Neuroscience & Medicine, 2013, 4, 123-133
http://dx.doi.org/10.4236/nm.2013.43020 Published Online September 2013 (http://www.scirp.org/journal/nm)
123
Neuropeptide Y Increases Both Ingestion of a
Self-Selection Macronutrient Diet and Fos Expression in
the Medial Amygdala of Rats*
Bruna Mombach Dietrich1, Marli Sita Scalcon1, Franklin Back2, Bárbara B. Philippi Martins2,
Elisa Cristiana Winkelmann-Duarte2, Alberto A. Rasia-Filho1,3#
1Graduation Course in Pathology, Federal University of Health Sciences of Porto Alegre, Porto Alegre, Brazil; 2Departments of
Pharmacology and Morphological Sciences, Federal University of Santa Catarina, Florianopolis, Brazil; 3Department of Basic Sci-
ences/Physiology, Federal University of Health Sciences of Porto Alegre, Porto Alegre, Brazil.
Received June 14th, 2013; revised July 4th, 2013; accepted July 20th, 2013
Copyright © 2013 Bruna Mombach Dietrich et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The rat posterodorsal medial amygdala (MePD) is responsive to the orexigenic neuropeptide Y (NPY) and is a putative
candidate to participate in neural circuits that modulate feeding behavior. Here, we studied the effects of intracere-
broventricular (icv) microinjection of NPY on the appetitive and food intake behaviors of rats under the paradigm of the
self-selection macronutrient isolated diets [high-carbohydrate (high-CHO), high-protein and high-lipid food pellets]. At
the same time, Fos expression was also evaluated in the MePD as a marker of local cellular activation. Adult male rats
received icv microinjections of NPY (1 g and 10 g/5 L, n = 10 and 8, respectively) whereas the control groups ei-
ther received icv microinjection of artificial cerebrospinal fluid (5 L, n = 8) or underwent sham procedure (n = 8). The
data were obtained after a fasting protocol. Feeding behavior was evaluated during a 2 h test period of free access to the
selective diets. Rats in all groups preferred the high-CHO diet. Compared to controls, both doses of NPY increased the
appetitive behaviors (searching for food and the frequency of attempts to eat any diet) and the percentage of animals
eating high-CHO diet. However, only NPY at a dose of 1 μg led to a significant increase in food intake and showed a
strong positive correlation with Fos expression in the MePD (p < 0.05 in all cases). These new data reveal a biphasic
effect of NPY on the appetite and food intake behaviors and suggest that the MePD participates in the NPY-induced
feeding behavior in rats.
Keywords: Central Control of Appetite; Extended Amygdala; Feeding Behavior; Food Intake Behavior; Motivation
1. Introduction
Multiple neural systems control food intake, body weight
and energy homeostasis in rats [1-4]. The central organi-
zation of feeding behavior—cue-induced feeding, asso-
ciative learning, decision-making, reward and feed-back
modulation of appetite, eating and satiety—involves vari-
ous classical neurotransmitters, neuropeptides and hor-
mones for motivation [3-6]. For example, glutamate, the
main excitatory neurotransmitter, increases food intake
when microinjected in the lateral nucleus of the hypo-
thalamus [7,8], and the activation of glutamatergic
NMDA receptors is needed for the orexigenic effects of
neuropeptide Y (NPY) in this area [9]. Leptin and NPY
show opposing actions on appetitive and food intake be-
havior [5].
The integration of feeding behavior with emotional
processing and associative learning has been centered on
some hypothalamic and extra-hypothalamic areas, in-
cluding the “amygdala” [10,11]. The role of the different
amygdaloid nuclei in feeding behavior is still under study
[e.g., 6,10,12-14]. While basolateral, basomedial and la-
teral amygdaloid nuclei are involved in cue-driven intake,
the central nucleus (CeA) is decisive for feeding cessa-
tion by an aversive conditioned stimulus in male rats [re-
viewed in 6,10]. Studies using large electrolytic lesion
have shown that a wide area, named the “posterodorsal
region” of the amygdala, could alter feeding behavior
*Conflict of Interest: Authors declare no actual or potential conflict o
f
interest.
#Corresponding author.

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
124
and induce hyperphagia and obesity in female rats [15,
16]. More specifically, damage of posterodorsal medial
amygdala (MePD) and intra-amygdaloid bed nucleus of
the stria terminalis (and also the globus pallidus) were
mainly associated with an excess of weight gain in these
female rats [17].
The MePD is one of the four major subnuclei of the
medial amygdaloid nucleus (MeA) and is a complex part
of the forebrain, subcortical extended amygdala in rats
[18-22]. More recently, it was demonstrated that gluta-
mate microinjection in the MePD induced only a subtle
increase in the intake of a three-choice of macronutrient
self-selection diet in male rats [14]. This finding is in-
triguing once other experimental approaches suggested
that the MePD might be a modulatory area for feeding
behavior in rats [7,17,23]. Moreover, the MePD affects
relevant visceral reflex responses [24,25] and innervates,
directly or via the stria terminalis (ST), the hypothalamic
preoptic area, the ventromedial, the anteroventral peri-
ventricular, the arcuate, the paraventricular and the ven-
tral premammillary nuclei [26-28]. All of them integrate
various sensory inputs to control adaptive behaviors,
including food intake and energy balance [1,2].
It would be interesting to examine whether other neu-
rotransmitter or neuropeptide would affect the MePD
function and the modulation of goal-oriented food intake
[14]. NPY, one of the most widely expressed neuropep-
tides in the rat brain [29-31], is a putative candidate to
elicit such feeding responses since it can induce both
appetitive activities for food searching and has an intense
orexigenic effect [5,32-35]. There is a moderate concen-
tration of NPY-immunoreactive perikarya, but a high
density of fibers, in the dorsal half of the MeA [36], es-
pecially in the MePD of rats [37]. Although the mRNA
encoding NPY Y5 receptor is weakly observed in the
MeA of rats [33], a prominent population of neurons
expressing NPY Y1 receptor [38] and the NPY Y2 re-
ceptor [39] are found in this nucleus. These data are rele-
vant given that NPY Y1 and Y5 receptors basically me-
diate the effects of NPY on central feeding behavior [40].
Here, we tested the effects of intracerebroventricular
(icv) microinjections of NPY on the appetite and food
intake behaviors of rats under the paradigm of major
macronutrient-specific dietary selection. Also, Fos im-
munolabeling was evaluated in the MePD as a marker of
local cells activation [according to 22,26] following the
same rationale of previous studies on the central effects
of NPY or other orexigenic neuropeptides [4,6,29,30]. We
tested the correlation between the occurrence of feeding
behavior and the number of Fos-immunoreactive cells in
the MePD.
2. Materials and Methods
The present methodological approach was adapted from
Rosa et al. [14].
2.1. Animals
All animals were adult male Wistar rats (N = 34, 3 - 4
months old; body weight, mean ± SD = 234.1 ± 9.8
grams) initially housed in groups with standard lab chow
(Bio Base/Bio-Tec, Brazil) and water ad libitum, at room
temperature of 22˚C and a 12 h light/dark cycle (lights on
at 6 a.m.). All efforts were made to minimize the number
of animals studied and their suffering. Rats were ma-
nipulated according to international laws for the ethical
care and use of laboratory animals (European Communi-
ties Council Directive of November 1986, 86/609/EEC).
The present project was approved by the local Ethics
Committee (Federal University of Health Sciences of
Porto Alegre, Brazil; protocol no. 563/09).
2.2. Experimental Procedure
The procedural design included adapting periods, and
control and test recordings along 16 experimental days
(EDs; Figure 1). In the first ED, each rat was placed in
an individual metabolic cage (21 × 24 × 21 cm3 of length,
size and height, respectively; in accordance with the
“Guide for the Care and Use of Laboratory Animals,”
National Academy of Science, USA, 1996). The rats had
a 4 days initial period of adaptation to its new environ-
ment. During this period, there was free access to food
pellets of a full nutrient diet based on the AIN-93M
(composition and kilocalories are shown in Table 1;
consumption values in Table 2, ED 4). Water was avail-
able during all the EDs. The urine aspect and volume did
not indicate signs of dehydration at any moment (data not
shown). In every ED, food intake was evaluated by the
difference of weight of the available food between the
initial and the end of the monitoring period. Spillage was
collected on paper towels and subtracted from food in-
take to correct the obtained values. Rat body weight was
evaluated each day along the experiment. No behavioral
test for depression or plasma stress hormones measure-
ments were performed to directly determine possible
side-effects of isolation to avoid undue stress to the rats.
Nevertheless, during this initial period of isolation, and
more notably thereafter, rats displayed a constant and
homogeneous increase in body weight and no signs of
illness or abnormal behaviors, as reported elsewhere
[14].
On the EDs 5 to 8, rats received a three-choice self-
selection macronutrient isolated diets [pellets of high-
carbohydrate (high-CHO), high-protein and high-lipid
diets; compositions and kilocalories as described in Ta-
ble 1]. Diets were offered in separate dishes attached to
the metabolic cage. The location of each dish inside

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
125
Figure 1. Experimental design (in days) and behavioral test schedule.
Table 1. Diet compositions (adapted from Rosa et al., 2011).
Diets
Nutrients* AIN-93M (Adapted) High-CHO High-PTN High-LIP
Carboxymethylcellulose
Casein
Celullose
Citric acid
Cornstarch
Food dye
Hidrogenated vegetable fat
L-cystine
Mineral mix**
Soybean oil
Sugar
Vitamin mix**
1.0
14.0
5.0
0.01
60.96
0.1
2.0
0.18
3.5
2.0
10.0
1.25
2.0
-
5.0
0.01
78.14
0.1
-
-
3.5
-
10.0
1.25
2.0
87.96
5.0
0.01
-
0.1
-
0.18
3.5
-
-
1.25
2.0
-
5.0
0.01
-
0.1
79.39
-
7.0
4.0
-
2.5
* Nutrients expressed as percent by weight diet. ** The vitamin and mineral mixes contain 97.47% and 21% cellulose respectively, according to AIN -93M.
Table 2. Diet intake along different experimental days (EDs) and procedures.
Groups EDs Diet intake (g)
AIN-93M Adapted High-CHO High-PTN High-LIP
Sham
4
8
10
16
16.6 (15.9/17.1)
12.2 (11.3/13.4)
0.2 (0/1.1)
0.2 (0/1.1)
3.7 (3.2/4.2)
0 (0/0)
0 (0/0)
0.6 (0.3/0.7)
0 (0/0)
0 (0/0)
aCSF
4
8
10
16
17.6 (16.5/18.1)
13.0 (11.6/14.4)
0.2 (0/1.1)
0.2 (0/1.1)
3.5 (2.2/4.5)
0 (0/0)
0 (0/0)
1.0 (0.6 /1.1)
0 (0/0)
0 (0/0)
NPY
1 ug
4
8
10
16
11.2 (8.2/16.7)
13.6 (13.1/15.3)
0 (0/0.5)
1.7 (1.1/1.9)**
4.6 (3.6/4.8)
0 (0/0.1)
0 (0/0)
1.0 (0.9/1.1)
0 (0/0.3)
0 (0/0.1)
NPY
10ug
4
8
10
16
11.5 (8.4/16.9)
13.6 (10.9/13.6)
0 (0/0.6)
1.1 (1.0/2.0)
3.7 (3.1/4.1)
0 (0/0)
0 (0/0.1)
0.5 (0.1/1.0)
0 (0/0)
0 (0/0)
*

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
126
the cage was changed daily to avoid biases resulting from
a conditioned place preference. The “palatability” of the
high-CHO diet was not intentionally enhanced by adding
sweet monosaccharides and disaccharides. Although it is
possible that few corn starch had been broken in less
complex sugars, heating temperature that induce the
formation of a final high amount of dextrin was avoided
during the diet preparation [c.f., 14]. Values of daily in-
take of these selective diets are shown in Table 2 (on ED
8).
On the ED 10, rats underwent food deprivation for 4 h
(from 8 a.m. to 12 a.m.) and, thereafter, allowed to eat
the macronutrient isolated diets during 2 hr. During this
period, the appetitive and the food intake behaviors were
observed. The parameters to evaluate the appetitive be-
haviors included, the number of attempts to reach each
macronutrient isolated diet and the frequency of attempts
to eat a pellet of any diet. The amount of food intake of
each self-selection diet in grams was indicative of food
intake behavior. The data obtained from ED 10 was the
baseline (Table 2, on ED 10) and was compared with the
data obtained on the upcoming test day. The importance
of this comparison was addressed previously [14,22].
On the ED 11 (beginning at 8 a.m.), rats were anesthe-
tized with ketamine and xylazine (80 and 10 mg/kg, i.m.,
respectively) and stereotaxically implanted with a stain-
less steel guide cannula (0.6 mm of outer diameter, OD)
in the right lateral ventricle of the brain. The stereotaxic
coordinates were: 1.0 mm posterior to the bregma, 1.5
mm lateral to the midsagital suture and 3.5 mm below the
dura-mater [adapted from 41]. The upper incisor bar was
positioned horizontally to the interaural line.
The animals were allowed to recover from surgery for
2 days (EDs 12 and 13) and had free access to the iso-
lated macronutrient diet. On the EDs 14 and 15, rats were
gently manipulated to simulate the icv microinjection
procedure (described below) and were again allowed to
eat the isolated macronutrient diets until the next day.
On the ED 16, rats were deprived of food for 4 h and,
thereafter, the feeding tests were initiated at 12 am for all
the experimental groups to avoid circadian effects on the
results. Rats were randomly assigned to receive artificial
cerebrospinal fluid (aCSF, 5 L, n = 8) or NPY (1 g or
10 g/5 L diluted in aCSF, n = 10 and 8, respectively).
The aCSF composition was: NaCl 126 mM, KCl 2.5 mM,
NaH2PO4 1.25 mM, MgCl2 2 mM, CaCl2 2 mM, Na-
HCO3 26 mM, and glucose 10 mM [42]. NPY was pur-
chased from Sigma Chemicals Co. (USA). Both doses of
NPY were previously reported as able to promote
marked behavioral effects, some lasting from 1 to 24 h
[5,43]. The microinjection needle (0.3 mm OD) was in-
troduced by the stereotaxic guide cannula for the icv
microinjections and the displacement of liquid and an air
bubble inside the catheter connecting to a 10 L Hamil-
ton microsyringe (USA) was used to monitor the proce-
dure. This lasted for 1 minute and the needle remained
inside the cannula for an additional minute to avoid re-
flux [14,25]. No obvious alterations in behavior were
observed after microinjections. An additional control
group for the experimental procedures was assigned for
sham microinjection (n = 8), i.e., these rats were anesthe-
tized and had an icv cannula, the needle was inserted and
rats were manipulated as the other experimental groups,
but did not receive any treatment.
Immediately afterwards, rats returned to their meta-
bolic cage and had access to the three-choice macronu-
trient self-selection diet. The intake of each diet was
evaluated at the end of 2h following microinjection
(shown in Ta ble 2, on ED 16). It was observed the num-
ber of the appetitive behaviors and the ingested amount
of each isolated macronutrient diet taken. The percentage
of animals that had taken the selective diets in each ex-
perimental group was also calculated.
At the end of the experiment, rats were deeply anes-
thetized as above-mentioned and icv microinjected with
methylene blue to identify the location of the microinjec-
tion site. Rats were transcardially perfused with phos-
phate buffer solution (PBS, 0.1 M, pH 7.4) followed by
4% paraformaldehyde diluted in PBS. Brains were re-
moved from the skull, remained in the same fixative so-
lution for 12 hr, transferred to a 30% sucrose solution at
4˚C for at least 1 week and, thereafter, were sectioned
using a vibratome (Leica, Germany; 50 m-thick coronal
sections) to identify the path of the icv cannula. Animal
with signals of large mechanical lesions or brain hemor-
rhage was excluded of the studied groups. The same
brains were used to study of Fos expression in the MePD.
The MePD was recognized by its ventral location to the
ST and lateral position to the optic tract in the rat ventral
forebrain, 2.76 to 3.48 mm posterior to the bregma [41].
Both hemispheres were studied. Brain sections were ini-
tially coded and kept immersed in an anti-freeze solution
(PBS, distilled water, sucrose, and propylene glycol) and
stored at 70˚C in biofreezer until further processing.
The Fos immunohistochemistry was carried out using
the avidin-biotin peroxidase method as previously de-
scribed [44]. Free-floating brain sections were washed in
PBS and incubated in a solution containing PBS, Triton
X-100 (Sigma Chemicals Co., USA), goat serum and
anti-Fos antiserum raised in rabbit (Ab-5; Calbiochem,
USA) diluted 1:20.000 for 48 h at 4˚C and continuous
shaking. Sections were then incubated during 90 min at
room temperature in a solution of biotinylated goat
anti-rabbit IgG (1:200; Vector, USA) and then placed in

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
127
the a mixed avidin–biotin horseradish peroxidase com-
plex solution (1:200; ABC Elite Kit; Vector, USA) for
the same time. The peroxidase complex was visualized
after a 10 min exposure to a chromogen solution con-
taining 0.02% 3,30-diaminobenzidine tetrahydrochloride
(Sigma Chemicals Co., USA) with 0.3% nickel ammo-
nium sulfate in 0.05 M Tris-buffer (pH 7.6), followed by
incubation for 5 min in chromogen solution with glucose
oxidase (Glucose Oxidase Type VII from Aspergillus
Niger, 0.01%, Sigma Chemicals Co., USA) and 10% p-
D-glucose (Sigma Chemicals Co., USA). Sections were
rinsed in PBS, dehydrated in ethanol, washed with xy-
lene and covered with DPX (distyrene/plasticizer/xylene)
synthetic balsam and coverslips. All experimental groups
were processed altogether to avoid possible unspecific
variations in the procedures and their results. To control
the enzymatic activity, some random sections were per-
formed omitting the primary antiserum, which provided
no detectable Fos-immunostaining and a “negative” back-
ground aspect (data not shown).
The images of the cellular Fos expression were similar
to those published elsewhere [22,30,44] and staining
pattern was empirically considered adequate for further
comparisons among groups. Quantification of the num-
ber of Fos immunoreactive (Fos-ir) cells in the MePD
was made according to previous reports [22,45]. The
criteria to identify and to calculate Fos-ir cells in the
MePD included the following: the cells must a) be within
the including borders of an overlaid rectangle named
here as the “area of interest” [0.36 mm², Figure 2(A)]; b)
have a staining color notably contrasting with the back-
ground; and, c) have identifiable nuclear cell borders
[Figure 2(B)]. All microscopic images lightning and
contrast conditions were maintained identical during the
experimental data acquisition. Five sections per rat were
studied (n = 5 rats for the sham and NPY 10 g groups, n
= 6 rats for the aCSF and NPY 1 g groups). Direct cal-
culation of Fos-ir cells in the MePD were performed by
two independent observers using an optic microscope
(400 x; Olympus BX-61, Japan) coupled to a high-per-
formance CCD DP72 camera (Olympus, Japan) and an
image processing software (Image Pro Plus 7.0, Media
Cybernetics, USA). Fos-ir cells were also observed in the
hypothalamic periventricular nucleus (PVN) and the dor-
somedial nucleus (DMH), previously described as criti-
cally involved with food intake behavior [1,30,40] and
food-anticipatory activity [46,47], respectively, as well as
with the central effects of NPY [29, 30,40]. It was not the
scope of the present work to depict the presence of Fos-ir
in the different hypothalamic nuclei, which was already
done indeed [29,30]. Nevertheless, the presence of Fos-ir
cells in these above-mentioned hypothalamic nuclei serv-
ed as additional “internal” control data for the Fos ex-
pression in our studied animals.
Figure 2. A. Schematic diagram of a coronal brain section
showing the right and left posterodorsal medial amygdala
(MePD, approximately 3.0 mm posterior to the bregma, as
an example of the sampled sections studied here). The
overlaid rectangle (the “area of interest” of 0.36 mm²)
represents the site where Fos-immunoreactive cells were
calculated. Both hemispheres were studied. opt = optic tract,
st = stria terminalis. Adapted from [41]. B. Reconstructed
photomicrographs of Fos immunolabeling in the MePD of
NPY icv microinjection group (1 g or 10 g/5 L diluted in
aCSF, n = 10 and 8, respec tively) and control groups [sham
(n = 8) or after icv microinjection of artificial cerebrospinal
fluid (aCSF, 5 L, n = 8)]. Background and contrast were
adjusted (Adobe Photoshop 7.0, USA), and the same criteria
to calculate the cells were adopted for all experimental
groups (see text for further details). Arrows point to some
Fos-immunoreactive cell. Scale bar = 20 µm. Spatial coor-
dinates are: D = dorsal, L = lateral, M = medial, V = ventral.
C. Values are mean ± standard error of the mean of Fos-ir
cells in the MePD of the studied experimental groups. *p <
0.05 compared to all other groups.
2.3. Statistical Analysis
Analysis of variance (ANOVA) test for repeated meas-
ures followed by the Tukey test was used to evaluate the
weight gain of the rats during the EDs 1, 10 and 16.

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
128
After logarithmic transformation, the searching for
food and frequency of attempts to eat a pellet of any diet
and the amount of the high-CHO diet intake on EDs 10
and 16 were compared among groups using ANOVA test
for repeated measures followed by the Newman-Keuls
test for multiple comparisons. Fisher exact test was used
to compare the percentage of rats of each group that have
taken high CHO diet intake on ED 16. The low intake of
the high-protein and the high-lipid selective diets pre-
vented further statistical comparisons among groups.
After square root transformation, Fos expression data
were compared among groups by the one-way ANOVA
test followed by the Tukey test. The nonparametric
Spearman rank test was used to correlate the frequency
of the appetitive behaviors and the amount of food intake
with the number of Fos-ir cells counted in the MePD. In
all cases, results were considered significant when P <
0.05.
3. Results
3.1. Body Weight
Animals in all groups showed increased values of body
weight along the EDs 1, 10 and 16 [mean ± standard de-
viation (SD), 234.1 ± 9.8; 241.9 ± 9.8; and, 261.1 ± 15.6
grams, respectively], which were statistically significant
[ANOVA test, F (2,98) = 99.61; P < 0.01] and different
from each other (Tukey test, P < 0.01 in all cases).
3.2. Appetitive Behaviors and Selective Diet
Intake
Data are shown in Tables 2 and 3. Animals in all groups
showed a similar intake of AIN-93M diet during the first
EDs (see mean values on ED 4). When rats had free ac-
cess to the self-selection macronutrient diet, all groups
showed a clear preference for the high-CHO diet when
compared to the other diets (see mean values on ED 8).
Following 4 h of food deprivation and the subsequent 2 h
of free access to the 3-choice self-selection macronutrient
diet, rats of all groups also had taken more high-CHO
diet (see mean values on ED 10) and .small amount of
high-protein and high-lipid diets (Table 2).
There was a significant difference among experimental
groups in the appetite behaviors of searching for food
dishes [ANOVA test, F (3,29) = 11.66; P < 0.01] and the
frequency of eating the pellets of any selective diet on
the ED 16 [ANOVA test, F (3,29) = 10.32; P < 0.01].
There was no difference between sham and aCSF micro-
injected groups (Newman-Keuls test, P > 0.05), whereas
both doses of NPY increased these appetitive behaviors
when compared to the control groups (Newman-Keuls
test, P < 0.05; Table 3).
There was also a significant difference in the intake of
the high-CHO diet between groups [ANOVA test, F
(7,55) = 3.54; P < 0.01]. The Newman-Keuls post hoc
test showed that all groups had a similar intake of this
diet on the control ED 10 (P > 0.05), whereas differences
among groups were found on the test day, ED 16. Data
from the sham and the aCSF microinjected groups were
not statistically different (P > 0.05). On the other hand,
the microinjection of NPY 1 μg led to an increased intake
of the high-CHO diet, which became different from the
values obtained in this same group on the ED 10 (P <
0.01) and when compared to both sham and aCSF data
on the ED 16 (P < 0.05). This same effect was not ob-
served in the group microinjected with NPY 10 μg (P >
0.05; Table 2).
There was no significant difference in the percentage
of rats that had taken the high-CHO diet on the EDs 10
and 16 for sham and aCSF microinjected groups (per-
centages ranging from 50% to 75% in both groups; Fisher
test, P > 0.6). On the other hand, all tested rats had taken
the high-CHO diet after icv microinjection of both doses
of NPY on EDs 10 and 16 (Fisher test, P < 0.03).
3.3. Fos Expression
Results are shown in Figure 2(C). There was a signifi-
cant difference in the number of Fos-ir cells in the MePD
among experimental groups [ANOVA test, F (3,17) =
11.18; P < 0.01]. Additionally, the animals receiving
NPY 1 μg had higher number of Fos-ir cells when com-
pared to the sham, the aCSF and the NPY 10 μg experi-
mental groups (Tukey test, P < 0.05 in all cases). No
statistical difference was found among these three latter
groups (Tukey test, P > 0.05).
Table 3. Appetitive behaviors.
Groups Frequency of Searching for Food Frequency of Attempts to Eat
High-CHO High-PTN High-LIP High-CHO High-PTN High-LIP
Sham 1.6 ± 0.7 1.3 ± 0.5 0.8 ± 0.7 1.4 ± 1.3 0.6 ± 0.7 0 ± 0
aCSF 1.6 ± 1.3 1.3 ± 0.9 1.0 ± 1.0 0.9 ± 0.9 0.4 ± 0.7 0.1± 0.3
NPY 1 ug 8.9 ± 6.2* 7.5 ± 7.0* 4.7 ± 2.4* 6.9 ± 3.8* 0.9 ± 1.9* 0.4 ± 0.7*
NPY 10 ug 5.8 ± 3.8* 4.1 ± 2.5* 3.0 ± 2.7* 5.5 ± 4.4* 0 ± 0 0.1 ± 0.3

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
129
The correlation between the number of MePD Fos-ir
cells, the appetitive behaviors display and the amount of
high-CHO diet intake was tested specifically in the group
receiving icv microinjection of NPY 1 μg. There was no
correlation between the number of Fos-ir cells in the
MePD and the frequency of searching for the food dishes
[Spearman test, P = 0.24; Figure 3(A)] or the frequency
of eating pellets of any selective diet [Spearman test, P =
Figure 3. Relationship (Spearman’s correlation) between
the number of Fos-immunoreactive cells in the rat postero-
dorsal medial amygdala and the number of attempts of the
appetitive behaviors of (A) searching for food and (B) fre-
quency of attempts to eat the available self-selection mac-
ronutrient diets or (C) the consummatory behavior of tak-
ing the high-CHO selective diet (intake in grams) during a
test period of 2 hours after the central microinjection of
NPY (1 g/5 L, icv, n = 6). A highly significant correlation
was only found in (C), as evidenced by the respective r and
P values.
0.10; Figure 3(B)]. However, there was a highly signifi-
cant and positive correlation between the amount of
high-CHO diet consumption and the number of MePD
Fos-ir cells [Spearman test, P < 0.01; Figure 3(C)].
Finally, as control data, Fos expression in the PVN re-
sembled that found in the MePD, with higher amount of
Fos-ir cells in the NPY 1 μg microinjected group when
compared with the others (data not shown). In the DMH,
Fos-ir in the MePD appeared to be similar for control and
NPY microinjected groups (data not shown).
4. Discussion
In this study, we showed that the rats were well adapted
to the experimental protocol, exhibited a continuous
body weight gain along the EDs and had a preference for
the high-CHO selective diet. Sham and aCSF microin-
jected groups had similar results and served as control.
NPY increased the food intake behavior with a biphasic
effect under the present behavioral paradigm. That is,
microinjection of NPY 1 μg was able to stimulate the
searching for food, the frequency of attempts to eat any
selective diet and induced a higher CHO intake. The
NPY-mediated orexigenic effect (on food intake behav-
ior rather than on appetitive behaviors) has a strong cor-
relation with the number of Fos-ir cells in the MePD. On
the other hand, NPY 10 μg stimulated appetitive behav-
iors, but did not consistently affect the food intake be-
havior or the Fos expression in the MePD when com-
pared to the aCSF microinjected group.
Previous data showed that NPY can direct the atten-
tion towards food in a dose-dependent manner [5] and/or
can increase the motivation for food intake by modulat-
ing appetite and hunger [32]. The enhanced high-CHO
diet intake described here agrees with other reported ef-
fects of centrally administered NPY in rats [34,43,48,49].
However, under the present behavioral test, lower and
higher doses of NPY did not promote quite related results
on food intake behavior and MePD Fos-ir cells number.
Recent findings made clear that, indeed, the MePD re-
sponded differently to increase amount of neurotransmi-
ters and neuromodulators locally microinjected, likely
coding for distinct synaptic strength and for the specific
“fine-tuning” of behavioral displays [further discussed in
14,22,24,25,50; see conceptual comments in 4,51].
Our study of Fos expression was based on previous
similar procedures that revealed the functional roles of
the MePD on social/motivated behaviors [22,45,52,53]
and the activation of brain areas following NPY action
[29,30]. Here, control observations of Fos-ir in the PVN
and DMH occurred as expected for their function. In
addition, Fos expression was similar to that described
previously by others [29,30,40,46,47]. It is worth to
mention that the enhanced Fos-ir in the MePD observed

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
130
following NPY microinjection could be a direct effect of
NPY signaling on this subnucleus, an indirect conse-
quence of the activation of interconnected neural net-
works for feeding behavior. Alternatively, it could be
secondary to the different amount of food intake. There
are evidences in favor of all these possibilities. First,
NPY innervation and receptors are found in the MePD
[36-38] and icv microinjected NPY could reach the
MePD caudal part of the wall of the lateral ventricle
[18,41]. Second, the MePD has direct and indirect con-
nections with hypothalamic areas that modulate feeding
behavior [17,28], and are sensitive to centrally adminis-
tered NPY. These areas also show marked Fos-ir in rats
allowed to feed after NPY microinjection [30], such as
the PVN and the DMH. However, lesions of the entire
MeA or ST did not reduce the density or the distribution
of NPY immunoreactive terminal fields in the basal
forebrain or in the hypothalamus, suggesting that immu-
noreactive neurons in the MeA did not contribute signifi-
cantly to these innervations [36]. On the other hand,
widespread neurons that synthesized or responded to
NPY can really establish little functional dependence among
them and represented redundant neuronal mechanisms
for feeding behavior [additional comments in 23]. Third,
there was a positive correlation between Fos-ir in the
MePD and the amount of food intake following icv
microinjection of NPY 1 μg. Clearly, these are not mutu-
ally exclusive possibilities and, in common, they indicate
that the rat MePD is activated during feeding behavior.
This is a new an interesting venue revealed by the pre-
sent data.
Furthermore, it would be also possible that other
physiological factors could account for the Fos-ir in the
MePD following NPY icv microinjection. The MePD
can elicit homeostatic or adaptive general adjustments in
the context of emotional/social stimuli processing [21,22,
26,54] and NPY Y1 receptors in the whole amygdala are
important to generate anxiolytic effects seen in rats tested
in the elevated plus maze test [55]. The NPY Y1 receptor
was notably found in the MeA subnuclei [38] and the
present icv microinjection of NPY would have stimulated
both food intake and anticonflict/anxiolytic-like effects
in rats. However, ibotenic acid neurotoxic damage of the
MePD was not able to modify any parameter of anxiety-
like behavior in lesioned rats studied in the same plus
maze test [22]. The CeA is another component of the ex-
tended amygdala along with the MeA subnuclei [18], and
local microinjections of NPY (0.1, 0.3 and 1.0 μg in 1 μl
of saline) after overnight fasting increased high-CHO
food intake and reduced high fat diet at 24h post-injec-
tion in rats [34]. NPY or a selective NPY Y1 receptor
agonist microinjected directly in the CeA reduced anxi-
ety without increasing meal consumption [56]. Altoge-
ther, these findings indicate a likely dissociation between
anxiolytic-like actions and food intake effects in the
MePD and CeA of rats. Novel, conflictive or stressful
experimental condition appears not to be responsible for
marked Fos-ir cells in the MePD, as evidenced by the
lower Fos expression in the control groups (sham and icv
aCSF microinjection) subjected to the same paradigm.
Also, there are no direct data that prove the involvement
of the MePD in other aspects of feeding behavior, such
as learning of appetitive motivated tasks [reviewed in 57].
Other studies are needed to test if NPY action in the
MePD relates to food-anticipatory responses and prepa-
ration for scheduled feeding [58] or to prepare the animal
to consume a calorically large meal [59]. Currently, evi-
dent experimental differences (e.g., Long-Evans adult
females vs Wistar adult males) hamper the direct com-
parison of our data with large electrolytic lesions of the
“posterodorsal region” of the amygdala, which induced
hyperphagia and obesity in lesioned female rats [14-16],
even more because the MePD is sexually dimorphic and
affected by ovarian steroid fluctuations along the estrous
cycle [19,21; and references therein].
Finally, some important issues should be mentioned to
direct future efforts. Here we show the correlation be-
tween NPY-induced selective high-CHO diet intake and
Fos expression in the MePD. These data suggest the par-
ticipation of this subnucleus in goal-oriented feeding
behavior and highlight the MePD as a subcortical com-
ponent of different social behavior neural networks [22,
based on 26]. The dual effects of NPY can also add to the
scholarly debate about the actual distinction between
appetitive vs consummatory behaviors [see critical com-
ments in 60]. In addition, from the insightful proposition
of Zeltser et al. [4] on brain regions that participate in
energy homeostasis, the MePD shows notable dendritic
spines and synaptic plasticity [21], modulates reproduc-
tion [22] and can be part of dynamic neuronal circuits
that regulate the balance between energy intake and ex-
penditure. Then, future research can involve the direct
microinjection of NPY in the MePD to test feeding be-
havior and compare the present results with those to be
obtained with this additional experimental approach.
Other amygdaloid nuclei should be tested as well. It is
also highly deserved to reveal whether NPY acting in the
MePD would play a role in pathological conditions that
involve feeding behavior disturbances. These are opened
possibilities now awaiting further advancements.
5. Acknowledgements
Authors are thankful to M.Sc. Carolina Böetge Rosa,
M.Sc. Daniela Haas and Ms. Renata V. de Souza (Brazil)
for their contribution to the experimental protocol. Also,
to Dr. Maria Elisa Calcagnotto (Brazil) for her sugges-
tions. Grants from the Brazilian Founding Agency CNPq.

Neuropeptide Y Increases Both Ingestion of a Self-Selection Macronutrient
Diet and Fos Expression in the Medial Amygdala of Rats
131
AARF is a CNPq researcher.
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