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1.
Int J Mol Sci ; 20(18)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546771

RESUMO

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aß oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas PrPC/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doença de Alzheimer/patologia , Animais , Ácido Glutâmico/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Glutamato/metabolismo
2.
Int J Mol Sci ; 20(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500132

RESUMO

Parkinson's disease is a progressive neurodegenerative disorder resulting from the degeneration of pigmented dopaminergic neurons in the substantia nigra pars compacta. It induces a series of functional modifications in the circuitry of the basal ganglia nuclei and leads to severe motor disturbances. The amino acid glutamate, as an excitatory neurotransmitter, plays a key role in the disruption of normal basal ganglia function regulated through the interaction with its receptor proteins. It has been proven that glutamate receptors participate in the modulation of neuronal excitability, transmitter release, and long-term synaptic plasticity, in addition to being related to the altered neurotransmission in Parkinson's disease. Therefore, they are considered new targets for improving the therapeutic strategies used to treat Parkinson's disease. In this review, we discuss the biological characteristics of these receptors and demonstrate the receptor-mediated neuroprotection in Parkinson's disease. Pharmacological manipulation of these receptors during anti-Parkinsonian processes in both experimental studies and clinical trials are also summarized.


Assuntos
Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Receptores de Glutamato/metabolismo , Animais , Ensaios Clínicos como Assunto , Descoberta de Drogas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Humanos , Terapia de Alvo Molecular , Neurotransmissores/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Receptores de Glutamato/genética , Transdução de Sinais/efeitos dos fármacos , Substância Negra/metabolismo , Transmissão Sináptica , Resultado do Tratamento
3.
Brain Struct Funct ; 224(8): 2733-2756, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31392403

RESUMO

The macaque monkey superior parietal lobule (SPL) is part of a neuronal network involved in the integration of information from visual and somatosensory cortical areas for execution of reaching and grasping movements. We applied quantitative in vitro receptor autoradiography to analyse the distribution patterns of 15 different receptors for glutamate, GABA, acetylcholine, serotonin, dopamine, and adenosine in the SPL of three adult male Macaca fascicularis monkeys. For each area, mean (averaged over all cortical layers) receptor densities were visualized as a receptor fingerprint of that area. Multivariate analyses were conducted to detect clusters of areas according to the degree of (dis)similarity of their receptor organization. Differences in regional and laminar receptor distributions confirm the location and extent of areas V6, V6Av, V6Ad, PEc, PEci, and PGm as found in cytoarchitectonic and functional studies, but also enable the definition of three subdivisions within area PE. Receptor densities are higher in supra- than in infragranular layers, with the exception of kainate, M2, and adenosine receptors. Glutamate and GABAergic receptors are the most expressed in all areas analysed. Hierarchical cluster analyses demonstrate that SPL areas are organized in two groups, an organization that corresponds to the visual or sensory-motor characteristics of those areas. Finally, based on present results and in the framework of our current understanding of the structural and functional organization of the primate SPL, we propose a novel pattern of homologies between human and macaque SPL areas.


Assuntos
Neurônios/citologia , Neurônios/metabolismo , Lobo Parietal/citologia , Lobo Parietal/metabolismo , Receptores de Neurotransmissores/metabolismo , Animais , Autorradiografia , Macaca fascicularis , Masculino , Receptores Colinérgicos/metabolismo , Receptores Dopaminérgicos/metabolismo , Receptores de GABA/metabolismo , Receptores de Glutamato/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores de Serotonina/metabolismo
4.
Nat Commun ; 10(1): 3622, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399584

RESUMO

Caspase-2 is the most evolutionarily conserved member in the caspase family of proteases and is constitutively expressed in most cell types including neurons; however, its physiological function remains largely unknown. Here we report that caspase-2 plays a critical role in synaptic plasticity and cognitive flexibility. We found that caspase-2 deficiency led to deficits in dendritic spine pruning, internalization of AMPA receptors and long-term depression. Our results indicate that caspase-2 degrades Rictor, a key mTOR complex 2 (mTORC2) component, to inhibit Akt activation, which leads to enhancement of the GSK3ß activity and thereby long-term depression. Furthermore, we found that mice lacking caspase-2 displayed elevated levels of anxiety, impairment in reversal water maze learning, and little memory loss over time. These results not only uncover a caspase-2-mTORC2-Akt-GSK3ß signaling pathway, but also suggest that caspase-2 is important for memory erasing and normal behaviors by regulating synaptic number and transmission.


Assuntos
Caspase 2/metabolismo , Cognição/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de AMPA/metabolismo , Transdução de Sinais/fisiologia , Animais , Ansiedade , Comportamento Animal , Proteínas de Transporte/metabolismo , Caspase 2/genética , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/genética , Depressão Sináptica de Longo Prazo/fisiologia , Masculino , Aprendizagem em Labirinto , Transtornos da Memória/metabolismo , Camundongos , Camundongos Knockout , Plasticidade Neuronal , Neurônios/metabolismo , Receptores de Glutamato/metabolismo
5.
Methods Enzymol ; 622: 411-430, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155063

RESUMO

Investigating functions of membrane-bound receptors provides invaluable information about cellular signaling and physiological events. Recently, chemical genetic methods to design chemical switches on the target proteins have intensely been developed for interrogation of the cellular signaling of individual receptor proteins. We recently reported coordination chemistry-based chemogenetics to allosterically activate two types of neurotransmitter receptors, ionotropic and metabotropic glutamate receptors, in living cells. Based on their well-studied structure-activity relationships, we semi-rationally incorporated two His mutations into glutamate receptors near ligand binding pockets as an allosteric site. The engineered glutamate receptors could be allosterically activated upon treatment of Pd(bpy) complex (bpy: 2,2'-bipyridine) through stabilization of the activated conformation in mammalian cells and cultured neurons. Here, we describe the detailed protocol of our approach including the receptor design and activation of the His-engineered receptors and the downstream of the signal transduction cascade in living cells.


Assuntos
Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Imagem Óptica/métodos , Engenharia de Proteínas/métodos , Ratos , Receptores de Glutamato/química , Receptores de Glutamato/genética
6.
Food Chem ; 293: 263-270, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151610

RESUMO

Gray mold caused by Botrytis cinerea is the most important disease in postharvest tomato fruit. Inducing resistance to fungal pathogens in the harvested fruit and vegetable is a promising approach to control postharvest losses. In the present study, the effect of l-glutamate on induction of resistance to B. cinerea and the underlying mechanisms were investigated. The results indicated that l-glutamate at 100 ppm was effective in reducing the gray mold of tomatoes after inoculation of the pathogen. Gene expressions of nine glutamate receptors, four pathogenesis-related proteins and the content of amino acids were affected by l-glutamate treatment. Furthermore, the metabolites of l-glutamate, including GABA, Met, Lys and Arg, could also induce significant resistance against B. cinerea in tomato fruit. Our findings suggested that l-glutamate treatment may represent a promising method for managing postharvest decay of tomato fruit.


Assuntos
Aminoácidos/metabolismo , Botrytis/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Lycopersicon esculentum/imunologia , Micoses/prevenção & controle , Doenças das Plantas/prevenção & controle , Receptores de Glutamato/metabolismo , Resistência à Doença , Frutas/química , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Micoses/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Regulação para Cima/efeitos dos fármacos
7.
Ecotoxicology ; 28(6): 650-657, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31197614

RESUMO

Glutamate receptors (GLRs) are ligand-gated Ca2+-permeable channels that govern and modulate the dynamic influx of cytosolic Ca2+ in plants. The present study investigated the interaction of OsGLR3 gene expression with subcellular Ca distribution in rice seedlings exposed to chromium (Cr) solution containing Cr(III) or Cr(VI). The results displayed that the accumulation of Ca was evaluated or higher in shoots compared to roots under Cr exposure, and a similar pattern of subcellular Ca distribution was observed between rice tissues exposed to Cr(III) and Cr(VI). Real-time quantitative polymerase chain reaction (qRT-PCR) analysis revealed that eight OsGLR3 isogenes were distinctly expressed in different rice tissues at different levels of Cr exposures. This differential expressions could possible be due to the uptake variations, subcellular distribution and chemical speciation of the two Cr species. Notably, distinct expression patterns of OsGLR3 genes were found between Cr(III) and Cr(VI) exposures, suggesting that different regulation strategies are used to mediate Ca influx in rice materials under different Cr exposures. These results demonstrated a full picture of Cr-induced transcriptional alterations in OsGLR3 expression levels in rice seedlings, and provided suggestive evidence for further investigation on specific OsGLR3 genes participated in the regulation of cytosolic Ca2+ concentrations under Cr exposure.


Assuntos
Cálcio/metabolismo , Cromo/efeitos adversos , Oryza/genética , Proteínas de Plantas/genética , Receptores de Glutamato/genética , Poluentes do Solo/efeitos adversos , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Glutamato/metabolismo , Plântula/genética , Plântula/metabolismo
8.
Cell Mol Neurobiol ; 39(7): 1039-1049, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31197744

RESUMO

Serotonin (5-HT) has been recognized as a neurotransmitter in the vertebrate retina, restricted mainly to amacrine and bipolar cells. It is involved with synaptic processing and possibly as a mitogenic factor. We confirm that chick retina amacrine and bipolar cells are, respectively, heavily and faintly immunolabeled for 5-HT. Amacrine serotonergic cells also co-express tyrosine hydroxylase (TH), a marker of dopaminergic cells in the retina. Previous reports demonstrated that serotonin transport can be modulated by neurotransmitter receptor activation. As 5-HT is diffusely released as a neuromodulator and co-localized with other transmitters, we evaluated if 5-HT uptake or release is modulated by several mediators in the avian retina. The role of different glutamate receptors on serotonin transport and release in vitro and in vivo was also studied. We show that L-glutamate induces an inhibitory effect on [3H]5-HT uptake and this effect was specific to kainate receptor activation. Kainate-induced decrease in [3H]5-HT uptake was blocked by CNQX, an AMPA/kainate receptor antagonist, but not by MK-801, a NMDA receptor antagonist. [3H]5-HT uptake was not observed in the presence of AMPA, thus suggesting that the decrease in serotonin uptake is mediated by kainate. 5-HT (10-50 µM) had no intrinsic activity in raising intracellular Ca2+, but addition of 10 µM 5-HT decreased Ca2+ shifts induced by KCl in retinal neurons. Moreover, kainate decreased the number of bipolar and amacrine cells labeled to serotonin in chick retina. In conclusion, our data suggest a highly selective effect of kainate receptors in the regulation of serotonin functions in the retinal cells.


Assuntos
Ácido Caínico/farmacologia , Retina/metabolismo , Serotonina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Embrião de Galinha , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Neurotransmissores/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Ácido Caínico/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Retina/embriologia , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/metabolismo , Trítio/metabolismo
9.
Int J Mol Sci ; 20(12)2019 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-31208140

RESUMO

Nicotine causes tobacco dependence, which may result in fatal respiratory diseases. The striatum is a key structure of forebrain basal nuclei associated with nicotine dependence. In the striatum, glutamate release is increased when α7 nicotinic acetylcholine receptors expressed in the glutamatergic terminals are exposed to nicotine, and over-stimulates glutamate receptors in gamma amino-butyric acid (GABA)ergic neurons. These receptor over-stimulations in turn potentiate GABAergic outputs to forebrain basal nuclei and contribute to the increase in psychomotor behaviors associated with nicotine dependence. In parallel with glutamate increases, nicotine exposure elevates brain-derived neurotrophic factor (BDNF) release through anterograde and retrograde targeting of the synapses of glutamatergic terminals and GABAergic neurons. This article reviews nicotine-exposure induced elevations of glutamatergic neurotransmission, the bidirectional targeting of BDNF in the striatum, and the potential regulatory role played by BDNF in behavioral responses to nicotine exposure.


Assuntos
Comportamento , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/fisiologia , Nicotina/administração & dosagem , Transmissão Sináptica , Animais , Ácido Glutâmico/biossíntese , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Fatores de Crescimento Neural/metabolismo , Receptor trkB/metabolismo , Receptores de Glutamato/metabolismo , Transdução de Sinais , Receptor Nicotínico de Acetilcolina alfa7/agonistas
10.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075835

RESUMO

Neuronal calcium (Ca2+) influx has long been ascribed mainly to voltage-gated Ca2+ channels and glutamate receptor channels. Recent research has shown that it is also complemented by stromal interaction molecule (STIM) protein-mediated store-operated Ca2+ entry (SOCE). SOCE is described as Ca2+ flow into cells in response to the depletion of endoplasmic reticulum Ca2+ stores. The present review summarizes recent studies that indicate a relationship between neuronal SOCE that is mediated by STIM1 and STIM2 proteins and glutamate receptors under both physiological and pathological conditions, such as neurodegenerative disorders. We present evidence that the dysregulation of neuronal SOCE and glutamate receptor activity are hallmarks of acute neurodegenerative diseases (e.g., traumatic brain injury and cerebral ischemia) and chronic neurodegenerative diseases (e.g., Alzheimer's disease and Huntington's disease). Emerging evidence indicates a role for STIM proteins and glutamate receptors in neuronal physiology and pathology, making them potential therapeutic targets.


Assuntos
Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Animais , Ácido Glutâmico/metabolismo , Humanos , Modelos Biológicos , Molécula 1 de Interação Estromal/metabolismo
11.
Eur J Pharmacol ; 855: 149-159, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31063776

RESUMO

Levodopa remains to be the mainstay for treatment of Parkinson disease (PD). Long-term levodopa treatment bears a risk for developing levodopa-induced dyskinesia (LID). LID significantly overshadows patients' quality of life and therapeutic efficacy of levodopa. Pre- and post-synaptic changes in dopamine secretion and signaling, along with altered glutamate receptor expression and glutamatergic signaling in striatal neurons, and the resulting disinhibition-like changes in the corticostriatal circuitry, lead to aberrant activity of motor cortex and formation of LID. Research has highlighted the role of group I metabotropic glutamate receptors especially the metabotropic glutamate receptor 5 (mGlu5) in formation of LID through potentiating of ionotropic glutamate NMDA receptors and dopamine D1/D5 receptors in direct pathway. Accordingly, MTEP and MPEP were the first mGlu5 receptor antagonists which were shown to attenuate LID in animal models through suppression of downstream signaling cascades involving mitogen-activated protein kinase (MAPK) and FosB/delta FosB activation, as well as modulation of prodynorphinegic, preproenkephalinergic, and GABA-ergic neurotransmission systems. Beneficial effects of other mGlu5 receptor antagonists such as AFQ056/mavoglurant and ADX48621/dipraglurant in amelioration of LID has been shown not only in animal models but also in clinical trials. Considering the presence of mGlu receptor dysregulation in rapid eye movement (REM) sleep behavior disorder and depression, which are prodromal signs of PD, along with the neuroprotective effects of mGlu receptor antagonists, and their cognitive benefits, potential effectiveness of mGlu receptor antagonists in early prevention of PD remains to be investigated.


Assuntos
Discinesia Induzida por Medicamentos/tratamento farmacológico , Discinesia Induzida por Medicamentos/fisiopatologia , Levodopa/efeitos adversos , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Animais , Discinesia Induzida por Medicamentos/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Receptores de Glutamato/metabolismo
12.
Int J Mol Sci ; 20(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30978987

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive malignant tumor of the central nervous system, with poor survival in both treated and untreated patients. Recent studies began to explain the molecular pathway, comprising the dynamic structural and mechanical changes involved in GBM. In this context, some studies showed that the human glioblastoma cells release high levels of glutamate, which regulates the proliferation and survival of neuronal progenitor cells. Considering that cancer cells possess properties in common with neural progenitor cells, it is likely that the functions of glutamate receptors may affect the growth of cancer cells and, therefore, open the road to new and more targeted therapies.


Assuntos
Neoplasias do Sistema Nervoso Central/patologia , Glioblastoma/patologia , Invasividade Neoplásica/patologia , Receptores de Glutamato/metabolismo , Animais , Fenômenos Biomecânicos , Movimento Celular , Neoplasias do Sistema Nervoso Central/metabolismo , Glioblastoma/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Canais Iônicos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais
13.
Mol Autism ; 10: 19, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31011411

RESUMO

Background: Autism spectrum disorders (ASD) affect around 1.5% of people worldwide. Symptoms start around age 2, when children fail to maintain eye contact and to develop speech and other forms of communication. Disturbances in glutamatergic and GABAergic signaling that lead to synaptic changes and alter the balance between excitation and inhibition in the developing brain are consistently found in ASD. One of the hallmarks of these disorders is hypersensitivity to sensory stimuli; however, little is known about its underlying causes. Since the retina is the part of the CNS that converts light into a neuronal signal, we set out to study how it is affected in adolescent mice prenatally exposed to valproic acid (VPA), a useful tool to study ASD endophenotypes. Methods: Pregnant female mice received VPA (600 mg/kg, ip) or saline at gestational day 11. Their male adolescent pups (P29-35) were behaviorally tested for anxiety and social interaction. Proteins known to be related with ASD were quantified and visualized in their retinas by immunoassays, and retinal function was assessed by full-field scotopic electroretinograms (ERGs). Results: Early adolescent mice prenatally exposed to VPA displayed impaired social interest and increased anxiety-like behaviors consistent with an ASD phenotype. The expression of GABA, GAD, synapsin-1, and FMRP proteins were reduced in their retinas, while mGluR5 was increased. The a-wave amplitudes of VPA-exposed were smaller than those of CTR animals, whereas the b-wave and oscillatory potentials were normal. Conclusions: This study establishes that adolescent male mice of the VPA-induced ASD model have alterations in retinal function and protein expression compatible with those found in several brain areas of other autism models. These results support the view that synaptic disturbances with excitatory/inhibitory imbalance early in life are associated with ASD and point to the retina as a window to understand their subjacent mechanisms.


Assuntos
Transtorno do Espectro Autista/fisiopatologia , Retina/metabolismo , Potenciais de Ação , Animais , Transtorno do Espectro Autista/etiologia , Transtorno do Espectro Autista/metabolismo , Endofenótipos , Feminino , Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Retina/fisiopatologia , Comportamento Social , Sinapsinas/genética , Sinapsinas/metabolismo , Ácido Valproico/toxicidade
14.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897826

RESUMO

The appropriate display of social behavior is critical for the well-being and survival of an individual. In many psychiatric disorders, including social anxiety disorder, autism spectrum disorders, depression and schizophrenia social behavior is severely impaired. Selective targeting of metabotropic glutamate receptors (mGluRs) has emerged as a novel treatment strategy for these disorders. In this review, we describe some of the behavioral paradigms used to assess different types of social behavior, such as social interaction, social memory, aggressive behavior and sexual behavior. We then focus on the effects of pharmacological modulation of mGluR1-8 on these types of social behavior. Indeed, accumulating evidence indicates beneficial effects of selective ligands of specific mGluRs in ameliorating innate or pharmacologically-induced deficits in social interaction and social memory as well as in reducing aggression in rodents. We emphasize the importance of future studies investigating the role of selective mGluR ligands on different types of social behavior to provide a better understanding of the neural mechanisms involved which, in turn, might promote the development of selective mGluR-targeted tools for the improved treatment of psychiatric disorders associated with social deficits.


Assuntos
Receptores de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Agressão/fisiologia , Animais , Ansiedade/fisiopatologia , Humanos , Roedores , Comportamento Social
15.
J Plant Physiol ; 236: 1-6, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30836205

RESUMO

Early stages of plant development are highly susceptible to environmental cues, and seedlings have to develop sophisticated mechanisms to sense and respond to abiotic stresses. We have previously identified that abscisic acid (ABA), nitric oxide (NO) and modulation of nitrogen metabolism are involved in adaptive responses in Medicago truncatula seedlings under water deficit stress. Here, we investigated whether glutamate receptor-like channels (GLRs) played a role in the developmental physiological processes of Medicago seedlings during post-germination after a short-term water deficit stress. Twenty-nine independent MtGLR genes have been identified and then divided into four clades following a phylogenetic analysis; seventeen of them exhibited specific domains which are characteristic of animal ionotropic glutamate receptors. Under drought stress, ABA-induced NO accumulation was significantly reduced in presence of a GLR competitive antagonist, suggesting that this water deficit-induced endogenous NO production was mediated through a MtGLR-dependent pathway. Water deficit-induced inhibition of embryo axis elongation was strongly reduced whereas loss of water content was alleviated when MtGLRs were inhibited. These results suggest that glutamate receptors-like channels are required, through their involvement in NO production, in adaptive responses under short-term water-deficit stress during Medicago seedling establishment.


Assuntos
Medicago truncatula/metabolismo , Óxido Nítrico/biossíntese , Proteínas de Plantas/metabolismo , Receptores de Glutamato/metabolismo , Desidratação , Genes de Plantas/genética , Genes de Plantas/fisiologia , Germinação , Medicago truncatula/genética , Medicago truncatula/fisiologia , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Receptores de Glutamato/genética , Receptores de Glutamato/fisiologia , Plântula/metabolismo , Alinhamento de Sequência
16.
Neuropharmacology ; 149: 35-44, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30731135

RESUMO

Ketamine is a fast acting experimental antidepressant with significant therapeutic potential for emotional disorders such as major depressive disorder and alcohol use disorders. Of particular interest is binge alcohol use, which during intermittent withdrawal from drinking involves depressive-like symptoms reminiscent of major depressive disorder. Binge drinking has been successfully modeled in mice with the Drinking in the Dark (DID) paradigm, which involves daily access to 20% ethanol, for a limited duration and selectively during the dark phase of the circadian light cycle. Here we demonstrate that DID exposure reduces the cell surface expression of NMDA- and AMPA-type glutamate receptors in the prelimbic cortex (PLC) of female but not male mice, along with reduced activity of the mammalian target of rapamycin (mTOR) signaling pathway. Pretreatment with an acute subanesthetic dose of ketamine suppresses binge-like ethanol consumption in female but not male mice. Lastly, DID-exposure reduces spontaneous glutamatergic synaptic transmission in the PLC of both sexes, but synaptic transmission is rescued by ketamine selectively in female mice. Thus, ketamine may have therapeutic potential as an ethanol binge suppressing agent selectively in female subjects.


Assuntos
Bebedeira/metabolismo , Bebedeira/terapia , Ácido Glutâmico/metabolismo , Ketamina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Córtex Pré-Frontal , Receptores de Glutamato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Serina-Treonina Quinases TOR/metabolismo
17.
Methods Mol Biol ; 1941: 3-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707423

RESUMO

Single-molecule fluorescence energy transfer methods allow us to determine the complete structural landscape between the donor and acceptor fluorophores introduced on the protein of interest. This method is particularly attractive to study ion channel proteins as single-molecule current recordings have been used to study the function of these proteins for several decades. Here we describe the smFRET method used to study glutamate receptors.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Receptores de Glutamato/metabolismo , Imagem Individual de Molécula/métodos , Fluorescência , Humanos
18.
Methods Mol Biol ; 1941: 17-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707424

RESUMO

A thorough understanding of the synaptic ultrastructure is necessary to bridge our current knowledge gap about the relationship between neuronal structure and function. Recent development of focused ion beam scanning electron microscopy (FIB/SEM) has made it possible to image neuronal structures with high speed and efficiency. Here, we present our routine protocol for correlative two-photon microscopy and FIB/SEM imaging of glutamatergic synapses. Femtosecond-pulsed near-infrared laser was used to create fiducial marks around the dendrite of interest in aldehyde-fixed tissues. Thereafter, samples were subjected to en bloc staining with rOTO (reduced osmium tetroxide-thiocarbohydrazide-osmium tetroxide), followed by lead aspartate and uranyl acetate to enhance tissue contrast. Reliable detection of postsynaptic density (PSD) and plasma membrane contours by the sample preparation protocol optimized for FIB/SEM allows us to precisely evaluate morphological features that shape glutamatergic synaptic transmission.


Assuntos
Espinhas Dendríticas/ultraestrutura , Ácido Glutâmico/metabolismo , Microscopia Eletrônica de Varredura/métodos , Receptores de Glutamato/metabolismo , Sinapses/ultraestrutura , Animais , Espinhas Dendríticas/metabolismo , Sinapses/metabolismo , Fixação de Tecidos/métodos
19.
Methods Mol Biol ; 1941: 47-54, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707426

RESUMO

Glutamate receptors (GluRs) located primarily on the membranes of neurons and glial cells are responsible for excitatory synaptic transmission in the central nervous system. The transport of GluRs to the cell surface is a highly regulated dynamic process that determines neuronal excitability and synaptic responses. The molecular and cellular mechanisms of GluR trafficking are often studied in cell cultures. These studies require sensitive techniques that allow the measurement of total and surface-expressed GluRs in cell populations. The cell-based enzyme-linked immunosorbent assay (cell-ELISA) combines steps of direct immunochemical labelling of cell cultures and ELISA. It can be used for quantitative comparisons of surface-expressed and total protein contents of various cell cultures. While several cell-ELISA protocols are available for different cell types, in this chapter we describe the procedure that we have applied for the investigation of quantitative changes in the cell surface expression of recombinant ionotropic glutamate receptors (iGluRs) in adherent human embryonic kidney 293 (HEK293) cells and endogenous iGluR proteins in primary neuronal cultures.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Proteínas Recombinantes/metabolismo , Transmissão Sináptica , Células HEK293 , Humanos
20.
Methods Mol Biol ; 1941: 57-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707427

RESUMO

Organotypic slice cultures enable the study of glutamate receptors in an environment closely related to the in vivo situation but with easy access to genetic manipulation of the receptors and regulatory signaling cascades as well as a more precise pharmacological intervention. We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes, and culture medium is allowed to form an interface. This method preserves the functionality and gross architecture of the hippocampus for up to 2 weeks in culture.


Assuntos
Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Técnicas de Cultura de Órgãos/métodos , Receptores de Glutamato/metabolismo , Animais
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