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Endocrinology Vol. 144, No. 1 75-83
Copyright © 2003 by The Endocrine Society

ARTICLE

Immunolesion of Norepinephrine and Epinephrine Afferents to Medial Hypothalamus Alters Basal and 2-Deoxy-D-Glucose-Induced Neuropeptide Y and Agouti Gene-Related Protein Messenger Ribonucleic Acid Expression in the Arcuate Nucleus

G. S. Fraley1 and S. Ritter

Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington 99164-6520

Address all correspondence and requests for reprints to: Dr. S. Ritter, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520. E-mail: sjr{at}vetmed.wsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptide Y (NPY) and agouti gene-related protein (AGRP) are orexigenic peptides of special importance for control of food intake. In situ hybridization studies have shown that NPY and AGRP mRNAs are increased in the arcuate nucleus of the hypothalamus (ARC) by glucoprivation. Other work has shown that glucoprivation stimulates food intake by activation of hindbrain glucoreceptor cells and requires the participation of rostrally projecting norepinephrine (NE) or epinephrine (E) neurons. Here we determine the role of hindbrain catecholamine afferents in glucoprivation-induced increase in ARC NPY and AGRP gene expression. The selective NE/E immunotoxin saporin-conjugated antidopamineß-hydroxylase (anti-dßh) was microinjected into the medial hypothalamus and expression of AGRP and NPY mRNA was analyzed subsequently in the ARC under basal and glucoprivic conditions using 33P-labeled in situ hybridization. Saporin-conjugated anti-dßh virtually eliminated dßh-immunoreactive terminals in the ARC without causing nonspecific damage. These lesions significantly increased basal but eliminated 2-deoxy-D-glucose-induced increases in AGRP and NPY mRNA expression. Results indicate that hindbrain catecholaminergic neurons contribute to basal NPY and AGRP gene expression and mediate the responsiveness of NPY and AGRP neurons to glucose deficit. Our results also suggest that catecholamine neurons couple potent orexigenic neural circuitry within the hypothalamus with hindbrain glucose sensors that monitor brain glucose supply.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDE Y (NPY) AND agouti gene-related protein (AGRP) are neuropeptides that have been strongly implicated in the control of appetite. Though neurons producing NPY are widely distributed throughout the central and peripheral nervous system, the highest concentration of NPY cell bodies is in the arcuate nucleus of the hypothalamus (ARC) (1). AGRP, an endogenous melanocortin receptor antagonist, is also found in many tissues throughout the body. However, within the central nervous system, AGRP is produced exclusively by ARC cell bodies that coexpress NPY (2). Both NPY and AGRP are present in dense terminal networks throughout the medial hypothalamus, including the paraventricular hypothalamic nucleus (PVH) (2, 4, 5). NPY mRNA levels (6) and NPY release (7) are increased in the hypothalamus by fasting. In addition, exogenous NPY administered into the PVH or surrounding hypothalamic sites produces potent orexigenic effects (8, 9, 10). Repeated (11) or continuous (12) administration of NPY into hypothalamic sites produces obesity. Similarly, intracerebral injection of AGRP causes pronounced and prolonged elevation of food intake in rats (13, 14), with the most potent site of action being the PVH (15). Overexpression of AGRP is associated with obesity (16, 17).

Both NPY and AGRP appear to be involved in the stimulation of food intake induced by glucose deficit (glucoprivic feeding). Feeding induced by 2-deoxy-D-glucose (2DG), a nonmetabolizable glucose analog that competitively inhibits glucose utilization (18), is attenuated by immunoneutralization of endogenous NPY in the PVH (19). In addition, NPY peptide levels (20) and levels of both NPY mRNA (21) and AGRP mRNA (21, 22) are increased in the ARC by 2DG. The effect of 2DG on NPY and AGRP neurons appears to be specifically related to glucose deficit because ß-mercaptoacetate, which blocks fatty acid oxidation and stimulates food intake, does not produce these effects (20, 21).

The responsiveness of AGRP and NPY neurons to glucose deficit is a highly significant observation because it identifies a specific endogenous signal that drives these neurons, presumably engaging them in a stimulatory control of appetite. Thus, the responsiveness of AGRP and NPY neurons to glucose deficit may be one mechanism that couples appetite to metabolic need. However, the basis for the responsiveness of these neurons to glucose deficit is not known. One possibility is that the activity of NPY and AGRP neurons is directly altered by glucose availability. Neurons of unknown function that change their electrical activity in response to alterations in glucose availability have been described in the hypothalamus and elsewhere in the brain (23, 24, 25, 26). Alternatively, the responsiveness of AGRP and NPY neurons to glucoprivation may be determined by their afferent inputs. In the present experiment, we test the hypothesis that the response of AGRP and NPY neurons to glucoprivation requires afferent input from hindbrain catecholamine neurons. The ARC is innervated heavily by norepinephrine (NE) and epinephrine (E) neurons (26A ). These neurons are activated by glucoprivation (27, 28, 29, 30, 31) and pharmacological blockade of their receptors impairs glucoprivic feeding (32).

Recent results using the ribosomal toxin, saporin (SAP), conjugated to a monoclonal antibody against dopamineß-hydroxylase (dßh), confirm and extend previous evidence indicating a crucial role for NE and E neurons in glucoregulatory responses, including glucoprivic feeding (33). Anti-dßh-SAP (DSAP) selectively targets NE and E neurons, which uniquely express the dßh enzyme (34, 35, 36, 37). When injected into NE and E terminal areas, the immunotoxin is selectively internalized by NE and E terminals and retrogradely transported to their cell bodies in the hindbrain (33, 37), destroying the cell bodies and their processes. We have found that this can be accomplished without producing detectable neurological impairment, nonspecific damage at the injection site, or destruction of catecholamine cell bodies that do not innervate the injection site (33, 38). Results from our laboratory also have shown that PVH microinjection of DSAP profoundly reduces feeding, corticosterone secretion, and Fos-immunoreactivity (ir) in the PVH and ARC in response to glucoprivation, demonstrating the dependence of these responses on the rostral projections of hindbrain NE or E neurons. In comparison, microinjections of DSAP into the spinal cord selectively lesion spinally projecting, but not PVH projecting, catecholamine neurons. Glucoprivic stimulation of the adrenal medulla, which is not impaired by PVH injections, is profoundly reduced by spinal cord DSAP injections (33). These findings strongly indicate that catecholamine neurons transmit information from hindbrain glucoreceptive sites (39) that is essential for elicitation of a number of glucoregulatory responses, including feeding. Therefore, DSAP is an effective and selective method of eliminating these neurons and the responses they mediate.

In the present experiment, we used DSAP immunolesions to examine the role of NE/E neurons in glucoprivation-induced increases in expression of AGRP and NPY mRNA in ARC cell bodies. Microinjections were made into the ventromedial hypothalamus just dorsal to the ARC, rather than into the PVH, to directly target the NE and E innervation of the area containing the NPY and AGRP cell bodies. The effects of the DSAP lesion on basal and 2DG-induced expression of AGRP and NPY mRNA in ARC cell bodies were analyzed subsequently using in situ hybridization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats weighing approximately 300 g were purchased from Simonsen Laboratories (Gilroy, CA) and housed individually in an animal care facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Rats were maintained on a 12-h light, 12-h dark cycle with ad libitum access to food and tap water. All experimental procedures were approved by Washington State University Institutional Animal Care and Use Committee, which conforms to NIH Guidelines.

Intracranial injections
For intracranial injections, the rats were anesthetized using ip injections of chloropent (3 ml/kg, ip). Chloropent anesthesia was made by combining 21.25 g of chloral hydrate, 10.6 g of magnesium sulfate, 4.43 g pentobarbital sodium, 75.26 ml ethyl alcohol, and 169.00 ml propylene glycol, brought to 500 ml with sterile double distilled H2O and filtered. Intracranial injections of saporin conjugated to a monoclonal antibody against dßh (DSAP, Chemicon International, Inc., Temecula, CA; 42 ng/200 nl in phosphate buffer, pH 7.4) or unconjugated saporin control solution (SAP, Advanced Targeting Systems, Carlsbad, CA; 20 ng/200 nl in phosphate buffer, pH 7.4) were delivered bilaterally using a Picospritzer through a pulled glass capillary pipette (30-µm tip diameter) positioned just dorsal to the targeted site in the ARC (33). The amount of unconjugated SAP in the control solution approximated the amount of SAP present in the DSAP conjugate (21%), as indicated in the manufacturer’s product information. Previous work comparing SAP and uninjected controls demonstrated that SAP did not produce behavioral or histological signs of toxicity (33). A 3-wk interval was allowed to elapse between the DSAP injections and further experimentation to permit retrograde transport of the toxin and complete degeneration of lesioned neurons (33, 36, 37).

Glucoprivic feeding
DSAP and SAP rats were tested for 2DG-induced feeding as a means of assessing the efficacy of the DSAP injections (33). For this test, rats were injected sc (1 ml/kg) with 0.9% saline or 2DG (Sigma, St. Louis, MO; 200 mg/kg) and returned to their cages with a weighed quantity of food. Food intake was measured at 1, 2, and 5 h after 2DG or saline injection by weighing remaining food and spillage. The 2DG test was conducted 48 h after the saline baseline test.

Tissue preparation
One week following the final feeding test, half of the subjects from each pretreatment group were injected sc with saline (SAL, 0.9%) and half with 2DG (200 mg/kg) and returned to their home cages without food for 2 h, at which time they were killed by deep anesthesia (100 mg/kg pentobarbital) and perfused transcardially with physiologic saline (0.9% NaCl, pH 7.4) followed by fresh 4% paraformaldehyde (pH 7.4, 4 C). The numbers of rats in each group were as follows: DSAP-2DG, n = 5; SAP-2DG, n = 5; DSAP-SAL, n = 5; and SAP-SAL, n = 5. After perfusion, brains were rapidly removed and placed in fresh 4% paraformaldehyde at 4 C for 12 h, then into diethylpyrocarbonate (Aldrich, Inc., St. Louis, MO)-treated cryoprotectant (20% sucrose in 0.1 M phosphate buffer, pH 7.4) for 24 h. The hypothalami were sectioned at 20 µm and collected into five sets of serial sections which were direct mounted onto SuperFrost Plus slides (Fisher Scientific, Los Angeles, CA). Sections were stored in desiccated slide boxes at -80 C until processed for in situ hybridization for NPY and AGRP mRNA as described below. For hindbrain immunohistochemical analysis of the DSAP lesion, serial 40-µm tissue sections were collected and processed for detection of dßh-ir.

Immunohistochemistry
One set of mounted 20-µm hypothalamic sections and the serial 40-µm free-floating hindbrain sections were washed three times in 0.1 M phosphate buffer (pH 7.4) then placed into 50% ethanol for 15 min and room temperature. Sections were then washed in 0.1 M Tris PBS (TPBS, pH 7.4) twice at room temperature before being incubated in blocking solution (10% normal horse serum in TPBS) at room temperature for 1 h. Sections were then placed into TPBS with 1% normal horse serum and mouse monoclonal anti-dßh (Chemicon International, 1:40,000) with agitation for 48 h at room temperature. After three washes in TPBS at room temperature, sections were incubated with agitation in TPBS with 1% normal horse serum and 1:500 biotinylated antimouse IgG (Peninsula Laboratories, Inc., Belmont, CA) overnight at room temperature. Again, after three 15-min washes in TPBS, sections were incubated overnight in TPBS with 1:1500 ExtraAvidin peroxidase (Sigma) at room temperature. After three 15-min washes in TPBS, dßh-ir was visualized using a glucose oxidase reaction with nickel-diaminobenzadine as the chromagen. Sections were mounted, air dried, dehydrated in serial ethanols, cleared in Citrasol (VWR Scientific Products Inc., Seattle, WA), and coverslipped with DPX (dibutyl pthalate and xylene) (VWR Scientific Products Inc.).

Qualitative analysis of dßh-ir was done under light microscopy. Hypothalamic sections were analyzed for the presence of dßh-ir fibers and terminals. Hindbrain sections were examined for the presence of dßh-ir cell bodies in rostrally and spinally projecting catecholaminergic cell groups.

In situ hybridization
Plasmids containing cDNA for either NPY (gift of Dr. Barry Levin, Veterans Administration Medical Center, East Orange, NJ) or AGRP (gift of Dr. Michael Schwartz, Veterans Administration Medical Center, Seattle, WA) were obtained. Linearized cDNA was transcribed for antisense riboprobes with T7 RNA polymerase (Life Technologies, Inc., Gaithersburg, MD) in the presence of 33P-uridine 5'-triphosphate (Perkin-Elmer, Indianapolis, IN). Sense transcriptions were carried out with either T3 RNA polymerase (NPY; Life Technologies, Inc.) or SP6 RNA polymerase (AGRP; Life Technologies, Inc.). Appropriate riboprobe length was determined with formaldehyde gel electrophoresis. Riboprobes were quantified following purification in a scintillation counter.

One set of 20-µm hypothalamic sections per probe was removed from the freezer. They were immediately placed into slide racks, rapidly dried, and placed into fresh 4% paraformaldehyde (pH 7.4) at 4 C for 5 min. All subsequent washes during the tissue pretreatment were performed at room temperature. Following two 5-min washes in 0.1 M phosphate buffer (pH 7.4), sections were dipped in diethylpyrocarbonate-treated water then washed in 0.1 M triethanolamine (Aldrich, Inc.) with 250 µl/ml acetic anhydride (Aldrich Inc.) for 10 min. Sections were then rinsed in 2x SSC (sodium citrate, sodium chloride; Ambion, Inc., Austin, TX) for 3 min then dehydrated in graded ethanols (3 min each) and delipidated in chloroform for 5 min. Sections were then washed in 100% then 95% ethanol (3 min each) and allowed to air dry before the hybridization procedure.

The final volume of probe mix plus hybridization buffer was equal to 100 µl/slide. The volume of probe was calculated (1 x 106 cpm/slide) and allowed to thaw on ice. The probe was combined with 1/20 vol Torula RNA (Sigma) and 1 M dithiothreitol (Sigma) in 0.1 M Tris/ 0.01 M EDTA (pH 8.0) to produce the probe mix. Probe mix was heat denatured by placing into boiling water for 3 min then returned to ice for 5 min. The denatured probe mix was added to prewarmed hybrdization buffer at a ratio of 5:20. After adding the hybridization mix to the slides, the sections were covered with Parafilm coverslips that were then sealed with rubber cement. Slides were placed in humidity chambers and placed into ovens at calculated hybridization temperatures (NPY = 55 C; AGRP = 52 C) for 16 h.

Following hybridization, humidity chambers were removed from the ovens and allowed to return to room temperature. Coverslips were removed and the slides were returned to slide racks and washed twice in 4x SSC for 15 min each at room temperature. Slides were then placed into ribonuclease (RNase) (Roche Molecular Biochemicals, Indianapolis, IN; 50 mg/ml RNase in 0.15 M sodium chloride; 10 mM Tris; 1 mM EDTA, pH 8.0) for 30 min at 37 C, then in RNase buffer without RNase at 37 C for another 30 min. After a 3-min wash in 2x SSC at room temperature, slides were washed with agitation in 0.1x SSC at 62 C for 30 min, then again for 15 min in fresh 0.1 M SSC. After a 3-min room temperature wash in 0.1x SSC, sections were dehydrated in graded ethanols (3 min each) and allowed to air dry before visualization.

Slides were dipped in Kodak NTB3 emulsion (VWR Scientific Products Inc.) and after 2–3 wk, slides were removed under safe light conditions and developed and counterstained in 0.5% cresyl violet (Sigma) in acetate buffer, dehydrated in graded ethanols and cleared in Citrasol (VWR Scientific Products Inc.) and coverslipped with DPX.

Quantification and analysis
The hybridization signal for NPY and AGRP mRNA was quantified in the ARC for all rats in each of the four treatment groups. Quantification was conducted using digitizing images obtained with a black and white video camera at x400 magnification under bright field microscopic conditions. The total number of hybridized cells was determined by counting all cells with visible stained nucleoli underlying tight groupings of silver grains greater than x5 background. Images were captured using NIH Image Analysis for the Macintosh computer by averaging 30 integrated frames. The cresyl violet staining was subtracted from this process by use of an NB3 filter (Edmund Scientific, Tonawanda, NY). The captured black and white images were made binary and the total number of pixels representing silver grains over each cell was computed, using previously described methods (40, 41, 42). A minimum of 60 positively hybridized cells were analyzed in this way unilaterally for each probe. Anatomically matched sections representing the entire rostral-caudal extent of the ARC were analyzed. Cells were analyzed only if silver grains were observed overlying nucleoli. Data are presented as the average number of silver grain pixels per cell in the ARC for each probe. All data from this experiment were analyzed using ANOVA with a Tukey/Kramer posthoc test using GB-Stat Statistical software for the Macintosh computer (General Dynamic, Inc., Bethesda, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glucoprivic feeding
Figure 1Go shows that the total 5-h food intake of DSAP rats in response to 2DG (2.7 ± 0.40 g) was not significantly different than their intake after saline injection (1.6 ± 0.38 g, P > 0.05), whereas SAP rats ate significantly more after 2DG (5.9 ± 0.60 g) than after saline control injection (2.1 ± 0.41 g, P < 0.05). Furthermore, DSAP-treated rats ate significantly less than SAP-treated rats at 1 and 2 h following 2DG injection, when intakes were also measured (P < 0.001). Body weights of DSAP and SAP rats did not differ significantly either at the beginning of the experiment (361.4 ± 8.21 g and 357.9 ± 7.57 g, respectively) or at the completion of the experiment 5 wk later (414.2 ± 3.66 g and 395.8 ± 11.37 g, respectively).



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  		Figure 1. Feeding induced by 2DG in rats injected previously into the ARC with the immunotoxin, DSAP, or unconjugated SAP control solution. Bars indicate amount of pelleted rat chow consumed during 5-h tests after injection of saline (0.9%) or 2DG (200 mg/kg). SAP rats, but not DSAP rats, increased their food intake significantly above their baseline in response to 2DG. *, P < 0.001, SAP SAL vs. SAP 2DG.

 
dßh immunocytochemistry
DSAP injections did not cause significant nonspecific disruption at the injection sites. In all but three cases, the injection site could not be located in either the immunoreacted or hybridized cresyl violet stained sections. In three cases (shown in Fig. 2Go), small hemocydarin deposits permitted the identification of the injection site. However, DSAP treatment caused a nearly complete loss of dßh-ir fibers in the ARC and median eminence (Fig. 3Go). The pattern of dßh-ir cell loss in the hindbrain catecholamine cell groups was consistent with their known projections (26A, 43, 44, 45). DSAP treatment caused a profound reduction in dßh-ir cell bodies within the A1/C1 overlap, where nearly all catecholamine neurons project to the medial hypothalamic structures, and more modest reductions in the A2, C2, and C3 cell groups, where cells are more heterogeneous with regard to their projections to the injection site. Cell groups A5, A7, and subcoeruleus, which do not project to the medial hypothalamus, did not appear to be not altered.



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  		Figure 2. Photomicrographs showing DSAP and SAP microinjection sites in 20-µm sections from the ventromedial hypothalamus just dorsal to the ARC in three rats. In most rats, the injection site and cannula tract cannot be detected due to the use of micocapillary pipettes for injection and the absence of nonspecific tissue disruption from the DSAP or SAP injections. In the rats shown in this figure, small hemosidarin deposits at the cannula tip enable visualization of the injection site. Calibration bar, 50 µm.

 


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  		Figure 3. Photomicrographs showing dßh-ir (black precipitate) in 20-µm sections from the ARC and median eminence of rats approximately 5 wk after bilateral microinjection of SAP (left) or DSAP (right) into the medial hypothalamus just dorsal to the ARC. Fiber and terminal staining present in SAP-treated rats is almost completely absent in DSAP-treated rats. Calibration bar, 200 µm, top row; 50 µm, bottom row.

 
In situ hybridization
Initial analyses of the riboprobe transcripts confirmed the appropriate size of the probes. 33P-uridine 5'-triphosphate transcriptions typically yielded approximately 1.5 x 106 cpm/microliter. Further preliminary formaldehyde gels run with RNA sizing ladders confirmed the appropriate length of the individual 33P-labeled riboprobes. Sections of ARC revealing the AGRP mRNA hybridization signal for DSAP and SAP rats treated with 2DG or saline are shown in Fig. 4Go. Hybridization results for NPY mRNA are shown in Fig. 5Go. No silver grains were observed in sections incubated with hybridization media containing 33P-labeled sense probes for AGRP or NPY.



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  		Figure 4. Dark field photomicrographs of ARC showing AGRP mRNA hybridization signal in rats give bilateral microinjection of SAP or DSAP into the medial hypothalamus and subsequently injected sc with 2DG (200 mg/kg) or saline (0.9%) 2 h before death. Panels show SAP-SAL (A); SAP-2DG (B); DSAP-SAL (C); DSAP-2DG (D); sense control (E). Calibration bar, 100 µm.

 


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  		Figure 5. Dark field photomicrographs of ARC showing NPY mRNA hybridization signal in rats give bilateral microinjection of SAP or DSAP into the medial hypothalamus and subsequently injected sc with 2DG (200 mg/kg) or saline (0.9%) 2 h before death. Panels show SAP-SAL (A); SAP-2DG (B); DSAP-SAL (C); DSAP-2DG (D); sense control (E). Calibration bar, 100 µm.

 
Quantification of mRNA hybridization signals are shown in Figs. 6Go and 7Go. Figure 6Go shows that there were no significant differences among treatment groups in the number of NPY or AGRP hybridized cells within the ARC (P = 0.1 and 0.07, respectively). Figure 7Go shows that 2DG-induced glucoprivation significantly increased the AGRP mRNA hybridization signal (number of silver grain pixels per cell) within the ARC in SAP controls (SAP-SAL = 130.7 ± 7.08 pixels; SAP-2DG = 219.8 ± 23.84 pixels; P < 0.01), but not in DSAP-treated rats (P > 0.1). In DSAP rats injected with saline, basal levels of hybridized AGRP mRNA were significantly increased compared with basal levels in SAP rats (P < 0.01), but levels were not elevated further in these rats by 2DG (DSAP-SAL = 247.7 ± 13.72 pixels; DSAP-2DG = 254.3 ± 35.9 pixels). A similar response was seen with NPY mRNA hybridization in the ARC (Fig. 7Go): glucoprivation significantly increased the NPY mRNA hybridization signal in SAP controls (SAP-SAL = 235.9 ± 9.17 pixels; SAP-2DG = 336.7 ± 28.93 pixels; P < 0.01), but not in DSAP-treated rats (P > 0.1). In DSAP rats injected with saline, basal levels of hybridized NPY mRNA were significantly increased compared with basal levels in SAP rats (P < 0.01), but levels were not significantly elevated above basal values by 2DG (DSAP-SAL = 379.2 ± 36.46 pixels; DSAP-2DG = 308.6 ± 33.6 pixels; P > 0.1).



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  		Figure 6. Total number of cells in the ARC containing AGRP or NPY mRNA hybridization signal in rats given DSAP or SAP injections into the medial hypothalamus and injected 2 h before death with 2DG (200 mg/kg, sc) or saline (0.9%). Numbers were obtained by counting all cells with visible stained nucleoli underlying tight groupings of silver grains greater than x5 background. The number of cells containing the AGRP or NPY hybridization signal was not altered by any of the treatments.

 


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  		Figure 7. Amount of AGRP and NPY mRNA hybridization signal (number of pixels containing silver grains per cell) in arcuate cells in DSAP- and SAP-treated rats given 2DG or saline 2 h before death. The number of silver grain pixels per cell was significantly increased in DSAP-treated rats injected with saline, compared with SAP-treated rats injected with saline. 2DG increased the hybridization signal above saline baseline levels in the SAP but not in the DSAP rats. Letters (a, NPY; b, AGRP) indicate statistical significance from SAP-SAL, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The potency of NPY and AGRP in stimulating food intake is widely recognized (see Ref. 46 for review). The involvement of NPY in the glucoprivic stimulation of feeding (19) and the responsiveness of NPY and AGRP neurons to glucose deficit (20, 21, 22) demonstrate that the orexigenic drive generated by NPY and AGRP neurons is controlled in part by glucose deficit. However, DSAP lesions, which produced virtually complete destruction of dßh-ir terminals in the ARC and median eminence, abolished the responsiveness of ARC NPY and AGRP mRNA to glucose deficit. Therefore, NPY and AGRP neurons do not sense glucose deficit directly. Rather, their responsiveness to this signal requires the afferent innervation of the medial hypothalamus by hindbrain catecholamine neurons. These results couple two important findings reported previously: that glucose deficit does not stimulate feeding either when catecholamine afferents to the hypothalamus are destroyed (33, 47) or pharmacologically blocked (32) or when NPY is acutely immunoneutralized (19). The fact that DSAP lesions significantly elevated basal levels of both AGRP mRNA and NPY mRNA, indicates that hindbrain catecholamine neurons also contribute in some way to basal levels of gene expression in AGRP and NPY neurons, as suggested previously for NPY (48).

Hindbrain catecholamine neurons conveying signals generated by glucoprivation may influence NPY and AGRP neurons in the ARC by direct synaptic contact. Innervation of the ARC by hindbrain catecholamine neurons has been studied in detail and appositions between tyrosine hydroxylase-ir terminals and NPY cell bodies have been identified by electron microscopy (49). Because a high percentage of C1–C3 neurons that innervate medial hypothalamic structures coexpress NPY (50, 51, 52, 53), it is also of interest that appositions between NPY-immunoreactive terminals and NPY cell bodies have also been observed in the ARC (49). Furthermore, functional interactions between catecholaminergic neurons and NPY neurons have been reported in numerous studies. Notably, Li and Pelletier (54) reported a marked increase in NPY-ir in the ARC after blockade of catecholamine synthesis with ß-methylparatyrosine. Although some of the catecholamine terminals in the ARC identified with tyrosine hydroxylase-ir are derived potentially from hypothalamic dopamine neurons in cell groups A11–A13, which may also interact with NPY neurons (49), we noted specifically in our previous work that DSAP did not appear to damage the hypothalamic dopamine neurons (33). Therefore, it is not likely that the interactions we observed in this study were due to destruction of dopamine neurons.

Removal of catecholamine terminals in medial hypothalamic structures may also have consequences that contribute indirectly to the changes in ARC NPY and AGRP mRNA that we observed. Corticosterone is one possible endocrine intermediary that is potentially altered by this catecholamine denervation and that might in turn alter peptide mRNA expression. Interactions between NPY and corticosterone have been widely reported (55, 56, 57, 58, 59, 60, 61). We do not know whether corticosterone secretion was altered by our ventromedial hypothalamic DSAP injections. However, catecholamine neurons potently influence neuroendocrine CRH neurons, and in other work we have shown that PVH DSAP injections profoundly reduce the corticosterone response to insulin-induced hypoglycemia and 2DG (38). Although the same PVH DSAP injections do not impair the corticosterone response to swim stress or the basal circadian rhythm of corticosterone secretion, the loss of modulation of corticosterone secretion in response to glucose fluxes could conceivably alter either basal or glucoprivation-induced NPY and mRNA gene expression. Similarly, DSAP lesions may alter interactions between leptin and NPY/AGRP neurons that contribute to basal levels of gene expression. Interruption of ascending NE and E fibers by midbrain injection of 6-hydroxydopamine has been shown to disrupt the dark phase up-regulation of leptin mRNA in epididymal fat (48), which may in turn reduce a signal that normally restrains AGRP and NPY mRNA expression.

Nonspecific damage of ARC neurons probably did not contribute to the loss of responsiveness of NPY and AGRP neurons to glucoprivation. Firstly, the numbers of NPY and AGRP cell bodies did not appear to differ between SAP and DSAP rats. Secondly, the cresyl violet-stained sections did not reveal cellular disruption either within the ARC or dorsal to it where the microcapillary injection pipettes were targeted. Thirdly, the responses of NPY and AGRP mRNA to glucoprivation in SAP-injected controls were similar to the responses reported previously in untreated rats (21). It is possible that the elevated basal mRNA expression in DSAP-treated rats may have increased the threshold for responding to 2DG such that the dose we used was not effective in these rats. However, the dose of 2DG used in this study is well above the threshold for eliciting glucoregulatory responses in normal rats. Therefore, it seems unlikely that further elevations would provide meaningful data. Another possible caveat is that the elevated basal mRNA in DSAP rats reflects a ceiling effect for mRNA expression that precluded additional elevation by 2DG. We cannot exclude this possibility from our data.

Previously, we analyzed the effects of DSAP injections on 2DG-induced changes in hypothalamic AGRP and NPY mRNA levels using Northern blots (22). Northern blots revealed effects similar to those reported here for AGRP mRNA. That is, we found that 2DG increased expression of AGRP mRNA in SAP, but not in DSAP rats, and that DSAP elevated basal AGRP mRNA expression. However, blots from the same rats failed to reveal any differences in NPY mRNA due to 2DG or DSAP treatment. Although we speculated that this result was due to the limitations of our Northern blot protocol in which homogenates of whole hypothalamus were used, it raised the important possibility that NPY and AGRP mRNAs may be differentially controlled by glucose deficit or by catecholamine neurons. The present study was initiated in part to investigate that issue further using a technique that provides resolution at the cellular level. In addition, because we wished to focus on the cells in the ARC, we targeted the ARC rather than the PVH with our DSAP injections. Using in situ hybridization, we were able in this study to detect treatment effects on both NPY and AGRP mRNA and can discount the possibility that these peptide mRNAs are differentially regulated by glucose availability. We attribute the differences in the NPY mRNA results between the two experiments to the differences in technical approach. Differences are less likely to be attributable to differences in the DSAP injection site because dßh-ir is eliminated from the ARC by PVH (38), as well as by ventromedial hypothalamic DSAP injections.

The present finding that DSAP injections impair glucoprivic feeding and responsiveness of ARC NPY and AGRP gene expression to glucose deficit contributes to a rich literature supporting a role for NPY and AGRP in feeding (19, 46, 62, 63, 64) and provides support for their specific involvement in glucoprivic feeding. Nevertheless, it is prudent to recall previous work showing that neither large electrolytic lesions destroying the ventromedial hypothalamus between the fornices and the base of the brain in rats (65) nor gold thioglucose lesions of the ARC in mice (66) abolish glucoprivic feeding. Similarly, destruction of the PVH, where many ARC NPY and AGRP neurons terminate, does not impair glucoprivic feeding (67, 68), although DSAP injections into either the PVH or ventromedial hypothalamic area abolish this response (33). These findings raise important issues regarding the interpretation of the present data. First, they indicate that the role of hindbrain NE/E neurons in eliciting glucoprivic feeding is not simply to stimulate NPY and AGRP neurons or to lower the response threshold of glucose-sensitive neurons in the ventromedial hypothalamic area (24, 25) because the feeding response occurs even when this brain area is destroyed. Indeed, even decerebrate rats increase their food intake in response to glucoprivation, if fed intraorally (69). Second, these findings suggest that the essential role of the hindbrain catecholamine neurons in glucoprivic feeding is dependent on their innervation of distributed neural circuits, which together are required for the expression of the feeding response. The extensive collateralization and widespread distribution of the hindbrain NE and E neurons that suit them for such a role are well known (70, 71, 72). Stimulation of NPY and AGRP neurons may be one aspect of the complex circuitry influenced by hindbrain NE and E neurons to elicit glucoprivic feeding, but these peptidergic neurons are not required for the feeding response. Therefore, the effectiveness of the medial hypothalamic DSAP injection in impairing glucoprivic feeding has more to do with its destruction of the entire collateral network of the particular NE and E neurons that innervate the injection site, than with its ability to denervate the ARC.

Although the focus of this study is on the hindbrain control of ARC NPY and AGRP neurons, it is important to note that a significant proportion of the hypothalamic NPY innervation originates from hindbrain catecholamine neurons (73). This proportion has been estimated to be about 40–50% (74, 75). The DSAP lesion, therefore, eliminates a substantial proportion of hypothalamic NPY terminals, in addition to eliminating virtually all of the dßh-ir terminals. And as noted above, appositions between NPY-immunoreactive terminals and NPY cell bodies have been observed in the ARC (49). Therefore, it is important to resist the attribution of all the functional deficits we observe in DSAP-treated rats either to changes in the ARC NPY/AGRP neurons or to the loss the control exerted on them by NE or E terminals. The distinct contributions of the hindbrain NPY system, also eliminated by the DSAP lesion, must also be considered.

The fact that DSAP lesions significantly elevated basal levels of both AGRP mRNA and NPY mRNA is a major finding of the present study, indicating that a role for hindbrain catecholamine neurons in determining basal levels of gene expression in AGRP and NPY neurons. Increased NPY mRNA has been reported previously in rats after injection of the catecholamine neurotoxin 6-hydroxy-dopamine into the ventral mesencephalic NE and E bundle (48). The elevated basal gene expression is particularly interesting with respect to control of body weight because repeated (76) or continuous (12) administration of NPY and overexpression of the AGRP gene (16) are associated with obesity. Rats in the present study did not become obese. Because retrograde transport of DSAP and neuronal loss requires approximately 2 wk, it is possible that the 5-wk post lesion survival time allowed in our study was not sufficient for the development of the obesity. This seems likely for two reasons. First, we previously reported that PVH DSAP lesions, which also reduced dßh-ir terminals throughout ARC and medial hypothalamus, produced a significant, but slowly developing obesity during the 6-month post-lesion survival time of the lesioned rats (33), whereas rats with the same lesion killed after 5 wk were not obese (22). Secondly, interruption of the ventral NE and E bundle by midbrain injection of the catecholamine toxin, 6-hydroxydopamine, which also increases NPY gene expression, as well as up-regulating NPY Y5 receptors (48), produces a gradually developing obesity (77, 78, 79). On the other hand, a variety of results demonstrate that NPY neurons are disparate in function and subject to a variety of controls (50, 80, 81, 82, 83). Therefore, it should not be surprising that elevation of NPY and AGRP mRNA or increased peptide levels are not always associated with increased body weight or with feeding (83). For example, it has been reported recently that a conditional knockout of NPY Y2 receptors increased ARC NPY and AGRP mRNA and food intake but caused a loss in body weight (84). It has also been demonstrated that during severe dehydration NPY mRNA is elevated in the hypothalamus even though the dehydrated rats are strongly anorexic (85).

Many investigators and a wealth of data (2, 21, 46, 62, 63, 86) have suggested that AGRP and NPY neurons in the ARC contribute to an orexigenic circuitry that is controlled by a variety of afferent inputs. The present results suggest that NE or E projections from hindbrain glucoreceptive sites exert a major influence on this orexigenic circuitry and thereby may control its responsiveness to metabolic state, and particularly to glucose availability. Loss of these hindbrain catecholamine neurons results in a failure to increase food intake in response to acute glucoprivation that may be associated with alteration of AGRP and NPY gene expression in the ARC.


   Acknowledgments
 
We thank Drs. Barry Levin and Michael Schwartz for NPY and AGRP cDNA, Dr. Heiko Jansen for help with hybridization protocols, and Thu Dinh and Yubei Zhang for technical assistance.


   Footnotes
 
This work was supported by the Juvenile Diabetes Foundation and Public Health Service Grant 40498 (to S.R.).

1 Current address: Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, Washington 98195-7290. Back

Abbreviations: AGRP, Agouti gene-related protein; ARC, arcuate nucleus of the hypothalamus; dßh, dopamine-ß-hydroxylase; 2DG, 2-deoxy-D-glucose; DSAP, anti-dßh-SAP; E, epinephrine; ir, immunoreactivity; NE, norepinephrine; NPY, neuropeptide Y; PVH, paraventricular hypothalamic nucleus; RNase, ribonuclease; SAL, 0.9% sodium chloride solution; SAP, saporin; SSC, sodium citrate, sodium chloride; TPBS, Tris PBS.

Received June 28, 2002.

Accepted for publication October 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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B. E Levin
Metabolic imprinting: critical impact of the perinatal environment on the regulation of energy homeostasis
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A.-J. Li, Q. Wang, and S. Ritter
Differential Responsiveness of Dopamine-{beta}-Hydroxylase Gene Expression to Glucoprivation in Different Catecholamine Cell Groups
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K. Toshinai, H. Yamaguchi, Y. Sun, R. G. Smith, A. Yamanaka, T. Sakurai, Y. Date, M. S. Mondal, T. Shimbara, T. Kawagoe, et al.
Des-Acyl Ghrelin Induces Food Intake by a Mechanism Independent of the Growth Hormone Secretagogue Receptor
Endocrinology, May 1, 2006; 147(5): 2306 - 2314.
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E. Watson, S. Hahm, T. M. Mizuno, J. Windsor, C. Montgomery, P. E. Scherer, C. V. Mobbs, and S. R. J. Salton
VGF Ablation Blocks the Development of Hyperinsulinemia and Hyperglycemia in Several Mouse Models of Obesity
Endocrinology, December 1, 2005; 146(12): 5151 - 5163.
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K. Bugarith, T. T. Dinh, A.-J. Li, R. C. Speth, and S. Ritter
Basomedial Hypothalamic Injections of Neuropeptide Y Conjugated to Saporin Selectively Disrupt Hypothalamic Controls of Food Intake
Endocrinology, March 1, 2005; 146(3): 1179 - 1191.
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G. N. Wade and J. E. Jones
Neuroendocrinology of nutritional infertility
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2004; 287(6): R1277 - R1296.
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D. K. Sindelar, L. Ste. Marie, G. I. Miura, R. D. Palmiter, J. E. McMinn, G. J. Morton, and M. W. Schwartz
Neuropeptide Y Is Required for Hyperphagic Feeding in Response to Neuroglucopenia
Endocrinology, July 1, 2004; 145(7): 3363 - 3368.
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N. M. Sanders, A. A. Dunn-Meynell, and B. E. Levin
Third Ventricular Alloxan Reversibly Impairs Glucose Counterregulatory Responses
Diabetes, May 1, 2004; 53(5): 1230 - 1236.
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X. Wu, J. Gao, J. Yan, C. Owyang, and Y. Li
Hypothalamus-Brain Stem Circuitry Responsible for Vagal Efferent Signaling to the Pancreas Evoked By Hypoglycemia in Rat
J Neurophysiol, April 1, 2004; 91(4): 1734 - 1747.
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R. Burcelin, M. Uldry, M. Foretz, C. Perrin, A. Dacosta, M. Nenniger-Tosato, J. Seydoux, S. Cotecchia, and B. Thorens
Impaired Glucose Homeostasis in Mice Lacking the {alpha}1b-Adrenergic Receptor Subtype
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L. Rinaman
Hindbrain Noradrenergic Lesions Attenuate Anorexia and Alter Central cFos Expression in Rats after Gastric Viscerosensory Stimulation
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H. I'Anson, L. A. Sundling, S. M. Roland, and S. Ritter
Immunotoxic Destruction of Distinct Catecholaminergic Neuron Populations Disrupts the Reproductive Response to Glucoprivation in Female Rats
Endocrinology, October 1, 2003; 144(10): 4325 - 4331.
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S. Ritter, A. G. Watts, T. T. Dinh, G. Sanchez-Watts, and C. Pedrow
Immunotoxin Lesion of Hypothalamically Projecting Norepinephrine and Epinephrine Neurons Differentially Affects Circadian and Stressor-Stimulated Corticosterone Secretion
Endocrinology, April 1, 2003; 144(4): 1357 - 1367.
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