demonstrate that repeated cocaine exposure alters the membrane trafficking of a voltage-sensitive ion channel.

Figure 1

Repeated cocaine administration induces behavioral sensitization. A challenge injection of cocaine (COC; 15 mg/kg) elicited a significantly greater locomotor response (measured as ambulation counts) in COC-pretreated compared to SAL-pretreated rats after a 3-day withdrawal (rmANOVA, p<0.05).

Figure 2

Repeated cocaine treatment increases PKA activity in the rat mPFC. A. Shaded areas indicate brain regions used for tissue harvesting. B. PKA activity (reflected by phosphorylation of kemptide) was significantly enhanced in the cocaine-exposed mPFC after a 3-day or 21-day withdrawal (*p<0.05). C. PKA activity in the motor cortex did not differ between SAL- and COC-pretreated rats (all p>0.05). p-Kemptide indicates phosphorylated Kemptide (see Materials and Methods for details).

Figure 3

Repeated cocaine administration produces a time-dependent increase in protein levels and/or surface expression of Ca_v1.2 α_1C and Ca_v1.3 α_1D L-type Ca²⁺ channels in the mPFC. Unmodified (intracellular) forms of these channels were detected as bands of ~210 and ~250 kDa, respectively, consistent with many prior studies (Ca_v1.2:Davare et al., 2001; Haller et al., 2008; Tippens et al., 2008; Ca_v1.3: Qu et al., 2005; Kim et al., 2007; Yu et al., 2008). A. Preabsorption of antibodies with their respective peptide antigens eliminated specific staining assessed at exposure times equivalent to those utilized below. B-C. Western blots of non-crosslinked (N) and BS³-crosslinked (X) tissues. Repeated cocaine significantly increased total and intracellular levels of HVA-Ca_v1.2 α1 subunits in mPFC after a 3-day withdrawal (**p<0.01). Total and surface expression of Ca_v1.2 α_1C subunit increased after a 21-day withdrawal (*p<0.05). D-E. Expression of Ca_v1.3 α_1D subunits in mPFC was unchanged after a 3-day withdrawal from cocaine (p>0.05), while delayed increases in total and intracellular levels occurred after a 21-day withdrawal (*p<0.05, **p<0.01).