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Ultrastructural analysis of phosphoinositides in nerve terminals: distribution, dynamics and physiological roles in synaptic transmission

Periodic Reporting for period 1 - ALPINE (Ultrastructural analysis of phosphoinositides in nerve terminals: distribution, dynamics and physiological roles in synaptic transmission)

Reporting period: 2018-04-01 to 2020-03-31

Phosphoinositides (PIs) are minor components on cytoplasmic sides of eukaryotic cell membranes, but they play important roles in many aspects of cellular functions, including synaptic transmission in neurons. Many of proteins contributing synaptic transmission has binding domains to one or some stereoisomers of PIs, indicating a potential role of PIs as an anchor or a modulator for the proteins to determine these localization and dynamics during synaptic transmission. Although it is important to know the distribution pattern of PIs and also their dynamics on presynaptic terminal membranes to understand the molecular machinery of synaptic transmission, the most of knowledge about the distribution pattern of PIs was speculated in speculated in nerve terminals from the observations in non-neuronal cells such as yeast and endocrine cells. This project was aimed to investigate the nanoscale distribution pattern and activity-dependent dynamics of PIs on nerve terminal membranes, and their physiological roles in synaptic transmission to understand the mechanisms of synaptic transmission.
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.
The results from this project identified the nanoscale distribution of PI(4,5)P2 in AZs of presynaptic nerve terminals. The results also suggest the physiological roles of PI(4,5)P2 on the dynamics of presynaptic proteins during synaptic transmission. This project will contribute to a better understanding of molecular machinery in synaptic transmission. Furthermore, it has been known that mutations of PI-metabolic enzymes cause neuronal disorders e.g. Parkinson’s disease, Alzheimer’s disease and dementia. Thus, the results from this project are also important to understand the pathological mechanisms in synapses under neuronal disorders.
In this project, I found that acute brain slices prepared at physiological temperature enhanced the quality of the preparations for investigation of synaptic functions rather than the conventional brain slice method preparing at ice-cold temperature. The “warm-cutting” method also provides us an additional advantage: skipping the recovery time after slicing. In conventional “cold-cutting” method, brain tissues need a recovery time (~60 min) in buffer at warm temperature before using in following experiments. However, the warm-cutting method does not require this recovery step, and experimenters can use the brain slices after slicing immediately. This is a huge advantage especially for experiments of long-term plasticity induced by in vivo behavior experiments. I have demonstrated that long-term depression (LTD) of electrophysiological synaptic events induced by a motor learning at cerebellum was more detectable in the warm-cut slices than the cold-cut slices, because of the shortening of the duration between the training and the recording. These advantages of the warm-cutting method for acute brain slice preparation will help experimenters in a wide range of neuroscience research, especially for synaptic plasticity induced in vivo.
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