Using a specific probe to PI(4,5)P2 and SDS-digested freeze-fracture replica labeling (SDS-FRL) method, I visualized nanoscale distribution of PI(4,5)P2 on parallel fiber (PF) boutons of mouse cerebellum. I found that PI(4,5)P2 localized at active zones (AZs) of PF boutons. Although the density of PI(4,5)P2 was not changed during synaptic transmission, the inhibition of PI(4,5)P2 production using the PI4K inhibitor caused the strong reduction of PI(4,5)P2 after synaptic transmission. These results suggest that PI(4,5)P2 density is equilibrated during synaptic transmission. Presynaptic CaV2.1 a major Ca2+ source to trigger neurotransmitter release in PF boutons, was co-localized with PI(4,5)P2, and its dynamics during synaptic transmission was inhibited by the application of PI4K inhibitor. These results indicates that PIs on presynaptic nerve terminal membrane has roles on synaptic transmission and regulates spatial positioning of presynaptic proteins. These results are intended to be published in the near future.
As an offshoot of this project, I found that acute brain slices prepared at physiological temperature enhanced the quality of the brain slices compared to the conventional brain slicing method preparing at ice-cold temperature. Using electron microscopic, super-resolution microscopic and electrophysiological approaches, I found that the acute cerebellar slice preparation at ice-cold temperature alters several crucial parameters of synapses including dendritic spine formation, synaptic protein distribution and synaptic vesicle distribution in AZs. In contrast, brain slices prepared at physiological temperature showed no alterations of the synaptic properties shown in “cold-cut” slices, means this “warm-cutting” method improves the slice quality for the following experiments. This result has been published in an open access scientific journal (Frontiers in Cellular Neuroscience) to be known the advantages of the warm-cutting method.
