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NEUROENDOCRINOLOGY |
Graduate Groups of Psychology and Neuroscience (H.J.G., J.M.K., J.S.F., D.G.B), University of Pennsylvania, Philadelphia, Pennsylvania 19104; and Veterans Affairs, Puget Sound Health Care System (D.G.B.), Seattle, Washington 98108, and Departments of Medicine (M.W.S., J.B., D.G.B.) and Biological Structure (D.G.B.), University of Washington, Seattle, Washington 98195
Address all correspondence and requests for reprints to: Harvey Grill, University of Pennsylvania, Department of Psychology, 3815 Walnut Street, Philadelphia, Pennsylvania 19104. E-mail: grill{at}psych.upenn.edu
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Introduction |
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-MSH (derived from POMC) and agouti-related protein. The
projections of these leptin-sensitive neurons (e.g., arcuate
to paraventricular nucleus and arcuate to lateral hypothalamus) are
central to the contemporary hypothalamic control model
(e.g., 12, 13, 14). Although evidence has mounted
supporting the hypothalamus as an important site for leptin’s action
on food intake, it is also recognized that leptin is potentially
available to all brain areas via the circulation (15), and
evidence suggests that leptin receptors are expressed at different
levels of the neuraxis (2). It seems premature, therefore,
to rule out possible functional contributions of neuronal leptin
receptors outside the hypothalamus. The focus of the present study is the hypothesis that Ob-Rb within the caudal brainstem (CBS) contributes to the anorexic effects of central leptin delivery. A growing literature has established a role for the CBS in the control of energy balance with respect to: 1) mediation of a variety of relevant autonomic reflexes (16, 17); 2) mechanisms or receptors that trigger ingestive effects of a number of pharmacologic and metabolic treatments (18, 19, 20, 21); and 3) the ingestive behavioral competence observed in the chronically maintained decerebrate rat (22, 23). There is as yet no direct evidence for intake-relevant action of leptin in the CBS, although two physiological studies suggest that the hindbrain contains a population of neurons that expresses functional leptin receptors. Smedh et al. (24) reported that fourth intracerebroventricular (i.c.v.) injection of leptin suppressed gastric emptying in rats, and Zhou and Schneider (25) showed that fourth-i.c.v. leptin reversed fasting-induced anestrus in the hamster. The location of CBS leptin receptors that mediate these effects, however, is not known. Evidence for leptin receptor expression in the dorsal vagal complex (DVC), a region of clear importance to meal size control, has been reported in several immunocytochemical studies (24, 26, 27). The antibodies used in these studies, however, did not distinguish Ob-Rb from other leptin receptor isoforms, such as Ob-Ra, which have markedly attenuated signaling capability. In situ hybridization studies have yielded inconsistent results with respect to Ob-Rb mRNA expression in the DVC (2, 28, 29, 30). One study reported negative results for the DVC (28) whereas another group showed Ob-Rb expression in the nucleus of the solitary tract of the mouse but much weaker expression in rat nucleus of the solitary tract (NTS), Ob-Rb expression in rat NTS (29). Elmquist et al. (2) found widespread Ob-Rb mRNA expression in the CBS including all three divisions of the DVC (NTS, area postrema [AP], dorsal motor nucleus [DMX]), and parabrachial nucleus, but the density of hybridization signal in these brain stem structures was reported to be relatively low and inconsistent across animals. There is therefore some uncertainty about the location of neurons that could mediate the direct action of leptin in the CBS.
We combined anatomical and functional analyses to address a CBS contribution to the ingestive effects of leptin. We reevaluated the expression of Ob-Rb in the CBS using fluorescence in situ hybridization (FISH) (31) and immunocytochemistry with a polyclonal antibody specific to human Ob-Rb (7). We found substantial Ob-Rb expression in the DVC and in other CBS structures. We found significant food intake and body weight reductions after leptin administration to the fourth ventricle and, at a low dose, to the DVC parenchyma. The results taken together are consistent with the hypothesis that brain stem Ob-Rb, perhaps in combination with leptin action in the hypothalamus, contribute to the intake-inhibitory effect of central leptin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization. Brains for in situ hybridization were removed from the cranium of anesthetized rats between 0900 and 1100 h and immediately frozen on dry ice. Coronal cryostat slices (14 µm thick) of brain stem were stored at -70 C. The riboprobe used for specific Ob-Rb mRNA in situ hybridization is complementary to the coding region for the c-terminus of the rat Ob-Rb and does not recognize mRNA for any other known leptin receptor splice variants (4, 7). Details of the preparation and labeling of the Ob-Rb mRNA riboprobes used for FISH are described elsewhere (5, 31). Briefly, the Ob-Rb mRNA riboprobes were prepared with biotin-UTP by transcribing cDNA containing the specific coding region for the cytoplasmic tail of the Ob-Rb spice variant (7). The probe was purified using a Qiaquick nucleotide removal kit (QIAGEN, Valencia, CA). To increase the sensitivity of FISH for detection of Ob-Rb mRNA, FISH was amplified by combining the enzyme-labeled fluorescent substrate (ELF) method (Molecular Probes, Inc., Eugene, OR) with tyramide signal amplification (TSA indirect kit, NEN Life Science Products, Boston, MA) (31, 32), in which Ob-Rb mRNA hybridization was visualized in a two-step process. In the first step, the biotin moiety was amplified using a Renaissance TSA indirect kit (NEN Life Science Products) (streptavidin-horseradish peroxidase 1:100) for 30 min at room temperature, followed by biotinyl tyramide (1:50) for 10 min at room temperature. The second step used an ELF-97 mRNA in situ hybridization kit no. 2 (Molecular Probes, Inc.). Slides were placed in blocking buffer containing 30 mM Tris (pH 7.4), 150 mM NaCl, 1% BSA, 0.5% Triton X-100, and 1 mM levamisole (Sigma, St. Louis, MO) for 30 min at room temperature, followed by the streptavidin-alkaline phosphatase conjugate (1:50) for 30 min at room temperature. The ELF-AP substrate was prepared according to manufacturer’s instructions and applied to each slide for exactly 10 min at room temperature.
Immunocytochemistry. Rats were perfused transcardially under Equithesin anesthesia with cold 4% paraformaldehyde fixative in 0.1 M phosphate buffer, pH 7.4. Brains were removed from the cranium and placed in fresh fixative overnight in the refrigerator, immersed in 0.05 M phosphate buffer containing 25% (wt/vol) sucrose for 1 d, and then frozen on dry ice and sectioned at 5 µm by cryostat. Slide-mounted sections were incubated overnight in a polyclonal antibody directed against the C-terminal region of human Ob-Rb (Linco Research, Inc., St. Charles, MO), diluted 1:1000 in 0.05 M phosphate buffer (pH 7.4, 4 C). Fluorescent visualization of the Ob-Rb antibody binding was done with donkey antigoat IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories, Inc., West Chester, PA) (7). Immunostaining controls included: 1) omission of primary Ob-Rb antibody; 2) replacement of Ob-Rb antibody with normal rabbit serum at the same concentration; 3) comparison to hypothalamic staining pattern produced by another Ob-Rb antibody that was characterized by immunoblots and immunostaining in a previous study (5); and 4) analysis of immunostaining of the choroid plexus, which expresses Ob-Ra but not Ob-Rb. Absorption controls for immunocytochemistry were not required (33) because the specificity of the antibody for Ob-Rb was shown by Western immunoblots of adipocytes (manufacturer’s literature) and rat brain (data not shown).
Microscopy and imaging. Visualization of FISH and immunostaining was done with a Axioplan fluorescence microscope (Carl Zeiss, Thornwood, NY). The red Cy3 fluorescence (immunostaining) was visualized with a conventional rhodamine filter set, whereas the green fluorescence of the ELF alkaline phosphatase substrate reaction product (FISH) was observed with a 365-nm excitation filter, 400-nm dichroic long pass filter, and a 515-nm emission filter (Chroma Technology, Brattleboro, VT). Digital RGB pseudocolored images (10 bit) of the fluorescence preparations were acquired with a C4880 fast-cooled CCD camera (Hamamatsu Corp., Bridgewater, NJ) and the MCID imaging system (Imaging Research, St. Catharines, Ontario) and were exported to Adobe Photoshop as 300 dpi tiff RGB files. The images were processed with pseudocolor palettes that closely matched the intensity and contrast present in the original preparations; no selective contrast enhancement of specific areas or cells was applied to the images.
Exp 2: Delivery of leptin to the fourth and lateral ventricles
Subjects. Male Sprague Dawley rats (n = 57) from
Charles River Laboratories, Inc. (Wilmington, MA) weighed
between 375 and 525 g at the start of testing. Rats were
maintained on a 12-h light, 12-h dark schedule, with food and water
available ad libitum except for the 75 min before the onset
of the dark period. All procedures conformed to standards for animal
care at the University of Pennsylvania.
Cannula placement. Rats were anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, im). A guide cannula (22 G, PlasticsOne, Roanoke, VA) was stereotaxically positioned 2 mm above the injection site in either the fourth or the left lateral ventricle. For the fourth i.c.v. placement, the cannula tip was positioned on the midline, 2.5 mm anterior to the occipital suture and 4.5 mm below the dura. The lateral i.c.v. cannula tip was placed 1.6 mm lateral to the midline, 0.9 mm posterior to bregma, and 2.0 mm below the dura. The cannula was fixed to the skull with jeweler’s screws and dental cement, and an obturator was inserted. Approximately 2 wk after surgery, cannula placement was verified by measurement of plasma glucose immediately before and 1 h after injection of 5-thio-D-glucose (210 µg in 3 µl isotonic saline). The criterion for an effective cannula was the sympathoadrenal hyperglycemic response (34) of a doubling of plasma glucose concentration above the preinjection baseline level.
Injections. Murine recombinant leptin (Peprotech, Roden Hill, NJ) was dissolved in 5 mM sodium citrate buffer, pH 4. The protein concentration of the solution was determined with BCA colorimetric assay (Pierce Chemical Co., Rockford, IL). The solution was aliquoted and stored at 4 C until time of use, when pH was adjusted to 7.5–8.0 by addition of sodium hydroxide. The leptin concentration of the aliquot was adjusted by the addition of vehicle (1:1 sodium citrate, pH 4: sodium hydroxide, pH 12) to yield leptin doses of 0.03, 0.277, and 1.67 µg/µl. A 3-µl injection volume was delivered via a 28-gauge injector. Injections into either the lateral or the fourth ventricle were driven by an infusion pump over a 4-min period, 1 min after which the injector was withdrawn and the obturator replaced. Injections were delivered an average of 30 min before the beginning of the dark.
Measurement of food intake and body weight. Rats were removed from their cages and weighed 30 min before the dark period. At the same time, the food dishes and water bottles were weighed. Food, water, and rat were returned to the cage at the beginning of the dark period. On injection days, food intake was also measured 2 and 4 h after lights out.
Experimental design. Testing began between 3 and 4 wk after surgery. Six weight- and age-matched groups of rats (n = 9–11) with fourth or lateral i.c.v. cannula were subjected twice to a 3-d injection protocol during which a given dose of leptin (0.1, 0.83, or 5.0 µg) or vehicle was delivered once daily. For each group, half of the rats received leptin injection for 3 d and then vehicle injections for 3 d. The remaining rats were tested first under vehicle injection conditions followed by leptin treatment. Leptin and vehicle treatment blocks were separated by 3 d without i.c.v. injection.
Data analysis. The fourth-i.c.v. results were analyzed by 3-way, mixed-model, ANOVA (drug [within-subject: leptin vs. vehicle] x day within treatment condition [within-subject: d 1, 2, 3] x dose [between-subject: 0.1, 0.83, 5 µg]). A separate ANOVA was run for each of four dependent measures (cumulative intake measures at 2, 4, and 24 h after lights-out, and the 24-h change in body weight). For each ANOVA, an effect of leptin is indicated by a significant main effect of the drug factor. The dose relatedness of the leptin response is indicated by a significant drug x dose interaction. Such interactions were explored via separate two-way ANOVA (drug x day) run at each dose. Comparison between fourth and lateral i.c.v. results was accomplished via four-way ANOVA, with ventricle as the added fourth (between-subjects) factor.
Exp 3: Delivery of leptin to the DVC
Surgery. Subject strain and source, maintenance, and
anesthesia were identical to that of Exp 2. Anesthetized male rats
(n = 13) were positioned in a stereotaxic instrument. A guide
cannula (26 G PlasticsOne) tip was placed 13.8 mm
posterior to bregma, 0.5 mm lateral to midline, and 3.7 mm dorsal to
the interaural line (a placement 2 mm above the injection site). The
cannula was fixed to the skull with jeweler’s screws and dental
cement, and an obturator was inserted. All procedures conformed to
standards for animal care at the University of Pennsylvania.
Verification of parenchymal placements. India ink (0.2 µl) was injected 1.5 mm ventral to the drug injection site just before transcardial perfusion with saline followed by 10% formalin. Brains were then removed and postfixed in a 10% sucrose-formalin solution. Coronal sections (50 µm) were stained with cresyl violet. Where possible, the presence of gliosis was used to determine the site of the injector. In cases in which glial damage was not apparent or determinate, the position of injector placement was assessed in relation to the dye reference. Data from only those animals with verified placements were included in the analysis.
Injections. Leptin and vehicle preparation was the same as described above. A 33-gauge injector (2 mm longer than the guide cannula) was inserted, and a 0.5-µl volume of leptin solution or vehicle was injected over 5 min using a microsyringe pump (Harvard, Holliston, MA). The injector was removed after 1 min. Injections were delivered 30 min before the beginning of the dark period.
Experimental design and analysis. Each rat was subjected twice to a 3-d injection protocol during which leptin (0.1 µg) or vehicle was delivered once daily. Condition (three leptin or three vehicle injections) testing order was counterbalanced across rats. Three days without DVC injection separated the two treatment blocks. A two-way ANOVA (drug x day) was performed for each of four evaluated parameters (2-, 4-, and 24-h intakes, and body weight change).
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Results |
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, A, B, and C).
Many cells in the AP were heavily labeled with a bright, fluorescent
FISH signal for Ob-Rb. DMX neurons also showed strong Ob-Rb FISH signal
in most cells. With comparable magnification, the FISH signal appeared
visibly weaker in the NTS than in the DMX although hybridization to
Ob-Rb in the NTS was clearly observed. To gauge the relative abundance
of NTS neurons with the potential to respond directly to leptin, we
counted Ob-Rb mRNA-positive cells in six to eight sections of the NTS
from four rats. The results showed that 29.3 ± 4.8% of the
neuronal cell bodies in the NTS contained Ob-Rb mRNA FISH signal at
detectable levels. Other CBS structures that were observed to express
hybridization signal in many neurons included the medial parabrachial
nucleus (Fig. 1D), hypoglossal nucleus (Fig. 1E), trigeminal motor
nucleus (Fig. 1F), locus ceruleus (Fig. 1G), cochlear nucleus (Fig. 1H), and lateral reticular nucleus (Fig. 1I).
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Immunocytochemistry of CBS sections with antibodies specific for the
cytoplasmic tail of the Ob-Rb splice variant of the leptin receptor
produced immunofluorescence in the same regions that were found to
contain FISH for Ob-Rb mRNA, including the AP (Fig. 1J
), NTS (Fig. 1K),
and DMX (Fig. 1L). Similar to the FISH results, the immunostaining
was primarily observed in neuronal cell bodies. Visual analysis
indicated the numbers of neurons that were Ob-Rb positive in these
regions were similar to those observed with Ob-Rb mRNA FISH.
Immunostaining was absent in negative control sections that were
treated with normal rabbit serum in place of the Ob-Rb antiserum or on
which primary antiserum was omitted.
Delivery of leptin to the fourth and lateral ventricles
Fourth i.c.v. injection. Leptin significantly suppressed
intake at 2, 4, and 24 h after injection into the fourth ventricle
and reduced body weight change, relative to vehicle injection
conditions (Fig. 2). At the 2-h food
intake, there was an overall effect of leptin (F[1,24] = 19.59,
P < 0.0002) that was dose related (F[2,24] = 8.44,
P < 0.002), with post hoc analysis showing
a significant difference between leptin and vehicle conditions only at
the 5.0-µg dose (mean intake: vehicle = 5.8 g; leptin
= 4.1 g). For this parameter, as for the 4- and 24-h intake
measurements, the magnitude of the drug effect did not vary across the
3 d of the injection protocol. At the 4-h food intake, intake was
significantly suppressed by leptin [drug factor (F[1,24] = 26.09,
P < 0.00004; Fig. 2]. A significant effect of dose
was obtained (F[2,24] = 3.56, P < 0.05) along with a
marginally significant (F[2,24] = 3.33, P = 0.052)
drug x dose interaction, reflecting the greater intake
suppression with leptin (vs. vehicle) at the two higher
doses. This observation was substantiated by significant post
hoc results for the 0.83- and 5.0-µg doses (vs. the
respective vehicle conditions). At the 24-h food intake, the leptin
effect on intake (F[1,24] = 41.21, P < 0.00001) was
dose related (F[2,24] = 8.73, P < 0.002), reflecting
significant effects at the two highest doses (Fig. 2, middle
graph). For body weight change, a significant main effect of
leptin to reduce body weight was obtained (F[1,24] = 9.76,
P < 0.005). Dose was not significant as a main effect,
but the dose x drug interaction was significant (F[2,24] =
4.29, P < 0.03). This finding reflects a lowering of
body weight at the two higher doses but not at the lowest dose of
leptin (Fig. 2, bottom). We also found a significant main
effect of day (F([2,48] = 14.88, P < 0.00001). This
reflected a loss of weight (-4 g) on d 1, followed by small gains in
average weight for d 2 (1 g) and 3 (1.3 g), averaging across drug and
dose conditions. There were no significant interactions between day and
the drug or dose factors.
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). In the overall
ANOVA, there was no main effect of ventricle and no significant
interaction involving the ventricle factor.
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). The
behavioral results are shown in Fig. 5.
DVC injection of a dose of leptin that was a subthreshold for food
intake suppression when given into either the lateral or fourth
ventricle significantly decreased food intake at 2 h (by 24%
averaged across days; F[1,12] = 8.52, P < 0.02), at
4 h (by 12%; F[1,12] = 7.21, P < 0.02), and at
24 h after injection (by 11%; F[1,12] = 17.08,
P < 0.002). Leptin also caused significant reductions
in daily body weight change (F[1,12] = 21.95, P <
0.0006). Relative to vehicle, rats receiving leptin microinjection into
the DVC lost a mean of 3.3 g/d, resulting in a total weight loss of
9.8 g for the 3-d treatment period. For each parameter, there was
no day effect and no drug x day interaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The behavioral results reported indicate that delivery of leptin to the CBS gives rise to significant suppression of intake and body weight. We found that fourth i.c.v. leptin administration produced a degree of food intake suppression that is similar to that obtained here and in earlier studies in which lateral i.c.v. effects were evaluated (35, 36). We also sought to determine whether leptin microinjection into a specific CBS site would recapitulate the effects of fourth i.c.v. administration. Because we showed the DVC to contain a relatively high abundance of cells expressing Ob-Rb mRNA and because of its well-documented role in food intake control and autonomic physiology (e.g. 19, 21, 37, 38, 39, 40, 41, 42, 43, 44, 45), we selected this site as the target for our initial parenchymal injection study. We found significant reductions in short- and longer-term food intake following unilateral DVC injections of leptin at a dose (0.1 µg) that we determined to be subthreshold for the i.c.v. effect. This result is consistent with the hypothesis that leptin is capable of reducing food intake via a direct action in or near the DVC. In addition, given the proximity of the DVC to the ventricle, it seems reasonable to suggest that stimulation of Ob-Rb in this region also contributes to the effects of fourth i.c.v. leptin delivery. The suggestion could be evaluated in subsequent experiments assessing the consequences of fourth i.c.v. leptin administration for intracellular signals linked to Ob-Rb stimulation (e.g. SOCS3 mRNA induction, STAT3 nuclear translocation, PI3K expression) in DVC neurons. Such intracellular signal analyses would also begin to address the possibility that other CBS structures containing Ob-Rb contribute to the effects of fourth i.c.v. leptin.
These results support the hypothesis that widely separated populations of neurons that express Ob-Rb can give rise to similar effects on feeding behavior and body weight. One argument for this contention is the striking similarity of the fourth and lateral i.c.v. response to leptin. If one region were responsible for the i.c.v. effects, we would have expected, rather, the dose-response curve for the more distal placement to be right shifted, with different thresholds and response magnitudes. A more direct source of support for a functionally distributed leptin receptor system is provided by results from parenchymal injection experiments. The anorexic effect we observed following microinjection of leptin into the DVC is similar to that induced by intrahypothalamic leptin administration as reported by Jacob et al. (46). They found that local administration of leptin into the ventromedial hypothalamic nucleus, at a daily dose of 0.1 µg bilaterally, reduced intake and body weight but was without effect when delivered to the lateral ventricle. It would appear highly unlikely that the low dose delivered to the DVC could via diffusion have a site of action as distant as the hypothalamus or conversely that a brain stem substrate underlies the effects of hypothalamic microinjection. Taken together, the present and cited results indicate that more than one site in the brain, apparently including at least one in the CBS and one in the hypothalamus can give rise to significant leptin effects on food intake and body weight in the rat.
The results reported here are consistent with the hypothesis that leptin’s action in CBS neurons contributes to energy homeostasis. The hypothesis, if correct, is a counterpoint to the currently most widely accepted model for leptin action that focuses almost exclusively on Ob-Rb in the hypothalamus, particularly in the arcuate nucleus. Analysis must now commence addressing the phenotypes of neurons in the brain stem that express Ob-Rb. Given the central role in the hypothalamic model of leptin-sensitive POMC and NPY neurons, it will be interesting to determine whether the NTS POMC and NPY neurons also express Ob-Rb and whether their projections contribute to ingestive effects observed following local administration of leptin. We know that there are neurons in the brain stem that are sensitive to other blood born signals of relevance to the control of energy homeostasis, including cells that express insulin receptor and cells sensitive to changes in blood glucose (37, 47, 48). It will be interesting in this light to determine whether, as in the hypothalamus (49, 50), there are populations of individual neurons that respond to leptin as well as to other interoceptive signals. There are, of course, limits to the parallels that can be drawn between local circuits in the CBS and hypothalamus that are engaged by leptin. Thus, for example leptin receptors are found on agouti-related protein neurons in the arcuate nucleus, whereas in the CBS, receptors but apparently not cell bodies for this peptide have been identified (40). Conversely, whereas Ob-Rb is expressed on GLP-1 neurons in NTS (30), GLP-1 neurons are not found in the hypothalamus (51). In any event, future work in this area will no doubt argue for an expanded model for the feeding-related actions of leptin that encompasses interactions between brain stem and hypothalamic structures and redundant as well as unique processing functions performed at different levels of the central control system.
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Acknowledgments |
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Footnotes |
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Abbreviations: AP, Area postrema; CBS, caudal brain stem; DMX, dorsal motor nucleus; DVC, dorsal vagal complex; ELF, enzyme-labeled fluorescent; FISH, fluorescence in situ hybridization; i.c.v., intracerebroventricular; NTS, nucleus of the solitary tract; Ob-Rb, long-form leptin receptor.
Received June 6, 2001.
Accepted for publication September 24, 2001.
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