Made available in DSpace on 2015-11-04T13:36:09Z (GMT). No. of bitstreams: 2 debora_amorim_ioc_mest_2014.pdf: 1324925 bytes, checksum: ef5bea54f46d162d48e15d4a0b299f08 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A sepse constitui importante problema de saúde pública. A incidência de sepse grave vem aumentando nas unidades de Terapia Intensiva, estando associada à alta morbimortalidade. Muitos pacientes evoluem para prótese ventilatória, precisando de drogas sedativas e analgésicas (como o midazolam e a morfina). Os sobreviventes podem apresentar disfunções cognitivas e comportamentais tardias. Estes déficits podem se dar tanto pelas alterações inflamatórias encontradas na sepse, como também por efeitos imunológicos e neurológicos de medicações utilizadas. Tentamos mimetizar este cenário clínico, usando o modelo experimental de Ligadura do Ceco e Perfuração (CLP de 2 furos e 4 furos) em camundongos suíços anestesiados com isoflurano inalatório. Após 5 h da cirurgia, eles receberam tratamento com salina 0,5 ml, midazolam 40 mg/kg ou morfina 80 mg/kg IP. Avaliamos escores de sedação, sinais clínicos de sepse, sobrevida (7-15 dias) e parâmetros neuro-inflamatórios 24 h após o CLP. Encontramos melhora dos índices de sobrevida nos grupos tratados com morfina, sobretudo no modelo de CLP de 4 furos (p<0,05) Observamos melhora dos escores clínicos de 24 h nos animais tratados com morfina e submetidos ao CLP de 4 furos (p<0,05). Houve tendência de diminuição das citocinas inflamatórias IL-1\03B2, IL-6 e de MCP-1 no lavado peritoneal, medidas por ELISA, nos grupos CLP 2 furos tratados com midazolam e morfina. Esta redução também foi verificada no córtex cerebral retirado pós perfusão. A redução de IL-6 foi significativa nos 2 grupos CLP tratados e de MCP-1, no grupo CLP tratado com midazolam (p<0,05). Esses resultados foram acompanhados pelo aumento da proteína pós-sináptica PSD95, no hipocampo dos animais dos grupos CLP tratados. Tais efeitos poderiam indicar um benefício antiinflamatório no uso de midazolam e da morfina, nas fases iniciais de sepse. Apesar dos indícios de melhorias neuroinflamatórias, ainda são necessários mais estudos para relacionarmos estas alterações a possíveis repercussões clínicas / Sepsis is a main problem in Public Health . The incidence of se vere sepsis is rising and is associated with high morbimortality indices. Many patients will need any kind of ventilatory assistance, sedative and analgesic drugs (e.g. m idazolam and m orphine). Survivals may present late cognitive and neurocomportamen tal dysfunctions . Sepsis inflammatory alterations , as well as neurologic and immunomodulatory effects mediated by these drugs may cause the deficits . We tried to simulate this clinical setting using the animal model of Cecal Ligature and Puncture (CLP), with 2 and 4 perf ora tions . Swiss mice were submitted to inalatory anesthesia with i soflurane for the surgery. After 5 h , they received s aline 0,5 ml, m idazolam 40 mg/kg or m orphine 80 mg/kg IP. We quantified sedation score, sepsis score , survival (7 - 1 5 days) and inflammatory parameters in 24 h after CLP . We found better survival taxes in the CLP group treated with m orphine , e specially in CLP with 4 perforations (p<0,05) . There were also better sepsis clinical scores 24 h and 48 h after CLP with 4 perforations, treated with m orphine (p<0,05) . The inflammatory cytokines IL - 1β, IL - 6 and MCP - 1 were lower in peritoneal lavage of CLP with 2 perforations treated with morphine and midazolam , measured by ELISA . This reduct ion w as also found in cerebral cortex, dissected after perfusion . IL - 6 reduction in cortex was important in both treated groups (CLP+Morp h ine and CLP+Midazolam) and MCP - 1 reduction in CLP+Midazolam group (p<0,05) . Postsynaptic p rotein PSD95 was augmented in hip p ocampal of CLP treated group. The results may point to anti - inflammatory benefits in using midazolam and morphine in initial phases of s epsis. Nevertheless, m ore studies are necessary to relate the potential neuroinflammatory benefits of these drugs with clinical repercussions

abstract: Tobacco and alcohol are the most commonly abused drugs worldwide. Many people smoke and drink together, but the mechanisms of this nicotine (NIC) -ethanol (EtOH) dependence are not fully known. EtOH has been shown to affect some nicotinic acetylcholine receptors (nAChRs), which potentially underlies NIC-EtOH codependence. Ventral Tegmental Area (VTA) dopamine (DA) and &#947;-aminobutyric acid (GABA) neurons express different nAChR subtypes, whose net activation results in enhancement of DA release in the Prefrontal Cortex (PFC) and Nucleus Accumbens (NAc). Enhancement of DA transmission in this mesocorticolimbic system is thought to lead to rewarding properties of EtOH and NIC, clarification of which is relevant to public health and clinical diseases. The aim of this study was to elucidate pharmacological mechanisms of action employed by both NIC and EtOH through nAChRs in VTA neurons by evaluating behavioral, network, synaptic and receptor functions therein. It was hypothesized that VTA GABA neurons are controlled by &#945;7 nAChRs on presynaptic GLUergic terminals and &#945;6 nAChRs on presynaptic GABAergic terminals. NIC and EtOH, via these nAChRs, modulate VTA GABA neuronal function. This modulation may underlie NIC and EtOH reward and reinforcement, while pharmacological manipulation of these nAChRs may be a therapeutic strategy to treat NIC or EtOH dependence. This data demonstrates that in VTA GABA neurons, &#945;7 nAChRs on GLUergic terminals play a key role in the mediation of local NIC-induced firing increase. &#945;6*-nAChRs on GABA terminals enhances presynaptic GABA release, and leads to greater inhibition to VTA GABA neurons, which results in an increase VTA DA neuron firing via a disinhibition mechanism. Genetic knockout of these nAChRs significantly prevents EtOH-induced animal conditioned place preference (CPP). Furthermore, levo-tetrahydropalmadine (l-THP), a compound purified from natural Chinese herbs, blocks nAChRs, prevents NIC-induced DA neuronal firing, and eliminates NIC CPP, suggesting it as a promising candidate in a new generation of interventions for smoking cessation. Improved understanding of underlying mechanisms and development of new drugs will increase the number of successful quitters each year and dramatically improve the quality of life for millions suffering from addiction, as well as those around them. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2015

Submitted by Inae Braga (inae_braga@yahoo.com.br) on 2018-05-04T20:01:34Z No. of bitstreams: 1 TESE INAÊ 2018 versão final Pós Defesa1.pdf: 6269053 bytes, checksum: 6263bd7a8bfc567a741ee29acc62e4a8 (MD5) / Approved for entry into archive by Ana Paula Santulo Custódio de Medeiros null (asantulo@rc.unesp.br) on 2018-05-07T12:34:39Z (GMT) No. of bitstreams: 1 braga_i_dr_rcla.pdf: 4231965 bytes, checksum: 91f9a4741e13f51a9d7686b64f56a083 (MD5) / Made available in DSpace on 2018-05-07T12:34:39Z (GMT). No. of bitstreams: 1 braga_i_dr_rcla.pdf: 4231965 bytes, checksum: 91f9a4741e13f51a9d7686b64f56a083 (MD5) Previous issue date: 2018-03-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A soja (Glycine max (L.) Merrill), uma das principais culturas oleaginosas, é muito sensível a fatores ambientais, sendo a deficiência hídrica um fator particularmente limitante para seu desenvolvimento e produção. Algumas substâncias podem ser utilizadas para minimizar os efeitos danosos do estresse hídrico. Bioreguladores, tais como ácido γ-aminobutírico (GABA) e Acetilcolina (Ach) atuam na defesa de plantas em respostas ao estresse, desempenhando um papel osmoprotetor e na regulação do movimento estomático, respectivamente. O objetivo deste estudo foi de analisar diferentes modos de aplicação exógena desses dois bioreguladores (isoladamente ou em combinação) e estudar o seu efeito na regulação dos processos de resposta de soja ao deficit hídrico. Para tanto, foi realizada a aplicação combinada dos dois bioreguladores na dose de 2,0 mM em plantas de soja cv. Intacta sob diferentes regimes hídricos. Os fatores estudados foram: 1) da aplicação dos bioreguladores (i) nas sementes- S (ii) via foliar F; (iii) nas semente e via foliar - SF; (iv) controle sem aplicação - C; e 2) regimes hídricos: (i) 100% da capacidade de campo (CC) e (ii) suspensão da irrigação (SI). Foram realizadas análises de trocas gasosas foliares e biomassa para caracterização fisiológica das plantas, das atividades das enzimas anti-oxidantes (CAT e APX) e das alterações nas respostas transcricionais dos genes induzidos por deficiência hídrica, P5CS, LEA e ABA2. Para os tratamentos de déficit hídrico, foi observada uma queda de 93% da capacidade fotossíntécia (PN) no sexto dia após a suspensão da irrigação, assim como diminuição da biomassa, além de apresentar maior expressão dos genes induzidos por deficiência hídrica. O tratamento da combinação das substâncias GABA e Ach aplicados na semente e na folha sob déficit hídrico, destacou-se por apresentar menor queda da fotossíntese, assim como, aumento para condutância estomática e biomassa. Para as análises transcricionais a aplicação dacombinação de GABA e Ach em semente e folha apresentaram menor expressão dos genes P5CS, LEA e ABA2 e maior atividade das enzimas SOD, CAT e APX, em relação ao tratamento controle Em nosso estudo foi possível observar que a aplicação combinada das substâncias GABA e Ach na semente e folha a 2,0 mM promoveu uma melhora do desempenho das plantas por meio da açãode Ach promovendo um aumento sobre a condutância estomática e um efeito osmoprotetor de GABA sobre as plantas em situação de estresse. / Soybean (Glycine max (L.) Merrill), a major oil crop, is very sensitive to environmental factors, and water deficiency is a particularly limiting factor for its development and production. Some substances can be used to minimize the harmful effects of water stress. Bioregulators, such as γ-aminobutyric acid (GABA) and Acetylcholine (Ach) act in the defense of plants in response to stress, playing an osmoprotective role and regulating stomatal movement, respectively. The objective of this study was to analyze different modes of exogenous application of these two bioregularators (alone or in combination) and to study their effect on the regulation of soybean response processes to the water deficit. Therefore, it was used in combination of two bioregulator dose of 2.0 mM in soybean plants cv. Intact under different water regimes. The factors studied were: 1) the application of bioregulators (i) in seeds- S (ii) in leaf L ; (iii) in the seed and leaf - SL; (iv) control without application - C; and 2) water regimes: (i) 100% of field capacity (CC) and (ii) suspension of irrigation (SI). Leaf gas exchange and biomass analyzes were performed for the physiological characterization of plants, the activities of the antioxidant enzymes (CAT and APX) and the changes in the transcriptional responses of genes induced by water deficiency, P5CS, LEA and ABA2. For water deficit treatments, a 93% decrease in the photosynthetic capacity (PN) was observed on the sixth day after the suspension of irrigation, as well as a decrease in biomass, in addition to a greater expression of genes induced by water deficiency. The treatment of the combination of the GABA and Ach substances applied to the seed and the leaf under water deficit was highlighted by lower photosynthesis decrease, as well as an increase in stomatal conductance and biomass. For the transcriptional analysis the application of GABA and Ach in seed and leaf showed lower expression of the genes P5CS, LEA and ABA2 and greater activity of the SOD, CAT and APX enzymes, in relation to the control treatment. In our study it was possible to observe that the combined application of the GABA and Ach substances in the seed and leaf at 2.0 mM promoted an improvement of the performance of the plants through the action of Ach promoting an increase on the stomatal conductance and an osmoprotective effect of GABA on plants in a situation of stress.

abstract: Cognitive function declines with normal age and disease states, such as Alzheimer's disease (AD). Loss of ovarian hormones at menopause has been shown to exacerbate age-related memory decline and may be related to the increased risk of AD in women versus men. Some studies show that hormone therapy (HT) can have beneficial effects on cognition in normal aging and AD, but increasing evidence suggests that the most commonly used HT formulation is not ideal. Work in this dissertation used the surgically menopausal rat to evaluate the cognitive effects and mechanisms of progestogens proscribed to women. I also translated these questions to the clinic, evaluating whether history of HT use impacts hippocampal and entorhinal cortex volumes assessed via imaging, and cognition, in menopausal women. Further, this dissertation investigates how sex impacts responsiveness to dietary interventions in a mouse model of AD. Results indicate that the most commonly used progestogen component of HT, medroxyprogesterone acetate (MPA), impairs cognition in the middle-aged and aged surgically menopausal rat. Further, MPA is the sole hormone component of the contraceptive Depo Provera, and my research indicates that MPA administered to young-adult rats leads to long lasting cognitive impairments, evident at middle age. Natural progesterone has been gaining increasing popularity as an alternate option to MPA for HT; however, my findings suggest that progesterone also impairs cognition in the middle-aged and aged surgically menopausal rat, and that the mechanism may be through increased GABAergic activation. This dissertation identified two less commonly used progestogens, norethindrone acetate and levonorgestrel, as potential HTs that could improve cognition in the surgically menopausal rat. Parameters guiding divergent effects on cognition were discovered. In women, prior HT use was associated with larger hippocampal and entorhinal cortex volumes, as well as a modest verbal memory enhancement. Finally, in a model of AD, sex impacts responsiveness to a dietary cognitive intervention, with benefits seen in male, but not female, transgenic mice. These findings have clinical implications, especially since women are at higher risk for AD diagnosis. Together, it is my hope that this information adds to the overarching goal of optimizing cognitive aging in women. / Dissertation/Thesis / Ph.D. Psychology 2012

Made available in DSpace on 2014-06-12T22:59:51Z (GMT). No. of bitstreams: 2 arquivo3884_1.pdf: 972393 bytes, checksum: b6a9eb05522515e024ec6c5fa9b28d34 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / O aminoácido glutamina (Gln) atua como precursor dos neurotransmissores glutamato e ácido gama-amino-butírico (GABA). Portanto, supõe-se que a sua suplementação dietética possa exercer influência sobre a excitabilidade cerebral. Esta dissertação originou o artigo intitulado L-glutamine supplementation during the lactation period facilitates cortical spreading depression in well-nourished and early-malnourished rats, atualmente submetido para publicação, e que está apresentado às páginas 25 &#150; 48. O objetivo foi caracterizar em ratos recém-desmamados, bem-nutridos (N) e desnutridos (D), os efeitos da suplementação enteral com Gln durante o período de desenvolvimento do cérebro sobre a depressão alastrante cortical (DAC). Ratos Wistar machos, nas condições N e D, receberam Gln (500mg/Kg/dia) por gavagem do 7º ao 27º dia de vida pós-natal. Dos 30 aos 40 dias de vida, foram submetidos ao registro da DAC durante 4 horas, em dois pontos da superfície cortical parietal do hemisfério direito. A velocidade de propagação foi calculada a partir do tempo requerido para uma onda da DAC atravessar a distância inter-eletrodos. Nas duas condições nutricionais, os ratos suplementados com Gln apresentaram velocidades de propagação da DAC mais elevadas (p<0,05; N-Gln = 4.22 ± 0.23; D-Gln = 4.51 ± 0.27 mm/min), quando comparados a um grupo controle tratado com o veículo (água) usado para dissolver a Gln (N-V = 3.77 ± 0.21; D-V = 4.15 ± 0.18 mm/min). Este último grupo, não diferiu de um outro grupo controle &#147;ingênuo&#148; (I), que não foi submetido ao procedimento de gavagem (N-I = 3.71 ± 0.16; D-I = 4.10 ± 0.11 mm/min). Um quarto grupo, tratado com um aminoácido &#147;placebo&#148; (glicina; Gly), também apresentou velocidade de propagação que não diferiu do controle tratado com água (N-Gly = 3.59 ± 0.24; D-Gly = 4.15 ± 0.18 mm/min). A suplementação com Gln não alterou os pesos corporais e encefálicos dos animais em nenhuma das condições nutricionais estudadas. Os resultados indicam que a suplementação com Gln durante o período de desenvolvimento do cérebro facilita a propagação da DAC e que este efeito não é alterado pela desnutrição. Supõe-se que a Gln exerce um papel sobre a excitabilidade cerebral, provavelmente resultante da modulação da síntese de neurotransmissores

Epilepsi är ingen speciell sjukdom utan ett symtom på en hjärnskada eller störd nervcellsfunktion i hjärnan. Epileptiska anfall beror på abnorm urladdning i hjärnans nervceller. Idag lever omkring 60 000 d.v.s. 0,5-1 % av Sveriges befolkning med epilepsi. Risken att drabbas är störst under det första levnadsåret och efter 65-årsålder då risken att drabbas av stroke är som störst. Behandling av epilepsi används i syfte att hindra uppkomst av anfall och göra det möjligt för den drabbade att leva ett relativt normalt liv. Antiepileptika dämpar aktiviteten i hjärnan och reducerar därmed risken för anfall. Under flera år har man försökt utveckla nya antiepileptika mot andra möjliga targets än de som finns idag, bl.a. GABA-transporthämmare. Det enda förekommande läkemedlet med GABA transporthämmande effekt är tiagabin men detta är inte registrerat som läkemedel i Sverige. Syftet med denna studie var att undersöka om GABA-transporthämmare skulle kunna användas som läkemedel mot epilepsi. Metoden som användes var en litteraturstudie där vetenskapliga artiklar hämtades från PubMed, ELIN, Cochrane och Google Scholar. Arbetet baseras på 4 experimentella originalartiklar och en metaanalys. Artiklarna beskriver antiepileptiska effekter och/eller relaterade egenskaper för olika substanser med hämmande effekter på olika GABA- transportörer. Dessa hämmare, ensamma eller i kombination, visades ge kramplösande effekt i olika djurmodeller av epilepsi. Hämmare av olika GABA-transportörer, till exempel tiagabin och EF1502, gav synergistisk effekt, medan hämmare av samma GABA-transportör, till exempel tiagabin och LU-32-176B, resulterade i additiv effekt. Hämning av olika GABA-transportörer i olika celltyper i och runt synapsklyftan verkar därför kunna ge synergistisk effekt. Ingen synergistisk effekt observerades för toxiska effekter. Det finns anledning att tro att ytterligare läkemedel med effekter på GABA-transportörer kan komma att finnas i framtiden för behandling av epilepsi. / Epilepsy is not a specific disease but a symptom of brain injury or impaired nerve cell function in the brain. Epileptic seizures are symptoms of abnormal activity in the brain neurons. Today, about 60 000 i.e. 0.5-1% of the Swedish population live with epilepsy. The risk of being affected is greatest during the first year of life and after the age of 65 years when the risk for stroke is greatest. The treatment of epilepsy is used in order to prevent the onset of seizures and to allow the patient to live a relatively normal life. Anticonvulsants dampen the activity in the brain and thus reduce the risk of seizures. During many years, attempts have been made to develop new anticonvulsants against other potential targets than those that exist today, for example GABA-transporter inhibitors. The only presently used medicine with GABA-transporter inhibiting effect is tiagabine, but this is not licensed as a pharmaceutical drug in Sweden. The aim of this study was to investigate whether GABA-transport inhibitors could be used as medication for epilepsy. The method that was used was a literature study in which scientific articles were chosen from PubMed, ELIN, Cochrane and Google Scholar. The work is based on 4 original research articles and one meta-analysis. The articles describe antiepileptic effects and/or related properties of various substances with inhibitory actions on different GABA-transporters. These inhibitors, alone or in combination, were shown to have anticonvulsant effects in several different animal models of epilepsy. Inhibitors of different GABA transporters, such as tiagabine and EF1502, resulted in synergistic effects, while inhibitors of the same GABA transporter, such as tiagabine and LU-32-176 B, resulted in additive effects. Inhibition of various GABA transporters in different cell types in and around synapses therefore seems to provide synergistic effects. No synergistic effect was observed for toxic effects. There is reason to believe that additional drugs with effects on GABA transporters may be used in the future for the treatment of epilepsy.

Cognitive dysfunction is a core symptom of schizophrenia, which is poorly treated by current antipsychotic medication. Deficits in the GABAergic system, as demonstrated by convergent genetic and [125I]-iomazenil imaging evidence from patients, are thought to underlie these cognitive deficits. The sub-chronic PCP rodent model was used as it shows cognitive and behavioural parallels to schizophrenia and therefore provides a translational model for some aspects of the disease. However the neurobiological mechanisms responsible for the behavioural alterations in this model have not been fully elucidated. The main aim of the studies presented in this thesis was to investigate the construct validity of the sub-chronic PCP model in relation to the GABAergic and sigma-1 (σ1) receptor systems. Transcriptional changes in gene markers were studied using qRT-PCR and proteomic alterations were investigated using radioligand binding, autoradiography and Western blotting. Finally, the cognitive enhancing potential of σ1 receptor modulators was tested using the novel object recognition (NOR) task. Data presented in chapter 3 shows that sub-chronic PCP treatment in rats produces an increase in GABAA receptor α5-subunit mRNA and a decrease in α3 and δ subunit mRNA levels. No differences were observed in the mRNA levels of the other studied GABAA receptor subunits (α1, α2, α4 or γ2). No alterations in benzodiazepine site- or α5-subunit-containing GABAA receptors were seen following a 7-day washout period, although increased frontal cortical levels of α5-subunit protein were observed prior to the washout period. This suggests that sub-chronic PCP treatment affects extrasynaptic cortical GABAA receptor expression, as shown by the alterations in α5- and δ-subunits, which may contribute to the cognitive deficits observed in this model. Studies in chapter 4 showed that sub-chronic PCP administration causes frontal cortical reductions in parvalbumin, GAD67, GABA transporter-1 and calretinin mRNA levels. No alterations were observed for somatostatin, GAD65, or GABA transporter-3 mRNA, although changes in the mRNA levels for the astrocytic marker glial fibrillary acidic protein were observed in the cerebellum, frontal cortex and hippocampus of sub-chronic PCP-treated animals. No differences in the frontal cortical protein levels of GAD67, GAT-1 and calretinin were observed, suggesting that any proteomic differences in these markers which are present in the sub-chronic PCP model, they are limited in a layer- or cell-type-specific manner. The NOR task is a translational cognitive test that measures recognition memory, which is known to be impaired in schizophrenia. Data in chapter 5 of this thesis showed that sub-chronic PCP-induced and delay-induced recognition memory deficits were ameliorated by acute administration of the σ1receptor agonist (PRE-084) at 1 and 3mg/kg and by the σ1receptor antagonist (NE-100) at 1mg/kg. NE-100 at 3mg/kg proved effective at ameliorating delay-, but not PCP-induced memory deficits. No procognitive effect was observed at lower concentrations of either compound or by co-administration of both compounds. These observations suggest that the improvement of recognition memory deficits is mediated, in part, by σ1 receptors in female rats. The overall results of these studies suggest that sub-chronic PCP administration causes frontal cortical transcriptional alterations in GABAergic neuronal markers which correlate to clinical findings in schizophrenia patients, although these alterations were not observed at the proteomic level following the washout period. These findings also suggest that the σ1 receptor is a potential therapeutic target for recognition memory deficits in schizophrenia, as well as other disorders.

The main goal of this study was to further explore the hypothesis that experience-dependent neural network activity and neurotransmission can modulate adult OPC proliferation and differentiation. More specifically, we used stereology to establish whether extensive reference memory training and system-wide administration of GABAergic agonists and antagonists could influence the proliferation and differentiation of adult OPCs, as well as the prevalence of OPC-neuron pairs. Analysis of the effects of reference memory training on OPC proliferation and differentiation corresponds to experiment 2, analysis of the effects of GABAergic agents on OPC proliferation and differentiation corresponds to experiment 3, and analysis of the effects of both reference memory training and GABAergic agents on OPC-neuron pairs, as well as an histological analysis of these closely apposed cells, corresponds to experiment 4.

Synaptic plasticity is the process whereby connections between neurons can be altered in an experience dependent manner. For example, drugs of abuse alter plasticity in the ventral tegmental area (VTA) of the midbrain. A large amount of research has been applied to uncovering the mechanism whereby synapses on the reward signaling dopamine cells is altered, however, less is known regarding the VTA inhibitory GABA neurons. Our objective was to examine the ability of GABA neurons to exhibit plasticity, and determine how drugs of abuse could influence it. Here we report a novel type of plasticity of excitatory neurotransmission onto VTA GABA cells. This plasticity is dependent on the metabotropic glutamate receptor 5, to signal for diacylglycerolipase alpha to make the endocannabinoid 2-arachadonoyl glycerol to signal via cannabinoid receptor 1 (CB1). Marijuana and cocaine are drugs of abuse that have been shown to alter the endocannabinoid system. Tetrahydrocannabinol is the active ingredient in marijuana, and is a known agonist of CB1, and cocaine is able to attenuate endocannabinoid signals. We tested the effects of these drugs on VTA GABA plasticity and found that it can be blocked by chronic injections of tetrahydrocannabinol, as well as acute and chronic injections of cocaine. If VTA GABA neurons are depressing excitatory inputs, that could lead to less inhibition onto VTA dopamine cells, and therefore, more reward signaling in the brain. This new type of plasticity could be an additional mechanism whereby cocaine and marijuana exert their rewarding and addictive effects. Another brain structure known to exhibit use-dependent plasticity is the hippocampus, which is involved in learning and memory. The stratum oriens is a layer of inhibitory interneurons in the hippocampus that is involved in feedback inhibition onto the principle excitatory cells in the stratum pyramidale. Our goal was to determine whether oriens interneurons were capable of producing an endocannabinoid signal, and if so, whether they could influence plasticity. We identified 2 major subtypes of oriens interneurons, oriens lacunosum-moleculare cells, and parvalbumin-positive basket cells, which are capable of receiving and producing an endocannabinoid signal. Furthermore, we demonstrated that one such endocannabinoid, anandamide, is responsible for signaling for synaptic plasticity. This plasticity is also dependent on CB1, and is unique in that there are few examples of CB1 signaling for potentiation rather than depression. Collectively, these experiments demonstrate two mechanisms of endocannabinoid mediated synaptic plasticity, which could influence reward signaling, addiction and memory.

Afin d’identifier de nouveaux régulateurs impliqués dans le contrôle du nombre des RGABAs à la synapse, nous avons utilisé la jonction neuromusculaire GABAergique du nématode Caenorhabditis elegans comme système modèle. Après mutagénèse aléatoire d’une souche knock-in exprimant les RGABAs tagués avec une protéine fluorescente (TagRFP), nous avons isolé plusieurs mutants présentant des défauts de localisation des récepteurs. Nous avons mis au point une nouvelle stratégie, basée sur l’analyse bio-informatique de données issues du séquençage du génome entier (WGS), en combinant identification et cartographie des mutations causales sans étape préalable de cartographie génétique. Sur 36 mutants analysés, nous avons retrouvé plusieurs gènes connus pour leur rôle dans la synaptogénèse GABAergique, validant ainsi notre approche. Nous avons initié la caractérisation fonctionnelle d’un nouveau gène candidat, provisoirement appelé nsp-3, qui code pour une protéine transmembranaire hautement conservée au cours de l’évolution. L’absence de nsp-3 induit la localisation ectopique de RGABAs au sein du muscle. Les récepteurs ectopiques colocalisent partiellement avec des marqueurs endosomaux. Des données d’électrophysiologie combinées à des analyses quantitatives du nombre de récepteurs synaptiques, montrent que NSP-3 régule la formation d’un pool de réserve de récepteurs sous-synaptiques. Des données pharmacologiques montrent que le recrutement de ce pool est essentiel dans la plasticité synaptique de la JNM GABAergique après un traitement aigu à l’aldicarbe, un inhibiteur de l’acétylcholine estérase (AChE). L’observation d’un reporteur transcriptionnel montre que nsp-3 est exprimé dans la plupart des tissus du vers. Des expériences de sauvetage phénotypique tissu-spécifiques et des données de colocalisation in vivo suggèrent que NSP-3 agit dans le muscle, à l’interface RE-Golgi, où elle régule le trafic des RGABAAs vers la surface. Cette étude décrit un rôle des nonaspanines dans un nouveau processus cellulaire où elles régulent le trafic des RGABAAs à la jonction neuromusculaire de C. elegans / To identify novel genes and mechanisms involved in the formation and regulation of inhibitory synapses, we used the inhibitory GABAergic neuromuscular junction of the nematode C. elegans as a genetically tractable model. After random mutagenesis of a knock-in strain expressing fluorescently tagged GABAA receptors (GABAAR), we screened for mutants with abnormal fluorescence pattern in vivo. We analyzed 36 mutant strains using a novel whole-genome sequencing strategy to simultaneously map and identify causative mutation without any prior time-consuming genetic mapping. We undertook the functional characterization of a non-characterized gene, tentatively named nsp-3, which encodes an evolutionarily conserved transmembrane protein. nsp-3 deletion using CRISPR technology causes ectopic localization of GABAAR in intracellular compartments of the muscle cell. We found partial colocalization of these ectopic receptors with endosomal markers. Interestingly, we observed a 50 % decrease of GABAAR at synapses while we saw no change in GABA neurotransmission by electrophysiology. These and additional data predict the presence of a subsynaptic pool of GABAARs, which is depleted in the absence of NSP-3. Additional pharmacological data set suggests that this pool of receptors is recruited for GABAergic synaptic plasticity upon acute aldicarb (acetylcholine esterase inhibitor) treatment. A transcriptional reporter of endogenous nsp-3 expression detected expression in most tissues of the worm. Tissue-specific rescue experiments and colocalization data show that NSP-3 functions in muscles at ER-Golgi interface to regulate GABAARs trafficking to cell surface. Our data identified a novel function of the nonaspanins in the traffic of neurotransmitter receptors in the nervous system