Post‐cocaine changes in regulator of G‐protein signaling (RGS) proteins in the dorsal striatum: Relevance for cocaine‐seeking and protein kinase C‐mediated phosphorylation

Abstract

Persistent cocaine-induced neuroadaptations within the cortico-striatal circuitry might be related to elevated risk of relapse observed in human addicts even after months or years of drug-free abstinence. Identification of these neuroadaptations may lead development of novel, neurobiologically-based treatments of relapse. In the current study, 12 adult male Sprague-Dawley rats self-administered cocaine (or received yoked-saline) for 2 weeks followed by 3 weeks of home-cage abstinence. At this point, we analyzed expression of proteins involved in regulation of Gαi- and Gαq-protein signaling in the dorsal striatum (dSTR). Animals abstinent from chronic cocaine showed decreased expression of regulator of G-protein signaling 2 (RGS2) and RGS4, as well as upregulation of RGS9. These data, together with the increased ratio of Gαq-to-Gαi proteins indicated, ‘sensitized’ Gαq signaling in the dSTR of abstinent cocaine animals. To evaluate activation of Gαq signaling during relapse, another group of abstinent cocaine animals (and yoked saline controls, 22 rats together) was re-introduced to the cocaine context and PKC-mediated phosphorylation in the dSTR was analyzed. Re-exposure to the cocaine context triggered cocaine seeking and increase in phosphorylation of cellular PKC substrates, including phospho-ERK and phospho-CREB. In conclusion, this study demonstrates persistent dysregulation of RGS proteins in the dSTR of abstinent cocaine animals that may produce an imbalance in local Gαq-to-Gαi signaling. This imbalance might be related to augmented PKC-mediated phosphorylation during relapse to cocaine-seeking. Future studies will address whether selective targeting of RGS proteins in the dSTR can be utilized to suppress PKC-mediated phosphorylation and relapse to cocaine-seeking.

Using rat operant delayed match-to-sample task to identify neural substrates recruited with increased working memory load

“…Finally, as PKC is the major cellular effector downstream from mGlu5 receptors implicated in WM (Dash et al 2007;Runyan et al 2005;Birnbaum et al 2004b), this study analyzed whether the activity of this kinase in the PrL tracks increased WM load (delay). The phosphorylation status of peptide sequences selectively recognized by the PKC served as a measure of PKC-mediated cellular phosphorylation, and ultimately, as an accurate indicator PKC tissue activity (Bilodeau and Schwendt 2016;Chopra et al;Kim et al 2010). We found that phosphorylation of both high-and low-molecularweight PKC substrates was increased in the 24s delay group.…”

“…To further explore possible neurobiological mechanisms recruited during WM task in the PrL, PKC activity in this brain region was evaluated using phospho-(Ser) PKC substrate antibody. This antibody selectively detects phosphorylation state of PKC consensus sites on many cellular proteins, providing an 8 indirect measure of tissue PKC activity (Chopra et al;Kim et al 2010;Bilodeau and Schwendt 2016).…”

“…The tissue protein lysates were prepared from the PrL as described previously (Bilodeau and Schwendt 2016). Total protein content was quantified using bicinchoninic acid (BCA) assay according to the manufacturer's instructions (BCA Protein Assay Kit, Thermo-Fisher, Waltham, MA).…”

Regulators of G Protein Signaling in Analgesia and Addiction

“…of a challenge cocaine dose promotes the expression of RGS4 transcript in the LC and in the dorsal central gray; however, it decreases in RGS4 mRNA levels in the red nucleus and in the reticulotegmental nucleus (Bishop, Cullinan, Curran, & Gutstein, 2002). Cocaine abstinence in rodents leads to a reduction in RGS4 and RGS2 expression in the dorsal striatum (Bilodeau & Schwendt, 2016;Schwendt, Hearing, See, & McGinty, 2007). Cocaine withdrawal does not alter RGS4 or RGS7 levels in the amygdala, the frontal cortex, or the hypothalamic paraventricular nucleus of rats (Carrasco et al, 2003).…”

Using rat operant delayed match-to-sample task to identify neural substrates recruited with increased working memory load

“…To further explore possible neurobiological mechanisms recruited during the WM task in the PrL, PKC activity in this brain region was evaluated using the phospho-(Ser) PKC substrate antibody. This antibody selectively detects the phosphorylation state of PKC consensus sites on many cellular proteins, providing an indirect measure of tissue PKC activity (Kim et al 2010;Bilodeau and Schwendt 2016;Chopra et al 2018). Due to different signal intensity, PKC-phosphorylated proteins with molecular weight >120 kDa (high kDa) were analyzed separately from the <120-kDa proteins (low kDa).…”