Lecture 4 Calcium Imaging

Question 1

What roles does calcium play in the brain? (4)

Answer 1

-exocytosis of synaptic vesicles
- synaptic plasticity
- gene transcription
- vasodilation

Question 2

how does intracellular free calcium concentration change during electrical activity

Answer 2

it rises transiently by 10-100 x higher

Question 3

development of calcium imaging involves 2 parallel processes:

Answer 3

1) Development and continuous improvement of fluorescent calcium indicators
2) development of appropriate microscopy techniques that can image calcium indicators

Question 4

contribution of neuronal Ca2+ arise from which 3 major sources?

Answer 4

• voltage gated calcium channels (VGCCs)
• NMDA receptors (spines)
• AMPA receptors (Aspiny neurons)

Question 5

discuss calcium transients and VGCCs

Answer 5

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)

Question 6

discuss calcium transients and NMDA receptors

Answer 6

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)

Question 7

discuss calcium transients and AMPA receptors

Answer 7

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

Question 8

describe overall calcium’s association with electrophysiological signals

Answer 8

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

Question 9

define calcium indicator

Answer 9

proteins that phosphoresce upon binding with calcium

Question 10

what technique was Ca2+ imaging originally based on?

Answer 10

injectable dyes

Question 11

describe fluorophores

Answer 11

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

Question 12

describe FURA-2 indicators

Answer 12

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.

Question 13

What are the 2 types of Genetically Encoded Calcium Indicators (GECIs)?

Answer 13

FRET-based and GCaMP based

Question 14

describe FRET-based GECIs

Answer 14

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

Question 15

describe GCaMP based GECI

Answer 15

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

Question 16

what are the 3 techniques for dye-loading?

Answer 16

dextran conjugate loading
electroporation
GECI in vitro

Question 17

describe dextran conjugate loading

Answer 17

dye is pressure injected in axonal pathways with retro- and antero-transport to soma/axons

inefficient and lacks specificity

Question 18

describe electroporation

Answer 18

voltage pulses disrupt tissue membrane and alternate with pulse that pushes dye into cells

also lacks specificity

Question 19

what’s the advantage of GECI dye loading compared to injection/electroporation?

Answer 19

it has higher specificity

Question 20

overall, how are calcium indicators imaged typically?

Answer 20

imaging devices with light source and detector scan laser beam over specimen and photomultiplier tubes record emitted light

Question 21

what is the premier technique of calcium imaging?

Answer 21

2-photon microscopy

Question 22

describe 2-photon microscopy

Answer 22

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

Question 23

describe the advantages of 2P- microscopy

Answer 23

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

Question 24

how is an image generated from 2-photon microscopy?

Answer 24

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