Male C57BL/6 J mice, age 6–8 weeks (22 ± 3 g), were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Mice were housed at 4–6 animals per cage, with food and water available ad libitum, in humidity- and temperature-controlled rooms on a 12-hour light/dark cycle (lights on/off at 8:00 am/20:00 pm each day). All animal experiments were performed according to the Animal Care and Use of Soochow University and protocols were proved by its Animal Welfare Committee according to Guidelines for the Care and Use of Laboratory Animals (Chinese National Research Council, 2006). All animals were studied at age 6–8 weeks, with every effort made to minimize animal suffering and the number of animals used.
PC12 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). YFP-tagged DAT stable expressed HEK-293T (DAT-YFP-HEK-293T) cells were prepared in the laboratory as previously described.65 Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
Drugs and reagents
Roxadustat (FG-4592) was purchased from Selleck (Shanghai, China) and dissolved in a mixed solvent containing 5% DMSO, 40% PEG-300, 5% Tween-80 and 50% ddH2O according to Selleck in vivo usage instructions for mouse intraperitoneal injection. Rox powder was stored at −20 °C in silver paper to avoid light and ensure dryness, with its solvent prepared immediately prior to use. Rox was dissolved in solvent to reach a concentration of 100 mM and then diluted to concentrations of 5–100 μmol/L with cell culture medium for cell assays. METH was obtained from Shanghai Standard Biotech Co., Ltd. (Shanghai, China) and diluted with saline for i.p. injection (2.0 mg/kg). Morphine was purchased from Shenyang First Pharmaceutical Factory (Shenyang, China) and dissolved in saline for i.p. injection (5.0 or 20.0 mg/kg). Deferiprone (DFP) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phenylmethanesulfonyl fluoride (PMSF) was purchased from Cell Signaling Technology (Danvers, MA, USA). Other common reagents were purchased from Sinopharm (Beijing, China).
Conditioned place preference
The CPP apparatus (Jiliang Ltd., Shanghai, China) contains two distinct visual and textural cue compartments: a dark plexiglass/polyvinyl chloride box with fine wire mesh floor, and a white plexiglass/polyvinyl chloride box with wide grid floor. A removable gate between the two boxes ensured that mice were free or restricted to cross the apparatus during different experimental periods. Time spent in each box was recorded and analyzed through infrared beams and an automated analysis system for baseline and test periods, as previously described.66 Animals were habituated in the test room for 60 min before beginning experiments. CPP test procedures were as previously described with minor modifications32,66,67 and involved three main phases: pre-test phase, conditioning phase, and post-test phase. In the pre-test phase (3 days), the gate between the two boxes was open and mice were allowed to cross freely between them: 900 s of free moving was recorded and analyzed for baseline preferences and mice that spent more than 630 s or less than 270 s in one side were excluded because of marked unconditioned preference; the remaining mice were divided into groups randomly according to the experimental design. In the conditioning phase, mice were limited to one side of the apparatus to perform conditioning training and received saline, Mor (20.0 mg/kg) or METH (2.0 mg/kg) in the drug-paired side in the morning and received saline in the opposite side in the afternoon, with at least a 6-hour interval between treatments. Mice were trained in the drug-paired box for 30 min during 7 consecutive days for Mor-CPP and 60 min during 4 consecutive days for METH-CPP. In the final phase, the gate between two boxes was removed to allow mice to cross freely. Mice were placed in the dark or light box randomly and time spent in each box was recorded during 900 s of free movement between them. Time spent in the drug-paired box was quantified by the automated analysis system, with preference score calculated as the difference in time spent in the drug-paired compartment between post- and pre-test.
Pattern 1 (training extinction): Mice were trained to establish a CPP model with the method in Conditioned place preference section. After the post-test phase, extinction training was performed as described previously.66 The gate between the two boxes was closed, mice were injected with saline and placed in the drug-paired box in the morning and then injected with saline and placed in the opposite box in the afternoon. Rox was administered 6 h before the morning training. As for the CPP extinction phase, each extinction training session lasted 30 min with two days of training for a cycle. The extinction session consisted of 3 cycles (Extin-1, Extin-2 and Extin-3). The day after each training cycle, the gate between the two boxes was removed and mice were placed in the dark or light box randomly and time spent in each box was recorded during 900 s of free movement. The time spent in the drug-paired box was quantified by the automated analysis system as preference score for extinction outcomes.
Pattern 2 (testing extinction): Mice were trained to establish a CPP model with the method in Conditioned place preference section. After the post-test phase, extinction testing was performed as described previously.67 The gate between the two boxes was removed, mice were reintroduced into the CPP apparatus randomly on each day for 900 s on 5 consecutive days (Test1–5) without saline or any drug injection and time spent in each box was recorded during 900 s of free movement. The time spent in the drug-paired box was quantified by the automated analysis system as preference score for extinction outcomes.
Following the training extinction period, mice received a single injection of saline or a low dose of Mor (5.0 mg/kg) to reinstate Mor-CPP. Mice were placed in the CPP apparatus 10 min following Mor injection and time spent in each box during 900 s of free movement was quantified by the automated analysis system. Preference score was calculated as the difference in time spent in the drug-paired compartment between the reinstatement test and pre-test.
Cells or animal tissues were lysed in RIPA buffer (Beyotime, Shanghai, China) with PMSF (1 mM, Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice and shocked every 10 min. Then, the samples were centrifuged at 12,500 g for 15 min at 4 °C and the supernatant was collected. BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to detect protein concentration. 5 × loading buffer was added to the remaining supernatant and incubated at 95 °C for 10 min to ensure complete denaturation of protein. Western blotting was performed with the Cell Signaling Technology standard protocol and blots were probed with primary antibodies: anti-HIF-1α (1:1000, Proteintech, Wuhan, China, 20960-1-AP), anti-p-CREB (1:1000, Cell Signaling Technology, Danvers, MA, USA, 9198 L), anti-CREB (1:1000, Cell Signaling Technology, Danvers, MA, USA, 9197 S), anti-Dopamine transporter (1:1000, Abcam, Cambridge, MA, USA, ab184451), anti-E-cadherin (1:5000, Proteintech, Wuhan, China,20874-1-AP), anti-Ubiquitin (1:1000, Santa Cruz, CA, USA, SC-8017), anti-Lamin B1 (1:1000, Proteintech, Wuhan, China, 12987-1-AP), anti-GAPDH (1:1000, Proteintech, Wuhan, China, 10494-1-AP), anti-α-Tubulin (1:10000, Sigma, St. Louis, MO, USA, T6074). After incubation with respective secondary antibodies (Rabbit anti mouse IgG, 1:20000, Sigma, St. Louis, MO, USA, A9044; Goat anti rabbit IgG, 1:20000, Sigma, St. Louis, MO, USA, A0545), the blots were captured by ClinxChemi-Capture 3300 Mini (Clinx Science Instrument, Shanghai, China). At least 3 independent experiments were conducted for each immunoblot assay and blot images were analyzed by Image J software (version 1.52 a).
Quantitative real time-PCR
Total RNA was extracted from cells using RNAiso Plus (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. RNA (1,000 ng) was reverse transcribed into cDNA using the TaRaKa reverse transcription kit with Oligo (dT) primer and cDNA was amplified using the specific primers in Supplemental Table S2. A StepOnePlusTM Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) was used to quantify mRNA expression using SYBR Premix II (TaKaRa, Tokyo, Japan). The parameters for quantitative real time-PCR were 30 s at 95 °C, 5 s at 95 °C and 30 s at 60 °C for 40 cycles. GAPDH was used as reference gene and relative quantification of target genes was determined using the 2-ΔΔCT formula. Results were expressed as fold changes relative to the control group as previously described.32
For mouse brain tissue, on completion of behavioral tests mice were anaesthetized with pentobarbital sodium (60 mg/kg i.p.) and then perfused with 40 mL 0.9% saline and subsequently with 40 mL 4% paraformaldehyde in 0.1 mol/L PBS (PH 7.4). Brains were transferred into a 10 mL centrifuge tube containing 4% PFA for a further 6 h for post-fixing at 4 °C and dehydration in a 30% sucrose solution for one week, with the sucrose solution renewed every two days. Dehydrated brains were cut into 20 μmol/L sections using a Leica freezing microtome (Wetzlar, Germany). For cell immunofluorescence, the staining protocol was as described previously.32 In brief, PC12 cells were washed 3 times with 0.01 mol/L PBS and fixed with iced methanol for 20 min. For immunofluorescence assay, the target brain sections or fixed PC12 cells were washed three times in 0.1 mol/L PBS. Then, samples were incubated in 0.01 mol/L PBS containing 3% BSA and 0.3% Triton X-100 for 2 h at room temperature. The samples were then incubated with anti-Δfos B antibody (1:1000, Santa Cruz, CA, USA, SC-398595) or anti-HIF-1α antibody (1:1000, Proteintech, Wuhan, China, 20960-1-AP) overnight at 4 °C and washed three times (10 min each time) in PBST (0.1% Triton X-100 in 0.01 mol/L PBS). After washing, samples were incubated with anti-Alexa Fluor 488 conjugated goat anti-rabbit IgG (1:500, Invitrogen, Carlsbad, CA, USA, A11001) or anti-Alexa Fluor 546 conjugated donkey anti-rabbit IgG (1:500, Invitrogen, Carlsbad, CA, USA, A10040) for two hours at room temperature and subsequently washed three times with PBST. Finally, samples were stained with DAPI (1:10000, Thermo Scientific, Waltham, MA, USA) for a further 30 min at 37 °C and again washed three times with PBS. Then, samples were fixed in glass slides and fluorescence observed and captured with a confocal microscope (Zeiss 271 LSM710 META, Jena, Germany).
Enzyme-linked immunosorbent assay (ELISA)
PC12 cells were homogenized ultrasonically in PBS and centrifuged at 5,000 g for 10 min 4 °C. Then, the supernatant of PC12 cell lysates was collected for measurement of hepcidin protein expression level. For mouse serum, mice were anaesthetized with pentobarbital sodium (60 mg/kg i.p.) and whole blood was collected from the orbit and centrifuged at 1,000 g for 20 min at 4 °C. Then, the supernatant was collected and moved into a new tube. For tissues, the samples were homogenized ultrasonically in 0.1 mol/L PBS containing PMSF (1 mM, Cell Signaling Technology, Danvers, MA, USA) and the supernatant was collected after centrifugation at 5,000 g for 10 min at 4 °C. Protein concentration was measured using BCA Protein Assay Kits (Beyotime, Shanghai, China). Hepcidin ELISA Kits were purchased from Elabscience Biotechnology Co., Ltd (TX, USA). The ELISA procedure was conducted in accordance with manufacturer’s instructions as previously described.32 Briefly, 100 μL of sample or standard was added to each ELISA well and incubated for 90 min at 37 °C. Then, the liquid was removed and 100 μL biotinylated detection antibody was added into each well for 60 min at 37 °C. After aspirating and washing three times (2 min each in wash buffer), HRP conjugate (100 μL) was added into each well and incubated for 30 min at 37 °C, followed by aspiration and washing five times with wash buffer. Substrate regent (90 μL) was added into each well and incubated for 15 min at 37 °C. Finally, stop solution (50 μL) was added and the OD values measured immediately at 450 nm using a Microplate Reader (Infinite M200 PRO, Tecan, Switzerland).
Establishment of iron deficiency mouse model
Low iron diet and normal diet were purchased from Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd (Jiangsu, China). Male C57BL/6 J mice (age 6–8 weeks) were fed with low or normal diet for 3-4 weeks to establish an iron deficiency mouse model68,69,70 before beginning CPP experiments.
Detection of iron content
Iron content of PC12 cells, mouse tissue or blood samples was detected as previous described.32 Briefly, mouse tissues were weighed wet and nitrified with a 5 mL mixed solution containing nitric acid, hydrochloric acid and perchloric acid (1:3:1, v- v- v). PC12 cells were collected and divided into two fractions: one fraction was homogenized ultrasonically in PBS and the protein concentration was detected by BCA Kits (Beyotime, Shanghai, China); the remaining fraction was nitrified by heating to 260 °C as for mouse tissue nitration. Finally, samples were measured by inductively coupled plasma mass spectrometry (ICP-MS) (Thermo Fisher Scientific, Waltham, MA, USA). Iron standard solution (1 mg iron/mL) was purchased from Guobiao Testing & Certification Co., Ltd (Beijing, China), with the standard curve prepared as described previously.32
Membrane protein extraction
Mem-PER™ Plus Membrane Protein Extraction Kit was purchased from Thermo Fisher Scientific Inc (Thermo Scientific, Waltham, MA, USA) and used according to manufacturer’s instructions. For PC12 cells, 5 × 106 cells were collected and washed using cell wash solution. 0.75 mL of Permeabilization Buffer was added to the cell pellet and incubated for 10 min at 4 °C with constant mixing. Collected cells were then centrifuged for 15 min at 16,000 g. Following the addition of 0.5 mL of solubilization buffer to the pellet and incubation at 4 °C for 30 min, with constant mixing, the samples were again centrifuged at 16,000 g for 15 min at 4 °C and the supernatant containing solubilized membrane and membrane-associated proteins was collected. For mouse tissue membrane protein collection, tissues were washed and cut into small pieces with a pair of scissors; 1 mL of permeabilization buffer was added to the tissue and homogenized with a grinding rod. Following addition of 1 mL permeabilization buffer and incubation for 10 min at 4 °C with constant mixing, the sample was centrifuged at 16,000 g for 15 min at 4 °C; the remaining procedures for preparation of cellular fractions were then as described above. Concentration of protein in each preparation was measured using BCA Protein Kits (Beyotime, Shanghai, China).
Nuclear and cytoplasmic protein extraction
PC12 cells were collected and washed with PBS. Cytoplasmic protein extraction solvent A (400 μL with 1 mM PMSF) was added and incubated for 10 min on ice, followed by addition of 20 μL cytoplasmic protein extraction solvent B for 1 min on ice. After centrifugation at 16,000 g for 5 min at 4 °C, the cytoplasmic protein supernatant was collected. The pellet was added to 100 μL nuclear protein extraction solvent (with 1 mM PMSF) and incubated for 30 min on ice with vortexing for 15 s every 2 min. Then, following centrifugation at 16,000 g for 10 min at 4 °C the nuclear protein supernatant was collected. Protein content of nuclear and cytoplasmic protein supernatants (5 μL) was detected using BCA Protein Kits (Beyotime, Shanghai, China).
Surgery, cannula implantation and local drug delivery
Mice were anesthetized using pentobarbital sodium (60 mg/kg i.p.) and fixed in a stereotaxic apparatus (RWD Life Science, Shenzhen, China). Mouse body temperature was maintained using a heating pad. Stereotaxic surgery was performed as described previously.71,72,73,74 Stainless steel guide cannulae was implanted according to the Paxinos and Watson Brain Atlas. Guide cannulae were 0.5 mm shorter than injection needles and placed 0.5 mm above the areas to be targeted. These areas had the stereotaxic coordinates (AP: anteroposterior, ML: mediolateral, DV: dorsoventral): PFC (anterior cingulate cortex, ML = ± 0.3 mm; AP = + 1.0 mm; DV = − 2.0 mm), NAc (nucleus accumbens shell, ML = ± 0.6 mm; AP = + 1.53 mm; DV = − 4.75 mm), STR (dorsomedial striatum, ML = ± 1.35 mm; AP = + 0.85 mm; DV = − 3.1 mm) and HIP (dorsal hippocampus, ML = ± 1.5 mm; AP = − 2.1 mm; DV = − 1.25 mm). The guide cannulas were kept patent with stylets and affixed to the skull using dental cement with two stainless steel screws serving as anchors. Following surgery, animals were housed for at least 3 days before CPP experiments. For Rox microinjection, Rox was dissolved in DMSO at a final concentration of 0–50 μmol/L and injected (2 μL) into the brain regions noted above at 2 h before CPP training. Deferiprone (DFP) was dissolved in PBS at a final concentration of 13 mg/mL and injected (2 μL) into PFC.75
Dopamine uptake assays
YFP-tagged DAT stable expressed HEK-293T (DAT-YFP-HEK-293T) cells were seeded in a 6-well plate and dopamine uptake assays performed as previously described76,77 Briefly, DAT-YFP-HEK-293T cells were washed twice with PBS, then 2 mL complete DMEM medium was added to each well, followed by addition of Rox at a final concentration of 10 µM or 100 µM and incubation for a further 24 h. Cells were washed 3 times with PBS and pre-incubated for 30 min in Krebs buffer (118.0 mM sodium chloride, 4.7 mM potassium chloride, 1.2 mM magnesium sulfate, 1.2 mM potassium dihydrogen phosphate, 2.25 mM calcium chloride, 3.25 mM sodium bicarbonate and 11.1 mM glucose, pH 7.2–7.4) containing 10 mM desipramine (to block the endogenous norepinephrine transporter). For uptake assays, [3H] DA (3,4-[Ring-2,5,6,-3H]-DA; Perkin-Elmer Life Sciences, Boston, MA, USA) was added at a final concentration of 50 nM per well and incubated at 37 °C for 5 min. DA uptake was terminated by immediate removal from the medium and washing 3 times with cold Krebs buffer on ice. Cells were dissolved in 1 mol/L NaOH for 15 min, with shaking, followed by addition of 1 mol/L HCl. Subsequently, all liquid content from each well was collected and radioactivity was determined by liquid scintillation counting (Beckman Instruments, Irvine, CA, USA) using Ecoscint A Cocktail (National Diagnostics, Atlanta, GA, USA). Cells in each well were measured by cell counter, with total protein content of cells measured by BCA Protein Kits (Beyotime, Shanghai, China).
HIF-1α siRNA was purchased from GenePharma Co., Ltd. (Shanghai, China) with the following sequences: sense 5’-CCAUGUGACCAUGAGGAAATT-3’, antisense 5’-UUUCCUCAUGGUCACAUGGTT-3’; negative control (NC): sense 5’-UUCUCCGAACGUGUCACGUTT-3’, antisense 5’-ACGUGACACGUUCGGAGAATT-3’. siRNA transfection was performed with Lipofectamine 2000 Transfection Reagent (Invitrogen) according to manufacturer’s instructions. PC12 cells were transfected with 30 pM siRNA for 48 h, followed by Rox treatment for another 24 h. Then, knockdown efficiency was validated by immunoblot analysis.
Stereotaxic injection of adeno-associated virus serotype 9 (AAV9) in mouse prefrontal cortex
AAV9-hSyn-EGFP-shHIF-1α and AAV9-hSyn-EGFP-NC virus particles were provided by Genechem Co., Ltd. (Shanghai, China). pAKD-CMV-bGlobin-mCherry-3*FLAG-WPRE-H1-shRNA and pAAV-CBG-mCherry-3*FLAG-WPRE-H1-shFPN1 virus particles were purchased from OBiO Technology Co., Ltd. (Shanghai, China). Surgery was performed under pentobarbital anaesthesia as described in Sugery, cannula implantation and local drug delivery section72 using the same PFC coordinates: ML = ± 0.3 mm; AP = + 1.0 mm; DV = − 2.0 mm. The virus (1 × 1012 viral particles/mL, 200 nL/side) was injected bilaterally into PFC areas at a flow rate of 0.1 μL/min, with the needle retained in position for 10 min after injection to limit viral leakage. CPP testing was performed 3 weeks after virus injection.
Data were analyzed using Graphpad Prism 8.0 (GraphPad Software, Inc. La Jolla, CA, USA). Values are presented as means ± SEM. Differences between multiple groups were determined using one-way or two-way ANOVA followed by Bonferroni-corrected tests. Differences between two groups were determined by two-tailed unpaired Student’s t-test. P < 0.05 was considered statistically significant.