Dopamine receptors are members of the G protein-coupled receptor superfamily and play important roles in neuronal transmission. A D1-D2 receptor heterooligomer generating a G-protein linked PLC-dependent intracellular calcium signal was previously identified. The discovery of this dopamine mediated calcium signal implicated a direct link between dopamine receptors and calcium generation, but its regulation remained to be elucidated. By measuring calcium signaling with Fluo-4 fluorescence or cameleon FRET, rapid desensitization of the calcium signal in heterologous cells and striatal neurons was demonstrated by pre-treatment with SKF 83959, which selectively activates D1-D2 receptor heteromers, or SKF 83822 which only activates D1 receptor homooligomers. Although SKF 83822 was unable to activate D1-D2 receptor heteromers, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without activation of the heteromer. BRET and co-immunoprecipitation studies indicated an agonist induced interaction between the D1-D2 receptor heteromer and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal and decreased expression of GRK2 led to an increased calcium signal. Expression of the catalytically inactive and RGS mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the D1-D2 receptor heteromer mediated calcium signal can occur by agonist occupancy even without activation and is regulated by two distinct functions of GRK2. Immunocytochemistry and calcium assays demonstrated that recycling of internalized D1 and D2 receptors and resensitization of the desensitized calcium signal occurred after dopamine pre-treatment but not SKF 83959, suggesting that the trafficking and resensitization response associated with the D1-D2 receptor heteromer is differentially regulated by specific ligands. Overall, these results suggest that D1-D2 receptor heterooligomers are uniquely regulated from their constituent receptors which are not coupled to Gq.

Dopamine receptors are members of the G protein-coupled receptor superfamily and play important roles in neuronal transmission. A D1-D2 receptor heterooligomer generating a G-protein linked PLC-dependent intracellular calcium signal was previously identified. The discovery of this dopamine mediated calcium signal implicated a direct link between dopamine receptors and calcium generation, but its regulation remained to be elucidated. By measuring calcium signaling with Fluo-4 fluorescence or cameleon FRET, rapid desensitization of the calcium signal in heterologous cells and striatal neurons was demonstrated by pre-treatment with SKF 83959, which selectively activates D1-D2 receptor heteromers, or SKF 83822 which only activates D1 receptor homooligomers. Although SKF 83822 was unable to activate D1-D2 receptor heteromers, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without activation of the heteromer. BRET and co-immunoprecipitation studies indicated an agonist induced interaction between the D1-D2 receptor heteromer and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal and decreased expression of GRK2 led to an increased calcium signal. Expression of the catalytically inactive and RGS mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the D1-D2 receptor heteromer mediated calcium signal can occur by agonist occupancy even without activation and is regulated by two distinct functions of GRK2. Immunocytochemistry and calcium assays demonstrated that recycling of internalized D1 and D2 receptors and resensitization of the desensitized calcium signal occurred after dopamine pre-treatment but not SKF 83959, suggesting that the trafficking and resensitization response associated with the D1-D2 receptor heteromer is differentially regulated by specific ligands. Overall, these results suggest that D1-D2 receptor heterooligomers are uniquely regulated from their constituent receptors which are not coupled to Gq.

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Doctor of Philosophy / Entomology / Yoonseong Park / Tick salivary secretion is crucial for survival and for successful feeding. Tick saliva includes excretory water/ions and bioactive components for compromising the hosts' immune responses, and provides a direct route for pathogen transmission. Control of the tick salivation involves autocrine/paracrine dopamine, the most potent stimulator of tick salivation. Our research group reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Dopamine-induced salivary secretion was orchestrated by two distinct physiological roles via activation of the two dopamine receptors (Chapter 2). Low concentration of dopamine activated D1 receptor on epithelial cells of salivary gland acini leading inward fluid transport. High concentration of dopamine activated InvD1L receptors on axonal projections innervating myoepithelial cells modulating pumping/gating actions for emptying luminal saliva into the main duct. Thus, ticks coordinated salivary secretion with duo dopamine receptors. Dopamine-mediated saliva production involves an important downstream component, Na/K-ATPase (Chapter 3). Na/K-ATPase was found in the epithelial cells of all types of acini. However, Na/K-ATPase had two different functions in salivary secretion in different acini: 1) dopamine-mediated production of primary saliva in distally located salivary gland acini type-2/- 3, and 2) dopamine-independent resorption in proximally located salivary gland acini type-1. Type-1 acini were also found to function in direct water absorption of off-host ticks, which could be a potential route for delivery of acaricides. Chapter 4 investigated the comparative transcriptomics of the lone star tick underlying the processes of pathogen acquisition. Differential expression analyses in pathogen-exposed ticks revealed a number of transcripts that are important in the tick-pathogen interaction. These included genes for tick immunity against pathogen and for modulation of tick physiology facilitating a pathogen’s invasion and proliferation. My study expanded the understanding of physiological mechanisms controlling tick salivation. In addition, transcriptomics of ticks in interaction with pathogen identified several genes that are relevant in vector/pathogen interactions. The knowledge obtained in my study will facilitate to the development of novel methods for the disruption of tick feeding and pathogen transmission.

No description available.

The aims of this thesis were (1) to learn about the identities of the molecules involved in the maintenance of long-term potentiation (LTP), and (2) to develop and test a behavioural paradigm capable of elucidating the interaction between these molecular processes and the persistence of long-term memories. By improving the stability of field recordings in in vitro electrophysiology, it was possible to investigate the molecular processes that determine the long-term changes in synaptic efficacy. In these experiments, the interactions between two convergent inputs onto the same neuronal population in the CA1 region of the hippocampus were monitored for over ten hours. Analytically powerful three-pathway protocols using sequential strong and weak tetanization in varying orders, and test stimulation over long periods of time after LTP-induction, enabled a pharmacological dissociation of potentially distinct roles of the calmodulin kinase (CaMK) pathways in LTP. This places constraints on the mechanisms by which synaptic potentiation, and possibly memories, become stabilized. The experiments show that tag setting is blocked by the CaMK inhibitor KN-93 that, at low concentration primarily blocks CaMKII, whereas a CaMKK inhibitor, STO-609, selectively limits the synthesis or the availability of plasticity related proteins (PRPs). To test whether memories can be subject to modulation by independent experiences, behavioural studies tested the possibility of lengthening the persistence of a relatively weak memory by pairing its induction with an event capable of inducing the synthesis of the required PRPs. Corticosterone-dependent stressful events like a cold swim proved to interfere and weaken spatial memories. On the other hand, the exploration of a novel environment succeeded in rescuing the decay of a weak memory. The effect of the exploration of the novel environment was dependent on NMDA and dopamine receptor activation, as well as protein synthesis. These results are discussed in relation to the synaptic tagging and capture hypothesis and a novel model of the neuronal mechanisms underlying synaptic plasticity is developed from them.

Made available in DSpace on 2016-08-19T18:16:06Z (GMT). No. of bitstreams: 1 Dissertacao Bruno.pdf: 2654529 bytes, checksum: 8a96ae87802691862664f63085d257f5 (MD5) Previous issue date: 2012-03-12 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / Pituitary tumors represent 15% of intracranial neoplasms and are usually benign. The treatment primary is surgical resection with exception for prolactinomas because dopamine agonists are very effective in the treatment these tumors. If surgery does not lead to healing, it is necessary other therapeutic strategy in a attempt to control hormone levels and tumor size reduction. The radiotherapy and medical treatment with somatostatin analogs and agonist dopamine are often used. The study aimed to analyze the presence, distribution and frequency of somatostatin receptor and dopamine receptor (DR2) in pituitary adenomas, and compared their mRNA expression and protein expression. We studied 38 patients with pituitary adenomas. The GH-secreting adenomas showed immunoreactivity more frequent SST2 and SST3 present in 100% of the tumors followed SST5, SST4 and SST1 respectively. Clinically nonfunctioning adenomas SST3 receptor was most widely distributed, present in 13 of 14 tumors followed SST2, SST4, SST1 and SST5 respectively. The mRNA expression of SST2 and SST5 receptor was present in all pituitary adenomas with higher expression of SST2. The DR2 receptor was present and 85% of samples analyzed. In conclusion, the high expression of SST2 in somatotropinomas support the possibility of the use of octreotide as an adjunct therapy in the treatment of acromegalic patients. Patients with clinically nonfunctioning adenomas showed expression of somatostatin receptor and dopamine which indicates the possibility of treating these patients with the somatostatin analogues and / or dopamine agonists. / Os tumores hipofisários correspondem por cerca de 15% das neoplasias intracranianas e são geralmente benignos. O tratamento primário mais comumente utilizado é a cirurgia com exceção para os prolactinomas, devido a eficácia do tratamento com a utilização de agonistas dopaminérgicos. Caso o resultado cirúrgico não leve a ressecção da doença, faz-se necessário o uso de outras modalidades terapêuticas, na tentativa de controlar os níveis hormonais e crescimento tumoral. A radioterapia e o tratamento medicamentoso com os análogos da somatostatina e/ou os agonistas dopaminérgicos são frequentemente utilizados como tratamento adjuvante. O presente estudo teve por objetivo analisar a presença, distribuição e frequência dos receptores da somatostatina e da dopamina (DR2) em adenomas hipofisários e comparar as suas expressões do mRNA e proteicas, através de testes por qPCR em tempo real e por imunohistoquímicos. Foram analisados 38 pacientes diagnosticados com adenomas hipofisários. Os adenomas secretores de GH apresentaram imunoreatividade mais frequente dos receptores SST2 e SST3, presentes em 100% dos tumores, seguidos pelos receptores SST5, SST4 e SST1, respectivamente. Nos adenomas clinicamente não funcionantes o receptor mais amplamente distribuído foi o SST3, presente em 13 dos 14 tumores seguidos pelo SST2, SST4, SST1 e SST5 respectivamente. A expressão do mRNA dos receptores SST2 e SST5 estava presente em todos os adenomas hipofisários estudados, com maior expressão do subtipo SST2. A expressão do mRNA do receptor DR2 foi encontrada em aproximadamente 85% das amostras analisadas. Em conclusão, a elevada expressão do SST2 nos somatotropinomas reforça a possibilidade do uso do octreotide como terapia complementar no tratamento dos pacientes acromegálicos. Os pacientes com adenomas clinicamente não funcionantes apresentaram expressão dos receptores da somatostatina e da dopamina o que indica a possibilidade de tratamento destes pacientes com os análogos da somatostatina e/ou agonistas dopaminérgicos.

This study analyzed the role of dopamine D1 and D2 receptors in methylphenidate (MPH) conditioned place preference (CPP) in adolescent male and female rats, in addition to the role of these receptors in the effects of MPH on brain-derived neurotrophic factor (BDNF) in the dorsal striatum and nucleus accumbens. Using a nonbiased CPP procedure, the animals were conditioned from postnatal day (PD) 33 to 37. On conditioning trials, animals were first administered saline or their respective antagonist (0.1 or 0.2 mg/kg SCH-23390; 0.01 or 0.03 mg/kg eticlopride HCl), followed by MPH (5 mg/kg). Approximately 10 min after MPH administration, the rats were placed into the paired context for a 10-min trial. One day after conditioning on PD38, a preference test was administered with dividers removed. One day following the preference test on PD39, brain tissue was removed, and the nucleus accumbens and striatum were analyzed for BDNF. Results revealed that MPH conditioning resulted in an increased preference that was blocked by either dose of SCH-23390, but generally not affected by either dose of eticlopride. Further, the higher dose of SCH-23390 resulted in a conditioned place aversion in males, presumably due to an increased number of dopamine D1 receptors in adolescent males. MPH produced a significant increase of striatal and accumbal BDNF alleviated by SCH-23390 or eticlopride. These results show that MPH results in CPP in adolescent male and female rats and these effects appear to be mediated by the dopamine D1 receptor, but the effects of MPH on BDNF appear to be mediated by both dopamine receptor families.

Many pathological processes are associated with excessive neurotransmitter release that leads to the over-stimulation of post-synaptic neurotransmitter receptors. Examples include excessive activation of glutamate receptors in ischemic stroke and hyper-dopaminergic state in schizophrenia and drug addiction. Thus, it would seem that simply antagonizing the involved receptors should be able to correct the pathological condition. In some instances, this strategy has been somewhat effective, such as with the use of dopamine D2 receptor antagonists as antipsychotics in the treatment of positive symptoms of schizophrenia despite severe side effect. However, clinical application of drugs antagonizing glutamate receptor in the treatment of stoke, although attracting intensive research effort, has been restricted by serious side effects caused by suppressing postsynaptic responses that are needed for normal brain function. As a consequence, it is important to develop novel therapeutics aiming at specific targets with minimized side effects. Numerous studies have suggested that the pathophysiology of neuropsychiatric disorders, drug addictions and stroke involves multiple neurotransmitter receptor systems such as the dopamine and glutamate systems. The activation or inhibition of one receptor can have cross-functional effect that will be better understood by investigating the functional and structural relationship between receptor systems. Thus, the present study has focused on characterizing receptor-receptor interactions associated with dopamine receptors and glutamate receptors, and to elucidate the physiological and pathological consequence of altered receptor interactions in schizophrenia, depression and ischemic stroke.