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

Abstract

Mammalian neuromuscular junctions are useful model synapses to study the relationship between synaptic structure and function, although these have rarely been studied together at the same synapses. To do this, we generated transgenic lines of mice in which the thy1.2 promoter drives expression of synaptopHluorin (spH) as a means of optically measuring synaptic vesicle distribution and release. SpH is colocalized with other synaptic vesicle proteins in presynaptic terminals and does not alter normal synaptic function. Nerve stimulation leads to readily detectable and reproducible fluorescence changes in motor axon terminals that vary with stimulus frequency and, when compared with electrophysiological recordings, are reliable indicators of neurotransmitter release. Measurements of fluorescence intensity changes reveal a surprising amount of heterogeneity in synaptic vesicle release throughout individual presynaptic motor axon terminals. Some discrete terminal regions consistently displayed a greater rate and extent of release than others, regardless of stimulation frequency. The amount of release at a particular site is highly correlated to the relative abundance of synaptic vesicles there, indicating that a relatively constant fraction of the total vesicular pool, ϳ30%, is released in response to activity. These studies reveal previously unknown relationships between synaptic structure and function at mammalian neuromuscular junctions and demonstrate the usefulness of spH expressing mice as a tool for studying neuromuscular synapses in adults, as well as during development and diseases that affect neuromuscular synaptic function.

Vesicular ATPase Inserted into the Plasma Membrane of Motor Terminals by Exocytosis Alkalinizes Cytosolic pH and Facilitates Endocytosis

“…For the stimulus trains applied here (200-1000 stimuli at 50 Hz) the half-time of decay of the post-stimulation alkalinization of motor terminal cytosol ranged from 30-150 s. This range is consistent with those reported for the half-time of endocytosis measured in mouse motor terminals (stimulated at 30-100 Hz) using other fluorescence-based techniques, including styryl dyes (~35 s, Zefirov et al, 2009) and synaptopHluorin (10-150 s, Tabares et al, 2007; ~30 s, Wyatt and Balice-Gordon, 2008). Rates of endocytosis measured using these other techniques became slower as the length of the stimulation train increased (Wu and Betz, 1996; Tabares et al, 2007).…”

Synaptic Vesicle Pools and Dynamics

“…Typical estimates of average R t P size range from 50% -85% of all vesicles (Harata et al 2001b;Fernandez-Alfonso and Ryan 2008; but see Ikeda and Bekkers 2009). A similar nonrecycling pool has been suggested at the calyx of Held (de Lange et al 2003), and NMJs of Drosophila (Poskanzer and Davis 2004) and mouse (Wyatt and Balice-Gordon 2008). Even in terminals said to be devoid of an R t P, 15% of vesicles are unlabeled in ultrastructural images of photoconverted boutons maximally loaded with FM dye (Richards et al 2003).…”

Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals

“…This type of membrane internalization was much less prevalent at 40 Hz. We were still able to identify spH spots during 40 Hz stimulation (albeit at a lower intensity; see also Wyatt and Balice-Gordon, 2008); therefore, tight spatial control over preferred exo- and endocytosis locations must not be required for all stimulation levels. The difference in spH spot intensity at 40 Hz (about half that at 100 Hz) may indicate the amount of synaptic vesicle membrane that can be efficiently recovered via non-FM 4-64 spot pathways.…”