Cocaine-induced behavioral sensitization is associated with changes in the expression of endocannabinoid and glutamatergic signaling systems in the mouse prefrontal cortex

Abstract

Background: Endocannabinoids modulate the glutamatergic excitatory transmission by acting as retrograde messengers. A growing body of studies has reported that both signaling systems in the mesocorticolimbic neural circuitry are involved in the neurobiological mechanisms underlying drug addiction.

Methods: We investigated whether the expression of both endocannabinoid and glutamatergic systems in the prefrontal cortex (PFC) were altered by an acute and/or repeated cocaine administration schedule that resulted in behavioral sensitization. We measured the protein and mRNA expression of the main endocannabinoid metabolic enzymes and the cannabinoid receptor type 1 (CB1). We also analyzed the mRNA expression of relevant components of the glutamate-signaling system, including glutamate-synthesizing enzymes, metabotropic receptors, and ionotropic receptors.

Results: Although acute cocaine (10 mg/kg) produced no significant changes in the endocannabinoid-related proteins, repeated cocaine administration (20 mg/kg daily) induced a pronounced increase in the CB1 receptor expression. In addition, acute cocaine administration (10 mg/kg) in cocaine-sensitized mice (referred to as cocaine priming) induced a selective increase in the endocannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). These protein changes were accompanied by an overall decrease in the ratios of endocannabinoid synthesis/degradation, especially the N-acyl phosphatidylethanolamine phospholipase D/FAAH and diacylglycerol lipase alpha/MAGL ratios. Regarding mRNA expression, while acute cocaine administration produced a decrease in CB1 receptors and N-acyl phosphatidylethanolamine phospholipase D, repeated cocaine treatment enhanced CB1 receptor expression. Cocaine-sensitized mice that were administered priming injections of cocaine mainly displayed an increased FAAH expression. These endocannabinoid changes were associated with modifications in glutamatergic transmission-related genes. An overall decrease was observed in the mRNA expression of the glutamate-synthesizing gene kidney-type glutaminase (KGA), the metabotropic glutamate receptors (mGluR3 and GluR), and subunits of NMDA ionotropic receptors (NR1, NR2A, NR2B and NR2C) after acute cocaine administration, while mice repeatedly exposed to cocaine only displayed an increase in NR2C. However, in cocaine-sensitized mice primed with cocaine, this inhibition was reversed and a strong increase was detected in the mGluR5, NR2 subunits, and both GluR1 and GluR3.

Conclusions: These findings indicate that cocaine sensitization is associated with an endocannabinoid downregulation and a hyperglutamatergic state in the PFC that, overall, contribute to an enhanced glutamatergic input into PFC-projecting areas.

Keywords: cannabinoid; cocaine; glutamate; prefrontal cortex.; sensitization.

Figure 1.

Acute and repeated cocaine injections on cocaine-induced sensitization in mice. (A) Schematic representation of the 5-phase-paradigm for acquisition and expression of cocaine-induced sensitization. (B) Effects of acute and repeated cocaine injections on the distance traveled by mice for 30min measured in the open field during cocaine sensitization. Bars represent the mean + SEM (n = 8 animals per group). Post hoc analysis: *p < 0.001 versus the vehicle-vehicle group; ^# p < 0.001 versus the vehicle-cocaine group; ^$ p < 0.001 versus the cocaine-vehicle group. OF: open field; CL: cocaine-conditioned locomotion; CS: cocaine-induced sensitization.

Figure 2.

Effects of cocaine-induced sensitization on the expression of endocannabinoid signaling-related proteins in the mouse PFC. (A) Representative immunoblots of endocannabinoid signaling-related proteins after repeated pretreatment (vehicle or cocaine) and acute treatment (vehicle or cocaine). Relative protein levels: (B) CB1 receptor; (C) NAPE-PLD; D) FAAH; (E) DAGLα; (F) DAGLβ; and (G) MAGL. Ratios between the synthesis and degradation enzymes: (H) NAPE-PLD/FAAH; (I) DAGLα/MAGL; and (J) DAGLβ/MAGL. Bars represent the mean + SEM (n = 8). Two-way ANOVA: ^& p < 0.05 and ^&& p < 0.01. Post hoc analysis: *p < 0.05 and **p < 0.01 versus the vehicle-vehicle group; ^# p < 0.05 versus the vehicle-cocaine group; ^$ p < 0.05 versus the cocaine-vehicle group. V-V: vehicle-vehicle group; V-C: vehicle-cocaine group; C-V: cocaine-vehicle group; C-C: cocaine-cocaine group; ns: non-significant.

Figure 3.

Effects of cocaine-induced sensitization on the gene expression of the endocannabinoid signaling-related components in the mouse PFC. Relative mRNA levels of the endocannabinoid signaling system were evaluated following a repeated pretreatment (vehicle or cocaine) and acute treatment (vehicle or cocaine): (A) CB1 receptor; (B) NAPE-PLD; (C) FAAH; (D) DAGLα; (E) DAGLβ; and (F) MAGL. Bars represent the mean + SEM (n = 8). Two-way ANOVA: ^& p < 0.05 and ^&& p < 0.01. Post hoc analysis: *p < 0.05 versus the vehicle-vehicle group; ^# p < 0.05 and ^## p < 0.01 versus the vehicle-cocaine group; ^$ p < 0.05 versus the cocaine-vehicle group. ns: non-significant.

Figure 4.

Effects of cocaine-induced sensitization on the gene expression of the glutamatergic signaling-related components in the mouse PFC. Relative mRNA levels of the glutamate signaling system were evaluated following a repeated pretreatment (vehicle or cocaine) and acute treatment (vehicle or cocaine): (A) LGA; (B) KGA; (C) mGluR3; (D) mGluR5; (E) NR1; (F) NR2A; (G) NR2B; (H) NR2C; (I) GluR1; (J) GluR2; (K) GluR3; and (L) GluR4. Bars represent the mean + SEM (n = 8). Two-way ANOVA: ^& p < 0.05 and ^&& p < 0.01. Post hoc analysis: *p < 0.05 and **p < 0.01 versus the vehicle-vehicle group; ^# p < 0.05 and ^## p < 0.01 versus the vehicle-cocaine group. ns: non-significant.