Mitragynine Attenuates Withdrawal Syndrome in Morphine-Withdrawn Zebrafish

A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway

Effect of two weeks chronic morphine treatment (1.5 mg/L), 24-h morphine withdrawal and morphine withdrawal in presence of mitragynine (1 mg/L or 2 mg/L) on whole-body cortisol (µg/g fish).

<p>Data are presented as mean ± SEM. (Control, n = 16; morphine treatments, n = 14; morphine withdrawal, n = 21; 1.0 mg/L Mitragynine, n = 23; 2 mg/L Mitragynine, n = 20). Mean values were subjected to one way ANOVA and where applicable, ranked by Tukey test (P<0.05); values with same letter are not significantly different.</p

Effect of two weeks chronic morphine treatment (1.5 mg/L), 24-h morphine withdrawal and morphine withdrawal in presence of 2 mg/L mitragynine on the mRNA expression of CRF-R1, CRF-R2 and PDYN in zebrafish.

<p>Data are presented as mean ± SEM of three independent experiments. Mean values were subjected to one way ANOVA and where applicable, ranked by Tukey test (P<0.05); mean percentage values with same letter are not significantly different.</p

Primer sequences used for semi-quantitative real time PCR gene expression study of corticotropin-releasing factor type 1 receptor, corticotropin-releasing factor type 2 receptor and prodynorphin.

<p>Each sequence is listed starting from the 5′-end and its corresponding sequence ID are provided. F, forward primer; R, reverse primer.</p

Effects of morphine treatment, morphine withdrawal and mitragynine on swimming behavior in adult zebrafish using the novel tank diving test.

<p>(A). Effect of two weeks chronic morphine treatment (1.5 mg/L), 24 h morphine withdrawal and morphine withdrawal in presence of 2 mg/L mitragynine on swimming behavior of zebrafish. (Control, n = 17; morphine treatments, n = 25; morphine withdrawal, n = 22; morphine withdrawal+mitragynine, n = 22). All data were presented in mean ± SEM. Mean values were subjected to one way ANOVA and where applicable, ranked by Tukey test (P<0.05); mean values with same letter are not significantly different. (B) Effect of 20 min acute mitragynine treatment (1 mg/L and 2 mg/L) on swimming behavior of zebrafish. (Control, n = 21; 1 mg/L mitragynine, n = 20; 2 mg/L mitragynine, n = 21). All data were presented in mean ± SEM. Mean values were subjected to one way ANOVA and where applicable, ranked by Tukey test (P<0.05); mean values with same letter are not significantly different.</p

Conditioned Place Preference (CPP) test for morphine in adult zebrafish.

<p>(A) Baseline preference of fish between dotted vs. white compartment as shown by percentage of initial time spend in compartment (n = 23). (B) Preference behavior responses in the CPP tanks for control group as shown by percentage of time spent in conditioning compartment before (white bar) and after (grey bar) conditioning. (n = 15). (C) Place preference behavior of adult zebrafish treated different morphine concentration (Control, n = 15; 0.5 mg/L, n = 18; 1.5 mg/L, n = 16; 3 mg/L, n = 15). Mean values were subjected to one way ANOVA and where applicable, ranked by Tukey test (P<0.05); mean percentage values with same letter are not significantly different.</p