- TBST: 1% (v/v) Tween-20 in TBS. Mix 100 mL TBS 10and
1 mL Tween-20. Make up to 1 L with distilled water. - Blocking solution (BSA-TBST): 3% (v/v) BSA in TBST. Mix
3 g BSA in 100 mL of TBST. Store at 4C.
2.5 Antigens
and Conjugates
- Primary antibody. Use a primary antibody that recognizes the
phosphorylation of AMPKαat Thr-172. Dilute the antibody in
BSA-TBST. As controls, antibodies against
non-phosphorylated AMPKα1 and AMPKα2 are used.β-actin
is used as loading control. - Secondary antibody. Use an HRP conjugated secondary anti-
body. Dilute the antibody in BSA-TBST. - Peroxidase substrate for enhanced chemiluminescence (ECL).
3 Methods
3.1 Induction
of Changes
in Hypothalamic AMPK
Phosphorylation
3.1.1 Feeding/Fasting
Experiments
Hypothalamic AMPK can be regulated by energetic status. In the
context of food deprivation (after 24-h fasting), increased ghrelin
levels stimulate the phosphorylation of hypothalamic AMPKαand
its downstream target ACCα, thus inducing feeding through the
modulation of hypothalamic fatty acid metabolism
[5, 29–35]. After an overnight fasting, refeeding reduces AMPK
phosphorylation in all hypothalamic nuclei [60].3.1.2 Administration
of Hormones, Peptides,
and Chemicals
Routes of Administration
1.ICV cannulae.This procedure is used in experiments that
require administration of substances in the brain ventricles,
such as the third ventricle (3V) and the lateral ventricle
[30, 32, 36, 42–45, 61]. The cannula consists in a polyethylene
tube of (rat/mice) 1.09/0.965 mm of external diameter and
0.38/0.58 mm of internal diameter. The length (rat/mouse) is
3.5/2.5 cm. In one of the extremes, cut a bevel of 0.5 mm, and
from this point, at 4/2.5 mm, insert a stopper that limits how
far the tube goes into the brain. Seal the other side until drug
administration. The ICV cannula is stereotaxically inserted
using the following coordinates: 1.5/1.2 mm lateral to sagittal
suture and 0.9/0.6 mm posterior to the bregma. Under keta-
mine/xylazine anesthesia, open the scalp (transversal cut) with
a surgical blade and identify the bregma. Using a surgical drill,
pierce the skull and insert the cannula until the stop, and glue it
to the skull with cyanoacrylate adhesive. Allow the animals to
recover 3–4 days before the injection of substances (seeNote 4).
2.Stereotaxic microinjection. This procedure is performed when it
is necessary to inject substances in a specific nucleus of the
hypothalamus. Place the animal in a stereotaxic frame under
ketamine/xylazine anesthesia. Open the scalp (transversal cut)Methods for Brain AMPK 437