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The intake of macronutrients is crucial for the fitness of any animal and is mainly regulated by peripheral signals to the brain. How the brain receives and translates these peripheral signals or how these interactions lead to changes in feeding behavior is not well-understood. We discovered that 2 crustacean cardioactive peptide (CCAP)-expressing neurons in Drosophila adults regulate feeding behavior and metabolism. Notably, loss of CCAP, or knocking down the CCAP receptor (CCAP-R) in 2 dorsal median neurons, inhibits the release of neuropeptide F (NPF), which regulates feeding behavior. Furthermore, under starvation conditions, flies normally have an increased sensitivity to sugar; however, loss of CCAP, or CCAP-R in 2 dorsal median NPF neurons, inhibited sugar sensitivity in satiated and starved flies. Separate from its regulation of NPF signaling, the CCAP peptide also regulates triglyceride levels. Additionally, genetic and optogenetic studies demonstrate that CCAP signaling is necessary and sufficient to stimulate a reflexive feeding behavior, the proboscis extension reflex (PER), elicited when external food cues are interpreted as palatable. Dopaminergic signaling was also sufficient to induce a PER. On the other hand, although necessary, NPF neurons were not able to induce a PER. These data illustrate that the CCAP peptide is a central regulator of feeding behavior and metabolism in adult flies, and that NPF neurons have an important regulatory role within this system., Competing Interests: The authors declare no competing interest.

New data have been accumulated in the scientific literature in recent years which allow a more adequate risk assessment of selenium with reference to human health. This new evidence comes from environmental studies, carried out in populations characterized by abnormally high or low selenium intakes, and from high-quality and large randomized controlled trials with selenium recently carried out in the US and in other countries. These trials have consistently shown no beneficial effect on cancer and cardiovascular risk, and have yielded indications of unexpected toxic effects of selenium exposure. Overall, these studies indicate that the minimal amount of environmental selenium which is source of risk to human health is much lower than anticipated on the basis of older studies, since toxic effects were shown at levels of intake as low as around 260 µg/day for organic selenium and around 16 µg/day for inorganic selenium. Conversely, populations with average selenium intake of less than 13-19 µg/day appear to be at risk of a severe cardiomyopathy, Keshan disease. Overall, there is the need to reconsider the selenium standards for dietary intake, drinking water, outdoor and indoor air levels, taking into account the recently discovered adverse health effects of low-dose selenium overexposure, and carefully assessing the significance of selenium-induced proteomic changes.

Background: G protein-coupled receptors (GPCRs) represent one of the largest families of transmembrane receptors and the most common drug target. The Adhesion subfamily is the second largest one of GPCRs and its several members are known to mediate neural development and immune system functioning through cell-cell and cell-matrix interactions. The distribution of these receptors has not been characterized in detail in the gastrointestinal (GI) tract. Here we present the first comprehensive anatomical profiling of mRNA expression of all 30 Adhesion GPCRs in the rat GI tract divided into twelve subsegments., Methods: Using RT-qPCR, we studied the expression of Adhesion GPCRs in the esophagus, the corpus and antrum of the stomach, the proximal and distal parts of the duodenum, ileum, jejunum and colon, and the cecum., Results: We found that twenty-one Adhesion GPCRs (70%) had a widespread (expressed in five or more segments) or ubiquitous (expressed in eleven or more segments) distribution, seven (23%) were restricted to a few segments of the GI tract and two were not expressed in any segment. Most notably, almost all Group III members were ubiquitously expressed, while the restricted expression was characteristic for the majority of group VII members, hinting at more specific/localized roles for some of these receptors., Conclusions: Overall, the distribution of Adhesion GPCRs points to their important role in GI tract functioning and defines them as a potentially crucial target for pharmacological interventions.

Aim: In order to increase the knowledge on the use of nanoparticles (NPs) in brain targeting, this article describes the conjugation of the sequence 12-32 (g21) of leptin to poly-lactide-co-glycolide NPs. The capability of these modified NPs to reach the brain was evaluated in rats after intravenous administration., Materials & Methods: The g21 was linked on the surface of NPs labeled with tetramethylrhodamine by means of the Avidin-Biotin technology. The g21-labeled NPs were injected into the tail vein of rats and, after animal sacrifice, the brain localization was evaluated by confocal microscopy, fluorescence microscopy and electron microscopy. Studies to evaluate the biodistribution of the g21-modified NPs in comparison to the unmodified NPs were also carried out. Moreover, to confirm the absence of any anorectic effect of g21 linked on the surface of NPs, appropriate studies were used to assess the rats., Results: After intravenous administration, the g21-modified NPs were able to cross the blood-brain barrier and to enter the brain parenchyma. The biodistribution studies of both unmodified and modified NPs pointed out an uptake at liver and spleen level, whereas only the g21-modified NPs showed brain localization. The food-intake experiments pointed out that the intravenous administration of g21 conjugated to the NP surface did not produce any anorectic effect in the rats., Conclusion: g21-modified NPs were able to cross the blood-brain barrier. These new modified NPs could be effectively considered as useful carrier systems for brain drug delivery.

Polymeric nanoparticles (Np) have been considered as strategic carriers for brain targeting. Specific ligands on the surface allowed the Np to cross the Blood-Brain Barrier (BBB) carrying model drugs within the brain district after their i.v. administration in experimental animals. It is known that sialic acid receptors are present in several organs, including in the brain parenchyma. Thus, in this paper, we prepared PLGA Np surface modified with a BBB-penetrating peptide (similopioid peptide) for BBB crossing and with a sialic acid residue (SA) for the interaction with brain receptors. This double coverage could allow to obtain novel targeted Np with a prolonged residence within the brain parenchyma, thus letting to reach a long-lasting brain delivery of drugs. The central analgesic activity of Loperamide (opioid drug, unable to cross the BBB) loaded in these novel Np was evaluated in order to point out the capability of the Np to reach and to remain in the brain. The results showed that the pharmacological effect induced by loaded Np administration remained significant over 24h. Using confocal and fluorescent microscopies, the novel Np were localized within the tissue parenchyma (brain, kidney, liver, spleen and lung). Finally, the biodistribution studies showed a localization of the 6% of the injected dose into the CNS over a prolonged time (24h). Notwithstanding an increased accumulation of SA-covered Np in those organs showing SA-receptors (liver, kidney, and lung), the pharmacological and biodistribution results are proofs of the ability of double targeted Np to enter the brain allowing the drug to be released over a prolonged time., (2010 Elsevier B.V. All rights reserved.)

The pharmacological treatment of neurological disorders is often complicated by the inability of drugs to pass the blood-brain barrier. Recently we discovered that polymeric nanoparticles (NPs) made of poly(D,L-lactide-co-glycolide), surface-decorated with the peptide Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-beta-D-glucose)-CONH2 are able to deliver, after intravenous administration, the model drug loperamide into the central nervous system (CNS). This new drug delivery agent is able to ensure a strong and long-lasting pharmacological effect, far greater than that previously observed with other nanoparticulate carriers. Here we confirmed the effectiveness of this carrier for brain targeting, comparing the effect obtained by the administration of loperamide-loaded NPs with the effect of an intracerebroventricular administration of the drug; moreover, the biodistribution of these NPs showed a localization into the CNS in a quantity about two orders of magnitude greater than that found with the other known NP drug carriers. Thus, a new kind of NPs that target the CNS with very high specificity was discovered., From the Clinical Editor: This paper discusses a nanoparticle-based technique of targeted drug delivery through the blood-brain barrier. The biodistribution of these novel nanoparticles showed two orders of magnitude greater efficiency compared to other known NP drug carriers.

The gender-specific expression pattern of aromatase and 5alpha-reductases (5alpha-R) during brain development provides neurons the right amount of estradiol and DHT to induce a dimorphic organization of the structure. Polychlorinated biphenyls (PCBs) are endocrine disruptive pollutants; exposure to PCBs through placental transfer and breast-feeding may adversely affect the organizational action of sex steroid, resulting in long-term alteration of reproductive neuroendocrinology. The study was aimed at: a) evaluating the hypothalamic expression of aromatase, 5alpha-R1 and 5alpha-R2 in fetuses (GD20), infant (PN12), weaning (PN21) and young adult (PN60) male and female rats exposed to PCBs during development; b) correlating these parameters with the time of testicular descent, puberty onset, estrous cyclicity and copulatory behavior; c) evaluating possible alterations of some non reproductive behaviors (locomotion, learning and memory, depression/anxiety behavior). A reconstituted mixture of four indicator congeners (PCB 126, 138, 153 and 180) was injected subcutaneously to dams at the dose of 10 mg/kg daily from GD15 to GD19 and then twice a week till weanling. The results indicated that developmental PCB exposure produced important changes in the dimorphic hypothalamic expression of both aromatase and the 5alpha-Rs, which were still evident in adult animals. We observed that female puberty onset occurs earlier than in control animals without cycle irregularity, while testicular descent in males was delayed. A slight but significant impairment of sexual behavior and an important alteration in memory retention were also noted specifically in males. We conclude that PCBs might affect the dimorphic neuroendocrine control of reproductive system and of other neurobiological processes.

The melanocortins (alpha, beta and gamma-melanocyte-stimulating hormones: MSHs; adrenocorticotrophic hormone: ACTH), a family of pro-opiomelanocortin (POMC)-derived peptides having in common the tetrapeptide sequence His-Phe-Arg-Trp, have progressively revealed an incredibly wide range of extra-hormonal effects, so to become one of the most promising source of innovative drugs for many, important and widespread pathological conditions. The discovery of their effects on some brain functions, independently made by William Ferrari and David De Wied about half a century ago, led to the formulation of the term "neuropeptide" at a time when no demonstration of the actual production of peptide molecules by neurons, in the brain, was still available, and there were no receptors characterized for these molecules. In the course of the subsequent decades it came out that melanocortins, besides inducing one of the most complex and bizarre behavioural syndromes (excessive grooming, crises of stretchings and yawnings, repeated episodes of spontaneous penile erection and ejaculation, increased sexual receptivity), play a key role in functions of fundamental physiological importance as well as impressive therapeutic effects in different pathological conditions. If serendipity had been an important determinant in the discovery of the above-mentioned first-noticed extra-hormonal effects of melanocortins, many of the subsequent discoveries in the pharmacology of these peptides (feeding inhibition, shock reversal, role in opiate tolerance/withdrawal, etc.) have been the result of a planned research, aimed at testing the "pro-nociceptive/anti-nociceptive homeostatic system" hypothesis. The discovery of melanocortin receptors, and the ensuing synthesis of selective ligands with agonist or antagonist activity, is generating completely innovative drugs for the treatment of a potentially very long list of important and widespread pathological conditions: sexual impotence, frigidity, overweight/obesity, anorexia, cachexia, haemorrhagic shock, other forms of shock, myocardial infarction, ischemia/reperfusion-induced brain damage, neuropathic pain, rheumathoid arthritis, inflammatory bowel disease, nerve injury, toxic neuropathies, diabetic neuropathy, etc. This review recalls the history of these researches and outlines the pharmacology of the extra-hormonal effects of melanocortins which are produced by an action at the brain level (or mainly at the brain level). In our opinion the picture is still incomplete, in spite of being already so incredibly vast and complex. So, for example, several of their effects and preliminary animal data suggest that melanocortins might be of concrete effectiveness in one of the areas of most increasing concern, i.e., that of neurodegenerative diseases.

Polymeric nanoparticles (Np) represent one of the most innovative non-invasive approaches for the drug delivery to the central nervous system (CNS). It is known that the ability of the Np to cross the Blood Brain Barrier (BBB), thus allowing the drugs to exert their pharmacological activity in the central nervous district, is linked to their surface characteristics. Recently it was shown that the biocompatible polyester poly(d,l-lactide-co-glycolide) (PLGA) derivatized with the peptide H(2)N-Gly-l-Phe-d-Thr-Gly-l-Phe-l-Leu-l-Ser(O-beta-d-Glucose)-CONH(2) [g7] was a useful starting material for the preparation of Np (g7-Np); moreover, fluorescent studies showed that these Np were able to cross the BBB. In this research, g-7 Np were loaded with Loperamide in order to assess their ability as drug carriers for CNS, and with Rhodamine-123, in order to qualitatively determine their biodistribution in different brain macro-areas. A pharmacological evidence is given that g7-Np are able to cross the BBB, ensuring, for the first time, a sustained release of the embedded drug, and that these Np are able to reach all the brain areas here examined. The ability to enter the CNS appears to be linked to the sequence of the peptidic moiety present on their surface.

Objective: To quantify and characterize the similarities and the differences between chronic migraine (CM) patients with medication overuse and episodic migraine (EM) patients with only occasional analgesic use., Background: Population-level epidemiology, characteristics, mechanisms of chronic daily headache, and medication-overuse headache have been widely studied but patient characteristics have received less attention. Methods.-We compared sociodemographic data, family history, physiological and medical history, health services utilized, drugs taken/prescribed, and outcome of 2 groups of subjects: 150 patients, suffering from CM, complicated by probable medication-overuse headache (CM group), consecutively admitted during 2005 to the inpatients' ward of the Headache Centre of the University Hospital of Modena and Reggio Emilia, Italy, to undergo withdrawal from their overused medications; 100 patients suffering from EM, uncomplicated by medication overuse (EM group), consecutively referred to the outpatients' ward of the Headache Centre during November and December 2005., Results: All sociodemographic characteristics were significantly different between the 2 groups. As a whole, the CM group began to suffer from migraine earlier than the EM group. Drug and/or alcohol abuse was significantly higher among first-degree relatives of CM (19%) than of EM (6%) patients. The most frequent comorbid disorders were psychiatric (67%) and gastrointestinal diseases (43%) in the CM group, and allergies in the EM group (31%). Seventy percent of CM patients and 42% of EM patients were taking daily at least another drug, besides those for headache treatment. Most overused medications in the CM group were triptans (43%); the EM group used above all single NSAIDs (56%). At 3-month follow-up, prophylactic treatments reduced, by at least 50%, the frequency of headache in about three-fourths of patients of both the groups; however, headache remained significantly more frequent in the CM than in EM group: only a minority (15%) of CM patients reverted to a headache frequency comparable to that of the EM group., Conclusions: CM patients present more multiple comorbid disorders, polypharmacy, and social impediments than EM patients. These associated conditions complicate CM clinical management. Even after withdrawal from medication overuse, CM could not be completely reverted by current prophylactic treatments.

Leptin produced by adipocytes controls body weight by restraining food intake and enhancing energy expenditure at the hypothalamic level. The diet-induced increase in fat mass is associated with the presence of elevated circulating leptin levels, suggesting the development of resistance to its anorectic effect. Rats, like humans, show different susceptibility to diet-induced obesity. The aim of the present study was to compare the degree of leptin resistance in obesity-prone (OP) vs obesity-resistant (OR) rats on a moderate high-fat (HF) diet and to establish if the effects of leptin on hypothalamo-pituitary endocrine functions were preserved. Starting from 6 weeks after birth, male Sprague-Dawley rats were fed on either a commercial HF diet (fat content: 20% of total calorie intake) or a standard pellet chow (CONT diet, fat content: 3%). After 12 weeks of diet, rats fed on HF diet were significantly heavier than rats fed on CONT diet. Animals fed on HF diet were ranked according to body weight; the two tails of the distribution were called OP and OR rats respectively. A polyethylene cannula was implanted into the right ventricle of rats 1 week before central leptin administration. After 12 weeks of HF feeding, both OR and OP rats were resistant to central leptin administration (10 mug, i.c.v.) (24 h calorie intake as a percent of vehicle-treated rats: CONT rats, 62 [50; 78]; OR, 93 [66; 118]; OP, 90 [70; 120] as medians and 95% confidence intervals (CIs) of six rats for each group). Conversely, after 32 weeks of diet both OR and OP rats were partially responsive to 10 mug leptin i.c.v. as compared with CONT rats (24 h calorie intake as a percent of vehicle-treated rats: CONT rats, 60 [50; 67]; OR, 65 [50; 80]; OP, 80 [60; 98] as medians and 95% CIs of six rats for each group); the decrease of food intake following 200 mug leptin i.p. administration was similar in all the three groups (calorie intake as a percent of vehicle-treated rats: 86 [80; 92] as median and 95% CI). The long-term intake of HF diet caused hyperleptinemia, hyperinsulinemia and higher plasma glucose levels in OP rats as compared with CONT rats. Plasma thyroxine (T4) was lower in all the rats fed the HF diet as compared with CONT. i.c.v. administration of leptin after 32 weeks of diet restored normal insulin levels in OP rats. Moreover, leptin increased plasma T4 concentration and strongly enhanced GH mRNA expression in the pituitary of OP as well as OR rats (180+/-10% vs vehicle-treated rats). In conclusion, long-term intake of HF diet induced a partial central resistance to the anorectic effect of leptin in both lean and fat animals; the neuroendocrine effects of leptin on T4 and GH were preserved.

Adenosine A2A receptors are a new target for drug development in Parkinson's disease. Some experimental and clinical data suggest that A2A receptor antagonists can provide symptomatic improvement by potentiating the effects of L-DOPA as well as a decrease in secondary effects such as L-DOPA-induced dyskinesia. L-DOPA-induced behavioral sensitization in unilateral 6-hydroxydopamine-lesioned rats is frequently used as an experimental model of L-DOPA-induced dyskinesia. In the present work this model was used to evaluate the effect of the A2A receptor agonist CGS 21680 and the A2A receptor antagonist MSX-3 on L-DOPA-induced behavioral sensitization and 6-hydroxydopamine-induced striatal dopamine denervation. L-DOPA-induced behavioral sensitization was determined as an increase in L-DOPA-induced abnormal involuntary movements and enhancement of apomorphine-induced turning behavior. Striatal dopamine innervation was determined by measuring tyrosine-hydroxylase immunoreactivity. Chronic administration of MSX-3 was not found to be effective at counteracting L-DOPA-induced behavioral sensitization. On the other hand, CGS 21680 completely avoided the development of L-DOPA-induced behavioral sensitization. The analysis of the striatal dopamine innervation showed that L-DOPA-CGS 21680 co-treatment conferred neuroprotection to the toxic effects of 6-hydroxydopamine. This neuroprotective effect was dependent on A2A and D2 receptor stimulation, since it was counteracted by MSX-3 and by the D2 receptor antagonist haloperidol. These results open new therapeutic avenues in early events in Parkinson's disease.

It has been previously described that gamma-hydroxybutyrate (GHB) provides significant protection against transient global cerebral ischemia in the rat (four vessel occlusion model), when given 30 min before or 10 min after artery occlusion. Here, we show that in the same rat model, significant protection can also be obtained when treatment is started 2 h after the ischemic episode. In saline-treated animals, 30 min of global ischemia followed by reperfusion caused a massive loss of neurons in the hippocampal CA1 subfield (examined 63 days after the ischemic episode), and an impairment of sensory-motor performance (tested on the 51st and 63rd days after ischemia) and of spatial learning and memory (evaluated starting 46 days after the ischemic episode). Treatment with GHB--300 mg/kg intraperitoneally (i.p.) 2 h after the ischemia-reperfusion episode, followed by 100 mg/kg i.p. twice daily for the following 10 days--afforded a highly significant protection, against both histological damage and sensory-motor and learning-memory impairments. These data further suggest the possible therapeutic effectiveness of GHB in brain ischemia, and indicate that the underlying mechanism of action involves non-immediate steps of the ischemia-induced cascade of events.

The present paper enlightens a new point of view on brain homeostasis and communication, namely how the brain takes advantage of different chemical-physical phenomena such as pressure waves, and temperature and concentration gradients to allow the homeostasis of the brain internal milieu as well as some forms of intercellular communications (volume transmission, VT) at an energy cost much lower than the classical synaptic transmission (the prototype of wiring transmission, WT). The possible melanocortin control of uncoupling protein 2 (UCP2) expression (hence of local brain temperature gradients) has been studied in relation to food intake in male Wistar rats. Osmotic minipumps were subcutaneously (sc) implanted in the midscapular region for intracerebroventricular (icv) infusion. The control rats received an icv infusion of 0.5 microl/h of artificial cerebrospinal fluid (ACSF), while experimental rats received either an icv infusion of 0.16 nmol/h of HS024 or of 0.16 nmol/h of adrenocorticotropin-(1-24) [ACTH-(1-24)]. The ACTH-treated group ate significantly less than the ACSF-treated group during the first three days of infusion, while, subsequently, food intake of the two groups was similar. On the other hand, the HS024-treated group ate significantly more (up to 153% of the control value) than ACSF- and ACTH-treated rats during the entire period. UCP2 mRNA analysis in arcuate nuclei of ACTH, HS024 and ACSF-treated animals showed a significant 75% decrease (p<0.05 vs saline) of the total specific mRNA level in the HS024-treated group vs ACSF-treated animals (control group), while no significant change was observed between ACTH- and ACSF-treated animals. Melanocortin antagonist HS024 via blockade of MCR4 increases food intake and via a reduction of UCP2 expression enhances the food consumption ratio. This result underlines the fact that UCP2 expression and food intake can be differentially regulated. In other words, via a peptidergic control the central nervous system (CNS) can modulate the energy stored from the amount of the food that the animal has eaten and also uncouple the thermal micro-gradients (dependent on UCP2 expression) and hence the VT-signal micro-migrations from the food intake. It should also be noticed that the control of the thermal gradients affects also the neuronal firing rate and hence the transmitter release (likely above all the release of peptides such as neuropeptide Y (NPY), melanin-concentrating hormone (MCH) and beta-endorphin, e.g., in the arcuate nucleus representing signals relevant to energy homeostasis). Thus, WT and VT are both modulated by peptidergic signals that affect thermal gradients.

The melanocortin (MC)-4 receptor participates in regulating body weight homeostasis. We demonstrated early that acute blockage of the MC-4 receptor increases food intake and relieves anorexic conditions in rats. Our recent studies show that 4-week chronic blockage of the MC-4 receptor leads to robust increases in food intake and development of obesity, whereas stimulation of the receptor leads to anorexia. Interestingly, the food conversion ratio was clearly increased by MC-4 receptor blockage, whereas it was decreased in agonist-treated rats in a transient manner. Chronic infusion of an agonist caused a transient increase in oxygen consumption. Our studies also show that the MC-4 receptor plays a role in luteinizing hormone and prolactin surges in female rats. The MC-4 receptor has a role in mediating the effects of leptin on these surges. The phylogenetic relation of the MC-4 receptor to other GPCRs in the human genome was determined. The three-dimensional structure of the protein was studied by construction of a high-affinity zinc binding site between the helices, using two histidine residues facing each other. We also cloned the MC-4 receptor from evolutionary important species and showed by chromosomal mapping a conserved synteny between humans and zebrafish. The MC-4 receptor has been remarkably conserved in structure and pharmacology for more than 400 million years, implying that the receptor participated in vital physiological functions early in vertebrate evolution.

Prolactin releasing peptide (PrRP) has been reported to reduce food intake in rats. We tested the effect of i.c.v. administration of PrRP-31 on food intake in both food deprived and free-feeding rats. We did not find any effect of PrRP-31 on food intake after single injections of up to an 8-nmol dose, but observed a marked decrease in food intake and body weight in rats that received a repeated twice daily administration of 8 nmol of PrRP-31. This effect was associated with an adverse behavioral pattern, indicating that the repeated high doses of the peptide caused non-specific effects inducing anorexia. We also tested several other behavioral parameters like locomotion and exploratory time, grooming and resting time, using lower doses of PrRP that did not cause the adverse behavior. Moreover, we carried out locomotor and sensory motor activity tests at the doses that exerted the most pronounced effect on the food intake. None of these tests suggested any specific behavioral effect of PrRP. We conclude that the behavioral pattern induced by PrRP is likely to be different from those induced by many other neuropeptides affecting food intake in rats.

The injection of a melanocortin peptide or of melanocortin peptide analogues into the cerebrospinal fluid or into the ventromedial hypothalamus in nanomolar or subnanomolar doses induces a long-lasting inhibition of food intake. The effect keeps significant for up to 9 h and has been observed in all animal species so far tested, the most susceptible being the rabbit. The anorectic effect of these peptides is a primary one, not secondary to the shift towards other components of the complex melanocortin-induced behavioral syndrome, in particular grooming. The site of action is in the brain, and the effect is not adrenal-mediated because it is fully exhibited also by adrenalectomized animals. It is a very strong effect, because the degree of feeding inhibition is not reduced in conditions of hunger, either induced by 24 h starvation, or by insulin-induced hypoglycemia, or by stimulation of gamma-aminobutyric acid (GABA), noradrenergic or opioid systems. The microstructural analysis of feeding behavior suggests that melanocortins act as satiety-inducing agents, because they do not significantly modify the latencies to start eating, but shorten the latencies to stop eating. The mechanism of action involves the activation of melanocortin MC(4) receptors, because selective melanocortin MC(4) receptor antagonists inhibit the anorectic effect of melanocortins, while inducing per se a strong stimulation of food intake and a significant increase in body weight. Melanocortins seem to play an important role in stress-induced anorexia, because such condition, in rats, is significantly attenuated by the blockage of melanocortin MC(4) receptors; such a role is not secondary to an increased release of corticotropin-releasing factor (CRF), because, on the other hand, the CRF-induced anorexia is not affected at all by the blockage of melanocortin MC(4) receptors. The physiological meaning of the feeding inhibitory effect of melanocortins, and, by consequence, the physiological role of melanocortins in the complex machinery responsible for body weight homeostasis, is testified by the hyperphagia/obesity syndromes caused by mutations in the pro-opiomelanocortin (POMC) gene, or in the melanocortin MC(4) receptor gene, or in the agouti locus. Finally, recent evidences suggest that melanocortins could be involved in mediating the effects of leptin, and in controlling the expression of neuropeptide Y (NPY).

It has been previously shown that long-term treatment with low doses of l-sulpiride is highly effective in rat models of depression and of anticipatory anxiety/panic behavior. The present study was aimed at investigating whether the same treatment can prevent the ulcerogenic effect of repeated inescapable stresses. In adult rats, the repeated (7 consecutive days) exposure to an uncontrollable stressful condition (inescapable 2.5 mA scrambled shock for 60 s) produced the development of gastric lesions (multiple punctiform telangiectasias in all rats, with superficial erosions or more severe ulcerations in 10 out 13 rats; score 4.67 +/- 0.44). l-sulpiride, intraperitoneally injected once a day at an antidepressant dose level (4 mg kg(-1) per day), starting 21 days before the beginning of the 7-day sequence of inescapable punishments ( = 28 daily treatments), almost completely prevented the stress-induced gastric injury (score 1.67 +/- 0.29; P< 0.001 vs saline-treated rats, Mann-Whitney U test). These results show that, in rats, a long-term treatment with low doses of l-sulpiride prevents the development of gastric lesions induced by chronic exposure to uncontrollable stress., (Copyright 2000 Academic Press.)

The effect of gamma-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia was studied in the four vessel occlusion rat model. In saline-treated animals, 30 min ischemia caused a massive loss of neurons in the hippocampal CA1 subfield (normal neurons: 14%, 5%, 23% and 30% on the 3rd, 10th, 15th and 65th day after ischemia, respectively). gamma-Hydroxybutyrate - 300 mg/kg intraperitoneally (i.p.) 30 min before or 10 min after arteries occlusion, followed by 100 mg/kg i.p. twice daily for the following 10 days - afforded a highly significant protection (normal neurons on the 3rd, 10th, 15th and 65th day after ischemia: 88% and 91%, 80% and 80%, 91% and 90%, 72% and 71% in rats receiving the first dose before or after arteries occlusion, respectively). The ischemia-induced sensory-motor impairment was significantly attenuated in rats receiving the first dose of gamma-hydroxybutyrate before arteries occlusion. Finally, the ischemia-induced impairment in spatial learning and memory, evaluated starting 27 days after the ischemic episode, was significantly attenuated by gamma-hydroxybutyrate, either injected first at 30 min before or 10 min after arteries occlusion. Lower doses of gamma-hydroxybutyrate had no significant effect. In conclusion, these results indicate that gamma-hydroxybutyrate provides significant protection against both histological and behavioral consequences of transient global cerebral ischemia in rats.

The melanocortin (ACTH/MSH) peptides exert a number of central effects. In the eighties, we described for the first time a role for melanocortins in the central control of appetite. We showed that the injection of ACTH-(1-24) into a brain lateral ventricle reduced food intake up to 76.6% in starved rats. Injections into the ventromedial hypothalamus during the nocturnal feeding phase also markedly inhibited food intake. These effects were also confirmed in mice and rabbits. Targeted disruption of the MC4 receptor resulting in obesity in mice explained the role of this receptor in mediating effects of melanocortins on food intake. Administration of MC4 receptor agonists leads to acute reduction in food intake and body weight, while the reverse effects are observed after administration of selective MC4 receptor antagonists, confirming the role of the melanocortins in mediating a tonic inhibition on feeding behavior. Moreover, immobilization stress-induced anorexia may be partially reversed by single and repeated intracerebroventricular administration of selective MC4 receptor antagonists. It is thus evident that MC4 receptor blockage can reduce stress-induced anorexia and that repeated injections of selective MC4 receptor antagonists have a sustained effect on food intake without any sign of tachyphylaxis. However, we have also shown that the behavioral effects of CRF (anorexia and grooming) are not influenced by MC4 receptor blockage. These effects of CRF are thus not due to an indirect mechanism caused by an increased release of melanocortins acting on the central MC receptors.

Melanocortin peptides are known to be extremely potent in causing the sustained reversal of different shock conditions, both in experimental animals and humans; the mechanism of action includes an essential brain loop. Three melanocortin receptor subtypes are expressed in brain tissue: MC(3), MC(4,) and MC(5) receptors. In a volume-controlled model of hemorrhagic shock in anesthetized rats, invariably causing the death of control animals within 30 min after saline injection, the i.v. bolus administration of the adrenocorticotropin fragment 1-24 (agonist at MC(4) and MC(5) receptors) at a dose of 160 microg/kg i.v. (54 nmol/kg) produced an almost complete and sustained restoration of cardiovascular and respiratory functions. An equimolar dose of gamma(1)-melanocyte stimulating hormone (selective agonist at MC(3) receptors) was completely ineffective. The selective antagonist at MC(4) receptors, HS014, although having no influence on cardiovascular and respiratory functions per se, dose-dependently prevented the antishock activity of adrenocorticotropin fragment 1-24, with the effect being complete either at the i.v. dose of 200 microg/kg or at the i.c.v. dose of 5 microg/rat (17-20 microg/kg). We concluded that the effect of melanocortin peptides in hemorrhagic shock is mediated by the MC(4) receptors in the brain.

We investigated the effects of selective melanocortin MC4 receptor blockage on immobilization stress-induced anorexia. Male rats were subjected to immobilization once a day for 4 days. Prior to each of the stress treatments, the rats were injected i.c.v. (intracerebroventricularly) with either saline or the melanocortin MC4 receptor antagonist HS014 (cyclic [AcCys11, D-Nal14, Cys18, Asp-(NH22)2]beta-MSH-(11-22) (melanocyte-stimulating hormone). Rats subjected to neither stress nor i.c.v. injections served as controls. The results showed that the cumulative food intake and body weight gain in the stressed group treated with HS014 was significantly higher than in the stressed group and significantly lower than in the control group. Repeated injections of the melanocortin MC4 receptor antagonist were effective and there were no signs of tachyphylaxis. This is the first report showing that melanocortin MC4 receptor blockage can relieve an anorectic condition, which may indicate that melanocortin MC4 receptor blockage is an effective way to treat anorectic disorders.

Antidepressant drugs are effective in anxiety states, including panic disorder. Both clinical and animal studies indicate that l-sulpiride, at low, non-neuroleptic doses, has antidepressant activity. The present study examined the effect of an antidepressant dose of l-sulpiride (4 mg/kg per day SC), compared with a well-established antidepressant drug (fluoxetine, 3 mg/kg per day SC), in a rat model of anticipatory anxiety/panic behavior: conditioned fear stress-induced freezing behavior. Long-term (26 days) administration of l-sulpiride almost completely abolished freezing, a similar effect being produced by fluoxetine (freezing duration, in seconds: controls, 148.1 +/- 29.6; l-sulpiride, 27.5 +/- 8.3; fluoxetine, 72.0 +/- 15.2). The same doses of l-sulpiride (4 mg/kg SC) and fluoxetine (3 mg/kg SC) had no effect when administered for shorter periods (1, 5, or 12 days). No effect was produced by the long-term (26 days) administration of a neuroleptic dose of l-sulpiride (20 mg/kg per day SC). These results demonstrate that long-term administration of low, non-neuroleptic doses of l-sulpiride, is highly effective in an animal model of anticipatory anxiety/panic behavior.

CRF and melanocortin (MSH/ACTH) peptides share a number of central effects including anorexia and grooming. The effects of CRF may be secondary, due to CRF's effects on melanocortin peptide release. We investigated if the newly discovered selective melanocortin 4 receptor antagonist HS014 could influence CRF induced anorexia and grooming. The data show that ICV administration of CRF (3 mg/rat), significantly reduced food intake, feeding time and feeding episodes whereas it increased grooming time and grooming episodes. HS014 (5 mg/rat), that previously has been shown to antagonize the anorectic effect and the excessive grooming induced by alpha-MSH, did however not influence any of the behavioral effects induced by CRF when the peptides were administered together. The data indicate that the anorectic and grooming effects of CRF are independent of pathways involving the MC4 receptors. These data suggest that the anorectic and grooming effect of CRF are not due to a secondary effect caused by increase in release of melanocortins acting on the central MC receptors.

We injected i.c.v. the natural agonist alpha-MSH (melanocyte-stimulating hormone) and the first selective melanocortin MC4 receptor antagonist HS014 (cyclic [AcCys11, D-Nal14, Cys18, Asp-NH(2)22]-beta-MSH(11-22) in rats and scored a number of behavioral effects which have been related to the melanocortic peptides. The results showed that HS014 (5 microg/rat) completely blocked alpha-MSH (3 and 5 microg/rat)-induced grooming, yawning and stretching. Penile erections induced by alpha-MSH were, however, only partially blocked by HS014. Injections of alpha-MSH decreased food intake in food-deprived rats, whereas HS014 increased food intake. When the peptides were given together, the food intake was similar to that of saline treated controls. Locomotion/exploration and resting were not influenced by either peptide. Our data show that exogenous beta-MSH decreases food intake, and that an endogenous central melanocortinergic inhibitory tone on feeding prevails which can be blocked with HS014, leading to an increase in food intake. Our data also provide evidence that grooming, stretching and yawning in rats may be mediated by the melanocortin MC4 receptor, whereas penile erections might perhaps be mediated by some other melanocortin receptor.

Since reduced levels of brain serotonin are known to cause behavioural abnormalities, to which diabetics are also prone, we investigated the effect, in rats, of chronic diabetes on brain serotonin concentration and on the numbers of 5-HT(1A) and 5-HT2 receptors in cerebral cortex and brainstem. Our data show that streptozotocin induces a longlasting hyperglicemia that is associated with a decrease in cerebral concentration of serotonin and with an accompanying increase in the maximum number of 5-HT(1A) and 5-HT2 receptors in the brain areas studied. Our results may suggest that changes in serotonergic transmission in the CNS play a role in diabetes-related behavioural abnormalities.

Hyperthyroidism is often associated with behavioral disorders, and thyroid hormones modify receptor sensitivity as well as the synthesis and/or turnover rate of many neurotransmitters. We evaluated the influence in adult rats of triiodothyronine (T3), administered s.c. (100 micrograms/kg) acutely (once only) or chronically (once a day for 3 or 7 consecutive days), on brain serotonin concentration and on the density and affinity of two brain serotonin (5-HT) receptor subtypes mainly involved in behavioral effects. After both acute and chronic T3 treatment, serotonin levels increased in the cerebral cortex but not in the hippocampus. The density and affinity of 5-HT1A receptors (using [3H]-8-OH-DPAT as ligand) were not affected, while there was a significant decrease in the number of 5-HT2 receptors in the cerebral cortex (using [3H]ketanserin as ligand). This observation might indicate that thyroid hormones enhance 5-HT concentration in certain brain areas, thus causing a down-regulation of 5-HT2 receptors. The serotonergic system could be involved in the complex brain-neurotransmitter imbalance underlying hyperthyroidism-linked behavioral changes.

It has been shown that long-term administration of l-sulpiride induces a down-regulation of beta receptor-associated adenylate cyclase activity in the frontal cortex of rats, and adaptive response that is typically associated with the chronic administration of antidepressants. Here we show that in two animal models of "depression-like" behavior (forced swim in rats and tail suspension in mice), the long-term (21 days) administration of l-sulpiride at a non-neuroleptic dose (2 mg/kg IP twice a day) significantly decreases the duration of immobility, the effect being similar to that of desipramine (20 mg/kg IP). The same dose (2 mg/kg) of l-sulpiride, acutely administered, has no effect at all. On the other hand, either chronic (21 days) or acute administration of neuroleptic doses of l-sulpiride have an opposite effect, and indeed increase the duration of immobility. These results are an in vivo support to the in vitro findings suggesting that low doses of l-sulpiride may have antidepressant-like activity.

In adult male rats, the intracerebroventricular (i.c.v.) injection of pinacidil, a potassium channel opener, at the doses of 100, 200 or 300 micrograms/rat, dose-dependently reduced the display of the most typical behavioral symptoms (excessive grooming, stretching, yawning, penile erections) induced by the i.c.v. administration of ACTH-(1-24) (4 micrograms/rat). These data indicate that the complex mechanism of the melanocortin-induced behavioral syndrome involves closure of potassium channels in target neurons, and provide further experimental support to the idea that melanocortins are functional antagonists of opioids.

In 28 month-old male rats, the i.c.v. injection of adrenocorticotropin [ACTH-(1-24)] (4 micrograms/rat) did not induce the typical behavioral syndrome (excessive grooming, stretching, yawning, penile erections). This indicates that the behavioral effects of melanocortins are age-dependent, suggesting either an aging-linked impairment of the nervous circuitries involved or a reduction of the number (or affinity, or both) of the brain melanocortin receptors in the elderly.

In intact adult male rats an inhibitor of aromatase and an inhibitor of 5 alpha-reductase did not change the characteristics of [3H]imipramine binding sites in cerebral cortex, hypothalamus, and hippocampus. Testosterone, estradiol and dihydrotestosterone prevented the effect of castration on the number of [3H]imipramine binding sites, but had no effect in non-castrated animals. These data suggest that testosterone and its major metabolites, estradiol and dihydrotestosterone, are equally effective with regard to imipramine binding sites.

The effect of a treatment with L-triiodothyronine (T3) or propylthiouracil (PTU) on the characteristics of benzodiazepine and chloride ion channel binding sites in rat hippocampus and cerebral cortex was studied using a radiolabelled technique. In our experimental conditions, neither hyper- nor hypothyroidism modified number and affinity of [3H]flunitrazepam or [3H]butylbicycloorthobenzoate (TBOB) binding sites. These data indicate that neither benzodiazepine nor chloride ionophore sites of the GABA complex are modified in an experimental condition of dysthyroidism.

High densities of atriopeptin-immunoreactive fibers and of highly specific and selective atriopeptin receptor sites are present in brain areas involved in animal behavior. The possible influence of these peptides on behavior was thus investigated in adult rats. The intracerebroventricular injection of atriopeptin II modified male sexual behavior (reduction in mount latency) at the dose of 5 micrograms/animal; lower and higher doses were ineffective. Open-field behavior was also modified by i.c.v. atriopeptin II at the doses of 5 and 10 micrograms/rat, which induced an increase in the number of external and internal crossings and of external rearings. Finally, in fasted rats, atriopeptin II, at the dose of 10 micrograms/rat, significantly increased the amount of food intake 30 and 60 min after injection. These findings indicate that atriopeptins may modify different animal behaviors.

The opening of K+ channels in the membrane of target neurons is a key mechanism of the effect of opioids. Here we show that the K+ channel opener, pinacidil, i.c.v. injected at doses of 50, 100 or 150 micrograms/rat, significantly increases and prolongs the effect of morphine on the thermal pain threshold (hot-plate and tail-flick tests). These data may suggest a novel approach to the management of pain.

In an experimental condition of hyperthyroidism, obtained by repeated administration of triiodothyronine in adult rats (100 micrograms/kg/day, sc for 7 consecutive days), there is an increased susceptibility to the convulsant effect of isoniazid (300 mg/kg, ip) and picrotoxin (4 mg/kg, ip). On the other hand, the characteristics of brain [3H] flunitrazepam binding sites are not modified. These data afford further experimental evidence of the influence of thyroid hormones on brain function.

In a model of haemorrhagic shock causing the death of all saline-treated rats within 25.8 +/- 2.7 min after treatment, the intravenous injection of thyrotropin-releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induces a prompt and sustained increase of arterial pressure and pulse amplitude, with survival of all rats. Bilateral vagotomy, atropine sulphate (2 mg/kg intraperitoneally) and hemicholinium-3 (20 micrograms/rat intracerebroventricularly) partially prevent the TRH-T-induced shock reversal, whereas atropine methylbromide has no effect. These data indicate that afferent vagal fibres, brain cholinergic neurons and central muscarinic receptors play a role in the mechanism of the anti-shock effect of TRH-T.

The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alpha 1, alpha 2, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [3H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [3H]prazosin, [3H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [3H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status.

The intracerebroventricular injection of the cholecystokinin-A receptor antagonist L-364,718, at the doses of 0.5, 5, 10 or 20 micrograms/mouse, while having no effect on pain threshold (hot plate, 51 degrees C), antagonized the analgesic activity of morphine (10 mg/kg i.p.). This effect was obtained with a dose of 10 micrograms/mouse and was associated with a reduction of brainstem opiate-binding sites.