Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Lippia alba (Mill.) N. E. Brown is an aromatic herb commonly used as sedative and for the treatment of gastrointestinal disturbs. Many literature data confirm the sedative property of the essential oil (EO) of this species; however, just recently its anesthetic activity was confirmed. The present study reports the isolation of two major chemical constituents of the EO of L. alba, linalool and eucalyptol, and also the evaluation of their anesthetic activity in juveniles of silver catfish (Rhamdia quelen). Additionally, linalool was defined as the major active compound of the EO with anesthetic property, and eucalyptol did not presented activity in the tested concentration. The involvement of GABAergic system on the mechanism of action of the EO and its major constituent was also studied. The results showed that the EO contains one or more compounds which act in synergism with linalool and also by different mechanism. Benzodiazepine like effect was detected for the EO of L. alba, but not for linalool. In addition, this work brings a case report of contact dermatitis caused by the leaves of L. alba chemotype linalool. Finally, to establish a good quality of the plant material, after yield and chemical composition analyses of the EO, 1,0 x 0,8 m was defined as the better spacing for cultivation of L. alba. The results are presented in three manuscripts. / Lippia alba (Mill.) N. E. Brown é uma planta odorífera utilizada popularmente com finalidade ansiolítica e para o tratamento de distúrbios gastrintestinais. Na literatura existem muitos trabalhos que confirmam a propriedade sedativa do seu óleo essencial (OE), porém, somente há pouco tempo a sua propriedade anestésica foi confirmada. Este trabalho apresenta o isolamento de dois constituintes majoritários do OE de L. alba, linalol e eucaliptol, bem como a avaliação de sua atividade anestésica geral em jundiás juvenis (Rhamdia quelen). Ainda, o linalol foi definido como o constituinte ativo majoritário do OE com potencial anestésico, enquanto que o eucaliptol não apresentou atividade nas concentrações testadas. Adicionalmente foi estudado o envolvimento do sistema GABAérgico no mecanismo de ação do OE e de seu constituinte ativo majoritário. Os resultados correspondentes aos estudos de mecanismo permitem afirmar que existe(m) um ou mais compostos presentes no OE, que atuam em sinergismo com o linalol e por mecanismos distintos. Enquanto que para o OE de L. alba foi detectado um mecanismo tipo benzodiazepínico, o linalol age por outro mecanismo ainda não esclarecido. Em adição, este trabalho relata pela primeira vez um caso de dermatite de contato causada pelas folhas frescas de L. alba quimiotipo linalol. Por fim, no sentido de estabelecer uma boa qualidade da matéria prima vegetal para posteriores trabalhos, após a análise de rendimento e composição química do OE, 1,0 x 0,8 m foi definido como melhor espaçamento para o cultivo de L. alba. O trabalho encontra-se na forma de manuscritos, contendo três artigos científicos.

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.

No description available.

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.

Η ΒΥΠ διαθέτει αντίτυπο της διατριβής σε έντυπη μορφή στο βιβλιοστάσιο διδακτορικών διατριβών που βρίσκεται στο ισόγειο του κτιρίου της. / Ο ιππόκαμπος στον αρουραίο είναι μια δομή σε C-σχήμα που εκτείνεται από τον διαφραγματικό πυρήνα προσθιοραχιαία έως τον κροταφικό λοβό οπισθιοκοιλιακά. Παρόλο που από παλιά θεωρείτο ως ομοιογενής δομή, έχουν παρατηρηθεί πολλές διαφορές σε όλα τα επίπεδα οργάνωσης μεταξύ του διαφραγματικού και του κροταφικού ιππόκαμπου. Οι βενζοδιαζεπίνες δρουν στις θέσεις δέσμευσης των βενζοδιαζεπινών, οι οποίες είναι αλλοστερικά συνδεδεμένες με το σύμπλοκο του GABAA, όπου ενισχύουν την δράση του GABA στους GABAA υποδοχείς, αυξάνοντας τη συχνότητα ανοίγματος του διαύλου των ιόντων χλωρίου (Cl-) και έχοντας μικρή μόνο επίδραση στο χρόνο ανοίγματος ή στην αγωγιμότητα του διαύλου. Προηγούμενη μελέτη υποδεικνύει ότι η έκφραση των α1-, β2- και γ2-υπομονάδων ήταν μικρότερη, ενώ αντίθετα η έκφραση των α2-, α5- και β1-υπομονάδων ήταν μεγαλύτερη στον κροταφικό ιπποκάμπο σε σύγκριση με τον διαφραγματικό ιππόκαμπο. Σύμφωνα με προηγούμενες μελέτες που αφορούν την συνέκφραση των υπομονάδων στο σύμπλοκο του GABAA υποδοχέα τα αποτελέσματα μας υποδηλώνουν ότι ο α1β2-υποτύπος του GABAA υποδοχέα επικρατεί στον διαφραγματικό ιππόκαμπο (ΔΙ), ενώ ο α2β1-υπότυπος κυριαρχεί στον κροταφικό ιππόκαμπο (ΚΙ). Επιπλέον, φαρμακολογικές μελέτες έχουν δείξει ότι η καταπραϋντική δράση της diazepam πραγματοποιείται μέσω των GABAA υποδοχέων, που περιέχουν την α1-υπομονάδα, ενώ η αγχολυτική δράση της πραγματοποιείται μέσω των υποδοχέων που φέρουν την α2-υπομονάδα. Επομένως, η μελέτη των φαρμακολογικών ιδιοτήτων των υποτύπων του GABAΑ υποδοχέα δίνει την δυνατότητα στο να σχεδιαστούν ειδικά φάρμακα τα οποία θα βελτιώνουν το κλινικό προφίλ ασθενειών, όπως το άγχος, η αϋπνία ή η επιληψία, δρώντας σε εξειδικευμένους υποτύπους του GABAΑ υποδοχέα. Ο στόχος της συγκεκριμένης εργασίας ήταν να μελετηθούν οι φαρμακολογικές ιδιότητες των υποτύπων του συμπλόκου του GABAA υποδοχέα/βενζοδιαζεπινών στον διαφραγματικό σε σύγκριση με τον κροταφικό ιππόκαμπο επίμυος. Έτσι, για τη μελέτη των θέσεων δέσμευσης των βενζοδιαζεπινών μεταξύ του διαφραγματικού και κροταφικού ιπποκάμπου χρησιμοποιήθηκε ο ευρέως φάσματος αγωνιστής των θέσεων δέσμευσης των βενζοδιαζεπινών, [3H]-flunitrazepam, σε μια αυτοραδιογραφική μελέτη. Επιπλέον, χρησιμοποιήθηκαν εξειδικευμένα φάρμακα, που δεσμεύονται σε συγκεριμένους υποτύπους του GABAA υποδοχέα, όπως το zolpidem (ειδικός αγωνιστής των GABAA υποδοχέων, που περιέχουν την α1-υπομονάδα), το etomidate (ειδικός θετικός αλλοστερικός ρυθμιστής των GABAA υποδοχέων, που περιέχουν τη β2-υπομονάδα) και το L-655,708 (ειδικός αντίστροφος αγωνιστής των GABAA υποδοχέων, που περιεχούν την α5-υπομονάδα) σε μια αυτοραδιγραφική κινητική μελέτη. Τα αποτελέσματα μας έδειξαν ότι οι τομές που προέρχονταν από τον ΚΙ συγκρινόμενες με αυτές από τον ΔΙ είχαν: Α) μειωμένη δέσμευση της [3H]-flunitrazepam στις CA1, CA3 και DG περιοχές, Β) μικρότερη χημική συγγένεια της δέσμευση της [3H]-flunitrazepam στη CA1 περιοχή, Γ) καμία διαφορά στον αριθμό των θέσεων δέσμευσης, Δ) μεγαλύτερες τιμές IC50 και EC50 για το zolpidem και το etomidate, αντίστοιχα και Ε) μικρότερες τιμές IC50 για το L-655,708. Συμπερασματικά, τα αποτελέσματά μας υποδεικνύουν διαφορετικές φαραμκολογικές ιδιότητες των υποτύπων του GABAΑ/ΒΖ υποδοχέα μεταξύ του ΔΙ και ΚΙ, ενώ επιβεβαιώνεται και ενισχύεται προηγούμενη μελέτη που υποδηλώνει ότι ο α1β2-υποτύπος του GABAA υποδοχέα επικρατεί στον διαφραγματικό ιππόκαμπο (ΔΙ), ενώ ο α2β1-υπότυπος κυριαρχεί στον κροταφικό ιππόκαμπο (ΚΙ). Η μελέτη αυτή είναι πιθανό να τυγχάνει ιδιαίτερης κλινικής αξίας, στην κατεύθυνση της ακριβέστερης ρύθμισης των αποτελεσματικών δόσεων των διάφορων μορίων που δρουν στις θέσεις δέσμευσης των βενζοδιαζεπινών των α1- και α2-υποτύπων του GABAA υποδοχέα. / The hippocampus in the rat appears grossly as an elongated structure with its long axis bending in a C-shaped manner from the septal nuclei rostrodorsally to the incipient temporal lobe caudoventrally. The long axis of the hippocampal formation is referred as the dorsoventral axis. Although hippocampus has been traditionally thought as a homogeneous structure, several studies have been demonstrated differences at several organization levels (from the behavioural to the cellular) between its dorsal (DH) and ventral (VH) pole. Pharmacological studies have shown that the α1-GABAA receptor subtype is associated with the sedative and anticonvulsant effects of benzodiazepines (BZs), whereas the α2-subtype is associated with the anxiolytic effects of BZs. Recent data have demonstrated a differential distribution of the GABAA receptor subunits between DH and VH, with the α1/β2 GABAA receptor subtype dominating in the DH and the α2/β1 subtype prevailing in the VH. We therefore study possible differences in the pharmacological properties and receptor binding parameters of the GABAA/BZ receptor subtypes between DH and VH, by examining: 1a) the specific binding of [3H]-flunitrazepam (BZ sites agonist), by using quantitative autoradiography, b) the kinetic parameters of [3H]-flunitrazepam specific binding, by using the “wipe off” technique and 2) the competitive displacement of [3H]-flunitrazepam binding by using zolpidem (a specific agonist of α1-subtype) and L-655,708 (a specific inverse agonist of α5-subtype) and the enhancement of [3H]-flunitrazepam binding by using etomidate (a selective positive modulator of β2- subunit), in an autoradiographical saturation kinetic study. Our results showed in VH compared to the DH: A) lower level of [3H]-flunitrazepam binding in CA1, CA3 and DG regions, B) higher KD value for [3H]-flunitrazepam specific binding in CA1 region and no differences in the Bmax value, C) higher IC50 and EC50 values for zolpidem and etomidate, respectively and D) lower IC50 values for L-655,708. In conclusion, the lower affinity of GABAA receptors for [3H]-flunitrazepam binding, the higher IC50 and EC50 values for zolpidem and etomidate, respectively, as well as the lower IC50 values for L-655,708 observed in VH compared to DH, support the evidence that the α1/β2-GABAA receptor subtype dominates in DH and the α2/β1-subtype prevails in VH and suggest differential pharmacological effects of the benzodiazepines in DH compared to VH, which could be of clinical relevence for the more accuate adjustment of the effective dose of α1- and α2-subtype specific BZ binding sites’ ligands.

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.

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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.

GABAA receptor binding is transiently increased in rat whisker barrels during the second postnatal week, at a time when neurons in the developing rat cortex are vulnerable to excitotoxic effects. To test whether these GABAA receptors might serve to protect neurons from excessive excitatory input, polymer implants containing the GABAA receptor antagonist bicuculline were placed over barrel cortex for a 4-day period in young (postnatal days 8 - 12) and adult rats. In the cortex of young, but not adult rats, the chronic blockade of GABAA receptors resulted in substantial tissue loss and neuron loss. The greater loss of neurons in young rats supports the hypothesis that a high density of GABAA receptors protects neurons from excessive excitatory input during a sensitive period in development.