(A) Adult hippocampus-derived neural progenitor cells were cultured in complete differentiation medium for 4 d, with or without the treatment of 1 μM morphine or 10 nM fentanyl, and 10 μM CTOP. Cells were stained with markers for neurons (Tuj1), astrocytes (GFAP), oligodendrocytes (O4) and with DAPI. Scale bar, 25 μm. Images are representative of at least three independent experiments with similar results.
(B) Quantification of cells stained with each marker, calculated as the percentage of the total number of cells stained with DAPI. Red: Tuj1; Green: GFAP; Purple: O4. *, p<0.05 between indicated groups. #, p<0.05 compared to Tuj1 in control group. Data are the mean ± SEM of at least three independent experiments.
(C) The expression of βIII-tubulin, GFAP and MBP were determined by real-time PCR after 4 d of differentiation. The results were normalized against those of GAPDH, and further normalized against the result obtained from untreated cultures in the control group. All data represent the mean ± SEM of four independent experiments. *, p<0.05; **, p<0.01 between indicated groups.
(D) CD-1 mice (8-week-old males) received daily i.p. injections of 300 mg/kg BrdU for 3 consecutive days, followed by CPP training with subcutaneous injection of 5 mg/kg morphine or an equal volume of saline. Brains were fixed at day 14 and then sectioned for BrdU staining and co-immunostaining with Tuj1 and GFAP antibodies, and DAPI. Cells co-stained with antibodies of both BrdU and Tuj1 or GFAP are indicated by white arrows. Scale bars, 50 μm.
(E) Total numbers of BrdU-labeled cells co-stained with Tuj1 or GFAP antibodies in mouse hippocampus sections were counted and compared. Data represent the mean ± SEM of six independent experiments. *, p<0.05 compared to the number of cells with the same positive marker in the saline group.

(A) The expression of Notch1 was determined by real-time PCR after 4 d of differentiation, in the presence of 1 μM morphine or 10 nM fentanyl. The results were normalized against those of GAPDH, and further normalized against the result obtained from the control group. *, p<0.05 compared to control.
(B) The expression of Notch1 was determined by real-time PCR after transfection with control and Notch1 siRNA. The results were normalized against those of GAPDH. **, p<0.01 compared to control siRNA transfected group with the same treatment; #, p<0.05 compared to the control siRNA transfected group without morphine treatment.
(C) Adult hippocampus-derived neural progenitor cells were transfected with control siRNA or Notch1 siRNA, and cultured in complete differentiation medium with 1 μM morphine for 4 d. Cells were stained with markers for neurons (Tuj1), astrocytes (GFAP) and with DAPI. Scale bar, 25 μm. Images are representative of at least three independent experiments with similar results.
(D) Quantification of cells stained with each marker, calculated as the percentage of the total number of cells stained with DAPI. Red: Tuj1; Green: GFAP. *, p<0.05 compared to the control siRNA group with the same treatment; #, p<0.05 compared to the control group transfected with control siRNA.
(E) The expression of βIII-tubulin and GFAP were determined by real-time PCR after 4 d of differentiation with indicated treatments. The results were normalized against those of GAPDH. *, p<0.05, **, p<0.01, compared to control-siRNA-transfected group with the same treatment. ##, p<0.01 compared to the control group transfected with control siRNA. All data represent mean ± SEM of four independent experiments.

(A) Transcriptional assays in HEK293T cells co-transfected with pcDNA3.0-Flag-mProx1 and the empty pcDNA3.0 vector, or control siRNA and Prox1 siRNA, along with luciferase reporter constructs containing human Notch1 and empty vector (PRless). *, p<0.05 between indicated groups.
(B–C) The binding of Prox1 to Notch1 promoter was determined by qRT-PCR after ChIP assay. No Ab (without Prox1 antibody) and IgG control (with an antibody of rabbit host that reacts with an irrelevant and non-nuclear antigen) were used to confirm that the binding is specific to Prox1. Real-time PCR performed for the inputs was used to confirm equal amounts of samples were used. The results were normalized against those in vector (B) or control (C) samples in each group. *, p<0.05; **, p<0.01; ***, p<0.001 compared to vector or control groups. All data represent mean ± SEM of four independent experiments.
(D) The expression of Prox1 was determined by real-time PCR after 4 d of differentiation, in the presence of 1 μM morphine or 10 nM fentanyl. The results were normalized against those of GAPDH, and further normalized against the result obtained from the control group. *, p<0.05 compared to control.
(E) The expression of Prox1 was determined by real-time PCR after transfection with vector or Prox1-expressing plasmid (left panel), or control or Notch1 siRNA (right panel). The results were normalized against those of GAPDH. **, p<0.01 compared to control siRNA transfected group with the same treatment; ***, p<0.001 compared to vector group with the same treatment; #, p<0.05 compared to the vector (left panel) or control siRNA (right panel) transfected group without morphine treatment.
(F) Adult hippocampus-derived neural progenitor cells were transfected with vector or Prox1-expressing plasmid (top panel), or control siRNA or Prox1 siRNA (bottom panel), and cultured in complete differentiation medium with 1 μM morphine for 4 d. Cells were stained with markers for neurons (Tuj1), astrocytes (GFAP) and with DAPI. Scale bar, 25 μm. Images are representative of at least three independent experiments with similar results.
(G) Quantification of cells stained with each marker, calculated as the percentage of the total number of cells stained with DAPI. Red: Tuj1; Green: GFAP. *, p<0.05, **, p<0.01, compared to vector or control siRNA transfected groups with the same treatment. #, p<0.05 compared to the control group transfected with vector or control siRNA.
(H) The expression of βIII-tubulin and GFAP were determined by real-time PCR after 4 d of differentiation with indicated treatments. The results were normalized against those of GAPDH. *, p<0.05, **, p<0.01, compared to vector or control siRNA transfected groups with the same treatment. ##, p<0.05 compared to the control group transfected with control siRNA. All data represent mean ± SEM of four independent experiments.

Mouse NPC primary cultures were transfected with miR-181a mimic control (double strand), miR-181a inhibitor control (single strand), miR-181a mimic, or miR-181a inhibitor by using Lipofectamine 2000. Two days after transfection, the mRNA (A) and protein (B–C) levels of Prox1 and Notch1 were determined by real-time PCR and western blot, respectively. The results were normalized against internal controls (GAPDH for mRNA and β-actin for protein). MC: miR-181a mimic control; IC: miR-181a inhibitor control; MM: miR-181a mimic; IH: miR-181a inhibitor. *, p<0.05, **, p<0.01 compared to cultures transfected with control RNA and with the same drug treatment. #, p<0.05, ##, p<0.01 compared to cultures transfected with control RNA without drug treatment. All data represent mean ± SEM of four independent experiments.

(A) Schematics of the Prox1 3′UTR and Prox1 3′UTR mutant reporters. The first nucleotide after the stop codon of mouse Prox1 mRNA is designated as number 1.
(B) HEK293T cells were transfected with one of the RNAs, one of the reporters, and the luciferase reporter system by using Lipofectamine 2000. RNAs included control RNA, miR-181a mimic and miR-181a inhibitor. Reporters included vector, Prox1 3′UTR, and Prox1 3′UTR mutant. The luciferase expression was determined as described under “Materials and Methods”. The results were normalized against internal control (R. reniformis luciferase) and further normalized against the results obtained from cultures transfected with control RNA in each group. MC: miR-181a mimic control; IC: miR-181a inhibitor control; MM: miR-181a mimic; IH: miR-181a inhibitor. *, p<0.05 compared to cells transfected with Prox1 3′UTR and control RNA, and treated with the same agonist. #, p<0.05 compared to cells transfected with Prox1 3′UTR and the same control RNA, and without agonist treatment. Data represent mean ± SEM of four independent experiments.

(A) Time-dependent abilities of morphine and fentanyl to modulate the expression of miR-181a-5p in mouse NPCs cultured in complete differentiation medium. Cultures were treated with 1 μM morphine or 10 nM fentanyl for indicated times with or without the treatment of 10 μM CTOP. The expression of miR-181a-5p was determined by real-time PCR and normalized against the mRNA level of GAPDH. The normalized results were further normalized against the results in untreated cultures (0 h). *, p<0.05, **, p<0.01 compared to untreated cultures in the absence of CTOP. #, p<0.05 compared to the morphine-treated group with the same time point or dose, but without CTOP treatment.
(B) Dose-dependent curves of morphine and fentanyl to modulate the expression of miR-181a-5p in mouse NPCs cultured in complete differentiation medium. Cultures were treated with indicated doses of agonists for 96 h with or without the treatment of 10 μM CTOP. The expression of miR-181a-5p was determined by qRT-PCR. *, p<0.05, **, p<0.01 compared to untreated cultures in the absence of CTOP. #, p<0.05 compared to the morphine-treated group with the same time point or dose, but without CTOP treatment.
(C) The abilities of morphine and fentanyl to modulate the expression of the four isoforms of miR-181 in mouse NPCs cultured in complete differentiation medium. Cultures were treated with 1 μM morphine or 10 nM fentanyl for 96 h. The expression of miR-181a-5p was determined by qRT-PCR. **, p<0.01 compared to control.
(D) Time-dependent abilities of morphine to modulate the expression of pri- and pre-miR-181a in mouse NPCs cultured in complete differentiation medium. Cultures were treated with 1 μM morphine for indicated times with or without the treatment of 10 μM CTOP. The expression of pri- and pre-miR-181a was determined by qRT-PCR. All data represent mean ± SEM of four independent experiments.

(A) Mouse NPC primary cultures were transfected with control RNA, miR-181a mimic, or miR-181a inhibitor by using Lipofectamine 2000, with or without the treatment of 1 μM morphine for 4 d. Cells were stained with markers for neurons (Tuj1), astrocytes (GFAP) and with DAPI. Scale bar, 25 μm. Images are representative of at least three independent experiments with similar results.
(B) Quantification of cells stained with each marker, calculated as the percentage of the total number of cells stained with DAPI. Red: Tuj1; Green: GFAP. *, p<0.05 compared to cultures transfected with control RNA with the same treatment. #, p<0.05 compared to the control group transfected with the control RNA. Data are the mean ± SEM of at least three independent experiments.
(C) The expression of βIII-tubulin and GFAP were determined by real-time PCR after 4 d of differentiation. The results were normalized against those of GAPDH, and further normalized against the results obtained from cultures transfected with control RNA in each group. *, p<0.05; **, p<0.01 compared to cultures transfected with control RNA and treated with the same agonist. #, p<0.05 compared to the control group transfected with control RNA. Data represent the mean ± SEM of four independent experiments.
(D) Schematic representation of the miR-181a/Prox1/Notch regulation pathway modulated by OPRM1 activation induced by morphine but not fentanyl.