Contrasting the Role of xCT and GLT-1 Upregulation in the Ability of Ceftriaxone to Attenuate the Cue-Induced Reinstatement of Cocaine Seeking and Normalize AMPA Receptor Subunit Expression

Abstract

Long-term treatment with ceftriaxone attenuates the reinstatement of cocaine seeking while increasing the function of the glutamate transporter 1 (GLT-1) and system xC- (Sxc) in the nucleus accumbens core (NAc). Sxc contributes the majority of nonsynaptic extracellular glutamate in the NAc, while GLT-1 is responsible for the majority of glutamate uptake. Here we used antisense to decrease the expression of GLT-1 and xCT (a catalytic subunit of Sxc) to determine the relative importance of both proteins in mediating the ability of ceftriaxone to prevent cue-induced reinstatement of cocaine seeking and normalize glutamatergic proteins in the NAc of rats. Intra-NAc xCT knockdown prevented ceftriaxone from attenuating reinstatement and from upregulating GLT-1 and resulted in increased surface expression of AMPA receptor subunits GluA1 and GluA2. Intra-NAc GLT-1 knockdown also prevented ceftriaxone from attenuating reinstatement and from upregulating xCT expression, without affecting GluA1 and GluA2 expression. In the absence of cocaine or ceftriaxone treatment, xCT knockdown in the NAc increased the expression of both GluA1 and GluA2 without affecting GLT-1 expression while GLT-1 knockdown had no effect. PCR and immunoprecipitation of GLT-1 revealed that ceftriaxone does not upregulate GLT-1 and xCT through a transcriptional mechanism, and their coregulation by ceftriaxone is not mediated by physical interaction. These data support important and distinct roles for xCT and GLT-1 in the actions of ceftriaxone and add to a body of literature finding evidence for coregulation of these transporters. Our results also point to xCT expression and subsequent basal glutamate levels as being a key mediator of AMPA receptor expression in the NAc. Ceftriaxone attenuates the reinstatement of cocaine, alcohol, and heroin seeking. The mechanism of action of this behavioral effect has been attributed to glutamate transporter 1 (GLT-1) and xCT (a catalytic subunit of Sxc)/Sxc upregulation in the nucleus accumbens core. Here we used an antisense strategy to knock down GLT-1 or xCT in the nucleus accumbens core and examined the behavioral and molecular consequences. While upregulation of both xCT and GLT-1 are essential to the ability of ceftriaxone to attenuate cue-induced reinstatement of cocaine seeking, each protein uniquely affects the expression of other glutamate receptor and transporter proteins. We also report that reducing basal glutamate levels through the manipulation of xCT expression increases the surface expression of AMPA receptor subunits, providing insight to the mechanism by which cocaine alters AMPA surface expression.

The effects of ceftriaxone on cue-primed reinstatement of cocaine-seeking in male and female rats: estrous cycle effects on behavior and protein expression in the nucleus accumbens

“…3 b, c). While we have consistently found cocaine-induced decreases in GLT-1 and xCT expression in male rats when blotting in a membrane fraction (Knackstedt et al 2010a; LaCrosse et al 2016; Sondheimer and Knackstedt 2011), we recently reported that in the total protein fraction, xCT and GLT-1a are only modestly decreased (Lacrosse et al 2017). While consistent with this previous report, we acknowledge that the lack of a significant reduction in xCT expression in males in the present work may also stem from insufficient sample size.…”

“…These receptors have been mechanistically linked to the reinstatement of cocaine-seeking as the CP-AMPA receptor antagonist Naspm attenuates relapse following both abstinence and extinction from cocaine self-administration (White et al 2016; Conrad et al 2008). We have also previously demonstrated that ceftriaxone normalizes surface GluA1 expression following extinguished cocaine-seeking in male rats and genetic manipulations which prevent ceftriaxone from normalizing GluA1 expression result in a loss of the ability of ceftri-axone to attenuate reinstatement (Lacrosse et al 2017). Surface expression of GluA1 increased during estrus compared to non-estrus, and ceftriaxone-treated rats in estrus at the time of biotinylation displayed increased surface GluA1, potentially explaining the failure of ceftriaxone to attenuate reinstatement.…”

“…We have previously demonstrated that following extinguished cocaine-seeking, male rats display increased surface GluA1 in the NAc in the absence of changes in surface GluA2 expression or total protein expression of either protein (Lacrosse et al 2017). This pattern of expression is characteristic of calcium-permeable (CP)-AMPA receptors (Liu and Zukin 2007), which have a higher conductance and are proposed to partially underlie the increased reactivity of NAc neurons to cocaine-associated cues (Conrad et al 2008).…”

“…Ceftriaxone has no effect on GLT-1 and xCT expression in drug-naïve rats (Knackstedt et al 2010a). Ceftriaxone normalizes the increased total protein expression of the AMPA receptor subunit GluA1 that results from cocaine self-administration followed by abstinence without extinction training (LaCrosse et al 2016), as well as the increased surface expression of GluA1 present 2–3 weeks after extinguished cocaine-seeking (Lacrosse et al 2017). The ability of ceftriaxone to normalize GluA1 expression is dependent on the upregulation of xCT (Lacrosse et al 2017).…”

Corticostriatal network dysfunction in Huntington's disease: Deficits in neural processing, glutamate transport, and ascorbate release

“…Ceftriaxone also improved the motor phenotype in multiple behavioral tests. Importantly, the ceftriaxone‐induced up‐regulation of GLT‐1, which does not involve a transcriptional mechanism, continues to be effective at later ages when R6/2 mice are strongly symptomatic . Thus, the mechanism for increasing GLT‐1 expression remains functional despite a rapidly advancing neuropathology, although it may be too late to rescue a rapidly advancing behavioral phenotype.…”