Since I am very new to this field, I kindly ask you to help me with determining the start points, from which I can push off for a further methodology development.

If you were faced these tasks, simply where would you start? (with which methods and tools, possibly):

Goal:

1) to test the hypothesis that AMPK is a key regulator of energy availability during synaptic transmission

2) to examine the effect of AMPK inhibition or activation on neuronal excitability and synaptic release during periods of high metabolic demand.

For the 1) I can think of:

- use the markers of AMPK activity to see how it regulates the availability of energy during synaptic transmission.

- The AMPK-activating/inhibiting (eg., Compound C) drugs can be also used for this purpose.

For the 2):

Probably, there are two approaches: morphological and electrophysiological.

-  First si to determine the type of the tissue/cell model I will work with. In my case, it is Calyx of Held.

- Therefore, this region undergoes surgical labeling of needed area in anesthetized animals.

- Then, AMPK activators/inhibitors can be applied via perfusion

- The next step is electrophysiological recordings (patch clamp-voltage-clamp/ field recording) in brain slices using different protocols. Depends on the needs and the features recorded.

We can also use imaging methods to investigate the changes in morphology.

Are these suggestions appropriate?

Thank you very much for your help!