La dépression majeure constitue aujourd’hui une maladie dévastatrice qui affecte jusqu’a 20% de la population mondiale et qui est classifiée comme une des causes principales des années de vie en handicap. Depuis les années ’50 et avec la serendipité des composés antidépresseurs, la physiopathologie et le traitement actuel de la dépression sont dominés par l’hypothèse monoaminergique. Malgré leur utilisation répandue encore aujourd’hui, les antidépresseurs qui ciblent le système monoaminergique sont inefficaces chez une proportion significative des patients et présentent un délai du début d’action prolongé. Dans l’optique d’approfondir l’action antidépressive, nombreuses études récentes ont démontré différents mécanismes à travers les quels ces composés restaurent la plasticité neuronale et la neurotransmission, qui sont trouvées perturbées dans les troubles de l’humeur et dans des modèles animaux de la dépression. La transmission glutamatergique, notamment le récepteur AMPA (AMPA R), et des cascades intracellulaires de signalisation ont été impliqués dans la physiopathologie de la dépression et l’action antidépressive. Au cours de cette étude, des potentiateurs AMPA R (modulateurs positives allostériques) ont montré des effets antidépresseurs dans un modèle chronique de dépression et des résultats préliminaires suggèrent un délai du début d’action précoce de ces composés. La voie de signalisation d’ERK/MAPK a été aussi étudiée, comme un intégrateur des modifications de la plasticité synaptique qui constitue le lien entre les signaux extracellulaires et la régulation de l’expression génique. On a utilisé une nouvelle classe d’inhibiteurs de la voie de signalisation d’ERK. La construction de ces molécules est basée sur la propriété particulière d’ERK à s’attacher à ses cibles à travers des domaines de liaison spécifiques. Une batterie de tests comportementaux, aigus et chroniques, ont apporté une somme de données convaincants sur l’action antidépressive de l’inhibition sélective du complexe de signalisation ERK/Elk1. L’ensemble des résultats de cette étude apportent de nouveaux éléments sur la physiopathologie de la dépression et révèlent des pistes thérapeutiques prometteuses. / Major depression is a devastating disease that affects up to 20% of world population and is classified today as a leading cause of disability-adjusted life years. Since late 50s with the serendipitous discovery of the first antidepressant agents, pathophysiology and therapeutics of depression are governed by the monoaminergic hypothesis. Monoaminergic-based treatment, although still in use today, was proven inefficient to treat a significant proportion of cases and presents a delayed onset of action. Recent research has unveiled an array of new mechanisms through which antidepressant medication helps restore neuronal plasticity and neurotransmission that is disrupted in mood disorders and in animal models of depression. Glutamatergic transmission, in particular AMPA receptor, and signal transduction cascades have been implicated both in antidepressant action and the pathophysiology of depression, as here and now regulators that mediate persistent changes. In this study, AMPA receptors positive modulators demonstrated antidepressant-like effects in a chronic model of depression and preliminary data suggest a faster onset of action than conventional antidepressants. The ERK/MAPK signaling pathway was also studied as a major integrator of synaptic plasticity modifications that links extracellular signals to gene expression regulation via its downstream molecular partners. We used a new class of inhibitors of the ERK pathway, whose design was based on the particular property of ERK to bind to its downstream targets via specific docking domains. A considerable amount of data provided evidence for an antidepressant action of selective inhibition of the ERK/Elk1 signaling complex in multiple animal models of depression. Overall, the findings of this work reveal novel, promising targets for the treatment of depression.

O córtex pré-frontal medial (PFCm) é caracterizado por entradas glutamatérgicas e dopaminérgicas que convergem sobre os mesmos neurônios alvos. Devido à escassa informação sobre as bases anatômicas das interações entre a dopamina (DA) e o glutamato (Glu), mapeamos a distribuição de subunidades (Su) de receptores (Rs) de Glu do tipo AMPA, NMDA e kainato no PFCm e investigamos a sua expressão em neurônios contendo a fosfoproteína DARPP-32 e em interneurônios. Os resultados mostram que as Su GluR2/3 dos Rs do tipo AMPA são as mais amplamente distribuídas no PFCm e expressas em todos os neurônios DARPP-32+. GluR2/3 é também amplamente co-localizado com as Su NMDAR1 dos Rs de Glu do tipo NMDA e GluR5/6/7 dos Rs do tipo kainato. Em contraste, as Su GluR1 e GluR4 são somente fracamente expressos no PFCm e não são co-localizados com DARPP-32, porém com GABA ou parvalbumina. Os resultados indicam que as Su GluR2/3, NMDAR1 e GluR5/6/7 são amplamente expressos em neurônios piramidais DARPP-32+ enquanto GluR1 e GluR4 são predominantemente expressos em interneurônios do PFCm. / The medial prefrontal cortex (PFCm) is characterized by glutamatergic and dopaminergic afferents that converge on the same target neurons. Since there is only limited information about the anatomical bases for interactions between dopamine (DA) and glutamate (Glu), we mapped the distribution of AMPA, NMDA and kainate Glu receptor (Rs) subunits (Su) in the PFcm and investigated their expression in neurons containing the phosphprotein DARPP-32 and in interneurons. Results show that the Su GluR2/3 of AMPA type Rs are the most prominently distributed in the PFCm and expressed in all neurons DARPP-32+. GluR2/3 is also widely co-localized with the NMDA type Su NMDAR1 and the Kainate Su GluR5/6/7. In contrast, the Su GluR1 and GluR4 are only weakly expressed in the PFCm and are not colocalized with DARPP-32 but with GABA or parvalbumin. Results indicate that the Su GluR2/3, NMDAR1, and GluR5/6/7 are prominently expressed in DARPP-32+ pyramidal neurons, whereas GluR1 and GluR4 are predominantly expressed by interneurons in the PFC.

O núcleo acumbens (Acb) é subdividido em core e shell (AcbSh). Há evidências que as divisões do Acb vão além da dicotomia core-shell e que regiões pobres em tirosina hidroxilase (TH) e calretinina (Calr) formam um sistema de corredores no AcbSh. Para detalhar melhor a organização do Acb investigamos a distribuição de TH, Calr, DARPP-32 e do transportador de dopamina (DAT). Em seguida, foi comparada a distribuição destes marcadores com a das subunidades GluA2/3 dos receptores de glutamato do tipo AMPA. Finalmente, exploramos se as aferências pré-frontais são direcionadas à distintos compartimentos do AcbSh. Nossos resultados revelaram que regiões que contém neurônios GluA2/3+ intensamente marcados formam um sistema de corredores no AbSh que coincide com áreas pobres em TH, Calr e DAT. Os experimentos de rastreamento anterógrado indicaram que somente uma pequena parte das aferências pré-frontais é direcionada aos corredores. Nossos resultados delinearam um sistema de corredores no AcbSh que provavelmente constitui um compartimento neuroquímico altamente especializado. / The nucleus accumbens (Acb) is subdivided in core and shell (AcbSh). There is evidence that accumbal subdivisions go beyond this core-shell dichotomy and that regions poor in tyrosine hydroxylase (TH) and calretinin (Calr) form a corridor system in the Acbsh. To better detail accumbal organization, we investigated the distribution of TH, Calr, DARPP-32 and of the dopamine transporter (DAT). Then we compared the distribution of these markers with that of the AMPA-type glutamate receptor subunits GluA2/3. Finally, we explored whether prefrontal afferents are directed to distinct AcbSh compartments. Our findings revealed that regions containing intensely labeled GluA2/3+ neurons form a corridor system in the AcbSh that coincides with regions poor in TH, Calr, and DAT. Anterograde tracing experiments indicated that only a small portion of the prefrontal afferents is specifically related to the corridors. Our findings delineated a complex corridor system in the AcbSh which might constitute a highly specialized neurochemical compartment.

Les récepteurs AMPA (rAMPA) sont les récepteurs ionotropiques du glutamate responsables de la majeure partie des courants excitateurs rapides dans la transmission synaptique rapide. Lors de la libération de glutamate, le rAMPA passe par 3 états conformationnels majoritaires : pore fermé/agoniste non lié, pore ouvert/agoniste lié et pore fermé/agoniste lié. Le contrôle du nombre et de l’organisation dans la synapse des rAMPA, via une combinaison de diffusion latérale et d’endo/exocytose, est essentiel à la régulation de l’intensité de la transmission synaptique. Les interactions existant entre les protéines de la densité post-synaptique et les protéines partenaires des récepteurs régulent la diffusion des récepteurs, contrôlant leur nombre et leur organisation à la post-synapse. Mon travail de thèse a consisté à étudier l’impact de l’activation des rAMPA sur leur mobilité et leur organisation à la post-synapse. En effet, la fixation de glutamate sur les récepteurs ainsi que leur désensibilisation entraînent des modifications structurales majeures affectant leurs interactions avec les protéines d’échafaudage et les protéines accessoires. L’impact de telles modifications sur les propriétés de diffusion et sur l’organisation sub-synaptique de ces rAMPA était jusqu’à présent inconnu. Mes travaux démontrent une mobilisation des rAMPA synaptiques consécutivement à leur activation par le glutamate. A l’échelle moléculaire, je propose que le passage de l’état activé à l’état désensibilisé des rAMPA entraîne un changement d’affinité de ces derniers pour une de leur protéine partenaire : la Stargazin. Cette régulation glutamate dépendante de la diffusion des rAMPA participe au maintien de la fidélité de la transmission synaptique rapide. / AMPA receptors (AMPAR) are ionotropic glutamate receptors which are responsible for the vast majority of fast excitatory synaptic currents in fast transmission. Upon release of glutamate, AMPAR undergo three main conformational states: pore closed/agonist unbound, pore open/agonist bound and pore closed/agonist bound. Controlling the number of AMPAR and their organization in the synapse, through a combination of lateral diffusion and endo/exocytosis, is essential to regulate the intensity of synaptic transmission. The interactions between proteins of the post-synaptic density and accessory receptor proteins regulate the distribution of receptors, controlling their number and organization in the post-synapse. During my PhD, I studied the impact of AMPAR activation on their mobility and organization in the post-synapse. Indeed, the binding of glutamate to AMPAR and their following desensitization lead to major structural changes on the receptor which impacts on their interactions with scaffolding proteins and accessory proteins. The impact of such modifications on the lateral diffusion and sub-synaptic organization of AMPAR was not known yet. My findings show a mobilization of synaptic AMPAR following their activation by glutamate. At the molecular level, I suggest that the transition from the activated state to the desensitized state of AMPAR leads to a change in affinity of the receptor for their partner protein: Stargazin. This glutamate dependent regulation of AMPAR diffusion participates in maintaining the fidelity of fast synaptic transmission.

Neste trabalho investigamos a interação entre ATP-glutamato na modulação de potenciais de ação e atividade sináptica de neurônios PVN-RVLM, além de avaliar se esta interação induziria mudanças na atividade simpática lombar (ANSL) por estímulo osmótico. Utilizamos de técnicas de imunohistoquímica, whole-cell patch clamp e registro eletroneurográfico. Observou-se que o ATP aumenta a frequência de potenciais de ação em neurônios PVN-RVLM, efeito bloqueado por acido quinurênico (KYN) e PPADS. A injeção de ATP no PVN aumenta a ANSL (25 nmol: 72%), um efeito atenuado por PPADS e/ou KYN, e também por CNQX. O ATP não afeta a função sináptica, mas aumenta correntes glutamatérgicas induzidas por aplicação AMPA em 52%, a qual foi bloqueada por PPADS ou por quelação de Ca2+ intracelular. Além disso, o estímulo osmótico ativa neurônios do PVN que expressam receptores P2X2 e potencia as correntes mediadas por AMPA (53%), um efeito bloqueado por PPADS. Finalmente, demonstrou-se que receptores P2 no PVN são importantes na simpatoexcitação induzida por estímulo osmótico agudo. / In the present study we investigate the interaction of ATP-glutamate on the firing activity and synaptic function in PVN-RVLM neurons, besides whether that interaction would be translated in changes on sympathetic nerve activity (SNA) induced by osmotic stimulus. Immunohistochemistry, whole-cell patch clamp and electroneurography technical approaches were used. Our data have shown that ATP increases firing rate of PVN-RVLM neurons, an effect blocked by kynurenic acid (KYN) or PPADS. ATP injection into the PVN enhanced SNA (72%), which was attenuated by PPADS and/or KYN, or CNQX. ATP did not affect synaptic function but, glutamatergic currents evoked by AMPA application were augmented with ATP (AMPA area: 52%), blocked by PPADS and chelation of intracellular Ca2+. In addition, we observed that acute osmotic stimulus activates P2X2 expressing neurons in the PVN. Moreover, an osmotic challenge potentiated AMPA responses (53%), an effect blocked by PPADS. Finally, we demonstrated that P2 receptors in the PVN are important for osmotically-driven sympathoexcitation.

Le synaptic scaling est une forme de plasticité homéostatique par lequel les synapses ajustent leur efficacité pour compenser des variations normales ou pathologiques de l'activité neuronale notamment lors des maladies neurodégeneratives ou suite à la perte d’afférences sensorielles après une lésion. Dans un modèle expérimental classique, le traitement chronique des neurones primaires avec la tétrodotoxine (TTX) pour bloquer la propagation des potentiels d'action présynaptiques induit une augmentation significative de l'amplitude des courants miniatures excitateurs transmis par les récepteurs du glutamate AMPA postsynaptiques. Plusieurs voies de signalisation ont été proposées, dont celle impliquant les microARNs (miRs), de petits ARN non-codants qui inhibent la traduction des protéines en se liant aux ARN messagers cibles. Dans ce contexte, nous avons exploré l'hypothèse que le microARN, miR-124, fortement exprimé dans le cerveau, pourrait être un régulateur important de l'homéostasie synaptique en contrôlant l'expression de la protéine synaptopodine, une protéine structurante des épines dendritiques et indispensable à l'expression du synaptic scaling.En combinant des approches de RTq-PCR, d'immunocytochimie et d'électrophysiologie in vitro, nous avons montré dans un premier temps que la privation globale de l'activité des neurones primaires d’hippocampe diminuait le niveau d'expression de miR-124 et augmentait celui de la synaptopodine et des récepteurs AMPA dont la sous-unité GluA2 est une autre cible de miR-124. Par ailleurs, en rendant des synapses individuelles inactives via l’expression présynaptique de la toxine tétanique, nous avons observé que le recrutement synaptique des récepteurs AMPA et de la synaptopodine était spécifique de ces synapses, suggérant une régulation homéostatique locale. Dans un deuxième temps, nous avons trouvé que la surexpression de miR-124 ou l’inhibition de son interaction avec l’ARNm de la synaptopodine ou de GluA2 bloquaient la réponse synaptique homéostatique induite par le traitement TTX. Enfin, des expériences de FRAP ont suggéré que la synaptopodine influençait le trafic des récepteurs AMPA à la membrane probablement en les stabilisant à la synapse, ce qui expliquerait ainsi son rôle pendant la plasticité homéostatique. / Synaptic scaling is a form of homeostatic plasticity where synapses adjust their own efficacy to compensate for normal or pathological variations in neuronal activity such as neurodegenerative disorders or sensory deprivation after a lesion. In a well-established paradigm, the chronic application of tetrodotoxin (TTX) in primary neurons, to block presynaptic action potential propagation, induces a significant upscaling of miniature excitatory postsynaptic currents mediated-AMPA receptors. Numerous regulators of this plasticity have been identified including microRNAs (miR), which are small endogenous non-coding RNAs, inhibiting protein translation by binding to mRNA targets. This led us to hypothesize that the most highly expressed microRNA in the brain, miR-124, could be an important regulator of homeostatic scaling by controlling the expression of synaptopodin, a structural protein of dendritic spines playing a crucial role in homeostatic plasticity.By combining qRT-PCR, immunocytochemistry and in vitro electrophysiology approaches, first we showed that a global 48hrs TTX treatment in hippocampal primary neurons led to a decrease in miR-124 level and an increase in the expression of synaptopodin and synaptic AMPA receptors containing the GluA2 subunit which is another miR-124 target. Moreover, we observed that the synaptic accumulation of AMPA receptors and synaptopodin could be synapse-specific by expressing the tetanus toxin to block the activity of individual presynapses, which suggested a local homeostatic regulation. Importantly, we found that overexpressing miR-124 or inhibiting its interaction with synaptopodin or GluA2 mRNAs blocked the synaptic homeostatic response. In addition, FRAP experiments suggested that synaptopodin controlled AMPA receptor trafficking at the membrane by probably retaining them in dendritic spines, which could explain its role during homeostatic plasticity.

No description available.

O núcleo acumbens (Acb) é subdividido em core e shell (AcbSh). Há evidências que as divisões do Acb vão além da dicotomia core-shell e que regiões pobres em tirosina hidroxilase (TH) e calretinina (Calr) formam um sistema de corredores no AcbSh. Para detalhar melhor a organização do Acb investigamos a distribuição de TH, Calr, DARPP-32 e do transportador de dopamina (DAT). Em seguida, foi comparada a distribuição destes marcadores com a das subunidades GluA2/3 dos receptores de glutamato do tipo AMPA. Finalmente, exploramos se as aferências pré-frontais são direcionadas à distintos compartimentos do AcbSh. Nossos resultados revelaram que regiões que contém neurônios GluA2/3+ intensamente marcados formam um sistema de corredores no AbSh que coincide com áreas pobres em TH, Calr e DAT. Os experimentos de rastreamento anterógrado indicaram que somente uma pequena parte das aferências pré-frontais é direcionada aos corredores. Nossos resultados delinearam um sistema de corredores no AcbSh que provavelmente constitui um compartimento neuroquímico altamente especializado. / The nucleus accumbens (Acb) is subdivided in core and shell (AcbSh). There is evidence that accumbal subdivisions go beyond this core-shell dichotomy and that regions poor in tyrosine hydroxylase (TH) and calretinin (Calr) form a corridor system in the Acbsh. To better detail accumbal organization, we investigated the distribution of TH, Calr, DARPP-32 and of the dopamine transporter (DAT). Then we compared the distribution of these markers with that of the AMPA-type glutamate receptor subunits GluA2/3. Finally, we explored whether prefrontal afferents are directed to distinct AcbSh compartments. Our findings revealed that regions containing intensely labeled GluA2/3+ neurons form a corridor system in the AcbSh that coincides with regions poor in TH, Calr, and DAT. Anterograde tracing experiments indicated that only a small portion of the prefrontal afferents is specifically related to the corridors. Our findings delineated a complex corridor system in the AcbSh which might constitute a highly specialized neurochemical compartment.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glyphosate [N-(phosphonomethyl) glycine] is the most widely used pesticide in the world for controlling weeds in agriculture. High amounts of this herbicide and its major metabolite, aminomethylphosphonic acid (AMPA), have been found in water due to intensive use. The Ministry of Health 2914 establishes a maximum value allowed for the sum of glyphosate and AMPA at 0.5 mg L-1. The most comum method to determine these compounds requires a derivatization step for fluorescence detection. Thus, this study aimed to develop two simple and rapid methods for the direct determination of glyphosate and AMPA in water without treatment or sample derivatization steps, by capillary ion chromatography (Capillary IC) and liquid chromatography tandem mass spectrometry (LC-MS/MS). The Capillary IC method employed a capillary IonPac AS19 column (250 x 0.4 mm, 7.5 μm), a gradient elution with KOH a conductivity detector. The LC-MS/MS method used a Pursuit XRS C18 column (150 x 2 mm ID, 5 μm), an aqueous solution of ammonium hydroxide 0.01% (v/v) as mobile phase and a triple-quadrupole MS detector, operating in a selected reaction monitoring (SRM) mode. The method validation was performed by recovery experiments, evaluating accuracy, precision, linearity, limits of detection (LOD) and of quantification (LOQ). The analytical curves for glyphosate and AMPA obtained for the concentration levels 25, 50, 100, 250 and 500 μg L-1 by Capillary IC and LC-MS/MS showed r2 >0.99 for both compounds. For spiked levels of 25, 50, 250 and 500 μg L-1 recoveries were between 70 -113%, with RSD <10% for Capillary IC and 79-105%, with RSD <14% for LC-MS/MS. The method limits of detection for both glyphosate and AMPA was 8 μg L-1 and of quantification was 25 μg L-1, in both chromatographic techniques. Both methods showed good selectivity and sensitivity. Determination by direct injection of the sample, using capillary IC or LC-MS/MS proved to be efficient, simple and cost-effective tools for analysis, making it possible to monitor at levels below the maximum allowed limits for drinking water. / O glifosato [N-(fosfonometil)glicina] é o agrotóxico mais utilizado na agricultura mundial no controle de plantas daninhas. Altas quantidades desse herbicida e seu principal metabólito, o ácido aminometilfosfônico (AMPA), vem sendo encontrados em água devido ao uso intensivo. A portaria do Ministério da Saúde nº 2914 estabelece o valor máximo permitido para a soma de glifosato e AMPA em 0,5 mg L-1. O método amplamente utilizado para a determinação desses compostos é a HPLC sendo imprescindível uma etapa de derivatização para a detecção por fluorescência. Assim, este trabalho teve como objetivo desenvolver métodos simples e rápidos para a determinação direta de glifosato e AMPA em água para consumo humano, sem etapa de tratamento de amostra ou derivatização, empregando a cromatografia iônica capilar (CIC) e cromatografia líquida acoplada à espectrometria de massas em série (LC-MS/MS). O método por cromatografia iônica capilar empregou coluna capilar IonPac AS19 (250 x 0,4 mm; 7,5 μm), gradiente de eluição com gerador de KOH e detector de condutividade. Para LC-MS/MS utilizou-se coluna Pursuit XRs C18 (150 x 2 mm d.i.; 5 μm), solução aquosa de hidróxido de amônio 0,01% (v/v) como fase móvel e detector triplo quadrupolo (TQ) operando no modo de monitoramento de reações selecionadas (SRM). A validação dos métodos foi realizada através de ensaios de recuperação, avaliando exatidão, precisão, linearidade, limites de detecção (LD) e de quantificação (LQ). As curvas analíticas para glifosato e AMPA obtidas para os níveis 25; 50; 100; 250 e 500 μg L-1 por CIC e LC-MS/MS apresentaram coeficiente de determinação (r2) >0,99 para ambos os compostos. Para as fortificações nos níveis de 25, 50, 250 e 500 μg L-1 os compostos apresentaram recuperações de 70 a 113%, com RSD <10% por CIC e recuperações foram de 79 a 105%, com RSD <14% por LC-MS/MS. Os limites de detecção do método para AMPA e glifosato foi 8 μg L-1 e o de quantificação foi de 25 μg L-1, em ambas as técnicas cromatográficas. Ambos os métodos apresentaram boa seletividade e sensibilidade. A determinação, através da injeção direta da amostra, de glifosato e AMPA utilizando cromatografia iônica capilar ou LC-MS/MS demonstrou ser uma ferramenta eficiente, simples e economicamente viável para análise, permitindo o monitoramento em níveis abaixo do valor máximo permitido em água potável.

Neste trabalho investigamos a interação entre ATP-glutamato na modulação de potenciais de ação e atividade sináptica de neurônios PVN-RVLM, além de avaliar se esta interação induziria mudanças na atividade simpática lombar (ANSL) por estímulo osmótico. Utilizamos de técnicas de imunohistoquímica, whole-cell patch clamp e registro eletroneurográfico. Observou-se que o ATP aumenta a frequência de potenciais de ação em neurônios PVN-RVLM, efeito bloqueado por acido quinurênico (KYN) e PPADS. A injeção de ATP no PVN aumenta a ANSL (25 nmol: 72%), um efeito atenuado por PPADS e/ou KYN, e também por CNQX. O ATP não afeta a função sináptica, mas aumenta correntes glutamatérgicas induzidas por aplicação AMPA em 52%, a qual foi bloqueada por PPADS ou por quelação de Ca2+ intracelular. Além disso, o estímulo osmótico ativa neurônios do PVN que expressam receptores P2X2 e potencia as correntes mediadas por AMPA (53%), um efeito bloqueado por PPADS. Finalmente, demonstrou-se que receptores P2 no PVN são importantes na simpatoexcitação induzida por estímulo osmótico agudo. / In the present study we investigate the interaction of ATP-glutamate on the firing activity and synaptic function in PVN-RVLM neurons, besides whether that interaction would be translated in changes on sympathetic nerve activity (SNA) induced by osmotic stimulus. Immunohistochemistry, whole-cell patch clamp and electroneurography technical approaches were used. Our data have shown that ATP increases firing rate of PVN-RVLM neurons, an effect blocked by kynurenic acid (KYN) or PPADS. ATP injection into the PVN enhanced SNA (72%), which was attenuated by PPADS and/or KYN, or CNQX. ATP did not affect synaptic function but, glutamatergic currents evoked by AMPA application were augmented with ATP (AMPA area: 52%), blocked by PPADS and chelation of intracellular Ca2+. In addition, we observed that acute osmotic stimulus activates P2X2 expressing neurons in the PVN. Moreover, an osmotic challenge potentiated AMPA responses (53%), an effect blocked by PPADS. Finally, we demonstrated that P2 receptors in the PVN are important for osmotically-driven sympathoexcitation.