1. Методы стимуляциии проблемы имиджинга Алексей Васильевич Семьянов

2. Induction of Ca 2+ 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untagonist NaATP – nonselective purinergic receptor agonist MRS2578 – P2Y6 receptor antagonist Lebedinskiy et al., unpublished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 Ca 2+ signal chemical stimulation (bath application) depolarization of neurons in whole cell configuration (axonal and dendritic action potential mediated Ca 2+ transients)

5. Use of DIC for cell identification CA1 regionpyramidal cells Interneuron

6. 30  m Two-photon imaging of Ca 2+ transients in dendrites of CA1 pyramidal cells 5  m Two-photon excitation x=810 nm Fluo 4 (100  M) antidromic AC 100 ms 50%  F/F

7. Induction of Ca 2+ signal chemical stimulation (bath application) depolarization of excitable cell in whole cell configuration (axonal and dendritic action potential mediated Ca 2+ transients) stimulation of presynaptic fibres (Ca 2+ transients due to EPSP/C)

8. Measurement of changes in Ca 2+ evoked by synaptic stimulation Yasuda et al., Sci. STKE, 2004

9. Troubleshooting an absence of Ca 2+ transient in response to synaptic stimulation Yasuda et al., Sci. STKE, 2004

10. Induction of Ca 2+ signal chemical stimulation (bath application) depolarization of excitable cell in whole cell configuration (axonal and dendritic action potential mediated Ca 2+ transients) stimulation of presynaptic fibres (Ca 2+ transients due to EPSP/C) pressure or iontoforetic application of receptor agonists (e.g. glutamate, acetylcholine)

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

12. Ca 2+ response in astrocytes evoked by 1 mM glutamate puff application Lebedinskiy et al., unpublished Response depends on agonist concentration pressure duration of puff Can be blocked by antagonists

13. Induction of Ca 2+ signal chemical stimulation (bath application) depolarization of excitable cell in whole cell configuration (axonal and dendritic action potential mediated Ca 2+ transients) stimulation of presynaptic fibres (Ca 2+ transients due to EPSP/C) pressure or iontoforetic application of receptor agonists (e.g. glutamate, acetylcholine) uncaging of receptor agonists or intracellular Ca 2+

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 Caged glutamate free glutamate (inactive) (active) UV

17. 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 Ca 2+ buffering by indicators and interaction with endogenous buffers reducing indicator concentration to minimise its buffering capacity increases signal-to-noise ratio where: [Ca 2+ ] T –total Ca 2+, [BCa 2+ ] - Ca 2+ bound to endogenous buffers [dyeCa 2+ ] - Ca 2+ bound to dye molecules K B and K dye – Ca 2+ binding ratios Yasuda et al., Sci. STKE, 2004

22. Problems with imaging Ca 2+ 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 K d which corresponds to concentration of Ca 2+, too high K d (low affinity) gives bad signal-to-noise ratio

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

24. Problems with imaging Ca 2+ 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 K d which corresponds to concentration of Ca 2+, too high K d (low affinity) gives bad signal-to-noise ratio photobleaching of indicator –reduce intensity of laser light and exposure –use Ca 2+ indicators with lower photobleaching rate –use ratiometric dyes

25. Phototoxic damage 5.3 mW, 75 fs10-12 mW, 75 fs Basal dendrite, layer 5 pyramidal cell, OGB-1 (100 mM), 400 s light exposure, l x =800 nm (Koester et al., 1999) Local irreversible increase in baseline fluorescence Decrease in relative  F/F signal Local swelling of cell processes Local destruction of plasmalemma

26. Problems with imaging Ca 2+ 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 K d which corresponds to concentration of Ca 2+, too high K d (low affinity) gives bad signal-to-noise ratio photobleaching of indicator –reduce intensity of laser light and exposure –use Ca 2+ 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/ www.probes.com/handbook/