Experimental models and mouse genetics

All experiments were approved by and conducted in accordance with the regulations of the local Animal Care and Use Committee (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, LAVES). WT C57BL/6N (Charles River), NexCreERT221, R26R-tdTomato50, Thy1-YFPH (Jackson Laboratory, 003782), CAGCreERT2-ODD28, NexCre51, and EPOR-floxed (EPORfl/fl) mice were used for the experiments. Juvenile (P23) and adult (3 months old) mice were used in this study. For genetic labeling of projection neurons, NexCreERT2 mice were bred with Rosa26 floxed-stop tdTomato (R26R-tdTomato) reporter mice to generate NexCreERT2:R26R-tdTomato mice. For labeling cells undergoing hypoxia, CAGCreERT2-ODD mice were employed28. Briefly, a fusion protein consisting of the ODD domain of Hif-1α and a ubiquitous CAGCreERT2 is expressed upon tamoxifen induction. CAGCreERT2-ODD were bred with R26R-tdTomato to generate CAGCreERT2-ODD:tdTomato reporter mice. Upon tamoxifen induction, cells are irreversibly labeled with tdTomato containing stabilized Hif-1α. NexCre::Eporfl/fl mice were generated to specifically delete Epor in projection neurons. For the generation of Eporfl/fl mice, embryonic stem cells (ES) harboring an engineered allele (Eportm1a(KOMP)Wtsi) of the Epor gene were acquired from the Knockout Mouse Project (KOMP, University of California, Davis CA 95618, USA). ES cells were microinjected into blastocysts derived from C57BL/6N mice and the embryos were transferred to pseudopregnant foster mothers, yielding chimeric males. For ES clone EPD0316_5_A03, germline transmission was achieved upon breeding with C57BL/6N females, generating mice harboring the Eportm1a(KOMP)Wtsi allele (termed EporlacZ-neo). The lacZ-neo cassette was excised in vivo upon interbreeding with mice expressing FLIP recombinase (129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J; backcrossed into C57BL/6N), yielding mice carrying the Eportm1c(KOMP)Wtsi allele (termed Eporflox). To recombine the Epor gene specifically in projection neurons, exons 3–6 were excised in vivo upon appropriate interbreedings of Eportm1c(KOMP)Wtsi mice with mice expressing Cre recombinase under control of the Nex1/Neurod6 promoter51, generating mice carrying the Eportm1d(KOMP)Wtsi allele (NexCre::Eporfl/fl mice).

All mice were housed in a temperature controlled environment (21 ± 2 °C) on a 12 h light–dark cycle with food and water available ad libitum. Male mice of the same age were used for the experiments, unless stated otherwise. All animals were genotyped before the start of each experiment. Detailed PCR protocols are available on request.

Mouse treatment

Tamoxifen: Tamoxifen solution (10 mg/ml) was freshly prepared by dissolving tamoxifen freebase (Sigma) in corn oil (Sigma) at room temperature (RT) for 45 min. Postnatal CreERT2 activity in NexCreERT2 mice was induced by a total of five i.p. injection of 100 mg/kg tamoxifen over the course of 3 days in juvenile mice. For CreERT2 induction in adult NexCreERT2 mice, a total of ten i.p. injections of 100 mg/kg tamoxifen were administered over the course of ten consecutive days. For the desired induction of CreERT2 in CAGCreERT2-ODD mice, a single i.p. injection of 100 mg/kg tamoxifen was sufficient.

EPO: Male mice were i.p. injected with 5000IU/kg rhEPO (NeoRecormon, Roche) or placebo (solvent solution, 0.01 ml/g). At 48 h after the last tamoxifen injection, EPO/placebo treatment was initiated in P28 or 3-month-old NexCreERT2, Thy1-YFPH and also in P28 old WT and NexCre::EPORfl/fl mice, and carried out every other day for 3 weeks. For DropSeq analysis, EPO was administered once followed by tissue collection 6 h later. Additionally, for labeling of proliferating cells, NexCreERT2 mice obtained EdU (0.2 mg/ml; ThermoFisher) via drinking water (exchanged on all alternate days).

Hypoxia: The hypoxia chamber was designed in cooperation with Coy Laboratory Products Inc. (Grass Lake, MI, USA) with the dimensions 164 cm × 121 cm × 112 cm. The system includes an air filtration system consisting of carbolime and activated charcoal. The oxygen and carbon dioxide levels were constantly detected and controlled via online monitoring. A gradual reduction of 3% oxygen per day resulted in 12% oxygen level in 3 days and was maintained until the end of the experiment. During the course of the experiment, mice were treated with EdU via drinking water as described above.

Complex running wheels (CRW)

Post weaning, mice were single-housed and tamoxifen treatment was initiated for NexCreERT2::tdTomato mice (described above). NexCreERT2::tdTomato and NexCre::EpoRfl/fl mice were divided into four groups and monitored for 17 days. Groups included (1) normoxic room conditions (at 21% O 2 ) in standard cages, (2) normoxic conditions with voluntary running on CRW, (3) hypoxic conditions (hypoxia chamber to 12% O 2 ) in standard cages, and (4) hypoxic conditions with voluntary running on CRW. CRW (TSE Systems, Bad Homburg, Germany) is characterized by randomized missing bars as previously described26,41. The testing period of 17 days was followed by 1 week of normal conditions for all groups (no running, normoxia). The mice that were previously running (in normoxic or hypoxic conditions) were finally exposed again to CRW for 4 h (as cFos inducing challenge) before being sacrificed (Fig. 4f). For ISH experiments, male WT mice (P55) were exposed to 5, 9, or 13 h of CRW (Fig. 3a). For the experiment involving CAGCreERT2-ODD::tdTomato mice, animals were exposed to overnight complex wheel running (Fig. 3e). Running was voluntary at all times with ad libitum access to food and water. Control mice (no running) were housed in standard cages. Mice were sacrificed, perfused, and brains collected as described below. Running was tracked automatically via Phenomaster software (TSE Systems, Germany) for the whole day. Since mice are mainly night-active (dark phase), the total distance run between 6 p.m. and 6 a.m. was summarized for every individual animal. The total distance run on each night was normalized to the average distance of each animal run in the first 3 nights (data expressed as % performance in relation to the first 3 nights). For the experiments involving EPO treatment, mice were treated with EPO (5000IU/kg, 11 i.p. injections on alternate days for 3 weeks), followed by 1-week break. They were then exposed to 12 h of CRW overnight (Fig. 4a–c). Data for this experiment are expressed as distance run summarized for every 30 min.

Immunohistochemistry (IHC)

Mice were anesthetized and perfused transcardially with 4% cold formaldehyde. Dissected brains were post fixed in 4% formaldehyde at 4 °C and equilibrated in 30% sucrose dissolved in phosphate-buffered saline (PBS) at 4 °C overnight. Brains were then embedded in cryoprotectant (O.C.T.TM Tissue-Tek, Sakura) and stored at −80 °C. Whole mouse brains were cut into 30 μm thick coronal sections (coordinates from bregma: −1.34 to −2.54 mm posterior) using a cryostat (Leica) and stored in a cryoprotective solution (25% ethylene glycol and 25% glycerol in PBS) at −20 °C until further use. For analysis of dendritic spines and Map2 dendrites, the right hemisphere, destined to the neuronal structural analysis, was cut in 100 μm coronal sections with a vibratome (Leica VT 1000E, Leica), collected in three subseries and stored at 4 °C in PB 0.1 M with sodium azide (0.05%).

For IHC, sections were permeabilized in PBS containing 0.3% Triton X-100 and blocked in 5% horse serum for 1 h at RT. Brain sections were incubated with primary antibodies in blocking solution overnight at 4 °C, followed by 2 h incubation of appropriate fluorophore-conjugated secondary antibodies in blocking solution containing 3% horse serum (or 5% normal donkey serum) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were then mounted on SuperFrostPlus Slides (ThermoFisher) with Aqua-Poly/mount (Polysciences, Inc). Primary antibodies used were: anti-Ctip2 (1:500; Guinea pig polyclonal; SYSY 325005), anti-Map2 (1:1000; mouse monoclonal; Sigma M9942), anti-Tbr1 (1:200; rabbit monoclonal; Abcam ab183032), anti-Tle4 (1:200; rabbit polyclonal; Abcam ab64833), anti-NeuN (1:500; mouse monoclonal; Millipore MAB377), and anti-cFos (1:1000; rabbit polyclonal; SYSY 226003). Secondary antibodies used were: Alexa488 anti-Guinea pig (1:500; Jackson Immuno Research 706-548-148) and Alexa635 anti-mouse (1:400; ThermoFisher A31575). Depending on the need for a triple or a quadruple staining, Alexa405 anti-rabbit (1:1000; Abcam 175652) or Alexa647 anti-rabbit (1:500; ThermoFisher A31573) were used. Following IHC, sections were stained for EdU using Click-iTTM EdU Alexa FluorTM 647 Imaging kit (ThermoFisher E10415), according to the manufacturer’s instructions.

Golgi–Cox staining

Mice were anesthetized and decapitated. The brain was removed, shortly rinsed in PBS, and processed with the ND Rapid GolgiStainTM Kit (FDNeuroTechnologies, PK401) according to the manufacturer’s instructions. After impregnation, the brains were embedded in 2% Agar Agar in PBS and cut in 100 µm coronal sections with a vibratome (Leica, VT 1000 S) and placed on gelatin-coated glass slides (FDNeuroTechnologies, PO101). The slices were stained and dehydrated as described in the kit protocol. Slices were mounted with Perimount (ThermoFisher).

Imaging and analysis

Pyramidal neurons: Imaging was performed using the Andor Eclipse TiE microscope system (Nikon, Tokyo, Japan) with a 40× objective (Plan Apo λ 40×, NA = 0.95) to image the hippocampal layers. Ctip2+ cells among total neuron numbers were manually counted. Ctip2+ and tdTomato− cells were characterized as newly generated neurons. Quantifications are expressed as number of newly generated neurons divided by the total area of CA1 stratum pyramidale (mm2). For Ctip2 staining, a total of 16 hippocampi with 8 sections per animal were used. Quadruple (tdTomato, Ctip2, cFos, and EdU) staining was acquired with a TCS SP5-II System (Leica) equipped with a 20× objective (NA = 0.70). For total Ctip2+ counts, six hippocampi with three sections per animal were used. Quantifications for CRW mice were expressed as percentage of Ctip2+ cells normalized to their respective non-running controls. Whole hippocampi were imaged and analyzed by Fiji software. Neurons showing positive immunoreactivity for cFos were identified and quantified manually. These neurons were further sub-categorized into pre-existing (cFos+, tdTomato+, Ctip2+) or newly generated (cFos+, tdTomato−, Ctip2+) neurons. Again, quantifications are indicated as cFos+ cells divided by the total area of the CA1 stratum pyramidale (mm2). For cFos staining, a total of eight hippocampi with four sections per animal were used. For quantification of hypoxic neurons, whole hippocampus sections of CAGCreERT2-ODD::tdTomato mice were imaged. The tdTomato+ cells were characterized as neurons undergoing hypoxia and manually quantified. Quantifications are expressed as number of hypoxic neurons divided by the total area of CA1 stratum pyramidale (mm2). A total of ten hippocampi with five sections per animal were used. The cFos+ cells were also analyzed as described above. For Map2+ dendritic density analysis, the stratum radiatum of the CA1 region was imaged using a laser scanning confocal microscope (Leica TCS SPE). Per animal, three dorsal hippocampal slices were selected starting at bregma −1.58 mm. Confocal z-stacks (0.38 µm intervals) of whole sections were taken using a 63× objective (NA = 1.40). Per hippocampus, three images were captured, and four fields of the dimension 36.67 µm × 36.67 µm were analyzed in each image (total 36 fields per animal). Images were processed using Fiji software and Map2+ principal apical dendrites were manually quantified. The quantifications are expressed as number of Map2+ dendrites divided by the area of each image (1344.7 µm2).

Dendritic spines: Images were captured using a 63× oil immersion objective (NA = 1.40) and a 3.5× additional digital zoom to investigate the first 200 µm of the principal apical dendrite in segments of 50 µm. Confocal z-stacks (0.38 µm intervals) of whole sections were performed. Dendrites included in the study were at least 200 µm in length. Per animal, six such dendrites of six different Thy1-YFPH expressing pyramidal neurons were randomly selected from the CA1 region. The stitching plugin in Fiji software (2.0.0) was used to reconstruct three-dimensional images of the apical dendrites. The spines were further sub-categorized as proximal (0–50 µm), medial (50–100 µm), medial-distal (100–150 µm), and distal (150–200 µm) segments of the dendrite, depending on their distance from the soma. The total density of spines was also analyzed. Based on their morphology23,52, the spines were manually divided into (1) stubby, i.e., length of the protrusion was <1 µm and no neck is observed; (2) mushroom, when a clear head-like structure could be observed (maximum diameter of the head was at least 1.5 times the average length of the neck) and the total length of the protrusion was <1.5 µm; and (3) thin, i.e., the length of the protrusion was >1.5 µm or the length was between 1 and 1.5 µm and a clear head-like structure could not be distinguished.

For dendritic spine quantification using the Golgi–Cox method: Images were acquired using the Nikon Ti2 using a 100× objective (NA = 1.45). Stretches of mid-apical dendrites in CA1 pyramidal neurons were recorded with a z-stack size of 0.3 µm. Per animal, ten dendrites of ten different cells were quantified for the number of spines (calculated as spines per µm dendrite).

RNAscope in-situ hybridization (ISH): RNAscope® 2.5 HD Brown Reagent Kit (CatNo.322300), Advanced Cell Diagnostics (ACD), Hayward, CA, USA was used for the detection of EPO and EPOR mRNA. ISH was performed according to the manufacturer’s instructions. Briefly, coronal cryosections of 15 µm thickness were mounted on SuperFrostPlus Slides, dried, and stored at −80 °C. Sections were then pretreated by dropwise addition of hydrogen peroxide and incubated for 10 min at RT. Slides were immersed in boiling target retrieval buffer for 15 min, followed by incubation with protease plus for 30 min at 40 °C. Sections were then hybridized with the corresponding target probe Mm-Epo-01 (CatNo.444941) or Mm-Epor (CatNo.412351) for 2 h at 40 °C, followed by a series of amplification and washing steps. Chromogenic signal detection was performed with 3,3′-diaminobenzidine (DAB) incubation for 20 min at RT. Sections were counterstained with 50% Mayer’s hemalum (Merck) and mounted with EcoMount (BioCare Medical). Brown punctate dots in the CA1 were counted in a total of 12 hippocampi with 6 sections per animal using a light microscope (Olympus BX-50, Tokyo, Japan) equipped with a 100× oil immersion objective (NA = 1.35) and normalized to the area of the respective region (mm²). Sagittal 15 µm sections from kidney (P55) and heart (E11.5) of WT mice were used as positive controls for EPO and EPOR, respectively. According to manufacturer’s instructions, each dot represents a single molecule of mRNA in these sections. The quantification is normalized and presented as percentage of the mean dot number of groups and time points [% value = (number/mm²)/(mean dot number/mm²) × 100].

Drop sequencing

Tissue dissociation: Juvenile male WT mice (P28; three mice/group to allow biological replication) were injected i.p. with placebo or EPO and sacrificed after 6 h. The hippocampi were dissected and sliced into 600 µm sections using McIlwain Tissue chopper (Cavey Laboratory Engineering Co. Ltd). The CA1 region was digested with a working solution of Papain/DNaseI in Earle’s Balanced Salt Solution, according to manufacturer’s instructions (Worthington Biochemical Corp). The samples were then incubated at 37 °C for 40 min with constant agitation before gentle manual trituration. The samples were centrifuged for 10 min at 200 × g at 4 °C. After discarding the Papain/DNaseI supernatant, cells were resuspended in 1 mL of sterile DMEM/F12 (Sigma) without phenol-red containing 3% fetal bovine serum (FBS; Life Technologies) and the suspension was passed through a 40 µm strainer cap (Corning) to yield a uniform single-cell suspension.

Single-cell barcoding and library preparation: Barcoded single cells, or STAMPs (single-cell transcriptomes attached to microparticles), and cDNA libraries were generated following the protocol53. Briefly, single-cell suspensions (100 cells/μl), droplet generation oil (Bio-Rad), and barcoded microparticles (ChemGenes; 120 beads/μl) were co-flowed through a FlowJEM aquapel-treated DropSeq microfluidic device (FlowJEM) and droplets were generated for 15 min. Captured RNA on the bead surface was recovered by washing the beads in saline-sodium citrate buffer and perflurooctanol solutions, and then reverse transcribed using Maxima H minus reverse transcriptase kit (ThermoFisher). Excess primer on the surface of the bead uncaptured by an RNA molecule was digested using Exonucleases I kit (ThermoFisher). A cDNA amplification PCR was performed using 10 µM SMART PCR primer and 2X Kapa HiFi HotStart ReadyMix (Kapa Biosystems) with 5000 beads per tube, and amplified for nine PCR cycles. The resulting samples were purified using AMPureXP beads (Beckman Coulter) and the quality and concentration of the cDNA was assessed using High-sensitivity DNA bioanalyzer (Agilent Technologies). Library sizes were adjusted using the Nextera Amplicon Tagmentase enzyme and DNA was amplified for 14 cycles using 10 µM New-P5-SMART PCR hybrid oligo, 10 µM Nextera Index, and the Nextera PCR mix (Nextera XT DNA Library Preparation kit; Illumina). Tagmented libraries were again purified (AMPureXP), quality controlled (high-sensitivity DNA Bioanalyzer), quantified (Qubit dsDNA HS assay kit; Life Technologies), and sequenced (Illumina Hi-seq 2500). All assays mentioned above have been performed according to manufacturer’s protocol.

Single-cell RNA-seq processing: Unique molecular identifier (UMI) gene counts for each group (placebo or EPO) was imported into R (v3.4.1). Seurat (v2.3.0) function within the R environment was used for filtering, normalization, canonical correlation analyses (CCA), unsupervised clustering, visualization, and differential expression analyses.

Filtering and data normalization: Cells with minimum and maximum of 1000 and 8000 genes expressed (≥1 count), respectively, and the genes that were expressed in at least three cells were retained. Cells with >40% of counts on mitochondrial genes were excluded (Supplementary Fig. 1). After filtering, there were 14,061 genes in 390 cells from placebo and 14,971 genes in 583 cells from EPO group.

Gene UMI counts for each cell were normalized via natural-log normalization of gene UMI counts divided by total UMI counts per cell and scaled by 10,000. After normalization, scaled expression (z-scores for each gene) for downstream analyses was calculated.

Canonical correlation analysis: Integration of scRNA-seq data from the two groups (placebo and EPO) was performed using CCA. Top 1000 highly variable genes from each group were used to calculate canonical correlation vectors (reduced dimension) and subsequently, first 20 vectors were aligned using dynamic time warping.

Clustering, visualization, and differential expression: Clustering was performed using “FindClusters” function with default parameters with resolution set to 1 and first 20 CCA aligned dimensions were used in the construction of the shared-nearest neighbor graph and to generate two-dimensional embeddings for data visualization using tSNE. Based on the visualization the glutamatergic cluster 0 and 1 were merged manually to represent a single cluster. The percentages of ‘immature glutamatergic cells’ for each mouse were: placebo 1.6%, 2.3%, and 4.3%; EPO 3.7%, 6.8%, and 7.0%. We used the “FindAllMarkers” function with default parameters and tested genes with a detected threshold of minimum of 25% of cells in either of the two clusters. Genes with an adjusted p < 0.01 were considered to be differentially expressed (for a list of cluster markers see Source Data File). A heatmap showing the top ten marker genes, i.e., differentially expressed genes per cluster is provided in the Supplementary Fig. 2.

Cell trajectory (pseudotime) analysis: Trajectory analysis of cells from the ‘Immature Glutamatergic’ and ‘Mature Glutamatergic1’ clusters (n = 502) was performed in Monocle225. The trajectory was constructed according to the documentation of Monocle2. Prior to cell ordering, reclustering was performed to confirm robust detection of the immature cluster across methods, which revealed three clusters (one cluster largely corresponding to Seurat’s ‘Immature Glutamatergic’ cluster and two corresponding to Seurat’s ‘Mature Glutamatergic1’ cluster; Supplementary Fig. 3a). Subsequently, dimension reduction using the ‘DDRTree’ method was performed. Differentially expressed genes (q < 0.01) between the three clusters obtained by reclustering in Monocle2 were used as input for pseudotemporal ordering.

Quantification and statistical analysis

All statistical analysis was performed using GraphPad Prism 5. For comparisons across multiple groups, a two-way analysis of variance (ANOVA) was used. For comparisons across two groups, an unpaired Student’s t-test was performed. A p < 0.05 is considered statistically significant. Variance was similar between compared groups for their respective experiments. All values represent mean ± SEM (standard error of the mean). All analysis and quantification were performed in a double-blinded fashion.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.