Dopamine (DA) is a key striatal neuromodulator central to normal functioning of the basal ganglia. Identifying and characterizing circuits governing striatal DA transmission is necessary for understanding DA involvement in adaptive behaviour and pathology. Properties of evoked striatal DA release can be examined using fast-scan cyclic voltammetry at carbon fibre microelectrodes, a technique enabling live monitoring of transmitter release events with sub-millisecond resolution. Experimental work presented in this thesis employed this approach to study regulation of striatal DA by acetylcholine (ACh) in health and disease in acute brain slices. Synchronous activity in a small population of striatal cholinergic interneurons (ChIs) was previously shown to directly drive striatal DA release. Here using optogenetic approach I explore physiological relevance of ChI-evoked drive of striatal DA by examining whether corticostriatal and thalamostriatal afferents to ChIs can trigger ACh-evoked DA events. Following floxed vector injections in motor cortex or caudal intralaminar thalamus of CaMK2a-Cre mice I examine the properties of evoked DA upon light activation of channelrhodopsin-2-transduced inputs to striatal ChIs. These experiments revealed that both cortical and thalamic afferents are capable of driving ACh-evoked DA release, but operate using a different complement of post-synaptic ionotropic glutamate receptors and display distinct release recovery profiles. I further explore if rebound excitation in a population of striatal ChIs could drive DA events by examining whether ACh-evoked DA release follows optical inhibition of striatal ChIs selectively expressing hyperpolarizing halorhodopsin 3.0 or archaerhodopsin 3.0 in ChAT-Cre mice. This work showed that hyperpolarizing ion pumps were not successful in triggering ChI-evoked DA release. I also investigate whether cholinergic brainstem innervation of striatum could contribute to or drive ACh-evoked striatal DA events in ChAT-Cre rat, concurrently showing that ChI-evoked DA release is not a species artefact, and is present in mouse and rat alike. Current results also suggest that cholinergic brainstem afferents do not drive or contribute to striatal ACh-evoked DA events. Close interaction between DA and ACh systems further indicates that ACh could impact dopaminergic dysfunction. To explore this I examined the state of ACh transmission in a mouse model of Parkinson’s disease overexpressing human wild type alpha–synuclein protein. These animals present with impaired striatal DA release from young age, but DA deficits could be mediated by changes in ACh tone. Here I show that impaired striatal DA release is the results of primary DA axon dysfunction, although in ventral striatum DA release deficits could be partially compensated by increased ACh tone at nicotinic receptors. I further show that the functional state of muscarinic ACh receptors in not altered following decreased DA transmission, although the data from aged animals suggest that alpha–synuclein-dependent changes in vesicle handling could contribute to impaired DA releasability. Finally, I show that vesicle handling may indeed be altered in this mouse model as impaired DA release is evident with short stimulation protocols, while with prolonged depolarization of DA axon terminals alpha–synuclein-overexpressor mice are better able to sustain evoked DA release. Overall, the main body of work presented in this thesis examined the processes regulating striatal DA transmission via ACh system. In particular, I show that ChI-evoked drive of striatal DA release can be recruited physiologically and further establish that changes in ACh transmission are not the primary drivers of impaired DA releasability in a mouse model of Parkinson’s disease overexpressing human alpha–synuclein protein.

Dopamine (DA) is a key neuromodulator in the striatum, and is important for action selection and reinforcement learning. Dysfunctions in striatal DA signalling contribute to numerous disorders including Parkinson’s disease (PD) and drug addiction. Midbrain DA neurons switch from low to high frequency firing in response to reward-related events, which is proposed to increase striatal DA release. However, in addition to DA neuron firing pattern, striatal DA signalling depends upon the short-term plasticity of DA release, which is controlled by presynaptic and local network factors. This thesis uses fast-scan cyclic voltammetry, in murine striatal slices, to detect subsecond changes in extracellular DA, and investigate the presynaptic control of striatal DA release and release plasticity. Acetylcholine from striatal cholinergic interneurons, acting at nicotinic receptors (nAChRs) on DA terminals, is one factor that strongly influences DA release. This thesis particularly explores how presynaptic factors interact with nAChRs to control DA release. Firstly, release probability, a key determinant of release plasticity at many synapses, was found to be only weakly related to DA release plasticity, and only when nAChRs are inactive. Secondly, a direct role of the DA uptake transporter (DAT) in controlling DA release plasticity was identified, when nAChRs are inactive. Thirdly, regional differences were identified in the role of the DAT in controlling DA release via control of D2 receptor activation, when nAChRs are active. Finally, mutant α-synuclein, which causes PD in humans, was found to only subtly affect striatal DA release. These data suggest that the control of striatal DA release differs substantially from other central transmitters. Release probability and α-synuclein play only minor roles, but nAChRs and the DAT significantly control DA release plasticity. These findings review our understanding of striatal DA release and may have implications for understanding the actions of drugs of abuse and early PD pathogenesis.

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Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-28T14:50:20Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) ATIVIDADE ANTICOLINESTERÁSICA DE PLANTAS DA CAATINGA COM INDICAÇÃO POPULAR PARA DISTÚRBIOS DO SIS.pdf: 10498637 bytes, checksum: 6697d682e5713ad3939431b53b38ec0b (MD5) / Made available in DSpace on 2017-07-28T14:50:20Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) ATIVIDADE ANTICOLINESTERÁSICA DE PLANTAS DA CAATINGA COM INDICAÇÃO POPULAR PARA DISTÚRBIOS DO SIS.pdf: 10498637 bytes, checksum: 6697d682e5713ad3939431b53b38ec0b (MD5) Previous issue date: 2016-03-18 / FACEPE / No Brasil e no mundo há um crescimento na população de idosos e conforme as pesquisas demográficas recentes, esse aumento se explica como consequência de uma redução da fertilidade e mortalidade observada ao longo do século XX. A idade pode ser considerada como um dos fatores de risco melhor aceito para o desenvolvimento de diversas doenças, destacando-se entre elas a Doença de Alzheimer (DA). No entanto até hoje, não existe uma causa única que pode levar ao desenvolvimento da DA, mas sim um conjunto de fatores, a exemplo do estresse, danos físicos (traumas ou pancadas), déficit no metabolismo e vascularização cerebral, que associados a hipótese colinérgica, poderiam explicar melhor o surgimento desta doença. Atualmente, a terapêutica disponível para a DA baseia-se principalmente nesta hipótese, não sendo possível a cura, mas apenas o retardo da evolução da doença bem como o alívio nos sintomas que a mesma apresenta, que de certa forma contribui com a qualidade de vida dos pacientes. No entanto, estão em fase não clínica de testes alguns candidatos que poderiam atuar sobre a formação das placas amilóides e diminuição dos agregados protéicos insolúveis, o que poderia evitar a evolução da doença para etapas mais severas. Na perspectiva de busca de novos fármacos, os produtos naturais apresentam relevância e as endemias de algumas espécies vegetais as deixam com composição química características daquele local específico. A Caatinga, por exemplo, é um bioma dominado por um dos tipos de vegetação cuja distribuição é totalmente restrita ao Brasil. Diante disto, o presente trabalho teve como objetivo buscar novas substâncias com potencial atividade anticolinesterásica e que possam servir como alternativa para o tratamento da DA, levando em consideração a hipótese colinérgica como uma das principais causas da doença. Partindo de uma pesquisa etnofarmacológica com indicação de plantas medicinais utilizadas para patologias que pudessem ter relação ao sistema nervoso, foram coletadas 23 espécies, sendo duas partes diferentes de duas espécies particularmente, e obtidos extratos com três polaridades para cada uma, gerando 75 amostras para estudo. Apenas 26,7% apresentaram resultado positivo no pre-teste qualitativo de inibição da enzima AChE. Quando realizado o teste falso positivo, este número diminuiu para 30% do resultado positivo, sendo representado por 8% do total de amostras analisadas. Das 30 amostras analisadas quantitativamente, apenas 13,3% apresentaram fraca atividade inibitória da AChE, 43,3% apresentaram moderada atividade e 43,3%, apresentaram potente atividade, sempre levando-se em consideração a menor concentração estudada (0,5 mg/mL). Destacaram-se com melhores resultados Citrus limonum, Ricinus communis e Senna occidentalis, com porcentagem de inibição da atividade anticolinesterásica variando de 50% até 65%, na menor concentração. Estas espécies tiverem seus extratos de média e baixa polaridade submetidas a Cromatografia Gasosa-Espectrometria de Massa. Foi possível perceber a presença de classes de compostos em comum entre as amostras, como os derivados de terpenoides e cumarinas, que em alguns estudos na literatura científica são citados com a atividade anticolinesterásica. Com maior frequência entre os melhores resultados, C. limonum foi escolhida para os testes de isolamento e doseamento de cumarinas, para o qual foram encontrados teores de 688,57 ±3,94 mg/g de extrato para fração de diclorometano. Ao ser particionada, esta amostra apresentou resultados de porcentagem de inibição anticolinesterásica entre 74,6% a 94,4%, sendo possível isolar o composto caracterizado como 2H-1-Benzopiran-2-ona-5,7-dimetoxi. As frações da amostra de metanol apresentaram inibição da AChE entre 92,3% e 100%. Os resultados obtidos para C. limonum mostram o potencial desta espécie para o tratamento da DA. / In Brazil, as well as in the world, there is an increase in the elderly population, and according to recent demographic research this increase is explained as a consequence of decreased fertility and mortality observed throughout the twentieth century. Age can be considered as one of the most accepted risk factors for the development of several diseases, for example Alzheimer's disease (AD). However, until today, a single cause cannot lead to the development of AD, but a set of factors, such as stress, physical injury (trauma or strokes), deficits in metabolism and brain vasculature that associated with cholinergic hypothesis could better explain the emergence of this disease. Currently, the treatment available for AD is mainly based on this assumption, it is not possible to cure, but the disease progression can be delayed, relieving the symptoms presented which in a certain way contributes to the patients‟ quality of life. However, in preclinical testing there are some candidates that could act on the formation of amyloid plaques and reduction of insoluble protein aggregates, which could prevent the disease progression to more severe stages. In the search for new drugs, natural products have relevance, and the endemic of some species of plants let them with anatomy and chemistry characteristics of that specific location. The Caatinga, for example, is a biome dominated by one type of vegetation which distribution is completely restricted to Brazil. Therefore, this study was aimed at seeking new substances with potential acetylcholinesterase activity, which could serve as an alternative for the treatment of AD, taking into consideration the cholinergic hypothesis as one of the main causes of the disease, starting from an ethnopharmacological research with medicinal plants used for diseases that could be related to the nervous system. Twenty-three species were collected, and it was obtained extracts with three polarities for each one, generating 75 samples to study. Only 26.7% were positive in the qualitative test of AChE enzyme. When the false positive test is performed this number decreases to 30% of the positive results, being represented by 8% of the total samples. From the 30 samples analyzed quantitatively, only 13.3% had weak inhibitory activity of AChE, 43.3% had moderate activity and 43.3% showed potent activity, always taking into account the lowest concentration studied (0.5 mg / mL). Those with the best results were Citrus limonum, Ricinus communis and Senna occidentalis, with percentage of inhibition of acetylcholinesterase activity varying from 50% to 65%, at lowest concentration. These species had their average and low polarity extracts subjected to Gas Chromatography-Mass Spectrometry. It was possible to perceive the presence of classes of compounds in common between the species, such as terpenes and coumarin, which in some studies in the scientific literature are cited with the activity in question. With a higher frequency among the best results, C. limonum was chosen for the isolation and dosing tests of Coumarins, in which were found levels of 688.57 ± 3.94 mg / g for fraction of extract of dichloromethane. By being partitioned, this sample showed results of acetylcholinesterase inhibition percentage of 74.6% to 94.4%, being possible to isolate the compound described as 2H-1-benzopyran-2one-5,7-dimethoxy. The fractions of methanol sample showed inhibiting between 92.3% and 100%. The results for C. limonum showed the potential of this species for the treatment of AD.

No description available.

Proper synaptic connectivity is critical for communication between cells and information processing in the brain. Neurons are highly interconnected, forming synapses with multiple partners, and these connections are often refined during the course of development. While decades of research have elucidated many molecular players that regulate these processes, understanding their specific roles can be difficult due to the large number of synapses and complex circuitry in the brain. In this thesis, I investigate mechanisms that establish neural circuits in the simple organism C. elegans, allowing us to address this important problem with single cell resolution in vivo. First, I investigate remodeling of excitatory synapses during development. I show that the immunoglobulin domain protein OIG-1 alters the timing of remodeling, demonstrating that OIG-1 stabilizes synapses in early development but is less critical for the formation of mature synapses. Second, I explore how presynaptic excitatory neurons instruct inhibitory synaptic connectivity. My work shows that disruption of cholinergic neurons alters the pattern of connectivity in partnering GABAergic neurons, and defines a time window during development in which cholinergic signaling appears critical. Lastly, I define novel postsynaptic specializations in GABAergic neurons that bear striking similarity to dendritic spines, and show that presynaptic nrx-1/neurexin is required for the development of spiny synapses. In contrast, cholinergic connectivity with their other postsynaptic partners, muscle cells, does not require nrx-1/neurexin. Thus, distinct molecular signals govern connectivity with these two cell types. Altogether, my findings identify fundamental principles governing synapse development in both the developing and mature nervous system.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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The high rate of relapse often seen in breast cancer patients has been suggested to be the result of a small subset of chemotherapy-resistant cancer stem cells (CSCs), believed to be responsible for initiating tumor formation. These CSCs possess the capability to self-renew and give rise to a hierarchy of cells which makes up the bulk of a tumor. Neurotransmitters have been suggested to influence CSC self-renewal and proliferation capabilities, and antagonists of neurotransmission pathways have been implicated as possible treatment methods for chemo-resistant tumors. Using nicotinic acetylcholine receptor (nAChR) antagonists in sphere-forming assays, we have identified a very promising candidate compound: MG624. We found this compound to have a high selectivity for sphere-forming cells over non-sphere-forming cells in vitro, in a dose-dependent relationship, across a panel of cell lines as well as in patient-derived xenograft cells. This was validated in two ex vivo assays, where tumor formation was significantly delayed in mice injected with MG624-treated HCC1954 cells at both the IC50 and IC90 of the compound, indicating that MG624 does indeed target functional BTICs. MG624 was also found to synergize with both taxotere and doxorubicin chemotherapies in vitro, and shrink tumors in NOD/SCID mice when combined with taxotere in vivo. MG624 in combination with taxotere was found to induce apoptosis, and prevent cells from entering into the M-phase of the cell cycle. Interestingly, MG624 was found to eliminate intratumoral fibroblasts in combination with taxotere, despite taxotere being found to recruit fibroblasts to the tumor site when used on its own. Most importantly, the combination of MG624 and taxotere was found to significantly delay tumor progression/relapse in mice, indicating that MG624 may be an excellent candidate compound to one day be combined with chemotherapy to provide durable remission to breast cancer patients. / Thesis / Master of Science (MSc)

Agonist-stimulated phosphoinositide (PPI) hydrolysis is a major signal transduction pathway in brain. These studies investigated neurotransmitter stimulated PPI hydrolysis in postmortem human brain. Preliminary studies using rat brain suggested that moderate postmortem delay has little effect on PPI hydrolysis and that human tissue might be reliably studied for differences in receptor-PLC coupling. Studies in human brain membranes (frontal cortex) indicated that the time course for GTPγS and carbachol/GTPγS-stimulated PPI hydrolysis was linear for at least 20 min. GTPγS-stimulated [3H] inositol phosphate (InsP) formation was enhanced by carbachol (232%) and 5-Hydroxytryptamine (5HT - 147%). SAX-HPLC seperation of [3H] inositol polyphosphates indicated that the major isomer of inositol trisphosphate (InsP3) was Ins(1,4,5)P3, the expected product of PtdIns(4,5)P2 hydrolysis. Ca2+ increased PPI hydrolysis progressively from 100 nM through 50 μM and synergistically enhanced carbachol/GTPγS stimulation. Comparisons of age-matched controls with Alzheimer's patients indicated that GTPγS, carbachol/GTPγS, and 5HT/GTPγS-stimulation of PPI hydrolysis is reduced approximately 50% in membranes prepared from Alzheimer's patients. Ca2+ stimulation of PPI hydrolysis was not different between controls and Alzheimer's patients suggesting that muscarinic cholinergic and serotonergic receptors are uncoupled from PLC in Alzheimer's disease. These studies indicate that there are changes in cholinergic and serotonergic signal transduction in Alzheimer's disease. Further, this method can be used to study signal transduction events in postmortem human brain.