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Molecular biology of glutamate receptors in the central nervous system and their role in excitotoxicity, oxidative stress and aging
Abstract
Forty years of research into the function of L-glutamic acid as a neurotransmitter in the vertebrate central nervous system (CNS) have uncovered a tremendous complexity in the actions of this excitatory neurotransmitter and an equally great complexity in the molecular structures of the receptors activated by L-glutamate. L-Glutamate is the most widespread excitatory transmitter system in the vertebrate CNS and in addition to its actions as a synaptic transmitter it produces long-lasting changes in neuronal excitability, synaptic structure and function, neuronal migration during development, and neuronal viability. These effects are produced through the activation of two general classes of receptors, those that form ion channels or "ionotropic" and those that are linked to G-proteins or "metabotropic". The pharmacological and physiological characterization of these various forms over the past two decades has led to the definition of three forms of ionotropic receptors, the kainate (KA), AMPA, and NMDA receptors, and three groups of metabotropic receptors. Twenty-seven genes are now identified for specific subunits of these receptors and another five proteins are likely to function as receptor subunits or receptor associated proteins. The regulation of expression of these protein subunits, their localization in neuronal and glial membranes, and their role in determining the physiological properties of glutamate receptors is a fertile field of current investigations into the cell and molecular biology of these receptors. Both ionotropic and metabotropic receptors are linked to multiple intracellular messengers, such as Ca2+, cyclic AMP, reactive oxygen species, and initiate multiple signaling cascades that determine neuronal growth, differentiation and survival. These cascades of complex molecular events are presented in this review.
... For instance, systemic administration of KA to rats led to the cell death of AMPA and KA receptor-equipped pyramidal neurons in the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) regions of the hippocampus, while granule cells of the dentate gyrus (DG) were resistant [21]. Since excitatory stress and the resulting intracellular ROS are considered important drivers of neurological aging [22,23], KA-induced hippocampal damage in rodents is a well-known model for human aging and neurodegenerative disorders [23][24][25]. To counteract the damage caused by ROS, cells possess an antioxidant defense system which can be distinguished by enzymatic and nonenzymatic antioxidants [10]. ...
... For instance, systemic administration of KA to rats led to the cell death of AMPA and KA receptor-equipped pyramidal neurons in the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) regions of the hippocampus, while granule cells of the dentate gyrus (DG) were resistant [21]. Since excitatory stress and the resulting intracellular ROS are considered important drivers of neurological aging [22,23], KA-induced hippocampal damage in rodents is a well-known model for human aging and neurodegenerative disorders [23][24][25]. To counteract the damage caused by ROS, cells possess an antioxidant defense system which can be distinguished by enzymatic and nonenzymatic antioxidants [10]. ...
... Within this study, we treated ex vivo-cultured mouse organotypic hippocampal slices with human blood serum and its most abundant protein HSA to investigate potential neuroprotective activities against KA-mediated oxidative stress. Since excitatory stress and the resulting intracellular ROS are considered important drivers of neurological aging [22,23], KA-induced hippocampal damage in rodents is a well-known model for human aging and neurodegenerative disorders [23][24][25]. KA-induced hippocampal damage is also widely used to model epilepsy in animal models [40]. ...
Neuroprotection from oxidative stress is critical during neuronal development and maintenance but also plays a major role in the pathogenesis and potential treatment of various neurological disorders and neurodegenerative diseases. Emerging evidence in the murine system suggests neuroprotective effects of blood plasma on the aged or diseased brain. However, little is known about plasma-mediated effects on human neurons. In the present study, we demonstrate the neuroprotective effect mediated by human plasma and the most abundant plasma–protein human serum albumin against oxidative stress in glutamatergic neurons differentiated from human neural crest-derived inferior turbinate stem cells. We observed a strong neuroprotective effect of human plasma and human serum albumin against oxidative stress-induced neuronal death on the single cell level, similar to the one mediated by tumor necrosis factor alpha. Moreover, we detected neuroprotection of plasma and human serum albumin against kainic acid-induced excitatory stress in ex vivo cultured mouse hippocampal tissue slices. The present study provides deeper insights into plasma-mediated neuroprotection ultimately resulting in the development of novel therapies for a variety of neurological and, in particular, neurodegenerative diseases.
... Dentro del sistema nervioso central el glutamato no solo se limita a mediar la neurotransmisión excitatoria, sino que presenta un gran número de funciones adicionales (Michaelis, 1998): ...
... En la membrana de las vesículas se conocen entre otras, la sinaptobrevina, la sinaptofisina y la sinaptotagmina. En el citoplasma está presente la alfa-SNAP, mientras que en la membrana presináptica se localizan la SNAP-25 (proteína asociada a la sinapsis) y la fisofilina (Michaelis, 1998). ...
... Las vesículas que se ligan al citoesqueleto por medio de la proteína sinapsina se liberan por la activación de la enzima calcio-calmodulina-kinasa (CaM-Kinasa) en respuesta al ingreso de calcio a la terminal presináptica del citoplasma y de la terminal presináptica, a su vez inducido por la llegada de un potencial de activación y organización de las proteínas de anclaje vesicular. La interacción de estas proteínas forma un núcleo o eje que permite la fusión de la vesícula con la membrana presináptica, lo cual antecede a la exocitosis (Michaelis, 1998 En la membrana vesicular, la SNPT actúa como sensor de Ca + en la vesícula, la SNPB se encarga de reconocer la membrana presináptica y la SNPF establece un poro de fusión con la membrana. En el citoplasma se encuentra la proteína de fusión, que requiere la SNAP para su acople al eje. ...
The effect of glutamate supply upon the onset of puberty and possible links to changes in serum concentrations of total protein (TP) and urea (U) was evaluated. The study was conducted in prepuberal female goats (n = 18; 3 mo. old, 7/8 Saanen-Alpine, 1/8 Criollo) randomly assigned to two experimental groups: 1) Excitatory amino acids group (AAE, n = 10; 16.52±1.04 kg live weight (LW), 3.4±0.12 body condition score (BCS)) receiving an intravenous infusion of 7 mg kg-1 live weight of L-glutamate, and 2) Control group (CONT, n = 8; 16.1±1.04 kg LW, 3.1±0.12 BCS) receiving saline. The content of serum TP, U and progesterone (P4) was evaluated. The general average for LW (23.2±0.72 kg), BCS (3.37±0.10 units), and global averages for serum TP (65.28 ± 2.46 mg mL-1) and U (23.42±0.95 mg mL-1) did not differ (P> 0.05) among treatments. In addition, serum TP and U did not differ (P>0.05) across time between treatments. However, a treatment effect (P <0.05) was detected regarding both the onset (207±9 vs. 225±12 days) and the percentage (70 vs. 25%) of females showing puberty, favoring to the AAE group. These results confirm that exogenous administration of glutamate promotes an earlier onset while an increased percentage of puberty in goats. The last suggest that such physiological scenario seems to involve a total protein-independent mechanism while a possible direct effect of glutamate supplementation as activator of the hypothalamic-hypophyseal-gonadal axis in peripuberal goats.
... However, glutamate does not act individually as the presence of glycine is required to activate the NMDA receptor [22][23][24] studies concerning the NMDA receptor, the molecular structure has not been quite elucidated. It has been proposed that it can form tetra and pentameric structures [25]. This receptor is formed by different subunits: NMDAR1 (NR1), NMDAR2 (NR2) and NMDAR3 (NR3) 2 , which together form a Ca 2+ permeable ion channel. ...
... The existing information regarding the interaction of memantine with the NMDA receptor, cell membrane molecular models and human red blood cells is presented in this mini-review. One of the aspects that could eventually improve the effectiveness of memantine would be the union of the drug to the NMDA receptor, although its structure has not been completely elucidated [25]. The binding sites of memantine to the NMDA receptor have been reported in different experimental and theoretical studies concluding that memantine binds to the receptor in the inter membrane domain of the receptor [35]. ...
... Regarding the interaction of memantine with human red blood cells, the information is limited. In some reports, the presence and importance of NMDA receptors in the control of intracellular calcium flow in erythrocytes has been described [20,25,31]. In addition, a slight influence of memantine on the rheological properties (aggregation and deformity) of human red blood cells has been reported [19]. ...
Memantine is an NMDA receptor antagonist clinically used for the treatment of moderate to severe Alzheimer's disease. Currently, it is the only NMDA receptor antagonist drug marketed against this disease. Despite the large number of publications regarding its clinical and therapeutic use, studies related to its mechanism of action are still inconclusive. Knowledge of drug interactions with cell membranes may lead to the development of novel drugs for neurodegenerative diseases. The present mini-review aims to give an overview of the latest findings regarding the interaction of memantine with cell membranes, specifically with that of the human erythrocyte.
... Excitotoxic cell death has been implicated in SCI, stroke, TBI, epilepsy, MS, AD, ALS, PD and Huntington disease [144]. Excessive glutamate and over-activation of glutamate receptors consequently impair calcium buffering, and promote free radical formation and mitochondrial permeability that collectively lead to cell death [145,146]. Glutamate excitotoxicity results from impaired uptake of excess glutamate from extracellular space following neurotransmission. Following injury and depolarization of cell membrane, glutamate uptake pattern is reversed and glutamate is released from Na + dependent glutamate transporter-1 (GLT1), glutamate/aspartate transporter (GLAST) and EAAC1 (EAAT3) located on glia and neurons [147]. ...
... Activation of two general classes of receptors, "ionotropic" and G-proteins linked receptors or "metabotropic" are implicated in these effects. Kainate (KA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), and N-methyl-D-aspartate (NMDA) receptors are different groups of ionotropic receptors involved in excitotoxicity [145,150]. Glutamate receptors are widely expressed by soma, axons and dendrites; therefore, neurons are potentially susceptible to glutamate excitotoxicity in their entirety. Accordingly, although glutamate excitotoxicity is considered primarily a somato-dendritic insult, it also occurs in WM myelinated tracts as their axons express AMPA and KA receptors [151,152]. ...
White matter degeneration is an important pathophysiological event of the central nervous system that is collectively characterized by demyelination, oligodendrocyte loss, axonal degeneration and parenchymal changes that can result in sensory, motor, autonomic and cognitive impairments. White matter degeneration can occur due to a variety of causes including trauma, neurotoxic exposure, insufficient blood flow, neuroinflammation, and developmental and inherited neuropathies. Regardless of the etiology, the degeneration processes share similar pathologic features. In recent years, a plethora of cellular and molecular mechanisms have been identified for axon and oligodendrocyte degeneration including oxidative damage, calcium overload, neuroinflammatory events, activation of proteases, depletion of adenosine triphosphate and energy supply. Extensive efforts have been also made to develop neuroprotective and neuroregenerative approaches for white matter repair. However, less progress has been achieved in this area mainly due to the complexity and multifactorial nature of the degeneration processes. Here, we will provide a timely review on the current understanding of the cellular and molecular mechanisms of white matter degeneration and will also discuss recent pharmacological and cellular therapeutic approaches for white matter protection as well as axonal regeneration, oligodendrogenesis and remyelination.
... However, cysteine as well as other sulfur-containing derivatives and quinolinic acid are also endogenous agonists. The pharmacology of these receptors has revealed at least five groups of glutamate receptors in the vertebrate CNS (for review see Ozawa et al., 1998 andMichaelis, 1998). Three of these are linked directly to ion channels (ionotropic) and have been classified into N-methyl-D-aspartate (NMDA)-and non-NMDA receptors (for review see Nakanishi et al., 1998;Fletcher and Lodge, 1996). ...
... The AP4 receptor seems to be an autoreceptor whose function is to inhibit glutamate release. Different subtypes of glutamate receptors can coexist in one cell (Michaelis, 1998). ...
In the developing CNS the majority of neurons migrate to their final position, further differentiate and form synapses one with another. This highly orchestrated process requires intercellular communication from earliest times. This thesis investigates the appearance of amino acid transmitter release, the expression of amino acid transmitter receptors and gap junctional connections by ganglion cells in the embryonic chick retina. The release of the amino acid transmitters glutamate, aspartate, GABA and glycine from the retina was investigated using high performance liquid chromatography (HPLC). These experiments show the release of all four transmitters can be detected by HPLC from embryonic day four (E4) onwards, although in the case of GABA such release was only detectable when extracellular potassium was raised. The ratio of release under normal ionic conditions to that evoked by raising extracellular potassium increased towards synaptogenesis. High potassium evoked both Ca2+-dependent and Ca2+-independent release of transmitter. The developmental profile of the appearance of receptors for the four amino acid transmitters in ganglion cells was established in situ by whole-cell patch- clamping. The results show that ganglion cells respond to GABA and glycine at E6, while responses to AMPA and NMDA appear at E6 and E8, respectively. The time course of the development of gap junctional coupling between ganglion cells and other cells has been established using whole-cell patch- clamping as a means to introduce a mixture of gap junction permeant and impermeant dyes into their cytoplasm. Confocal microscopy of these preparations shows a general increase in the extent of coupling towards the time of synaptogenesis (~E12), which is followed by a marked decrease at E14. Throughout the period examined (E5-E14) some ganglion cells were coupled to cells that traverse the retina, which may represent neurons in the process of migration to their final destination.
... They are made up of a number of subunits, GluRl-4, arranged in a heterotetrameric fashion, each of which is expressed as at least 2 splice variants. The inclusion of particular subunits can drastically alter the functional properties of the receptor, for example by allowing the channel to be permeable to calcium as well as sodium ions (Hollmann et al, 1991; and see below) or by altering its ability to open at certain membrane potentials (Michaelis, 1998;Dingledine et a l, 1999). ...
... The KA receptor family is made up of 5 subunits, GluR5, GluR6, GluR7, KAl and KA2, with each receptor consisting of four subunits arranged in a tetrameric fashion (Michaelis, 1998). In situ hybridisation studies have shown the presence of KA receptors in the spinal cord at embryonic day (E) 12 (Bahn et al, 1994), although binding does not occur until E l4. ...
The dorsal horn of the spinal cord undergoes alterations during early postnatal life. For example, the primary afferent terminations from low-threshold Aβ fibres reorganise, and the interneurones increase their axodendritic elaborations. Many of these changes require activation of the NMDA receptor. The aim of this thesis was to examine the role of a major downstream signalling protein- calcium/calmodulin-dependent kinase II- in the activity-dependent development of the dorsal horn. After confirming the presence of the kinase in the spinal cord from birth, and elucidating its localisation within this region, I obtained a transgenic mouse in which a point mutation of the T286A site (T286A) prevents the kinase from entering its autophosphorylated form, and thus from remaining active after dissipation of the calcium stimulus. Using Dil labelling of Aβ fibres, it was shown the postnatal reorganisation that normally occurs during development, but is prevented by chronic NMDA receptor blockade, was not affected by the mutation, suggesting that CaMKII autophosphorylation is not required for normal A fibre development. Electrophysiological analysis of the dorsal horn using in vitro whole-cell patch clamp and in vivo extracellular recordings revealed decreases in polysynaptic Aβ input to the superficial dorsal horn, combined with reduced C fibre input and increased neuronal receptive field sizes in the mutant compared with wild-type littermates. This suggests a role for CaMKII in the normal postnatal development of the dorsal horn intemeuronal connectivity. Finally, the pain-processing ability of the adult mutant mouse was examined using behavioural and electrophysiological techniques. It was found that, while the baseline sensory processing was unaffected in the mutant, there was a decrease in pain behaviour in response to intraplantar injection of formalin, accompanied by an increase in pain behaviour in response to nerve injury. These findings highlight the different mechanisms that control pain processing in the spinal cord, and suggest different roles for CaMKII in each of these mechanisms.
... To measure its ligand activity, rat GRINA was cloned in Escherichia coli and purified from the bacterial extracts by affinity chromatography on glutamate-treated columns, displaying an estimated dissociation constant of 263 nM for glutamate [2]. According to the authors, it was part of an NMDA receptor-like complex formed by 4 subunits (the glutamate-binding protein corresponding to GRINA, the glycine-binding protein, the carboxypiperazinylphosphonate-binding protein, and the phencyclidine-binding protein) [4,5]. To evaluate the formation of ion channels, they reconstituted the protein complex (previously isolated from rat brain synaptic vesicles) into liposomes and measured their activity using voltage clamp techniques following their fusion with planar lipid bilayer membranes. ...
... When the mRNA levels of GRINA were examined by in situ hybridization in the rat brain, they showed a similar expression pattern to the NMDAR1 receptor, except for the hypothalamus, where GRINA was not expressed, and the parafascicular nucleus of the thalamus, where NMDAR1 was not present [7]. After immunocytochemistry with antibodies developed by their own team, staining was observed in the cell body and dendrites of pyramidal neurons from the hippocampus and layers II, III, and V/VI of the cerebral cortex, as well as granule cells from the dentate gyrus, cerebellar cortex, and the olfactory bulb, also including the mitral cells of the olfactory bulb [4,8]. However, unlike in situ hybridization, no staining was observed in Purkinje cells [9]. ...
The Glutamate Receptor Ionotropic NMDA-Associated Protein 1 (GRINA) belongs to the Lifeguard family and is involved in calcium homeostasis, which governs key processes, such as cell survival or the release of neurotransmitters. GRINA is mainly associated with membranes of the endoplasmic reticulum, Golgi, endosome, and the cell surface, but its presence in the nucleus has not been explained yet. Here we dissect, with the help of different software tools, the potential roles of GRINA in the cell and how they may be altered in diseases, such as schizophrenia or celiac disease. We describe for the first time that the cytoplasmic N-terminal half of GRINA (which spans a Proline-rich domain) contains a potential DNA-binding sequence, in addition to cleavage target sites and probable PY-nuclear localization sequences, that may enable it to be released from the rest of the protein and enter the nucleus under suitable conditions, where it could participate in the transcription, alternative splicing, and mRNA export of a subset of genes likely involved in lipid and sterol synthesis, ribosome biogenesis, or cell cycle progression. To support these findings, we include additional evidence based on an exhaustive review of the literature and our preliminary data of the protein-protein interaction network of GRINA.
... The GlyR and the NMDA-R display selective clustering at the postsynaptic neuronal membrane. 111,112 Abs that bind to the GlyR are associated with PERM (progressive encephalomyelitis with rigidity and myoclonus) and stiff-person syndrome 113 and Abs against the NMDAR are found mainly in younger women with a complex form of encephalitis. 114 Although both antibodies can bind divalently to their targets on the cell surface, and cause internalization and degradation of the receptors, anti-GlyR-Abs can also activate the complement system. ...
... 114 Although both antibodies can bind divalently to their targets on the cell surface, and cause internalization and degradation of the receptors, anti-GlyR-Abs can also activate the complement system. 112,115 Finally, another channel protein, the water channel aquaporin-4 (AQP4), which is a glial surface membrane protein expressed at the end feet of astrocytes in the CNS, also displays clustered forms, 116 is the target for antibodies that act via complement-mediated and also complement-independent pathways in neuromyelitis optica spectrum disorders. 117 ...
The nicotinic acetylcholine receptor (nAChR) family, the archetype member of the pentameric ligand-gated ion channels, is ubiquitously distributed in the central and peripheral nervous systems and its members are the targets for both genetic and acquired forms of neurological disorders. In the central nervous system nAChRs contribute to the pathological mechanisms of neurodegenerative disorders, such as Alzheimer and Parkinson diseases. In the peripheral nervous system, antibody-mediated autoimmune diseases such as “classical” myasthenia gravis (MG) and related disorders can affect the neuromuscular synapse. In MG, antibodies to the nAChR bind to the postsynaptic receptors and activate the classical complement pathway culminating in the formation of the membrane attack complex, with the subsequent destruction of the postsynaptic apparatus. Divalent nAChR-antibodies also cause internalization and loss of the nAChRs. Loss of receptors by either mechanism results in the muscle weakness and fatigability that typify the clinical manifestations of the disease. Other targets for antibodies, in a minority of patients, include muscle specific kinase (MuSK) and low-density lipoprotein related protein 4 (LRP4). This brief review analyzes the current status of muscle-type nAChR in relation to the pathogenesis of autoimmune diseases affecting the peripheral cholinergic synapse.
... El glutamato como sus receptores, están localizados en una gran cantidad de tejido neuroendocrino y en una gran variedad de núcleos hipotalámicos como los núcleos paraventriculares (NPV), en el núcleo ventromedial (NVM), arqueado (ARC) y supra óptico (NSO), en la eminencia media (EM) y en el tallo Infundibular importantes en la función reproductiva (Estienne et al., 2000;Mahesh y Brann, 2005;Durand et al., 2008). Además, este aminoácido no esencial tiene una infinidad de funciones entre especies, tanto dentro como fuera del SNC (Michaelis, 1998;Spitzer et al., 2016): La señalización del glutamato está comúnmente asociada con la transmisión sináptica. La sinapsis, definida por un sitio presináptico y un sitio postsináptico separado por la hendidura sináptica, es el centro primario de información neuronal. ...
... En la membrana de las vesículas se conocen entre otras, la sinaptobrevina, la sinaptofisina y la sinaptotagmina. En el citoplasma está presente la α-SNAP, mientras que en la membrana presináptica se localizan la SNAP-25 (proteína asociada a la sinapsis) y la fisofilina (Michaelis, 1998). para formar la sinapsis axón-OPC. ...
El objetivo del presente trabajo fue evaluar el efecto de la administración de glutamato y/o testosterona sobre los indicadores de la actividad reproductiva y del comportamiento sexual en ovinos jóvenes y adultos de la raza Dorper, además de la capacidad de estos machos para incrementar la respuesta sexual de las ovejas nulíparas anovulatorias Dorper a través del efecto macho durante fotoperiodos crecientes. Se realizaron 3 experimentos en el norte de México (25°LN y 103°LO) durante 2014 y 2015 al inicio de la primavera bajo condiciones naturales de luz. De manera general a través de los experimentos realizados se revela un posible estímulo sinérgico proporcionado por la administración de glutamato + testosterona al incrementar el comportamiento sexual de los carneros jóvenes y adultos de la raza Dorper y, a su vez, el rendimiento reproductivo de las ovejas nulíparas anovulatorias expuestas a estos carneros previamente tratados. En el mismo sentido, los resultados de esta investigación revelan al glutamato como una molécula con efectos alentadores que puede tener aplicaciones transmisibles a otras especies y a otros sistemas de producción pecuaria.
... ). This coupling maintains a transmembrane L-Glu concentration gradient ([Glu]in/[Glu]out) exceeding 10 6 -fold under physiological conditions [8], i.e., the [Glu]in of astrocytes is approximately 2-3 mM L-Glu in their cytoplasm, and [Glu]out under resting-state conditions is approximately 25 nM [9]. Transport against the concentration gradient uses ATP derived from the Na + /K + -ATPase pathway [10]. ...
In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development.
... is the most widespread excitatory neurotransmitter in the vertebrate central nervous system (CNS) with a multiplicity of functions, including regulating neurite elongation and synapse formation, especially during early CNS development (Artola & Singer, 1987;Balazs et al., 1988;Bear & Abraham, 1996;Brewer & Cotman, 1989;Cotman et al., 1988;Komuro & Rakic, 1993;Michaelis, 1998;Rojas, 2014;Soto et al., 2014). Neurite elongation and synapse formation requires microtubule and actin rearrangements, organelle and protein transport, and macromolecular complex formation, particularly at sites of new synapse formation, and these processes are responsive to changes in neuronal activity (Chang & De Camilli, 2001;Conde & Caceres, 2009;Sabo & McAllister, 2003;Sceniak et al., 2012). ...
The excitatory neurotransmitter glutamate has a role in neuronal migration and process elongation in the central nervous system (CNS). The effects of chronic glutamate hyperactivity on vesicular and protein transport within CNS neurons, that is, processes necessary for neurite growth, have not been examined previously. In this study, we measured the effects of lifelong hyperactivity of glutamate neurotransmission on axoplasmic transport in CNS neurons. We compared wild‐type (wt) to transgenic (Tg) mice over‐expressing the glutamate dehydrogenase gene Glud1 in CNS neurons and exhibiting increases in glutamate transmitter formation, release, and synaptic activation in brain throughout the lifespan. We found that Glud1 Tg as compared with wt mice exhibited increases in the rate of anterograde axoplasmic transport in neurons of the hippocampus measured in brain slices ex vivo, and in olfactory neurons measured in vivo. We also showed that the in vitro pharmacologic activation of glutamate synapses in wt mice led to moderate increases in axoplasmic transport, while exposure to selective inhibitors of ion channel forming glutamate receptors very significantly suppressed anterograde transport, suggesting a link between synaptic glutamate receptor activation and axoplasmic transport. Finally, axoplasmic transport in olfactory neurons of Tg mice in vivo was partially inhibited following 14‐day intake of ethanol, a known suppressor of axoplasmic transport and of glutamate neurotransmission. The same was true for transport in hippocampal neurons in slices from Glud1 Tg mice exposed to ethanol for 2 h ex vivo. In conclusion, endogenous activity at glutamate synapses regulates and glutamate synaptic hyperactivity increases intraneuronal transport rates in CNS neurons. image
... In the CNS, there is a neuron-specific cell death mechanism called excitotoxicity, in which excessive concentrations of the extracellular excitatory neurotransmitter L-glutamate (L-Glu) activate N-methyl-D-aspartate receptors (NMDARs) and cause acute Ca 2+ influx, leading to neuronal death [7]. The machinery necessary to reproduce excitotoxicity should therefore be installed in the CNS safety/toxicity assessment systems. ...
Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 μM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 μM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 μM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.
... In the present study, there was an increase in NO levels in the brains and hippocampi of epileptic animals compared to control animals, with a possible protective action resulting from the reduction in NO after the use of exogenous antioxidants. Another relevant point of exacerbated NMDA receptor activation is the production of RS, which can occur through NADPH oxidase [73] and metabolic stress in mitochondria [74,75]. NO can react with superoxides from NADPH oxidase and mitochondria to produce peroxynitrite, which can be decomposed to form hydroxyl radicals that are highly toxic to cells [76]. ...
Epilepsy is a neurological disorder characterized by epileptic seizures resulting from neuronal hyperexcitability, which may be related to failures in Na,K-ATPase activity and oxidative stress participation. We conducted this study to investigate the impact of antioxidant therapy on oxidative stress, Na,K-ATPase activity, seizure factors, and mortality in rodent seizure/epilepsy models induced by pentylenetetrazol (PTZ), pilocarpine (PILO), and kainic acid (KA). After screening 561 records in the MEDLINE, EMBASE, Web of Science, Science Direct, and Scopus databases, 22 were included in the systematic review following the PRISMA guidelines. The meta-analysis included 14 studies and showed that in epileptic animals there was an increase in the oxidizing agents nitric oxide (NO) and malondialdehyde (MDA), with a reduction in endogenous antioxidants reduced glutathione (GSH) and superoxide dismutase (SO). The Na,K-ATPase activity was reduced in all areas evaluated. Antioxidant therapy reversed all of these parameters altered by seizure or epilepsy induction. In addition, there was a percentage decrease in the number of seizures and mortality, and a meta-analysis showed a longer seizure latency in animals using antioxidant therapy. Thus, this study suggests that the use of antioxidants promotes neuroprotective effects and mitigates the effects of epilepsy. The protocol was registered in the Prospective Register of Systematic Reviews (PROSPERO) CRD42022356960.
... Here we found ON and OFF UBC in ipsilateral flocculus might be crucial during the acute phase instead of the chronic phase ( Figure 6). mGluR1α is a G-protein coupled receptor mediates glutamatergic transmission transduced into intracellular secondary messengers, particularly calcium signaling [32]. After UL, MF stimulation leads to a large increase of calcium released from intracellular stores within the brush dendrites of the UBCs, which occurs because of prolonged neuron depolarization that may spread to the cell body [33]. ...
Vestibular compensation is a natural behavioral recovery process following unilateral vestibular injury. Understanding the mechanism can considerably enhance vestibular disorder therapy and advance the adult central nervous system functional plasticity study after injury. The cerebellum, particularly the flocculonodular lobe, tightly modulates the vestibular nucleus, the center for vestibular compensation; however, it is still unclear if the flocculus on both sides is involved in vestibular compensation. Here we report that the unipolar brush cells (UBCs) in the flocculus are modulated by unilateral labyrinthectomy (UL). UBCs are excitatory interneurons targeting granule cells to provide feedforward innervation to the Purkinje cells, the primary output neurons in the cerebellum. According to the upregulated or downregulated response to the mossy fiber glutamatergic input, UBC can be classified into ON and OFF forms of UBCs. Furthermore, we discovered that the expression of marker genes of ON and OFF UBCs, mGluR1α and calretinin, was increased and decreased, respectively, only in ipsilateral flocculus 4–8 h after UL. According to further immunostaining studies, the number of ON and OFF UBCs was not altered during UL, demonstrating that the shift in marker gene expression level in the flocculus was not caused by the transformation of cell types between UBCs and non-UBCs. These findings imply the importance of ipsilateral flocculus UBCs in the acute response of UL, and ON and OFF UBCs may be involved in vestibular compensation in opposite directions.
... Glutamatergic pathway is involved in learning, memory formation/storage and synaptic plasticity [23]. It is also well known that aging is associated with cognitive deficits/decline via diverse mechanisms, including the change/impairment of glutamatergic pathway [5,24] and also affecting receptors' binding and density [25][26][27]. Changes in the expression of the ionotropic receptors N-methyl-D-aspartate receptor (NMDARs) have been Biology 2023, 12,196 3 of 28 previously described in rat and murine hippocampus during aging or disease [28,29]. The G protein-coupled metabotropic glutamate receptors (mGluRs) are enriched in the hippocampal formation and interact physically with other proteins, including glutamate ionotropic receptors, to ensure the maintenance of cognitive performance. ...
Simple Summary
With the increase in the geriatric population worldwide, the promotion of healthy aging arose as a key issue and, hence, the scientific community dedicates huge effort to counteract age-related impairments. Brain aging is a crucial risk factor for several neurodegenerative disorders and dementia. One of the most affected cognitive functions is recognition memory. Inflammation and oxidative stress play a key role in pathogenesis of cognitive impairments, and a link exists between frailty, oxidative stress, and inflammaging. Medicinal mushrooms represent a source to develop new therapeutic strategy, and among them Hericium erinaceus (He) displays several actions ranging from boosting immune system to fighting senescence, due to its active ingredients/metabolites. Among these, Ergothioneine (ERGO) is known as the longevity vitamin. Currently, when monitoring physiological aging in mice, we demonstrated the selective, preventive and neuroprotective effect of an ERGO-rich He primordium extract in hippocampus, preventing recognition memory decline during aging, decreasing key markers of inflammation and oxidative stress, and increasing the expression of glutamate receptors, which are crucially involved in glutamatergic neurotransmission.
Abstract
Brain aging is a crucial risk factor for several neurodegenerative disorders and dementia. The most affected cognitive function is memory, worsening early during aging. Inflammation and oxidative stress are known to have a role in pathogenesis of cognitive impairments, and a link exists between aging/frailty and immunosenescence/inflammaging. Based on anti-aging properties, medicinal mushrooms represent a source to develop medicines and functional foods. In particular, Hericium erinaceus (He) displays several actions ranging from boosting the immune system to fighting senescence, due to its active ingredients/metabolites. Among these, Ergothioneine (ERGO) is known as the longevity vitamin. Currently, we demonstrated the efficacy of an ERGO-rich He primordium extract (He2) in preventing cognitive decline in a murine model of aging. We focused on recognition memory deterioration during aging, monitored through spontaneous behavioral tests assessing both memory components and frailty index. A parallel significant decrease in key markers of inflammation and oxidative stress, i.e., IL6, TGFβ1, GFAP, Nrf2, SOD1, COX2, NOS2, was revealed in the hippocampus by immunohistochemistry, accompanied by an enhancement of NMDAR1and mGluR2, crucially involved in glutamatergic neurotransmission. In summary, we disclosed a selective, preventive and neuroprotective effect of He2 on aged hippocampus, both on recognition memory as well on inflammation/oxidative stress/glutamate receptors expression.
... Among these changes are alterations to the release, uptake, and clearance of glutamate, as well as changes in the functions and subunit composition of its receptors [11]. Glutamate, being the major excitatory neurotransmitter in the CNS, is primarily released from presynaptic vesicles and acts on different types of postsynaptic glutamate receptors [12,13]. ...
Glutamate is the major excitatory neurotransmitter in the central nervous system and is intricately linked to learning and memory. Its activity depends on the expression of AMPA and NMDA receptors and excitatory amino transporters on neurons and glial cells. Glutamate transporters prevent the excess accumulation of glutamate in synapses, which can lead to aberrant synaptic signaling, excitotoxicity, or cell death. Neuroinflammation can occur acutely after surgical trauma and contributes to the development of perioperative neurocognitive disorders, which are characterized by impairment in multiple cognitive domains. In this review, we aim to examine how glutamate handling and glutamatergic function are affected by neuroinflammation and their contribution to cognitive impairment. We will first summarize the current data regarding glutamate in neurotransmission, its receptors, and their regulation and trafficking. We will then examine the impact of inflammation on glutamate handling and neurotransmission, focusing on changes in glial cells and the effect of cytokines. Finally, we will discuss these changes in the context of perioperative neuroinflammation and the implications they have for perioperative neurocognitive disorders.
... Excitotoxicity plays a significant role in the development and progression of AD and other neurodegenerative disorders [8,9]. A disruption of calcium ion homeostasis acts excitotoxically, leading to the process of neurodegeneration in AD and contributing to nerve cell death [10][11][12]. ...
(1) Background: The use of uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists results in neuroprotective benefits in patients with moderate to severe Alzheimer’s disease. In this study, we demonstrated mathematical and computer modelling of the excitotoxicity phenomenon and performed virtual memantine therapy. (2) Methods: A computer simulation environment of the N-methyl-D-aspartate receptor combining biological mechanisms of channel activation by means of excessive extracellular glutamic acid concentration in three models of excitotoxicity severity. The simulation model is based on sliding register tables, where each table is associated with corresponding synaptic inputs. Modelling of the increase in extracellular glutamate concentration, through over-stimulation of NMDA receptors and exacerbation of excitotoxicity, is performed by gradually increasing the parameters of phenomenological events by the power function. Pathological models were virtually treated with 3–30 µM doses of memantine compared to controls. (3) Results: The virtual therapy results of memantine at doses of 3–30 µM in the pathological models of excitotoxicity severity show statistically significant neuroprotective benefits in AD patients with moderate severity, 1.25 (95% CI, 1.18–1.32) vs. 1.76 (95% CI, 1.71–1.80) vs. 1.53 (95% CI, 1.48–1.59), (p < 0.001), to severe, 1.32 (95% CI, 1.12–1.53) vs. 1.77 (95% CI, 1.72–1.82) vs. 1.73 (95% CI, 1.68–1.79), (p < 0.001), in the area of effects on memory. A statistically significant benefit of memantine was demonstrated for all neuronal parameters in pathological models. In the mild severity model, a statistically significant increase in frequency was obtained relative to virtual memantine treatment with a dose of 3 µM, which was 23.5 Hz (95% CI, 15.5–28.4) vs. 38.8 Hz (95% CI, 34.0–43.6), (p < 0.0001). In the intermediate excitotoxicity severity model, a statistically significant increase in frequency was obtained relative to virtual memantine therapy with a 3 µM dose of 26.0 Hz (95% CI, 15.7–36.2) vs. 39.0 Hz (95% CI, 34.2–43.8) and a 10 µM dose of 26.0 Hz (95% CI, 15.7–36.2) vs. 30.9 Hz (95% CI, 26.4–35.4), (p < 0.0001). A statistically significant increase in frequency was obtained in the advanced excitotoxicity severity model as in the medium. (4) Conclusions: The NMDA antagonist memantine causes neuroprotective benefits in patients with moderate to severe AD. One of the most important benefits of memantine is the improvement of cognitive function and beneficial effects on memory. On the other hand, memantine provides only symptomatic and temporary support for AD patients. Memantine is prescribed in the US and Europe if a patient has moderate to severe AD. Memantine has also been approved for mild to moderate AD patients. However, its very modest effect provides motivation for further research into new drugs in AD. We are the first to present a mathematical model of the NMDA receptor that allows the simulation of excitotoxicity and virtual memantine therapy.
... Glutamate is the major excitatory neurotransmitter in mammalian CNS and a key player in excitotoxicity (Nakanishi and Masu, 1994;Michaelis, 1998;Ferraguti et al., 2008). Glutamate binds to two types of glutamate receptors: ionotropic and metabotropic glutamate receptors (mGluRs) (Conn and Pin, 1997). ...
Gyroxin is a thrombin-like toxin obtained from the venom of the South American rattlesnake, Crotalus durissus terrificus. Literature has reported “gyroxin syndrome” characterized, in mice, as series of aberrant motor behavior, known as barrel rotation, mainly after intraperitoneal administration. Despites several studies, a physiological mechanism of “gyroxin syndrome” are still not completely understood. In this context, alterations on the central nervous system (CNS), especially causing neurotoxic events, are pointed out as likely candidates. Then, we decided to investigate whether gyroxin induces alterations in glutamate release, one of the most important neurotransmitter involved in neurotoxicity. For that, we performed all experiments, in vitro, using a model of mice brain cortical synaptosomes. Notably, our results indicate that the administration of gyroxin on purified presynaptic brain cortical terminals resulted in an extracellular Ca²⁺- dependent raise in glutamate release. Indeed, our results also showed that gyroxin increases intrasynaptosomal calcium (Ca²⁺) levels through acting on voltage gated calcium channels (VGCC), specifically N and P/Q subtypes. Moreover, our data show that gyroxin increases exocytosis rate. Interestingly, these data suggest that gyroxin might induce neurotoxicity by increasing glutamate levels. However, future investigations are needed in order to elucidate the nature of the following events.
... L-glutamate is a ubiquitous neurotransmitter in the vertebrate central nervous system, and it also plays a decisive role in the synaptic circuitry of the retina [1]. The broad diversity of postsynaptic glutamate receptors is key for transmission of intricate signal properties, especially in sensory systems like the retina. ...
Glutamate is an essential neurotransmitter for signal processing in the vertical pathway of the mammalian retina, where it is involved in the distribution of visual information into several parallel channels. The excitatory effects of glutamate are mediated by AMPA-, kainate-, and NMDA-type ionotropic glutamate receptors (iGluRs). The expression patterns of these receptors in the vertebrate retina have been investigated so far with mainly immunocytochemical, in-situ hybridization, and electrophysiological/pharmacological techniques. Here, we have used scRNA sequencing data from chicken, mouse, macaque, and human retina to describe and compare the profile of iGluR expression in major retinal cell types across species. Our results suggest that major retinal cell types each express a unique set of iGluRs with substantial differences between non-mammalian and mammalian retinae. Expression of iGluRs has been investigated in more detail for amacrine and bipolar cell types of the human retina, each showing minor variations of a common pattern. The differential expression of iGluRs is likely to convey unique signal processing properties to individual elements of the retinal circuitry.
... Previous studies have shown that one of the most critical mechanisms leading to DND is the release of large amounts of excitatory Glu, which leads to the opening of glutamate ion channels (NMDA, AMPA, and KA receptors, mainly NMDA receptors) [16,17]. Numerous studies have shown that Glu is the central link of cerebral ischemic injury. ...
Ischemic stroke is the most serious disease that harms human beings. In principle, its treatment is to restore blood flow supply as soon as possible. However, after the blood flow is restored, it will lead to secondary brain injury, that is, ischemia-reperfusion injury. The mechanism of ischemia-reperfusion injury is very complicated. This study showed that P2X4 receptors in the pyramidal neurons of rat hippocampus were significantly upregulated in the early stage of ischemia-reperfusion injury. Neurons with high expression of P2X4 receptors are neurons that are undergoing apoptosis. Intraventricular injection of the P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) and PSB-12062 can partially block neuronal apoptosis, to promote the survival of neurons, indicating that ATP through P2X4 receptors is involved in the process of cerebral ischemia-reperfusion injury. Therefore, identifying the mechanism of neuronal degeneration induced by extracellular ATP via P2X4 receptors after ischemia-reperfusion will likely find new targets for the treatment of ischemia-reperfusion injury, and will provide a useful theoretical basis for the treatment of ischemia-reperfusion injury.
... Glutamate is a excitatory neurotransmitter in the central nervous system [1]. It plays an essential role in learning and memory, synaptic plasticity, and cytoskeleton formation [2,3]. ...
Glutamate induces neurotoxicity during brain development, causing nerve damage. Protein phosphatase 2A (PP2A) is a type of serine/threonine phosphatase that regulates various biological functions. Among the PP2A subunit types, subunit B is abundant in brain tissue and plays an essential role in the nervous system. This study investigated changes in PP2A subunit B expression through glutamate exposure in the cerebral cortex of newborn rats. Sprague-Dawley rat pups (7 days after birth) were injected intraperitoneally with vehicle or glutamate (10 mg/kg). After 4 h of drug treatment, the brain tissue was isolated and fixed for morphological study. In addition, the cerebral cortex was collected for RNA and protein works. We observed severe histopathological changes including swollen neuron and atrophied dendrite in the glutamate exposed cerebral cortex. Glutamate exposure leads to a decrease in PP2A subunit B. Reverse-transcription PCR and Western blot analyses confirmed that glutamate induces a decrease of PP2A subunit B in the cerebral cortex of newborn rats. Moreover, immunohistochemical study showed a decrease in PP2A subunit B positive cells. The reduction of PP2A subunit B expression is considered an indicator of neurodegenerative damage. These results suggest that glutamate exposure causes neuronal damage in the cerebral cortex of new born rats through a decrease in PP2A subunit B.
... increases the strength of the synaptic transmission. As the NMDA receptor allows the intracellular increase of Ca 2+ , the surge of LTP increases the density of the AMPA and NMDA receptors in the membrane (Bashir et al., 1991;Mattson, 2008;Michaelis, 1998). ...
Advances in the understanding of genetic and molecular mechanisms and imaging technologies have opened a new window of research possibilities to address dynamic processes associated with neuroplasticity in physiologically intact models of neurodegenerative diseases. This review aims to: (i) establish the most relevant molecular mechanisms, as well as cellular and structural biomarkers in the study of neuroplasticity; (ii) introduce different neurodegenerative diseases in animal models that contribute to our knowledge of neuroplasticity; and (iii) illustrate the capabilities and limitations of current diffusion magnetic resonance imaging techniques to study cortical plasticity, as well as the use of alternative diffusion models.
... lonotropic receptors are primarily located at postsynaptic sites but can also be found at presynaptic terminals, whereas mGluRs are distributed pre-and postsynaptically. Additionally, glutamate receptors can be found in glial cells (see Mayer and Westbrook, 1987a;Collingridge and Lester, 1989;McBain and Mayer, 1994;Michaelis, 1998;Ozawa et al., 1998;Dingledine et al., 1999 for reviews on glutamate receptors). ...
The properties of synaptic transmission may be modulated by transporters which regulate neurotransmitter and ion concentrations, and by proteins which interact with ion channels and transporters. I have investigated this for inhibitory and excitatory synapses in the retina and the cerebellum, using electrophysiological (patch-clamp) techniques. For inhibitory synaptic transmission, I have (1) discovered that GABAC receptors in retinal bipolar cells are modulated by the intracellular cytoskeletal protein MAP-1B, and shown that disrupting the interaction between MAP-1B and the GABAC receptor increases the sensitivity of the receptor to GABA, which is expected to alter the duration of the inhibitory postsynaptic current in these cells; and (2) studied the possibility that chloride transporters maintain a non-uniform intracellular chloride distribution in retinal bipolar cells, which determines the direction and magnitude of the GABA evoked membrane potential changes in the cell. For excitatory synaptic transmission I have (1) studied glutamatergic synaptic transmission in the cerebellum of transgenic mice lacking either of the glutamate transporters GLT-1 or GLAST, and demonstrated that GLAST knockout prolongs the synaptic current at the parallel fibre to Purkinje cell synapse, but that knocking out GLT-1 or GLAST does not alter the mossy fibre to granule cell synaptic current; (2) studied the effect of glycine on mossy fibre to granule cell synaptic transmission in the cerebellum of the rat, showing that the NMDA receptor glycine site is saturated even when no glycine is added to the superfusing solution; and (3) studied the properties of the LIM protein Ajuba, which interacts with the major glial glutamate transporter GLT-1, and shown that Ajuba does not modulate the transporter's glutamate sensitivity, its associated anion channel, or the number of transporters in the plasma membrane.
... Excessive release of glutamate is harmful for animal survival, since it leads to the synchronous discharge of neuronal activity and excitotoxic damage to the brain (Michaelis, 1998). Since GLT-1 plays a primary role in >90% of extracellular glutamate uptake (Haugeto et al., 1996;Tanaka, 1997), homozygotes of the germ-line GLT-1 knockout allele showed lethal seizure and selective neuronal degeneration in many brain regions such as hippocampus and cerebral cortex (Kiryk et al., 2008;Tanaka, 1997). ...
Cortical spreading depression (CSD) is a pathological neural excitation that underlies migraine pathophysiology. Since glutamate receptor antagonists impair CSD propagation, susceptibility to CSD might be determined by any of the neuronal (excitatory amino acid carrier 1 [EAAC1]) and glial (GLutamate ASpartate Transporter [GLAST] and glial glutamate transporter 1 [GLT‐1]) glutamate transporters, which are responsible for clearing extracellular glutamate. To investigate this hypothesis, we performed electrophysiological, hemodynamic, and electrochemical analyses using EAAC1‐ (EAAC1 KO), GLAST‐ (GLAST KO), and conditional GLT1‐1‐knockout mice (GLT‐1 cKO) to assess altered susceptibility to CSD. Despite the incomplete deletion of the gene in the cerebral cortex, GLT‐1 cKO mice exhibited significant reduction of GLT‐1 protein in the brain without apparent alteration of the cytoarchitecture in the cerebral cortex. Physiological analysis revealed that GLT‐1 cKO showed enhanced susceptibility to CSD elicited by chemical stimulation with increased CSD frequency and velocity compared to GLT‐1 control. In contrast, the germ‐line EAAC1 and GLAST KOs showed no such effect. Intriguingly, both field potential and cerebral blood flow showed faster dynamics with narrower CSD than the controls. An enzyme‐based biosensor revealed more rapid accumulation of glutamate in the extracellular space in GLT‐1 cKO mice during the early phase of CSD than in GLT‐1 control, resulting in an increased susceptibility to CSD. These results provided the first evidence for a novel role of GLT‐1 in determining susceptibility to CSD. Mice defective of glutamate transporter glial glutamate transporter 1 (GLT‐1) but not GLutamate ASpartate Transporter or excitatory amino acid carrier 1 exhibited enhanced susceptibility to the cortical spreading depression (CSD). An enzyme‐based biosensor revealed more rapid accumulation of extracellular glutamate in GLT‐1 mutant during CSD than in controls.
... Glutamate is an excitatory neurotransmitter in the central nervous system [1]. It contributes to various physiological functions including memory and learning, synaptic transmission, and plasticity [2,3]. ...
Glutamate is a representative excitatory neurotransmitter. However, excessive glutamate exposure causes neuronal cell damage by generating neuronal excitotoxicity. Excitotoxicity in neonates caused by glutamate treatment induces neurological deficits in adults. The 14-3-3 family proteins are conserved proteins that are expressed ubiquitously in a variety of tissues. These proteins contribute to cellular processes, including signal transduction, protein synthesis, and cell cycle control. We proposed that glutamate induces neuronal cell damage by regulating 14-3-3 protein expression in newborn animals. In this study, we investigated the histopathological changes and 14-3-3 proteins expressions as a result of glutamate exposure in the neonatal cerebral cortex. Rat pups at post-natal day 7 were intraperitoneally administrated with vehicle or glutamate (10 mg/kg). Animals were sacrificed 4 h after treatment, and brain tissues were fixed for histological study. Cerebral cortices were isolated and frozen for proteomic study. We observed serious histopathological damages including shrunken dendrites and atypical neurons in glutamate-treated cerebral cortices. In addition, we identified that 14-3-3 family proteins decreased in glutamate-exposed cerebral cortices using a proteomic approach. Moreover, Western blot analysis provided results that glutamate treatment in neonates decreased 14-3-3 family proteins expressions, including the β/α, ζ/δ, γ, ε, τ, and η isoforms. 14-3-3 proteins are involved in signal transduction, metabolism, and anti-apoptotic functions. Thus, our findings suggest that glutamate induces neonatal neuronal cell damage by modulating 14-3-3 protein expression.
... Despite the fact that fast excitation is mediated by ionic movement rather than slow-metabotropic actions, the latter mechanism is a key component of the kainate-mediated transmission (see for example, Valbuena and Lerma, 2016). All iGluRs members are formed by proteins structured as tetramers with a vast molecular diversity widely studied (Michaelis, 1998;Traynelis et al., 2010). ...
Metabotropic glutamate receptors (mGluRs) are a group of G protein-coupled receptors that exert a broad array of modulatory actions at excitatory synapses of the central nervous system. In the hippocampus, the selective activation of the different mGluRs modulates the intrinsic excitability, the strength of synaptic transmission, and induces multiple forms of long-term plasticity. Despite the relevance of mGluRs in the normal function of the hippocampus, we know very little about the changes that mGluRs functionality undergoes during the non-pathological aging.
Here, we review data concerning the physiological actions of mGluRs, with particular emphasis on hippocampal area CA3. Later, we examine changes in the expression and functionality of mGluRs during the aging process. We complement this review with original data showing an array of electrophysiological modifications observed in the synaptic transmission and intrinsic excitability of aged CA3 pyramidal cells in response to the pharmacological stimulation of the different mGluRs.
... Glutamate acts as a critical excitatory neurotransmitter in central nervous system [1]. It plays an important role in synaptic maintenance and plasticity, learning and memory, and cytoskeleton formation [2]. ...
Glutamate leads to neuronal cell damage by generating neurotoxicity during brain development. The objective of this study is to identify proteins that differently expressed by glutamate treatment in neonatal cerebral cortex. Sprague-Dawley rat pups (post-natal day 7) were intraperitoneally injected with vehicle or glutamate (10 mg/kg). Brain tissues were isolated 4 h after drug treatment and fixed for morphological study. Moreover, cerebral cortices were collected for protein study. Two-dimensional gel electrophoresis and mass spectrometry were carried out to identify specific proteins. We observed severe histopathological changes in glutamate-exposed cerebral cortex. We identified various proteins that differentially expressed by glutamate exposure. Identified proteins were thioredoxin, peroxiredoxin 5, ubiquitin carboxy-terminal hydrolase L1, proteasome subunit alpha proteins, isocitrate dehydrogenase, and heat shock protein 60. Heat shock protein 60 was increased in glutamate exposed condition. However, other proteins were decreased in glutamate-treated animals. These proteins are related to anti-oxidant, protein degradation, metabolism, signal transduction, and anti-apoptotic function. Thus, our findings can suggest that glutamate leads to neonatal cerebral cortex damage by regulation of specific proteins that mediated with various functions.
... NMDA receptors modulate longer lasting synaptic events in response to glutamate release and are non-selective cation channels allowing influx of Ca 2+ together with Na + and efflux of K + ions (Daw et al. 1993;Scheetz and Constantine-Paton 1994;Michaelis 1998). NMDA receptors also exist as heterotetrameric structures that require that assembly of the obligatory NR1 subunits (encoded by the Grin1 gene) with either NR2(A,B,C,D) subunits or with a combination of NR2 subunits with NR3(A,B) subunits (Paoletti et al. 2013). ...
The diaphragm muscle comprises various types of motor units that are recruited in an orderly fashion governed by the intrinsic electrophysiological properties (membrane capacitance as a function of somal surface area) of phrenic motor neurons (PhMNs). Glutamate is the main excitatory neurotransmitter at PhMNs and acts primarily via fast acting AMPA and N‐methyl‐D‐aspartic acid (NMDA) receptors. Differences in receptor expression may also contribute to motor unit recruitment order. We used single cell, multiplex fluorescence in situ hybridization to determine glutamatergic receptor mRNA expression across PhMNs based on their somal surface area. In adult male and female rats (n = 9) PhMNs were retrogradely labeled for analyses (n = 453 neurons). Differences in the total number and density of mRNA transcripts were evident across PhMNs grouped into tertiles according to somal surface area. A ~ 25% higher density of AMPA (Gria2) and NMDA (Grin1) mRNA expression was evident in PhMNs in the lower tertile compared to the upper tertile. These smaller PhMNs likely comprise type S motor units that are recruited first to accomplish lower force, ventilatory behaviors. In contrast, larger PhMNs with lower volume densities of AMPA and NMDA mRNA expression presumably comprise type FInt and FF motor units that are recruited during higher force, expulsive behaviors. Furthermore, there was a significantly higher cytosolic NMDA mRNA expression in small PhMNs suggesting a more important role for NMDA‐mediated glutamatergic neurotransmission at smaller PhMNs. These results are consistent with the observed order of motor unit recruitment and suggest a role for glutamatergic receptors in support of this orderly recruitment.
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Cover Image for this issue: doi: 10.1111/jnc.14747.
... One of its receptors, N-methyl D-aspartate (NMDA), has been implicated in longterm potentiation, which is the neuronal mechanism responsible for learning and memory (Katsuki et al., 1997;Rondi-Reig et al., 2001). Too much stimulation, however, leads to excitotoxicity due to high intracellular concentrations of calcium, which causes neuronal dysfunction and death (Michaelis, 1998), effects involved in the pathogenic cascade of AD. ...
... Glutamate is one of the excitatory neurotransmitters, which is involved in various physiological functions of the brain, including synaptic plasticity. The glutamate action is mediated by ionotropic and metabotropic receptors (Greenamyre and Porter, 1994;Michaelis, 1998). α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is one of ligand-gated ion channels for glutamate (Traynelis et al., 2010). ...
To elucidate the pharmacological properties of perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile, a novel non-competitive antagonist of AMPA receptor], we investigated its effects on the up-stream regulatory pathways of GluA1 phosphorylation including protein kinase C (PKC), Ca2+-calmodulin-dependent protein kinase II (CAMKII), protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), protein phosphatase (PP) 1, PP2A, and PP2B in normal and pilocarpine-induced epileptic rat model using Western blot analysis. In normal animals, perampanel affected GluA1 expression/phosphorylation, PKC, CAMKII, PKA, ERK1/2, JNK, and PPs activities. In epileptic rats, perampanel effectively inhibited spontaneous seizure activities. Perampanel enhanced phospho (p)-GluA1-S831 and -S845 ratios (phosphoprotein/total protein), while it reduced GluA1 expression. Perampanel also increased pCAMKII and pPKA ratios, which phosphorylate GluA1-S831 and -S845 site, respectively. Perampanel elevated pJNK and pPP2B ratios, which phosphorylates and dephosphorylates both GluA1-S831 and -S845 sits. Perampanel also increased pERK1/2 ratio in epileptic animals, while U0126 (an ERK1/2 inhibitor) did not affect pGluA1 ratios. Perampanel did not influence PKC, PP1, and PP2A expression levels and their phosphorylation ratios. In addition, perampanel did not have a detrimental impact on cognitive abilities of epileptic and normal rats in Morris water maze test. These findings suggest that perampanel may regulate AMPA receptor functionality via not only blockade of AMPA receptor but also the regulations of multiple molecules (CAMKII, PKA, JNK, and pPP2B)-mediated GluA1 phosphorylations without negative effects on cognition, although the effects of perampanel on PKC, PP1, and PP2A activities were different between normal and epileptic rats.
... En ellos se distinguen los de tipo AMPA, kainato y NMDA, según el nombre del agonista más afín. El otro tipo son los llamados receptores metabotrópicos, una familia de ocho miembros conocidos que -como su nombre indica-se relacionan con cambios metabólicos más que con conductancias iónicas [79,80]. Los receptores AMPA/kainato son los responsables de la transmisión sináptica normal, pues median corrientes de sodio que despolarizan la membrana post-sináptica. ...
... N-Methyl-Daspartate receptors (NMDARs) are the primary glutamate receptors in the central nervous system (CNS) (Iacobucci and Popescu, 2017;Ozawa et al., 1998)], including both the outer and inner primate retina with most robust expression in the latter (Shen et al., 2006). Excessive stimulation of NMDARs initiates a massive intracellular influx of Ca 2+ that consequently leads to mitochondrial damage, oxidative stress and cell death, a process termed excitotoxicity (LUCAS and NEWHOUSE, 1957;Michaelis, 1998;Olney, 1969). N-Methyl-D-aspartate (NMDA), the agonist molecule of glutamate that binds selectively to NMDARs, has been extensively used in the experimental model of NMDA-induced retinal excitotoxicity, an acute form of retinal injury, that results in severe and rapid cell death of RGCs and the inner retina. ...
N-methyl-D-aspartate (NMDA)-induced excitotoxicity is an acute form of experimental retinal injury as a result of overactivation of glutamate receptors. NLRP3 (nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain containing-3) inflammasome, one of the most studied sensors of innate immunity, has been reported to play a critical role in retinal neurodegeneration with controversial implications regarding neuroprotection and cell death. Thus far, it has not been elucidated whether NMDA-mediated excitotoxicity can trigger NLRP3 inflammasome in vivo. Moreover, it is unknown if NLRP3 is beneficial or detrimental to NMDA-mediated retinal cell death. Here, we employed a murine model of NMDA-induced retinal excitotoxicity by administering 100 nmoles of NMDA intravitreally, which resulted in massive TUNEL⁺ (TdT-dUTP terminal nick-end labelling) cell death in all retinal layers and especially in retinal ganglion cells (RGCs) 24 h post injection. NMDA insult in the retina potentiates macrophage/microglia cell infiltration, primes the NLRP3 inflammasome in a transcription-dependent manner and induces the expression of interleukin-1β (IL-1β). However, despite NLRP3 inflammasome upregulation, systemic deletion of Nlrp3 or Casp1 (caspase-1) did not significantly alter the NMDA-induced, excitotoxicity-mediated TUNEL⁺ retinal cell death at 24 h (acute phase). Similarly, the deletion of the two aforementioned genes did not alter the survival of the Brn3a⁺ (brain-specific homeobox/POU domain protein 3A) RGCs in a significant way at 3- or 7-days post injection (long-term phase). Our results indicate that NMDA-mediated retinal excitotoxicity induces immune cell recruitment and NLRP3 inflammasome activity even though inflammasome-mediated neuroinflammation is not a leading contributing factor to cell death in this type of retinal injury.
... Glutamatergic neurotransmission, altered in normal aging and disease, is critically involved in facilitating neuronal plasticity and modulating both LTP and long-term depression (LTD) [294][295][296][297]. Aging has been associated with a decline of glutamate content in the prefrontal cortex and hippocampus, a reduction in high-affinity glutamate transporters and glutamatergic receptors, and reduced glutamate uptake capacity [298][299][300]. Expression of the NMDA receptor class is particularly influenced by advanced age [200,298,301,302]. As neuronal plasticity is in large part dependent on synaptic NMDAR activation, a decrease in synaptic NMDAR expression may functionally contribute to the age-related in memory and cognition [295,[303][304][305]. ...
Physical activity plays an essential role in maintaining a healthy body, yet it also provides unique benefits for the vascular and cellular systems that sustain a healthy brain. While the benefit of exercise has been observed in humans of all ages, the availability of preclinical models has permitted systematic investigations into the mechanisms by which exercise supports and protects the brain. Over the past twenty-five years, rodent models have shown that increased physical activity elevates neurotrophic factors in the hippocampal and cortical areas, facilitating neurotransmission throughout the brain. Increased physical activity (such as by the voluntary use of a running wheel or regular, timed sessions on a treadmill) also promotes proliferation, maturation and survival of cells in the dentate gyrus, contributing to the process of adult hippocampal neurogenesis. In this way, rodent studies have tremendous value as they demonstrate that an ‘active lifestyle’ has the capacity to ameliorate a number of age–related changes in the brain, including the decline in adult neurogenesis. Moreover, these studies have shown that greater physical activity may protect the brain health into advanced age through a number of complimentary mechanisms: in addition to upregulating factors in pro-survival neurotrophic pathways and enhancing synaptic plasticity, increased physical activity promotes brain health by supporting the cerebrovasculature, sustaining the integrity of the blood–brain barrier, increasing glymphatic clearance and proteolytic degradation of amyloid beta species, and regulating microglia activation. Collectively, preclinical studies demonstrate that exercise initiates diverse and powerful neuroprotective pathways that may converge to promote continued brain health into old age. This review will draw on both seminal and current literature that highlights mechanisms by which exercise supports the functioning of the brain, and aids in its protection.
... La interacción del Glu con sus diferentes receptores genera una amplia gama de respuestas en las sinapsis glutamatérgicas. Esta diversidad se incrementa tanto por la generación de variantes postranscripcionales de las subunidades a través de la edición y el empalme alternativo del mRNA, como por mecanismos de modificación postraduccional como la fosforilación y la desfosforilación de los receptores y la regulación de su expresión en la membrana 1 La composición de los NMDARs no se conoce con precisión. Se ha demostrado que los receptores formados por 4 subunidades NR1 (homoméricos) son funcionales. ...
The multiple actions of glutamate in the retina, as a neurotransmitter, a trophic factor or a neurotoxin, are directly related to the heteromeric composition of the NMDA type of glutamate receptors present in this tissue. The tetrameric NMDARs include two NR1 subunits which form the calcium-permeable cation channel, in addition to NR2 and NR3 subunits which modulate its activity. Recent results demonstrate that retinal NMDARs might include tissue-specific NR1 splice variants and NR2 subunits, in both mature and embryonic tissue, which determine structural and functional differences with brain receptors. As a consequence, drugs shown to protect brain neurons from glutamate-induced neuronal death, are ineffective in the retina. The clarification of the precise subunit composition of NMDARs in the retina will allow the future design of drugs aimed to prevent neuronal loss in pathological conditions such as glaucoma, vascular occlusion, optic neuropathy and ischemia, in which glutamate excitotoxicity seems to be involved.
... This evidence sustains a prevalent inhibitory effect of indicaxanthin on discharge activity of neurons in rat brain ( Table 1). As for the exclusive excitatory effect observed in the striatum, in order to explain this different result, it should be noted that all the other structures examined are mainly based on glutamatergic or GABAergic transmission, while in the striatum a relevant dopaminergic influence integrate glutamatergic input and contribute to the neuronal excitability (Parent and Hazrati, 1995;Michaelis, 1998). Therefore, the modulatory action of indicaxanthin occurred in a peculiar synaptic environment, more finely regulated, possibly underlying the increase in discharge frequency. ...
Several studies have recently investigated the role of nutraceuticals in complex pathophysiological processes such as oxidative damages, inflammatory conditions and excitotoxicity. In this regard, the effects of nutraceuticals on basic functions of neuronal cells, such as excitability, are still poorly investigated. For this reason, the possible modulation of neuronal excitability by phytochemicals (PhC) could represent an interesting field of research given that excitotoxicity phenomena are involved in neurodegenerative alterations leading, for example, to Alzheimer’s disease. The present study was focused on indicaxanthin from Opuntia ficus indica, a bioactive betalain pigment, with a proven antioxidant and anti-inflammatory potential, previously found to cross blood-brain barrier (BBB) and to modulate the bioelectric activity of hippocampal neurons. On this basis, we aimed at detecting the specific brain areas where indicaxanthin localizes after oral administration at dietary-achievable amounts and highlighting eventual local effects on the excitability of single neuronal units. HPLC analysis of brain tissue 1 h after ingestion of 2 μmol/kg indicaxanthin indicated that the phytochemical accumulates in cortex, hippocampus, diencephalon, brainstem and cerebellum, but not in the striato-pallidal complex. Then, electrophysiological recordings, applying the microiontophoretic technique, were carried out with different amounts of indicaxanthin (0.34, 0.17, 0.085 ng/neuron) to assess whether indicaxanthin influenced the neuronal firing rate. The data showed that the bioelectric activity of neurons belonging to different brain areas was modulated after local injection of indicaxanthin, mainly with dose-related responses. A predominating inhibitory effect was observed, suggesting a possible novel beneficial effect of indicaxanthin in reducing cell excitability. These findings can constitute a new rationale for exploring biological mechanisms through which PhC could modulate neuronal function with a relapse on complex cognitive brain process and related neurodegenerative conditions.
... Glutamate is the major excitatory neurotransmitter in the brain, and it mediates learning and memory under normal physiological conditions. There are two kinds of glutamate receptors; ionotropic glutamate receptors (iGluR) and metabotropic glutamate receptors (mGluRs) (Michaelis, 1998). iGluRs are ligand-gated ion channels and mediate the rapid synaptic responses to glutamate (Kew and Kemp, 2005). ...
The aim of the present study was to evaluate functional changes of mGluR5 expression in advanced Alzheimer's disease (AD) using positron emission tomography (PET) with an mGluR5 specific radiotracer ([18F]FPEB) in 5xFAD AD model mice. Subsequently, in the same animal, mGluR5 expression was quantified by immunoassay techniques. The non-displaceable binding potential values for mGluR5 was estimated by the Logan's graphical analysis. Brain PET imaging revealed that radioactivities in the hippocampus and the striatum were significantly lower in 5xFAD rats compared to control animals. Binding values were also significantly lowered in 5xFAD mice. This decline was validated by immunoblotting of protein isolates from brain tissues, as the mean band density for 5xFAD mice had a lower mGluR5 intensity than for wild type mice. These results indicated that mGluR5 levels in 5xFAD mice were down regulated in the limbic system.
Introduction: Monosodium glutamate (MSG) is a flavor enhancer commonly used worldwide. Studies haveshown that high dose of MSG could act as neurotoxic or excitotoxic agent for neurons in the central nervoussystem. The aim of this study was to determine the effect of MSG on neuron changes of hippocampusanimalmodel.Materials and Methods: A total of 25 white male Sprague-Dawleyrats, aged 8-10 weeks,were dividedinto5 groups (2 control groups (i.e., none and solvent group) and3 treatment groups that received2, 4and 6 mg/gram MSG orallyfor 30 days). Afterfour weeks on treatment, all animals were sacrificed and the entire braintissueswere removed and immediately fixed in formalin for hematoxylin and eosin (H&E) staining.Results:The percentage of damaged neurons in three Cornuammonisareas of hippocampus was higher inanimal supplemented with MSG compared to controls. At the highest MSG concentration (6 mg/gram),52.1%, 55.2% and 66.0% of neurons from Cornuammonis 1, 2, and 3, respectively were damaged. Thepercentage of neuron damages in hippocampus was in dose-dependent manner.Conclusion: Our data suggested that high dose of MSG increased thehippocampus neuron damages in dosedependenteffect. This suggests the neurotoxicity effect of high dose of MSG.
Alzheimer’s disease (AD) is the most common type of dementia that characterizes the accumulation of amyloid-β plaques and neurofibrillary tangles in the brain. AD is the most common cause of death and accounts for approximately 60–70% of the total cases imposing greater socio-economic burden across the globe. Despite extensive research, the mechanisms underlying AD are unknown. There are many hypotheses that define the pathogenesis of Alzheimer’s disease among which the amyloid cascade hypothesis is widely accepted. The hypothesis discusses the role of amyloid-β in causing alteration in calcium homeostasis. Amyloid-β-mediated dysregulation of calcium ions homeostasis has been extensively studied to understand the underlying mechanisms of neurodegeneration observed in AD. Amyloid-β and tau interact with the calcium and calcium ion channels on the plasma membrane, endoplasmic reticulum, and mitochondria of the neuronal cells to form the vicious circle of calcium load and amyloid-β accumulation. The disruption in calcium homeostasis accompanies changes as observed in the whole brain pathology of AD. This includes synaptic dysfunction, impaired cognition, mitochondrial dysfunction, oxidative stress, inflammation, and cell apoptosis. This presents an immense need for a variety of potential therapeutic targets that could prevent or slow the progression of AD. This book chapter deals with the comprehensive source of information about mechanisms through which amyloid-β and tau interact with various calcium ion channels and the beneficial effect of calcium ion channel modulator in AD.KeywordsAlzheimer’s diseaseCalcium dyshomeostasisAmyloid-βTauCognitive dysfunction
d-Galactose (d-gal) and l-glutamate (l-glu) impair learning and memory. The mechanism of interaction between the gut microbiome and brain remains unclear. In this study, a model of cognitive impairment was induced in tree shrews by intraperitoneal (ip) injection of d-gal (600 mg/kg/day), intragastric (ig) administration with l-glu (2000 mg/kg/day), and the combination of d-gal (ip, 600 mg/kg/day) and l-glu (ig, 2000 mg/kg/day). The cognitive function of tree shrews was tested by the Morris water maze method. The expression of Aβ1-42 proteins, the intestinal barrier function proteins occludin and P-glycoprotein (P-gp), and the inflammatory factors NF-κB, TLR2, and IL-18 was determined by immunohistochemistry. The gut microbiome was analyzed by 16SrRNA high-throughput sequencing. After administering d-gal and l-glu, the escape latency increased (p < .01), and the times of crossing the platform decreased (p < .01). These changes were greater in the combined administration of d-gal and l-glu (p < .01). The expression of Aβ1-42 was higher in the perinuclear region of the cerebral cortex (p < .01) and intestinal cell (p < .05). There was a positive correlation between the cerebral cortex and intestinal tissue. Moreover, the expression of NF-κB, TLR2, IL-18, and P-gp was higher in the intestine (p < .05), while the expression of occludin and the diversity of gut microbes were lower, which altered the biological barrier of intestinal mucosal cells. This study indicated that d-gal and l-glu could induce cognitive impairment, increase the expression of Aβ1-42 in the cerebral cortex and intestinal tissue, decrease the gut microbial diversity, and alter the expression of inflammatory factors in the mucosal intestines. The dysbacteriosis may produce inflammatory cytokines to modulate neurotransmission, causing the pathogenesis of cognitive impairment. This study provides a theoretical basis to explore the mechanism of learning and memory impairment through the interaction of microbes in the gut and the brain.
p>Intracellular consequences of glutamate receptor activation were investigated in mouse hippocampal neurons using fluorescent probes for the measurement of Ca<sup>2+</sup> (Fluo-3 AM; Fluo-5N AM), mitochondrial DΨ (TMRE) and superoxide (Het) in conjunction with confocal laser scanning microscopy (CLSM). The excessive activation of glutamate receptors is thought to be a common feature of neuronal death in a wide spectrum of neurological disorders. The intracellular events that link toxic glutamate receptor activation to eventual cell death are not well understood, but are likely to involve to loss of Ca<sup>2+</sup> homeostasis, mitochondrial dysfunction and free radical damage.
Glutamate acts on a large number of ionotropic and metabotropic receptor subtypes. The selective activation of NMDA receptors was particularly damaging, causing neuronal cell death in over 80% of cultured mouse hippocampal neurons 24 hours after drug application. In contrast, agonists at other glutamate receptor subtypes including kainate and ACPD caused only 14% and 7% cell death respectively. Doses of glutamate receptor agonist were selected to elicit equivalent "early" neuronal Ca<sup>2+</sup> increases are measured by Fluo-3 AM in the first 200 seconds following drug addition. Early neuronal Ca<sup>2+</sup> increases did not correlate with eventual neuronal death, suggesting that differential routes of Ca<sup>2+</sup> increase could have different pathological consequences for neurons. Experiments were then conducted to investigate intracellular events downstream of glutamate receptor activation that could lead to neuronal death.
The same doses of glutamate receptor agonists had profoundly different effects on mitochondrial DΨ, which correlated to neuronal death, NMDA caused a robust increase in TMRE fluorescence indicative of mitochondrial depolarization, whereas kainate, ACPD, AMPA and quisqualate had no significant effect on mitochondrial DΨ despite causing equivalent early Ca<sup>2+</sup> increases. All the glutamate receptor agonists caused increased neuronal superoxide production as measured by increased Het fluorescence.</p
p>I used Electrophysiological recording techniques to investigate the effects of nociceptin (Noc) in various models of epileptiform activity, in acute hippocampal slices taken from adult rats. I subsequently looked at the effects of Noc in slices taken from an epileptic (EL) mouse model and in organotypic hippocampal slice cultures (OHSC’s). In some models I compare these effects to the action of Neuropeptide Y (NPY) as this is a well studied anti-convulsant neuropeptide.
I found that Noc caused a blockade of the bursting activity seen in the 10μM Biculculline (Bic) and 0 Mg<sup>++</sup> models of epileptiform activity. I also found that Noc did not cause a change in the bursting activity seen in the High K<sup>+</sup> (8.5mM) or 100μM 4-Aminopyridine (4-AP) models.
When I looked at the Noc effects on the Bic and 0 Mg++ models in EL mice I found that Noc showed a greater reduction in bursting activity when applied to slices taken from ‘sensitised’ mice as compared to ‘non-sensitised’. I found that Noc showed no effects on synaptic transmission or paired-pulse inhibition in OHSC’s. The same result was seen with NPY. I showed through Western Blotting that the OP4, Y1, Y2 and Y5 receptors are present in OHSC’s. I then showed that Noc caused a reduction in bursting activity in the Bic model in OHSC’s and that no effect was seen when the experiment was repeated with NPY. I also experienced problems with the 4-AP model in OHSC’s and subsequently showed that 4-AP causes neuronal death in the CA1 sub-field of OHSC’s.
This data shows that targets of the OP4 receptor pathway could prove a useful experimental tool, and potential future anticonvulsant treatment.</p
N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors, highly important in regulating substantial physiologic processes in the brain and the nervous system, and disturbance in their function could contribute to different pathologies. Overstimulation and hyperactivity of NMDARs, termed as glutamate toxicity, could promote cell death and apoptosis. Meanwhile, their blockade could lead to dysfunction of the brain and nervous system as well. A growing body of evidence has demonstrated the prominent role of NMDARs in demyelinating disorders and anti-NMDAR encephalitis. Herein, we provide an overview of the role of NMDARs’ dysfunction in the physiopathology of demyelinating disorders such as multiple sclerosis and neuromyelitis optica spectrum disorders.
Objectives
The effects of intra-ventral hippocampal memantine administration in male NMRI stressed mice were studied.
Methods
Two stainless steel gauge 23 guide cannulas were placed in the middle part of the mice ventral hippocampus using stereotaxic coordination. Seven days later, the animals were undergone to the stress protocol as follows: They experience four consecutive electro-foot shock stress sessions lasting for 10 min. Five or 30 min before each stress session, the animals received intra-ventral hippocampal (0.1, 1 and, 5 µg/mouse) or intraperitoneal (1, 5, and 10 mg/kg) memantine respectively. Eight days after stress termination, the animals were tested either for the maintenance of either anxiety (elevated plus maze) or depression (forced swimming test).
Results
Animals show anxiety eight days after stress termination. Intra-ventral hippocampal infusion of memantine (5 µg/mouse) 5 min before stress inhibited the anxiety-like behaviors. However, other doses of the drug exacerbate the stress effect. The drug, when injected peripherally exacerbated the stress effect in all doses. The drug by itself had no effect. In addition, animals also show depression nine days after stress termination and memantine (0.1, 1, and 5 µg/mouse) reduced the stress effect. The drug (0.1 µg/mouse) by itself induced depression in the animals. However, the drug when injected peripherally reduced the stress effect in all doses.
Conclusions
It could be concluded that NMDA glutamate receptors in the ventral hippocampus may play a pivotal role in the mediation of maintenance of anxiety and depression induced by stress in the mice.
Visceral pain describes pain originating from the internal organs and is often variable in its experience, poorly localized and difficult to treat. Despite its prevalence, chronic visceral pain is clinically challenging to delineate its etiology based on symptoms. Although conventional analgesics offer relief for acute visceral pain, prolonged use is associated with tolerance, unwanted side effects and hyperalgesia. Chronic visceral pain is characterized by hypersensitivity to stimuli such as inflammation or mechanical distension may arise due to a peripheral augmentation of primary afferent signals, or may be due to central facilitation at spinal and supraspinal levels affecting descending nociceptive modulation. Abdominal pain as a central defining feature of common pathological conditions; in this review we focus in abdominal pain originating from the colon and the bladder, as observed in patients with irritable bowel syndrome, inflammatory bowel disease, and painful bladder syndrome. We discuss the neural innervation of the colon and bladder with attention to the nerves involved in visceral nociception. We discuss the etiology of chronic visceral hypersensitivity and the use of animal model in the identification of mediators and novel molecular mechanisms that may serve as novel targets in the development of better pharmacological approaches to treat chronic abdominal pain.
Introduction
The role of extra-hypothalamic thyrotropin-releasing hormone (TRH) has been investigated by pharmacological studies using TRH or its analogues and found to produce a wide array of effects in the central nervous system.
Methods
Immunofluorescence, In situ labeling of DNA (TUNEL), in situ hybridization chain reaction and quantitative real-time polymerase chain reaction were used in this study.
Results
We found that the granular cells of the dentate gyrus expressed transiently a significant amount of TRH-like immunoreactivity and TRH mRNA during the 6–24 h period following global cerebral ischemia/reperfusion injury. TUNEL showed that apoptosis of neurons in the CA1 region occurred from 48 h and almost disappeared at 7 days. TRH administration 30 min before or 24 h after the injury could partially inhibit neuronal loss, and improve the survival of neurons in the CA1 region.
Conclusion
These data suggest that endogenous TRH expressed transiently in the dentate gyrus of the hippocampus may play an important role in the survival of neurons during the early stage of ischemia/reperfusion injury and that delayed application of TRH still produced neuroprotection. This delayed application of TRH has a promising therapeutic significance for clinical situations.
Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes problems with memory, thinking, and behavior. Currently, there is no drug that can reduce the pathological events of this degenerative disease but symptomatic relief is possible that can abate the disease condition. N-methyl-D-aspartate (NMDA) receptors exert a critical role for synaptic plasticity as well as transmission. Overstimulation of glutamate receptors, predominantly NMDA type, may cause excitotoxic effects on neurons and is recommended as a mechanism for neurodegeneration. Atypical activation of the NMDA receptor has been suggested for AD by synaptic dysfunction. NMDA receptor antagonists especially memantine block the NMDA receptor and can reduce the influx of calcium (Ca2+) ions into neuron, thus, toxic intracellular events are not activated. This review represents the role of NMDA receptors antagonists as potential therapeutic agents to reduce AD. Moreover, this review highlights the repositioning of memantine as a potential novel therapeutic multitargeting agent for AD.
The glutamatergic system is the main excitatory system in the central nervous system (CNS). This chapter focuses on the structure and function of all different assemblies involved in glutamate neurotransmission at the CNS level. Glutamate ionotropic (N‐methyl‐d‐aspartate [NMDA] and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid [AMPA], but not kainic) and metabolomic (mGlu) receptors located in the CNS are treated in detail. The structure and function of the transporters are also discussed. Enzymes processing glutamate are treated in a further section.
Dexamethasone (DEX) is a synthetic agonist of glucocorticoid receptors that has been associated with neurotoxicity and neuropsychiatric diseases. (p-ClPhSe)2 is an organoselenium compound reported to have antioxidant, antidepressant-like, and neuroprotective actions. This study investigated whether antioxidant activity and modulation of the glutamatergic system contribute to the antidepressant-like effect of (p-ClPhSe)2 in mice subchronically exposed to DEX. Swiss mice received intraperitoneal injections of DEX (2 mg/kg) or saline (vehicle) once a day for 21 days. After, the mice received (p-ClPhSe)2 (1-10 mg/kg) or mineral oil (vehicle) by the intragastric route (i.g.) for 7 days. The mice exposed to DEX were treated with fluoxetine (20 mg/kg, i.g.) once a day for 7 days. 24 h after the last treatment, the animals performed the locomotor activity (LMA), tail suspension, and forced swimming tests. Ex vivo assays were performed in samples of prefrontal cortex (PFC). The results show that (p-ClPhSe)2 reversed depressive-like behavioral phenotype induced by DEX without affecting LMA. Further, (p-ClPhSe)2 at all doses reduced ROS levels and increased CAT activity in the PFC of DEX-exposed mice. The highest dose of (p-ClPhSe)2 was effective against the decrease of SOD activity in the PFC of mice exposed to DEX. (p-ClPhSe)2 increased the [3H] glutamate uptake/release and decreased the Na+/K+-ATPase activity as well as the EAAT1 and NMDA R2A protein contents in the PFC of DEX-exposed mice. Regarding the NMDA R2B levels, there was no difference among experimental groups. In conclusion, this study reveals the effectiveness of (p-ClPhSe)2 in reversing the depressive-like phenotype of DEX-exposed mice. In addition, (p-ClPhSe)2 modulated oxidative stress and glutamate neurotransmission in the PFC of mice subchronically exposed to DEX.
Over the past two decades, driven by the enormous public health importance of bipolar disorder, research initiatives have begun to elucidate the pathophysiology of this prevalent and debilitating condition. These research initiatives have led to breakthroughs in our understanding of causation, and now promise to foster the development of novel treatments. This new edition presents contributions from the leaders at the forefront of these areas of research, and includes chapters on the groundbreaking advances in the fields of genetics, neuroimaging, neuropsychopharmacology, oxidative stress and neuronal resilience, inflammatory mechanisms, psychosocial factors, childhood onset and late-life bipolar disorder, and many other important areas. Throughout, the therapeutic implications and potential of this new understanding are emphasized. This will be essential reading for those interested in the neurobiology of mental illness, and will be of interest to mental health practitioners more generally.
Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.
The molecular mechanism by which interleukin (IL)-1 inhibits insulin secretion and ultimately causes destruction of the pancreatic beta-cell remains unknown. Evidence is presented which suggests that IL-1 beta-induced inhibition of insulin secretion is dependent on the metabolism of L-arginine to nitric oxide. NG-Monomethylarginine, a competitive inhibitor of the L-arginine-dependent enzyme nitric oxide synthase, completely prevents IL-1-induced inhibition of glucose-stimulated insulin secretion as well as nitrite production by islets. It is further shown that IL-1 beta induces nitric oxide formation in islets as evidenced by an electron paramagnetic resonance feature at g = 2.04 which is similar to previously reported iron-nitrosyl complexes formed from the destruction of iron-sulfur centers by nitric oxide. Inhibition of the nitric oxide synthase by NG-monomethylarginine completely prevents the formation of this EPR signal in islets. These results show that IL-1-induced inhibition of insulin secretion is mediated through formation of nitric oxide and suggest that the generation of nitric oxide may represent the cellular mechanism responsible for beta-cell destruction.
Neurexins are neuronal cell surface proteins with hundreds of isoforms. In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, we identified a single interacting protein called CASK. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 and that contains DHR-, SH3-, and guanylate kinase domains. CASK is enriched in brain in synaptic plasma membranes but is also detectable at low levels in all tissues tested. The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction. In neurexin I, this interaction is dependent on the C-terminal three residues. Thus, CASK is a membrane-associated protein that combines domains found in Ca2+ - activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins.
In addition to its functions as a neuronal messenger molecule, nitric oxide (NO) has also been implicated in playing a major role in ischemic damage and glutamate neurotoxicity. Using primary cortical cultures from transgenic neuronal NO synthase (NOS) null (nNOS-) mice, we definitively establish NO as a mediator of NMDA and hypoxic neurotoxicity. Neurotoxicity elicited by NMDA is markedly attenuated in nNOS- cortical cultures compared with wild-type cultures. The NOS inhibitor nitro-L-arginine is neuroprotective in wild-type but not nNOS- cultures, confirming the role of nNOS-derived NO in glutamate neurotoxicity. Confirming that the nNOS- cultures lack NMDA-stimulated nNOS activity, NMDA did not stimulate the formation of cGMP in nNOS- cultures, but markedly elevates cGMP in wild-type cultures. Both wild- type and nNOS- cultures are sensitive to toxicity induced by NO donors, indicating that pathways stimulated by NO that result in neuronal cell death are still intact in the transgenic mice. Superoxide dismutase is neuroprotective against NMDA and NO neurotoxicity in both wild-type and nNOS- cultures, highlighting the importance of superoxide anion in subsequent neuronal damage. The unknown cellular factors that endow differential resistance to NMDA neurotoxicity and differential susceptibility to quisqualate neurotoxicity remain intact in the nNOS- cultures, because the response of somatostatin-immunopositive neurons in nNOS- cultures to high-dose NMDA and low-dose quisqualate is identical to the response of NOS-immunopositive neurons in the wild- type cultures. There is no difference in susceptibility to kainate neurotoxicity between nNOS- and wild-type cultures and only a modest resistance to quisqualate neurotoxicity, confirming observations that NO-mediated neurotoxicity is associated primarily with activation of the NMDA receptor. The nNOS- cultures are markedly protected from 60 min of combined oxygen-glucose deprivation neurotoxicity compared with wild-type cultures. Wild-type cultures are protected from neuronal cell death by the NMDA receptor antagonist MK-801 and the NOS inhibitor L- nitroarginine methyl ester, but not its inactive stereoisomer D- nitroarginine methyl ester. nNOS- cultures were not additionally protected. These data confirm that activation of NMDA receptors and production of NO are primary mediators of neuronal damage after ischemic insult.
We developed a new approach to study single- and double-stranded DNA breaks during chronic, moderate excitotoxicity resulting from the inhibition of the glutamate transporter in cerebellar granule cell primary cultures. A 24 hr treatment of 2-week-old cultures with L-alpha-amino adipate (LAA), an inhibitor of the cerebellar glutamate uptake transporter, caused a gradual extracellular accumulation of endogenous glutamate that induced reversible morphological change of granule neurons but no neuronal cell death despite sustained, but moderate, elevations of the free intracellular calcium concentrations. Nick translation experiments on isolated nuclei or cells from cerebellar cultures chronically exposed to LAA revealed increased radioactive nucleotide incorporation indicative of DNA nicking. This LAA effect was dose-dependent and suppressed by NMDA receptor antagonists. Cultures treated for 24 hr with LAA and subjected to in situ nick translation showed an intense nuclear labeling of neurons but not glia, which could be abolished by MK801. A similar labeling was also observed in altered nuclei of granule neurons acutely exposed to high glutamate concentrations or undergoing an apoptotic cell death. Although the TUNEL labeling method detected no DNA double-strand breaks in LAA-treated cerebellar cultures, it displayed clear evidence of DNA damage during acute glutamate excitotoxicity or during apoptosis. However, Southern blot analysis of nuclear DNA revealed a DNA laddering only in apoptotic cell death. Our results demonstrate that DNA damage, characterized by DNA single-strand breaks, is an early event in chronic, moderate excitotoxicity. This type of DNA degradation, which appears before any nuclear morphological changes, is distinct from the massive DNA single- and/or double-strand damages observed during acute glutamate excitotoxicity or apoptosis.
A novel metabotropic glutamate receptor, mGluR8, was identified by screening a mouse retina cDNA library. This receptor is most related to mGluR4, mGluR7, and mGluR6 (74%, 74%, and 70% identical amino acid residues, respectively). Similar to these receptors, stimulation by L-glutamate or L-2-amino-4-phosphonobutyrate (L-APB) of Chinese hamster ovary (CHO) cells stably transfected with mGluR8 result in the inhibition of forskolin-stimulated adenylyl cyclase. In situ hybridization studies revealed a strong expression of the mGluR8 gene in the olfactory bulb, accessory olfactory bulb, and mammillary body. A weaker expression was found in the retina, and in scattered cells in the cortex and hindbrain. During development, the distribution of mGluR8 expression was more widespread. These results extend the diversity of metabotropic glutamate receptors in the CNS. Because at least two APB receptors are expressed in the retina, the use of this drug to block selectively the ON pathway needs to be reconsidered. The pharmacology and expression of mGluR8 in mitral/tufted cells suggest it could be a presynaptic receptor modulating glutamate release by these cells at their axon terminals in the entorhinal cortex.
The relationship between four pharmacologically distinct NMDA receptor subtypes, identified in radioligand binding studies, and the recently identified NMDA receptor subunits (NR1a-g, NR2A-D) has not been determined. In this report, we demonstrate that the anatomical distribution of the four NMDA receptor subtypes strikingly parallels the distribution of mRNA encoding NR2A-D subunits. The distribution of NR2A mRNA was very similar to that of "antagonist-preferring" NMDA receptors [defined by high-affinity 3H-2-carboxypiperazine-4-yl-propyl-1-phosphonic (3H-CPP) binding sites; correlation coefficient = 0.88]. Agonist-preferring NMDA receptors localized to brain regions expressing both NR2B mRNA and NR1- mRNA (NR1 splice variant lacking insert 1). NR2C mRNA was largely restricted to the cerebellar granule cell layer, a region that displays a unique pharmacological profile. NR2D mRNA localized exclusively to those diencephalic nuclei that have a fourth, distinct pharmacological profile (typified by the midline thalamic nuclei). The pharmacology of native NMDA receptors was compared to that of heteromeric NMDA receptors expressed in Xenopus oocytes (NR1/NR2A, NR1/NR2B, NR1/NR2C). The oocyte-expressed NR1/NR2A receptor displayed a higher affinity for antagonists and a slightly lower affinity for agonists than the NR1/NR2B receptor. These patterns are analogous to those found for radioligand binding to native receptors in the lateral thalamus and medial striatum, respectively. NMDA receptors in the lateral thalamus (with a high density of NR2A subunit mRNA) displayed higher affinity for antagonists and a lower affinity for agonists than did NMDA receptors of the medial striatum (a region rich in NR2B mRNA). Relative to the NR1/NR2A and NR1/NR2B receptors, oocyte-expressed NR1/NR2C receptors had a lower affinity specifically for both D-3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid (D-CPPene) and homoquinolinate (HQ). This pattern was identical to that observed for cerebellar (NR2C-containing) versus forebrain (NR2A- and NR2B-containing) NMDA receptors. Taken together, the data in this report suggest that the four previously identified native NMDA receptor subtypes differ in their NR2 composition. Furthermore, the NR2 subunits significantly contribute to the anatomical and pharmacological diversity of NMDA receptor subtypes.
A single site in recombinant glutamate receptor channels of the GluR1- GluR4 family has been previously identified as a key regulator of ion permeation. The natural amino acid at this position (arginine in GluR2 but glutamine in GluR1, GluR3, and GluR4) determines both the ability to pass outward current and the divalent cation permeability of kainate- activated receptor channels. By mutagenesis of GluR6, we demonstrated that the same site also controls the ability to pass outward current in another non-NMDA receptor family. Additional mutations at and near this site in GluR3 indicated that the position of the arginine is critical to function, that the ability to pass outward current is not necessarily linked to low barium permeability, and that the size as well as the charge of the side chain at this position influences barium permeation. These results provide evidence that this site forms part of the selectivity filter of glutamate receptor channels.
Excessive stimulation of excitatory amino acid (EAA) receptors and abnormal production of oxygen-derived free radicals have repeatedly been implicated in the series of events linking brain hypoxia or ischemia to neuronal death. We report here that in rat hippocampal slices the KCl-stimulated output of labeled D-3H aspartate or of endogenous aspartate and glutamate significantly increased under in vitro simulated hypoxic, hypoglycemic, or ischemic conditions. In particular, when the slices were incubated for 10 min at 32 degrees C under “ischemic” conditions (namely, lack of oxygen and glucose), endogenous aspartate and glutamate in the supernatant increased by 10 and 20 times, respectively. Since radical scavengers (D-mannitol), drugs reducing free radical formation (indomethacin, corticosteroid), or enzymes able to metabolize them (catalase and superoxide dismutase) significantly reduced this output, it was supposed that free radicals caused EAA release. A direct demonstration of this concept was obtained by showing a significant release of EAA after incubation of hippocampal slices with enzymes and substrates known to cause the formation of free radicals, such as xanthine plus xanthine oxidase or arachidonic acid plus prostaglandin synthase. Neither ischemia nor the enzymatic reactions leading to free radical production increased the activity of the cytoplasmic enzyme lactate dehydrogenase in the incubation medium, thus ruling out a nonspecific cellular lysis. It appears therefore that during ischemic states, brain production of reactive molecules (free radicals) causes an increased output of EAA. This may trigger a series of events which could help to explain the delayed loss of neurons after a transient ischemic period.
A complementary DNA encoding the key subunit of the human N-methyl-D-aspartate (NMDA) receptor (NMDAR1) has been cloned using a probe derived from the rat NMDAR1 cDNA. The cDNA encodes a 938-amino acid protein, which shows 99% amino acid homology with the rat counterpart. Of the 7 of 938 amino acids which are different, three occur in the region of the signal peptide and the others in the extracellular amino-terminal domain preceding the 4 putative transmembrane segments. Expression in Xenopus oocytes demonstrated that the single protein encoded by the cloned cDNA possesses the electrophysiological and pharmacological properties characteristic of the NMDA receptor, including Ca2+ permeability, voltage-dependent Mg2+ block, and inhibition by selective antagonists such as Zn2+ and channel blockers. The high evolutionary conservation in the structure and properties of NMDAR1 argues strongly for the importance of this receptor in functions of glutamate neurotransmission. RNA blot analysis showed abundant expression of mRNA whose size is about 4.5 and 4.8 kilonucleotides. The human gene encoding the NMDAR1 subunit has been mapped to chromosome 9q34.3 by the analyses of blot hybridization of a DNA panel of human/hamster somatic cell hybrids and fluorescence in situ hybridization of human chromosomes.
The acetylcholine (ACh) receptors in muscle have the composition alpha(2) beta gamma delta and contain two ACh binding sites. One is formed between an alpha subunit and the gamma subunit, and the other is formed between an alpha subunit and the delta subunit, Among the residues in the ACh binding sites are alpha Cys-192 and alpha Cys-193. The negatively charged delta Asp-180 is at an appropriate distance from alpha Cys-192/193 also to be in the ACh binding site and to interact electrostatically with the positively charged ammonium group common to agonists and competitive antagonists. Mutation to Asn of either delta Asp-180 or the aligned residue in the gamma subunit, gamma Asp-174, decreased the affinities of three agonists, acetylcholine, tetramethylammonium, and succinyldicholine 170-560-fold. By contrast, these mutations decreased the affinities of three competitive antagonists, (+)-tubocurarine, hexamethonium, and dihydro-beta-erythroidine, only 2-15-fold. Agonists, but not antagonists, promote the transitions of the receptor from the resting state to the higher affinity active and desensitized states, and the greater effects of the mutations of gamma Asp-174 and delta Asp-180 on the apparent affinities of agonists could reflect the involvement of these residues in the conformational changes of the receptor corresponding to its transitions to higher affinity states. In these transitions, one possibility is that gamma Asp-174 and delta Asp-180 move closer to bound agonist.
The cellular mechanisms by which excess exposure to the excitatory neurotransmitter glutamate can produce neuronal injury are unknown. More than a decade ago it was hypothesized that glutamate neurotoxicity (GNT) is a direct consequence of excessive neuronal excitation (“excitotoxicity” hypothesis); more recently, it has been hypothesized that a Ca influx triggered by glutamate exposure might mediate GNT (Ca hypothesis). A basic test to discriminate between these hypotheses would be to determine the dependence of GNT on the extracellular ionic environment. The excitotoxicity hypothesis predicts that GNT should depend critically on the presence of extracellular Na, since that ion appears to mediate glutamate neuroexcitation in the CNS; the Ca hypothesis predicts that GNT should depend critically on the presence of extracellular Ca. The focus of the present experiments was to determine the effects of several alterations in the extracellular ionic environment upon the serial morphologic changes that occur after mouse neocortical neurons in cell culture receive toxic exposure to glutamate. The results suggest that GNT in cortical neurons can be separated into 2 components distinguishable on the basis of differences in time course and ionic dependence. The first component, marked by neuronal swelling, occurs early, is dependent on extracellular Na and Cl, can be mimicked by high K, and is thus possibly “excitotoxic.” The second component, marked by gradual neuronal disintegration, occurs late, is dependent on extracellular Ca, can be mimicked by A23187, and is thus possibly mediated by a transmembrane influx of Ca. While either component alone is ultimately capable of producing irreversible neuronal injury, the Ca-dependent mechanism predominates at lower exposures to glutamate. Glutamate exposure likely leads to a Ca influx both through glutamate-activated cation channels and through voltage- dependent Ca channels activated by membrane depolarization. Addition of 20 mM Mg, however, did not substantially block GNT; this finding, together with the observation that GNT is largely preserved in sodium- free solution, supports the notion that the activation of voltage- dependent Ca channels may not be required for lethal Ca entry. The possibility that N-methyl-D-aspartate receptors may play a dominant role in mediating glutamate-induced lethal Ca influx is discussed.
Antibodies were made to synthetic peptides corresponding to sequences specific to the glutamate receptor (GluR) subunits, GluR1-4. The specificity of the antibodies was established by Western blotting using membranes of simian kidney cells (COS-7) transfected with GluR subunit DNA. Four antibodies were found to be selective for each of the four GluR subunits, and a fifth antibody recognized both GluR2 and 3. All five antibodies immunoadsorbed Triton X-100-solubilized rat brain [H-3]AMPA binding activity and labeled an M(r) = 108,000 band in samples of rat brain. The structure of the Triton X-100-solubilized GluR was studied using subunit-specific antibodies covalently attached to protein A-agarose and analyzing GluR subunits bound to the antibodies by Western blotting. Each of the four subunit-specific antibodies immunoadsorbed its respective GluR subunit as well as the other three forms of GluR, showing that the detergent solubilized GluR exists as hetero-oligomers composed of two or more of the four subunits. Evidence supporting a similar structure for membrane bound GluR was obtained using synaptic membranes chemically cross-linked with dithiobis(succinimidylpropionate). GluR was immunoaffinity-purified using the GluR2 and 3-selective antibody. This antibody, covalently attached to protein A-agarose, adsorbed 55% of [H-3]AMPA binding activity, and after elution with 1 M KSCN, 22-37% of the binding activity was recovered. Analysis of the purified product showed a major immunoreactive band at M(r) = 108,000, and silver staining identified the same major band and no additional polypeptides. The GluR receptor complex, therefore, appears to be made up exclusively of GluR1-4. In the purified GluR preparation, in addition to the M(r) = 108,000 band, three higher molecular weight immunoreactive components were also detected. These bands migrated at M(r) = 325,000, 470,000, and 590,000. Similar sized proteins were seen in the cross-linked synaptic membrane sample, with the M(r) = 590,000 component being substantially enriched after cross-linking. The M(r) = 590,000 band is the largest component detected, and it has a size consistent with its being a pentamer of the M(r) = 108,000 protein.
The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical
events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound
conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that
p21ras is essential for NO-induced downstream signaling, such as NF-κB activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of NO in T cells and suggest that NO activates p21ras by an action which mimics that of guanine nucleotide exchange factors.
As progress in cell developmental biology carries on at a breakneck speed, new techniques constantly arise to plug the gaps left by traditional strategies. Cellular Interactions in Development provides detailed discussion and protocols of some of these new techniques, which allow the manipulation of developing organisms such as Drosophila or plants, when and where cells interact with each other to influence their development. The book looks at the really exciting innovations of the identification and functional test of molecules which control these cellular behaviours. The book also describes a number of new ways of hunting for these important proteins involved in cellular communication. A fully comprehensive manual which will prove indispensable to researchers in the fields of cell, developmental, and molecular biology.
Oxidant-mediated toxicity resulting from acute pulmonary inflammation has been demonstrated in acute lung injury. A potent biological oxidant, peroxynitrite, is formed by the near diffusion-limited reaction of nitric oxide with Superoxide. In addition to having hydroxyl radical-like oxidative reactivity, peroxynitrite is capable of nitrating phenolic rings, including protein-associated tyrosine residues. Nitric oxide does not directly nitrate tyrosine residues, therefore, demonstration of tissue nitrotyrosine residues infers the action of peroxynitrite or related nitrogen-centered oxidants. Lung tissue was obtained from formalin-fixed, paraffinembedded autopsy specimens, and specific polyclonal and monoclonal antibodies to nitrotyrosine were visualized by diaminobenzidene-peroxidase staining. Acute lung injury resulted in intense staining throughout the lung, including lung interstitium, alveolar epithelium, proteinaceous alveolar exudate, and inflammatory cells. In addition, staining of the vascular endothelium and subendothelial tissues was present in those patients with sepsis-induced acute lung injury. Antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. Reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. In control specimens with no overt pulmonary disease, there was only slight staining of the alveolar septum. These results demonstrate that nitrogen-derived oxidants are formed in human acute lung injury and suggest that peroxynitrite may be an important oxidant in inflammatory lung disease.
The proposal that nitric oxide (NO) or its reactant products mediate toxicity in brain remains controversial in part because of the use of nonselective agents that block NO formation in neuronal, glial, and vascular compartments. In mutant mice deficient in neuronal NO synthase (NOS) activity, infarct volumes decreased significantly 24 and 72 hours after middle cerebral artery occlusion, and the neurological deficits were less than those in normal mice. This result could not be accounted for by differences in blood flow or vascular anatomy. However, infarct size in the mutant became larger after endothelial NOS inhibition by nitro-L-arginine administration. Hence, neuronal NO production appears to exacerbate acute ischemic injury, whereas vascular NO protects after middle cerebral artery occlusion. The data emphasize the importance of developing selective inhibitors of the neuronal isoform.
The responses of vertebrate neurones to glutamate involve at least three receptor types. One of these, the NMDA receptor (so called because of its specific activation by N-methyl-D-aspartate), induces responses presenting a peculiar voltage sensitivity. Above resting potential, the current induced by a given dose of glutamate (or NMDA) increases when the cell is depolarized. This is contrary to what is observed at classical excitatory synapses, and recalls the properties of 'regenerative' systems like the Na+ conductance of the action potential. Indeed, recent studies of L-glutamate, L-aspartate and NMDA-induced currents have indicated that the current-voltage (I-V) relationship can show a region of 'negative conductance' and that the application of these agonists can lead to a regenerative depolarization. Furthermore, the NMDA response is greatly potentiated by reducing the extracellular Mg2+ concentration [( Mg2+]o) below the physiological level (approximately 1 mM). By analysing the responses of mouse central neurones to glutamate using the patch-clamp technique, we have now found a link between voltage sensitivity and Mg2+ sensitivity. In Mg2+-free solutions, L-glutamate, L-aspartate and NMDA open cation channels, the properties of which are voltage independent. In the presence of Mg2+, the single-channel currents measured at resting potential are chopped in bursts and the probability of opening of the channels is reduced. Both effects increase steeply with hyperpolarization, thereby accounting for the negative slope of the I-V relationship of the glutamate response. Thus, the voltage dependence of the NMDA receptor-linked conductance appears to be a consequence of the voltage dependence of the Mg2+ block and its interpretation does not require the implication of an intramembrane voltage-dependent 'gate'.
There is an increasing amount of experimental evidence that oxidative stress is a causal, or at least an ancillary, factor in the neuropathology of several adult neurodegenerative disorders, as well as in stroke, trauma, and seizures. At the same time, excessive or persistent activation of glutamate-gated ion channels may cause neuronal degeneration in these same conditions. Glutamate and related acidic amino acids are thought to be the major excitatory neurotransmitters in brain and may be utilized by 40 percent of the synapses. Thus, two broad mechanisms--oxidative stress and excessive activation of glutamate receptors--are converging and represent sequential as well as interacting processes that provide a final common pathway for cell vulnerability in the brain. The broad distribution in brain of the processes regulating oxidative stress and mediating glutamatergic neurotransmission may explain the wide range of disorders in which both have been implicated. Yet differential expression of components of the processes in particular neuronal systems may account for selective neurodegeneration in certain disorders.
A new fluorescence method using the dye FM1-43 was used to examine exocytotic release from hippocampal synaptosomes. Nitric oxide caused a marked transient stimulation of vesicle release. Several structurally unrelated nitric oxide donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, 3-morpholino-sydnonimine, and acidified sodium nitrite, were effective. Release stimulated by nitric oxide and KCI were comparable in time course, using both the fluorescence assay and [3H]l-glutamate to monitor neurotransmitter release. Activation of guanylyl cyclase was not responsible for nitric oxide-stimulated release. Unlike release stimulated by KCl or A23187, nitric oxide-stimulated release was found to be independent of a rise in intrasynaptosomal Ca2+. Indo-1/AM measurements indicated that nitric oxide actually decreased intracellular Ca2+, and the Ca2+ channel blocker Cd2+ did not affect nitric oxide-stimulated vesicle release. Nitric oxide does, however, appear to act on the Ca2+-sensitive pool of vesicles. Nitric oxide may be the first physiological mediator that induces vesicle exocytosis without raising Ca2+ and may provide an interesting new tool for the study of molecules involved in vesicle exocytosis.
1) High concentration of sodium glutamate (as well as sodium aspartate) which is applied into grey matters of motor cortex in dogs, monkeys and men, generates clonic convulsions with very short latent periods.2) Small dose of sodium glutamate which is introduced into circulation in dogs improves the differentiation of conditioned salivary reflexes during 20 minutes after its administration, and continues its action for some hours.3) The above two aspects of the effect of sodium glutamate must be attributable to the direct physiological action of the chemicals on the central nervoos system in higher animals.
In rat brain, the cellular localization of a phosphoinositide-linked metabotropic glutamate receptor (mGluR1 alpha) was demonstrated using antibodies that recognize the C-terminus of the receptor. mGluR1 alpha, a 142 kd protein, is enriched within the olfactory bulb, stratum oriens of CA1 and polymorph layer of dentate gyrus in hippocampus, globus pallidus, thalamus, substantia nigra, superior colliculus, and cerebellum. Lower levels of mGluR1 alpha are present within neocortex, striatum, amygdala, hypothalamus, and medulla. Dendrites, spines, and neuronal cell bodies contain mGluR1 alpha. mGluR1 alpha is not detectable in presynaptic terminals. mGluR1 alpha and ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits show differential distributions, but in Purkinje cells, mGluR1 alpha and specific AMPA receptor subunits colocalize. The postsynaptic distribution of mGluR1 alpha is consistent with postulated physiological roles of this subtype of glutamate receptor.
Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.
We have examined glutamate receptor desensitization in voltage-clamped embryonic chicken spinal cord neurons and postnatal rat hippocampal neurons maintained in culture. Rapid currents that rose in 0.8-3.6 msec were evoked when glutamate was ionophoresed with 0.5- to 1.0-msec pulses. With prolonged pulses or brief, repetitive pulses, glutamate-evoked currents decayed rapidly in a manner that was independent of holding potential. A similar desensitization occurred following close-range pressure ejection of glutamate. The rapid, desensitizing glutamate current exhibited a linear current-voltage relation and it was not blocked by 2-amino-5-phosphonovalerate, suggesting that it was mediated by N-methyl-D-aspartate-insensitive (G2) receptors. Desensitization of G2 receptors may be agonist-dependent: currents evoked by kainate, a selective G2 agonist, did not decay, whereas prior application of glutamate did reduce the size of kainate responses. The appearance of the rapid current depended critically on the position of the ionophoretic pipette. Such glutamate-receptor "hot spots" often corresponded to points of contact with neighboring neurites, which raises the possibility that they are located at synapses.
The cerebroprotective effects of noncompetitive N-methyl-D-aspartate (NMDA) antagonists are well documented in animal focal ischemia models when the agents are administered prior to or just after ischemia begins. However, studies on the delayed administration of these drugs beyond the very acute stage of infarction are few and the results have varied. We assessed the extent of infarction and neurological outcome in 28 rats treated with either CNS-1102 (n = 14), a noncompetitive NMDA antagonist, or saline vehicle (n = 14), 1 h after ischemic onset using an intraluminal occlusion model of stroke. CNS-1102 significantly reduced lesion volume as assessed by 2,3,5,-triphenyltetrazolium chloride staining at 24 h compared to saline-treated rats (120 ± 30 vs. 251 ± 20 mm3, mean ± SEM; p < 0.001). The neurological outcome at 24 h was also significantly better in the CNS-1102-treated animals (p < 0.04). The extent of the infarction was correlated with neurological outcome (p < 0.01); larger lesions were associated with a poorer outcome. The results indicate that the time window for treatment with CNS-1102 is at least 1 h in this rat stroke model. The potential clinical efficacy of this compound is apparent but utility will depend, in part, on its side effect profile.Copyright © 1994 S. Karger AG, Basel
Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'14C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [14C]succinate was rapid, as shown by the incorporation of 14C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [14C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-14C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points.
Incorporation of 14C into GABA was very low (< 1 per cent of the amino acid -14C) after the injection of [1.4-14C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-14C]succinate 2 min beforehand. Since the oxidation of [l,4-14C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce 14C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD+ ratios and [14C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.
Endothelium-derived relaxing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhydryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism of NO and raise the possibility of an additional type of cellular regulatory mechanism.
