RESUMEN
The retina is the sensory tissue responsible for the first stages of visual processing, with a conserved anatomy and functional architecture among vertebrates. To date, retinal eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, and others, affect nearly 170 million people worldwide, resulting in vision loss and blindness. To tackle retinal disorders, the developing retina has been explored as a versatile model to study intercellular signaling, as it presents a broad neurochemical repertoire that has been approached in the last decades in terms of signaling and diseases. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres and/or as organoids, are valuable to understand both neuronal and glial compartments, which have contributed to revealing roles and mechanisms between transmitter systems as well as antioxidants, trophic factors, and extracellular matrix proteins. Overall, contributions in understanding neurogenesis, tissue development, differentiation, connectivity, plasticity, and cell death are widely described. A complete access to the genome of several vertebrates, as well as the recent transcriptome at the single cell level at different stages of development, also anticipates future advances in providing cues to target blinding diseases or retinal dysfunctions.
Asunto(s)
Enfermedades de la Retina , Animales , Humanos , Ceguera , Estado de Salud , Neuroglía , Neuronas , RetinaRESUMEN
Synaptic strength is altered during synaptic plasticity by controlling the number of AMPA receptors (AMPARs) at excitatory synapses. During long-term potentiation and synaptic upscaling, AMPARs are accumulated at synapses to increase synaptic strength. Neuronal activity leads to phosphorylation of AMPAR subunit GluA1 (also known as GRIA1) and subsequent elevation of GluA1 surface expression, either by an increase in receptor forward trafficking to the synaptic membrane or a decrease in receptor internalization. However, the molecular pathways underlying GluA1 phosphorylation-induced elevation of surface AMPAR expression are not completely understood. Here, we employ fluorescence recovery after photobleaching (FRAP) to reveal that phosphorylation of GluA1 serine 845 (S845) predominantly plays a role in receptor internalization, rather than forward trafficking, during synaptic plasticity. Notably, internalization of AMPARs depends upon the clathrin adaptor AP2, which recruits cargo proteins into endocytic clathrin-coated pits. In fact, we further reveal that an increase in GluA1 S845 phosphorylation upon two distinct forms of synaptic plasticity diminishes the binding of the AP2 adaptor, reducing internalization and resulting in elevation of GluA1 surface expression. We thus demonstrate a mechanism of GluA1 phosphorylation-regulated clathrin-mediated internalization of AMPARs.
Asunto(s)
Clatrina , Receptores AMPA , Clatrina/metabolismo , Hipocampo/metabolismo , Potenciación a Largo Plazo , Fosforilación , Receptores AMPA/genética , Receptores AMPA/metabolismo , Sinapsis/metabolismoRESUMEN
Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A1R and A2R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca2+ homeostasis. We found that caffeine (30-200 µM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A1Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca2+ transients in neuronal cell culture, an action mimicked by the A1R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 µM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca2+ homeostasis.
Asunto(s)
Cafeína/farmacología , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Antagonistas de Receptores Purinérgicos P1/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Glutamina , Hipocampo/metabolismo , Masculino , Ratones , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Purified retina glial Müller cells can express the machinery for dopamine synthesis and release when maintained in culture. Dopamine is detected in cell extracts of cultures exposed to its precursor, L-DOPA. A large portion of synthesized dopamine is recovered in the superfusing medium showing the tendency of the accumulated dopamine to be released. Müller cells purified from developing chick and mouse retinas express L-DOPA decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; EC 4.1.1.28) and the dopamine transporter DAT. The synthesis of dopamine from L-DOPA supplied to Müller cultures is inhibited by m-hydroxybenzylhydrazine, a DDC inhibitor. Dopamine release occurs via a transporter-mediated process and can activate dopaminergic D(1) receptors expressed by the glia population. The synthesis and release of dopamine were also observed in Müller cell cultures from mouse retina. Finally, cultured avian Müller cells display increased expression of tyrosine hydroxylase, under the influence of agents that increase cAMP levels, which results in higher levels of dopamine synthesized from tyrosine. A large proportion of glial cells in culture do express Nurr1 transcription factor, consistent with the dopaminergic characteristics displayed by these cells in culture. The results show that Müller cells, deprived of neuron influence, differentiate dopaminergic properties thought to be exclusive to neurons.
Asunto(s)
Diferenciación Celular/fisiología , Dopamina/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Retina/metabolismo , Animales , Inhibidores de Descarboxilasas de Aminoácidos Aromáticos , Biomarcadores/metabolismo , Células Cultivadas , Embrión de Pollo , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Dopa-Decarboxilasa/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes , Ratones , Neuroglía/citología , Neuronas/citología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fenotipo , Receptores de Dopamina D1/metabolismo , Retina/citología , Factores de Transcripción/metabolismo , Tirosina/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The role of dopamine in the retina has been studied for the last 30 years and there is now increasing evidence that dopamine is used as a developmental signal in the embryonic retina. Dopamine is the main catecholamine found in the retina of most species, being synthesized from the L-amino acid tyrosine. Its effects are mediated by G protein coupled receptors constituting the D(1) (D(1) and D(5)) and D(2) (D(2), D(3) and D(4)) receptor subfamilies that can be coupled to adenylyl cyclase in opposite manners. Dopamine-mediated cyclic AMP (cAMP) accumulation, via D(1)-like receptors, is observed very early during retina ontogeny, before synaptogenesis and, in some species, before the expression of tyrosine hydroxylase (TH), the enzyme that characterizes the neuronal dopaminergic phenotype. D(2)-like receptors appear in the tissue days after D(1)-like activity is detected. In the embryonic avian retina, before the tissue is capable of synthesizing its own dopamine via TH, dopamine synthesis is observed from L-DOPA supplied to the neuroretina from retina pigmented epithelium which results in dopaminergic communication in the embryonic tissue before TH expression. Müller cells, the main glia type found in the retina, seem to actively contribute to dopaminergic activity in the retinal tissue. Understanding the dopaminergic role during retina development may contribute to novel strategies approaching certain visual dysfunctions such as those found in ocular albinism.
Asunto(s)
Dopamina/biosíntesis , Neuronas/metabolismo , Retina/embriología , Retina/crecimiento & desarrollo , Transducción de Señal/fisiología , Animales , AMP Cíclico/metabolismo , Vías Nerviosas/embriología , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/metabolismo , Neuroglía/metabolismo , Receptores Dopaminérgicos/metabolismo , Retina/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212-2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.
RESUMEN
Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB1 and CB2), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB1 and CB2 receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l-Aspartate (L-ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212-2 (WIN, a CB1/CB2 agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca2+ levels evoked by ATP, but had no effect in Ca2+ shifts in neuronal cells activated by KCl. Finally, WIN inhibited [3H]-GABA release induced by KCl or L-ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation.
Asunto(s)
Neuroglía/metabolismo , Neuronas/metabolismo , Receptor Cannabinoide CB1/fisiología , Receptor Cannabinoide CB2/fisiología , Retina/embriología , Retina/metabolismo , Analgésicos/farmacología , Animales , Benzoxazinas/farmacología , Embrión de Pollo , Técnicas de Cocultivo , Morfolinas/farmacología , Naftalenos/farmacología , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as ßIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 µM and MK-801 20 µM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit.
Asunto(s)
Calcio/metabolismo , Glutatión/farmacología , Neuroglía/efectos de los fármacos , Retina/citología , Animales , Apoptosis/efectos de los fármacos , Proteínas Aviares/metabolismo , Células Cultivadas , Embrión de Pollo , Pollos , Relación Dosis-Respuesta a Droga , Disulfuro de Glutatión/farmacología , Microscopía Fluorescente , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores Purinérgicos P2X7/metabolismo , Retina/embriología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [(3)H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10min of cocaine administration and lasted for up to 30min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC.
Asunto(s)
Ácido Aspártico/metabolismo , Cocaína/administración & dosificación , Inhibidores de Captación de Dopamina/administración & dosificación , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Receptores de Dopamina D1/metabolismo , Adenilil Ciclasas/metabolismo , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 3 de Aminoácidos Excitadores/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Ácido Glutámico/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
BACKGROUND: Maternal alcohol use during pregnancy causes a continuum of long-lasting disabilities in the offspring, commonly referred to as fetal alcohol spectrum disorder (FASD). Attention-deficit/hyperactivity disorder (ADHD) is possibly the most common behavioral problem in children with FASD and devising strategies that ameliorate this condition has great clinical relevance. Studies in rodent models of ADHD and FASD suggest that impairments in the cAMP signaling cascade contribute to the hyperactivity phenotype. In this work, we investigated whether the cAMP levels are affected in a long-lasting manner by ethanol exposure during the third trimester equivalent period of human gestation and whether the acute administration of the PDE1 inhibitor vinpocetine ameliorates the ethanol-induced hyperactivity. METHODS: From postnatal day (P) 2 to P8, Swiss mice either received ethanol (5g/kg i.p.) or saline every other day. At P30, the animals either received vinpocetine (20mg/kg or 10mg/kg i.p.) or vehicle 4h before being tested in the open field. After the test, frontal cerebral cortices and hippocampi were dissected and collected for assessment of cAMP levels. RESULTS: Early alcohol exposure significantly increased locomotor activity in the open field and reduced cAMP levels in the hippocampus. The acute treatment of ethanol-exposed animals with 20mg/kg of vinpocetine restored both their locomotor activity and cAMP levels to control levels. CONCLUSIONS: These data lend support to the idea that cAMP signaling system contribute to the hyperactivity induced by developmental alcohol exposure and provide evidence for the potential therapeutic use of vinpocetine in FASD.
Asunto(s)
Consumo de Bebidas Alcohólicas/patología , Trastorno por Déficit de Atención con Hiperactividad/tratamiento farmacológico , Trastornos del Espectro Alcohólico Fetal/tratamiento farmacológico , Actividad Motora/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Alcaloides de la Vinca/farmacología , Consumo de Bebidas Alcohólicas/efectos adversos , Animales , Trastorno por Déficit de Atención con Hiperactividad/fisiopatología , Depresores del Sistema Nervioso Central/efectos adversos , Depresores del Sistema Nervioso Central/sangre , Depresores del Sistema Nervioso Central/toxicidad , AMP Cíclico/análisis , Modelos Animales de Enfermedad , Esquema de Medicación , Evaluación Preclínica de Medicamentos , Etanol/efectos adversos , Etanol/sangre , Etanol/toxicidad , Femenino , Humanos , Masculino , Ratones , Inhibidores de Fosfodiesterasa/administración & dosificación , Inhibidores de Fosfodiesterasa/efectos adversos , Inhibidores de Fosfodiesterasa/uso terapéutico , Embarazo , Complicaciones del Embarazo/inducido químicamente , Método Simple Ciego , Tasa de Supervivencia , Alcaloides de la Vinca/administración & dosificación , Alcaloides de la Vinca/efectos adversos , Alcaloides de la Vinca/uso terapéutico , Aumento de Peso/efectos de los fármacosRESUMEN
Exposure to cocaine during the fetal period can produce significant lasting changes in the structure and function of the brain. Cocaine exerts its effects on the developing brain by blocking monoamine transporters and impairing monoamine receptor signaling. Dopamine is a major central target of cocaine. In a mouse model, we show that cocaine exposure from embryonic day 8 (E8) to E14 produces significant reduction in dopamine transporter activity, attenuation of dopamine D1-receptor function and upregulation of dopamine D2-receptor function. Cocaine's effects on the D1-receptor are at the level of protein expression as well as activity. The cocaine exposure also produces significant increases in basal cAMP levels in the striatum and cerebral cortex. The increase in the basal cAMP levels was independent of dopamine receptor activity. In contrast, blocking the adenosine A2a receptor downregulated the basal cAMP levels in the cocaine-exposed brain to physiological levels, suggesting the involvement of adenosine receptors in mediating cocaine's effects on the embryonic brain. In support of this suggestion, we found that the cocaine exposure downregulated adenosine transporter function. We also found that dopamine D2- and adenosine A2a-receptors antagonize each other's function in the embryonic brain in a manner consistent with their interactions in the mature brain. Thus, our data show that prenatal cocaine exposure produces direct effects on both the dopamine and adenosine systems. Furthermore, the dopamine D2 and adenosine A2a receptor interactions in the embryonic brain discovered in this study unveil a novel substrate for cocaine's effects on the developing brain.
Asunto(s)
Adenosina/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Dopamina/metabolismo , Antagonistas del Receptor de Adenosina A2 , Animales , Encéfalo/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/embriología , Cuerpo Estriado/metabolismo , AMP Cíclico/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Femenino , Técnicas In Vitro , Ratones , Proteínas de Transporte de Nucleósidos/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal , Receptor de Adenosina A2A/metabolismo , Receptores de Dopamina D1/antagonistas & inhibidores , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
DOPA decarboxylase (DDC; aromatic-l-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre-incubated with l-DOPA for 1 h, increased 250- and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mm l-DOPA, washed and followed by incubation in the presence of 0.5 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with l-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 micro m). The incubation of retinas in medium previously conditioned by retina-pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l-DOPA is supplied to the tissue. It also shows that RPE is a potential source of l-DOPA early in development.