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[This corrects the article on p. 513 in vol. 9, PMID: 26793064.].
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The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).
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Células Amacrinas , Técnicas de Placa-Clamp , Retina , Animales , Técnicas de Cultivo de Célula , Ratones , Neuronas , Células Ganglionares de la RetinaRESUMEN
It has been shown in rd1 and rd10 models of photoreceptor degeneration (PD) that inner retinal neurons display spontaneous and rhythmic activities. Furthermore, the rhythmic activity has been shown to require the gap junction protein connexin 36, which is likely located in AII amacrine cells (AII-ACs). In the present study, an autosomal dominant PD model called rhoΔCTA, whose rods overexpress a C-terminally truncated mutant rhodopsin and degenerate with a rate similar to that of rd1, was used to investigate the generality and mechanisms of heightened inner retinal activity following PD. To fluorescently identify cholinergic starburst amacrine cells (SACs), the rhoΔCTA mouse was introduced into a combined ChAT-IRES-Cre and Ai9 background. In this mouse, we observed excitatory postsynaptic current (EPSC) oscillation and non-rhythmic inhibitory postsynaptic current (IPSC) in both ON- and OFF-SACs. The IPSCs were more noticeable in OFF- than in ON-SACs. Similar to reported retinal ganglion cell (RGC) oscillation in rd1 mice, EPSC oscillation was synaptically driven by glutamate and sensitive to blockade of NaV channels and gap junctions. These data suggest that akin to rd1 mice, AII-AC is a prominent oscillator in rhoΔCTA mice. Surprisingly, OFF-SAC but not ON-SAC EPSC oscillation could readily be enhanced by GABAergic blockade. More importantly, weakening the AII-AC gap junction network by activating retinal dopamine receptors abolished oscillations in ON-SACs but not in OFF-SACs. Furthermore, the latter persisted in the presence of flupirtine, an M-type potassium channel activator recently reported to dampen intrinsic AII-AC bursting. These data suggest the existence of a novel oscillation mechanism in mice with PD.
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Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.
Asunto(s)
Expresión Génica/fisiología , Proteínas RGS/metabolismo , Recuperación de la Función/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Animales , Western Blotting/métodos , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Expresión Génica/efectos de la radiación , Técnicas In Vitro , Luz , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación/efectos de la radiación , Estimulación Luminosa/métodos , Proteínas RGS/genética , Recuperación de la Función/genética , Retina/citología , Factores de Tiempo , Visión Ocular/fisiologíaRESUMEN
Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L. An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtalphaQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtalpha, the GtalphaQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtalphaQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtalpha, and apparently, do not require Gtalpha interaction with RGS9 and PDE6.
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Luz , Células Fotorreceptoras Retinianas Bastones/metabolismo , Transducina/metabolismo , Adaptación Ocular/fisiología , Animales , Anticuerpos Monoclonales/genética , Transporte Biológico , Adaptación a la Oscuridad/fisiología , Dipéptidos/inmunología , Técnica del Anticuerpo Fluorescente , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Ácido Glutámico/inmunología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Hidrólisis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutagénesis , Proteínas RGS/deficiencia , Proteínas RGS/fisiología , Retina/química , Transducina/deficiencia , Transducina/genéticaRESUMEN
Retinal photoreceptors are highly differentiated postmitotic neurons that transduce photons into electrical signals. While the functions of many photoreceptor-specific genes can be evaluated by direct gene targeting, here we facilitate the studies of nonphotoreceptor-specific genes in these cells by developing an Opsin-iCre transgenic mouse line, iCre-75, in which a 4-kb mouse rod opsin promoter drives the expression of bacteriophage P1 Cre recombinase. Immunohistochemical analysis demonstrated that Cre recombinase is present exclusively in the outer nuclear layer of iCre75 mouse retina. Cre expression is found only in rods and not in cones. The expression level reached 188+/-44 ng per retina at postnatal day (pnd) 11 and increased to 687+/-56 ng at 2 months and older. Cre-mediated excision of floxed genomic DNA was absent at pnd 4, became detectable at pnd 7, and was completed by pnd 18. Retinal morphology and electroretinograms were normal in 8-month-old transgenic animals. The iCre-75 transgenic mice are thus suitable for future genetic studies of essential genes in retinal rod photoreceptors.
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Integrasas/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/genética , Proteínas Virales/genética , Animales , Secuencia de Bases , ADN/genética , Electrorretinografía , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Operón Lac , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Retina/crecimiento & desarrollo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/crecimiento & desarrollo , Opsinas de Bastones/genéticaRESUMEN
RGS (regulator of G protein signaling) proteins containing the G protein gamma-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein beta-subunit family, Gbeta5. This interaction is necessary for the stability of both the RGS protein and for Gbeta5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that Gbeta5 levels may be limiting. To examine further the interactions of Gbeta5 and the GGL domain-containing RGS proteins, we inactivated the Gbeta5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of Ggamma2 and RGS4 proteins remain normal in the absence of Gbeta5. The homozygous Gbeta5 knockout (Gbeta5-/-) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the Gbeta5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of Gbeta5 and all four RGS proteins that form protein complexes with Gbeta5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.