David G. Ho

Composite synthetic lethal identification of membrane traffic inhibitors

Small molecule inhibitors provide powerful tools to characterize highly dynamic and complex eukaryotic cell pathways such as those mediating membrane traffic. However, a lack of easy and generalizable assays has constrained identification of novel inhibitors despite availability of diverse chemical libraries. Here, we report a facile growth-based strategy in yeast to screen for pathway-specific inhibitors. The approach uses well characterized synthetic genetic growth defects to guide design of cells genetically sensitized for inhibition of chosen pathways. With this strategy, we identified a family of piperazinyl phenylethanone compounds as inhibitors of traffic between the trans-Golgi network (TGN) and endosomes that depends on the clathrin adaptor complex AP-1. The compounds did not significantly alter other trafficking pathways involving the TGN or endosomes, indicating specificity. Compound treatment also altered localization of AP-1 in mammalian cells. These previously uncharacterized inhibitors will be useful for future studies of clathrin-mediated transport in yeast, and potentially in other organisms. Furthermore, the easily automated technology should be adaptable for identification of inhibitors of other cellular processes.

Dynamics of cortical dendritic membrane potential and spikes in freely behaving rats

Dendrites are more active than expected Dendrites occupy more than 90% of neuronal tissue. However, it has not been possible to measure distal dendritic membrane potential and spiking in vivo over a long period of time. Moore et al. developed a technique to record the subthreshold membrane potential and spikes from neocortical distal dendrites in freely behaving animals. These recordings were very stable, providing data from a single dendrite for up to 4 days. Unexpectedly, distal dendrites generated action potentials whose firing rate was nearly five times greater than at the cell body. Science, this issue p. eaaj1497 In vivo recordings over several days from neuronal dendrites in the brain’s neocortex reveal disproportionate dendritic spiking. INTRODUCTION Neurons are large, tree-like structures with extensive, branch-like dendrites spanning >1000 μm, but a small ~10-μm soma (figure). Dendrites receive inputs from other neurons, and the electrical activity of dendrites determines synaptic connectivity, neural computations, and learning. The prevailing belief has been that dendrites are passive; they merely send synaptic currents to the soma, which integrates the inputs to generate an electrical impulse, called an action potential or somatic spike, thought to be the fundamental unit of neural computation. These ideas have not been directly tested because traditional electrodes, which puncture the dendrite to measure dendritic voltages in vitro, do not work in vivo due to constant movement of the animals that kills the punctured dendrites. Hence, the voltage dynamics of distal dendrites, constituting the vast majority of neural tissue, is unknown during natural behavior. RATIONALE Tetrodes are a bundle of four fine electrodes, commonly used for measuring somatic spikes from a distance, that is, extracellularly. Hence, they work well in freely behaving animals. However, tetrodes do not measure the membrane voltages of soma, let alone dendrites. Chronically implanted tetrodes also elicit a naturally occurring immune response, where glial cells encapsulate the tetrode and shield it from the extracellular medium. We tested the hypothesis that a segment of dendrite could get trapped between the tetrode tips before this glial encapsulation occurred (figure). This would enable us to measure the dendritic membrane voltage without penetrating it in freely behaving subjects. RESULTS Despite low success rate, we measured the putative, distal-dendritic membrane potential in freely behaving rats for long periods, up to 4 days. These data showed frequent occurrence of spikes that were quite different from somatic spikes, but they closely resembled the waveforms of spikes generated within the distal dendrites in vitro (figure). This finding was further confirmed by computational modeling. The glial seal mechanism was verified using immunohistochemistry, predicted shielding of extracellular spikes during the dendritic measurements, in vivo impedance spectroscopy, and modeling. The identity of the general cell type from which the dendrite spikes were measured was confirmed using the analysis of short-term plasticity, suggesting that most of the dendritic measurements were from pyramidal neurons. The dendritic spike rates, however, were fivefold greater than the somatic spike rates of pyramidal neurons during slow-wave sleep and 10-fold greater during exploration. The high stability of dendritic signals suggested that these large rates are unlikely to arise due to the injury caused by the electrodes. The data also showed large subthreshold membrane voltage fluctuations. Their magnitude was always larger than that of dendritic spikes. This is the converse of comparable measurements in the soma. The dendritic spikes and subthreshold voltages contained significant information about the rat’s exploratory behavior, which is comparable to somatic spikes. CONCLUSION The glial sheath method can be used for measuring dendritic membrane potential in freely behaving animals for long periods. The large subthreshold voltage fluctuations in dendrites, which modulate the dendritic firing rates, indicate a hybrid, analog-digital code in the dendrites. These dendritic dynamics could profoundly influence synaptic plasticity and neural computations. The dendrites generated several-fold more spikes than the soma. The large dendritic spike rates could be responsible for the seemingly weak correlations between the somatic spikes across neurons. This requires a revision of many prevailing beliefs, for example, the dendrites are largely passive in vivo, and the somatic spike is the fundamental unit of neural computation. Glial sheath mechanism of measuring dendritic membrane potential in vivo. Top left shows a pyramidal neuron with soma (blue) and extensive dendrites (red). Top right shows the tetrode (gold) and glial cells (green) that trap a dendritic branch. Bottom left shows the extracellular measurements from the soma (blue), showing few, small-amplitude, downward-going spikes. Bottom right shows membrane potential from the dendrite, showing many, large-amplitude, upward-going spikes. Neural activity in vivo is primarily measured using extracellular somatic spikes, which provide limited information about neural computation. Hence, it is necessary to record from neuronal dendrites, which can generate dendritic action potentials (DAPs) in vitro, which can profoundly influence neural computation and plasticity. We measured neocortical sub- and suprathreshold dendritic membrane potential (DMP) from putative distal-most dendrites using tetrodes in freely behaving rats over multiple days with a high degree of stability and submillisecond temporal resolution. DAP firing rates were several-fold larger than somatic rates. DAP rates were also modulated by subthreshold DMP fluctuations, which were far larger than DAP amplitude, indicating hybrid, analog-digital coding in the dendrites. Parietal DAP and DMP exhibited egocentric spatial maps comparable to pyramidal neurons. These results have important implications for neural coding and plasticity.

Reaction of singlet oxygen with Ir(I) and Rh(I) thiolato complexes: oxidative addition vs. S-oxidation

Singlet oxygen reacts with Ir(I) and Rh(I) thiolato complexes to form the corresponding Ir(III) and Rh(III) peroxo thiolato complexes which do not undergo intramolecular oxidation of the thiolate moiety.