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1.
Invest Ophthalmol Vis Sci ; 60(13): 4360-4377, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31634394

RESUMO

Purpose: To investigate the neuroprotective properties of creatine in the retina using in vitro and in vivo models of injury. Methods: Two different rat retinal culture systems (one containing retinal ganglion cells [RGC] and one not) were subjected to either metabolic stress, via treatments with the mitochondrial complex IV inhibitor sodium azide, or excitotoxic stress, via treatment with N-methyl-D-aspartate for 24 hours, in the presence or absence of creatine (0.5, 1.0, and 5.0 mM). Neuronal survival was assessed by immunolabeling for cell-specific antigens. Putative mechanisms of creatine action were investigated in vitro. Expression of creatine kinase (CK) isoenzymes in the rat retina was examined using Western blotting and immunohistochemistry. The effect of oral creatine supplementation (2%, wt/wt) on retinal and blood creatine levels was determined as well as RGC survival in rats treated with N-methyl-D-aspartate (NMDA; 10 nmol) or high IOP-induced ischemia reperfusion. Results: Creatine significantly prevented neuronal death induced by sodium azide and NMDA in both culture systems. Creatine administration did not alter cellular adenosine triphosphate (ATP). Inhibition of CK blocked the protective effect of creatine. Retinal neurons, including RGCs, expressed predominantly mitochondrial CK isoforms, while glial cells expressed exclusively cytoplasmic CKs. In vivo, NMDA and ischemia reperfusion caused substantial loss of RGCs. Creatine supplementation led to elevated blood and retinal levels of this compound but did not significantly augment RGC survival in either model. Conclusions: Creatine increased neuronal survival in retinal cultures; however, no significant protection of RGCs was evident in vivo, despite elevated levels of this compound being present in the retina after oral supplementation.


Assuntos
Creatina/farmacologia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Células Cultivadas , Creatina Quinase/metabolismo , Eletrorretinografia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Isoenzimas/metabolismo , N-Metilaspartato/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Retina/enzimologia , Retina/fisiopatologia , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Azida Sódica/farmacologia , Estresse Fisiológico
2.
Croat Med J ; 60(4): 352-360, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31483121

RESUMO

AIM: To analyze the effects of glutamatergic agonists and antagonists on the activation of the A1 and A2 noradrenergic neurons localized in caudal ventrolateral medulla and nucleus tractus solitarii, respectively. METHODS: Rats were injected with glutamatergic agonists - kainic acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or N-methyl-D-aspartic acid (NMDA), and the brain sections were prepared for immunohistochemistry. Before agonist injections, antagonists - 6-cyano-7-nitroquinoxaline-2,3-dione or dizocilpine were administered. The expression of c-Fos, as the neuronal activation marker, and tyrosine hydroxylase (TH), as the marker of noradrenergic neurons was assessed with dual immunohistochemistry. The percentage of c-Fos-positive noradrenergic neurons relative to all TH-positive neurons in the respective areas of the brain stem was calculated. RESULTS: All three glutamatergic agonists significantly increased the number of the c-Fos-positive noradrenergic neurons in both the A1 and A2 area when compared with control animals. Kainic acid injection activated about 57% of TH-positive neurons in A1 and 40% in A2, AMPA activated 26% in A1 and 38% in A2, and NMDA 77% in A1 and 22% in A2. The injections of appropriate glutamatergic antagonists greatly decreased the number of activated noradrenergic neurons. CONCLUSION: Our results suggest that noradrenergic neurons are regulated and/or activated by glutamatergic system and that these neurons express functional glutamate receptors.


Assuntos
Neurônios Adrenérgicos/efeitos dos fármacos , Tronco Encefálico/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/agonistas , Fármacos Atuantes sobre Aminoácidos Excitatórios/antagonistas & inibidores , Animais , Feminino , Imuno-Histoquímica , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/biossíntese , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
3.
Exp Eye Res ; 187: 107756, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31421136

RESUMO

Endoplasmic reticulum (ER) stress is recognized as a contributing factor to various ocular neurovascular pathologies including retinitis pigmentosa, glaucoma, and diabetic retinopathy (DR). ER stress in particular is implicated in the development of DR, which is significantly influenced by inflammation driven retinal vascular degeneration and dysfunction. Ultimately, loss of vision occurs if left untreated. However, the identity of the target cells and their temporal involvement in diabetes-mediated dysfunction need further investigation. Early diabetes-induced stress in photoreceptor cells is proposed as the driver of inflammatory mediated neurovascular changes during diabetes. Although tunicamycin induced ER stress results in photoreceptor loss, its consequences for retinal vascular degeneration and retinal ganglion (RGC) and pigment epithelium (RPE) cell loss remains unclear. Here we show intravitreal delivery of tunicamycin primarily induced ER stress in photoreceptor cells resulting in their loss by apoptosis. This was concomitant with induced expression of the unfolded protein response marker CHOP in these cells. We also demonstrated significant degeneration of retinal capillaries following the loss of photoreceptor cells with minimal impact on loss of RGC and RPE cells. However, activation of retinal microglial and Muller cells were noticeable. Thus, our data support the notion that ER stress mediated dysfunction and/or loss of photoreceptor cells in response to inflammation and oxidative stress could precede retinal vascular and neuronal dysfunction and degeneration.


Assuntos
Antibacterianos/farmacologia , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologia , Epitélio Pigmentado da Retina/patologia , Vasos Retinianos/patologia , Tunicamicina/farmacologia , Animais , Atrofia , Capilares/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Marcação In Situ das Extremidades Cortadas , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Metilaspartato/farmacologia , Estresse Oxidativo , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
4.
Neurochem Res ; 44(9): 2068-2080, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31317507

RESUMO

The mechanisms underlying chronic and neuropathic pain pathology involve peripheral and central sensitisation. The medial prefrontal cortex (mPFC) seems to participate in pain chronification, and glutamatergic neurotransmission may be involved in this process. Thus, the aim of the present work was to investigate the participation of the prelimbic (PrL) area of the mPFC in neuropathic pain as well as the role of N-methyl D-aspartate (NMDA) glutamate receptors in neuropathic pain induced by a modified sciatic nerve chronic constriction injury (CCI) protocol in Wistar rats. Neural inputs to the PrL cortex were inactivated by intracortical treatment with the synapse blocker cobalt chloride (CoCl2, 1.0 mM/200 nL) 7, 14, 21, or 28 days after the CCI or sham procedure. The glutamatergic agonist NMDA (0.25, 1 or 4 nmol) or the selective NMDA receptor antagonist LY235959 (2, 4 or 8 nmol) was microinjected into the PrL cortex 21 days after surgery. CoCl2 administration in the PrL cortex decreased allodynia 21 and 28 days after CCI. NMDA at 1 and 4 nmol increased allodynia, whereas LY235959 decreased mechanical allodynia at the highest dose (8 nmol) microinjected into the PrL cortex. These findings suggest that NMDA receptors in the PrL cortex participate in enhancing the late phase of mechanical allodynia after NMDA-induced increases and LY235959-induced decreases in allodynia 21 days after CCI. The glutamatergic system potentiates chronic neuropathic pain by NMDA receptor activation in the PrL cortex. Mechanism of neuropathic pain. The infusion of CoCl2, a synapse activity blocker, into the prelimbic (PrL) division of the medial prefrontal cortex (mPFC) decreased the severity of mechanical allodynia, showing the late participation of the limbic cortex. The glutamatergic system potentiates chronic neuropathic pain via NMDA receptor activation in the PrL cortex.


Assuntos
Neuralgia/metabolismo , Nervos Periféricos/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cobalto/farmacologia , Hiperalgesia/tratamento farmacológico , Isoquinolinas/farmacologia , Masculino , N-Metilaspartato/farmacologia , Neuralgia/tratamento farmacológico , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Córtex Pré-Frontal/efeitos dos fármacos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos
5.
Life Sci ; 232: 116612, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31260687

RESUMO

AIMS: Accumulating evidence suggest that endoplasmic reticulum (ER) stress is an important mechanism underlying the development of diabetes. We have reported that sustained treatment with N-methyl-d-aspartate (NMDA) results in apoptotic ß-cell death and impairs insulin secretion. However, the molecular mechanism responsible for NMDA-induced ß-cell dysfunction remains largely obscure. Thus, this study aimed to determine whether sustained activation of NMDA receptors (NMDARs) causes ß-cell dysfunction through ER stress. MAIN METHODS: Primary mouse islets and MIN6 mouse pancreatic ß-cells were treated with NMDA for 24 h or high-glucose for 72 h. After the treatment, glucose-stimulated insulin secretion (GSIS) and the expression of ER stress markers were measured, respectively. In vivo, the expression of ER stress markers was measured in the pancreas of diabetic mice treated with or without NMDARs inhibitor Memantine. KEY FINDINGS: NMDA treatment caused an increase in the expression of ER stress markers (ATF4, CHOP, GRP78, and Xbp1s) in primary islets. While, tauroursodeoxycholic acid (TUDCA), an inhibitor of ER stress, significantly attenuated NMDA-induced ß-cell dysfunction, including the loss of glucose-stimulated insulin secretion and reduction of pancreas duodenum homeobox factor-1 (Pdx-1) mRNA expression, a transcription factor regulating insulin synthesis. Besides, NMDA-induced ER stress strongly promoted pro-inflammatory cytokines synthesis (IL-1ß and TNF-α) in ß cells. Interestingly, knockdown of CHOP attenuated ß-cell dysfunction evoked by NMDA. Furthermore, we demonstrated that blockade of NMDARs ameliorated high-glucose-induced ER stress in vitro and in vivo. SIGNIFICANCE: This study confirms that ER stress is actively involved in the activation of NMDARs-related ß-cell dysfunction.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/metabolismo , Fator de Transcrição CHOP/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo
6.
Exp Eye Res ; 186: 107710, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31254512

RESUMO

Quantifying the number of axons in the optic nerve is of interest in many research questions. Here, we show that a stereological method allows simple, efficient, precise and unbiased determination of the total axon number in the murine optic nerve. Axons in semi-thin optic nerve cross sections from untreated eyes (n = 21) and eyes subjected to retinal damage by intravitreous NMDA injections (n = 32) or PBS controls (n = 5) were manually identified, counted and digitally labeled by hand. A stereological procedure was empirically tested with systematic combinations of different sampling methods (simple random sampling without replacement, systematic uniform random sampling, stratified random sampling) and sampling parameters. Extensive numerical Monte Carlo experiments were performed to evaluate their large-sample properties. Our results demonstrate reliable determination of total axon number and superior performance compared to other methods at a small fraction of the time required for a full manual count. We specify suitable sampling parameters for the adoption of an efficient stereological sampling scheme, give empirical estimates of the additionally introduced sampling variance to facilitate experimental planning, and offer AxonCounter, an easy-to-use plugin implementing these stereological methods for the multi-platform image processing application NIH ImageJ.


Assuntos
Contagem de Células/métodos , Técnicas Citológicas , Nervo Óptico/citologia , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Endogâmicos BALB C , N-Metilaspartato/farmacologia , Nervo Óptico/efeitos dos fármacos
7.
Neuropharmacology ; 151: 64-73, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30943384

RESUMO

Behavioral studies using pharmacological tools have implicated histamine H1 receptors in cognitive function via their interactions with N-methyl-D-aspartate receptors (NMDARs) in the hippocampus. However, little is known about the neurophysiological mechanism that underlies the interaction between H1 receptors and NMDARs. To explore how H1 receptor activation affects hippocampal excitatory neurotransmission and synaptic plasticity, this study aimed to examine the effect of H1 receptor ligands on both NMDAR-mediated synaptic currents and long-term potentiation (LTP) at synapses between Schaffer collaterals and CA1 pyramidal neurons using acute mouse hippocampal slices. We found that the H1 receptor antagonist/inverse agonists, pyrilamine (0.1 µM) and cetirizine (10 µM), decreased the NMDAR-mediated component of stimulation-induced excitatory postsynaptic currents (EPSCs) recorded from CA1 pyramidal neurons without affecting the AMPA receptor-mediated component of EPSCs and its paired pulse ratio. Pretreatment of slices with either the glial metabolism inhibitor, fluoroacetate (5 mM), or D-serine (100 µM) diminished the pyrilamine- or cetirizine-induced attenuation of the NMDAR-mediated EPSCs. Furthermore, the LTP of field excitatory postsynaptic potentials induced following high frequency stimulation of Schaffer collaterals was attenuated with application of pyrilamine or cetirizine. Pretreatment with D-serine again attenuated the pyrilamine-induced suppression of LTP. Our data suggest that H1 receptors in the CA1 can undergo persistent activation induced by their constitutive receptor activity and/or tonic release of endogenous histamine, resulting in facilitation of the NMDAR activity in a manner dependent of astrocytes and the release of D-serine. This led to the enhancement of NMDA-component EPSC and LTP at the Schaffer collateral-CA1 pyramidal neuron synapses.


Assuntos
Astrócitos/efeitos dos fármacos , Região CA1 Hipocampal/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H1/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Receptores Histamínicos H1/metabolismo , Serina/farmacologia , Animais , Astrócitos/metabolismo , Região CA1 Hipocampal/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Camundongos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pirilamina/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Valina/análogos & derivados , Valina/farmacologia
8.
Neurosci Lett ; 705: 33-38, 2019 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-31004707

RESUMO

Noradrenergic projections from the nucleus tractus solitarius (NTS) to the hypothalamic paraventricular nucleus (PVN) are involved in nicotine (Nic) dependence. Nic induces hypothalamic norepinephrine (NE) release through N-methyl-d-aspartate receptors (NMDARs) and nitric oxide in the NTS. However, acupuncture attenuates Nic withdrawal-induced anxiety. Therefore, this study investigated the effects of acupuncture on Nic-induced hypothalamic NE release. Rats received an intravenous infusion of Nic (90 µg/kg, over 60 s) and extracellular NE levels in the PVN were determined by in vivo microdialysis. Immediately after Nic administration, the rats were bilaterally treated with acupuncture at acupoint HT7 (Shen-Men) or PC6 (Nei-Guan), or a non-acupoint (tail) for 60 s. Acupuncture at HT7, but not at PC6 or the tail, significantly reduced Nic-induced NE release. However, this was abolished by a post-acupuncture infusion of either NMDA or sodium nitroprusside into the NTS. Additionally, acupuncture at HT7, but not the control points, prevented Nic-induced plasma corticosterone secretion and inhibited Nic-induced increases in the phosphorylation of neuronal nitric oxide synthase (nNOS) and endothelial NOS in the NTS. These findings suggest that acupuncture at HT7 reduces Nic-induced NE release in the PVN via inhibition of the solitary NMDAR/NOS pathway.


Assuntos
Terapia por Acupuntura , Nicotina/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Norepinefrina/metabolismo , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Corticosterona/sangue , Infusões Intravenosas , Masculino , Microdiálise , N-Metilaspartato/administração & dosagem , N-Metilaspartato/farmacologia , Nicotina/administração & dosagem , Nicotina/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/administração & dosagem , Nitroprussiato/farmacologia , Fosforilação/efeitos dos fármacos , Ratos
9.
Int J Mol Sci ; 20(8)2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31010057

RESUMO

The interactions between neuronal, glial, and vascular cells play a key role in regulating blood flow in the retina. In the present study, we examined the role of the interactions between neuronal and glial cells in regulating the retinal vascular tone in rats upon stimulation of retinal neuronal cells by intravitreal injection of N-methyl-d-aspartic acid (NMDA). The retinal vascular response was assessed by measuring the diameter of the retinal arterioles in the in vivo fundus images. Intravitreal injection of NMDA produced retinal vasodilation that was significantly diminished following the pharmacological inhibition of nitric oxide (NO) synthase (nNOS), loss of inner retinal neurons, or intravitreal injection of glial toxins. Immunohistochemistry revealed the expression of nNOS in ganglion and calretinin-positive amacrine cells. Moreover, glial toxins significantly prevented the retinal vasodilator response induced by intravitreal injection of NOR3, an NO donor. Mechanistic analysis revealed that NO enhanced the production of vasodilatory prostanoids and epoxyeicosatrienoic acids in glial cells in a ryanodine receptor type 1-dependent manner, subsequently inducing the retinal vasodilator response. These results suggest that the NO released from stimulated neuronal cells acts as a key messenger in neuron-glia signaling, thereby causing neuronal activity-dependent and glial cell-mediated vasodilation in the retina.


Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , Vasos Retinianos/metabolismo , Transdução de Sinais , Animais , Gangliosídeos/metabolismo , Hidroxilaminas , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Modelos Biológicos , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Prostaglandinas/metabolismo , Ratos Wistar , Vasos Retinianos/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
10.
Neurochem Int ; 126: 59-63, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30858017

RESUMO

We investigated the impact of the prolonged exposure of rat hippocampal synaptosomes to CXCL12 (3 nM) on the NMDA-mediated release of [3H]D-aspartate ([3H]D-Asp) or [3H]noradrenaline ([3H]NA). Synaptosomes were stimulated twice with NMDA/CXCL12 and the amount of the NMDA-evoked tritium release (S1 and S2) quantified to calculate the S2/S1 ratio. The S2/S1 ratio for both transmitters was drastically decreased by 3 nM CXCL12 between the two stimuli (CXCL12-treated synaptosomes) in a AMD3100-sensitive manner. The phosphorylation of the GluN1 subunit in Ser 896 was reduced in CXCL12-treated synaptosomes, while the overall amount of GluN1 and GluN2B proteins as well as the GluN2B insertion in synaptosomal plasmamembranes were unchanged. We conclude that the CXCR4/NMDA cross-talk is dynamically regulated by the time of activation of the CXCR4s. Our results unveil a functional cross-talk that might account for the severe impairments of central transmission that develop in pathological conditions characterized by CXCL12 overproduction.


Assuntos
Hipocampo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores CXCR4/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinaptossomos/metabolismo , Animais , Quimiocina CXCL12/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sinaptossomos/efeitos dos fármacos
11.
Int J Mol Sci ; 20(6)2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871175

RESUMO

N-arachidonoyl glycine (NAGly) is an endocannabinoid involved in the regulation of different immune cells. It was shown to activate the GPR18 receptor, which was postulated to switch macrophages from cytotoxic to reparative. To study GPR18 expression and neuroprotection after NAGly treatment we used excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). The effect of NAGly was also tested in isolated microglia and astrocytes as these cells play a crucial role during neuronal injury. In the present study, the GPR18 receptor was found in OHSC at mRNA level and was downregulated after N-Methyl-D-aspartate (NMDA) treatment at a single time point. Furthermore, treatment with NAGly reduced neuronal damage and this effect was abolished by GPR18 and cannabinoid receptor (CB)2 receptor antagonists. The activation but not motility of primary microglia and astrocytes was influenced when incubated with NAGly. However, NAGly alone reduced the phosphorylation of Akt but no changes in activation of the p44/42 and p38 MAPK and CREB pathways in BV2 cells could be observed. Given NAGly mediated actions we speculate that GPR18 and its ligand NAGly are modulators of glial and neuronal cells during neuronal damage.


Assuntos
Ácidos Araquidônicos/farmacologia , Glicina/análogos & derivados , Fármacos Neuroprotetores/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Endocanabinoides/farmacologia , Glicina/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptores de Canabinoides/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Pharm Biol ; 57(1): 1-7, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30734636

RESUMO

CONTEXT: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities. OBJECTIVE: The effects of fucoidan on Ca2+ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined. MATERIALS AND METHODS: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca2+ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5 mg/mL or 1.5 mg/mL was applied for 3 min to determine its effects on Ca2+ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5 mg/mL for 3 h. RESULTS: The Ca2+ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca2+ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca2+ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca2+ channels, PR1/PR2. DISCUSSION AND CONCLUSIONS: Our data indicate that fucoidan suppresses the intracellular Ca2+ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca2+ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Neurônios/efeitos dos fármacos , Polissacarídeos/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Células Cultivadas , Córtex Cerebelar/citologia , Córtex Cerebelar/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/biossíntese , Receptores de N-Metil-D-Aspartato/metabolismo
13.
Behav Brain Res ; 359: 298-303, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30428335

RESUMO

It has been shown that drug addiction and memory system are related but the signaling cascades underlying this interaction is not completely revealed yet. It has been demonstrated that binding of Calcium-calmodulin-dependent protein kinase II (CaMKII) to NMDA receptor is important in the memory process. The main objective of the study was to evaluate the role of CaMKII on the spatial memory of rats which previously were sensitized by morphine. The effect of CaMKII inhibitor (KN-93) on memory changes was investigated by hippocampal microinjection of KN-93 on the morphine-sensitized rats. Also, the role of the NMDA receptor in memory retention by KN-93 on the morphine sensitized rat was investigated with NMDA agonist and antagonist. Sensitization was induced by morphine injection (once daily for 3 days) followed by 5 days free of the drug before the trial phase. For the evaluation of spatial memory, the Morris Water Maze test (MWM) was used. Results showed that pre-trial administration of morphine, induced amnesia in MWM (p < 0.05). Also, three days pretreatment with morphine (20 mg/kg) followed by five days washout period, caused to enhance memory retrieval in confront with a pre-trial challenging dose of morphine (5 mg/kg). In addition, KN-93 administration during induction phase in morphine sensitization phenomena facilitated morphine-induced memory retention. In addition, inhibition of the NMDA receptor and KN-93 during the induction phase did not improve memory. However; intra-CA1 co-administration of KN-93 and NMDA during the induction phase of morphine sensitization resulted in improving spatial memory. It can be concluded that the effect of CaMKII on memory retention in morphine-sensitized rats depends on NMDA receptor.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transtornos da Memória/etiologia , Morfina/farmacologia , Entorpecentes/farmacologia , Transtornos Relacionados ao Uso de Opioides/enzimologia , Memória Espacial/efeitos dos fármacos , Animais , Benzilaminas/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/enzimologia , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacologia , Nootrópicos/farmacologia , Transtornos Relacionados ao Uso de Opioides/psicologia , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Memória Espacial/fisiologia , Sulfonamidas/farmacologia
14.
Transl Stroke Res ; 10(2): 204-215, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29687302

RESUMO

Cerebral preconditioning (PC) confers endogenous brain protection after stroke. Ischemic stroke patients with a prior transient ischemic attack (TIA) may potentially be in a preconditioned state. Although PC has been associated with the activation of pro-survival signals, the mechanism by which preconditioning confers neuroprotection is not yet fully clarified. Recently, we have described that PC-mediated neuroprotection against ischemic insult is promoted by p53 destabilization, which is mediated by its main regulator MDM2. Moreover, we have previously described that the human Tp53 Arg72Pro single nucleotide polymorphism (SNP) controls susceptibility to ischemia-induced neuronal apoptosis and governs the functional outcome of patients after stroke. Here, we studied the contribution of the human Tp53 Arg72Pro SNP on PC-induced neuroprotection after ischemia. Our results showed that cortical neurons expressing the Pro72-p53 variant exhibited higher PC-mediated neuroprotection as compared with Arg72-p53 neurons. PC prevented ischemia-induced nuclear and cytosolic p53 stabilization in Pro72-p53 neurons. However, PC failed to prevent mitochondrial p53 stabilization, which occurs in Arg72-p53 neurons after ischemia. Furthermore, PC promoted neuroprotection against ischemia by controlling the p53/active caspase-3 pathway in Pro72-p53, but not in Arg72-p53 neurons. Finally, we found that good prognosis associated to TIA within 1 month prior to ischemic stroke was restricted to patients harboring the Pro72 allele. Our findings demonstrate that the Tp53 Arg72Pro SNP controls PC-promoted neuroprotection against a subsequent ischemic insult by modulating mitochondrial p53 stabilization and then modulates TIA-induced ischemic tolerance.


Assuntos
Isquemia Encefálica/genética , Hipóxia Celular/genética , Precondicionamento Isquêmico/métodos , Neurônios/patologia , Polimorfismo de Nucleotídeo Único/genética , Proteína Supressora de Tumor p53/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/genética , Arginina/genética , Isquemia Encefálica/prevenção & controle , Caspase 3/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Estudos de Coortes , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Embrião de Mamíferos , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Glucose/deficiência , Humanos , Masculino , Potenciais da Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , N-Metilaspartato/farmacologia , Prolina/genética , Frações Subcelulares/metabolismo , Frações Subcelulares/patologia
15.
Behav Brain Res ; 357-358: 71-81, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-28736332

RESUMO

It has been established that chemical stimulation of the inferior colliculus (IC) of laboratory animals evokes fear-related defensive responses, which are considered panic attack-like behaviours. In addition, there is evidence that defensive reactions provoked by chemical stimulation of midbrain tectum neurons may induce an antinociceptive response. Morphologically, the IC receives projections from other mesencephalic structures, such as the dorsal raphe nucleus (DRN), a region rich in serotonergic neurons that play a critical role in the control of defensive behaviours. Moreover, this monoaminergic brainstem reticular nucleus is suggested to comprise the endogenous pain modulatory system. The aim of the present study was to investigate the role of DRN 5-hydroxytryptamine 2A (5-HT2A) receptors in Wistar rats by local microinjection of R-96544 (a selective antagonist of the 5-HT2A receptor) at doses of 5, 10 or 15 nM on defensive reactions and fear-induced antinociception evoked by chemical stimulation of the central nucleus of the IC with NMDA (6, 9 or 12 nmol). Behavioural responses were analysed for 10 min, and then the nociceptive threshold was measured at 10 min intervals for 70 min. The dose of 12 nmol of NMDA was the most effective in causing panic attack-like defensive behaviours and much higher hypoalgesia. In addition, both effects were attenuated by pretreatment of the DRN with R-96544. These findings suggest the critical participation of DRN 5-HT2A receptors in the modulation of panic attack-like defensive behaviour and unconditioned fear-induced antinociception organised by neurons in the central nucleus of the IC.


Assuntos
Medo/psicologia , Colículos Inferiores/citologia , Neurônios/fisiologia , Nociceptividade/fisiologia , Dor/psicologia , Receptor 5-HT2A de Serotonina/metabolismo , Animais , Modelos Animais de Doenças , Núcleo Dorsal da Rafe , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/farmacologia , Reação de Congelamento Cataléptica/efeitos dos fármacos , Masculino , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Dor/tratamento farmacológico , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Pirrolidinas/farmacologia , Ratos , Ratos Wistar , Estatísticas não Paramétricas
16.
Elife ; 72018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30575520

RESUMO

The piriform cortex (PCx) receives direct input from the olfactory bulb (OB) and is the brain's main station for odor recognition and memory. The transformation of the odor code from OB to PCx is profound: mitral and tufted cells in olfactory glomeruli respond to individual odorant molecules, whereas pyramidal neurons (PNs) in the PCx responds to multiple, apparently random combinations of activated glomeruli. How these 'discontinuous' receptive fields are formed from OB inputs remains unknown. Counter to the prevailing view that olfactory PNs sum their inputs passively, we show for the first time that NMDA spikes within individual dendrites can both amplify OB inputs and impose combination selectivity upon them, while their ability to compartmentalize voltage signals allows different dendrites to represent different odorant combinations. Thus, the 2-layer integrative behavior of olfactory PN dendrites provides a parsimonious account for the nonlinear remapping of the odor code from bulb to cortex.


Assuntos
Potenciais de Ação/efeitos dos fármacos , N-Metilaspartato/farmacologia , Córtex Piriforme/fisiologia , Animais , Cálcio/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/fisiologia , Feminino , Ácido Glutâmico/metabolismo , Masculino , Modelos Neurológicos , Dinâmica não Linear , Condutos Olfatórios/efeitos dos fármacos , Condutos Olfatórios/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinapses/fisiologia
17.
Sci Rep ; 8(1): 17700, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30531887

RESUMO

Retinal degenerative diseases, due to the lack of regeneration systems and self-renewable cells, often lead to visual impairment. Pax6 is a pleiotropic transcription factor and its expression level determines self-renewal status or differentiation of retinal cells. Here, we investigated the fate of simultaneous induction of retinal ganglion cell death and Pax6 overexpression in retro-differentiation of retinal cells and their commitment to re-enter into the cell cycle. Induction of acute retinal ganglion cell death and generation of mouse experimental model was performed by N-methyl D-aspartic acid (NMDA) injection. Recombinant AAV2 virus harboring PAX6 cDNA and reporter gene was injected into untreated and model mouse eyes. Histological analyses, including IHC and retinal flatmounts immunostaining were performed. The number of Ki67+ cells was clearly increased in model mice, presumably due to NMDA treatment and regardless of Pax6 over-expression. Unlike previous studies, Ki67+ cells were found in GCL layer and interestingly ONL cells expressed Sox2 stemness marker after NMDA cytotoxicity. The potential of retinal cells for robust Ki67 expression, after injury, and expression of Sox2, confirmed their intrinsic plasticity and made a vivid prospect for retinal regenerative medicine.


Assuntos
Proliferação de Células/efeitos dos fármacos , N-Metilaspartato/farmacologia , Fator de Transcrição PAX6/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células HEK293 , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Modelos Animais , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo
18.
Biochem Biophys Res Commun ; 506(3): 648-652, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30454701

RESUMO

Homocysteine (HCY) induced neurotoxicity largely depends on interaction of this endogenous amino acid with glutamate NMDA receptors (NMDARs). This receptor type is composed by GluN1 and different GluN2 (A, B, C or D) subunits. However, the receptor activity of HCY in brain regions which differ in relative contribution of GluN2 subunits was not tested so far. In the current study, we explored the action of HCY on cerebellar neurons which natively express GluN2C and GluN2D subunits of NMDARs and compared this with the action of HCY on cortical neurons which are mainly composed by GluN2A and GluN2B subunits. To validate obtained results, we also studied the responses to HCY in recombinant GluN1/2C and GluN1/2D NMDARs expressed in HEK293T cells. Responses to HCY were compared to membrane currents evoked by glutamate or by the specific agonist NMDA. First, we found that on HEK cells expressing GluN1/2C or GluN1/2D NMDARs, HCY was full agonist producing membrane currents similar in amplitude to currents induced by glutamate. The EC50 values for these particular receptor subtype activation were 80 µM and 31 µM, respectively. Then, we found that HCY similarly to NMDA, evoked large slightly desensitizing membrane currents in native NMDARs of cerebellar and cortical neurons. In cortical neurons, the ratio of the respective currents (IHCY/INMDA) was 0.16 and did not significantly change during in vitro maturation. In sharp contrast, in cerebellar neurons, the ratio of currents evoked by HCY and NMDA was dramatically increased from 0.31 to 0.72 from 7 to 21 day in culture. We show that least 75% of HCY-induced currents in cerebellum were mediated by GluN2C- or GluN2D-containing NMDARs. Thus, our data revealed a large population of cerebellar NMDA receptors highly sensitive to HCY which suggest potential vulnerability of this brain region to pathological conditions associated with enhanced levels of this neurotoxic amino acid.


Assuntos
Cerebelo/citologia , Homocisteína/farmacologia , Neurônios/metabolismo , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/agonistas
19.
Can J Neurol Sci ; 45(6): 675-681, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30430968

RESUMO

BACKGROUND: We have previously shown that low-intensity ultrasound (LIUS), a noninvasive mechanical stimulus, inhibits brain edema formation induced by oxygen and glucose deprivation (OGD) or treatment with glutamate, a mediator of OGD-induced edema, in acute rat hippocampal slice model in vitro. METHODS: In this study, we treated the rat hippocampal slices with N-methyl-d-aspartic acid (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) to determine whether these different glutamate receptor agonists induce edema. The hippocampal slices were then either sonicated with LIUS or treated with N-methyl-d-aspartic acid receptor (NMDAR) antagonists, namely, MK-801 and ketamine, and observed their effects on edema formation. RESULTS: We observed that treatment with NMDA, an agonist of ionotropic glutamate receptors, induced brain edema at similar degrees compared with that induced by OGD. However, treatment with DHPG, an agonist of metabotropic glutamate receptors, did not significantly induce brain edema. Treatment with the NMDAR antagonists MK-801 or ketamine efficiently prevented brain edema formation by both OGD and NMDA in a concentration-dependent manner. N-Methyl-d-aspartic acid-induced brain edema was alleviated by LIUS in an intensity-dependent manner when ultrasound was administered at 30, 50, or 100 mW/cm2 for 20 minutes before the induction of the edema. Furthermore, LIUS reduced OGD- and NMDA-induced phosphorylation of NMDARs at Y1325. CONCLUSION: These results suggest that LIUS can inhibit OGD- or NMDA-induced NMDAR activation by preventing NMDAR phosphorylation, thereby reducing a subsequent brain edema formation. The mechanisms by which LIUS inhibits NMDAR phosphorylation need further investigation.


Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Ultrassonografia , Animais , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Ratos Sprague-Dawley , Receptores de Aminoácido/efeitos dos fármacos , Receptores de Aminoácido/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Ultrassonografia/efeitos adversos
20.
Cell Physiol Biochem ; 51(1): 97-112, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30439717

RESUMO

BACKGROUND/AIMS: The N-methyl-D-aspartic acid receptor (NMDAR) has been extensively studied for its important roles in synaptic plasticity and learning and memory. However, the effects of microwave radiation on the subunit composition and activity of NMDARs and the relationship between NMDARs and microwave-induced synaptic plasticity have not been thoroughly elucidated to date. MATERIALS: In our study, primary hippocampal neurons were used to evaluate the effects of microwave radiation on synaptic plasticity. Structural changes were observed by diolistic (Dil) labeling and scanning electron microscopy (SEM) observation. Functional synaptic plasticity was reflected by the NMDAR currents, which were detected by whole cell patch clamp. We also detected the expression of NMDAR subunits by real-time PCR and Western blot analysis. To clarify the effects of microwave radiation on NMDAR-induced synaptic plasticity, suitable agonists or inhibitors were added to confirm the role of NMDARs on microwave-induced synaptic plasticity. Dil labeling, SEM observation, whole cell patch clamp, real-time PCR and Western blot analysis were used to evaluate changes in synaptic plasticity after treatment with agonists or inhibitors. RESULTS: Our results found that microwave exposure impaired neurite development and decreased mRNA and protein levels and the current density of NMDARs. Due to the decreased expression of NMDAR subunits after microwave exposure, the selective agonist NMDA was added to identify the role of NMDARs on microwave-induced synaptic plasticity injuries. After adding the agonist, the expression of NMDAR subunits recovered to the normal levels. In addition, the microwave-induced structural and functional synaptic plasticity injuries recovered, including the number and length of neurites, the connections between neurons, and the NMDAR current. CONCLUSION: Microwave radiation caused neuronal synaptic plasticity injuries in primary hippocampal neurons, and NMDARs played protective roles on the damage process.


Assuntos
Micro-Ondas , Plasticidade Neuronal/efeitos da radiação , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Microscopia Confocal , N-Metilaspartato/farmacologia , Neuritos/fisiologia , Neuritos/efeitos da radiação , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/agonistas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética
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