As previously described,
29 six weeks after injections, mice were euthanized using carbon dioxide, the eyes were enucleated, and the cornea and lens were removed to form an eyecup. The eyecup was incubated in cold extracellular solution with hyaluronidase (800 U/mL, 20 minutes) and washed with cold extracellular solution, and the retina was removed. For slice preparations, the retina was trimmed into a rectangle, mounted onto nitrocellulose filter paper (0.45-µm; Millipore, Billerica, MA, USA), transferred to a hand chopper and sliced (250 µm), rotated 90°, and mounted onto glass coverslips using vacuum grease. Whole mounts were prepared according to previous methods.
30,31 The retina was cut into four equal quadrants and mounted photoreceptor side down onto a trimmed cell culture insert (1 mm height; Millipore Sigma, Burlington, MA, USA). All dissections and light response recording procedures were performed under infrared illumination to preserve the light sensitivity.
Extracellular solution was bubbled with a mixture of 95% O2–5% CO2 (pH to ∼7.4) and contained (in mM) the following: 125.00 NaCl, 2.50 KCl, 1.00 MgCl2, 1.25 NaH2PO4, 20.00 glucose, 26.00 NaHCO3, and 2.00 CaCl2. Intracellular solution in the recording pipette contained (in mM) the following: 120.00 CsOH, 120.00 gluconic acid, 1.00 MgCl2, 10.00 HEPES, 10.00 EGTA, 10.00 tetraethylammonium-Cl, 10.00 phosphocreatine-Na2, 4.00 Mg-ATP, 0.50 Na-GTP, and 0.1% sulforhodamine-B dissolved in water (pH 7.2 with CsOH). To selectively activate D4Rs, the D4R agonist PD-168077-maleate (PD, 500 nM; solubilized in DMSO Waltham, Massachusetts, USA) was diluted in extracellular solution and applied to the recording bath by a gravity-driven superfusion system (Cell Microcontrols, Norfolk, VA, USA; ∼1 mL/min). The perfusate had a final DMSO concentration of less than 0.0025%. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.
Responses were recorded in a control dark-adapted state, agonist was applied for 5 minutes, and then responses were recorded in the continuous presence of agonist. Retinal slices on glass coverslips or whole-mount preps were placed in a custom chamber and heated to 32° (TC-324 temperature controller with SH-27B inline heater; Warner Instruments, Hamden, CT, USA). Whole-cell voltage-clamp recordings of light-evoked (L-) and spontaneous (s) excitatory postsynaptic currents (EPSCs) were made from ON-s ganglion cells voltage clamped at –60 mV, the reversal potential for Cl− currents. Series resistance was uncompensated. Electrodes with resistances of 3 to 7 MΩ were pulled (borosilicate glass; World Precision Instruments, Sarasota, FL, USA) using a P97 Flaming/Brown puller (Sutter Instruments, Novato, CA, USA). Calculated liquid junction potentials of 20 mV (Clampex; Molecular Devices, Sunnyvale, CA, USA), were corrected before recording. Recordings were sampled at 10 kHz, filtered at 6 kHz (Bessel filter, Multi-Clamp 700B amplifier; Molecular Devices), and digitized with a Digidata 1140 and Clampex software (Molecular Devices).
During whole-cell recordings, cells were passively filled with sulforhodamine-B included in the intracellular solution. ON-s ganglion cells were targeted by their large soma size (>15 µM diameter).
32 Similar to a previous report,
13 confirmation of ganglion cell morphology and presence of an axon was done at the end of each recording using an Intensilight fluorescence lamp and Digitalsight camera controlled by Elements software (Nikon Instruments, Tokyo, Japan). Ganglion cells were further characterized by their light-evoked EPSC resulting from a 500-ms duration 9.5·10
5 photons·µm
−2·s
−1 flash of light. They were classified as ON-sustained if a light-evoked EPSC coincided with the onset of light, did not return to baseline until after light offset, and did not possess a distinct OFF response as well.