Testing optokinetic head or eye movements is an established method to determine visual performance of laboratory animals, including chickens, guinea pigs, mice, or fish. It is based on the optokinetic reflex which causes the animals to track a drifting stripe pattern with eye and head movements. We have developed an improved version of the optomotor test with better control over the stimulus parameters, as well as a high degree of automation. The stripe pattern is presented on computer monitors surrounding the animal. By tracking the head position of freely moving animals in real time, the visual angle under which the stripes of the pattern appeared was kept constant even for changing head positions. Furthermore, an algorithm was developed for automated evaluation of the tracking performance of the animal. Comparing the automatically determined behavioral score with manual assessment of the animals' tracking behavior confirmed the reliability of our methodology. As an example, we reproduced the known contrast sensitivity function of wild type mice. Furthermore, the progressive decline in visual performance of a mouse model of retinal degeneration, rd10, was demonstrated.
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.
Systemic injection of sodium iodate has considerable effects on RPE, photoreceptors, and inner nuclear layer neurons, and provides a model to assay reconstitution and maturation of RPE cell transplants. The availability of an RPE-free Bruch's membrane in this model likely allows the unprecedented formation of extensive polarized cell monolayers from donor hESC-derived RPE cell suspensions.
Temporal resolution is a basic property of the visual system and critically depends upon retinal temporal coding properties which are also of importance for directional coding. Whether the temporal coding properties for directional coding derive form inherent properties or critically depend upon the temporal coding mechanisms is unclear. Here, the influence of acetylcholine and GABA upon photopic temporal coding was investigated in goldfish, using flicker stimuli, in a behavioral and an electrophysiological (ERG) approach. The goldfish temporal resolution ability decreased from more than 90% correct choices at 20 Hz flicker frequency to about 65% at 45 Hz flicker frequency with a flicker fusion frequency of approximately 39 Hz. Blockade of GABAa-receptors reduced the flicker fusion frequency to about 23 Hz, not affecting temporal resolution below 20 Hz flicker frequency. Partial blockade of nicotinic acetylcholine receptors reduced the flicker fusion frequency slightly and lowered the temporal resolution ability in the 25-30 Hz range. Blockade of muscarinic acetylcholine receptors had a smaller effect than the partial blockade of nicotinic acetylcholine receptors. In ERG-recordings, blocking GABAa-receptors increased the a- and b-wave amplitude, induced a delay, an increase and a slow fall-off of the d-wave. Blocking GABAc-receptors had little effect. Blocking GABAa- or GABAa/c-receptors changed the temporal resolution, when expressed as a linear filter, from a 3rd degree filter with resonance to a low order low-pass filter with a low upper limit frequency. The temporal transfer properties were barely changed by blocking either nicotinic or muscarinic acetylcholine receptors, although ERG-components increased in amplitude to varying degrees. The behavioral and electrophysiological data indicate the important role of GABA for temporal processing but little involvement of the cholinergic system. It is proposed that the interaction of the GABAergic amacrine cell network and bipolar cells determines the gain of the retinal temporal coding in the upper frequency range.
The Jimpy mutant mouse has a point mutation in the proteolipid protein gene (plp1). The resulting misfolding of the protein leads to oligodendrocyte death, myelin destruction, and failure to produce adequately myelinated axons in the central nervous system (CNS). It is not known how the absence of normal myelination during development influences neural function. We characterized the Jimpy mouse retina to find out whether lack of myelination in the optic nerve during development has an effect on normal functioning and morphology of the retina. Optokinetic reflex measurements showed that Jimpy mice had, in general, a functional visual system. Both PLP1 antibody staining and reverse transcriptase-polymerase chain reaction for plp1 mRNA showed that plp1 is not expressed in the wild-type retina. However, in the optic nerve, plp1 is normally expressed, and consequently, in Jimpy mutant mice, myelination of axons in the optic nerve was mostly absent. Nevertheless, neither axon count nor axon ultrastructure in the optic nerve was affected. Physiological recordings of ganglion cell activity using microelectrode arrays revealed a decrease of stimulus-evoked activity at mesopic light levels. Morphological analysis of the retina did not show any significant differences in the gross morphology, such as thickness of retinal layers or cell number in the inner and outer nuclear layer. The cell bodies in the inner nuclear layer, however, were larger in the peripheral retina of Jimpy mutant mice. Antibody labeling against cell type-specific markers showed that the number of rod bipolar and horizontal cells was increased in Jimpy mice. In conclusion, whereas the Jimpy mutation has dramatic effects on the myelination of retinal ganglion cell axons, it has moderate effects on retinal morphology and function.
Little is known about the function of the auxiliary α2δ subunits of voltage-gated calcium channels in the retina. We investigated the role of α2δ-3 (Cacna2d3) using a mouse in which α2δ-3 was knocked out by LacZ insertion. Behavior experiments indicated a normal optokinetic reflex in α2δ-3 knockout animals. Strong expression of α2δ-3 could be localized to horizontal cells using the LacZ-reporter, but horizontal cell mosaic and currents carried by horizontal cell voltage-gated calcium channels were unchanged by the α2δ-3 knockout. In vivo electroretinography revealed unaffected photoreceptor activity and signal transmission to depolarizing bipolar cells. We recorded visual responses of retinal ganglion cells with multi-electrode arrays in scotopic to photopic luminance levels and found subtle changes in α2δ-3 knockout retinas. Spontaneous activity in OFF ganglion cells was elevated in all luminance levels. Differential response strength to high-and low-contrast Gaussian white noise was compressed in ON ganglion cells during mesopic ambient luminance and in OFF ganglion cells during scotopic and mesopic ambient luminances. In a subset of ON ganglion cells, we found a sharp increase in baseline spiking after the presentation of drifting gratings in scotopic luminance. This increase happened after gratings of different spatial properties in knockout compared to wild type retinas. In a subset of ON ganglion cells of the α2δ-3 knockout, we found altered delays in rebound-like spiking to full-field contrast steps in scotopic luminance. In conclusion, α2δ-3 seems to participate in shaping visual responses mostly within brightness regimes when rods or both rods and cones are active.
Zusammenfassung Bei neurodegenerativen Erkrankungen der Netzhaut sind die lichtempfindlichen Zellen, die Photorezeptoren, oft als Erstes betroffen. Die Optogenetik ist ein vielversprechender Ansatz, die Netzhaut wieder lichtempfindlich zu machen und dadurch das Sehvermögen wiederherzustellen. Bei der Optogenetik werden lichtempfindliche Proteine über gentechnische Methoden in die Netzhaut eingebracht; die Aktivität der Zielzellen wird durch diese Behandlung durch Licht beeinflussbar. Dieser Einfluss kann die direkte lichtinduzierte Änderung des Membranpotenzials sein (sowohl hemmend als auch erregend) oder die lichtinduzierte Aktivierung intrazellulärer Signalkaskaden. Dies hat zur Folge, dass das Zielgewebe, die Netzhaut, wieder auf Licht reagiert. Diese Übersicht beschreibt die Prinzipien der Optogenetik und den gegenwärtigen Stand im Hinblick auf ihre Anwendung zur Behandlung von Blindheit.
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