Flashcards in deck_4990198 Deck (54)
what is the definition of optogenetics?
sensitising neurons to light, then manipulating neural activity in precise spatiotemporal patterns to answer questions regarding neural circuits and behaviour. or The use of genetically encoded light-activated proteins for manipulation of cells in an almost noninvasive way by light
what was indirect approach to optogenetics first discovered in?
green algae, the wild type moves towards the sun
what is the structure and functional morphology of channel rhodopsin (1 and 2)?
7 pass transmembrane protein, it binds all-trans-retinal, it is directly light gated, cation selective membrane channel, 1 is a proton channel, 2 is a cation channel.
how was channelrhodopsin expression in cells first shown to be able to induce cell depolarisation?
the CHR2 was expressed in an oocyte and HEK-293 cell and when a blue light was shown, the cells demonstrated an inward current within milliseconds
what was the second example of CHR2 being used to control cell voltage but more importantly, the first example of neurons being controlled optogenetically? what was a bonus observation from this experiment?
hippocampal neurons expressed ChR2 via a promoter which was hippocampal specific promoter i assume. They showed that they could induce an inward current and that a pulsed application of light generates more reliable firing pattern
what is general protocol for inducing the expression of light sensitive channels into mice?
1. produce a genetic construct that contains a promoter to drive expression of your channel and the gene encoding ChR2/1 downstream. 2. insert this construct intro a virus 3. inject virus into animal brain and opsin is expressed in targeted neurons.4. insert "optrode" fibre optic cable plus electrode. 5. laser light from fibre optic cable stimulates ion channel opening6. neuron is depolarised.
other than expression light sensitive channels in neurons, what is the direct approach to optogenetics?
They tried to harness the components of the phototransduction response and express these in cells. Zemelman (2002) expressed photoreceptor genes encoding arresting-2, rhodopsin (formed by liganding opsin with retinal) and the alpha subunit of the cognate hetertrimeric G-protein. They showed this made neurons sensitive to light. The neurons that express these components are known as charged neurons
what is the significance of the label "charged" neurons
the ARG stands for arrestin, rhodopsin, and g-protein
how does the activation of chARGed neurons in flies work?
rhodopsin is stimulated by light which turns it into metarhodopsin, this activates the Gq sub unit which catalyses the conversion of PIP2 to IP3 and DAG. This releases calcium stores and and opens cation influx channels. Arrestin inactivates metarhodopsin
from what animal process was the CHARGED neuron concept taken from?
phototransduction in the fly
what was the first evidence that CHARGED neurons could be used in animals other than flies?
Zemmelmann transferred the fly phototransduction pathway into vertebrate neurons. They found that NineE, arrestin and Galpha protein are sufficient to make cells light sensitive . NinaE encodes an opsin. the NinaE required all-trans retinal to be added to enable phototransduction. They made xenopus oocyte light sensitive
what are the down sides of "charged" neurons compared to those that express ChR2/1?
they have a slower response and there is more variable spike latency compared to ChR2
describe a way of incorporating ATP cages when using optogenetics
There are no P2X2 cation channels not present in drosophila. You can express this channel in neurons that you want to stimulate. This is done by having a line of P2X2 under the control of UAS and cross it with a tissue specific Gal4 driver. then inject caged ATP into these areas. When you shine light on the cages they release ATP which bind to P2X channels and open them, allowing cations.
describe an experiment which used caged ATP and P2X2 to use light to stimulate neurons at the neuromuscular junction?
- a group expressed P2X2 in cholinergic neurons, using the Cha promoter to drive Gal4. Then they put an electrode in the muscle and stimulate the neuromuscular junction. They found that caged ATP could stimulate the muscle
why can the ATP P2X2 ontogenetic mechanism be used in flies?
because they dont express P2X2 so it can be expressed in specific neurons- this is not the case for vertebrates
describe an experiment which used P2X2 in flies to mediate behaviour.
They targeted the neuronal circuit which stimulate fly jumping and flying. They targeted the neurons in the head using a c17 driven Ga;4 and in the thorax with a ShakB driven Gal4. for each of these they injected ATP and then shone line. They showed that in both the head and the thorax the fly flew or jumped. To show this wasn't due to sensing the light they carried out the same experiment in a fly with a eye mutant making it blind. To take this even further they cut off the head and the stimulation go the thorax got the same effect.
what are the two channels that can be use dot activate and silence neurons?
ChR2 to activate or Halorhodopsin. halorodopsin is a chloride channel which is activated by light and allows chloride to move into the cell and silences it. i
what is halorhodopsin?
halorodopsin is a chloride channel which is activated by light and allows chloride to move into the cell and silences it. i
how and why can ChR2 and halorhodopsin be used together?
halorhodopsin is activated by higher wavelength lights so you can both silence and activate a neuron in the same experiment.
how can you combine ATP/P2X stimulation with calcium and cAMP imaging to map functional connectivity in the fly brain? describe an example of where this was done.
If you predict or what to see if some neurons connect to another neuronal type of region of the brain, you can stimulate the first set of neurons using the P2X2 and ATP system and then express a calcium activated fluorescence protein to report the stimulation of the down stream neuron. You can also put electrodes into each neuronal subtype to record voltage changes. This was done to show that the Bolwigs Organ in the fly (?) connected to clock neurons.
what are the 6 components of the drosophila tool kit to visualise and manipulate neurons and their activity?
you have a neuron specific enhancer driving Gal4 (or another transcription factor with an activation domain), you then have a number of options that you can choose. You have a UAS site (or another TF binding site) that can activate any other the following downstream genes: - GFP to visualise expression of the certain gene or neuronal type - Synapto-pHluorin to a genetically encoded optical indicator of vesicle release and recycling. It is used in neuroscience to study transmitter release. It consists of a pH-sensitive form of green fluorescent protein (GFP) fused to the luminal side of a vesicle-associated membrane protein (VAMP). Or GCAMP which is a calcium indicator. - Shibire or halorhodopsin to block neural transmission -the first being temperature sensitive which can be useful - P2X or ChR2 to activate - genes or RNAi
what can you use Cryptochromes for?
they are proteins that bind when exposed to light. So you can fuse them to you target protein and when you shine light the target protein will be brought near and activate each other. They need blue light.
how can you use the PhyB and PIF light sensitive proteins- give a real example
- need to ass PCB chromophore, far-red light leads to PhyB binding to PIF within seconds and can be reversed by infrared light. in one paper they used this phytochrome B-PIF system to control protein localisation in zzebrafish embryos. They expressed PHYB in the membrane and thn tagged it to a reporter cherry ptoeint. Then they used this to recruit PIF6 (which was also labelled) to the membrae (with the additon of of PCB chromophore because that is what is needed for this reaction) then they . the pictures show that when you shine 650nm the PIF6 in the membrane and when at 750nm it is the in the cytoplams- easy stwitch.this was a proof of principle- they fuse a protein of interest to the pif protein and then this will be recruite to were PHYB is. Pard3 was bound to PIF6 and when you shine a light you see a massive recruitment to the membrane. the protein called pard 6 which normaly reacts with pard 3 is then consequently recruited to pard 3- so you can recruit proteins that bind to portein of interest also. Then they showed that PARD3 is normally expressed in the localised to the centre of the cell and is equally seperated between the two daughters cells and this is importnant for neuonal development. Then they trapped pard 3 on the right side of the cell using this medhtod so that all of the PARD3 goes into one cell and this is not an equal distibution.
what are three classical approaches used by neuroscientists to work out how the brain works?
-record electrophysiologpically from the cells of the brain -they image the structure of cells and brain regions-they lesion areas of the brain to see if they are important
what is wrong with the classical approaches of neuroscience?
blind to the cell types that you are dealing with- you just see areas of cells that are active or do certain things but you dont actually know what these cells are.
how can you circumvent the issues with classical approaches used by neuroscientists (lack of ability to distinguish cell types)?
- tools to determine the identity of different neurons - tools to investigate the function of these identified cells in unprecendeted detail
what are the key questions neuroscientist want to answer?
- analysis of cell to cell connectivity - measurement of activity during behaviour and dynamic properties- loss anf gain of function- necessity and sufficiency
once you have a certain area of the brain that you want to understand how can you go about understanding it?
You can identify the cell types within the brain. you can do this by analysing the expression profiles of the neurons within the area of interest and identifying specific sub types of neurons. Once you have done this you can hijack the promoters of these genes and carry out many different experiments.
what is the most effective way of expressing a gene (such as a reporter) under the control of the regulatory elements of another gene?
Back transgenics- using a modified BAC from a BAC library