Calcium imaging

Question 1

What is the role of calcium in the nervous system?

Answer 1

Exocytosis of synaptic vesicles
Synaptic plasticity
Gene transcription
Vasodilation

Question 2

What is the intracellular free Ca2+ concentration at rest?

Answer 2

Approx. 50-100nM at rest
Rises transiently during electrical activity to levels that are 10 to 100 times higher
Observable with Ca2+ indicators

Question 3

What 2 parallel processes were involved in the development of calcium imaging?

Answer 3

1. The development and the continuous improvement of fluorescent calcium indicators
2. The development of appropriate microscopy techniques that can image calcium indicators

Question 4

What 3 major sources contribute to neuronal Ca2+ signals?

Answer 4

1. Voltage-gated calcium channel
   - Soma and spines
2. NMDA receptors
   - Spines
3. AMPA receptors
   - Aspiny transmission

Question 5

What do voltage-gated calcium channels allow for?

Answer 5

Allow for influx of Ca2+ following compartmentalised voltage changes
- Action potential backpropogation
- Depolarisation of dendrites
Main determinant of somatic calcium signals
- Allows for monitoring of action potentials
Blockade of Na+ disrupts action potential initiation and conduction

Question 6

How are NMDA receptors involved in calcium imaging?

Answer 6

NMDA receptors mediate postsynaptic calcium signalling in dendritic spines

Ionotropic glutamate receptors - widely expressed e.g., hippocampal and pyramidal neurons

Transient rise in Ca2+ levels in spines is essential for the induction of activity dependent synaptic plasticity

NMDARs mediate a major part of the postsynaptic Ca2+ influx in dendritic spines

NMDAR antagonism significantly reduces amplitude of Ca2+ transients

Question 7

How are AMPA receptors involved?

Answer 7

Calcium-permeable AMPA receptors have been identified in many aspiny neurons e.g., GABA striatal fast spiking interneurons
2P Ca2+ imaging has shown that activation of single synapses creates highly localised dendritic Ca2+ signals

Question 8

How is calcium associated with electrophysiological signals?

Answer 8

In vitro and in vivo work shows somatic Ca2+ signals are highly correlated with action potentials
- Most likely backpropogation
- Models support that somatic Ca2+ imaging accurately predict spikes

Early calcium imaging focused on imaging of dendritic spines
- Demonstrated that Ca2+ signals can be restricted to single dendritic spines
Current in vivo Ca2+ imaging techniques can isolate single response to sensory events

Question 9

What does this knowledge of calcium mean for imaging?

Answer 9

Transient Ca2+ signals arise in discrete cellular sub compartments
Signals are time locked with electrophysiological responses
== If we can image changes in free Ca2+ concentration we can monitor the firing of individual neurons in real time

Question 10

How do we image calcium signalling?

Answer 10

Required collaborative developments from other fields
Calcium indicators = proteins that phosphoresce upon binding with Ca2+
Growth of calcium imaging initially based on injectable dyes
Increasing move towards genetically encoded calcium indicators (GECIs)

Question 11

What are fluorophores?

Answer 11

Fluorescent chemical compound which absorbs light energy of a specific wavelength and emits light of a different wavelength
Exist in ground and excited state - photon absorption provides energy for transition from ground to excited state, photons emitted as it returns to ground state

Question 12

What is Fura-2?

Answer 12

An early fluorescent calcium indicator
Combination of calcium chelator and fluorophore
The binding of calcium ions causes conformational changes that alters fluorescence dynamics
Fluorescence dynamics change upon calcium elevation
Excitable by ultraviolet light; photon emittance in range of 505-520mm

Question 13

What are FRET based calcium indicators?

Answer 13

Genetically encoded calcium indicators - FRET based
Based on Forster resonance energy transfer (FRET) between an excited donor fluorophore and an acceptor fluorophore

Enhanced cyan fluorescent protein (ECFP) as donor and Venus protein as acceptor

Calcium binding reduces the spatial distance between the two fluorescent proteins

Therefore, the calcium signal detected is the ratio between Venus and ECFP fluorescence

Question 14

What are GCaMP based calcium indicators?

Answer 14

The current premier in vivo calcium indicator
GCaMPs comprise of enhanced green fluorescent protein (EGFP) and a CaM-M13 sequence
In presence of Ca2+, CaM-M13 interactions elicit conformational changes - increase in emitted fluorescence

Question 15

How do you load up the dye?

Answer 15

Traditional techniques
- Dextran-conjugate loading - pressure injected into axonal pathways, retro- and anterograde transport to soma and axons
-Electroporation - voltage pulses disrupt plasma membrane creating pores, pushes charged molecules into cell, suitable for single cell loading, network loading and for transmission of GECIs

Recent introduction of transgenic models which express GCaMP in specific neurons

Question 16

Why is GCaMP better than electroporation?

Answer 16

GCaMP good as you can select specific targets rather than electroporation which creates non specific pores and consequently causes dye to enter several areas

Question 17

How is calcium imaged in vivo?

Answer 17

Imaging devices typically consists of a light source and a detector
Images are generated by scanning a laser beam over the specimen and using a photomultipler tube to record emitted light
Two-photon microscopy currently the premier technique
Recent advancements allow for imaging of awake behaving animals

Question 18

How does two photon microscopy work?

Answer 18

Fluorescence imaging technique that allows imaging of living tissue up to about 1mm in thickness

Penetration depth of traditional confocal microscopy limited by Ca2+ indicator absorption range

2 photon microscopy uses two lower-energy higher wavelength photons
Double the wavelength = half the energy –> allows for higher tissue penetration
Multiple low energy photons arriving within a femtosecond summate - provides same energy as a photon within indicator absorption range

Question 19

What is a major benefit of 2P microscopy?

Answer 19

Precise confocal excitation

Almost exclusively excites fluorophores in a diffraction-limited focal volume
Signal is exclusively generated from the region of interest

Question 20

How do you generate 2 photon images?

Answer 20

Create image by scanning laser point over sample and recording intensity at each spot
Raster scan tightly focused beam across sample - record total emitted light emitted from sample
Comparable to voxels in fMRI

Question 21

What are the absorption spectras in 2-photon microscopy?

Answer 21

2-photon imaging allows for excitation of dyes with broad absorption ranges within a single recording session

Photomultiplier tubes comprise specific compartments tuned to detect photons of discrete wavelengths

Loading of multiple dyes into specific neuronal populations allows us to detect complex phenomena