Методы стимуляциии проблемы имиджинга

1. Методы стимуляциии проблемы имиджинга Алексей Васильевич Семьянов

2. Induction of Ca2+ signal • chemical stimulation (bath application)

3. Bath application of receptor agonists Stimulation of calcium activity in astrocytes trans-ACPD – group I/II mGluR agonist (RS)-MCPD – nonselective mGluR untagonist NaATP – nonselective purinergic receptor agonist MRS2578 – P2Y6 receptor antagonist Lebedinskiy et al., unpublished

4. Induction of Ca2+ signal • chemical stimulation (bath application) • depolarization of neurons in whole cell configuration (axonal and dendritic action potential mediated Ca2+ transients)

5. Use of DIC for cell identification Interneuron CA1 region pyramidal cells

6. 5 mm Two-photon imaging of Ca2+ transients in dendrites of CA1 pyramidal cells Two-photon excitation lx=810 nm Fluo 4 (100 mM) 50% DF/F 100 ms antidromic AC 30 mm

7. Induction of Ca2+ signal • chemical stimulation (bath application) • depolarization of excitable cell in whole cell configuration (axonal and dendritic action potential mediated Ca2+ transients) • stimulation of presynaptic fibres (Ca2+ transientsdue to EPSP/C)

8. Measurement of changes in Ca2+ evoked by synaptic stimulation Yasuda et al., Sci. STKE, 2004

9. Troubleshooting an absence of Ca2+ transient in response to synaptic stimulation Yasuda et al., Sci. STKE, 2004

10. Induction of Ca2+ signal • chemical stimulation (bath application) • depolarization of excitable cell in whole cell configuration (axonal and dendritic action potential mediated Ca2+ transients) • stimulation of presynaptic fibres (Ca2+ transientsdue to EPSP/C) • pressure or iontoforetic application of receptor agonists (e.g. glutamate, acetylcholine)

11. Synaptic and extrasynaptic parts of astrocyte Oregon Green AM Amplifier, fiber volley 275 mM Puff CA3 sulforhodamine 101 Confocal imaging of astrocytes (Oregon Green AM)

12. Ca2+ response in astrocytes evoked by 1 mM glutamate puff application Response depends on agonist concentration pressure duration of puff Can be blocked by antagonists Lebedinskiy et al., unpublished

13. Induction of Ca2+ signal • chemical stimulation (bath application) • depolarization of excitable cell in whole cell configuration (axonal and dendritic action potential mediated Ca2+ transients) • stimulation of presynaptic fibres (Ca2+ transientsdue to EPSP/C) • pressure or iontoforetic application of receptor agonists (e.g. glutamate, acetylcholine) • uncaging of receptor agonists or intracellular Ca2+

14. Photoactivation (1) Kinetics –photorelease ligands from’caged’precursors at intracellular or extracellular receptors. Overcomes diffusional barriers -‘unstirred layers’ in isolated tissue or slices -intracellular receptors and enzymes (2) Spatially resolved kinetics - photorelease localised by point excitation or imaging of local responses with uniform excitation. (3) Labelling and tracking Photoactivation or photorelease of fluorophores for cell lineage studies cytoskeletal rearrangements, organelle trafficking (4) Compartmentalisation – diffusional exchange between compartments

15. Photoactivation ‘Caged’ amino acid neurotransmitters Nitroindolinyl -L-glutamate (NI-glutamate) 4-methoxynitroindolinyl-L-glutamate (MNI-glutamate) •Chemically stable carboxyl group cage •Efficient near-UV photolysis – Extinction 4300 M-1 cm-1, Q= 0.085 •near UV Flashlamp conversion MNI - glu~35% •Fast dark reaction– half-time 0.2 μs Physiological controls: •Caged glutamate at 1mM does not activate or block AMPAR, NMDAR, mGluR, transporters. •No effect of photolysis of NI-caged phosphate on cerebellar climbing fibre transmission or short term plasticity. However: NI-caged GABA and glycine are antagonists at respective receptors

16. Use of two-scanner system for simultaneous imaging and uncaging UV Caged glutamate free glutamate (inactive) (active)

17. Voltage clamp, 2P imaging and 1P uncaging

18. Voltage clamp, 2P imaging and 1P uncaging

19. Specificity of 1P uncaging Works only with superficial cells. For deep cells 2P uncaging is required.

20. Calcium uncaging in astrocytes

21. Problems with imaging • Ca2+ buffering by indicators and interaction with endogenous buffers where: [Ca2+]T –total Ca2+, [BCa2+] - Ca2+ bound to endogenous buffers [dyeCa2+] - Ca2+ bound to dye molecules KB and Kdye – Ca2+ binding ratios Yasuda et al., Sci. STKE, 2004 • reducing indicator concentration to minimise its buffering capacity increases • signal-to-noise ratio

22. Problems with imaging • Ca2+ buffering by indicators and interaction with endogenous buffers • reducing indicator concentration to minimise its buffering capacity increases signal-to-noise ratio • dye fluorescence saturation • use indicator with Kd which corresponds to concentration of Ca2+, too high Kd (low affinity) gives bad signal-to-noise ratio

23. Photobleaching of indicators Light-induced change in a fluomophore, resulting in the loss of its absorption of light of a particular wave length. Useful for FRAP (fluorescence recovery after photobleching) technique

24. Problems with imaging • Ca2+ buffering by indicators and interaction with endogenous buffers • reducing indicator concentration to minimise its buffering capacity increases signal-to-noise ratio • dye fluorescence saturation • use indicator with Kd which corresponds to concentration of Ca2+, too high Kd (low affinity) gives bad signal-to-noise ratio • photobleaching of indicator • reduce intensity of laser light and exposure • use Ca2+ indicators with lower photobleaching rate • use ratiometric dyes

25. 5.3 mW, 75 fs 10-12 mW, 75 fs Phototoxic damage • Local irreversible increase in baseline fluorescence • Decrease in relative DF/F signal • Local swelling of cell processes • Local destruction of plasmalemma Basal dendrite, layer 5 pyramidal cell, OGB-1 (100 mM), 400 s light exposure, lx=800 nm (Koester et al., 1999)

26. Problems with imaging • Ca2+ buffering by indicators and interaction with endogenous buffers • reducing indicator concentration to minimise its buffering capacity increases signal-to-noise ratio • dye fluorescence saturation • use indicator with Kd which corresponds to concentration of Ca2+, too high Kd (low affinity) gives bad signal-to-noise ratio • photobleaching of indicator • reduce intensity of laser light and exposure • use Ca2+ indicators with lower photobleaching rate • use ratiometric dyes • phototoxic damage • reduce intensity of laser light • reduce exposure

27. References • Imaging in Neuroscience and Development Rafael Yuste (Editor), Arthur Konnerth (Editor) Cold Spring Harbor Laboratory Pr  /  2005 • Yasuda et al., Imaging calcium concentration dynamics in small neuronal compartments.Sci STKE. 2004 • Handbook of Fluorescent Probes and Research Products www.probes.com/handbook/