Homeostatic Plasticity of the Mammalian Neuromuscular Junction

Abstract

The mammalian neuromuscular junction (NMJ) is an ideal preparation to study synaptic plasticity. Its simplicity- one input, one postsynaptic target- allows experimental manipulations and mechanistic analyses that are impossible at more complex synapses. Homeostatic synaptic plasticity attempts to maintain normal function in the face of perturbations in activity. At the NMJ, 3 aspects of activity are sensed to trigger 3 distinct mechanisms that contribute to homeostatic plasticity: Block of presynaptic action potentials triggers increased quantal size secondary to increased release of acetylcholine from vesicles. Simultaneous block of pre- and postsynaptic action potentials triggers an increase in the probability of vesicle release. Block of acetylcholine binding to acetylcholine receptors during spontaneous fusion of single vesicles triggers an increase in the number of releasable vesicles as well as increased motoneuron excitability. Understanding how the NMJ responds to perturbations of synaptic activity informs our understanding of its response to diverse neuromuscular diseases.

Keywords: Acetylcholine receptor; Endplate; Homeostatic; Motoneuron; Muscle; Neuromuscular junction; Plasticity; Trophic.

Fig. 1

The mammalian NMJ. (a) Shown is neurofilament staining of an axon entering the NMJ from the top left (in green) and SV2 staining of synaptic vesicles at the NMJ (also in green) as well as α-bungarotoxin (BTX) staining of acetylcholine receptors (AChRs, in red). When the green and red images are superimposed the alignment of presynaptic vesicles with AChRs is apparent. (b) Shown are voltage clamp recordings from a single NMJ of both a spontaneous miniature endplate current (MEPC) and the endplate current (EPC) evoked by nerve stimulation. The miniature end-plate current is thought to result from release of acetylcholine from a single vesicle. The endplate current is triggered by a nerve action potential and is roughly 50 times larger than the spontaneously occurring miniature endplate current, suggesting release of acetylcholine from 50 vesicles. The stimulus artifact preceding the endplate current is caused by the current pulse used to trigger the nerve action potential

Fig. 2

Determination p and n using analysis of variance of quantal content at the NMJ. To determine p and n at the NMJ one can plot the variance (standard deviation squared) of quantal content versus quantal content. Each parabola represents the plot for a given n as p is increased from 0 to 1.0, assuming uniform p for all synaptic sites. Included in the plot shown are the parabolas for n = 20 up to n = 200. All the parabolas start with Var(m) = 0 when m = 0 and increase to a maximum Var(m) when p = 0.5. When p reaches 1.0 for each n, Var(m) again equals 0 and n = m as every vesicle that can be released is released. Intersecting the parabolas are straight lines representing the theoretical plot for a given p as n is increased. Each point on the plot has a unique p and n allowing for simultaneous measurement of both parameters. One limitation of this analysis is that when p is low, the parabolas for various values of n run together, making determination of p and n difficult. Superimposed on the theoretical lines for values of p and n are the mean value and standard error of Var(m) plotted versus m for control NMJs, NMJs in which pre- and postsynaptic action potentials were blocked by placement of a tetrodotoxin (TTX) containing cuff on the sciatic nerve for 1 week and NMJs in which AChRs were partially blocked with α-bungarotoxin (BTX) for 4–5 days. All recordings were performed in solution containing 1 mM Ca²⁺. Following block of action potentials there was an increase in m that was accompanied by reduction in Var(m): indicating an increase in p while n remained constant. Following block of AChRs there was an increase in m that was accompanied by a dramatic increase in Var(m): indicating p may have decreased slightly while n increased dramatically. (Wang et al. 2010b)

Fig. 3

Homeostatic synaptic plasticity at the mouse NMJ triggered by prolonged block of pre- and postsynaptic action potentials. In the top row, synaptic function is shown in solution containing normal external Ca²⁺. Following block of action potentials there are two changes in synaptic function. The first is an increase in the release of acetylcholine during fusion of individual synaptic vesicles (illustrated as increased acetylcholine in the synaptic cleft). The second change is an increase in probability of release of vesicles that appears to be due to increased entry of Ca²⁺ into the presynaptic terminal during an action potential. When extracellular Ca²⁺ is normal, the increase in Ca²⁺ entry has no significant effect on the number of synaptic vesicles released (quantal content) as each releasable vesicle is already released with each action potential. In the bottom row is shown the situation when extracellular Ca²⁺ is lowered. In this case Ca²⁺ entry during the action potential limits quantal content. When Ca²⁺ entry is increased following block of action potentials, there is an increase in quantal content. In addition, there is increased acetylcholine release from each vesicle. This combines with the increase in quantal content to cause a dramatic increase in synaptic strength. This is illustrated as an increase from 2 to 8 acetylcholine molecules in the synaptic cleft. AChR = acetylcholine receptor. (This figure is reproduced from Wang and Rich 2018)

Fig. 4

The increase in n following block of AChRs. In the top row is shown synaptic function in solution containing normal external Ca²⁺. Two types of synaptic vesicles are present. The blue vesicles represent normal vesicles that participate in synaptic transmission at baseline. The red vesicles represent a special pool of synaptic vesicles that normally do not play an important role in synaptic transmission. A few vesicles in this pool are released late in the course of the synaptic current (indicated by the long dotted red arrow). Following partial block of AChRs, the special pool of synaptic vesicles is released more rapidly (synchronous with the normal pool). In addition, the special pool of vesicles rapidly recycles (indicated by the curved red arrow) to increase quantal content. In solution containing low extracellular Ca²⁺, Ca²⁺ entry during action potentials is insufficient for the special pool of vesicles to participate in release. (This figure is reproduced from Wang and Rich 2018)

Fig. 5

Summary of homeostatic synaptic plasticity and the mammalian NMJ. Block of action potentials in the presynaptic axon, which also causes block of muscle action potentials, triggers to an increase in quantal amplitude as well as an increase in p. Block of postsynaptic AChRs on muscle causes a retrograde signal(s) that increases both n at the nerve terminal and an increase in motoneuron excitability that is present in the cell body in the spinal cord