A Doença de Parkinson, uma condição neurodegenerativa e progressiva, está relacionada à morte de neurônios localizados na Substância Negra compacta, um dos componentes dos Núcleos da Base. Quando há a morte de neurônios dopaminérgicos nigrais, esta via modulatória é perdida, levando ao desequilíbrio entre as vias direta e indireta, esta última tendo sua atividade aumentada em detrimento da outra. O estriado tem um papel importante no recebimento e filtração de sinais motores corticais e talâmicos e suas maiores populações neuronais são GABAérgicas, demonstrando a importância do neurotransmissor GABA nesta modulação. O estriado recebe projeções dopaminérgicas vindas da Substância Negra compacta e, na falta desta aferentação, surgem os sintomas e sinais da Doença de Parkinson. Nosso objetivo é caracterizar a liberação de GABA nesta estrutura, avaliando os efeitos de outros transmissores e também o papel de alguns sinalizadores intracelulares neste processo. Para isto, empregamos o método de superfusão e liberação de GABA radiomarcado, previamente carregado, em tecido picado in vitro. A lesão nigral é produzida por cirurgia estereotáxica e microinjeção de 6-OHDA no feixe medial prosencefálico (mfb). Diversas drogas foram utilizadas para avaliarmos diferentes passos na liberação do transmissor. Concluímos que a liberação é fortemente dependente de cálcio e segue o modelo de exocitose vesicular, além de a subpopulação neuronal GABAérgica estrital estudada sofrer pouca influência de aferências glutamatérgicas e colinérgicas. No entanto, drogas dopaminérgicas regulam complexamente a liberação de GABA no estriado e ela também é bastante dependente de calmodulina. Conjecturamos se algumas drogas antipsicóticas que agem sobre calmodulina devem seu efeito terapêutico, ou parte dele, a esta ação e se, no modelo de DP de lesão unilateral por 6-OHDA, há comunicação entre os hemisférios lesado e não lesado após o estabelecimento da lesão e processo de rearranjo neuronal / Parkinsons disease, a progressive and neurodegenerative condition, is related to the death of neurons located in Substantia Nigra compacta, a component of Basal Ganglia. When nigral dopaminergic neurons die, this modulatory pathway is lost leading to imbalance between direct and indirect pathways, the latter having its activity increased over the former. Striatum has an essential role in receiving and filtering motor signals from cortex and thalamus and its major neuronal populations are composed by GABAergic neurons, showing how important is GABA in this modulation. Striatum receives dopaminergic projections from Substantia Nigra compacta and in its absence the typical signals and symptoms of the disease arise. We aimed to characterize GABA relase at this structure, assessing the effect of other transmitters as well the role of some intracellular signaling molecules in this process. For that, we employed the superfusion method and release of preloaded radiolabeled GABA from chopped striatal tissue. Nigral injury was produced by stereotaxic surgery and 6-OHDA microinjection at medial forebrain bundle (mfb). Several drugs were used to evaluate different steps in transmitter release. We concluded that the release is strongly calcium-dependent and follows vesicular exocytosis model; in addition the striatal GABAergic subpopulation of neurons studied here undergo little influence of glutamatergic and cholinergic afferents. However, dopaminergic drugs complexly regulate striatal GABA release and it also shows high involvement of calmodulin. We wonder if some antipsychotic drugs that act over calmodulin owe their therapeutical effects, or at least part of it, to this activity and if in 6-OHDA unilateral lesion parkinsonism model there is communication between injuried and healthy hemispheres after the establishment of the injury and neuronal rearrangement process

Quiescent neural stem cells support continuous, lifelong neurogenesis in specific regions of the adult mammalian brain. The largest adult neurogenic region is the ventricular-subventricular zone (V-SVZ), which lines the entire lateral wall of the lateral ventricles. Quiescent neural stem cells (qNSCs) enter the cell cycle (activate) and give rise to new neurons during homeostasis and regeneration, suggesting they can potentially be harnessed for regenerating the brain after neurodegenerative disease, stroke, and injury. Defining the signals that regulate NSC quiescence and activation is essential to unlock their potential for regenerative medicine. NSCs residing in specific regions of the V-SVZ give rise to distinct subtypes of olfactory bulb interneurons. It is unknown whether quiescence-regulating signals map onto the regional heterogeneity of NSCs, and might thereby underlie the production of distinct interneuron subtypes. A major limitation to our understanding of the regulation of NSC quiescence has been the lack of specific markers to identify qNSCs, and prospectively purify them from their in vivo niche. Using a novel fluorescence-activated cell sorting (FACS) strategy that allows the purification of qNSCs from the adult mouse V-SVZ niche for the first time, I performed in vitro screens for quiescence-regulating signals. Unexpectedly, neurotransmitters emerged as the main class of qNSC-activating signals, including dopamine, GABA, serotonin, acetylcholine, and opioids. Local and long-range neurons that use these neurotransmitters innervate the V-SVZ in unique regional patterns, suggesting these signals map onto the regional heterogeneity of NSCs. Consistent with this hypothesis, infusions of cholinergic agonist and antagonists into the lateral ventricle resulted in regional changes in NSC proliferation. Moreover, cholinergic antagonists blocked the activation of qNSCs during regeneration, providing evidence that neurotransmitter signaling activates qNSCs in vivo. I then showed that hypothalamic Pomc-expressing neurons innervate the anterior-ventral V-SVZ and promote the activation of Nkx2.1+ qNSCs. Ablation of Pomc+ neurons resulted in decreased proliferation of NSCs in the anterior-ventral, but not anterior-dorsal, V-SVZ. Moreover, both the activity of Pomc+ neurons, and the proliferation of Nkx2.1+ NSCs in the anterior-ventral V-SVZ decreased in fasted animals, suggesting that hunger and satiety states regulation the generation of a single olfactory bulb interneuron subtype. Indeed, ablation of Pomc+ neurons resulted in a loss of the subtype of olfactory bulb interneuron that is generated by Nkx2.1+ NSCs. Together, my findings suggest that both local and long-range neurons regionally innervate the V-SVZ and mediate neural stem cell activation from the quiescent state.

P/Q-type voltage-gated calcium channels (CaV2.1) are expressed in both central and peripheral nervous systems, where they play a critical role in neurotransmitter release. Mutations in the pore-forming α1 subunit of CaV2.1 can cause neurological disorders such as episodic ataxia type 2, familial hemiplegic migraine type 1 and spinocerebellar ataxia type 6. Interestingly, a 190-kDa fragment of CaV2.1 was found in mouse brain tissue and cultured mouse cortical neurons, but not in heterologous systems expressing full-length CaV2.1. In the brain, the 190-kDa species is the predominant form of CaV2.1, while in cultured cortical neurons the amount of the 190-kDa species is comparable to that of the full-length channel. The 190-kDa fragment contains part of the II-III loop, repeat III, repeat IV and the C-terminal tail. A putative complementary fragment of 80-90 kDa was found along with the 190-kDa form. Moreover, preliminary data show that the abundance of the 190-kDa species and the 80-90-kDa species relative to the full-length channel is upregulated by increased intracellular Ca²⁺ concentration. Truncation mutations in the P/Q-type calcium channel have been found to cause the neurological disease episodic ataxia type 2. Some of the disease-causing truncations resemble the 190-kDa truncated channel that we found. Three pairs of truncated versions of CaV2.1 were engineered to resemble putative products of proteolytic cleavage in the three intracellular loops. Electrophysiological properties of these truncated channels were studied. The truncated channel corresponding to the N-terminal fragment produced by cleavage in the II-III loop has a suppressive effect on full-length P/Q channel currents, resembling the effects of several truncation mutants that cause episodic ataxia type 2. The complementary pair of truncated channels created by a truncation site in the I-II loop forms a functional channel when coexpressed. These results shed light on the functional effects of proteolytic cleavage in the intracellular loops of the P/Q channel.

One strategy to repair an injured or degenerated retina is to stimulate the replacement of damaged or dead neurons with cells derived from endogenous stem- or progenitor cells. A successful strategy requires knowledge about how the proliferation and differentiation of the endogenous cells are regulated. In particular, this knowledge will be important in the establishment of protocols that produce sufficient numbers of specific neurons. The main aim of this thesis was to find and characterise factors regulating the proliferation and differentiation of retinal progenitor cells (RPCs) and hence, contribute to the knowledge of how to use progenitor cells for retinal repair.    The major result in this thesis is that GABA contributes to and maintains RPC proliferation. Inhibition of GABAA receptors decreases the proliferation of non-pigmented ciliary epithelial (NPE) cells and RPCs in the intact retina. We propose that this effect is mediated through changes in the membrane potential and voltage-gated calcium channels, which in turn regulate components of the cell cycle. Furthermore, we show that one of the endogenous RPC sources, the Müller cells, consists of two subpopulations based on Pax2 expression. This is interesting because Pax2 may suppress the neurogenic potential, characterised by de-differentiation and proliferation, in Müller cells. Finally, we show that over-expression of FoxN4 induces differentiation-associated transcription factors in the developing chick retina. However, FoxN4 over-expression did not trigger differentiation of NPE cells. These results indicate that the intrinsic properties of the RPCs are determinant for FoxN4-induced differentiation. The results presented in this thesis advance our understanding of how specific cells may be generated from different sources of RPCs. Our results show that the different sources are highly diverse in their potential to proliferate and produce neurons. GABA, Pax2 and FoxN4 may be factors to consider when designing strategies for retinal repair. However, the results indicate that the specific responses to these factors are highly associated with the specific properties of the progenitor cells. / <p>Doctor of Philosophy <strong>(Faculty of Medicine)</strong></p>

The gaseous messenger, nitric oxide (NO), has emerged as a key component of olfactory systems. Localization and imaging studies in the moth, Manduca sexta, suggest that NO may affect the excitability of olfactory neurons by modifying neuronal membrane properties through sGC-dependent mechanisms. This hypothesis was tested using a multidisciplinary approach, including two types of physiological recording techniques and immunocytochemical analysis of sGC antibody expression in the Manduca brain. The excitability of large populations or individual antennal lobe (AL) neurons was monitored with in vivo physiological recordings while various NO pharmacological agents were bath applied to the brain. To examine possible targets of NO, the binding site of sGC was blocked and the results were compared to NO blockade. Finally, sGC immunocytochemistry was used to also determine possible targets of NO.Two NO synthesis inhibitors and a sGC blocker were potent effectors of resting, baseline activity in the Manduca brain. Blocking NO synthesis caused significant decreases in AL neuron conductance. This conductance decrease led to changes in baseline activity, including the appearance of bursts in some neurons, and increased and decreased firing rates in other neurons. Further, the neurons had a decreased responsiveness and excitability to presynaptic input. Blocking the sGC binding site caused similar effects in most neurons, which indicates that NO likely acts through sGC-dependent signaling to exert its effects in at least a subset of neurons. However, some neurons had different responses to NO and sGC blockade, which indicates that NO may act through other signaling mechanisms in some neurons. Further examination using sGC immunocytochemistry revealed that only about 90% of projection neurons (PNs) and 30% of local neurons (LNs) contained sGC immunoreactivity.The results in this dissertation indicate that NO performs a global function in the antennal lobe to maintain the resting membrane conductance of AL neurons. NO likely exerts its effects through both sGC-dependent and sGC-independent mechanisms. Finally, these results have major implications for odor coding in all species, as NO has been found in the olfactory systems of every animal examined thus far.

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.

No description available.

Los pesticidas organoclorados (POCs) son compuestos altamente persistentes en el medio ambiente y dada su capacidad de acumularse en los tejidos grasos animales se biomagnifican a lo largo de la cadena trófica. Estos contaminantes ambientales son antagonistas del receptor GABAA, lo que les confiere sus propiedades convulsivas tras una intoxicación aguda. En este estudio hemos planteado la hipótesis de que la exposición prolongada al pesticida dieldrín en cultivos primarios de neuronas corticales podría alterar la funcionalidad, expresión o localización de los receptores de glutamato como consecuencia del bloqueo mantenido del receptor GABAA. La exposición durante 6 días in vitro a una concentración no citotóxica (60nM) de dieldrín produjo la internalización de los receptores NMDA que contienen la subunidad NR2B (NR1/NR2B), pero no NR2A, y disminuyó también los niveles del receptor metabotrópico de glutamato 5 (mGluR5). El tratamiento con los neuroesteroides estradiol o alopregnanolona previno la reducción de los receptores NR1/NR2B en la membrana inducida por el dieldrín. Además, la exposición prolongada a 200nM dieldrin disminuyó la expresión génica de NR2A, efecto que fue revertido sólo por alopregnanolona. En neuronas corticales maduras, el dieldrín causó el truncamiento de NR2B mediado por las calpaínas y redujo la interacción de ésta subunidad con SAP102, efecto de nuevo prevenido por alopregnanolona. A pesar de que ambas hormonas restauraron la funcionalidad del receptor NMDA, alopregnanolona (pero no estradiol) revirtió tanto la inhibición del receptor GABAA como la muerte neuronal causadas por el dieldrín. Posteriormente, estudiamos los efectos de dieldrín, endosulfán y lindano sobre los receptores de estrógenos (ER) en cultivos primarios de neuronas corticales y de células granulares de cerebelo (CGC). Todos ellos inhibieron la unión de [3H]-estradiol en ambos tipos neuronales, siendo el dieldrín en CGC aquel con mayor afinidad sobre el ER. Además, dieldrín y endosulfán causaron un aumento de la fosforilación de Akt en neuronas corticales, efecto que fue inhibido por el antagonista de ERβ, PHTPP. En cambio, la fosforilación de Akt y ERK1/2 inducida por dieldrín en CGC fue mediada por la activación múltiple de ERα, ERβ, y GPR30. El lindano inhibió el efecto del estradiol sobre estas kinasas. En neuronas corticales, los tres pesticidas activaron ERK1/2 a través de los receptores GABAA y de glutamato. La exposición a largo plazo a los POCs redujo los niveles de ERα pero no de ERβ. Finalmente, comparamos la neurotoxicidad de los POCs en CGC y neuronas corticales. El dieldrín y el endosulfán fueron más tóxicos que el lindano en ambos cultivos. Se observó también que las CGC maduras son más sensibles que las neuronas corticales a la neurotoxicidad inducida por los POCs. Por el contrario, las neuronas corticales inmaduras, especialmente las glutamatérgicas, fueron más susceptibles a la exposición prolongada a concentraciones bajas de estos pesticidas. Además, la fosforilación de Akt y ERK1/2 se vio disminuida tras 24 horas de exposición a dieldrín o endosulfan en CGC. El tratamiento con estradiol e insulina previno la muerte apoptótica mediada por caspasa-3 causada por estos pesticidas a través de la activación conjunta de ERK1/2 y Akt. Este trabajo confirma que el bloqueo permanente del receptor GABAA por dieldrín provoca una reducción de la neurotransmisión glutamatérgica, lo que podría afectar a funciones cerebrales como la memoria y el aprendizaje. Además, los POCs son capaces de mimetizar o inhibir algunas de las acciones no genómicas del estradiol, lo que puede interferir con la señalización fisiológica de esta hormona en el cerebro. Este estudio revela también la susceptibilidad específica de las neuronas glutamatérgicas frente a la exposición prolongada a estos contaminantes y desvela los mecanismos moleculares por los cuales los POCs producen la muerte de estas neuronas.

Signal processing in the visual system is mediated by classic neurotransmission and neuropeptidergic modulatory pathways. In Dipteran insects, especially in the fruitfly Drosophila melanogaster, the morphology of the visual system is very well described. However neurotransmitter and neuropeptidergic circuits within the optic lobe neuropil are only partially known. Using several transgenic fly lines and antibodies we determined the localization of the classical neurotransmitters GABA, acetylcholine and glutamate in the visual system, and their putative targets via detecting several neurotransmitter receptors. We paid particular attention to the peripheral neuropil layer called the lamina, where the light signals are filtered, channeled and amplified (Paper I). We discovered four new types of efferent tangential neurons branching distally to the lamina. Among them was the first neuropeptidergic neuron (LMIo) in this region of Drosophila. The LMIo expresses myoinhibitory peptide (MIP) and has its cell body located close to the main lateral clock neurons that express the neuropeptide pigment-dispersing factor (PDF)(Paper II). Since in other Dipteran species PDF is expressed in processes distally to the lamina, we performed comparative anatomical studies of the MIP, PDF, Ion Transport Peptide (ITP) and serotonin (5-HT) distribution in the visual system of the flies Drosophila and Calliphora. Our data suggest that PDF signaling distal to the lamina of the blowfly might be replaced by MIP signaling in the fruitfly, while ITP and 5-HT expression is conserved in the two species (Paper III). Serotonin is crucial in light adaptation during the daily light-dark cycles. We analyzed putative serotonergic circuits in the lamina. We found that LMIo neurons express the inhibitory receptor 5-HT1A, while 5-HT1B and 5-HT2 are both expressed in the epithelial glia of the lamina. Another novel wide-field neuron with lamina branches expresses the excitatory serotonin receptor 5-HT7. Our studies have identified a fairly complex neuronal circuitry in the tangential plexus above the lamina. (Paper IV). Finally we tested circadian locomotor activity rhythms in flies with the GABAB receptor knocked down on the lateral PDF-expressing clock neurons. We observed significant changes in the activity periods and diminished strength of rhythmicity during DD suggesting a modulatory role of GABA in clock function (Paper V). / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.