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 ...

Humboldt-Universität zu Berlin and Berlin Institute of Health

Serotonin and cholecystokinin (CCK) play a role in the short-term inhibition of food intake. It is known that peripheral injection of CCK increases c-Fos-immunoreactivity (Fos-IR) in the nucleus of the solitary tract (NTS) in rats, and injection of the serotonin antagonist ondansetron decreases the number of c-Fos-IR cells in the NTS. This supports the idea of serotonin contributing to the effects of CCK. The aim of the present study was to elucidate whether peripherally injected CCK-8S modulates the concentration of serotonin in brain feeding-regulatory nuclei. Ad libitum fed male Sprague-Dawley rats received 5.2 and 8.7 nmol/kg CCK-8S (n=3/group) or 0.15M NaCl (n=3-5/group) injected intraperitoneally (ip). The number of c-Fos-IR neurons, and the fluorescence intensity of serotonin in nerve fibers were assessed in the paraventricular nucleus (PVN), arcuate nucleus (ARC), NTS and dorsal motor nucleus of the vagus (DMV). CCK-8S increased the number of c-Fos-ir neurons in the NTS (mean±SEM: 72±4, and 112±5 neurons/section, respectively) compared to vehicle-treated rats (7±2 neurons/section, P<0.05), but did not modulate c-Fos expression in the DMV or ARC. Additionally, CCK-8S dose-dependently increased the number of c-Fos-positive neurons in the PVN (218±15 and 128±14, respectively vs. 19±5, P<0.05). In the NTS and DMV we observed a decrease of serotonin-immunoreactivity 90 min after injection of CCK-8S (46±2 and 49±8 pixel/section, respectively) compared to vehicle (81±8 pixel/section, P<0.05). No changes of serotonin-immunoreactivity were observed in the PVN and ARC. Our results suggest that serotonin is involved in the mediation of CCK-8's effects in the brainstem.

Peripheral injected cholecystokinin-8S modulates the concentration of serotonin in nerve fibers of the rat brainstem.

It has been shown that dopamine antagonists suppress the ghrelin-induced increased motivation to work for food. The aim of this study was to investigate the influence of the dopamine antagonist flupentixol on ghrelin-induced food intake. Ad libitum fed male Sprague-Dawley (SD) rats were injected intraperitoneally (ip) with vehicle plus vehicle, vehicle plus ghrelin (13 μg/kg), 0.25mg/kg or 0.5mg/kg flupentixol plus ghrelin, or 0.25mg/kg or 0.5 mg/kg flupentixol plus vehicle. In a second experiment, intracerebroventricularly (icv) cannulated rats received an ip injection of vehicle (0.15M NaCl) or flupentixol (0.25mg/kg) and 20 min later an icv injection of vehicle or ghrelin (1 μg/rat). Both experiments were performed twice: first, rats were offered only standard chow, while in the second experiment they could choose between standard chow and a palatable/preferred chow. Cumulative light phase food intake was assessed for 7h. Ip as well as icv injected ghrelin reliably increased intake of standard chow. Flupentixol did not affect ghrelin-induced intake of standard chow. Ip injected ghrelin failed to increase the intake of palatable chow, whereas icv injected ghrelin did. This effect was not blocked by ip flupentixol. In summary, ip administered ghrelin did not increase the intake of chow the rats preferred; whereas icv injected ghrelin further stimulated the intake of preferred chow suggesting a direct central mediation of this effect. Our results show that the dopamine antagonist flupentixol does not influence ghrelin-induced feeding in our choice paradigm.

The dopamine antagonist flupentixol does not alter ghrelin-induced food intake in rats.

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.

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

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.

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

Activity-based anorexia activates CRF immunoreactive neurons in female 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-1-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-1 (0.1, 0.3, or 0.9 nmol/rat) or vehicle 30 min before testing. Nesfatin-1 at a dose of 0.3 nmol reduced sucrose consumption in the sucrose preference test in NW rats compared to vehicle (⁻33%,  < 0.05), indicating depression-like/anhedonic behavior. This dose was used for all following experiments. Nesfatin-1 also reduced cookie intake during the novelty-induced hypophagia test (-62%,  < 0.05). Moreover, nesfatin-1 reduced the number of entries into the center zone in the open field test (-45%,  < 0.01) and the visits of open arms in the elevated zero maze test (-39%,  < 0.01) in NW rats indicating anxiety. Interestingly, DIO rats showed no behavioral alterations after the injection of nesfatin-1 ( > 0.05). These results indicate an implication of nesfatin-1 in the mediation of anxiety and depression-like behavior/anhedonia under normal weight conditions, while in DIO rats, a desensitization might occur.

Peter Kobelt

Humboldt-Universität zu Berlin and Berlin Institute of Health

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