Basomedial Hypothalamic Injections of Neuropeptide Y Conjugated to Saporin Selectively Disrupt Hypothalamic Controls of Food Intake

http://www.100md.com 内分泌学杂志 2005年第3期

     Programs in Neuroscience, Washington State University, Pullman, Washington 99164-6520

    Address all correspondence and requests for reprints to: Sue Ritter, Programs in Neuroscience, Washington State University, Pullman, Washington 99164-6520. E-mail: sjr@vetmed.wsu.edu

    Abstract

    Neuropeptide Y (NPY) conjugated to saporin (NPY-SAP), a ribosomal inactivating toxin, is a newly developed compound designed to selectively target and lesion NPY receptor-expressing cells. We injected NPY-SAP into the basomedial hypothalamus (BMH), just dorsal to the arcuate nucleus (ARC), to investigate its neurotoxicity and to determine whether ARC NPY neurons are required for glucoprivic feeding. We found that NPY-SAP profoundly reduced NPY Y1 receptor and MSH immunoreactivity, as well as NPY, Agouti gene-related protein (AGRP), and cocaine and amphetamine-related transcript mRNA expression in the BMH. NPY-SAP lesions were localized to the injection site with no evidence of retrograde transport by hindbrain NPY neurons with BMH terminals. These lesions impaired responses to intracerebroventricular (icv) leptin (5 μg/5 μl·d) and ghrelin (2 μg/5 μl), which are thought to alter feeding primarily by actions on ARC NPY/AGRP and proopiomelanocortin/cocaine and amphetamine-related transcript neurons. However, the hypothesis that NPY/AGRP neurons are required downstream mediators of glucoprivic feeding was not supported. Although NPY/AGRP neurons were destroyed by NPY-SAP, the lesion did not impair either the feeding or the hyperglycemic response to 2-deoxy-D-glucose-induced blockade of glycolysis use. Similarly, responses to glucagon-like peptide-1 (GLP-1, 5 μg/3 μl icv), NPY (5 μg/3 μl icv), cholecystokinin octapeptide (4 μg/kg ip), and ?-mercaptoacetate (68 mg/kg ip) were not altered by the NPY-SAP lesion. Thus, NPY-SAP destroyed NPY receptor-expressing neurons in the ARC and selectively disrupted controls of feeding dependent on those neurons but did not disrupt peptidergic or metabolic controls dependent upon circuitry outside the BMH.

    Introduction

    INCREASED FOOD INTAKE is an important component of the total glucoregulatory response normally elicited by central glucose deficit (glucoprivation) (1). Although the neural pathway for this response is not fully understood, a number of findings suggest that neurons in the arcuate nucleus (ARC) of the hypothalamus that coexpress neuropeptide Y (NPY) and Agouti gene-related protein (AGRP) may be involved. Electrophysiological studies have reported the presence of glucose sensing neurons in the ARC (2, 3, 4), some of which respond to NPY (5). The paraventricular nucleus of the hypothalamus (PVH), which is heavily innervated by NPY/AGRP nerve terminals (6, 7, 8), is a sensitive site for elicitation of feeding by the administration of exogenous NPY (9), and injection of NPY antibodies into the PVH attenuates glucoprivic feeding (10). Expression of NPY and AGRP mRNAs is increased in the ARC by systemic glucoprivation (11, 12, 13). Finally, deletion of the NPY gene impairs glucoprivic feeding (14).

    In the present experiment, we further examine the importance of NPY/AGRP neurons and the basomedial hypothalamus (BMH) neurons they innervate for glucoprivic feeding. We use a novel targeted toxin, saporin conjugated to NPY (NPY-SAP), designed to lesion NPY receptor-expressing neurons. Saporin, a type 1 ribosomal inactivating protein (15), can be targeted to destroy specific populations of neurons by conjugation with substances that are selectively internalized by the targeted cell population (16, 17). A number of saporin conjugates have been useful as selective neurochemical lesioning agents, including conjugates to substance P (18), corticotropin-releasing factor (19), 192-IgG (20), and orexin (21). Because agonist-driven internalization has been demonstrated for NPY receptors (22, 23, 24, 25, 26), we predicted that the NPY-SAP conjugate would bind to NPY receptors, resulting in its selective internalization. Therefore, we injected NPY-SAP into the BMH. Injections were directed specifically at the dorsal aspect of the ARC to lesion NPY receptor-expressing neurons in the ARC, including those coexpressing NPY/AGRP and those coexpressing proopiomelanocortin (POMC) and cocaine and amphetamine-related transcript (CART) (27, 28, 29). Lesions were characterized anatomically using in situ hybridization and immunohistochemistry and behaviorally by examining effects on spontaneous feeding and body weight and on feeding in response to glucoprivation, lipoprivation, leptin, ghrelin, cholecystokinin (CCK), NPY, and glucagon-like peptide-1 (GLP-1). Results characterize this targeted toxin as a useful lesioning agent and provide new information regarding the neural circuitry underlying glucoprivic feeding.

    Materials and Methods

    Animals

    Adult male Sprague Dawley rats weighing approximately 320 g (Simonsen Laboratories Inc., Gilroy, CA) were housed individually in suspended wire mesh cages in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care (Rockville, MD). Rats were on a 12-h light (0600–1800 h), 12-h dark cycle with ad libitum access to food and tap water. Except as noted, experiments used six to nine rats per group. Washington State University Institutional Animal Care and Use Committee approved all experimental animal protocols, which conform to National Institute of Health guidelines.

    Preliminary studies

    Because there were no published studies reporting the lesioning properties of NPY-SAP, preliminary studies were conducted to establish dosing and injection parameters. Doses between 12 and 250 ng (50- to 300-nl injection volume) were examined for effects on ARC tissue. The size of the NPY-SAP lesion was dose-related, with the highest doses and volumes producing more extensive damage than required to test our hypotheses. Doses of 90 ng and above caused significant tissue destruction, possibly including nonspecific damage. We adopted a standard dose of 48 ng in 100 nl per side for all of the studies reported here, with the exception of the dose-response curve described below. With these injection parameters, loss of NPY-Y1 receptor immunoreactivity could be detected throughout the extent of the ARC. We also examined the time course (5, 10, 13, and 21 d after injection) of NPY-SAP effects on NPY terminals in the hypothalamus to determine when to initiate behavioral testing. We found a significant reduction of terminals was present by d 10. Therefore, feeding tests were conducted beginning approximately 2 wk after NPY-SAP injection. In addition, we examined the hindbrain cell groups A1 and C1 in rats with BMH NPY-SAP injections. These cell groups are the major hindbrain sources of NPY innervation of the BMH (30), and loss of these cells would indicate that the NPY-SAP is retrogradely transported from the injection site. However, no reduction in the number of TH-immunoreactive cells was apparent, as shown below.

    Receptor binding

    Competitive binding studies were conducted to evaluate the binding of NPY-SAP to homogenates of forebrain tissue. For these studies, rats were killed by decapitation; the forebrain was rapidly removed, weighed, and homogenized in 50 mM Tris-HCl. The homogenate was centrifuged at 48,000 x g for 20 min and the supernatant discarded. The pellet was resuspended in 50 mM Tris-HCl and recentrifuged. The supernatant was discarded, and the pellet containing the cell membranes was resuspended in 25 mM HEPES buffer. One hundred and fifty microliters of 125I-NPY were added to each of 24 tubes. To this, 20 μl of different concentrations (10–5 to 10–9 M) of competing ligand (NPY or NPY-SAP) or buffer and 30 μl tissue homogenate were added. Nonspecific binding was determined using 1 μM NPY. The tubes were incubated at room temperature for 1 h to allow the radioligand to bind. During this time, radioactivity in each tube was measured to precisely determine the amount of radioligand in each tube. The reaction was then stopped by filtration through Schleicher & Schuell (Dassel, Germany) no. 34 glass fiber filters, and the filters were extensively washed and counted in a -counter. The data were analyzed using Microsoft Excel to calculate specific binding (total minus nonspecific) and GraphPad Prism (one-site competition model) to obtain the IC50.

    Intracranial injections

    For intrahypothalamic administration of NPY-SAP and control solution, rats were anesthetized using 0.1 ml/100 g body weight of ketamine/xylazine/acepromazine cocktail (5 ml ketamine HCl, 100 mg/ml, Fort Dodge Animal Health, Fort Dodge, IA; 2.5 ml xylazine, 20 mg/ml, Vedco, Inc., St. Joseph, MO; 1 ml acepromazine, 10 mg/ml, Vedco, Inc.; and 1.5 ml 0.9% saline solution). Intracranial injections of saporin conjugated to NPY (NPY-SAP, gift of Dr. Douglas A. Lappi, Advanced Targeting Systems, San Diego, CA) or the equivalent amount of a blank saporin control (B-SAP, Advanced Targeting Systems, San Diego, CA) were delivered bilaterally (100 nl/side) just dorsal to the ARC (2.8 mm caudal and 0.4 mm lateral to bregma, 8.6 mm ventral to dura) (31). B-SAP is a control conjugate of saporin with a nontargeted peptide with no known biological binding site or function. B-SAP has the same molecular weight as the NPY-SAP. Injections were made through a stereotaxically positioned drawn glass capillary micropipette (30-μm tip diameter) connected to a microinjector (Picospritzer, General Valve Corp., Fairfield, NJ) with polyethylene tubing. The solution was delivered slowly over a 5-min period and was monitored microscopically. Because the injection site is often difficult to detect in histological sections, colloidal gold was coinjected with the NPY-SAP in some rats to permanently mark the injection site. During the same surgical procedure, a permanent 26-gauge stainless steel lateral ventricle guide cannula was permanently implanted (1.0 mm caudal and 1.5 mm lateral to bregma, 3.9 mm ventral to dura) (31) for subsequent administration of peptides into the lateral ventricle. Cannulas were implanted stereotaxically and secured to the skull using three stainless steel screws and methyl methacrylate bone cement. The cannulas accepted 30-gauge obturators and injectors that extended 0.5 mm beyond the guide cannula tip. At least 2 wk was allowed for recovery from surgery before initiation of behavioral studies.

    Dose-response effects of NPY-SAP on spontaneous feeding and body weight

    To examine the effect of NPY-SAP dose on food intake and body weight, 12, 24, or 48 ng of NPY-SAP or B-SAP was injected bilaterally (100 nl per side) into the BMH. Daytime and overnight food intake and body weights were measured 8 wk after surgery.

    Feeding tests

    All animals were handled extensively and habituated to experimental procedures before the start of the feeding tests. Except for leptin, all other substances were tested acutely between 0800 and 1300 h of the light phase of the animals’ light-dark cycle. Feeding tests used the rats’ standard pelleted chow diet and spillage was accounted for during the tests. Doses of injected substances that have been shown to be effective in altering food intake using similar protocols and routes of administration were chosen from published work. Injections into the lateral cerebral ventricle [intracerebroventricular (icv)] were performed manually using a glass 10-μl syringe (Hamilton, Reno, NV) connected by polyethylene tubing (PE-10) to a stainless steel injector, whose tip extended 0.5 mm beyond that of the guide cannula. Injections were given over a 60-sec period, and the injector was left in place for an additional 30 sec to allow diffusion of the injectate away from the cannula tip. Murine leptin (5 μg, Calbiochem, San Diego, CA) in 5 μl of artificial cerebrospinal fluid (aCSF) was administered into the lateral ventricle. Before leptin injection, animals were injected icv with 5 μl aCSF for 3 consecutive days to establish baseline feeding and body weights. Leptin was then administered daily for 4 d, followed by an additional 8 recovery days in which aCSF was administered. Body weights and 24-h food intake were measured daily throughout the experiment. The ability of icv ghrelin (2 μg/5 μl; Sigma Chemical Co., St. Louis, MO) to stimulate food intake was assessed during the 4 h immediately after the injection. To test for the suppression of feeding by GLP-1, rats were injected icv with 5 μg/5 μl GLP-1 (Bachem, Torrance, CA) after an overnight (18 h) fast, and food intake was measured in a 60-min test. Effects of NPY (5 μg/3 μl, Phoenix Pharmaceuticals, San Diego, CA) on feeding were assessed in a 2-h test immediately after the icv injection. Baseline tests using aCSF injection were conducted for each of these conditions.

    2-Deoxy-D-glucose (2DG; Sigma), 2-mercaptoacetate (MA; thioglycolic acid, Sigma), and sulfated CCK (Peptides International, Inc., Louisville, KY) were dissolved in 0.9% sterile saline for peripheral administration. For tests of glucoprivic feeding, rats were injected sc with 100, 200, and 400 mg/kg 2DG or saline (0.9%), and food intake was measured during the subsequent 4 h. One week was allowed to elapse between 2DG tests. Blood glucose responses were measured in a separate test in the absence of food. Food was removed 2 h before the 2DG injection at time 0 (200 mg/kg, sc), and tail blood was sampled at –45, 0, 30, 60, 90, and 120 min for assay using the glucose oxidase method (32). ?-MA blocks mitochondrial acyl-CoA dehydrogenases, thereby reducing ?-oxidation of fatty acids (33, 34) and producing a stimulus for feeding (35, 36, 37). For the assessment of MA-induced feeding, rats were given an ip injection of MA (68 mg/kg, 1 ml/kg) or an equal volume of saline and food intake measured over a 4-h period. To test suppression of feeding in response to CCK, animals were fasted overnight. In the morning, they were injected ip with CCK (4 μg/kg, 1 ml/kg), and food intake was measured in a 30-min test. Saline tests were conducted in the same way 3 d before the CCK test.

    In situ hybridization

    Riboprobes complementary to rat NPY, AGRP, and CART mRNAs were prepared and used for hybridization after labeling with 33P-uridine 5'-triphosphate as previously described (38). Briefly, plasmids containing cDNA for either NPY (gift of Dr. Barry Levine, Veterans Administrations Medical Center, East Orange, NJ), AGRP, or CART (gifts of Dr. Streamson Chua, Department of Pediatrics, Columbia University, New York, NY, and Dr. Kellie Tamashiro, Neuroscience Program, University of Cincinnati Medical Center, Cincinnati, OH) were linearized and transcribed for antisense riboprobes with T7 (for NPY and CART) or T3 (for AGRP) RNA polymerase in the presence of 33P-uridine 5'-triphosphate (Perkin-Elmer Life Sciences, Boston, MA), using a MAXIscript kit (Ambion, Austin, TX). Sense transcriptions were then carried out with T3 (for NPY), T7 (for AGRP), or SP6 (for CART) RNA polymerase (Life Technologies, Inc., Gaithersburg, MD). Hybridization controls, using the sense probe, showed no hybridization signal.

    After testing was completed, animals were rapidly killed by a lethal dose of pentobarbital and transcardially perfused with diethylpyrocarbonate (Sigma)-treated 0.1 M PBS (PBS, pH 7.4) followed by fresh 4% paraformaldehyde (pH 7.4, 4 C). The brains were removed and postfixed in fresh 4% paraformaldehyde for 8 h. Brains were then cryoprotected overnight in diethylpyrocarbonate-treated cryoprotectant containing 20% sucrose in 0.1 M PBS (pH 7.4) and sectioned on a cryostat. Coronal cryostat sections of the hypothalamus (20 μm) were collected into five sets of serial sections, direct mounted onto Superfrost Plus slides (Fisher Scientific, Los Angeles, CA), and stored in desiccated slide boxes at –80 C until they were processed for in situ hybridization.

    Sections were removed from the freezer and allowed to return to room temperature. After being placed into slide racks, sections were first dipped in diethylpyrocarbonate-treated water, followed by 0.1 M triethanolamine (Sigma-Aldrich Inc.). Thereafter, the sections were washed in 0.1 M triethanolamine with 250 μl/ml acetic anhydride (Sigma-Aldrich Inc.) for 10 min. Sections were then rinsed two times in 2x sodium citrate, sodium chloride (SSC; Ambion, Inc.) for 3 min each, dehydrated in a graded series of ethanol (70, 90, and 100%; 3 min each), and allowed to air dry before the hybridization procedure.

    The volume of the probe was calculated (1.5 x 106 cpm/slide) and allowed to thaw on ice. The probe mix was prepared by combining the probe with 1/20 vol Torula RNA (Sigma-Aldrich Inc.) and 0.1 M Tris/0.01 M EDTA (pH 8.0) and then mixed with hybridization buffer [6.25% deionized formamide, 12.5% dextran sulfate, 0.375 M NaCl, 10 mM Tris (pH 8.0), 1.6 mM EDTA, 1.25x Denhardt’s solution, and 10 mM dithiothreitol] at a ratio of 1:3. The probe mix was then heat-denatured by placing into a 65 C water bath for 3 min. After adding 150 μl of the hybridization mix per slide (equivalent to 1.5 x 106 cpm/slide), the sections were covered with Parafilm coverslips and incubated at calculated hybridization temperatures (NPY, 52 C; AGRP, 50 C; and CART, 50 C) overnight (16–17 h) in a humid chamber.

    After hybridization, coverslips were removed and slides were washed twice in 2x SSC for 30 min each at room temperature. Sections were then incubated in ribonuclease (RNase) buffer containing RNase A (Roche Molecular Biochemicals, Indianapolis, IN; 0.02 mg/ml RNase A in 10 mM Tris, 0.5 M NaCl, and 1 mM EDTA [pH 8.0]) for 30 min at 37 C, then in RNase buffer without RNase A at 37 C for another 10 min. Slides were then washed for 5 min in 2x SSC at room temperature, followed first by a 30-min wash in 0.1x SSC at 62 C for NPY and at 60 C for AGRP and CART, then by another 30-min wash in fresh 0.1x SSC at the same temperatures. Thereafter, sections were washed twice for 5 min in 0.1x SSC at room temperature and dehydrated in a graded series of ethanol (50, 70, 90, and 100%, 3 min each). Finally, the sections were air-dried and exposed to hyperfilm (Kodak, Eastman Kodak Co., Rochester, NY) at –80 C for 3 d for NPY and CART and 5 d for AGRP.

    After the film was developed, the signal on the film was quantified using densimetric analysis (AIS, Imaging Research, St. Catherines, Ontario, Canada) according to a previously reported method (39). Briefly, to quantify, an analog camera was used to capture and magnify the image of each section, with the lighting and magnification kept constant for the entire study. The area to be sampled in the ARC was established and kept constant for all sections. A threshold OD reading for the selected region was established and kept constant for the measurement of all brains. OD values below this level were not selected. Three sections from each brain were sampled, and for each section, the OD of the region, subtracting out background, and the sample area were recorded. The peptide mRNA values in the ARC for each experiment are in arbitrary units of density x area; therefore they have been converted to a percentage of the respective peptide mRNA measured in control animals.

    Immunohistochemistry

    At the conclusion of the experiments, rats were killed using a lethal dose of pentobarbital sodium (Abbott Laboratories, North Chicago, IL; 300 mg/kg) and perfused transcardially with PBS (pH 7.4) and then with fresh 4% formalin solution prepared with phosphate buffer (PB) (pH 7.4). Brains were removed, postfixed at room temperature for 4 h in 4% formalin, and cryoprotected overnight in 25% sucrose solution. Coronal cryostat sections (40 μm) through the length of the ARC and medulla oblongata were collected in multiple sets. Sections were placed in 0.1 M PB (pH 7.4) and processed using previously described immunohistochemical techniques (40). After pretreatment with 50% ethanol for 20 min, sections were washed (3 x 5 min) in 0.1 M PB and incubated for 1 h in 10% normal horse serum [made in Tris sodium PBS (TPBS) containing 0.05% thimerosol]. The blocking solution was removed from the tissue was incubated in the respective primary antibody; rabbit anti-NPY-YI (generous gift of CURE, University of California Los Angeles; 1:25,000), rabbit anti-NPY (Chemicon, 1: 50,000), sheep -MSH (1:25,000, Chemicon), and mouse monoclonal anti-tyrosine hydroxylase (TH; Roche Diagnostics, Mannheim, Germany; 1:1000) made up in 10% normal horse serum-TPBS. After 48 h, the primary antibody was removed, the sections washed (3 x 10 min) in TPBS, and then incubated in a secondary antibody (biotinylated donkey antirabbit for Y1 receptor and NPY and biotinylated donkey antimouse for TH, 1:500, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) made in 1% normal horse serum-TPBS. After 24 h, the tissue was washed (3 x 10 min) in TPBS, incubated with Extravidin-peroxidase (1:1500 in TPBS, Sigma-Aldrich Inc.) overnight, washed again (3 x 10 min), and reacted for visualization of NPY-Y1 receptors, NPY, -MSH, and TH immunoreactivity by using nickel-intensified diaminobenzidine in the peroxidase reaction to produce a black reaction product. Sections were then mounted on slides and cover-slipped for microscopic evaluation. Antibodies were titrated before use to determine optimal concentrations. Standard controls for the specificity of primary antibodies were used, including the incubation of the tissue with normal instead of immune serum and preincubation of the immune serum with the targeted antigen before its application to tissue. The immunohistochemical findings were not quantified.

    Statistical analysis

    A two-way ANOVA followed by Bonferroni’s pair-wise multiple comparison test was used to analyze the effect of different doses of NPY-SAP on body weight. Feeding responses were analyzed by repeated measures ANOVA followed by the Bonferroni pair-wise multiple comparisons procedure. Differences were considered significant if P 0.05. Values for GLP-1, NPY, CCK, ghrelin, 2DG, and MA are reported as grams of food consumed. For leptin-treated animals, results for food intake are represented as percent change from baseline intake. Similarly, body weight changes in leptin-treated animals are represented as percent change over baseline body weight. Density values for in situ hybridization studies are reported for the NPY-SAP group as a percentage of the density value obtained for the corresponding brain region in the B-SAP group. Groups were compared using the Student’s t test.

    Results

    Receptor binding

    Results of the competitive binding study using I125-NPY and rat forebrain homogenates indicate that NPY-SAP binds to and has a higher binding affinity than NPY for the NPY receptor (Fig. 1).

    FIG. 1. Competitive binding of NPY and NPY-SAP with I125-NPY in rat forebrain tissue homogenates. Duplicate determinations were made for each concentration. Bars below show the IC50 for NPY and NPY-SAP binding. Data show that NPY-SAP has a binding affinity for NPY receptors that is equal to or greater than NPY at the concentrations examined.

    Effect of NPY-SAP dose on body weight and spontaneous daily food intake

    Basomedial hypothalamic injections of NPY-SAP (12, 24, and 48 ng) produced dose-dependent increases in body weight (Fig. 2). Eight weeks after injections into the BMH, B-SAP and NPY-SAP groups, respectively, weighed 400.9 ± 9.4 vs. 456.9 ± 20.4 g, P = 0.03, for 12 ng; 448.7 ± 4.8 vs. 512 ± 17.1 g, P = 0.005, for 24 ng; and 407 + 8.8 vs. 512.8 + 29.6 g, P = 0.005, for 48 ng. Expressed as percent change from preinjection body weights, the changes 8 wk after each of the three doses, respectively, were 23, 30, and 35% in the NPY-SAP rats, compared with 14, 19, and 13% in the B-SAP rats.

    FIG. 2. Body weights of rats 8 wk after bilateral injection of B-SAP control or NPY-SAP (12, 24, or 48 ng/100 nl per side) into the BMH just dorsal to the ARC. Body weights were significantly increased in the NPY-SAP rats, compared with the B-SAP rats, at both the 24- and 48-ng doses (P < 0.01 and P < 0.001, respectively).

    The effects of NPY-SAP on daytime and nighttime feeding are shown in Fig. 3. In animals injected with NPY-SAP in the BMH, the 12-ng dose of NPY-SAP had no effect on daytime feeding (B-SAP, 4.7 ± 0.4 g; NPY-SAP, 6.1 ± 0.7 g; P = 0.1) or nighttime feeding (B-SAP, 18.5 ± 0.6 g; NPY-SAP, 16.4 ± 1.1 g; P = 0.2). However, both 24 and 48 ng NPY-SAP produced significant increases in daytime feeding without altering nighttime feeding. After 24 ng NPY-SAP, daytime food intake (B-SAP, 5.9 ± 0.5 g; NPY-SAP, 9.7 ± 0.6 g; P < 0.001) was almost twice that of control animals. Similarly, 48 ng of NPY-SAP resulted in an almost doubling of daytime food intake (B-SAP, 5.7 ± 0.4 g; NPY-SAP, 9.2 ± 0.8 g; P = 0.002). In contrast, NPY-SAP did not have a significant effect on nighttime feeding after either 24 (B-SAP, 19.6 ± 0.7 g; NPY-SAP, 21.0 ± 1.5 g; P = 0.4) or 48 (B-SAP, 16.5 ± 1.1 g; NPY-SAP, 18.4 ± 2.8 g; P = 0.5) ng of NPY-SAP.

    FIG. 3. Daytime (top) and nighttime (bottom) food intake of rats injected bilaterally into the BMH with B-SAP control or NPY-SAP (12, 24, or 48 ng/100 nl per side). Daytime food intake of rats injected with 24 or 48 ng NPY-SAP was nearly doubled, compared with that of B-SAP-injected rats (P < 0.001 and P < 0.002). These doses of NPY-SAP did not alter food intake during the dark period.

    Feeding responses to peptides, MA and 2DG

    Food intake was not significantly reduced by leptin treatment in NPY-SAP rats (P = 1 vs. aCSF baseline) (Fig. 4). In contrast, after the first of four leptin injections, the 24-h food intake in B-SAP rats was reduced by 23.9 ± 3.6% (P < 0.001 vs. aCSF baseline). This trend continued and by the fourth day of leptin treatment B-SAP rats had decreased their food intake 35.2 + 3.3% of control values. Food intake of B-SAP rats rapidly returned to baseline levels when leptin treatment was stopped, and intake remained at this level for the remainder of the experiment. Similarly, body weights of NPY-SAP-injected rats did not differ from baseline levels after leptin injections. Compared with their baseline weights, B-SAP animals lost 3.1 ± 1.1% (P = 0.04) by d 3 and 5.0 ± 1.2% (P < 0.001) by d 4. Body weights of the B-SAP rats then steadily increased until reaching baseline levels 6 d later.

    FIG. 4. Food intake (top) and body weights (bottom) of rats injected bilaterally into the BMH with B-SAP control or NPY-SAP (48 ng/100 nl) and subsequently treated with leptin. Rats were injected into the lateral ventricle with aCSF (5 μl) each day for 3 d followed by four daily injections of leptin (5 μg in 5 μl aCSF) and 8 additional recovery d of aCSF. Average 24-h food intake and body weight during the three control days was determined for each rat and changes during the subsequent leptin and aCSF postleptin recovery days were calculated as percentage change from that average control value. Days on which leptin were injected are indicated by the bar above the graphs (d 1–4). Food intake was significantly reduced from baseline on all four leptin treatment days in B-SAP animals (*, P < 0.001) and then rapidly returned to baseline after termination of leptin treatment. In contrast, food intake of NPY-SAP-injected rats was not reduced by leptin. Leptin significantly reduced body weight in B-SAP rats from their control level, with the nadir of weight loss during the day after the last leptin injection, followed by recovery (*, P < 0.001). Leptin did not reduce body weight of NPY-SAP-injected rats.

    Feeding responses of NPY-SAP and B-SAP rats to ghrelin, GLP-1, NPY, CCK, and MA are shown in Fig. 5.

    FIG. 5. Feeding responses of rats injected bilaterally into the BMH with B-SAP or NPY-SAP (48 ng/100 nl). The various graphs show responses to ghrelin, GLP-1, CCK, MA, and NPY. All tests were conducted with pelleted rodent chow during the light period. Ghrelin, GLP-1, and NPY were injected into the lateral cerebral ventricle in 3–5 μl of aCSF vehicle. Feeding responses to ghrelin (2 μg) were measured in a 4-h test immediately after the injection. Effects of GLP-1 (5 μg) were measured in a 1-h test after overnight food deprivation. CCK (4 μg, 1 ml/kg) was injected ip after overnight food deprivation, and food intake was measured in a 30-min test. ?-MA was injected ip (68 mg/kg, 1 ml/kg), and food intake was measured in a 4-h test. The baseline intakes in the GLP-1 and CCK tests also indicate that responses of NPY-SAP rats to overnight food deprivation did not differ from those of the B-SAP rats. Effects of NPY (5 μg) were measured in a 2-h test immediately after the injection. Differential impairment of the response to leptin (Fig. 4), and ghrelin may be due to their greater dependence on neural circuits within the ARC that were disrupted by the NPY-SAP injections. In contrast, responses to GLP-1, CCK, MA, and NPY, which are mediated in large part by extrahypothalamic neural circuits, were not impaired by the NPY-SAP injections. **, P < 0.001, B-SAP vs. NPY-SAP; *, P < 0.001, vs. SALINE or aCSF and P > 0.1, B-SAP vs. NPY-SAP.

    Ghrelin.

    Injections of NPY-SAP abolished the feeding response to icv ghrelin administration (Fig. 5). B-SAP and NPY-SAP animals ate 1 ± 0.5 vs. 1.8 ± 0.4 g (P = 0.2) after aCSF and 3.1 ± 0.1 vs. 1.9 ± 0.4 g (P < 0.001) after ghrelin, respectively.

    GLP-1.

    GLP-1 suppressed deprivation-induced feeding to the same extent in both NPY-SAP- and B-SAP-injected animals. Animals ate 6.1 ± 0.8 vs. 5.6 ± 0.8 g (P = 0.707) after CSF and 3.8 ± 0.5 vs. 3.4 ± 0.5 g (P = 0.6) after GLP-1 for B-SAP- and NPY-SAP-injected rats, respectively.

    NPY.

    NPY-induced feeding was present in both NPY-SAP and B-SAP rats and did not differ significantly between groups. B-SAP and NPY-SAP rats ate 3.2 ± 0.9 and 3.6 ± 0.9 g above baseline intakes, respectively, in the 2-h test.

    CCK.

    Deprivation-induced feeding was suppressed to the same extent in both NPY-SAP and control B-SAP animals. Rats ate 5.1 ± 0.2 vs. 4.8 ± 0.2 g (P = 0.4) after saline and 1.9 ± 0.2 g vs. 2.2 ± 0.3 g (P = 0.4) after CCK octapeptide for B-SAP and NPY-SAP rats, respectively.

    MA.

    MA significantly stimulated food intake in both NPY-SAP and B-SAP control rats. B-SAP and NPY-SAP rats ate 2.6 ± 0.3 vs. 2.5 ± 0.4 g (P = 0.7) after saline and 4.3 ± 0.3 vs. 5.5 ± 0.4 g (P = 0.040) after MA, respectively.

    2DG.

    Injections of NPY-SAP into the BMH did not impair 2DG-induced feeding or hyperglycemic responses (Fig. 6). B-SAP-injected rats ate 1.1 ± 0.3 g of food in response to saline injection and 3.6 ± 0.4, 4.6 ± 0.5, and 6.8 ± 0.4 g in response to 100, 200, and 400 mg/kg 2DG, respectively. NPY-SAP rats ate 2.3 ± 0.4 g in response to saline injection and 3.8 ± 0.8, 4.6 ± 0.5, and 6.2 ± 0.5 g in response to 100, 200, and 400 mg/kg 2DG, respectively. For each group, food intake was increased significantly above baseline by 2DG (P < 0.05), and there were no differences between B-SAP and NPY-SAP at any dose. Similarly, the blood glucose response to 2DG (200 mg/kg) did not differ between groups. Basal values were similar and the peak response occurred at 60 or 90 min after 2DG for all rats.

    FIG. 6. Feeding (top) and hyperglycemic responses (bottom) to glucoprivation in rats injected bilaterally into the BMH with B-SAP or NPY-SAP (48 ng/100 nl). Glucoprivation was induces by sc administration of 2DG. Food intake was measured in 4-h tests beginning immediately after 2DG injection (100, 200, or 400 mg/kg). Plasma glucose was measured from tail blood at the times indicated. Food was removed 2 h before the injection of 2DG (200 mg/kg) and was withheld until the last blood sample was taken. +, P < 0.05 vs. B-SAP SAL; *, P < 0.05 vs. NPY-SAP SAL.

    In situ hybridization

    Figure 7 shows the results of the quantification of gene expression in the BMH. The OD of mRNA after bilateral NPY-SAP injection is expressed as a percentage of the values for the same mRNA from the anatomically equivalent area of B-SAP controls. NPY-SAP caused a significant reduction in NPY mRNA expression (B-SAP, 100 ± 15.4%; NPY-SAP, 9.4 ± 1.7%; P < 0.001), AGRP mRNA expression (B-SAP, 100 ± 16.0%; NPY-SAP, 11.9 ± 1.2%; P < 0.001) and CART mRNA expression (B-SAP, 100 ± 9.1%; NPY-SAP, 1 ± 0.3%; P < 0.001). Figure 8 provides a pseudocolor representation of mRNA expression in the ARC in the same rats.

    FIG. 7. Drawing (top) adapted from Paxinos and Watson (31 ) shows approximate area (oval) within which NPY, AGRP, and CART mRNAs were quantified by densitometric analysis. Injections of NPY-SAP (48 ng/100 nl) or B-SAP control were directed bilaterally into the BMH just dorsal to the ARC at 2.8 mm caudal to bregma. Quantification was done at three levels of the ARC (2.80 ± 0.5 mm caudal to bregma). Bars (bottom) show NPY, AGRP, and CART mRNA hybridization signal in the BMH of animals injected with NPY-SAP, as measured by OD. OD for each peptide mRNA is expressed as percentage of the OD of the same peptide mRNA from the anatomically equivalent areas taken from controls injected with B-SAP. See also Fig. 8.

    FIG. 8. Pseudocolor representation of OD of NPY, AGRP, and CART mRNA expression in the ARC after bilateral injection of B-SAP (left) or NPY-SAP (right) into the BMH. See Fig. 7 legend for more information. NPY-SAP significantly reduced NPY, AGRP, and CART hybridization signal in the BMH.

    Immunohistochemistry

    In preliminary studies, we observed a significant reduction of NPY-immunoreactivity 10 d after NPY-SAP injection in a number of sites known to be innervated by ARC NPY neurons, including the PVH (Fig. 9), suprachiasmatic nucleus, and paraventricular nucleus of the thalamus. However, most animals used in this study were killed for immunohistochemical analysis at the conclusion of behavioral studies, at which time the NPY-immunoreactivity in the NPY-SAP rats did not appear to differ from B-SAP controls in any of the previously affected areas, as shown in Fig. 10 for the ARC and PVH. However, apparent in Fig. 10 and in cresyl violet-stained sections (Fig. 11), the NPY-SAP injection caused a persisting loss of cellularity in the ARC and a dramatic reduction in ARC NPY Y1 and -MSH immunoreactive neurons (Fig. 12). This evidence of neuronal destruction is consistent with the loss of CART, AGRP, and NPY gene expression. TH immunoreactivity in the hindbrain catecholamine cell groups with projections to the hypothalamus did not appear to be reduced at any time point by NPY-SAP. Particular attention was paid to the area of overlap of cell groups A1 and C1 (Fig. 13). Because nearly all of the catecholamine neurons in this particular area coexpress NPY and project to the medial hypothalamus, results suggest that NPY-SAP was not retrogradely transported.

    FIG. 9. Photomicrographs showing NPY-immunoreactive fibers and terminals in the PVH 10 d after microinjection of the BMH with B-SAP (left) or NPY-SAP (right). At this early postinjection time point, NPY immunoreactivity appeared to be reduced in NPY-SAP-injected rats, compared with B-SAP-injected rats. Calibration bar, 100 μm.

    FIG. 10. Coronal sections of rat ARC (A and B) and PVH (C and D) showing NPY immunoreactivity several months after bilateral BMH injection of B-SAP (left) and NPY-SAP (right). The arrow in A indicates the location of the microcapillary injector pipette tip from a rat injected with B-SAP control solution. The arrow in B indicates deposits of colloidal gold (black particulate), coinjected with NPY-SAP to mark the injection site. At the conclusion of behavioral testing, NPY immunoreactive fibers and terminals did not appear to differ between rats injected with B-SAP and NPY-SAP.

    FIG. 11. Coronal cryostat sections stained with cresyl violet showing effects of B-SAP (left) and NPY-SAP (right) on cytoarchitecture at injection sites in the ARC. The arrow in B indicates particles of colloidal gold, which was coinjected with NPY-SAP in some animals to mark the injection site. A loss of cellularity is apparent in the NPY-SAP injection field. Calibration bar, 100 μm.

    FIG. 12. Coronal sections showing effects of B-SAP (left) and NPY-SAP (right) injection on NPY Y1 receptor (A and B) and MSH (C and D) immunoreactivity in the ARC. B-SAP and NPY-SAP were microinjected into the BMH just dorsal to the ARC. The arrow (B) indicates particles of colloidal gold, coinjected with NPY-SAP in some animals to mark the injection site. NPY-SAP caused profound reduction of MSH and NPY-Y1 immunoreactivity throughout the ARC.

    FIG. 13. Coronal sections showing TH immunoreactivity in the ventrolateral medullary catecholamine cell column from rats injected bilaterally into the BMH with NPY-SAP (48 ng in 100 nl) or an equivalent amount of B-SAP control solution. The area shown is the area of overlap of A1 and C1 cell groups, where nearly all TH-ir neurons coexpress NPY and innervate medial hypothalamic structures. The number of immunoreactive cells did not appear to differ between NPY-SAP- and B-SAP-injected rats, indicating that NPY-SAP was not retrogradely transported in these neurons and therefore did not destroy them.

    Discussion

    Competitive binding studies showed that NPY-SAP had a higher affinity for NPY receptors than NPY itself, suggesting that agonist-driven receptor internalization (22, 23, 24, 25, 26) is the probable mechanism for selective internalization of this toxin. Accordingly, NPY/AGRP and POMC/CART neurons in the ARC, both of which express NPY receptors (41, 42), were destroyed by NPY-SAP. Compared with the B-SAP controls, NPY and AGRP mRNA hybridization signals in the ARC were reduced by 90% in rats injected with NPY-SAP, and CART mRNA hybridization signal was reduced by 99%. Although not quantified, it was apparent that expression of CART mRNA was also reduced profoundly in the dorsomedial hypothalamus. Presumably, the latter area was within the diffusion radius of the NPY-SAP injection, which was directed at a point just dorsal to the ARC. Immunoreactive -MSH, a peptide cleaved from the POMC precursor molecule, and NPY Y1 receptor immunoreactivity were also reduced in the area of the injection. B-SAP, which is not an NPY receptor ligand, did not cause comparable destruction in the ARC.

    NPY cell bodies in the ARC contribute importantly to the innervation of hypothalamic nuclei (7, 8). Although immunoreactive NPY terminals were reduced in the hypothalamus soon after the NPY-SAP injection, NPY-terminal immunoreactivity appeared to be similar to control at the conclusion of the experiments, several weeks or months later. Because our results leave no doubt that ARC NPY cell bodies were destroyed by the NPY-SAP lesion, the apparent recovery of NPY terminals is intriguing. One possible explanation of this recovery is that surviving NPY neurons outside the ARC that innervate hypothalamic nuclei may extend new processes into areas partially denervated by destruction of ARC NPY cell bodies. Hindbrain NPY/catecholamine coexpressing neurons could potentially be involved in such a process because they innervate many of the same sites innervated by ARC NPY neurons (30, 43) and were not destroyed by the NPY-SAP lesion. A systematic analysis of terminal loss and regrowth will be required to resolve this issue.

    The fact that the cell bodies of hindbrain catecholamine/NPY neurons did not appear to be reduced in number by the hypothalamic NPY-SAP injections is noteworthy. Presumably, the hindbrain NPY neurons were not destroyed because the saporin was not internalized by their hypothalamic terminals or because the saporin, once internalized, was not transported retrogradely to the soma where it exerts its toxic action. The lack of retrograde transport has been observed for some other SAP-peptide conjugates (44) and distinguishes these conjugates from the immunotoxin, anti-dopamine-?-hydroxylase conjugated to saporin, which is retrogradely transported (38, 40, 45).

    Disruption of NPY/AGRP and POMC/CART circuitry in the ARC by NPY-SAP impaired responses to intraventricular ghrelin and leptin. These impairments are consistent with results showing that leptin exerts a major influence on food intake and body weight through its actions within the ARC. Destruction of circumventricular organs, including the ARC, by systemic treatment of neonatal rats with monosodium glutamate, attenuates leptin-mediated effects (46, 47). In addition, a direct effect of leptin on NPY, POMC, and CART expression in ARC neurons has been demonstrated (29, 41, 48, 49). Electrophysiological evidence has shown that leptin directly stimulates POMC neurons by depolarization through a nonspecific cation channel and indirectly stimulates them by inhibiting the adjacent NPY/-aminobutyric acid neurons (29).

    NPY-SAP treatment also abolished stimulation of food intake by ghrelin. Ghrelin is the natural ligand for the growth hormone secretagogue receptor 1a, which is expressed by neurons in the hypothalamus and pituitary, including NPY/AGRP, POMC, GHRH, and somatostatin neurons of the ARC (50). Numerous studies have shown that both peripheral and central administration of ghrelin stimulate food intake and increase body weight in rats (51, 52, 53). Although a single report has suggested that the orexogenic effects of ghrelin are vagally mediated (54), other investigators have been unable to substantiate vagal participation in ghrelin’s action (55). On the other hand, it is well established that the orexigenic actions of ghrelin are mediated primarily by its actions on ARC NPY/AGRP neurons (56, 57, 58, 59). Moreover, a selective NPY Y1 receptor antagonist attenuated the orexigenic effect of ghrelin in a dose-dependent fashion, further supporting the role of the ARC NPY system in mediating the orexigenic effect of ghrelin (56). Finally, lesion of the ARC by systemic neonatal monosodium glutamate administration abolished the orexigenic effect of ghrelin (60). The loss of responsiveness to leptin and ghrelin in the present study therefore provides functional confirmation of the NPY-SAP lesion because compelling evidence demonstrates that these peptides influence food intake primarily, although perhaps not exclusively, by action on the NPY/AGRP and POMC/CART neurons within the ARC.

    The present series of experiments was initiated to assess the importance of NPY/AGRP neurons in the ARC for control of glucoprivic feeding. The BMH is a site where glucose-excited and glucose-inhibited neurons have been identified using electrophysiological approaches (2, 3, 4) and where neural substrates for glucoregulatory responses have been proposed to reside (61, 62). Moreover, some NPY and POMC neurons in the ARC express glucokinase (63), a glycolytic enzyme with glucose-sensing properties in pancreatic ?-cells and some central neurons (64). In addition, glucoprivation increases the expression of the c-fos gene, an indicator of neuronal activation, in some hypothalamic NPY neurons (65), as well as the expression of both NPY and AGRP mRNA (12, 13, 66) and NPY peptide levels (11). Our own previous work using the retrogradely transported immunotoxin, anti-d?h- saporin, showed that destruction of hindbrain catecholamine neurons that innervate the medial hypothalamus both impaired the glucoprivic feeding response and eliminated the glucoprivation-induced increase in expression of AGRP and NPY mRNA in the ARC (40, 66). Together, these findings suggested that the AGRP/NPY neurons are major downstream components of the neural pathway controlling glucoprivic feeding. However, the present results do not support such a role for these neurons. Despite the destruction of NPY/AGRP neurons, NPY-SAP-injected rats increased their food intake normally across a range of 2DG doses. These findings are consistent with earlier reports that electrolytic lesion of the PVH, a major projection site for ARC NPY/AGRP neurons, does not impair glucoprivic feeding (67, 68, 69). Therefore, although NPY/AGRP neurons may contribute to glucoprivic feeding in intact rats, clearly they are not required for this response.

    Previous work has shown that NPY –/– mice have impaired feeding responses to glucoprivation (14). In light of the present results showing that lesion of ARC NPY neurons does not impair glucoprivic feeding, the deficit in this response in NPY null mice strongly suggests that glucoprivic feeding depends most heavily on hindbrain NPY neurons. In contrast to NPY/AGRP neurons, hindbrain NPY/catecholamine neurons do appear to be required for glucoprivic feeding. Hindbrain NPY mRNA is increased in response to glucoprivation (38). Furthermore, lesion of hindbrain NPY/catecholamine neurons using the immunotoxin, antidopamine ?-hydroxylase saporin, abolishes glucoprivic feeding (38, 40), although forebrain NPY neurons, including the ARC NPY/AGRP neurons, are not destroyed by this lesion.

    In addition to normal glucoprivic feeding responses, NPY-SAP-injected rats also had normal feeding responses to MA-induced blockade of fatty acid oxidation, systemic administration of CCK, and intraventricular administration of GLP-1. A feature shared by these latter three responses is that they appear to depend heavily upon neural circuits outside the ARC, including the vagus nerve or dorsal vagal complex and lateral parabrachial nucleus. Lesion and Fos mapping studies indicate that MA-induced feeding is largely mediated by a vagally driven pathway using the nucleus of the solitary tract, lateral parabrachial nucleus, and central nucleus of the amygdala (36, 37, 67, 70, 71, 72). Similarly, GLP-1 cell bodies are found in the nucleus of the solitary tract, and they extend axons into the hypothalamus and other brain regions (73, 74). Receptors for GLP-1 are present in the ARC, on cell types including POMC neurons, and in the PVH, supraoptic nucleus, and dorsal hindbrain (75). The effectiveness of intracerebroventricular injections of GLP-1 in suppressing food intake in intact rats is well-established (76, 77, 78). Our results showing that suppression of food intake by GLP-1 was not attenuated by NPY-SAP indicate either that GLP-1-responsive cells in the ARC are not lesioned by NPY-SAP or that the GLP-1-responsive neurons outside the hypothalamus are capable of independent mediation of GLP-1’s suppression of feeding. The observation that the innervation by hindbrain GLP-1 neurons is not restricted to the ARC (73, 74) and that hindbrain GLP-1-receptive sites appear to play a role in suppression of feeding (79, 80) would lend support to the latter possibilities. Finally, peripheral CCK-induced suppression of food intake requires vagal sensory neurons terminating in the nucleus of the solitary tract (81, 82, 83, 84). Although signals initiated by CCK may eventually contribute to integrative circuits in the hypothalamus (85), it is clear that CCK-induced satiety does not require the forebrain. When the hypothalamus and hindbrain are disconnected by a supracollicular decerebration, rats continue to suppress their intake in response to CCK (86). The present results showing preservation of responses to MA, GLP-1, and CCK therefore indicate that NPY-SAP did not produce widespread disruption of all controls of food intake but produced its effects through a localized toxic effect in the BMH and on controls of food intake dependent on neurons in this site.

    The fact that NPY-SAP did not impair NPY-induced feeding is understandable in light of the widespread distribution of NPY receptors throughout the brain (87, 88, 89, 90, 91). Based on our histological results, neurons that express NPY receptors but whose cell bodies lie outside the radius of diffusion of the NPY-SAP would be expected to remain intact and responsive to administration of exogenous NPY.

    NPY-SAP produced a significant increase in daily food intake, which could be accounted for almost entirely by increased feeding during the light phase of the circadian cycle, a time when normal rats eat very little. Daytime intakes were nearly doubled in NPY-SAP rats compared with controls. The disruption of circadian feeding patterns by NPY-SAP is similar to effects observed in animals with MSG or electrolytic lesion of the ARC (92, 93, 94, 95). There are neuronal connections between the suprachiasmatic nucleus, the dominant mammalian circadian pacemaker (96, 97), and a number of hypothalamic nuclei, including the ARC (98, 99, 100, 101). Thus, it is reasonable to speculate that the ARC may act in concert with the suprachiasmatic nucleus and other hypothalamic nuclei, such as the ventromedial nucleus (102), to integrate ingestive controls with the circadian cycle. The fact that NPY release in the hypothalamus follows a circadian pattern, with an increase in prepro-NPY mRNA expression in the basal hypothalamus just preceding the onset of the dark phase (103), suggests that ARC NPY neurons may be of particular importance in this process.

    In summary, anatomical and behavioral results reported here demonstrate that injection of NPY-SAP into the ARC produces a site-specific lesion of NPY-receptor-expressing neurons. This lesion clearly differentiates ingestive controls dependent on NPY-receptor expressing neurons in the ARC from those, such as glucoprivic feeding, that depend on hindbrain processes. A major goal of this work was to determine whether glucoprivic feeding requires NPY/AGRP neurons within the ARC. The results across a series of 2DG doses show that glucoprivic feeding does not require these neurons. In addition to these findings, the present report is the first to describe effects of NPY-SAP and to demonstrate that this novel toxin is a useful tool for chemical microdissection of circuits underlying physiological and behavioral responses that involve NPY-receptive neurons.

    Acknowledgments

    We thank Dr. Douglas A. Lappi of Advanced Targeting Systems, Inc. for donation of the NPY-SAP and Shayne Andrew and Shannon Roland for their excellent technical assistance.

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