Valeria P. Carlini

Ghrelin increases anxiety-like behavior and memory retention in rats

Ghrelin is a peptide found in the hypothalamus and stomach that stimulates food intake and whose circulating concentrations are affected by nutritional state. Very little is known about other central behavioral effects of ghrelin, and thus, we investigated the effects of ghrelin on anxiety and memory retention. The peptide was injected intracerebroventricularly in rats and we performed open-field, plus-maze, and step-down tests (inhibitory avoidance). The administration of ghrelin increased freezing in the open field and decreased the number of entries into the open spaces and the time spent on the open arms in the plus-maze, indicating an anxiogenic effect. Moreover, the peptide increased in a dose-dependent manner the latency time in the step-down test. A rapid and prolonged increase in food intake was also observed. Our results indicate that ghrelin induces anxiogenesis in rats. Moreover, we show for the first time that ghrelin increases memory retention, suggesting that the peptide may influence processes in the hippocampus.

Decreased memory for novel object recognition in chronically food-restricted mice is reversed by acute ghrelin administration

It has been demonstrated, in normal and aged rats and mice, that acute i.c.v. ghrelin (Ghr) administration increases memory retention. In order to evaluate if this treatment, restores memory retention in animals exhibiting impaired memory, in the present work we selected a chronic food restriction mouse model (since undernutrition prejudices higher nervous functions). We employed adult female mice with 28 days of 50% food restriction and evaluated: a) behavioral performance using novel object recognition test for memory, and plus maze for anxiety-like behavior, b) some morphometric parameters as body and hepatic weights and c) plasma Ghr levels. The animals with 50% food restriction showed an increase in plasma Ghr levels and a decrease in morphometric parameters and in the percentage of novel object recognition time. When the peptide was i.c.v. injected in food-restricted animals (0.03, 0.3 or 3.0 nmol/microl), memory increases in relation to food-restricted mice injected with vehicle, reaching a performance similar to controls.

Selective serotonin reuptake inhibitor (fluoxetine) decreases the effects of ghrelin on memory retention and food intake

Ghrelin (Ghr) is an appetite stimulating hormone that is produced peripherally, by the stomach, and centrally as well. Previous investigations show that Ghr increases food intake and memory retention in rats, and that extra-hypothalamic structures, such as the hippocampus, participate in these effects. In the present work we analyzed the effect on food intake and memory retention induced by Ghr after serotonin (5-HT) availability modification at the serotoninergic synapses. Animals only treated with a selective serotonin reuptake inhibitor (SSRI), fluoxetine (FLU) 5 mg/kg or clomipramine (CLO) 2.5 and 5 mg/kg, showed a significant reduction in both food intake and memory retention. On the contrary, Ghr administration induces a significant increase in food intake and a dose-dependent increase in short and long term memory retention. When the animals were treated with FLU prior to Ghr injection, the food intake induced, as well as the expression of short and long term memory retention, was decreased. In conclusion, evidence presented in this paper suggests that the effects of Ghr on both feeding and memory retention in extra-hypothalamic structures such as the hippocampus, could depend on the availability of 5-HT.

Ghrelin and memory: Differential effects on acquisition and retrieval

In a previous paper we have demonstrated that the orexigenic peptide Ghrelin (Ghr), increases memory retention in rats and mice. In the present work we evaluated the Ghr effect when it was administered previous the training session or previous the test session (24h after training) on the memory performance, using step-down test. The results showed that the intra-hippocampal Ghr administration previous the training session improved the long-term memory in this task, but did not modify the short-term memory. Nevertheless, when the Ghr was administrated previous the test session, no changes were observed in the memory performance. Taking into account these results and other previously published by our group, we could hypothesizes that Ghr may modulate specific molecular intermediates involved in memory acquisition/consolidation but not in the retrieval.

Central ghrelin increases anxiety in the Open Field test and impairs retention memory in a passive avoidance task in neonatal chicks.

Ghrelin (Grh) is an endogenous ligand for the growth hormone secretagogue receptor. Although Ghr stimulates feeding in rats, it inhibits feeding in neonatal chicks. However, little is known about other central behavioral effects of Ghr. Therefore, we investigated the Ghr effects, injected intracerebroventricularly, on anxiety and memory retention of neonatal chicks in an Open Field test and in a one-trial passive avoidance task, respectively. In the Open Field test, the administration of Ghr in a dose-dependent manner increased the latency to ambulate but decreased ambulation activity, indicating an anxiogenic effect. Furthermore, chicks trained on a passive avoidance task and injected with a dose of 30pmol of Ghr immediately after training showed an impairment of memory retention. However, there were no significant effects on the number of pecks during the pretraining, training, retention and discrimination. In addition, different doses of Ghr produced an inhibition in food intake at different times after injection. Our results indicate that Ghr induces anxiogenesis in chicks. Moreover, we have shown for the first time that Ghr can decrease memory retention in a non-mammalian species, suggesting that Ghr may play an important role in the processes of memory retention in birds.

Acute ghrelin administration reverses depressive-like behavior induced by bilateral olfactory bulbectomy in mice

This study aims to examine the antidepressant-like action of Ghrelin (Ghr), a hormone synthesized predominantly by gastrointestinal endocrine cells and released during periods of negative energy balance, in two behavioral models: tail suspension test (TST), a predictive model of antidepressant activity, and the olfactory bulbectomy (OB), an established animal model of depression. The reduction in the immobility time in the TST was the parameter used to assess antidepressant-like effect of Ghr. The depressive-like behavior in olfactory bulbectomized mice was inferred through the increase in the immobility time in the TST and the hyperlocomotor activity in the open-field test. Ghr produced antidepressant-like effect in TST (0.3 nmol/μl, i.c.v.), and reversed OB-induced depressive-like behavior. In conclusion, these results provide clear evidence that an acute administration of ghrelin produce antidepressant-like effect in the TST and OB.

Differential effects of fluoxetine and venlafaxine on memory recognition: possible mechanisms of action.

Serotonin-specific reuptake inhibitors (SSRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) are antidepressant drugs commonly used to treat a wide spectrum of mood disorders (Wong and Licinio, 2001). Although they have been clinically used for more than 50 years, the molecular and cellular basis for the action of SSRIs and SNRIs is not clear. Considering that the changes in gene expression involved in the action of antidepressant drugs on memory have not been identified, in this study we investigated the impact of chronic treatment with a SSRI (fluoxetine) and a SNRI (venlafaxine) on the mRNA expression of genes related to memory cascade in the mouse hippocampus, namely, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), nitric oxide synthase 1 (NOS1), neurotrophic tyrosine kinase receptor type 2 (TrKB), mitogen-activated protein kinases (MAPK/ERK) and serotonin transporter (SERT). Animals treated with fluoxetine 10 mg/Kg/day for 28 days showed a significant decrease in the percentage of time spent in the novel object recognition test (p≤0.005) and induced MAPK1/ERK2 down-regulation (p=0.005). Our results suggest that the effect on cognition could probably be explained by fluoxetine interference in the MAPK/ERK memory pathway. In contrast, chronic treatment with venlafaxine did not reduce MAPK1/ERK2 expression, suggesting that MAPK1/ERK2 down-regulation is not a common effect of all antidepressant drugs. Further studies are needed to examine the effect of chronic fluoxetine treatment on the ERK-CREB system, and to determine whether there is a causal relationship between the disruption of the ERK-CREB system and the effect of this antidepressant on memory performance.

Ghrelin effects expression of several genes associated with depression-like behavior

Ghrelin (Ghr) is an orexigenic peptide that is being investigated for its potential role in development of anxiety-like behavior and modulation of depressive-like symptoms induced by bilateral olfactory bulbectomy (OB) in rodents. Olfactory bulbectomy is an animal model useful to study of depression and Ghr could be an alternative therapeutic tool in depression therapy. We studied the effects of intracerebroventricular (i.c.v.) Ghr administration on the expression of hypothalamic genes related to depression and mood (delta opioid receptor (DOR), mu opioid receptor (MOR) and kappa opioid receptor (KOR), lutropin-choriogonadotropic hormone receptor (LHCGR), serotonin transporter (SERT), interleukin 1 beta (IL-1b), vasopressin (AVP) and corticotrophin releasing hormone (CRH)) in OB animals, as well as changes in plasma levels of AVP, CRH and adenocorticotropic hormone (ACTH). We found that acute Ghr 0.3 nmol/μl administration increases gene expression of DOR, SERT and LHCGR in OB mice and decreased expression of IL-1b, suggesting that these genes could be involved in the antidepressant-like effects of Ghr. In addition, OB animals exhibit high AVP gene expression and elevated plasma concentrations of AVP and ACTH and acute Ghr 0.3 nmol/μl administration reduces AVP gene expression and the concentration of these hormones, suggesting that peptide-effects on depressive-like behavior could be mediated at least in part via AVP. In conclusion, this study provides new evidence about genes, receptors and hormones involved in the antidepressant mechanism/s induced by Ghr in OB animals.

Ghrelin inhibited serotonin release from hippocampal slices.

Ghrelin (Ghr) is a peptide produced peripherally and centrally. It participates in the modulation of different biological processes. In our laboratory we have shown that (a) Ghr administration, either intracerebroventricular or directly into the hippocampus enhanced memory consolidation in a step down test in rats (b) the effect of Ghr upon memory decreases in animals pretreated with a serotonin (5-HT) reuptake inhibitor, Fluoxetine, suggesting that Ghr effects in the hippocampus could be related to the availability of 5-HT. It has been demonstrated that Ghr inhibits 5-HT release from rat hypothalamic synaptosomes. Taking in mint these evidences, we studied the release of radioactive 5-HT to the superfusion medium from hippocampal slices treated with two doses of Ghr (0.3 and 3 nm/μl). Ghr inhibited significantly the 5-HT release in relation to those superfused with artificial cerebrospinal fluid (ACSF) (H = 9.48, df = 2, p ≤ 0.05). In another set of experiments, Ghr was infused into the CA1 area of hippocampus of the rats immediately after training in the step down test and the 5-HT release from slices was studied 24h after Ghr injection showing that in this condition also the 5-HT release was inhibited (H = 11.72, df = 1, p ≤ 0.05). In conclusion, results provide additional evidence about the neurobiological bases of Ghr action in hippocampus.

Inhibitory effects of ghrelin on sexual behavior: role of the peptide in the receptivity reduction induced by food restriction in mice.

Ghrelin (Ghr) is a gut/hypothalamus peptide with inhibitory actions on reproductive physiology; however, there are no previous reports of its role on estrous behavior. Under the hypothesis that the increase of plasma Ghr during food restriction (FR) is responsible for receptivity reduction, we intended to evaluate the receptivity percentage of female mice subjected to: exp. 1) acute and chronic FR and Ghr administration (3 nmol/animal/day, s. c.) and exp. 2) the co-administration of a ghrelin antagonist [ant=(d-Lys3)-GHRP-6; 6 nmol/animal/day s. c.]. All females were ovariectomized, primed with steroids, trained, and randomly subjected every week to each one of several protocols, followed by a behavioral test. Experiment 1 (n=8): basal, no treatment; acute FR (aFR), 24-h fasting; chronic FR (cFR), 50% FR for 5 days; acute ghrelin (aGhr), Ghr 30 min before test and chronic ghrelin (cGhr), Ghr for 5 days. Except for cGhr, all treatments significantly decreased the percentage of receptivity (mean±SEM): basal 61.9±6.0, aFR 33.1±8.1, cFR 18.8±7.7, aGhr 45.6±10.6, p<0.05 vs. basal. In exp. 2 (n=11), except for cFR+ant (55.0±6.4) the co-administration of the antagonist reversed the deleterious effects detected in exp. 1: basal 70.9±5.4; aFR+ant 72.3±7.6; aGhr+ant 73.6±4.7. As expected, the administration of vehicle or antagonist alone did not modify receptivity. Besides, we found a significant correlation between percentage of body weight loss and percentage of receptivity reduction (r=0.62, p=0.0004). This is the first study demonstrating that ghrelin is able to inhibit female mice sexual behavior and that is involved, at least in part, in receptivity reduction after food scarcity.