http://www.pharmacol.usyd.edu.au/thesis This thesis is primarily concerned with a class of chemical compounds known as pyridazinediones, being 6-membered aromatic rings containing two adjacent nitrogen atoms (pyridazine), doubly substituted with oxygen. In particular, the work focuses on pyridazine-3,6-diones, derivatives of maleic hydrazide (1). Understanding of the chemistry of these compounds is extended, using theoretical and synthetic techniques. This thesis is also concerned with two very important classes of receptors which bind amino acids in the brain: firstly, the inhibitory GABA receptor, which binds g-aminobutyric acid (GABA) (2) in vivo, and for which muscimol (3) is an agonist of the GABAA subclass; secondly, Excitatory Amino Acid (EAA) receptors, which bind glutamate (4) in vivo, and in particular the AMPA subclass, for which (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) (5) is an agonist. The connection between pyridazinediones and amino acid receptors is the design, synthesis, and evaluation of structures based on pyridazinediones as potential GABA and EAA receptor ligands. Techniques of theoretical chemistry, molecular modelling, synthetic chemistry, and in vitro pharmacology are used to explore pyridazine-3,6-dione derivatives as ligands.

Le syndrome de Coffin-Lowry (CLS), une déficience intellectuelle liée à l'X, est causée par des mutations du gène RPS6KA3 codant pour la kinase RSK2 régulée par les facteurs de croissance.Pour comprendre les conséquences du déficit en RSK2 dans l'hippocampe nous avons effectué une comparaison des profils d'expression génique d'hippocampes de souris Rsk2-KO et WT. Elle a révélé l'expression différentielle de 100 gènes, codant pour des protéines agissant dans divers processus biologiques. Nous avons analysé les conséquences de la dérégulation de l'un de ces gènes Gria2 codant pour GluR2, une sous-unité du récepteur glutamate AMPA. Un niveau d'expression doublé de GluR2 a été relevé dans l'hippocampe des souris Rsk2-KO et les études électrophysiologiques y ont révélé une réduction des transmissions AMPAR et NMDAR. L’activité de ERK1/2 était aussi anormalement augmentée dans l'hippocampe des souris Rsk2-KO, ainsi que le niveau de P-Sp1. Ensemble, mes résultats ont suggéré que la surexpression de GluR2 dans les neurones déficients en RSK2, était causée par une augmentation de l'activité transcriptionnelle de Sp1 sur le gène Gria2, qui, elle-même, est le résultat de l’augmentation anormale de l’activité de ERK1 / 2. / Coffin–Lowry Syndrome (CLS), an X-linked form of intellectual disability, is caused by mutations of the RPS6KA3 gene encoding the growth factor regulated kinase RSK2. To understand the consequences of RSK2 deficiency in the hippocampus we performed a comparison of the hippocampal gene expression profiles from Rsk2-KO and WT mice. It revealed differential expression of 100 genes, encoding proteins acting in various biological pathways. We further analyzed the consequences of deregulation of one of these genes, Gria2 encoding GluR2, a subunit of the glutamate AMPAR. An abnormal two-fold increased expression of GluR2 was found in the hippocampus of Rsk2-KO mice. Electrophysiology studies showed a reduction of basal AMPAR and NMDAR mediated transmission, in the hippocampus of Rsk2-KO mice. Activity of ERK1/2 was also abnormally increased in the adult hippocampus of Rsk2-KO mice. P-Sp1 level was also significantly higher in RSK2 deficient cells. Together, my results suggested that over expression of GluR2 in RSK2 deficient cells, is caused by increased Sp1 transcriptional activity on the Gria2 gene, which, itself, is the result of ERK1/2 increased signaling.

Este estudo visou observar os efeitos plásticos induzidos pelo exercício a curto prazo em regiões motoras do encéfalo de ratos. Observou-se a expressão das subunidades GluR1 e GluR2/3. Os animais foram divididos em grupos de: 3 dias(COR3), 7 dias(COR7) e 15 dias(COR15); e um grupo controle(CONT). Empregaram-se as técnicas de imuno-histoquímica e immunoblotting. A expressão de GluR1 no cerebelo, demonstrou um decréscimo em COR3. No hipocampo houve uma queda na expressão em COR3(40%), retornando aos níveis basais em COR7. No córtex cerebral observou-se uma queda da expressão com máxima queda em COR7(52%), retornando à expressão basal em COR15. O estriado não sofreu alterações na expressão de GluR1 ao longo dos primeiros 7 dias, tendo um aumento em COR15(90%). A expressão de GluR2/3 não foi alterada, exceto no cerebelo, onde houve um decréscimo em dois momentos distintos, COR3(55%) e COR15(25%), retornando à expressão basal em COR7. Os nossos dados revelam que o exercício físico a curto prazo foi capaz de promover alterações plásticas ao longo do treinamento. / This study aimed at analyzing the plastic effects of the short-term exercise upon the rat motor area. We check the expression of GluR1 and GluR2/3. We divided into 3 experimental groups based on duration of exercise: 3 days(COR3), 7 days(COR7), and 15 days(COR15); and a control group(CONT). The experimental animals were subjected to a treadmill exercise protocol. The brains were subjected to the techniques of immunohistochemistry and immunoblotting. In the cerebellum, there was a decrease for COR3(17%). In the hippocampus, there was a decrease of the GluR1 expression for COR3 (40%). In the cerebral cortex there was a drop of GluR1 expression for COR3 and COR7(52%). In the striatum, there was no change of GluR1 expression during the first seven days, with a increase for COR15(90%). The GluR2/3 expression did not change in any brain structure analyzed, except in the cerebellum, where there was a significant decrease for two distinct groups, COR3(55%) and COR15(25%). Our data show that short-term physical exercise was able to promote plastic changes during training.

Lithium is a drug used to treat mood disorders and also has many side effects, including central nervous system (CNS) complications (such as cognitive dulling), associated with its use. The mechanism of its action still remains unknown. Over the years, many leads have started emerging. It has been shown to inhibit several enzymes in the cell and has been implicated in altering many neurotransmitter systems and signal transduction pathways (serotonin, dopamine and norepinephrine neurotransmissions). Effect of exposure to therapeutic levels of lithium on mature glutamatergic synapses is being studied and several changes in glutamate receptor subtypes have already been reported. Effects of lithium on developing glutamatergic synapses have not been studied. The thesis tries to document and understand the changes brought about by long term lithium treatment on developing glutamatergic synapses in vitro in hippocampal neuronal cultures. In the present work, patch clamp technique was used to monitor the changes in the postsynapse and fluorescence imaging to study the presynaptic changes. The hippocampal neuronal cultures were treated with 1 mM lithium for 6 days during the synaptogenesis stage (DIV 4-10) and termed as chronic Li treatment (CLi). Following CLi treatment the changes occurring in amplitude and rectification property of the AMPA receptor (AMPAR), a subtype of glutamate ionotropic receptor, mediated miniature excitatory postsynaptic currents (mEPSCs) have been reported (Chapter III). Lithium inhibits protein kinase A (PKA), glycogen synthase kinase–3β (GSK-3β) and glutamate reuptake. Effect of inhibiting PKA, GSK-3β and glutamate reuptake was also studied with a view to understand the molecular basis of lithium action on AMPAR mEPSCs (Chapter IV). It was found that chronic lithium treatment (CLi) caused a reduction in the mean amplitude of mEPSCs mediated by AMPARs and also changed the rectification property of these receptors from being more outwardly rectifying to being more inwardly rectifying, an indication probably of increase in contribution of Ca2+-permeable AMPARs to the synaptic events. AMPAR events in chronic lithium treated cultures were more sensitive to both N-acetyl spermine (NASPM) application and di-fluoro-methyl-ornithine (DFMO) treatment, both specific to Ca2+-permeable AMPARs, indicating that there was an increase in the contribution from Ca2+-permeable AMPARs to the synaptic events. PKA inhibition with H-89 treatment (starting from DIV 4 (for 6 days)) reduced the mean amplitude of AMPAR mEPSCs and increased the mean rectification index (RI). GSK-3β inhibition with SB415286 (starting from DIV 4 (for 6 days)) did not alter the mean mEPSC amplitude but reduced the mean RI. Transient (24 hrs) glutamate reuptake inhibition with threo-β-Hydroxy-Aspartic-Acid (THA) at DIV 4 followed by a period of recovery led to smaller amplitudes but no change in RI. The 24 hr glutamate reuptake block on DIV 4 had long term effects. It led to an increase in AMPAR mEPSC frequency while AMPAR mEPSC amplitudes were reduced. The mean RI decrease seen when glutamate reuptake was blocked for 24 hrs on DIV 10, was absent in DIV 4 THA treated neurons. However, when the neuronal cultures were maintained in the presence of PKA and GSK-3β inhibitors, the DIV 4 THA treated neurons showed AMPAR mEPSC characteristics similar to CLi neurons. Thus, it was seen that individual inhibition of PKA, GSK-3β and glutamate reuptake did not lead to changes in AMPAR mEPSCs similar to that seen in lithium treated neurons. The effect of lithium exposure during synapse development on AMPARs could be reproduced closely by co-inhibiting PKA, GSK-3β and glutamate reuptake. Using the styryl dye FM1-43, the changes induced in presynaptic release by a similar chronic lithium treatment was studied (Chapter V). It was found that lithium exposure (1 mM, DIV 4-10) brought down the extent of dye loading, destaining and also slowed down the rate of dye loss in response to high KCl stimulation (the τfast component of destaining was significantly slower). Minimum loading experiments did not reveal any difference in mode of exocytosis (kiss and run/full-collapse) in control and lithium treated cultures. Chlorpromazine treatment (that inhibits clathrin-mediated endocytosis) affected dye loading to a lesser extent in lithium treated cultures as compared to control. Surprisingly, exposure to hyperosmotic solution 10 minutes after dye wash out boosted the extent of dye loading and destaining in lithium treated cultures (a phenomenon not seen in control). This could happen if the FM1-43 is trapped away from the wash solution during the wash period. This would be possible if endocytosis in CLi takes place, differently from control, through a process involving membrane infoldings similar to bulk endocytosis albeit a slower/compromised one. Taken together, the data presented here indicates that lithium treatment during synaptogenesis affects vesicular recycling mostly at the endocytosis and docking/priming steps (mobilization of vesicles for release). Lithium treated cultures also did not show the high KCl associated presynaptic potentiation observed in control which is a significant finding. In conclusion, chronic lithium treatment affected both the presynaptic and postsynaptic compartments of the glutamatergic synapse. The effect of lithium on AMPAR mEPSC could not be reproduced by individual inhibitions of biochemical effectors but by multiple inhibitions. Thus, the study done here underscores the need to look at the manifold effect of lithium in an integrated way. The study also might have implications in understanding the CNS complications seen in patients taking lithium treatment and in babies perinatally exposed to lithium.

Les champs récepteurs corticaux se réorganisent en réponse aux changements de l'environnement. Par exemple, suite à une lésion périphérique, les modalités sensorielles préservées gagnent de l'espace cortical au détriment de celles lésées. L'étude du cortex somatosensoriel en tonneau des rongeurs a fourni des données importantes pour la compréhension des mécanismes synaptiques à l'origine de cette réorganisation corticale. En condition normale, les neurones de chaque colonne corticale répondent préférentiellement à la stimulation d'une seule vibrisse principale ("Principal Whisker, PW"). Au contraire, suite à l'amputation de l'ensemble des vibrisses sauf une ("Single Whisker Experience, SWE"), les neurones des colonnes associées aux vibrisses amputées répondent à la stimulation de la vibrisse conservée, à l'origine du renforcement et de l'expansion des représentations corticales des vibrisses conservées. Bien que des preuves indirectes aient révélées un rôle de la potentialisation à long terme ("Long-Term Potentiation, LTP") de synapses préexistantes dans la modification des cartes corticales, probablement via une augmentation du nombre des récepteurs AMPA (AMPARs) aux synapses, un lien direct entre la LTP, la réorganisation des cartes corticales, et l'adaptation des comportements sensori-moteurs suite à une altération des entrées sensorielles n'a pas encore été démontré. L'objectif de cette thèse a donc été de mettre en évidence cette relation de façon expérimentale et en condition physiologique. Pour cela, nous avons mis au point une stratégie in vivo combinant des enregistrements électrophysiologiques, de l'imagerie biphotonique et l'analyse du comportement d'exploration chez la souris contrôle ("Full Whisker Experience, FWE) et amputée de certaines vibrisses (SWE). Nous avons d'abord confirmé que la stimulation rythmique de la PW ("Rhytmic Whisker Swtimulation, RWS") renforce les synapses excitatrices (RWS-LTP) in vivo des souris anesthésiées FWE. Au contraire des souris FWE, les neurones pyramidaux des souris SWE présentent une augmentation de l'excitabilité neuronale et une absence de RWS-LTP, indiquant ainsi que les synapses corticales associées à la vibrisse intacte ont été potentialisées en réponse au protocole SWE. Pour mieux comprendre l'implication de la RWS-LTP dans la réorganisation des cartes corticales et l'adaptation des comportements sensori-moteurs, nous avons développé une nouvelle approche pour manipuler la LTP in vivo grâce à l'immobilisation des AMPARs par des anticorps extracellulaires ("cross-linking"). En effet, notre équipe a montré précédemment que le cross-linking des AMPARs empêche la LTP in vitro. Par ailleurs, une accumulation des AMPARs au niveau post-synaptique a été démontrée in vivo par imagerie biphotonique au cours d'une stimulation RWS, suggérant un rôle de la mobilité de ces récepteurs dans cette RWS-LTP. Au cours de cette thèse, nous avons démontré que le cross-linking des AMPARs in vivo bloque également l'expression de la RWS-LTP, mais sans affecter la transmission synaptique basale, ni l'induction de la RWS-LTP, indiquant ainsi que la mobilité des AMPARs est également fondamental pour l'expression de la LTP in vivo. De façon importante, le cross-linking des AMPARs de façon chronique, au cours du SWE, permet non seulement de rétablir la RWS-LTP et l'excitabilité neuronale, et donc de bloquer la réorganisation corticale, mais aussi de modifier les capacités de récupération sensori-motrices des souris amputées. Dans l'ensemble, nos données démontrent pour la première fois un rôle critique et direct de la RWS-LTP dans le réarrangement des circuits en réponse à l'amputation de certaines vibrisses. La réorganisation des cartes corticales serait ainsi assurée par le renforcement de la transmission synaptique, et constituerait alors un mécanisme compensatoire pour optimiser le comportement sensorimoteur de l'animal lors de l'altération des entrées sensorielles. / Neuronal receptive fields in the cerebral cortex change in response to peripheral injury, with active modalities gaining cortical space at the expense of less active ones. Experiments on the mouse whisker-to-barrel cortex system provided important evidences about the synaptic mechanisms driving this cortical remapping. Under normal conditions, neurons in each barrel-column have receptive fields that are strongly tuned towards one principal whisker (PW). However, trimming all the whiskers except one (single-whisker experience, SWE) causes layer (L) 2/3 pyramidal neurons located in the deprived and spared-related columns to increase their response towards the spared input. This results in a strengthening and expansion of the spared whisker representation within the barrel sensory map. Indirect evidences suggest that these cortical alterations might depend on the activity-dependent potentiation of pre-existing excitatory synapses (LTP), likely through increased levels of postsynaptic AMPA receptors (AMPARs). However, a clear link between LTP, cortical remapping, and the adaptation of sensorimotor skills following altered sensory experience has not yet convincingly been demonstrated. Here, we combined in vivo whole-cell recordings, 2-Photon calcium imaging and a whisker-dependent behavior protocol to directly demonstrate this relationship. It has been described that rhythmic whisker stimulation potentiates cortical synapses (RWS-LTP) in vivo. An accumulation of postsynaptic AMPARs during similar sensory stimulation was also reported by imaging evidences. Our data demonstrates that this potentiation is occluded by SWE, suggesting that cortical synapses are already potentiated by this trimming protocol. This is translated into an increased neuronal excitability in the spared column and sensorimotor recovery by the spared whisker. To better understand the implication of LTP in cortical remapping, we developed a novel approach to manipulate LTP in vivo without affecting overall circuit properties. Our team showed previously that the blockage of AMPARs synaptic recruitment by extracellular antibody cross-linking prevents LTP in vitro. Here, we report that in vivo cross-linking of AMPARs blocks the expression but not the induction of RWS-LTP, suggesting that the synaptic recruitment of AMPARs is fundamental for in vivo LTP as well. Moreover, chronic AMPAR cross-linking during SWE reverts RWS-LTP occlusion and the increased neuronal excitability caused by whisker trimming. As consequence, the sensorimotor performance by the spared whisker is permanently impaired by the blockage of cortical remapping. Altogether, these evidences led us to define a critical role for synaptic LTP on circuit re-arrangement after whisker trimming. Our data shows that LTP-driven cortical remapping is a compensatory mechanism to optimize animal’s sensorimotor behavior upon altered sensory experience.

Ionotrope Glutamatrezeptoren steuern exzitatorische Prozesse im Gehirn. Der AMPA-Rezeptor ist der schnellste Glutamatrezeptor und reguliert die Signaltransduktion in hochfrequenten Bereichen. Um die Konformationen des Rezeptors für die Aktivierung aufzuschlüsseln, wurden die Ligandenbindedomänen (LBD), mit künstlichen Metallbrücken verlinkt. Die tetramerische LBD-Schicht hat mehrere Freiheitsgrade. Sie reichen vom Schließen der LBD durch Ligandenbindung, bis hin zu den verschiedenen Konformationsmöglichkeiten innerhalb des Tetramers. Die bereits publizierte T1-Mutante wurde verlinkt und auf Einzelkanalebene elektrophysiologisch untersucht. Anschließend wurde T1 mit einer Mutante (DKD-3H) verglichen die durch molekulardynamische Simulationen identifiziert wurde. Die Eigenschaften von T1 und DKD-3H wurden mit einem natürlich vorkommenden AMPAR-Linker, namens Con ikot ikot, verglichen. Meeresschnecken der Gattung Conus setzen das Polypeptid zur Fischjagd ein. Zur Analyse wurde eine Software entwickelt, speziell für Ströme von ligandengesteuerten Ionenkanälen mit multiplen Leitfähigkeiten (www.github.com/AGPlested/ASCAM). Durch Verlinkung in T1 konnten drei präaktive Konformationen aufgeschlüsselt werden und die schnelle Aktivierung im sub-Millisekundenbereich verschwindet ganz. Obwohl die Metall-koordinierenden Residuen von T1 und DKD-3H nah beieinander liegen, konnte für DKD-3H nur eine einzelne präaktive Konformation gefunden werden. Es deutet darauf hin, dass die LBD-Schicht von AMPA-Rezeptoren hochdynamisch ist da die geringe Abweichung große Auswirkungen auf den Phänotypen des Rezeptors hat. LBD-Verlinkung mit Con-ikot-ikot ergab einen EC50 von ~5 nM. Alle drei Verlinkungsarten resultierten in einer reduzierten Offenwahrscheinlichkeit. Einzelkanalanalysen von T1 und Con-ikot-ikot-gebundenen AMPA-Rezeptoren unterstützen die Hypothese, dass die veränderten Phänotypen aus dem Wechselspiel der LBDs innerhalb der LBD-Schicht resultieren. / Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the mammalian brain. The AMPA-type receptor is the fastest iGluR member, activating at the sub-millisecond time scale. In this study, ligand-binding domains (LBDs), where AMPAR activation is initialised, were cross-linked with artificially introduced metal bridges to trap and investigate conformational states of the receptor before- and during activation. The tetrameric LBD layer has multiple degrees of freedom, from closure of individual clamshells and their conformational arrangement within the dimers and the tetramer. The previously studied T1 mutant was cross-linked at the single-channel level and compared to a second cross-linking mutant (DKD 3H) that was predicted on a molecular dynamics approach. T1 and DKD 3H were to the effects of a naturally occurring AMPAR-specific LBD cross-linker (Con ikot ikot) from a fish-hunting snail of the genus Conus. Single-channel recordings were analysed with a newly developed software dedicated to ligand-gated ion channels with conductances in the low pA range and multiple subconductance levels (www.github.com/AGPlested/ASCAM). Trapping of the T1 mutant revealed three pre-active conformational states that were not described for AMPARs before. Fast activation in the sub-millisecond range disappears fully indicating that these conformations proceed too fast in non-restricted wild-type receptors to be resolved. Although the metal-coordinating residues of T1 and DKD 3H are in close proximity, DKD 3H yielded one single pre-active state. A slightly distinct register of the bridge results in largely different phenotypes. Cross-linking with con ikot ikot identified an EC50 of ~5 nM. All three cross-linking approaches reduced open probability. The results support the hypothesis that the altered phenotypes are not a result of singly restricted LBDs but the dynamics of the tetrameric LBD-layer as one unit.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18222号 / 薬科博第26号 / 新制||薬科||4(附属図書館) / 31080 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 岡村 均, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20555号 / 理博第4313号 / 新制||理||1619(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 平野 丈夫, 教授 森 和俊, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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