Cocaine Self‐administration Modulates Brain Ceramide Metabolism

Abstract

Introduction Chronic cocaine exposure produces neuroadaptations of many plasma membrane integrated proteins including dopamine transporters that contribute to relapse; however, the mechanisms remain elusive. A few studies show that chronic cocaine exposure alters blood levels of lipids including fatty acids and cholesterol and these changes are correlated with vulnerability to relapse. To date, little information is available on chronic cocaine modulation of brain lipids. Neuronal membrane predominantly consists of sphingolipids along with cholesterol and glycerophospholipids. Among sphingolipids, ceramide is a key membrane structural component and highly concentrated. Ceramide not only influences membrane rigidity but also acts as a signaling molecule. A change in ceramide content in cultured cells alters the activity of dopamine transporters. The purpose of this project was to investigate if cocaine self-administration modulated brain ceramide content, which may underlie functional changes of dopamine transporters. Methods Male Sprague-Dawley rats were trained and allowed to self-administer cocaine (1.5 mg/kg/infusion, 40 injections/6hrs/day) for 5 days. Rat prefrontal cortex (PFC) and striatum were dissected 18 hrs after the last cocaine infusion along with saline controls. Samples underwent a Bligh & Dyer method of lipid extraction. A triple quadrupole mass spectrometer was used to measure the content of ceramide species (C16, C18, C20 and C24) in these brain regions. A 50 × 2.0 mm, 3 µm reversed-phase column was used with a flow rate set to 0.8 mL/min. Samples underwent a five minute gradient consisting of mobile phase A (0.1% formic acid and 25 mM ammonium acetate in water) and mobile phase B (100% acetonitrile). The C17 ceramide, not present in the brain, was used as an internal standard for data analysis. Moreover, the expression and activity of enzymes involved in ceramide metabolism including neural sphingomyelinase 2, which converts sphingomyelin to ceramide on the membrane, and ceramide synthesis 1 & 2, which are primarily responsible for the synthesis of brain ceramide species, were analyzed by western blot and activity assay. Results We optimized lipid extraction method and mass spectrometry condition so that ceramide species in the brain can be clearly identified and quantitated. We found that cocaine SA alters brain ceramide content in a brain region and species-dependent manner. The C18 ceramide was confirmed as the most abundant ceramide species compared to the others. Cocaine SA also changed the expression and/or the activity of sphingomyelinase 2, ceramide synthesis 1&2. The present study provides evidence that cocaine SA regulates brain ceramide content by influencing ceramide synthesis in both sphingomyelin hydrolysis and de novo synthesis pathways. Our data suggests that cocaine SA modulates brain ceramide homeostasis. Given the important role of ceramide in regulation of membrane fluidity and acting as a signaling molecule, brain ceramide remodeling highlights a potential new mechanism underlying chronic cocaine-mediated functional changes of membrane proteins.