Figure 5
From: Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse
Impairments of synaptic morphology and activity in Ptchd1−/y mice. (a) Altered morphology of primary embryonic hippocampal cultures from Ptchd1−/y embryos. Quantification of dendritic lengths, branching numbers and complexity from green fluorescent protein (GFP)-labelled hippocampal neurons at 18 days in vitro between wild-type (WT) and Ptchd1−/y littermate embryos. Scale bar: 10 μm. Data are presented as mean±s.e.m., n=21 WT and 20 Ptchd1−/y neurons. *P<0.05 and **P<0.01, Mann–Whitney (length of branches: P=0.005; and number of branches: P=0.0452) and two-way analysis of variance with Sidak's multiple comparisons (branching complexity: level 1, P>0.999; level 2, P>0.999; level 3, P=0.9668; level 4, P=0.4663; level 5, P=0.0119; level 6, P=0.0004) tests. (b) Reduced synaptic density in hippocampus from Ptchd1−/y mice. Representative electron micrographs (EM) of synaptic contacts from hippocampal sections. Scale bar: 200 nm. Quantification of postsynaptic densities (PSDs) performed on a defined plane of 65 μm2. A total of 3 WT and 3 Ptchd1−/y brains (31 sections for each genotype representing 8–13 sections per animal). Graphical data are presented as scatter plots representing sections analysed for each animal±s.e.m., **P<0.01 (exact P=0.0085, calculated using the mean values per animal); unpaired two-tailed Student’s t-test. (c) Altered morphology of the excitatory synapses in Ptchd1−/y mice. Higher magnification of EM is shown (white bars, dotted bars and white arrows indicate synaptic cleft width, PSD length and PSD width, respectively). All analysed excitatory synaptic junctions were defined by an asymmetric structural organization, including well-defined presynaptic (for example, vesicles) and postsynaptic (PSD) structures. Scale bars: 200 nm and 50 nm. (d) Comparison of the cumulative frequency for PSD length and width and for synaptic cleft width in WT and in Ptchd1−/y hippocampi. A total of 3 WT brains (86 synapses splitted in 19, 30 and 37 structures in the respective brains) and 3 Ptchd1−/y brains (79 synapses splitted in 17, 26 and 35 in the respective brains) were analysed. Graphical data are presented as mean cumulative frequency curves obtained for each genotype±s.e.m.. Kolmogorov–Smirnov test; PSD length, P=0.4154; PSD width, P=0.0310; synaptic cleft width, P=0.0008. (e–i) Reduced miniature excitatory postsynaptic current (mEPSC) frequency and increased release probability in Ptchd1−/y mice. Example traces (e) and quantification of mEPSC amplitude (f) and frequency (g). Example traces (h) and quantification (i) of paired pulse ratio. Sample size (n) is indicated in the bars as cells per animals. Data are presented as mean±s.e.m., **P<0.01. Student’s t-test. KO, knockout.
