Методы стимуляциии проблемы имиджинга
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Transcript of Методы стимуляциии проблемы имиджинга
Методы стимуляциии Методы стимуляциии проблемы имиджинга проблемы имиджинга
Алексей Васильевич Алексей Васильевич СемьяновСемьянов
Induction of CaInduction of Ca2+2+ signal signal
• chemical stimulation (bath application)
Bath application of receptor agonistsBath 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MRS2578 – P2Y6 receptor antagonistLebedinskiy et al., unpublished
Induction of CaInduction of Ca2+2+ signal signal
• chemical stimulation (bath application)
• depolarization of neurons in whole cell configuration (axonal and dendritic action potential mediated Ca2+ transients)
Use of DIC for cell identificationUse of DIC for cell identification
CA1 region pyramidal cells Interneuron
30 m
Two-photon imaging of CaTwo-photon imaging of Ca2+2+ transients in dendrites of transients in dendrites of CA1 pyramidal cellsCA1 pyramidal cells
5
m
Two-photon excitation x=810 nm Fluo 4 (100 M)
antidromic AC
100 ms
50%F/F
Induction of CaInduction of Ca2+2+ signal 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+ transients due to EPSP/C)
Measurement of changes in CaMeasurement of changes in Ca2+2+ evoked by evoked by synaptic stimulation synaptic stimulation
Yasuda et al., Sci. STKE, 2004
Troubleshooting an absence of CaTroubleshooting an absence of Ca2+2+ transient in transient in response to synaptic stimulation response to synaptic stimulation
Yasuda et al., Sci. STKE, 2004
Induction of CaInduction of Ca2+2+ signal 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+ transients due to EPSP/C)
• pressure or iontoforetic application of receptor agonists (e.g. glutamate, acetylcholine)
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
CaCa2+2+ response in astrocytes evoked by 1 mM response in astrocytes evoked by 1 mM glutamate puff applicationglutamate puff application
Lebedinskiy et al., unpublished
Response depends on agonist concentrationpressure duration of puff
Can be blocked by antagonists
Induction of CaInduction of Ca2+2+ signal 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+ transients due to EPSP/C)
• pressure or iontoforetic application of receptor agonists (e.g. glutamate, acetylcholine)
• uncaging of receptor agonists or intracellular Ca2+
PhotoactivationPhotoactivation
(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
PhotoactivationPhotoactivation
‘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
Use of two-scanner system for simultaneous Use of two-scanner system for simultaneous imaging and uncagingimaging and uncaging
Caged glutamate free glutamate
(inactive) (active)
UV
Voltage clamp, 2P imaging and 1P uncagingVoltage clamp, 2P imaging and 1P uncaging
Voltage clamp, 2P imaging and 1P uncagingVoltage clamp, 2P imaging and 1P uncaging
Specificity of 1P uncagingSpecificity of 1P uncaging
Works only with superficial cells. For deep cells 2P uncaging is required.
Calcium uncaging in astrocytesCalcium uncaging in astrocytes
Problems with imagingProblems with imaging
• Ca2+ buffering by indicators and interaction with endogenous buffers
reducing indicator concentration to minimise its buffering capacity increases
signal-to-noise ratio
dt
Cadkk
dt
dyeCad
dt
BCad
dt
Cad
dt
CaddyeB
T][
)1(][][][][ 22222
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
Problems with imagingProblems 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 indicatorsPhotobleaching 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.
Problems with imagingProblems 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 damagePhototoxic damage
5.3 mW, 75 fs 10-12 mW, 75 fs
Basal dendrite, layer 5 pyramidal cell, OGB-1 (100 mM), 400 s light exposure, lx=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
Problems with imagingProblems 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
ReferencesReferences
• 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/