The role of intragestational ghrelin on postnatal development and reproductive programming in mice

The purpose of this study was to evaluate the intragestational role of ghrelin in offspring development and reproductive programming in a mouse model of ghrelin imbalance during pregnancy. Female mice were injected with ghrelin (supraphysiological levels: 4 nmol/animal/day), antagonist (endogenous ghrelin inhibition with (D-Lys3)GHRP-6, 6 nmol/animal/day) or vehicle (control = normal ghrelin levels) throughout the pregnancy. Parameters evaluated in litters were growth, physical, neurobiological and sexual development and, at adulthood, reproductive function. Litter size and initial weight did not vary between treatments. Male pups from dams treated with ghrelin showed higher body weight increase until adulthood (31.7 ± 0.8 vs control = 29.7 ± 0.7, n = 11­14 litters/treatment; P < 0.05). Postnatal physical and neurobiological development was not modified by treatments. The antagonist accelerated male puberty onset, evidenced as earlier testis descent and increased relative testicular weight (antagonist = 0.5 ± 0.0% vs ghrelin = 0.4 ± 0.0% and control = 0.4 ± 0.0%, n = 5­10 litters/treatment; P < 0.05). At adulthood, these males exhibited lower relative testicular weight and reduced sperm motility (63.9 ± 3.6% vs control = 70.9 ± 3.3 and ghrelin = 75.6 ± 3.0, n = 13­15 animals; P < 0.05), without changes in plasma testosterone or fertility. Female pups intragestationally exposed to the antagonist showed earlier vaginal opening (statistically significant only at Day 25) and higher ovarian volume (antagonist = 1085.7 ± 64.0 mm3 vs ghrelin = 663.3 ± 102.8 mm3 and control = 512.3 ± 116.4 mm3; n = 4­6 animals/treatment; P < 0.05), indicating earlier sexual maturation. At adulthood, these females and those exposed to ghrelin showed a tendency to higher percentages of embryo loss and/or foetal atrophy. In conclusion, ghrelin participates in reproductive foetal programming: alterations in ghrelin activity during pregnancy modified body weight increase and anticipated puberty onset, exerting (or tending to) negative effects on adult reproductive function.

Hippocampal effects of neuronostatin on memory, anxiety-like behavior and food intake in rats.

A 13-amino acid peptide named neuronostatin (NST) encoded in the somatostatin pro-hormone has been recently reported. It is produced throughout the body, particularly in brain areas that have significant actions over the metabolic and autonomic regulation. The present study was performed in order to elucidate the functional role of NST on memory, anxiety-like behavior and food intake and the hippocampal participation in these effects. When the peptide was intra-hippocampally administered at 3.0 nmol/µl, it impaired memory retention in both, object recognition and step-down test. Also, this dose blocked the hippocampal long-term potentiation (LTP) generation. When NST was intra-hippocampally administered at 0.3 nmol/µl and 3.0 nmol/µl, anxiolytic effects were observed. Also, the administration in the third ventricle at the higher dose (3.0 nmol/µl) induced similar effects, and both doses reduced food intake. The main result of the present study is the relevance of the hippocampal formation in the behavioral effects induced by NST, and these effects could be associated to a reduced hippocampal synaptic plasticity.

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.

Different chronic cocaine administration protocols induce changes on dentate gyrus plasticity and hippocampal dependent behavior.

Hippocampus is a limbic structure that participates in learning and memory formation. Specifically the dentate gyrus has been described as a hippocampal subregion with high rates of plasticity and it is targeted by different psychoactive drugs modulating synaptic plasticity. Repeated cocaine administration induces sensitization to the locomotor effects and it is believed that sensitization involves the same mechanisms of drug seeking and relapse. Although, the mechanisms underlying sensitization is not fully understood. In this work we investigated the impact of repeated intraperitoneal administration of cocaine (15 or 20 mg/kg/day along 5 or 15 days respectively; and 15 mg/kg/day along 5 day followed by a challenge dose after three days of withdrawal) on the dentate gyrus synaptic plasticity, differentiating between sensitized and nonsensitized rats. Furthermore, we correlated changes on the hippocampal synaptic plasticity to memory retention. Our results revealed that the prevalence of cocaine sensitization (around 50%) was identical in all protocols used. The results found in the threshold to generate LTP were similar for all protocols used, being the threshold values cocaine-treated groups (sensitized and nonsensitized) significantly reduced compared to controls, observing the highest reduction in the sensitized group. Moreover, we observed a facilitated retention of recent memory formation only in sensitized animals the nonsensitized subjects remained at the control levels. In conclusion, sensitization to cocaine generates a high efficiency of hippocampal synaptic plasticity that may underlie the aberrant engagement of learning processes occurred during drug addiction.

Effects of hexarelin (a ghrelin analogue) on fertilisation and the pre- and postnatal development of mice.

Ghrelin (Ghr) has been associated with reproductive physiology and pre- and postnatal development. The objectives of the present study were to evaluate the effects of hexarelin (HEX; 100 or 200 microg kg(-1) day(-1)), a therapeutic Ghr analogue, on: (1) embryo development 60 h post ovulation, induced pharmacologically, in pregnant mice; (2) the physical, neurobiological and sexual development of offspring of female mice injected with HEX during the first, second or third week of pregnancy or throughout the entire pregnancy; and (3) adult memory acquisition in these offspring. We also evaluated the effects of chronic HEX administration on memory acquisition in adult mice. Treatment of non-pregnant female mice with HEX decreased ovulation rate. However, treatment of pregnant mice with HEX at any time during pregnancy tended to accelerate offspring maturation, regardless of bodyweight. This effect was only significant on neurobiological parameters following treatment during the first week. HEX treatment during the first week and/or throughout the entire pregnancy resulted in impaired memory acquisition in the offspring, with female mice being more susceptible to these effects. Similar results were observed for the effects of chronic HEX treatment on memory acquisition in adult mice. In conclusion, HEX seems to exert differential effects depending on when it is administered. Because HEX has started to be used therapeutically, its deleterious effects on ovulation and memory acquisition must be further evaluated.

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.

Huperzia saururus increases memory retention in rats.

Huperzia saururus is reported in Argentinean popular medicine as a memory improver. Chemical studies have shown that the main constituents of the species are Lycopodium alkaloids. On the basis of this information, a purified alkaloid extract was obtained by alkaline extraction of the aerial parts. The aim of this work was to investigate the effects of intra-hippocampal administration of the purified alkaloid extract (AE) on memory retention in vivo, using a step down test, in order to correlate with previous results obtained in vitro in an electrophysiological model. The AE administration significantly increased the latency time in comparison to control animals. For treated animals the latency time was 37.61 +/- 2.84, 80.94 +/- 2.37, and 180.00 +/- 5.74 s for 1, 5, and 10 ng/rat, respectively versus 14.89 +/- 2.38 s for controls. According to these results there is a good relationship between the ethnopharmacological use and the effects hereby showed.