To identify neuronal networks in which dual AMPK-α1/α2 deletion had been induced, mice with PNMT-Cre-driven AMPK-α1/α2 deletion and TH-Cre-driven AMPK-α1/α2 deletion were crossed with mice engineered for Cre-dependent expression of Rosa (tdTomato). These mice were deeply anaesthetised using 2 g/kg Pentobarbital Sodium (Merial), transcardially perfused with ice-cold heparinised saline and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Brains were extracted, post-fixed, and stored in 30% sucrose in 0.1 M PB at 4 °C. Thirty-micrometre sections of the brainstem were cut using a Frigomobil freezing microtome (Leica). Alternate sections were collected together and mounted onto glass slides, briefly air-dried, and coverslipped using Vision™ PermaFluor™ Aqueous Mounting Medium (Thermo Fisher).
Brainstem sections were imaged using a Nikon A1R + confocal system and tdTomato autofluorescence detected using an excitation wavelength of 554 nm and emission wavelength of 581 nm. Relevant regions of the caudal brainstem harbouring catecholaminergic neurons were identified using the mouse brain atlas [39 ].
Brainstem sections were imaged using a Nikon A1R + confocal system and tdTomato autofluorescence detected using an excitation wavelength of 554 nm and emission wavelength of 581 nm. Relevant regions of the caudal brainstem harbouring catecholaminergic neurons were identified using the mouse brain atlas [39 ].