www.elsevier.com/locate/brainres

Brain Research 1020

Research report

Amygdala enterostatin induces c-Fos expression in regions of

hypothalamus that innervate the PVN

Ling Lin*, David A. York

Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808, USA

Accepted 15 June 2004

Available online 21 July 2004

Abstract

Enterostatin selectively inhibits the intake of the dietary fat after both central and peripheral administration. Our previous studies have

shown that a central site of action is the central nucleus of amygdala. Serotonergic agonists administered into the paraventricular nucleus

(PVN) inhibit fat intake and serotonergic antagonists block the feeding suppression induced by amygdala enterostatin, suggesting that there

are functional connections between the PVN and amygdala that affect the feeding response to enterostatin. Our purpose was to identify the

anatomic and functional projections from the amygdala to the PVN and hypothalamic area that are responsive to enterostatin, by using a

retrograde tracer fluorogold (FG) and c-Fos expression. Rats were injected with fluorogold unilaterally into the PVN and a chronic amygdala

cannula was implanted ipsilaterally. After 10 days recovery, rats were injected with either enterostatin (0.1 nmol) or saline vehicle (0.1 Al)into the amygdala and sacrificed 2 h later by cardiac perfusion under anesthesia. The brains were subjected to dual immunohistochemistry to

visualize both FG and c-Fos-positive cells. FG/c-Fos double-labeled cells were found in forebrain regions including the PVN, amygdala,

lateral hypothalamus (LH), ventral medial hypothalamus (VMH) and arcuate nucleus (ARC). The data provides the first anatomical evidence

that enterostatin activates amygdala neurons that have functional and anatomic projections directly to the PVN and also activates neurons in

the arcuate, LH and VMH, which innervate the PVN.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Enterostatin; Amygdala; PVN; Retrograde tracer; c-Fos

1. Introduction

Enterostatin, the N-terminal pentapeptide derived from

the procolipase precursor protein, selectively inhibits the

intake of dietary fat in rodent models [4,13–16,18,28]. The

procolipase gene is expressed in the exocrine pancreas, the

stomach, the duodenal mucosa [20] and in specific brain

regions [17]. Enterostatin-like immunoreactivity has been

identified at similar locations suggesting that procolipase

is processed to colipase and enterostatin in these other

sites in addition to the exocrine pancreas and gastric

mucosa [29,30]. The response to enterostatin is dependent

upon the previous ingestion of dietary fat. Diet-switch

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.06.029

* Corresponding author. Tel.: +1 225 763 2559; fax: +1 225 763 2525.

E-mail address: linl@pbrc.edu (L. Lin).

studies suggest that there is an adaptive period of fat

ingestion before the response to enterostatin becomes

evident [11].

Like other gut peptides, enterostatin appears to have

both a peripheral and a central site of action. Peripherally,

it appears that enterostatin acts within the gastroduodenal

region to activate vagal fibers that communicate with the

central nervous system to affect food intake [16].

Centrally, intracerebroventriclular injections of enterostatin

and microinjection of enterostatin into the paraventricular

nucleus (PVN) and central nucleus of the amygdala all

suppress feeding [9,10,14]. However, the dose responses

and time courses suggest that the amygdala is the central

site of action of enterostatin [9]. The feeding response to

enterostatin is modulated through a pathway that involves

paraventricular serotonergic activity since a serotonergic

antagonist injected into the PVN blocked the feeding

(2004) 147–153

L. Lin, D.A. York / Brain Research 1020 (2004) 147–153148

suppression induced by injection of enterostatin into the

amygdala [12,33]. Peripheral injection of enterostatin

increased c-Fos expression in the brain nuclei, such as

the PVN, suprachiasmatic nucleus, lateral parabrachial

nucleus and nucleus tractus solitarius [32]. To date, the

central neuronal pathways involved in amygdala enter-

ostatin signaling have not been determined. It is well

known that the hypothalamus is the primary locus for

integration of signals that influence appetite and energy

expenditure. There may be anatomical and functional

connections between the amygdala and hypothalamus

involved in the response to enterostatin. The objective

of this study was to identify the neuronal populations of

the hypothalamus in response to the injection of enter-

ostatin into the amygdala. This was accomplished by

combining c-Fos expression with a neuroanatomical

retrograde tracer fluorogold (FG) [24]. c-Fos is an

immediate early gene protein product and a marker for

neuronal activation which is widely used as a functional

anatomical mapping tool to identify cells and extended

circuitries that respond to various stimuli [8]. Injection of

the retrograde tracer FG into the PVN was based on

previous observation that enterostatin induced the c-Fos

expression in this area [32].

2. Materials and methods

2.1. Animals

Male Sprague–Dawley rats (Harlan–Sprague–Dawley,

Indianapolis, IN, USA, weighing 300 g, n=12) were used

for this study. Rats were housed individually in a humidity

and temperature controlled (20–22 8C) room with lights on

from 07:00 to 19:00 h. They had free access to water and a

high fat diet (4.78 kcal/g) as described previously [14]. The

protocol was approved by the Institutional Animal Care and

Use Committee (IACUC).

2.2. Retrograde tracer injections

Rats were anesthetized with a mixture of ketamine (80

mg/ml), ace-promazine (1.6 mg/ml) and xylazine (5 mg/

ml) (1.25 ml/kg body weight, subcutaneous injection) and

placed in a stereotaxic frame with the incisor bar 3.3 mm

below the intraaural line. A 33-gauge (OD: 0.2 mm (0.008

in.)) stainless steel injector connected with a polyethylene

tubing (PE-10) was inserted into the PVN unilaterally. The

coordinates of the PVN (referred to bregma) were: AP:

�1.9 mm, L: �0.4 mm, DV: �8 mm [21]. FG

(Fluorochrome, Denver, CO) was dissolved in 0.9% (w/

v) sterile saline as a 2% (w/v) solution. The FG (0.1 Al)was delivered into the PVN from a 0.5-Al Hamilton

syringe over a 30-s period. The injector was left in situ for

an additional 5 min to ensure maximum delivery and

prevent any back-leakage.

2.3. Chronic amygdala cannula

Immediately after the FG injection, a stainless steel

cannula (24-gauge, 17.5 mm long; Plastic One, Roanoke,

VA) was implanted ipsilaterally to the central nucleus of the

amygdala. The coordinates were: AP: �2.4 mm, L: �3.8

mm, DV: �6 mm to bregma [21]. The cannula was secured

in place with 3 anchor screws and dental acrylic and

occluded with a 31-gauge stylet. The animals were returned

to the home cages after recovery from the anesthesia and

maintained on the diet for 10 days to allow time for the

retrograde tracer to be transported throughout the brain, and

for rats to regain their preoperative weight.

2.4. Enterostatin administration into the central nucleus of

amygdala

This procedure was conducted on ad libitum fed and

freely moving rats in the early light period (08:00 h). Rats

were divided into two groups matched by their body weight

and received either vehicle (0.1 Al, 0.9% saline w/v) or

enterostatin (0.1 nmol in 0.1 Al vehicle) injection into the

amygdala over 30-s period through an injector (31-gauge)

that projected 2 mm beyond the guide cannula tip. Enter-

ostatin was synthesized by the Core Laboratory of the

Louisiana State University Medical Center (New Orleans,

LA). Rats were returned to their home cages and left

undisturbed with water but no food available. Two hours

after injection, rats were anesthetized with a mixture of

ketamine (80 mg/ml), ace-promazine (1.6 mg/ml) and

xylazine (5 mg/ml) (1.25 ml/kg body weight, subcutaneous

injection) and perfused transcardially with 100 ml hepari-

nized saline (20 U/ml) followed by fixative (300 ml ice-cold

4% paraformaldehyde in 0.1 M sodium phosphate buffer,

pH 7.4). The brains were removed and post-fixed overnight,

then blocked and cryoprotected in 25% sucrose.

2.5. Immunocytochemistry

The tissue blocks were embedded in O.C.T. compound

(Miles Elkhart, IN). Coronal (30 Am) sections were cut on a

cryostat and collected serially in five sets in multiwell

culture plates with cryoprotectant (5 mM phosphate-

buffered saline (PBS), pH 7.3, 30% ethylene glycol and

20% glycerol) and stored at �20 8C until further processing.

A sixth set of sections was thaw-mounted on glass slides to

visualize FG fluorescence (wavelength of excitation: 323

nm and emission: 620 nm). Only animals with FG

fluorescence limited to one side of the PVN (10 rats) were

used for further processing as described below. One set of

sections were removed from cryprotectant and rinsed in 0.01

M PBS, pH 7.3 prior to immunocytochemical procedures.

The sections were pre-treated with 1% NaBH4 for 30 min to

reduce any remaining fixative, and a solution of 1.5%

hydrogen peroxide, 20% methanol and 0.5 % Triton X-100

for 30 min to inactivate endogenous peroxidase. Tissue

L. Lin, D.A. York / Brain Research 1020 (2004) 147–153 149

sections were preincubated for 2 h in 5% normal goat serum

plus 1% of bovine serum albumin, 0.5% Triton X in PBS to

block non-specific binding of the primary antibody, then

incubated with a rabbit anti-cFos (1:30,000, Ab-5, Onco-

gene Research Products, San Diego, CA) overnight with

gentle agitation. After four rinses, sections were incubated

with a biotinylated secondary antibody (1:500, goat anti-

rabbit immunoglobulin G, Vector Lab, Burlingham, CA),

followed by reaction with an avidin-biotin complex (Vec-

tastain Elite ABC kit, Vector Lab). The antibody peroxidase

complex was visualized with a metal-enhanced DAB

substrate kit (0.5% 3.3V-diaminobenzidine tetrahydrochlor-

ide, 1% cobalt chloride and nickel chloride with stable

hydrogen peroxide; Pierce Chemical, Rockford, IL) for 5–

10 min to generate a blue-black c-Fos nuclear product. The

c-Fos-labeled sections were subsequently processed for

localization of FG using a FG antibody (1:35,000, Fluo-

rochrome). The remainder of the process was similar to that

described above. The DAB without metal was used to

produce a brown staining FG product, which was present in

the cytoplasm, axons and dendrites of the neurons. Brain

sections were mounted on microscope glass slides, air-dried,

dehydrated, cleared in xylene and cover slipped with

mounting medium.

2.6. Data analysis

Slides were observed under a ZeissAxiophat micro-

scope. The areas of interest were central nucleus of the

amygdala, PVN, lateral hypothalamus (LH), ventromedial

nucleus (VMH), arcuate nucleus (ARC) and mammillary

Fig. 1. The tracts of the PVN injector and amygdala cannula in rat brain diagrams

(bregma: �2.30 mm, B) [21]. Arrows in the photographs show the injector tract t

The photomicrographs at bottom show the higher power of the FG staining in P

amygdala (E, indicated by an arrow).

nucleus. Pictures were taken by a digital camera using the

computer software Spot Advance program (Diagnostic

Instruments, Sterling Heights, MI). Stained neurons in an

area of interest were counted using Image-Pro Plus software

(version 4.1, Media Cybernetics, Silver Spring, MD). All

sections were examined but counts were only performed on

the sections from three representative animals in each

treatment group. Counts from individual animal are the

average count of two adjacent sections. Data are expressed

as meansFstandard error by each treatment group. The

difference of treatment groups were compared using two

tailed Student’s t-test.

3. Results

3.1. Verification of injection sites

The site of FG injection to the PVN and cannula

placement into amygdala are shown in Fig. 1A,B. FG-

labeled cells in the region of the PVN were predominately

located ipsilateral to the site of injection (Fig. 1C,D) with

only a few scattered cells on the contralateral side. Chronic

cannulas towards the amygdala for enterostatin injection

were located just dorsal to the central nucleus of the

amygdala (Fig. 1B,E).

3.2. Fluorogold in rat forebrain

FG is a retrograde tracer and is incorporated into the cell

axons and then carried back to the parent cell body. In the

illustrate the PVN (bregam: �1.80 mm, A) and central nucleus of amygdala

o PVN (A) and cannula tract towards the amygdala (B) in coronal sections.

VN by fluorescent (C) and DAB (D) and the tip of the cannula above the

L. Lin, D.A. York / Brain Research 1020 (2004) 147–153150

current study, neurons containing FG were identified by

brown staining that resulted from DAB color reaction with

antibody peroxidase complex. This color indicates the

presence of the FG tracer and identifies the neurons with

projections to PVN. Ten days after unilateral injection of FG

Fig. 2. Photomicrographs showing fluorogold-positive cells (brown color)

in the forebrain. Left panel: schematic brain sections. The coordinates are

referred to Bregam [21]: (A) LH, (B) VMH, (C) central nucleus of the

amygdala (AMYG), (D) ARC, (E) premammillary nucleus, ventral part

(PMV), (F) lateral mammillary nucleus (LM) and medial mammillary

nucleus, median part (MM), (G) cortex and ependymal layer of third

ventricle. Middle panel: 10� photomicroimages correspond to the left

panel’s illustrations. Right panel: higher power photomicrographs (40�) of

the boxed area in the middle panel, respectively, except very bottom row

that shows the cortex and ventricle.

into the PVN, cells containing FG were found throughout

the forebrain with brown staining in cytoplasm, axons and

dendrites. Fig. 2 shows photomicrographs of coronal

sections from the bregma �1.8 to �4.52 mm. In the

forebrain region, most of the FG positive cells were found in

the hypothalamus, LH (Fig. 2A), VMH (Fig. 2B), ARC

(Fig. 2D) and ependymal layer of third ventricle (Fig. 2G).

Outside of the hypothalamus, scattered FG cells were

observed in the central nucleus of amygdala (Fig. 2C). In

addition, a high density of the FG cells were present both

ipsilateraly and contralaterally in the mammillary nucleus,

which are a caudal portion of the hypothalamus(Fig. 2E and

F). In contrast, the cortex had no FG-positive cells (Fig.

2G), which indicates that there is no direct anatomical

connection between the cortex and PVN.

3.3. Co-localization of fluorogold and c-Fos

c-Fos, an immediate early gene protein product, is a

marker for neuronal activation. c-Fos staining was limited to

neuronal nuclei and visible as black staining, round or oval

in appearance. Two hours after injection of enterostatin (0.1

nmol) into the amygdala, c-Fos expression was observed in

several regions of the brain that are involved in appetite

control, including the LH, VMH, ARC, PVN and amygdala,

indicating that those areas were activated in response to

enterostatin (Fig. 3). Fig. 3 also shows FG/c-Fos double-

labeled cells in those areas. There was a high density of FG/

c-Fos-labeled cells in the LH, VMH and PVN areas and

scattered double-labeled cells in the amygdala and arcuate

nucleus (Fig. 3, right panels). Only a few c-Fos or FG/c-Fos

double-labeled cells were found in saline vehicle-treated

animals (Fig. 3, left panel). In contrast, the cortex area had a

high number of the c-Fos positive cells but no FG-labeled

cells, whereas the piriform cortex had both high density of

single-labeled c-Fos and FG cells without colocalization

(data not shown). A summary of FG/c-Fos double-labeling

is provided in Table 1.

4. Discussion

The present study provides the first evidence of a link

between the amygdala and hypothalamus, through which

enterostatin acts on the neurons in amygdala to affect

neuronal activity in the PVN. In addition, the data show that

enterostatin also activates LH, ARC and VMH areas that

have direct innervations to the PVN. It suggests that

enterostatin may act in the amygdala to convey information

to the PVN via both direct and indirect neural pathways.

Together, these neural populations may be important in

regulating the inhibitory effect of enterostatin on feeding

and the ingestion of dietary fat.

In the present study, the majority of FG/c-Fos double-

labeled cells were found in the PVN, LH and ARC, all

regions recognized to have a role in the regulation of

Fig. 3. Colocalization of retrograde-labeled FG and c-Fos expression after enterostatin injected into central nucleus of the amygdala (AMYG). Left panel: saline

control; right panel: enterostatin injection in amygdala. The top row of the photos show the example the FG, c-Fos and double-labeled neuron as indicated in

the figure. There are more c-Fos-positive cells (dark, round nuclear label) in PVN, VMH, ARC, LH and AMYG areas with enterostatin treatment. Some of

those cells are surrounded by the brown cytoplasm or fiber staining (FG). All of the photos are 40�.

L. Lin, D.A. York / Brain Research 1020 (2004) 147–153 151

appetite and energy balance. Our previous study suggested

that the response to enterostatin is modulated through

functional connections between the amygdala and PVN

since a serotonergic antagonist into PVN reversed the

feeding suppression induced by injection of enterostatin

into the amygdala [12]. The data presented here clearly

demonstrate this anatomic connection with c-Fos-labeled

cells in the amygdala also containing FG retrogradely

transported from the PVN. In addition to the double-labeled

cells, FG- or c-Fos-single-labeled neurons were found in the

forebrain areas. In the current study, they either represent the

direct anatomic inputs to the PVN (FG labeling) or the

functional outputs from the amygdala (c-Fos staining),

whereas co-localization of both the FG and c-Fos identifies

which of the neuronal populations activated by the enter-

ostatin stimulus to amygdala project to the PVN. This study

Table 1

FG/c-Fos positive cells in brain areas after enterostatin injection into

amygdala

Areas Treatment Neurons labeled

with c-Fos only

Neurons labeled with

c-Fos and fluorogold

LH Saline 34F1 5F1

Enterostatin 98F9** 57F4**

VMH Saline 15F2 4F0

Enterostatin 71F33* 55F26*

PVN Saline 19F7 6F2

Enterostatin 99F34* 74F24*

ARC Saline 23F6 4F1

Enterostatin 140F48* 80F20*

AMYG Saline 26F5 2F0

Enterostatin 86F26* 24F7*

ARC: arcuate nucleus, AMYG: central bed of amygdala nucleus, PVN:

paraventricular nucleus of hypothalamus, VMH: ventromedial nucleus of

hypothalamus, LH: lateral hypothalamus.

* pb0.05 compared to the saline treatment group, respectively.

** pb0.01 compared to the saline treatment group, respectively.

L. Lin, D.A. York / Brain Research 1020 (2004) 147–153152

was focused on the co-localization that gives more precise

functional and anatomical pathways.

The central nucleus of the amygdala has complex

functions that may include not only behavioral roles but

also autonomic and neuroendocrine regulation. Likewise,

we have also shown that centrally injected enterostatin has

multiple effects in addition to those on feeding behavior,

including inhibition of insulin secretion, activation of

sympathetic drive to brown adipose tissue, regulation of

energy balance and bodyweight, and effects on the

hypothalamic–pituitary adrenal axis [14,19]. It is unlikely

that the c-Fos expression induced by amygdala enterostatin

is only limited to pathways affecting feeding. Thus, while

there is pharmacological evidence that the amygdala–PVN

pathway affected the enterostatin feeding response, it is

possible that the double-labeled cFos-FG cells identified in

other regions might be more involved in the autonomic and

endocrine responses to enterostatin.

It is interesting to note that a high density of FG was

present in the mammillary nucleus both ipsilateraly and

contralaterally. Although little is known about the function

of this nucleus in relation to appetite control and energy

balance, the presence of serotonergic axons, 5-HT receptors

and its mRNA, melanin-concentrating hormone (MCH)

fibers and leptin receptor mRNA in this nucleus [1,3,22,23]

might suggest a role in energy homeostasis.

c-Fos is a transcription factor and a functional marker of

activated neurons [8]. As an immediate-early gene, c-Fos is

the most widely used powerful tool to delineate individual

neurons as well as extended circuitries that are responsive to

wide variety of external stimuli. c-Fos protein is rapidly

induced by acute challenges and has a half life of

approximately 2 h. However, using c-Fos to map activated

neurons does not provide information on the transcriptional

consequence of this induction. Although significant c-Fos

expression was present in neurons of the LH, ARC and

VMH and PVN areas, the chemical phenotype of those cells

activated is not known yet. Neuropeptide Y (NPY), agouti-

related protein (AgRP), a-melanocyte-stimulating hormone

(aMSH), proopiomelanocortin (PMOC) and cocaine-

amphetamine-regulated transcript (CART) in the ARC and

orexin and MCH in the LH are known to influence feeding

behavior. In addition, adrenergic, dopaminergic, serotoner-

gic, histaminergic and GABAergic synaptic activity can

influence feeding [2,25]. AgRP and NPY, despite earlier

observations, have both been reported to have selective

effects on dietary fat intake when animals are allowed to

choose macronutrients [5,26,31]. Likewise, we have pre-

viously reported the presence of a serotonergic system

within the PVN that selectively attenuates the ingestion of

dietary fat and that serotonin inhibitions block the response

to enterostatin [12,27]. The recent demonstration of 5-HT

2C receptors on POMC neurons in the arcuate nucleus [6,7]

suggest that these are the potential target for afferent fibers

from the amygdala that are activated by enterostatin to

modulate the feeding response. This would be consistent

with previous behavioral studies and the current neuro-

anatomical data. The present study provides anatomical

evidence about the possible sites upon activation by the

amygdala enterostatin. An important next step is character-

ization of the phenotypes of the neurons in the areas

showing co-localization of the FG and c-Fos. It will

facilitate the understanding of the mechanism by which

the enterostatin stimulus activates the hypothalamic circuits.

References

[1] J.C. Bittencourt, L. Frigo, R.A. Rissman, C.A. Casatti, J.L. Nahon,

J.A. Bauer, The distribution of melanin-concentrating hormone in the

monkey brain (Cebus apella), Brain Res. 804 (1998) 140–143.

[2] J. Blundell, Pharmacological approaches to appetite suppression,

Trends Pharmacol. Sci. 12 (1991) 147–157.

[3] J.K. Elmquist, C. Bjorbaek, R.S. Ahima, J.S. Flier, C.B. Saper,

Distributions of leptin receptor mRNA isoforms in the rat brain,

J. Comp. Neurol. 395 (1998) 535–547.

[4] C. Erlanson-Albertsson, J. Mei, S. Okada, D. York, G.A. Bray,

Pancreatic procolipase propeptide, enterostatin, specifically inhibits

fat intake, Physiol. Behav. 49 (1991) 1191–1194.

[5] M.M. Hagan, P.A. Rushing, S.C. Benoit, S.C. Woods, R.J. Seeley,

Opioid receptor involvement in the effect of AgRP-(83–132) on food

intake and food selection, Am. J. Physiol., Regul. Integr. Comp.

Physiol. 280 (2001) R814–R821.

[6] L.K. Heisler, M.A. Cowley, L.H. Tecott, W. Fan, M.J. Low, J.L.

Smart, M. Rubinstein, J.B. Tatro, J.N. Marcus, H. Holstege, C.E. Lee,

R.D. Cone, J.K. Elmquist, Activation of central melanocortin path-

ways by fenfluramine, Science 297 (2002) 609–611.

[7] L.K. Heisler, M.A. Cowley, T. Kishi, L.H. Tecott, W. Fan, M.J. Low,

J.L. Smart, M. Rubinstein, J.B. Tatro, J.M. Zigman, R.D. Cone, J.K.

Elmquist, Central serotonin and melanocortin pathways regulating

energy homeostasis, Ann. N.Y. Acad. Sci. 994 (2003) 169–174.

[8] K.J. Kovacs, c-Fos as a transcription factor: a stressful (re)view from a

functional map, Neurochem. Int. 33 (1998) 287–297.

[9] L. Lin, D.A. York, Enterostatin actions in the amygdala and PVN to

suppress feeding in the rat, Peptides 18 (1997) 1341–1347.

[10] L. Lin, D.A. York, Changes in the microstructure of feeding after

administration of enterostatin into the paraventricular nucleus and the

amygdala, Peptides 19 (1998) 557–562.

L. Lin, D.A. York / Brain Research 1020 (2004) 147–153 153

[11] L. Lin, D.A. York, Chronic ingestion of dietary fat is a prerequisite for

inhibition of feeding by enterostatin, Am. J. Physiol. 275 (1998)

R619–R623.

[12] L. Lin, D.A. York, 5-HT 1b Antagonist in the PVN blocks the feeding

response to enterostatin in the amygdala, Obes. Res. 9 (2001) S56.

[13] L. Lin, S. Okada, D.A. York, G.A. Bray, Structural requirements for

the biological activity of enterostatin., Peptides 15 (1994) 849–854.

[14] L. Lin, J. Chen, D.A. York, Chronic ICV enterostatin preferentially

reduced fat intake and lowered body weight, Peptides 18 (1997)

657–661.

[15] L. Lin, M. Umahara, D.A. York, G.A. Bray, Beta-casomorphins

stimulate and enterostatin inhibits the intake of dietary fat in rats,

Peptides 19 (1998) 325–331.

[16] L. Lin, G.A. Bray, D.A. York, Enterostatin suppresses food intake in

rats after near-celiac and intracarotid arterial injection, Am. J. Physiol.,

Regul. Integr. Comp. Physiol. 278 (2000) R1346–R1351.

[17] L. Lin, H.D. Braymer, D.A. York, Procolipase gene and enterostatin

expression in the rat brain, FASEB J. (2002) A783.

[18] J. Mei, C. Erlanson-Albertsson, Role of intraduodenally administered

enterostatin in rats: inhibition of food, Obes. Res. 4 (1996) 161–165.

[19] H. Nagase, G.A. Bray, D.A. York, Effect of galanin and enterostatin

on sympathetic nerve activity to interscapular brown adipose tissue,

Brain Res. 709 (1996) 44–50.

[20] S. Okada, D.A. York, G.A. Bray, Procolipase mRNA: tissue

localization and effects of diet and adrenalectomy, Biochem. J. 292

(Pt. 3) (1993) 787–789.

[21] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 2nd

ed., Academic Press, Sydney, 1982.

[22] C.F. Phelix, D.M. Adai, C. Cantu, H. Chen, M.J. Wayner,

Immunohistochemical demonstration of serotonin-containing axons

in the hypothalamus of the white-footed mouse, Peromyscus

leucopus, Brain Res. 808 (1998) 197–219.

[23] M. Pompeiano, J.M. Palacios, G. Mengod, Distribution of the

serotonin 5-HT2 receptor family mRNAs: comparison between 5-

HT2A and 5-HT2C receptors, Brain Res. Mol. Brain Res. 23 (1994)

163–178.

[24] L.C. Schmued, J.H. Fallon, Fluoro-Gold: a new fluorescent retrograde

axonal tracer with numerous unique properties, Brain Res. 377 (1986)

147–154.

[25] M.W. Schwartz, S.C. Woods, D. Porte Jr., R.J. Seeley, D.G. Baskin,

Central nervous system control food intake, Nature 404 (2000)

661–671.

[26] B.K. Smith, H.R. Berthoud, D.A. York, G.A. Bray, Differential effects

of baseline macronutrient preferences on macronutrient selection after

galanin, NPY, and an overnight fast, Peptides 18 (1997) 207–211.

[27] B.K. Smith, D.A. York, G.A. Bray, Activation of hypothalamic

serotonin receptors reduced intake of dietary fat and protein but not

carbohydrate, Am. J. Physiol. 277 (1999) R802–R811.

[28] M. Sorhede, J. Mei, C. Erlanson-Albertsson, Enterostatin: a gut-brain

peptide regulating fat intake in rat, J. Physiol. (Paris) 87 (1993)

273–275.

[29] M. Sorhede, C. Erlanson-Albertsson, J. Mei, T. Nevalainen, A. Aho,

F. Sundler, Enterostatin in gut endocrine cells-immunocytochemical

evidence, Peptides 17 (1996) 609–614.

[30] M. Sorhede, H. Mulder, J. Mei, F. Sundler, C. Erlanson-Albertsson,

Procolipase is produced in the rat stomach—a novel source of

enterostatin, Biochim. Biophys. Acta 1301 (1996) 207–212.

[31] B.G. Stanley, K.C. Anderson, M.H. Grayson, S.F. Leibowitz,

Repeated hypothalamic stimulation with neuropeptide Y increases

daily carbohydrate and fat intake and body weight gain in female rats,

Physiol. Behav. 46 (1989) 173–177.

[32] Q. Tian, H. Nagase, D.A. York, G.A. Bray, Vagal-central nervous

system interactions modulate the feeding response to peripheral

enterostatin, Obes. Res. 2 (1994) 527–534.

[33] D.A. York, L. Lin, B.K. Smith, G.A. Bray, Enterostatin and 5HT:

modulation of fat intake, in: G.A. Bray, D.H. Ryan (Eds.), Nutrition,

genetics, and obesity, vol. 9, Louisiana State University Press, Baton

Rouge, 1999, pp. 246–266.