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

Abstract

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.