Region Specificity in Endogenous Opioid Peptides and Mu-opioid Receptor Gene Expression in Rat Brain Areas Involved in Addiction After Frequent Morphine Treatment

Backgrounds: Molecular mechanisms involved in adverse effects of morphine, including tolerance and dependence, have remained elusive. We examined possible alterations in the gene expression of proenkephalin (Penk), prodynorphin (Pdyn), and mu-opioid receptor (Oprm1) in reward brain areas following frequent morphine treatment. Methods: Two groups of male Wistar rats were used. The groups received either saline (1 mL/kg) or morphine (10 mg/kg) twice daily for eight days. On day 8, rats were decapitated, brain areas involved in addiction were dissected, including the midbrain, striatum, prefrontal cortex (PFC), hippocampus, and hypothalamus, and gene expression was evaluated with real-time PCR. Results: Prolonged morphine treatment decreased Penk, Pdyn, and Oprm1 gene expressions in the midbrain but upregulated them in the striatum compared to the control group treated with saline. Significant increases in Pdyn and Oprm1 gene expressions were detected in the PFC, but there was no significant difference in Penk gene expression between the two groups. Besides, Pdyn gene expression was decreased in the hippocampus and hypothalamus; however, no significant differences in Penk and Oprm1 gene expressions were detected between the groups in these areas. Conclusions: The expression of endogenous opioid peptides and receptors after frequent use of morphine follows a region specificity in brain areas involved in addiction. These alterations may result in new physiological setpoints outside the normal range, which need to be considered when using morphine in medicine.

Opioid Receptor-Mediated and Non-Opioid Receptor-Mediated Roles of Opioids in Tumour Growth and Metastasis

Opioids are administered to cancer patients in the period surrounding tumour excision, and in the management of cancer-associated pain. The effects of opioids on tumour growth and metastasis, and their consequences on disease outcome, continue to be the object of polarised, discrepant literature. It is becoming clear that opioids contribute a range of direct and indirect effects to the biology of solid tumours, to the anticancer immune response, inflammation, angiogenesis and importantly, to the tumour-promoting effects of pain. A common misconception in the literature is that the effect of opioid agonists equates the effect of the mu-opioid receptor, the major target of the analgesic effect of this class of drugs. We review the evidence on opioid receptor expression in cancer, opioid receptor polymorphisms and cancer outcome, the effect of opioid antagonists, especially the peripheral antagonist methylnaltrexone, and lastly, the evidence available of a role for opioids through non-opioid receptor mediated actions.

G protein signaling–biased mu opioid receptor agonists that produce sustained G protein activation are noncompetitive agonists

The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity, and biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor, which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein–biased mu opioid receptor (MOR) agonists SR-17018 and SR-14968 stabilize the MOR in a wash-resistant yet antagonist-reversible G protein–signaling state. Furthermore, we demonstrate that these structurally related biased agonists are noncompetitive for radiolabeled MOR antagonist binding, and while they stimulate G protein signaling in mouse brains, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR-17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long-lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression.

In Situ Analysis of the Mu-Opioid Receptor Splice Variants in Adult Rat Brain

<p>The original intention of this study was to exploit the specificity of circularisable ligation probes (CLiPs) in a unique approach of in situ genotyping the mu-opioid receptor (MOR) splice variants. CLiPs were designed to target a PCR generated MOR-1 template in vivo. The ligation results were consistent with circularised CLiPs, however due to the inherent limitations of this method the more conventional technique of fluorescent in situ hybridisation (FISH) was substituted for CLiPs to analyse to distribution of MOR splice variants in rat brain. Utilising FISH, the aim was to produce RNA probes (riboprobes) approximately the same size as the target specific region of CLiPs (~60-70 nt) to analyse the distribution patterns of MOR splice variants in rat brain. Five short (70-222 nt) riboprobes were generated to exons 1, 3, 4 and 9, and the 5' UTR + exon 1 of the Rattus norvegicus MOR gene (Oprm) to be utilised in FISH. The exon 1, 4 and 5' UTR + exon 1 riboprobes were shown to localise to MOR mRNA in brain structures previously reported to express MORs. These riboprobes also localised to mRNA within the Purkinje cells of the adult rat cerebellum, where it is generally accepted that only DOR is expressed in the rat cerebellum. MOR mRNA was visualised in many structures in the rat brain, including the dendate gyrus, inferior olive and spinal trigeminal nucleus. Riboprobes generated to the 5' UTR + exon 1 and exon 4 showed differential distribution patterns, the functional significance of this discovery is unknown, however these results implicate a role for FISH in tracking the distribution patterns of untranslated and translated mRNA. The use of novel new short riboprobes represents a technically difficult yet feasible technique for mapping MOR mRNA distribution in adult rat brain.</p>

In Situ Analysis of the Mu-Opioid Receptor Splice Variants in Adult Rat Brain

<p>The original intention of this study was to exploit the specificity of circularisable ligation probes (CLiPs) in a unique approach of in situ genotyping the mu-opioid receptor (MOR) splice variants. CLiPs were designed to target a PCR generated MOR-1 template in vivo. The ligation results were consistent with circularised CLiPs, however due to the inherent limitations of this method the more conventional technique of fluorescent in situ hybridisation (FISH) was substituted for CLiPs to analyse to distribution of MOR splice variants in rat brain. Utilising FISH, the aim was to produce RNA probes (riboprobes) approximately the same size as the target specific region of CLiPs (~60-70 nt) to analyse the distribution patterns of MOR splice variants in rat brain. Five short (70-222 nt) riboprobes were generated to exons 1, 3, 4 and 9, and the 5' UTR + exon 1 of the Rattus norvegicus MOR gene (Oprm) to be utilised in FISH. The exon 1, 4 and 5' UTR + exon 1 riboprobes were shown to localise to MOR mRNA in brain structures previously reported to express MORs. These riboprobes also localised to mRNA within the Purkinje cells of the adult rat cerebellum, where it is generally accepted that only DOR is expressed in the rat cerebellum. MOR mRNA was visualised in many structures in the rat brain, including the dendate gyrus, inferior olive and spinal trigeminal nucleus. Riboprobes generated to the 5' UTR + exon 1 and exon 4 showed differential distribution patterns, the functional significance of this discovery is unknown, however these results implicate a role for FISH in tracking the distribution patterns of untranslated and translated mRNA. The use of novel new short riboprobes represents a technically difficult yet feasible technique for mapping MOR mRNA distribution in adult rat brain.</p>

The Effect of Opiates on the Developing Cerebral Cortex

<p>Opiate drugs, such as codeine, morphine and heroin are powerful analgesics and drugs of abuse. The unborn child is invariably exposed to opiate drugs as a consequence of maternal use. Studies that have investigated the impact of opiate drugs demonstrated opioid system expression in proliferating regions of the developing brain, as well as on proliferative astroglia taken from the developing central nervous system. The effects of opiates on astroglial proliferation (largely mediated by the mu opioid receptor) are predominantly inhibitory, but are extremely context dependent. This context dependency exists because of the complexity resident within the opioid signalling system. However, since this previous research was conducted, there has been impressive progress made in the field of developmental neurobiology with the demonstration that cells of astrocytic lineage are responsible for the generation of the central nervous system. It was therefore the aim of the current research project to investigate the developmental impact of opiate exposure in the context of the foetal mouse cerebral cortex. This aim was divided into 3 separate aims that comprised of; determining the cellular localisation of the mu opioid receptor, the effects of opiate exposure on cortical progenitor cells, and to determine the effect of opiate exposure on the development of the cerebral cortex itself. The mu opioid receptor was expressed on proliferative radial glia of both the embryonic day 15.5 (neurogenic) and embryonic day 18.5 (gliogenic) ventricular zone of the dorsal forebrain. Interestingly and significantly, the mu opioid receptor-positive glia observed in the embryonic day 18.5 mouse forebrain were also observed at a comparable developmental stage in the foetal human forebrain. Morphine exposure slowed down G2 phase of the cell cycle at embryonic day 15.5 in the neurogenic murine cortical ventricular zone. This opiate-induced slowing in cell cycle progression was shown not to impact on proliferation in the ventricular zone, although future research should address whether this perturbation altered differentiation or developmental maturation of the radial glia. Morphine exposure throughout corticogenesis decreased levels of doublecortin expression (a migratory neuronal marker) at the end of gestation. Postnatally, mice exposed to morphine during corticogenesis also showed decreased numbers of neurons in layer V of the cerebral cortex. Collectively, this thesis presents the first evidence that shows morphine affects cortical progenitor cells in vivo. This research supports the possibility that the opioid system plays an endogenous role in corticogenesis. The clinical significance is morphine has the potential to perturb normal development of the cerebral cortex.</p>