Investigating the role of pH in presynaptic homeostatic potentiation at the vertebrate neuromuscular junction

Grinnell College, Grinnell IA

Investigators

Abstract

This project continues thirty-two years of research with undergraduate students at Grinnell College, who participate in all aspects of the research. My laboratory studies synaptic transmission at the vertebrate neuromuscular junction (NMJ). This project will focus on a phenomenon called presynaptic homeostatic potentiation (PHP), which refers to the increase in acetylcholine (ACh) release that follows the partial inhibition of postsynaptic acetylcholine receptors (AChRs). Although the phenomenon is well-known, the identity or nature of the feedback signal from the muscle to the nerve has eluded researchers for over 40 years. We recently uncovered evidence that protons in the synaptic cleft link the inhibition of AChRs to the increase in ACh release. Specifically, we found that (i) PHP requires functional acid sensing ion channels (ASICs), (ii) a decrease in extracellular pH increases quantal ACh release and precludes PHP, (iii) a strong extracellular pH buffer prevents PHP, (iv) inhibition of the plasma membrane Ca2+ ATPase (PMCA), which removes protons from the synaptic cleft, increases neurotransmitter release and precludes PHP, and (v) ASIC channels are present at the mouse NMJ. The projects described in this proposal will test the hypothesis that protons control PHP at the vertebrate NMJ. Our first goal is to measure the pH in the synaptic cleft of a mouse NMJ while the synapse is undergoing PHP. We plan to introduce fluorescent pH indicators into the cleft using AAV viruses. In preliminary experiments we successfully expressed pHusion-Ex and demonstrated its expected sensitivity to pH. We also plan to use a ‘kisser” probe that will allow us to examine pH in the immediate vicinity of the PMCA in the muscle membrane at the synapse. Our second goal is to understand the mechanism by which block of AChRs lowers the pH in the cleft. Our third goal is to characterize the effect of external pH in the synaptic cleft by applying acidic buffer via a micropipette and by photolytically uncaging NPE-caged-protons with a UV laser. Finally, we will explore PHP while allowing muscle action potentials to release internal calcium from the sarcoplasmic reticulum. PHP has never been investigated under these physiological conditions. Collectively, these studies will expand our knowledge of pH in the synaptic cleft at the mammalian NMJ, especially while undergoing PHP. Since protons are increasingly recognized as signaling molecules at a variety of synapses, what we learn at the NMJ about gain adjustments effected by postsynaptic detection of neurotransmitter efficacy could represent a simple and widespread mechanism for maintaining effective communication across synapses throughout the central and peripheral nervous systems. In addition to its scientific merit, this grant will directly support at least 12 undergraduate students in consequential research experiences. Most of these students, if not all, will matriculate in a Ph.D. program in a biomedical science or medical school.