Experiment Module: Confirming the Roles of an Ion, a Second Messenger, and an Enzyme in Long-Term Potentiation

Many methods have been used to determine the role of a particular ion, or a second messenger, or an enzyme in a synaptic process.

For example, the role of calcium in long-term potentiation (LTP) has been confirmed in a number of ways. One experiment involved injecting the postsynaptic neuron with chelating agents such as EGTA and BAPTA, two molecules that bind to calcium and render it inactive. As a result, it becomes impossible to induce LTP. he reverse procedure has also been used. Researchers have injected special molecules into the postsynaptic neuron and then illuminated them with UV light, thus causing them to release enough calcium to induce LTP in this neuron.

Another approach is to produce mutations that make a protein non-functional or that block its action with another molecule. Blocking CaM kinase II in this way prevents LTP from becoming established, which also demonstrates the central role of this protein.

Similarly, inhibiting PKA or CREB prevents LTP from reaching its second phase and being sustained.

In certain cases, researchers have even identified the one amino acid, among the hundreds of amino acids that make up a protein, whose phosphorylation is essential for LTP (Ser831 for the GluR1 sub-unit of the AMPA receptor and Thr286 for the autophosphorylation site of CaM kinase II).

Researchers have also shown that a mouse whose gene for the GluR1 sub-unit of the AMPA receptor had been knocked out could not have any LTP, thus confirming the role of CaM kinase II once again.

As for the mice for which site Thr286 in CaM kinase II was deactivated, their basic synaptic transmission was maintained, but LTP could no longer be induced in them, thus proving the need for CaM kinase II. The reverse procedure also led to the same conclusion: adding activated CaM kinase II to the pyramidal neurons of the hippocampus causes a potentiation phenomenon similar to LTP.