In a neuromuscular junction, synaptic vesicles in the motor neuron contain which neurotransmitter?

8.2 The neuromuscular junction is comprised of three cell types

The NMJ consists of the nerve ending from an alpha-motor neuron, a muscle fiber, and several nonmyelinating terminal/perisynaptic Schwann cells (T/PSCs) (Couteaux, 1947; Sanes and Lichtman, 1999; Darabid etal., 2014). Thus, neuromuscular synapses are tripartite (Fig.8.1), as are many synapses in the CNS that are composed of pre- and postsynaptic neurons and astrocytes (Araque etal., 1999). The presynaptic region is a highly branched structure located at the terminal of the alpha-motor neuron axon. Directly adjacent is the postsynapse, which is characterized as a shallow, gutter-like depression in the muscle fiber membrane containing nAChRs clustered at high density (Fig.8.2). Both the pre- and postsynapse are surrounded by nonmyelinating T/PSCs that monitor and modulate synaptic transmission. A synaptic potential, called an endplate potential, induces muscle contraction when nAChRs are activated by ACh released from the presynapse. To avoid the possibility of transmission failure, ACh released by the presynapse is 35 times greater than necessary to fully depolarize and cause contraction of a muscle fiber, a feature called the safety factor (Wood and Slater, 2001).

Figure8.1. Cellular components of adult neuromuscular junctions. An illustration of the major components and spatial arrangement of a typical neuromuscular synapse. The terminal of a motor neuron makes synaptic contact with a single skeletal muscle fiber. Terminal Schwann cells, a type of non-myelinating glia, cap the motor nerve terminal branches which are separated by the synaptic basal lamina, an extracellular matrix that envelopes the neuromuscular junction. Synaptic transmission begins when an action potential enters the terminal inducing the opening of voltage-gated calcium channels leading to the fusion of acetylcholine filled vesicles with the plasma membrane. Once in the synaptic cleft, acetylcholine binds to AChRs located along the crests of secondary synaptic folds allowing cation influx and localized depolarization of the postsynaptic muscle fiber membrane. This depolarization spreads to voltage-gated sodium channels located in the bottom of the each secondary synaptic folds further depolarizing the muscle fiber leading to a muscle fiber action potential. Synaptic transmission is terminated by the enzyme acetylcholinesterase, which hydrolyzes acetylcholine into recyclable choline and acetate.

Figure8.2. Cells that form the mouse neuromuscular junction. The expression of fluorescence proteins facilitates the visualization of alpha-motor neuron axons (green; A) and of SCs, including T/PSCs (green; B). nAChRs depicting the postsynaptic region (red in top and middle panels) of muscle fibers using fluorescently labeled alpha-bungarotoxin. Notice in the merged images colocalization between the presynaptic motor nerve terminal and T/PSCs (SCs, green cells in middle panel, specifically located at the NMJ) with the postsynaptic region. (C) Electron micrograph showing three elements of the mouse neuromuscular junction: (nt) motor nerve terminal, (mf) muscle fiber, (Sc) terminal Schwann cell process. The portion of nerve terminal shown contains several translucent acetylcholine filled synaptic vesicles (black arrowheads), the primary synaptic cleft (black arrow), and secondary folds (white arrows). Notice that the nerve terminal branch sits in a depression on the muscle fiber surface called the primary synaptic gutter which gives rise to several secondary gutters that appear as long invaginations that reticulate along the muscle fiber membrane. Located partway along the sides and crests of the secondary gutters are the AChRs (asterisks). (D) A scanning electron micrograph showing the 3D aspects of a mouse neuromuscular junction. In this example, the nerve terminal (nt) has been dislodged from the muscle fiber (mf) revealing the primary postsynaptic gutter (g) on which the motor nerve terminal normally resides. In addition, the body of a single terminal Schwann cell (Sc) is present on top of the nerve terminal.

The presynaptic region contains peptidergic vesicles, synaptic vesicles, mitochondria (Matteoli etal., 1988; Misgeld etal., 2007), and hundreds of sites along the plasma membrane that are specialized for neurotransmitter release called active zones (Chen etal., 2012). Visible as electron dense regions at the electron microscope level, active zones are characterized by accumulations of proteins, including voltage-gated calcium channels, vesicle docking proteins, and proteins involved in the synthesis and transport of ACh into synaptic vesicles (50 nm diameter). Among these, the active zone-specific protein Bassoon has been utilized to visualize the presynaptic region (tom Dieck etal., 1998). Work based on 3D reconstructions of Bassoon-labeled active zones have shown that the number of active zones increases proportionally as the presynapse expands, reaching a plateau in early adulthood.

The postsynaptic membrane is morphologically unique, exhibiting a large gutter-like depression and secondary junctional folds that are aligned with active zones located in the presynapse. At the crests of secondary junctional folds are high-density clusters of nAChRs, ca. 10,000 per μm2 (Salpeter and Loring, 1985), which become activated with the release of ACh into the synaptic cleft following an action potential. Much of what is known about the postsynaptic region including its initial formation, developmental changes, overall structure, and disruption has stemmed from the utilization of alpha-bungarotoxin (αBTX). A small 74 amino acid peptide isolated from the venom of the banded krait (Bungarus multicinctus; reviewed in Chang, 1999), αBTX, binds with high affinity and essentially irreversibly to nAChRs at NMJs in vertebrates and in the Torpedo electric organ (Lee etal., 1967). In addition to revealing cellular features of forming and maturing NMJs, αBTX has accelerated the identification of signaling molecules necessary for the clustering of postsynaptic components and the maintenance of postsynaptic specializations. These include low-density lipoprotein receptor-related protein 4 (Lrp4) (Weatherbee etal., 2006; Zhang etal., 2008), a receptor for agrin; tumorous imiginal disks 1 (Tid1) (Linnoila etal., 2008); muscle-specific kinase (MuSK), the signaling receptor for agrin (DeChiara etal., 1996; Glass etal., 1996a; Lin etal., 2001; Yang etal., 2001; Kim and Burden, 2008) and Wnts (Henríquez and Salinas, 2012; Koles etal., 2012); ErbB receptors, the signaling receptor for neuregulin (Trinidad etal., 2000; Falls, 2003; Trinidad and Cohen, 2004; Jaworski and Burden, 2006; Rimer, 2007; Schmidt etal., 2011); among many others (Simeone etal., 2010). It has also helped demonstrate that the neural cell adhesion molecule (NCAM), ankyrin, dystrophin, α-dystrobrevin-2, and β-spectrin localize at the bottom of junctional folds (Covault and Sanes, 1986; Flucher and Daniels, 1989; Bewick etal., 1996; Slater, 2003). The postsynaptic region is also highly enriched with voltage-sensitive sodium channels (Caldwell, 1986, 2000) that are spatially coupled with nAChRs. This proximity facilitates opening of voltage-gated sodium channels following the influx of cations through nAChRs bound to ACh. This close proximity between nAChRs and voltage-sensitive sodium channels contributes to the high efficiency of depolarization, initiation of an action potential, and ensuing contraction of a muscle fiber upon the release of ACh from the presynaptic region. The postsynaptic region is also unique because of theconcentration of several myonuclei, called subsynaptic myonuclei, underneath the postsynaptic membrane. Thesesubsynaptic myonuclei preferentially express a number of NMJ-associated genes (Kishi etal., 2005), making them transcriptionally distinct from all other myonuclei (Angus etal., 2005; Hippenmeyer etal., 2007; Haitao Wu etal., 2010) and accounting for the higher concentration of certain gene products at the NMJ. Several studies have found important roles for KASH domain-containing Syne-1/Nesprin-1 and SUN-domain containing proteins (SUN1 and SUN2) in the anchoring of subsynaptic myonuclei underneath the postsynaptic membrane (Apel etal., 2000; Ruegg, 2005; Zhang etal., 2007b; Lei etal., 2009). Thus, the postsynaptic region has a variety of structural and molecular features that readily distinguish it from other areas in a muscle fiber.

T/PSCs are integral components of the NMJ that play pivotal roles in the maturation and regeneration of NMJs as well as the facilitation of synaptic transmission. These nonmyelinating SCs ensheath the presynaptic and postsynaptic regions of the NMJ. T/PSCs are derived from SCs that migrate to the NMJ (citation needed). When SCs are individually labeled, it is easy to appreciate that each T/PSC occupies a precise territory that does not overlap with its neighbors (Brill etal., 2011). However, this is not the case at developing NMJs. During development immature T/PSCs and their processes extensively intermingle (Love and Thompson, 1998; Brill etal., 2011). Therefore, one interesting question raised by light and electron microscopic studies is how these cells become precisely aligned with and occupy well-defined territories on motor nerve terminal branches as the NMJ expands. Experiments that perturb T/PSC territories at adult NMJs using single-cell laser ablation, axon injury followed by regeneration, or blockade of soluble (N-ethylmaleimide sensitive factor) attachment protein receptor (SNARE)dependent secretion suggest that SC territories are highly dynamic. As NMJs mature during development and reform following injury, T/PSCs rapidly partition and become tiled over motor nerve terminal branches. These territories remain more or less stable for much of adult life. On the other hand, following the ablation of a single T/PSC at adult NMJs, the remaining T/PSCs phagocytose the remnants of the dead cell and quickly send processes to cover unoccupied motor nerve terminal branches while remaining tiled (Brill etal., 2011).

All three cells that make up the NMJ are physically separated by a synaptic basal lamina. The synaptic basal lamina extends into secondary junctional folds, separating the presynapse from the postsynapse by approximately 50nm, and surrounds T/PSCs. The synaptic basal lamina is contiguous with, but molecularly different from, the basal lamina that ensheathes the rest of the muscle fiber. The synaptic basal lamina contains AChE, a hydrolyzing enzyme that terminates synaptic transmission by degrading ACh into reusable acetate and choline (reviewed by Massoulié and Bon, 1982). In addition to collagen IV, laminin, and heparin sulfate proteoglycans (Patton, 2003; Rogers and Nishimune, 2017), the synaptic basal lamina is populated with signaling molecules necessary for NMJ formation and maintenance. Among these is z-agrin, originally purified from extracellular matrices collected from the electric organ of T.californica (Godfrey etal., 1984; Nitkin etal., 1987). Highlighting the importance of synaptic basal lamina proteins at NMJs, deletion of z-agrin prevents the maturation of nascent NMJs and results in the degeneration of mature NMJs (Gautam etal., 1996a; Burgess etal., 1999). The deletion of laminin α2, α4, α5, and β2 affects the morphology of the NMJ, including causing the misalignment of active zones (Patton, 2003). Thus, proteins of the synaptic basal lamina are regarded as essential structural and signaling elements at NMJs.

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