Fig 2: The size of presynaptic boutons is altered in layer 5 of the mPFC of Mecp2 KO mice.(A–B) Schematic and representative examples of AAV2-hSyn-mCherry injection sites for identifying vHIP axons (A) or contralateral mPFC axons (B). Scale bars 1 mm. (C) Schematic of vHIP axons in the mPFC. (D) Axonal swellings identified as presynaptic boutons co-labeled for mCherry and VGLUT1. Scale bar 2 µm. (E) Representative examples of mCherry-filled presynaptic boutons. Scale bar 2 µm. (F) Numerical densities of axonal boutons per length of vHIP and c-mPFC axons located in layer 5 of mPFC (n = 45 axons WT vHIP; n = 36 Mecp2 KO vHIP; n = 49 WT c-mPFC; n = 43 KO c-mPFC; WT vHIP vs. Mecp2 KO vHIP, p=0.9925; WT vHIP vs. WT c-mPFC, p=0.8317 Mecp2 KO vHIP vs. Mecp2 KO c-mPFC, p=0.6765; WT c-mPFC vs. Mecp2 KO c-mPFC, p=0.6765; Interaction p=0.0135; Axon p=0.9592; Genotype p=0.3102; Two-way ANOVA with Benjamini and Hochberg multiple comparisons). Mean ± SD. (G) Cumulative probability distributions of the estimated size of presynaptic boutons comparing vHIP and c-mPFC axons in mPFC layer 5 of WT and Mecp2 KO mice. (H) Average weighted bouton sizes (vHIP WT mice vs. Mecp2 KO mice, p<0.0001; c-mPFC WT mice vs. Mecp2 KO mice, p=2320; vHIP vs. c-mPFC WT mice, p<0.0001; vHIP vs. c-mPFC Mecp2 KO mice, p=0.0133; n = 993 WT vHIP boutons; n = 682 KO vHIP; n = 792 WT c-mPFC; n = 577 KO c-mPFC; Interaction p=0.1193; Axon p=0.3135; Genotype p=0.2967; Two-way ANOVA with Benjamini and Hochberg multiple comparisons). Mean ± SEM; *p<0.05, **p<0.01. Figure 7—source data 1. See also Figure 7—figure supplement 1.10.7554/eLife.44182.038Figure 7—source data 1.The size of presynaptic boutons is altered in layer 5 of the mPFC of Mecp2KO mice.

Fig 3: Paired-pulse stimulation reveals the absence of the slow phase of facilitation in VGluT1+/− mice. (a) Sample EPSCs recorded from a WT (top) and VGluT1+/− (bottom) relay cell during paired-pulse stimulation. CT inputs were stimulated with PPI as indicated (bars above). Current traces are averages of 3–5 repeats. Paired-pulse repeats were separated by 30 s to allow recovery from previous facilitation. Traces are aligned by the first pulse in each pair (*). For PPI > 500 ms, the time between EPSC1 and EPSC2 has been cropped for display purposes. The thick gray lines indicate the mean amplitude of EPSC1 across all PPI (EPSCm1) for each cell. (b) Mean facilitation values for each PPI are plotted for WT (n = 10; black dots) and VGluT1+/− (n = 9; gray dots). Error bars are SEM. For both genotypes, facilitation decreases exponentially with increasing PPI. WT data are best fit by a double exponential curve (black), while VGluT1+/− data only require a single exponent (gray) with a time course similar to the fast component of the WT data. Inset shows the shorter PPI on an expanded time scale.

Fig 4: Layers of the human hippocampus determined by VGLUT1 immunofluorescence and Nissl staining. (A) Lipofuscin (pigment, purple) and Nissl substance (red) were stained with aldehyde fuchsine and Darrow red in a human hippocampal section, occipital part. The asterisk marks adjoining parts of the gyrus fasciolaris. Scale bar, 700 µm. (B) Zoom-in of panel (A) to display layer boundaries. Alv = alveus, SO = stratum oriens, SP = stratum pyramidale, SR = stratum radiatum, SLM = stratum lacunosum-moleculare, oml = outer molecular layer, iml = inner molecular layer, PML = polymorphic layer, GCL = granule cell layer, CA4 = cornu Ammonis region 4. Scale bar, 500 µm. (C) A subsequent section from the tissue block used in panel (A) was immunostained with VGLUT1 and acquired at a confocal microscope. LAS-X Navigator was used to create and stitch the tilescan. Dashed squares (white) are shown in panel (E) as high-resolution z-stacks. The asterisk marks adjoining parts of the gyrus fasciolaris. Scale bar, 700 µm. (D) Zoom-in of panel (C) to display layer boundaries. The yellow arrows mark the borders of the stratum lucidum (SL), i.e., CA3. Scale bar, 500 µm. (E) Maximum intensity projections (MIPs) of high-resolution z-stacks (stack size = 2.64 µm) acquired in the SP of CA3 (1), SL and SR of CA3 (2), dentate gyrus (3), and CA4 (4). Numbers refer to position 1–4 in the tilescan from panel (C). Scale bar, 30 µm. Images in panels (A–E) are derived from case 1.

Fig 5: Proprioceptive inputs synapse onto a- but not ?-motor neuronsConfocal image of an a-motor neuron (left, CHAT+ (green), NeuN+ (blue)) and ?-motor neuron (right, CHAT+, NeuN-) of a P110 mouse. Proprioceptive synapses (VGluT1+, magenta) innervate only the a-motor neuron which is larger. Scale bar = 15 µm.