A Selective Activity-Dependent Requirement for Dynamin 1 in Synaptic Vesicle Endocytosis NOTES

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McGill University
Anatomy & Cell Biology
ANAT 365
Peter Mc Pherson

A Selective Activity-Dependent Requirement for Dynamin 1 in Synaptic Vesicle Endocytosis -its only required for endocytosis that is selective activity-dependent -dynamin is required for synaptic vesicle endocytosis -what is known at the time? what does it show ? -what is already known- that dynamin is required for endocytosis, this paper does not really show it -there is an activity dependent endocytosis, is a better answer -what did the paper do? it made a dynamin 1 knockout mice for the first time, -there was alot of model system to disrupt the dynamin, and this blocks endocytosis -this is NOT the first paper to show that it blocks endocytosis -this is the first time they did a knock out mice for dynamin (close to human)...dynamin 1, dynamin 2, dynamin 3 -knocking out a selective gene, from the time of conception...one gene that is missing...what changes in the animal? -three genes make dynamin...there was originally one ancesterol dynamin, and then with evolution formed this -dynamin 1 was expressed only in the nervous system -dynamin 2 is expressed ubiquitously almost every tissue -dynamin 3 - mostly in the brain, also in the testis and other tissues -expression level of dynamin 1 is high compared to dynamin 2, 3 because it is located in the brain, because there are alot of synapses -if you knock out dynamin 1, the animal is born at the normal mendalien ratio, there is NO EMBRYONIC LETHALITY, like a wildtype-surprise, but not really, because everything is supplied by placenta, you don't brain to develop in utero, -animal is born and survive for 2 weeks -new born mouse pup needs a functional nervous system to live... in that sense its surprising -heterozygous- has one good copy dynamin knockout- doesn't suckle as well to get milk, and it dies -maybe its not a dynamin knockout -they do western blot, they have antibodies are specific for 1,2,3 and they have antibody that recognizes all three dynamin well, 70% amino acids are same in the three dynamins, when you make antibodies specific for 1,2,3 you are targetting that is different for each -they are using neuronal culture from brain, there is very little dynamin left for total dynamin -for specific antibody for dynamin 1 , there is none in knockout -total dynamin, in the knock out went down very dramatically because it is mostly made of dynamin 1(total of dynamin 2, 3 was much less)--> proof that dynamin 1 was the most abundant--- its not the first proof, but the strongest evidence today (adapter proteins, and clathirin remained same) -the western blot, made sure it dynamin 1 knockout, and its mainly dynamin that would be in the brain -what effect did knocking out dynamin 1 have on neurotransmission -you have to endocytose synaptic vessicle, to reuse -to measure exocytosis, is an indirect measurement of endocytosis -they measured synaptic transmission: (figure 2) -if you disrupt dynamin 1, we might see decrease in ability of synaptic vessicle to fuse, and we see decrease in neurotranmitter, because there is less synaptic vessicle reuptaken -they did low density culture, they took brains from the pups, and create cortical neuron culture -if you do in a low density culture, you can stick electrode into one neuron, and then trigger synaptic vessicle release and measure it in the other neuron -measure miniature excitatory postsynaptic curve, and IPSC (inhibitory) - measure of synaptic vessicle release -if it was excitatory- it was glutamate -inhibitory- it was gaba -they measure amplitude of the post synaptic signal -without stimulation: the knockouts shifted to the right...there is bigger release of neurotransmitter, this was counterintuitive, this is an activity independent type of measure -synaptic vessicles are bigger when you knock out dynamin 1 -with stimulation...what happens when you compare wild type to knockout, they do a single stimulus, first one is excitatory, and second one excitatory, in both cases the knock out the peak amplitude of release is much reduced for excitatory knockout and much more reduced for inhibitory -dynamin is much more important in inhibitory neurons there is less synaptic vessicle or less neurotransmitter, could be reduced response postsynaptically -E important figure...tells us more direct info about dynamin measure IPSC measur
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