Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 Jan 3;15:725195. doi: 10.3389/fncel.2021.725195

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2022 Olmsted, Stigliano, Marzullo, Cibelli, Horner and Paluh.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

PMC Copyright notice

NMP-derived SMNs are positive for axon initial segment proteins, assemble neuromuscular junctions, and develop characteristic synaptic profiles. (A) Maturing SMN-NS cultures exhibit short segments immunopositive for AIS proteins Ankyrin-G (AnkG) and βIV-Spectrin at day 35. Top: AnkG/TUJ1 counterstained with DAPI (nuclei). High-magnification image depicts AnkG+ staining distal to the soma. Bottom-left: AnkG immunopositive region along TUJ1+ filaments. Bottom-right: βIV-Spectrin immunopositive region along TUJ1+ filaments. (B) Length of AnkG (32 ± 2 μm) and βIV-Spectrin (34 ± 1 μm) segments by day 35 (two-tailed t test, t = 0.7082, df = 18, P = 0.4879, n = 10 segments measured per antibody). (C) Time course normalized expression of AIS-related voltage gated ion channels Na_v1.6 and K_v1.2 by RNA-Seq. IF showing cells positive for K_v1.2 on right. (D) Rat L6 multinucleated skeletal myotubes for NMJ formation assays. (E) Day 4 co-culture of SMN-NS (day 42) seeded onto rat myotubes. Top: TUJ1+ filament termination onto αBTX+ junctions (nAChR). Bottom: SYN1/αBTX. (F) SMI312 axon termination in Desmin+ myotubes resembling early motor end plates. (G) Co-localization of cholinergic terminals (ChAT, bottom) with SYN1 puncta along axons (SMI312, top). (H) Cartoon diagram of ChAT/SYN1 pre-synaptic ACh terminals and nAChR post-synaptic machinery. (I) IF image and histogram of ChAT/SYN1 (64.7 ± 13.1%, n = 118 puncta) and SYN1/αBTX co-localization in the absence (0%, n = 48 puncta) or presence (50.7 ± 13.8%, n = 69 puncta) of L6 myotubes (two-tailed t test, t = 10.36, df = 14, ****P < 0.0001). N = 8 fields were averaged for each condition. (J) Heatmap of glutamate ionotropic receptor expression (AMPA_R, NMDA_R, Kainate) during SMN differentiation. (K) Histogram of normalized gene expression for pre-synaptic vesicular transporters (VGlut1 excitatory, VGAT inhibitory, VAChT cholinergic) and post-synaptic density proteins/excitatory receptors. (L) Co-localization of AMPA_R GluR1 (top) and NMDA_R GluN2A (bottom) with scaffolding protein PSD-95. Individual channels provided as inverted LUT. Data are reported as mean ± s.e.m. Scale bars are 50 μm, and 10 μm in (A) high magnification images (bottom). n.s., not significant.