Impact of three-month morphine withdrawal on rat brain cortex, hippocampus, striatum and cerebellum: proteomic and phosphoproteomic studies
Introduction
Among opioid drugs, morphine is considered one of the most powerful painkillers used for clinical pain treatment (Listos et al., 2019). Repeated use of opioids leads to adaptations in the brain and other organs, such as heart, gastrointestinal and respiratory system (Kosten et al., 2019). Abrupt cessation of morphine administration results in development of characteristic withdrawal symptoms: abdominal pain, respiratory depression, cramps, tremor, insomnia, sweating, heart pounding, diarrhea, and emesis (Kosten et al., 2019; Listos et al., 2019).
While there are many studies regarding chronic morphine treatment and short-term drug abstinence (Bierczynska-Krzysik et al., 2006; Li et al., 2006; Ujcikova et al., 2014), only little is known about protracted opioid withdrawal and its symptoms which may last for weeks or even longer. Moreover, experimental data focused on the effect of protracted morphine withdrawal on the protein expression in different brain regions are completely lacking.
Our previous work was focused on identification of morphine-altered proteins in rats treated with morphine for 10 days (groups + M10, −M10) and in rats treated with morphine for 10 days and subsequently nurtured for 20 days without this drug (groups + M10/−M20, −M10/−M20). In the forebrain cortex, the number of altered proteins decreased from 28 (±M10) to 14 (±M10/−M20) when determined in CBB-stained 2D gels, or from 113 to 19 when determined by label-free quantification (LFQ), (Ujcikova et al., 2016). Importantly, in the hippocampus, we obtained the opposite results (Ujcikova et al., 2020). The number of proteins with significantly changed expression level increased from 6 (±M10) to 13 (±M10/−M20), when determined by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), or from 19 to 20, when determined by LFQ analysis (Ujcikova et al., 2020). These data indicated that different brain regions are diversely affected by chronic morphine treatment followed by 20-day drug abstinence.
As outlined above, there is a substantial research gap in proteome changes proceeding during the long-lasting opioid withdrawal. Therefore, the aim of our present work was to study the effect of 3-month morphine withdrawal on the protein composition in the rat brain cortex, hippocampus, striatum, and cerebellum, as these brain regions represent the previously well-studied targets of morphine action (Bierczynska-Krzysik et al., 2006; Shen et al., 2016; Miquel et al., 2016). In parallel samples, we characterized the change in the spectrum of phosphorylated proteins, because up-to-now, no data have been published on phosphoproteome profiling in different rat brain regions during protracted morphine withdrawal. Phosphorylated proteins were separated by 2D electrophoresis (2D-ELFO) and detected by the Pro-Q® Diamond Phosphoprotein Gel Stain (Invitrogen™), which is suitable for the fluorescence detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in SDS-PAGE gels (Schulenberg et al., 2004).
Section snippets
Chemicals
Acrylamide and bis-acrylamide were from SERVA (Heidelberg, Germany). Immobiline DryStrips, Pharmalyte buffer (broad pH range 3–10) and Immobiline DryStrip cover fluid were purchased from GE Healthcare (Piscataway Township, NJ). Pro-Q® Diamond Phosphoprotein Gel Stain was from Invitrogen™ (Carlsbad, CA, USA). All other chemicals were of the highest purity available and purchased from Sigma-Aldrich (St. Louis, USA).
Morphine treatment and withdrawal of experimental animals
Young adult male Wistar rats (weighting 250–280 g) were kept on 12/12 light/dark
Phosphoprotein separation and Pro-Q® diamond staining
Images of 2D gels were acquired on a laser scanner (Molecular Imager Fx, Bio-Rad) following Pro-Q® Diamond Phosphoprotein Gel Stain (Invitrogen™) as described in Methods. The PDQuest™ software (Bio-Rad) was applied to analyze 2D gels. The Student's t-test was used to calculate significant differences in relative abundances of protein spots in the samples. Protein levels showing significant quantitative difference (p ≤ 0.05) were selected for mass spectrometric analysis. P-values were calculated
Discussion
Protracted abstinence can be associated with persisting behavioral and neurobiological abnormalities that are responsible for drug-seeking reinstatement (Stinus et al., 2000, 2012). However, these issues have not been addressed systematically before. Therefore, in this work, we studied protein alterations in the selected brain regions (rat brain cortex, hippocampus, striatum and cerebellum) 3 months after cessation of 10-day morphine treatment. In parallel, we applied Pro-Q® Diamond staining to
Conclusions
To the best of our knowledge, this is the first complex proteomic analysis performed simultaneously in the rat brain cortex, hippocampus, striatum, and cerebellum after 3 months since morphine withdrawal. Analysis of phosphoproteome revealed by far the largest change in the hippocampus – 14 phosphoproteins. The number of altered phosphoproteins in the forebrain cortex (5), striatum (4) and cerebellum (3) was much lower. The total protein composition determined in 2D gels and by subsequent LFQ
Author statement
Hana Ujcikova: Conceptualization, Investigation, Methodology, Formal analysis, Visualization, Writing-original draft Lucie Hejnova: Investigation, Formal analysis Adam Eckhardt: Investigation, Formal analysis Lenka Roubalova: Formal analysis Jiri Novotny: Conceptualization, Project administration, Supervision Petr Svoboda: Project administration, Supervision, Writing-original draft.
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the Czech Science Foundation GA CR [19-03295S] and by the Institute of Physiology of the Czech Academy of Sciences [RVO:67985823]. The authors thank to Karel Harant and Pavel Talacko (from the Proteomics Core Facility, BIOCEV, Faculty of Science, Charles University in Prague) for performing label-free quantification. This work used instruments provided by C4Sys infrastructure.
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