Interaction between effects of genes coding for dopamine and glutamate transmission on striatal and parahippocampal function

The genes for the dopamine transporter (DAT) and the D-Amino acid oxidase activator (DAOA or G72) have been independently implicated in the risk for schizophrenia and in bipolar disorder and/or their related intermediate phenotypes. DAT and G72 respectively modulate central dopamine and glutamate transmission, the two systems most robustly implicated in these disorders. Contemporary studies have demonstrated that elevated dopamine function is associated with glutamatergic dysfunction in psychotic disorders. Using functional magnetic resonance imaging we examined whether there was an interaction between the effects of genes that influence dopamine and glutamate transmission (DAT and G72) on regional brain activation during verbal fluency, which is known to be abnormal in psychosis, in 80 healthy volunteers. Significant interactions between the effects of G72 and DAT polymorphisms on activation were evident in the striatum, parahippocampal gyrus, and supramarginal/angular gyri bilaterally, the right insula, in the right pre-/postcentral and the left posterior cingulate/retrosplenial gyri (P < 0.05, FDR-corrected across the whole brain). This provides evidence that interactions between the dopamine and the glutamate system, thought to be altered in psychosis, have an impact in executive processing which can be modulated by common genetic variation.

Post-translational regulation of an Aplysia glutamate transporter during long-term facilitation.

Regulation of glutamate transporters accompanies plasticity of some glutamatergic synapses. The regulation of glutamate uptake at the Aplysia sensorimotor synapse during long-term facilitation (LTF) was investigated. Previously, increases in levels of ApGT1 (Aplysia glutamate transporter 1) in synaptic membranes were found to be related to long-term increases in glutamate uptake. In this study, we found that regulation of ApGT1 during LTF appears to occur post-translationally. Serotonin (5-HT) a transmitter that induces LTF did not increase synthesis of ApGT1. A pool of ApGT1 appears to exist in sensory neuron somata, which is transported to the terminals by axonal transport. Blocking the rough endoplasmic reticulum-Golgi-trans-Golgi network (TGN) pathway with Brefeldin A prevented the 5-HT-induced increase of ApGT1 in terminals. Also, 5-HT produced changes in post-translational modifications of ApGT1 as well as changes in the levels of an ApGT1-co-precipitating protein. These results suggest that regulation of trafficking of ApGT1 from the vesicular trafficking system (rough endoplasmic reticulum-Golgi-TGN) in the sensory neuron somata to the terminals by post-translational modifications and protein interactions appears to be the mechanism underlying the increase in ApGT1, and thus, glutamate uptake during memory formation.

Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia.

An increase in transmitter release accompanying long-term sensitization and facilitation occurs at the glutamatergic sensorimotor synapse of Aplysia. We report that a long-term increase in neuronal Glu uptake also accompanies long-term sensitization. Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent, high-affinity Glu transport. Different treatments that induce long-term enhancement of the siphon-withdrawal reflex, or long-term synaptic facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a treatment that induces long-term facilitation, also produced a long-term increase in Glu uptake in cultures of sensory neurons. The mechanism for the increase in uptake is an increase in the V(max) of transport. The long-term increase in Glu uptake appeared to be dependent on mRNA and protein synthesis, and transport through the Golgi, because 5,6-dichlorobenzimidazole riboside, emetine, and brefeldin A inhibited the increase in Glu uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into Aplysia prevented long-term sensitization. Synthesis of Glu itself may be regulated during long-term sensitization because the same treatments that produced an increase in Glu uptake also produced a parallel increase in Gln uptake. These results suggest that coordinated regulation of a number of different processes may be required to establish or maintain long-term synaptic facilitation.

CONFORMATIONAL CHANGES IN THE EXTRACELLULAR DOMAIN OF GLUTAMATE RECEPTORS

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning. Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic neuron. They receive the burst of neurotransmitters and respond by fielding the neurotransmitters and opening ion channels. Glutamate receptors have been implicated in a number of neuropathologies like ischemia, stroke and amyotrophic lateral sclerosis. Specifically, the NMDA subtype of glutamate receptors has been linked to the onset of Alzheimer’s disease and the subsequent degeneration of neuronal cells. While crystal structures of AMPA and kainate subtypes of glutamate receptors have provided valuable information regarding the assembly and mechanism of activation; little is known about the NMDA receptors. Even the basic question of receptor assembly still remains unanswered. Therefore, to gain a clear understanding of how the receptors are assembled and how agonist binding gets translated to channel opening, I have used a technique called Luminescence Resonance Energy Transfer (LRET). LRET offers the unique advantage of tracking large scale conformational changes associated with receptor activation and desensitization. In this dissertation, LRET, in combination with biochemical and electrophysiological studies, were performed on the NMDA receptors to draw a correlation between structure and function. NMDA receptor subtypes GluN1 and GluN2A were modified such that fluorophores could be introduced at specific sites to determine their pattern of assembly. The results indicated that the GluN1 subunits assembled across each other in a diagonal manner to form a functional receptor. Once the subunit arrangement was established, this was used as a model to further examine the mechanism of activation in this subtype of glutamate receptor. Using LRET, the correlation between cleft closure and activation was tested for both the GluN1 and GluN2A subunit of the NMDA receptor in response to agonists of varying efficacies. These investigations revealed that cleft closure plays a major role in the mechanism of activation in the NMDA receptor, similar to the AMPA and kainate subtypes. Therefore, suggesting that the mechanism of activation is conserved across the different subtypes of glutamate receptors.

Glutamate receptors expressed in {\it Xenopus\/} oocytes: Studies in function and regulation

The amino acid glutamate is the primary excitatory neurotransmitter for the CNS and is responsible for the majority of fast synaptic transmission. Glutamate receptors have been shown to be involved in multiple forms of synaptic plasticity such as LTP, LTD, and the formation of specific synaptic connections during development. In addition to contributing to the plasticity of the CNS, glutamate receptors also are involved in, at least in part, various pathological conditions such as epilepsy, ischemic damage due to stroke, and Huntington's chorea. The regulation of glutamate receptors, particularly the ionotropic NMDA and AMPA/KA receptors is therefore of great interest. In this body of work, glutamate receptor function and regulation by kinase activity was examined using the Xenopus oocyte which is a convenient and faithful expression system for exogenous proteins. Glutamate receptor responses were measured using the two-electrode voltage clamp technique in oocytes injected with rat total forebrain RNA. NMDA elicited currents that were glycine-dependent, subject to block by Mg$\sp{2+}$ in a voltage-dependent manner and sensitive to the specific NMDA antagonist APV in a manner consistent with those types of responses found in neural tissue. Similarly, KA-evoked currents were sensitive to the specific AMPA/KA antagonist CNQX and exhibited current voltage relationships consistent with the calcium permeable type II KA receptors found in the hippocampus. There is evidence to indicate that NMDA and AMPA/KA receptors are regulated by protein kinase A (PKA). We explored this by examining the effects of activators of PKA (forskolin, 1-isobutyl-3-methylxanthine (IBMX) and 8-Br-cAMP) on NMDA and KA currents in the oocyte. In buffer where Ca$\sp{2+}$ was replaced by 2 mM Ba$\sp{2+},$ forskolin plus IBMX and 8-Br-cAMP augmented currents due to NMDA application but not KA. This augmentation was abolished by pretreating the oocytes in the kinase inhibitor K252A. The use of chloride channel blockers resulted in attenuation of this effect indicating that Ba$\sp{2+}$ influx through the NMDA channel was activating the endogenous calcium-activated chloride current and that the cAMP mediated augmentation was at the level of the chloride channel and not the NMDA channel. This was confirmed by (1) the finding that 8-Br-cAMP increased chloride currents elicited via calcium channel activation while having no effect on the calcium channels themselves and (2) the fact that lowering the Ba$\sp{2+}$ concentration to 200 $\mu$M abolished the augmentation NMDA currents by 8-Br-cAMP. Thus PKA does not appear to modulate ionotropic glutamate receptors in our preparation. Another kinase also implicated in the regulation of NMDA receptors, calcium/phospholipid-dependent protein kinase (PKC), was examined for its effects on the NMDA receptor under low Ba$\sp{2+}$ (200 $\mu$M) conditions. Phorbol esters, activators of PKC, induced a robust potentiation of NMDA currents that was blockable by the kinase inhibitor K252A. Furthermore activation of metabotropic receptors by the selective agonist trans-ACPD, also potentiated NMDA albeit more modestly. These results indicate that neither NMDA nor KA-activated glutamate receptors are modulated by PKA in Xenopus oocytes whereas NMDA receptors appear to be augmented by PKC. Furthermore, the endogenous chloride current of the oocyte was found to be responsive to Ba$\sp{2+}$ and in addition is enhanced by PKA. Both of these latter findings are novel. In conclusion, the Xenopus oocyte is a useful expression system for the analysis of ligand-gated channel activity and the regulation of those channels by phosphorylation. ^

Glutamate iontophoresis induces long-term potentiation in the absence of evoked presynaptic activity

$\rm\underline{L}$ong-$\rm\underline{t}$erm $\rm\underline{p}$otentiation (LTP) is a candidate cellular mechanism underlying mammalian learning and memory. Protocols that induce LTP typically involve afferent stimulation. The experiments described in this dissertation tested the hypothesis that LTP induction does not require presynaptic activity. The significance of this hypothesis is underscored by results suggesting that LTP expression may involve activity-dependent presynaptic changes. An induction protocol using glutamate iontophoresis was developed that reliably induces LTP in hippocampal slices without afferent stimulation (ionto-LTP). Ionto-LTP is induced when excitatory postsynaptic potentials are completely blocked with adenosine and $\rm\underline{t}$etrodo$\rm\underline{t}$o$\rm\underline{x}$in (TTX). These results suggest constraints on the involvement of presynaptic mechanisms and putative retrograde messengers in LTP induction and expression; namely, these processes must function without many forms of activity-dependent presynaptic processes.^ In testing the role of pre-and postsynaptic mechanisms in LTP expression whole-cell recordings were used to examine the frequency and amplitude of $\rm\underline{s}$pontaneous $\rm\underline{e}$xcitatory $\rm\underline{p}$o$\rm\underline{s}$ynaptic $\rm\underline{c}$urrents (sEPSCs) in CA1 pyramidal neurons. sEPSCs where comprised of an equal mixture of TTX insensitive miniature EPSCs and sEPSCs that appeared to result from spontaneous action potentials (i.e., TTX sensitive EPSCs). The detection of all sEPSCs was virtually eliminated by CNQX, suggesting that sEPSCs were glutamate mediated synaptic events. Changes in the amplitude and frequency sEPSCs were examined during the expression of ionto-LTP to obtain new information about the cellular location of mechanisms involved in synaptic plasticity. The findings of this dissertation show that ionto-LTP expression results from increased sEPSC amplitude in the absence of lasting increases in sEPSC frequency. Potentiation of sEPSC amplitude without changes in sEPSC frequency has been previously interpreted to be due to postsynaptic mechanisms. Although this interpretation is supported by findings from peripheral synapses, its application to the central nervous system is unclear. Therefore, alternative mechanisms are also considered in this dissertation. Models based on increased release probability for action potential dependent transmitter release appear insufficient to explain our results. The most straightforward interpretation of the results in this dissertation is that LTP induced by glutamate iontophoresis on dendrites of CA1 pyramidal neurons is mediated by postsynaptic mechanisms. ^

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

Metabotropic glutamate receptor 5 binding in patients with obsessive-compulsive disorder

Obsessive-compulsive disorder (OCD) is a disabling, mostly chronic, psychiatric condition with significant social and economic impairments and is a major public health issue. However, numerous patients are resistant to currently available pharmacological and psychological interventions. Given that recent animal studies and magnetic resonance spectroscopy research points to glutamate dysfunction in OCD, we investigated the metabotropic glutamate receptor 5 (mGluR5) in patients with OCD and healthy controls. We determined mGluR5 distribution volume ratio (DVR) in the brain of ten patients with OCD and ten healthy controls by using [11C]ABP688 positron-emission tomography. As a clinical measure of OCD severity, the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) was employed. We found no significant global difference in mGluR5 DVR between patients with OCD and healthy controls. We did, however, observe significant positive correlations between the Y-BOCS obsession sub-score and mGluR5 DVR in the cortico-striatal-thalamo-cortical brain circuit, including regions of the amygdala, anterior cingulate cortex, and medial orbitofrontal cortex (Spearman's ρ's⩾ = 0.68, p < 0.05). These results suggest that obsessions in particular might have an underlying glutamatergic pathology related to mGluR5. The research indicates that the development of metabotropic glutamate agents would be useful as a new treatment for OCD.

Coreleased orexin and glutamate evoke nonredundant spike outputs and computations in histamine neurons.

Stable wakefulness requires orexin/hypocretin neurons (OHNs) and OHR2 receptors. OHNs sense diverse environmental cues and control arousal accordingly. For unknown reasons, OHNs contain multiple excitatory transmitters, including OH peptides and glutamate. To analyze their cotransmission within computational frameworks for control, we optogenetically stimulated OHNs and examined resulting outputs (spike patterns) in a downstream arousal regulator, the histamine neurons (HANs). OHR2s were essential for sustained HAN outputs. OHR2-dependent HAN output increased linearly during constant OHN input, suggesting that the OHN→HAN(OHR2) module may function as an integral controller. OHN stimulation evoked OHR2-dependent slow postsynaptic currents, similar to midnanomolar OH concentrations. Conversely, glutamate-dependent output transiently communicated OHN input onset, peaking rapidly then decaying alongside OHN→HAN glutamate currents. Blocking glutamate-driven spiking did not affect OH-driven spiking and vice versa, suggesting isolation (low cross-modulation) of outputs. Therefore, in arousal regulators, cotransmitters may translate distinct features of OHN activity into parallel, nonredundant control signals for downstream effectors.

Association of long-term nicotine abstinence with normal metabotropic glutamate receptor-5 binding

BACKGROUND Nicotine addiction is a major public health problem and is associated with primary glutamatergic dysfunction. We recently showed marked global reductions in metabotropic glutamate receptor type 5 (mGluR5) binding in smokers and recent ex-smokers (average abstinence duration of 25 weeks). The goal of this study was to examine the role of mGluR5 downregulation in nicotine addiction by investigating a group of long-term ex-smokers (abstinence >1.5 years), and to explore associations between mGluR5 binding and relapse in recent ex-smokers. METHODS Images of mGluR5 receptor binding were acquired in 14 long-term ex-smokers, using positron emission tomography with radiolabeled [11C]ABP688, which binds to an allosteric site with high specificity. RESULTS Long-term ex-smokers and individuals who had never smoked showed no differences in mGluR5 binding in any of the brain regions examined. Long-term ex-smokers showed significantly higher mGluR5 binding than recent ex-smokers, most prominently in the frontal cortex (42%) and thalamus (57%). CONCLUSIONS Our findings suggest that downregulation of mGluR5 is a pathogenetic mechanism underlying nicotine dependence and the high relapse rate in individuals previously exposed to nicotine. Therefore, mGluR5 receptor binding appears to be an effective biomarker in smoking and a promising target for the discovery of novel medication for nicotine dependence and other substance-related disorders.

The role of metabotropic glutamate receptor 5 in the pathogenesis of mood disorders and addiction: combining preclinical evidence with human Positron Emission Tomography (PET) studies.

In the present review, we deliver an overview of the involvement of metabotropic glutamate receptor 5 (mGluR5) activity and density in pathological anxiety, mood disorders and addiction. Specifically, we will describe mGluR5 studies in humans that employed Positron Emission Tomography (PET) and combined the findings with preclinical animal research. This combined view of different methodological approaches-from basic neurobiological approaches to human studies-might give a more comprehensive and clinically relevant view of mGluR5 function in mental health than the view on preclinical data alone. We will also review the current research data on mGluR5 along the Research Domain Criteria (RDoC). Firstly, we found evidence of abnormal glutamate activity related to the positive and negative valence systems, which would suggest that antagonistic mGluR5 intervention has prominent anti-addictive, anti-depressive and anxiolytic effects. Secondly, there is evidence that mGluR5 plays an important role in systems for social functioning and the response to social stress. Finally, mGluR5's important role in sleep homeostasis suggests that this glutamate receptor may play an important role in RDoC's arousal and modulatory systems domain. Glutamate was previously mostly investigated in non-human studies, however initial human clinical PET research now also supports the hypothesis that, by mediating brain excitability, neuroplasticity and social cognition, abnormal metabotropic glutamate activity might predispose individuals to a broad range of psychiatric problems.

Role of calcium, glutamate and NMDA in major depression and therapeutic application

Major depression is a common, recurrent mental illness that affects millions of people worldwide. Recently, a unique fast neuroprotective and antidepressant treatment effect has been observed by ketamine, which acts via the glutamatergic system. Hence, a steady accumulation of evidence supporting a role for the excitatory amino acid neurotransmitter (EAA) glutamate in the treatment of depression has been observed in the last years. Emerging evidence indicates that N-methyl-D-aspartate (NMDA), group 1 metabotropic glutamate receptor antagonists and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) agonists have antidepressant properties. Indeed, treatment with NMDA receptor antagonists has shown the ability to sprout new synaptic connections and reverse stress-induced neuronal changes. Based on glutamatergic signaling, a number of therapeutic drugs might gain interest in the future. Several compounds such as ketamine, memantine, amantadine, tianeptine, pioglitazone, riluzole, lamotrigine, AZD6765, magnesium, zinc, guanosine, adenosine aniracetam, traxoprodil (CP-101,606), MK-0657, GLYX-13, NRX-1047, Ro25-6981, LY392098, LY341495, D-cycloserine, D-serine, dextromethorphan, sarcosine, scopolamine, pomaglumetad methionil, LY2140023, LY404039, MGS0039, MPEP, 1-aminocyclopropanecarboxylic acid, all of which target this system, have already been brought up, some of them recently. Drugs targeting the glutamatergic system might open up a promising new territory for the development of drugs to meet the needs of patients with major depression.

Activation of metabotropic glutamate 5 and NMDA receptors underlies the induction of persistent bursting and associated long-lasting changes in CA3 recurrent connections.

The aim of this study was to describe the induction and expression mechanisms of a persistent bursting activity in a horizontal slice preparation of the rat limbic system that includes the ventral part of the hippocampus and the entorhinal cortex. Disinhibition of this preparation by bicuculline led to interictal-like bursts in the CA3 region that triggered synchronous activity in the entorhinal cortex. Washout of bicuculline after a 1 hr application resulted in a maintained production of hippocampal bursts that continued to spread to the entorhinal cortex. Separation of CA3 from the entorhinal cortex caused the activity in the latter to become asynchronous with CA3 activity in the presence of bicuculline and disappear after washout; however, in CA3, neither the induction of bursting nor its persistence were affected. Associated with the CA3 persistent bursting, a strengthening of recurrent collateral excitatory input to CA3 pyramidal cells and a decreased input to CA3 interneurons was found. Both the induction of the persistent bursting and the changes in synaptic strength were prevented by antagonists of metabotropic glutamate 5 (mGlu5) or NMDA receptors or protein synthesis inhibitors and did not occur in slices from mGlu5 receptor knock-out mice. The above findings suggest potential synaptic mechanisms by which the hippocampus switches to a persistent interictal bursting mode that may support a spread of interictal-like bursting to surrounding temporal lobe regions.

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.