Neuromuscular Junction

Twelve mice (six male, six female) were selected at random from a single litter of CD1 wild-type mice at six weeks of age. Following euthanasia by overdose of inhaled isoflurane, each mouse was weighed, and the chosen muscles from each side were dissected out, within 30 min post-mortem. Muscles were immediately fixed in 4% paraformaldehyde for 30 min, then washed in 1% phosphate buffered saline (PBS). All remaining connective tissue was then removed. Cranial and lumbrical muscles intended for whole-mount were immediately prepared for immunohistochemistry (see below). The triceps and quadriceps were cryoprotected by immersion in 30% sucrose overnight; 100 µm sections were then obtained on a Thermo Scientific Microm HM 450/KS 34 freezing microtome.
Neuromuscular junctions were immunohistochemically labelled using a standard laboratory protocol for visualizing pre-synaptic 2H3/SV2 and post-synaptic AChRs [26 (link)]. Muscle preparations were placed in the following sequence of solutions (made up in 1% PBS unless otherwise specified; antibodies and their concentrations are listed below): α-bungarotoxin (BTX) for 30 min to label post-synaptic AChRs; 4% Triton X for 90 min; a blocking solution of 4% bovine serum albumin (BSA) and 2% Triton X for 30 min; the primary antibodies (made up in blocking solution) for 72 h at 4°C; 1% PBS for 80 min; 4% BSA for 30 min; the secondary antibodies (made up in 1% PBS) for 150 min; 1% PBS for 80 min. Finally, muscles preparations were mounted on glass slides in Mowiol, and stored at −20°C. At all stages, samples were protected from excessive light exposure prior to imaging.

Female virgin flies with the genotype nSyb-Gal4>UAS-Act5C::GFP were crossed to UAS-YARS1, UAS-PLS3, or UAS-CD8::RFP male flies. Third instar larvae were dissected in HL3.1, fixed in 4% PFA solution (in HL3.1) for 10 min, washed three times for 10 min with 1XPBS, blocked with 2%BSA/Normal Goat Serum, 0.1% Triton-X 1X PBX, incubated with α-GFP nanobodies (Nanotag Biotechnologies) to amplify the Act5C::GFP signal and HRP-Rhodamin red to label the neuronal membrane (both at 1:250 in blocking solution) for 2 h at room temperature, washed three times for 10 min in 0.1% PBX and mounted on glass microscopic slide in Diamond ProLong. NMJs on muscle 6/7 on both sides of abdominal segment A3 and A4 were imaged using a Nikon Ni-E upright microscope equipped with a Yokogawa CSU-W1 spinning-disk head, an Andor iXon 897U EMCCD camera and Nikon Elements AR software. A 60× (NA 1.4) oil immersion objective was used to image the NMJs. Image acquisition settings were identical for all images. Act5C::GFP-labeled actin assemblies were assessed on maximum-intensity projection images. To analyze actin in boutons, boutons were cropped, and actin assemblies were analyzed with the pipeline described below. The Trainable Weka Segmentation (TWS) machine-learning tool^{59 (link)} in Fiji was used to manually annotate GFP-positive actin assemblies with different fluorescence intensities, and to train a classifier that will automatically segment these structures. The segmented objects were subjected to Huang auto thresholding to obtain binary masks. Next, we applied a Watershed processing on the binary image, to improve the isolation of individual neighboring objects from the diffraction-limited images. We performed particle analysis on the segmented actin assemblies to obtain their number and area. Mean fluorescence intensity of individual actin assemblies was measured by applying the mask on the original image. The number of actin assemblies was normalized to the bouton area. To determine the bouton area using TWS, we developed different classifier by annotating the Act5C-positive bouton (Gaussian blur 2).

Synapsin-1 was localized in the levator auris longus (LAL) muscle by immunohistochemistry (IHC) in accordance with the protocol presented in Cilleros-Mañé et al. [41 (link)]. LAL muscles from the same animals that we used for western blot were used to perform this technique (n = 3).
As a control, primary antibodies were omitted from some muscles during the immunohistochemical procedures. These control muscles never exhibited positive staining. In double-staining protocols, omitting either one of the two primary antibodies completely abolished the corresponding staining and there was no cross-reaction with the other primary antibody. At least three muscles were used as negative controls.
A laser-scanning confocal microscope (Nikon TE2000-E) was used to study immunolabeled NMJs from the whole-mount muscles [41 (link)]. FIJI (ImageJ) software was used to perform 3D colocalization analyses from confocal stacks. The Pearson correlation coefficient (r) was used for quantitative analysis of colocalization. This statistic coefficient provides the overall association of two probes in an image. Images were assembled using Adobe Photoshop software (Adobe Systems, San Jose, CA), and neither the contrast nor brightness was modified.

C5–C7 spinal segments or musculocutaneous nerves were cut into 15-µm-thick frozen sections for immunostaining. The blocking buffer was composed of 5% goat serum and 3% bovine serum albumin diluted in 0.1 M phosphate buffer saline (PBS). Signal was detected with Alexa fluor 546 or 488 coupled secondary antibodies (1:1000, Invitrogen). Primary antibodies were: goat anti- choline acetyltransferase (ChAT, 1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Calretinin (1:300, ab702, Abcam), mouse anti-Parvalbumin (1:1000, Mab1572, Millipore), rabbit anti-CAMKII (1:500, ab104224, Abcam), rabbit anti-vesicular GABA transporter (VGAT; 1:800, NO131013, Synaptic Systems), mouse anti-vesicular glutamate transporter 1 (vGlut1; 1:1000, Mab5502, Millipore), rat anti-major histocompatibility complex 1 (MHC1; 1:300, sc-59199, Santa Cruz), rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, AB7260, Abcam), rabbit anti-Iba1(1:1000, 019–19,741, Wako), and rabbit anti-Oligo2 (1:500, ab9610, Merck Millipore).
On day 50 after BPA, the biceps were collected and 7-µm horizontal sections were prepared with a sliding microtome (Leica, Germany) and double stained with rabbit anti-NF200 (1:500, n4142, Sigma) and α-BT (1:1000, Molecular probes, USA) to visualize neuromuscular junctions (NMJs).