Rezeptorinduzierte PI(4,5)P2-Dynamik in hippocampalen CA1-Pyramidenneuronen

The membrane lipid phosphatidylinositol(4,5)bisphosphate (PIP2) regulates many cellular processes. Yet the dynamics of PIP2 concentration changes in living cells in their native environment are virtually unknown. However, knowledge of these dynamics is prerequisite to understanding the physiological functions of PIP2. Recent development of PIP2-probes made of PIP2-binding domains and fluorescent proteins has enabled optical measurements of PIP2 dynamics in living cells. Previous studies in heterologous expression systems and isolated neurons showed that activation of Gáq coupled receptors can deplete the PIP2 content in the plasma membrane. This PIP2 reduction results in a decrease of lipid-protein interactions and consequently to a modulation of the activity and localization of PIP2-sensitive proteins (effectors). In neurons, PIP2 depletion is thought to modulate various ion channels and thus electric excitability. In this study, I have characterized the Gq-induced PIP2 depletion in CA1 hippocampal neurons in situ in acute brain slices. Measurement of PIP2 dynamics was achieved by means of confocal microscopy of PIP2 dependent membrane association of the tubby domain. Comparative measurements of PIP2- and potentially IP3-sensitive PLCd1-PH domain translocation largely resembled the tubby data but showed a slower recovery especially upon strong translocation. Pharmacological activation of both muscarinic acetylcholine (mAch) and metabotropic glutamate (mGlu) receptors (R) induced a robust depletion of PIP2. The amplitude of mAchR induced PIP2 depletion was significantly larger compared to mGluR (type I). This difference in amplitudes was more pronounced in the main apical dendrite compared to the soma. The average time course of PIP2 dynamics was similar for both receptors and showed an initial peak and a lower plateau phase, revealing desensitization. The phasic-tonic time course according to desensitization was more pronounced for mAchR activation. Furthermore, the data obtained here show for the first time that in some neurons Gáq coupled receptor activation induces oscillations of membrane PIP2 concentrations. Moreover, oscillations demonstrate that neurons can rapidly recover PIP2 during prolonged presence of agonists. Application of other Gq coupled receptor agonists did not induce a measureable PIP2 depletion in soma and main apical dendrite. Taken together the results support a physiological function of PIP2 dynamics and correlate well with the general time course of electrophysiological responses to the agonists. A physiological role of PIP2-dynamics is further supported by results of electric stimulation in the area of afferent fibers in the stratum oriens. The data implicate that PIP2 depletion can be induced by synaptic receptor activation, but the pharmacology needs further verification. Data from dentate gyrus granule cells show that PIP2 depletion is not only a receptor but also neurontype specific process, as application of mAchR and mGluR agonists did not induce a measureable PIP2-depletion here.