The retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and thalamic relay neurons, undergoes extensive remodeling and refinement in the first few postnatal weeks. While many studies have focused on this process, little is known about the factors that influence excitatory transmission during this dynamic period. A major goal of my dissertation research was to identify mechanisms that regulate glutamate release and clearance at the developing synapse. First, we investigated the role of glutamate transporters and metabotropic glutamate receptors (mGluRs) in shaping excitatory transmission. Early in development, we found presynaptic group II/III mGluRs are present and are activated by glutamate released from RGCs following optic tract stimulation at natural frequencies. This response was found to diminish with age, but glutamate transporters continued to shape synaptic currents throughout development. The finding that glutamate is able to escape the synaptic cleft and bind extrasynaptic high-affinity mGluRs led us to speculate that glutamate might also diffuse to neighboring synapses and bind ionotropic glutamate receptors opposing quiescent release sites. Excitatory currents recorded from immature, but not mature, retinogeniculate synapses display a prolonged decay timecourse. We found evidence that both asynchronous release of glutamate as well as spillover of glutamate between neighboring synapses contributes to these slowly decaying synaptic currents. Furthermore, we uncovered and characterized a novel, purely spillover-mediated current from immature relay neurons, which strongly supports the presence of glutamate spillover between boutons of different RGCs. The results of my studies indicate that far more RGCs contribute to relay neuron firing than would be predicted by the anatomy alone. Finally, in an ongoing study, we investigated the functional role of the neuronal glutamate transporter GLT-1 at the immature retinogeniculate synapse. While GLT-1 has been found in both neurons and glia, excitatory currents at the retinogeniculate synapse were largely unaffected in mice lacking neuronal GLT-1, suggesting non-neuronal glutamate transporters are responsible for the majority of glutamate removal from the developing synapse. Taken together, these results provide insight into the synaptic environment of the developing retinogeniculate synapse and identify a number of mechanisms that shape excitatory transmission during this period of synaptic maturation and refinement.

Στον αμφιβληστροειδή επιτελείται η μετατροπή δηλαδή της φωτεινής ενέργειας σε ηλεκτρικό ερέθισμα. Κύριος διεγερτικός διαβιβαστής στον αμφιβληστροειδή είναι το γλουταμινικό οξύ του οποίου η δράση επιτελείται μέσω ιοντοτρόπων, NMDA και μη NMDA, και μεταβοτρόπων υποδοχέων. Οι υποδοχείς ΝΜDA παρουσιάζουν ιδιαίτερα χαρακτηριστικά, όπως μεγάλη αγωγιμότητα ασβεστίου και τασεοεξαρτώμενη αναστολή από το Μg, ενώ φαίνεται να παίζουν ιδιαίτερο ρόλο σε διαδικασίες συναπτικής πλαστικότητας, στην ανάπτυξη του νευρικού συστήματος καθώς και στην διεγερσιτοξικότητα του γλουταμινικού. Στον αμφιβληστροειδή εντοπίζονται κυρίως στα γαγγλιακά και βραχύϊνα κύτταρα αλλά και σε διάμεσους νευρώνες. Οι υποδοχείς NMDA είναι ετερομερή που αποτελούνται από τις υπομονάδες NR1, NR2 και NR3. Το γονίδιο της βασικής λειτουργικής υπομονάδας NR1 περιέχει τρία εξόνια τα οποία υφίστανται εναλλακτικό μάτισμα προς δημιουργία οχτώ ισομορφών. Οι ισομορφές που περιλαμβάνουν το εξόνιο 21 ή C1 στο καρβοξυτελικό άκρο έχουν την χαρακτηριστική ιδιότητα φωσφορυλίωσης στην θρεονίνη 879 και στις σερίνες 890, 896 και 897. Η φωσφορυλίωση αποτελεί έναν από τους κυριότερους μηχανισμούς ρύθμισης των υποδοχέων του γλουταμινικού οξέος επηρεάζοντας τις ιδιότητες τους, την μεταφορά τους προς την κυτταρική μεμβράνη, αλλά και την υποκυτταρική κατανομή των υπομονάδων τους. Στόχος της εργασίας είναι η μελέτη της φωσφορυλιωμένης υπομονάδας NR1 του υποδοχέα NMDA στις θέσεις σερίνης 896 και 897 (NR1-Ser896 και NR1-Ser897) κατά την ανάπτυξη στον αμφιβληστροειδή. Για τον σκοπό αυτό χρησιμοποιήσαμε επίμυες Wistar ηλικιών 9, 14, 21, 35 και 60 ημερών. Με την μέθοδο ανοσοαποτύπωσης κατά Western μελετήσαμε τα επίπεδα της φωσφορυλιωμένης πρωτείνης. Η ΝR1-Ser897 παρουσιάζει ένα πρότυπο αύξησης μέχρι και την ηλικία των 35 ημερών με επακόλουθη πτώση σε αυτήν των 60 ημερών. Η NR1-Ser896 αυξάνεται μέχρι την ηλικία των 14 ημερών όπου και παρουσιάζει μια σταθερή πορεία μέχρι την ηλικία των 60 ημερών. Συμπερασματικά, οι φωσφορυλιώσεις στις διαφορετικές σερίνες της υπομονάδας NR1 του υποδοχέα NMDA παρουσιάζουν διαφορετικό αναπτυξιακό προφίλ και γενικότερα η φωσφορυλίωση αυτής της υπομονάδας φαίνεται ότι ρυθμίζεται αναπτυξιακά στον αμφιβληστροειδή. / The retina is responsible for the light conversion into nerve signals. Glutamate is the major excitatory neurotransmitter in the retina. Its actions are mediated by glutamate ionotropic (NMDA and non NMDA) and metabotropic receptors. The NMDA receptors (NMDARs) are permeable to Ca++ and are unique among glutamate receptors in that they are blocked by Mg++ in a voltage dependent manner. These receptors also seem to play an important role in the development of the nervous system, in synaptic plasticity as well as in glutamate neurotoxicity. In the retina they are expressed in many ganglion and amacrine cells and occasionally in horizontal and glial cells. Functional NMDARs are heteromers composed of the NR1 NR2 and NR3 subunits. The gene of the NR1 subunit has three exons which undergo alternative splicing to generate theoretically eight NR1 splice variants. Half of them include the exon 21 or C1 in carboxy-terminus domain which can be phosphorylated in the following residues: threonine 879 and serines 890,896 and 897. Protein phosphorylation has been recognized as a major mechanism for the regulation of glutamate receptor function, changing their properties and because of its proposed role in trafficking and targeting of the NMDARs, as well as in clustering NR1 subunits into receptor-rich domains. The aim of the present work is to study the phosphorylated subunit NR1 at the serine residues 896 and 897 of the NMDAR (NR1-Ser896 and NR1-Ser897) during retinal development. Wistar rats at postnatal days 9, 14, 21, 35 and 60 are used for the developmental studies. The protein levels of the phosphorylated NR1 subunit were evaluated in Western blots. NR1-Ser897 increased gradually with a peak value observed at postnatal day 35, followed by a decrease at P60. NR1-Ser 896 was also increased to its peak level at the age of 14 and its levels sustained until the age of 60.These data reveal that the developmental profiles of the phoshorylated NR1 subunits at the serine residues 896 and 897 are different, and that the phosphorylation of the NR1 subunit is, in general, subject to regulation during development of the retina

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references.

Aromatase is an enzyme that converts androgens to estrogens. In teleosts, brain aromatase, also known as aromatase B (cy19a1b), is only expressed in radial glial cells (RGCs). This is in contrast to aromatase A, which is expressed in gonads. Estrogens such as estradiol (E2) modulate neurogenesis in the adult teleost brain. Recent studies show that E2 also differentially regulates aromatase B expression in goldfish RGCs. As a result, teleost RGCs are suggested to be involved in regulating neurogenesis. In addition, aromatase B expression in goldfish RGC is under the control of dopamine suggesting that neurons and neurotransmitters can regulate RGC function. Interestingly, goldfish RGC transcriptome data shows the expression of one group of metabotropic glutamate receptors (mGluRs), group III mGluRs, which suggests that glutamate may affect RGC function. In this thesis, I present my findings regarding potential glutamatergic regulation of RGCs. Firstly, I investigated the distribution of glutamatergic synaptic vesicles and RGCs in the female goldfish forebrain. Double-staining immunohistochemistry shows that vesicular glutamate transporter (vGLUT) 1/2-labelled glutamatergic synaptic vesicles are in close anatomical proximity to aromatase B-labelled RGCs, which suggests potential regulation of RGCs by glutamate. Glutamatergic regulation of cyp19a1b, cyclin D1 (ccnd1), cyclin A2 (ccna2), mGluR6b (grm6b), mGluR7 (grm7), and mGluR8b (grm8b) expression in cultured adult female goldfish RGCs was also examined. Results from pharmacological manipulations and qPCR data analysis show that selective activation of group III mGluRs decreased cyp19a1b, ccnd1, and ccna2 mRNA via inhibition of cAMP/PKA signalling. Furthermore, grm7 mRNA is positively regulated by cAMP-dependent signalling. The glutamate analog L-glutamic acid decreased cyp19a1b mRNA and increased ccnd1 and grm6b mRNA in a dose-dependent manner. This suggests that ccnd1 and grm6b expression may be regulated by glutamate receptors other than group III mGluRs, for example, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are expressed in cultured goldfish RGCs. It was found that E2 upregulated cyp19a1b, ccnd1 and grm7 mRNA. However, selective activation of group III mGluRs decreases the stimulatory effect of E2 on ccnd1 expression. My findings show that glutamate finely regulates RGC neurogenic and steroidogenic genes, which may implicate glutamate in the regulation of RGC differentiation, RGC proliferation, and neurogenesis in surrounding cells.

We employed imaging, electrophysiological, and molecular techniques with the genetically tractable model organism Drosophila melanogaster to unravel fundamental biological and genetic underpinnings regulating synaptic function and plasticity. Using a forward genetic screen, we identified mutations in the Drosophila ortholog of a human WD40 repeat-containing protein termed DDB1 and CUL4 associated factor 12 (DCAF12). We show that DCAF12 likely serves as an adaptor protein for the DDB1-Cul4 E3 ubiquitin ligase complex by recruiting specific target proteins for ubiquitination. DCAF12 is expressed in neurons, muscles, and glia. In mitotically active cells such as muscles, DCAF12 is localized to nuclei and co-localizes in distinct foci with CUL4, suggesting that DCAF12 mediates a nuclear role for the CUL4 E3 ubiquitin ligase complex. In neurons, DCAF12 is localized to both cytoplasmic and nuclear compartments of motor neuron cell bodies, where it colocalizes with Cul4 in nuclei. DCAF12 is also expressed at the periactive zone of presynaptic terminals, but does not distinctly associate with DDB1 or Cul4 at this region. Evoked neurotransmitter release at larval NMJs is significantly reduced in DCAF12 mutants. These defects are rescued by presynaptic expression of wild-type DCAF12, demonstrating that DCAF12 is required presynaptically and serves as an important component of the machinery that facilitates evoked release. In addition, our studies show that DCAF12 is required for the differential expression of glutamate receptor subunits at the larval NMJ through transcriptional and post-translational mechanisms. GluRIID subunit mRNA levels and GluRIIA/C/D subunit protein levels are increased at DCAF12 mutant NMJs. Normal GluRIIA subunit levels can be restored by postsynaptic expression of wild-type DCAF12, but not with a truncated DCAF12 protein lacking a nuclear localization signal (∆NLS-DCAF12). Furthermore, DCAF12 overexpression in muscle nuclei reduces synaptic GluRIIA levels, an effect that can be suppressed by removing a copy of Cul4. These data strongly indicate that DCAF12 in muscle nuclei is required for GluRIIA expression and/or function in a Cul4-dependent manner. Moreover, homozygous DCAF12-GluRIIA double mutants show a strong synthetic lethality phenotype, providing further support for the hypothesis that GluRIIA directly or indirectly requires DCAF12. Mutations in glutamate receptors at larval NMJs trigger a retrograde trans-synaptic signal that leads to a compensatory increase in presynaptic release, which precisely restores the normal efficacy of synaptic transmission and muscle excitation. Reducing the gene dosage of DCAF12 by one gene copy suppresses the initiation and maintenance of GluRIIA-mediated synaptic homeostatic potentiation. This block of synaptic homeostatic potentiation can be rescued by presynaptic expression of DCAF12. In our studies, we determined that DCAF12 is critical for 3 distinct synaptic mechanisms: evoked neurotransmitter release, neurotransmitter reception by regulation of GluR subunit composition, and retrograde synaptic homeostatic signaling. Future research will strive to identify presynaptic and postsynaptic protein targets of DCAF12 and the Cul4 E3 ubiquitin ligase complex and the role of ubiquitination in regulating these synaptic processes.

A doença de Chagas, também conhecida como tripanossomíase americana é causada pelo protozoário flagelado Trypanosoma cruzi e afeta aproximadamente 10 milhões de pessoas em áreas endêmicas do México, America central e do Sul. A quimioterapia disponível atualmente limita-se a dois compostos: Nifurtimox e o Benzonidazol. Ambos os fármacos reduzem os sintomas da doença e a mortalidade das pessoas infectadas, quando utilizadas na fase aguda, mas sua eficácia na fase crônica é controversa. Além do mais apresentam vários efeitos colaterais. O T. cruzi é capaz de utilizar carboidratos e aminoácidos como fonte de energia e carbono. O nosso grupo tem estudado o envolvimento do glutamato na resistência do parasita ao estresse térmico, oxidativo e metabólico como também a importância do aminoácido GABA no metabolismo do T. cruzi, visto que este também é transportado pelo parasita. Nesse contexto, o objetivo desse trabalho foi avaliar a interferência de fármacos, que atuam via receptores ou transportadores de GABA e glutamato, no ciclo de vida do T. cruzi. Os fármacos selecionados foram: Vigabatrina, Pregabalina, MK-801, MTEP e Memantina. Os resultados demonstram que apenas Memantina apresentou um melhor efeito tripanocida. Inibiu a proliferação de formas epimastigotas, interferiu na metaciclogênese, como também afetou o metabolismo energético do parasita com a diminuição dos níveis de ATP, além de desencadear mecanismos que levam a apoptose das formas epimastigotas. Memantina também interferiu no ciclo intracelular do parasita, mais especificamente no estágio amastigota. Interessantemente as fases do ciclo do parasita que são mais afetadas, são as fases que requerem mais energia: estágios replicativos (amastigota e epimastigota), como também os processos de diferenciação e invasão celular. Apesar de apresentar valores de IC50 que podem ser considerados altos em relação aos valores descritos para formas epimastigotas tratadas com Benzonidazol, esses resultados mostram perspectivas promissoras, visto que o Memantina poderá ser utilizado como composto líder para o desenho de derivados com atividade tripanocida otimizada. / Chagas disease, also known as American trypanosomiasis is caused by the flagellate protozoan Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico, Central America and South America, with about 25 million people living in areas at risk of infection. The currently available chemotherapy is limited to two compounds, which are: Nifurtimox and Benznidazole. Both drugs reduce symptoms of the disease and mortality of infected people when used in the acute phase, but its efficacy in chronic phase (phase in which the majority of cases are diagnosed) is still controversial. Moreover these drugs have several side effects. T. cruzi is able to utilize carbohydrate and amino acids as carbon and energy source. Our group has studied the involvement of glutamate in parasite resistance to thermal, oxidative and metabolic stress, as well as the importance of the amino acid GABA metabolism in T. cruzi. The aim of this study was to evaluate the interference of drugs, that act in GABA and glutamate transporters or receptors, in the life cycle of T. cruzi. The drugs selected were: vigabatrin, pregabalin, MK-801, memantine and MTEP. The results showed that only memantine has a better trypanocidal effect. Memantine inhibited the proliferation of epimastigotes, interfered in metacyclogenesis, and affected the energy metabolism of the parasite with decreased levels of ATP, and trigger mechanisms that lead to apoptosis of epimastigotes. Moreover interferes with intracellular cycle of the parasite, specifically in amastigote stage. Interesting that stages of the parasite that require more energy are more affected: replicative stages (epimastigote and amastigote) as well as the processes of differentiation and cell invasion. IC50 value is more than that described to epimastigotes treateds with Benznidazole, but Memantine could be used as leader compound for the design of drugs with optimized trypanocidal activity.

Schizophrenia is a neurological disorder with a complex psychopathology, which is far from fully elucidated. In the patients with this disorder, changes on anatomical, cellular, and neurotransmitter level have been found. The aim of this work is to elucidate the function of specific ionotropic glutamate receptors in NG2 glia in the hippocampus of a mouse model of schizophrenia. For this purpose, a mouse model of schizophrenia was generated and validated using immunohistochemistry and behavioural testing. Mice with NG2 glia labelled by a fluorescent protein with a calcium indicator also in NG2 glia were used to observe the activity of glutamate channels and the properties of the extracellular space in these mice. Changes were found in the schizophrenic animals when compared to control animals in the numbers of hippocampal oligodendrocyte lineage cells, in prepulse inhibition and in both volume fraction and tortuosity of the extracellular space in hippocampus. Moreover, the percentage of cells responding to glutamate receptor agonists in NG2 glia in hippocampus also differed significantly between the schizophrenic and the control animals. In conclusion, it can be said that we were able to observe significant changes in the mouse model of schizophrenia that we generated in comparison to control...

'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.