Spiperone

Intravitreal injections were performed using a 0.5-mL insulin syringe (30-gauge needle, BD Medical, Le Pont deClaix, France). They consisted of atropine sulfate monohydrate (250 µg/360 nMol, > 97%, Sigma-Aldrich, Deisenhofen, Germany) in 25 µL saline, or spiperone (500 µMol, Sigma-Aldrich, Deisenhofen, Germany) in 25 µL ascorbic acid (1 mg/mL, pH 7.4), or a combination of both atropine and spiperone in 25 µL ascorbic acid, while the fellow eye received 25 µL saline or 25 µL ascorbic acid, respectively. Alternatively, both eyes received 25 µL saline. The atropine dose was selected based on previous experiments [21 (link)], where daily injections completely blocked myopia induced by wearing − 7D lenses. The dose of spiperone was also selected based on previous experiments. Thomson et al. [17 (link)] had shown that daily injections of 500 µMol of spiperone suppressed the protective effects of dopamine or a dopamine agonist against the development of both deprivation myopia and lens-induced myopia. Ashby and Schaeffel [23 (link)] had found that 500 µMol spiperone blocked the protective effect of bright light on deprivation myopia in chickens. All injections were performed under mild ether anesthesia. The intravitreal injection procedure in the chicken is well established (e.g., [11 (link), 24 (link)–26 (link)]) and, as in previous studies, we never observed ocular inflammations.

LCLs of the study sample were derived at Rutgers University Cell and DNA Repository (RUCDR)^{36 (link)}. For each LCL, we measured EBV (viral) load (copy number), viable cell count (to index growth rate), and ATP level (to index energy status) at cell harvest (for use as covariates in expression analyses), which are known to have an effect on gene expression in LCLs^{41 (link)}. For the initially processed 515 SZ cases and 692 controls, RNAseq was carried out in five large batches; further detailed methodology was previously described^{37 (link)}. For DA perturbation (the pilot on four LCLs and the large-scale RNAseq samples), we grew cells in independent wells (on 6-well plates) in the presence or absence of DA at indicated concentrations. DA perturbation lasted 24 h. To block DA effects, we pre-treated the cells with the DA receptor antagonists for 6 h before adding DA to the cell culture medium. These DA blockers included: D1-like receptor (D1 or D5) antagonist SCH23390 (200 nM; ~100-fold saturation concentration^{42 (link)}) and D2-like receptor (D2, D3, or D4) antagonist spiperone (200 nM; ~100-fold saturation concentration^{43 (link)}). We purchased DA, SCH23390, and spiperone from Sigma-Aldrich. We included batch as a possible confounding variable in the analysis, i.e., as a covariate.