Genetic Tools to Study Neural Circuits Flashcards
Disadvantage of traditional transgenic animals
viability - deletion may be fatal
non selective = not site specific
present from birth - not good for developmental adaption
Conditional gene targetting
allows inactivation of a gene restricted to an organ and/or developmental stage
how many mouse strains needed for cre loxp
2
how many bp is loxP?
34
how can cre-lox P result in inversion?
loxP sites face eachother and flank gene
cre lox mediated activation
lox P sites flank poly A which prevent transcription - delete then gene left active
3 parts of viral vector
virus particle
envelope
capsid
what does envelope determine?
tropism
what does capsid do?
enclose genetic material
3 types of virus used
adenovirus - high immunogenicity, dsDNA
retrovirus/lentivirus
AAV - very low immunogenicity - ssdna
virus construct creloxp
gene inverted beside lox p sites
specific areas with cre will be switched on to become active
what can enhance cre-lox usefulness?
reporter
examples of previous reporters and problems?
luciferase, beta galactosidase
require exogenous substrates - no good for living tissue
reporter
labels cell - can see cells infected with virus
now use genetically encoded fluorescent proteins
GFP
discovere 1960s in jellyfish
238 amino acids
concentration of calcium at rest
50-100nM
what does calcium activity correlate with?
electrical activity
firing frequency and fluorescence - direct correlation
chemical calcium indicator
fura-2
genetically encoded calcium indicators
GCamp in GFP cells - change fluorescence depending on calcium around indicator
GFP, calcmodulin and M13
single cell loading of calcium indicator
sharp electrode, whole cell patch clamp or single cell electroporation
in vivo photometry
mouse - GCaMP expressing cells in part of brain
implant small optic fibre - light source and collect emitted light
photoreceiver output voltage = average light intensity
light intensity = cell activity eg can get animal to run on treadmill
In vivo calcium imaging
GCamp in group of cells
put in lens - see when particular cell active or not
graded refractive index lens
tracers
compounds transported along axons away from injection site
what is neural tracing often combined with?
immunohistochemistry or insitu hybridisation
example of anterograde tracers
rhodamine isothiocyanate
[3H] amino acids
explain anterograde tracers
from brain area A to B and C
example of retrograde tracers
latex spheres
fast blue
fluorogold
explain retrograde tracers
B receives inputs from A and C so B transports it back to A and C
polysynaptic tracer
pseudorabies
taken up by cell but moves back to connected cells
hard to tell if connected directly or neurones inbetween
monosynpatic tracer
AAV
modified rabies virus
taken up by cell - direct connection
how to find what cells are directly connected
cre in subset of neurons inject virus into 2 brain areas expression - green 2 days later inject with rabies virus, red fluorescent cannot jump synapses
Channel rhodopsin 2
light sensitive cation channel
naturally occur in unicellular green algae
3 other opsins
bacteriorhodopsin
halorhodopsin
channelrhodopsin
in vivo optogenetics
chr2 in some cells - optic fibre and light source
ex vivo optogenetics
inject virus - chr2 in gabaergic neurons with YFP
label other par of brain with RFP
light stimulus affect other cells
record electrophysiology
DREADDS
designer receptors exclusively activated by designer drugs
get excitable and inhibitory ones
what are DREADDs mutated forms of?
human muscarinic receptors
hm4d
what activates DREADDS?
inert ligand clozapine-N-oxide (CNO)
not Ach
what binds DREADDS?
metabolite clozapine - not really inert
DREADD expressed in 2 parts of hypothalamus - LHA and PVH
LHA - increase food intake with cno
PVH decreased food intake with cno
what toxin used in toxin receptor mediated cell KO?
diptheria toxin
diptheria toxin
exotoxin made by bacterium corynebacterium diptheriae
mice and rats and DT
resistant
conditional cell ablation to mice expressing human DTR in specific cell population
