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

Abstract

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].

In vivo two-photon imaging of the mouse retina

“…This was done in conjunction with electrophysiology and patch-clamp methods for recording functional signals from the retina [20,21]. Recently, in vitro two-photon imaging of neurons labeled with G-CaMP3 has also been demonstrated [22], which offers the possibility of simultaneously recording functional responses from many cells.…”

Morphological and physiological properties of enhanced green fluorescent protein (EGFP)‐expressing wide‐field amacrine cells in the ChAT‐EGFP mouse line

“…Patch-clamp recordings were made as reported previously Pottek et al, 2011). Briefly, animals were dark-adapted for 3 h prior to the experiment, and all of the above-mentioned preparation steps were performed under dim red light illumination (> 700 nm).…”

“…The light stimulation system is described in full detail elsewhere Pottek et al, 2011). In short, optical stimuli of varying geometrical dimensions and light intensity were generated on a CRT computer screen using a visual stimulus software (QDS; Thomas Euler, Center for Integrative Neurosciences, T€ ubingen, Germany) and were projected through the condenser to the object plane of the microscope allowing to pass the glass bottom of the recording chamber and reaching the retina from the photoreceptor side.…”

“…Due to their large dendritic tree size, wide-field amacrine cells often present a rather low cell density and are therefore hard to study systematically. To overcome this drawback, fluorescent protein expression under the control of cell-specific promoters is now employed, allowing for the visual identification of special amacrine cell populations that can easily be targeted by microelectrodes for electrophysiological characterization and dye injection (Sarthy et al, 2007;Zhang et al, 2007;Cederlund et al, 2011;Knop et al, 2011;Pottek et al, 2011).…”

Marking Cells with Infrared Fluorescent Proteins to Preserve Photoresponsiveness in the Retina

“…These GFP-labeled mouse lines have also contributed to morphological studies (5,6). However, because the light (wavelength ∼500 nm) used to identify GFP-marked cells bleaches photoreceptors (Figure 1A), recording of retinal light responses is only possible by using two-photon laser microscopy, which preserves the light responsiveness of the photoreceptors (7,8). Although the two-photon microscope has become more popular, it is still an expensive state-of-the-art tool and carries a high maintenance cost.…”