Ghrelin treatment leads to dendritic spine remodeling in hippocampal neurons and increases the expression of specific BDNF-mRNA species.

Ghrelin (Gr) is an orexigenic peptide that acts via its specific receptor, GHSR-1a distributed throughout the brain, being mainly enriched in pituitary, cortex and hippocampus (Hp) modulating a variety of brain functions. Behavioral, electrophysiological and biochemical evidence indicated that Gr modulates the excitability and the synaptic plasticity in Hp. The present experiments were designed in order to extend the knowledge about the Gr effect upon structural synaptic plasticity since morphological and quantitative changes in spine density after Gr administration were analyzed "in vitro" and "in vivo". The results show that Gr administered to hippocampal cultures or stereotactically injected in vivo to Thy-1 mice increases the density of dendritic spines (DS) being the mushroom type highly increased in secondary and tertiary extensions. Spines classified as thin type were increased particularly in primary extensions. Furthermore, we show that Gr enhances selectively the expression of BDNF-mRNA species.

Ghrelin increases memory consolidation through hippocampal mechanisms dependent on glutamate release and NR2B-subunits of the NMDA receptor.

RATIONALE: Ghrelin (Ghr) is a peptide that participates in the modulation of several biological processes. Ghr administration into the hippocampus improves learning and memory in different memory tests. However, the possible mechanisms underlying this effect on memory have not yet been clarified. OBJECTIVE: The purpose of the present work is to add new insights about the mechanisms by which Ghr modulates long-term memory consolidation in the hippocampus. We examined Ghr effects upon processes related to increased synaptic efficacy as presynaptic glutamate release and changes in the expression of the NR2B-subunits containing n-methyl-d-aspartate receptors (NMDAR), which are critical for LTP induction. We also attempted to determine the temporal window in which Ghr administration induces memory facilitation and if the described effects depend on GHS-R1a stimulation. RESULTS: The present research demonstrated that Ghr increased glutamate release from hippocampal synaptosomes; intra-hippocampal Ghr administration increased NR2B-subunits expression in CA1 and DG subareas and also reversed the deleterious effects of the NR2B-subunit-specific antagonist, Ro 25-6981, upon memory consolidation and LTP generation in the hippocampus. These effects are likely to be the consequence of GHS-R1a activation. CONCLUSION: According to the results above mentioned and previous findings, we can hypothesize some of the mechanisms by which Ghr modulates memory consolidation. At presynaptic level, Ghr stimulates glutamate release, probably by enhancing [Ca(2+)]i. At postsynaptic level, the glutamate released activates NMDAR while Ghr also mediates effects directly activating its specific receptors and increases NR2B-subunit expression.

Gabaergic control of anxiety-like behavior, but not food intake, induced by ghrelin in the intermediate medial mesopallium of the neonatal chick.

Ghrelin (Grh) is an endogenous ligand of the growth hormone secretagogue receptor. In neonatal chicks, central Ghr induces anxiogenic-like behavior but strongly inhibits food intake. The intermediate medial mesopallium (IMM) of the chick forebrain has been identified to be a site of the memory formation, and the modulation of the GABAA receptors that are present here modifies the expression of behavior. Thus, the GABAergic system may constitute a central pathway for Ghr action in regulating the processes of food intake and stress-related behaviors. Therefore, we investigated if the effect of systemic administration of bicuculline (GABAA receptor antagonist) and diazepam (benzodiazepine receptor agonist) on the anxiety in an Open Field test and inhibition in food intake induced by Grh (30pmol) when injected into IMM, were mediated by GABAergic transmission. In Open Field test, bicuculline was able to block the anxiogenic-like behavior induced by Ghr, whereas diazepam did not produce it. However, the co-administration of bicuculline or diazepam plus Ghr did not show any change in food intake at 30, 60 and 120min after injection compared to Ghr alone. Our results indicate for the first time that Ghr, injected into the forebrain IMM area, induces an anxiogenic-like behavior, which was blocked by bicuculline but not diazepam, thus suggesting that Ghr plays an important role in the response pattern to acute stressor, involving the possible participation of the GABAergic system. Nevertheless, as neither drug affected the hypophagia induced by intra-IMM Ghr, this suggests that it may be mediated by different mechanisms.

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.

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.

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.

Anxiogenesis induced by nitric oxide synthase inhibition and anxiolytic effect of melanin-concentrating hormone (MCH) in rat brain.

In this study, the involvement of nitric oxide (NO) in the mechanism of anxiety was investigated. The rats received an intraamygdaline or intrahippocampal injection of the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine (L-NOARG), and were then tested in the plus-maze test. L-NOARG induced a decrease in the time spent by rats in the open arms. Conversely, the administration of the melanin-concentrating hormone (MCH) into these structures increased the number of entries into the open arms as well as the time spent on them. MCH injected in rats pretreated with L-NOARG also was able to revert the anxiogenic effects of L-NOARG in amygdala.

Interaction between alpha-MSH and acetylcholinergic system upon striatal cAMP and IP(3) levels.

The interaction between the neuropeptide alpha-MSH and the acetylcholinergic system as reflected by changes in cAMP and inositol 1-3-5 triphosphate(IP(3))production was investigated in an in vitro model of striatal slices. The possible involvement of D(1) receptors in cholinergic and alpha-MSH- stimulated cAMP and IP(3) production in slices of rat striatum was also examined, because it has been demonstrated that acetylcholinergic drugs induce endogenous dopamine release in the striatum. alpha-MSH, pilocarpine(PL) and the selective muscarinic M1 agonist McN-A-343 increased cAMP and IP(3) striatal levels, effects blocked by the D(1) antagonist SCH-23390, except for the effects of alpha-MSH on IP(3). The muscarinic M(2) antagonist gallamine (GL) brought about an increase in cAMP levels, an effect blocked by SCH-23390. The M(1) antagonist pirenzepine (Pz) induced a decrease both in cAMP and IP(3) content, and the nicotinic antagonist di-hydro-beta-eritroidine(DBE) only diminished cAMP production. When alpha-MSH and cholinergic agents were simultaneously added, cAMP and IP(3) levels were modified with respect to the values reached when these agents were added alone. An interaction between the acetylcholinergic system and alpha-MSH through M(1) and nicotinic receptors was also observed. These results suggest that the intracellular signaling pathways related to cAMP and IP(3) production gated by alpha-MSH and these cholinergic receptors are probably related. alpha-MSH striatum cAMP IP(3) muscarinic and nicotinic receptors an in vitro model.

Response to novelty after i.c.v. injection of melanin-concentrating hormone (MCH) in rats.

Some behavioral response of rats to spatial novelty after i.c.v. administration of melanin-concentrating hormone (MCH) were evaluated. To this purpose, an open-field test was used, as well as an elevated plus-maze to study the possible anxiolytic effect of this peptide. In the open field, the frequency of exploratory components (locomotion and rearing) increased after MCH administration in comparison to controls. Moreover, in the plus-maze, MCH increased the number of entries into the open arms as well as the time spent on them, whereas no changes in the number of entries onto the closed arms were found. The data indicate that MCH exerts an anxiolytic effect, and suggests a physiological role for this.

Melanin-concentrating hormone (MCH) modifies memory retention in rats.

The purpose of the present study was to evaluate the possible effect of melanin-concentrating hormone (MCH) on learning and memory by using the one-trial step-down inhibitory avoidance test in rats. The peptide was infused into hippocampus, amygdala, and entorhinal cortex. MCH caused retrograde facilitation when given at 0 or 4 h post-training into hippocampus, but only at 0 h into amygdala. From these results, it seems that MCH modulates memory early after training by acting on both the amygdala and hippocampus and, 4 h after training, on the hippocampus.

Interaction between alpha-MSH and gabaergic agents upon striatal cAMP levels: an in vitro model.

We have tried to investigate the possible interaction between the gabaergic system and alpha-MSH at a cellular level in an in vitro model of male albino rats tissue slices containing accumbens and caudate-putamen nuclei. Alpha-MSH alone increases cAMP levels, as does diazepam and phaclofen; however, these effects were blocked by SCH-23390. Both flumazenil and baclofen induced a decrease in the cAMP content. When both alpha-MSH and gabaergic agents were incubated together, cAMP levels were modified. It can be assumed that cAMP production by the neuropeptide and the gabaergic agents could be linked to the activation of dopaminergic D1 receptors. The latter receptors had no prominent effect on the interaction between alpha-MSH and the GABA agonists and antagonists. In summary, our results suggested that alpha-MSH and GABA system could be biochemically linked to produce a cellular effect.

Effect of alpha-MSH upon cyclic AMP levels induced by the glutamatergic agonists NMDA, quisqualic acid, and kainic acid.

This study was carried out to investigate possible interactions between some glutamatergic agonists and the peptide alpha-MSH upon the cyclic AMP levels. We used an in vitro tissue slice preparation incubated in the presence of different glutamatergic agonists such as N-methyl-D-aspartic acid (NMDA), quisqualic acid (QUIS), kainic acid (KA), and the peptide alpha-MSH together with each agonist. Slices containing caudate putamen and accumbens were chosen according to neurochemical data indicating that the striatum contains a moderate amount of MSH binding sites and also receives glutamatergic innervation. Exposure of these slices to either MSH or to the agonists NMDA or QUIS resulted in an increase in the cAMP levels in relation to controls. Nevertheless, incubation with KA resulted in no changes in the nucleotide levels. The combination of MSH/NMDA induced a reduction of cAMP levels in relation to those obtained with NMDA alone. The combinations of QUIS/MSH or KA/MSH also induced variations in the values of nucleotide in relation to the those obtained with the peptide alone or with the corresponding agonist; these changes were related to the dose of agonist used in each case. The results obtained in these experiments suggest the existence of some interaction between the peptide and the agonist used.

alpha-MSH-induced behavior: changes after diazepam and baclofen administration related with cyclic AMP levels.

The present work was performed to evaluate the participation of the benzodiacepinic GABAA and GABAB components upon excessive grooming, locomotion, rearing, and stretching/yawning syndrome induced by the intracerebroventricularly alpha-MSH administration by using GABAA and GABAB agonists. It also aims at evaluating possible relation between changes in cAMP levels in caudate-putamen and accumbens nuclei and the behavioral responses. Injection of diazepam or baclofen reduced the total behavioral scores in a dose-related manner as well as the cAMP levels with respect to the control values (animals treated with artificial cerebrospinal fluid). When diazepam was tested in animals simultaneously injected with alpha-MSH, behavioral scores decreased with respect to those treated with the peptide alone. Cyclic AMP also decreased after combined treatment (MSH + diazepam).

alpha-MSH changes cyclic AMP levels in rat brain slices by an interaction with the D1 dopamine receptor.

The exposure of rat brain slices containing caudate putamen and accumbens nuclei to alpha-MSH or dopamine (DA) results in an increase in cyclic AMP (cAMP) levels. When tissues are compared with those containing both alpha-MSH and DA, a reduction in the cyclic nucleotide is observable. This study was carried out to determine whether variations in tissular cAMP levels induced by alpha-MSH might be explained by an interaction between the peptide and some dopaminergic receptors. Therefore, we measured cAMP in tissues and medium in response to alpha-MSH in the presence of haloperidol, the selective D1 (SCH 23390) or D2 (sulpiride) antagonists, or the selective D1 (SKF 38393) or D2 (bromocriptine) agonists. Haloperidol by itself induced no changes either in the cAMP content or in the cAMP efflux to the medium. When slices were exposed to alpha-MSH and haloperidol, the latter blocked the alpha-MSH effect of inducing an increase in the content of cAMP. None of the specific antagonists (at the administered doses) induced changes in the content of cAMP when compared with the control group. The presence of SCH 23390 in the incubation medium together with alpha-MSH yielded a reduction in cAMP levels compared with those incubated with alpha-MSH. A slight stimulatory effect on cAMP formation was observed when the dopaminergic agonists (SKF 38393 10 microM) were used. We conclude that alpha-MSH interacts with the D1 dopamine receptor, changing the cAMP levels in striatum and accumbens nuclei.

Independent and simultaneous effects of alpha-MSH and dopamine on cyclic AMP levels in rat brain slices.

This study was carried out to explore whether the postulated interaction between alpha-MSH and dopamine (DA) could be explained on the basis of variations in tissular cyclic AMP (cAMP) levels. We used slices containing caudate putamen and accumbens nuclei incubated in the presence of alpha-MSH, DA, or both simultaneously. Exposure of slices to alpha-MSH or DA resulted in an increase in cAMP levels. The simultaneous presence of alpha-MSH and DA resulted in a reduction in the accumulation of the cyclic nucleotide in the tissues as compared with those treated only with DA or alpha-MSH. The effect of alpha-MSH and DA on cAMP efflux was also tested. Incubation of slices with DA led to a marked increase in cAMP efflux; this efflux could be prevented if alpha-MSH was present in the medium. The results suggest that the inhibition by alpha-MSH of DA-induced striatal cAMP accumulation and efflux are a consequence of an interaction between the peptide and the neurotransmitter; DA and alpha-MSH transducing mechanisms could be biochemically linked.

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine on Trypanosoma cruzi.

The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes of Trypanosoma cruzi, at concentrations lower than 100 microM, and motility and infectivity of the bloodstream trypomastigote form at 200 microM. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 microM TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.

Alpha MSH-induced excessive grooming behavior involves a GABAergic mechanism.

It has been shown that MSH administered in the ventral tegmental area (VTA) elicits excessive grooming behavior (EGB) by stimulating an acetylcholinergic pathway. The present work was performed in order to evaluate the possible participation of the GABAergic system in this behavior. VTA injection of GABA antagonist bicuculline stimulated the EGB (55.5 +/- 2.4). In contrast, this effect disappeared if the animals were pretreated with atropine (33.1 +/- 1.5). When bicuculline was injected before a 200 ng/microliters dose of MSH, the EGB increased (87.6 +/- 4.4) in comparison to MSH-treated rats (46.5 +/- 3.2). Our results suggest that GABA, ACh, and MSH interact in the VTA in the induction of EGB; an increase in MSH levels appears to stimulate cholinergic neurons. GABAergic fibers probably modulate the cholinergic discharge at the presynaptic level.

Effects of helio-neon laser radiation upon cellular cycle in a plant model.

The scope of this study was to investigate possible relationships between He-Neon laser radiation and mitotic and phase indices in meristematic cells of Allium cepa L. bulbs. Our results indicate that mitotic index increased after irradiation depending this modification on the time exposure and the potency of the He-Neon beam. Phase indices were also modified: frequency of prophase increased, while inter- meta- and anaphase decreased: telophases remain unchanged. These variations were significative only when the preparations were irradiated a) with 5 mW for 10 min. or more, b) with 10 mW or c) when the preparations were processed 60 min. after irradiation. These findings could not be attributed to thermal changes. Modifications in RNA or protein synthesis could be responsible.