Synaptic Vesicles

The analysis of microglial cell bodies and processes comprised several ultrastructural measures of morphology, phagocytic activity, cellular stress, and physiological function. Experimenters were blinded to the experimental conditions throughout the analysis. Size and shape descriptors were determined using ImageJ. For each microglial cell body and process, phagocytic inclusions (appearing electron-lucent and named “empty” versus containing materials and named “non-empty”), lysosomes (primary, secondary, versus tertiary), lipid bodies, fibrillar materials, ER dilation, vacuoles (diameter < 100 nm), and extracellular space pockets containing debris were counted, according to the quantitative code 0, 1, 2, and 3+ (designating 3 and more occurrences). Lysosomes were identified by their dense, heterogeneous contents enclosed by a single membrane [49 (link)]. Primary lysosomes possessed a homogenous granular content and their diameter ranged from 0.3 to 0.5 μm [50 (link)]. Secondary lysosomes were 1 to 2 μm across, and their content was heterogeneous showing fusion with vacuoles. Tertiary lysosomes ranged in diameter between 1.5 and 2.5 μm, and they were usually fused to one or two vacuoles associated with lipofuscin granules, as well as lipidic inclusions showing signs of degradation [51 (link)]. Lipidic inclusions were identified as the clustering of round organelles with an electron dense, either opaque or limpid, cytoplasm enclosed by a single membrane. Lipofuscin granules were identified by their oval or round shapes, finely granular composition, and associated amorphous materials [51 (link)]. Extracellular fAβ was identified as densely packed fibrils and filaments, according to previous ultrastructural descriptions [52 (link)]. ER dilation was recognized by a swelling of the cisternal space ranging from 50 to 300 nm [53 (link)]. Extracellular space pockets containing debris, which could result from “exophagy” (degradation of cellular constituents by lysosomal enzymes released extracellularly), exocytosis (the process of expelling the contents of a membrane-bound vesicle into the extracellular space, often lysosomal and in preparation for phagocytosis; [54 (link)]), or pinocytosis (also named bulk-phase endocytosis, by which cells can take up extracellular contents in a non-phagocytic manner; [55 (link)]) was defined by the appearance of degraded materials (including cellular membranes and organelles) or debris in the extracellular space juxtaposing the microglia [54 (link)].
For microglial processes, their encirclement of neuropil compartments (axon terminals, dendritic spines, synapses between axon terminals and dendritic spines, cellular elements with signs of degradation) was also quantified. Encirclement was defined as microglial interactions with these neuropil compartments that displayed at least two points of contact, sometimes extending over several hundreds of nanometers. They were scored using the quantitative code 0, 1, 2, and 3+ (designating 3 and more elements). The encircled elements were identified according to the following criteria: axon terminals contained synaptic vesicles and were frequently seen branching from axons or making synapses onto dendritic branches and spines, and dendritic spines were identified as extensions from dendrites often forming synapses where a postsynaptic density was observed. Moreover, microglial encirclement of extracellular debris was determined.

4dpf-fish were fixed overnight in 4% paraformaldehyde and washed in 1X PBS containing 1%BSA, 1% DMSO, 0.5% Triton X-100. Primary antibodies were incubated overnight at 4°C in 5% normal goat serum, 1%BSA, 1% DMSO, 0.5% Triton X-100. For muscle and connective tissue staining MY1H (DSHB MF20 at 2 ug/mL) and Thbs4b (GeneTex-GTX125869 at 1:500 dilution) antibodies were used. To investigate the neuromuscular junctions, Synaptic vesicle glycoprotein 2 A (DSHB SV2 at 5 ug/mL) and α-Bungarotoxin (Alexa Fluor 647 conjugated-B35450 at 1:250 dilution) were used to label the presynaptic and postsynaptic termini, respectively. Anti-RFP (DSHB) and anti-mCherry (DHSB 3A11) at 2 ug/mL were used to label nerves and connective tissues in isl1:RFP and scxa:mCherry fish. Anti-Kaede antibody (MBL M106-3M) at 1:250 dilution was used to label skeleton in sox10:Kaede fish. Secondary antibodies (1:500 dilution) were also incubated overnight at 4°C in 5% normal goat serum, 1%BSA, 1% DMSO, 0.5% Triton X-100. Following incubation, antibody solution was removed, the fish were washed three times in 1X PBSTx (0.5% Triton X-100).