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

Le récepteur du glutamate de type NMDA (NMDAR) est l'un des deux principaux récepteurs glutamatergiques, et donc un des principaux mediateurs de la neurotransmission excitatrice dans le système nerveux central. Les NMDARs sont impliqués dans la plasticité synaptique, le corrélat cellulaire de l'apprentissage et de la mémoire. Actuellement, la technologie de pointe permettant l'investigation des propriétés des récepteurs synaptiques dans leur environnement natif est la photolyse de composés chimiques cagés, mais cet outil a des limitations concernant sa capacité à stimuler des NMDAR de manière très précise spatiellement et temporellement, à cause de la limite de diffraction de la lumière, qui définit le volume minimal de décageage duquel les molécules diffusent, et à cause de la nature irréversible de la réaction de décageage. En revanche, les molécules photoréversibles peuvent être activées et désactivées rapidement et de manière repétée, ce qui permet d'éviter les limitations de la diffusion afin d'accomplir une stimulation plus précise au niveau spatial et temporel. J'ai établi une collaboration autour du premier composé chimique photoréversible spécifique pour les NMDAR, et de plus, le premier qui est inactif dans son état le plus stable : l'azobenzene triazole glutamate (ATG). J'ai caracterisé ce composé chimique par activation un-photon et bi-photon, par l'application en bain et l'application locale, et dans plusieurs paradigmes expérimentaux. Dans ma thèse, je décris le fonctionnement de ce composé chimique, ses avantages et ses inconvénients, et certaines modifications à considérer pour l'optimisation future des composés chimiques photoréversibles. / The NMDA-type glutamate receptor (NMDAR) is one of two principal glutamate receptors, the main mediators of excitatory neurotransmission in the central nervous system. NMDARs are critically implicated in synaptic plasticity, the cellular correlate of learning and memory. Although significant advances have been made in understanding the behavior of this receptor, many questions remain. Currently, the state-of-the-art technology for investigating receptor properties in the native environment is caged compounds, which are restricted in their ability to precisely control the spatial and temporal activation of NMDAR due to the diffraction limit of light, which defines the minimum volume of uncaging from whence uncaging molecules diffuse, and the irreversible nature of uncaging. Photoswitchable molecules, by contrast, can rapidly and repeatedly be switched on and off, circumventing the diffusion limitation to permit fine spatial and temporal control of receptor activation. With this in mind, I formed a collaboration with a team of chemists to characterize a novel compound, azobenzene triazole glutamate (ATG), the first photoswitchable compound specific for NMDAR and biologically inert in its thermally stable state. Such a tool holds great promise for finely probing receptor behavior in its native environment. I characterized this compound using one- and two-photon activation, through bath and local application, and through a variety of different experimental paradigms. I demonstrate in detail the properties of this novel compound, propose potential applications of ATG as a novel tool, and suggest possible modifications to optimize future photoswitchable compound design.

Hyper-activation of N-methyl-D-aspartate receptors (NRs) is associated with excitotoxic cell death during secondary injury following traumatic brain injury (TBI). The efficiency of the NR is dependent on the location of receptors in membrane raft microdomains that provide a platform for coupling of NRs and effector proteins. In many neurodegenerative diseases, activation of the autophagy pathway has been suggested to contribute to glutamate excitotoxicity, but whether increased autophagy signaling contributes to pathology after TBI has not been defined. In these studies, I investigate whether membrane rafts mediate NR signaling and autophagy in cortices of adult male rats subjected to moderate TBI and in sham-operated controls. These studies demonstrate that membrane rafts of the normal rat cortex contain a novel multi-protein signaling complex that links the NR2B glutamate receptor and the autophagic protein Beclin 1. TBI caused a rapid disruption of this complex in which NR2B and pCaMKII were recruited to membrane microdomains. Alteration in NR2B-Beclin 1 association in membrane rafts resulted in activation of autophagy as demonstrated by increased expression of key autophagic proteins Beclin 1, ATG 5 and ATG 7, and significant increases in autophagic vacuoles in neurons of traumatized brains. Administration of the NR2B antagonist RO 25-6981 significantly blocked TBI-induced redistribution of NR2B signaling intermediates and Beclin 1 and delayed the increase in autophagy protein expression in traumatized cortices. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin 1 in membrane rafts after TBI.

Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine that orchestrates key events of development, disease and repair in the central nervous system (CNS). To investigate the effects of chronically producing TGF-β1 on synaptic structure and synaptic transmission, I performed immunohistochemistry and immunoblot of brain tissues from transgenic mice (TGF-β1 mice) that over-express active form of TGF-β1 from astrocytes in the CNS. Immunohistochemical assays showed that synaptophysin increased in the CA3 subfield whereas calbindin-D28K decreased in the mossy fibres. Immunoblot analysis revealed that several α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunit proteins were up-regulated in the hippocampus of TGF-β1 mice. To examine the direct effect of TGF-β1 alone on glutamatergic synaptic activity, cultured hippocampal neurons were treated with or without TGF-β1. Electrophysiological recordings displayed that TGF-β1 significantly increased the amplitude of glutamate-evoked current (p<0.05). Taken together, these data suggest that TGF-β1 modulates hippocampal glutamatergic synaptic protein expression and regulates synaptic transmission.

Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine that orchestrates key events of development, disease and repair in the central nervous system (CNS). To investigate the effects of chronically producing TGF-β1 on synaptic structure and synaptic transmission, I performed immunohistochemistry and immunoblot of brain tissues from transgenic mice (TGF-β1 mice) that over-express active form of TGF-β1 from astrocytes in the CNS. Immunohistochemical assays showed that synaptophysin increased in the CA3 subfield whereas calbindin-D28K decreased in the mossy fibres. Immunoblot analysis revealed that several α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunit proteins were up-regulated in the hippocampus of TGF-β1 mice. To examine the direct effect of TGF-β1 alone on glutamatergic synaptic activity, cultured hippocampal neurons were treated with or without TGF-β1. Electrophysiological recordings displayed that TGF-β1 significantly increased the amplitude of glutamate-evoked current (p<0.05). Taken together, these data suggest that TGF-β1 modulates hippocampal glutamatergic synaptic protein expression and regulates synaptic transmission.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 104-118).

Schizophrenia is a serious psychiatric disorder that affects 1% of the population. Current theories implicate NMDA receptor hypofunction as a contributor to the symptomology and pathological alterations in schizophrenia. Cognitive impairments are increasingly recognized as not only fundamental to schizophrenia, but the strongest predictor of patient functional outcomes. Current antipsychotics do not improve the cognitive symptoms of the disorder; however, recent efforts have resulted in the identification of novel drug targets. One target is metabotropic glutamate receptors as they interact with and modulate NMDA receptors. Another approach focuses on dopamine, the neurotransmitter system targeted by traditional antipsychotics. Tetrahydroprotoberberines, such as D- and L-govadine, are synthetic compounds derived from traditional medicine that have demonstrated efficacy in treating schizophrenic symptoms. The present study assessed the effects of CDPPB (a metabotropic glutamate receptor 5 positive allosteric modulator), D- and L-govadine, and the typical antipsychotic haloperidol on the Paired Associates Learning (PAL) task in rats. The PAL task is impaired in patients with schizophrenia, has been adapted for use with rodents using touchscreen-equipped operant chambers, and has been promoted by MATRICS as a promising behavioural task with the potential to further translational health research in schizophrenia. The objectives of this study were: 1) examine the effects of acute NMDA receptor antagonism with MK-801 as a model for schizophrenia on performance of the PAL task; 2) test the effects of the putative antipsychotics, CDPPB and D- and L-govadine on reversing the effects of NMDA receptor antagonism on the task; and 3) to compare these novel therapeutics to a classic antipsychotic. Two squads of male Long-Evans rats were trained to perform the PAL task in touchscreen-equipped operant chambers. After the rats reached criterion the following treatment schedules were divided between the two squads: 1) vehicle (10% cyclodextrin; i.p.), and CDPPB (1.0, 3.0, and 10.0 mg/kg, i.p.); or 2) vehicle (10% cyclodextrin; i.p.), CDPPB (3.0 mg/kg, i.p.), the NMDA receptor antagonist MK-801 (0.15 mg/kg, i.p.), and CDPPB with MK-801; or 3) vehicle (50% DMSO; s.c.), MK-801, D-govadine (1.0 mg/kg; s.c.), L-govadine (1.0 mg/kg; s.c.) and MK-801 with each isomer of govadine; or 4) vehicle (sodium acetate and acetic acid, pH 5.0, s.c.), and haloperidol (0.05 and 0.1 mg/kg, s.c.). Acute MK-801 significantly reduced the number of trials completed, impaired accuracy, and increased the number of errors in the PAL task. CDPPB had no effect on the PAL task and did not improve the MK-801 induced impairments. Administration of L-govadine, but not D-govadine, prior to MK-801 improved accuracy and reduced errors compared to MK-801 alone. L-govadine alone, but not D-govadine, reduced total responding compared to vehicle. Haloperidol caused a dose-dependent decrease in all activity in the task confounding interpretation of the results in regard to cognition. These data establish disruptive effects of acute MK-801 treatment on PAL task performance and demonstrate that L-govadine is capable of cognitive enhancement in a rodent model of schizophrenia.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 98-123).

Glutamate is the major excitatory neurotransmitter in the mammalian brain. It binds to three different subclasses of ionotropic glutamate receptors (iGluRs): AMPA, kainate and NMDA receptors, and triggers a cation influx that generates synaptic currents crucial to brain function. Significantly, iGluRs are implicated in various neurological disorders, such as depression, schizophrenia, Alzheimer’s and Parkinson’s diseases, autism, seizure, and stroke. Several crystal structures for intact iGluRs in various functional states (i.e. closed, activated and desensitized) have now been reported. The receptors have also been studied using single-particle cryo-electron microscopy. Together, these studies provide fascinating ‘snap-shots’ of the receptors as they transition between different states. What is lacking, so far, is information about the kinetics underlying these structural transitions, because the techniques used lack time resolution. I have used fast-scan atomic force microscopy (AFM), in some cases in combination with UV photolysis of caged L-glutamate, to study activation-induced structural changes in GluK2 kainate receptors and GluA2 AMPA receptors. AFM provides single-molecule resolution under fluid, permitting the imaging of proteins ‘in action’. Receptors were purified from transfected cells by immunoaffinity chromatography and imaged after integration into supported lipid bilayers. Activation of both receptors caused a rapid ~1-nm vertical compression of the receptor. In both cases, the height reduction did not occur in the presence of receptor antagonists. Further, the D776K mutant of the kainate receptor, which does not desensitize, did not undergo the height change, and cyclothiazide, which blocks desensitization of the AMPA receptor, also blocked the height change. I conclude, therefore, that the vertical compression is associated with receptor desensitization, and suggest that it may reflect a weakening of the interaction between receptor subunits at the LBD dimer interface. When imaged from the ‘top’ by AFM, the receptors appeared as double-blob structures, with each blob representing a pair of ATDs. By measuring the distance between the centres of the blobs in successive AFM images, I was able to monitor the mobility of the ATDs relative to each other before and during receptor stimulation. I found that for both kainate and AMPA receptors, the relative mobility of the ATDs became greater after stimulation. Further, at low glutamate concentrations, the ATDs of the (rapidly desensitizing) flop splice variant of the AMPA receptor were more mobile than those of the (more slowly desensitizing) flip splice variant. I suggest that the greater mobility of the flop splice variant might be connected with its more short-lived functional response to activation. In a final series of experiments, in collaboration with two other groups, I used AFM to measure conformational changes induced by allosterically-bound halide ions. We found that anion substitution (i.e. chloride to bromide, or chloride to iodide) produced vertical compression of AMPA receptors prior to agonist binding, and also (in electrophysiological experiments conducted by collaborators) altered the duration of agonist-evoked channel activity. The anion binding site was identified (in X-ray crystal structures obtained by collaborators) within the ligand binding domain, where flip-flop alternative splicing occurs. Interestingly both anion effects were isoform-dependent.

This thesis describes a novel reconstitution of &alpha;-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) for the purposes of structural characterization by atomic force microscopy (AFM) and functional characterization by electrical recordings of lipid bilayers. AMPARs are glutamate gated ion channels, ubiquitous in the vertebrate central nervous system where they mediate fast excitatory neurotransmission. In a healthy brain, AMPARs are involved in memory formation and learning and their dysfunction has been related to numerous neurological disorders such as Alzheimer's disease, epilepsy, schizophrenia and many others. AMPARs were reconstituted at high and low densities. Densely reconstituted samples contained &GT;100 receptors per &mu;m2, a value comparable to the AMPAR density at synapses. This allowed, for the first time, the imaging of full length tetrameric AMPARs in native-like conditions and with clearly assigned domains: the extracellular domains extended 14 nm above the membrane in agreement with electron microscopy (EM) and X-ray crystallography data. Lipid-protein interactions were studied in samples with low protein density with the receptors showing preference for lipids in the liquid crystalline phase. The activity of the reconstituted receptors was confirmed through single-channel recordings. This is the first case in which an AMPAR has been reconstituted and given (a) single-channel recordings with (b) physiologically plausible conductance levels and (c) pharmacological and no-protein controls and (d) structure. As a result, previously reported biochemistry and EM are now for the first time available in concert with AFM and single-channel recordings for a purified AMPAR of known composition.