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Vollum Institute (C.H.-L., R.D.C.), Oregon Regional Primate Research Center (P.C., C.L., M.S.S., J.C.) Oregon Health Sciences University, Portland, Oregon 97201; and Phoenix Pharmaceuticals, Inc. (K.C.), Mountain View, California 94043
Address all correspondence and requests for reprints to: Dr. Roger D. Cone, Vollum Institute L-474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: cone{at}ohsu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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-MSH, ß-MSH, 
-MSH, and ACTH), is expressed in the
brain exclusively in neuronal cell bodies throughout the rostrocaudal
extent of the arcuate nucleus (ARC) and periarcuate area of the
hypothalamus and within the caudal half of the commissural nucleus of
the solitary tract (1, 2, 3, 4, 5). Proceeding from rostral to caudal, arcuate
POMC cells send a dense bundle of fibers ventral to the anterior
commissure, to a number of nuclei in the septal region, including the
bed nucleus of the stria terminalis and lateral septal nucleus (LS), as
well as to the nucleus accumbens in the caudate putamen. More caudally,
fibers are seen projecting to the periventricular region of the
thalamus and to the medial amygdala. Within the hypothalamus, the
densest fibers project to the periventricular nucleus, the
paraventricular nucleus (PVH), and the perifornical region, with some
fibers seen in almost all hypothalamic regions. Finally, arcuate POMC
fibers also project to several regions of the brain stem, including the
periaquaductal gray, reticular formation, and parabrachial nucleus.
In situ binding studies using
[125I]Nle4,D-Phe7--MSH
have demonstrated that -MSH binding sites correlate well with POMC
terminal fields in the forebrain (6) and brain stem (7). Furthermore,
the distribution of expression of the MC3-R and MC4-R in the rat
central nervous system (CNS), determined by in situ
hybridization (8, 9, 10, 11), can easily account for the majority, if not all,
of the melanocortin binding demonstrated in the CNS.
The central melanocortin system, as defined above by POMC neurons and the central MC3-R and MC4-R receptors, was recently given an added level of complexity by the discovery of agouti-related protein (AGRP) (12, 13). This 132-amino acid peptide is a homolog of the skin agouti peptide. The skin agouti peptide has previously been demonstrated to be an antagonist of the melanocortin-1 receptor (14) on melanocytes, where it is involved in the inhibition of eumelanin, or brown-black pigment, synthesis. The existence of a brain-specific agouti was suggested by the observation that the skin agouti peptide was also a high affinity antagonist of the neural MC4-R receptor (14) and caused an obesity syndrome when expressed ectopically in mouse strains containing certain dominant alleles of the gene (15, 16, 17). This obesity syndrome appears to result specifically from blockade of MC4-R signaling, as shown both pharmacologically and by gene knockout of the MC4-R (18, 19), suggesting that one function of POMC neurons involves the control of energy homeostasis.
In addition to being expressed in the arcuate nucleus, AGRP is also a potent antagonist of both the MC3-R and MC4-R (13, 20). As might be predicted, ubiquitous expression of a ß-actin promoter/AGRP gene in transgenic mice (13, 21) results in an obesity syndrome comparable to that seen in mice with dominant agouti alleles such as AVY (22) and in MC4-RKO mice (19). POMC neurons and the MC4-R have been demonstrated to be involved in a number of processes other than energy homeostasis, however, including grooming behavior (23), control of fever (24), and cardiovascular homeostasis (25). Thus, determination of the potential physiological roles for AGRP requires characterization of the distribution of this protein in the CNS to identify MC3-R- and MC4-R-containing sites that may be coregulated by melanocortin agonists and the AGRP antagonist. Using a specific antibody to the C-terminal portion of this protein, we show here the distribution of AGRP immunoreactivity in the rat and rhesus monkey.
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Materials and Methods |
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Perfusion and tissue sectioning: Rats were anesthetized with a lethal dose of pentobarbital (125 mg/kg BW, ip) and perfused transcardially with 150 ml 2% sodium nitrite in saline followed by 150 ml 2.5% acrolein (EM grade, Polysciences, Warrington, PA) in phosphate-buffered 4% paraformaldehyde (pH 7.4). The brain was removed, blocked, and immersed in 25% sucrose at 4 C overnight. Coronal sections (25 µm) were cut through the whole brain on a sliding microtome and collected in a one in six series. The tissue sections were stored at -20 C in multiwell tissue culture plates containing cryoprotectant until use.
Rhesus monkeys. Hypothalamic and brain stem tissues from adult male rhesus monkeys (Macaca mulatta), weighing 8.9–10.2 kg, were used for this study. Monkeys were maintained in single animal cages in a room with a controlled lighting schedule (lights on, 0700–1900 h) and were fed one meal a day at approximately 1100 h of about 600 Cal high protein monkey chow (Ralston Purina Co., St. Louis, MO), with water available at all times. At least 1 month before death, monkeys had indwelling iv and intragastric catheters implanted using sterile surgical techniques, as previously described (26, 27). Catheters exited the body at the midscapular region of the back, and monkeys were maintained in jacket/tether/swivel systems (27), with the catheters running from the top of the swivel through a hole in the wall behind the monkey’s cage to enter an adjacent room. Catheter patency was maintained by a constant saline infusion through the catheters of 100 ml/day, with the venous drip containing 400 U sodium heparin. Before death, all monkeys had been fasted at least one time and had received at least one meal via the gastric cannula, and blood samples had been collected and assayed for insulin and T3 to ensure that the metabolic changes expected to occur with fasting and feeding were detectable. This study was approved by the institutional animal care and use committee of the University of Pittsburgh.
Perfusion and tissue sectioning: Monkeys were fasted the day
before death, and on the morning of death a fed monkey received an
infusion of 200 ml nutrients (containing 600 Cal, with the same
percentage of protein, carbohydrate, and fat as the standard monkey
chow) (26) via the gastric cannula from 0630–0730 h, whereas the
fasted animal did not. At 1025 h monkeys were sedated with
ketamine HCl (10 mg/kg, im), and deeply anesthetized with sodium
pentobarbital (
15 mg/kg, iv). The chest was then opened by a midline
incision, and each monkey was perfused transcardially with 0.9% NaCl
containing 2% sodium nitrite (800–1000 ml) to flush blood from the
vascular system, followed by 4% paraformaldehyde in 0.1 M
potassium phosphate buffer solution (KPBS; pH 6.8–7.2; 1000–1200 ml).
The brain and upper portion of the spinal cord were removed,
hypothalamic and brain stem tissue blocks were cut, and blocks were
immersed in a postperfusion fixative of 2.5% acrolein and 4%
paraformaldehyde-KPBS solution for 2 h at 25 C, followed by
placement in a 25% sucrose solution in distilled water at 4 C for 4–6
days until they sunk to the bottom of the sucrose solution. Fresh
sucrose solution was replaced daily. The hypothalamus was sectioned
(30-µm sections) on a coronal plane using a freezing microtome, and
sections were stored in cryoprotectant at -20 C until
immunocytochemical and in situ hybridization procedures were
performed. Due to the limited number of animals available for this
study, the data presented here result from analysis of one fasted and
one fed rhesus monkey.
Immunohistochemistry
Both monkey and rat sections were processed at the same time to
ensure uniformity of immunostaining. Tissue sections were removed from
cryoprotectant and rinsed in 0.05 M KPBS followed by
treatment with 1% NaBH4-KPBS solution (Sigma Chemical Co., St. Louis, MO). Sections were incubated in rabbit
anti-AGRP antibody (G-003–53; 1:35,000; Phoenix Pharmaceuticals, Inc., Mountain View, CA) in KPBS with 0.4% Triton X-100 at 4 C
for 48 h. Control sections were preabsorbed with 20 µg
AGRP-(83–132) peptide to 1 µg antibody to test the specificity of
the antibody. After rinsing in KPBS, the sections were incubated for
1 h at room temperature in biotinylated goat antirabbit IgG
(1:600, Vector Laboratories, Inc., Burlingame, CA),
followed by 10-min incubation in tyramide signal amplification solution
according to the manufacturer’s instruction (TSA-Indirect kit, New
England Nuclear Life Science, Boston, MA). The antibody-peroxidase
complex was stained with a mixture of 3,3'-diaminobenzidine and
H2O2 in 0.05 M Tris buffer-saline
solution. After the staining, tissue sections were mounted on
gelatin-coated glass slides and air-dried. The 3,3'-diaminobenzidine
staining on the tissue sections was further enhanced by osmium
tetroxide (OsO4), as described previously (28), before
being coverslipped with Histomount (Fisher Scientific,
Pittsburgh, PA).
In situ hybridization
Monkey brain. A 399-bp human AGRP complementary DNA was
generated by PCR and subcloned into the pBSII SK(±) vector
(Stratagene, La Jolla, CA). The human (h) AGRP
complementary RNA (cRNA) probe was transcribed from the vector with
40% of the UTP provided in a 33P-labeled form (New England
Nuclear Life Science, Boston, MA) (29). The specific activity of the
probe ranged from 1–2 x 109 dpm/µg. Brain sections
were rinsed in PBS and treated with 1% NaBH4-PBS solution.
After washing with 0.25% acetic anhydride in 0.1 M
triethanolamine (pH 8.0) and in 2 x SSC, sections were incubated
in prehybridization solution containing 50% formamide, 6.25% dextran
sulfate, 0.7% Ficoll, 0.7% polyvinyl pyrolidone, and 2 mg/ml yeast
transfer RNA for 2 h at 55 C. Subsequently, sections were
incubated in the same solution, with the addition of 1.5 x
105 cpm/µl antisense (or sense) cRNA probe for 15 h
at 55 C. After hybridization, sections were washed in 4 x SSC
(standard saline citrate), digested with ribonuclease A for 30 min at
37 C, and washed through decreasing concentrations of SSC to a final
stringency of 0.1 x SSC at 60 C for 30 min. Sections were mounted
on gelatin-coated glass slides and allowed to dry. The slides were
dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester,
NY), exposed for 7–8 days at 4 C, and developed. After development,
the slides were dehydrated, counterstained with cresyl violet, and
coverslipped in Histomount.
Rat brain. Human AGRP cRNA probe was synthesized and used for in situ hybridization as described above. Briefly, the hAGRP cRNA probe was transcribed from a 399-bp complementary DNA in which 40% of the UTP was 35S labeled (New England Nuclear Life Science, Boston, MA). The saturating concentration of the probe used in the assay was 1.0 µg/ml. The specific activity of the probe ranged from 8–9 x 108 dpm/µg. In situ hybridization was performed as described previously (29). Frozen brain sections (20 µM) were fixed in 4% paraformaldehyde and treated with a fresh solution containing 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), followed by a rinse in 2 x SSC, dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air-dried. The slides were exposed to the hAGRP cRNA probe overnight in moist chambers at 55 C. After incubation, the slides were washed in ribonuclease-containing SSC that increased in stringency up to a wash in 0.1 x SSC at 60 C, then dehydrated through a graded series of alcohols and air-dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 7–8 days at 4 C, and developed. After development, the slides were coverslipped with Histomount.
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Results |
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). AGRP-positive signals in the rhesus
were detected in the comparable structure in the primate, known as the
infundibular nucleus, in the fasted animal (Fig. 1b). No signal was
detected in tissue from the fed animal (not shown).
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, a and b). The specificity of the
staining was confirmed by preabsorbing the hAGRP antibody with the
peptide corresponding to the C-terminal residues 83–132 before the
antibody was incubated with tissue sections (Fig. 2c). Dense AGRP
fibers were found projecting from the arcuate to only two divisions of
the neuraxis, the hypothalamus and the septal region. In the
hypothalamus, the densest fiber staining was observed originating in
the arcuate and proceeding along the third ventricle as well as in the
anterioperiventricular preoptic area, the periventricular nucleus, the
parvocellular portion of the PVH, the dorsomedial nucleus (DMH), and
the rostral end of the posterior nucleus (PH). Dense fiber staining was
seen throughout the lateral and medial ARC. Scattered fiber staining
was observed in the anterior and lateral hypothalamus (data not shown).
Hypothalamic areas devoid of AGRP-positive fibers include the
magnocellular portion of the PVH, the supraoptic nucleus, the
suprachiasmatic nucleus (SCN), the ventromedial nucleus, and the
compact zone of the DMH. The main extrahypothalamic projections led to
the bed nucleus of the stria terminalis and the ventral part of the LS.
Scattered fiber staining was also observed in the paraventricular
nucleus of thalamus. No fibers were detected in the corpus striatum or
the rostral portion of the brain stem. The caudal portion of the rat
brain stem was not examined. Table 1
summarizes the regions in the CNS of the diestrous rat containing AGRP
immunoreactivity.
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) and more caudally in the DMH (Fig. 3b). Also, as in the rat, a dense caudal fiber bundle was found in the
septal region, predominantly in the stria terminalis (Fig. 4a)
and LS region (Fig. 4b), passing
through the medial preoptic area (Fig. 4c). In the rhesus, very dense
fiber staining could be seen in the infundibulum (equivalent to the
rodent median eminence, Fig. 4d), and cell bodies could be detected in
the infundibular nucleus (Fig. 4e). Table 2 summarizes the AGRP-immunoreactive
fibers distributed throughout the hypothalamus of the fasted male
rhesus monkey. AGRP antiserum preabsorbed with control peptide,
corresponding to the amino acids 83–132 mapping to the C-terminus of
hAGRP, abolished all immunoreactivity (data not shown).
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Discussion |
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). Within the rat
hypothalamus, POMC fibers have a much wider distribution than AGRP,
with moderate fiber density seen in just about every nucleus, with the
possible exceptions of the VMH and supraoptic nucleus. A notable lack
of AGRP fibers was observed in the VMH. These initial studies will, of
course, need to be tempered by studies using additional antibodies, as
it is possible that smaller amounts of immunoreactivity may be below
the sensitivity of detection here, that the carboxyl-terminal epitope
is specifically blocked in some brain regions, or that the
immunoreactive epitope is unrecognizable in some brain regions as a
consequence of proteolytic processing. Additionally, although the
distributions reported here in the diestrous rat and fasted male rhesus
are highly conserved, it will be important to determine whether the
same distribution of immunoreactivity in the diestrous rat is seen in
the fasted rat.
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-binding sites in the infundibulum proper, as high levels of
[125I]NDP--MSH binding are observed in the rat median
eminence (6) and thus may exist in the primate as well.
The overall distribution of AGRP-immunoreactive fibers represents a
subset of regions that contain POMC projections and express MC3-R and
MC4-R receptor mRNA hybridization (8, 9). These data thus further
support the general hypothesis that AGRP serves as a locally delivered
functional antagonist of the MC3-R and MC4-R (8, 9, 34). Although MC4-R
is very widely distributed in the brain and has been demonstrated to be
involved in feeding behavior and metabolism (18, 19), control of
somatic growth (19), grooming behavior (23), control of the
hypothalamic-pituitary-adrenal axis (23), febrile responses (24), and
cardiovascular homeostasis (25), the data shown here suggest that AGRP
may serve as a counterregulatory force in a more limited subset of
these functions. In particular, the dense expression of AGRP in the PVH
and DMH strongly imply a role for the peptide in energy homeostasis, as
further supported by the observation that AGRP mRNA levels are
regulated by leptin. In fact, preliminary data suggest that the
melanocortin antagonist AGRP is much more robustly regulated by
metabolic state than is the source of melanocortin agonist, POMC. For
example, AGRP mRNA is up-regulated 5- to 10-fold in the
ob/ob mouse (12, 13) compared with the 40–60%
down-regulation reported for POMC mRNA in this model (35, 36). It is
thus tempting to speculate that the main site for hormonal input to the
melanocortin system in regard to regulation of energy homeostasis is at
the AGRP neuron rather than the POMC neuron. A comparable example is
seen in the pigmentary system, in which variable levels of agouti gene
expression in the hair follicle act to modulate the effects of
constitutive -MSH on eumelanin synthesis in the melanocyte.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this manuscript.
Received August 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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-Melanocyte
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brainstem baroreceptor areas of the rat. Brain Res 436:323–328[CrossRef][Medline]
-melanocyte stimulating hormone. Brain Res 536:124–132[CrossRef][Medline]
-MSH and other proopiomelanocortin
peptides in the hypothalamus and limbic system. Proc Natl Acad Sci USA 90:8856–8860- and
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