Allergic airway inflammation induces migration of mast cell populations into the mouse airway.

Mast cells (MCs) and airway nerves play an important role in allergic asthma. However, little is known about the MCs and their interaction with airway nerves during allergic airway inflammation. This study aims to investigate the distribution and proliferation of MC populations in different lung compartments, along with the association of mast cells with nerve endings, using a house dust mite (HDM) model for allergic airway inflammation. BALB/c mice were exposed to HDM extract intranasally (25 µg/50 µl) for 5 consecutive days a week over 7 weeks. Immunofluorescence and Edu stains were used to examine the colocalisation of MCs and nerves and the proliferation of MCs, respectively. HDM treatment caused an increased migration of MCs into bronchi, alveolar parenchyma and airway vessels. The proportions of tryptase-chymase expressing MC (MCTC) increased significantly in the bronchi and the alveolar parenchyma but not in the vascular tissues, by allergic airway inflammation. The association of MCs with nerves was found only in the bronchi and there were no changes in comparison of controls to HDM-treated animals. The present study shows a strong migration of tryptase expressing MC (MCT) and MCTC into the bronchi and the alveolar parenchyma, as well as of MCT in the vascular compartment under HDM treatment. This supports the hypothesis that these mast cell populations may contribute to allergic airway inflammation.

CXCR4(+) dendritic cells promote angiogenesis during embryo implantation in mice.

Early pregnancy is characterized by decidual adaption to the developing embryo involving angiogenesis and vascular growth. Failure of decidual vascular expansion is linked to diseases of pregnancy. Dendritic cells (DC) have been associated with vascular growth during early gestation, though it is unknown whether their capacity to modulate angiogenesis is ubiquitous to all DC subsets. Here, we show that DC normally found associated with the decidual vasculature co-express the C-X-C chemokine receptor type 4 (CXCR4). In addition, we demonstrate that impaired homing of CXCR4(+)DC during early gestation provoked a disorganized decidual vasculature with impaired spiral artery remodeling later in gestation. In contrast, adoptive transfer experiments provided evidence that CXCR4(+)DC are able to rescue early pregnancy by normalizing decidual vascular growth and delivery of pro-angiogenic factors, which results in adequate remodeling of the spiral arteries during placental development. Taken together, our results indicate an important role of CXCR4(+)DC in the regulation of decidual angiogenesis and highlight the importance of the CXCL12/CXCR4 pathway during this process, suggesting that this may represent a key pathway to evaluate during pregnancy pathologies associated with impaired vascular expansion.

Ghrelin and NUCB2/nesfatin-1 are expressed in the same gastric cell and differentially correlated with body mass index in obese subjects.

The orexigenic peptide ghrelin and the anorexigenic peptide nesfatin-1 are expressed by the same endocrine cell of the rat stomach, the X/A-like cell. However, data in humans are lacking, especially under conditions of obesity. We collected gastric tissue of obese patients undergoing sleeve gastrectomy and investigated the expression of nesfatin-1 and ghrelin in the gastric oxyntic mucosa by immunofluorescence. Nesfatin-1 immunoreactivity was detected in the human oxyntic mucosa in cells with an endocrine phenotype. A major portion of nesfatin-1 immunoreactive cells (78 %) co-localized with ghrelin indicating the occurrence in human X/A-like cells. In patients with very high body mass index (BMI 55-65 kg/m(2)), the number of nesfatin-1 immunoreactive cells/low-power field was significantly higher than in obese patients with lower BMI (40-50 kg/m(2), 118 ± 10 vs. 82 ± 11, p < 0.05). On the other hand, the number of ghrelin immunoreactive cells was significantly reduced in obese patients with higher compared to lower BMI (96 ± 12 vs. 204 ± 21, p < 0.01). Also the ghrelin-acylating enzyme ghrelin-O-acyltransferase decreased with increasing BMI. In conclusion, nesfatin-1 immunoreactivity is also co-localized with ghrelin in human gastric X/A-like cells giving rise to a dual role of this cell type with differential effects on stimulation and inhibition of appetite dependent on the peptide released. The expression of these two peptides is differentially regulated under obese conditions with an increase of nesfatin-1 and a decrease of ghrelin immunoreactivity with rising BMI pointing towards an adaptive change of expression that may counteract further body weight increase.

Abdominal surgery increases activity in several phoenixin immunoreactive nuclei.

BACKGROUND: Research on the peptide phoenixin has increased in recent years and greatly widened the known scope of its functions since its discovery in 2013. Involvement of phoenixin has since been shown in anxiety, food intake, reproduction as well as emotional and immunological stress. To further evaluate its involvement in stress reactions, this study aims to investigate the effects of abdominal surgery, a well-established physical stressor, on the activity of phoenixin-immunoreactive brain nuclei. METHODS: Male Sprague-Dawley rats (n = 6/group) were subjected to either an abdominal surgery stress protocol or a sham operation. Animals in the verum group were anesthetized, the abdominal cavity opened and the cecum palpated, followed by closing of the abdomen and recovery. Sham operated animals only received inhalation anesthesia and time for recovery. All animals were subsequently sacrificed and brains processed and evaluated for c-Fos activity as well as phoenixin density. RESULTS: Compared to control, abdominal surgery significantly increased c-Fos activity in the paraventricular nucleus (PVN, 6.4-fold, p < 0.001), the medial part of the nucleus of the solitary tract (mNTS, 3.8-fold, p < 0.001), raphe pallidus (RPa, 3.6-fold, p < 0.001), supraoptic nucleus (SON, 3.2-fold, p < 0.001), ventrolateral medulla (VLM, also called A1C1, 3.0-fold, p < 0.001), dorsal motor nucleus of vagus (DMN, 2.9-fold, p < 0.001), locus coeruleus (LC, 1.8-fold, p < 0.01) and Edinger-Westphal nucleus (EW, 1.6-fold, p < 0.05), while not significantly altering c-Fos activity in the amygdala (CeM, 1.3-fold, p > 0.05). Phoenixin immunoreactivity was not significantly affected by abdominal surgery (p > 0.05). CONCLUSION: The observed abdominal surgery-related increase in activity in phoenixin immunoreactive nuclei compared to sham surgery controls supports the hypothesis of an involvement of phoenixin in stress reactions. Interestingly, various psychological and physical stressors lead to specific changes in activity and immunoreactivity in phoenixin-containing nuclei, giving rise to a stressor-specific involvement of phoenixin.

Inflammatory Stress Induced by Intraperitoneal Injection of LPS Increases Phoenixin Expression and Activity in Distinct Rat Brain Nuclei.

Due to phoenixin's role in restraint stress and glucocorticoid stress, as well as its recently shown effects on the inflammasome, we aimed to investigate the effects of lipopolysaccharide (LPS)-induced inflammatory stress on the activity of brain nuclei-expressing phoenixin. Male Sprague Dawley rats (n = 6/group) were intraperitoneally injected with either LPS or control (saline). Brains were processed for c-Fos and phoenixin immunohistochemistry and the resulting slides were evaluated using ImageJ software. c-Fos was counted and phoenixin was evaluated using densitometry. LPS stress significantly increased c-Fos expression in the central amygdaloid nucleus (CeM, 7.2-fold), supraoptic nucleus (SON, 34.8 ± 17.3 vs. 0.0 ± 0.0), arcuate nucleus (Arc, 4.9-fold), raphe pallidus (RPa, 5.1-fold), bed nucleus of the stria terminalis (BSt, 5.9-fold), dorsal motor nucleus of the vagus nerve (DMN, 89-fold), and medial part of the nucleus of the solitary tract (mNTS, 121-fold) compared to the control-injected group (p < 0.05). Phoenixin expression also significantly increased in the CeM (1.2-fold), SON (1.5-fold), RPa (1.3-fold), DMN (1.3-fold), and mNTS (1.9-fold, p < 0.05), leading to a positive correlation between c-Fos and phoenixin in the RPa, BSt, and mNTS (p < 0.05). In conclusion, LPS stress induces a significant increase in activity in phoenixin immunoreactive brain nuclei that is distinctively different from restraint stress.

Circulating Spexin Is Associated with Body Mass Index and Fat Mass but Not with Physical Activity and Psychological Parameters in Women across a Broad Body Weight Spectrum.

Spexin (SPX) is a novel, widely expressed peptide, with anorexigenic effects demonstrated in animal models and negatively correlated with body mass index (BMI) in humans. It increases locomotor activity in rodents and is elevated in human plasma following exercise. Studies have also shown an effect of stress and anxiety on SPX's expression in different brain structures in animals. The relationships between plasma SPX and physical activity, body composition, and patient-reported outcomes such as perceived stress, depressiveness, anxiety, and eating behaviors are unknown and were examined in this study over a wide BMI range. A total of 219 female (n = 68 with anorexia nervosa; n = 79 with obesity; n = 72 with normal weight) inpatients were enrolled. Perceived stress (PSQ 20), anxiety (GAD 7), depressiveness (PHQ 9), and eating disorder pathology (EDI 2), as well as BMI, bioimpedance analysis, and accelerometry, were measured cross-sectionally at the beginning of treatment and correlated with plasma SPX levels (measured by ELISA) obtained at the same time. Plasma SPX levels were negatively associated with BMI (r = −0.149, p = 0.027) and body fat mass (r = −0.149, p = 0.04), but did not correlate with perceived stress, anxiety, depressiveness, eating behavior, energy expenditure, and physical activity (p > 0.05). The results replicate the negative correlation of SPX with BMI and fat mass, but do not support the hypothesis that peripheral SPX plays a role in the regulation of stress, depressiveness, anxiety, eating behavior, or physical activity.

Cholecystokinin and bombesin activate neuronatin neurons in the nucleus of the solitary tract.

Neuronatin (Nnat) is involved in the regulation of cellular molecular signaling and appears to be also linked to metabolic processes. The gastrointestinal peptides cholecystokinin (CCK) and bombesin (BN) have an effect on the short-term inhibition of food intake and induce neuronal activation in different brain nuclei, prominently in the nucleus of the solitary tract (NTS) involved in the modulation of food intake. The aim of the study was to examine if Nnat immunoreactivity is detectable in the NTS, and whether peripheral CCK-8S or BN cause c-Fos activation of Nnat neurons. Non-fasted male Sprague-Dawley rats received an intraperitoneal (i.p.) injection of 5.2 or 8.7 nmol CCK-8S/kg or 26 or 32 nmol BN/kg (n = 4 all groups) or vehicle solution (0.15 M NaCl; n = 7). The number of c-Fos neurons was determined 90 min post injection in the NTS and dorsal motor nucleus of the vagus (DMV). We observed Nnat immunoreactive neurons in the NTS and DMV. CCK-8S (25-fold and 51-fold, p = 0.025 and p = 0.001) and BN (31-fold and 59-fold, p = 0.007 and p = 0.001) at both doses increased the number of c-Fos positive neurons in the NTS. CCK and BN did not show a significant effect in the DMV. Both doses of CCK-8S (24-fold and 48-fold p = 0.011 and p = 0.001) and bombesin (31-fold and 56-fold, p = 0.002 and p = 0.001) increased the number of activated Nnat neurons in the NTS (p = 0.001) compared to the vehicle group, while in the DMV no significant increase of c-Fos activation was detected. In conclusion, i.p. injected CCK-8S or BN induce an increased neuronal activity in NTS Nnat neurons, giving rise that Nnat may play a role in the regulation of food intake mediated by peripheral CCK-8S or BN.

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Corticotropin-releasing factor-overexpressing mice exhibit reduced neuronal activation in the arcuate nucleus and food intake in response to fasting.

Corticotropin-releasing factor (CRF) overexpressing (OE) mice are a genetic model that exhibits features of chronic stress. We investigated whether the adaptive feeding response to a hypocaloric challenge induced by food deprivation is impaired under conditions of chronic CRF overproduction. Food intake response to a 16-h overnight fast and ip injection of gut hormones regulating food intake were compared in CRF-OE and wild type (WT) littermate mice along with brain Fos expression, circulating ghrelin levels, and gastric emptying of a nonnutrient meal. CRF-OE mice injected ip with saline showed a 47 and 44% reduction of 30-min and 4-h cumulative food intake response to an overnight fast, respectively, compared with WT. However, the 30-min food intake decrease induced by ip cholecystokinin (3 microg/kg) and increase by ghrelin (300 microg/kg) were similar in CRF-OE and WT mice. Overnight fasting increased the plasma total ghrelin to similar levels in CRF-OE and WT mice, although CRF-OE mice had a 2-fold reduction of nonfasting ghrelin levels. The number of Fos-immunoreactive cells induced by fasting in the arcuate nucleus was reduced by 5.9-fold in CRF-OE compared with WT mice whereas no significant changes were observed in other hypothalamic nuclei. In contrast, fasted CRF-OE mice displayed a 5.6-fold increase in Fos-immunoreactive cell number in the dorsal motor nucleus of the vagus nerve and a 34% increase in 20-min gastric emptying. These findings indicate that sustained overproduction of hypothalamic CRF in mice interferes with fasting-induced activation of arcuate nucleus neurons and the related hyperphagic response.

Peripheral obestatin has no effect on feeding behavior and brain Fos expression in rodents.

Obestatin is produced in the stomach from proghrelin by post-translational cleavage. The initial report claimed anorexigenic effects of obestatin in mice. Contrasting studies indicated no effect of obestatin on food intake (FI). We investigated influences of metabolic state (fed/fasted), environmental factors (dark/light phase) and brain Fos response to intraperitoneal (ip) obestatin in rats, and used the protocol from the original study assessing obestatin effects in mice. FI was determined in male rats injected ip before onset of dark or light phase, with obestatin (1 or 5 micromol/kg), CCK8S (3.5 nmol/kg) or 0.15 M NaCl, after fasting (16 h, n=8/group) or ad libitum (n=10-14/group) food intake. Fos expression in hypothalamic and brainstem nuclei was examined in freely fed rats 90 min after obestatin (5 micromol/kg), CCK8S (1.75 nmol/kg) or 0.15 M NaCl (n=4/group). Additionally, fasted mice were injected ip with obestatin (1 micromol/kg) or urocortin 1 (2 nmol/kg) 15 min before food presentation. No effect on FI was observed after obestatin administration during the light and dark phase under both metabolic conditions while CCK8S reduced FI irrespectively of the conditions. The number of Fos positive neurons was not modified by obestatin while CCK8S increased Fos expression in selective brain nuclei. Obestatin did not influence the refeeding response to a fast in mice, while urocortin was effective. Therefore, peripheral obestatin has no effect on FI under various experimental conditions and did not induce Fos in relevant central neuronal circuitries modulating feeding in rodents.

Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats.

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 microg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 microg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12h. Ghrelin and desacyl ghrelin (64 microg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.

Peripheral injection of ghrelin induces Fos expression in the dorsomedial hypothalamic nucleus in rats.

Peripheral ghrelin has been shown to act as a gut-brain peptide exerting a potent orexigenic effect on food intake. The dorsomedial nucleus of the hypothalamus (DMH) is innervated by projections from other brain areas being part of the network of nuclei controlling energy homeostasis, among others NPY/AgRP-positive fibers arising from the arcuate nucleus (ARC). The aim of the study was to determine if peripherally administered ghrelin affects neuronal activity in the DMH, as assessed by Fos expression. The number of Fos positive neurons was determined in the DMH, paraventricular nucleus of the hypothalamus (PVN), ARC, ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS) and in the area postrema (AP) in non-fasted Sprague-Dawley rats in response to intraperitoneally (ip) injected ghrelin (3 nmol/rat) or vehicle (0.15 M NaCl). Peripheral ghrelin induced a significant increase in the number of Fos-ir positive neurons/section compared with vehicle in the ARC (mean+/-SEM: 49+/-2 vs. 23+/-2 neurons/section, p=0.001), PVN (69+/-5 vs. 34+/-3, p=0.001), and DMH (142+/-5 vs. 83+/-5, p<0.001). Fos-ir positive neurons were mainly localized within the ventral part of the DMH. No change in Fos expression was observed in the VMH (53+/-8 vs. 48+/-6, p=0.581), NTS (42+/-2 vs. 40+/-3, p=0.603), and in the AP (7+/-1 vs. 5+/-1, p=0.096). Additional double-labelling with anti-Fos and anti-AgRP revealed that Fos positive neurons in the DMH were encircled by a network of AgRP-ir positive fibers. These data indicate that peripheral ghrelin activates DMH neurons and that NPY-/AgRP-positive fibers may be involved in the response.

Activity-based anorexia activates CRF immunoreactive neurons in female rats.

Activity-based anorexia (ABA) is a well-established animal model mimicking the eating disorder anorexia nervosa (AN). Since the pathophysiology of AN is yet poorly understood and specific drug treatments are lacking so far, animal models might be useful to further understand this disease. ABA consists of time-restricted access to food for 1.5 h/day and the possibility to exercise in a running wheel for 24 h/day. This combination leads to robust body weight loss as observed in AN. Here, we investigated the activation of brain corticotropin-releasing factor (CRF) neurons, a transmitter involved in the response to stress, emotional processes and also food intake. After development of ABA, rat brains were processed for c-Fos and CRF double immunohistochemistry. ABA increased the number of c-Fos/CRF double labeled neurons in the paraventricular nucleus (PVN) and the dorsomedial hypothalamic nucleus (DMH) compared to the ad libitum (AL, ad libitum fed, no running wheel) and activity (AC, ad libitum fed, running wheel, p < 0.05) but not to the restricted feeding (RF, food for 1.5 h/day, no running wheel, p > 0.05) group. Also the number of CRF neurons was increased in the DMH of ABA rats compared to AL and AC (p < 0.05). In the Edinger-Westphal nucleus (EW) the number of c-Fos positive neurons was increased in ABA and RF compared to AC (p < 0.05), while the number of double labeled neurons was not different (p > 0.05). Taken together, brain CRF activated under conditions of ABA might play a role in the development and maintenance of this animal model and possibly also in human AN.

Nesfatin-130-59 Injected Intracerebroventricularly Increases Anxiety, Depression-Like Behavior, and Anhedonia in Normal Weight Rats.

Nesfatin-1 is a well-established anorexigenic peptide. Recent studies indicated an association between nesfatin-1 and anxiety/depression-like behavior. However, it is unclear whether this effect is retained in obesity. The aim was to investigate the effect of nesfatin-130-59-the active core of nesfatin-1-on anxiety and depression-like behavior in normal weight (NW) and diet-induced (DIO) obese rats. Male rats were intracerebroventricularly (ICV) cannulated and received nesfatin-130-59 (0.1, 0.3, or 0.9 nmol/rat) or vehicle 30 min before testing. Nesfatin-130-59 at a dose of 0.3 nmol reduced sucrose consumption in the sucrose preference test in NW rats compared to vehicle (⁻33%, p < 0.05), indicating depression-like/anhedonic behavior. This dose was used for all following experiments. Nesfatin-130-59 also reduced cookie intake during the novelty-induced hypophagia test (-62%, p < 0.05). Moreover, nesfatin-130-59 reduced the number of entries into the center zone in the open field test (-45%, p < 0.01) and the visits of open arms in the elevated zero maze test (-39%, p < 0.01) in NW rats indicating anxiety. Interestingly, DIO rats showed no behavioral alterations after the injection of nesfatin-130-59 (p > 0.05). These results indicate an implication of nesfatin-130-59 in the mediation of anxiety and depression-like behavior/anhedonia under normal weight conditions, while in DIO rats, a desensitization might occur.

Phoenixin-14 injected intracerebroventricularly but not intraperitoneally stimulates food intake in rats.

Phoenixin, a recently discovered 20-amino acid peptide was implicated in reproduction. However, the expression in food intake-regulatory nuclei such as the paraventricular nucleus, the arcuate nucleus and the nucleus of the solitary tract suggests an implication of phoenixin in food intake regulation. Therefore, we investigated the effects of phoenixin-14, the shorter form of phoenixin, on food intake following intracerebroventricular (icv) and intraperitoneal (ip) injection in ad libitum fed male Sprague-Dawley rats. Phoenixin-14 injected icv (0.2, 1.7 or 15nmol/rat) during the light phase induced a dose-dependent increase of light phase food intake reaching significance at a minimum dose of 1.7 nmol/rat (+72%, p<0.05 vs. vehicle) used for all further analyses. Assessment of the food intake microstructure showed an icv phoenixin-14-induced increase in meal size (+51%), meal duration (+157%), time spent in meals (+182%) and eating rate (+123%), while inter-meal intervals (-42%) and the satiety ratio (-64%) were decreased compared to vehicle (p<0.05). When injected icv during the dark phase, no modulation of food intake was observed (p>0.05). The light phase icv phoenixin-14-induced increase of water intake did not reach statistical significance compared to vehicle (+136%, p>0.05). The increase of food intake following icv phoenixin-14 was not associated with a significant alteration of grooming behavior (0.4-fold, p=0.377) or locomotion (6-fold, p=0.066) compared to vehicle. When injected ip at higher doses (0.6, 5nmol/kg or 45nmol/kg body weight) during the light phase, phoenixin-14 did not affect food intake (p>0.05). In summary, phoenixin-14 exerts a centrally-mediated orexigenic effect.

Activity-based anorexia activates nesfatin-1 immunoreactive neurons in distinct brain nuclei of female rats.

Activity-based anorexia (ABA) is an established animal model for the eating disorder anorexia nervosa (AN). The pathophysiology of AN and the involvement of food intake-regulatory peptides is still poorly understood. Nesfatin-1, an anorexigenic peptide also involved in the mediation of stress, anxiety and depression might be a likely candidate involved in the pathogenesis of AN. Therefore, activation of nesfatin-1 immunoreactive (ir) brain nuclei was investigated under conditions of ABA. Female Sprague-Dawley rats were used and divided into four groups (n=6/group): activity-based anorexia (ABA), restricted feeding (RF), activity (AC) and ad libitum fed (AL). After the 21-day experimental period and development of ABA, brains were processed for c-Fos/nesfatin-1 double labeling immunohistochemistry. ABA increased the number of nesfatin-1 immunopositive neurons in the paraventricular nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, locus coeruleus and in the rostral part of the nucleus of the solitary tract compared to AL and AC groups (p<0.05) but not to RF rats (p>0.05). Moreover, we observed significantly more c-Fos and nesfatin-1 ir double-labeled cells in ABA rats compared to RF, AL and AC in the supraoptic nucleus (p<0.05) and compared to AL and AC in the paraventricular nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, dorsal raphe nucleus and the rostral raphe pallidus (p<0.05). Since nesfatin-1 plays a role in the inhibition of food intake and the response to stress, we hypothesize that the observed changes of brain nesfatin-1 might play a role in the pathophysiology and symptomatology under conditions of ABA and potentially also in patients with AN.

Peripheral injection of CCK-8S induces Fos expression in the dorsomedial hypothalamic nucleus in rats.

Peripheral cholecystokinin (CCK) plays a physiological role in the regulation of food intake. The dorsomedial hypothalamic nucleus (DMH) has been implicated in the brain regulation of food intake and satiety. The aim of this study was to determine if peripherally administered CCK affects neuronal activity in the DMH, as assessed by Fos expression. Density of Fos-positive neurons was determined in the DMH, paraventricular nucleus of the hypothalamus (PVN), arcuate nucleus of the hypothalamus (ARC) and ventromedial hypothalamic nucleus (VMH) in non-fasted Sprague-Dawley rats in response to intraperitoneally (ip) injection of CCK-8S (2 microg/kg, n=6) or vehicle (0.15 M NaCl; n=6). CCK-8S increased Fos immunoreactivity in the DMH (mean+/-SEM; cells/section: 108+/-10 versus 54+/-6, p<0.001) and PVN (120+/-12 versus 20+/-3, p<0.001) compared to the vehicle group while not influencing Fos expression in the ARC and VMH. Double labeling showed that 27.4+/-6.4% (n=3) of Fos-positive neurons induced by CCK-8S were positive for corticotropin-releasing factor immunoreactivity, that were mainly localized in the ventral part of the DMH, and encircled in a network of tyrosine-hydroxylase-immunoreactive positive fibers. These data indicate that in addition of the PVN, peripheral CCK increases neuronal activity in the DMH suggesting a possible role in this hypothalamic nucleus in the satiating effect of the peptide.

Cerebral processing of auditory stimuli in patients with irritable bowel syndrome.

AIM: To determine by brain functional magnetic resonance imaging (fMRI) whether cerebral processing of non-visceral stimuli is altered in irritable bowel syndrome (IBS) patients compared with healthy subjects. To circumvent spinal viscerosomatic convergence mechanisms, we used auditory stimulation, and to identify a possible influence of psychological factors the stimuli differed in their emotional quality. METHODS: In 8 IBS patients and 8 controls, fMRI measurements were performed using a block design of 4 auditory stimuli of different emotional quality (pleasant sounds of chimes, unpleasant peep (2000 Hz), neutral words, and emotional words). A gradient echo T2*-weighted sequence was used for the functional scans. Statistical maps were constructed using the general linear model. RESULTS: To emotional auditory stimuli, IBS patients relative to controls responded with stronger deactivations in a greater variety of emotional processing regions, while the response patterns, unlike in controls, did not differentiate between distressing or pleasant sounds. To neutral auditory stimuli, by contrast, only IBS patients responded with large significant activations. CONCLUSION: Altered cerebral response patterns to auditory stimuli in emotional stimulus-processing regions suggest that altered sensory processing in IBS may not be specific for visceral sensation, but might reflect generalized changes in emotional sensitivity and affective reactivity, possibly associated with the psychological comorbidity often found in IBS patients.

Peripheral injection of bombesin induces c-Fos in NUCB2/nesfatin-1 neurons.

As anorexigenic hormones bombesin and nucleobindin2 (NUCB2)/nesfatin-1 decrease food intake in rodents. Both hormones have been described in brain nuclei that play a role in the modulation of hunger and satiety, like the paraventricular nucleus of the hypothalamus (PVN) and the nucleus of the solitary tract (NTS). However, the direct interaction of the two hormones is unknown so far. The aim of study was to elucidate whether bombesin directly interacts with NUCB2/nesfatin-1 neurons in the PVN and NTS. Therefore, we injected bombesin intraperitoneally (ip) at two doses (26 and 32nmol/kg body weight) and assessed c-Fos activation in the PVN, arcuate nucleus (ARC) and NTS compared to vehicle treated rats (0.15M NaCl). We also performed co-localization studies with oxytocin or tyrosine hydroxylase. Bombesin at both doses increased the number of c-Fos positive neurons in the PVN (p<0.05) and NTS (p<0.05) compared to vehicle, while in the ARC no modulation was observed (p>0.05). In the PVN and NTS the number of c-Fos positive neurons colocalized with NUCB2/nesfatin-1 increased after bombesin injection compared to vehicle treatment (p<0.05). Moreover, an increase of activated NUCB2/nesfatin-1 immunoreactive neurons that co-expressed oxytocin in the PVN (p<0.05) or tyrosine hydroxylase in the NTS (p<0.05) was observed compared to vehicle. Our results show that peripherally injected bombesin activates NUCB2/nesfatin-1 neurons in the PVN and NTS giving rise to a possible interaction between bombesin and NUCB2/nesfatin-1 in the modulation of food intake.

Activity-Based Anorexia Reduces Body Weight without Inducing a Separate Food Intake Microstructure or Activity Phenotype in Female Rats-Mediation via an Activation of Distinct Brain Nuclei.

Anorexia nervosa (AN) is accompanied by severe somatic and psychosocial complications. However, the underlying pathogenesis is poorly understood, treatment is challenging and often hampered by high relapse. Therefore, more basic research is needed to better understand the disease. Since hyperactivity often plays a role in AN, we characterized an animal model to mimic AN using restricted feeding and hyperactivity. Female Sprague-Dawley rats were divided into four groups: no activity/ad libitum feeding (ad libitum, AL, n = 9), activity/ad libitum feeding (activity, AC, n = 9), no activity/restricted feeding (RF, n = 12) and activity/restricted feeding (activity-based anorexia, ABA, n = 11). During the first week all rats were fed ad libitum, ABA and AC had access to a running wheel for 24 h/day. From week two ABA and RF only had access to food from 9:00 to 10:30 a.m. Body weight was assessed daily, activity and food intake monitored electronically, brain activation assessed using Fos immunohistochemistry at the end of the experiment. While during the first week no body weight differences were observed (p > 0.05), after food restriction RF rats showed a body weight decrease: -13% vs. day eight (p < 0.001) and vs. AC (-22%, p < 0.001) and AL (-26%, p < 0.001) that gained body weight (+10% and +13%, respectively; p < 0.001). ABA showed an additional body weight loss (-9%) compared to RF (p < 0.001) reaching a body weight loss of -22% during the 2-week restricted feeding period (p < 0.001). Food intake was greatly reduced in RF (-38%) and ABA (-41%) compared to AL (p < 0.001). Interestingly, no difference in 1.5-h food intake microstructure was observed between RF and ABA (p > 0.05). Similarly, the daily physical activity was not different between AC and ABA (p > 0.05). The investigation of Fos expression in the brain showed neuronal activation in several brain nuclei such as the supraoptic nucleus, arcuate nucleus, locus coeruleus and nucleus of the solitary tract of ABA compared to AL rats. In conclusion, ABA combining physical activity and restricted feeding likely represents a suited animal model for AN to study pathophysiological alterations and pharmacological treatment options. Nonetheless, cautious interpretation of the data is necessary since rats do not voluntarily reduce their body weight as observed in human AN.