Synaptic Vesicle Distribution and Release at Rat Diaphragm Neuromuscular Junctions

Abstract

Rowley KL, Mantilla CB, Ermilov LG, Sieck GC. Synaptic vesicle distribution and release at rat diaphragm neuromuscular junctions. J Neurophysiol 98: 478 -487, 2007. First published May 9, 2007 doi:10.1152/jn.00251.2006. Synaptic vesicle release at the neuromuscular junction (NMJ) is highly reliable and is vital to the success of synaptic transmission. We examined synaptic vesicle number, distribution, and release at individual type-identified rat diaphragm NMJ. Three-dimensional reconstructions of electron microscopy images were used to obtain novel measurements of active zone distribution and the number of docked synaptic vesicles. Diaphragm musclephrenic nerve preparations were used to perform electrophysiological measurements of the decline in quantal content (QC) during repetitive phrenic nerve stimulation. The number of synaptic vesicles available for release vastly exceeds those released with a single stimulus, thus reflecting a relatively low probability of release for individual docked vesicles and at each active zone. There are two components that describe the decline in QC resulting from repetitive stimulation: a rapid phase (Ͻ0.5 s) and a delayed phase (Ͻ2.5 s). Differences in the initial rapid decline in QC were evident across type-identified presynaptic terminals (fiber type classification based on myosin heavy chain composition). At terminals innervating type IIx and/or IIb fibers, the initial decline in QC during repetitive stimulation matched the predicted depletion of docked synaptic vesicles. In contrast, at terminals innervating type I or IIa fibers, a faster than predicted decline in QC with repetitive stimulation suggests that a decrease in the probability of release at these terminals plays a role in addition to depletion of docked vesicles. Differences in QC decline likely reflect fiber-type specific differences in activation history and correspond with welldescribed differences in neuromuscular transmission across muscle fiber types.

Heterogeneity in Synaptic Vesicle Release at Neuromuscular Synapses of Mice Expressing SynaptopHluorin

“…Previous studies using electron microscopy to examine the distribution of synaptic vesicles in mammalian neuromuscular terminals have demonstrated that although vesicles are primarily clustered at active zones, they are also present throughout the terminal (Rowley et al, 2007). Consistent with this, we found that the addition of NH 4 Cl to reveal the total intracellular vesicle pool led to fluorescence increases over the entire presynaptic terminal.…”

Neuromuscular transmission modulation by adenosine upon aging

“…Whether such differences might influence the presently reported effects of adenosine upon aging, needs to be explored at the light of the results obtained by Rowley et al (2007) and Ermilov et al (2007). However, in a previous work (see Ginsborg and Hirst, 1972) the initial quantal content of EPPs (either being low, in experiments where muscle contractions were paralyzed by high magnesium, or high in the experiments where muscle contractions were paralyzed by tubocurarine) did not influence the inhibitory effects of adenosine at the rat diaphragm neuromuscular junction.…”

“…Neuromuscular junctions differ in quantal content, safety factor, and adaptation to repetitive activation (see Ermilov et al, 2007;Rowley et al, 2007). Whether such differences might influence the presently reported effects of adenosine upon aging, needs to be explored at the light of the results obtained by Rowley et al (2007) and Ermilov et al (2007).…”

Key aspects of phrenic motoneuron and diaphragm muscle development during the perinatal period

“…Neuromuscular junctions at fibers expressing MHC 2X and/or MHC 2B are larger and more complex than those at fibers expressing MHC Slow or MHC 2A isoforms (74). Synaptic vesicle density at active zones is lower in axon terminals at fibers expressing MHC 2X and/or MHX 2B isoforms than at those expressing MHC Slow or MHC 2A isoforms (66,79). In addition, mitochondria, rough endoplasmic reticulum, free polysomes, and nuclei are frequently interposed between postsynaptic specializations and myofibrils at fibers expressing MHC 2X and/or MHX 2B isoforms, not at fibers expressing MHC Slow or MHC 2A isoforms.…”

“…The safety factor for neuromuscular transmission (defined as the ratio of the excitatory postsynaptic potential amplitude to activation threshold of a muscle fiber) varies across fiber types, being larger for type IIb DIAm fibers than for type I or IIa fibers (25). With repeated activation, the amplitude of excitatory postsynaptic potentials declines to a greater extent at terminals of type I or IIa fibers than at type IIb fibers (79). Taken together, these findings suggest that the probability of synaptic vesicle release is reduced at type I or IIa fibers, which may serve to limit the depletion of synaptic vesicles from these terminals during repeated activation.…”