Glucose and the constituents of the nutrient solution used for retinal superfusion were purchased from Merck (p.a. grade). For the preparation of the murine retina  a superfine scissor (WPI, Nr. 501839), and an ultrafine suturing forceps (WPI, Nr. 555063FT) were used. Further, a 27-gauge needle (Sterican, size 20: 0.4 mm × 20 mm Bl/LB) was used to punch a hole into the cornea of the extirpated eye bulb. The receptor antagonists UBP 301 and CNQX were purchased from Sigma Aldrich (Munich, Germany).
In order to compare ERG responses from mice deficient of the voltage-gated Ca2+-channel Cav2.3 (R-type), we used control mice with an identical genetic background. Both mouse lines were generated and bred in our animal facility. Cav2.3-deficient animals and control mice were kept as separate mouse lines derived from heterozygous parents (fourth backcrossing into C57Bl/6). Homozygous littermates were regularly interbred with each other and back-bred into C57Bl/6 (for further information on Cav2.3-deficient generation see [23, 24]). In short, the cacna1e gene encoding Cav2.3 was disrupted in vivo by agouti-colored Cav2.3(fl|+) and deleter mice expressing Cre-recombinase constitutively . Thus, exon 2 was ablated by Cre-mediated recombination. Cav2.3-deficient mice were fertile, exhibited no obvious behavioral abnormalities and had the same lifespan as control mice. The Cav2.3-deficient mouse line, which was generated in the Cologne lab was transferred to the Mutant Mouse Resource & Research Centers (MMRRC) with the strain name B6J.129P2(Cg)-Cacna1etm1.1Tsch/Mmjax.
Adult male mice were used at the age of 12 to 18 month and kept at 20 to 22°C in makrolon type II cages under a 12h light-dark cycle (7:00 a.m./p.m.) with food and water provided ad libitum. All animal experiments were in line with the European Communities Council Directive 2010/63/EU for the care and use of laboratory animals as described in the UFAW handbook on the care and management of laboratory animals. All experiments were approved by the local institutional committee on animal care (UniKöln_Anzeige§4.17.007).
In order to reach maximum transretinal signaling realized by a full ERG, the bovine  and the murine retina  had to be superfused under different conditions (Tab. 1).
Bovine eyes were received from a local slaughterhouse and immediately stored in a nutrient solution (Sickel medium), which was aerated with pure oxygen, consisting of the following (in mM): NaCl (120), KCl (2.0), CaCl2 (0.15), MgCl2 (0.1), NaH2PO4 (1.5), Na2HPO4 (13.5) and glucose (5) with a final pH of 7.8. The bovine retina was isolated as described in detail by Luke et al. 2005 . After isolation, a plain retina segment was transferred into the recording chamber described below, which is placed in an electrically and optically isolated air thermostat. From the dark-adapted retina and in response to a single white flash, electroretinograms were recorded at intervals of 5 minutes at 30 °C, with a constant superfusion at 1 ml/min controlled by a roll pump. The flash intensity was set to 6.3 mlux, with the duration of light stimulation at 500 ms .
After reaching a stable equilibrium of the light-evoked ERG responses, 5 mM tricine (tricine, Sigma # RES3077T-A701X) was added to the nutrient solution with 10 mM (HEPES) (Carl Roth, p.A., # H3375) and superfused for 30 min. Washout was started thereafter with Sickel medium.
Murine retinas were isolated from mice of our animal facility department, in which the light–dark regime was 12:12 hr, and the light intensity between 5 and 10 lx at the surface of the animal cages.
DNA-containing tissue samples were collected from tail biopsies. DNA was extracted and used as template for genotyping. Transcripts of Cav2.3 were detected by RT-PCR (RT, reverse transcriptase) using primers, which flanked the deleted exon 2 and exon 3 region . In short, contaminating protein and RNA were enzymatically digested by protease and RNAse, respectively. For the PCR amplification of indicative Cav2.3 DNA-fragments, about 1 µg DNA was introduced and amplified with the forward primer (B45Hilx1) 5’- AAA AAC AGC CGG GGA AAG CTT AT-3‘ and the reverse primer (a1eb45r) 5‘-ctg ccc ttt ctt ctt gcc tga c-3‘. The sizes of DNA fragments expected are 1056 bp for the WT and 86 bp for the Cav2.3-KOs. PCRs for the genotyping were performed using a DNAEngine Peltier thermal cycler (BioRad, Germany) or a PTC-200 Peltier thermal cycler (MJ Research, Biozym Diagnostik, Germany) with the initial denaturation (94°C for 10 min) followed by 34 cycles (denaturation at 94°C for 60 sec, annealing at 60°C for 90 sec, extension at 72°C 4 min) and final extension at 72°C for 10 min. The PCR products were separated by agarose gel electrophoresis and fluorescent bands were detected on a Herolab UVT-28M transilluminator by UV irradiation (312 nm excitation wave length).
For Cav2.3, mouse lines were used as separate inbred strains for Cav2.3(+|+) and Cav2.3(-|-), each after the fourth backcrossing in C57Bl ⁄ 6 mice.
The mice used for the retina isolation were dark adapted overnight, sacrificed by cervical dislocation under dim red light and the eyes were extirpated immediately. Enucleated eyes were protected from light and transferred into carbogen (95% O2 / 5% CO2)-saturated modified Ames medium respectively . The isolation of the murine retina was started immediately post mortem and carried out under dim red light. The complete retina was transferred to the recording chamber  and the electroretinogram was recorded via two silver/silver-chloride electrodes on either side of the isolated retina. The recording chamber containing the retina was placed in an electrically and optically isolated air thermostat. From the dark-adapted retina responses to a single white flash were recorded at intervals of 3 minutes at 27.5 °C and with a constant superfusion at 2 ml/min controlled by a roller pump. The duration of light stimulation was 500 ms, controlled by a timer operating a mechanical shutter system. The pre-stimulus delay was 380 ms. Unless noted otherwise, the flash intensity was set to 63 mlux at the retinal surface using calibrated neutral density filters.
As soon as the isolated retina was placed into the recording chamber, it was equilibrated for about 60 min in modified Ames-solution (n=20 ERGs) (Tab. 1). After reaching a stable equilibrium of the light-evoked ERG responses, 100 nM CuCl2 was added to the modified Ames-medium and superfused for 30 min (n=10 ERGs). Washout was started thereafter with Cu2+-free modified Ames-medium. Each ERG response contains 239 data points.
The ERG was amplified and bandpass-limited between 0.3 and 300 Hz (PowerLab 8/35; Animal Bio Amp FE136, ADIntruments, Oxford, UK). Light flash, heating unit, fan and roller pump were automatically controlled by National-Instruments (BNC-2120; DASY-Lab V8.0). For each experiment, a new retina was transferred to the recording chamber. The retina was superfused with nutrient solution and stimulated repetitively until the responses had reached a stable level (usually after 60 minutes of perfusion). Switching from one solution to another was performed with a three-way valve to prevent disturbance of the experimental conditions. Experimental protocols for the isolation, storage and incubation of the vertebrate retina can markedly alter phototransduction and transretinal signaling . To quantify such changes, we evaluated the a- and b-wave amplitudes and their implicit times.
1.4 Data analysis
The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. After reaching the equilibrium, the initial b-wave amplitude was set to 100 % (= Pre) resulting preferably illustration of ERGs after treatment with drugs as well as after washout. Quantitative normally distributed data are presented as mean ± standard error of mean (SEM) and as percentage. Nonparametric tests are demonstrated as median [1. quartile – 3. quartile]. Two-sided, paired Student t-test was used for comparison of quantitative parameters in case of normal distribution. If not applicable, Wilcoxon test was used instead.
All analyses were performed with IBM® SPSS® Statistics V22.0 (IBM, Chicago, Illinois, USA).