Development and plasticity of the Drosophila larval neuromuscular junction

Abstract

The Drosophila larval neuromuscular system is relatively simple, containing only 32 motor neurons in each abdominal hemisegment, and its neuromuscular junctions (NMJs) have been studied extensively. NMJ synapses exhibit developmental and functional plasticity while displaying stereotyped connectivity. Drosophila Type I NMJ synapses are glutamatergic, while the vertebrate NMJ uses acetylcholine as its primary neurotransmitter. The larval NMJ synapses use ionotropic glutamate receptors (GluRs) that are homologous to AMPA-type GluRs in the mammalian brain, and they have postsynaptic scaffolds that resemble those found in mammalian postsynaptic densities. These features make the Drosophila neuromuscular system an excellent genetic model for the study of excitatory synapses in the mammalian central nervous system. The first section of the review presents an overview of NMJ development. The second section describes genes that regulate NMJ development, including: (1) genes that positively and negatively regulate growth of the NMJ, (2) genes required for maintenance of NMJ bouton structure, (3) genes that modulate neuronal activity and alter NMJ growth, (4) genes involved in transsynaptic signaling at the NMJ. The third section describes genes that regulate acute plasticity, focusing on translational regulatory mechanisms. As this review is intended for a developmental biology audience, it does not cover NMJ electrophysiology in detail, and does not review genes for which mutations produce only electrophysiological but no structural phenotypes.

Figure 1. Growth of the larva and its neuromuscular system

(A) A dissected late stage embryo ‘fillet’ stained with anti-horseradish peroxidase (anti-HRP), which stains neuronal membranes. The bright structure in the center is the ventral nerve cord (VNC), with the brain at the top and the ladder-like axon array extending downward from the brain. Extending outwards from each segment of the VNC are the motor and sensory axon tracts. (B) A third instar larval fillet expressing green fluorescent protein (GFP) in all neurons. The brain and VNC are now located at the anterior end, and the motor/sensory nerves run posteriorly from the VNC to reach each body segment. Note the stereotyped array of nerve endings in each segment. The inset in B shows the embryo from A at the same scale as the larva, illustrating the dramatic growth of the animal during larval life (the embryo is about the same size as a newly hatched first instar larva). (C) Innervation pattern of the intersegmental nerve (ISN) and its ISNb and ISNd branches in a third instar larva. This is a composite of many confocal images of GFP-labeled neurons in an abdominal hemisegment. Numbers indicate muscles innervated by the different branches of the ISN. (D) A schematic representing the three nerve roots: ISN, the segmental nerve (SN), and the transverse nerve (TN), and their respective innervation patterns. For clarity, not all muscles and nerve branches are shown. For the SN root, we show only the SNa nerve; SNc is not depicted. The ISNd branch of the ISN root, visible in C, has also been omitted from the diagram. The dashed lines are used to indicate the sections of the nerves that lie under (ventral to) the muscle(s). Scale bars in A and B are 100 μm and 250 μm, respectively.

Figure 2. NMJ expansion and synaptic growth

(A) A cartoon depicting patterned growth of the type 1b boutons on muscles 6 and 7 NMJ during larval development from the first instar (left panel) to the third instar (right panel). As the muscles increase in size, the NMJs add more branches and boutons. (B) During NMJ growth, new boutons are added by any of the these mechanisms: 1) asymmetric budding of a pre-existing bouton, similar to cell division in yeast, 2) symmetric division of a bouton, 3) de novo formation of a bouton from the axonal membrane (adapted from Zito et al.).

Figure 3. Types and structure of boutons at the Drosophila larval NMJ

(A) Type Ib, Is, II, and III boutons on muscle 12 are indicated by arrows. Type Ib and type 1s boutons differ in size, morphology, physiology, and the amount of subsynaptic reticulum (SSR) that surrounds them. The SSR is stained by Discs-Large (Dlg) antibody, which labels both Type Ib boutons and type Is boutons. Type Ib boutons are surrounded by more SSR membrane as compared to the Type Is boutons, resulting in the differential staining of the two types. Dlg is absent from type II and III boutons. Anti-HRP labels the presynaptic neuronal membrane and allows visualization of all bouton types. (B) A cartoon depicting the differences in the bouton types seen in (A). Other than the size and morphological differences, the boutons also differ in the neurotransmitter utilized. (C) A schematic showing a neuromuscular junction on arbitrary muscles labeled 1 and 2. One of the branches of the neuromuscular junction (NMJ) is indicated. Active zones localized inside each bouton are represented as pink dots. The subsynaptic reticulum (SSR), which consists of postsynaptic muscle membrane surrounding each bouton, is shown in blue. (D) Immunohistological staining showing type Ib boutons on muscle 4 stained with a Bruchpilot (Brp) antibody that labels the presynaptic active zones, which are visualized as punctate structures. Also stained with a Dlg antibody to show the SSR. Scale bars are 5 μm.

Figure 4. Examples of NMJ phenotypes

(A,B,E,F) Muscle 4 NMJs; (C,D,G,H,I) Muscle 6-7 NMJs. (A-F) are labeled with anti-HRP; (G-I) are double-labeled with anti-HRP and anti-Dlg. (A) Wild-type; the entire NMJ is shown. (B) A mutant NMJ with boutons that are greater in number but smaller in size than in wild-type. Entire muscle 4 NMJs are shown. (C) Wild-type. (D) A mutant NMJ with boutons that are fewer in number but larger in size than in wild-type. Partial muscle 6-7 NMJs are shown. (E) Wild-type. (F) An NMJ with a satellite bouton phenotype. Satellite boutons (arrows) resemble ‘budding’ structures, and are seen here on the terminal parent boutons and on the branching bouton. (G-I) An NMJ with ghost boutons. Ghost boutons (arrow in I) appear as boutons that have the presynaptic marker, anti-HRP (G; red in I), but lack postsynaptic markers, such as Dlg (H, green in I). Scale bar is 5 μm.

Figure 5. A schematic diagram of the NMJ depicting some of the translational regulatory mechanisms that function in the postsynaptic muscle or in the motor neuron, possibly in its presynaptic terminal

In the presynaptic motor neuron, dFMR1 (indicated by the dark oval) binds to futsch mRNA and inhibits its translation. In the postsynaptic muscle, mRNAs are shown that are being actively translated (indicated by the ribosome), regulated by either microRNA and the RISC complex (indicated by base-paired complementary strand and light oval) or by translational regulatory proteins (indicated by dark oval). miR-8 regulates translation of enabled and other target mRNAs. The translational repressors Pum and Nos regulate expression of GluRIIA and GluRIIB, respectively, and Pum binds directly to GluRIIA mRNA. dFMR1 represses expression of both GluRII subunits. RISC: RNAi-silencing complex, SSR: subsynaptic reticulum.

Figure 6. A diagram depicting the actions of translational regulatory proteins on GluRIIA, GluRIIB, and eIF-4E mRNAs

GluRIIA is repressed by Pum, miR-284a, and dFMR1. However, only Pum has been shown to directly bind to the 3′UTR of GluRIIA mRNA. Pum also binds to eIF-4E and nos mRNAs. GluRIIB expression is repressed by Nos, miR-284a and dFMR1. GluRIIA and GluRIIB compete for synaptic occupancy, so that GluRIIA represses GluRIIB expression and vice versa.