with a surgical blade and identify the bregma. Target the spe-
cific nucleus bilaterally using a 25 gauge needle connected to a
Hamilton 1μL syringe using specific coordinates. In rat, for the
VMH, perform two injections in each cerebral hemisphere
(2.4–3.2 mm posterior to bregma, 0.6 mm lateral to midline,
10.1 mm deep); for the ARC, perform one injection in each
hemisphere (2.8 mm posterior to bregma, 0.3 mm lateral to
midline, 10.2 mm deep); and for the LHA, 2.9 mm posterior
to bregma, 2 mm lateral to midline, and 8.1 mm deep. In mice,
perform one injection in each cerebral hemisphere: for the
VMH, 1.7 mm posterior to bregma, 0.5 mm lateral to midline,
and 5.5 mm deep; for the ARC, 1.5 mm posterior to bregma,
0.2 mm lateral to midline, and 6 mm deep; and for the LHA,
1.3 mm posterior to bregma, 1.1 mm lateral to midline, and
5.2 mm deep. Using a surgical drill, pierce the skull in each
point. The substances should be delivered at a rate of 200 nL/
min during 5 min for rats and 10 min for mice. The entire
injector system needs to be left in place for an additional 5 min
after the injections are completed. Close the cut with silk
suture.
3.Stereotaxic implantation of nucleus-specific cannulae. For
chronic administration of substances in specific nuclei of the
rat hypothalamus, insert bilaterally a 28 gauge stainless steel
cannula with different length and gap between the injectors for
each hemisphere, according to the coordinates for each specific
nuclei: for VMH, 2.8 mm posterior to bregma, 0.6 mm lateral
to midline, and 10.1 mm ventral; for ARC, 2.8 mm posterior
to bregma, 0.3 mm lateral to midline, and 10.2 mm ventral;
and for LHA, 2.9 mm posterior to bregma, 2 mm lateral to
midline, and 8.1 mm ventral [44]. Using a surgical drill, pierce
the skull in each point. Insert the cannula and glue it to the
skull with cyanoacrylate adhesive. Connect a catheter tube
from each infusion cannula to an osmotic minipump flow
moderator, which should be inserted in a subcutaneous pocket
on the dorsal surface created using blunt dissection. Close the
cut with silk suture.
4.Oral gavage administration. Restrain the animal grasping skin
over the shoulders to immobilize the head, and thus the fore-
legs are extended. Check the length of the gavage needle by
measuring the tip of the nose to the last rib. Hold the animal in
a vertical position and introduce the gavage needle following
the roof of the mouth, moving the needle toward the esopha-
gus. The animal will naturally gag and have a swallow reflex.
After the needle pass to the correct length, the compound may
be injected [62].438 Patricia Seoane-Collazo and Miguel Lo ́pez