Abstract
Purpose:
Wild-type and genetically modified mice are widely used to study retinal physiology and disease. An important method to assess the function of the retina in different mouse models is the In Vivo electroretinogram (ERG) for which commercial systems are available. However, studying photoreceptor physiology with In Vivo ERG is hampered by inner retinal signals that mask the photoreceptor-originating fast PIII component. To facilitate 1) studies of photoreceptor function and 2) use of pharmacology in the mouse retina, we aimed to develop and test an affordable and easy way to adapt a commercial mouse In Vivo ERG system to do Ex Vivo ERG.
Methods:
We performed Ex Vivo ERG from isolated WT and GNAT1-/- mouse retinas. The specimen holder was mounted on the heating pad of a commercial mouse In Vivo ERG system (LKC Technologies Ltd.). DC-ERG signal was recorded across the retina with Ag/AgCl pellet electrodes connected to LKC system’s amplifier (UBA-4200). Retina wase perfused with Ames medium heated to 37 oC by a custom-made heat exchanger placed on the LKC system’s heating pad and regulated by its heat controller. The fast PIII component was isolated by adding 20 μM D, L-AP4 and 50 - 100 μM BaCl2 to the perfusion. Data acquisition (fs = 1000 Hz, fc = 300 Hz) and green light stimulation (LKC Bigshot LED ganzfeld) were performed with the LKC system.
Results:
Stable a- and b-wave responses could be recorded for several hours with significantly better signal-to-noise ratio than typical In Vivo responses. Average maximum a- and b-wave amplitudes (rsat) were 590 ± 120 μV and 742 ± 190 μV for rods (n = 3) and 23 ± 3 μV and 75 ± 23 μV for cones (GNAT1-/-, n = 3), respectively. Half-saturating flash strength (Q½ in log Cdsm-2) for the rod a- and b-wave were -1.4 ± 0.1 and -3.6 ± 0.1 (n = 3) and for cones -0.0 ± 0.1 and -0.7 ± 0.1 (n = 3). For comparison, In Vivo WT rod recordings yielded Q½ of -0.3 ± 0.1 for the a-wave. For pharmacologically isolated rod fast PIII rsat was 350 ± 70 μV and Q½ was -2.7 ± 0.1 (n = 3). Recordings with the same specimen holder in similar conditions with calibrated light stimulation showed Q½ of 34 ± 2 R* per rod (n = 5).
Conclusions:
Commercial In Vivo ERG systems can be adapted easily and with low cost to perform Ex Vivo ERG. This innovation should facilitate physiological studies of the retina and especially photoreceptor cells. In particular, it will be useful in studying mammalian cone physiology.
Keywords: 508 electrophysiology: non-clinical •
510 electroretinography: non-clinical •
648 photoreceptors