Nasal Chemosensory-Stimulation Evoked Activity Patterns in the Rat Trigeminal Ganglion Visualized by In Vivo Voltage-Sensitive Dye Imaging

Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, the olfactory and the trigeminal system. Within the early olfactory system, spatiotemporal responses of the olfactory bulb to various odorants have been mapped in great detail. In contrast, far less is known about the representation of volatile chemical stimuli at an early stage in the trigeminal system, the trigeminal ganglion (TG), which contains neurons directly projecting to the nasal cavity. We have established an in vivo preparation that allows high-resolution imaging of neuronal population activity from a large region of the rat TG using voltage-sensitive dyes (VSDs). Application of different chemical stimuli to the nasal cavity elicited distinct, stimulus-category specific, spatiotemporal activation patterns that comprised activated as well as suppressed areas. Thus, our results provide the first direct insights into the spatial representation of nasal chemosensory information within the trigeminal ganglion imaged at high temporal resolution

High-Throughput Screening of Australian Marine Organism Extracts for Bioactive Molecules Affecting the Cellular Storage of Neutral Lipids

Mammalian cells store excess fatty acids as neutral lipids in specialised organelles called lipid droplets (LDs). Using a simple cell-based assay and open-source software we established a high throughput screen for LD formation in A431 cells in order to identify small bioactive molecules affecting lipid storage. Screening an n-butanol extract library from Australian marine organisms we identified 114 extracts that produced either an increase or a decrease in LD formation in fatty acid-treated A431 cells with varying degrees of cytotoxicity. We selected for further analysis a non-cytotoxic extract derived from the genus Spongia (Heterofibria). Solvent partitioning, HPLC fractionation and spectroscopic analysis (NMR, MS) identified a family of related molecules within this extract with unique structural features, a subset of which reduced LD formation. We selected one of these molecules, heterofibrin A1, for more detailed cellular analysis. Inhibition of LD biogenesis by heterofibrin A1 was observed in both A431 cells and AML12 hepatocytes. The activity of heterofibrin A1 was dose dependent with 20 µM inhibiting LD formation and triglyceride accumulation by ∼50% in the presence of 50 µM oleic acid. Using a fluorescent fatty acid analogue we found that heterofibrin A1 significantly reduces the intracellular accumulation of fatty acids and results in the formation of distinct fatty acid metabolites in both cultured cells and in embryos of the zebrafish Danio rerio. In summary we have shown using readily accessible software and a relatively simple assay system that we can identify and isolate bioactive molecules from marine extracts, which affect the formation of LDs and the metabolism of fatty acids both in vitro and in vivo