Lecture 9 Optogenetics and GECIs in NV research Flashcards
what does optogenetics bypass
the need for sensory stimulation or direct electrical stimulation
overall how does optogenetics work
- insertion of opsins (which are protein ion channels) into a target cell which respond to light of a particular wavelength to open channel
can be excitatory or inhibitory
is channelrhodopsin inhibitory or excitatory?
excitatory (depolarises)
what could light does channelrhodopsin respond to?
blue
is halorhodopsin inhibitory or excitatory?
inhibitory (hyperpolarises)
what is often done alongside insertion of opsin?
insertion of fluorescent protein- so the opsin cells can be identified
how in general, do opsins get into cells?
through adeno-associated viruses (AAVs)
what are the 4 methods of getting opsin into target cells?
direct injection into WT animal
direct injection into cre-recombinase transgenic animal
no injection- breeding transgenic mouse strains
in vitro electroporation
describe direct injection into WT animal (optogenetics)
virus targets one type of cell my expressing promoters that are only expressed in cell population of choice
e.g. CaMKIIa - biased to cortical cells
describe no injection breeding transgenic mouse strains
avoids injection (neuroinflammation)
cross mouse with cre-recombinase in a specific cell type- heterozygous
with channelrhodopsin and fluorescent protein when exposed to cre-recombinase homozygous mouse
50% will have cre-combinase and respond to light signals, 50% won’t- controls
describe direct injection into cre-recombinase transgenic animals
transgenic mouse that only expresses cre-recombinase enzyme in a specific set of neurons
the AAV will go into all neurons, but will only express opsins in cre-recombinase cells
describe in vitro electroporation- optogenetics
injecting genetic material to embryo in vitro brain, voltage put across pushing DNA into the cells that are dividing at that time. if timing is right, transfected cells can be targeted (by point in embryonic development)
what did Lee et al 2010 find (optogenetics BOLD)
injected AAV- CaMKIIa-expressing excitatory neurons into rat motor cortex and did BOLD fMRI with laser to simulate cells. when shining light at certain wavelength, neurons fire, they got robust BOLD signalls alongside firing via light activation- first time showing this.
they found the same for deeper cortical layers
parvalbumin interneurons showed the central positive BOLD with negative surround
discuss interneuron role in BOLD
one study used parvalbumin in slice using light stimulation, which caused a constriction of BVs- perhaps interneurons involved in negative BOLD
another study used VGAT to label all GABA interneurons in brain, when activated, it inhibited APs and using speckle imaging, found large increase in BF- opposite result. They then used glutamate inhibitors, which reduced neuronal response but did not change blood flow- posited NO involvement in interneuron dilation
Anna Devor’s group used whisking stimulation and found sometimes there was a BOLD undershoot and other times there wasn’t. Found a particular population of interneurons was causing the post-stimulus undershoot
research into optogenetics in astrocytes
2015 study- channel rhodopsin with stabilised step function (blue light opens channels, orange closes). They found astrocyte activation, caused vasodilation and increased BF and inactivation caused vasoconstriction and reduced BF
has not been repeated
