Erythropoietin restrains the inhibitory potential of interneurons in the mouse hippocampus

Abstract

Severe psychiatric illnesses, for instance schizophrenia, and affective diseases or autism spectrum disorders, have been associated with cognitive impairment and perturbed excitatory-inhibitory balance in the brain. Effects in juvenile mice can elucidate how erythropoietin (EPO) might aid in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, conceivably explaining mitigation of neuropsychiatric diseases. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing ~12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.

Fig. 1. Single nuclei landscape of interneuronal lineages from 12 murine hippocampi.

A Violin plots illustrating the expression dynamics of cell type specific marker genes (two each) of interneurons, oligodendrocytes, glial (microglia and astrocytes), vascular (endothelial and pericytes) and pyramidal lineages across all obtained clusters. The 15 clusters are sublineages of mature interneurons, each cluster is color-coded as in (B). B Two-dimensional Uniform Manifold Approximation Plot (UMAP) resolving ∼8000 single nuclei into 15 distinct clusters, merged from 12 adult hippocampi of mice treated with either rhEPO (N = 6) or PL (N = 6). The clusters affiliate with interneuron subtypes, validated by testing co-expression of Gad1 and Gad2 genes (A). Absence of transcriptional markers from the rest of hippocampal lineages was also confirmed. Colors indicate an unbiased classification of nuclei via graph-based clustering, where each dot represents a single nucleus. C Dotplot illustrating cluster-specific expression of top marker genes for interneuronal populations identified from the above data. Cluster numbers derived from (B) are given on the left of the plot. Marker gene names are presented at the bottom of the plot. Colors represent an average Log2 expression level scaled to the number of unique molecular identification (UMI) values in single nuclei. The color scale ranges from light blue to pink, corresponding to lower and to higher expression. Dot size is proportional to the percent of cells expressing the corresponding gene.

Fig. 2. rhEPO modulates differential expression of synapse-associated genes in interneuron lineages.

A Heatmap representing the differential expression of genes between rhEPO and PL samples in individual lineages shown on the bottom. Only those genes with adjusted p-value < 0.01 in any of the comparisons were used for plotting. The number of detected differentially expressed genes in each lineage is shown at the right side of the heatmap. The genes with insignificant differential expression are colorless in this figure. Annotation of gene names are the top DEGs in the given lineage (red, upregulated; blue, downregulated). B Barplot showing the gene ontologies in which genes that are differentially expressed between rhEPO and PL cells are enriched. The strength of enrichment is labeled on the X-axis. C Violin plots showing the expression distribution of Bmpr1a, Gabra2, Pde1c, Filip1, and Jak2 genes between rhEPO and PL samples in a pairwise fashion among the individual lineages of PVALB, CCK and SST interneurons. Adjusted p-values are obtained by Benjamini-Hochberg (BH) correction.

Fig. 3. Modification by rhEPO of cell-to-cell interactions between interneuronal and pyramidal lineages and expression of EPOR in interneuronal subpopulations.

Cell-to-cell communication analysis predicts source/target of cross-talks between pyramidal and interneuronal lineages among rhEPO and PL samples. These communications are demonstrated as an array of dotplots. The color scale ranges from dark blue to yellow-green, corresponding to lower and to higher co-expression magnitude. Dot size is proportional to the intensity of their interactions, specificity given in CellPhoneDB [85]. A Baiting mature CA1 pyramidal lineages as a source, the dotplot shows the differential interaction of ligands Ptn, Ncam1, Sema3e, with Ptprz1, or Nrp2 receptors, respectively, encoded by distinct lineages of SST interneurons. B, C Similarly, while the Calm2 interaction with Pde1c is diminished in CCK-dendrite targeting lineage, PVALB basket cells lack this interaction completely in rhEPO samples. D The dotplot as above shows the differential interaction of newly formed pyramidal neurons with SST interneurons between rhEPO and PL samples. While Nrg1-Gpc1 appears in O-LM, the Nrxn1-Nlgn3 interaction is lost in the Backprojection lineages in rhEPO samples. E Pdgfc-Pdgfrb, and Nrg1-Gpc1 interactions emerge between CCK-dendrite targeting cells and newly formed pyramidal neurons in rhEPO samples. F Curiously though, Nectin3-Nectin2 interaction is shifted from PVALB bistratified cells to basket cells upon comparison of gain and loss of interactions between PL to rhEPO samples. However, the Calm1-Oprm1 interaction is lost in basket cells of rhEPO samples. G Graph showing the percentage of Gad1+ and Pvalb+ interneurons expressing Epor in CA1 hippocampal region. H Representative FISH image from CA1 demonstrating Epor mRNA expression in Gad1+ and Pvalb+ interneurons. Magnification of focal planes show co-expression of Epor (red) with Gad1 (green) and Pvalb (blue) (H1-H2). Scale bar: 35 µm for overview in (H) and 16 µm for focal planes.

Fig. 4. Treatment with rhEPO decreases the structural complexity of SST O-LM cells and PV-expressing interneurons in the hippocampal CA1 region.

A Representative hippocampal image of GAD-EGFP mice showing a schematic of different structural analyses performed. The majority of GAD-EGFP cells correspond to SST O-LM interneurons. B Treatment scheme and hippocampal area of analysis (CA1 region) illustrated with different interneuronal subtypes and their communication with pyramidal neurons. C Analysis of GAD-EGFP positive EPB (arrowheads) in the stratum lacunosum-moleculare. Confocal 2D projections show EGFP-positive axons of rhEPO and PL treated mice. Graph represents the changes in the density of EPB, expressed as boutons per micron. D1, D2 - H Analysis of dendritic spine density in GAD-EGFP expressing cells located in stratum oriens. Representative fluorescent images of spiny dendrites are shown for rhEPO and PL treated mice (D1). Dendritic spines are indicated by arrowheads. Magnified segments on the left are used to show the different spine subtypes (D2): mushroom (m), stubby (s) and thin (t). E Graphs showing changes in total density of dendritic spines corresponding to the first 150 μm from soma and in distinct segments stablished (0–50 μm, 50–100 μm and 100-150 μm). Graphs showing total spine density considering whole length (150 µm) and in specific segments of 50 µm for mushroom (F), stubby (G) and thin spines (H). I, J Structural Sholl analysis of GAD-EGFP and PV expressing interneurons in the stratum oriens. The images show 2D projection of a Z confocal stack of GAD-EGFP (I) and PV (J) positive interneurons. I1-2 and J1-2: Representative 3D reconstructions of dendritic arbors from rhEPO and PL treated mice. Graphs show changes in total number of intersections with Sholl spheres and in number of intersections as function of distance from soma. All graphs show mean ± SEM; N numbers depicted as dots in the bars; unpaired two-tailed Student’s t-test. All analyses were conducted following treatment scheme in (B), at same age, and same area evaluated. Scale bar: 5 μm for C and D; 6.5 μm for 3D projections in (I, J) and 20 μm for 3D reconstructions in (I, J).

Fig. 5. Treatment with rhEPO slightly affects the E:I ratio and decreases the density of inhibitory perisomatic puncta on excitatory hippocampal neurons.

A Analysis of excitatory/inhibitory balance in CA1. Single confocal planes with magnified insets show expression of VGLUT1 (blue) and VGAT (red) immunoreactive puncta from rhEPO and PL treated mice in the CA1 strata: oriens (A1), pyramidale (A2), radiatum (A3) and lacunosum-moleculare (L-M; A4). B–E Graphs showing density of puncta expressed as number of puncta per micron and E/I ratio in the different layers. F–I Analysis of the density of perisomatic inhibitory puncta on excitatory neurons. Schematic illustration shows perisomatic input that pyramidal neurons receive from both types of basket cells, PVALB and CCK basket cells (PV+ and CB1r+ puncta, respectively). F, G Panoramic and single confocal views of CA1 stratum pyramidale showing PV and CB1r expressing puncta (red), surrounding the soma of CAMKII+ excitatory neurons (green). Graphs present changes in density of perisomatic puncta expressing PV (H) and CB1r (I). J Density of perisomatic inhibitory puncta in CA1 of older GAD-EGFP mice. Graphs show changes in density of perisomatic PV+ puncta and in percentage of area covered by PV expressing puncta. All graphs show mean ± SEM; N numbers depicted as dots in the bars; unpaired two-tailed Student’s t-test. All analyses were conducted following treatment scheme in Fig. 4B, except F that followed the 3-week PL/rhEPO treatment at older age (P90). Scale bar: 5 μm for A; 25 μm for F, G panoramic views and 5 μm for insets.

Fig. 6. rhEPO produces a shift towards excitation and decreases the metabolic activity of hippocampal interneurons.

A Scheme of experimental timeline and diagram of recording electrode location in the dorsal region of CA1 (dCA1). B Representative power spectrum and raw LFP signal obtained during recording. After filtering, theta cycles and their coupled gamma activity are highlighted with black and green lines, respectively. C Power spectrum highlighting the mean slope of the gamma activity of interest (30–50 Hz) with a red line. D Non-linear regression model of normalized power spectrum showing a decrease in the mean slope of the 30-50 Hz activity band of rhEPO treated mice. E Boxplot of slope values obtained in the 30-50 Hz band showing a decrease in the slope of rhEPO treated mice. F Schematic of NanoSIMS experiment after ¹⁵N-leucine-enriched food, provided for the same 3-week duration as PL/rhEPO treatment (N = 4 mice/group), starting at P28. Representative images and calculated ¹⁵N/¹⁴N ratio in PV+ cells as measure of ¹⁵N-leucine incorporation (normalized to PV- areas). Electrophysiological statistical analysis explained in text; for NanoSIMS: 2-tailed Mann–Whitney U test; scale bar: 15 μm for F.

Fig. 7. Treatment with rhEPO decreases the expression of plasticity-related molecules in the hippocampus.

A Densitometric analysis of polySia-NCAM expression in CA1. Microphotographs from conventional light microscope compare the expression of polySia-NCAM in the CA1 strata (oriens, pyramidale, radiatum and lacunosum-moleculare) in rhEPO and PL treated mice. B Graph representing changes in the grey levels of polySia-NCAM immunoreactivity. C Expression of PV, PNNs and their co-localization after rhEPO treatment. Confocal plane focused on stratum pyramidale shows distribution of PV+ interneurons (red) and PNNs (blue) in rhEPO and PL groups. The squared area (C1) shows a single PV immunoreactive neuron surrounded by a PNN. D Graph presenting changes in total number of PV+ cells and PNNs, and the total number of PV+ neurons surrounded by PNNs. E Graph showing the volume of CA1 in rhEPO and PL treated mice. All graphs show mean ± SEM; N numbers depicted as dots in the bars; unpaired two-tailed Student’s t-test (except 7E, one-tailed). All analyses were conducted following treatment scheme in Fig. 4B, at same age, and same area evaluated; scale bar: 60 μm for A; 12.5 μm for overview in C and 9 μm for inset magnifications.