The hippocampus is an integral brain region for affective disorders. HCN1 protein shows age-dependent increase in expression resulting in an increase in I[subscript h] in the dorsal hippocampal CA1 region. TRIP8b knockout mice lacking functional HCN channels as well as both HCN1 and HCN2 knockout mice have been shown to display antidepressant-like behaviors. The mechanisms or brain regions involved in these alterations in behavior, however, are not clear. We developed a lentiviral shRNA system to examine whether knockdown of HCN1 protein, and therefore h-channels, in the dorsal hippocampal CA1 region is sufficient to produce antidepressant-like effects. We found that silencing of HCN1 gene resulted in physiological changes consistent with a reduction of I[subscript h] and increased cellular excitability of CA1 pyramidal neurons. Rats infused with lentiviral-shRNA-HCN1 in the dorsal hippocampal CA1 region displayed antidepressant- and anxiolytic-like behaviors. Using voltage-sensitive dye imaging, we found that knockdown of HCN1 in the dorsal hippocampal CA1 region led to enhancement of hippocampal activity in large regions of the dorsal hippocampus. Our results demonstrate that changed hippocampal network activity by local manipulation of HCN1 channels in dorsal hippocampus led to anxiolytic- and antidepressant-like behaviors and suggest that HCN1 channels could be a potential target for treatment of anxiety and depression. / text

Elevations in extracellular potassium (K+e) accompany seizure-like events (SLEs), but elevated K+ may also participate in seizure cessation. The objective of this thesis was to investigate the possibility that K+ may undergo a pro- to anti-convulsant switch in the epileptic juvenile (postnatal day 17-21) rat hippocampus. Field recordings were performed in the CA1 pyramidal layer. SLEs and primary afterdischarges (PADs) were induced with 0.25 mM Mg/5 mM K+ perfusion or tetanic stimulation of the Schaffer collaterals respectively. In these seizure models, elevating [K+]e beyond 7.5 mM showed anticonvulsant properties. The addition of ZD7288, a blocker of the hyperpolarization activated nonspecific cationic current (Ih) and allowed SLEs to continue even in elevated [K+]e. This suggests that [K+]e switches from being pro- to anti-convulsant, in part due to an elevated [K+]e-induced potentiation of Ih. Ih likely contributes to this anticonvulsant behavior by decreasing membrane resistance and subsequently attenuating summation of incoming EPSPs.

Elevations in extracellular potassium (K+e) accompany seizure-like events (SLEs), but elevated K+ may also participate in seizure cessation. The objective of this thesis was to investigate the possibility that K+ may undergo a pro- to anti-convulsant switch in the epileptic juvenile (postnatal day 17-21) rat hippocampus. Field recordings were performed in the CA1 pyramidal layer. SLEs and primary afterdischarges (PADs) were induced with 0.25 mM Mg/5 mM K+ perfusion or tetanic stimulation of the Schaffer collaterals respectively. In these seizure models, elevating [K+]e beyond 7.5 mM showed anticonvulsant properties. The addition of ZD7288, a blocker of the hyperpolarization activated nonspecific cationic current (Ih) and allowed SLEs to continue even in elevated [K+]e. This suggests that [K+]e switches from being pro- to anti-convulsant, in part due to an elevated [K+]e-induced potentiation of Ih. Ih likely contributes to this anticonvulsant behavior by decreasing membrane resistance and subsequently attenuating summation of incoming EPSPs.

Voltage-dependent channels on the dendrites of motoneurons provide additional current that amplifies or dampens synaptic current en route to the soma. The specific consequences will depend on the density and distribution of the voltage-dependent channels. HCN channels generate a positive inward current in response to hyperpolarization. HCN channels are responsible for a resonance phenomenon in motoneurons where inputs of certain frequencies are preferentially amplified. Modelling studies indicate that this resonance behaviour only occurs if HCN channels are uniformly distributed on the dendritic tree. However, the distribution of HCN channels on the dendrites of motoneurons is unknown. Furthermore, current techniques for measuring channel density on dendrites suffer from methodological limitations that prevent sampling on a scale necessary to map the distribution of voltage-dependent channels across the dendritic tree. The goal of the present study is to develop a high throughput method for measuring the membrane-associated density of voltage-dependent channels using immunohistochemical, confocal and three-dimensional image analysis techniques. Secondly, the proximal to distal distribution of membrane-associated channels formed by the HCN1-subunit on the dendritic trees of trapezius motoneurons in the adult feline will be compared. Antidromically identified motoneurons innervating the trapezius muscle were intracellularly stained in order to visualize the entire dendritic tree. HCN1-subunit containing channels were labeled with a specific antibody. Dendritic segments (n=27 to 92) of ten trapezius motoneurons at different distances from the soma were acquired using confocal microscopy and rendered into a three-dimensional volume. The perimeter of the intracellular stain was used to define the membrane-cytoplasmic border. Analysis of the HCN1 immunoreactivitywas constrained to this perimeter. This technique provides a means of extracting membrane-associated HCN1 labeling and therefore the functional distribution of HCN1 channels. The density of membrane-associated HCN1 immunoreactvity across the dendritic tree was either uniform or increased with distance from the soma among the ten trapezius motoneurons. The increase in HCN1 density with distance from the soma was inversely related to the density of HCN1 on the soma and proximal dendrites. Lastly, the change in HCN1 density with distance from the soma was inversely related to the total input conductance of the motoneuron. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-08-01 13:59:32.403

Universidade Federal do Pará, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. / The brazilian Amazon region maintains the largest variety of arboviruses and the state of Pará is responsible for 26% of this territory, so the major goal of this work was to determine the prevalence and distribution of HI antibodies to 19 domestic arbovirus in domestic herbivores in the state of Pará and to standardize an indirect sandwich IgG ELISA test to serum samples of equines, cattle, buffaloes and sheep. In all species studied and throughout the State of Pará a large prevalence of HI antibodies to all arbovirus analyzed was observed and SLEV, ILHV, EEEV, MAGV and WEEV, showed higher prevalence, where the SLEV was the most prevalent. Regarding the virus families HI antibodies to MAGV was the most prevalent Bunyaviridae in all species, the most prevalent Flaviviridae was SLEV in all species and in the family Togaviridae the EEEV was more prevalent in horses. In order to analyze the prevalence of HI antibodies by animal species was observed that horses did not show significant differences compared to buffaloes, however, showed significant difference compared to cattle and sheep; there was not observed significant difference between the ruminant species. Using sandwich indirect IgG ELISA a large number of crossed reactions were found. This enzymatic test can be used to detect IgG antibodies among families of arboviruses studied. / A região Amazônica brasileira mantém a maior variedade de arbovírus e o estado do Pará corresponde a 26% desse território, o presente trabalho teve como objetivo determinar a prevalência e a distribuição de anticorpos detectados por inibição de hemaglutinação (IH) para 19 arbovírus em herbívoros domésticos no estado do Pará e padronizar testes de ELISA sanduíche indireto em equinos, bovinos, bubalinos e ovinos. Em todas as espécies de animais estudadas e em todo estado do Pará ocorreu detecção de anticorpos para todos os arbovírus analisados dentre os quais os SLEV, ILHV, EEEV, MAGV e WEEV apresentaram maior prevalência de anticorpos IH, sendo o SLEV o mais prevalente. Na detecção de anticorpos para diferentes famílias de arbovírus o MAGV foi o mais prevalente da família Bunyaviridae em todas as espécies, o SLEV foi o mais prevalente da família Flaviviridae em todas as espécies, na família Togaviridae o EEEV foi mais prevalente em equinos. Ao analisar a prevalência de anticorpos IH por espécie animal foi observado que os equinos não apresentaram diferença significativa em relação aos bubalinos, porém, apresentaram diferença significativa maior em comparação aos bovinos e ovinos, não havendo diferença significativa entre as espécies de ruminantes. O uso de ELISA IgG sanduíche indireto apresentou grande frequência de reações sorológicas cruzadas entre as famílias de arbovírus estudadas.

Pendant des décennies, la plasticité synaptique a été considérée comme le substrat principal de la plasticité fonctionnelle cérébrale. Récemment, plusieurs études expérimentales indiquent que des régulations à long terme de l’excitabilité intrinsèque participent à la plasticité dépendante de l’activité. En effet, la modulation des canaux ioniques dépendants du potentiel, lesquels régulent fortement l’excitabilité intrinsèque et l’intégration des entrées synaptiques, a été démontrée essentielle dans les processus d’apprentissage. Cependant, la régulation, dépendante de l’activité, du courant ionique activé par l’hyperpolarisation (Ih) et ses conséquences sur l’induction de futures plasticités reste à éclaircir, tout comme la présence d’une régulation de conductances dépendantes du potentiel dans les neurones inhibiteurs. Dans la première partie de ma thèse, nous caractérisons les mécanismes d’induction et d’expression de la plasticité à long terme de l’excitabilité (LTP-IE) dans les interneurons en panier de la région CA1 exprimant la parvalbumine. Dans une seconde partie, le rôle de Ih dans la régulation homéostatique de l’excitabilité neuronale induite par des manipulations de l’activité neuronale dans sa globalité a été étudié. Dans la troisième étude, nous montrons que la magnitude de la Dépression à Long Terme (LTD) détermine le sens de la régulation de Ih dans les neurones pyramidaux de CA1. En conclusion, cette thèse montre qu’à la fois dans les neurones excitateurs et inhibiteurs, les régulations des conductances dépendantes du potentiel aident à maintenir une relative stabilité dans l’activité du réseau. / Synaptic plasticity has been considered for decades as the main substrate of functional plasticity in the brain. Recently, experimental evidences suggest that long-lasting regulation of intrinsic neuronal excitability may also account for activity-dependent plasticity. Indeed, voltage-dependent ionic channels strongly regulate intrinsic excitability and inputs integration and their regulation was found to be essential in learning process. However, activity-dependent regulation of the hyperpolarization-activated ionic current (Ih) and its consequences for future plasticity remain unclear, so as the presence of any voltage-dependent conductances regulation in inhibitory neurons. In the first part of this thesis, we report the characterization of the induction and expression mechanisms of Long-Term Potentiation of Intrinsic Excitability (LTP-IE) in CA1 parvalbumin-positive basket interneurons. In a second part, the role of Ih in the homeostatic regulation of intrinsic neuronal excitability induced by global manipulations of neuronal activity was reported. In the third experimental study, we showed that the magnitude of Long-term Depression (LTD) determines the sign of Ih regulation in CA1 pyramidal neurons. In conclusion, this thesis shows that in both excitatory and inhibitory neurons, activity-dependent regulations of voltage-dependent conductances help to maintain a relative stability in the network activity.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21692号 / 医科博第96号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 渡邉 大, 教授 伊佐 正, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Les canaux nucléotidiques cycliques activés par hyperpolarization (HCN) sont un type de canaux ioniques voltage-dépendants qui contrôlent l'activité rythmique et la plasticité synaptique dans le cœur et le cerveau. Ces canaux permettent aux ions K+ et Na+ de passer, créant ainsi un courant entrant lors de l'hyperpolarization de la membrane. En raison de ses propriétés biophysiques inhabituelles, ce courant est appelé courant «If» ou courant d'hyperpolarization «Ih». Des anomalies du courant Ih sont associées à des arythmies et des troubles neurologiques, y compris l'épilepsie. On constate que différentes molécules modulent ce courant. Des résultats expérimentaux ont montré que les lipides jouent un rôle dans le déplacement de la dépendance en tension des canaux HCN vers des tensions plus positives ou dépolarisées. Le phosphatidylinositol 4,5-bisphosphate de phospholipide endogène et exogène, ou PI (4,5) P2, régule les canaux HCN en déplaçant l'ouverture du canal vers une tension plus dépolarisée. Cette modulation est supposée être par interaction directe de PI (4,5) P2 avec le canal HCN. Ici, nous utilisons la dynamique moléculaire et l'amarrage pour explorer et identifier le site de liaison grâce à l'analyse des contacts et de la stabilité des liaisons hydrogène impliquées dans les molécules de phsiphoinositide et l'interaction des canaux HCN. Nous proposons LYS et ARG du domaine HCN et S3 pour être des résidus clés dans le site de liaison à travers lequel les molécules de phosphoinositide peuvent potentiellement activer le canal. / Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a type of voltage gated ion channels that control rhythmic activity and synaptic plasticity in the heart and brain. These channels allow K+ and Na+ ions to pass, thereby creating an inward current upon hyperpolarization of the membrane. Due to its unusual biophysical properties, this current is called funny « If» or hyperpolarization « Ih » current. Abnormalities in Ih current are associated with arrythmia and neurological disorders including epilepsy. Different molecules are found to modulate this current. Experimental results have shown that lipids play a role in shifting the voltage dependence of HCN channels to more positive, or depolarized voltages. Both endogenous and exogenous phospholipid phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P2, regulates HCN channels by shifting the opening of the channel to a more depolarized voltage. This modulation is postulated to be through direct interaction of PI(4,5)P2 with the HCN channel. Here, we use molecular dynamics and docking to explore and identify the binding site through analysis of the contacts and stability of the hydrogen bonds involved in phosphoinositide molecules and HCN channel interaction. We propose LYS and ARG residues of the HCN domain and S3 to be key residues in the binding site through which phosphoinositide molecules can potentially activate the channel.

Il a été préalablement montré dans les neurones pyramidaux de CA1 qu'en plus d'une plasticité synaptique à long terme, les protocoles de tétanisation des afférences (HFS/LFS) induisent une plasticité synergique de l'intégration des messages synaptiques. Dans ce contexte, nous avons abordé les questions suivantes: 1) des changements d'intégration dendritiques sont-ils associés à la STDP ? 2) quels sont les mécanismes d'expression de la facilitation de l'intégration observée après la LTP ? et 3) dans quelle mesure l'activité synaptique induit des changements persistants de l'excitabilité des interneurones GABAergiques ?<br />Nos résultats montrent que la règle préétablie de STDP pour la plasticité synaptique est aussi valide pour la plasticité de l'intégration et décrit aussi parfaitement les changements de la relation amplitude/pente des PPSE observés en parallèle. Ces changements sont spécifiques de la voie synaptique activée et nécessitent l'activation des récepteurs NMDA. <br />La plasticité de l'intégration met en jeu une régulation des conductances voltage-dépendantes présentes à la surface neuronale. Une réduction locale dendritique, NMDAR-dépendante du courant Ih est observée lors de l'augmentation d'intégration associée à la LTP. En présence de bloqueurs pharmacologiques du courant Ih, la LTP est toujours présente mais la facilitation de l'intégration n'est plus observée. Finalement, une facilitation de l'intégration similaire à celle observée après induction de la LTP est induite quand la conductance h est réduite dans la dendrite par la technique de courant imposé dynamique en temps réel.<br />Des changements de l'excitabilité neuronale ne sont pas restreints aux neurones pyramidaux et nous montrons qu'une augmentation de l'excitabilité intrinsèque, dépendante des récepteurs mGluR de type I est également observée dans certains interneurones GABAergiques de CA1 après une HFS. Cette augmentation d'excitabilité pourrait permettre de maintenir l‘équilibre entre excitation et inhibition au sein du réseau hippocampique en facilitant le recrutement des interneurones à la suite d'épisodes d'hyperactivité.<br />Nos résultats montrent donc qu'en parallèle des modifications de l'efficacité synaptique, des modifications de l'excitabilité intrinsèque des neurones peuvent participer au processus de stockage de l'information et permettent également de maintenir l'activité neuronale à un niveau physiologique.