Analysis of rat brain lipids and metabolites after antidepressant drug treatment

Depression is a major debilitating disorder and the key aim of the current project was to investigate some of the molecular/biochemical mechanisms of antidepressant drugs with an emphasis on the relatively unexplored role of sphingolipids. For this purpose, the current study used two antidepressant drugs: the tricyclic antidepressant desipramine, and the selective serotonin re-uptake inhibitor paroxetine. The effects of the drugs in rat brain regions implicated in depression: the prefrontal cortex (PFC), hippocampus (HP) and striatum (ST) were investigated both acutely (single administration) and chronically (daily treatment for 15 days). In Chapter Three, ¹H NMR spectroscopy was used to explore the metabolic response of acute and chronic administration of desipramine and paroxetine. These experiments showed significant changes in a number of water-soluble metabolites (i.e. N-acetylaspartylglutamate, glutamate, glutamine, lactate and creatine) following acute but not chronic treatment of the drugs. Sphingolipids including ceramide and its main metabolite sphingosine are key modulators of numerous cellular functions and in Chapter Four, it was shown by using liquid chromatography with mass spectrometry (LC-MS) that chronic but not acute administration of the antidepressant drugs decreased sphingosine levels in the HP and PFC but not in the ST. The effect of the drugs (e.g. paroxetine) on ceramide levels was also tested (HP only) by benzoylation of ceramide using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and in Chapter Four, it was shown that hippocampal levels of ceramide were as for sphingosine decreased by chronic paroxetine treatment. This chapter also demonstrated a highly significant correlation for the two sphingolipids in both controls and drug-treated animals. In Chapter Five , the effect of chronic paroxetine and desipramine administration was investigated on gene expression for two key enzymes of the brain sphingolipid pathway namely, acid sphingomyelinase (ASM) and acid ceramidase (AC). By using real-time quantitative polymerase chain reaction (RT-qPCR) it was shown that paroxetine and desipramine significantly reduced mRNA levels of ASM in the HP while effects in the PFC and ST did not reach significance. Similar effect was seen for desipramine but not paroxetine on mRNA levels for AC in the HP. Recent studies indicate that ceramide modifies monoaminergic neurotransmission. In Chapter Six, the effect of carmofur, a potent inhibitor of acid ceramidase (AC) was investigated on monoamine neurotransmitters levels and their corresponding metabolites in rat brain regions by using HPLC with electrochemical detection (HPLC-ECD). Carmofur significantly increased 5-HT and decreased its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in tissue samples from the PFC, HP and ST. In contrast, carmofur failed to significantly alter brain levels of dopamine, noradrenaline and the dopamine metabolite 3, 4- dihydroxyphenylacetic acid (DOPAC). In conclusion, findings of this project are supportive of a putative role for sphingolipids in the mechanism of action by antidepressant drugs. The potential clinical significance of these findings requires further studies.