In vitro and postmortem studies of the brain opioid system : association to opiate dependence

Abstract

Opioids such as heroin are highly addictive illicit drugs. The μ opioid receptor (MOR) plays a central role in mediating the effects of opiates and related opioid. The interaction between opioids and MOR activate and regulate multiple cell signaling pathways associated with expression of a number of genes which in rum lead to acute and chronic disturbances in central neuronal system (CNS). The opioid system including, proenkephlin (PENK) and prodynorphin (PDYN), are highly expressed in mesolimbic circuits and striatum, which are key neural systems implicated in opiate dependence.

The aim of this thesis was to investigate the effect of opiates on the opioid system in both cellular models and post-mortem human brain specimens in an attempt to understand specific opioid system events induced by opioid exposure.

Rat pheochromocytoma (PC12) recombinant cell line was found to express MORs, which were predominantly located in the plasma membrane. Potential interactions between MOR receptors and cell signaling were determined using cAMP assays. Forskolin-induced cAMP formation was inhibited by DAMGO, a specific MOR agonist, whereas naloxone, a specific MOR antagonist, completely abolished this effect. Using cDNA arrays, changes in gene expression were detected after cellular treatment with DAMGO (3 h). Annexin V, an apoptosis-induced gene and RGS4 were prominently up-regulated in this cell line.

Cultured rat adult hippocampal progenitors (AHPs) were shown to release beta-endorphin and express MOR and delta-opioid receptors (DOR). Incubation with MOR and DOR antagonists for 48 hours (h) reduced signaling of mitogen-activated protein kinase (MAPK) pathway. Cultures incubated with naloxone for 10 days resulted in an approximately threefold increase in neurogenesis which suggest an impairment of cellular functioning after long-term effect of opiate on opioid system. To investigate the possible effect of heroin on the PENK and PDYN systems, post-mortem amygdala and caudal putamen specimens from human heroin abusers were studied. Heroin abusers had significantly reduced PDYN mRNA expression in the periamygdaloid cortex.

PENK mRNA expression was found to be significantly elevated in the central nucleus. MOR agonist-stimulated [35S]GTPγS binding studies revealed reduced MOR coupling in the cortical amygdala region, but elevated binding in the accessory basal nucleus of heroin subjects. These findings suggest impairment in PDYN/KOR and PENK systems in discrete amygdala neuronal populations in association with heroin abuse. In the caudal putamen, although a similar reduction of the opioid gene expression was observed in this study, differential regulation and potential imbalance of MOR and KOR function existed in the caudal putamen of heroin abusers. Finally, the relevance of human MOR gene polymorphism (OPRMI) was investigated since approximately 90% of our heroin subjects carried the 11 8G allele.

The findings suggest that the OPRMI genotype significantly influenced the PDYN/KOR system in amygdala of heroin abuser in this study. The nociceptin (N-OFQ)/opioid receptor like-1 (ORL-1) system belong also to the opioid system and counteract the effects of opioids such as morphine. Based on the limited information known about the anatomical organization of the NOFQ/ORL-1 system in the human CNS, the regional mRNA expression and receptor localization were analyzed in the postmortem human brain. The results, demonstrated a specific presence of the N-OFQ/ORL-1 system in the cerebral cortex, central and medial amygdala, discrete ventral thalamic nuclear group and paraventricular hypothalamus.

The anatomical distribution N-OFQ/ORL-1 pattern in the human brain resembles the general pattern observed in other species and substantiates a potential role in functions related to cognition and stress/anxiety that can be explored in opiate abusers. Overall, these finding indicate that opiates produce impairments in different aspects of the opioid system in the brain. Specific alterations in amygdala dynorphinergic and enkephalinergic systems may underlie disturbances in mood and anxiety disorders apparent in opiate abusers.