D9628

Extracellular matrix (ECM) immobilization with S-S treatment was performed according to a published protocol [16 ]. Briefly, 50 mg of S-S (catalog no. 22589, Thermo Fisher Scientific) was dissolved in 1 ml of dimethyl sulfoxide (DMSO) and diluted 25 times in Milli-Q water, resulting in an S-S solution (2 mg/ml). Three hundred microliters of this solution was added to the gel surface and placed under an ultraviolet light in a biological safety cabinet for 30 min. Gels were then washed twice with PBS and incubated with the desired ECM (0.2% gelatin in PBS; G1890, Sigma-Aldrich) overnight at 4 °C.
Gels were prepared for cell culture by washing once in PBS, rinsing in 70% ethanol for 2 min, washing in PBS twice, ultraviolet-sterilizing for 30 min, and incubating with culture medium for 1 h. For ECM conjugation using L-DOPA (3,4-dihydroxy-l-phenylalanine; D9628, Sigma-Aldrich) [17 (link)], L-DOPA solution (2 mg/ml) was prepared using 10 mM tris-HCl buffer (pH balanced to 10.2 using 1 M NaOH) by mixing gently on a roller shaker for 30 min under no-light conditions. The solution was filtered to remove undissolved L-DOPA, distributed over the gels, and then incubated for 30 min under no-light conditions. Gels were then conjugated with ECM and prepared for cell culture as per the procedure described for S-S above.

The solutions were freshly prepared every week in vehicle (sucrose 5% in mineral water Vittel^®) and 200 ml were poured on a Kimwipe paper (1.5 cm × 3.5 cm) deposited in a 15 ml Falcon tube. Nine 1-day-old virgin females were introduced in the tube for the length of the treatment, at 18°C, 12 h:12 h light:dark cycle. To explore dopamine effect, flies were fed with 3-IY (10 mg/ml, Sigma I8250) and/or L-DOPA (1 mg/ml, Sigma D9628) for 36–40 h (Bainton et al., 2000 (link); Seugnet et al., 2008 (link)). To explore serotonin effect, flies were fed with PCPA (10 mg/ml, Sigma C6506) and/or 5-HTP (16 mg/ml, Sigma H9772) for 3 days, with papers being changed once during the 3 days period (Dierick and Greenspan, 2007 (link); Plaçais et al., 2012 (link)). We used a high 5-HTP concentration (30% more than in Dierick and Greenspan, 2007 (link)) to ensure rescued serotonin levels in PCPA-treated flies in our conditions. This concentration did not affect mate-copying in flies fed with 5-HTP (Figure 2). The treatment “vehicle” consisted of vehicle solution given during 36–40 h or 3 days.

The effect of retinal DA replacement was investigated by treating Gnat1^rd17 mice with L-DOPA (3,4-dihydroxy-L-phenylalanine, D9628, Sigma-Aldrich, Darmstadt, Germany) using different administration routes: i.p. injection and topical application of eye drops. For i.p. administration, L-DOPA was dissolved in 0.9% saline just prior to injection and was given in a dose of 25 mg/kg body weight per day at 7:30 a.m. from day 9 to day 14 after immunization. A control group received equal amounts of vehicle solution (0.9% saline). To increase the bioavailability of topically applied ophthalmic drugs, L-DOPA was dissolved to 15 mM in 1× phosphate-buffered saline (PBS, pH 5.5) with 10% (v/v) dimethyl sulfoxide (DMSO), a well-tolerated corneal-penetration enhancer [105 (link)]. Mice received topical L-DOPA treatment in the form of two 10 μL eye drops in the left eye and an equal volume of vehicle solution (1× PBS, pH 5.5 with 10% DMSO) in the right eye twice daily (at 7:30 a.m. and 7:00 p.m.) for a period of six days starting from day 9 after immunization. The last dose of L-DOPA eye drops or i.p. injection was administered at 7:30 a.m. on day 14 after immunization. The optomotor response test on day 14 was conducted 1 h after L-DOPA or vehicle injection. The animals were then examined for fundus changes and immediately sacrificed to collect neuroretinas for flow cytometry analysis.