Understanding GABAa receptors Flashcards
Why do we need to create a better anxiolytic?
Because they have side effects such as sedation which we don’t want, we need to find out which subtype of GABAa receptors cause the sedative effect and which part of the brain it is
GABAa receptor distribution in the mouse brain
Give diazepam, can see where the GABAa receptors are:
a1 - everywhere, high in cortex (60%) brain
a2 - everywhere (but less) limbic structure, hippocampus, amygdala
a3 - brain stem, basal forebrain, spinal cord
a5 - hippocampus, spinal cord
By looking at the distribution of GABAa receptors in the mouse brain, which subunit do we think is involved in anxiolytic effects?
A2 because it is in the amygdala
What does the elevated maze study show?
Put a mouse or rat in the middle and measure how long it spends in different compartments
anxiety: degree to which animal avoids the open arms of the maze
anxious: spends more time in the closed arm
effect of diazepam: increase the amount spent in the aversive open arms of the maze, checking surroundings, less anxious
What happens in the elevated maze study when you give different drug doses?
Give TPA023 (agonist at a2/a3 subunit containing GABAa receptors) vs CDP (benzodiazepine)
% spent in open arms: when given CDP compared to control, spend more time in open arms
TPA is just as equivalent or maybe even more of an antiolyic effect - just by activating A2 - shows the A2 is the one mediating anxiety
What is the conditioned emotional response test?
Train mice to lever press for food
CS (bell) paired with footshock
CS (light) paired with no consequence
put them back in box with levers.. played cues
Change in lever pressing recorded as suppression ratio - measure lever pressing during CS presentation and then measure it again after once they have learnt
What happens when you give different doses of drugs in the conditioned emotional response test?
L-838417 - agonist at a2/3 subunit
If you give them 0mg, suppress the response
if you give the more, they stop suppressing so are pressing more, less scared, reduction in anxiety response
What happens to learning if we remove the receptor a2 in the conditioned emotional response test?
Genetically manipulate the mice by taking away alpha 2 - alpha2 subunit knockout mouse
look at how well they learn the test
no difference between wild type and knockout mice in acquisition or lever pressing for food
Why would you knock out a receptor in a mouse?
To see if it is important in behaviour
What happens to anxiety if we remove the receptor a2 in the conditioned emotional response test?
The wild type - don’t care at the start but then they start showing the fear response
Knockout - starting to show suppression at an earlier time point, they have an increased fear response, more anxiety
What do benzodiapines do in the wildtype vs knockout mouse in the conditioned emotional response test?
In the wild type, it reduces the suppression, less bothered about the shock, less anxious
In the knockout, there is no effect, doesn’t reduce anxiety at all
What is the structure of the GABAa receptor?
Protein structure
5 protein subunits which form a doughnut structure around the Cl- channel
Each consists of 4 transmembrane domains
What is the problem with knockout mice?
A2 hasn’t been there through all of development, which could’ve effected the changes later on, can’t show that it is totally due to A2 receptors as they have been gone forever
How can we overcome the problem of knockout mice?
By changing the property of a receptor - genetic manipulation by changing the amino acids: turning of the benzodiazepine binding sites
How do we turn of the benzodiazepine binding site?
In the extracellular domain of all GABAa receptors that bind benzodiazepines, have a histodine
a4 and a6 (non BZD) have arginine at this site
Can make knock in mice with BZD-insensitive a1, a2, a3 or a5 subunit - change it to arginine
determine if different subtypes of receptor regulate different behaviours
