Subjects recruitments and Samplings periods:
The present study is six months follow-up of addicts who registered for taper treatments in congress 60. In the beginning, 200 male subjects with opium addiction that were registered in congress 60, have been recruited. At the end of the study, 79 opium-addicted individuals who completed all the examinations before and after six months period of taper off-treatment were included in the data analysis. Twenty non-psychiatric male subjects from family members of addicts were recruited for the control group. The Control group completed only the first time examinations. Subjects with IQ total score (Wechsler Abbreviated Intelligence Scale, 1999) lower than < 80 were excluded from the study. The control group selected with age, race, somatic properties, socioeconomic situation, familial situation, and education matched to patients group and with no history of any psychological or somatic problem in their family. None of the subjects had a current or history of any severe medical condition, neurological disorder, history of head trauma with loss of consciousness, and any psycho-stimulant drug abuse and alcohol dependence. For better presentation of data test groups shoed by this order: N (normal group), C (addicts before admission in congress 60), and PC (addicts after six months of treatment in congress 60). All subjects have explained the purpose of the study and next, written informed consent has been provided.
N-Back and spatial N-Back:
The n-back and spatial n-Back tasks are a continuous performance task that is commonly used to the examination of working memory and spatial working memory respectively. The n-back was introduced in 1958 and meta-analysis have shown that it could be used to analysis of brain region (dorsal cingulate and medial, premotor cortex, lateral posterior parietal, etc.) activities (21). In recent decades spatial n-Back a novel and simple task designed to measure spatial WM in adults and children (22). Participants were presented with a sequence of stimuli, and the task consisted of indicating when the current stimulus was matched the one from n steps earlier in the sequence.
The digit-span task is one of the subsets of the Wechsler Adult Intelligence Scale (WAIS) and was commonly used to measure working memory's number storage capacity in psychiatric disorders and addiction (23, 24). All Subjects were sitting in an isolated room and hear a sequence of numerical digits and asked to recall the sequence correctly, with increasingly longer sequences being tested in each trial. Digit-span tasks were performed forwards and backward, to evaluate the verbal and spatial working memory respectively.
GO no GO:
Go/no Go testing refers to a pass/fail test to evaluate sustained attention and decision-making abilities (25). A computational form of the test had performed and subjects were using binary classification. The test is passed only when the Go condition is met and also the No go condition fails.
Gene expression analysis by quantitative PCR:
Blood (5 ml) was collected from the cubital vein without a tourniquet between 8.00 and 10.00 AM. Total RNA was extracted from peripheral blood samples according to standard protocols using by RNA Purification kit (GeneJET™ RNA Purification Kit#K0732, Thermo scientific, Latvia). The cDNA was synthesized using a Transcription First Strand cDNA Synthesis Kit (RevertAid Premium First Strand cDNA Synthesis Kit #K1652, Thermo scientific, Latvia) according to the manufacturer’s protocol. We performed quantitative RT-PCR by using CFX96 Touch Real-Time PCR Detection System (BIO-RAD, California, United States) and SYBR green kit (Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix (2X) #K0221, Thermo scientific, Latvia) according to a previous study (26). Specific primers for target and reference genes were designed by "oligo7" software and blasted on the NCBI website. The GAPDH gene was used for normalization as an internal control gene.
Lymphocyte pellets were thawed on ice and immediately lyzed in VRL buffer: 50 mM HEPES (pH 7.5), 250 mM sucrose, 5 mM MgCl2,100 mM KAc, 2 mM PMSF (all Sigma–Aldrich, Germany), 2x Pro-tease Inhibitor (Roche, Germany) supplemented with 1% TritonX-100, 1 mM PMSF (Sigma–Aldrich, Germany) and 40 U/mL DNaseI (Roche, Germany). After 40 min on ice, for DNA digestion the lysate was incubated for 40 min at 37°C. Then, the protein content of the lysate was detected by using the DC Protein Assay Kit (Bio-Rad, Germany) and for each sample, 30 micrograms of total protein were loaded onto 10% SDS-PAGE gels. After blotting, the membranes were directly treated with 100 mM KOH for 5 min at room temperature and subsequently blocked with 5% milk in PBS with 0.05% Tween-20 (PBST). Immunoreactivity was tested with the human anti-BDNF Antibody (#ANT-010) and human anti-Serotonin Transporter (SERT) (#AMT-004) that are highly specific antibodies. Analysis was done blind with regard to diagnosis. Protein level analysis was conducted based on previous studies (27).
Descriptive data are expressed as mean ± SD (range) and the level of statistical significance was set at P < 0.05. Compliance with normal distribution for continuous variables was assessed via the Kolmogorov-Smirnov test by using SPSS version 23. One-way ANOVA and Pearson correlation analysis was conducted to determine the relationship between the 2 independent variables.