RESUMO
Cost-effective and efficient, the fruit fly (Drosophila melanogaster) has been used to make many key discoveries in the field of neuroscience and to model a number of neurological disorders. Great strides in understanding have been made using sophisticated molecular genetic tools and behavioral assays. Functional analysis of neural activity was initially limited to the neuromuscular junction (NMJ) and in the central nervous system (CNS) of embryos and larvae. Elucidating the cellular mechanisms underlying neurological processes and disorders in the mature nervous system have been more challenging due to difficulty in recording from neurons in adult brains. To this aim we developed an ex vivo preparation in which a whole brain is isolated from the head capsule of an adult fly and placed in a recording chamber. With this preparation, whole cell recording of identified neurons in the adult brain can be combined with genetic, pharmacological and environmental manipulations to explore cellular mechanisms of neuronal function and dysfunction. It also serves as an important platform for evaluating the mechanism of action of new therapies identified through behavioral assays for treating neurological diseases. Here we present our protocol for ex vivo preparations and whole-cell recordings in the adult Drosophila brain.
RESUMO
The value of induced pluripotent stem cells (iPSCs) within regenerative medicine is contingent on predictable and consistent iPSC differentiation. However, residual influence of the somatic origin or reprogramming technique may variegate differentiation propensity and confound comparative genotype/phenotype analyses. The objective of this study was to define quality control measures to select iPSC clones that minimize the influence of somatic origin on differentiation propensity independent of the reprogramming strategy. More than 60 murine iPSC lines were derived from different fibroblast origins (embryonic, cardiac, and tail tip) via lentiviral integration and doxycycline-induced transgene expression. Despite apparent equivalency according to established iPSC histologic and cytomorphologic criteria, clustering of clonal variability in pluripotency-related gene expression identified transcriptional outliers that highlighted cell lines with unpredictable cardiogenic propensity. Following selection according to a standardized gene expression profile calibrated by embryonic stem cells, the influence of somatic origin on iPSC methylation and transcriptional patterns was negated. Furthermore, doxycycline-induced iPSCs consistently demonstrated earlier differentiation than lentiviral-reprogrammed lines using contractile cardiac tissue as a measure of functional differentiation. Moreover, delayed cardiac differentiation was predominately associated with upregulation in pluripotency-related gene expression upon differentiation. Starting from a standardized pool of iPSCs, relative expression levels of two pluripotency genes, Oct4 and Zfp42, statistically correlated with enhanced cardiogenicity independent of somatic origin or reprogramming strategy (R(2) = 0.85). These studies demonstrate that predictable iPSC differentiation is independent of somatic origin with standardized gene expression selection criteria, while the residual impact of reprogramming strategy greatly influences predictable output of tissue-specification required for comparative genotype/phenotype analyses.
