Calcium Imaging Flashcards
Calcium signalling
Calcium is a versatile intracellular messenger and its role includes:
Exocytosis of synaptic vesicles
Synaptic plasticity
Gene transcription
Vasodilation
All of these involve calcium levels changing at specific neurons or in specific parts of the brain
Intracellular free calcium concentration is low at rest and massively increases when activated. This is observable with calcium indicators so get something to bind and show where the calcium is we can strongly correlate with specific events
Synaptic plasticity
Happens at the level of the dendritic spines of postsynaptic neurons
Strengthens neuronal cinnections
Gene transcription
Happens at the soma of postsynaptic neurons
Calcium imaging
Happens in discrete sub compartments within cells and in discrete time frames.
Intracellular calcium signals regulate processes which operate over a wide time range.
Development of calcium imaging involved 2 parallel processes:
The development and continuous improvement of fluorescent calcium indicators and the development and continuous improvement of appropriate microscopy techniques that can image calcium indicators.
These parallel developments have allowed calcium imaging to get to where it is.
Calcium transmission
Calcium signals are widely used experimentally to monitor somatic and dendritic events.
Contribution of neuronal calcium signals come from:
Voltage gated calcium channels
NMDA receptors
AMPA receptors
So if we understand what drives activity at these channels/receptors then we can use calcium changes to inform us of neural events.
Voltage gated calcium channels
These channels open following voltage changes happening primarily through back propagation of APs (only significant event which changes voltage) - AP initiated at level of soma shoot’s up and down neuron, opening VGCCs expressed along body AP is initiated.
VGCCs are main determinant of somatic calcium signals = allowing for monitoring of APs.
Blockade of sodium disrupts AP initiation and conduction; stopping VGCCs from opening.
Good evidence solidifying if we monitor calcium changes at soma they can tell us when APs occur.
NMDA receptors
Ionotropic glutamate receptors - purely this involved in calcium; in the spines.
See a transient increase in calcium following synaptic activity so thought these to be very important in plasticity.
NMDARs = antagonist which mediate a major part of the postsynaptic calcium influx in dendritic spines (significantly reduced amplitude)
AMPA receptors
Calcium-permeable AMPA receptors have been identified in many aspiny neurons.
Well localised calcium transients as shown by 2-photon calcium imagining - activation of single synapses creates highly localised dendritic calcium signals.
Calcium association with electrophysiology signals
Both in vitro and in vivo work shows somatic calcium signals are highly correlated with APs.
Imaging on dendritic spines demonstrated that calcium signals can be restricted to single dendritic spines.
Current in vivo calcium imaging techniques can isolate single spine response to sensory events.
How do we image calcium signalling?
Calcium indicators
These are proteins which phosphoresce upon calcium binding.
Increasing move towards GECIs - these can genetically encode calcium indicators to only express in a particular subtype of interest - allowed for higher stability indicators and more specific recordings of targets.
Fluorophores
Absorb light energy of a specific wavelength and emit light of a different wavelength - principle which underlies calcium imaging as each fluorophore will have a specific absorption and emission rate.
They exist in ground and excited state - photon of a specific wavelength will excite them and there are photons of a specific different wavelength are emitted as it returns to ground state.
Basis of contemporary calcium indicators cos calcium alters the fluorophore in some way.
Fura-2
Early fluorescent calcium indicator.
Fluoresce dynamics change upon calcium elevation - by having things that respond to different wavelengths and varying these wavelengths of light we can calculate how much free calcium there is in a specific part of the cell
GECIs - FRET based
Forster resonance energy transfer between an excited donor fluorophore and a lower energy acceptor fluorophore.
Calcium binding reduces spatial distance between the two fluorescent proteins so the previously lower energy fluorophore fluoresces more.
Know the calcium is there cos it facilitates the difference in light shone - only calcium can bind the two together and facilitate the energy transfer so know its present.
GECIs - GCaMP based
Ongoing development of these so they’re stable for a longer time to allow longer imaging - current premier in vivo calcium indicator.
Changes intensity of emitted light when calcium is in the system.
Comprised of enhanced green fluorescent protein and CaM-M13 sequence. In presence of calcium, everything binds together and increases the fluorescent emitted as there’s now the electrons to fluoresce.
Dye loading
Basic techniques = patch comping = injecting dyes into single cells or blast them down white matter tracks hoping it hits a certain neural population.
Quite non-selective - issue cos want to be quite precise in the cells that are going to express the indicators and these techniques allow you to get dye into lots of cells just not necessarily the ones you want to get it into.
Had transgenic models introduced and allowed for animals to express GCaMP only in specific populations of interest.
Viral transduction - load multiple dyes to multiple different cell types allowing you to look at multiple different neuronal populations.
Imaging calcium in vivo - 2 photon microscopy process!
Typically have light source and detector
Looking at photons emitted by sample following excitation as it returns to ground state.
2-photon microscopy is the premier technique.
Light bounced off a scanner and through beam splitters, into mouse, calcium indicators may change the fluorescence dynamics, the emitted light from the fluorophore reflects off the dichroic mirror and is detected by photomultiplier tube.
Higher calcium concentrations = higher intensity of fluorescence.
Why 2 photon?
2 photons - lower energy higher wavelength (red as blue doesn’t have the penetration ability) - so need 2 to have the same energy as blue with added penetration depth.
Blue is normal range of calcium indicators fluorescence - cant get far into body but red can so use two of red.
Precise and accurate cos need 2 photons at same position for it to work and no out of focus light.
Absorption spectra in 2-photon microscopy
Diff fluorophores in system and to detect them at the same time use the long pass beam splitters
Whichever one reflects the light rather than it passing through (less than the criteria, if it’s longer it passes through)
This allows you to split up wavelengths being emitted from the sample = allows you to separately measure effects of different somethings.
