P: 74 Cholestasis Decreases Dendritic Spine Density in a Rat Hippocampal Organotypic Culture

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BACKGROUND: Executive functioning impairment in children with cholestatic liver disease is increasingly recognized. Injury to developing neuronal networks could be an underlying mechanism. Given that the CSF of rat pups following bile duct ligation is highly concentrated in ammonia, murocholic and taurocholic acids, we hypothesized that cholestasis may significantly alter density of synaptic contacts onto pyramidal neurons, known to be essential in the development of executive functioning. METHODS: Transverse hippocampal organotypic slice (400 µm thick) were harvested from 6–7-day-old Wistar rats. They were maintained for 15 days in a CO2-incubator (33°C). pc-DNA3.1-EGFP plasmid biolistic transfection was performed 7 days after harvesting. 3 days following transfection, control medium or experimental medium containing 100 µM α-murocholic-acid, 200 µM taurocholic-acid and 2.5 mM ammonium was added to the culture (day 0 of exposure, MIX condition). Confocal microscopy using imageJ was used for to manually count CA1 pyramidal neuron dendritic spines as proxies for excitatory synapses. Static analysis quantified dendritic spines density each day. Dynamic analysis quantified dendritic spines turnover (loss and neo-formation) for each 24 h time-window. Statistical analysis was conducted using PRISM software for multiple t-test or mixed-effect ANOVA. RESULTS: Static analysis showed a biphasic profile in MIX condition. During early phase (first 3 days of exposure), we observed >50% decrease in dendritic spine density compared to control (cf Figure 1, P < 0.001). On days 3 to 4), spine density recovered to reach control value. Dynamic analysis showed 15% loss in dendritic spines stability during the early phase of exposure to MIX condition, compared to controls, with comparable low rates of spine turnover. During the late phase of MIX exposure, spine turnover increased significantly in favor of spine neo-formation: spine neo-formation was 10 times higher (0.280 vs 0.023 spines.µm-1, P = 0.033) than controls, while spine loss was 6 times higher in neurons exposed to MIX (0.154 vs 0.026 spines.µm-1, P = 0.0026) than controls. CONCLUSIONS: We demonstrate here that mimicking cholestasis ex vivo leads to a biphasic response in spine density of rat hippocampal CA1 pyramidal neurons. Spine density decreases during the first 3-days of exposure to a mix of bile acids and ammonium chloride-owing to decreased in spine stability. This is followed by increased spine turnover with spine neo-formation in excess of spine loss, yielding a final spine density similar to control animals. Whether these new spines are functionally equivalent to controls remains to be determined.