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1.
Life Sci ; 233: 116729, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31386876

RESUMO

AIMS: Glutamatergic receptors are important targets of ethanol. Intake of ethanol may produce analgesic effects. The present study examined the effects of ethanol on the activity of ionotropic glutamate receptors in spinal cord substantia gelatinosa (SG) neurons, critical neurons involved in nociceptive transmission. MAIN METHODS: Whole-cell recordings were made from SG neurons of the lumbar spinal cord slices from 15 to 20-day-old rats. Ethanol and glutamate receptor agonists or antagonists were applied by superfusion. KEY FINDING: Ethanol (50 and 100 mM) applied by superfusion for 5 min dose-dependently decreased the amplitude of evoked excitatory postsynaptic potential in SG neurons. Superfusion of ethanol (100 mM) for 15 min consistently inhibited NMDA- or AMPA-induced depolarizations in SG neurons. Ethanol (100 mM) also inhibited the depolarizations induced by glutamate. However, ethanol inhibition of glutamate-induced responses significantly decreased at 10-15 min following continuous superfusion, suggesting the development of acute tolerance to the inhibition during prolonged exposure. Application of MPEP hydrochloride (an antagonist of metabotropic glutamate receptor [mGluR] 5) or GF109203X (a protein kinase C [PKC] inhibitor), together with ethanol significantly blocked the tolerance. The inhibition by ethanol of the NMDA-induced, but not AMPA-induced, depolarizations significantly decreased at 15 min during continuous superfusion while ACPD (a mGluR agonist) was co-applied with ethanol. SIGNIFICANCE: The results suggest that (1) ethanol exposure may inhibit ionotropic glutamate receptor-mediated neurotransmission; (2) regulation of NMDA receptor function by mGluR5/PKC pathways may be involved in the development of the tolerance to ethanol inhibition of glutamate-induced responses during prolonged exposure in SG neurons.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Neurônios/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Substância Gelatinosa/metabolismo , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores , Potenciais da Membrana , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Substância Gelatinosa/citologia , Substância Gelatinosa/efeitos dos fármacos , Transmissão Sináptica
2.
Neuron ; 103(4): 617-626.e6, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31257103

RESUMO

The autism-associated synaptic-adhesion gene Neuroligin-4 (NLGN4) is poorly conserved evolutionarily, limiting conclusions from Nlgn4 mouse models for human cells. Here, we show that the cellular and subcellular expression of human and murine Neuroligin-4 differ, with human Neuroligin-4 primarily expressed in cerebral cortex and localized to excitatory synapses. Overexpression of NLGN4 in human embryonic stem cell-derived neurons resulted in an increase in excitatory synapse numbers but a remarkable decrease in synaptic strength. Human neurons carrying the syndromic autism mutation NLGN4-R704C also formed more excitatory synapses but with increased functional synaptic transmission due to a postsynaptic mechanism, while genetic loss of NLGN4 did not significantly affect synapses in the human neurons analyzed. Thus, the NLGN4-R704C mutation represents a change-of-function mutation. Our work reveals contrasting roles of NLGN4 in human and mouse neurons, suggesting that human evolution has impacted even fundamental cell biological processes generally assumed to be highly conserved.


Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Transtorno Autístico/genética , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Córtex Cerebral/fisiologia , Células-Tronco Embrionárias/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Genes Reporter , Ácido Glutâmico/fisiologia , Humanos , Camundongos , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Mutação de Sentido Incorreto , Neurogênese , Neurônios/fisiologia , Fenótipo , Receptores de Glutamato/fisiologia , Especificidade da Espécie , Sinapses/química
3.
Neuron ; 103(3): 432-444.e3, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31221559

RESUMO

Subtypes of nucleus accumbens medium spiny neurons (MSNs) promote dichotomous outcomes in motivated behaviors. However, recent reports indicate enhancing activity of either nucleus accumbens (NAc) core MSN subtype augments reward, suggesting coincident MSN activity may underlie this outcome. Here, we report a collateral excitation mechanism in which high-frequency, NAc core dopamine 1 (D1)-MSN activation causes long-lasting potentiation of excitatory transmission (LLP) on dopamine receptor 2 (D2)-MSNs. Our mechanistic investigation demonstrates that this form of plasticity requires release of the excitatory peptide substance P from D1-MSNs and robust cholinergic interneuron activation through neurokinin receptor stimulation. We also reveal that D2-MSN LLP requires muscarinic 1 receptor activation, intracellular calcium signaling, and GluR2-lacking AMPAR insertion. This study uncovers a mechanism for shaping NAc core activity through the transfer of excitatory information from D1-MSNs to D2-MSNs and may provide a means for altering goal-directed behavior through coordinated MSN activity.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Potenciação de Longa Duração/fisiologia , Núcleo Accumbens/fisiologia , Substância P/metabolismo , Potenciais de Ação/fisiologia , Animais , Aprepitanto/farmacologia , Sinalização do Cálcio/fisiologia , Neurônios Colinérgicos/fisiologia , Neurônios Dopaminérgicos/efeitos da radiação , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Interneurônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Motivação , Antagonistas do Receptor de Neuroquinina-1/farmacologia , Núcleo Accumbens/citologia , Estimulação Luminosa , Piperidinas/farmacologia , Receptor Muscarínico M1/fisiologia , Receptores de AMPA/fisiologia , Receptores de Dopamina D1/análise , Receptores de Dopamina D2/análise , Receptores da Neurocinina-1/fisiologia
4.
Life Sci ; 231: 116567, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202839

RESUMO

AIMS: Metabotropic glutamate receptor 5 (mGluR5), a member of group I mGluR, exerts its effect via elevation of intracellular Ca2+ level. We here characterized Ca2+ signals in the tsA201 cells transfected with mGluR5 and investigated the role of passages for mGluR5-induced Ca2+ signals in synaptic plasticity. MAIN METHODS: Using a genetically encoded Ca2+ indicator, GCamp2, Ca2+ signals were reliably induced by bath application of (S)-3,5-dihydroxyphenylglycine, the group I mGluR agonist, in the tsA201 cells transfected with mGluR5. Using whole-cell recordings in the substantia gelatinosa (SG) neurons of the spinal trigeminal subnucleus caudalis (Vc), excitatory postsynaptic currents were recorded by stimulating the trigeminal tract. KEY FINDINGS: Ca2+ signals were mediated by "classical" or "canonical" transient receptor potential (TRPC) channels, particularly TRPC1/3/4/6, but not TRPC5, naturally existing in the tsA201 cells. Interestingly, the induction of Ca2+ signals was independent of the phospholipase C signaling pathway; instead, it critically involves the cyclic adenosine diphosphate ribose/ryanodine receptor-dependent signaling pathway and only partially protein kinase C. On the other hand, both TRPC3 and TRPC4 mediated mGluR1/5-induced long-lasting potentiation of excitatory synaptic transmission from the trigeminal primary afferents to the SG neurons of the Vc. SIGNIFICANCE: This study demonstrates that endogenous TRPC channels contribute to mGluR5-induced Ca2+ signals in tsA201 cells and synaptic plasticity at excitatory synapses.


Assuntos
Sinalização do Cálcio/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Receptor de Glutamato Metabotrópico 5/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Nervo Trigêmeo/metabolismo , Núcleo Espinal do Trigêmeo/metabolismo
5.
Neuron ; 103(1): 66-79.e12, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31104951

RESUMO

The precision and reliability of synaptic information transfer depend on the molecular organization of voltage-gated calcium channels (VGCCs) within the presynaptic membrane. Alternative splicing of exon 47 affects the C-terminal structure of VGCCs and their affinity to intracellular partners and synaptic vesicles (SVs). We show that hippocampal synapses expressing VGCCs either with exon 47 (CaV2.1+47) or without (CaV2.1Δ47) differ in release probability and short-term plasticity. Tracking single channels revealed transient visits (∼100 ms) of presynaptic VGCCs in nanodomains (∼80 nm) that were controlled by neuronal network activity. Surprisingly, despite harboring prominent binding sites to scaffold proteins, CaV2.1+47 persistently displayed higher mobility within nanodomains. Synaptic accumulation of CaV2.1 was accomplished by optogenetic clustering, but only CaV2.1+47 increased transmitter release and enhanced synaptic short-term depression. We propose that exon 47-related alternative splicing of CaV2.1 channels controls synapse-specific release properties at the level of channel mobility-dependent coupling between VGCCs and SVs.


Assuntos
Canais de Cálcio/genética , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio/efeitos da radiação , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Células HEK293 , Humanos , Luz , Neurotransmissores/metabolismo , Optogenética , Gravidez , Isoformas de Proteínas/genética , Ratos , Vesículas Sinápticas/fisiologia
6.
Int J Mol Sci ; 20(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970677

RESUMO

This study investigated whether metabotropic glutamate receptor (mGluR) 5 and 8 are involved in the effect of ultramicronizedpalmitoylethanolamide (um-PEA) on the cognitive behavior and long term potentiation (LTP) at entorhinal cortex (LEC)-dentate gyrus (DG) pathway in mice rendered neuropathic by the spare nerve injury (SNI). SNI reduced discriminative memory and LTP. Um-PEA treatment started after the development of neuropathic pain had no effects in sham mice, whereas it restored cognitive behavior and LTP in SNI mice. 2-Methyl-6-(phenylethynyl) pyridine (MPEP), a selective mGluR5 antagonist, improved cognition in SNI mice and produced a chemical long term depression of the field excitatory postsynaptic potentials (fEPSPs) in sham and SNI mice. After theta burst stimulation (TBS) MPEP restored LTP in SNI mice. In combination with PEA, MPEP antagonized the PEA effect on discriminative memory and decreased LTP in SNI mice. The (RS)-4-(1-amino-1-carboxyethyl)phthalic acid (MDCPG), a selective mGluR8 antagonist, did not affect discriminative memory, but it induced a chemical LTP and prevented the enhancement of fEPSPs after TBS in SNI mice which were treated or not treated with PEA. The effect of PEA on LTP and cognitive behavior was modulated by mGluR5 and mGluR8. In particular in the SNI conditions, the mGluR5 blockade facilitated memory and LTP, but prevented the beneficial effects of PEA on discriminative memory while the mGluR8 blockade, which was ineffective in itself, prevented the favorable action of the PEA on LTP. Thus, although their opposite roles (excitatory/inhibitory of the two receptor subtypes on the glutamatergic system), they appeared to be required for the neuroprotective effect of PEA in conditions of neuropathic pain.


Assuntos
Etanolaminas/administração & dosagem , Neuralgia/tratamento farmacológico , Ácidos Palmíticos/administração & dosagem , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Modelos Animais de Doenças , Etanolaminas/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos , Neuralgia/etiologia , Neuralgia/metabolismo , Córtex Olfatório/efeitos dos fármacos , Córtex Olfatório/metabolismo , Ácidos Palmíticos/farmacologia , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/metabolismo , Piridinas/administração & dosagem , Piridinas/farmacologia
7.
Nat Commun ; 10(1): 1201, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867415

RESUMO

Evaluating odor blends in sensory processing is a crucial step for signal recognition and execution of behavioral decisions. Using behavioral assays and 2-photon imaging, we have characterized the neural and behavioral correlates of mixture perception in the olfactory system of Drosophila. Mixtures of odors with opposing valences elicit strong inhibition in certain attractant-responsive input channels. This inhibition correlates with reduced behavioral attraction. We demonstrate that defined subsets of GABAergic interneurons provide the neuronal substrate of this computation at pre- and postsynaptic loci via GABAB- and GABAA receptors, respectively. Intriguingly, manipulation of single input channels by silencing and optogenetic activation unveils a glomerulus-specific crosstalk between the attractant- and repellent-responsive circuits. This inhibitory interaction biases the behavioral output. Such a form of selective lateral inhibition represents a crucial neuronal mechanism in the processing of conflicting sensory information.


Assuntos
Drosophila melanogaster/fisiologia , Odorantes , Bulbo Olfatório/fisiologia , Condutos Olfatórios/fisiologia , Percepção Olfatória/fisiologia , Animais , Animais Geneticamente Modificados , Antenas de Artrópodes/inervação , Comportamento Animal/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Neurônios GABAérgicos/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Optogenética , Receptores Odorantes/genética , Receptores Odorantes/fisiologia
8.
eNeuro ; 6(1)2019.
Artigo em Inglês | MEDLINE | ID: mdl-30847389

RESUMO

While the primary role of vesicular transporters is to load neurotransmitters into synaptic vesicles (SVs), accumulating evidence suggests that these proteins also contribute to additional aspects of synaptic function, including vesicle release. In this study, we extend the role of the VAChT to include regulating the transmitter content of SVs. We report that manipulation of a C-terminal poly-glutamine (polyQ) region in the Drosophila VAChT is sufficient to influence transmitter content, and release frequency, of cholinergic vesicles from the terminals of premotor interneurons. Specifically, we find that reduction of the polyQ region, by one glutamine residue (13Q to 12Q), results in a significant increase in both amplitude and frequency of spontaneous cholinergic miniature EPSCs (mEPSCs) recorded in the aCC and RP2 motoneurons. Moreover, this truncation also results in evoked synaptic currents that show increased duration: consistent with increased ACh release. By contrast, extension of the polyQ region by one glutamine (13Q to 14Q) is sufficient to reduce mEPSC amplitude and frequency and, moreover, prevents evoked SV release. Finally, a complete deletion of the polyQ region (13Q to 0Q) has no obvious effects to mEPSCs, but again evoked synaptic currents show increased duration. The mechanisms that ensure SVs are filled to physiologically-appropriate levels remain unknown. Our study identifies the polyQ region of the insect VAChT to be required for correct vesicle transmitter loading and, thus, provides opportunity to increase understanding of this critical aspect of neurotransmission.


Assuntos
Acetilcolina/metabolismo , Proteínas de Drosophila/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Neurônios Motores/metabolismo , Optogenética , Técnicas de Patch-Clamp , Proteínas Vesiculares de Transporte de Acetilcolina/genética
9.
Proc Natl Acad Sci U S A ; 116(12): 5737-5746, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819889

RESUMO

In spike-timing-dependent plasticity (STDP), the direction and degree of synaptic modification are determined by the coherence of pre- and postsynaptic activities within a neuron. However, in the adult rat hippocampus, it remains unclear whether STDP-like mechanisms in a neuronal population induce synaptic potentiation of a long duration. Thus, we asked whether the magnitude and maintenance of synaptic plasticity in a population of CA1 neurons differ as a function of the temporal order and interval between pre- and postsynaptic activities. Modulation of the relative timing of Schaffer collateral fibers (presynaptic component) and CA1 axons (postsynaptic component) stimulations resulted in an asymmetric population STDP (pSTDP). The resulting potentiation in response to 20 pairings at 1 Hz was largest in magnitude and most persistent (4 h) when presynaptic activity coincided with or preceded postsynaptic activity. Interestingly, when postsynaptic activation preceded presynaptic stimulation by 20 ms, an immediate increase in field excitatory postsynaptic potentials was observed, but it eventually transformed into a synaptic depression. Furthermore, pSTDP engaged in selective forms of late-associative activity: It facilitated the maintenance of tetanization-induced early long-term potentiation (LTP) in neighboring synapses but not early long-term depression, reflecting possible mechanistic differences with classical tetanization-induced LTP. The data demonstrate that a pairing of pre- and postsynaptic activities in a neuronal population can greatly reduce the required number of synaptic plasticity-evoking events and induce a potentiation of a degree and duration similar to that with repeated tetanization. Thus, pSTDP determines synaptic efficacy in the hippocampal CA3-CA1 circuit and could bias the CA1 neuronal population toward potentiation in future events.


Assuntos
Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Potenciais de Ação/fisiologia , Animais , Região CA1 Hipocampal/fisiologia , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/fisiologia , Masculino , Neurônios/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Sinapses/fisiologia , Lobo Temporal
10.
Nat Neurosci ; 22(4): 556-564, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911184

RESUMO

Heterozygous loss-of-function mutations in SHANK2 are associated with autism spectrum disorder (ASD). We generated cortical neurons from induced pluripotent stem cells derived from neurotypic and ASD-affected donors. We developed sparse coculture for connectivity assays where SHANK2 and control neurons were differentially labeled and sparsely seeded together on a lawn of unlabeled control neurons. We observed increases in dendrite length, dendrite complexity, synapse number, and frequency of spontaneous excitatory postsynaptic currents. These findings were phenocopied in gene-edited homozygous SHANK2 knockout cells and rescued by gene correction of an ASD SHANK2 mutation. Dendrite length increases were exacerbated by IGF1, TG003, or BDNF, and suppressed by DHPG treatment. The transcriptome in isogenic SHANK2 neurons was perturbed in synapse, plasticity, and neuronal morphogenesis gene sets and ASD gene modules, and activity-dependent dendrite extension was impaired. Our findings provide evidence for hyperconnectivity and altered transcriptome in SHANK2 neurons derived from ASD subjects.


Assuntos
Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Dendritos/patologia , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Transtorno do Espectro Autista/metabolismo , Técnicas de Cocultura , Dendritos/metabolismo , Potenciais Pós-Sinápticos Excitadores , Técnicas de Inativação de Genes , Haploinsuficiência , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Plasticidade Neuronal , Neurônios/metabolismo , Transcriptoma
11.
Mol Cell Neurosci ; 95: 71-78, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30738184

RESUMO

Interleukin 16 (IL-16) is a cytokine that is primarily associated with CD4+ T cell function, but also exists as a multi-domain PDZ protein expressed within cerebellar and hippocampal neurons. We have previously shown that lymphocyte-derived IL-16 is neuroprotective against excitotoxicity, but evidence of how it affects neuronal function is limited. Here, we have investigated whether IL-16 modulates neuronal excitability and synaptic activity in mouse primary hippocampal cultures. Application of recombinant IL-16 impairs both glutamate-induced increases in intracellular Ca2+ and sEPSC frequency and amplitude in a CD4- and CD9-independent manner. We examined the mechanisms underlying these effects, with rIL-16 reducing GluA1 S831 phosphorylation and inhibiting Na+ channel function. Taken together, these data suggest that IL-16 reduces neuronal excitability and synaptic activity via multiple mechanisms and adds further evidence that alternative receptors may exist for IL-16.


Assuntos
Potenciais Pós-Sinápticos Excitadores , Interleucina-16/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de AMPA/metabolismo , Canais de Sódio/metabolismo , Animais , Antígenos CD4/metabolismo , Cálcio/metabolismo , Células Cultivadas , Ácido Glutâmico/toxicidade , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação , Tetraspanina 29/metabolismo
12.
Mol Pain ; 15: 1744806919832718, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30717631

RESUMO

The neuropeptide of calcitonin gene-related peptide (CGRP) plays critical roles in chronic pain, especially in migraine. Immunohistochemistry and in situ hybridization studies have shown that CGRP and its receptors are expressed in cortical areas including pain perception-related prefrontal anterior cingulate cortex. However, less information is available for the functional roles of CGRP in cortical regions such as the anterior cingulate cortex (ACC). Recent studies have consistently demonstrated that long-term potentiation is a key cellular mechanism for chronic pain in the ACC. In the present study, we used 64-electrode array field recording system to investigate the effect of CGRP on excitatory transmission in the ACC. We found that CGRP induced potentiation of synaptic transmission in a dose-dependently manner (1, 10, 50, and 100 nM). CGRP also recruited inactive circuit in the ACC. An application of the calcitonin receptor-like receptor antagonist CGRP8-37 blocked CGRP-induced chemical long-term potentiation and the recruitment of inactive channels. CGRP-induced long-term potentiation was also blocked by N-methyl-D-aspartate (NMDA) receptor antagonist AP-5. Consistently, the application of CGRP increased NMDA receptor-mediated excitatory postsynaptic currents. Finally, we found that CGRP-induced long-term potentiation required the activation of calcium-stimulated adenylyl cyclase subtype 1 (AC1) and protein kinase A. Genetic deletion of AC1 using AC1-/- mice, an AC1 inhibitor NB001 or a protein kinase A inhibitor KT5720, all reduced or blocked CGRP-induced potentiation. Our results provide direct evidence that CGRP may contribute to synaptic potentiation in important physiological and pathological conditions in the ACC, an AC1 inhibitor NB001 may be beneficial for the treatment of chronic headache.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Giro do Cíngulo/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , 2-Amino-5-fosfonovalerato/farmacologia , Adenilil Ciclases/deficiência , Adenilil Ciclases/genética , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Carbazóis/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Giro do Cíngulo/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , Pirróis/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Mol Pain ; 15: 1744806918824243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30799694

RESUMO

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a µ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating µ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.


Assuntos
Analgésicos Opioides/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Neurônios/efeitos dos fármacos , Substância Gelatinosa/citologia , Tramadol/análogos & derivados , Animais , Interações de Medicamentos , Fármacos atuantes sobre Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Masculino , Antagonistas de Entorpecentes/farmacologia , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/fisiologia , Neurônios/fisiologia , Norepinefrina/farmacologia , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Ratos , Serotonina/farmacologia , Tramadol/farmacologia
14.
Proc Natl Acad Sci U S A ; 116(11): 5160-5169, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30796190

RESUMO

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.


Assuntos
Antidepressivos/farmacologia , Ketamina/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Concentração Inibidora 50 , Ketamina/administração & dosagem , Ketamina/química , Masculino , Camundongos , N-Metilaspartato/metabolismo , Subunidades Proteicas/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Xenopus laevis
15.
Proc Natl Acad Sci U S A ; 116(12): 5727-5736, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30808806

RESUMO

Homeostatic synaptic scaling is a negative feedback response to fluctuations in synaptic strength induced by developmental or learning-related processes, which maintains neuronal activity stable. Although several components of the synaptic scaling apparatus have been characterized, the intrinsic regulatory mechanisms promoting scaling remain largely unknown. MicroRNAs may contribute to posttranscriptional control of mRNAs implicated in different stages of synaptic scaling, but their role in these mechanisms is still undervalued. Here, we report that chronic blockade of glutamate receptors of the AMPA and NMDA types in hippocampal neurons in culture induces changes in the neuronal mRNA and miRNA transcriptomes, leading to synaptic upscaling. Specifically, we show that synaptic activity blockade persistently down-regulates miR-186-5p. Moreover, we describe a conserved miR-186-5p-binding site within the 3'UTR of the mRNA encoding the AMPA receptor GluA2 subunit, and demonstrate that GluA2 is a direct target of miR-186-5p. Overexpression of miR-186 decreased GluA2 surface levels, increased synaptic expression of GluA2-lacking AMPA receptors, and blocked synaptic scaling, whereas inhibition of miR-186-5p increased GluA2 surface levels and the amplitude and frequency of AMPA receptor-mediated currents, and mimicked excitatory synaptic scaling induced by synaptic inactivity. Our findings elucidate an activity-dependent miRNA-mediated mechanism for regulation of AMPA receptor expression.


Assuntos
MicroRNAs/genética , Neurônios/metabolismo , Receptores de AMPA/genética , Regiões 3' não Traduzidas , Animais , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células HEK293 , Hipocampo/metabolismo , Homeostase , Humanos , MicroRNAs/metabolismo , Plasticidade Neuronal/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de AMPA/metabolismo , Receptores de AMPA/fisiologia , Sinapses/metabolismo
16.
Brain Struct Funct ; 224(3): 1279-1290, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30701309

RESUMO

BACE1 is a ß-secretase involved in the cleavage of amyloid precursor protein and the pathogenesis of Alzheimer's disease (AD). The entorhinal cortex and the dentate gyrus are important for learning and memory, which are affected in the early stages of AD. Since BACE1 is a potential target for AD therapy, it is crucial to understand its physiological role in these brain regions. Here, we examined the function of BACE1 in the dentate gyrus. We show that loss of BACE1 in the dentate gyrus leads to increased granule cell excitability, indicated by enhanced efficiency of synaptic potentials to generate granule cell spikes. The increase in granule cell excitability was accompanied by prolonged paired-pulse inhibition, altered network gamma oscillations, and impaired synaptic plasticity at entorhinal-dentate synapses of the perforant path. In summary, this is the first detailed electrophysiological study of BACE1 deletion at the network level in vivo. The results suggest that BACE1 is important for normal dentate gyrus network function. This has implications for the use of BACE1 inhibitors as therapeutics for AD therapy, since BACE1 inhibition could similarly disrupt synaptic plasticity and excitability in the entorhinal-dentate circuitry.


Assuntos
Secretases da Proteína Precursora do Amiloide/deficiência , Ácido Aspártico Endopeptidases/deficiência , Relógios Biológicos/genética , Giro Denteado/citologia , Plasticidade Neuronal/genética , Via Perfurante/citologia , Potenciais de Ação/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Biofísica , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/genética , Masculino , Camundongos , Camundongos Knockout , Neurônios , Tempo de Reação/genética , Fatores de Tempo
17.
Stroke ; 50(3): 728-737, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30727847

RESUMO

Background and Purpose- Stroke is a major public health concern worldwide. Although clinical treatments have improved in the acute period after stroke, long-term therapeutics remain limited to physical rehabilitation in the delayed phase. This study is aimed to determine whether nNOS (neuronal NO synthase)-CAPON (carboxy-terminal postsynaptic density-95/discs large/zona occludens-1 ligand of nNOS) interaction may serve as a new therapeutic target in the delayed phase for stroke recovery. Methods- Photothrombotic stroke and transient middle cerebral artery occlusion were induced in mice. Adeno-associated virus (AAV)-cytomegalovirus (CMV)-CAPON-125C-GFP (green fluorescent protein)-3Flag and the other 2 drugs (Tat-CAPON-12C and ZLc-002) were microinjected into the peri-infarct cortex immediately and 4 to 10 days after photothrombotic stroke, respectively. ZLc-002 was also systemically injected 4 to 10 days after transient middle cerebral artery occlusion. Grid-walking task and cylinder task were conducted to assess motor function. Western blotting, immunohistochemistry, Golgi staining, and electrophysiology recordings were performed to uncover the mechanisms. Results- Stroke increased nNOS-CAPON association in the peri-infarct cortex in the delayed period. Inhibiting the ischemia-induced nNOS-CAPON association substantially decreased the number of foot faults in the grid-walking task and forelimb asymmetry in the cylinder task, suggesting the promotion of functional recovery from stroke. Moreover, dissociating nNOS-CAPON significantly facilitated dendritic remodeling and synaptic transmission, indicated by increased dendritic spine density, dendritic branching, and length and miniature excitatory postsynaptic current frequency but did not affect stroke-elicited neuronal loss, infarct size, or cerebral edema, suggesting that nNOS-CAPON interaction may function via regulating structural neuroplasticity, rather than neuroprotection. Furthermore, ZLc-002 reversed the transient middle cerebral artery occlusion-induced impairment of motor function. Conclusions- Our results reveal that nNOS-CAPON coupling can serve as a novel pharmacological target for functional restoration after stroke.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Plasticidade Neuronal/genética , Óxido Nítrico Sintase Tipo I/genética , Acidente Vascular Cerebral/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Edema Encefálico/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Dendritos/patologia , Potenciais Pós-Sinápticos Excitadores , Infarto da Artéria Cerebral Média/genética , Camundongos , Óxido Nítrico Sintase Tipo I/metabolismo , Densidade Pós-Sináptica , Desempenho Psicomotor , Recuperação de Função Fisiológica , Transmissão Sináptica
18.
Nat Commun ; 10(1): 386, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30674877

RESUMO

Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynapse. However, the molecular mechanisms that underlie excitatory synaptogenesis are only partially resolved, in part because the internal machinery of developing synapses is largely unknown. To address this, we apply a chemicogenetic approach, in vivo biotin identification (iBioID), to discover aspects of the proteome of nascent synapses. This approach uncovered sixty proteins, including a previously uncharacterized protein, CARMIL3, which interacts in vivo with the synaptic cytoskeletal regulator proteins SrGAP3 (or WRP) and actin capping protein. Using new CRISPR-based approaches, we validate that endogenous CARMIL3 is localized to developing synapses where it facilitates the recruitment of capping protein and is required for spine structural maturation and AMPAR recruitment associated with synapse unsilencing. Together these proteomic and functional studies reveal a previously unknown mechanism important for excitatory synapse development in the developing perinatal brain.


Assuntos
Citoesqueleto/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteoma/metabolismo , Proteômica , Sinapses/metabolismo , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Animais , Biotina , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas do Citoesqueleto/metabolismo , Espinhas Dendríticas/metabolismo , Proteínas Ativadoras de GTPase , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Proteoma/genética , Sinapses/genética
19.
Muscle Nerve ; 59(4): 509-516, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30677146

RESUMO

INTRODUCTION: The aim of this study was to compare the effects of adenosine-5'-triphosphate (ATP) and adenosine on the contractility of rodent extensor digitorum longus (EDL) muscle at normal and low temperatures. METHODS: Contractions of rat and mouse isolated EDL were induced by either electrical stimulation (ES) or exogenous carbachol and recorded in the presence of ATP or adenosine (both at 100 µM). RESULTS: ATP at all temperatures caused a decrease of the contractions induced by carbachol in rat and mouse EDL and ES-induced contractions in rat EDL, while it potentiated the ES-induced contractions of mouse EDL. Adenosine reduced the contractility of rat and mouse EDL evoked by ES and did not affect the carbachol-induced contractions of rat and mouse EDL at any temperature. DISCUSSION: Under various temperature conditions, ATP inhibits pre- but potentiates postsynaptic processes in the mouse EDL; in the rat EDL ATP causes only inhibition of neuromuscular conduction. Muscle Nerve 59:509-516, 2019.


Assuntos
Trifosfato de Adenosina/farmacologia , Contração Muscular/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Animais , Carbacol/farmacologia , Temperatura Baixa , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Camundongos , Agonistas Muscarínicos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Fármacos Neuromusculares não Despolarizantes/farmacologia , Agonistas Purinérgicos/farmacologia , Ratos , Ratos Wistar , Tubocurarina/farmacologia
20.
Cell Mol Neurobiol ; 39(2): 287-300, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30607810

RESUMO

The mechanisms of impairment in long-term potentiation after status epilepticus (SE) remain unclear. We investigated the properties of LTP induced by theta-burst stimulation in hippocampal slices of rats 3 h and 1, 3, and 7 days after SE. Seizures were induced in 3-week old rats by a single injection of pentylenetetrazole (PTZ). Only animals with generalized seizures lasting more than 30 min were included in the experiments. The results revealed that LTP was strongly attenuated in the CA1 hippocampal area after PTZ-induced SE as compared with that in control animals. Saturation of synaptic responses following epileptic activity does not explain weakening of LTP because neither the quantal size of the excitatory responses nor the slopes of the input-output curves for field excitatory postsynaptic potentials changed in the post-SE rats. After PTZ-induced SE, NMDA-dependent LTP was suppressed, and LTP transiently switched to the mGluR1-dependent form. This finding does not appear to have been reported previously in the literature. An antagonist of NMDA receptors, D-2-amino-5-phosphonovalerate, did not block LTP induction in 3-h and 1-day post-SE slices. An antagonist of mGluR1, FTIDS, completely prevented LTP in 1-day post-SE slices; whereas it did not affect LTP induction in control and post-SE slices at the other studied times. mGluR1-dependent LTP was postsynaptically expressed and did not require NMDA receptor activation. Recovery of NMDA-dependent LTP occurred 7 day after SE. Transient switching between NMDA-dependent LTP and mGluR1-dependent LTP could play a role in the pathogenesis of acquired epilepsy.


Assuntos
Região CA1 Hipocampal/fisiopatologia , Região CA3 Hipocampal/fisiopatologia , Potenciação de Longa Duração , N-Metilaspartato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Sinapses/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores , Aprendizagem em Labirinto , Pentilenotetrazol , Ratos Wistar , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/induzido quimicamente , Memória Espacial
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