Article Figures & Data
Figures
- Figure 1.
PE increases inhibitory and excitatory drive in the DG. A, Illustration of the SH and PE. Mice were raised either in SH or PE for 3 weeks after birth. Schematic illustration of the hippocampus with a representative whole-cell patch clamp recording of a granule cells in the DG. B, Representative mEPSC traces recorded from mature GCs (P19–21) in acute brain slice from SH and PE. C, D, Cumulative distribution of the interevent intervals (C) and amplitude (D) of the mEPSC recorded in GCs from SH and PE (P19–P21). Insets, Mean frequency and amplitude. *p < 0.05 by a two-sample t test, SH: a/n = 3/12, PE: a/n = 3/13. E–G, Same as B to D but for mIPSC. Insets: mean frequency and amplitude; **p < 0.01 by a two-sample t test, SH: a/n = 4/21, PE: a/n = 3/15. H, Representative images of excitatory synaptic terminals labeled with antibodies against VGLUT1 (green) in the ML. I, Quantification of VGLUT1 puncta in the ML of brain section from mice raised in SH (3–4 hemispheres from a total of 7 mice) and PE mice (3–4 hemispheres from a total of 11 mice). PE data were normalized to SH control. Statistical comparisons were performed with a two-sample t test (***p < 0.001). Data represent mean ± SEM. The number of animals and images used are reported within each bar (a/n). All scale bars in μm.
- Figure 2.
PE induces a perisomatic increase of inhibitory synapses on GCs. A, Schematic representation of interneuron connectivity in the DG. PP(EC2), Projection from layer 2 entorhinal cortex neurons; PV, parvalbumin interneuron; CCK, cholecystokinin interneuron; SOM, somatostatin interneuron. Dendrites are segmented based on their location within the different laminae; distal dendrites (DD) in the ML, medial dendrites (MD) in the inner ML, proximal dendrites (PD) in the GC layer (GCL) and axon initial segment (AIS) in the hilus. B, Confocal images of the DG from SH (left) and PE (right) mice stained with anti-VGAT (green) antibody and DAPI (blue). Box with dashed line indicates the region acquired at high magnification for VGAT puncta density quantification. C, Left, representative images of the VGAT labeling for SH and PE mice. Right, quantification of VGAT puncta density in all laminae of the DG. Non-normalized data are presented in Table 1. D. Top, Schematic of dual AAV injection with AAV-hSyn-Gephyrin-GFP (green) and AAV-Ef1α-tdT-F in neonatal pups at P1. Bottom, Tracing and segmentation of GC dendrites tracing with Imaris is performed using anatomic cue (GC nuclei) as well as with the help of the labeling of EC2 fibers with tdT-F to localize the separation between inner and outer molecular layer (iML and ML). E, Representative images of gephyrin-GFP fluorescence along the somatodendritic axis of GCs from SH and PE mice. Dashed lines represent the contour of the cell soma. The quantifications of the density of GFP clusters along the segments of the dendrites and the AIS are reported as number of clusters per µm. Data are presented in Table 2. F, Representative images of GC soma labeled with gephyrin-GFP and quantification of the number of cluster per soma. Data represent mean ± SEM. Statistical comparison between SH and PE in individual layer or segment was performed by a two-sample t test; *p < 0.05, **p < 0.01, and ***p < 0.001. Data represent mean ± SEM. The number of animals and images used are reported within each bar (a/n). All scale bars in μm.
- Figure 3.
PE enhances CCK+ somatic synapses in the DG. A, Representative images of the GC layer stained with either antibodies against VGAT (magenta) and CB1R (green) or Syt2 (green) from brain sections of SH and PE mice. Scale bars in μm. B, Corresponding quantification of CCK + basket cell synapses (overlap of VGAT and CB1R) and PV+ synapses (Syt2) in the GC layer. Statistic by a two-sample t test, *p < 0.05. Non-normalized data are presented in Table 1. C, Representative images of the iML and the GC layer stained with antibodies against VGAT (magenta) and VGLUT3 (green) from brain sections of SH and PE mice. The dashed line represents boundary of iML and GCL; box with dashed line indicates the region at high magnification shown below. Scale bars in μm. D, Corresponding quantification of CCK + basket cell synapses labeled with VGAT and VGLUT3 in the iML and GC layer. Statistic by a two-sample t test, p < 0.01. Non-normalized data are presented in Table 1. E, Representative mIPSC traces recorded from mouse GCs (P19–21) in the absence or presence of 1 μm extracellular ω-conotoxin GVIA from SH and PE mice. F, Summary graphs of the average frequency. All data were normalized to SH control. Statistical comparisons were performed with a one-way ANOVA (*p < 0.05) followed by Tukey's honestly significant difference post hoc test. Data represent mean ± SEM. The number of animals and the number of images or of recorded cells used are reported within each bar (a/n).
- Figure 4.
EE increases GABA release and magnitude of DSI by CCK+ inputs. All whole-cell recordings were performed from GCs of SH and PE mice (P19–21). A, Representative mIPSC traces recorded in the absence or presence of 5 μm extracellular WIN 55 212-2 (WIN). B, Summary graphs of the average frequency. All data were normalized to SH control. Statistical comparisons were performed with a one-way ANOVA (*p < 0.05) followed by Tukey's honestly significant difference post hoc test. C, Representative example of traces of the evoked IPSC recorded before and after WIN. D, Summary graphs of the average amplitudes. Statistical comparisons were performed with a one-way ANOVA (*p < 0.05) followed by Tukey's honestly significant difference post hoc test. E. Example of paired-pulse eIPSC traces recorded with a 50 ms interstimulus interval (ISI) before and after WIN. F, Summary graph of the averaged PPRs for ISIs of 50 ms, 100 ms, 250 ms, 500 ms, and 900 ms. Statistical comparisons were performed with a one-way ANOVA. Data are presented in Table 3. G, Representative spontaneous IPSC traces recorded before and after depolarization-induced DSI (+70 mV, truncated). H, DSI magnitude expressed as percentage of the reduction in charge transfer after DSI induction in all four groups. Statistical comparisons were performed with a two-sample t test (***p < 0.001). Data represent mean ± SEM. The number of animals and recorded cells used are reported within each bar (a/n).
- Figure 5.
Inhibition of EC2 afferents using DREADD receptor hM4Di. A, Diagram representing the experimental paradigm used to induce c-fos expression in response to the exploration of a novel environment (NE), or in home cage (HC), in mice injected with AAV-hSyn-hM4D(Gi)-mCHerry in the EC. B, Left, Representative images of the EC-DG circuit from brain sections labeled with RFP antibody to amplify mCherry signal. Right, Quantification of mCherry expressing cells in the medial EC2. C, Left, Representative images of the DG from brain sections labeled with an antibody against c-fos. The projections from EC2 neurons are labeled with mCherry, and nuclei were labeled with DAPI. Right, Quantification of c-fos-expressing cells in the GCL. Number is compared in sections obtained from HC group or mice expressing hM4Di and placed in NE. Mice expressing hM4Di received an IP with either normal saline solution (NE-hM4D-NS) or CNO (NE-hM4D-CNO). All data were normalized to the control HC values. Statistical comparisons were performed with a one-way ANOVA followed by Tukey's honestly significant difference post hoc test (***p < 0.001). Graph bars represent mean ± SEM. The number of animals and images used are reported within each bar (a/n).
- Figure 6.
Experience-dependent remodeling of CCK+ basket cell inputs is controlled by EC2 neuron activity. A, Left, Experimental paradigm for chemogenetic inhibition of excitatory afferents from EC2 in the DG circuit. Right, Representative image of the perforant path fibers in the DG (afferents from the EC2) labeled with mCherry (red) following neonatal injection of AAV-hSyn-hM4D(Gi)-mCherry in the EC. B, D, F, Representative images of the GCL and ML stained with antibodies against either, VGAT (magenta; B, D), CB1R (green; D), or Syt2 (green; F) from brain sections of SH mice injected or not with hM4Di-expressing virus and treated with CNO (SH-hM4D-CNO and SH-CNO, respectively). C, E, G, Corresponding quantification of GABAergic terminals (VGAT; C) in the ML, CCK+ synapses (VGAT/CB1R; E), and PV+ synapses (Syt2; G) in the GCL. All data were normalized to SH-CNO control. Statistical comparisons were performed with a two-sample t test (*p < 0.05 and ***p < 0.001). Graph bars represent mean ± SEM. The number of animals and images used are reported within each bar (a/n). All scale bars are in μm.
- Figure 7.
Density of presynaptic GABAergic and glutamatergic terminals are regulated by the activity of the EC2 afferents. A–C, Representative images of GCL and ML stained with antibody against either VGAT (magenta; A, B), CB1R (green; A), or VGLUT1 (green; C) from brain sections of SH and PE mice. Chronic inhibition of the EC2 afferents was done by injecting hM4Di expressing virus and CNO treatment (PE-hM4D-CNO) and compared with values obtained from SH and PE mice also injected with the virus but received normal saline (NS) treatment (SH-hM4D-NS and PE-hM4D-NS). An additional control group consisted of PE mice not injected with the virus but received the CNO injection (PE-CNO). D–F, Corresponding quantification of CCK+ synapses in the GC layer, and GABAergic or glutamatergic terminals in the ML for each condition. All data were normalized to SH-hM4D-NS. Statistical comparisons were performed with a one-way ANOVA (**p < 0.01, ***p < 0.001) followed by Tukey's honestly significant difference post hoc test. Graph bars represent mean ± SEM. The number of animals and images are reported for each bar (a/n). All scale bars are in μm.
Tables
Layer Puncta density (/μm3)a tb pb Puncta density (%SH)a tc pc VGAT ML SH 0.287 ± 0.013 2.405 0.019* 100 ± 3.89 2.940 0.0046** PE 0.332 ± 0.012 115.61 ± 3.32 iML SH 0.168 ± 0.014 0.608 0.548 100 ± 8.34 0.608 0.55 PE 0.179 ± 0.012 106.86 ± 7.08 GCL SH 0.100 ± 0.005 3.401 0.001** 100 ± 4.42 3.746 0.0004*** PE 0.128 ± 0.006 129.15 ± 6.41 Hilus SH 0.066 ± 0.003 0.635 0.532 100 ± 4.03 0.635 0.53 PE 0.063 ± 0.003 94.90 ± 4.84 CB1R-VGAT GCL SH 0.0070 ± 0.0007 2.439 0.021* 100 ± 10.02 2.439 0.021* PE 0.0088 ± 0.0003 126.10 ± 5.31 Syt2 GCL SH 0.0325 ± 0.0027 0.175 0.862 100 ± 2.50 0.986 0.33 PE 0.0331 ± 0.0023 103.86 ± 2.57 VGLUT3-VGAT iML SH 0.0126 ± 0.0015 0.381 0.706 100 ± 11.73 0.381 0.71 PE 0.0082 ± 0.0009 165.86 ± 15.96 PE 0.0134 ± 0.0014 106.34 ± 11.75 GCL SH 0.0050 ± 0.0004 3.702 0.002** 100 ± 7.85 3.702 0.002** The density of the synaptic puncta in the different layers of the DG were quantified in SH and PE mice. Puncta densities are reported as number of puncta per cubic micrometer (/μm3) or as percentage of control (%SH).
↵aAll data are represented as mean ± SEM
↵bStatistical comparisons for each layer were performed for the original data of puncta density (/μm3), with a two-sample t test.
↵cStatistical comparisons for each layer were performed for the normalized data of puncta density (%SH), with a two-sample t test. Asterisks indicate significant difference between groups (*p < 0.05, **p < 0.01,***p < 0.001).
- Table 2.
Density of the gephyrin-GFP clusters along the somatodendritic axis of the GCs in SH and PE mice
Puncta density (/μm3)a tb pb DD SH 0.281 ± 0.014 0.304 0.762 PE 0.275 ± 0.012 MD SH 0.247 ± 0.015 0.809 0.422 PE 0.232 ± 0.011 PD SH 0.338 ± 0.035 2.107 0.039* PE 0.433 ± 0.029 AIS SH 0.393 ± 0.031 0.07 0.945 PE 0.388 ± 0.053 Soma# SH 34.50 ± 1.677# 3.108 0.0029** PE 44.42 ± 2.716# Density of tde gephyrin-GFP clusters are reported as number of cluster per micrometer along the DDs in the ML, medial dendrites (MDs) in the inner ML, the proximal dendrites (PDs) in the GCL and the axon initial segment (AIS) in the hilus. For the soma, data are reported as number of gephyrin-GFP clusters per soma (#).
↵aAll data are represented as mean ± SEM.
↵bStatistical comparisons for each segment were performed for original data of puncta density (/μm3), with a two-sample t test. Asterisks indicate significant difference between groups (*p < 0.05, **p < 0.01).
ISIs (ms) SHa PEa Fb pb Before WIN After WIN Before WIN After WIN 900 0.87 ± 0.089 0.79 ± 0.038 0.78 ± 0.029 0.81 ± 0.037 0.489 0.691 500 0.78 ± 0.049 0.87 ± 0.072 0.74 ± 0.040 0.97 ± 0.012 1.771 0.162 250 0.84 ± 0.056 0.90 ± 0.131 0.68 ± 0.023 0.85 ± 0.063 1.486 0.227 100 0.85 ± 0.046 0.82 ± 0.037 0.72 ± 0.044 0.77 ± 0.031 1.789 0.159 50 0.89 ± 0.077 0.72 ± 0.045 0.70 ± 0.067 0.73 ± 0.043 2.110 0.108 Evoked inhibitory postsynaptic currents recorded from the granular cells were evoked at different interstimulus intervals before and after bath application of WIN. ISI, Interstimulus interval. The corresponding pair-pulse ratios are reported for SH and PE mice.
↵aAll data are represented as mean ± SEM.
↵bStatistical comparisons were performed with a one-way ANOVA.
