The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated glutamatergic ion channels that mediate most excitatory neurotransmission in the brain. Alterations in AMPAR synaptic accumulation mediate synaptic plasticity, including long-term potentiation, long-term depression and homeostatic synaptic plasticity. AMPAR abundance in neurons is determined by balanced processes of protein translation and degradation. Changes in AMPAR function and trafficking have direct impacts on synaptic transmission and cognitive functions. However, the molecular mechanisms regulating AMPAR expression and dynamics in neurons remain largely unknown. In this thesis, two molecular mechanisms that regulate AMPAR translation and protein stability through two different signaling pathways, 5' adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 2 (SIRT2), are described. It is shown that SIRT2, a NAD+-dependent protein deacetylase, directly controls AMPAR stability by regulating AMPAR acetylation. For the first time, we discovered that AMPARs are subject to lysine acetylation, a novel form of post-translational modification for glutamate receptors. Under basal conditions, AMPARs are highly acetylated at their intracellular C termini, which protects against ubiquitination to antagonize AMPAR endocytosis and degradation, leading to prolonged receptor half-life. SIRT2 is also identified as the enzyme responsible for AMPAR deacetylation. Knockdown of SIRT2 led to elevated AMPAR acetylation and reduced ubiquitination, and consequently, increased AMPAR levels and synaptic transmission. SIRT2 knockout mice displayed weakened synaptic plasticity and impaired learning and memory. Resveratrol is a phytoalexin that has been shown to increase AMPAR expression and synaptic accumulation in neurons. The resveratrol effect on AMPAR expression is independent of sirtuin 1, the conventional target of resveratrol, but rather is mediated by AMPK and its downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Application of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), to neurons mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis through the AMPK-PI3K pathway activation. These studies describe novel regulatory mechanisms responsible for the control of AMPAR protein amount and subcellular distribution in neurons, providing insights into our understanding of synaptic plasticity, brain function and neurological disorders. / 2017-12-06T00:00:00Z

The evolution of synapses from early proto-synaptic protein complexes in unicellular eukaryotes to sophisticated machines comprising thousands of proteins parallels the emergence of finely tuned synaptic plasticity, a molecular correlate for memory and learning. Phenotypic change in organisms is ultimately the result of evolution of their genotype at the molecular level. Selection pressure is a measure of how changes in genome sequence that arise though naturally occurring processes in populations are fixed or eliminated in subsequent generations. Inferring phylogenetic information about proteins such as the variation of selection pressure across coding sequences can provide valuable information not only about the origin of proteins, but also the contribution of specific sites within proteins to their current roles within an organism. Recent evolutionary studies of synaptic proteins have generated attractive hypotheses about the emergence of finely-tuned regulatory mechanisms in the post-synaptic proteome related to learning, however, these analyses are relatively superficial. In this thesis, I establish a scalable molecular phylogenetic modelling framework based on three new inference methodologies to investigate temporal and spatial aspects of selection pressure changes for the whole human proteome using protein orthologs from up to 68 taxa. Temporal modelling of evolutionary selection pressure reveals informative features and patterns for the entire human proteome and identifies groups of proteins that share distinct diversification timelines. Multi-ontology enrichment analysis of these gene cohorts was used to aid biological interpretation, but these approaches are statistically under powered and do not capture a clear picture of the emergence of synaptic plasticity. Subsequent pathway-centric analysis of key synaptic pathways extends the interpretation of temporal data and allows for revision of previous hypotheses about the evolution of complex synaptic function. I proceed to integrate inferred selection pressure timeline information in the context of static protein-protein interaction data. A network analysis of the full human proteome reveals systematic patterns linking the temporal profile of proteins’ evolution and their topological role in the interaction graph. These graphs were used to test a mechanistic hypothesis that proposed a propagating diversification signal between interactors using the temporal modelling data and network analysis tools. Finally, I analyse the data of amino-acid level spatial modelling of selection pressure events in Arc, one of the master regulators of synaptic plasticity, and its interactors for which detailed experimental data is available. I use the Arc interactome as an example to discuss episodic and localised diversifying selection pressure events in tightly coupled complexes of protein and showcase potential for a similar systematic analysis of larger complexes of proteins using a pathway-centric approach. Through my work I revised our understanding of temporal evolutionary patterns that shaped contemporary synaptic function through profiling of emergence and refinement of proteins in multiple pathways of the nervous system. I also uncovered systematic effects linking dependencies between proteins with their active diversification, and hypothesised about their extension to domain level selection pressure events.

The nervous system requires rapid, efficient, and accurate transmission between cells for proper functioning. Synapses are the predominant structures through which such vital communication occurs. How synapses are formed, maintained, and eliminated are questions of fundamental importance. At the nerve-muscle synapse, formation of the postsynaptic apparatus is directed by agrin. The hallmark activity of agrin is the aggregation of acetylcholine receptors (AChRs) into dense clusters opposite the presynaptic nerve terminal. Early events in the agrin signal transduction cascade include activation of the receptor tyrosine kinase MuSK and tyrosine phosphorylation of AChRs, but how these events lead to AChR cluster formation is unknown. Using the calcium buffer BAPTA, we demonstrate that intracellular calcium fluxes are necessary for agrin-induced formation of AChR clusters. However, clamping calcium fluxes before agrin stimulation does not alter agrin-induced phosphorylation of either MuSK or AChRs, indicating that this calcium-dependent step occurs downstream of both MuSK and AChR phosphorylation. These results identify a new step in the agrin signaling pathway required for the formation of AChR clusters. We show that intracellular calcium fluxes also play an important role in stabilizing AChR clusters. Clamping intracellular calcium fluxes results in rapid dispersal of AChR clusters and dephosphorylation of both MuSK and AChRs, even if agrin is continually present. Furthermore, the protein tyrosine phosphatase inhibitor pervanadate inhibits both the dispersal and dephosphorylation, indicating a role for a tyrosine phosphatase in AChR cluster dispersal. Our data indicate that AChR clusters are maintained by agrin/MuSK-induced intracellular calcium fluxes that tonically inhibit a tyrosine phosphatase localized to AChR clusters. Our findings also show that distinct molecular mechanisms mediate the formation and the dispersal of agrin-induced AChR clusters. The work presented here expands our understanding of synaptic differentiation in several ways. First, I characterized a new, calcium-dependent step required for the formation of agrin-induced AChR clusters. Next, I showed that postsynaptic specializations must be actively maintained, and describe a molecular mechanism that stabilizes AChR clusters. Finally, dispersal and formation of AChR clusters occurs by distinct pathways. Our understanding of the mechanisms regulating the formation and modulation of synapses will help us to better understand how the nervous system develops and responds to the world around us.

Neste trabalho foi investigado o efeito da administração crônica de prolina sobre o conteúdo total de gangliosídios, fosfolipídios e de colesterol, assim como, sobre o perfil de gangliosídios no córtex, no hipocampo, no hipotálamo e no cerebelo de ratos. Também, foi avaliado o conteúdo e o perfil de gangliosídios nas frações solúvel e resistente a detergente obtidas de membranas sinápticas de córtex. Ratos Wistar foram divididos em dois grupos: 1) injetados subcutaneamente com solução 0,9% de NaCl (animais controle) e 2) injetados subcutaneamente com solução de prolina, em concentrações adequadas ao peso corporal (animais hiperprolinêmicos). Tanto a solução de prolina quanto a salina foram administradas do 6° ao 28° dia pós-natal. Doze horas após a última administração, os animais foram sacrificados mediante decapitação sem anestesia. As estruturas cerebrais foram dissecadas e em seguida homogeneizadas em clorofórmio:metanol na proporção 1:1 vpara a extração lipídica. As membranas sinápticas foram obtidas através de centrifugação diferencial e as frações solúvel e resistente a detergente foram isoladas através de tratamento das membranas com Triton X-100 a 4°C para investigação de microdomínios de membrana. Após a realização das análises, os resultados mostraram que os animais submetidos ao tratamento crônico com prolina apresentaram um marcado aumento no conteúdo de gangliosídios no córtex cerebral e no hipocampo, enquanto os conteúdos de fosfolipídios e de colesterol aumentaram somente no hipocampo. Além disso, os conteúdos destes compostos não foram alterados no hipotálamo e no cerebelo de animais hiperprolinêmicos. Por outro lado, o conteúdo de gangliosídios diminuiu nas frações solúvel e resistente a detergente obtidas de membranas sinápticas de córtex de animais hiperprolinêmicos. Embora os perfis de gangliosídios não tenham sido aparentemente modificados, as quantidades absolutas das espécies foram alteradas tanto no extrato total, como nos microdomínios de membrana obtidos do córtex. Estes dados revelam que o tratamento crônico com prolina afeta de forma distinta as diferentes regiões cerebrais quanto à composição lipídica das membranas celulares, refletindo-se sobre a distribuição de lipídios nos microdomínios de membrana do córtex. Entre as conseqüências destes fenômenos poderiam ser sugeridas modulações diferentes nas transmissões sinápticas que contribuiriam para o déficit cognitivo e/ou outras disfunções neurológicas presentes em pacientes com hiperprolinemia tipo II. / In the present work we investigated the effects of chronic proline administration on ganglioside, cholesterol and phospholipid total contents, as well as on ganglioside profile in cerebral cortex, hippocampus, hypothalamus and cerebellum of rats. We also evaluated the ganglioside content and profile in detergent- soluble and resistant fractions isolated from synaptic membranes obtained from cerebral cortex. Wistar rats were divided into two groups: 1) saline (control) and 2) proline injected (hyperprolinemic). Proline solution or saline were administered from 6th to 28th postnatal day, according to body weight. Twelve hours after the last injection, the animals were sacrificed by decapitation without anesthesia. Brain structures were homogenized with chlorophorm:methanol 1:1 for lipid extraction. Synaptic membrane was extracted by differential centrifugation and detergent- soluble and resistant fractions were isolated by cold Triton X-100 treatment. Results showed that rats subjected to chronic proline treatment presented a significant increase of ganglioside content on cortex and hippocampus, while phospholipid and cholesterol contents only increased in hippocampus. However, the content of these components were not altered in hypothalamus and cerebellum of hyperprolinemic rats. On the other hand, ganglioside content decreased in detergent- soluble and resistant fractions isolated from synaptic membrane obtained from hyperprolinemic cortex. Although ganglioside profiles were apparently not modified, the individual absolute quantities were altered in cortex total lipid extract and membrane microdomains obtained from cerebral cortex. Our findings suggest that chronic proline treatment affects, in a distinct manner, different cerebral regions concerning the lipid composition of the cell membranes, reflecting on its distribution in the cortex membrane microdomains. Among these phenomena consequences, different modulations in synaptic transmission may be suggested which may contribute to the impairment in cognition and/or other neurological disfunctions found in hyperprolinemia type II patients.

COSTA FILHO, Geraldo Pedro da. Socialização: fronteira entre a sociologia e a neurociência. 2016. 255f. – Tese (Doutorado) – Universidade Federal do Ceará, Programa de Pós-graduação em Sociologia, Fortaleza (CE), 2016. / Submitted by Márcia Araújo (marcia_m_bezerra@yahoo.com.br) on 2016-08-08T11:39:52Z No. of bitstreams: 1 2016_tese_gpcfilho.pdf: 2525597 bytes, checksum: 9f6468b494feab68628ec0db8131d365 (MD5) / Approved for entry into archive by Márcia Araújo (marcia_m_bezerra@yahoo.com.br) on 2016-08-08T11:43:16Z (GMT) No. of bitstreams: 1 2016_tese_gpcfilho.pdf: 2525597 bytes, checksum: 9f6468b494feab68628ec0db8131d365 (MD5) / Made available in DSpace on 2016-08-08T11:43:16Z (GMT). No. of bitstreams: 1 2016_tese_gpcfilho.pdf: 2525597 bytes, checksum: 9f6468b494feab68628ec0db8131d365 (MD5) Previous issue date: 2016 / This thesis studies the possible interface between sociology and evolutionary biology (represented by neuroscience). To this end, we made the reconstitution of the affinities between areas since the birth of both the nineteenth century, with Durkheim and Darwin until the appearance of sociobiology and neuroscience, whose leitmotif was the relationship between natural selection and adaptive cultural selection and brain evolution. First, we seek to answer key research questions about the intersection between areas and the relationship between the process of socialization and the synaptic exuberance of the human brain. Second, we seek to identify the empirical expression of these processes among adolescents/young people are completing primary school. Finally, we evaluate how the concept of socialization is being worked on in high school, after the return of compulsory sociology study in 2008, taking into account the public this level of education is experiencing intense brain restructuring that prepares you for adulthood. The theoretical support of the thesis was based on authors who in their areas sought convergence of sciences, among them: Richard Dawkins and the theory of "memes" as cultural replicators, that based what we call adaptive cultural selection; and Edgar Morin and the theory of "epistemological junction" between the social sciences and the disciplines derived from evolutionary biology. / A presente tese estuda a possível interface entre a sociologia e a biologia evolutiva (representada pela neurociência). Com esse objetivo, fizemos a reconstituição das afinidades entre as áreas desde o nascimento de ambas, no século XIX, com Durkheim e Darwin, até o aparecimento da sociobiologia e da neurociência, cujo fio condutor foi a relação entre a seleção natural e cultural adaptativa e a evolução do cérebro. Em primeiro lugar, procuramos responder as questões principais de pesquisa sobre a intersecção entre as áreas e sobre a relação entre o processo de socialização e o de exuberância sináptica do cérebro humano. Em segundo lugar, procuramos identificar a expressão empírica destes processos entre os adolescentes/jovens que estão concluindo o ensino fundamental. Finalmente, avaliamos como o conceito de socialização está sendo trabalhado no ensino médio, após o retorno da obrigatoriedade do ensino de sociologia em 2008, levando em consideração que o público desse nível de ensino está vivendo intensa reestruturação cerebral que o prepara para a vida adulta. A sustentação teórica da tese se fundamentou em autores que nas áreas respectivas buscaram a convergência entre as ciências, dentre os quais: Richard Dawkins e a teoria dos “memes” como replicadores culturais, que embasou o que denominamos seleção cultural adaptativa; e Edgar Morin e a teoria da “junção epistemológica” entre as ciências sociais e as disciplinas derivadas da biologia evolutiva.

A clear definition of the mechanisms involved in synaptic transmission is of paramount importance for the understanding of the processes governing synaptic efficacy. Despite decades of intense study, these mechanisms remain poorly understood. The work contained in this thesis examines several such mechanisms using the hypothalamic-neurohypophysial system (HNS), a classical preparation for the study of Ca2+-dependent neuropeptide release. The first portion of this thesis is comprised of my efforts to define the cellular machinery essential for the exocytosis of secretory granules isolated from peptidergic neurohypophysial terminals of the HNS. Here, using the planar lipid bilayer model system, I have been able to show that syntaxin alone in the target membrane is sufficient to elicit fusion of modified neurohypophysial secretory granules. Surprisingly, SNAP-25 does not appear to be necessary for this process. This suggests that syntaxin may be able to substitute for SNAP-25 to form functional non-cognate fusion complexes. Additionally, the coupling of amperometric detection with the planar lipid bilayer system has allowed me to confirm these results using native, unmodified secretory granules, and also provides some insight into the kinetics of release in this reconstituted system. This model system should provide a convenient means for the study of additional regulatory factors believed to be involved in secretory vesicle exocytosis. The second and third sections of this thesis involve my examination of the role of presynaptic Ca2+ stores in neuropeptide secretion from isolated peptidergic neurohypophysial terminals (NHT). I initially examined the source of recently discovered ryanodine-sensitive Ca2+ stores in this system. Using Immuno-electron microscopy I have found that ryanodine receptor (RyR) labeling appears to co-localize with large dense core granules. Additionally, I have shown that a large conductance cation channel, with similarities to the RyR, found in the membrane of these granules has the same characteristic response to pharmacological agents specific for the RyR. Further, application of RyR agonists modulates basal neuropeptide release from NHT. These results suggest that the large dense core granules of NHT serve as the source of a functional ryanodine-sensitive Ca2+store. Recent work has revealed that spark-like Ca2+ transients, termed syntillas, can be observed in NHT. These syntillas arise from ryanodine-sensitive intracellular stores. In other neuronal preparations, similar Ca2+ transients have been suggested to affect spontaneous transmitter release. However, such a role for syntillas had yet to be examined. To assess if syntillas could directly trigger spontaneous release from NHT, I used simultaneous Ca2+imaging along with amperometric detection of release. Amperometry was adapted to this system via a novel method of false-transmitter loading. Using this approach I have found no apparent correlation between these two events, indicating that syntillas are unable to directly elicit spontaneous transmitter release. As this finding did not rule out an indirect modulatory role of syntillas on release, I additionally present some preliminary studies examining the ability of ryanodine-sensitive Ca2+ release to modulate vesicular priming. Using immunocytochemistry, I have shown that RyR agonist treatment shifts the distribution of neuropeptides toward the plasma membrane in oxytocinergic NHT, but not in vasopressinergic NHT. RyR antagonists have the opposite affect, again only in oxytocinergic NHT. Further, I have found that application of RyR agonists result in a facilitation of elicited release in NHT using membrane capacitance recording. This facilitation appears to be due primarily to an increase in recruitment of vesicles to the readily-releasable pool. These findings suggest that ryanodine-sensitive Ca2+stores may be involved in vesicular priming in NHTs. Taken together, the work presented in this thesis provides some new and interesting insights into the underlying mechanisms and modulation of transmitter release in both the HNS and other CNS terminals.

Neste trabalho foi investigado o efeito da administração crônica de prolina sobre o conteúdo total de gangliosídios, fosfolipídios e de colesterol, assim como, sobre o perfil de gangliosídios no córtex, no hipocampo, no hipotálamo e no cerebelo de ratos. Também, foi avaliado o conteúdo e o perfil de gangliosídios nas frações solúvel e resistente a detergente obtidas de membranas sinápticas de córtex. Ratos Wistar foram divididos em dois grupos: 1) injetados subcutaneamente com solução 0,9% de NaCl (animais controle) e 2) injetados subcutaneamente com solução de prolina, em concentrações adequadas ao peso corporal (animais hiperprolinêmicos). Tanto a solução de prolina quanto a salina foram administradas do 6° ao 28° dia pós-natal. Doze horas após a última administração, os animais foram sacrificados mediante decapitação sem anestesia. As estruturas cerebrais foram dissecadas e em seguida homogeneizadas em clorofórmio:metanol na proporção 1:1 vpara a extração lipídica. As membranas sinápticas foram obtidas através de centrifugação diferencial e as frações solúvel e resistente a detergente foram isoladas através de tratamento das membranas com Triton X-100 a 4°C para investigação de microdomínios de membrana. Após a realização das análises, os resultados mostraram que os animais submetidos ao tratamento crônico com prolina apresentaram um marcado aumento no conteúdo de gangliosídios no córtex cerebral e no hipocampo, enquanto os conteúdos de fosfolipídios e de colesterol aumentaram somente no hipocampo. Além disso, os conteúdos destes compostos não foram alterados no hipotálamo e no cerebelo de animais hiperprolinêmicos. Por outro lado, o conteúdo de gangliosídios diminuiu nas frações solúvel e resistente a detergente obtidas de membranas sinápticas de córtex de animais hiperprolinêmicos. Embora os perfis de gangliosídios não tenham sido aparentemente modificados, as quantidades absolutas das espécies foram alteradas tanto no extrato total, como nos microdomínios de membrana obtidos do córtex. Estes dados revelam que o tratamento crônico com prolina afeta de forma distinta as diferentes regiões cerebrais quanto à composição lipídica das membranas celulares, refletindo-se sobre a distribuição de lipídios nos microdomínios de membrana do córtex. Entre as conseqüências destes fenômenos poderiam ser sugeridas modulações diferentes nas transmissões sinápticas que contribuiriam para o déficit cognitivo e/ou outras disfunções neurológicas presentes em pacientes com hiperprolinemia tipo II. / In the present work we investigated the effects of chronic proline administration on ganglioside, cholesterol and phospholipid total contents, as well as on ganglioside profile in cerebral cortex, hippocampus, hypothalamus and cerebellum of rats. We also evaluated the ganglioside content and profile in detergent- soluble and resistant fractions isolated from synaptic membranes obtained from cerebral cortex. Wistar rats were divided into two groups: 1) saline (control) and 2) proline injected (hyperprolinemic). Proline solution or saline were administered from 6th to 28th postnatal day, according to body weight. Twelve hours after the last injection, the animals were sacrificed by decapitation without anesthesia. Brain structures were homogenized with chlorophorm:methanol 1:1 for lipid extraction. Synaptic membrane was extracted by differential centrifugation and detergent- soluble and resistant fractions were isolated by cold Triton X-100 treatment. Results showed that rats subjected to chronic proline treatment presented a significant increase of ganglioside content on cortex and hippocampus, while phospholipid and cholesterol contents only increased in hippocampus. However, the content of these components were not altered in hypothalamus and cerebellum of hyperprolinemic rats. On the other hand, ganglioside content decreased in detergent- soluble and resistant fractions isolated from synaptic membrane obtained from hyperprolinemic cortex. Although ganglioside profiles were apparently not modified, the individual absolute quantities were altered in cortex total lipid extract and membrane microdomains obtained from cerebral cortex. Our findings suggest that chronic proline treatment affects, in a distinct manner, different cerebral regions concerning the lipid composition of the cell membranes, reflecting on its distribution in the cortex membrane microdomains. Among these phenomena consequences, different modulations in synaptic transmission may be suggested which may contribute to the impairment in cognition and/or other neurological disfunctions found in hyperprolinemia type II patients.

Made available in DSpace on 2014-12-17T14:03:42Z (GMT). No. of bitstreams: 1 LarissaMA_DISSERT.pdf: 2537553 bytes, checksum: 12f02428f3ae0866d5e225dc96048ea4 (MD5) Previous issue date: 2013-05-20 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Dopamine (DA) is known to regulate both sleep and memory formations, while sleep plays a critical role in the consolidation of different types of memories. We believe that pharmacological manipulation of dopaminergic pathways might disrupt the sleep-wake cycle, leading to mnemonic deficits, which can be observed in both behavioral and molecular levels. Therefore, here we investigated how systemic injections of haloperidol (0.3 mg/kg), immediately after training in dark and light periods, affects learning assessed in the novel object preference test (NOPT) in mice. We also investigated the hippocampal levels of the plasticity-related proteins Zif-268, brain-derived neurotrophic factor (BDNF) and phosphorylated Ca2+/calmodulin-dependent protein kinases II (CaMKII-P) in non-exposed (na?ve), vehicle-injected controls and haloperidol-treated mice at 3, 6 and 12 hours after training in the light period. Haloperidol administration during the light period led to a subsequent impairment in the NOPT. In contrast, preference was not observed during the dark period neither in mice injected with haloperidol, nor in vehicle-injected animals. A partial increase of CaMKII-P in the hippocampal field CA3 of vehicle-injected mice was detected at 3h. Haloperidol-treated mice showed a significant decrease in the dentate gyrus of CaMKII-P levels at 3, 6 and 12h; of Zif-268 levels at 6h, and of BDNF levels at 12h after training. Since the mnemonic effects of haloperidol were only observed in the light period when animals tend to sleep, we suggest that these effects are related to REM sleep disruption after haloperidol injection / A dopamina (DA) regula tanto o sono quanto o processo de forma??o de mem?rias, enquanto o sono desempenha uma fun??o essencial na consolida??o de diferentes tipos de mem?rias. Acreditamos que a manipula??o farmacol?gica das vias dopamin?rgicas possa afetar o ciclo sono-vig?lia, acarretando d?ficits mnem?nicos, os quais podem ser observados tanto em n?vel comportamental quanto molecular. Dessa forma, no presente estudo, investigamos o efeito do haloperidol (halo - 0,3 mg/kg; i.p.), administrado imediatamente ap?s sess?es de treino no per?odo da manh? e da noite, sobre o desempenho de camundongos no teste de prefer?ncia por objetos novos (TPON). Observamos que esse tratamento causou um preju?zo mnem?nico apenas nos camundongos testados pela manh?. Devido ? maior ocorr?ncia de epis?dios de sono durante a fase clara dos roedores, acreditamos que esse d?ficit pode estar associado a uma cascata de plasticidade hipocampal, a qual envolve a express?o de diferentes mol?culas em per?odos distintos ap?s a exposi??o ao est?mulo mnem?nico inicial. Para testar tal hip?tese, realizamos imunohistoqu?mica (IHQ) em tecidos de animais na?ve n?o expostos (NV), bem como de animais expostos aos objetos com posterior inje??o de halo ou salina (ve?culo - VH), perfundidos 3, 6 e 12h ap?s o est?mulo inicial. Quantificamos a express?o hipocampal (CA1, CA3 e GD) das prote?nas CaMKII-P, Zif-268 e BDNF, atrav?s de densitometria e contagem de c?lulas (somente para Zif-268). Observamos um aumento parcial na express?o de CaMKII-P em animais VH 3h, na regi?o CA3. Quanto ao grupo halo, houve uma redu??o dos n?veis de CaMKII-P nos tr?s hor?rios analisados; de Zif-268 em 6h, tanto em CA1 quanto CA3; e de BDNF em 12h, apenas no GD. Considerando-se o melhor desempenho diurno na consolida??o de mem?rias, associado ? fun??o do halo na regula??o do sono, propomos que a redu??o de plasticidade sin?ptica e os d?ficits no processo mnem?nicoocorreram devido ? supress?o do sono REM mediada pelo tratamento com haloperidol

We employed imaging, electrophysiological, and molecular techniques with the genetically tractable model organism Drosophila melanogaster to unravel fundamental biological and genetic underpinnings regulating synaptic function and plasticity. Using a forward genetic screen, we identified mutations in the Drosophila ortholog of a human WD40 repeat-containing protein termed DDB1 and CUL4 associated factor 12 (DCAF12). We show that DCAF12 likely serves as an adaptor protein for the DDB1-Cul4 E3 ubiquitin ligase complex by recruiting specific target proteins for ubiquitination. DCAF12 is expressed in neurons, muscles, and glia. In mitotically active cells such as muscles, DCAF12 is localized to nuclei and co-localizes in distinct foci with CUL4, suggesting that DCAF12 mediates a nuclear role for the CUL4 E3 ubiquitin ligase complex. In neurons, DCAF12 is localized to both cytoplasmic and nuclear compartments of motor neuron cell bodies, where it colocalizes with Cul4 in nuclei. DCAF12 is also expressed at the periactive zone of presynaptic terminals, but does not distinctly associate with DDB1 or Cul4 at this region. Evoked neurotransmitter release at larval NMJs is significantly reduced in DCAF12 mutants. These defects are rescued by presynaptic expression of wild-type DCAF12, demonstrating that DCAF12 is required presynaptically and serves as an important component of the machinery that facilitates evoked release. In addition, our studies show that DCAF12 is required for the differential expression of glutamate receptor subunits at the larval NMJ through transcriptional and post-translational mechanisms. GluRIID subunit mRNA levels and GluRIIA/C/D subunit protein levels are increased at DCAF12 mutant NMJs. Normal GluRIIA subunit levels can be restored by postsynaptic expression of wild-type DCAF12, but not with a truncated DCAF12 protein lacking a nuclear localization signal (∆NLS-DCAF12). Furthermore, DCAF12 overexpression in muscle nuclei reduces synaptic GluRIIA levels, an effect that can be suppressed by removing a copy of Cul4. These data strongly indicate that DCAF12 in muscle nuclei is required for GluRIIA expression and/or function in a Cul4-dependent manner. Moreover, homozygous DCAF12-GluRIIA double mutants show a strong synthetic lethality phenotype, providing further support for the hypothesis that GluRIIA directly or indirectly requires DCAF12. Mutations in glutamate receptors at larval NMJs trigger a retrograde trans-synaptic signal that leads to a compensatory increase in presynaptic release, which precisely restores the normal efficacy of synaptic transmission and muscle excitation. Reducing the gene dosage of DCAF12 by one gene copy suppresses the initiation and maintenance of GluRIIA-mediated synaptic homeostatic potentiation. This block of synaptic homeostatic potentiation can be rescued by presynaptic expression of DCAF12. In our studies, we determined that DCAF12 is critical for 3 distinct synaptic mechanisms: evoked neurotransmitter release, neurotransmitter reception by regulation of GluR subunit composition, and retrograde synaptic homeostatic signaling. Future research will strive to identify presynaptic and postsynaptic protein targets of DCAF12 and the Cul4 E3 ubiquitin ligase complex and the role of ubiquitination in regulating these synaptic processes.

O córtex visual desempenha papel essencial no processamento de informação visual. A primeira região do córtex a receber estímulos visuais é o córtex visual primário (V1) e pode ser subdividida anatomicamente em seis camadas, onde cada camada contém diferentes tipos e números de neurônios. Entender a forma como a informação é processada entre as diferentes camadas envolve o estudo da dinâmica dos padrões coletivos de atividade neural quando a rede é exposta a diferentes situações e como esses padrões relacionam-se com a organização estrutural e funcional da rede cortical. Essa dinâmica é afetada por mecanismos de plasticidade sináptica, de maneira que modelos computacionais que busquem capturá-la devem incluir tais mecanismos. Neste trabalho foi construído um modelo computacional de uma rede neural com 4000 neurônios baseada em informações sobre a estrutura local das conexões em V1 disponíveis na literatura neurobiológica. O modelo contém características estruturais consideradas fundamentais tais como: proporção entre neurônios inibitórios e excitatórios e probabilidades de conexões entre neurônios de diferentes populações em diferentes camadas. Os neurônios foram descritos pelo modelo de Izhikevich, reproduzindo três classes eletrofisiológicas mais abundantes no córtex: neurônios de disparo regular, para os excitatórios; neurônios de disparo rápido e baixo limiar de disparo, para os inibitórios. A regra de plasticidade sináptica utilizada foi do tipo plasticidade dependente dos tempos dos disparos neuronais (STDP em inglês), que pode fortalecer ou enfraquecer a força da conexão entre dois neurônios dependendo dos instantes dos seus disparos. Foram utilizadas versões diferentes dessa regra de plasticidade para sinapses excitatórias (STDPe) e inibitórias (STDPi). Foram simuladas situações com e sem plasticidade e alterando o tipo de neurônio inibitório presente na rede. Para cada uma, três protocolos de estimulação da rede foram utilizados: 1 estimulação por trens de disparos poissonianos aplicada a neurônios da camada 4 (simulando entradas talâmicas) e aplicada aleatoriamente aos neurônios da rede como ruído de fundo; 2 - pulsos aplicados a neurônios da camada 4 simulando estimulação visual com barras luminosas com diferentes orientações angulares; 3 - similar ao segundo protocolo, porém, estimulando a rede com dois pulsos alternantes de diferentes ângulos. Os parâmetros do modelo foram ajustados para que a atividade neural tivesse baixas frequências de disparos coerentes com dados experimentais. Esse ajuste foi mais fácil nos casos em que os neurônios inibitórios eram do tipo FS e havia STDPi. Os resultados mostraram que, de modo geral, os neurônios do tipo LTS contribuem para a formação de atividade síncrona na rede e este efeito foi amplificado com a STDPe. Para todos os protocolos, a STDPe aumentou a frequência média de disparos da rede e, para o segundo experimento, apesar da seletividade à orientação dos neurônios não ter sido alterada significativamente, houve mudanças visíveis na formação de assembleias funcionais. A competição da atividade dos neurônios no experimento 3 na presença da STDPe foi intensificada fortalecendo respostas funcionais de neurônios que não respondiam a ambos os estímulos. O balanço entre os dois tipos de regra de STDP manteve o equilíbrio entre as forças das conexões excitatórias e inibitórias. / The visual cortex plays essential role in the processing of visual information. The first region of the cortex that receives visual stimuli is the primary visual cortex (V1) or striate cortex, which can be anatomically divided into six layers, where each layer has different types and numbers of neurons. Understanding the way in which information is processed by the different layers involves the study of the dynamics of collective patterns of neural activity when the network is exposed to different situations, and how these patterns are related with the structural and functional organization of cortical network. This dynamics is affected by mechanisms of synaptic plasticity, so computational models which seek to capture it should include them. In this project a computational model of a neural network was built with 4000 neurons based on information on local connectivity in V1 from the neurobiological literature. The model has realistic structural characteristics such as the proportion between inhibitory and excitatory neurons and the connection probabilities among neurons from different populations of different layers. Neurons were described by the Izhikevich model, reproducing the three most abundant electrophysiological classes in cortex: RS, for the excitatory ones; FS and LTS, for the inhibitory neurons. The synaptic plasticity rule used was spike-timing dependent plasticity (STDP), whereby the synaptic strength between two neurons can increase or decrease depending on the timing of their spikes. Were used different versions of this plasticity rule to synapses made by excitatory neurons (STDPe) and by inhibitory neurons (STDPi). Different scenarios were simulated with and without plasticity and changing the type of inhibitory neuron present in the network. For each configuration, three network stimulation protocols were used: 1 - stimulation applied to layer 4 neurons (simulating thalamic inputs) modeled by Poissonian spike trains and background noise applied to all network neurons modeled in a similar manner; 2 - pulses applied to layer 4 neurons simulating visual stimulation with light bars at different angular directions; 3 - similar to the second protocol, however, stimulating the network with two alternating pulses of different angles. The parameters of the model were adjusted so that neural activity had low spike frequencies consistent with experimental data. This adjustment was easier in cases where inhibitory neurons were of FS type and had STDPi. The results showed that, in general, LTS neurons contribute to the formation of synchronous activity in the network and this effect was amplified with the insertion of STDPe. For all protocols, the STDPe increased the average firing frequency of the network. For Experiment 2, although the orientation selectivity of the neurons did not change significantly, there have been noticeable changes in the formation of functional assemblies. The competition of the activity of neurons in Experiment 3 in the presence of STDPe strengthened functional responses of neurons that do not respond to both stimuli. The balance between the two types of STDP rule maintained the equilibrium between excitatory and inhibitory connections.