Proteomic analysis of protein composition of rat hippocampus exposed to morphine for 10 days; comparison with animals after 20 days of morphine withdrawal

Abstract

Opioid addiction is recognized as a chronic relapsing brain disease resulting from repeated exposure to opioid drugs. Cellular and molecular mechanisms underlying the ability of organism to return back to the physiological norm after cessation of drug supply are not fully understood. The aim of this work was to extend our previous studies of morphine-induced alteration of rat forebrain cortex protein composition to the hippocampus. Rats were exposed to morphine for 10 days and sacrificed 24 h (groups +M10 and −M10) or 20 days after the last dose of morphine (groups +M10/−M20 and −M10/−M20). The six altered proteins (�2-fold) were identified in group (+M10) when compared with group (−M10) by twodimensional fluorescence difference gel electrophoresis (2D-DIGE). The number of differentially expressed proteins was increased to thirteen after 20 days of the drug withdrawal. Noticeably, the altered level of α-synuclein, β-synuclein, α-enolase and glyceraldehyde-3phosphate dehydrogenase (GAPDH) was also determined in both (±M10) and (±M10/ −M20) samples of hippocampus. Immunoblot analysis of 2D gels by specific antibodies oriented against α/β-synucleins and GAPDH confirmed the data obtained by 2D-DIGE analysis. Label-free quantification identified nineteen differentially expressed proteins in group (+M10) when compared with group (−M10). After 20 days of morphine withdrawal (±M10/−-M20), the number of altered proteins was increased to twenty. We conclude that the morphine-induced alteration of protein composition in rat hippocampus after cessation of drug supply proceeds in a different manner when compared with the forebrain cortex. In forebrain cortex, the total number of altered proteins was decreased after 20 days without morphine, whilst in hippocampus, it was increased.

Identification of Morphine and Heroin-Treatment in Mice Using Metabonomics

“…Opiate-use disorders include dependence and addiction, and addiction represents the most serious form of the disorder [ 12 ]. In addition to changes in the basal nucleus and brain regions such as the nucleus accumbens [ 13 , 14 ], hippocampus [ 15 , 16 ] and amygdala [ 17 , 18 ], heroin or morphine addiction also involves reward [ 19 , 20 ], motivation [ 21 , 22 ], learning and memory [ 23 , 24 ] and changes the coupling among salience, default mode, and executive control networks [ 25 , 26 ]. However, heroin or morphine addiction is an uncontrolled, chronic, and recurrent encephalopathy that lacks specific and characteristic biomarkers for diagnosis and treatment.…”

Repeated Administration of Clinically Relevant Doses of the Prescription Opioids Tramadol and Tapentadol Causes Lung, Cardiac, and Brain Toxicity in Wistar Rats

“…Other opioid exposure-studies reiterate such inconsistencies, as α-synuclein protein levels decrease in human serum [ 133 ], but increase in brain paranigral nucleus and substantia nigra ventral part after chronic heroin use [ 134 ]. In turn, α-synuclein protein levels increase in neuroblastoma cells after chronic exposure to morphine [ 135 ], as well as in rat forebrain cortex upon a 10-day exposure to the same drug [ 136 ], but decrease in rat hippocampus under the same conditions [ 137 ]. Therefore, a combination of brain area-specific phenomena and posttranslational mechanisms regulating protein stability might account for discrepancies between α-synuclein gene and protein expression levels.…”

Alterations in the Proteome and Phosphoproteome Profiles of Rat Hippocampus after Six Months of Morphine Withdrawal: Comparison with the Forebrain Cortex

“…Rats (8 weeks of age) were exposed to increasing doses of morphine (dissolved in 0.9% NaCl) for 10 days (10–50 mg/kg) in parallel with corresponding control animals according to our previously established protocols [ 5 , 12 , 14 , 15 , 16 , 17 , 28 ] approved by the Ministry of Education, Youth and Sports of the Czech Republic (license number MSMT-1479/2019–6). Male Wistar rats were housed in the group of 3 per plastic cage on a 12/12 light/dark cycle.…”

“…We applied 2D electrophoretic proteomic approach accompanied by label-free quantification to analyze the altered proteins in the rat brain after 10-day morphine administration followed by protracted drug abstinence [ 14 , 15 , 16 , 17 ]. The identified proteins were mainly involved in the change of energy metabolism, regulation of the cytoskeleton, signal transduction, oxidative stress pathways, and apoptotic pathways.…”

“…Interestingly, we obtained the opposite results in the rat brain cortex and hippocampus. The number of altered proteins was decreased in the cortical samples and increased in the hippocampus after 20 days of morphine withdrawal [ 16 ]. Proteomic and phosphoproteomic comparison of the four rat brain parts (cortex, hippocampus, striatum, and cerebellum) isolated from animals after 3 months of drug abstinence demonstrated that withdrawal symptoms may last for weeks or even longer [ 17 ].…”