In Situ Hybridization Nanoprobes to Investigate Mu Opioid Receptor Alternative Splicing

• Main Author: Langenbacher, Rachel E
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2021
• Subjects: Immunology; Medical imaging; Nanotechnology; Neurosciences
• ISBN: 9798492740672

Fluorescence in situ hybridization (FISH) is a powerful tool for investigating mRNA expression in fixed tissues and cells while preserving structural information. Such in situ detection is of particular importance in the brain, where complex function arises from strict regional and temporal control of gene expression. This includes alternative splicing, a process by which different exons are included or excluded from the final mRNA, producing unique protein products or differentially impacting expression, stability, protein localization, and protein-protein interactions. The mu opioid receptor (Oprm1) undergoes extensive alternative splicing, producing several highly conserved splice variants of biological relevance in mouse, rat, and human. The function and expression profiles of most alternatively spliced Oprm1 products remain unknown, in part due to limitations of current FISH techniques. Low levels of mRNA expression, high autofluorescence of brain, and physical limitations of fluorescent dyes used to construct FISH probes hinder the detection of these splice variants by FISH. The simultaneous, multiplexed detection of low-abundance exon targets required for mRNA splice variant resolution is made difficult by these limitations and by the intricate hybridization, processing, and wash steps required for adequate signal detection. We used semiconducting single-walled carbon nanotubes (SWCNTs) to develop novel FISH probes due to their inherent fluorescence in the near-infrared tissue-transparent window where autofluorescence is low. Single-nanotube resolution is possible and SWCNTs have high multiplexing potential due to narrow absorbance and emission peaks. NT-FISH probes were constructed to investigate the mRNA expression of four clinically relevant exons of the mouse mu opioid receptor. Sensitivity compared favorably to CARD-FISH for target detection in a cell model of endogenous Oprm1 expression. Moreover, the NT-FISH signal was compatible with IHC co-staining, allowing for simultaneous in situ assessment of target mRNA expression of Oprm1 exon 1, 4, 7, and 11 in cell populations expressing GAD1 or GFAP in mouse brains sections. NT-FISH detection was consistent with reported expression in inhibitory neurons and lent insight into astrocyte expression of Oprm1 splice variants, portending NT-FISH probe utility for conducting high-resolution, multi-omic expression investigation in situ.