Psychostimulant dependence remains a major health and economic problem, leading to premature death and costing $181 billion annually in health care, crime, and lost productivity costs. Currently, no pharmacotherapies are available to effectively treat psychostimulant dependence. Psychostimulants cause changes in neural circuits involved in reward and affect, but addiction neurocircuitry is incompletely understood and new targets for therapeutic intervention are needed. Lateral hypothalamic orexins (hypocretins) have been shown to have functional roles in arousal, reward processing, attention, motivation, and impulsivity. The opioid peptide dynorphin, co-localized with orexin, has critical roles in producing negative affective emotion states through interactions with, among others, stress circuitry. Orexin-dynorphin neurons project to neural substrates governing positive and negative motivated behavior, including the bed nucleus of the stria terminalis (BNST), amygdala, locus coeruleus and ventral tegmental area (VTA). Orexin and dynorphin modulate post-synaptic membrane activity through opposing signaling mechanisms; while orexins bind to predominantly excitatory orexin-1 and -2 G-protein coupled receptors, dynorphins bind to predominantly inhibitory G-protein coupled kappa opioid receptors (KORs). Multiple psychopathologies, including anxiety and substance abuse disorders, are characterized in part by alterations in orexin-dynorphin signaling. While these peptides have been shown to co-localize within single presynaptic vesicles and exert opposing effects on post-synaptic membrane potentials, the utility of producing oppositely-behaving peptides and the implications on psychopathologies remains unknown. The present studies were conducted to explore the role of orexin and dynorphin activity in cocaine’s rewarding effects as well as the negative effects of withdrawal. To accomplish this, we measured 1. Effects of orexin and cocaine administration on impulsive behaviors that increase the likelihood of psychostimulant addiction, using 5-choice serial reaction time task in concert with systemic and site directed pharmacology. 2. Effects of orexin and dynorphin on motivation for cocaine administration and intracranial self-stimulation. Using immunohistochemistry, ultrasonic vocalizations, and fast scan cyclic voltammetry we explored possible dopaminergic mechanisms of orexin and dynorphin contributions to reward. Lastly 3. Effects of orexin, dynorphin and chronic cocaine on withdrawal-induced anhedonia using intracranial self-stimulation, elevated plus maze, and correlations with immunohistochemistry and plasma corticosterone levels to explore further mechanisms. The results of this dissertation support our hypothesis that orexin receptor activity contributes to cocaine-induced impulsivity, motivation to self-administer cocaine and the reinforcing effects of psychostimulants. Dynorphin activity contributes to anhedonia and anxiety seen during drug abstinence after chronic exposure. Orexin and dynorphin exert these effects, in part, by modulating activity of dopaminergic neurons projecting from the ventral tegmental area to the nucleus accumbens. / Biomedical Sciences

Millions of people in the US currently suffer from chronic pain but available therapeutics do not provide effective chronic pain treatment. Opiate therapy is still the gold standard for chronic pain management with detrimental side effects, such as tolerance, addiction, constipation, and respiratory depression that limit their therapeutic potential. Opiates exert their positive and negative effects by activating the μ opioid receptor (MOR). Conversely, the κ opioid receptor (KOR) has been shown to modulate the tolerance and addiction produced by MOR agonists and is also involved in mood modulation (anxiety and depression). Therefore, blocking KOR activation results in positive effects against opiate side effects and stress-related depression. Dynorphin A (Dyn A) is the endogenous opioid peptide for the KOR. Structure-activity relationship (SAR) studies were carried out to develop a KOR selective antagonist based on the Dyn A structure. A minimum Dyn A pharmacophore with improved stability, no cell toxicity, and antagonist activity was discovered. Peptidomimetic enkephalin analogues previously developed in our group as MOR and δ opioid receptor (DOR) agonists have shown multifunctional activity, with MOR/DOR agonist and KOR antagonist activities. To our knowledge, this finding is first of its class for the opioid receptors. Novel design and synthesis of KOR selective ligands based on our multifunctional enkephalin analogues was done. Successful peptide synthesis resulted in analogues with high stability in rat plasma and no cell toxicity.

Made available in DSpace on 2016-06-02T19:23:02Z (GMT). No. of bitstreams: 1 6776.pdf: 1764671 bytes, checksum: 0befe5018516a7acffe79be054e73ae7 (MD5) Previous issue date: 2014-07-07 / Universidade Federal de Minas Gerais / Adolescence marks a period of increased vulnerability to drug abuse. Early initiation of tobacco use is strictly associated with alcohol use and dependence in adolescence and adulthood. The present study aims to investigate: 1) changes in anxiogenic-like effects and on operant ethanol self-administration in adult rats exposed to nicotine during adolescence; 2) changes on pro-dynorphin (PDYN) gene expression in the nucleus accumbens (NAc) on late nicotine withdrawal and after ethanol self-administration; 3) which brain areas are activated on short or long-term nicotine withdrawal during adolescence. Our results showed that pre-exposure to nicotine during ten days (3mg/kg/day; s.c) in adolescent rats, did not change anxiogenic-like effects in the elevated plus maze, 15 days after the treatment cessation [postnatal day (PND) 52], progressive ratio (PND 75-79) and binge (PND 81-84) in the operant ethanol selfadministration protocol. However, animals pre-treated with nicotine displayed increased motivation to saccharin seeking. No changes were observed on gene expression of PDYN 15 days after chronic nicotine treatment cessation or 24 hours after the last binge session of ethanol self-administration. Concerning the areas involved on nicotine withdrawal, we observed that adolescent rats exposed to nicotine through the minipumps during 10 days (PND 28-37), showed enhanced Fos activation in the medial prefrontal cortex (mPFC) 3 days after nicotine treatment cessation (PND 40). The percentage of neuronal activation in this group was 22.03%, of those activated neurons, 28.5% were GABAergic and only 2.5% were glutamatergic neurons. Meanwhile, 15 days after the mini-pumps were removed, no difference was observed on Fos activation in the different brain areas analyzed. We concluded that: a) administration of nicotine during adolescence was not able to modify anxiety-like behaviors and ethanol seeking behavior in adulthood; b) late nicotine withdrawal did not change gene expression of PDYN in NAc, as well as, did not change Fos protein neuronal activation in the regions of mPFC, NAc and habenula; c) short-term nicotine withdrawal involves the activation of neural ensembles in the mPFC. / A adolescência é o período de maior vulnerabilidade ao abuso de drogas. O início precoce do uso de tabaco está significativamente associado ao abuso e dependência de etanol na adolescência e na vida adulta. O presente trabalho teve como objetivo investigar: 1) as alterações nos comportamentos relacionados a ansiedade e na autoadministração operante de etanol em ratos adultos expostos a nicotina durante a adolescência; 2) as alterações na expressão gênica da pró-dinorfina (PDYN) no núcleo acumbens (NAc) na abstinência tardia à nicotina e ao final da autoadministração de etanol; 3) quais as áreas encefálicas ativadas na abstinência, imediata e tardia, à nicotina durante a adolescência. Nossos resultados mostraram que a administração de nicotina durante 10 dias (3mg/kg/dia; s.c), em ratos adolescentes [dia pós-natal (DPN) 28-37], não causou alterações nos comportamentos relacionados a ansiedade no labirinto em cruz elevado 15 dias após a término do tratamento (DPN 52), assim como não alterou a autoadministração operante de etanol na razão progressiva (DPN 75-79) e no binge (DPN 81-84). Contudo, animais pré-tratados com nicotina apresentaram aumento da motivação para a busca de sacarina. Não foram observadas alterações na expressão gênica de PDYN 15 dias após a suspensão do tratamento crônico com nicotina ou 24 horas após a última sessão do binge de etanol. Quanto às áreas envolvidas na abstinência à nicotina, observamos que ratos adolescentes expostos à nicotina, por meio de mini-bombas, durante 10 dias (DPN 28-37), apresentaram maior ativação de Fos no córtex pré-frontal medial (CPFm) 3 dias após o término do tratamento. A porcentagem de ativação neuronal nesse grupo, foi de 22,03%, sendo que 28,5% desses neurônios ativados eram GABAérgicos e apenas 2,5% eram glutamatérgicos. Entretanto, 15 dias após a retirada das mini-bombas não houve diferença na ativação de Fos nas diferentes áreas encefálicas. Concluímos que: a) a administração de nicotina, durante a adolescência, não foi capaz de alterar os comportamentos relacionados a ansiedade e o comportamento de busca pelo etanol na idade adulta; b) a retirada tardia da nicotina não promoveu alterações na expressão gênica de PDYN no NAc, assim como não promoveu alterações na ativação neuronal da proteína Fos nas regiões do CPFm, NAc e habenula; c) a retirada da nicotina a curto prazo envolve a ativação de grupamentos neurais no CPFm.

This study was designed to explore the hormonal regulation of CNS opioid peptide levels in female Sprague Dawley rats. Forty-eight animals were divided into 2 equal groups for acute and chronic studies. Each group was further divided into 4 subgroups, each containing 6 animals. Each rat in the control group received an inert pill (in 0.25 ml corn oil daily by gavage); the second group, 15 μg norethindrone (NE, a potent progestin present in the oral contraceptive Micronor®); the third group, 15 μg NE and 1 μg ethinyl estradiol, EE2 (present in the oral contraceptive Modicon®) and the fourth group, 10 times the dose of the third group. Rats were treated either acutely for 5 days or chronically for 7 weeks. Opioid peptides were estimated by radioimmunoassay. Acute administration of 150 μg NE + 10 μg EE2 decreased the levels of methionine-enkephalin (ME), leucine-enkephalin (LE), dynorphin (DYN) and β-endorphin like immunoreactivity (β-EI) by about 50% in the pituitary. The same dose on chronic administration also decreased DYN, but increased the levels of ME and LE in the pituitary by 331 and 69%, respectively. In the hypothalamus, chronic administration of NE + EE2 increased the level of ME (155%) and LE (87%) as well as of DYN (97%). In the striatum, the levels of LE (33%) and DYN (115%) were elevated during chronic administration. It is concluded that the acute administration of NE + EE2, in general, reduces the levels of ME, LE, DYN and β-EI. The extent of this decrease is about the same in the pituitary, hypothalamus and striatum. Chronic administration of these hormones, however, results in a reversal of this decrease (except for β-EI) and actually can increase the levels of ME, LE and DYN in all three tissues.

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An investigation of the interaction of stable opioid/ligands and unstable opioid peptides with opioid receptors in guinea pig brain, guinea pig myenteric plexus and mouse vas deferens has been carried out. The initial aim of the study was to further characterise K opioid receptors, using binding assays and isolated tissue bioassays. The second aim was to determine the true affinity and potency of small dynorphin peptides for the K opioid receptor and to determine if metabolism of the peptides to non K opioid receptor-preferring products contributes to their observed in vitro pharmacology.

My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles). The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I). Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV. The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency. Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs. Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Early-life experience has profound effects on the individual’s neurobiology and behaviour later in life. The rodent animal experimental model maternal separation (MS) was used to study this more in detail. The MS model involves short and prolonged postnatal separations simulating an emotionally safe and stressful environment, respectively. The aims of the thesis were to examine the impact of individual MS on ethanol consumption and on brain dopamine and serotonin systems in adult male rats. Furthermore, the influence of separation conditions on the short- and long-term consequences of MS on several neurotransmitter systems was examined. Rat pups were assigned to either litter-wise MS for 15 or 360 minutes (MS15l or MS360l) or individual MS for 15 or 360 minutes (MS15i or MS360i). Control rats were subjected to conventional animal facility rearing (AFR). Ethanol intake was assessed in a two-bottle free-choice paradigm. Neuropeptides were analyzed with radioimmunoassay, monoamines and metabolites with electrochemical detection and gene expression with qPCR. Using the MSi paradigm, minor effects on voluntary ethanol consumption were observed. However, the monoaminergic responses elicited by ethanol were dependent on the early-life environment. Furthermore, short- and long-term consequences of MS on serotonin, opioid, oxytocin and vasopressin systems were studied. Multiple neurobiological measurements in one and the same rat offered a unique possibility to examine the effects of duration (MS15 versus MS360) and condition (l versus i) of MS. Time-, region-, sex- and transmitter-specific effects were observed. More pronounced differences were seen in serotonin measures and oxytocin in young rats. In adults these differences in basal levels were normalized. Opioid peptides differed in stress-related brain areas in young rats and in limbic areas in adults. Rats subjected to the MS15l environment that relates to natural conditions generally exhibited a different neurobiological profile than other groups. AFR rats, i.e. conventional control rats, were more similar to the putative most stressful condition MS360. Taken together, the networks examined in the present thesis are important for the establishment of normal social behaviour and derangements in these systems may result in neurobiological changes leading to the susceptibility for psychopathological conditions later in life.

The interaction of a diverse set of opioid alkaloids and peptides with various opioid receptors has been examined using biochemical and pharmacological techniques. Structural information on the compounds was obtained from single crystal X-ray diffraction and nuclear magnetic resonance studies, and modelled by computational methods. The introduction of a dithiocarbazate moiety into the 7a-position of a bridged thebaine was shown to afford a degree of μ selectivity in this class of nonselective compounds. X-ray diffraction analysis of this compound and comparison with the structure of [Met5]enkephalin showed the importance of the sulphydryl moiety. The conformation of [Leu5]enkephalin, in which the amino acid methionine is replaced by leucine, at the same receptor is unlikely to be similar. A series of morphinan derivatives which had been developed as μ-antagonists were evaluated. Substitution patterns of the morphinan ring nucleus and their effect upon activity were examined. X-ray analysis of several key compounds was performed. Unexpectedly a 3-hydroxymorphinan-6-one analogue showed an ability to differentiate apparently similar opioid Kreceptors. The implications in terms of K-receptor subtypes are discussed. The opioid receptor binding characteristics of structurally diverse K-receptor ligands were examined in two different buffer systems. Electrostatic modelling of the K-ligands, based upon crystal structure coordinates, was performed. From electrostatic potential maps a requirement for ligands acting at Kreceptors is postulated. Solution conformations of the endogenous K-ligand, dynorphin A(1-8), were determined by nuclear magnetic resonance studies and compared with the wo preferring [Leu5]enkephalin. Models were proposed based upon dihedral angles determined from HCtl-NH coupling constants, amide proton-deuteron exchange and amide proton temperature coefficient data. Candidate conformations were shown to be stable under dynamic simulation conditions. Electrostatic modelling of a chosen dynorphin An-8) conformation gave results comparable with the observed electrostatic model of the K-ligands. The proposed model is discussed in terms of its suitability as a retro-model for the active site ofthe K-opioid receptor.