Journal: Brain : a journal of neurology

Article Title: NPRL3 loss alters neuronal morphology, mTOR localization, cortical lamination and seizure threshold.

doi: 10.1093/brain/awac044

Figure Lengend Snippet: Figure 5 Focal Nprl3 KO in vivo results in lamination defects, increased soma diameter, increased cortical excitability, and reduced seizure threshold. (A and B) GFP+ cells observed in the subcortical white matter (bottom arrow) and in layers IV-VI in Nprl3 KO cortex at P3. In contrast, all GFP+ cells in scramble control specimens (n = 5 per groups; P < 0.001) were observed in the birthdate appropriate layer II/III. GFP+ neurons deep layers in Nprl3 KO specimens did not express CTIP2 indicating that these cells had failed to attain their appropriate laminar destination in layers II/III. GFP+ cells in layer II/III in Nprl3 KO pups were larger than scramble cells in the layer II/III. Rapamycin treatment prevented laminar defects (A and B) and soma diameter increases (C; each box represents mean, SEM, whiskers represent 5–95% confidence intervals). (D) Section from a surgical epilepsy FCD specimen (NeuN-labelled, from Subject 18 in Fig. 1) shows heterotopic white matter neurons (arrows) similar to what was observed in mouse (A and E, arrows). (F) Five weeks after in utero electroporation, mice were implanted with dural EEG electrodes and recorded for 48 h. A representative line length analysis of the 48 h EEG recording shows increased line length (a proxy for spike amplitude) in the electroporated cortex suggesting a hyperexcitable cortex (n = 6 per group) compared with the contralateral cortex and wild-type (WT) mouse cortices, where line lengths in the right and left hemispheres were simi lar. Total average EEG power also showed increased power in Nprl3 KO cortices compared to the contralateral cortex and control cortices (G). Rapamycin treatment after Nprl3 KO rescued increased line length and EEG power (F and G). Mice were treated with 55 mg/kg PTZ and EEGs were recorded. (H) Representative EEG tracings in Nprl3 KO, Nprl3 +rapamycin and wild-type mice. (I) After PTZ injection, Nprl3 KO mice had a statistically significant average decrease (ANOVA; P < 0.05) in latency to seizure (77.2 s) compared with wild-type mice (168 s). Rapamycin treated Nprl3 KO mice had seizure latencies that did not differ from control mice (178 s). ***P < 0.001, *P < 0.05. Calibration mark in A = 200 µM.

Article Snippet: Whole brains were post-fixed in 4% PFA overnight [12 h for postnatal Day 3 (P3) pups, 24 h for adult mice], paraffin embedded, microtome sectioned at 8 μm and probed with CTIP2 (1:500; rat D ow nloaded from https://academ ic.oup.com /brain/article/145/11/3872/6524443 by guest on 30 August 2024 monoclonal; Abcam), GFP (1:1000; chicken polyclonal; Abcam), MAP2 (1:100; goat polyclonal; Abcam), PS6 (240/244) or ribosomal S6 protein (mouse monoclonals; 1:2000; Cell Signaling) primary antibodies and then secondary antibodies Alexa488, 647 and 594, respectively.

Techniques: In Vivo, Control, In Utero, Electroporation, Injection