Abstract
Every year, approximately 30 children are born from mothers who receive treatment for opioid dependence. Opioid exposure of the foetus during pregnancy has been linked to several effects on the central nervous system, such as developing autism and ADHD, but the exact mechanisms of neuronal development need more research. Pregnant women are rarely included in clinical studies, as they are considered a vulnerable group. Therefore, it is important to use good animal models for safety-pharmacological research. In vitro studies were conducted to study the effects of methadone and morphine on the neuronal development of chicken cerebellar granule neurons and PC12 cells. Viability was studied using an MTT assay, and the effects of the opioids were studied in the presence and absence of ANA-12 and TAT-Pep5, which are inhibitors of important signalling pathways promoting neuronal survival and apoptosis. Neurite outgrowth and synaptogenesis were studied using live-cell imaging and high-content imaging. The expressions of the genes MOR, DOR, KOR, PENK, PDYN, BDNF, CREB1, GluN2B and CYP3A4 were studied using real- time qPCR. MOR, DOR, KOR and GluN2B encode receptors and receptor subunits involved in opioid signalling and are linked to processes in neurodevelopment. PENK and PDYN encode precursors of endogenous opioid peptides. BDNF and CREB1 are involved in signalling through the TrkB receptor, which is linked to important processes in neurodevelopment. CYP3A4 encode the main metabolising enzyme of methadone. In ovo injections were conducted to study in vivo distribution of methadone and its main metabolite EDDP in the brain, lungs, and yolk of the chicken embryo. This was done to advance our understanding of the distribution in the chicken model, with the goal of enhancing its utility in safety-pharmacological studies. Neurite outgrowth was unaffected by methadone and morphine at therapeutic concentrations, but 100 μM methadone was toxic for both the PC12 cells and the chicken granule neurons. In the presence of ANA-12, the toxic effect of methadone was exacerbated. Therapeutic concentrations of the opioids did not affect the expression of the genes studied. However, 100 μM methadone increased the expression of MOR, PDYN, and CYP3A4, and decreased the expression of PENK and GluN2B. These findings should be validated by conducting western blots and additional real-time qPCR in future research, as these changes can cause neurodevelopment consequences. In vivo, the uptake of methadone into the brain, lungs and V yolk happened rapidly, and the main metabolite, EDDP, reached the lungs at higher concentrations than the brain initially. Additionally, there seemed to be an accumulation of methadone and EDDP in the yolk.