Download Imaging Neuronal Activity with Voltage- and Calcium

Imaging Neuronal Activity with
Voltage- and Calcium-Sensitive
Dyes
Ryota Homma
Yale University School of Medicine
[email protected]
in vivo optical imaging
Tools for recording population activity
Population averaged
signal: each pixel
represents many neurons
(macroscopic)
Multiple Individual
neurons
(single cell resolution)
Grinvald and Hildesheim (2004)
Nat. Rev. Neurosci. 5:874
Ohki et al., (2006) Nature, 442:925
Voltage- v.s. calcium-sensitive
dyes
• Voltage-sensitive dye:
– binds to cell membranes
– changes its fluorescence depending
on membrane potentials of the cell
• Calcium-sensitive dye
– works on the inside of cell
– changes its fluorescence depending
on intracellular calcium level
Voltage- v.s. calcium-sensitive
dyes
• Voltage-sensitive dye:
population average measurement
– Smaller signal
– Relatively easy staining
• Calcium-sensitive dye:
individual cell measurement
– Larger signal
– Dye loading is more difficult.
Population activity recording
using voltage-sensitive dye
Emergence of the iso-orientation domains
Grinvald and Hildesheim (2004)
Nat. Rev. Neurosci. 5: 874
in vivo VSD signal mainly reflects
subthreshold synaptic potentials.
Whole-cell recordings from
layer 2/3 pyramidal neurons
of rats barrel cortex and the
local VSD signals.
Petersen, Grinvald, and Sakmann (2003) J. Neurosci. 23: 1298
Imaging synaptic potentials
Visually evoked response in Monkey IT cortex
Imaging synaptic potentials
Visually evoked response
in Monkey IT cortex
Tsunoda et al. (2001)
Nat. Neurosci. 4: 832
Loading neurons with calciumsensitive dyes in vivo
Multi-cell bolus loading
Stosiek et al., (2003) PNAS 100, 7319
Two-photon calcium imaging
Three types of odor-evoked response in the
interneuron of the mouse olfactory bulb
ON
INHIBITED
OFF
Recording from multiple individual cells using
Calcium-sensitive dye and 2-photon microscope
Cells responding to
“2-hexanone”
ON response
Cells responding to
“isoamyl acetate”
INHIBITED response
“2-hexanone”
ON response
“isoamyl acetate”
INHIBITED response
OFF response
Calcium response observed in cell soma is
correlated with action potentials.
Kerr, Greenberg, and Helmchen (2005) PNAS 102: 14063
Why using dyes?
You will know…
– what you measure.
Dyes monitor membrane potential or intracellular
calcium level.
– when it happens.
Dyes follow the change of membrane potential or
calcium level with a fast enough response time.
– where the signals come from.
Signal is strictly localized within a neuron.
Targeted labeling: Recording from axonterminal population using calcium dye
Wachowiak and Cohen (2001) Neuron 32: 723
Toward target labeling of any cell –
genetically encoded indicators
Voltage-sensitive proteins
VSFP2.1
Dimitrov et al., PLoS ONE (2007) 2: e440
Mermaid
Tsusui et al., (2008) Nat Methods 5: 683
Calcium-sensitive proteins
GCaMP2
Nakai, Ohkura, and Imoto (2001)
Nat. Biotech 19: 137
CerTN-L15
Garaschuk, Griesbeck, and Konnerth
(2007) Cell Calcium 42: 351
Acknowledgements
• Manabu Tanifuji
RIKEN Brain Science Institute
• Larry Cohen
Yale University School of Medicine
• Olga Garaschuk
University Tubingen / Technical University Munich
• Arthur Konnerth
Technical University Munich