Retinal function of the voltage-gated calcium channel subunit α2δ-3 / Light-dependent effects in α2δ-3 mutant and in wild type retina

The retina employs a large number of cell types that fulfill a broad spectrum of computations. It comes as no surprise that this complex network would make use of an equal diversity of molecular tools, such as voltage-gated calcium channels (VGCC). In fact, all pore-forming α1 subunits of VGCC and modulatory β and auxiliary α2δ subunits were found in the retina. Yet, little detail is known about the functional implementation of individual VGCC subunits in the retinal circuitry. My work described in part 1 focused on the retinal expression and function of one VGCC subunit, called α2δ-3, employing an α2δ-3 knockout mouse. I found transcription of all α2δ subunit genes throughout postnatal retinal development and strong expression of α2δ-3 in horizontal cells. Yet, in my patch-clamp recordings from isolated horizontal cells I did not find an impact on their somatic calcium currents, leaving a possible involvement of α2δ-3 in the horizontal cell axon-to-rod connection. Outer retina function, determined by electroretinogram, and optokinetic reflex behavior was normal in α2δ-3 knockout animals. However, I discovered changes to the retinal output in micro-electrode array recordings of ganglion cell responses. I applied a paradigm of light stimulation at different ambient luminance levels that revealed effects of the α2δ-3 knockout only in scotopic and mesopic light levels. In summary, α2δ-3 is a candidate for horizontal cell axon-specific calcium signal modulation and exerts its function in non-photopic regimes. The retina constantly adapts to features of the current visual environment, most prominently, the ambient light intensity or luminance. These adaptations are based on mechanisms throughout the retinal network. Adaption is commonly considered to keep signal processing within the dynamic range of the system as well as keep the retinal output stable across changing conditions, such as the light intensity. The results of part 1 show that different building blocks of retinal circuits - here the α2δ-3 subunit - can contribute to retinal function at distinct light level regimes. In part 2, we looked more generally at the output of the retina (responses of ganglion cells) across different levels of ambient luminance. We found that ganglion cell responses were not stable across luminance levels, neither in single ganglion cell types nor in the ganglion cell population, but that they changed their responses qualitatively. These response changes were also reflected downstream in the activity of the lateral geniculate nucleus. Furthermore, we observed that rod photoreceptors could drive visual responses of ganglion cells in photopic luminance levels, where they were commonly thought to be saturated. While experiencing initial incremental saturation upon stepping to photopic luminance, rods recovered responsiveness at all light levels tested, but the rate of recovery was faster with brighter ambient light intensity. Computational modeling suggested adaptive translocation of elements of the signal transduction cascade as potential explanations for rod signaling at high light intensities. The photopic rod activity dynamics have important implications for the interpretation of experimental data and for the question of rod photoreceptor contributions to daylight vision. In summary, while some circuitry elements associated with luminance regimes are known (e.g. rod and cone pathways), details on the underlying molecular mechanisms are scarce. My data suggests α2δ-3 as a promising candidate for a molecular determinant of light adaptation that could exert its function within horizontal cells in an axonal compartment-specific way. It will be interesting to pinpoint the exact role of α2δ- 3 in retinal light adaptation and to determine what (sub-)cellular function this protein serves in horizontal cells

Rods progressively escape saturation to drive visual responses in daylight conditions

Rod and cone photoreceptors support vision across large light intensity ranges. Rods, active under dim illumination, are thought to saturate at higher (photopic) irradiances. The extent of rod saturation is not well defined; some studies report rod activity well into the photopic range. Using electrophysiological recordings from retina and dorsal lateral geniculate nucleus of cone-deficient and visually intact mice, we describe stimulus and physiological factors that influence photopic rod-driven responses. We find that rod contrast sensitivity is initially strongly reduced at high irradiances, but progressively recovers to allow responses to moderate contrast stimuli. Surprisingly, rods recover faster at higher light levels. A model of rod phototransduction suggests that phototransduction gain adjustments and bleaching adaptation underlie rod recovery. Consistently, exogenous chromophore reduces rod responses at bright background. Thus, bleaching adaptation renders mouse rods responsive to modest contrast at any irradiance. Paradoxically, raising irradiance across the photopic range increases the robustness of rod responses.Peer reviewe

Step-By-Step Instructions for Retina Recordings with Perforated Multi Electrode Arrays

Multi-electrode arrays are a state-of-the-art tool in electrophysiology, also in retina research. The output cells of the retina, the retinal ganglion cells, form a monolayer in many species and are well accessible due to their proximity to the inner retinal surface. This structure has allowed the use of multi-electrode arrays for high-throughput, parallel recordings of retinal responses to presented visual stimuli, and has led to significant new insights into retinal organization and function. However, using conventional arrays where electrodes are embedded into a glass or ceramic plate can be associated with three main problems: (1) low signal-to-noise ratio due to poor contact between electrodes and tissue, especially in the case of strongly curved retinas from small animals, e.g. rodents; (2) insufficient oxygen and nutrient supply to cells located on the bottom of the recording chamber; and (3) displacement of the tissue during recordings. Perforated multi-electrode arrays (pMEAs) have been found to alleviate all three issues in brain slice recordings. Over the last years, we have been using such perforated arrays to study light evoked activity in the retinas of various species including mouse, pig, and human. In this article, we provide detailed step-by-step instructions for the use of perforated MEAs to record visual responses from the retina, including spike recordings from retinal ganglion cells and in vitro electroretinograms (ERG). In addition, we provide in-depth technical and methodological troubleshooting information, and show example recordings of good quality as well as examples for the various problems which might be encountered. While our description is based on the specific equipment we use in our own lab, it may also prove useful when establishing retinal MEA recordings with other equipment

Recording stability.

<p><b>A)</b> Responses of one ganglion cell to a step in contrast over 6 hours. A two second light decrement step has been shown >120 times over a period of 6 hours. Each dot in the raster plot represents one spike produced by the ganglion cell. The ganglion cell stably responded to the stimulus during the whole recording time. Changes in latency and number of spikes are due to different mean brightness levels used during the experiment. <b>B)</b> Receptive field of one ganglion cell calculated from checkerboard stimuli. 15×15 checkers out of 40×40 shown here. The stimulus has been repeated approximately every 90 minutes. Time above each receptive field map: presentation time of the checkerboard stimulus (0 min = beginning of experiment). The receptive field location and shape was stable during the whole 8 hours, indicating that the retina did not move significantly.</p

Additional steps for <i>in vitro</i> ERG recordings.

<p><b>A) Additions to Step 1:</b> The AgCl reference is positioned over the MEA by a reference electrode holder and is attached to pin 15 (REF) by a wire ferrule insulated by shrink-on tubing (asterisk). <b>B) Additions to Step 5:</b> Schematic of the reference electrode and its holder as shown in A. Note the optical shield needed to avoid photoelectric artifacts resulting from light hitting the reference electrode.</p

Experimental procedure Step 1: Filling of MEA chamber.

<p><b>Step 1a)</b> Placing the MEA chamber on the baseplate. <b>Step</b><b>1b)</b> Preparation of perfusion and vacuum. <b>Step</b><b>1c)</b> Filling the MEA. Detailed description is given in the text.</p

Setup for pMEA recordings.

<p>Our MEA setup consists of two perfusion loops. Solution is supplied to the MEA chamber from the top through the upper perfusion (A) and excessive solution is removed by the suction (B). The necessary negative pressure is supplied by the additional perfusion, consisting of the lower perfusion (C) and a vacuum (D). Details are given in the following text and figures.</p

Experimental procedure Steps 5 and 6: Recording data (<i>in vitro</i> ERG recordings).

<p><b>A₁)</b> Snapshot of the Longterm Data Display (raw data) from MC_Rack. Note that on most electrodes the ganglion cell spikes mask the <i>in vitro</i> ERG responses (e.g. the electrode marked in orange). Only the highest contrast flash elicits a response that is visible on most electrodes (red asterisks), while on some electrodes without ganglion cell spikes the <i>in vitro</i> ERG responses are clearly visible (electrode marked in blue). Reference electrode 15 (REF) is on the left. <b>A₂)</b> Zoomed view of the electrode marked in blue from panel A₁ showing the responses to flash stimuli of different contrast (highest two contrasts marked with red asterisks). The low-pass filtered data around the time highlighted by the box is shown in B₁+B₂. <b>B₁)</b> Data Display with 200 Hz low-pass filter applied. There is a clear response on almost all electrodes. Not all spikes get filtered out by the low-pass filter. Note the different time scale than in A₁. <b>B₂)</b> Zoomed view of the electrode marked in blue from panel B₁ that shows a very clear low frequency <i>in vitro</i> ERG response without contamination by ganglion cell spikes.</p

Experimental procedure Steps 5 and 6: Recording data (Spike recordings).

<p><b>A)</b> Snapshot of a 500 Hz high-pass filtered MC_Rack display. Spiking activity with good signal-to-noise is visible on many electrodes. <b>B)</b> Snapshot of MC_Rack display after overflow. Noise with amplitudes of 200 to over 1000 µV due to wet electronics is visible on most electrodes. <b>C)</b> Snapshot of MC_Rack display several hours after strong overflow. Slow noise on many electrodes is visible either if the electronics is not fully dry yet or when the electronics has been irreversibly harmed. <b>D)</b> Snapshot with slow fluctuations and spike-like noise peaks (red asterisks). See text (Step 5 and 6, troubleshooting) for details.</p