Splice-dependent trans-synaptic PTPδ-IL1RAPL1 interaction regulates synapse formation and non-REM sleep (2020)
Figure 2

Figure 2. Ptprd deletion leads to input-specific suppression of excitatory synapse density and strength in the hippocampal SLM layer.

(A) Strong PTPδ-tdTomato (red) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus (2 months), revealed by quantitative analysis of PTPδ-tdTomato fluorescence across hippocampal lamina. DAPI was used for nuclear staining. CC, corpus callosum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare; MO, molecular layer; SG, granule cell layer; PO, polymorph layer. Scale bar 500 μm.

(B) Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer. Micro-dissected SLM and SR lysates (2 months) were immunoblotted for PTPδ (C-terminal antibodies) and α-tubulin (control). The asterisk indicates a non-specific band. Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR. (n = 4 mice for SR and SLM, mean ± SEM, ***p < 0.001, Student's t-test; See Dataset EV2 for statistical details for these and all following results).

(C) Schematic diagram for global Ptprd KO in mice, targeting exon 3. HTNC (His-tagged, TAT-fusion Cre with nuclear localization signal), carrying a membrane-permeable Cre recombinase, was used to remove exon 13 in fertilized eggs (see Methods for details). Sites on the PTPδ protein corresponding to epitopes recognized by anti-PTPδ antibodies (Ig1 domain [exon 13]) and N- and C-termini) are indicated.

(D) Gene dose-dependent reductions in PTPδ protein levels in whole brains of Ptprd+/- (HT) and Ptprd-/- (KO) mice relative to Ptprd+/+ (WT) mice (P21), shown by immunoblot analyses. Note that the C-terminal antibody also detects a non-specific band of similar size indicated by an asterisk, possibly either PTPσ or LAR.

(E-J) (E, G, and I) Suppressed basal excitatory synaptic transmission in the Ptprd-/- hippocampal SLM layer, but not SO or SR layer (2 months; male), as shown by the I/O ratio (n = 14 slices from 5 mice [WT-SO], 14, 5 [KO-SO], 11, 4 [WT-SR], 12, 5 [KO-SR], 14, 6 [WT-SLM], 12, 6 [KO-SLM], mean ± SEM, *p < 0.05; **p < 0.01, two-way repeated-measures/RM ANOVA, Holm-Sidak post-hoc multiple comparisons test). (F, H, and J) Normal presynaptic release in the Ptprd-/- hippocampal SLM, SO, and SR layers (P22-27; male), as shown by the PPF ratio (n = 13 slices from 5 mice [WT-SO], 13, 5 [KO-SO], 10,4 [WT-SR], 12, 5 [KO-SR], 15, 6 [WT-SLM], 14, 6 [KO-SLM], mean ± SEM, ns, not significant, two-way RM ANOVA).

(K-O) Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs. (n = 18 images from 3 mice [WT and KO], ***p < 0.001, ns, not significant, Student's t-test for L, O-R, Mann-Whitney U test for M, N). Scale bar, 500 nm.

(P-R) Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd-/- SLM (P21; male), revealed by EM. (n = 3 mice [WT and KO], mean ± SEM, ***p < 0.001, ns, not significant, Student's t-test).