Transcript Document

Методы стимуляциии
проблемы имиджинга
Алексей Васильевич
Семьянов
Induction of Ca2+ signal
•
chemical stimulation (bath application)
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Lebedinskiy et al., unpublished
Induction of Ca2+ 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 identification
CA1 region
pyramidal cells
Interneuron
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
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+ transients due to EPSP/C)
Measurement of changes in Ca2+ evoked by
synaptic stimulation
Yasuda et al., Sci. STKE, 2004
Troubleshooting an absence of Ca2+ transient in
response to synaptic stimulation
Yasuda et al., Sci. STKE, 2004
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+ transients due to EPSP/C)
•
pressure or iontoforetic application of receptor agonists (e.g. glutamate,
acetylcholine)
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)
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Lebedinskiy et al., unpublished
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+ transients due to EPSP/C)
•
pressure or iontoforetic application of receptor agonists (e.g. glutamate,
acetylcholine)
•
uncaging of receptor agonists or intracellular Ca2+
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
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
Use of two-scanner system for simultaneous
imaging and uncaging
UV
Caged glutamate
(inactive)
free glutamate
(active)
Voltage clamp, 2P imaging and 1P uncaging
Voltage clamp, 2P imaging and 1P uncaging
Specificity of 1P uncaging
Works only with superficial cells. For deep cells 2P uncaging is required.
Calcium uncaging in astrocytes
Problems with imaging
•
Ca2+ buffering by indicators and interaction with endogenous buffers
d[Ca 2 ]T
dt
d[Ca 2 ] d[ BCa2 ] d[dyeCa2 ]
d[Ca 2 ]



 (1  k B  kdye )
dt
dt
dt
dt
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
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 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
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
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 DF/F signal
Local swelling of cell processes
Local destruction of
plasmalemma
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
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/