Assessment of ventral hippocampus activation after intranasal
infusion
Animals were randomly allocated into five experimental groups: (1)
Control: distilled water; (2) M1145- treated group (132 µg); (3) Y1R
agonist-treated group receiving the Y1R agonist
[Leu31- Pro34]NPY (132 µg); (4)
M1145+Y1R: group administered with both substances; (5) M1145+Y1R+M871:
group treated with M1145, [Leu31-
Pro34]NPY and the GALR2 antagonist (M871; 132 µg)
(N=4 in each group). The doses indicated are based on previously
published protocols (Borroto-Escuela et
al., 2022; Serova et al., 2017).
Twenty-four hours after the after the i.n. administration, rats were
deeply anesthetized with pentobarbital (Mebumal, 100 mg/kg, i.p.) and
transcardially perfused with 4% PFA (para-formaldehyde (wt./vol, Sigma
Aldrich, St. Louis, MI, USA)). Using a Cryostat (HM550, Microm
International, Walldorf, Germany) the brains were coronally sliced (30
μm-thick) through the ventral hippocampus (anterior in primates) (-5.20
to -6.72 Bregma; (Paxinos & Watson,
2006)).
We used the c-Fos immunohistochemistry, as an indirect marker of
neuronal activation. Free-floating sections were incubated for
antigenical retrieval at 65 °C during 90 min in saline sodium citrate
buffer (pH 6; 10 nM sodium citrate). After this procedure to remove
endogenous peroxidases, the slices were treated 30 min in 0.6%
H2O2. Then, slices were incubated at 4
°C overnight with a primary antibody mouse anti-c-Fos protein (1:800
sc-271243, Santa Cruz Biotechnology, CA) in 2.5% donkey serum. After
several washes with PBS, the slices were incubated with a secondary
antibody for 90 min (biotinylated anti-mouse IgG, 1:300, B8520, Sigma,
St. Louis, MO, USA). Then, ExtrAvidin peroxidase (1:100, Sigma, St.
Louis, MO, USA) was used to amplify the specific signal for one hour at
room temperature in darkness. Detection was performed with 0.05%
diaminobenzidine (DAB; Sigma) and 0.03%
H2O2 in PBS. After several washes,
slices were mounted on gelatin-coated slides, dehydrated in graded
alcohols, and cover-slipped with DePeX mounting medium (Merck Life
Science S.L.U., Darmstadt, Germany). C-Fos-labeled cells were studied
using the optical fractionator method in unbiased stereological
microscopy (Olympus BX51 Microscope, Olympus, Denmark), as previously
described (Borroto-Escuela et al., 2022;
Mirchandani-Duque et al., 2022;
Narvaez et al., 2018) (see Supplementary
Materials for details).