Ion imaging in neurons

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Ion imaging in neurons

• Nathan Urban
• CMU Biological Sciences

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Choices for imaging neuronal activity

• Voltage sensitive dye imaging
• Ion imaging (calcium/sodium/zinc)
• Intrinsic optical imaging
• fMRI

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Outline

• The basics how indicator dyes work
• The examples
• Small molecule dyes in vitro
• Small molecule dyes in vivo

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Ion imaging

• Why ions?
• Ionic fluxes occur during neuronal activity
• In some cases they are causally related to
  activity
• Good dyes for ions have been created
• Which ions?
• Calcium is most popular
• Very low resting calcium concentration mean many
  fold changes with activity
• Good dyes exist for calcium
• Calcium is also involved in processes such as
  transmitter release and plasticity
• Sodium imaging is also possible

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Fura-2 one of the first calcium indicators
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Fura-2 one of the first calcium indicators
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How calcium dyes work
kon

• B Ca ? BCa
• Ftotal FBB FBCaBCa FAuto

koff
Neher and Augustine 1992
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Converting fluorescence to calcium concentration

• Thus, calcium transients are often reported as
  DF/F.
• However in some circumstances DF/F can change
  because of other factors, e.g. swelling of cells.
  
• For some dyes, one can correct for this and
  obtain the actual change in Ca. Most
  commonly this is does with dyes that are calcium
  insensitive in their fluorescence when excited at
  a particular wavelength. Changes in fluorescence
  at this wavelength are used to correct for other
  sources of change in fluorescence.
• The most common dyes of this sort are fura
  derivatives
• These dyes are ratiometric dyes because the ratio
  of fluorescence at two wavelengths is
  proportional to the calcium concentration.

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Helmchen Imoto Sakmann
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Problems with calcium dyes

• Dyes have a dynamic range and are subject to
  saturation
• Dyes report volume averaged calcium
• Dyes are also buffers of calcium

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Effects of calcium dyes/buffers on calcium
transients
Helmchen Imoto Sakmann
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Types of calcium dyes

• Small molecule organic dyes
• Protein-based dyes (genetically encoded)

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Delivery of calcium dyes

• Dyes must get cross the cells membrane
• Injection (via small glass electrodes)
• Difficult in vivo
• Bulk loaded
• AM-ester forms of dyes
• Local electroporation
• Genetically encoded

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AOB mitral cell dendrites support backpropagating
spikes and local tuft calcium spikes
Urban and Castro 2005
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In vivo single cell calcium imaging
Helmchen et al Nature Neuroscience 1999
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Bulk loading of calcium indicator
Stosiek et al PNAS 2003
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Input (presynaptic calcium transients) ouput
(pyramidal neuron spiking) and glial activity can
be measured simultaneously
Kerr Greenberg Helmchen PNAS 2005
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Imaging cortical activity at the single cell
level in vivo
Ohki et al Nature 2005
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Ohki et al Nature 2005
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Ohki et al Nature 2005
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Correspondence between physiology and imaging
Ohki et al Nature 2005
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Local electroporation of calcium indicator and
other dyes
Nagayama et al 2007
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Nagayama et al Neuron 2007
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Nagayama et al Neuron 2007
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Rapid 3D scanning of neuronal populations
Goebel Kampe and Helmchen 2007
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Genetically encoded calcium indicators
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Advantages of genetically encoded calcium
indicators

• Cells come pre-loaded
• Genetically identified cell types can be loaded
  selectively
• Indicator can be localized to specific cell
  compartments
• In principle indicators of other cellular
  processes may be generated as well

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Genetically-encoded calcium indicators
Hasan et al 2004
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Light Evoked Responses in Retina Measured with
Genetically Encoded Calcium Dye
Hasan et al 2004