Hypothalamic Cocaine- and Amphetamine-Regulated Transcript (CART) and Agouti-Related Protein (AgRP) Neurons Coexpress the NOP1 Receptor and
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内分泌学杂志 2005年第8期
Department of Metabolic Medicine (G.A.B., W.S.D., S.J.D., K.G.M., J.V.G., P.H.J., W.M.K., M.A.G., S.R.B.), Division of Investigative Science, Hammersmith Campus, Imperial College London, London W12 0NN, United Kingdom
Address all correspondence and requests for reprints to: Professor S. R. Bloom, Department of Metabolic Medicine, Division of Investigative Science, Imperial College London, 6th Floor Commonwealth Building, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: s.bloom@imperial.ac.uk.
Abstract
Nociceptin or orphanin FQ (N/OFQ) and its receptor NOP1 are expressed in hypothalamic nuclei involved in energy homeostasis. N/OFQ administered by intracerebroventricular or arcuate nucleus (ARC) injection increases food intake in satiated rats. The mechanisms by which N/OFQ increases food intake are unknown. We hypothesized that N/OFQ may regulate hypothalamic neurons containing peptides involved in the control of food intake such as cocaine- and amphetamine-regulated transcript (CART), MSH, neuropeptide Y (NPY), and agouti-related protein (AgRP). We investigated the ability of N/OFQ to alter the release of CART, MSH, NPY, and AgRP using ex vivo medial basal hypothalamic explants. Incubation of hypothalamic explants with N/OFQ (1, 10, 100 nM) resulted in significant changes in CART and AgRP release. One hundred nanomoles N/OFQ caused a 33% decrease in release of CART (55–102) immunoreactivity (IR) and increased release of AgRP-IR to 163% but produced no change in either MSH-IR or NPY-IR. Double immunocytochemistry/in situ hybridization demonstrated that CART-IR and NOP1 mRNA are colocalized throughout the hypothalamus, in particular in the paraventricular nucleus, lateral hypothalamus, zona incerta, and ARC, providing an anatomical basis for N/OFQ action on CART release. Dual in situ hybridization demonstrated that AgRP neurons in the ARC also express the NOP1 receptor. Our data suggest that nociceptin via the NOP1 receptor may increase food intake by decreasing the release of the anorectic peptide CART and increasing the release of the orexigenic peptide AgRP.
Introduction
NOCICEPTIN/ORPHANIN FQ (N/OFQ) has structural homology to the opioid peptides, in particular dynorphin A. It was discovered to be the endogenous ligand for the orphan G protein-coupled receptor, NOP1, which is structurally similar to the -opioid receptor (1, 2, 3). Despite similarities between the two systems, N/OFQ lacks the N-terminal tyrosine required for activation of the opioid receptors (4). N/OFQ and NOP1 are widely distributed in the central nervous system (CNS). N/OFQ is involved in the modulation of pain (5), anxiety and stress (6), reward and dependence (7), memory and learning (8, 9), and hearing (10).
N/OFQ and NOP1 are present in hypothalamic nuclei that regulate the control of food intake, namely the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and the lateral hypothalamus (LH) (11, 12). Such a distribution suggested that N/OFQ might play a role in the hypothalamic regulation of food intake. Intracerebroventricular (icv) injection of N/OFQ dose dependently increased food intake in satiated rats over a 1- to 10-nmol range (13). N/OFQ-induced hyperphagia could be blocked by treatment with antisense oligonucleotides directed against each of the three exons of the NOP1 receptor (14). This suggests that the hyperphagic effect of N/OFQ is mediated through its action at the NOP1 receptor.
Several peptide agonists and antagonists of NOP1 have been synthesized. NC(1–13)NH2,[Nphe(1)]NC(1–13)NH2, a NOP1 antagonist, when injected icv almost completely abolished the orexigenic effect of icv N/OFQ. The same dose of this NOP1 antagonist administered icv to rats also significantly reduced feeding induced by food deprivation but not the hyperphagic effect of neuropeptide Y (NPY) (15). In addition, the selective Y1 receptor antagonist BIBO 3304 (16) markedly inhibited NPY-induced feeding but did not significantly modify food intake induced by N/OFQ when administered icv (15). This suggests that the N/OFQ system exerts its effects on food intake via pathways that do not involve the NPY system.
Microinjection studies have revealed the areas involved in mediating the orexigenic action of N/OFQ within the CNS. Intranuclear injections of N/OFQ at doses of 2.5–25 nmol into either the VMH or the shell of the nucleus accumbens increased food intake (17). However, the ARC has been shown to be the most sensitive site of action for N/OFQ, where bilateral injections of N/OFQ (0.21 nmol/rat) increased food intake 6-fold (18). The mechanisms of action of N/OFQ’s hyperphagic effect are unknown. N/OFQ, like opioids, exerts an inhibitory action on ARC neurons (19), and it has been postulated that N/OFQ could act by inhibiting known pathways involved in food intake, i.e. inhibiting the release of proopiomelanocortin (POMC) peptides and/or cocaine- and amphetamine-regulated transcript (CART) (20). Because the ARC is the most sensitive site of N/OFQ’s orexigenic action, we hypothesized that N/OFQ may affect the release of ARC neuropeptides involved in the regulation of appetite. Four ARC neuropeptides thought to control food intake are agouti-related protein (AgRP) and NPY, which are orexigenic, and CART and MSH, which have been suggested to be anorexigenic (for reviews see Refs. 21 and 22).
We investigated the ability of N/OFQ to alter the release of CART, MSH, NPY, and AgRP using ex vivo medial basal hypothalamic explants and dual immunocytochemistry (ICC) and in situ hybridization (ISH) to examine the expression of the NOP1 receptor in CART and AgRP hypothalamic neurons.
Materials and Methods
Materials
N/OFQ was purchased from Bachem (St. Helens, Merseyside, UK), and CART (55–102) was purchased from Peptide Institute Inc. (Osaka, Japan). All the CART peptides referred to in this paper are numbered as if they are products of the longer 102-residue peptide unless otherwise stated (23). CART (55–102) was used in this study as it is the bioactive C-terminal fragment of CART (24). Reagents for basal hypothalamic explant experiments were supplied by BDH (Poole, Dorset, UK).
Animals
Male Wistar rats weighing 170–200 g and female BALB/C mice weighing 20–25 g (specific pathogen free, Imperial College School of Medicine, London, UK) were maintained in individual cages under controlled temperature (21–23 C) and light (12 h light, 12 h dark cycle; lights on at 0700 h) with ad libitum access to food (RM1 diet, SDS Ltd., Witham, UK) and water. Animal procedures were approved by the British Home Office Animals Scientific Procedures Act 1986 (project licenses 90/1077 and 70/04326).
Food intake
Animals were anesthetized by ip injection of a mixture of Ketalar (ketamine HCl 60 mg/kg; Parke-Davis, Pontypool, UK) and Rompun (xylazine 12 mg/kg; Bayer UK Ltd., Bury St. Edmonds, UK) and placed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Permanent 22-gauge stainless steel guide cannulae (Plastics One Inc., Roanoke, VA) were stereotaxically placed into the lateral ventricle of the brain. Fed animals received either saline or 4.2 nmol/rat nociceptin administered in 5 μl and food intake measured in the first hour post injection. The dose of nociceptin used in this study was chosen based on published data demonstrating that this dose of nociceptin robustly increases food intake when injected into the lateral ventricle of rats (18).
Static incubation of hypothalamic explants
The static incubation system used was as previously described (25). Briefly, male Wistar rats were killed by decapitation and the whole brain immediately removed. The brain was mounted with ventral surface uppermost and placed in a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A horizontal 1.7-mm slice was taken from the basal hypothalamus and blocked lateral to the Circle of Willis. The hypothalamic nuclei included in the hypothalamic explant were the medial preoptic area, supraoptic nucleus (SON), anterior hypothalamic area, PVN, DMH, VMH, LH, ARC, and posterior hypothalamus (PH) (26). The hypothalamic slice was incubated in individual chambers containing 1 ml artificial cerebrospinal fluid (aCSF) (20 mM NaHCO3, 126 mM NaCl, 0.09 mM Na2HPO4, 6 mM KCl, 1.4 mM CaCl2, 0.09 mM MgSO4, 5 mM glucose, 0.18 mg/ml ascorbic acid, and 100 μg/ml aprotinin) equilibrated with 95% O2-5% CO2. The tubes were placed on a platform in a water bath maintained at 37 C. After an initial 2-h equilibration period, the hypothalami were incubated for 45 min in 600 μl aCSF (basal period) before being challenged with 0.1, 1, 10, or 100 nM N/OFQ in a 45-min test period. The viability of the tissue was verified by a final 45 min of exposure to aCSF containing 56 mM KCl; isotonicity was maintained by substituting K+ for Na+. Hypothalamic explants that failed to show peptide release above that of basal in response to aCSF containing 56 mM KCl were excluded from the data analysis (less than 10%). At the end of each period, the aCSF was collected and stored at –20 C until measurement of CART-immunoreactivity (IR), MSH-IR, NPY-IR, and AgRP-IR by RIA.
RIAs
RIAs for CART-IR, MSH-IR, NPY-IR, and AgRP-IR were performed using established methods (27, 28, 29). The CART antibody was produced in-house and has been previously described (30). The CART antibody was used at a final dilution of 1:28,000. Commercially available CART fragments were tested for cross-reactivity. The assay showed 20% cross-reaction with CART (61–102) and CART (62–102) and less than 0.1% cross-reaction with CART (1–39) (Phoenix Pharmaceuticals, Belmont, CA). The assay also showed less than 0.1% cross-reaction with 1 nmol/tube or less of AgRP, arginine vasopressin, bombesin, brain natriuretic peptide, calcitonin gene-related peptide, FSH, galanin, glucagons, GHRH, NPY, oxytocin, prolactin, prolactin-releasing peptide, somatostatin, and substance P. The CART assay had a sensitivity of 1.25 ± 0.5 (SE) fmol/tube (n = 4) with 95% confidence interval. The midrange was 16.2 ± 0.9 fmol/tube (n = 8). The specific activity of freshly prepared CART (55–102) peptide label, as estimated by self-displacement in the assay, was 42 Bq/fmol. The intra- and interassay variation was 8 and 9%, respectively.
The NPY antibody was produced in-house and has been previously characterized (31). The final dilution of antibody was 1:24,000 and showed no cross-reactivity with porcine pancreatic polypeptide or porcine peptide YY in concentrations up to 100 pmol/tube. The specific activity of iodinated NPY as measured by self-displacement was 40 Bq/fmol. The intra- and interassay variation was 7 and 8%, respectively.
The rabbit AgRP antiserum was a gift from G. S. Barsh (Stanford University, Stanford, CA) and was used at a final dilution of 1:200,000. Cross-reactivity for this antiserum has been characterized by Li et al. (32). No cross-reactivity was observed with recombinant agouti protein, MSH, ACTH, [Nle4,D-Phe7], leptin, orexin B, or NPY. However, 0.0038% cross-reactivity was noted for orexin A. The sensitivity of the assay was 1 fmol/ml, and the minimal detectable amount was 2 fmol/explant. Synthetic AgRP-(83–132) (Bachem) was used for the assay standard. The intra- and interassay variation was 9 and 8%, respectively.
The MSH antibody was obtained from Chemicon International (Temecula, CA; catalog no. AB5087) and was used at a final dilution of 1:320,000. This antibody has been previously characterized by Elias et al. (33). Briefly, the antiserum is specific for MSH with no cross-reactivity with melanin-concentrating hormone and is dependent on the amidated C-terminal region for recognition. Cross-reactivities of the antiserum with related peptides are desacetyl-MSH, 82–100%; MSH-free acid, 0.018%; ?MSH (monkey), 0.0018%; MSH (11–13), 1MSH, and 2MSH (each undetectable at 1 μM); ACTH (1–10), 0.018%; ACTH (1–24), 0.02%; ACTH (1–39) (human), 0.022%; ?-lipotropin (human), 0.022%; and ?-endorphin (human) and -endorphin (each undetectable at 1 μM). The sensitivity of the assay was 1 fmol/ml. The specific activity of iodinated MSH as measured by the self-displacement assay was 55 Bq/fmol. The intra- and interassay variation was 7 and 8%, respectively.
Ribonuclease protection assay (RPA)
Ad libitum-fed male rats were administered a single lateral ventricle injection of nociceptin (4.2 nmol/rat) or saline (n = 10/group). Four hours after injection, rats were killed and hypothalami collected. Total RNA was extracted from the hypothalami using Tri-Reagent (Helena Biosciences, Sunderland, UK) following the manufacturer’s protocol. Hypothalamic AgRP, POMC, NPY, and CART (all 5 μg) mRNA were quantified by RPA (RPA III kit, Ambion Inc., Austin, TX) using in-house probes. AgRP corresponded to nucleotides 17–353 (accession no. XM226404), NPY corresponded to nucleotides 81–538 (accession no. NM_012614), POMC corresponded to nucleotides 185–674 (accession no. NM_139326), and CART corresponded to nucleotides 218–533 (accession no. U10071). Rat ?-actin was used as an internal control (Ambion). cDNAs corresponding to the above probes were made by PCR and cloned into pBluescript. Linearized cDNAs were transcribed using T3 polymerase (Promega, Madison, WI) to produce antisense riboprobes labeled with 32P CTP (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK). RNA was hybridized overnight at 42 C and separated on a 5% polyacrylamide gel. The dried gel was exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) overnight and protected RNA hybrids quantified using ImageQuant software (Molecular Dynamics). For each neuropeptide, the ratio of the OD of the band of neuropeptide mRNA to that of ?-actin was calculated and expressed in relative units (34, 35, 36).
Monoclonal antibody (Mab) production
Rat CART peptide (55–102) was conjugated to BSA via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. BALB/c mice were injected ip with the conjugate (100 μg) in emulsion with Freund’s complete adjuvant (1:1 ratio) and boosted twice with the conjugate (50 μg) in Freund’s incomplete adjuvant (1:1 ratio). Spleen cells from a CART antibody-positive mouse were fused by the polyethylene glycol method with Sp2/o-Ag14 myeloma cell line (European Collection of Cell Cultures, Wiltshire, UK). Supernatants from the resulting hybridoma lines were screened in a RIA by using 125I CART (55–102). Positive hybridoma lines were cloned by limiting dilution. The Mab (from clone CART G3 G1 A9 F8 H12) was purified using Hitrap protein G (Amersham Pharmacia) affinity chromatography.
Electrophoresis
CART (55–102) standard (Bachem) over a concentration 1.9–500 ng was used. The samples were loaded. Mark 12 standards (Novex, San Diego, CA) were run to determine molecular weight. Samples were electrophoresed at 125 V i on a precast 16% tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA). Proteins were transferred at 77 V for 1 h in a minitransblot cell (HIS TE series transphor electrophoresis unit, Hoefer Scientific Instruments, San Francisco, CA).
Western blot
Membranes were washed three times for 10 min in PBS-Tween 20. Nonspecific binding sites were blocked by incubation with nonfat diluent (Marvel, Premier International Foods Ltd., Lincs, UK) (5%) in PBS-Tween 20. Membranes were then incubated overnight at 4 C with unconcentrated CART monoclonal antibody tissue culture supernatant (TCSN) (approximately 40 μg/ml). Membranes were washed and then incubated for 1 h with horseradish peroxidase-linked sheep antimouse Ig (Amersham Biosciences) diluted (1:2000) in blocking diluent. The immunoreactivity was revealed after incubation with SuperSignal West Pico chemiluminescent reagent (Pierce, Rockford IL), and then the membranes were apposed to film for several exposures of varying duration.
ICC
Rats were decapitated, the brains quickly removed, and a block of tissue encompassing the hypothalamus fixed in Bouin’s fixative for 24 h before being transferred to 70% ethanol. The brains were paraffin embedded, 5-μm sections cut using an AS500 microtome (Anglia Scientific, Cambridge, UK), and mounted on poly-lysine slides (BDH).
Paraffin sections were dewaxed in xylene and rehydrated through graduated ethanol to water. Endogenous peroxidase was blocked by incubation for 15 min with a solution of 1% hydrogen peroxide in methanol. Antigen retrieval was performed by microwaving sections in 0.01 M citrate buffer (pH 6.0) for 10 min at 800 W. Sections were incubated for 10 min at room temperature with normal donkey serum diluted 1:20 in 0.01 M PBS, 0.1% Na azide, and 0.1% BSA (Jackson ImmunoResearch Inc., West Grove, PA). The CART monoclonal antibody was diluted 1:100 in 0.01 M PBS, 0.1% Na azide, and 0.1% BSA and sections incubated overnight at 4 C. Negative controls used the primary antibody saturated with CART (55–102). Sections were then incubated in donkey antimouse antibody diluted 1:50 for 30 min at room temperature before being rinsed in Tris-buffered saline and incubated with mouse monoclonal alkaline phosphatase anti-alkaline phosphatase (Dako, High Wycombe, UK) diluted 1:50 in 50 mM Tris-HCl (pH 7.6) at room temperature for 30 min. Sections were rinsed in Tris-buffered saline and the secondary and tertiary antibody layers repeated to further amplify the signal. The alkaline phosphatase activity was developed with naphthol ASMX phosphate as substrate and fast blue as a chromogen (Sigma Chemical Co., Poole, UK).
ISH
ISH for NOP1 was performed using a riboprobe corresponding to nucleotides 1065–1355 of the rat sequence of NOP1 (accession no. NM031569). ISH for AgRP used a probe corresponding to nucleotides 17–353 of the rat sequence (accession no. XM226404). Linearized cDNAs were transcribed using T7 polymerase and T3 polymerase (Promega) to produce antisense and sense riboprobes, respectively, labeled with 35S CTP (Amersham Biosciences UK) for NOP1 and digoxigenin (DIG)-CTP for the AgRP probe (DIG RNA labeling mix; Roche Diagnostics, Mannheim, Germany).
Immediately after chromagen development in the ICC protocol, the slides were washed in 0.1 M PBS, incubated in 1 μg/ml proteinase K for 30 min at 37 C, acetylated in 0.25% acetic anhydride for 10 min at room temperature, and dried under vacuum. Sections were hybridized at 60 C with 35S-NOP1 riboprobes (14 x 106 counts/ml) overnight in a humidified chamber. Sections were then washed four times in 4x saline sodium citrate (SSC), and treated with 0.02 mg/ml RNase A at 37 C. After RNase A treatment, sections were washed twice in 2x SSC for 5 min, once in 1x SSC for 10 min, once in 0.5x SSC for 10 min, and finally 0.1x SSC at 60 C for 30 min. Sections were then dried and dipped in Ilford Scientific G.5 emulsion in gel form (Ilford Imaging UK Ltd., Mobberley, Cheshire, UK). Sections were exposed for 10 d at 4 C before being developed using Ilford Phenisol and fixed using Ilford Hypam both following the manufacturer’s instructions. Slides were coverslipped and observed for colocalization under a microscope.
For dual ISH for AgRP and NOP1, brains were removed and snap frozen in isopentane before 12-μm sections were cut on a cryostat (Bright, Huntingdon, UK). Slides were initially fixed in 4% formalin in 0.01 M PBS for 20 min on ice before acetylation in 0.25% acetic anhydride for 10 min at room temperature, dehydration in 70% ethanol, and delipidation in chloroform for 5 min at room temperature. Vacuum-dried sections were hybridized at 60 C with a mixture of 35S-NOP1 riboprobe (14 x 106 counts/ml) and DIG-AgRP (2 ng/ml) overnight, then washed and RNase treated as described above, and blocked in 5% fetal bovine serum (Invitrogen) in 0.1 M Tris, 0.15 M NaCl, and 0.05% Tween 20 for 2 h. Slides were then incubated with anti-DIG conjugated with alkaline phosphatase Fab fragments (Roche Diagnostics) in blocking buffer for 2 h at a 1:2000 dilution. After washing, the alkaline phosphatase was developed overnight with an 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indoyl-phosphate, 4-toluidine salt kit (Vector Laboratories, Burlingame, CA); sections were then dried and dipped in Ilford Scientific G.5 emulsion and exposed for 10 d at 4 C before being developed as described above.
Data analysis
All data are expressed as mean effect of treatment ± SEM as a percentage of basal, which is expressed as 100% ± SEM and also mean value ± SEM. Data from hypothalamic explant release experiments were compared by paired t test between the basal period and test period (Systat, Evanston, IL). P < 0.05 was considered to be statistically significant. Brain sections were analyzed qualitatively with an Eclipse E800 microscope (Nikon, Tokyo, Japan) for all single and double-ICC studies. For the dual-ISH and -ICC protocol, slides were analyzed with a Nikon Eclipse E800 microscope linked to a Xillix Microimager PMI (Richmond, Canada) and Image Pro-Plus 4.0 software (Media Cybernetics, Silver Spring, MD). Total numbers of cells were counted in the relevant nuclei, and colocalized cells were expressed as a percentage of the total number of NOP1 and CART neurons per section per nucleus. Cells were considered colocalized if they exhibited silver grain densities of 5 x background. Background densities were calculated for each section as the mean of 10 density readings using methods described by Elias et al. (37, 38). As with all colocalization studies, cell numbers are representative and not meant to give absolute cell counts.
Results
Effect of nociceptin administration to hypothalamic explants on the release of CART (55–102)-IR, MSH-IR, NPY-IR, and AgRP-IR
Incubation of hypothalamic explants with 1, 10, and 100 nM of N/OFQ produced significant reductions in basal CART (55–102)-IR release from hypothalamic explants of 28, 28, and 33%, respectively (Fig. 1A and Table 1). N/OFQ (1, 10, and 100 nM) significantly increased AgRP-IR release from hypothalamic explants to 176, 166, and 163% of basal levels, respectively (Fig. 1B and Table 1). A dose of 0.1 nM failed to either increase AgRP or decrease CART release from the preparations. N/OFQ had no effect on the basal release of MSH-IR or NPY-IR from the same ex vivo hypothalamic explants (Table 1).
FIG. 1. Effect of nociceptin (0.1, 1, 10, and 100 nM) on CART and AgRP release from hypothalamic explants. White bars, basal; light gray bars, 0.1 nM; gray bars, 1 nM; dark gray bars, 10 nM; very dark gray bars, 100 nM. The effect of a 45-min exposure to 0.1, 1, 10, and 100 nM of N/OFQ on the release of CART-IR (A) and AgRP-IR (B) (percent of basal ± SEM) from hypothalamic explants. Significance values for each group are indicated. **, P < 0.001 vs. basal (n = 20 per group).
TABLE 1. Effects of nociceptin (0.1, 1, 10, and 100 nM) on the release of neuropeptides from hypothalamic explants
Food intake
Administration of nociceptin (4.2 nmol/rat) into the lateral ventricle (LV) of ad libitum-fed rats significantly increased food intake in the first hour, compared with saline-injected animals (saline: 1.6 ± 0.25 g; nociceptin: 2.42 ± 0.29 g) (Fig. 2). There was no difference in food intake at 2, 4, 8, and 24 h post injection between the two groups (data not shown).
FIG. 2. Effect of nociceptin (4.2 nmol) on food intake in fed rats. Nociceptin (4.2 nmol/rat) administered into the LV significantly increases food intake in fed rats during the first hour post injection (saline injected 1.6 ± 0.25 g vs. nociceptin injected 2.42 ± 0.29 g; *, P < 0.05).
RPA
Administration of 4.2 nmol/rat of nociceptin into the LV had no effect on POMC or NPY hypothalamic mRNA levels (60 ± 3 saline vs. 61 ± 3 nociceptin and 107 ± 5.6 saline vs. 111 ± 5.8 nociceptin, respectively). This dose of nociceptin produced a nonsignificant 10% increase in hypothalamic AgRP mRNA (5.2 ± 0.1 saline vs. 5.7 ± 0.1 nociceptin) and also produced a nonsignificant 15% reduction in CART hypothalamic mRNA levels (43.3 ± 2.1 saline vs. 37.0 ± 1.4 nociceptin; Table 2).
TABLE 2. Effect of nociceptin (4.2 nmol/rat) on the mRNA levels of neuropeptides in the hypothalamus
Characterization of CART Mab
Single bands corresponding to approximately 6 kDa were detected in lanes run with 250 ng (47.5 pmol) and 500 ng (95 pmol) of CART (55–102) standard (Fig. 3A). The single-band detection was demonstrated as specific for CART Mab because the band was not detected when the TCSN Mab was preincubated with the immunizing peptide CART (55–102) (Fig. 3C), but CART (55–76) fragment did not block Mab binding (Fig. 3B), suggesting the CART antibody targets epitopes on the CART peptide, which include amino acid residues between 77 and 102.
FIG. 3. Immunoblotting of CART (55–102). A, Standard curve (1.9–500 ng) immunostained with unconcentrated CART Mab TCSN. B, Standard, immunostaining with CART Mab TCSN preincubated with CART (55–76). C, Standard, immunostaining CART Mab TCSN preincubated with CART (55–102).
NOP1 and CART dual labeling in controls
CART-IR in the PVN and ARC is shown in Fig. 4, A–D. ARC staining was abolished by preabsorption with a 5-fold excess full-length CART peptide (Fig. 4E). ISH using a partial cDNA probe corresponding to nucleotides 1119–1425 of the NOP1 gene (accession no. NM031569) produced specific labeling of N/OFQ mRNA (Fig. 4F), whereas the use of a sense probe produced no specific signal (Fig. 4G). Tissue sections hybridized with the 35S-labeled NOP1 mRNA sense probe exhibited silver grains that were diffuse and did not form clusters (see Figs. 5A and 6F). Sections labeled with the antisense probe exhibited a distinct pattern of silver grains on development (Fig. 5B and 6E).
FIG. 4. CART and N/OFQ distribution and controls. Series of photomicrographs (A–D) demonstrate CART-IR (blue) in the hypothalamus. A, PVN and ZI are shown. B–D, ARC is shown. E, Abolition of CART-IR in the ARC by preabsorption with a 5-fold excess of full-length CART peptide. G, Autoradiograph of ISH using a radiolabeled antisense NOP1 probe at the level of the midhypothalamus. F, Autoradiograph of ISH using an NOP1 sense control probe. Magnification, x40 (A); x100 (B); x200 (D and E); x400 (C). 3V, Third cerebral ventricle.
FIG. 5. Colocalization of CART peptide and NOP1 mRNA in the hypothalamus. Dual labeling using ICC for the CART antigen and ISH for NOP1 mRNA was performed. A series of photomicrographs demonstrate CART-IR (blue) with either a sense control NOP1 probe (A) or an antisense probe (B; silver grains). CART neurons are shown to coexpress NOP1 in the ARC (C and D) and LH (E and F). The green arrow indicates a cell expressing only NOP1 mRNA; the red arrows indicate dual-labeled cells. Magnification, x400 (A, B, D, and F); x200 (C); x100 (E). 3V, Third cerebral ventricle.
FIG. 6. Colocalization of AgRP and NOP1 mRNA in the ARC. Dual ISH for AgRP and NOP1 mRNA was performed. A series of photomicrographs demonstrates that AgRP-expressing neurons coexpress NOP1 mRNA in the ARC. AgRP expression uses a DIG-labeled antisense probe (A and B, purple) and a sense probe (C). D and E, Dual labeling in the ARC uses an AgRP DIG-labeled antisense probe and an NOP1 antisense radiolabeled probe (silver grains). The green arrows indicate cells expressing only NOP1 mRNA; the red arrows indicate dual-labeled cells. F, Dual labeling uses an AgRP antisense probe and an NOP1 sense probe. Magnification, x40 (A); x200 (B and C); x100 (D); x400 (E and F). 3V, Third cerebral ventricle.
NOP1 and CART dual labeling in the hypothalamus
The pattern of expression of N/OFQ mRNA and CART-IR observed in this study was consistent with previously published reports (12, 40) (Fig. 4, A, B, and F). Dual labeling using ICC for the CART antigen and ISH for the NOP1 receptor revealed coexpression in many areas of the hypothalamus. Colocalization was evident in the zona incerta (ZI), LH, SON, ARC, PVN, DMH, and PH (Table 3). NOP1 expression was found in 100% of CART-expressing neurons in the ZI, 89% of CART neurons in the LH (Fig. 5, E and F), 75% of SON CART neurons, 73% of ARC CART neurons (Fig. 5, C and D), 61% of PVN CART neurons, 54% of DMH neurons, and 41% of PH CART neurons. In the LH, double-labeled cells were present in the caudal areas of this nucleus, and in particular colocalized cells were found in the perifornical regions. The lateral aspects of the ARC exhibited double labeling throughout its rostrocaudal extent, and NOP1 mRNA appeared to be highly expressed in this nucleus. In the PVN, dual-labeled cells were found in the anterior, medial, and posterior parvicellular parts of the nucleus, and NOP1 expression appeared to be expressed at higher levels in the more anterior sections of the PVN. Coexpressing neurons were evident in the medial areas of the DMH and PH.
TABLE 3. Expression of NOP1 in CART neurons of the hypothalamus
NOP1 and AgRP dual labeling in controls
The pattern of expression of the AgRP antisense DIG-labeled RNA probe in this study was similar to previously published results being present exclusively in the ARC (41) (Fig. 6, A and B). This specific pattern was not evident when a sense DIG-labeled probe was used (Fig. 6C).
NOP1 and AgRP dual labeling in the hypothalamus
Dual-labeling ISH revealed coexpression of AgRP with the NOP1 receptor in the ARC (Table 4). Seventy-eight percent of AgRP neurons throughout the rostrocaudal extent of the ARC expressed the NOP1 receptor (Fig. 6, D and E).
TABLE 4. Expression of NOP1 in AgRP neurons of the hypothalamus
Discussion
It has been postulated that N/OFQ may mediate its orexigenic effect by modulating neuropeptides implicated in the control of food intake, i.e. by inhibiting anorectic neuropeptides (i.e. CART and MSH) or stimulating orexigenic neuropeptides (i.e. NPY and AgRP) (23, 42, 43, 44, 45). We have shown that N/OFQ significantly reduces the hypothalamic basal release of CART and also increases the release of AgRP from ex vivo hypothalamic slices but has no effect on the release of MSH or NPY. These effects appear to be all or nothing rather than showing a dose-dependent effect. In our experience, this is often the case using the hypothalamic explant system (28, 46). However, it is difficult to ascertain whether this effect is due to difficulties in tissue penetration or more complex effects on neuronal activation.
We have also shown that nociceptin 4.2 nmol/rat given into the LV increases food intake in the first hour after administration in accordance with previously published data (18). We found that this dose also decreased in vivo hypothalamic mRNA levels of CART and increased AgRP mRNA. Whereas the effects were not significant, the trends were in agreement with our peptide release data.
NOP1 ISH/CART ICC demonstrated that CART neurons in the hypothalamus express the NOP1 receptor. Colocalization was observed in the ARC, PVN, SON, DMH, PH, LH, and ZI. Expression of NOP1 by the majority of CART-staining neurons in these nuclei suggests that CART neurons may be one of the targets of the N/OFQ system.
The ARC exhibited a high percentage of CART neurons expressing the NOP1 receptor. It is reported that CART polyclonal antibodies when administered icv increase food intake suggesting CART exerts an inhibitory tone on food intake (23). N/OFQ elicits its most potent effect on food intake when administered into the ARC (18) and has been shown to have an inhibitory action on ARC neurons by activation of inward K+ currents (19). N/OFQ may therefore influence CART’s effect on food intake by attenuating its release from ARC neurons via the NOP1 receptor.
The PVN exhibited 60% coexpression of CART and the NOP1 receptor. Forty percent of CART neurons in the PVN coexpress TRH (40). N/OFQ dose-dependently increases TRH release from the hypothalamus and as a consequence increases circulating TSH (48). Olszewski and Levine (49) recently reported that N/OFQ reduces feeding induced c-fos expression in PVN oxytocin-expressing neurons. Thus, the PVN may be a region involved in mediating the effects of N/OFQ on appetite.
In the SON, 75% of CART neurons were found to express the NOP1 receptor, suggesting CART neurons in this nucleus may also be a target for the N/OFQ system. Intracerebroventricular administration of N/OFQ induces c-fos expression in the SON (50). It has recently been demonstrated that there is colocalization of CART in oxytocin and vasopressin-containing neurons in the SON (51). N/OFQ activates inwardly rectifying K+ channels to inhibit vasopressin neurons (52). In addition, icv administered N/OFQ has been shown to inhibit oxytocin and vasopressin neurons in the SON (53, 54). Because there is colocalization of CART in vasopressinergic and oxytocinergic neurons, it is possible that N/OFQ could inhibit CART release from the SON. The SON has been implicated in the control of food intake and therefore reduced CART release from this nucleus could increase food intake.
Recently CART has been shown to elicit an orexigenic effect when administered directly into the ARC, VMH, SON, DMH, and lateral hypothalamic area (55). It has been suggested that there may be two CART feeding circuits within the hypothalamus, one that is orexigenic, the other anorexigenic (55). N/OFQ’s effect on appetite may involve a reduction in CART release.
The lack of effect of N/OFQ on MSH appears to contrast with recently reported data in a review article published by Olszewski and Levine (49). They suggested N/OFQ may act via MSH to increase food intake because N/OFQ administered icv reduced feeding-induced c-fos activation in MSH neurons in the ARC. However, the level of c-fos expression does not provide information on neuropeptide release. In addition, more than 90% of CART-expressing neurons in the ARC also express MSH (56). Therefore, it is possible that the N/OFQ-induced attenuation of feeding-induced c-fos expression seen by Olszewski and Levine may result in reduced CART release without an effect on MSH release.
AgRP is a well-known orexigenic factor exclusively expressed in the ARC. It is the endogenous antagonist of the melanocortin-3 and -4 receptors. AgRP attenuates the powerful anorexigenic effects of MSH, the putative endogenous agonist of the melanocortin-4 receptor. N/OFQ significantly increases AgRP release and therefore may increase food intake by increasing antagonism at the melanocortin-4 receptor by AgRP. Dual ISH demonstrated that 78% of AgRP neurons in the ARC expressed the NOP1 receptor, providing an anatomical basis for N/OFQ’s effect on AgRP release from hypothalamic slices.
Similarly to N/OFQ’s proposed selective effects on CART release, N/OFQ appears to alter the release of AgRP but not NPY from the AgRP/NPY containing neurons of the ARC. The differential release of colocalized neuropeptides has previously been reported. It has been proposed that differential sorting and targeting of neuropeptides in the CNS allows their independent regulation within the same cell. For example, vasopressin and galanin localized to different processes could be released by different stimuli (39). In addition, NPY has recently been shown to be differentially released from the same secretory granule in which there are other costored peptides (47). The cellular mechanisms involved in this selective release of colocalized neuropeptides warrant further investigation. The lack of effect of N/OFQ on NPY release from hypothalamic explants is consistent with a previous report suggesting that the orexigenic effects of N/OFQ are not mediated by pathways involving NPY (15).
Our data suggest that N/OFQ may stimulate food intake through the inhibition of CART release and the stimulation of AgRP release from hypothalamic neurons. Previously published data indicate the orexigenic effect of N/OFQ is mediated via the NOP1 receptor, and here we have shown both CART and AgRP neurons coexpress this receptor. Because N/OFQ produces its most potent effect on food intake when injected directly into the ARC, it is possible that N/OFQ-induced hyperphagia may be mediated via the orexigenic AgRP and the anorexigenic CART neurons of the ARC.
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Address all correspondence and requests for reprints to: Professor S. R. Bloom, Department of Metabolic Medicine, Division of Investigative Science, Imperial College London, 6th Floor Commonwealth Building, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: s.bloom@imperial.ac.uk.
Abstract
Nociceptin or orphanin FQ (N/OFQ) and its receptor NOP1 are expressed in hypothalamic nuclei involved in energy homeostasis. N/OFQ administered by intracerebroventricular or arcuate nucleus (ARC) injection increases food intake in satiated rats. The mechanisms by which N/OFQ increases food intake are unknown. We hypothesized that N/OFQ may regulate hypothalamic neurons containing peptides involved in the control of food intake such as cocaine- and amphetamine-regulated transcript (CART), MSH, neuropeptide Y (NPY), and agouti-related protein (AgRP). We investigated the ability of N/OFQ to alter the release of CART, MSH, NPY, and AgRP using ex vivo medial basal hypothalamic explants. Incubation of hypothalamic explants with N/OFQ (1, 10, 100 nM) resulted in significant changes in CART and AgRP release. One hundred nanomoles N/OFQ caused a 33% decrease in release of CART (55–102) immunoreactivity (IR) and increased release of AgRP-IR to 163% but produced no change in either MSH-IR or NPY-IR. Double immunocytochemistry/in situ hybridization demonstrated that CART-IR and NOP1 mRNA are colocalized throughout the hypothalamus, in particular in the paraventricular nucleus, lateral hypothalamus, zona incerta, and ARC, providing an anatomical basis for N/OFQ action on CART release. Dual in situ hybridization demonstrated that AgRP neurons in the ARC also express the NOP1 receptor. Our data suggest that nociceptin via the NOP1 receptor may increase food intake by decreasing the release of the anorectic peptide CART and increasing the release of the orexigenic peptide AgRP.
Introduction
NOCICEPTIN/ORPHANIN FQ (N/OFQ) has structural homology to the opioid peptides, in particular dynorphin A. It was discovered to be the endogenous ligand for the orphan G protein-coupled receptor, NOP1, which is structurally similar to the -opioid receptor (1, 2, 3). Despite similarities between the two systems, N/OFQ lacks the N-terminal tyrosine required for activation of the opioid receptors (4). N/OFQ and NOP1 are widely distributed in the central nervous system (CNS). N/OFQ is involved in the modulation of pain (5), anxiety and stress (6), reward and dependence (7), memory and learning (8, 9), and hearing (10).
N/OFQ and NOP1 are present in hypothalamic nuclei that regulate the control of food intake, namely the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and the lateral hypothalamus (LH) (11, 12). Such a distribution suggested that N/OFQ might play a role in the hypothalamic regulation of food intake. Intracerebroventricular (icv) injection of N/OFQ dose dependently increased food intake in satiated rats over a 1- to 10-nmol range (13). N/OFQ-induced hyperphagia could be blocked by treatment with antisense oligonucleotides directed against each of the three exons of the NOP1 receptor (14). This suggests that the hyperphagic effect of N/OFQ is mediated through its action at the NOP1 receptor.
Several peptide agonists and antagonists of NOP1 have been synthesized. NC(1–13)NH2,[Nphe(1)]NC(1–13)NH2, a NOP1 antagonist, when injected icv almost completely abolished the orexigenic effect of icv N/OFQ. The same dose of this NOP1 antagonist administered icv to rats also significantly reduced feeding induced by food deprivation but not the hyperphagic effect of neuropeptide Y (NPY) (15). In addition, the selective Y1 receptor antagonist BIBO 3304 (16) markedly inhibited NPY-induced feeding but did not significantly modify food intake induced by N/OFQ when administered icv (15). This suggests that the N/OFQ system exerts its effects on food intake via pathways that do not involve the NPY system.
Microinjection studies have revealed the areas involved in mediating the orexigenic action of N/OFQ within the CNS. Intranuclear injections of N/OFQ at doses of 2.5–25 nmol into either the VMH or the shell of the nucleus accumbens increased food intake (17). However, the ARC has been shown to be the most sensitive site of action for N/OFQ, where bilateral injections of N/OFQ (0.21 nmol/rat) increased food intake 6-fold (18). The mechanisms of action of N/OFQ’s hyperphagic effect are unknown. N/OFQ, like opioids, exerts an inhibitory action on ARC neurons (19), and it has been postulated that N/OFQ could act by inhibiting known pathways involved in food intake, i.e. inhibiting the release of proopiomelanocortin (POMC) peptides and/or cocaine- and amphetamine-regulated transcript (CART) (20). Because the ARC is the most sensitive site of N/OFQ’s orexigenic action, we hypothesized that N/OFQ may affect the release of ARC neuropeptides involved in the regulation of appetite. Four ARC neuropeptides thought to control food intake are agouti-related protein (AgRP) and NPY, which are orexigenic, and CART and MSH, which have been suggested to be anorexigenic (for reviews see Refs. 21 and 22).
We investigated the ability of N/OFQ to alter the release of CART, MSH, NPY, and AgRP using ex vivo medial basal hypothalamic explants and dual immunocytochemistry (ICC) and in situ hybridization (ISH) to examine the expression of the NOP1 receptor in CART and AgRP hypothalamic neurons.
Materials and Methods
Materials
N/OFQ was purchased from Bachem (St. Helens, Merseyside, UK), and CART (55–102) was purchased from Peptide Institute Inc. (Osaka, Japan). All the CART peptides referred to in this paper are numbered as if they are products of the longer 102-residue peptide unless otherwise stated (23). CART (55–102) was used in this study as it is the bioactive C-terminal fragment of CART (24). Reagents for basal hypothalamic explant experiments were supplied by BDH (Poole, Dorset, UK).
Animals
Male Wistar rats weighing 170–200 g and female BALB/C mice weighing 20–25 g (specific pathogen free, Imperial College School of Medicine, London, UK) were maintained in individual cages under controlled temperature (21–23 C) and light (12 h light, 12 h dark cycle; lights on at 0700 h) with ad libitum access to food (RM1 diet, SDS Ltd., Witham, UK) and water. Animal procedures were approved by the British Home Office Animals Scientific Procedures Act 1986 (project licenses 90/1077 and 70/04326).
Food intake
Animals were anesthetized by ip injection of a mixture of Ketalar (ketamine HCl 60 mg/kg; Parke-Davis, Pontypool, UK) and Rompun (xylazine 12 mg/kg; Bayer UK Ltd., Bury St. Edmonds, UK) and placed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Permanent 22-gauge stainless steel guide cannulae (Plastics One Inc., Roanoke, VA) were stereotaxically placed into the lateral ventricle of the brain. Fed animals received either saline or 4.2 nmol/rat nociceptin administered in 5 μl and food intake measured in the first hour post injection. The dose of nociceptin used in this study was chosen based on published data demonstrating that this dose of nociceptin robustly increases food intake when injected into the lateral ventricle of rats (18).
Static incubation of hypothalamic explants
The static incubation system used was as previously described (25). Briefly, male Wistar rats were killed by decapitation and the whole brain immediately removed. The brain was mounted with ventral surface uppermost and placed in a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A horizontal 1.7-mm slice was taken from the basal hypothalamus and blocked lateral to the Circle of Willis. The hypothalamic nuclei included in the hypothalamic explant were the medial preoptic area, supraoptic nucleus (SON), anterior hypothalamic area, PVN, DMH, VMH, LH, ARC, and posterior hypothalamus (PH) (26). The hypothalamic slice was incubated in individual chambers containing 1 ml artificial cerebrospinal fluid (aCSF) (20 mM NaHCO3, 126 mM NaCl, 0.09 mM Na2HPO4, 6 mM KCl, 1.4 mM CaCl2, 0.09 mM MgSO4, 5 mM glucose, 0.18 mg/ml ascorbic acid, and 100 μg/ml aprotinin) equilibrated with 95% O2-5% CO2. The tubes were placed on a platform in a water bath maintained at 37 C. After an initial 2-h equilibration period, the hypothalami were incubated for 45 min in 600 μl aCSF (basal period) before being challenged with 0.1, 1, 10, or 100 nM N/OFQ in a 45-min test period. The viability of the tissue was verified by a final 45 min of exposure to aCSF containing 56 mM KCl; isotonicity was maintained by substituting K+ for Na+. Hypothalamic explants that failed to show peptide release above that of basal in response to aCSF containing 56 mM KCl were excluded from the data analysis (less than 10%). At the end of each period, the aCSF was collected and stored at –20 C until measurement of CART-immunoreactivity (IR), MSH-IR, NPY-IR, and AgRP-IR by RIA.
RIAs
RIAs for CART-IR, MSH-IR, NPY-IR, and AgRP-IR were performed using established methods (27, 28, 29). The CART antibody was produced in-house and has been previously described (30). The CART antibody was used at a final dilution of 1:28,000. Commercially available CART fragments were tested for cross-reactivity. The assay showed 20% cross-reaction with CART (61–102) and CART (62–102) and less than 0.1% cross-reaction with CART (1–39) (Phoenix Pharmaceuticals, Belmont, CA). The assay also showed less than 0.1% cross-reaction with 1 nmol/tube or less of AgRP, arginine vasopressin, bombesin, brain natriuretic peptide, calcitonin gene-related peptide, FSH, galanin, glucagons, GHRH, NPY, oxytocin, prolactin, prolactin-releasing peptide, somatostatin, and substance P. The CART assay had a sensitivity of 1.25 ± 0.5 (SE) fmol/tube (n = 4) with 95% confidence interval. The midrange was 16.2 ± 0.9 fmol/tube (n = 8). The specific activity of freshly prepared CART (55–102) peptide label, as estimated by self-displacement in the assay, was 42 Bq/fmol. The intra- and interassay variation was 8 and 9%, respectively.
The NPY antibody was produced in-house and has been previously characterized (31). The final dilution of antibody was 1:24,000 and showed no cross-reactivity with porcine pancreatic polypeptide or porcine peptide YY in concentrations up to 100 pmol/tube. The specific activity of iodinated NPY as measured by self-displacement was 40 Bq/fmol. The intra- and interassay variation was 7 and 8%, respectively.
The rabbit AgRP antiserum was a gift from G. S. Barsh (Stanford University, Stanford, CA) and was used at a final dilution of 1:200,000. Cross-reactivity for this antiserum has been characterized by Li et al. (32). No cross-reactivity was observed with recombinant agouti protein, MSH, ACTH, [Nle4,D-Phe7], leptin, orexin B, or NPY. However, 0.0038% cross-reactivity was noted for orexin A. The sensitivity of the assay was 1 fmol/ml, and the minimal detectable amount was 2 fmol/explant. Synthetic AgRP-(83–132) (Bachem) was used for the assay standard. The intra- and interassay variation was 9 and 8%, respectively.
The MSH antibody was obtained from Chemicon International (Temecula, CA; catalog no. AB5087) and was used at a final dilution of 1:320,000. This antibody has been previously characterized by Elias et al. (33). Briefly, the antiserum is specific for MSH with no cross-reactivity with melanin-concentrating hormone and is dependent on the amidated C-terminal region for recognition. Cross-reactivities of the antiserum with related peptides are desacetyl-MSH, 82–100%; MSH-free acid, 0.018%; ?MSH (monkey), 0.0018%; MSH (11–13), 1MSH, and 2MSH (each undetectable at 1 μM); ACTH (1–10), 0.018%; ACTH (1–24), 0.02%; ACTH (1–39) (human), 0.022%; ?-lipotropin (human), 0.022%; and ?-endorphin (human) and -endorphin (each undetectable at 1 μM). The sensitivity of the assay was 1 fmol/ml. The specific activity of iodinated MSH as measured by the self-displacement assay was 55 Bq/fmol. The intra- and interassay variation was 7 and 8%, respectively.
Ribonuclease protection assay (RPA)
Ad libitum-fed male rats were administered a single lateral ventricle injection of nociceptin (4.2 nmol/rat) or saline (n = 10/group). Four hours after injection, rats were killed and hypothalami collected. Total RNA was extracted from the hypothalami using Tri-Reagent (Helena Biosciences, Sunderland, UK) following the manufacturer’s protocol. Hypothalamic AgRP, POMC, NPY, and CART (all 5 μg) mRNA were quantified by RPA (RPA III kit, Ambion Inc., Austin, TX) using in-house probes. AgRP corresponded to nucleotides 17–353 (accession no. XM226404), NPY corresponded to nucleotides 81–538 (accession no. NM_012614), POMC corresponded to nucleotides 185–674 (accession no. NM_139326), and CART corresponded to nucleotides 218–533 (accession no. U10071). Rat ?-actin was used as an internal control (Ambion). cDNAs corresponding to the above probes were made by PCR and cloned into pBluescript. Linearized cDNAs were transcribed using T3 polymerase (Promega, Madison, WI) to produce antisense riboprobes labeled with 32P CTP (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK). RNA was hybridized overnight at 42 C and separated on a 5% polyacrylamide gel. The dried gel was exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) overnight and protected RNA hybrids quantified using ImageQuant software (Molecular Dynamics). For each neuropeptide, the ratio of the OD of the band of neuropeptide mRNA to that of ?-actin was calculated and expressed in relative units (34, 35, 36).
Monoclonal antibody (Mab) production
Rat CART peptide (55–102) was conjugated to BSA via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. BALB/c mice were injected ip with the conjugate (100 μg) in emulsion with Freund’s complete adjuvant (1:1 ratio) and boosted twice with the conjugate (50 μg) in Freund’s incomplete adjuvant (1:1 ratio). Spleen cells from a CART antibody-positive mouse were fused by the polyethylene glycol method with Sp2/o-Ag14 myeloma cell line (European Collection of Cell Cultures, Wiltshire, UK). Supernatants from the resulting hybridoma lines were screened in a RIA by using 125I CART (55–102). Positive hybridoma lines were cloned by limiting dilution. The Mab (from clone CART G3 G1 A9 F8 H12) was purified using Hitrap protein G (Amersham Pharmacia) affinity chromatography.
Electrophoresis
CART (55–102) standard (Bachem) over a concentration 1.9–500 ng was used. The samples were loaded. Mark 12 standards (Novex, San Diego, CA) were run to determine molecular weight. Samples were electrophoresed at 125 V i on a precast 16% tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA). Proteins were transferred at 77 V for 1 h in a minitransblot cell (HIS TE series transphor electrophoresis unit, Hoefer Scientific Instruments, San Francisco, CA).
Western blot
Membranes were washed three times for 10 min in PBS-Tween 20. Nonspecific binding sites were blocked by incubation with nonfat diluent (Marvel, Premier International Foods Ltd., Lincs, UK) (5%) in PBS-Tween 20. Membranes were then incubated overnight at 4 C with unconcentrated CART monoclonal antibody tissue culture supernatant (TCSN) (approximately 40 μg/ml). Membranes were washed and then incubated for 1 h with horseradish peroxidase-linked sheep antimouse Ig (Amersham Biosciences) diluted (1:2000) in blocking diluent. The immunoreactivity was revealed after incubation with SuperSignal West Pico chemiluminescent reagent (Pierce, Rockford IL), and then the membranes were apposed to film for several exposures of varying duration.
ICC
Rats were decapitated, the brains quickly removed, and a block of tissue encompassing the hypothalamus fixed in Bouin’s fixative for 24 h before being transferred to 70% ethanol. The brains were paraffin embedded, 5-μm sections cut using an AS500 microtome (Anglia Scientific, Cambridge, UK), and mounted on poly-lysine slides (BDH).
Paraffin sections were dewaxed in xylene and rehydrated through graduated ethanol to water. Endogenous peroxidase was blocked by incubation for 15 min with a solution of 1% hydrogen peroxide in methanol. Antigen retrieval was performed by microwaving sections in 0.01 M citrate buffer (pH 6.0) for 10 min at 800 W. Sections were incubated for 10 min at room temperature with normal donkey serum diluted 1:20 in 0.01 M PBS, 0.1% Na azide, and 0.1% BSA (Jackson ImmunoResearch Inc., West Grove, PA). The CART monoclonal antibody was diluted 1:100 in 0.01 M PBS, 0.1% Na azide, and 0.1% BSA and sections incubated overnight at 4 C. Negative controls used the primary antibody saturated with CART (55–102). Sections were then incubated in donkey antimouse antibody diluted 1:50 for 30 min at room temperature before being rinsed in Tris-buffered saline and incubated with mouse monoclonal alkaline phosphatase anti-alkaline phosphatase (Dako, High Wycombe, UK) diluted 1:50 in 50 mM Tris-HCl (pH 7.6) at room temperature for 30 min. Sections were rinsed in Tris-buffered saline and the secondary and tertiary antibody layers repeated to further amplify the signal. The alkaline phosphatase activity was developed with naphthol ASMX phosphate as substrate and fast blue as a chromogen (Sigma Chemical Co., Poole, UK).
ISH
ISH for NOP1 was performed using a riboprobe corresponding to nucleotides 1065–1355 of the rat sequence of NOP1 (accession no. NM031569). ISH for AgRP used a probe corresponding to nucleotides 17–353 of the rat sequence (accession no. XM226404). Linearized cDNAs were transcribed using T7 polymerase and T3 polymerase (Promega) to produce antisense and sense riboprobes, respectively, labeled with 35S CTP (Amersham Biosciences UK) for NOP1 and digoxigenin (DIG)-CTP for the AgRP probe (DIG RNA labeling mix; Roche Diagnostics, Mannheim, Germany).
Immediately after chromagen development in the ICC protocol, the slides were washed in 0.1 M PBS, incubated in 1 μg/ml proteinase K for 30 min at 37 C, acetylated in 0.25% acetic anhydride for 10 min at room temperature, and dried under vacuum. Sections were hybridized at 60 C with 35S-NOP1 riboprobes (14 x 106 counts/ml) overnight in a humidified chamber. Sections were then washed four times in 4x saline sodium citrate (SSC), and treated with 0.02 mg/ml RNase A at 37 C. After RNase A treatment, sections were washed twice in 2x SSC for 5 min, once in 1x SSC for 10 min, once in 0.5x SSC for 10 min, and finally 0.1x SSC at 60 C for 30 min. Sections were then dried and dipped in Ilford Scientific G.5 emulsion in gel form (Ilford Imaging UK Ltd., Mobberley, Cheshire, UK). Sections were exposed for 10 d at 4 C before being developed using Ilford Phenisol and fixed using Ilford Hypam both following the manufacturer’s instructions. Slides were coverslipped and observed for colocalization under a microscope.
For dual ISH for AgRP and NOP1, brains were removed and snap frozen in isopentane before 12-μm sections were cut on a cryostat (Bright, Huntingdon, UK). Slides were initially fixed in 4% formalin in 0.01 M PBS for 20 min on ice before acetylation in 0.25% acetic anhydride for 10 min at room temperature, dehydration in 70% ethanol, and delipidation in chloroform for 5 min at room temperature. Vacuum-dried sections were hybridized at 60 C with a mixture of 35S-NOP1 riboprobe (14 x 106 counts/ml) and DIG-AgRP (2 ng/ml) overnight, then washed and RNase treated as described above, and blocked in 5% fetal bovine serum (Invitrogen) in 0.1 M Tris, 0.15 M NaCl, and 0.05% Tween 20 for 2 h. Slides were then incubated with anti-DIG conjugated with alkaline phosphatase Fab fragments (Roche Diagnostics) in blocking buffer for 2 h at a 1:2000 dilution. After washing, the alkaline phosphatase was developed overnight with an 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indoyl-phosphate, 4-toluidine salt kit (Vector Laboratories, Burlingame, CA); sections were then dried and dipped in Ilford Scientific G.5 emulsion and exposed for 10 d at 4 C before being developed as described above.
Data analysis
All data are expressed as mean effect of treatment ± SEM as a percentage of basal, which is expressed as 100% ± SEM and also mean value ± SEM. Data from hypothalamic explant release experiments were compared by paired t test between the basal period and test period (Systat, Evanston, IL). P < 0.05 was considered to be statistically significant. Brain sections were analyzed qualitatively with an Eclipse E800 microscope (Nikon, Tokyo, Japan) for all single and double-ICC studies. For the dual-ISH and -ICC protocol, slides were analyzed with a Nikon Eclipse E800 microscope linked to a Xillix Microimager PMI (Richmond, Canada) and Image Pro-Plus 4.0 software (Media Cybernetics, Silver Spring, MD). Total numbers of cells were counted in the relevant nuclei, and colocalized cells were expressed as a percentage of the total number of NOP1 and CART neurons per section per nucleus. Cells were considered colocalized if they exhibited silver grain densities of 5 x background. Background densities were calculated for each section as the mean of 10 density readings using methods described by Elias et al. (37, 38). As with all colocalization studies, cell numbers are representative and not meant to give absolute cell counts.
Results
Effect of nociceptin administration to hypothalamic explants on the release of CART (55–102)-IR, MSH-IR, NPY-IR, and AgRP-IR
Incubation of hypothalamic explants with 1, 10, and 100 nM of N/OFQ produced significant reductions in basal CART (55–102)-IR release from hypothalamic explants of 28, 28, and 33%, respectively (Fig. 1A and Table 1). N/OFQ (1, 10, and 100 nM) significantly increased AgRP-IR release from hypothalamic explants to 176, 166, and 163% of basal levels, respectively (Fig. 1B and Table 1). A dose of 0.1 nM failed to either increase AgRP or decrease CART release from the preparations. N/OFQ had no effect on the basal release of MSH-IR or NPY-IR from the same ex vivo hypothalamic explants (Table 1).
FIG. 1. Effect of nociceptin (0.1, 1, 10, and 100 nM) on CART and AgRP release from hypothalamic explants. White bars, basal; light gray bars, 0.1 nM; gray bars, 1 nM; dark gray bars, 10 nM; very dark gray bars, 100 nM. The effect of a 45-min exposure to 0.1, 1, 10, and 100 nM of N/OFQ on the release of CART-IR (A) and AgRP-IR (B) (percent of basal ± SEM) from hypothalamic explants. Significance values for each group are indicated. **, P < 0.001 vs. basal (n = 20 per group).
TABLE 1. Effects of nociceptin (0.1, 1, 10, and 100 nM) on the release of neuropeptides from hypothalamic explants
Food intake
Administration of nociceptin (4.2 nmol/rat) into the lateral ventricle (LV) of ad libitum-fed rats significantly increased food intake in the first hour, compared with saline-injected animals (saline: 1.6 ± 0.25 g; nociceptin: 2.42 ± 0.29 g) (Fig. 2). There was no difference in food intake at 2, 4, 8, and 24 h post injection between the two groups (data not shown).
FIG. 2. Effect of nociceptin (4.2 nmol) on food intake in fed rats. Nociceptin (4.2 nmol/rat) administered into the LV significantly increases food intake in fed rats during the first hour post injection (saline injected 1.6 ± 0.25 g vs. nociceptin injected 2.42 ± 0.29 g; *, P < 0.05).
RPA
Administration of 4.2 nmol/rat of nociceptin into the LV had no effect on POMC or NPY hypothalamic mRNA levels (60 ± 3 saline vs. 61 ± 3 nociceptin and 107 ± 5.6 saline vs. 111 ± 5.8 nociceptin, respectively). This dose of nociceptin produced a nonsignificant 10% increase in hypothalamic AgRP mRNA (5.2 ± 0.1 saline vs. 5.7 ± 0.1 nociceptin) and also produced a nonsignificant 15% reduction in CART hypothalamic mRNA levels (43.3 ± 2.1 saline vs. 37.0 ± 1.4 nociceptin; Table 2).
TABLE 2. Effect of nociceptin (4.2 nmol/rat) on the mRNA levels of neuropeptides in the hypothalamus
Characterization of CART Mab
Single bands corresponding to approximately 6 kDa were detected in lanes run with 250 ng (47.5 pmol) and 500 ng (95 pmol) of CART (55–102) standard (Fig. 3A). The single-band detection was demonstrated as specific for CART Mab because the band was not detected when the TCSN Mab was preincubated with the immunizing peptide CART (55–102) (Fig. 3C), but CART (55–76) fragment did not block Mab binding (Fig. 3B), suggesting the CART antibody targets epitopes on the CART peptide, which include amino acid residues between 77 and 102.
FIG. 3. Immunoblotting of CART (55–102). A, Standard curve (1.9–500 ng) immunostained with unconcentrated CART Mab TCSN. B, Standard, immunostaining with CART Mab TCSN preincubated with CART (55–76). C, Standard, immunostaining CART Mab TCSN preincubated with CART (55–102).
NOP1 and CART dual labeling in controls
CART-IR in the PVN and ARC is shown in Fig. 4, A–D. ARC staining was abolished by preabsorption with a 5-fold excess full-length CART peptide (Fig. 4E). ISH using a partial cDNA probe corresponding to nucleotides 1119–1425 of the NOP1 gene (accession no. NM031569) produced specific labeling of N/OFQ mRNA (Fig. 4F), whereas the use of a sense probe produced no specific signal (Fig. 4G). Tissue sections hybridized with the 35S-labeled NOP1 mRNA sense probe exhibited silver grains that were diffuse and did not form clusters (see Figs. 5A and 6F). Sections labeled with the antisense probe exhibited a distinct pattern of silver grains on development (Fig. 5B and 6E).
FIG. 4. CART and N/OFQ distribution and controls. Series of photomicrographs (A–D) demonstrate CART-IR (blue) in the hypothalamus. A, PVN and ZI are shown. B–D, ARC is shown. E, Abolition of CART-IR in the ARC by preabsorption with a 5-fold excess of full-length CART peptide. G, Autoradiograph of ISH using a radiolabeled antisense NOP1 probe at the level of the midhypothalamus. F, Autoradiograph of ISH using an NOP1 sense control probe. Magnification, x40 (A); x100 (B); x200 (D and E); x400 (C). 3V, Third cerebral ventricle.
FIG. 5. Colocalization of CART peptide and NOP1 mRNA in the hypothalamus. Dual labeling using ICC for the CART antigen and ISH for NOP1 mRNA was performed. A series of photomicrographs demonstrate CART-IR (blue) with either a sense control NOP1 probe (A) or an antisense probe (B; silver grains). CART neurons are shown to coexpress NOP1 in the ARC (C and D) and LH (E and F). The green arrow indicates a cell expressing only NOP1 mRNA; the red arrows indicate dual-labeled cells. Magnification, x400 (A, B, D, and F); x200 (C); x100 (E). 3V, Third cerebral ventricle.
FIG. 6. Colocalization of AgRP and NOP1 mRNA in the ARC. Dual ISH for AgRP and NOP1 mRNA was performed. A series of photomicrographs demonstrates that AgRP-expressing neurons coexpress NOP1 mRNA in the ARC. AgRP expression uses a DIG-labeled antisense probe (A and B, purple) and a sense probe (C). D and E, Dual labeling in the ARC uses an AgRP DIG-labeled antisense probe and an NOP1 antisense radiolabeled probe (silver grains). The green arrows indicate cells expressing only NOP1 mRNA; the red arrows indicate dual-labeled cells. F, Dual labeling uses an AgRP antisense probe and an NOP1 sense probe. Magnification, x40 (A); x200 (B and C); x100 (D); x400 (E and F). 3V, Third cerebral ventricle.
NOP1 and CART dual labeling in the hypothalamus
The pattern of expression of N/OFQ mRNA and CART-IR observed in this study was consistent with previously published reports (12, 40) (Fig. 4, A, B, and F). Dual labeling using ICC for the CART antigen and ISH for the NOP1 receptor revealed coexpression in many areas of the hypothalamus. Colocalization was evident in the zona incerta (ZI), LH, SON, ARC, PVN, DMH, and PH (Table 3). NOP1 expression was found in 100% of CART-expressing neurons in the ZI, 89% of CART neurons in the LH (Fig. 5, E and F), 75% of SON CART neurons, 73% of ARC CART neurons (Fig. 5, C and D), 61% of PVN CART neurons, 54% of DMH neurons, and 41% of PH CART neurons. In the LH, double-labeled cells were present in the caudal areas of this nucleus, and in particular colocalized cells were found in the perifornical regions. The lateral aspects of the ARC exhibited double labeling throughout its rostrocaudal extent, and NOP1 mRNA appeared to be highly expressed in this nucleus. In the PVN, dual-labeled cells were found in the anterior, medial, and posterior parvicellular parts of the nucleus, and NOP1 expression appeared to be expressed at higher levels in the more anterior sections of the PVN. Coexpressing neurons were evident in the medial areas of the DMH and PH.
TABLE 3. Expression of NOP1 in CART neurons of the hypothalamus
NOP1 and AgRP dual labeling in controls
The pattern of expression of the AgRP antisense DIG-labeled RNA probe in this study was similar to previously published results being present exclusively in the ARC (41) (Fig. 6, A and B). This specific pattern was not evident when a sense DIG-labeled probe was used (Fig. 6C).
NOP1 and AgRP dual labeling in the hypothalamus
Dual-labeling ISH revealed coexpression of AgRP with the NOP1 receptor in the ARC (Table 4). Seventy-eight percent of AgRP neurons throughout the rostrocaudal extent of the ARC expressed the NOP1 receptor (Fig. 6, D and E).
TABLE 4. Expression of NOP1 in AgRP neurons of the hypothalamus
Discussion
It has been postulated that N/OFQ may mediate its orexigenic effect by modulating neuropeptides implicated in the control of food intake, i.e. by inhibiting anorectic neuropeptides (i.e. CART and MSH) or stimulating orexigenic neuropeptides (i.e. NPY and AgRP) (23, 42, 43, 44, 45). We have shown that N/OFQ significantly reduces the hypothalamic basal release of CART and also increases the release of AgRP from ex vivo hypothalamic slices but has no effect on the release of MSH or NPY. These effects appear to be all or nothing rather than showing a dose-dependent effect. In our experience, this is often the case using the hypothalamic explant system (28, 46). However, it is difficult to ascertain whether this effect is due to difficulties in tissue penetration or more complex effects on neuronal activation.
We have also shown that nociceptin 4.2 nmol/rat given into the LV increases food intake in the first hour after administration in accordance with previously published data (18). We found that this dose also decreased in vivo hypothalamic mRNA levels of CART and increased AgRP mRNA. Whereas the effects were not significant, the trends were in agreement with our peptide release data.
NOP1 ISH/CART ICC demonstrated that CART neurons in the hypothalamus express the NOP1 receptor. Colocalization was observed in the ARC, PVN, SON, DMH, PH, LH, and ZI. Expression of NOP1 by the majority of CART-staining neurons in these nuclei suggests that CART neurons may be one of the targets of the N/OFQ system.
The ARC exhibited a high percentage of CART neurons expressing the NOP1 receptor. It is reported that CART polyclonal antibodies when administered icv increase food intake suggesting CART exerts an inhibitory tone on food intake (23). N/OFQ elicits its most potent effect on food intake when administered into the ARC (18) and has been shown to have an inhibitory action on ARC neurons by activation of inward K+ currents (19). N/OFQ may therefore influence CART’s effect on food intake by attenuating its release from ARC neurons via the NOP1 receptor.
The PVN exhibited 60% coexpression of CART and the NOP1 receptor. Forty percent of CART neurons in the PVN coexpress TRH (40). N/OFQ dose-dependently increases TRH release from the hypothalamus and as a consequence increases circulating TSH (48). Olszewski and Levine (49) recently reported that N/OFQ reduces feeding induced c-fos expression in PVN oxytocin-expressing neurons. Thus, the PVN may be a region involved in mediating the effects of N/OFQ on appetite.
In the SON, 75% of CART neurons were found to express the NOP1 receptor, suggesting CART neurons in this nucleus may also be a target for the N/OFQ system. Intracerebroventricular administration of N/OFQ induces c-fos expression in the SON (50). It has recently been demonstrated that there is colocalization of CART in oxytocin and vasopressin-containing neurons in the SON (51). N/OFQ activates inwardly rectifying K+ channels to inhibit vasopressin neurons (52). In addition, icv administered N/OFQ has been shown to inhibit oxytocin and vasopressin neurons in the SON (53, 54). Because there is colocalization of CART in vasopressinergic and oxytocinergic neurons, it is possible that N/OFQ could inhibit CART release from the SON. The SON has been implicated in the control of food intake and therefore reduced CART release from this nucleus could increase food intake.
Recently CART has been shown to elicit an orexigenic effect when administered directly into the ARC, VMH, SON, DMH, and lateral hypothalamic area (55). It has been suggested that there may be two CART feeding circuits within the hypothalamus, one that is orexigenic, the other anorexigenic (55). N/OFQ’s effect on appetite may involve a reduction in CART release.
The lack of effect of N/OFQ on MSH appears to contrast with recently reported data in a review article published by Olszewski and Levine (49). They suggested N/OFQ may act via MSH to increase food intake because N/OFQ administered icv reduced feeding-induced c-fos activation in MSH neurons in the ARC. However, the level of c-fos expression does not provide information on neuropeptide release. In addition, more than 90% of CART-expressing neurons in the ARC also express MSH (56). Therefore, it is possible that the N/OFQ-induced attenuation of feeding-induced c-fos expression seen by Olszewski and Levine may result in reduced CART release without an effect on MSH release.
AgRP is a well-known orexigenic factor exclusively expressed in the ARC. It is the endogenous antagonist of the melanocortin-3 and -4 receptors. AgRP attenuates the powerful anorexigenic effects of MSH, the putative endogenous agonist of the melanocortin-4 receptor. N/OFQ significantly increases AgRP release and therefore may increase food intake by increasing antagonism at the melanocortin-4 receptor by AgRP. Dual ISH demonstrated that 78% of AgRP neurons in the ARC expressed the NOP1 receptor, providing an anatomical basis for N/OFQ’s effect on AgRP release from hypothalamic slices.
Similarly to N/OFQ’s proposed selective effects on CART release, N/OFQ appears to alter the release of AgRP but not NPY from the AgRP/NPY containing neurons of the ARC. The differential release of colocalized neuropeptides has previously been reported. It has been proposed that differential sorting and targeting of neuropeptides in the CNS allows their independent regulation within the same cell. For example, vasopressin and galanin localized to different processes could be released by different stimuli (39). In addition, NPY has recently been shown to be differentially released from the same secretory granule in which there are other costored peptides (47). The cellular mechanisms involved in this selective release of colocalized neuropeptides warrant further investigation. The lack of effect of N/OFQ on NPY release from hypothalamic explants is consistent with a previous report suggesting that the orexigenic effects of N/OFQ are not mediated by pathways involving NPY (15).
Our data suggest that N/OFQ may stimulate food intake through the inhibition of CART release and the stimulation of AgRP release from hypothalamic neurons. Previously published data indicate the orexigenic effect of N/OFQ is mediated via the NOP1 receptor, and here we have shown both CART and AgRP neurons coexpress this receptor. Because N/OFQ produces its most potent effect on food intake when injected directly into the ARC, it is possible that N/OFQ-induced hyperphagia may be mediated via the orexigenic AgRP and the anorexigenic CART neurons of the ARC.
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