High-frequency stimulus and high-K+ solution cause kiss-and-run fusion at the crayfish neuromuscular junction

Abstract

The conventional method of exocytosis in synaptic cells, full collapse fusion, occurs when the vesicles containing neurotransmitters collapse entirely into the presynaptic membrane. Kiss-and-run fusion (KR), an alternative to full collapse fusion, suggests that instead of fully collapsing into the membrane, vesicles briefly open a pore into the cleft and retain their spherical shape so they can be quickly reused by the cell. KR has been discovered at some synapses, but the existence of KR at the neuromuscular junction (NMJ) is not definitive, and the effects of variables such as stimulus frequency on KR fusion are subject to further research. We contribute to these areas of interest by determining the rate of KR fusion at the NMJ and exploring the effects of two kinds of nerve stimulation (100Hz, and high-potassium solution). We studied KR by loading the fluorescent dye FM1-43, which allows us to see and measure fluorescence intensity, along with the quencher bromophenol blue (BPB), which partially suppresses fluorescence, into NMJ vesicles and recorded the fluorescence by taking photographs and comparing fluorescence levels using the program MetaMorph. Our experiment was based on the premise that the rate of KR would manifest in an increase of fluorescence (signaling that the quencher BPB, which has more hydrophilic properties than FM1-43, had left the vesicle through the vesicle pore). We hypothesized that KR would occur in baseline conditions and would occur at a lower rate during depolarization caused by electrical stimulation and high-potassium. We found that KR is not present in the NMJ under normal conditions but can be caused by both electrical stimulation and high-potassium solutions. We speculate that residual calcium from these depolarizing influences is causally responsible for the increased rate of KR.