RESUMEN
To describe the effect of myopic eye growth on the structure and distribution of astrocytes, vasculature, and retinal nerve fiber layer thickness, which are critical for inner retinal tissue homeostasis and survival. Astrocyte and capillary distribution, retinal nerve fiber (RNFL), and ganglion cell layer (GCL) thicknesses were assessed using immunochemistry and spectral domain optical coherence tomography on eleven retinas of juvenile common marmosets (Callithrix Jacchus), six of which were induced with lens-induced myopia (refraction, Rx: -7.01 ± 1.8D). Five untreated age-matched juvenile marmoset retinas were used as controls (Rx: -0.74 ± 0.4D). Untreated marmoset eyes grew normally, their RNFL thickened and their astrocyte numbers were associated with RNFL thickness. Marmosets with induced myopia did not show this trend and, on the contrary, had reduced astrocyte numbers, increased GFAP-immunopositive staining, thinner RNFL, lower peripheral capillary branching, and increased numbers of string vessels. The myopic changes in retinal astrocytes, vasculature, and retinal nerve fiber layer thickness suggest a reorganization of the astrocyte and vascular templates during myopia development and progression. Whether these adaptations are beneficial or harmful to the retina remains to be investigated.
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Miopía , Células Ganglionares de la Retina , Humanos , Neuroglía , Retina , Vasos Retinianos , Tomografía de Coherencia Óptica/métodosRESUMEN
Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.
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Astrocitos/metabolismo , Conexina 43/metabolismo , Regeneración , Vasos Retinianos/fisiología , Vitreorretinopatía Proliferativa/metabolismo , Animales , Apoptosis , Astrocitos/patología , Quinasa Idelta de la Caseína/metabolismo , Hipoxia de la Célula , Supervivencia Celular , Femenino , Masculino , Ratones , Fosforilación , Vitreorretinopatía Proliferativa/patología , Vitreorretinopatía Proliferativa/fisiopatologíaRESUMEN
BACKGROUND/AIMS: Maxi-anion channel (Maxi-Cl) is ubiquitously expressed and involved in a number of important cell functions especially by serving as an ATP release pathway. We recently identified SLCO2A1 as its essential core component. However, the regulatory component required for the channel activation/inactivation remains unidentified. METHODS: In the present study, to identify the regulatory component, we made genome-wide analysis combined with siRNA screening and performed patch-clamp studies and ATP release assay after gene silencing and overexpression. RESULTS: Comparative microarray analysis between Maxi-Cl-rich C127 and -deficient C1300 cells revealed highly differential expression not only of SLCO2A1 but also of four annexin family members. Gene silencing study showed that Anxa2 is involved in Maxi-Cl activity. The Maxi-Cl events appeared in C1300 cells by overexpression of Slco2a1 and more efficiently by that of Slco2a1 plus Anxa2. Immunoprecipitation assay supported the interaction between ANXA2 and SLCO2A1. Suppressive effects of overexpression of a phospho-mimicking mutant of Anxa2, Anxa2-Y23E, indicated that protein tyrosine dephosphorylation dependence of Maxi-Cl is conferred by ANXA2. Maxi-Cl activity was suppressed by gene silencing of S100A10, a binding partner of ANXA2, and by applying a synthetic ANXA2 peptide, Ac-(1-14), which interferes with the ANXA2-S100A10 complex formation. Intracellular Ca2+ dependence of Maxi-Cl activity was abolished by S100a10 knockdown. CONCLUSION: The ANXA2-S100A10 complex represents the regulatory component of Maxi-Cl conferring protein tyrosine dephosphorylation dependence and intracellular Ca2+ sensitivity on this channel.
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Anexina A2/metabolismo , Calcio/metabolismo , Transportadores de Anión Orgánico/metabolismo , Proteínas S100/metabolismo , Tirosina/metabolismo , Animales , Aniones , Anexina A2/genética , Línea Celular Tumoral , Silenciador del Gen , Células HEK293 , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/fisiología , Fosforilación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas S100/genética , Regulación hacia ArribaRESUMEN
KEY POINTS: Retinal ganglion cells (RGCs) in dark-adapted retinas show a range of threshold sensitivities spanning â¼3 log units of illuminance. Here, we show that the different threshold sensitivities of RGCs reflect an inhibitory mechanism that masks inputs from certain rod pathways. The masking inhibition is subserved by GABAC receptors, probably on bipolar cell axon terminals. The GABAergic masking inhibition appears independent of dopaminergic circuitry that has been shown also to affect RGC sensitivity. The results indicate a novel mechanism whereby inhibition controls the sensitivity of different cohorts of RGCs. This can limit and thereby ensure that appropriate signals are carried centrally in scotopic conditions when sensitivity rather than acuity is crucial. ABSTRACT: The responses of rod photoreceptors, which subserve dim light vision, are carried through the retina by three independent pathways. These pathways carry signals with largely different sensitivities. Retinal ganglion cells (RGCs), the output neurons of the retina, show a wide range of sensitivities in the same dark-adapted conditions, suggesting a divergence of the rod pathways. However, this organization is not supported by the known synaptic morphology of the retina. Here, we tested an alternative idea that the rod pathways converge onto single RGCs, but inhibitory circuits selectively mask signals so that one pathway predominates. Indeed, we found that application of GABA receptor blockers increased the sensitivity of most RGCs by unmasking rod signals, which were suppressed. Our results indicate that inhibition controls the threshold responses of RGCs under dim ambient light. This mechanism can ensure that appropriate signals cross the bottleneck of the optic nerve in changing stimulus conditions.
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Antagonistas del GABA/farmacología , Potenciales de la Membrana/efectos de los fármacos , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Luz , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estimulación Luminosa/métodos , Receptores de GABA/metabolismo , Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Sinapsis/metabolismo , Vías Visuales/efectos de los fármacos , Vías Visuales/metabolismoRESUMEN
Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors.
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Uniones Comunicantes/efectos de los fármacos , Fototransducción/efectos de los fármacos , Degeneración Retiniana/genética , Potenciales de Acción/efectos de los fármacos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Potenciales Postsinápticos Excitadores , Uniones Comunicantes/fisiología , Técnicas In Vitro , Luz , Fototransducción/genética , Ácido Meclofenámico/farmacología , Ácido Meclofenámico/uso terapéutico , Ratones , Ratones Transgénicos , Células Fotorreceptoras/fisiología , Degeneración Retiniana/tratamiento farmacológico , Células Ganglionares de la Retina/fisiología , Visión Ocular/efectos de los fármacos , Visión Ocular/genéticaRESUMEN
The aim of this article is to describe sustained myopic eye growth's effect on astrocyte cellular distribution and its association with inner retinal layer thicknesses. Astrocyte density and distribution, retinal nerve fiber layer (RNFL), ganglion cell layer, and inner plexiform layer (IPL) thicknesses were assessed using immunochemistry and spectral-domain optical coherence tomography on seventeen common marmoset retinas (Callithrix jacchus): six induced with myopia from 2 to 6 months of age (6-month-old myopes), three induced with myopia from 2 to 12 months of age (12-month-old myopes), five age-matched 6-month-old controls, and three age-matched 12-month-old controls. Untreated marmoset eyes grew normally, and both RNFL and IPL thicknesses did not change with age, with astrocyte numbers correlating to RNFL and IPL thicknesses in both control age groups. Myopic marmosets did not follow this trend and, instead, exhibited decreased astrocyte density, increased GFAP+ spatial coverage, and thinner RNFL and IPL, all of which worsened over time. Myopic changes in astrocyte density, GFAP+ spatial coverage and inner retinal layer thicknesses suggest astrocyte template reorganization during myopia development and progression which increased over time. Whether or not these changes are constructive or destructive to the retina still remains to be assessed.
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Miopía , Células Ganglionares de la Retina , Animales , Astrocitos , Fibras Nerviosas , Retina , Tomografía de Coherencia Óptica/métodos , CallithrixRESUMEN
The retinal vasculature supplies oxygen and nutrition to the cells and is crucial for an adequate retinal function. In myopia, excessive eye growth is associated with various anatomical changes that can lead to myopia-related complications. However, how myopia-induced ocular growth affects the integrity of the aged retinal microvasculature at the cellular level is not well understood. Here, we studied how aging interacts with myopia-induced alteration of the retinal microvasculature in fourteen marmoset retinas (Callithrix jacchus). String vessel and capillary branchpoint were imaged and quantified in all four capillary plexi of the retinal vasculature. As marmosets with lens-induced myopia aged, they developed increasing numbers of string vessels in all four vascular plexi, with increased vessel branchpoints in the parafoveal and peripapillary retina and decreased vessel branchpoints in the peripheral retina. These myopia-induced changes to the retinal microvasculature suggest an adaptive reorganization of the retinal microvascular cellular structure template with aging and during myopia development and progression.
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Purpose: Retinal astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and unopposed hemichannels. While it is well established that Cx43 is upregulated in retinal injuries, it is unclear whether astrocytic Cx43 plays a role in retinal ganglion cell (RGC) loss associated with injury. Here, we investigated the effect of astrocyte-specific deletion of Cx43 (Cx43KO) and channel inhibitors on RGC loss in retinal ischemia/reperfusion (I/R) injury and assessed changes in expression and GJ channel and hemichannel function that occur in I/R injury. The effect of Cx43 deletion on neural function in the uninjured retina was also assessed. Methods: Cx43 expression, astrocyte density and morphology, and RGC death in wild-type and Cx43KO mice after I/R injury were determined using immunohistochemistry and Western blotting. Visual function was assessed using ERG recordings. GJ coupling and hemichannel activity were evaluated using tracer coupling and uptake studies, respectively. Results: Loss of RGCs in I/R injury was accompanied by an increase of Cx43 expression in astrocytes. Functional studies indicated that I/R injury augmented astrocytic GJ coupling but not Cx43 hemichannel activity. Importantly, deletion of astrocytic Cx43 improved neuronal survival in acute ischemia but did not affect RGC function in the absence of injury. In support, pharmacologic inhibition of GJ coupling provided neuroprotection in I/R injury. Conclusions: The increase in Cx43 expression and GJ coupling during acute I/R injury exacerbates RGC loss. Inhibition of astrocytic Cx43 channels might represent a useful strategy to promote RGC survival in pathologic conditions.
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Astrocitos/metabolismo , Conexina 43/genética , Uniones Comunicantes/metabolismo , Regulación de la Expresión Génica/fisiología , Neuroglía/metabolismo , Daño por Reperfusión/metabolismo , Células Ganglionares de la Retina/patología , Animales , Biotina/análogos & derivados , Biotina/farmacología , Western Blotting , Supervivencia Celular , Electrorretinografía , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión/patología , Células Ganglionares de la Retina/metabolismo , Ácidos Triyodobenzoicos/farmacologíaRESUMEN
The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5'-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP)zeta, activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTPzeta gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTPzeta in mouse fibroblasts, but not in C127 cells.
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Canales Iónicos/metabolismo , Tirosina/metabolismo , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/fisiología , Adenilil Imidodifosfato/farmacología , Adenilil Imidodifosfato/fisiología , Animales , Aniones/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Activación del Canal Iónico , Magnesio , Ratones , Fosforilación , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/fisiología , Transducción de SeñalRESUMEN
Cellular communication through chemical synapses is determined by the nature of the neurotransmitter and the composition of postsynaptic receptors. In the excitatory synapse between bipolar and ganglion cells of the retina, postsynaptic AMPA receptors mediate resting activity. During evoked response, however, more abundant and sustained levels of glutamate also activate GluN2B-containing NMDA receptors (NMDARs). This phasic recruitment of distinct glutamate receptors is essential for visual discrimination; however, the fidelity of this basic mechanism under elevated glutamate levels due to aberrant activity, a common pathophysiology, is not known. Here, in both male and female mice with retinal degeneration (rd10), a condition associated with elevated synaptic activity, we reveal that changes in synaptic input to ganglion cells altered both composition and activation of NMDARs. We found that, in contrast to wild type, the spontaneous activity of rd10 cells was largely NMDAR-dependent. Surprisingly, this activity was driven primarily by atypical activation of GluN2A -containing NMDARs, not GluN2B-NMDARs. Indeed, immunohistochemical analyses and Western blot showed greater levels of the GluN2A-NMDAR subunit expression in rd10 retina compared to wild type. Overall, these results demonstrate how aberrant signaling leads to pathway-specific alterations in NMDAR expression and function.
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Receptores de N-Metil-D-Aspartato/metabolismo , Retina/metabolismo , Degeneración Retiniana/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Potenciales Postsinápticos Excitadores/fisiología , Ganglios de Invertebrados/metabolismo , Ácido Glutámico/metabolismo , Ratones , Sinapsis/fisiologíaRESUMEN
Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.
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Conexinas/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/fisiología , Animales , Electrorretinografía , Potenciales Evocados Visuales , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-ClampRESUMEN
PURPOSE: Interactions between vasculature and neurons provide important insight into the function of the nervous system, as well as into neurological diseases wherein these interactions are disrupted. This study characterizes a previously unreported retinal vascular plexus and examines potential sites of neurovascular interaction. METHODS: Vascular, neuronal, and glial elements were visualized using immunohistochemical markers. The distribution of vascular layers was measured and compared across eccentricities. Intensity profiles were calculated from confocal image reconstructions to reveal the proximity of vasculature to neuronal and glial processes. RESULTS: Retinal vasculature forms a plexus that coincides with the dendritic processes of OFF cholinergic amacrine cells within the inner plexiform layer. Across eccentricities, this plexus comprises approximately 8% of the total length of horizontally running blood vessels in the retina. Processes of Müller glia and OFF cholinergic amacrine cells colocalize with the blood vessels that form the intersublaminar plexus. CONCLUSIONS: In the retina, vasculature lacks autonomic control, but shows efficient local regulation. Although the source of this regulation is unclear, these results suggest that cholinergic amacrine cells and Müller glia may interact with the intersublaminar plexus to influence vasomotor activity. This may indicate a key role in modulating reciprocal interactions between neuronal activity and blood flow.
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Neuronas/citología , Vasos Retinianos/inervación , Vasos Retinianos/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiologíaRESUMEN
Retinal degeneration describes a group of disorders which lead to progressive photoreceptor cell death, resulting in blindness. As this occurs, retinal ganglion cells (RGCs) begin to develop oscillatory physiological activity. Here we studied the morphological and physiological properties of RGCs in rd1 mice, aged 30-60 days, to determine how this aberrant activity correlates with morphology. Patch-clamp recordings of excitatory and inhibitory currents were performed, then dendritic structures were visualized by infusion of fluorescent dye. Only RGCs with oscillatory activity were selected for further analysis. Oscillatory frequency and power were calculated using power spectral density analysis of recorded currents. Dendritic arbor stratification, total length, and area were measured from confocal microscope image stacks. These measurements were used to sort RGCs by cluster analysis using Ward's Method. This resulted in a total of 10 clusters, with monostratified and bistratified cells having five clusters each. Both populations exhibited correlations between arbor stratification and aberrant inhibitory input, while excitatory input did not vary with arbor distribution. These findings illustrate the relationship between aberrant activity and RGC morphology at early stages of retinal degeneration.
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Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/fisiología , Sinapsis/fisiología , Animales , Análisis por Conglomerados , Dendritas/patología , Dendritas/fisiología , Modelos Animales de Enfermedad , Femenino , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones Transgénicos , Microscopía Confocal , Mutación , Técnicas de Placa-ClampRESUMEN
Spontaneous rhythmic activity is a hallmark feature of the developing retina, where propagating retinal waves instruct axonal targeting and synapse formation. Retinal waves cease around the time of eye-opening; however, the fate of the underlying synaptic circuitry is unknown. Whether retinal waves are unique to the developing retina or if they can be induced in adulthood is not known. Combining patch-clamp techniques with calcium imaging, we demonstrate that propagative events persist in adult mouse retina when it is deprived of inhibitory input. This activity originates in bipolar cells, resembling glutamatergic stage III retinal waves. We find that, as it develops, the network interactions progressively curtail this activity. Together, this provides evidence that the correlated propagative neuronal activity can be induced in adult retina following the blockade of inhibitory interactions.
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Calcio/metabolismo , Retina/fisiología , Animales , Canales de Calcio/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neurogénesis/fisiología , Periodicidad , Células Bipolares de la Retina/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing. Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.
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Inmunohistoquímica/métodos , Retina/anatomía & histología , Retina/química , Fijación del Tejido/métodos , Animales , Carbodiimidas/química , Colorantes Fluorescentes/química , Formaldehído/química , Membranas Artificiales , Ratones , Polímeros/química , Politetrafluoroetileno/química , Retina/metabolismo , Sinapsis/química , Sinapsis/metabolismoRESUMEN
In this manuscript, we describe a protocol for capturing both physiological and structural properties of living neuronal tissue. An essential aspect of this method is its flexibility and fast turnaround time. It is a streamlined process that includes recording of electrophysiological neuronal activity, calcium imaging, and structural analysis. This is accomplished by placing intact tissue on a modified Millicell Biopore insert. The Biopore membrane suspends the tissue in the perfusion solution, allowing for complete access to nutrients, oxygen, and pharmacological agents. The ring that holds the membrane ensures its structural stability; forceps can be used to grip the ring without contacting the filter or the tissue, for easy transfer between multiple setups. We show that tissue readily adheres to the surface of the membrane, its entire surface is visible in transmitted light and accessible for recording and imaging, and remains responsive to physiological stimuli for extended periods of time.
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Electrofisiología/instrumentación , Retina/fisiología , Técnicas de Cultivo de Tejidos/instrumentación , Compuestos de Anilina/análisis , Animales , Proteínas Bacterianas/genética , Encéfalo/fisiología , Encéfalo/ultraestructura , Calcio/análisis , Costos y Análisis de Costo , Medios de Cultivo , Electrofisiología/métodos , Fluoresceínas/análisis , Colorantes Fluorescentes/análisis , Genes Reporteros , Proteínas Luminiscentes/genética , Membranas Artificiales , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Técnicas de Placa-Clamp , Células Fotorreceptoras de Vertebrados/fisiología , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Politetrafluoroetileno , Retina/efectos de la radiación , Células Bipolares de la Retina/fisiología , Células Bipolares de la Retina/efectos de la radiación , Células Ganglionares de la Retina/fisiología , Células Ganglionares de la Retina/efectos de la radiación , Vasos Retinianos/ultraestructura , Rodaminas , Factores de Tiempo , Técnicas de Cultivo de Tejidos/economíaRESUMEN
Neural oscillations play an important role in normal brain activity, but also manifest during Parkinson's disease, epilepsy, and other pathological conditions. The contribution of these aberrant oscillations to the function of the surviving brain remains unclear. In recording from retina in a mouse model of retinal degeneration (RD), we found that the incidence of oscillatory activity varied across different cell classes, evidence that some retinal networks are more affected by functional changes than others. This aberrant activity was driven by an independent inhibitory amacrine cell oscillator. By stimulating the surviving circuitry at different stages of the neurodegenerative process, we found that this dystrophic oscillator further compromises the function of the retina. These data reveal that retinal remodeling can exacerbate the visual deficit, and that aberrant synaptic activity could be targeted for RD treatment.
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In the present study, we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress. We first examined the expression of mRNAs for proteins constituting possible ATP-releasing pathways that have been suggested over the past several years. In RT-PCR analysis using both control and osmotically swollen astrocytes, amplification of cDNA fragments of expected size was seen for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), the P2X7 receptor, MRP1 and MDR1, but not CFTR. Inhibitors of exocytotic vesicular release, gap junction hemi-channels, CFTR, MRP1, MDR1, the P2X7 receptor, and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes. In contrast, the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium (50 muM), an inhibitor of the maxi-anion channel, which has recently been shown to serve as a pathway for ATP release from several other cell types. Thus, we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.