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APPETITE, OBESITY AND METABOLISM
1Division of Endocrinology/Metabolism, Veterans Affairs Puget Sound Health Care System, Seattle 91808; and 2Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, and 3Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington 98195
Submitted 17 October 2003 ; accepted in final form 22 March 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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brain stem; adiposity; satiety; food intake
These findings are consistent with the hypothesis that leptin-activated neurons in the hypothalamus converge on NTS circuits that regulate meal size, although the identities of the neurons comprising such a pathway are not well established. An increasing body of evidence suggests that neurons in the hypothalamic PVN are responsive to leptin and play a role in the relay of anorectic leptin signaling from the arcuate nucleus in the hypothalamus to the NTS and that the effect of this signaling is to enhance satiety signals. For example, PVN neurons express Fos proteins following leptin administration (13, 15), and PVN lesions cause hyperphagia and obesity (23) characterized by attenuated satiety effects of peripherally administered CCK (10). The PVN also contains a diverse population of neuronal cell types expressing an array of peptides that influence energy balance (51). Nevertheless, oxytocin neurons located in the parvocellular subdivisions of the PVN (pPVN) are particularly promising candidates for mediating leptin-anorectic signals to the brain stem, as some pPVN oxytocin neurons project directly to the NTS (38, 39) and all oxytocin fibers in the NTS reportedly originate in the pPVN (36). Furthermore, several reports indicate that centrally administered oxytocin reduces food intake (3, 4, 24, 29) and activates Fos protein expression by neurons in NTS subdivisions that are activated by peripheral administration of CCK-8 (31). We recently reported that the oxytocinergic innervation of the NTS is anatomically correlated with the medial (mNTS) and gelatinosus (gNTS) subdivisions of the NTS, which are particularly sensitive to peripheral CCK-8 treatment (8) and express oxytocin receptors. Furthermore, injection of oxytocin receptor antagonists into a lateral ventricle (3, 4, 24) or the fourth ventricle (4V) (9) stimulates food intake and blunts the satiety effect of CCK-8 (8).
Thus leptin on the one hand has been shown to potentiate the response of the mNTS to CCK satiety signaling, resulting in smaller meals, reduced food intake, and augmented mNTS Fos activation, and pPVN oxytocin neurons are implicated as mediators in this mechanism by both anatomic and pharmacological data. To validate this hypothesis, we investigated three critical questions related to components of the proposed mechanism. First, we conducted immunocytochemical staining studies to demonstrate the feasibility of the hypothesis that 3V leptin administration activates Fos expression in oxytocin neurons in the pPVN. Second, we combined immunocytochemical staining and neuronal retrograde tracing methods to demonstrate that a subset of leptin-activated oxytocin neurons in the pPVN project to the NTS region of the CBS. Third, we determined that blockade of oxytocin action by intracerebroventricular injection of an oxytocin receptor antagonist attenuated leptin's ability both to reduce food intake and to augment the Fos expression in NTS neurons induced by peripheral CCK-8 administration.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Surgical and stereotaxic procedures. The procedure for implantation of the guide cannulas into specific brain sites has been described previously (45). For the immunocytochemistry studies on leptin induction of Fos in pPVN oxytocin neurons, animals were anesthetized with equithesin (3.4 ml/kg ip) (33). In other studies, animals were anesthetized with a standard ketamine cocktail (1 ml/kg body wt ip) (7). For mounting of cannulas, animals were placed into a stereotaxic apparatus with the incisor bar positioned 3.3 mm below the interaural line. When mounting the 3V cannulas, a 26-gauge guide cannula (18 mm; Small Parts, Miami Lakes, FL) was stereotaxically positioned 1 mm dorsal to the 3V (6.8 mm anterior to the interaural line, 0.05 mm lateral to the midline, and 7.6 mm ventral to the skull surface); for 4V cannulas the coordinates were 3.5 mm posterior to the interaural line, 1.4 mm lateral to the midline, and 6.2 mm ventral to the skull surface. Cannulas were fastened to the surface of the skull with denture acrylic and stainless steel screws. A 33-gauge obturator was inserted into the guide cannula to maintain patency. Ceftriaxone (25 mg/kg; Roche Laboratories, Nutley, NJ) and buprenorphine hydrochloride (0.3 mg/kg im; Reckett & Colman Pharmaceuticals, Richmond, VA) were administered at completion of surgery.
Injections of retrograde tracer (cholera toxin subunit B) into CBS. The fluorescent retrograde tracer cholera toxin subunit B (CTB) conjugated to Alexa 488 (Molecular Probes, Eugene, OR) was used to trace PVN projections to the CBS. The Alexa 488-CTB was injected (n = 3) unilaterally into the CBS region occupied by the NTS, dorsal motor nucleus of the vagus (DMV), and area postrema (AP). The goal was to target the mNTS region by using stereotaxic coordinates of –5.5 mm posterior to interaural line, +0.7 mm to the cerebral vein, and –7.1 mm ventral to the surface of the skull. These coordinates for the mNTS were modified from those in the brain atlas of Paxinos et al. (32) on the basis of our postmortem analysis of the injection sites in preliminary studies. A 0.5-µl volume of 0.2% Alexa 488-CTB in saline was injected via a 33-gauge injector connected by PE-20 tubing to a 10-µl Hamilton syringe. Unilateral injections of the Alexa 488-CTB tracer were completed over 60 s; the injector was held stationary for another 60 s and slowly removed over the following 60 s. The guide cannula was held stationary for an additional 5 min and slowly removed during the following 5 min. Each of these animals also received a 3V cannula for leptin injection, as described above. We waited 4 days for retrograde transport of the Alexa 488-CTB to the PVN (12) before injecting the animals with either leptin or vehicle.
Injections into 3V and 4V.
Rats were injected through chronic 3V or 4V cannulas, as described above, with the exception that the 33-gauge injector was held stationary at the end of the injection for 
30 s and the intracerebroventricular injection volume was 2 µl (4V) or 2.5 µl (3V). Animals were immediately returned to their home cages at the conclusion of each injection protocol. Leptin (Peprotech, Rocky Hill, NJ) was solubilized in a 4 mM sodium citrate (pH 4.0)-saline cocktail and was always given into the 3V 1 h before the start of the dark cycle. OVT (Bachem-Penninsula Laboratories, San Carlos, CA) dissolved in saline was administered into the 3V 30–45 min before leptin administration, or it was given immediately before the start of the dark cycle into either the 3V or 4V, depending on the design of the particular study. Food intake was measured at 0.5, 1, 2, and 4 h after the start of the dark cycle and access to food. Visual examination of the cage bottom revealed negligible spillage, and there were no apparent differences in spillage within or between treatment groups. The results obtained from either leptin or CCK injections from these studies are similar to what others have reported, suggesting that the spillage had minimal impact on the results. Food intake was also measured at 24 h to verify that the animals responded normally to leptin (42).
3V injections of OVT before leptin. Food intake was measured following 3V leptin injection (3.5 µg/2.5 µl) given 1 h before the start of the dark cycle in either the presence or absence of 3V pretreatment with OVT (9,300 pmol given 30–45 min before administration of either leptin or vehicle). This dose of leptin produces a 23–40% reduction in food intake during the 1st h, comparable to the effect that others have reported using a similar paradigm (42). The effects of OVT (0, 93, 930, 9,300 pmol), administered into the 3V under identical conditions to those above (minus the leptin treatment) were examined 48 h before this study to verify that all doses of OVT had no effect on food intake for up to 4 h when administered alone (n = 27 total, 5–8/group). In the subsequent experiment, animals were divided into groups (n = 29 total, 6–9/group) that were matched for body weights and age. Each group received one of four 3V treatments in a 2 x 2 matrix. Group I received the OVT vehicle (saline; i.e., no OVT) before leptin vehicle (i.e., no leptin). Group II received OVT vehicle (no OVT) before leptin administration. Group III received a dose of OVT before the leptin vehicle (i.e., no leptin). Group IV received OVT before leptin (i.e., received both OVT and leptin). Because interpretation of the results of the OVT administration would be ambiguous if the rats failed to show a response to leptin (which occasionally occurred), we established an inclusion requirement of a 20% reduction of food intake within 24 h after leptin treatment in the absence of the OVT either in the actual experiment or in a follow-up study whereby animals in a cross-over within-subjects design received leptin and vehicle. At the completion of this study, all six animals that received vehicle only, five of the eight animals that were initially injected with leptin in the absence of OVT, five of the seven animals that were initially injected with leptin in the presence of OVT, and six of the eight animals that were initially injected with OVT in the absence of leptin responded to leptin at the conclusion of the study or during the experiment and were used in the final data analysis. Food intake was measured at 0.5, 1, 2, 3, and 4 h after the start of the dark cycle and access to food.
Injection of OVT before leptin and CCK-8. To determine whether endogenous oxytocin signaling contributed to the effect of leptin to potentiate CCK-8-induced Fos expression in NTS neurons, we administered OVT to fasted rats 30–45 min before 3V leptin, which was given 1 h before injections of CCK-8 (ip), given immediately before the start of the dark cycle and access to food. Fos expression was measured by counting Fos-positive neurons within specific regions of the dorsal vagal complex: the gNTS, mNTS, and AP. These regions of the dorsal vagal complex have oxytocin innervation in close vicinity to neurons activated by CCK-8 (8). These four groups were weight- and age-matched and categorized as follows: (group 1) OVT vehicle (saline) before leptin vehicle (no leptin) and CCK-8; (group II) OVT vehicle (no OVT) before leptin (3.5 µg/2.5 µl) and CCK-8 (1.2 nmol = 1.4 µg ip); (group III) OVT (9,300 pmol) before leptin and CCK-8; (group IV) OVT vehicle (no OVT) before leptin vehicle (no leptin) and CCK-8 vehicle (no CCK-8) (n = 18 total, n = 5–7 rats/group, with exception of vehicle-alone group, which had n = 2 rats). OVT and leptin were administered in a manner identical to that of the previous study, and CCK-8 was given 1 h after leptin administration. The timing of these treatments was designed so that CCK-8 was administered immediately before the start of the dark cycle, when the animals normally begin eating and when CCK-8 has a potent effect on reducing food intake. At 90 min following CCK-8 or vehicle injections, animals were perfused with 4% paraformaldehyde, as described in tissue collection for immunostaining below.
Effect of 3V and 4V administration of OVT. After a 1- to 2-wk period of adaptation to the cannulas and handling, weight- and age-matched groups of animals were fasted for 6 h and received 3V (n = 22 rats) or 4V (n = 20 rats) injections of OVT over a range of doses from 0 (saline control) to 9,300 pmol administered in 2 µl (4V) or 2.5 µl (3V) immediately before the start of the dark cycle (n = 5–7/dose). Food intake was measured at 1, 2, 3, 4, and 24 h after the start of the dark cycle and access to food.
Behavioral assessment of cannula placement. In behavioral studies, the placement of 3V cannulas was validated by injecting 10 ng of angiotensin II in 1 µl of saline (Sigma, St. Louis, MO) at least 48 h before the start of the feeding experiments. All animals used in the subsequent analysis of the data drank at least 5 ml of water over 30 min. Assessment of 4V cannula placement was done with injections of 15 pmol/2 µl bombesin (Bachem-Peninsula) immediately before the start of the dark cycle in rats that were fasted for 6 h. All animals used in the subsequent analysis of the data reduced their food intake by at least 20% within 1 h, an established criterion for correct placement of these cannulas (2).
Tissue collection for immunostaining. Animals used for immunocytochemistry were returned to their cages immediately after injections. Food was removed to prevent stimulation of brain stem neurons by gastric distension (14, 47). At the conclusion of each study (2–2.5 h after leptin injections and 1–1.5 h from the beginning of the dark cycle), rats were anesthetized with equithesin (3.4 ml/kg ip) and transcardially exsanguinated with saline followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. Brains were removed and stored overnight in fresh fixative at 4°C and then transferred to 0.1 M phosphate buffer containing 25% sucrose for 24 h. Brains were then frozen by submersion for 20–30 s in isopentane and placed under crushed dry ice. Coronal cryostat sections (14 µm) of the hypothalamus and brain stem were mounted on slides and stored at –80°C. Representative sections for immunocytochemical staining of Fos and oxytocin were selected at 126-µm intervals in the classic pPVN region and at 240-µm intervals in the NTS. Sections were selected with the objective of sampling the rostrocaudal extent of these nuclei at several coronal levels anterior (PVN: 6.88, 7.2, 7.6, 7.7, 8.08 mm) (33) or posterior (NTS: 3.38, 3.63, 3.87, 4.11, 4.34, 4.58, and 4.83 mm) to the interaural line (32).
Immunocytochemical staining. Slides were washed with 10 mM PBS at room temperature followed by blocking buffer (5% normal goat serum in 10 mM PBS) for 90 min and additional buffer washes. Primary antibodies were 1) rabbit polyclonal anti-Fos, 1:5,000 dilution (Ab-5, Oncogene, San Diego, CA) (28, 46); 2) mouse monoclonal anti-oxytocin, 1:200 dilution (gift of Dr. Ann-Judith Silverman, Columbia University, NY) (20). Oxytocin and Fos antibodies were diluted in 10 mM PBS as a cocktail mixture. After an overnight incubation in primary antibodies at 4°C, slides were washed in 10 mM PBS followed by 1 h in fluorescent second antibodies diluted 1:200 in 10 mM PBS: 1) goat anti-rabbit IgG-Alexa 488 (Molecular Probes, Eugene, OR) to detect Fos antibodies; 2) goat anti-mouse IgG-Cy3 (Jackson ImmunoResearch, West Grove, PA) to detect oxytocin monoclonal antibody. Slides were washed in 10 mM PBS and coverslipped using an antifading glycerol-based mounting media. Immunostaining specificity controls included replacement of the primary antibody with normal rabbit serum (for polyclonal antibodies) or mouse serum (for the monoclonal antibody) at the same dilutions as the respective primary antibodies. Furthermore, immunocytochemical staining of the hypothalamus with the oxytocin antibody revealed brightly immunofluorescent cell bodies and axons in the PVN and the supraoptic nucleus (SON), appropriate to the known anatomic distribution of oxytocin cell bodies and axons. Under conditions used in the immunocytochemical staining protocol, immunoreactive Fos protein was concentrated in the nuclei of labeled cells. Specificity of the Fos immunocytochemistry was also confirmed by the highly reproducible increase in nuclear immunoreactive Fos cells in the PVN and SON of rats that were dehydrated for 24 h.
Immunocytochemical data analysis. Slides were analyzed with a Zeiss Axioplan fluorescence microscope, and all measurements were made with a x40 objective lens. Identification of anatomic landmarks was assisted by staining cell nuclei with Hoechst 33258 (Sigma), which was added to the mounting medium and observed with a conventional DAPI filter set. The blue fluorescence of the Hoechst 33258 did not interfere with any fluorochromes or tracers used in the staining. Digital RGB images of the fluorescent preparations were acquired with a Hamamatsu (C4880; Tokyo, Japan) fast-cooled charge-coupled device camera and the MCID imaging system (Imaging Research, St Catherines, ON, Canada) and were exported to Photoshop (Adobe, Tucson, AZ). Measurements of Fos expression in oxytocin neurons in classically defined regions of the pPVN, specifically the dorsal cap and medial pPVN regions, were made unilaterally on 6–9 coronal sections per brain (with the exception of one leptin-injected animal that used only four coronal sections because of tissue loss). The analyzed sections, each separated by 126 µm, were located between atlas levels 6.88 and 8.08 mm anterior to the interaural line (33). The classically defined magnocellular PVN was not included in this analysis, because the magnocellular oxytocin neurons project mainly to hypophysiotropic sites and the neurohypophysis. A neuron was considered to be double labeled if the Fos-positive nucleus was located within the boundary of the oxytocin-stained cytoplasm, as determined by focusing the microscope. On each slide all oxytocin-positive neurons in the pPVN were counted and averaged across all of the PVN sections from each brain. The mean values for each brain were averaged to obtain the mean of the treatment group. The data are expressed as mean numbers of cells counted in microscopic fields (cells/field) sampled with a x40 microscope objective. The objective was placed visually so as to include all of the oxytocin-positive cells in the pPVN region. This required counting several fields for each pPVN, but an effort was made to avoid counting the same cells in separate fields. The relative change of Fos expression in leptin-treated vs. control cells was calculated as the percentage increase in the number of Fos-positive oxytocin neurons counted within the 6–9 anatomically matched pPVN levels. Analysis of Fos immunostaining in the NTS following OVT, leptin, and CCK-8 was assessed similarly but bilaterally and at constant magnification using a x20 objective.
Statistical analysis. One-way analysis of variance (ANOVA) was used to evaluate the effects of leptin on short-term food intake and on Fos expression within the pPVN oxytocin-positive neurons, the effects of 3V administration of OVT on leptin's inhibition of food intake, the effects of OVT on leptin's potentiation of CCK-8-induced Fos within the NTS, and the effects of 4V administration of OVT on food intake. Analyses were performed using the statistical program SYSTAT. Data are expressed as means per group ± SE. Differences were considered significant at P < 0.05. Animals that did not have proper cannula placement were eliminated from the analysis.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3V injections of OVT before leptin. To determine whether endogenous oxytocin signaling influences the anorectic effect of leptin, we administered OVT or vehicle to 6-h-fasted rats 30–45 min before 3V leptin or vehicle, which was given 1 h before the start of the dark cycle and access to food. If oxytocin signaling contributes to the ability of leptin to reduce food intake, it is expected that the oxytocin receptor antagonist OVT will blunt the effects of leptin to reduce food intake in these animals (Fig. 2). In the absence of OVT pretreatment (group II), leptin treatment decreased cumulative food intake by 61, 64, 48, and 37% during the first 0.5, 1, 4, and 24 h, respectively (P < 0.05), compared with vehicle-injected animals (group 1). Leptin administered in the presence of OVT (group IV) did not significantly attenuate cumulative food intake at 0.5, 1, and 4 h (P > 0.05). Administration of OVT followed by the leptin vehicle (group III) had no effect on cumulative food intake (Fig. 2). These results reproduced an earlier study under identical conditions in these animals whereby OVT attenuated the effect of leptin (group IV) to reduce food intake during the first 0.5 h compared with leptin alone (group II) (1.6 ± 0.4 vs. 3.2 ± 0.4 g, group II vs. group IV, P < 0.01). Therefore, the between-subjects comparisons showed significant differences between groups II and IV in both studies. A within-subjects analysis of the 0.5-h data from the initial study suggests a similar conclusion (P = 0.089).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Similarly, the effect of leptin to enhance CCK-8-induced activation of NTS neurons was also attenuated by pretreatment with OVT. Specifically, this effect of OVT was detected in the medial NTS, an area that is richly innervated by oxytocin fibers from the PVN but was not seen in adjacent CBS areas that had relatively less oxytocin innervation [e.g., gNTS, AP (8)]. Moreover, we demonstrated that injections of OVT into the 4V stimulated short-term food intake, but 3V injections were ineffective in this same dose range and paradigm. Collectively, these findings support the hypothesis that hypothalamic activation of oxytocin neurons by leptin enhances the efficacy of gut-derived satiety signals to activate key neurons within the NTS that control food intake during individual meals. Thus oxytocin neurons may be a critical link between leptin signaling within the hypothalamus and CBS neurons that regulate meal size.
The findings suggest that oxytocin neurons within the dorsal and medial subdivisions of the PVN are activated by 3V leptin. Our morphological quantitative method sampled a relatively small portion of the pPVN, but the procedure was applied uniformly for all of the animals, so the results can be considered a reflection of the relative change across the pPVN among the experimental groups. In our analysis, we observed many oxytocin neurons in the PVN (including magnocellular neurons) that expressed Fos irrespective of leptin treatment, although only the oxytocin neurons in the pPVN showed a significant increase in Fos expression following 3V treatment. This finding is consistent with a previous study by Elmquist et al. (13), which reported leptin stimulation of Fos expression in parvocellular subdivisions of the PVN. It is possible that some magnocellular PVN neurons were included in this present analysis. However, Fos activation was not detected in magnocellular oxytocin neurons within the SON of leptin-treated animals, a finding consistent with the model of PVN-CBS leptin signaling and with evidence that PVN oxytocin neurons provide the sole source of oxytocin innervation of the NTS (36).
A primary goal of this study was to establish whether it is feasible to detect the presence of pPVN oxytocin neurons that are activated by leptin and, if so, whether any of these neurons project to the NTS. The results support this hypothesis, although future studies are needed to quantify the observation and determine the extent to which this pathway plays a physiological role in food intake and body weight regulation. This is particularly the case for the pPVN oxytocin neuronal population that showed a positive Fos response to leptin and projected to the NTS. An important limitation of the study is that the fluorescent CTB tracer was certainly not confined to the dorsal vagal complex, as the injected CTB certainly spread slightly outside its boundaries (which we confirmed by examining sections of the brain stem injection site with fluorescence microscopy). Thus some of the leptin-activated pPVN oxytocin neurons that contained the CTB tracer may have originated outside the NTS. Future quantitative studies employing this experimental paradigm are needed to characterize the precise brain stem projections of the leptin-activated oxytocin neurons in the pPVN.
These findings do not preclude the possibility that oxytocin-independent signaling pathways contribute to leptin's downstream signaling to the CBS. Indeed, our observation that leptin's inhibition of food intake was attenuated but not completely blocked by OVT pretreatment is an indication that nonoxytocinergic neurons may be involved in mediating the feeding effects of leptin. Such nonoxytocin pathways might include other catabolic peptides that are made within the PVN and reported to be activated by leptin, such as corticotropin-releasing hormone (CRH), gastrin-releasing peptide (GRP), and thyrotropin-releasing hormone (TRH). TRH neurons and GRP neurons were reported to be activated after leptin administration into the 3V (1, 18, 22). We reported previously that 3V leptin treatment increased CRH mRNA content in rat PVN (40). Moreover, 3V infusion of a CRH receptor antagonist blocked the effect of leptin to reduce food intake and body weight (48). Interestingly, the potentiation of CCK-induced Fos expression in the NTS by leptin was completely blocked by OVT treatment, a finding that contrasts with the partial blockade of OVT on leptin’s reduction of food intake. The neural circuits that mediate the response of these nonoxytocin PVN neurons to leptin's anorexic effects require investigation.
In our study, OVT was effective at stimulating food intake when infused into the 4V at doses as low as 9.3 pmol in rats fasted for 6 h, but not in the 3V. In contrast, previous studies in rats found that OVT doses as high as 9,300 pmol were required to stimulate food intake following injections into the lateral ventricles (3, 4). In the latter studies, rats were fasted for 21 h. Furthermore, in our protocol, all doses of OVT, including 9,300 pmol, were ineffective in the 3V when rats were fasted for only 6 h, but both 930 and 9,300 pmol doses were effective at stimulating food intake when administered in the 3V if the rats had been fasted for 16 h. Thus the oxytocin receptor antagonist appears to be more effective in an extended fast, when the animals presumably would be in a state of prolonged leptin deficiency (although leptin levels were not measured in these studies) and, therefore, leptin-stimulated release of oxytocin would also be expected to be reduced, although more studies are required to verify this inference. One limitation of the studies that examined the effects of OVT on leptin's inhibition of food intake and potentiation of CCK-induced Fos in the NTS was that OVT was administered into the 3V rather than the 4V, closer to the hypothesized site of action of oxytocin within the NTS. Consequently, the role of forebrain oxytocin receptor-driven signaling mechanisms cannot be ruled out in mediating the effects of leptin on food intake or on leptin's interaction with CCK on Fos induction within the NTS. However, the 4V appears to be more sensitive than the 3V to the effects of OVT on food intake, as none of the OVT doses that we used (up to 9,300 pmol) were effective in the 3V of rats fasted for 6 h whereas doses as low as 9.3 pmol were effective in the 4V. Clearly, additional analyses are required to validate the 4V as the primary site of action of oxytocin, but the initial findings are supportive of this hypothesis.
Recent reports demonstrate that leptin may act directly within the dorsal vagal complex to reduce food intake (17, 19). These reports raise the possibility that oxytocin's interactions within leptin could take place directly in the brain stem in addition to the hypothalamus. Thus it is possible that leptin could influence the CBS response to satiety signals such as CCK via a direct brain stem action, independent of hypothalamic input, although physiological evidence for such a mechanism remains to be demonstrated. It should also be noted that a transgenic knockout OT–/– mouse, which has loss of function mutation in the oxytocin gene, reportedly shows normal reduction in food intake in response to CCK-8 administered intraperitoneally (1, 3, and 10 µg/kg) (26), although the interaction of leptin with CCK signaling has not been evaluated. Nevertheless, this finding suggests that nonoxytocin pathways may be able to compensate for the developmental absence of oxytocin.
The findings of the present investigation are consistent with a well-documented role in autonomic regulation for pPVN neurons, along with evidence that NTS neuronal terminals containing oxytocin appear to arise solely from the pPVN (36). Although magnocellular oxytocin neurons contribute to neurohypophysial hormone secretion, they have not been implicated in the direct control of autonomic function. Moreover, oxytocin receptors (25) and oxytocin receptor mRNA are also found in the NTS (5) and adjacent areas such as the DMV (49, 50, 52). Although it remains to be determined whether these CBS oxytocin receptors are expressed by neurons activated by CCK-8, central administration of oxytocin has been shown to induce Fos protein in the same subdivisions of the NTS that are sensitive to peripheral injections of CCK-8 (31). Furthermore, the satiety effects of peripherally injected CCK-8 are attenuated following administration of OVT given intracerebroventricularly (30), and, like CCK-8, oxytocin administration inhibits food intake (29). Our findings that oxytocin receptor antagonism blocks the ability of leptin to potentiate CCK's activation of Fos within NTS neurons and that food intake increases after administration of OVT into the fourth ventricle are consistent with a model in which the CBS is a key site mediating the inhibitory effects of oxytocin on food intake by acting on neurons that are also sensitive to input from CCK. Taken together, the data suggest that oxytocin-containing neurons are key components of a descending pathway from the pPVN to the NTS, connecting leptin-sensitive hypothalamic neurons to CBS areas that process input from meal-related signals. We propose that this signaling system is a critical component of the mechanism whereby leptin enhances the ability of satiety signals to activate hindbrain neurons that control meal size.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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R. Malcher-Lopes, S. Di, V. S. Marcheselli, F.-J. Weng, C. T. Stuart, N. G. Bazan, and J. G. Tasker Opposing crosstalk between leptin and glucocorticoids rapidly modulates synaptic excitation via endocannabinoid release. J. Neurosci., June 14, 2006; 26(24): 6643 - 6650. [Abstract] [Full Text] [PDF]
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J. A. Amico, R. R. Vollmer, H.-m. Cai, J. A. Miedlar, and L. Rinaman Enhanced initial and sustained intake of sucrose solution in mice with an oxytocin gene deletion Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1798 - R1806. [Abstract] [Full Text] [PDF]
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E. E. Ladenheim, M. Emond, and T. H. Moran Leptin enhances feeding suppression and neural activation produced by systemically administered bombesin Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2005; 289(2): R473 - R477. [Abstract] [Full Text] [PDF]
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W. A. Cupples Physiological regulation of food intake Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1438 - R1443. [Full Text] [PDF]
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D. Porte Jr., D. G. Baskin, and M. W. Schwartz Insulin Signaling in the Central Nervous System: A Critical Role in Metabolic Homeostasis and Disease From C. elegans to Humans Diabetes, May 1, 2005; 54(5): 1264 - 1276. [Abstract] [Full Text] [PDF]
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