Glutamate receptors at Drosophila neuromuscular junctions (NMJs).

(A) Phylogeny of 16 glutamate receptors in the Drosophila genome and their reported expression at the larval NMJ (based on Li et al., 2016). (B) Schematic of the larval Drosophila fly NMJ (top) and composition of glutamate receptors (bottom). Larval GluRs occur as two distinct tetrameric complexes, each with the same auxiliary subunit (Neto). (C) Example fluorescence images of larval and adult NMJs. Motor neurons are labeled by driving GFP with vGlut-Gal4 (green). Active zones are labeled with an antibody against Brp (magenta). Muscle is labeled with phalloidin (blue).

GluR subunit expression mapping in larval and adult muscles.

(A) Representative confocal images showing GFP expression driven by a GAL4 reporter line (GluRIIC-GAL4) in larval and adult muscles. (B) Representative images showing antibody staining of GluRIIC at larval and adult neuromuscular junctions. (C) Summary table of Gal4 expression/antibody staining for all GluRs in larval and adult muscles. Note that antibodies and tagged proteins are combined in a single immunohistochemistry (IHC) column. Note that this initial screen focused on the coxa segment of the adult leg. See supplemental figures for representative images and methods for a complete list of reagents screened. (D) Representative images showing the GluR auxiliary subunit Neto-β tagged with GFP at larval and adult neuromuscular junctions.

Differential GluRIIB vs GluRIIC subunit expression in adult muscle subtypes.

(A) Schematic of the fly’s front leg with annotations of three key muscle groups in the femur. (B) Femur with Netoβ::sfGFP (green) in NMJs (Brp, magenta) in all femur muscle types (phalloidin, blue). (C) 3D rendering of a confocal stack of femur muscles (phalloidin, blue) and GluRIIB GAL4> UAS-GFP (green). GFP is expressed specifically in tibia extensor muscle fibers. (D) Representative antibody staining images showing presence of GluRIIB in a tibia extensor muscle (left) and absence of GluRIIB in an accessory tibia flexor muscle (right). (E) 3D rendering of a confocal stack of femur muscles (phalloidin, blue) and GluRIIC GAL4> UAS-GFP (green). GFP is expressed specifically in accessory tibia flexor muscle fibers. (F) Representative antibody staining images showing absence of GluRIIC in a tibia extensor muscle (left) and presence of GluRIIC in an accessory tibia flexor muscle (right).

GluClα is expressed extrasynaptically in adult fly muscles.

(A) Single-nuclei RNA-seq data shows strong expression of GluClα in adult leg muscles. (B) A Gal4 reporter for GluClα labels muscles in adult legs but not larval muscles. Expression in the larva is mainly in the nervous system, as expected. (C) Representative images showing antibody staining of endogenously-tagged GluClα:V5 in larval and adult muscles. Co-staining with an antibody against Brp reveals that GluClα does not co-localize with active zones. Labeling in larval muscle is likely from motor neurons (Li et al., 2021). (D) Labeling of GluClα protein with a V5 tag shows extrasynaptic expression throughout the muscle fiber, whereas active zones (magenta) are localized.