Genetic Tools to Study Neural Circuits

Question 1

Disadvantage of traditional transgenic animals

Answer 1

viability - deletion may be fatal
non selective = not site specific
present from birth - not good for developmental adaption

Question 2

Conditional gene targetting

Answer 2

allows inactivation of a gene restricted to an organ and/or developmental stage

Question 3

how many mouse strains needed for cre loxp

Answer 3

2

Question 4

how many bp is loxP?

Answer 4

34

Question 5

how can cre-lox P result in inversion?

Answer 5

loxP sites face eachother and flank gene

Question 6

cre lox mediated activation

Answer 6

lox P sites flank poly A which prevent transcription - delete then gene left active

Question 7

3 parts of viral vector

Answer 7

virus particle
envelope
capsid

Question 8

what does envelope determine?

Answer 8

tropism

Question 9

what does capsid do?

Answer 9

enclose genetic material

Question 10

3 types of virus used

Answer 10

adenovirus - high immunogenicity, dsDNA
retrovirus/lentivirus
AAV - very low immunogenicity - ssdna

Question 11

virus construct creloxp

Answer 11

gene inverted beside lox p sites

specific areas with cre will be switched on to become active

Question 12

what can enhance cre-lox usefulness?

Answer 12

reporter

Question 13

examples of previous reporters and problems?

Answer 13

luciferase, beta galactosidase

require exogenous substrates - no good for living tissue

Question 14

reporter

Answer 14

labels cell - can see cells infected with virus

now use genetically encoded fluorescent proteins

Question 15

GFP

Answer 15

discovere 1960s in jellyfish

238 amino acids

Question 16

concentration of calcium at rest

Answer 16

50-100nM

Question 17

what does calcium activity correlate with?

Answer 17

electrical activity

firing frequency and fluorescence - direct correlation

Question 18

chemical calcium indicator

Answer 18

fura-2

Question 19

genetically encoded calcium indicators

Answer 19

GCamp in GFP cells - change fluorescence depending on calcium around indicator
GFP, calcmodulin and M13

Question 20

single cell loading of calcium indicator

Answer 20

sharp electrode, whole cell patch clamp or single cell electroporation

Question 21

in vivo photometry

Answer 21

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

Question 22

In vivo calcium imaging

Answer 22

GCamp in group of cells
put in lens - see when particular cell active or not
graded refractive index lens

Question 23

tracers

Answer 23

compounds transported along axons away from injection site

Question 24

what is neural tracing often combined with?

Answer 24

immunohistochemistry or insitu hybridisation

Question 25

example of anterograde tracers

Answer 25

rhodamine isothiocyanate | [3H] amino acids

Question 26

explain anterograde tracers

Answer 26

from brain area A to B and C

Question 27

example of retrograde tracers

Answer 27

latex spheres fast blue fluorogold

Question 28

explain retrograde tracers

Answer 28

B receives inputs from A and C so B transports it back to A and C

Question 29

polysynaptic tracer

Answer 29

pseudorabies taken up by cell but moves back to connected cells hard to tell if connected directly or neurones inbetween

Question 30

monosynpatic tracer

Answer 30

AAV modified rabies virus taken up by cell - direct connection

Question 31

how to find what cells are directly connected

Answer 31

``` 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 ```

Question 32

Channel rhodopsin 2

Answer 32

light sensitive cation channel | naturally occur in unicellular green algae

Question 33

3 other opsins

Answer 33

bacteriorhodopsin halorhodopsin channelrhodopsin

Question 34

in vivo optogenetics

Answer 34

chr2 in some cells - optic fibre and light source

Question 35

ex vivo optogenetics

Answer 35

inject virus - chr2 in gabaergic neurons with YFP label other par of brain with RFP light stimulus affect other cells record electrophysiology

Question 36

DREADDS

Answer 36

designer receptors exclusively activated by designer drugs | get excitable and inhibitory ones

Question 37

what are DREADDs mutated forms of?

Answer 37

human muscarinic receptors | hm4d

Question 38

what activates DREADDS?

Answer 38

inert ligand clozapine-N-oxide (CNO) | not Ach

Question 39

what binds DREADDS?

Answer 39

metabolite clozapine - not really inert

Question 40

DREADD expressed in 2 parts of hypothalamus - LHA and PVH

Answer 40

LHA - increase food intake with cno | PVH decreased food intake with cno

Question 41

what toxin used in toxin receptor mediated cell KO?

Answer 41

diptheria toxin

Question 42

diptheria toxin

Answer 42

exotoxin made by bacterium corynebacterium diptheriae

Question 43

mice and rats and DT

Answer 43

resistant | conditional cell ablation to mice expressing human DTR in specific cell population