ABSTRACT
Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.
Subject(s)
Amygdala/metabolism , Depressive Disorder, Major/metabolism , Enkephalins/physiology , Heroin Dependence/metabolism , Protein Precursors/physiology , Adult , Amygdala/chemistry , Amygdala/diagnostic imaging , Animals , Clozapine/analogs & derivatives , Clozapine/pharmacology , Corticosterone/blood , Depressive Disorder, Major/genetics , Designer Drugs/pharmacokinetics , Enkephalins/analysis , Enkephalins/biosynthesis , Enkephalins/deficiency , Enkephalins/genetics , Female , Fluorine Radioisotopes , Fluorodeoxyglucose F18 , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Heroin Dependence/genetics , Humans , Hungary , Limbic System/chemistry , Limbic System/diagnostic imaging , Limbic System/metabolism , Male , Middle Aged , Neuroimaging/methods , Neurons/metabolism , Positron-Emission Tomography/methods , Protein Precursors/analysis , Protein Precursors/biosynthesis , Protein Precursors/deficiency , Protein Precursors/genetics , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Radiopharmaceuticals , Rats , Rats, Long-Evans , Recombinant Fusion Proteins/metabolism , United StatesABSTRACT
It has been postulated that opiates induce addictive behaviour via changes in gene expression. PC12 cells were stably transfected with the recombinant human mu-opioid receptor (MOR) to study opioid-induced gene expression. Expression was verified by binding assay, immunocytochemistry, and immunblotting experiments. Forskolin-induced cAMP formation was inhibited by [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO 1 microM), a specific MOR agonist. This effect was completely antagonized by naloxone. By using cDNA arrays, including approximately 1,200 well-defined genes normally expressed in neural tissue, we monitored semi-quantitative changes in gene expression after 3 h short-term exposure to DAMGO. Incubation with DAMGO increased mRNA levels for 13 genes and down-regulated 13 other genes. Annexin V, RGS4 and CREB genes showed pronounced increase in expression after stimulation with DAMGO. Quantitative RT-PCR confirmed that DAMGO increased mRNA levels of Annexin V, an apoptosis-induced gene. We suggest that the PC12 cell transfected with the recombinant human MOR is a useful tool for identification of opioid-induced genes that may provide information on opiate effects of relevance for dependence.
Subject(s)
Gene Expression Regulation/drug effects , Narcotics/pharmacology , Receptors, Opioid, mu/metabolism , Analgesics, Opioid/pharmacology , Animals , Annexin A5/genetics , Annexin A5/metabolism , Blotting, Western/methods , Colforsin/pharmacology , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Gene Expression Regulation/physiology , Humans , Immunohistochemistry/methods , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Oligonucleotide Array Sequence Analysis/methods , PC12 Cells , Protein Binding/drug effects , Protein Binding/physiology , RNA, Messenger/metabolism , Radioligand Assay/methods , Rats , Receptors, Opioid, mu/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection/methodsABSTRACT
Opioids have previously been shown to affect proliferation and differentiation in various neural cell types. In the present study, cultured rat adult hippocampal progenitors (AHPs) were shown to release beta-endorphin. Membrane preparations of AHPs were found to bind [125I]beta-endorphin, and immunoreactivity for mu- and delta-opioid receptors (MORs and DORs), but not for kappa-opioid receptors (KORs), was found on cells in culture. Both DNA content and [3H]thymidine incorporation were reduced after a 48-h incubation with 100 microM naloxone, 10 micro m naltrindole or 10 microM beta-funaltrexamine, but not nor-binaltorphimine, suggesting proliferative actions of endogenous opioids against MORs and DORs on AHPs. Furthermore, analysis of gene and protein expression after incubation with MOR and DOR antagonists for 48 h using RT-PCR and Western blotting suggested decreased signalling through the mitogen-activated protein kinase (MAPK) pathway and lowered levels of genes and proteins that are important in cell cycling. Cultures were incubated with naloxone (10 or 100 microM) for 10 days to study the effects on differentiation. This resulted in an approximately threefold increase in neurogenesis, a threefold decrease in astrogliogenesis and a 50% decrease in oligodendrogenesis. In conclusion, this study suggests that reduced signalling through MORs and DORs decreases proliferation in rat AHPs, increases the number of in vitro-generated neurons and reduces the number of astrocytes and oligodendrocytes in culture.
