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. 2016 Jan 27;36(4):1386-400.
doi: 10.1523/JNEUROSCI.3535-15.2016.

Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons

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Free PMC article

Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons

Martin Kruse et al. J Neurosci. .
Free PMC article

Abstract

In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca(2+) upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons.

Significance statement: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons.

Keywords: M-current; PI(4,5)P2; excitability; phosphoinositide metabolism; superior cervical ganglion neurons.

Figures

Figure 1.
Figure 1.
Faster kinetics of IKCNQ2/3 and PI(4,5)P2 recovery in SCG neurons versus tsA201 cells. A, Overview of the analyzed reactions of PI metabolism. Red, Enzymes. B, Schematic of closure of KCNQ2/3 channels due to PI(4,5)P2 hydrolysis. Left, Activation of phospholipase C after binding of the ligand Oxo-M to M1R. Right, Activation of a VSP by membrane depolarization. C, Normalized KCNQ2/3 tail current amplitude upon activation of M1 receptors by 10 μm Oxo-M measured with whole-cell recordings from SCG neurons and tsA201 cells. D, Time constants of tail current amplitude recovery after removal of Oxo-M. Numbers in brackets indicate number of experiments. E, Normalized KCNQ2/3 tail current amplitude upon activation of M1 receptors by 1 μm Oxo-M measured with whole-cell recordings from SCG neurons 1 h after isolation. F, Time constants of tail current amplitude recovery after activation of M1Rs with 1 μm Oxo-M at different time points after isolation of SCG neurons (black) or percentage inhibition of M-current (red). G, Same as in C but with activation of VSP. H, Time constants of tail current amplitude recovery after VSP activation. I, SCG neurons were transfected by intranuclear injection with plasmids encoding TubbyR332H-YFP and CAAX-CFP to measure changes in plasma membrane PI(4,5)P2 levels with ratiometric FRETr recordings. Top, Schematic of FRETr measurements before and after depletion of PI(4,5)P2. Bottom, Negative contrast confocal image of fluorescence intensity distribution of TubbyR332H before (left) and after (right) application of Oxo-M. J, Line scans of TubbyR332H distribution before (black) and after (orange) application of Oxo-M along the lines indicated in I. K, Normalized FRETr between TubbyR332H-YFP and CAAX-CFP upon application of Oxo-M to SCG neurons and tsA201 cells. L, Negative contrast confocal images of PH-PLCδ1-YFP fluorescence in a SCG neuron before and 20 s after stimulation with 10 μm Oxo-M. M, Line scans showing fluorescence intensity distribution of PH-PLCδ1-YFP along lines indicated in L before (black) and after (orange) application of Oxo-M. N, Mean normalized FRETr from SCG neurons transfected with PH-PLCδ1-CFP and -YFP upon stimulation of cells with Oxo-M.
Figure 2.
Figure 2.
Recovery of PI(4)P is faster in SCG neurons than in tsA201 cells. A, Negative contrast confocal images of the PI(4)P indicator P4M-GFP in a SCG neuron before (left) and after (right) application of 10 μm Oxo-M. B, Line scan showing P4M distribution along lines indicated in (A) before (black) and after (orange) Oxo-M application. C, Normalized FRETr measured from SCG neurons (black) and tsA201 cells (blue) transfected with P4M-YFP and CAAX-CFP upon application of Oxo-M for 20 s.
Figure 3.
Figure 3.
Estimation of PLC and M1R expression levels in SCG neurons. A, Western blot of cytoplasmic lysates of tsA201 cells and SCG neurons with an anti-PLCβ1 antibody. Protein amounts of the two lysates were normalized for equal amounts; percentages indicate dilution steps. “PLCβ1” indicates detection of a recombinant PLCβ1 protein used as a positive control for the primary antibody. B, Percentage of PLCβ1 in SCG neurons compared with tsA201 cells as measured by analysis in A. Number in brackets indicates number of experiments. C, Left, Time course of KCNQ2/3 tail current amplitude recorded from a SCG neuron upon application of indicated concentrations of Oxo-M. Right, Current traces corresponding to time points indicated by letters on the left. Arrow indicates tail currents. D, IKCNQ2/3 inhibition evoked by 20 s applications of different concentrations of Oxo-M in SCG neurons (black) or tsA201 cells (blue). E, Modeled IKCNQ2/3 inhibition evoked by 20 s applications of different concentrations of Oxo-M for indicated densities of M1R. Dotted line indicates M1R density of 16 molecules μm−2. F, Comparison of experimental (black) and modeled (red) dose–response curves for I KCNQ2/3 inhibition by Oxo-M in SCG neurons. G, IC50 values for I KCNQ2/3 inhibition by Oxo-M in SCG neurons as measured experimentally (n = 5–25) and from our model.
Figure 4.
Figure 4.
Phosphoinositide levels determined by mass spectrometry. A, Ratio of total PI to total PIP as measured from SCG and tsA201 cells. B, Levels of PIP and PIP2 relative to controls from SCG and tsA201 cells after 1 min application of 10 μm Oxo-M. C, Relative amounts of total PI, PIP, and PIP2 as percentages of total phosphoinositides for SCG and tsA201 cells. Number in brackets indicates number of experiments.
Figure 5.
Figure 5.
Modeling PI metabolism of SCG neurons requires faster lipid 4-kinase. A, Time course of normalized IKCNQ2/3 amplitude upon stimulation of SCG neurons with Oxo-M. Comparison of experimental data from whole-cell recordings (black) and normalized IKCNQ2/3 amplitude predicted by simulation (red). Traces were segmented into onset and recovery phases and normalized to basal levels to correct for partial recovery. B, Same as in A, but for activation of VSP. C, Time course of normalized FRETr between TubbyR332H-YFP and CAAX-CFP upon stimulation of SCG neurons with Oxo-M as measured experimentally (black) and as predicted by simulation (red). Traces were segmented into onset and recovery phases and normalized to basal levels to correct for partial recovery. D, Same as in C, but for PH-PLCδ1 domains as FRETr reporters. The prediction plotted in red assumes no binding of PH-PLCδ1 domains to IP3. Note that the modeled traces approximate changes in FRETr as a cooperative square law of the membrane-bound fraction of PH-PLCδ1 domains as recently described (Itsuki et al., 2014). E, Comparison of rate constant of synthesis of PI(4)P and PI(4,5)P2 between SCG neurons and tsA201 cells, expressed as fold-changes, SCG/tsA201. F, Simulated time courses of normalized levels of PI(4)P in SCG neurons (red, “neuron model”) and tsA201 cells (blue, “tsA201 model”) upon stimulation with Oxo-M.
Figure 6.
Figure 6.
Kinetics of IP3 and DAG signals after M1R activation. A, Schematic for detection of DAG and IP3 by FRET recordings. B, Mean normalized FRETr of LIBRAvIII in response to 10 μm Oxo-M in SCG neurons (inverted scale, FCFP/FYFP). C, D, Overlay of modeled LIBRAvIII response (red) on the time course of normalized inverted LIBRAvIII-FRETr evoked by stimulation of SCG neurons with Oxo-M (black). Traces were segmented into onset (C) and recovery (D) phases and normalized to basal levels to correct for partial recovery. E, Same as in B, but SCG neurons were transfected with C1-YFP and CAAX-CFP to measure changes in DAG levels (FYFP/FCFP). Note different time scale. F, G, Same as in C, D, but for FRETr data and modeled C1-YFP/CAAX-CFP signal.
Figure 7.
Figure 7.
Summary comparison of modeled phosphoinositide metabolism and signaling in SCG neurons and tsA201 cells. A, Modeled time courses of IP3 in SCG neurons and tsA201 cells in response to a 20 s long 10 μm Oxo-M application. BD, As in A, but for DAG (B), PI(4,5)P2 (C), and IKCNQ2/3 (D).
Figure 8.
Figure 8.
Ca2+ signals after M1R activation in SCG neurons. A, Cytoplasmic Ca2+ in SCG neurons loaded with fura-2-AM and stimulated with 10 μm Oxo-M. Fura-2 signals, measured in the presence of 100 nm TTX. B, Same as in A, but with 100 μm CdCl2 as indicated instead of TTX. C, Western blot of membrane preparations of tsA201 cells and SCG neurons with anti-IP3R1 (top left) and anti-IP3R2 (bottom left) antibodies. Protein amounts of the samples were normalized for equal amounts. Right, Percentage of IP3R in SCG neurons compared with tsA201 cells. Number in brackets indicates number of experiments. D, Steady-state IP3 profile in a two-dimensional model of the cytoplasm of a neuron. IP3-production is assumed to occur at the origin. Concentrations at different distances depend on the diffusion coefficient of IP3 as well as the modeled activity of an IP3-5-phosphatase. E, Calculated cytoplasmic IP3 concentrations in one plane of a three-dimensional model of a SCG neuron upon uniform activation of M1Rs for the indicated time on the cell surface with a saturating concentration of Oxo-M. F, Calculated IP3 concentrations at two selected regions-of-interest (ROI) in the 3-D diffusion model in E. G, Western blot of lysate of SCG neurons with anti-IRBIT antibody. H, Scheme of the components of the model involved in Ca2+-release from intracellular stores. Gray, Components included in tsA201 cell model; Red, additional components added in SCG model. I, Overlay of modeled fura-2 response (red) on the time course of normalized fura-2 signal evoked by stimulation of SCG neurons with Oxo-M (black) in the presence of 100 nm TTX. J, Same as in I, but for simulation of fura-2 signal measured in the presence of 100 μm CdCl2 instead of TTX.
Figure 9.
Figure 9.
Gradients of IP3 during local activation of M1R in a simulated SCG neuron. A, The image shows one plane of a three-dimensional spatial model using the geometry of a SCG neuron. Outlined are the plasma membrane as well as the nuclear membrane. The red field is the area in which the activating ligand 10 μm Oxo-M is present throughout the simulation. B, Map of cytoplasmic IP3 concentrations for several time points during the simulation. The dark area inside the cytoplasm is the cell nucleus. C, Time course of IP3 concentrations for two regions-of-interest (ROI). Inset, Localization of ROIs. D, Simulated relative levels of Ca2+-bound and Ca2+-unbound NCS-1 isoforms in response to a simulated application of 10 μm Oxo-M. Simulations were performed for presence (black) or absence (red) of IRBIT. E, Simulated increase in Ca2+-bound NCS-1 upon muscarinic stimulation in the presence or absence of IRBIT.

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