Spontaneously Recycling Synaptic Vesicles Constitute Readily Releasable Vesicles in Intact Neuromuscular Synapses.

Area of Science:

• Neuroscience
• Cell Biology
• Synaptic Transmission

Background:

• Spontaneous synaptic transmission is crucial for neuronal function, mediated by presynaptic mechanisms distinct from action potential (AP)-evoked release.
• The segregation of synaptic vesicle (SV) populations for spontaneous versus AP-evoked release remains debated, with conflicting data from cultured neurons.

Purpose of the Study:

• To investigate whether synaptic vesicle populations are segregated for spontaneous and AP-evoked transmission in intact neuromuscular synapses.
• To characterize the origin and properties of SVs involved in spontaneous release.

Main Methods:

• Utilized transgenic zebrafish larvae expressing pHluorin and HaloTag in SVs at neuromuscular synapses.
• Quantified recycled SV fractions using a novel measurement technique.
• Manipulated AP firing and temperature, and used tetanus toxin to differentiate SV populations.

Main Results:

• High-frequency AP firing mobilized approximately 85% of SVs.
• Spontaneous recycling involved a limited SV fraction (<8%) with a slow time constant (45 min at 25°C).
• Prolonged AP inhibition revealed distinct early-onset (temperature-insensitive, tetanus toxin-resistant) and late-onset (temperature-sensitive, tetanus toxin-inhibited) spontaneous fusion populations.
• The early-onset spontaneous SV population matched the characteristics of the readily releasable pool and was involved in hypertonic stimulation and initial AP-evoked release.

Conclusions:

• Genuine physiological spontaneous SV fusion arises from a highly limited fraction (<8%) of the readily releasable pool.
• Prolonged AP inhibition can activate a distinct, late-onset SV fusion population requiring separate molecular machinery.
• The readily releasable pool serves a dual role, contributing to both spontaneous and AP-evoked neurotransmission.