Transcript mapping of neuronal systems in the rat brainstem using a combination of HCR-FISH and iDISCO tissue clearing method
Kumar V, Krolewski DM, Aydin C, Akil H, Watson SJ Jr
Society for Neuroscience. 2018.
Hybridization chain reaction (HCR) based fluorescent in situ hybridization (FISH) in iDISCO processed tissues, has potential to provide vital spatiotemporal information for molecular characterization and mapping of heterogeneous neuronal populations in the brain. HCR-FISH relies on binding of cDNA probes on target mRNAs to initiate chain reactions in which metastable fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. HCR method significantly improves sensitivity, efficiency, and amplifies the FISH signal. In our experience, iDISCO clearing method has shown the best compatibility for HCR-FISH with fresh frozen tissues, later has been preferentially used as the starting material for the radioactive/digoxigenin based in situ hybridization. We are using HCR-FISH and iDISCO together to map behaviorally relevant key transcripts in the brainstem region. A great deal of study has shown that noradrenergic (NE) and GABAergic system play a significant role in the pathophysiology of depression, however much of the underlying specific mechanisms induced by the stress, still need to be understood. A circuit level investigation of gene expression would help us better understand the neurobiological mechanisms underlying such disorders. To achieve an optimal transcript signal for this mapping study, we first successfully optimized the probe hybridization efficiency and permeability in the fresh frozen tissues processed with modified iDISCO protocol. In the next step, we chose a few of the predominant genes from the NE/GABAergic systems- dopamine-beta-hydroxylase (Fig.1), somatostatin (Fig.2) and parvalbumin (Fig.3) to test and validate the mapping efficiency of HCR-FISH/iDISCO combination. By using light sheet theta microscope, we anticipate to capture a detailed transcript map of NE and GABAergic systems in the brainstem. A possible implication would be to utilize this developed system in behaviorally relevant animal model and assess the qualitative/quantitative molecular adaptations in the neuronal circuits.