Brucella est l'agent causal de la brucellose, une zoonose bactérienne répandue à l'échelle mondiale. Durant les dernières années, de nouvelles souches et espèces de Brucella (dont Brucella microti) ont été isolées de l’environnement et d’animaux sauvages. Ces souches, phylogénétiquement anciennes, sont plus acido-résistantes que les espèces classiques, plus récentes et inféodées aux animaux domestiques et à l’homme. Chez Escherichia coli, le système glutamate décarboxylase (GAD) et le système glutaminase (AR2_Q), basés respectivement sur la décarboxylation du glutamate et la déamination de la glutamine, sont les systèmes d’acido-résistance (AR) les plus efficaces. Notre équipe a démontré que le système GAD (GadB et GadC) est fonctionnel seulement dans les nouvelles souches et espèces de Brucella, et participe à la réussite de l’infection des souris par voie orale. Dans cette thèse, le rôle de nouveaux facteurs et les mécanismes moléculaires impliqués dans l’acido-résistance ont été explorés. Premièrement, nous avons montré que GlsA et GadC sont les deux protéines structurales du système AR2_Q qui, avec le système GAD, joue un rôle essentiel dans l’AR de ces nouvelles souches. De plus, chez ces mêmes souches, le système uréase intervient également dans la survie en milieu acide.Nos résultats suggèrent que les systèmes GAD, AR2_Q et uréase, en fonction de la disponibilité des substrats, pourraient contribuer à améliorer l’adaptation des nouvelles espèces dans les environnements acides naturels et/ou dans le tractus gastro-intestinal de leurs hôtes. / Brucella is the etiological agent of brucellosis, a worldwide bacterial zoonosis. In the last ten years, new and atypical strains of Brucella (among which Brucella microti) were isolated from the environment and wild hosts. These strains, of ancient origin, are considered more environmental and acid resistant than classical Brucella species, which are mostly pathogenic for livestock and humans. In Escherichia coli, the glutamate decarboxylase (GAD)-dependent system and the glutaminase (AR2_Q) system, based on the decarboxylation of glutamate and on the deamination of glutamine, respectively, are most efficient in conferring acid resistance (AR). Our team has previously demonstrated that in Brucella the GAD system (GadB and GadC) is functional only in new/atypical strains and contributes to murine infection by oral route. In this thesis, novel molecular mechanisms and factors involved in specific AR of new/atypical Brucella species were explored. Firstly, we have shown that in these strains, GlsA and GadC are the two structural proteins of the AR2_Q system, which, in concert with the GAD system, plays an essential role in AR. In addition, the functionality and role of the urease system in AR was also demonstrated in these strains.Our results suggest that the GAD, AR2_Q and urease systems may participate in a better adaptation of new Brucella species to certain natural acidic habitats and/or to the gastrointestinal tract of their hosts, depending on substrate availability.

Les récepteurs de type kaïnate (rKA) appartiennent, avec les récepteurs de type NMDA (rNMDA) et les recepteurs de type AMPA (rAMPA), à la famille des récepteurs canaux glutamatergiques (iGluR). Les propriétés fonctionelles des rKA contenant la sous-unité GluK3 en font des récepteurs tout à fait singuliers. Une étude réalisée dans le laboratoire a montré que la faible sensibilité de ces récepteurs au glutamate est liée à une entrée très rapide dans l’état désensibilisé et que la fonction de ces récepteurs pourrait être amplifiée par des modulateurs endogènes.Parmi les modulateurs potentiels de la fonction des rKA pré-synaptiques, nous avons choisi d’étudier le zinc, en raison de sa concentration importante dans les vésicules des terminaisons des axones des cellules granulaires du gyrus denté (fibres moussues). En dépit du rôle proposé des rKA contenant la sous unité GluK3 dans la régulation pré-synaptique aux synapses MF-CA3, la modulation de ces récepteurs par le zinc n’a jamais été étudiée.Grâce à l’enregistrement électrophysiologique des courants GluK3 exprimés dans les cellules HEK-293, nous avons montré que le zinc facilite les courants des récepteurs contenant la sous-unité GluK3, activés par le glutamate. L’analyse des cinétiques, ainsi que la modélisation, montrent que l’effet facilitateur du zinc est dû à la réduction de l’entrée dans l’état désensibilisé des récepteurs GluK3. Grâce à la mutagénèse dirigée et l’étude cristallographique, nous avons pu déterminer le site de liaison du zinc, constitué de l’aspartate 759, de l’histidine 762 et de l’aspartate 730, et localisé dans l’interface de dimérisation du domaine de liaison de l’agoniste (LBD).Cette étude décrit pour la première fois un nouveau site de modulation positive de la fonction des rKA. / Glutamate released at excitatory synapses acts on ligand-gated ionotropic receptors which fall into three classes: NMDA, AMPA and kainate receptors.At hippocampal mossy fiber synapses onto CA3 pyramidal cells, KARs are present both at the pre- and postsynaptic levels. Postsynaptic KARs are composed of the GluK2, GluK4 and GluK5 subunits, whereas presynaptic KARs are thought to comprise the GluK2 and GluK3 subunits. The functional properties of GluK3 (and GluK2/GluK3) receptors set it apart from the other ionotropic glutamate receptors. In particular, its sensitivity to glutamate is the lowest of all known ionotropic glutamate receptors, due in large part to fast desensitization of receptors with one or two bound glutamate molecules. The low agonist sensitivity of this receptor raises questions about its relevance for synaptic function. Therefore, it is possible that endogenous modulators may potentiate its function.Among potential endogenous modulators of KAR function, we chose to address the role of zinc, because of the large amounts contained in mossy fiber terminals. Zinc is thought to be accumulated into synaptic vesicles, and is co-released with glutamate in the extracellular milieu during neuronal activity. Zinc has been reported to inhibit most of native and recombinant KARs. Despite the proposed role of at hippocampal mossy fiber synapses, although modulation of GluK3-containing KARs by zinc has not yet been addressed.In this study, we show that zinc greatly potentiates recombinant GluK3 receptor currents evoked by glutamate. Zinc markedly slows receptor desensitization and increases apparent affinity for glutamate. Crystallographic studies and analysis of chimeric GluK2/GluK3 KARs and of GluK3 bearing selected point mutations, allowed us to identify the zinc binding domain defined by D759, H762, Q756 and D730, and localized in a region forming the interface between two GluK3 subunits in an LBD dimer assembly. Based on these structure-function studies and on modeling of KAR activity, we show that zinc plays a very distinct role on GluK3-KARs by stabilizing the interaction between dimers of LBD thereby reducing desensitization.Given the proposed localization of GluK3 close to zinc containing synaptic vesicles, zinc may be an endogenous allosteric modulator for native GluK3-KARs, and its binding site a new pharmacological target.

'Excitotoxicity' represents the excitatory amino acid mediated degeneration of neurons. Glutamate is the major excitatory neurotransmitter in the brain. Glutamate excitotoxicity has been implicated in a number of neurodegenerative disorders like Stroke, Epilepsy, Alzheimer's disease and traumatic brain injury. This neurotoxicity is summed up by the 'glutamate hypothesis' which describes the cause of neuronal cell death as an excessive release of glutamate causing over excitation of the glutamate receptors and subsequent increase in influx of calcium leading to cell death. An effort to counteract this neurotoxicity has lead to the development of glutamate receptor antagonists that can effectively serve as neuroprotective agents. Nevertheless, the downside to these drugs has been the side effects observed in clinical trial patients due to their disruptive action on the physiological function of these receptors like learning and memory. This work was undertaken to identify targets that can effectively be used to treat excitotoxicity without affecting any normal physiological functions. In one approach, (chapter I) we have identified the KATP channels as an effective modulator of epileptogenesis. In another approach, (Chapter II) we show that targeting the AMPA receptor subunit GluR2 is a practical strategy for stroke therapy. KATP channels that are gated by intracellular ATP/ADP concentrations are a unique subtype of potassium channels and play an essential role in coupling intracellular metabolic events to electrical activity. Opening of KATP channels during energy deficits in the central nervous system (CNS) induces efflux of potassium ions and in turn hyperpolarizes neurons. Thus, activation of KATP channels is thought to be able to counteract excitatory insults and protect against neuronal death. Here, we show that, functional Kir6.1 channels are located at excitatory pre-synaptic terminals as a complex with type-1 Sulfonylurea receptors (SUR1) in the hippocampus. The mutant mice with deficiencies in expressing the Kir6.1 or the SUR1 gene are more vulnerable to generation of epileptic form of seizures, compared to wild-type controls. Whole-cell patch clamp recordings demonstrate that genetic deletion of the Kir6.1/SUR1 channels enhances glutamate release at CA3 synapses. Hence, expression of functional Kir6.1/SUR1 channels inhibits seizure responses and possibly acts via limiting excitatory glutamate release. In addition to epilepsy, ischemic stroke is a leading cause of death in developed countries. A critical feature of this disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons, particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR) -1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Here we show that ischemic insults induce toxic Ca2+ entry through AMPA receptors into vulnerable neurons by modification of GluR2 RNA editing. Thus, targeting of GluR2 subunit can be considered as a promising target for stroke therapy.

No description available.

The main goal of this research was to develop an easy to prepare and sensitive biosensor that would be able to detect glutamate in solution using ionic self-assembly methods. This was accomplished by preparing an ionically-self-assembled monolayer that included an electron transport mediator and an enzyme that would generate a current proportional to the concentration of analytes in solution. Biosensors were produced for the detection of glucose and glutamate. Ferrocene poly(allylamine) (FePAA) was assembled on negatively charged self-assembled monolayer and shown to be electrostatically bound by cyclic voltammetry. Model films were made of FePAA and poly(styrenesulfonate) to determine if multilayer films could be assembled using electrostatic assembly. These experiments demonstrated that 7 bilayers is the maximum number of bilayers oxidizable by the heterogeneous reaction at the electrode surface. ISAMs were then assembled on a 2 mm gold electrode and on a gold fiber microelectrode using FePAA and glucose oxidase. Using cyclic voltammetry, these ISAMs were shown to be able to oxidize glucose in solution. The LOD was determined to be lower for the microelectrode than for the 2 mm gold electrode, which was expected, while both compared well to the literature. The Km? were found to be smaller than other glucose biosensors while the Icat increased with increasing number of bilayers. This demonstrated that the GluOx is making good electrical contact with the layer below. These glucose oxidase ISAMs, however, do not exhibit structural stability in flow-injection experiments. As a solution to the ISAM modified electrodes degrading in the flowing system, a covalently modified surface was developed. Using cyclic voltammetry, these covalently modified surfaces were shown to be able to oxidize glucose in solution. The LOD of the covalently modified 2 mm gold electrode was calculated to be lower than the 2 mm ISAM modified gold electrode, due to the fast heterogeneous kinetics on the covalently modified electrode surface. The Km? and Icat for the covalently modified 2 mm gold electrode were found to be the similar to the 2 mm ISAM modified gold electrode indicating that the covalently modified electrodes will be a suitable replacement. The covalently modified surfaces exhibit excellent structural stability and detect much lower glucose amounts in flow-injection experiments. ISAMs were subsequently assembled on gold fiber microelectrodes using FePAA and glutamate oxidase. Glutamate was able to be detected in solution at biologically significantly quantities using cyclic voltammetry. The Km? was shown to be comparable to literature values and Icat was shown to increase with increasing number of bilayers. These results demonstrate that an ISAM constructed using FePAA/GlutOx is a feasible way to detect glutamate in a system. / Ph. D.

Development of the rodent somatosensory cortex is well characterised and involves activity-dependent mechanisms that occur during the first postnatal week. Glutamate is a key neurotransmitter responsible for signalling events that result in formation of cortical barrels - aggregates of cells in the cortex corresponding to whiskers on the face pad. The molecular mechanisms that occur downstream of glutamate signalling are not fully understood and data here contributes to the unveiling of some of these mechanisms. Transgenic mice with deletions of genes that encode members of the post-synaptic complex associated with NMDARs were used to understand the role of individual genes in the formation of barrels. SynGAP, a ras GTPase activating protein (GAP) that negatively regulates the ERK-MAPK pathway downstream of NMDARs is required for the formation of barrels and data here agrees with other findings that the ras GAP NF1 has a similar role. Examination of RICS, a RhoGAP and Dusp6 - a phosphatase that inactivates ERK reveals that neither are necessary for the formation of barrels. This finding adds to previous data postulating that barrels form in an ERK-independent manner (Watson et al., 2006, Barnett et al., 2006). MAGUKs are important scaffolding molecules in the PSD and bind NMDARs to downstream signalling molecules such as SynGAP. Two of these MAGUKs SAP102 and PSD-95 have roles in hippocampal plasticity, and learning and memory and Sap102 mutations result in a form of X-linked human retardation (Tarpey et al., 2004). Deletion of either gene does not cause defects in the development of barrels, perhaps due to compensation mechanisms already described in hippocampus (Vickers et al., 2006 Cuthbert et al., 2007). Double knockout mice die by P3 and analysis of all other mutants revealed a defect in the formation of barrels and segregation of TCAs in Sap102-/y Psd-95+/-. Surprisingly this defect was not seen in Sap102+/- Psd-95-/- mice, agreeing with previous findings that SAP102 is better able to compensate for loss of PSD-95 (is up-regulated) than PSD-95 is for SAP102. An explanation for this effect may lie with the fact that Sap102 is X-linked and therefore females that are heterozygote for Sap102 are mosaic with a population of cells expressing SAP102 and a population not expressing SAP102. Using β-Galactosidase antibody to label one population of cells, female mice that had two populations of cells were examined. In these mice one population of cells were Sap102-Psd-95+/-, and did not previously segregate into normal barrels and the other population were Sap102+Psd-95+/- and should segregate normally. Both populations of cells segregated normally, indicating that the cells expressing SAP102 were rescuing the cells not expressing SAP102 by a cell non-autonomous mechanism. The final part of this thesis focuses on the role of glutamate-dependent signalling pathways in the regulation of CSPGs- key extracellular matrix proteoglycans that regulate the termination of the sensitive period. Analysis of 3 overlapping but distinct subsets of chondroitin-sulphate proteoglycans (CSPGs) reveals that expression of each of the three is different throughout development. After 2-3 weeks perineuronal nets (PNNs) labelled with Cat-315 and Cat-316 are visible and locate to specific regions within the cortical barrel-field. To determine whether the formation of PNNs is regulated by proteins involved in glutamate signalling, expression of the three CSPG subsets was analysed in mice with barrel defects due to mutations of Plcβ1, Mglur5, Syngap and Prkar2b. Interestingly, Prkar2b mutant adults but no other mutants have reduced Cat-315-PNNs, indicating that PKARIIβ regulates pathways that lead to formation of Cat-315-PNNs in adulthood. Cat-315 has previously been found to be regulated in the cortex of visually deprived cats and the cortex of whisker-trimmed mice, indicating that specific subsets of CSPGs are regulated by neuronal activity. Molecular pathways that lead to expression of Cat-315 positive PNNs involve PKARIIβ and the formation of PNNs may be an important step in the plasticity of circuits in barrels. Taken together, these results demonstrate that an important part of molecular signalling downstream of glutamate enabling barrels to form is played by molecules that maintain structure inside the synapse and outside the cell.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of the tuberculosis disease, is estimated to infect a third of the world’s population and is therefore, arguably, the most successful human pathogen in recorded history. Immense efforts to understand the genetic factors and biochemical processes underlying the complex interactions between M. tuberculosis and its host cells have delivered staggering insights into the profound proficiency by which this bacterium establishes and maintains an infection. It is now clear that M. tuberculosis can interfere with the immune responses initiated by host cells in such a manner as to subvert the various bactericidal conditions established by these cells and thus eliminate the tubercle bacilli that infect them. Specific characteristics of M. tuberculosis which provide it with this ability include a nearly impenetrable cell wall, secretion systems which secrete special factors which directly interact with host immune factors. This enables M. tuberculosis to modulate the activities of the host environment and unique metabolic adaptations of M. tuberculosis allows the organism to survive in the hypoxic, oxidative, nitrosative, acidic and nutrient poor environment of immune cell phagosomes and to persist for decades in a quiescent state in otherwise healthy people. New observations into the pathways which constitute energy, carbon and central nitrogen metabolism, among others, in M. tuberculosis, suggest that a carefully orchestrated homeostasis is maintained by the organism which may modulate the concentrations and ameliorate the effect of molecules that are important to defensive strategies employed by host cells. Here we discuss various recent studies as well as new information provided by this study, focusing on central metabolism and its regulation in M. tuberculosis. We aim to highlight the importance of nitrogen metabolism in the subversive response employed by M. tuberculosis to survive, colonise and persist in the host. We argue that the homeostatic regulation of nitrogen metabolism in M. tuberculosis presents a profound vulnerability in the pathogen which should be exploited with compounds that inhibit the activities of various effector proteins found in this pathway and that are unique to the organism. Such compounds may provide valuable novel chemotherapies to treat tuberculosis patients and may alleviate the burden of multiple drug resistance which plagues tuberculosis treatments. Specifically, in this study we investigate the role of M. bovis BCG glutamate dehydrogenase (GDH) and glutamate synthase (GltS) by subjecting knockout mutants of the aforementioned gene products to various cellular stress conditions. Furthermore, we investigated how the genomes of each M. bovis BCG strain was affected post deletion of the aforementioned protein products. The role of GDH was also tested in an murine macrophage model of infection to elucidate potential importance to colonisation and infection. This study provides novel results indicating an importance of GDH toward the resistance of nitrosative stress as well as a requirement for optimal persistence in RAW 264.7 macrophages. In addition, it was found that GltS is dispensable for resistance against nitrosative stress. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat die aansteeklike siekte tuberkulose veroorsaak, infekteer ongeveer ‘n dêrde van die wêreld populasie en is daarom, waarskynlik, een van die mees suksesvolle menslike patogene in geskiedenis. In die afgelope jare is daar noemenswaardige poging aangewend om genetiese faktore sowel as biochemiese prosesse te verstaan wat die komplekse interaksies tussen M. tuberculosis en sy gasheer selle verduidelik. Dit is nou voor die hand liggend dat M. tuberculosis kan inmeng met die reaksies van die immuun sisteem, om dus die bakteriosidiese omgewing wat geskep word deur die selle van hierde sisteem te vermy. Daar is spesifieke kenmerke van M. tuberculosis wat toelaat dat die bacilli so ‘n omgewing kan weerstaan. Hierdie kenmerke is, onder andere, ‘n byna ondeurdringbare selwand en uitskeiding sisteme wat spesiale faktore vrystel. Hierdie faktore het die vermoë om direk met die gasheer immuun sisteem ‘n interaksie te hê wat dus die immuun sisteem moduleer. Verder, is M. tuberculosis se metabolisme aan gepas om die organisme te help teen die lae suurstof, hoë oksidatiewe en stikstof stress, lae pH en lae voedingswaarde omgewing te oorleef. M. tuberculosis het ook die vermoë om vir ‘n onbeperkte tyd in ‘n statiese toestand te oorleef, in gashere wat toon as gesond. Nuwe waarnemings in die energie, koolstof en sentrale stikstof metaboliese paaie stel voor dat ‘n homeostase gehandhaaf word deur M. tuberculosis, wat die konsentrasies van verskeie molekules moduleer of die effek van molekules wat deur die gasheer vrygestel word as ‘n verdedigings meganisme versag. In hierdie dokument bespreek ons verskeie studies, asook nuwe inligting voortgebring deur hierdie studie, wat fokus op sentrale metabolisme en sy regulering in M .tuberculosis. Ons raak aan die vermoë van M. tuberculosis om intrasellulêr te oorleef, koloniseer en voort te bestaan in ‘n gasheer. Ons vemoed dat die homeostatiese regulering van stikstof metabolisme in M. tuberculosis n diepgaande kwesbaarheid in die patogeen skep wat die potentiaal het om uit gebuit te word. Molekules kan gesintiseer word wat die aktiwiteite van verskeie ensieme in hierdie padweg inhibeer en sodoende die organisme hinder. Sulke molekules mag dalk as waardevolle en oorspronklike medisynes ontwikkel word om tuberkulose patiënte meer suksesvol te behandel asook om die las van middelweerstandige bakterieë te verlig. Met betrokke tot hierdie spesifieke studie, het ons die rol van glutamaat dehidrogenase (GDH) en glutamaat sintase (GltS) van M. bovis BCG bestudeer deur om die uitslaan mutante van die genoemde geen produkte aan verskeie sellulêre stress toestande bloot te stel. Die effek van die verlore gdh en gltBD gene op die evolusie van die genome van elke M. bovis BCG uitslaan mutant ras ten opsigte van die wilde tipe was ook bestudeer. Die rol van GDH was getoets in ‘n muis makrofaag model van infeksie om te bepaal of GDH n funksie het in koloniseering en infeksie van M. bovis BCG. Hierdie studie het nuwe bevindinge voort gebring wat die belangrikheid van GDH in die weerstand teen stikstof oksied stress. Daar is verder bevind dat GDH n vereiste toon vir die suksessvolle oorlewing van M. bovis BCG in RAW 264.7 macrofage

Το ενδογενές σύστημα των κανναβινοειδών αποτελεί ένα πολύπλοκο ενδογενές σύστημα μεταγωγής σήματος το οποίο επηρεάζει ένα σημαντικό αριθμό φυσιολογικών διεργασιών και μεταβολικών μονοπατιών του οργανισμού (Cota and Woods, 2005). Απαρτίζεται από τους διαμεμβρανικούς υποδοχείς των κανναβινοειδών (CBR), τα ενδοκανναβινοειδή και τις πρωτεΐνες που είναι υπεύθυνες για την βιοσύνθεση και την αποικοδόμηση των δεύτερων. (Petrocellis et al., 2004). Τα κανναβινοειδή παρουσιάζουν ποικιλία επιπτώσεων, όπως δυσλειτουργία στη μάθηση και μνήμη, διαφοροποίηση των συναισθηματικών καταστάσεων, μείωση κινητικού ελέγχου και αναλγησία. Αναστέλλουν τη συναπτική διαβίβαση σε διάφορες περιοχές του εγκεφάλου όπως ο ιππόκαμπος, ο επικλινής πυρήνας και ο προμετωπιαίος φλοιός κυρίως μέσω προσυναπτικών μηχανισμών. Το γλουταμινικό οξύ είναι ο κύριος διεγερτικός νευροδιαβιβαστής στο Κεντρικό Νευρικό Σύστημα των θηλαστικών. Οι γλουταμινεργικοί νευρώνες διανέμονται ευρέως στο κεντρικό νευρικό σύστημα και παίζουν ρόλο σε πολλές βιολογικές διεργασίες, συμπεριλαμβανομένης της κωδικοποίησης των πληροφοριών, το σχηματισμό και την ανάκτηση των μνημών, τη χωρική αναγνώριση και τη διατήρηση της προσοχής (McEntee και Crook, 1993). Λόγω του ρόλου του στη συναπτική πλαστικότητα, το γλουταμινικό εμπλέκεται σε γνωσιακές λειτουργίες του εγκεφάλου, όπως η μάθηση και η μνήμη (McEntee και Crook, 1993). Το γλουταμινικό δρα στα μετασυναπτικά κύτταρα σε τρεις οικογένειες ιοντοτροπικών υποδοχέων τους NMDA, τους AMPA και τους καϊνικούς υποδοχείς, ενώ υπάρχουν, επίσης, και γλουταμινικοί μεταβολοτροπικοί υποδοχείς που συνδέονται με G πρωτεΐνες. Η αμυγδαλή εκτελεί ένα πρωταρχικό ρόλο στην επεξεργασία των συναισθηματικών αντιδράσεων και τη μνήμη, ενώ ο ιππόκαμπος έχει σημαντικό ρόλο στην εδραίωση των πληροφοριών από βραχυχρόνια μνήμη σε μακροχρόνια μνήμη και στη χωρική πλοήγηση. Οι δύο αυτές περιοχές είναι πλούσιες σε CB1 και NMDA υποδοχείς, ενώ τα τελευταία χρόνια, πληθώρα ερευνών υποδεικνύει ότι η έκθεση πειραματοζώων σε κανναβινοειδή επιφέρει σημαντικές αλλαγές σ’ αυτές τις δύο περιοχές. Στόχος της ερευνητικής εργασίας ήταν η μελέτη της επίδρασης των κανναβινοειδών στο γλουταμινεργικό σύστημα, στην αμυγδαλή και τον ιππόκαμπο επίμυων. Η μελέτη επικεντώθηκε στους NMDA υποδοχείς του γλουταμινικού οξέος με τη χρήση των μεθόδων της in situ υβριδοποίησης για το mRNA των NR1, NR2A και NR2B υπομονάδων του υποδοχέα και της ποσοτικής αυτοραδιογραφίας υποδοχέων, σε επίμυς που τους χορηγήθηκε WIN55212-2, ένας συνθετικός αγωνιστής του CB1 κανναβινοειδικού υποδοχέα, ο οποίος εμφανίζει παρόμοια δράση με την Δ9-τετραϋδροκανναβινόλη (THC), το φυσικό συστατικό του φυτού Cannabis sativa. Μελετήθηκαν τέσσερις ομάδες επίμυων όπου σε δύο από αυτές χορηγήθηκε WIN και στις υπόλοιπες δύο χορηγήθηκε ο διαλύτης του WIN, Vehicle. Στις δύο ομάδες που χορηγήθηκε WIN55212- 2 1mg/kg για 20 ημέρες, οι επίμυς της μίας ομάδας (ομάδα WIN) θανατώθηκαν 2 ώρες μετά από την τελευταία δόση ενώ οι επίμυς της άλλης ομάδας (ομάδα WIN+WITHD) θανατώθηκαν 7 ημέρες μετά την τελευταία δόση. Κατά το διάστημα αυτό δεν έγινε καμία χορήγηση ώστε να μελετηθεί αν οι επιπτώσεις της χρόνιας χορήγησης του WIN ήταν μόνιμες. Τα αποτελέσματά μας έδειξαν ότι μετά τη χρόνια χορήγηση του WIN τα επίπεδα έκφρασης των υπομονάδων του NMDA υποδοχέα μειώθηκαν στο ιππόκαμπο και την αμυγδαλή των επίμυων, ενώ μετά την διακοπή της χορήγησης του WIN, τα επίπεδα έκφρασης επανέχρονται. Είναι πιθανό ότι οι αλλαγές στην έκφραση και λειτουργία των υποδοχέων του γλουταμινικού που παρουσιάζονται ως προσαρμοστικές αλλαγές στο νευρικό σύστημα, να είναι ένας κοινός μηχανισμός με τον οποίο τα εθιστικά ναρκωτικά επηρεάζουν την νευρική λειτουργία. / The endocannabinoid system is a complicated endogenous signaling system that affects a variety of physiological processes and metabolic routes in human body (Cota and Woods, 2005). The endocannabinoid system includes the transmembrane cannabinoid receptors (CBR), the endocannabinoids and the enzymes that synthesize and degrade the endocannabinoids (Petrocellis et al., 2004). Cannabinoids have a variety of effects, such as impairment in learning and memory, modulation of emotional states, reduced motor control and analgesia. Cannabinoids inhibit synaptic transmission in several brain regions such as the hippocampus, nucleus accumbens and the prefrontal cortex mainly via presynaptic mechanisms. Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system. The glutamatergic neurons are widely distributed in the central nervous system and play a role in many biological processes, including the coding of information, the formation and recovery of memories, the spatial recognition and maintaining the attention (McEntee και Crook, 1993). Because of its role in synaptic plasticity, glutamate is involved in cognitive functions like learning and memory in the brain. (McEntee και Crook, 1993). Glutamate activates three families of ionotropic receptors in postsynaptic cells, those are NMDA, AMPA and kainate receptors, while there are also metabotropic G proteins coupled glutamate receptors. Amygdala has a primary role in the processing of emotional reactions and memory, whereas hippocampus has an important role in the consolidation of informations from short term memory into long term memory and spatial navigation. These two brain regions contain a large number of CB1 and NMDA receptors, while recently, many studies suggest that animals treated with cannabinoids display significant changes in these two areas. The aim of this research was to study the changes in glutamatergic system in the amygdala and hippocampus of rats treated with cannabinoids. We focused on NMDA glutamate receptors, using in situ hybridization for studying the expression of NR1, NR2A and NR2B subunits and quantitative receptor autoradiography, in rats treated with WIN55212-2, a synthetic agonist of the CB1 cannabinoid receptor, which shows similar effects with delta-9-tetrahydrocannabinol (THC), a natural component of the plant Cannabis sativa. Adult rats were injected with WIN55212-2 (1mg/kg) and Vehicle. Animals received repeated administrations of WIN55212-2 1mg/kg once a day for 20 days. Animals in group WIN were sacrificed 2 hours after the last administration whereas in group WIN + WITHD were sacrificed 7 days after the last administration. During this time there was no administration of WIN so we could study whether the effects of chronic exposure were permanent. Our results demonstrate that chronic exposure to WIN55212-2 produced significant decreases in the expression of NMDA receptor's subunits in hippocampus and amygdala. These changes were reversed one week after abstinence. These adaptive synaptic changes may share common mechanisms with addictive drugs in modifying neural circuitry.

The neurotransmitter dopamine (DA) represents a neural substrate for positive motivation as its spatiotemporal distribution across the brain is responsible for goaldirected behavior and learning reward associations. The critical determinant of DA release throughout the brain is the firing pattern of DA-producing neurons. Synchronized bursts of spikes can be triggered by sensory stimuli in these neurons, evoking phasic release of DA in target brain areas to drive reward-based reinforcement learning and behavior. These bursts are generated by NMDA-type glutamate receptors (NMDARs). This dissertation reports a novel form of long-term potentiation (LTP) of NMDARmediated excitatory transmission at DA neurons as a putative cellular substrate for changes in DA neuron firing during reward learning. Patch-clamp electrophysiological recording from DA neurons in acute brain slices from young adult rats demonstrated that synaptic NMDARs exhibit LTP in an associative manner, requiring coordinated pre- and postsynaptic burst firing. Ca2+ signals produced by postsynaptic burst firing needed to be amplified by preceding metabotropic neurotransmitter inputs to effectively drive plasticity. Activation of NMDARs themselves was also necessary. These two coincidence detectors governed the timingdependence of NMDAR plasticity in a manner analogous to the timing rule for cuereward learning paradigms in behaving animals. Further mechanistic study revealed that PKA, but not PKC, activity gated LTP induction by regulating the magnitude of Ca2+ signal amplification via the inositol 1,4,5-triphospate (IP3) receptor and release of Ca2+ from intracellular stores. Plasticity of NMDARs was input specific and appeared to be expressed postsynaptically, but was not associated with a change in NMDAR subunit stoichiometry. LTP of NDMARs was DA-independent, and was specific for NMDARs: the same induction protocol produced long-term depression of AMPA receptors. NMDARs that had undergone LTP could be depotentiated in a spike-conditional manner, consistent with active unlearning. Finally, repeated, in vivo amphetamine experience dramatically increased facilitation of spike-evoked Ca2+ signals, which in turn drove enhanced plasticity. NMDAR plasticity thus represents a potential neural substrate for conditioned DA neuron burst responses to environmental stimuli acquired during reward-based learning as well a novel therapeutic target for intervention-based therapy of addictive disorders. / text

The prefrontal cortex (PFC) is an area of the brain that is critically important for learning, memory, organization, and integration, and PFC dysfunction has been associated with pathologies including Alzheimer’s disease, schizophrenia, and drug addiction. However, there exists a paucity of information regarding neurochemical signaling in the distinct sub-regions of the PFC, particularly the medial prefrontal cortex (mPFC). The mPFC receives glutamatergic input from a number of brain areas, and functional glutamate signaling is essential for normal cognitive processes. To further understand glutamate neurotransmission, in vivo measurements of glutamate were performed in the cingulate cortex, prelimbic cortex, and infralimbic cortex of anesthetized rats using enzyme-based microelectrode array technology. Measurements of acetylcholine were also performed to examine the relationship between glutamate and other neurotransmitters in the mPFC. The described studies revealed a homogeneity of glutamate and acetylcholine signaling in the mPFC sub-regions, indicating somewhat uniform tonic and phasic levels of these two transmitters. In the infralimbic mPFC of awake freely-moving rats, rapid, phasic glutamate signaling events, termed “transients” were observed and in vivo glutamate signaling was successfully monitored over 24 hour time periods. The effects of methylphenidate (MPH), a stimulant medication with abuse potential that is used in the treatment of attention-deficit hyperactivity disorder, were measured in mPFC sub-regions of anesthetized rats. Data revealed similar tonic and phasic glutamate levels between chronic MPH-treated rats and controls in all sub-regions. Locomotor data from the chronic treatment period supported the behavioral sensitization effects of multiple MPH treatments. Significant effects were observed in locomotor activity, resting levels of glutamate, and glutamate uptake rates in the infralimbic mPFC of awake, freely-moving animals that received chronic MPH treatment. Taken together, this body of work characterizes glutamate signaling in the rat mPFC to a degree never before reported, and serves to report for the first time the effects of MPH on glutamate signaling in the mPFC.