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Molecular Neuroscience > Conditional Knockout of genes and the Morris water maze > Flashcards

Conditional Knockout of genes and the Morris water maze Flashcards

0
Q

describe the glutamate ionotropic receptors…

A 
mutli subunit ion channels 
activated by ligands 
classified into 3 types based on pharmacology
- NMDA
- AMPA
- Kainate
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1
Q

What does the knockout mouse project aim to generate ?

A

a comprehensive and public resource comprised of mousse embryonic stem cells containing a null mutation in every gene in the mouse genome

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2
Q

what is the obligate subunit in NMDA receptors ?

A

NR1

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3
Q

what are the rules of assembly of AMPA receptors ?

A

homomultimers- 2 or more identical components

heteromultimers- 2 or more different components

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4
Q

what are the rules of assembly for kainate receptors ?

A

GluR5,6 AND 7 are homomultimers

heteromultimers

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5
Q

what are the rules of assembly of NMDA receptors ?

A

heteromultimers
NR1 must partners with one or more NR2 subunits
assemble as hetero-tetramersof 2 obligatory NR1 subunits and 2 regionally localised NR2 subunits

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6
Q

what binds to the NR1 and NR2 subunits of glutamate receptors ?

A

NR1 binds the coagonist glycine

NR2 binds glutamate

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7
Q

where is the greater expression of NR1 expression in the mouse brain ?

A

hippocampus

- also relatively large expression in the cortex

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8
Q

where are NR2A subunits highly expressed in the mouse brain ?

A

hippocampus

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9
Q

how was an NMDA receptor NR1 knockout produced?

A

exons of the gene were replaced with a neomycin resistance gene

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10
Q

what happened to the KO NR1 mice?

A

the homozygous KO mice were born so therefore the mutation does not result in embryonic fatality- body weights were normal so it didnt affect in utero development
BUT
10-20 hours after birth the homozygotes were dead or the mother had removed them from the nest

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11
Q

what was the KO mices coat colour and what does this indicate ?

A

flushed and had a reddish colouration like the wild type

- therefore their cardiovascular and respiratory systems are functionaal at least soo after birth

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12
Q

what was the difference shown between the normal pups and the mutant pups?

A

the mutant pups didnt have any milk in their stomachs

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13
Q

where was no abnormality detected in the mutant pups?

A

no abnormality was detected in the electrocardiographic pattern or the respiratory rhythm

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14
Q

what was seen in the mutant mice and what was seen several hourse after birth?

A

mutant animals were severly ataxic- couldnt support their body weight on their hindlimbs therefore they couldnt get milk to feed
increased apnea is seen several hours after birth and cyanosis occurs shortly before death indicating respiratory distress

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15
Q

what is cre-loxP recombination?

A

special type of site specific recombination

- involves targeting a specific sequence of DNA and splicing it with the help of the enzyme cre recombinase

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16
Q

what is the cre protein ?

A

it is a site specific DNA recombinase which catalyses the recombination of DNA between specific sites in the DNA - sites are known as loxP sequences- they contain specific binding sites for cre

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17
Q

what can occur if cells have loxP sites and express cre?

A

recombination event occurs between the loxP sites

- the cre protein cuts the DNA at both the loxP sites

18
Q

what are the 2 types of transgenic mice strains involved in the cre-loxP system ?

A

loxP-flanked strain: contains loxP sites flanking a critical portion of the target gene

cell-specific cre expressing strains: contains a transgene that expresses cre under control of a specific promoter- used to producce cell specific knockouts
- these are made by transgenic technology

19
Q

what strains of mice are mated in cell specifc cre-loxP mating scheme ?

A

strain carrying floxed alleles of the gene to be inactivated
x
strain expressing cre recombinase in aa tissue specific manner
= offspring with gene inactivated in cre expressing tissue

20
Q

in the cell specific cre-loxP mating scheme what would happen if the cre was under the control of a widespread promoter?

A

a general knockout would be produced

21
Q

what are examples of a widespread promoter?

A

beta-actin

CMV

22
Q

what is Flp?

A

flippase- recombinase enzyme

23
Q

what is flippase used for ?

A

it is used to remove neo cassette
the flippase recognition target = 34bp FRT is positioned to flank the neo so it can be removed
then the flippase cuts at this specific site to cause site specific excisional recombination- this is to produce a cleaner allele

24
Q

how was NR1 just KO in he CA1 region of the hippocampus?

A

by putting the cre transgene under the control of the aCaMK11 promoter becaause its expression is quite specific to the CA1 pyramidal cells

25
Q

what was trying to be tested by KO of the NR1 subunit in the CA1 region of th hippocampus?

A

to carry out memory and learning studies

26
Q

why are behaviour test used to understand brain function ?

A

because behaviour is the final output and visible manifestation of the nervous system

psychiatric and neurological disorders involve changes in behaviour

27
Q

what are some examples of different mouse behavioural tests ?

A 
startle chamber
operant chamber
cross maze
t maze
radial arm maze
morris water maze
these different tests can be used to test different parts of the brain
28
Q

who conceived the morris water maze and what is it used to test ?

A

1981 by proffessor richard morris

used to assay spatial cognition in rodents

29
Q

explain the morris water maze procedure …

A

the rodent is placed in a pool of opaque water at room temp in a random start location
rodents are trained over several days to find the platform located in the water pool
the latency to find the platform is recorded

30
Q

what happens in probe trials ?

A

the platform is removed and the % of time spent in the quadrant which contained the platform is recorded and compared to the time spent in the other quadrants

31
Q

what needs to be around the pool and how big is the platform ?

A

visual ques need to be around the pool

the platform is about 0.5cm-1cm from the surface

32
Q

what is tested 24hours after the mice has been trained?

A

the mices memory
the mice are placed back into the pool without the platform and the amount of times the mouse passes over the area where the platform used to be is recorded

33
Q

what is a limitation of the morris water maze?

A

the mouse has to be able to swim

34
Q

what are the terms used to describe the different search strategies of the mouse in the morris water maze?

A
  • thigmotaxic/wall hugging
  • random searching
  • selectively scanning the inner area
  • focal search of a target quadrant
  • focal search in an incorrect area of the pool
  • direct swimming to the platform
  • cycling behaviour
  • chaining response
  • chaining - crossing all possible platform locations
35
Q

what do the probe trial performances measure ?

A

they are occupancy based tests to asses the amount of time the animal spends in a virtual area

1) percent quadrant time
2) percent zone
3) crossings
4) proximity measure

36
Q

what is the percent quadrant time?

A

it is the amount of time mice searched a virtual quadrant which contained the platform during training

37
Q

what is percent zone?

A

amount of time the mice searched the virtual target zone within a specified distance radius centred on the location of the platform in 60s

38
Q

what is the crossings measurements ?

A

this is the number of times the mice cross the exact location of the platform (5cm in radius) during a 60s test
- it is a counting based measurement

39
Q

what is the proximity measure ?

A

also known as gallaghers measure
average distance in cm of mice from the centre of the platform location across the 60s test
it is an error based measure where the animals average distance from the former platform location is recorded

40
Q

what is the morris water maze sensitive to ?

A

manipulations of the hippocampus - if he hippocampus is damage then the mice perform poorly in the water maze

41
Q

what happened to spatial learning in CA1-KO mice in the water maze test ?

A

impaired spatial learning - mice are slower in learning the hidden platform

42
Q

what happens to memory of CA1-KO mice in the water maze test ?

A

impaired spatial memory- mice spend equal time in each of the quadrants in probe trials whereas normal mice spend significantly longer in the target quadrant

Molecular Neuroscience (12 decks)

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