Inputs Underlying the ON–OFF Light Responses of Type 2 Wide-Field Amacrine Cells in TH::GFP Mice

Knop, Gabriel; Feigenspan, Andreas; Weiler, Reto; Dedek, Karin

doi:10.1523/jneurosci.6235-10.2011

Cited by 28 publications

(75 citation statements)

References 55 publications

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“…The following results originate from a study on a mouse expressing green fluorescent protein (GFP) under the promoter for tyrosine hydroxylase 13,14 , the enzyme catalyzing the rate limiting step in catecholamine synthesis (TH::GFP mouse). Based on the brightness of the GFPsignal two distinct cell populations are distinguishable (Figure 2A).…”

Section: Representative Resultsmentioning

confidence: 99%

“…Cells expressing the higher GFP level possess cell bodies located in the inner nuclear layer (INL, Figure 2A) or displaced in the ganglion cell layer (GCL, Figure 2B) and stratify in the middle of the inner plexiform layer (IPL, Figure 2C). They were identified as type 2 cells [14][15][16] and could systematically be studied with respect to morphology ( Figure 3 …”

Section: Representative Resultsmentioning

confidence: 99%

“…Tune the stimulus intensity by inserting neutral density filters into the beam path. For calibration of monitor spectrum and intensity see Ref [14]. Choose a prolonged interstimulus interval (e.g.…”

Section: Recordingsmentioning

confidence: 99%

“…The usefulness of this approach is already demonstrated by studies on specific populations of amacrine cells 14,18 from which electrophysiological recordings otherwise would have been possible only by accident 12 . Finally, this powerful technique can further be extended by including a pharmacological approach 14 , Ca 2+ imaging 19 or by using injected cells for immunocytochemical studies or electron microscopy.…”

mentioning

confidence: 99%

“…Finally, this powerful technique can further be extended by including a pharmacological approach 14 , Ca 2+ imaging 19 or by using injected cells for immunocytochemical studies or electron microscopy. That way, the…”

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confidence: 99%

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Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Pottek

Knop

Weiler

et al. 2011

JoVE

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Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals 1 . Though being a crucial part of the visual processing by shaping the retinal output 2 , most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue 3,4 . These prelabeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage 5 . Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments 6 .In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels. Video LinkThe video component of this article can be found at https://www.jove.com/video/3457/ ProtocolThe following description assumes that the experimenter has a basic understanding of retinal structure, patch-clamp recording, and two-photon microscopy. For useful information on establishing and running a patch-clamp setup and two-photon imaging system see Refs [7][8][9][10][11][12].

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Section: Representative Resultsmentioning

confidence: 99%

Section: Representative Resultsmentioning

confidence: 99%

Section: Recordingsmentioning

confidence: 99%

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confidence: 99%

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Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Pottek

Knop

Weiler

et al. 2011

JoVE

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Age-dependent Müller glia neurogenic competence in the mouse retina

Löffler

Patrick

Völkner

et al. 2015

Glia

105

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The mechanisms limiting neuronal regeneration in mammals and their relationship with reactive gliosis are unknown. Müller glia (MG), common to all vertebrate retinas, readily regenerate neuron loss in some species, but normally not in mammals. However, experimental stimulation of limited mammalian retina regeneration has been reported. Here, we use a mouse retina organ culture approach to investigate the MG responses at different mouse ages. We found that MG undergo defined spatio-temporal changes upon stimulation. In EGF-stimulated juvenile postmitotic retinas, most MG upregulate cell-cycle regulators (Mcm6, Pcna, Ki67, Ccnd1) within 48 h ex vivo; some also express the neurogenic factors Ascl1, Pax6, and Vsx2; up to 60% re-enter the cell cycle, some of which delaminate to divide mostly apically; and the majority cease to proliferate after stimulation. A subpopulation of MG progeny starts to express transcription factors (Ptf1a, Nr4a2) and neuronal (Calb1, Calb2, Rbfox3), but not glial, markers, indicating neurogenesis. BrdU-tracking, genetic lineage-tracing, and transgenic-reporter experiments suggest that MG reprogram to a neurogenic stage and proliferate; and that some MG progeny differentiate into neuronal-like cells, most likely amacrines, no photoreceptors; most others remain in a de-differentiated state. The mouse MG regeneration potential becomes restricted, dependent on the age of the animal, as observed by limited activation of the cell cycle and neurogenic factors. The stage-dependent analysis of mouse MG revealed similarities and differences when compared with MG-derived regeneration in fish and chicks. Therefore, the mouse retina ex vivo approach is a potential assay for understanding and overcoming the limitations of mammalian MG-derived neuronal regeneration. Postmitotic MG in mouse retina ex vivo can be stimulated to proliferate, express neurogenic factors, and generate progeny expressing neuronal or glial markers. This potential regenerative competence becomes limited with increasing mouse age.

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Phenotyping of Gap-Junctional Coupling in the Mouse Retina

Meyer

Yadav

Dedek

2018

Methods in Molecular Biology

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In the mammalian retina, gap junctions, made of connexin proteins, are found in all neuronal cell types and are important for the transmission of rod photoreceptor signals, spike synchronization, noise reduction, and signal averaging. There are several methods available to assess gap junctional coupling in the retina: simultaneous electrical recordings from two adjacent cells, cut-loading, and intracellular injection of gap junction-permeable tracers. Here, we focus on the latter as it allows precise targeting of the cell of interest and is suitable to assess tracer coupling in a wide variety of retinal cell types, e.g., horizontal cells, amacrine cells, and ganglion cells. Tracer coupling experiments are usually performed in the intact retina and can provide information on the extent of coupling, the identity of synaptic partners, and (when combined with immunohistochemistry or pharmacology) the underlying connexin or the regulation of gap junctions.

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Inputs Underlying the ON–OFF Light Responses of Type 2 Wide-Field Amacrine Cells in TH::GFP Mice

Cited by 28 publications

References 55 publications

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Age-dependent Müller glia neurogenic competence in the mouse retina

Phenotyping of Gap-Junctional Coupling in the Mouse Retina

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