Response of Dopaminergic System to Cocaine Exposure, Recovery after Cocaine Abstinence, and Impact of a Long-acting Cocaine Hydrolase

Cocaine produces its physiological effects by targeting multiple proteins in the central nervous system, particularly by binding with dopamine transporter (DAT) to block the normal dopamine (DA) recycling process. This process along with the pleasure feeling would wear off shortly after the abstinence from the drug. However, chronic cocaine use is associated with neuroadaptations in the dopaminergic system, including increases in the density of DAT on plasma membrane of dopaminergic neurons. Given the critical role of DAT in neurotransmission, various studies have been carried out on animal models to learn its structure, function, and distribution. Long-standing evidence supports that DAT presentation on neuron cell surface is highly plastic and is subject to regulated endocytic trafficking – it constitutively internalizes and recycles during the neurotransmission process. Cocaine administration can significantly induce DAT trafficking to the plasma membrane, without significantly changing the total amount of DAT (which is a sum of the intracellular and plasma membrane DAT). One-time use of cocaine will increase the surface DAT expression (via DAT trafficking) for at least a month, while abstinence from chronic use of cocaine would take a longer period of time for striatal DAT distribution to recover in monkey models, as normalization of dopaminergic function is usually a slow process. However, detailed research into the correlation relationship between the DAT trafficking and cocaine dose has not been reported, and the specific role of cocaine abstinence in the dopaminergic pathway recovery was difficult to determine because of the lack of measurements in groups with different abstinence periods. The main purpose of this dissertation research is to determine the effects of cocaine at various doses (administered i.p.) on DAT trafficking to the plasma membrane in comparison with cocaine-induced hyperactivity and brain cocaine concentration in order to determine the threshold cocaine dose and the threshold of brain cocaine concentration that can significantly induce the DAT trafficking and hyperactivity in rats. Further, we studied the time-dependent distribution of striatal DAT in terms of intracellular and plasma membrane fractions after different cocaine abstinence periods following acute or chronic cocaine administrations. In our obtained data, the threshold (i.p.) dose of cocaine which can significantly induce DAT trafficking or hyperactivity should be between 5 and 10 mg/kg. When a cocaine dose is high enough to induce significant hyperactivity, it can also significantly induce DAT trafficking to the plasma membrane. Further, the threshold brain cocaine concentration required to induce significant hyperactivity and DAT trafficking was estimated to be ~2.0 ± 0.8 μg/g. In cocaine abstinence experiment, we found that after an acute cocaine exposure of 20 mg/kg (i.p.), the initial recovery process from days 1 to 15 in rats was relatively faster (from \u3e95% on Day 1 to ~35.4% on day 15), and the complete recovery of the striatal DAT distribution may take 60 days. In another situation, with repeated cocaine exposures for once every other day from days 0 to 32 for 17 doses of 20 mg/kg cocaine (i.p.) in total, the complete recovery of striatal DAT distribution may take an even longer time (about 90 days), which represents a consequence of the chronic cocaine use. In addition, to treat cocaine use disorders, in this study we also examined our previously demonstrated highly efficient Fc-fused cocaine hydrolases, for example, CocH3-Fc(M3) and CocH5-Fc(M6). The results show that CocH3-Fc(M3) is capable of effectively blocking cocaine from reaching the brain even with a lethal dose of 60 mg/kg cocaine (i.p.) and, thus, powerfully preventing cocaine-induced physiological effects such as the hyperactivity and DAT trafficking. CocH5-Fc(M6), on the other hand, can effectively block cocaine-induced hyperactivity and DAT trafficking with repeated cocaine exposures by maintaining a plasma CocH5-Fc(M6) concentration no less than 58.7 ± 2.9 nM in the blood system of rats, and thus helped the cocaine-altered DAT distribution to recover by protecting the dopaminergic system from further damage by cocaine for the rats being either naive or cocaine-exposed