RESUMEN
The complement C1q plays a critical role in microglial phagocytosis of glutamatergic synapses and in the pathogenesis of neuroinflammation in Alzheimer's disease (AD). We recently reported that upregulation of metabotropic glutamate receptor signaling is associated with increased synaptic C1q production and subsequent microglial phagocytosis of synapses in the rodent models of AD. Here, we explored the role of astrocytic glutamate transporter in the synaptic C1q production and microglial phagocytosis of hippocampal glutamatergic synapses in a rat model of AD. Activation of astrocyte and reduction glutamate transporter 1 (GLT1) were noted after bilateral microinjection of amyloid-beta (Aß1-40) fibrils into the hippocampal CA1 area of rats. Ceftriaxone is a ß-lactam antibiotic that upregulates GLT1 expression. Bilateral microinjection of ceftriaxone recovered GLT1 expression, decreased synaptic C1q production, suppressed microglial phagocytosis of glutamatergic synapses in the hippocampal CA1, and attenuated synaptic and cognitive deficits in rats microinjected with Aß1-40. In contrast, artificial suppression of GLT1 activity by DL-threo-beta-benzyloxyaspartate (DL-TBOA) in naïve rats induced synaptic C1q expression and microglial phagocytosis of glutamatergic synapses in the hippocampal CA1 area, resulting in synaptic and cognitive dysfunction. These findings demonstrated that impairment of astrocytic glutamate transporter plays a role in the pathogenesis of AD.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides/toxicidad , Astrocitos/efectos de los fármacos , Región CA1 Hipocampal/efectos de los fármacos , Trastornos del Conocimiento/inducido químicamente , Complemento C1q/fisiología , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Ácido Glutámico/fisiología , Microglía/fisiología , Neuronas/metabolismo , Fragmentos de Péptidos/toxicidad , Animales , Ácido Aspártico/farmacología , Astrocitos/metabolismo , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Ceftriaxona/farmacología , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Complemento C1q/biosíntesis , Complemento C1q/genética , Modelos Animales de Enfermedad , Transportador 2 de Aminoácidos Excitadores/biosíntesis , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/fisiología , Masculino , Prueba del Laberinto Acuático de Morris/efectos de los fármacos , Prueba del Laberinto Acuático de Morris/fisiología , Técnicas de Placa-Clamp , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/fisiología , Sinapsis/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Alterations of energy metabolism and of astrocyte number/function in ventral anterior cingulate cortex (vACC) have been reported in major depressive disorder (MDD) patients and may contribute to MDD pathophysiology. We recently developed a mouse model of MDD mimicking these alterations. We knocked down the astroglial glutamate transporters GLAST and GLT-1 in infralimbic cortex (IL, rodent equivalent of vACC) using small interfering RNA (siRNA). GLAST and GLT-1 siRNA microinfusion in IL evoked a depressive-like phenotype, associated with a reduced serotonergic function and reduced forebrain BDNF expression. Neither effect occurred after siRNA application in the adjacent prelimbic cortex (PrL), thus emphasizing the critical role of vACC/IL in MDD pathogenesis. Here we examined the cellular/network basis of the changes induced in IL using intracellular recordings of layer V pyramidal neurons from mice microinjected with siRNA 24â¯h before. We analyzed (i) the electrophysiological characteristics of neurons; (ii) the synaptic transmission properties, by monitoring miniature, spontaneous and evoked EPSCs, and (iii) the gliotransmission, by monitoring slow inward currents (SICs), mediated by astrocytic glutamate release and activation of extra-synaptic NMDA receptors. GLT-1 and GLAST knockdown led to a more depolarized membrane potential and increased action potential firing rate of layer V pyramidal neurons, and enhanced excitatory synaptic transmission, as shown by the enhanced amplitude/frequency of spontaneous EPSCs. Gliotransmission was also increased, as indicated by the enhanced SIC amplitude/frequency. Hence, the depressive-like phenotype is associated with IL hyperactivity, likely leading to an excessive top-down inhibitory control of serotonergic activity through IL-midbrain descending pathways.
Asunto(s)
Astrocitos/metabolismo , Transportador 1 de Aminoácidos Excitadores/fisiología , Transportador 2 de Aminoácidos Excitadores/fisiología , Giro del Cíngulo/metabolismo , Transmisión Sináptica/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Trastorno Depresivo Mayor/metabolismo , Trastorno Depresivo Mayor/fisiopatología , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Técnicas de Silenciamiento del Gen , Giro del Cíngulo/efectos de los fármacos , Lóbulo Límbico/efectos de los fármacos , Ratones , Microinyecciones , Prosencéfalo/metabolismo , Células Piramidales/fisiología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
The previous studies have shown that glial glutamate transporter-1 (GLT-1) participates in cerebral ischemic injury in rats. However, the mechanism involved remains to be elucidated. This study was undertaken to investigate whether p38 MAPK was involved in regulating GLT-1 in the process. At first, it was observed that global brain ischemia for 8 min led to obvious delayed neuronal death, GLT-1 down-regulation and p-p38 MAPK up-regulation in CA1 hippocampus in rats. Then, whether p-p38 MAPK was involved in regulating GLT-1 during cerebral ischemic injury was studied in vitro. Astrocyte-neuron co-cultures exposed to oxygen and glucose deprivation (OGD) were used to mimic brain ischemia. It was observed that lethal OGD (4-h OGD) decreased GLT-1 expression and increased p-p38 MAPK expression in astrocytes. The p-p38 MAPK protein rised from 0 min to 48 h that is the end time of the observation, and the peak value was at 12 h, which was 12.45 times of the control group. Moreover, pre-administration of p38 MAPK inhibitor SB203580 or its siRNA dose-dependently increased GLT-1 expression, and meanwhile alleviated the neuronal death induced by lethal OGD. The above results indicated that p38 MAPK signaling pathway participated in regulating GLT-1 during OGD injury in vitro. Finally, back to in vivo experiment, it was found that pre-administration of SB203580 by intracerebroventricular injection dose-dependently reversed the down-regulation of GLT-1 expression and attenuated the delayed neuronal death normally induced by global brain ischemia in CA1 hippocampus in rats. Taken together, it can be concluded that the mechanism of GLT-1 mediating cerebral ischemic injury depends on the activation of p38 MAPK.
Asunto(s)
Isquemia Encefálica/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Astrocitos/metabolismo , Isquemia Encefálica/fisiopatología , Región CA1 Hipocampal/metabolismo , Muerte Celular , Técnicas de Cocultivo , Transportador 2 de Aminoácidos Excitadores/fisiología , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas , Masculino , Neuronas/metabolismo , Oxígeno/metabolismo , Piridinas/farmacología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
AIM: Deficient glutamate reuptake occurs in the cerebral cortex of Huntington's disease (HD) patients and murine models. Here, we examine the effects of partial or complete blockade of glutamate transporters on excitatory postsynaptic currents (EPSCs) of cortical pyramidal neurons (CPNs). METHODS: Whole-cell patch clamp recordings of CPNs in slices from symptomatic R6/2 mice and wild-type (WT) littermates were used to examine the effects of selective or concurrent inhibition of glutamate reuptake transporters. RESULTS: Selective inhibition of the glial glutamate transporter 1 (GLT-1) or the glutamate aspartate transporter (GLAST) produced slight decreases in decay time of evoked EPSCs in CPNs from WT and R6/2 mice with no significant differences between genotypes. In contrast, concurrent inhibition of both transporters with DL-TBOA induced a significant increase in area and decay time and this effect was significantly greater in R6/2 CPNs. Furthermore, full blockade also reduced spontaneous EPSC frequency and exacerbated epileptiform activity in CPNs from symptomatic R6/2 mice. CONCLUSIONS: R6/2 CPNs are more sensitive to glutamate accumulation during full inhibition of both glutamate transporters, and these neurons have homeostatic mechanisms to cope with inhibition of GLT-1 or GLAST by a mechanism that involves upregulation of either transporter when the other is deficient.
Asunto(s)
Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Transportador 1 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Enfermedad de Huntington/fisiopatología , Animales , Ácido Aspártico/farmacología , Benzopiranos/farmacología , Transportador 1 de Aminoácidos Excitadores/fisiología , Transportador 2 de Aminoácidos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Transgénicos , Técnicas de Cultivo de ÓrganosRESUMEN
Gabapentinoids are effective in a wide range of animal pain models and in patients with neuropathic pain and has become one of first-line treatments for neuropathic pain. Because spinal plasticity and sensitization have been intensely studied in neuropathic pain, most laboratory studies have focused on actions of gabapentinoids in the spinal cord, where they reduce primary afferent traffic and excitation of spinal nociceptive neurons, via interaction with α2δ subunits of voltage-gated Ca2+ channels. However, a recent clinical study questioned the relevance of this in vitro and in vivo rodent studies by demonstrating a complete lack of clinical efficacy of intrathecal gabapentin in patients with chronic pain. Curiously, preclinical studies continue to focus on spinal cord actions of gabapentinoids despite this lack of translation to humans.We and others demonstrated that gabapentin inhibits presynaptic GABA release and induces glutamate release from astrocytes in the locus coeruleus (LC), thereby increasing LC neuron activity and spinal noradrenaline release, and that gabapentin relies on this action in the LC for its analgesia. We also recently discovered that, with prolonged time after neuropathic injury, noradrenergic neurons in the LC become less responsive to gabapentin, leading to impaired gabapentin analgesia, and that astroglial glutamate dysregulation is critical to this impaired LC response. The clinically available drug valproate increases glutamate transporter-1 (GLT-1) expression in the LC to restore this impaired gabapentin analgesia.
Asunto(s)
Analgesia , Gabapentina/farmacología , Locus Coeruleus/fisiología , Neuralgia/tratamiento farmacológico , Norepinefrina/fisiología , Animales , Transportador 2 de Aminoácidos Excitadores/fisiología , Ácido Glutámico/fisiología , Humanos , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Ácido gamma-AminobutíricoRESUMEN
We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate transporter in astrocytes [excitatory amino acid transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Astrocitos/fisiología , MicroARNs/metabolismo , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/fisiología , Animales , Astrocitos/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/fisiología , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/fisiología , Neuronas Motoras/metabolismo , Neuronas Motoras/fisiología , Neuroglía/metabolismoRESUMEN
The hypothalamus plays a central role in controlling poultry endocrine and reproductive activities. So far there is limited information focused on the proteome profiles of the hypothalamus from geese during different stages of the egg-laying cycle. In order to identify proteins regulating the egg-laying process of Huoyan geese, we investigated the proteome profiles of the hypothalamus from Huoyan geese during the laying period and pre-laying period by applying an isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic technology. A total number of 3,337 were identified and quantified, of which 18 were significantly up-regulated and 16 were significantly down-regulated. These differentially expressed proteins were subjected to bioinformatics analyses based on the Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway. Some of these were revealed to be involved in hormone and neurotransmitter secretion, exocytosis, calcium ion transport and synaptic transmission. Subsequently, excitatory amino acid transporter 2, complexin-1 and inositol 1,4,5-trisphosphate receptor, type 3 were confirmed at the messenger RNA level using quantitative real-time RT-PCR. Then, the abundance change of these proteins was verified further using Western blotting analysis. These data may aid in elucidating the molecular mechanism of higher laying performance in Huoyan geese.
Asunto(s)
Proteínas Aviares/genética , Proteínas Aviares/fisiología , Gansos/fisiología , Hipotálamo/química , Oviparidad/genética , Proteoma/genética , Proteómica/métodos , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Animales , Regulación hacia Abajo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/fisiología , Femenino , Hipotálamo/fisiología , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Proteoma/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
Objective To investigate the effect of dopamine (DA) on the glutamate (Glu)-uptake ability of astrocytes, and the role of trace amine-associated receptor 1-excitatory amino acid transporter 2 (TAAR1-EAAT2) signaling pathway in Glu uptake by astrocytes. Methods In the primary cultured astrocytes pretreated with DA, extracellular Glu levels were measured by the Amplex Red glutamic acid assay kit. The levels of TAAR1 and EAAT2 transcriptions were detected by reverse transcription PCR and their protein levels were analyzed by Western blotting. After TAAR1 plasmid and TAAR1 siRNA were separately transfected into the primary astrocytes pretreated by DA, Western blotting was performed to determine the level of EAAT2 and Amplex Red glutamic acid assay kit was used to analyze Glu uptake in primary cultured astrocyte supernatants. Results The expression of EAAT2 in the primary cultured astrocytes significantly decreased in response to DA, and the level of TAAR1 increased. DA significantly enhanced the Glu uptake in primary cultured astrocyte supernatants. After TAAR1 siRNA transfection, EAAT2 expression was upregulated by DA treatment and Glu content in the supernatants was downregulated. On the contrary, after TAAR1 plasmid transfection, EAAT2 expression descended and Glu level ascended in the supernatants. Conclusion DA reduces the Glu-uptake ability of astrocytes through TAAR1-EAAT2 signaling pathway, causes extracellular Glu accumulation, and ultimately destroys the function of astrocytes.
Asunto(s)
Astrocitos/efectos de los fármacos , Dopamina/farmacología , Transportador 2 de Aminoácidos Excitadores/fisiología , Ácido Glutámico/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Animales , Astrocitos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
KEY POINTS: Excitatory amino acid transporter 2 (EAAT2) is present on astrocytes in the nucleus tractus solitarii (nTS), an important nucleus in cardiorespiratory control. Its specific role in influencing nTS neuronal activity and thereby basal and reflex cardiorespiratory function is unknown. The specific role of nTS EAAT2 was determined via whole animal and brainstem slice patch clamp experiments. Astrocytic EAAT2 buffers basal glutamate activation of AMPA-type glutamate receptors and therefore decreases baseline excitability of nTS neurons. EAAT2 modulates cardiorespiratory control and tempers excitatory cardiorespiratory responses to activation of the peripheral chemoreflex. This study supports the concept that nTS astrocyte transporters influence sympathetic nervous system activity and cardiorespiratory reflex function in health and disease. ABSTRACT: Glutamatergic signalling is critical in the nucleus tractus solitarii (nTS) for cardiorespiratory homeostasis and initiation of sensory reflexes, including the chemoreflex activated during hypoxia. Maintenance of nTS glutamate concentration occurs in part through astrocytic excitatory amino acid transporters (EAATs). We previously established the importance of EAATs in the nTS by demonstrating their inhibition produced neuronal excitation to alter basal cardiorespiratory function. Since EAAT2 is the most expressed EAAT in the nTS, this study specifically determined EAAT2's role in nTS astrocytes, its influence on neuronal and synaptic properties, and ultimately on basal and reflex cardiorespiratory function. The EAAT2-specific antagonist dihydrokainate (DHK) was microinjected into the anaesthetized rat nTS or applied to rat nTS slices. DHK produced depressor, bradycardic and sympathoinhibitory responses and reduced neural respiration in the intact rat, mimicking responses to glutamate excitation. DHK also enhanced responses to glutamate microinjection. DHK elevated extracellular nTS glutamate concentration, depolarized neurons and enhanced spontaneous EPSCs. EAAT2 block also augmented action potential discharge in chemosensitive nTS neurons. Glial recordings confirmed EAAT2 is functional on nTS astrocytes. Neuronal excitation and cardiorespiratory effects following EAAT2 inhibition were due to activation of putative extrasynaptic AMPA receptors as their antagonism blocked DHK responses in the intact rat nTS and the slice. The DHK-induced elevation of extracellular glutamate and neuronal excitation augmented chemoreflex-mediated pressor, sympathoexcitatory and minute neural ventilation responses in the rat. These data shed new light on the important role astrocytic EAAT2 plays on buffering nTS excitation and overall cardiorespiratory function.
Asunto(s)
Transportador 2 de Aminoácidos Excitadores/fisiología , Neuroglía/fisiología , Respiración , Núcleo Solitario/fisiología , Potenciales de Acción , Animales , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacología , Masculino , Neuronas/fisiología , Ratas Sprague-Dawley , Receptores AMPA/fisiología , Reflejo/fisiología , Núcleo Solitario/metabolismoRESUMEN
Astrocytes, via excitatory amino-acid transporter type-2 (EAAT2), are the major sink for released glutamate and contribute to set the strength and timing of synaptic inputs. The conditions required for the emergence of Hebbian plasticity from distributed neural activity remain elusive. Here, we investigate the role of EAAT2 in the expression of a major physiologically relevant form of Hebbian learning, spike timing-dependent plasticity (STDP). We find that a transient blockade of EAAT2 disrupts the temporal contingency required for Hebbian synaptic plasticity. Indeed, STDP is replaced by aberrant non-timing-dependent plasticity occurring for uncorrelated events. Conversely, EAAT2 overexpression impairs the detection of correlated activity and precludes STDP expression. Our findings demonstrate that EAAT2 sets the appropriate glutamate dynamics for the optimal temporal contingency between pre- and postsynaptic activity required for STDP emergence, and highlight the role of astrocytes as gatekeepers for Hebbian synaptic plasticity.
Asunto(s)
Astrocitos/fisiología , Cuerpo Estriado/fisiología , Plasticidad Neuronal/fisiología , Potenciales de Acción , Animales , Astrocitos/efectos de los fármacos , Ceftriaxona/farmacología , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Transportador 2 de Aminoácidos Excitadores/agonistas , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/fisiología , Técnicas In Vitro , Potenciación a Largo Plazo , Depresión Sináptica a Largo Plazo , Masculino , Modelos Neurológicos , Ratas , Receptores de N-Metil-D-Aspartato/fisiología , Filtrado Sensorial , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
To maintain reliable signal transmission across a synapse, free synaptic neurotransmitters must be removed from the cleft in a timely manner. In the first visual synapse, this critical task is mainly undertaken by glutamate transporters (EAATs). Here we study the differential roles of the EAAT1, EAAT2 and EAAT5 subtypes in glutamate (GLU) uptake at the photoreceptor-to-depolarizing bipolar cell synapse in intact dark-adapted retina. Various doses of EAAT blockers and/or GLU were injected into the eye before the electroretinogram (ERG) was measured. Their effectiveness and potency in inhibiting the ERG b-wave were studied to determine their relative contributions to the GLU clearing activity at the synapse. The results showed that EAAT1 and EAAT2 plays different roles. Selectively blocking glial EAAT1 alone using UCPH101 inhibited the b-wave 2-24h following injection, suggesting a dominating role of EAAT1 in the overall GLU clearing capacity in the synaptic cleft. Selectively blocking EAAT2 on photoreceptor terminals had no significant effect on the b-wave, but increased the potency of exogenous GLU in inhibiting the b-wave. These suggest that EAAT2 play a secondary yet significant role in the GLU reuptake activity at the rod and the cone output synapses. Additionally, we have verified our electrophysiological findings with double-label immunohistochemistry, and extend the literature on the spatial distribution of EAAT2 splice variants in the mouse retina.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/fisiología , Transportador 2 de Aminoácidos Excitadores/fisiología , Transportador 5 de Aminoácidos Excitadores/fisiología , Glutamatos/metabolismo , Retina/fisiología , Transmisión Sináptica/efectos de los fármacos , Animales , Transporte Biológico , Adaptación a la Oscuridad/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Electrorretinografía/efectos de los fármacos , Transportador 1 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 5 de Aminoácidos Excitadores/metabolismo , Glutamatos/farmacología , Inmunohistoquímica , Inyecciones Intravítreas , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/metabolismo , Retina/efectos de los fármacos , Células Bipolares de la Retina/efectos de los fármacos , Células Bipolares de la Retina/metabolismoRESUMEN
Thiamine deficiency (TD) is the underlying cause of Wernicke's encephalopathy (WE), an acute neurological disorder characterized by structural damage to key periventricular structures in the brain. Increasing evidence suggests these focal histological lesions may be representative of a gliopathy in which astrocyte-related changes are a major feature of the disorder. These changes include a loss of the glutamate transporters GLT-1 and GLAST concomitant with elevated interstitial glutamate levels, lowered brain pH associated with increased lactate production, decreased levels of GFAP, reduction in the levels of glutamine synthetase, swelling, alterations in levels of aquaporin-4, and disruption of the blood-brain barrier. This review focusses on how these manifestations contribute to the pathophysiology of TD and possibly WE.
Asunto(s)
Astrocitos/fisiología , Deficiencia de Tiamina/fisiopatología , Sistema de Transporte de Aminoácidos X-AG/fisiología , Animales , Transporte Biológico , Barrera Hematoencefálica , Encéfalo/patología , Modelos Animales de Enfermedad , Transportador 2 de Aminoácidos Excitadores/fisiología , Ácido Glutámico/metabolismo , Humanos , Complejo Cetoglutarato Deshidrogenasa/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo , Piritiamina/toxicidad , Deficiencia de Tiamina/inducido químicamente , Deficiencia de Tiamina/metabolismo , Encefalopatía de Wernicke/etiología , Encefalopatía de Wernicke/metabolismo , Encefalopatía de Wernicke/fisiopatologíaRESUMEN
In recent years, interactions between neurons and glia have been evaluated as mediators of neuropsychiatric diseases, including drug addiction. In particular, compounds that increase expression of the astroglial glutamate transporter GLT-1 (N-acetylcysteine and ceftriaxone) can decrease measures of drug seeking. However, it is unknown whether the compounds that influence broad measures of glial physiology can influence behavioral measures of drug relapse, nor is it clear whether the upregulated GLT-1 is functionally important for suppressing of drug seeking. To address these questions, we sought to determine whether the glial modulator and neuroprotective agent propentofylline (PPF) modifies drug seeking in rats using a reinstatement model of cocaine relapse. We found that 7 days of chronic (but not acute) administration of PPF significantly decreased both cue- and cocaine-induced reinstatement of cocaine seeking. We next determined whether the effect of systemic PPF on reinstatement depended upon its ability to restore expression of GLT-1 in the nucleus accumbens. PPF restored the cocaine-induced decrease in GLT-1 in the accumbens core; then, using an antisense strategy against glutamate transporter GLT-1, we found that restored transporter expression was necessary for PPF to inhibit cue-primed cocaine seeking. These findings indicate that modulating glial physiology with atypical xanthine derivatives like PPF is a potential avenue for developing new medications for cocaine abuse, and support the hypothesis that neuron-glial interactions contribute to mechanisms of psychostimulant addiction, particularly via expression and function of astroglial glutamate transporters.
Asunto(s)
Conducta Adictiva/metabolismo , Conducta Adictiva/prevención & control , Cocaína/administración & dosificación , Transportador 2 de Aminoácidos Excitadores/fisiología , Xantinas/administración & dosificación , Animales , Conducta Adictiva/fisiopatología , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Núcleo Accumbens/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-L-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.
Asunto(s)
Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores/fisiología , Fibronectinas/química , Ácido Glutámico/metabolismo , Ácido Láctico/química , Polímeros/química , Animales , Astrocitos/citología , Adhesión Celular , Células Cultivadas , Poliésteres , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Andamios del TejidoRESUMEN
Relapse to cocaine-seeking behavior requires an increase in nucleus accumbens (NAc) core glutamate transmission. Decreased expression of glutamate type I transporter (GLT1), which is responsible for >90% of glutamate clearance, occurs in the core of rats withdrawn from cocaine self-administration, while treatment with ceftriaxone, a ß-lactam antibiotic previously shown to increase GLT1 expression and function in rodents, upregulates GLT1 and attenuates cue-induced cocaine reinstatement. Here, we tested the effects of increasing GLT1 expression on cue-induced cocaine seeking in rats exposed to either limited (2 h/d) or extended (6 h/d) cocaine access followed by short (2 d) or long (45 d) withdrawal periods. Treatment with ceftriaxone (200 mg/kg, i.p.) upregulated core GLT1 expression and attenuated cue-induced cocaine-seeking behavior only in rats exposed to long withdrawal periods, with a greater effect in the extended-access condition. Pearson's correlation revealed GLT1 expression in core to be inversely correlated with cue-induced cocaine-seeking behavior. To localize the effects of GLT1 upregulation within NAc, we tested the hypothesis that blockade of GLT1 in NAc core, but not shell, would reverse the ceftriaxone-mediated effect. Rats withdrawn from cocaine self-administration were treated with the same dose of ceftriaxone followed by intracore or intrashell infusions of one of two GLT1 blockers, dihydrokainic acid (500 µM) or DL-threo-ß-benzyloxyaspartate (250 µM), or saline. Our results reveal that the ceftriaxone-mediated attenuation of cue-induced cocaine reinstatement is reversed by GLT1 blockade in core, but not shell, and further implicate core GLT1 as a potential therapeutic target for cocaine relapse.
Asunto(s)
Trastornos Relacionados con Cocaína/psicología , Señales (Psicología) , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/fisiología , Núcleo Accumbens/fisiología , Análisis de Varianza , Animales , Ácido Aspártico/farmacología , Conducta Animal/efectos de los fármacos , Western Blotting , Ceftriaxona/farmacología , Trastornos Relacionados con Cocaína/fisiopatología , Condicionamiento Operante/efectos de los fármacos , Conducta Alimentaria/fisiología , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacología , Masculino , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/patología , Ratas , Ratas Sprague-Dawley , Recurrencia , Síndrome de Abstinencia a Sustancias/psicologíaRESUMEN
It is widely known that prenatal stress (PS) exposure causes depression-like behaviour to offspring, as well as maladaptive responses including neurobiological and physiological changes. However, the underlying mechanism of PS induced juvenile-onset depression remains largely unravelled. The inadequacies of monoamine deficiency hypothesis, the emerging evidence of altered glutamate neurotransmission in mood disorders, as well as our previous studies inspired us to assess the potential role of glutamatergic system in the pathogenesis of juvenile depression. In this research, we examined the expression of phosphorylated GluR1 subunit of ionotropic receptor alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), the Na+-dependent glutamate transporters excitatory amino acid transporter 2 (EAAT2) and EAAT3 in the hippocampus, striatum and frontal cortex of 1-month-old rat offspring after mid and late PS exposure. Prenatally stressed offspring rats showed significantly prolonged duration of immobility and shortened immobility latency in tail suspension test. We also detected that PS significantly altered the expression of glutamate receptor and glutamate transporters of these depressed rats. In brief, the changes of phosphorylated GluR1 subunit of AMPAR protein level in the hippocampus and frontal cortex, as well as markedly decreased EAAT2 mRNA expression in the hippocampus, striatum and frontal cortex and EAAT3 mRNA expression in the hippocampus of stressed rats were both observed. These results underpinned that glutamate receptors and glutamate transporters might be involved in the progress of depression-like behaviour in juvenile rat offspring induced by PS.
Asunto(s)
Depresión/psicología , Transportador 2 de Aminoácidos Excitadores/fisiología , Transportador 3 de Aminoácidos Excitadores/fisiología , Efectos Tardíos de la Exposición Prenatal/psicología , Receptores AMPA/fisiología , Animales , Western Blotting , Depresión/etiología , Femenino , Ácido Glutámico/fisiología , Suspensión Trasera , Hipocampo/metabolismo , Neostriado/metabolismo , Corteza Prefrontal/metabolismo , Embarazo , ARN/biosíntesis , ARN/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Restricción FísicaRESUMEN
A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABA(A)ergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABA(A) receptors (GABA(A)Rs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABA(A)Rs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR-GABA(A)R crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate-GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate-GABA interactions in MNCs, which involves crosstalk between NMDA and GABA(A) postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABA(A)Rs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure.
Asunto(s)
Astrocitos/fisiología , Hipotálamo/fisiología , Neuronas/fisiología , Sistemas Neurosecretores/fisiología , Receptor Cross-Talk/fisiología , Receptores de GABA-A/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Astrocitos/efectos de los fármacos , Señalización del Calcio/fisiología , Fenómenos Electrofisiológicos , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/fisiología , Agonistas del GABA/farmacología , Glutamatos/fisiología , Insuficiencia Cardíaca/fisiopatología , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Masculino , Muscimol/farmacología , Neuronas/efectos de los fármacos , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/efectos de los fármacos , Óxido Nítrico/fisiología , Técnicas de Placa-Clamp , Proteínas Quinasas/fisiología , Ratas , Ratas Wistar , Receptor Cross-Talk/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB) was identified as the selective blocker of volume-regulated anion channels (VRAC). VRAC are permeable to small inorganic and organic anions, including the excitatory neurotransmitter glutamate. In recent years DCPIB has been increasingly used for probing the physiologic and pathologic roles of VRAC and was found to potently suppress pathologic glutamate release in cerebral ischemia. Because ischemic glutamate release can be mediated by a plethora of mechanisms, in this study we explored the selectivity of DCPIB toward the majority of previously identified glutamate transporters and permeability pathways. l-[(3)H]glutamate, d-[(3)H]aspartate, and l-[(14)C]cystine were used to trace amino acid release and uptake. We found that in addition to its well-characterized effect on VRAC, DCPIB potently inhibited glutamate release via connexin hemichannels and glutamate uptake via the glutamate transporter GLT-1 in rat glial cells. In contrast, DCPIB had no direct effect on vesicular glutamate release from rat brain synaptosomes or the cystine/glutamate exchange in astrocytes. The compound did not affect the astrocytic glutamate transporter GLAST, nor did it block glutamate release via the P2X(7)/pannexin permeability pathway. The ability of DCPIB to directly block connexin hemichannels was confirmed using a gene-specific siRNA knockdown approach. Overall, our data demonstrate that DCPIB influences several glutamate transport pathways and that its effects on VRAC in vivo should be verified using additional pharmacological controls.
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Sistemas de Transporte de Aminoácidos/fisiología , Astrocitos/efectos de los fármacos , Ciclopentanos/farmacología , Ácido Glutámico/metabolismo , Indanos/farmacología , Microglía/efectos de los fármacos , Adenosina Trifosfato/farmacología , Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos y+/fisiología , Sistemas de Transporte de Aminoácidos/antagonistas & inhibidores , Sistemas de Transporte de Aminoácidos Acídicos , Animales , Astrocitos/metabolismo , Transporte Biológico , Células Cultivadas , Corteza Cerebral/citología , Conexinas/antagonistas & inhibidores , Conexinas/fisiología , Transportador 1 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 1 de Aminoácidos Excitadores/fisiología , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 2 de Aminoácidos Excitadores/fisiología , Microglía/metabolismo , Permeabilidad , Cultivo Primario de Células , Ratas , Receptores Purinérgicos P2X7/fisiología , Sinaptosomas/metabolismoRESUMEN
Glutamate transporter-1 (GLT-1) plays a dual role in glutamate transportation: both normally devotion to the clearance of glutamate and during some pathological conditions extruding glutamate to the extracellular space. Therefore, it is uncertain whether increased expression of GLT-1 will actually be helpful against glutamate excitotoxicity. In this study, GLT-1 up-regulation was induced by ceftriaxone, and L-glutamate was added to induce glutamate toxicity in primary cultured rat cortical cells. The results showed that up-regulated GLT-1 induced by 1 µM ceftriaxone for 2 days markedly increased cell viability, decreased apoptotic cell death and alleviated ultrastructural damage induced by 50 µM glutamate 15 min. as well as promoted L-[(3) H]-glutamate uptake in cultured cells. GLT-1 up-regulation had no effect on the intracellular free calcium concentration ([Ca(2+) ](i) ) in the resting situation, while relieved intracellular calcium overloading by reducing the elevation and promoting the recovery of [Ca(2+) ](i) following stimulation of 50 µM glutamate for 2 min. Applying 100 µM dihydrokainic acid (GLT-1 antagonist) 30 sec. before glutamate eliminated the above effect of GLT-1 up-regulation on [Ca(2+) ](i) . In conclusion, GLT-1 up-regulation induced by ceftriaxone plays a positive glutamate transporting role against glutamate toxicity in primary cultured rat cortical cells.
Asunto(s)
Calcio/metabolismo , Corteza Cerebral/efectos de los fármacos , Transportador 2 de Aminoácidos Excitadores/fisiología , Ácido Glutámico/toxicidad , Animales , Ceftriaxona/farmacología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
Both traumatic and slow-onset disc herniation can directly compress and/or chemically irritate cervical nerve roots, and both types of root injury elicit pain in animal models of radiculopathy. This study investigated the relative contributions of mechanical compression and chemical irritation of the nerve root to spinal regulation of neuronal activity using several outcomes. Modifications of two proteins known to regulate neurotransmission in the spinal cord, the neuropeptide calcitonin gene-related peptide (CGRP) and glutamate transporter 1 (GLT-1), were assessed in a rat model after painful cervical nerve root injuries using a mechanical compression, chemical irritation or their combination of injury. Only injuries with compression induced sustained behavioral hypersensitivity (p≤0.05) for two weeks and significant decreases (p<0.037) in CGRP and GLT-1 immunoreactivity to nearly half that of sham levels in the superficial dorsal horn. Because modification of spinal CGRP and GLT-1 is associated with enhanced excitatory signaling in the spinal cord, a second study evaluated the electrophysiological properties of neurons in the superficial and deeper dorsal horn at day 7 after a painful root compression. The evoked firing rate was significantly increased (p=0.045) after compression and only in the deeper lamina. The painful compression also induced a significant (p=0.002) shift in the percentage of neurons in the superficial lamina classified as low- threshold mechanoreceptive (sham 38%; compression 10%) to those classified as wide dynamic range neurons (sham 43%; compression 74%). Together, these studies highlight mechanical compression as a key modulator of spinal neuronal signaling in the context of radicular injury and pain.