Impact of three-month morphine withdrawal on rat brain cortex, hippocampus, striatum and cerebellum: proteomic and phosphoproteomic studies

https://doi.org/10.1016/j.neuint.2021.104975Get rights and content

Highlights

  • The effect of 3-month morphine withdrawal was tested in selected brain regions.

  • The rat brain cortex, hippocampus, striatum and cerebellum were analyzed.

  • The complex proteomic and phosphoproteomic analysis was performed.

  • The phosphoproteomic analysis revealed the largest changes in the rat hippocampus.

  • The majority of altered proteins were functionally related to energy metabolism.

Abstract

Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes.

The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus.

Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.

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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