Mariarosa Spina

Appetite-Suppressing Effects of Urocortin, a CRF-Related Neuropeptide

The neuropeptide corticotropin-releasing factor (CRF) is well known to act on the central nervous system in ways that mimic stress and result in decreases in exploration, increases in sympathetic activity, decreases in parasympathetic outflow, and decreases in appetitive behavior. Urocortin, a neuropeptide related to CRF, binds with high affinity to the CRF2 receptor, is more potent than CRF in suppressing appetite, but is less potent than CRF in producing anxiety-like effects and activation. Doses as low as 10 nanograms injected intracerebroventricularly were effective in decreasing food intake in food-deprived and free-feeding rats. These results suggest that urocortin may be an endogenous CRF-like factor in the brain responsible for the effects of stress on appetite.

Corticotropin-releasing factor antagonist attenuates the "anxiogenic-like" effect in the defensive burying paradigm but not in the elevated plus-maze following chronic cocaine in rats.

Rationale: Chronic cocaine abuse is associated with the development of anxiogenic states in humans. Corticotropin-releasing factor (CRF) is an endogenous neurotropic factor well known to modulate stress responses. It has been postulated that CRF is involved in the neurobiological mechanisms underlying the anxiety and/or stress responses associated with removal of cocaine after chronic administration. Objective: The present study investigated the role of endogenous CRF in mediating the “anxiety-like” effect 48 h after the cessation of saline or chronic cocaine treatment in rats, using the defensive burying paradigm and the elevated plus-maze. Methods: Rats received daily injections of cocaine (20 mg/kg IP, for 14 consecutive days) or vehicle. Forty-eight hours after the last injection, animals were tested in the plus-maze and then in the defensive burying paradigm. In a second experiment, intracerebroventricular (ICV) cannulae were implanted at the lateral ventricle. Animals were allowed a 1-week period for recovery before starting the chronic drug treatment. The defensive burying testing took place 48 h after cessation of the treatment. The CRF antagonist [DPhe12, Nle21,38, CαMe Leu37] r/h CRF(12–41), (also known as D-phe CRF(12–41)) (0.04, 0.2 and 1.0 μg/5 μl) was injected 5 min before the 15-min testing. Results: An “anxiogenic-like” effect following chronic cocaine treatment was demonstrated with the defensive burying paradigm, but not with the elevated plus-maze. This “anxiety-like” response was attenuated by ICV pretreatment with the CRF antagonist D-Phe CRF(12–41), with the highest dose of the CRF antagonist reversing the observed “anxiogenic-like” response. Conclusions: These data suggest that brain CRF may be substantially involved in the development of “anxiety-like” responses related to cocaine withdrawal and could be important for future drug dependence treatments.

Oxyresveratrol (trans-2,3′,4,5′-tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia

Oxidative stress is one of the major pathological factors in the cascade that leads to cell death in cerebral ischemia. Here, we investigated the neuroprotective effect of a naturally occurring antioxidant, oxyresveratrol, to reduce brain injury after cerebral stroke. We used the transient rat middle cerebral artery occlusion (MCAO) model of brain ischemia to induce a defined brain infarction. Oxyresveratrol was given twice intraperitoneally: immediately after occlusion and at the time of reperfusion. Oxyresveratrol (10 or 20 mg/kg) significantly reduced the brain infarct volume by approximately 54% and 63%, respectively, when compared to vehicle-treated MCAO rats. Also, the neurological deficits as assessed by different scoring methods improved in oxyresveratrol-treated MCAO rats. Histological analysis of apoptotic markers in the ischemic brain area revealed that oxyresveratrol treatment diminished cytochrome c release and decreased caspase-3 activation in MCAO rats. Also, staining for apoptotic DNA showed that the number of apoptotic nuclei in ischemic brain was reduced after oxyresveratrol treatment as compared to the vehicle-treated MCAO rats. This dose-dependent neuroprotective effect of oxyresveratrol in an in vivo stroke model demonstrates that this drug may prove to be beneficial for a therapeutic strategy to limit brain injury in acute brain ischemia.

Behavioral Effects of Central Administration of The Novel CRF Antagonist Astressin in Rats

Astressin, a novel corticotropin releasing factor (CRF) antagonist, has been found to be particularly potent at inhibiting the hypothalamo-pituitary-adrenal axis. The aim of the present study was to determine the effects in rats of astressin in attenuating the anxiogenic-like response produced by social stress and intracerebroventricular (ICV) CRF administration on the elevated plus-maze, and ICV CRF-induced locomotor activation in the rat. Astressin significantly reversed the anxiogenic-like response induced by both social stress and ICV rat/humanCRF (r/hCRF) on the elevated plus-maze, but failed to block the effects of r/hCRF-induced locomotor activity in a familiar environment. When these results were compared to previous studies performed with the same paradigms using other CRF antagonists, astressin showed effects similar to those of D-PheCRF(12-41) on plus-maze performance. However, contrary to α-helicalCRF(9-41) and D-PheCRF(12-41), astressin had no effect on CRF-induced locomotor activity. These results suggest that astressin may have a unique anti-CRF profile compared to previously tested antagonists.

Nociceptin fails to affect heroin self-administration in the rat

THE recently isolated peptide nociceptin has a primary structure similar to that of opioid peptides. Early functional studies suggested that it may act in opposition to opioid systems. To determine whether nociceptin influences the rewarding properties of heroin, nociceptin was given intracerebroventricularly (i.c.v.) to rats trained to self-administer heroin. Rats (n = 8) were given doeses of 0.01 μg, 0.1 μg 1.0 μg and 10.0 μg in a Latin square design. None of the doses significantly affected heroin self-administration rates compared to vehicle. The highest dose of nociceptin used inhibited spontaneous locomotor activity, evidence that the peptide retained its biological activity after i.c.v. infusion. These studies suggest that nociceptin does not affect the rewarding value of heroin.

Structural and Compositional Determinants of Cortistatin Activity

Cortistatin‐14 (CST‐14) is a putative novel neuropeptide that shares 11 of its 14 residues with somatostatin‐14 (SRIF‐14), yet its effects on sleep physiology, locomotor behavior and hippocampal function are different from those of somatostatin. We studied the structural basis for cortistatins distinct biological activities. As with SRIF‐14, CST‐14 does not show any preferred conformation in solution, as determined by circular dichroism and nuclear magnetic resonance. Synthetic cortistatin analogs were designed and synthesized based on the cyclic structure of octreotide. Biological assays were carried out to determine their binding affinities to five somatostatin receptors (sst1‐5) and their ability to produce changes in locomotor activity and to modulate hippocampal physiology and sleep. The results show that the compound with N‐terminal proline and C‐terminal lysine amide exhibits cortistatin‐like biological activities, including reduction of population spike amplitudes in the hippocampal CA1 region, decrease in locomotor activity and enhancement of slow‐wave sleep 2. These findings suggest that both proline and lysine are necessary for cortistatin binding to its specific receptor. J. Neurosci. Res. 56:611–619, 1999. 

Colocalization of urocortin and neuronal nitric oxide synthase in the hypothalamus and Edinger-Westphal nucleus of the rat.

Different lines of studies suggest that both the corticotropin‐releasing hormone‐related peptide Urocortin I (Ucn) and the neuromodulator nitric oxide (NO) are involved in the regulation of the complex mechanisms controlling feeding and anxiety‐related behaviors. The aim of the present study was to investigate the possible interaction between Ucn and NO in the hypothalamic paraventricular nucleus (PVN), an area known to be involved in the modulation of these particular behaviors. Therefore, we mapped local mRNA and peptide/protein presence of both Ucn and the NO producing neuronal NO synthase (nNOS). This investigation was extended to include the hypothalamic supraoptic nucleus (SON) and the Edinger‐Westphal nucleus area (EW), the latter being one of the major cellular Ucn‐expressing sites. Furthermore, we compared the two predominantly used laboratory rat strains, Wistar and Sprague‐Dawley. Ucn mRNA and immunoreactivity were detected in the SON and in the EW. A significant difference between Wistar and Sprague‐Dawley rats was found in mRNA levels in the EW. nNOS was detected in all brain areas analyzed, showing a significantly lower immunoreactivity in the PVN and EW of Sprague‐Dawley versus Wistar rats. Contrary to some previous reports, no Ucn mRNA and only a very low immunoreactivity were detectable in the PVN of either rat strain. Interestingly, double‐labeling immunofluorescence revealed that in the SON ∼75% of all cells immunoreactive for Ucn were colocalized with nNOS, whereas in the EW only ∼2% of the Ucn neurons were found to contain nNOS. These findings suggest an interaction between Ucn and NO signaling within the SON, rather than the PVN, that may modulate the regulation of feeding, reproduction, and anxiety‐related behaviors. J. Comp. Neurol. 479:271–286, 2004.

Nitric oxide is not involved in the control of vasopressin release during acute forced swimming in rats.

Summary.Neurons of the hypothalamo-neurohypophyseal system (HNS) are known to contain high amounts of neuronal nitric oxide (NO) synthase (nNOS). NO produced by those neurons is commonly supposed to be involved as modulator in the release of the two nonapeptides vasopressin (AVP) and oxytocin into the blood stream. Previous studies showed that forced swimming fails to increase the release of AVP into the blood stream while its secretion into the hypothalamus is triggered. We investigated here whether hypothalamically acting NO contributes to the control of the AVP release into blood under forced swimming conditions. Intracerebral microdialysis and in situ hybridization were employed to analyze the activity of the nitrergic system within the supraoptic nucleus (SON), the hypothalamic origin of the HNS. A 10-min forced swimming session failed to significantly alter the local NO release as indicated both by nitrite and, the main by-product of NO synthesis, citrulline levels in microdialysis samples collected from the SON. Microdialysis administration of NO directly into the SON increased the concentration of AVP in plasma samples collected during simultaneous forced swimming. In an additional experiment the effect of the defined stressor exposure on the concentration of mRNA coding for nNOS within the SON was investigated by in situ hybridization. Forced swimming increased the expression of nNOS mRNA at two and four hours after onset of the stressor compared to untreated controls. Taken together, our results imply that NO within the SON does not contribute to the regulation of the secretory activity of HNS neurons during acute forced swimming. Increased nNOS mRNA in the SON after forced swimming and the increase in AVP release in the presence of exogenous NO under forced swimming points to a possible role of NO in the regulation of the HNS under repeated stressor exposure.

Reduced urocortin 1 immunoreactivity in the non-preganglionic Edinger-Westphal nucleus during late pregnancy in rats.

Pregnancy is accompanied by an array of adaptive changes that play an important role in pre- and postnatal events. In rats, urocortin 1, a corticotropin-releasing factor-like peptide, is expressed mainly in the non-preganglionic Edinger-Westphal nucleus. We investigated the number of neurons immunoreactive for urocortin 1 at three different levels of the Edinger-Westphal nucleus in female rats by immunohistochemistry. The number of urocortin 1 immunoreactive cells was found to be decreased in pregnant rats compared to virgin rats. These results indicate that the hormonal status of the female rat affects urocortin 1 immunoreactive neurons in the non-preganglionic Edinger-Westphal nucleus and its signaling to target brain areas.

The effects of p-chloroamphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (ecstasy) on the gene expression of cytoskeletal proteins in the rat brain.

Repeated administration of β‐phenylalkylamines is known to produce neuronal changes in the central and peripheral nervous systems of mammals. It is suggested that various components of the cytoskeleton undergo profound alterations after amphetamine use and misuse, contributing to behavioral changes and neurotoxicity. Here we studied the expression of microtubule‐associated protein 2 (MAP2) and β‐actin after repeated intraperitoneal applications with equimolar doses of p‐chloroamphetamine (PCA), methamphetamine (METH) and 3,4‐methylenedioxymethamphetamine (MDMA) in the brain of male Wistar rats. Effective (molecular) pharmacological doses (ED) were derived and used for the calculation of (molecular) pharmacological indices (PI). Besides clear but different dose–response curves on the toxicity of the drugs, in situ hybridization and Western blot analysis revealed that repeated administration of these compounds resulted in different substance‐ and dose‐dependent changes in MAP2 gene expression, e.g. in the frontoparietal somatosensoric cortex. In contrast, the expression of β‐actin was not influenced by any of the compounds at the dose levels tested. Lethal doses were determined with 2.1 (PCA), >5.1 (METH) and 8.4 mg/kg/day (MDMA). Linear and non‐linear repeat‐dose lethality was observed for MDMA and PCA, respectively, whereas METH was non‐lethal in the dose range used. Values for EDMAP2 were 0.3, 0.52 and >16.8 mg/kg/day, and therefore those for PIMAP2 were 20, 4, and 0.5 for METH, PCA and MDMA, respectively. Although the results on mortality did not reflect changes in MAP2 gene expression, they suggest a remarkable difference for those amphetamines without substituents or with a halogen atom at the paraposition of the benzene ring, such as METH or PCA, when compared with MDMA‐like substances.