L-glutamate (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system. Monitoring extracellular Glu is critical to understanding Glu regulation to discriminate physiological and pathological roles. To overcome the limitations of previous in vivo extracellular Glu studies, we developed Glu selective microelectrode arrays with better spatial and temporal resolutions than commonly used techniques like microdialysis. We used these microelectrode arrays to characterize basal and potassium-evoked Glu neurotransmission in the normal rat brain. We then investigated disease-related Glu alterations in a rat model of Parkinson's disease and normal Glu regulation in young and aged rhesus monkeys. In the normal anesthetized rat striatum and frontal cortex, basal Glu was regulated by active release and uptake mechanisms, fully TTX-dependent, and measured at ~2 micromolar levels. Potassium-evoked Glu kinetics were fast, concentration-dependent, and rapidly reproducible at 15-20 seconds intervals. In the unilateral 6-hydroxydopamine-lesioned rat, there were significant bilateral increases in potassium-evoked Glu release in the striatum and frontal cortex compared to hemisphere-matched non-lesioned rats. Ipsilateral striatal effects may have been related to DA loss, while contralateral striatal effects and the bilateral frontal corticaleffects may have resulted from parkinsonian neurotransmitter changes or bilateral neuranatomical connectivity, especially in the cortex. There were also alterations in Glu kinetics in the nucleus accumbens in both non-lesioned and lesioned rats. With appropriate technological and methodological modifications, we successfully recorded normal Glu signaling in anesthetized nonhuman primates in the operating room. Fast potassium-evoked Glu signals were recorded in the motor cortex of all monkeys, and Glu ejections showed robust Glu uptake in the motor and frontal cortices of all monkeys. These findings are comparable to initial rat studies. Slow evoked Glu kinetics and high basal Glu levels with oscillatory behavior were recorded in the frontal cortex. The primary age-related differences between monkeys were the nearly ten-fold increases in the volumes of Glu ejected needed in the aged monkey to achieve amplitude-matched signals in the motor and frontal cortices and a decreased uptake rate in the motor cortex. Preliminary work with excised human tissue and future plans for patient-oriented research and clinical applications are discussed.

No description available.

No description available.

Fibromyalgia is a chronic pain disorder characterized by multiple symptoms including severe fatigue, headache, muscle pain, cognitive dysfunction, and paresthesias. Up to 81% of patients with fibromyalgia (FM) also suffer from irritable bowel syndrome (IBS). The objectives of this study were: 1) to evaluate the effect of a 4-week excitotoxin additive free diet on symptoms of fibromyalgia (FM) and irritable bowel syndrome (IBS), and then further, to use a randomized double blind crossover challenge to determine: 2) whether FM symptoms would return more frequently when subjects were challenged with MSG as compared to placebo, and 3) whether IBS symptoms returned upon MSG challenge more frequently than placebo. Subjects were recruited from the Portland, OR area, and attended a 2-hour group diet training session and individual clinic appointment before starting a one-month excitotoxin additive free diet. At the end of the month, subjects reporting greater than 30% symptom improvement went onto a 2-week double blind crossover challenge period where they were randomized to receive either MSG in juice for 3 days or placebo for 3 days. The following week they received whatever they did not receive the first week. Eighty-four percent of those who finished the diet reported >30% symptom improvement and pre-post diet analysis demonstrated highly significant difference scores for all major outcome measures. Total symptom scores (11.4, p<0.0001), fibromyalgia impact questionnaire-revised scores (FIQR) (22, p<0.0001), and IBS quality of life (IBS-QOL) questionnaire scores (11, p<0.0001) were all significantly reduced, as were visual analog pain (VAS) change scores for FM (5.4, p<0.0001) and IBS (4.6, p<0.0001). Challenge results demonstrated that diet responders got significantly worse when challenged with MSG as compared to placebo in most measures (total symptom score, p<0.02; FIQR, p<0.03; and IBS-QOL, p<0.05). VAS for IBS and FM both worsened, but to a lesser degree (mean change of 2.1 (p<0.19) and 2.5 (p<0.07) respectively). The majority of responders were still following the diet at 2 months post study which suggests feasibility and benefit. Results suggest that the excitotoxin additive free dietary intervention may provide significant symptom relief equal to or greater than current pharmacological strategies for fibromyalgia patients with IBS.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Developmental nicotine exposure (DNE) is associated with increased risk of cardiorespiratory, intellectual, and behavioral abnormalities in neonates, and is a risk factor for apnea of prematurity, altered arousal responses and Sudden Infant Death Syndrome. Alterations in nicotinic acetylcholine receptor signaling (nAChRs) after DNE lead to changes in excitatory neurotransmission in neural networks that control breathing, including a heightened excitatory response to AMPA microinjection into the hypoglossal motor nucleus. Here, we report on experiments designed to probe possible postsynaptic and presynaptic mechanisms that may underlie this plasticity. Pregnant dams were exposed to nicotine or saline via an osmotic mini-pump implanted on the 5th day of gestation. We used whole-cell patch clamp electrophysiology to record from hypoglossal motoneurons (XIIMNs) in thick medullary slices from neonatal rat pups (N = 26 control and 24 DNE cells). To enable the translation of our findings to breathing-related consequences of DNE, we only studied XIIMNs that were receiving rhythmic excitatory drive from the respiratory central pattern generator. Tetrodotoxin was used to isolate XIIMNs from presynaptic input, and their postsynaptic responses to bath application of L-glutamic acid (glutamate) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were studied under voltage clamp. DNE had no influence on inward current magnitude evoked by either glutamate or AMPA. However, in cells from DNE animals, bath application of AMPA was associated with a right shift in the amplitude distribution (P = 0.0004), but no change in the inter-event interval distribution of miniature excitatory postsynaptic currents (mEPSCs). DNE had no influence on mEPSC amplitude or frequency evoked by glutamate application, or under (unstimulated) baseline conditions. Thus, in the presence of AMPA, DNE is associated with a small but significant increase in quantal size, but no change in the probability of glutamate release.

Priority tasks of the present thesis were to generate various enantiopure C-3-substituted pyroglutamates as well as C-3-substituted glutamates, and furthermore to ameliorate the serious drawback of the bad atom-economy in the reaction sequence of previously published silylether-mediated procedures. To meet these requirements, the ortho ester functionality (OBO ester) developed by Corey was introduced. According to the plan of synthesis, the starting material, non-racemic (S)-pyroglutamic acid, was converted to the corresponding oxetane ester via a DCC-mediated esterification. The latter was N-protected to provide N-acceptor substituted pyroglutamic acid oxetane esters (Acceptor=Boc,Cbz,CO2Me). After rearrangement with boron trifluoride, the ortho ester derivatives (Acceptor=Cbz,CO2Me) were at hand and exclusively the N-Cbz derivative was converted to the corresponding alpha,beta-unsaturated lactam via a syn-elimination reaction. The formation of the C-3-substituted ortho ester compounds (R=methyl,ethyl,butyl,allyl,phenyl,4-chlorophenyl,biphenyl,naphthyl) was performed via a copper-mediated conjugate addition to the alpha,beta-enone system of the N-Cbz-alpha,beta-unsaturated lactam. The OBO functionality hence was envisaged to support perfect trans selectivity in this cuprate addition to the Michael system of the N-Cbz-alpha,beta-unsaturated lactam. Spectroscopic NMR-data, on the basis of 1H-, 13C- and DEPT spectra, proved the assumption that the C-3-substituted ortho ester derivatives exclusively are trans-configurated, i.e. the alkyl derivatives (R=methyl,ethyl,butyl,allyl) are (2S,3S)-configurated and the aryl derivatives (R=phenyl,4-chlorophenyl,biphenyl,naphthyl) are (2S,3R)-configurated). The C-3-substituted ortho ester derivatives were completely deprotected to yield the C-3-substituted pyroglutamates (R=ethyl,phenyl,4-chlorophenyl,naphthyl). Finally, ring opening reaction via route A-2 lead to the desired enantiopure C-3-substituted glutamates. Alternatively, latter preferably were reacted via route A-1 to yield the C-3-substituted glutamates (R=methyl,ethyl,butyl,phenyl,4-chlorophenyl,naphthyl). Their (2S,3R)-configuration (R=aryl) and (2S,3S)-configuration (R=alky), respectively, unambiguously was proved on the basis of available spectroscopic NMR-data. To ensure this assumption, diastereomeric (2S,3R)-3-methyl glutamic acid (i.e. cis-configurated) examplarily was synthesized via route A-3 and spectroscopic NMR-data was compared to that of (2S,3S)-3-methyl glutamic acid (i.e. trans-configurated). Conclusively, there can be recorded the fact that the serious drawback of the bad atom-economy in the reaction sequence previously used can be circumvented by the introduction of the OBO functionality, so the concept of an improved atom-economy is achieved. Additionally, in comparison to the silyl-ether-mediated synthesis, the OBO functionality provided crystalline ortho ester derivatives, which facilitated their purification as well as characterization. / Schwerpunkte dieser Arbeit lagen in der Synthese verschiedener enantiomerenreiner C-3-substituierter Pyroglutamate als auch Glutamate und desweiteren darin, die unzulängliche Wirtschaftlichkeit der bereits bekannten Silylether-vermittelten Methode zu verbessern. Um diesen Anforderungen gerecht zu werden, wurde die von Corey entwickelte Orthoester-Funktionalität (OBO Ester) eingeführt. Dem Syntheseplan entsprechend wurde die Ausgangskomponente, nicht-razemische (S)-Pyroglutaminsäure, über eine DCC-vermittelteVeresterung zum entsprechenden Oxetanester umgesetzt. Dieser wurde daraufhin N-geschützt, wobei die N-Akzeptor-substituierten Pyroglutaminsäureoxetanester(Akzeptor=Boc,Cbz,CO2Me) erhalten wurden. Nach der Umlagerung dieser Ester mit Bortrifluorid wurden die Orthoester-Derivate (Akzeptor=Cbz,CO2Me) erhalten, wobei sich ausschliesslich das N-Cbz Derivat über eine syn-Eliminierungsreaktion zum entsprechenden alpha,beta-ungesättigten Lactam umsetzen liess. Die C-3-substituierten Orthoester-Derivate (R = Methyl,Ethyl,Butyl,Allyl,Phenyl,4-Chlorphenyl,Biphenyl,Naphthyl) wurden über eine kupferkatalysierte 1,4-Addition an das alpha,beta-Enon-System des alpha,beta-ungesättigten Orthoester-Derivats erhalten. Hiebei war zu erwarten, dass die OBO-Funktionalität ausschliesslich trans-Selektivität bei der Cuprat-Addition an das Michael System dieses Derivats gewährleistet. 1H-, 13C- und DEPT-Spektren und die daraus erhaltenen Daten lieferten den Beweis, dass die C-3-substituierten Orthoester-Derivate ausschliesslich trans-Konfiguration aufwiesen, d.h. die Alkylderivate (R=Methyl,Ethyl,Butyl,Allyl) sind (2S,3S)-konfiguriert bzw. die Arylderivate (R=Phenyl,4-Chlorphenyl,Biphenyl,Naphthyl) (2S,3R)-konfiguriert. Die C-3-substituierten Orthoester-Derivate wurden vollständig entschützt, um die C-3-substituierten Pyroglutamate (R=Ethyl,Phenyl,4-Chlorphenyl,Naphthyl) zu erhalten. Schliesslich führte eine Ringöffnungsreaktion über Route A-2 zu den gewünschten enantiomerenreinen C-3-substituierten Glutamaten. Alternativ wurden diese Glutamate (R=Methyl,Ethyl,Butyl, Phenyl,4-Chlorphenyl,Naphthyl) auf eine einfachere Art und Weise über Route A-1 aus den entsprechenden C-3-substituierten Orthoester-Derivaten gewonnen. Deren (2S,3R)-Konfiguration (R=aryl) bzw. (2S,3S)-Konfiguration (R=alkyl) wurde anhand verfügbarer spectroskopischer NMR-Daten überprüft. Um diese Vermutung zu untermauern, wurde beispielhaft (2S,3R)-3-Methylglutaminsäure (i.e. cis-konfiguriert) über Route A-3 synthetisiert und deren spectroskopische NMR-Daten mit denen von (2S,3S)-3-Methylglutaminsäure (i.e. trans-konfiguriert) verglichen. Zusammenfassend lässt sich feststellen, dass der Nachteil der unzulänglichen Wirtschaftlichkeit früherer Synthesen durch die Einführung der OBO-Funktionalität umgangen werden kann, so dass letztendlich das Konzept einer verbesserten „atom-economy“ erreicht wurde. Zusätzlich lieferte die OBO-Funktionalität im Vergleich mit Silylether-Synthesen den Vorteil kristalliner Orthoester-Produkte, was sowohl die Aufarbeitung als auch deren Charakterisierung erleichterte.

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

Alcoholism is a devastating chronic relapsing disorder with significant costs to individuals and society. The mesolimbic dopamine (DA) system plays an important role in regulating reward and addiction. GABA neurons located in the ventral tegmental area (VTA) regulate VTA DA neuron activity, and are a relevant target for alcohol in the brain. VTA GABA neurons exhibit marked hyperexcitability during withdrawal from ethanol. Past research has demonstrated that the motivational effects of opiates cause a change in VTA GABA(A) receptors in opiate-dependent animals, which switch from a GABA-induced hyperpolarization of GABA neurons to a GABA-induced depolarization. The focus of this study was to characterize excitatory and inhibitory synaptic activity in VTA GABA neurons during withdrawal from acute and chronic alcohol, and to evaluate the function of the GABA(A) receptor in the pathway to dependence. Animals were either given injections of ethanol or saline, or were kept in ethanol vapor or air chambers for three weeks. We used standard whole-cell, perforated patch, and cell-attached mode electrophysiological techniques and pharmacology to obtain recordings of cellular activity. Results for excitatory and inhibitory synaptic events were somewhat mixed and inconclusive. There is evidence for a shift in function of the GABA(A) receptor after exposure to ethanol. We found that after a single injection of ethanol (4.0 g/kg) or a chronic intermittent ethanol vapor exposure, VTA GABA neuron firing rate is less sensitive to muscimol's inhibitory effects. The neural substrates of addiction studied here are important steps in the road to alcohol dependence, and a better understanding of them may lead to novel therapies.

Abstract not available