A cerebellar-thalamocortical pathway drives behavioral context-dependent movement initiation

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A Modified Dominici Technic for Autoradiography: Acidified Eosin-Orange and Alcoholic Toluidine Blue

Mouse lymph nodes were fixed in Bouin-Hollande solution, processed through paraffin embedding, sectioned and coated with Kodak NTB3 liquid emulsion. After development, the autoradiographs were stained 10 min in a 0.5% eosin Y-orange G mixture acidified to pH 4.4, differentiated in 0.5% acid alcohol and counterstained 10 sec with 0.25% toluidine blue O in 50% ethanol. The technic yielded distinct differentiation of neutrophils, eosinophils, blast cells, plasmacytes, mast cells, macrophages, reticular cells, histiocytes, lymphocytes and other lymphoid cells. The method also provided sensitive definition of the cytoplasmic and nuclear characteristics of all cell types.     

The Circadian Clock Protein Timeless Regulates Phagocytosis of Bacteria in Drosophila

Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection.