The experimental protocols were approved by the Animal Ethics Committee of the Zhongshan Ophthalmic Center, Sun Yat-sen University. All experimental procedures involving animals adhered to the Association Research in Vision and Ophthalmology. Generally, one eye of each animal was sutured during the critical period of vision development, and kept till the critical period ended.

To establish the primate model of amblyopia, 1-month-old baby cynomolgus monkeys were examined under optical coherence tomography, electroretinogram, fundus photography, and A-ultrasound to ensure that both eyes were well-developed, and there were no organic or functional defects in the eyes. After the examination, the left eye was sutured. Three months later, the monkeys were euthanized, and the dLGNs were isolated for RNA sequencing.

To establish the rodent model of amblyopia, the left eyes of postnatal day 18 C57/Bl6 mice were sutured. Twenty-four days later, the total LGN was isolated for mRNA and protein sample preparation. Alternatively, brain sections were prepared for immunofluorescence staining.

Total RNA was extracted using a Trizol reagent kit (Invitrogen, Carlsbad, CA, United States). The RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States) and checked using RNase free agarose gel electrophoresis. The mRNA was enriched by Oligo -deoxythymine beads, and processed for fragmentation and reverse transcription into cDNA with random primers. Second-strand cDNAs were synthesized and the cDNA fragments were purified, end repaired, A base added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co., (Guangzhou, China).

After removing the low-quality reads by fastp (version 0.18.0) (Chen et al., 2018), the clean reads were aligned with the reference genome [Macaca_fascicularis_5.0 in National Center for Biotechnology Information (NCBI) database] using HISA T2 (version 2.1.0) (Kim et al., 2015). Transcripts were reconstructed with software Stringtie (version 1.3.4) and their abundances were quantified by software StringTie in a reference-based approach (Pertea et al., 2015, 2016). The data are available in the Gene Expression Omnibus database GSE189488.

Gene Ontology (GO) enrichment analysis was performed for the functional classification of candidate genes (Ashburner et al., 2000). Pathway-based analysis was performed using the KEGG database (Kanehisa and Goto, 2000).

Quantitative polymerase chain reaction was performed to quantify the relative expression level of genes as previously described (Zhang et al., 2017). The monkey and mouse primers are listed in Tables 1, 2, respectively.

Mice were transcardially perfused with cold saline for 2 min followed by 4% paraformaldehyde (PFA) for 30 min. Brains were isolated and immersed in 4% PFA solution for 24 h. The fixed brains were dehydrated in 20%, followed by 30% sucrose solution till they sank down to the bottom of the containers, and then embedded in optimal cutting temperature compound (Tissue-Tek, United States), cut into 30-μm thick frozen sections.

Immunofluorescence staining was performed as described previously (Zhou et al., 2019). Briefly, brain sections were air-dried for 30 min at room temperature (RT), rinsed in 0.01 M phosphate buffered saline (PBS), and permeabilized with PBS supplemented with 0.5% Triton X-100 and 5% goat serum for 15 min at RT. Then, the sections were incubated in the following order: Primary antibodies against DRD1 (GeneTex, GTX100355) overnight at 4°C, Alexa Fluor 488-conjugated secondary antibody for 1 h at RT, and 0.1% DAPI for 5 min at RT, with washing in PBS 3 times after each incubation. The sections were examined under a confocal microscope (Nikon Eclipse Ni-E).

The immunofluorescence density measurement was carried out in accordance with the previous studies (Hanke, 2000; Torborg and Feller, 2004; Ogawa et al., 2005; Liu et al., 2020). For a semiquantitative estimation of DRD1 in the dLGN, a coronal section was selected in the middle of dLGN that was close to Figure 50 of The Mouse Brain atlas (Paxinos and Franklin, 2001). The images were captured after the laser intensity and gain were optimized in each channel. The boundary of the dLGN was identified and tracked with dotted lines. A large rectangle was drawn within the core dLGN along its long axis (from dorsomedial to ventrolateral), which was further divided into six areas, each corresponding to a region of interest (ROI). For the measurement, the background of each picture was adjusted to be consistent across blinded investigators. The fluorescence intensity of a neighboring region in the hippocampus was considered a control to be consistent in all samples. The color images were converted into binary images in ImageJ software (National Institutes of Health, Bethesda, MD, United States). The threshold percentage used for the conversion was also determined by the blinded investigators to ensure that the threshold percent value best represented the observed signal. The average fluorescence density was obtained by ImageJ.

Statistical analysis was carried out using the SPSS 20.0 statistical software package. All data were tested for normality and homogeneity of variance. The data with a normal distribution and homogeneity of variance were expressed as the mean and standard deviation (SD). Single factor analysis of variance (ANOVA) was used for comparison among groups and minimum significant difference (least significant difference, LSD). The t-test was used for pairwise comparisons between the groups. The difference was set to be statistically significant when p < 0.05.

To explore the etiology of amblyopia, we established a primate model of monocular visual deprivation by suturing the left eyes of baby monkeys. Three months later, brain regions involved in the central visual system were isolated for RNA sequencing. Here, we followed the mRNA sequencing results of the dorsal lateral geniculate nucleus (dLGN). Three samples from the untreated control monkeys and two samples from the ipsilateral (left) dLGN of amblyopia monkeys were qualified for sequencing (Figure 1A). We identified 818 differentially expressed genes (DEGs) that were regulated more than 2 times (Figure 1B). Of them, 492 were upregulated, and 326 were downregulated.

Next, we performed the enrichment analysis. The GO enrichment demonstrated that the most enriched categories were related to nervous system development. In the biological process (BP) category, many DEGs were related to cell–cell signaling or communication, neurogenesis, and neural development (Figure 2A). In the cell component (CC) category, many DEGs were related to synapse, membrane, and projection (Figure 2B). The KEGG enrichment analyses identified pathways related to synapse, nervous system development, and neurogenesis (Figure 2C). Thirteen DEGs were grouped in the dopaminergic pathway (Figure 2C). Dopamine receptor D1 (DRD1) is a member of this pathway. A list of DEGs classified by a biological process is available in the supplementary.

Thus, the mRNA sequencing results demonstrated that visual deprivation leads to the regulation of neural developmental factors in the monkey dLGN.

Because synaptic receptors play pivotal roles in neural plasticity and functions, we selected six receptors from the DEG list for further verification (Table 3). First, we checked the expression of these receptors in monkey LGN by qPCR. The up- or downregulation of these receptors was verified to be the same as the results from mRNA sequencing (Figure 3A).

Next, we examined the expression of these receptors by qPCR in amblyopia mouse samples. As shown in Figure 3B, Drd1 and Gabrb2 were downregulated in both ipsi- and contralateral LGNs in the mouse model, which was consistent with the corresponding regulation in the monkey model. The other four selected receptors were not regulated in the same ways as they were in the monkey model.

Of the six receptors under investigation thus far, the regulation of Drd1 was consistent in mRNA sequencing and in the qPCR study. In addition, we know that levodopa, a precursor of dopamine, has been under clinical trials for the treatment of amblyopia for years. Therefore, Drd1 was selected for further investigation. Thus, we checked the protein expression of DRD1 in the LGN of the mouse amblyopia model by Western blot. The semiquantitative study showed a 15–20% downregulation of expression in both ipsilateral and contralateral LGNs (Figure 4).

Next, we examined the distribution of DRD1 in the subregions of the mouse dLGN by immunostaining. In the coronal sections of intact (control) mouse brains, DRD1 immunoreactive (IR) cells and fibers were identified in the dLGN. A strong IR was detected in the outer shell portion and the ventrolateral region of the inner core portion. Light IR was detected in the dorsomedial region of the inner core portion (Figures 5A1–A2,B1–B2).

The pattern of DRD1 IR in the dLGN of the amblyopic mice was similar to that in the control group. However, lighter staining was identified in the dorsomedial region of the ipsilateral dLGN (Figures 5A3,B3) and in the ventrolateral region of the contralateral dLGN (Figures 5A4,B4) compared to the intact group.

To obtain a semiquantitative estimation of the intensity of DRD1 staining, we analyzed its regional expression. Green DRD1 IR was converted to gray. The core portion of the dLGN was divided into six rectangles along its long axis, each corresponding to a region of interest (ROI). The average staining intensity in each ROI was calculated. As shown in Figure 5C, in the ipsilateral dLGN, DRD1 IR was at a similar level as that of the control in ROIs 1–3 but was downregulated to less than half of the level of the control group in ROIs 5–6. In the contralateral dLGN, DRD1 IR was downregulated to approximately two-third of the level of the control group in ROIs 1–3 but not in ROIs 4–6. Both regions of downregulation were exactly the targets of the projections from the amblyopic eye. These data demonstrated the downregulation of DRD1 in the dLGN in the target regions of the amblyopic eye.