Figure S1. Galanin Receptor 1 and Galanin Receptor 2 colocalizes in neurons of rat
dorsal Hippocampus and dorsal Raphe. Double-immunolabeled for Galanin receptor 1
(GalR1) (green, arrows) and Galanin receptor 2 (GalR2) (red, arrowhead) in (A) dorsal
Hippocampus and (B) dorsal Raphe. The colocalization of GalR1 and GALR2 is shown in
the merge ( orange-yellow, rhombus). Escale bar, 20 μm.
Figure S2. Time course of Galanin receptor 2 (GalR2) protein expression following a
single i.c.v. injection of siRNA GalR2 or vehicle into the rat brain. (A,B) Quantification of
Immunohistochemical staining for GalR2 [GalR2 immunoreactivity was used as a
marker for GalR2 protein expression] in hippocampus and Piriform Cortex in rat brains
injected i.c.v. with siRNA GalR2 or vehicle at different times behind injection. GalR2
immunoreactivity was determined after immunohistochemical by measuring the
Optical Density of the cells. Vertical bars represents mean ± SEM of percentage of
change from respective controls. *p<0.05; **p<0.01 and ***p<0.001 versus vehicle
group according to one-way ANOVA followed by Newman Keuls Multiple Comparison
Test. (C) Representatives photographs for GalR2 immunohistochemical in dorsal
Hippocampus are presented. Magnifications of selected portions of these images are
shown in the insets. Scale bar, 50 μm.
Figure S3. Expression levels of GalR2 mRNA in dorsal hippocampus. A Expression levels
of mRNA GalR2 (4 rats per group) measured on different days after administration of
the Acell smart pool siRNA for GalR2. The quantitative PCR results were normalized to
the expression levels of GAPDH and expressed as arbitrary units. *p<0.05 vs Vehicle,
Student´s t test versus respective control. B Image of an agarose gel with the products
of the quantitative PCR of vehicle and Day 4.
Figure S4. Representative laser scanning confocal micrographs illustrating the decrease
of Galanin receptor 2 (GalR2) immunoreactivity in GalR2 knockdown rat at dorsal
hippocampus and dorsal Raphe. Double-immunolabeled for Galanin receptor 1 (GalR1)
(green) and Galanin receptor 2 (GalR2) (red) in rat (A) dorsal Hippocampus and (B)
dorsal Raphe, 9 days after of single icv injection of siRNA GalR2 or vehicle. Escale bar,
20 μm.
Figure S5. Analysis of Galanin(1-15) [Gal15] in GalR1 knockdown rats in the Forced
Swimming Test (FST) and Open field test. Behavioural effects of effective dose of Gal15
(3nmol/rat) in GalR1 knockdown in FST (A,B,C) and Open field test (D,E). Treatments
were injected via chronical cannula into lateral ventricle 15 min prior the tests. siRNA
control injected rats were used as control group. (A,B,C) Data represents mean ± SEM
of immobility, climbing and swimming time in FST and (D,E) entries and time in center
in open field test during the 5 min test period (6-8 rats per group). No differences were
found according to one-way ANOVA followed by Newman Keuls Multiple Comparison
Test. (F) Expression levels of mRNA GalR1 (4 rats per group) measured on different days
after administration of the Acell smart pool siRNA for GalR1. The quantitative PCR
results were normalized to the expression levels of GAPDH and expressed as arbitrary
units. Values are indicated as mean ± S.E.M. *p<0.05, **p<0.01 vs vehicle group
according to one-way ANOVA followed by Newman Kleus Multiple Comparison Test.