Sialic acid and glycopeptides conjugated PLGA nanoparticles for central nervous system targeting: In vivo pharmacological evidence and biodistribution.

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.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat Part 2: Effects on reproductive parameters, on sex behavior, on memory retention and on hypothalamic expression of aromatase and 5alpha-reductases in the offspring.

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.

Targeting the central nervous system: in vivo experiments with peptide-derivatized nanoparticles loaded with Loperamide and Rhodamine-123.

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.

Characterization of the resistance to the anorectic and endocrine effects of leptin in obesity-prone and obesity-resistant rats fed a high-fat diet.

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.

Energy gradients for VT-signal migration in the CNS: studies on melanocortin receptors, mitochondrial uncoupling proteins and food intake.

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.

Role of melanocortins in the central control of feeding.

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).

Chronic melanocortin 4 receptor blockage causes obesity without influencing sexual behavior in male rats.

We investigated the effects of continuous intracerebroventricular infusion of a melanocortin 4 receptor antagonist HS014 (cyclic [AcCys11, D-Nal14, Cys18, Asp-NH2(22)]beta-MSH-(11-22)) over 12 days and a subsequent 12-day recovery period on food intake, body weight and copulatory behavior in male rats. The results show that the food intake increased immediately after the start of the infusion of HS014 (0.16 nmol/h) and progressively increased thereafter. No tachyphylaxis was observed. When the infusion of HS014 was terminated, the food-intake levels dropped. The body weights of the rats had increased by 17% by the end of the study, compared with controls. During the recovery period, the body weight decreased towards the levels of the control rats. These results indicate that overeating and the subsequent increases in body weight caused by blockage of the melanocortin 4 (MC4) receptor are reversible when the blockage is ended. We also tested the copulatory behavior of vigorous male rats in the presence of female rats in estrous. We registered mount latency, the number of mounts, the intromission latency, the number of intromissions, the ejaculation latency and the post-ejaculatory interval three times during the study and also after acute administration of HS014 and alpha-MSH. The sexual behavior of the male rats was not affected. These results indicate that the MC receptors, in particular the MC4 receptor, may not be a major mediator of effects on copulatory behavior in male rats.

L -sulpiride, at antidepressant dosage, prevents conditioned-fear stress-induced gastric lesions in rats.

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.

Neuroprotective effect of gamma-hydroxybutyrate in transient global cerebral ischemia in the rat.

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.

Melanocortins and feeding behavior.

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.

Evidence that melanocortin 4 receptor mediates hemorrhagic shock reversal caused by melanocortin peptides.

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.

Corticotropin-releasing factor (CRF) induced anorexia is not influenced by a melanocortin 4 receptor blockage.

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.

l-Sulpiride, at a low, non-neuroleptic dose, prevents conditioned fear stress-induced freezing behavior in rats.

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.

Selective melanocortin MC4 receptor blockage reduces immobilization stress-induced anorexia in rats.

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.

Differential influence of a selective melanocortin MC4 receptor antagonist (HS014) on melanocortin-induced behavioral effects in rats.

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.

Streptozotocin-induced diabetes provokes changes in serotonin concentration and on 5-HT1A and 5-HT2 receptors in the rat brain.

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.

Effect of acute and chronic treatment with triiodothyronine on serotonin levels and serotonergic receptor subtypes in the rat brain.

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.

Chronic administration of l-sulpiride at non-neuroleptic doses reduces the duration of immobility in experimental models of "depression-like" behavior.

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.

Opening of brain potassium-channels inhibits the ACTH-induced behavioral syndrome in the male rat.

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.

Old rats are unresponsive to the behavioral effects of adrenocorticotropin.

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.