Optogenetics and dyes

• FLUORESCENT RECEPTOR DYES

  • fluorescents --> where a substance absorbs a photon of light and emits light at a longer wavelength
  • absorption spectrum --> frequency or wavelength of light which the flurophore absorbs
  • emission spectrum  light which is emitted when fluorescent occur
  • electrons at a excited state  and loses energy and emits it at a lower energy
  • electrophysiology is an invasive technique --> don’t have
  • spatial resolution of electrophysiology is usually 

  1. local but have a very high arch --> limits what we can patch onto
  2. global (whole neuron, circuit)
• FLURESCENT RECEPTOR DYES cont.

  • electrophysiology usually does one cell/ circuit at a time

  1. activity receptor can tell where within a neuron, across a whole neuron and when in multiple neurons stimulantoeus

  • ion move when ion channels open

  1. movement o calcium ions
  2. calcium has high conc in extracellular + stores and low conc in cytoplasm (easy to maintain)
  3. calcium moving out of stores is hard to measure with electrophysiology
• CALCIUM SENSITIVE DYE

  • types used depends on the cell types
  • many are excite by UV light and some are excited by visible light (under scanning caser confocal microscopy)
  • when calcium binding occurs --> disotrition changes excitation wavelength
  • shift in absorption spectrum --> allows it to be used to measure conc of calcium
  • ratio between the two emiemission will be directly proportional of the conc of calcium
  • PROBLEMS: polar

  1. can’t easily move into the membranes
• NEXT GENERATION INDICATOR DYES (GINAS)

  • hard to target them to specific cells
  • genetrically encoded calcium indicators (gcamp)
  • can be targeted ti individual cells and tissues
  • can have temportal control  turn on at a particular time
  • gcamp- fusion protein

  1. combination of a fluorescent proteins + calmodulin + myosin light chain kinase (m13)
  2.  calmodulin --> calcium binding protein
  3. weak signal without calcium, strong signal with calcium due to a conformational chain

  • transgenic animal is set up to express the reporter
  • it will target to tissue/ cell specific promoter
• FLUORESCENT RESONANCE ENERGY TRANSFER (FRET)

  • two fluorophores are required

  1. a donor and an acceptor
  2. both needs to be excited and emit at different wavelength
  3. when brought to close proximity --> donor is excited and transfer energy to acceptor
• OPTOGENETICS

  • involved manipulating neuronal behaviour using light sensitive ion channels + ion pumps
  • pioneered by georg nagel, peter hegemann and ernst bamberg (1990s)
  • animals have light-sensitive proteins --> opsins (gpcrs)
  • single cell green algae (chlamydomonas) --> channelrhodopsin  (transmembrane protein)

  1. responds to blue light
  2. non-selective light gated cation channels

  • halobacterin --> halorhodpsin

  1. responds to yellow or orange light
  2. chloride pump powered by light
• OPTOGENETICS cont.

  • channel rhodopsin 2

  1. deplolarisation causes a flux of ions
  2. causes an excitation effect

  • halorhopsin

  1. hyperpolarization occurs
  2. causes an inhibition effect

  • delivering light into the therapeutic system

  1. fibre-optic probes are used to activate channels
  2. uses a viral vector
  3. a flux of actions will depolarise --> and dire action potential
  4. chloride causes halorhodpsin ro hyperpolarisee

  • optogenetics control gpcr

  1. many signalling pathways in excitable cells activate gpcrs rather than ion channels
  2. optoxr-  chimeric proteins that increase camp for gs
• RADIOLIGAND BINDING

  • METHOD:

  1. make a radiolablled version of the drug, neurotransmitter or other ligands
  2. mix with tissue of interest
  3. measure how much radioactivity has bound to the tissue

  • APPLICATION

  1. adding radioligand to intact tissue --> tells you where in the tissue the radioligand binds to
  2. measuring the amount of binding at different concentration to show how tightly it binds to radioligand
  3. using non-radioactive drug --> stop radioligand binding + gives info on how tightly the non-radioactive drug binds
• RADIOLIGAND BINDING cont.

  • ADVANTAGES:

  1. easy to set up
  2. easily scaled up to look at large number of drugs
  3. cheap

  • DISADVANTAGES:

  1. doesn't tell us what the drug is doing to the receptor
  2. doesn't have good time resolution
  3. uses hazardous materials and waste