O transtorno do pânico (TP) é um transtorno de ansiedade caracterizado por ataques de pânico recorrentes e inesperados, com um prognóstico crônico. Entre as drogas utilizadas no tratamento do TP, os benzodiazepínicos (BZs) de alta potência, como o alprazolam e o clonazepam, apresentam a vantagem de serem eficazes logo no início do tratamento. Assim como outras drogas BZs, tais como o diazepam e o flurazepam, estes compostos também são empregados como ansiolíticos no tratamento de pacientes com transtorno de ansiedade generalizada. O mecanismo da ação primária dessas drogas ocorre pela interação com os sítios de ligação BZs presentes nos receptores do ácido gama-aminobutírico do tipo A (GABAA), facilitando a neutotransmissão GABAérgica. Entretanto, ainda permanecem desconhecidos os substratos neurais envolvidos no efeito panicolítico causado pelos BZs. Dentre os substratos em potencial, a substância cinzenta periaquedutal dorsal (SCPD), uma estrutura mesencefálica criticamente relacionada à fisiopatogênica do TP, apresenta alta densidade de receptores GABAA e de sítios de ligação BZs. Neste trabalho avaliamos o envolvimento do complexo receptor GABAA/BZ presente na SCPD no efeito panicolítico promovido pela administração sistêmica de alprazolam em ratos Wistar. Para isso, empregamos o labirinto em T elevado (LTE), que além da resposta de fuga, que é associada ao pânico, também permite avaliar a resposta de esquiva inibitória, associada à ansiedade. Neste modelo, o alprazolam inibe a expressão da resposta de fuga, indicando efeito panicolítico e inibe a aquisição da esquiva inibitória, sugestivo de efeito ansiolítico. Além do LTE, também empregamos os modelos experimentais da hipóxia e o de Vogel, associados ao pânico e a ansiedade, respectivamente. Os resultados obtidos mostraram que o efeito panicolítico promovido pela administração sistêmica de alprazolam, observado na resposta de fuga do LTE, foi bloqueado pela administração intra-SCPD de flumazenil, antagonista dos sítios de ligação BZs, ou de bicuculina, antagonista dos receptores 10 GABAA. No teste da hipóxia, o efeito panicolítico causado pela administração sistêmica de alprazolam foi inibido, porém não significativamente bloqueado, pela administração intra-SCPD de bicuculina. Já o efeito ansiolítico, observado na resposta de esquiva do LTE e no teste do beber punido de Vogel, não foi bloqueado pela administração intra-SCPD de flumazenil ou de bicuculina. No conjunto, nossos resultados sugerem que o complexo receptor GABAA/BZ da SCPD está envolvido no efeito panicolítico, mas não ansiolítico, promovido pela administração sistêmica de alprazolam. / Panic Disorder (PD) is an anxiety disorder characterized by recurrent and unexpected panic attacks with a chronic prognosis. Among the drugs used to treat PD, highpotency benzodiazepines (BZs), such as alprazolam and clonazepam, have the advantage of causing significant effects early in the treatment. Like others BZs, such as diazepam and flurazepam, these compounds are also used as anxiolytics in the treatment of patients with generalized anxiety disorder. The primary mechanism of action of these drugs is the interaction with BZs binding sites present at gammaaminobutyric acid type A receptors (GABAA), facilitating GABAergic neurotransmission. However, it remains yet unknown the neural substrates involved in the panicolytic-like action caused by BZs. Among the potential substrates, the dorsal periaqueductal gray matter (DPAG), a mesencephalic structure critically associated with the physiopathology of PD, presents a high density of GABAA receptors and of BZs binding sites. In this work, we evaluated the participation of the GABAA/BZ receptor complex present in the DPAG in the panicolytic-like effect caused by systemic administration of alprazolam in Wistar rats. For this, we use the elevated T-maze (ETM), that besides the escape response which is associated with panic, also allows the measurement of inhibitory avoidance acquisition, which has been related to anxiety. In this model, alprazolam inhibits the expression of escape, indicating a panicolytic-like effect and inhibits the acquisition of inhibitory avoidance, suggestive of an anxiolytic effect. In addition to the ETM, animals were also tested in the hypoxia and Vogel\'s conflict tests, which have been associated with panic and anxiety, respectively. The results showed that the panicolytic-like effect caused by alprazolam in ETM\'s escape response was blocked by intra-DPAG injection of flumazenil, a BZs binding site antagonist, or bicuculline, a GABAA receptor antagonist. In the hypoxia test, the panicolytic-like effect caused by alprazolam was inhibited, but not significantly blocked, by intra-DPAG injection of bicuculline. The anxiolytic effect observed in the 12 ETM\'s avoidance task or in the Vogel\'s conflict test was not blocked by intra-DPAG injection of flumazenil or bicuculline. Taken together, our results suggest that the GABAA/BZ receptor complex located in the DPAG is involved in the panicolytic, but not anxiolytic, effect caused by systemic administration of alprazolam.

Selon l'OMS, les troubles dépressifs majeurs (TDM) seront la 2ème cause d'incapacité dans le monde en 2020 et deviendront la 1ère en 2030. Les antidépresseurs classiques ont des effets thérapeutiques retardés et de nombreux patients sont résistants. La kétamine, antagoniste du récepteur N-methyl-D-aspartate (R-NMDA) du L-glutamate, possède un effet antidépresseur rapide chez les patients résistants à un traitement classique. Le mécanisme de cette activité étonnante n'est pas bien compris. En couplant la microdialyse intracérébrale à un test comportemental prédictif d'une activité antidépressive dans un modèle de souris BALB/cJ de phénotype anxieux, nous montrons que cette activité de la kétamine dépend de la balance excitation-inhibition entre les systèmes glutamate/R-NMDA et R-AMPA, GABA/R-GABAA, sérotonine du circuit cortex préfrontal/noyau du raphé. Nos résultats suggèrent également que ce serait la combinaison [kétamine-(2R,6R)-hydroxynorkétamine, son principal métabolite cérébral] qui porterait l'effet antidépresseur. Mes travaux de thèse contribuent à une meilleure compréhension de l'effet rapide antidépresseur de la kétamine. / According to the WHO, major depressive disorder (MDD) will be the second leading cause of disability in the world in 2020 and will become the first in 2030. Conventional antidepressant drugs have delayed therapeutic effects and many patients are resistant. Ketamine, an N-methyl-D-aspartate (NMDA-R) receptor antagonist of L-glutamate, exerts a rapid antidepressant effect in patients who are resistant to standard therapy. The mechanism of this amazing activity is not well understood. By coupling intracerebral microdialysis to a predictive behavioral test of antidepressant activity in a BALB/cJ mouse model with an anxious phenotype, we show that this ketamine activity is dependent on the excitation-inhibition balance between glutamate/NMDA-R and AMPA-R, GABA/GABAA-R, serotonin systems in the prefrontal cortex/raphe nucleus circuit. Our results also suggest that it would be the combination [ketamine-(2R,6R)-hydroxynorketamine, its main brain metabolite] that would carry the antidepressant effect. My thesis work pave the way for the development of new fast-acting antidepressant drugs.

Yes / The number of benzodiazepines appearing as new psychoactive substances (NPS) is continually increasing. Information about the pharmacological parameters of these compounds is required to fully understand their potential effects and harms. One parameter that has yet to be described is the blood-to-plasma ratio. Knowledge of the pharmacodynamics of designer benzodiazepines is also important, and the use of quantitative structure–activity relationship (QSAR) modelling provides a fast and inexpensive method of predicting binding affinity to the GABAA receptor. Methods: In this work, the blood-to-plasma ratios for six designer benzodiazepines (deschloroetizolam, diclazepam, etizolam, meclonazepam, phenazepam, and pyrazolam) were determined. A previously developed QSAR model was used to predict the binding affinity of nine designer benzodiazepines that have recently appeared. Results: Blood-to-plasma values ranged from 0.57 for phenazepam to 1.18 to pyrazolam. Four designer benzodiazepines appearing since 2017 (fluclotizolam, difludiazepam, flualprazolam, and clobromazolam) had predicted binding affinities to the GABAA receptor that were greater than previously predicted binding affinities for other designer benzodiazepines. Conclusions: This work highlights the diverse nature of the designer benzodiazepines and adds to our understanding of their pharmacology. The greater predicted binding affinities are a potential indication of the increasing potency of designer benzodiazepines appearing on the illicit drugs market. / Engineering and Physical Sciences Research Council. / Research Development Fund Publication Prize Award winner, Feb 2022.

No description available.

The overall aim of the present thesis is to clarify the control of intracellular chloride homeostasis in central neurons, because of the critical role of chloride ions (Cl–) for neuronal function. Normal function of the central nervous system (CNS) depends on a delicate balance between neuronal excitation and inhibition. Inhibition is, in the adult brain, most often mediated by the neurotransmitter γ-aminobutyric acid (GABA). GABA may, however, in some cases cause excitation. GABA acts by activating GABA type A receptors (GABAARs), which are ion channels largely permeable to Cl–. The effect of GABAAR-mediated neuronal signaling - inhibitory or excitatory - is therefore mainly determined by the Cl– gradient across the membrane. This gradient varies with neuronal activity and may be altered in pathological conditions. Thus, understanding Cl– regulation is important to comprehend neuronal function. This thesis is an attempt to clarify several unknown aspects of neuronal Cl– regulation. For such clarification, a sufficiently sensitive method for measuring the intracellular Cl– concentration, [Cl–]i, is necessary. In the first study of this thesis, we examined two electrophysiological methods commonly used to estimate [Cl–]i. Both methods, here called the interpolation and the voltage-ramp method, depend on an estimate of the Cl– equilibrium potential from the current-voltage relation of GABA- or glycine-evoked Cl– currents. Both methods also provide an estimate of the membrane Cl– conductance, gCl. With a combination of computational and electrophysiological techniques, we showed that the most common (interpolation) method failed to detect changes in [Cl–]i and gCl during prolonged GABA application, whereas the voltage-ramp method accurately detected such changes. Our analysis also provided an explanation as to why the two methods differ. In a second study, we clarified the role of the extracellular matrix (ECM) for the distribution of Cl– across the cell membrane of neurons from rat brain. It was recently proposed that immobile charges located within the ECM, rather than as previously thought cation-chloride transporter proteins, determine the low [Cl–]i which is critical to GABAAR-mediated inhibition. By using electrophysiological techniques to measure [Cl–]i, we showed that digestion of the ECM decreases the expression and function of the neuron-specific K+ Cl– cotransporter 2 (KCC2), which normally extrudes Cl- from the neuron, thus causing an increase in resting [Cl–]i. As a result of ECM degradation, the action of GABA may be transformed from inhibitory to excitatory. In a third study, we developed a method for quantifying the largely unknown resting Cl– (leak) conductance, gCl, and examined the role of gCl for the neuronal Cl– homeostasis. In isolated preoptic neurons from rat, resting gCl was about 6 % of total resting conductance, to a major part due to spontaneously open GABAARs and played an important role for recovery after a high Cl– load. We also showed that spontaneous, impulse-independent GABA release can significantly enhance recovery when the GABA responses are potentiated by the neurosteroid allopregnanolone. In a final commentary, we formulated the mathematical relation between Cl– conductance, KCC2-mediated Cl– extrusion capacity and steady-state [Cl–]i. In summary, the present thesis (i) clarifies how well common electrophysiological methods describe [Cl–]i and gCl, (ii) provides a novel method for quantifying gCl in cell membranes and (iii) clarifies the roles of the ECM, ion channels and ion transporters in the control of [Cl–]i homeostasis and GABAAR-mediated signaling in central neurons.

Zentralnervöse Lernvorgänge hängen wesentlich von der Plastizität der synaptischen Übertragung ab. Synapsen verändern ihre Effizienz sowohl durch Änderungen in der Freisetzungswahrscheinlichkeit von Neurotransmittern als auch durch Variabilität der postsynaptischen Rezeptorausstattung. Darüberhinaus gibt es seit einiger Zeit Hinweise, dass auch die Konzentration des Neurotransmitters in synaptischen Terminalen variieren kann und dadurch die Freisetzungswahrscheinlichkeit von Vesikeln verändert wird. Um den Zusammenhang zwischen der Füllung synaptischer Vesikel und ihrer Dynamik im Vesikelzyklus besser zu verstehen, habe ich zunächst ein Computermodell entwickelt. Ich habe gefunden, dass nur eine Modifikation des Nachschubs von Vesikeln aus der Reservepopulation in die Population unmittelbar freisetzbarer Vesikel die experimentell gemessene Abhängigkeit der Freisetzungswahrscheinlichkeit von der präsynaptischen Transmitterkonzentration reproduzieren kann, nicht jedoch ein direkter Effekt auf die Freisetzungsrate. Einer der im Modell simulierten Mechanismen für den beobachteten Effekt des vesikulären Füllungszustandes auf die Vesikelfreisetzung besteht in einer Rückwirkung des freigesetzten Transmitters auf ionotrope Autorezeptoren. Diesen Mechanismus habe ich anschliessend an GABAergen Synapsen in der CA3-Region des Hippocampus untersucht. Tatsächlich habe ich durch patch-clamp Messungen herausgefunden, dass sowohl die Antwortwahrscheinlichkeit auf extrazelluläre Stimulation als auch die Frequenz von spontanen Vesikelfreisetzungen nach Applikation des GABAA Rezeptor-Agonisten Muscimol signifikant verringert ist. Diese Befunde weisen darauf hin, dass GABA seine eigene Freisetzung über ionotrope Autorezeptoren hemmt. Direkte Messungen der Vesikelfreisetzungen sind an GABAergen Synapsen nur durch bildgebende Verfahren möglich. Mit Hilfe von Zwei-Photonen Mikroskopie ist es mir erstmalig gelungen, im Hirnschnitt die spontane Fusion von Vesikeln zu untersuchen. Diese Methode könnte für eine Reihe von Fragestellungen relevant sein. Dabei hat sich gezeigt, dass nach Applikation von Muscimol die spontane Abnahme der Fluoreszenz vorher angefärbter synaptischer Vesikel ausschliesslich in perisomatischen (überwiegend GABAergen) synaptischen Boutons signifikant verringert ist. Zusammenfassend habe ich bei der experimentellen Prüfung von Vorhersagen eines Computermodells durch patch-clamp Untersuchungen und durch eine neue Methode mit funktioneller Bildgebung Hinweise auf funktionelle präsynaptische GABAA Rezeptoren an GABAergen Terminalen der CA3-Region des Hippocampus gefunden, die eine negative Rückwirkung auf die Freisetzung von Vesikeln ausüben. / Learning processes depend on synaptic plasticity. Synaptic efficacy depends both on the probability of transmitter release and on the variability of postsynaptic receptors. Furthermore, recent data suggest that the transmitter concentration in presynaptic terminals may be an additional variable influencing the release probability of synaptic vesicles. To better understand the relationship between vesicular filling and vesicular dynamics, I first developed a computer model. I found that only a modification of the replenishment of the readily releasable pool from a reserve pool reproduces the experimentally observed dependence of vesicular release on the presynaptic transmitter concentration, but not a direct effect on the release rate. One of the simulated mechanisms consists in a feedback of released transmitter on ionotropic autoreceptors. I investigated this mechanism in the CA3 region of the hippocampus. Indeed, using patch-clamp recordings I observed that application of the GABAA receptor agonist muscimol decreases significantly the response probability to extracellular stimulation as well as the frequency of spontaneous transmitter release. These results suggest that GABA inhibits its own release via ionotropic autoreceptors. Direct measurements of GABAergic vesicular release are only possible by imaging techniques. Using two-photon microscopy, I was (to my knowledge) the first one to investigate spontaneous vesicle fusion in brain slices. This method could be relevant to a variety of topics. I found out that application of muscimol leads to a significant decrease of fusion-associated fluorescence decay in perisomatic (mostly GABAergic) synaptic boutons. Taken together, I used patch-clamp recordings and a new application of two-photon microscopy to verify predictions of a computer model. The results suggest a negative feedback of GABA release via presynaptic GABAA receptors in the CA3 region of the hippocampus.

Κύριος στόχος της εργασίας ήταν η συγκριτική μελέτη της παλίνδρομης αναστολής μεταξύ του ραχιαίου και του κοιλιακού πόλου του ιππόκαμπου αρουραίου. Χρησιμοποιήθηκε η μεθοδολογία της in vitro διατήρησης τομών ιππόκαμπου και εξωκυττάριων καταγραφών προκλητών δυναμικών πεδίου. Τα αποτελέσματά μας έδειξαν ότι η GABAA εξαρτώμενη παλίνδρομη αναστολή είναι ασθενέστερη, έχει μικρότερη διάρκεια και φθίνει πιο γρήγορα στον κοιλιακό σε σχέση με το ραχιαίο ιππόκαμπο. Χρησιμοποιώντας διάφορα φάρμακα που δρουν ενισχυτικά στον GABAA υποδοχέα δείξαμε ότι υπάρχει λειτουργική διαφοροποίηση του GABAA εξαρτώμενου ανασταλτικού μηχανισμού μεταξύ των δύο πόλων του ιππόκαμπου, ενισχύοντας την υπόθεση της λειτουργικής διαφοροποίησης στο επίπεδο του υποδοχέα μεταξύ των δύο πόλων. Στην in vivo μελέτη, χρησιμοποιώντας το μοντέλο επαγωγής επιληπτικών κρίσεων με χορήγηση πεντυλενοτετραζόλης, δείξαμε ότι η ενίσχυση της GABAA εξαρτώμενης αναστολής απο τα κατασταλτικά φάρμακα συσχετίζεται με το μέγεθος της αντιεπιληπτικής τους δράσης. Επιπλέον, η βιταμίνη D δεν παρουσίασε αντιεπιληπτική δράση στους C57BL/6J μύες, ούτε ενίσχυσε την αναστολή, κάτι που δείχνει ότι δεν έχει επίδραση στον GABAA υποδοχέα ή, τουλάχιστον, στους υπότυπούς του στον ιππόκαμπο. / The major aim of this work was the comparative study of recurrent inhibition between the dorsal and ventral pole of the rat hippocampus. We used the methodology of in vitro maintenance of hippocampal slices and recording of evoked field potentials. We showed that the GABAA mediated recurrent inhibition is weaker, lasts less and decays faster in ventral than in dorsal hippocampus. Using various drugs that act as positive allosteric modulators of the GABAA receptor, we showed that there is a functional differentiation of the GABAA inhibitory mechanism between the two hippocampal poles, strengthening the hypothesis of the functional differentiation at the level of the receptor between the two poles. In the in vivo study, using the pentylenetetrazole model for inducing epileptic seizures, we showed that the enhancement of the GABAA mediated recurrent inhibition correlates with the strength of antiepileptic action of the sedative drugs used. In addition vitamin D did not show antiepileptic action in C57BL/6J mice. Moreover it didn’t enhance recurrent inhibition, showing that it doesn’t have any action on the GABAA receptor or, at least, on its subtypes in hippocampus.

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels. The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin. Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery. AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction. The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

Made available in DSpace on 2015-07-22T20:50:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-26. Added 1 bitstream(s) on 2015-08-11T03:25:54Z : No. of bitstreams: 1 Publico-11764a.pdf: 1760987 bytes, checksum: 26371946d909a5525c0bd6c7cc6d7c33 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:55Z : No. of bitstreams: 2 Publico-11764a.pdf: 1760987 bytes, checksum: 26371946d909a5525c0bd6c7cc6d7c33 (MD5) Publico-11764b.pdf: 969495 bytes, checksum: d87ae7194b036aaff67581e32d434f64 (MD5) / Objetivo: Verificar se indivíduos susceptíveis e não-susceptíveis às convulsões clônicas induzidas pelo DMCM, um agonista inverso benzodiazepínico, diferem: 1) na sensibilidade ao efeito hipnótico induzido pelo diazepam e por outros moduladores alostéricos positivos do receptor GABAA; 2) na marcação auto-radiográfica com o [3H]- flunitrazepam ao longo do encéfalo; 3) na marcação de [3H]-L-glutamato e do [3H]-MK 801 em membranas de regiões encefálicas; e 4) na atividade da enzima Na+/K+- ATPase, bem como na marcação da [3H]-ouabaína às isoenzimas Na+/K+- ATPase de alta e de baixa afinidade ao radioligante em membranas de regiões encefálicas. Métodos: Ratos Wistar, machos, adultos foram administrados intraperitonealmente duas vezes com uma DC50 de DMCM (com intervalo de uma semana entre as administrações), obtendo-se dois grupos distintos: o grupo susceptível às convulsões (SC), que apresentou convulsões clônicas em ambas as exposições à droga, e o grupo não-susceptível às convulsões (NSC), que não apresentou alterações motoras em ambas as exposições. Após cerca de 25 dias da segunda administração de DMCM, os grupos selecionados foram submetidos aos experimentos com os hipnóticos diazepam, pentobarbital e etanol, nos quais foram registrados o tempo e a latência de sono ou foram sacrificados e seus encéfalos retirados para os seguintes ensaios bioquímicos: 1) auto-radiografia com o [3H]-flunitrazepam; 2) marcação de [3H]-L-glutamato e de [3H]- MK 801 em membranas neuronais; e 3) atividade enzimática da Na+/K+- ATPase e marcação de [3H]-ouabaína em enzimas de alta e baixa afinidade em membranas neuronais. Resultados: O grupo SC apresentou menor tempo de sono induzido pelo diazepam com relação ao grupo NSC, embora não tenham se distinguindo no tempo de sono induzido pelo pentobarbital e pelo etanol. Com relação aos experimentos bioquímicos, observou-se uma menor marcação de [3H]-flunitrazepam na região CA2 ventral do hipocampo no grupo SC. Quanto à ligação de [3H]-L-glutamato foi menor no grupo SC nas regiões do córtex frontal, amígdala + córtex límbico e hipocampo, enquanto que a ligação de [3H]-MK 801 foi menor no córtex frontal, hipocampo e estriado. Embora os grupos não tenham se diferenciado na atividade enzimática da Na+/K+- ATPase, o grupo SC apresentou uma menor marcação da [3H]-ouabaína em isoenzimas de alta afinidade nas regiões do tronco encefálico, córtex frontal e hipocampo, bem como uma menor marcação de [3H]-ouabaína nas regiões do tronco encefálico e córtex frontal em isoenzimas de baixa afinidade. Conclusão: As diferenças entre os grupos quanto à sensibilidade ao efeito convulsivante do DMCM, à ansiedade observada em experimentos anteriores, bem como à sensibilidade ao efeito hipnótico do diazepam podem estar associadas a uma diferença nos sítios benzodiazepínicos da região CA2 ventral do hipocampo, na ix atividade glutamatérgica e em isoformas específicas da Na+/K+- ATPase em determinadas regiões encefálicas. / Objective: The aim of this work was to verify if rats susceptible and non-susceptible to clonic convulsions induced by DMCM, a benzodiazepine inverse agonist, differ: 1) in the sensitivity to the hypnotic effect induced by diazepam and by others positive allosteric modulators of GABAA receptors; 2) in auto-radiographical analysis of [3H]-flunitrazepam binding along the brain; 3) in the binding of [3H]-L-glutamate and of [3H]-MK 801 in membranes from discrete brain regions; and 4) in the Na+/K+-ATPase activity, as well as in the binding of [3H]-ouabain to Na+/K+-ATPase isoenzimes with high and low affinity to the radioligand in membranes from discrete brain regions. Methods: Adult, male, Wistar rats were administered with two intraperitoneal injections of a convulsant dose 50% (CD50) of DMCM (one-week interval between them), resulting in two distinct groups: the group susceptible to clonic convulsions (SC), which presented clonic convulsions in both the expositions to the drug, and the group nonsusceptible to clonic convulsions (NSC), which did not present any motor disturbance in both the expositions. After 25 days from the second exposition to DMCM, the selected groups were submitted to the experiments with the hypnotics diazepam, pentobarbital and ethanol, in which were registered the latency and the time of sleep or they were sacrified and their brains were removed to carry out the following assays: 1) autoradiography with [3H]-flunitrazepam; 2) binding with the [3H]-L-glutamate and with the [3H]-MK 801 in neuronal membranes; 3) enzymatic activity of Na+/K+-ATPase and binding of [3H]-ouabain to the isoenzimes with high and low affinity in neuronal membranes. Results: The SC group presented a lower sleeping time induced by diazepam compared to the NSC group, and did not differ in the sleeping time induced by pentobarbital and ethanol. Concearning the biochemical experiments, it was observed a lower binding of [3H]-flunitrazepam in the CA2 subregion of ventral hippocampus in the SC group. A lower binding of [3H]-L-glutamate was also observed in the SC group in the frontal cortex, amygdala plus limbic cortex and hippocampus, whereas the binding of [3H]-MK 801 was lower in the frontal cortex, hippocampus and striatum compared to the NSC group. Althougt the groups did not differ in the enzymatic activity of Na+/K+- ATPase, the SC group presented a lower binding of [3H]-ouabain to the high-affinity isoenzimes in the brainstem, frontal cortex and hippocampus, as well as a lower binding of [3H]-ouabain to the low-affinity isoenzimes in the brainstem and in the frontal cortex compared to the NSC group. Conclusion: The differences between the groups concerning the sensitivity to the convulsant effect of DMCM, the level of anxiety previously observed, as well as the sensitivity to the hypnotic effect of diazepam may be associated with the GABAA/benzodiazepine site in CA2 subregion of ventral hippocampus, with glutamatergic activity and with specific isoforms of Na+/K+-ATPase in rat brain regions. / TEDE / BV UNIFESP: Teses e dissertações

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Na+,K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where serves as the principal regulator of intracellular ion homeostasis. Na+,K+-ATPase activity is activated by Na+ and K+ and current evidence indicates that total Na+,K+-ATPase activity is, in general, inhibited by anions. However, the effect of pharmacologically-induced Cl- flux on α1- and α2/3-subunit containing Na+,K+-ATPase activity is not established. In this study we investigated the effect of diazepam, a GABAA receptor positive allosteric modulator, on α1- and α2/3-subunit containing Na+,K+-ATPase activity. Hippocampal and cortical slices were incubated with diazepam (0, 0.05, 0.15 or 0.5 μM) and/or flumazenil (0, 0.005, 0.015, 0.05, 0.15, 0.5 or 1.5 μM) for 10 minutes. After incubation the slices were homogenized and α1 and α2/3 Na+,K+-ATPase activity were assayed using ouabain 3 μM (that inhibits α2/3-subunit containing Na+,K+-ATPase) and 4 mM (that inhibits both isoforms). Diazepam caused a 50% decrease of α2/3-subunit containing Na+,K+-ATPase activity in the hippocampus, but did not alter enzyme activity in the entorhinal cortex. The effect of diazepam was prevented by flumazenil, indicating that the decrease of Na+,K+-ATPase was involved GABAA receptors. Furthermore, a low chloride medium abolished the diazepam-induced decrease of Na+,K+-ATPase activity. Our data suggests that Na+,K+-ATPase in the hippocampus is sensitive to the pharmacological effects of a benzodiazepine by GABAA receptor-mediated mechanisms. Keywords: sodium pump. GABAA receptor. diazepam. flumazenil. chloride ion. hippocampus. entorhinal córtex. / A enzima Na+,K+-ATPase, ou bomba de sódio, é expressa na membrana plasmática de células eucarióticas, onde atua como principal regulador da homeostase iônica intracelular. A enzima Na+,K+-ATPase é ativada pelos íons Na+ and K+ e evidências indicam que a atividade total da enzima Na+,K+-ATPase é inibida por ânions. Entretanto, o efeito do fluxo de cloreto induzido farmacologicamente sobre a atividade das subunidades α1 e α2/3 da enzima Na+,K+-ATPase ainda não foi investigado. Neste estudo, nós investigamos o efeito do diazepam, um modulador alostérico positivo do receptor GABAA na atividade específica das subunidades α1 e α2/3 da Na+,K+-ATPase. Fatias de hipocampo e de córtex entorrinal foram incubadas com diazepam (0; 0,05; 0,15 ou 0,5 μM) e/ou flumazenil (0; 0,005, 0,015; 0,05; 0,15; 0,5 ou 1,5 μM) por 10 minutos. Após a incubação, as fatias foram homogeneizadas e a atividade das subunidades α1 e α2/3 da enzima Na+,K+-ATPase foi determinada. Diazepam diminuiu 50% a atividade da subunidade α2/3 da Na+,K+-ATPase no hipocampo, mas não alterou a atividade da enzima em córtex entorrinal. O efeito do diazepam foi prevenido por flumazenil, indicando que a diminuição da atividade da Na+,K+-ATPase envolveu a ativação dos receptores GABAA. Além disso, a baixa concentração de cloreto no meio de incubação aboliu a diminuição da atividade enzimática induzida por diazepam. Nossos dados sugerem que a enzima Na+,K+-ATPase no hipocampo é sensível a efeitos farmacológicos dos benzodiazepínicos por meio de mecanismos ativados por receptores GABAérgicos.