Lecture 4 Calcium Imaging Flashcards
What roles does calcium play in the brain? (4)
-exocytosis of synaptic vesicles
- synaptic plasticity
- gene transcription
- vasodilation
how does intracellular free calcium concentration change during electrical activity
it rises transiently by 10-100 x higher
development of calcium imaging involves 2 parallel processes:
1) Development and continuous improvement of fluorescent calcium indicators
2) development of appropriate microscopy techniques that can image calcium indicators
contribution of neuronal Ca2+ arise from which 3 major sources?
- voltage gated calcium channels (VGCCs)
- NMDA receptors (spines)
- AMPA receptors (Aspiny neurons)
discuss calcium transients and VGCCs
VGCCs allow an influx of Ca2+ into the cell following compartmentalised voltage changes (AP backpropagation/dendritic depolarisation)
VGCCs are the main controlled of somatic calcium levels and are involved in AP signalling (evidenced by the blockade of Na+ to disrupt AP depresses calcium- based fluorescence changes)
discuss calcium transients and NMDA receptors
ionotropic glutamate receptors, widely expressed in dendritic spines across brain. NMDARs mediate a major part of postsynaptic Ca2+ influx in dendritic spines, which is essential for the induction of LTP
(NMDAR involvement evidenced when antagonists significantly recued amplitude of Ca2+ transients)
discuss calcium transients and AMPA receptors
calcium-permeable AMPAR have been identified in many aspiny neurons, with 2-photon Ca2+ imaging showing that activation of single synapses creates localised Ca2+ signals
describe overall calcium’s association with electrophysiological signals
transient Ca2+ signals arise in discrete cellular sub-compartments with signals time locked with electrophysiological responses, therefore imaging changes in free Ca2+ can indicate firing of individual neurons in real time
define calcium indicator
proteins that phosphoresce upon binding with calcium
what technique was Ca2+ imaging originally based on?
injectable dyes
describe fluorophores
they start in ground state, then absorb light energy in the form of photons of a specific wavelength, putting them in excited state, then emit light photons of a differing wavelength taking them back to ground state
Ca2+ binding to fluorophores alters the emitted light
describe FURA-2 indicators
an early indicator, still used in slice work/ex-vivo
it is made up of a chelator and a fluorophore which binds calcium, causing conformational change and change in wavelength causing excitation. When light is shone at alternating wavelengths between the original and calcium-bound wavelengths amount of calcium present can be calculated by the wavelength proportions emitted.
What are the 2 types of Genetically Encoded Calcium Indicators (GECIs)?
FRET-based and GCaMP based
describe FRET-based GECIs
the mechanism is based of Forster resonance energy transfer (FRET) between an excited donor and an acceptor fluorophore
Enhanced Cyan Fluorescent Protein (ECFP) donor is bound to Acceptor Venus protein, connected by CaM-M13 (CaM calmodulin-binds calcium and M13- Peptide which binds calmodulin). At rest ECFP is excited at 440nm and emits light 480 (cyan), when calcium binds the space between ECFP and Venus reduces, enabling energy transfer between cyan to Venus which emits 530nm wavelength (yellow light). So when Ca2+ binds blue fluorescence reduces and yellow increases
describe GCaMP based GECI
it is the current major indicator in-vivo
constant development to improve GCaMP proteins (make them more stable etc.)
GCaMP also has CaM-M13 sequence bound, which binds Ca2+ and when 485 light is shone, 515nm at an increased intensity is emitted. So calcium increases intensity of green light
what are the 3 techniques for dye-loading?
dextran conjugate loading
electroporation
GECI in vitro
describe dextran conjugate loading
dye is pressure injected in axonal pathways with retro- and antero-transport to soma/axons
inefficient and lacks specificity
describe electroporation
voltage pulses disrupt tissue membrane and alternate with pulse that pushes dye into cells
also lacks specificity
what’s the advantage of GECI dye loading compared to injection/electroporation?
it has higher specificity
overall, how are calcium indicators imaged typically?
imaging devices with light source and detector scan laser beam over specimen and photomultiplier tubes record emitted light
what is the premier technique of calcium imaging?
2-photon microscopy
describe 2-photon microscopy
using low-energy, high-wavelength pair of photons (allowing deeper penetration than confocal) which arrive almost at the same moment into targeted tissue, to excite fluorophore
describe the advantages of 2P- microscopy
deeper penetration
high spatial resolution (single photon-confocal- microscopy excites entire sample, whereas bifocal only excites where photons meet)
this can also prevent fluorophore bleaching
able to use multiple dyes in a single imaging session to look at differing wavelength responses of different cell types with photomultiplier tubes detecting discrete wavelengths allowing complex phenomena to be detected
how is an image generated from 2-photon microscopy?
laser scans point by point over sample and records the intensity of emission at each spot- the spots of emitted light recorded are analogous to voxels in MRI
