the rats receive 1μL of 1 nM 17β-estradiol in each bilateral
injection (total 2μL in the ARC/4μL in the VMH). Rats are
sacrificed after 3–12 h [40].
3.Liraglutide. The clinically used glucagon-like peptide
1 (GLP1) agonist liraglutide stimulates BAT thermogenesis
and adipocyte browning independent from nutrient intake
and by the inhibition of hypothalamic AMPK [42]. Liraglutide
is given at a dose of 3μg in mouse and 10μg in rat either ICVor
in the VMH [42]
4.Olanzapine. Animals treated sub-chronically by oral gavage
with olanzapine present decreased hypothalamic AMPK phos-
phorylation. To deliver the drug (3 mg/kg) into the stomach,
use a gavage needle attached to a syringe twice daily (total daily
dose, 6 mg/kg) at 9 a.m. and 3 p.m. for 6 days and sacrifice on
day 7 after an overnight fasting. Dissolve the drug in 0.1 M
HCl and adjust to pH 5.5 using 0.1 M NaOH. Prepare a stock
solution of 1.5 mg/mL and administer the appropriate volume
to the rats via gavage (actual volume is modified for variation in
body weight so that for each rat, each of the two daily doses is
3 mg/kg) [62].
5.Compound C.Also known as dorsomorphin, it is a nonspecific
AMPK inhibitor (seeChapter 12) [67]. When treated ICV,
compound C decreases AMPK phosphorylation in the hypo-
thalamus. The dose for ICV treatment (2 h) in rats is 10μgin
5 μL of DMSO [30].3.1.3 Administration
of Adenovirus
AMPK phosphorylation can be modulated by genetic inhibition or
overexpression in specific nuclei. This can be achieved through
stereotaxic microinjection of adenoviral expression vectors expres-
sing dominant-negative (DN) or constitutively active
(CA) isoforms of AMPKα[30, 36, 40, 42–45]. To induce the
overexpression of AMPK, inject following specific coordinates
1 μL of a solution of adenoviral vectors that overexpress the isoform
AMPKα1 constitutively active protein (AMPKα1-CA) with a con-
centration of 1 1012 pfu/mL diluted 1:50 in PBS. To induce
inhibition of AMPK, inject following specific coordinates 1μLofa
mix of adenoviral particles overexpressing 1:1 of AMPKα1-DN and
AMPKα2-DN with a concentration each of 1.2 1012 pfu/mL.
For control group, inject 1μL of adenoviral particles expressing
green fluorescent protein (GFP) during 5–7 days (seeNote 5).
Infection efficiency is demonstrated by GFP expression by the
adenoviral construct specifically in the targeted nucleus (use GFP
immunohistochemistry or direct GFP immunofluorescence for
detection) and by the corroboration of pACCαlevels by Western
blot (seeNote 6).440 Patricia Seoane-Collazo and Miguel Lo ́pez