Iemolo et al., Anorectic effect of CeA PACAP
Pituitary adenylate cyclase-activating peptide in the Central Amygdala Causes Anorexia
and Body Weight Loss via the Melanocortin and the TrkB Systems
Attilio Iemolo, Ph.D.1; Antonio Ferragud, M.S.1, Pietro Cottone, Ph.D.1, *; Valentina Sabino,
Ph.D.1, *.
1
Laboratory of Addictive Disorders, Department of Pharmacology and Experimental
Therapeutics and Department of Psychiatry, Boston University School of Medicine, Boston, MA
02118
* These two authors contributed equally to the work.
Correspondence and requests for materials should be addressed to:
Valentina Sabino (vsabino@bu.edu)
Laboratory of Addictive Disorders
Boston University School of Medicine
72 E Concord St, R-612
Boston, MA 02118 USA
Phone: 617-638-4327, Fax: 617-638-5668
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Iemolo et al., Anorectic effect of CeA PACAP
Supplemental Materials and Methods
Subjects
Male Wistar rats, weighing 301-325g at arrival (Charles River, Wilmington, MA), were
single-housed in wire-topped, plastic cages, in a 12h:12h reverse light cycle (lights off at 9:00
am) AAALAC-approved humidity- (60%) and temperature-controlled (22 C) vivarium. Rats had
access to corn-based chow (Harlan Teklad LM-485 Diet 7012) and water ad libitum at all times
unless otherwise specified. The number of rats for each experiment was as follows: doseresponse studies, n= 11 (CeA) and n= 8 (BlA); motor activity, n= 9; meal microstructure, n= 8;
SHU 9119 blockade, n= 11; k252a blockade, n= 9; D-Phe-CRF(12-41) blockade, n= 15.
Procedures adhered to the National Institutes of Health Guide for the Care and Use of
Laboratory Animals and the Principles of Laboratory Animal Care, and were approved by
Boston University Medical Campus Institutional Animal Care and Use Committee (IACUC).
Drugs
PACAP (PACAP-38) was purchased from the American Peptide Company (Sunnyvale,
CA); SHU-9119 was purchased from R&D Systems (Minneapolis, MN); k252a was purchased
from Alomone Labs (Jerusalem, Israel); D-Phe-CRF(12-41) was kindly provided by Dr. Jean
Rivier (The Salk Institute, La Jolla, CA). Doses were given within-subject in a balanced Latin
square design with at least three intervening treatment-free days. All drugs were dissolved in
isotonic saline in the presence of 1% bovine serum albumin except for k252a which was
dissolved in DMSO 50% in saline. PACAP and/or antagonists (which were administered
together with PACAP as a cocktail in a single injection) were administered in a volume of 0.5
µl/side. Drug pretreatment time was 30 min in all experiments.
Intracranial surgeries, microinfusion procedure and cannula placement
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Iemolo et al., Anorectic effect of CeA PACAP
Intracranial surgeries. Rats were stereotaxically implanted with bilateral, intracranial
cannulas as described previously (Cottone et al, 2007a; Iemolo et al, 2012; Sabino et al, 2007).
Briefly, stainless steel, guide cannulas (24 gauge, Plastics One, Inc., Roanoke, VA) were
lowered bilaterally 2.0 mm above the CeA or the BlA. Four stainless steel jeweler’s screws were
fastened to the rat’s skull around the cannula. Dental restorative filled resin (Henry Schein Inc.,
Melville, NY) and acrylic cement were applied, forming a pedestal firmly anchoring the cannula.
The cannula coordinates from bregma used for the CeA were: AP -2.64, ML ±4.2, DV -6.2 (from
skull); coordinates for the BlA were: AP -2.64, ML ±4.8, DV -6.5 (from skull), according to
Paxinos and Watson (Paxinos and Watson, 2007). A dummy stylet (Plastics One, Inc.,
Roanoke, VA) maintained patency of the cannula.
Microinfusion procedure. Drug was microinfused in the brain of rats as previously
described (Blasio et al, 2013; Dore et al, 2013; Iemolo et al, 2013). The dummy stylet was
removed from the guide cannula and replaced with a 31-gauge stainless steel injector projecting
2 mm beyond the tip of the guide cannula; the injector was connected via PE 20 tubing to a
Hamilton microsyringe (Hamilton Company, Reno, NV) driven by a multi-syringe microinfusion
pump (KD Scientific/Biological Instruments, Holliston, MA). Microinfusions were performed in
0.5 μl volume per side delivered over 2 min; injectors were left in place for 1 additional min to
minimize backflow.
Cannula placement verification. Cannula placement was verified at the conclusion of all
testing. Subjects were sacrified under anesthesia (isoflurane, 2–3% in oxygen) and intra-CeA or
intra-BlA microinjections of India ink were performed. Brains were removed and frozen in an
isobutane/dry ice bath and stored at -80˚C. Coronal sections of 40 µm were collected using a
cryostat and placements verified under a microscope.
Home cage food intake and body weight determinations
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Iemolo et al., Anorectic effect of CeA PACAP
Pre-weighed food was provided at the beginning of the dark cycle, and recorded 1h, 3h,
6h and 24h later. Rat body weights were assessed right before drug administration and 24h
later.
Meal pattern, within meals microstructure and return map analyses.
Apparatus. To study the microstructure of ingestion, rats were individually housed during
the 12 hours of their active dark cycle in Plexiglas test cages (30×24×29 cm) equipped for this
purpose (Velazquez-Sanchez et al, 2014). Rats obtained nosepoke-contingent food and water
on a fixed ratio 1 (FR1) continuous schedule of reinforcement. 45 mg pellets (5TUM diet, 65.5%
[kcal] carbohydrate, 10.4% fat, 24.1% protein, metabolizable energy 330 kcal/100 g; TestDiet,
Richmond, IN) were delivered from an automated dispenser (Med Associates, St. Albans VT).
Water reinforcers (0.1 ml) were delivered by a solenoid into a liquid cup nosepoke receptacle.
All responses were recorded automatically by a computer with 10 msec resolution. Testing
began once food intake had stabilized (<15% variation across 3 consecutive days); PACAP-38
(0, 1 μg/rat) was administered 30 min prior to dark cycle onset, which corresponded to the
beginning of the food self-administration session.
Meal pattern analysis. Microstructure analysis was performed as previously described
(Cottone et al, 2007b; Zorrilla et al, 2005). Meals were defined as consecutive responses for
food or water that contained at least five food-directed responses (0.225 g) and in which
ingestive responses occurred within 5 min of the previous response (Fekete et al, 2011).
Microstructure parameters included the average size and duration of meals, the number of
meals and the average inter-meal interval. Meal size for eating was calculated as the average
number of food-directed responses during meals, duration of eating within the meal was
calculated as the sum of the durations of bursts of eating where each burst’s duration was
defined as the interval from the first to last of consecutive responses for food. The inter-meal
interval was defined as the interval from the last response of a meal to the first response of the
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Iemolo et al., Anorectic effect of CeA PACAP
next meal. The eating rate was calculated by dividing each meal size by its respective duration.
Because measures of meal duration reflect not only the time spent in the actual act of ingestion
(i.e. chewing, licking, swallowing), but also the quantity of intra-meal pausing, calculated
measures of eating/drinking rate reflect whether ingestion was sustained by food or water within
meals and not only the mechanical act of ingestion.
Within-meal microstructure analysis. To identify the effects of PACAP-38 on the rate and
regularity of sustained eating within meals, analysis of the ln-transformed duration of
consecutive (uninterrupted by drinking) within-meal interfeeding-intervals (IFIs) was performed
(Cottone et al, 2007a; Cottone et al, 2012). Histograms were constructed from ln-transformed
IFI durations that fell from e-1 to e3 sec (~0.4-20.1 sec), with a bin width of e0.2 (Cottone et al,
2007a; Cottone et al, 2012). The mean, entropy kurtosis, and skewness of the ln-transformed
duration of each subject’s consecutive IFIs was individually determined and then averaged
across subjects. The normalized frequency histogram entropy (H) is a measure of categorical
variability in the rate of ingestion (Shannon C.E., 1949) , and was computed as follows:
n
H 
p
i
i
log 2  pi 
log 2 n 
H is scaled between 0 and 1, with the denominator determined by the number of
possible bins in the histogram (n) and the numerator a function of the proportion of observations
that fall within a given histogram bin (pi). Minimal (H=0) entropy occurs when all observations
occur within a single histogram bin, whereas maximal entropy (H=1) occurs when each
histogram bin has an equal probability, or a flat uniform joint density distribution. Significant
decreases in the mean indicate an increased eating rate; significant increases in the histogram
entropy indicate a decreased regularity of intake. Conversely, a decrease in the kurtosis of the
inter food interval distribution (a measure of the distribution's “peakedness”, reflected in a flatter
top and taller tails of the distribution), is consistent with a decreased regularity of pellet-to-pellet
feeding. Finally, a significant increase in the skewness (a measure of the distribution’s
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Iemolo et al., Anorectic effect of CeA PACAP
symmetry, reflected in a selective increase of the frequency of the inter food interval falling to
the left of the histogram) is consistent with a selective increase of the fast pellet-to-pellet
responding.
Return map analysis. Return maps, or joint inter-event interval plots, are a nonlinear
method of time series analysis that reveals the serial temporal organization of discrete events in
time (Dekhuijzen and Bagust, 1996; Szucs et al, 2003). For return map analysis, each IFI in the
time-series was scatterplotted against its subsequent inter-feeding interval (IFI+1) in a Cartesian
plane. The sampling resolution of the probability density distribution was e0.05 sec. In such an
analysis, increased irregularity of serial events is observed as an increased spread/diffuseness,
of the return map, whereas regular and rhythmic events are observed as densely focused
clusters of points. Joint probability density distributions were constructed from return maps and
appear in gray scale format with darker shades indicating higher local probabilities (a colorcoded form with warmer colors -toward red- indicating higher local probabilities in the color
figure).
Motor Activity
Motor activity of rats in a familiar environment (home cage) was measured as described
in Dore et al (2013) using an Opto-M3 activity system (Columbus Instruments, Columbus, OH);
activity was recorded by a computer using the Multi Device Interface software over a 12h period
(dark cycle). Food, water and white noise were present.
Statistical analysis
All studies were performed within-subject. PACAP dose-response curve data at 1h, 3h
and 6h were analyzed using two-way repeated measure analyses of variance (ANOVAs); 24h
food intake and body change were analyzed using one-way repeated measure ANOVAs. Motor
activity data were analyzed using a two-way repeated measure ANOVA and meal
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Iemolo et al., Anorectic effect of CeA PACAP
microstructure analyses were performed using Wilcoxon matched pairs tests. Food intake and
body weight data from the SHU 9119, k252a and D-Phe-CRF(12-41) blockade experiments
were analyzed using two-way repeated measure ANOVAs. Pairwise post-hoc comparisons
were made using Newman-Keuls test. Significance was set at p≤0.05. The software/graphic
packages used were Systat 11.0, SigmaPlot 11.0, InStat 3.0, and Statistica 7.0.
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Iemolo et al., Anorectic effect of CeA PACAP
Supplemental Figure 1 Legend
Drawing of coronal rats’ brain slices. Dots represent the injection sites in the CeA (A),
and BlA (B) included in the data analysis.
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Iemolo et al., Anorectic effect of CeA PACAP
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