Regulation Of Autophagy Level In Dorsal Striatum Affects Cocaine-taking Behavior In Rats

Part I Effects of regulation of autophagy in dorsal striatum on cocaine-taking behavior in ratsAim: Drug addiction is a chronic recurrent brain disease characterized by compulsive drug seeking and long-lasting adaptive changes in the central nervous system.Dorsal striatum,which is an important brain area for regulating compulsive drug seeking,plays a key role in mediating the shift from recreational drug use to compulsive drug abuse.Autophagy is the process of cell lysosomal degradation and recycling of excessive or damaged proteins and organelles in vivo.There is strong evidence to suggest that autophagy is widely involved in regulating the pathophysiological processes of a variety of neurodegenerative diseases.Several literatures showed that high dose of cocaine induced a large number of autophagosomes in glial cells of rats.On this basis,we herein attempt to investigate whether autophagy involves in regulating compulsive-like cocaine-taking bebavior in rats to further elucidating the potential effect of autophagy on cocaine addiction.Methods: We first established a rat model of self-administration by intrajugular catheter implantations.Rats were tested in standard operant chambers(Ani Lab)during cocaine self-administration training,with bundled software controlling program and collecting data and recording cocaine-taking behavior.We used drug intracerebral microinfusion techniques to administrate MG132 into the dorsal striatum of rats to inhibit the ubiquitin-protease pathway.We used gene-silencing technology to silence Atg5 in the dorsal striatum of rats to inbibit autophagy.Results: After chronic cocaine self-administration,we reported that(1)chronic treatment with MG132 had no effect on cocaine-taking behavior;(2)Atg5 knockdown in dorsal striatum increased cocaine-taking behavior.Conclusion: The autophagy-lysosomal pathway in the dorsal striatum region is involved in cocaine-taking in rats,whereas the ubiquitin-proteasome pathway does not participate in it.Part Ⅱ The underlying mechanism of regulation of autophagy in dorsal striatum in cocaine-taking behavior in ratsAim: Protein 62(p62)is an ubiquitin-binding protein encoded by SQSTM1 gene.p62 functions as a specific autophagy receptor which binds to autophagic substrates(such as damaged proteins)and targets them into the autophagosome for degradation.Moreover,p62 interacts with microtubule-associated protein 1 light chain 3(LC3)to participate in the formation of autophasomes.AMPA receptors in dorsal striatum play an important role in cocaine addiction.Strong evidence has accumulated to support that p62 is involved in the degradation of tau proteins in Alzheimer’s disease(AD)and has a protective effect against AD.However,there are few reports about the role of p62 in cocaine addiction.We aim to examine the effect of intervention of p62 binding to target protein and regulation of p62 expression levels on cocaine-taking behavior in rats.Methods: We established a rat model of self-administration by intrajugular catheter implantations.Rats were tested in standard operant chambers(Ani Lab)during cocaine self-administration training,with bundled software controlling program and collecting data and recording cocaine-taking behavior.We used drug intracerebral microinfusion techniques to administrate polypeptide TAT-gkglhrehskli into the dorsal striatum of rats to specifically interfere the binding of p62 and Glu A1.Western blotting was used to verify LV-sqstm1-sh RNA and LV-sqstm1 in vivo.Gene regulation technology was used to silence and overexpress p62 in the dorsal striatum of rats before cocaine self-administration.Results:(1)After two weeks of microinjection of the LV-sqstm1-sh RNA into the dorsal striatum,the expression of p62 was decreased.(2)After three weeks of microinjection of the LV-sqstm1 into the dorsal striatum,the expression of p62 was increased.(3)After chronic cocaine self-administration,we found that chronic treatment with polypeptide increased cocaine-taking behavior.(4)p62 knockdown in dorsal striatum increased cocaine-taking behavior.(5)p62 overexpression in dorsal striatum decreased cocaine-taking behavior.Conclusions: The autophagic substrate p62 in the dorsal striatum region played an important role in cocaine intake in rats.