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MRS2578 – P2Y6 receptor antagonistLebedinskiy et al., unpublished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Lebedinskiy et al., unpublished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/