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 (Munic, 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. Meanwhile, they can also be ordered from MMRRC (ID 50523).
Cav2.3-KO 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 knock out generation see [18, 19]). 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).
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 using primers, which flanked the deleted exon 2 and exon 3 region . 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 (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. Washout was started thereafter with Cu2+-free modified Ames-medium.
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.
The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Quantitative normally distributed data are presented as mean ± standard error of mean (SEM) and as percentage. Student’s t-test was used for comparison of quantitative parameters in case of normal distribution. All analyses were performed with IBM® SPSS® Statistics V22.0 (IBM, Chicago, Illinois, USA).