Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

What the brain stem tells the frontal cortex. II. Role of the SC-MD-FEF pathway in corollary discharge.

One way we keep track of our movements is by monitoring corollary discharges or internal copies of movement commands. This study tested a hypothesis that the pathway from superior colliculus (SC) to mediodorsal thalamus (MD) to frontal eye field (FEF) carries a corollary discharge about saccades made into the contralateral visual field. We inactivated the MD relay node with muscimol in monkeys and measured corollary discharge deficits using a double-step task: two sequential saccades were made to the locations of briefly flashed targets. To make second saccades correctly, monkeys had to internally monitor their first saccades; therefore deficits in the corollary discharge representation of first saccades should disrupt second saccades. We found, first, that monkeys seemed to misjudge the amplitudes of their first saccades; this was revealed by systematic shifts in second saccade end points. Thus corollary discharge accuracy was impaired. Second, monkeys were less able to detect trial-by-trial variations in their first saccades; this was revealed by reduced compensatory changes in second saccade angles. Thus corollary discharge precision also was impaired. Both deficits occurred only when first saccades went into the contralateral visual field. Single-saccade generation was unaffected. Additional deficits occurred in reaction time and overall performance, but these were bilateral. We conclude that the SC-MD-FEF pathway conveys a corollary discharge used for coordinating sequential saccades and possibly for stabilizing vision across saccades. This pathway is the first elucidated in what may be a multilevel chain of corollary discharge circuits extending from the extraocular motoneurons up into cerebral cortex.