Indeed, significant spine loss and morphological change was observed after 63 days of culture, 6 weeks after initial PrPScdetection. morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seenin vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during contamination, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis. == Introduction == Prion diseases are fatal neurodegenerative diseases of humans (Creutzfeldt-Jakob disease (CJD)), cattle (bovine spongiform encephalopathy (BSE)), cervids (chronic wasting disease (CWD)) and sheep (scrapie). They arise when the normally expressed alpha-helical prion protein (PrPC) misfolds and aggregates as a beta sheet-rich form (PrPSc)[1],[2]. PrPScthen seeds the conversion of more PrPCinto PrPSc, leading to the hallmark neuropathology of prion diseases, PrPScaccumulation, neuronal loss, spongiform change and astrogliosis. The earliest pathological changes, occurring before neuronal loss, involve neuronal synapses, specifically the loss of dendritic spines and a concomitant loss of long term potentiation[3][7]. These changes have been observed in animal models of prion contamination, but not in cell culture models of prion contamination. Organotypic cultures have advantages over simple cell cultures in that they largely recapitulate thein vivomicroenvironment[8],[9]while providing a system which is usually more open Enalaprilat dihydrate to experimental manipulation than the whole animal. The prion organotypic slice culture assay[10],[11](POSCA) was developed as an assay for the detection andex vivoreplication of prion infectivity, using a slice culture from a neonatal mouse cerebellum. These cultures can be infected with a number of different prion strains, can be maintained for many weeks, and can generate Rabbit polyclonal to ARHGAP15 and accumulate PrPScat a much faster rate thanin vivo, with PrPScdetectable within 21 days. However, the fact that a cultured brain slice can replicate PrPScdoes notde factoindicate that prion infection specific pathological changes will also develop within Enalaprilat dihydrate that slice. One study has reported some prion-specific neuropathological changes detectable by electron microscopy in these infected cultures, specifically the development of small vacuolar degeneration and tubulovesicular bodies[12], but to our knowledge, quantification of dendritic spine density has not been performed. While the development of dendritic varicosities and loss of dendritic spines does co-localize to areas of vacuolar and prion protein pathology[13], spine loss in hippocampal neuronsin vivois not observed until 45 weeks after the initial detection of PrPScat day 70 post-inoculation[3]. Given the accelerated replication of PrPScin POSCA, detectable by day 21, we hypothesized that spine loss might also be accelerated, and thus detectable within the lifespan of the culture. Within the cerebellum, Purkinje cells are readily identifiable by post-natal day ten and have elaborate dendritic trees amenable to analysis; they are also known to undergo dendritic disintegrationin vivowhen infected by prion strains targeting the cerebellum[14]. As such, we chose to Enalaprilat dihydrate analyze Purkinje cell dendritic spine density over the course of prion infection in POSCA. We demonstrate that Purkinje cell dendritic spine loss does occur in POSCA, within a timeframe similar to pathogenesisin vivo. In addition, we found that Purkinje cell spine density increased transiently during early infection, at the time PrPScwas first detectable. == Materials and Methods Enalaprilat dihydrate == == Ethics statement == This study was carried out in strict accordance with the recommendations in the Canadian Council on Animal Care, as approved by the Animal Care and Use Committee (ACUC) of the University of Alberta Enalaprilat dihydrate (Study ID AUP00000335; Animal Welfare Assurance Number: #A5070-01). Mice were bred.