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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Exp Physiol. 2013 Jun 28;98(10):1495–1504. doi: 10.1113/expphysiol.2013.073726

Oxytocin projections to the nucleus of the solitary tract contribute to the increased meal-related satiety responses in primary adrenal insufficiency

Ernane Torres Uchoa 1, Daniel S Zahm 2, Beatriz de Carvalho Borges 1, Rodrigo Rorato 1, Jose Antunes-Rodrigues 1, Lucila L K Elias 1
PMCID: PMC3786458  NIHMSID: NIHMS500242  PMID: 23813803

Abstract

Anorexia is a common clinical manifestation of primary adrenal gland failure. Adrenalectomy (ADX)-induced hypophagia is reversed by oxytocin (OT) receptor antagonist and is associated with increased activation of satiety-related responses in the nucleus of the solitary tract (NTS). This study evaluated OT projections from the paraventricular nucleus of the hypothalamus (PVN) to NTS after ADX and the effect of pretreatment with intracerebroventricular injection of OT receptor antagonist ([d(CH2)5,Tyr(Me)2,Orn8]-vasotocin, OVT) on the activation of NTS neurons induced by feeding in adrenalectomized rats. Adrenalectomized animals showed higher OT labeling in the NTS than sham and ADX with corticosterone replacement (ADX+B) groups. Adrenalectomized animals exhibited co-localization of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and OT in axons in the NTS as well as OT fibers apposing NTS neurons activated by refeeding. After vehicle pretreatment, compared to fasting, refeeding increased the numbers of Fos− and Fos+TH-immunoreactive neurons in the NTS in sham, ADX and ADX+B groups, with a higher number of these immunolabeled neurons in adrenalectomized animals. Compared to fasting condition, refeeding also increased the activation of NTS neurons in OVT pretreated sham, ADX and ADX+B groups, however there was no difference among the three experimental groups. These data demonstrate that OT is up-regulated in projections to the NTS following ADX and that OT receptor antagonist reverses the greater activation of NTS neurons induced by feeding after ADX. The data indicate that OT pathways to the NTS contribute to higher satiety-related responses and, thus, to reduce meal size in primary adrenal insufficiency.

Keywords: Adrenalectomy, oxytocin, nucleus of the solitary tract

INTRODUCTION

Primary adrenal insufficiency in humans is associated with marked hypophagia and body weight loss (Oelkers, 1996). ADX in rodents is a well-established experimental model to investigate the mechanisms underlying these effects (Freedman et al., 1985, Uchoa et al., 2009a, Uchoa et al., 2009b). On the other hand, glucocorticoid replacement to adrenalectomized animals reverses the reduction of food intake (Freedman et al., 1985, Uchoa et al., 2009a, Uchoa et al., 2009b). The hypophagic effect induced by ADX is associated with increased expression of the anorexigenic neuropeptides corticotrophin-releasing factor (CRF) and oxytocin (OT) in the paraventricular nucleus of the hypothalamus (PVN) (Uchoa et al., 2009b, Uchoa et al., 2010). Conversely, ADX reduced the expression of the orexigenic neuropeptides neuropeptide Y and agouti related protein in the arcuate nucleus of the hypothalamus (Uchoa et al., 2012).

In the nucleus of the solitary tract (NTS), which is involved in satiety-related responses (Smith and Ferguson, 2008), neuronal activity is enhanced by meal-related signals (Emond et al., 2001, Rinaman et al., 1998). Previous studies by our group showed that ADX-induced hypophagia is associated with increased Fos expression in the hypothalamus and NTS in response to feeding, indicating that ADX facilitates the activation of satiety-related responses (Uchoa et al., 2009a, b). These enhanced satiety-related responses following ADX involve catecholaminergic and non-catecholaminergic neurons in the NTS, as well as CRF and OT neurons in the PVN (Uchoa et al., 2009a, b).

OT participates in the regulation of energy homeostasis (Olszewski et al., 2010b). OT receptor-deficient mice exhibit late-onset obesity with increases in the size of abdominal fat pads and levels of fasting plasma triglyceride (Takayanagi et al., 2008). In addition, there is a decrease in food intake following central injection of OT that is completely prevented by pretreatment with OT receptor antagonist (Arletti et al., 1989 and 1990). Recent studies have also shown that peripheral injections of OT reduce food intake and obesity in rodents (Maejima et al., 2011, Morton et al., 2012), and that OT acts as carbohydrate-specific inhibitor of feeding (Olszewski et al., 2010a).

The present study was designed to evaluate the role of hypothalamic OT-expressing projections to the NTS in the satiety-related responses of adrenalectomized animals. Specifically, we investigated the effects of ADX on OT-expressing projections to the NTS. We also evaluated the effect of OT receptor antagonist on the activation of NTS neurons in response to feeding in adrenalectomized rats.

EXPERIMENTAL PROCEDURES

Animals

Male Wistar rats (Animal Facility of the Campus of Ribeirao Preto, University of Sao Paulo, Brazil) and male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA), weighing 230-300 g, were housed in individual cages at controlled temperature (23±2°C) with a fixed light-dark cycle (light from 6:00 AM to 6:00 PM). Animals had ad libitum access to pelleted rat chow and water, unless otherwise specified. To improve adaptation to the laboratory environment, the rats were handled daily during 14 days prior to experiments. All experimental procedures were conducted between 7:00 AM and 12:00 PM and were approved by the Ethical Committee for Animal Use of the School of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil and in accordance with guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

For bilateral ADX and sham surgeries, animals were deeply anesthetized with intraperitoneal (i.p.) injections of a mixture of ketamine (72 mg/kg) and xylazine (11.2 mg/kg), administered as a cocktail consisting of 45% ketamine (100 mg/ml), 35% xylazine (20 mg/ml) and 20% 0.15M NaCl at a dose of 0.16 ml/100g of body weight. Surgeries were performed via a dorsal midline approach with a single incision in the skin and small bilateral cuts through the muscle layer at the angle between the last rib and vertebral column. Sham-operated animals underwent similar surgical procedures but without removal of the adrenal glands and were given tap water with 0.5% ethanol to drink. Adrenalectomized animals were given 0.9% saline with 0.5% ethanol, without (ADX) or with corticosterone diluted in 0.5% ethanol at a concentration of 25 mg/L (ADX+B) (Uchoa et al., 2009a).

Tracer injections

Sprague-Dawley rats were deeply anesthetized with i.p. injection of the cocktail of ketamine and xylazine as described above and placed in a Kopf stereotaxic instrument with bregma and lambda in a horizontal plane. The skulls were exposed and a small bore hole was made to allow PVN to be targeted by filament-containing borosilicate glass pipettes (o.d. - 1.0 mm). The posterior parvocellular subdivision of the PVN was targeted using coordinates from the atlas of Paxinos and Watson (21): 2.2 mm caudal to bregma, 0.4 mm lateral to the venous sinus and 7.2 mm below the dura mater. Pipettes were pulled to tip diameters of 10μm (o.d.) and contained the anterograde tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L; Vector, Burlingame, CA, 2.5% in 0.01 M phosphate buffer). A silver wire inserted into the pipettes contacted the solution containing the tracer, which was injected ionotophoretically into the brain using positive current pulses of 4 μA (7 s on, 7 s off, for 15 minutes). After surgery the incisions were closed with wound clips and the rats were kept warm until they had recovered from anesthesia. A series of sections was processed for the PHA-L immunohystochemistry to verify the injection site of PHA-L (Zahm et al., 2013).

Intracerebroventricular (icv) cannulation

Wistar rats deeply anesthetized with i.p. injections of a cocktail of ketamine and xylazine as described above were placed in a stereotaxic instrument (Kopf, model 900) with bregma and lambda in a horizontal plane. A stainless steel guide cannula (10.0 mm long, 0.6 mm o.d., 0.4mm i.d.) was implanted into the right lateral ventricle using coordinates from the atlas of Paxinos and Watson (21): 0.6 mm caudal to bregma, 1.5 mm lateral to the mid-line and 3.5 mm below the dura mater. The cannula was fixed to the cranium using dental acrylic resin and two jeweller's screws. A 30-gauge metal obturator filled the cannula except during the injections. After surgery, the rats received a prophylactic injection of penicillin (50,000 U, i.m.) and were allowed to recover for 7 days, during which they were handled daily and habituated to the removal of the obturator from the guide cannula in order to minimize stress during the experimental procedure. Cannula placement was verified by sectioning the brains of all rats with the cryostat at the end the experiment.

Fos, TH and OT immunohistochemistry and immunofluorescence

Wistar rats were anesthetized with i.p. injections of 2.5% tribromoethanol (1 ml/100g of body weight) and transcardially perfused with 150 mL of cold 0.01M sodium phosphate buffer (PBS), pH 7.35, followed by 350 mL of cold 4% paraformaldehyde solution in 0.01 M PBS. The brains were removed, post-fixed for 1 hour in 4% paraformaldehyde solution and stored at 4°C in PBS containing 30% sucrose. Coronal sections of 30 μm were obtained with a cryostat (Microm) and preserved at −20°C in cryoprotectant solution until further processing.

One series of sections was first processed for Fos immunoreactivity (ir) by incubating overnight at room temperature using an anti-Fos antibody raised in rabbit (Ab-5, Oncogene Science, Manhasset, NY, USA) and diluted 1:10,000 in 0.1 M phosphate buffer (PB) containing 2% normal goat serum and 0.3% Triton X-100. Free-floating sections were washed with PB and incubated first with biotin-labeled anti-rabbit immunoglobulin (Vector Laboratories Inc., Burlingame, CA, USA, 1:200 dilution in 1.5% normal goat serum-PB) and then with avidin-biotin-peroxidase complex (Vector Laboratories Inc., Burlingame, CA, USA, 1:200 in PB) for 1 hour at room temperature. Immunoperoxidase labeling was detected using diaminobenzidine hydrochloride (DAB, Sigma Chemical Co., St. Louis, MO, USA) intensified with 1% cobalt chloride and 1% nickel ammonium sulfate, which generates a blue-black reaction product. Next, the sections were incubated for 48 hours at 4°C with monoclonal anti-tyrosine hydroxylase antibody (anti-TH) raised in mouse (MAB318, Merck Millipore, Billerica, MA, USA; 1:1,000). Then, sections were rinsed and subjected to the protocol described above, except that biotinylated goat anti-mouse IgG (Vector, 1:200) and non-intensified DAB, which generates an insoluble brown reaction product, were used. The sections were mounted on gelatinized slides, air-dried overnight, dehydrated, cleared in xylene and placed under a cover slip with Entellan (New Jersey, USA).

The NTS (−13.68 to −14.08 mm from the bregma) was identified with the aid of the atlas of Paxinos and Watson (16). Numbers of immunoreactive (ir) neurons were counted with the aid of a Leica microscope equipped with a DC 200 Leica digital camera attached to a contrast enhancement device. Fos-ir neurons exhibited a conspicuous blue-black immunoreaction product in the cell nucleus. TH labeling comprised a brown cytoplasmic reaction. Sections were counted bilaterally in 3-5 sections in 4-9 animals from each experimental group by participants blind to the experimental protocols.

A separate series of sections was processed for qualitative immunofluorescence to show triple labeling of Fos, TH and OT. Briefly, sections were rinsed with Tris and co-incubated for 48 hours with rabbit anti-c-Fos (Ab-5, Oncogene Science, Manhasset, NY, USA; 1:20,000), mouse anti-TH (MAB318, Merck Millipore, Billerica, MA, USA; 1:1,000) and guinea pig anti-OT (T-5021, Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:80,000). The sections were immersed in biotin-SP-conjugated donkey anti-guinea pig IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:200), rinsed, and then immersed in Tris containing Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes; 1:250), AMCA-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:250) and Alexa Fluor 488-conjugated Streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:250). The sections were rinsed, mounted using Fluormount G (SouthernBiotech, Birmingham, AL) and analyzed with a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).

OT and PHA-L immunohistochemistry and immunofluorescence

Sprague-Dawley rats were deeply anesthetized with i.p. injections of the mixture of ketamine and xylazine as described above and transcardially perfused with 0.01 M phosphate buffer (PB; pH 7.4) containing 0.9% sodium chloride and 2.5% sucrose followed by 0.1 M PB (pH 7.4) containing 4% paraformaldehyde and 2.5% sucrose. The brains were removed, post-fixed, infiltrated with 25% sucrose, sectioned frozen at 50 μm and stored at −20° C in cryoprotectant solution.

Prior to processing with immunohistochemical or immunofluorescence reagents the sections were pretreated by immersion in 1% aqueous sodium borohydride for 15 minutes followed by rinsing with PB. One series of sections of all animals of the three experimental groups was immersed in PB containing 0.1% Triton X-100 (PB-t) and polyclonal antibody raised against OT made in rabbit (Bachem Lab. Inc., USA; 1: 5,000). The following day, after rinsing in PB-t, the sections were immersed for 1 hour in PB-t containing biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:200). Afterward, the sections were rinsed with 0.1M PB-t and then immersed for 1 hour in 0.1M PB containing avidin–biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, USA; 1:200). After rinsing with 0.1M PB, the sections were immersed in 0.1M PB containing 0.05% 3,3′-diaminobenzidine and 0.003% hydrogen peroxide, which generates an insoluble brown reaction product. The sections were then mounted on gelatin-coated slides and coverslipped with Permount (Fisher, Pittsburgh, PA, USA).

For the quantitative analysis of OT labeling in the NTS (−13.68 to −14.08 mm from the bregma), two sections from each of 5-7 animals per experimental group were used. Photomicrographs were captured in dark field illumination at 20× magnification with the aid of a Leica microscope and a Leica DC 200 digital camera equipped with a contrast enhancement device. All images were captured in similar illumination conditions in a 1392×1040 pixel field. Quantification of OT labeling was accomplished with the aid of ImageJ software (National Institutes of Health), which features a thresholding function allowing the level of signal regarded as positive to be set by the operator (Willemse et al., 1994). Briefly, the yellow-gold hue of the dark-field representation of OT immunolabeling comprises a unique set of RGB (red, green and blue) intensities, which the system can be set to recognize. This was done in six randomly chosen sections to establish a standard (fixed) threshold for specific labeling in order to avoid erroneously including background variations as significant levels of labeling. The NTS was circumscribed and the ratio between the total area of particles labeled for OT within the NTS and area of the NTS was calculated, thus providing an index of OT labeling, which was used in the statistical analysis.

For qualitative analysis of the co-localization of OT and PHA-L, another series of sections with PHA-L injections in the posterior parvocellular subdivision of PVN was processed for PHA-L and OT immunofluorescence. For this, sections were immersed in PB-t containing polyclonal antibody raised in goat against PHA-L (Vector Laboratories, Burlingame, CA, USA; 1:5,000). The following day, after rinsing with PB-t, the sections were immersed for 1 hour in PB-t containing biotinylated donkey anti-goat antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:200). Afterward, the sections were rinsed with PB and then immersed in PB containing streptadivin DyLight 594 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:200). The sections then were rinsed with 0.1M PB and immersed overnight in 0.1M PB containing anti-OT (1:5,000). The following day, the sections were rinsed with 0.1M PB and immersed for 1 hour in 0.1M PB containing CY2 conjugated to anti-rabbit IGg made in donkey (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 1:100). The sections were rinsed with 0.1M PB and mounted and coverslipped with ProLong Gold antifade reagent (Invitrogen). Sections were viewed with an Olympus BX51 microscope, and digital micrographs were generated using a DVC2000C-00-GE-MBF digital camera.

Experimental Protocols

Experiment 1: OT and PHA-L immunoreactivities in the NTS after ADX

On day 1, Sprague-Dawley rats were subjected to sham and ADX surgeries to generate the following experimental groups: sham, ADX and ADX+B. On day 7, animals received PHA-L injections into the PVN. On day 13 the rats were fasted for 16 hours, and on day 14, 2 hours after refeeding, they were anesthetized and perfused. Brain tissue was collected and processed as described for immunohistochemistry/immunofluorescence.

Experiment 2: Effects of pretreatment with OT receptor antagonist on NTS neuronal activation in sham, ADX and ADX+B groups in the fasting-refeeding regimen

On day 1, Wistar rats subjected to sham and ADX surgeries were formed into sham, ADX and ADX+B groups. On day 7, cannulas aimed at the lateral ventricle were implanted. Six days later, the animals were fasted for 16 hours, and on day 14 they were infused through the implanted cannulas with [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OVT, Penninsula/Bachem, 5μg/5μl icv) or vehicle (0.9% NaCl/5 μL). Fifteen minutes following the injections, half of the animals were given access to food and, 2 hours later, all were anesthetized and perfused. Brain tissue was collected and processed for immunohistochemistry/immunofluorescence as described.

Statistical analysis

The data were expressed as means ± SEM, which were tested using one-way (experiments 1) and three-way (experiment 2) ANOVAs, followed by the Newmann-Keuls post hoc test. Differences were considered significant at P<0.05.

RESULTS

ADX increases OT labeling in the NTS

The amount of OT immunolabeling in the NTS was increased in adrenalectomized animals as compared to sham (P<0.05) and ADX+B (P<0.05) groups (Fig. 1A). Representative photomicrographs of OT immunoreactivity in the NTS from the three experimental groups are shown in Figure 1B.

Figure 1. OT immunoreactivity in the NTS.

Figure 1

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A, Index of OT labeling in the NTS of refed sham, ADX and ADX+B groups (n=6-7 rats/group). Index represents the ratio between the area of particles labeled for OT (μm2) and the area of the NTS (μm2). *P<0.05 vs. sham and ADX+B groups. B, Representative photomicrographs of coronal sections, showing OT axonal projections to the NTS of refed sham, ADX and ADX+B groups. Photomicrographs are presented at 10× (left panels) and 20× (right panels) magnifications.

To evaluate qualitatively if axons in the NTS that exhibit increased OT labeling following ADX originate in the posterior parvocellular subdivision of PVN, another series of sections with PHA-L injections in the posterior parvocellular subdivision of PVN was processed for PHA-L and OT immunofluorescence. We observed that PHA-L was co-localized with OT in neurons in the PHA-L injection site in the PVN (Fig. 2A) and in axons in the NTS (Fig. 2B) after ADX.

Figure 2. PHA-L and OT labelings in the PVN and NTS after ADX.

Figure 2

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Representative photomicrographs (20× magnification) of coronal sections, showing PHA-L (red) and OT (green) immunoreactivities in the posterior parvocellular subdivision of the PVN (A) and NTS (B) of the same refed adrenalectomized animal. The right panels present the merging of OT and PHA-L labeling (yellow). Scale bar, 100μm. Each inset depicts the area where the photomicrograph was taken at 10× magnification.

OT receptor antagonist reverses the increased activation of NTS neurons induced by refeeding after ADX

In fasted groups, the numbers of Fos-ir (Fig. 3A) and Fos+TH-ir (Fig. 3B) neurons in the NTS were similar in all vehicle and OVT groups. In the vehicle-pretreated rats, refeeding increased (P<0.05) the numbers of Fos-ir (Fig. 3A) and Fos+TH-ir (Fig. 3B) neurons in the NTS in sham, ADX and ADX+B groups, with a greater (P<0.05) number of these neurons in adrenalectomized animals. After pretreatment with OVT, refeeding also augmented (P<0.05) the number of Fos-ir (Fig. 3A) and Fos+TH-ir (Fig. 3B) neurons in the NTS in the three groups, with no difference among sham, ADX and ADX+B groups. However, pretreatment with OVT decreased (P<0.05) the number of Fos-ir (Fig. 3A) and Fos+TH-ir (Fig. 3B) neurons after refeeding in adrenalectomized rats when compared to the ADX/vehicle group, with no effects in the sham and ADX+B groups. Representative photomicrographs of Fos expression and Fos+TH double labeling in the NTS are shown in Figure 4.

Figure 3. Fos and TH immunoreativities in the NTS.

Figure 3

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Number of Fos-ir (A) and Fos+TH-ir (B) neurons in the NTS of fasted and refed sham, ADX, and ADX+B groups (n=4-9 rats/group), pretreated with vehicle or [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OVT; 5μg/5μl icv). Data are shown as means±SEM. ND: not detectable. *P<0.05 vs. respective fasted group. # P<0.05 vs. refed sham/vehicle, refed ADX+B/vehicle and refed ADX/OVT groups.

Figure 4. Fos and TH labelings in the NTS.

Figure 4

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Representative photomicrographs (40× magnification) of coronal sections, showing Fos and TH immunoreactivities in the NTS of refed sham, ADX, and ADX+B groups, pretreated with vehicle or [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OVT; 5μg/5μl icv). The double labeled Fos-TH neurons are indicated by blue arrowheads and each inset depicts a double labeled Fos-TH neuron taken at 100× magnification.

Oxytocin fibers innervate NTS neurons activated by refeeding after ADX

To determine whether catecholaminergic (TH immunoreactive) and non-catecholaminergic neurons activated in response to feeding are innervated by OT neurons after ADX, triple labeling for Fos, TH and OT was performed in NTS sections. This is illustrated in Figure 5, which shows OT-ir fibers in close apposition to catecholaminergic and non-catecholaminergic neurons activated by food intake after ADX, as shown by Fos immunolabeling.

Figure 5. Fos, TH and OT labelings in the NTS after ADX.

Figure 5

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Confocal photomicrographs (40× magnification) of coronal sections, showing TH (A, blue), Fos (B, red), OT (C, green) immunoreactivities in the NTS of refed adrenalectomized animal. D, Merging of TH, Fos and OT labeling in the NTS of the same refed adrenalectomized animal. Scale bar, 50μm. E, The selected area (3× zoom at 40× magnification) of the triple labeling in the panel D, showing OT-ir fiber in contact with TH+Fos-ir neurons, indicated by the yellow arrow, and OT-ir fibers in close apposition to Fos-ir neurons, indicated by white arrows. Scale bar, 10μm.

DISCUSSION

The present study addressed whether OT-ir is modulated in projections to the NTS in conjunction with the increase in satiety-related responses following ADX. First, we observed that the ADX group had increased OT-ir in axons in the NTS. Then, we showed that OT-ir is present in NTS axons projecting from the posterior parvocellular subdivision of the PVN and that OT axons come into close apposition to NTS neurons activated by feeding after ADX. Finally, we showed that OT receptor antagonist reverses the increased activation of NTS neurons observed in adrenalectomized rats after refeeding.

OT projections to the NTS from the posterior parvocellular subdivision of the PVN have been demonstrated by the injection of retrograde tracer into the NTS (Rinaman, 1998, Sawchenko et al., 1982). Our study, however, is the first to demonstrate co-localization of OT in anterogradely labeled PVN projections to the NTS. The posterior parvocellular subdivision of the PVN was confirmed as the origin of these OT fibers to the NTS by the double-labeling of PHA-L and OT in this hypothalamic site. In addition, synthesis of OT in the PVN and its transport to the NTS have been demonstrated by White et al. (1984). Similarly, several authors have demonstrated OT-ir fibers in the NTS (Blevins et al., 2003, Llewellyn-Smith et al., 2012, Peters et al., 2008, Rinaman et al., 1998), suggesting that OT plays an important role in the different responses mediated by the NTS. In vivo experiments demonstrated that OT is released within the NTS after electrical stimulation of the PVN (Landgraf et al., 1990) and that NTS neurons are excited by local release of OT (McCann et al., 1990). Moreover, Peters et al. (2008) have shown that OT released from PVN axons facilitates the excitation of neurons in the NTS.

Our group recently showed that ADX-induced hypophagia is associated with increased activation of NTS neurons in response to refeeding despite the reduced food intake (Uchoa et al., 2009a), indicative of a greater responsiveness in adrenalectomized animals to physiological signals elicited by feeding in the form of enhanced satiety-related responses. Furthermore, we have shown the up-regulation of transcription of the OT gene in the PVN in adrenalectomized rats in fasted condition and after refeeding, by real time PCR (Uchoa et al., 2009). We also demonstrated in this previous study that ADX increases the activation of OT neurons in the posterior parvocellular subdivision of the PVN in response to food intake. These published results, together with the increased OT immunolabeling in the NTS following ADX, described for the first time in the present study, strongly suggest that OT is a mediator of the enhanced activation of NTS neurons induced by food intake after ADX. To test this, we used an OT receptor antagonist, OVT, which reversed the increased activation of NTS catecholaminergic and non-catecholaminergic neurons in response to feeding in adrenalectomized rats. In agreement with this new finding, OT receptor antagonist was also previously shown to reverse ADX-induced hypophagia (Uchoa et al., 2009b), indicating that OT inhibits food intake after ADX by increasing neuronal activation in the NTS. These data are consistent with previous reports showing that OT acting through NTS pathways is a key mediator of the anorexigenic effects of cholecystokinin (CCK) and leptin (Blevins et al., 2003 and 2004).

It is known that NTS catecholaminergic neurons are activated during meal (Rinaman et al., 1998), and the satiety-related signal, CCK, activates catecholaminergic neurons in the NTS that project to the PVN (Rinaman et al., 1995). Additionally, Rinaman et al. (2003) demonstrated that the lesion of NTS noradrenergic neurons attenuates CCK-induced hypophagia and CCK-induced OT neuron activation in the PVN. Furthermore, Sawchenko and Swanson (1982) described projections from parvocellular OT neurons to the NTS, and recently Llewellyn-Smith et al. (2012) reported the presence of OT fibers in close apposition to TH neurons. Therefore, these data give support to the present findings that augmented activation of descending OT neurons from the PVN could mediate the higher activation of TH neurons in the NTS observed in adrenalectomized animals after feeding, since OT receptor antagonist was also able to reverse this response.

OT effects in the NTS are supported by the increased neuronal activation in the NTS after central and peripheral injections of OT (Maejima et al., 2011, Morton et al., 2012, Olson et al., 1993), consistent with the identification of OT binding sites and OT receptors in the NTS (Gimpl and Fahrenholz, 2011, Loup et al., 1989). These data reinforce the idea that the NTS is a site of direct actions of OT to increase satiety responses after ADX. Indeed, cytotoxic lesions of cells that express OT receptors in the NTS attenuated the efficacy of CCK to reduce food intake (Baskin et al., 2010).

The functional modulation of OT on the activation of NTS neurons was further supported in the present study by demonstration of OT-ir fibers in close apposition to NTS neurons activated by feeding following ADX. Additionally, OT-ir fibers were also shown to be in contact with a catecholaminergic neuron activated by feeding after ADX. Blevins et al. (2003) demonstrated that OT innervates NTS neurons activated by CCK; likewise, OT-ir fibers were also identified in close apposition to neurons in the dorsal vagal complex (Llewellyn-Smith et al., 2012), while Geerling et al. (2010) demonstrated PVN axons in close appositions to TH neurons in the NTS. Consistent with these data, Singru et al. (2012) recently showed that refeeding-activated neurons of the PVN project to the dorsal vagal complex. Accordingly, an anorectic pathway from the PVN to the NTS was proposed by Maejima et al. (2009), who demonstrated that central administration of nesfatin-1 activates nesfatin-1 and OT neurons in the PVN, and nesfatin-1 neurons, once activated, could stimulate OT neurons in the PVN. OT signaling could be driven to POMC neurons in the NTS, causing melanocortin-dependent hypophagia. This study conducted by Maejima et al. (2009) shows the neural pathway for the anorexigenic response of OT and gives support to the proposed role of OT as a regulator of food intake. Collectively, these data strongly suggest that OT neurons in the PVN contribute to the enhanced activation of NTS neurons in meal-induced satiety responses after ADX.

CRF is also involved in the increased satiety-related responses elicited by ADX, since a CRF2 receptor antagonist abolished the anorexia and augmented activation of NTS neurons induced by feeding in adrenalectomized animals (Uchoa et al., 2010). CRF and OT might act through parallel pathways in the NTS, and/or OT might be a downstream mediator of CRF actions, as it was previously shown that an OT receptor antagonist reverses CRF-induced anorexia (Olson et al., 1991). Thus, the enhanced satiety-related responses after ADX seem to be mediated by central CRF and OT pathways to the NTS.

In summary, data obtained in the present study demonstrate that OT-ir is increased in projections to the NTS following ADX and OT receptor antagonist reverses the increased activation of NTS induced by feeding after ADX. Taken together, these functional and neuroanatomical observations strongly suggest that the enhanced OT projections from the PVN to the NTS contribute to satiety-related responses induced by ADX. Therefore, OT should be regarded as a mediator of hypophagia in primary adrenal insufficiency through its stimulatory effects on NTS pathways that mediate meal-induced satiety responses.

New findings.

  • What is the central question of this study?
    • Adrenalectomy-induced hypophagia is related with enhanced activation of satiety responses in nucleus of the solitary tract (NTS) and is reversed by oxytocin receptor antagonist. The potential role of hypothalamic oxytocin projections to the NTS in the satiety-related responses following adrenalectomy has not been reported.
  • What is the main finding and its importance?
    • Our study shows that adrenalectomy increases oxytocin projections in the NTS and oxytocin receptor antagonist reverses the increased activation of NTS neurons induced by feeding after adrenalectomy. These data indicate that oxytocin pathways to the NTS contribute to higher satiety-related responses, pointing oxytocin as a mediator of hypophagia following adrenalectomy through its stimulatory effects on NTS.

ACKNOWLEDGMENTS

The authors are grateful to Maria Valci dos Santos, Milene Mantovani Lopes, Antonio Renato Meirelles e Silva, Elizabete Rosa Milani, Kenneth P. Parsley and Rubens Fernando de Melo for their excellent technical assistance.

GRANTS

This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil, as well as by USPHS NIH NS-23805.

Footnotes

The authors have nothing to disclose.

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