Current Research Projects at the Lindgren Lab
Below are the titles and descriptions of our ongoing and planned projects. Explore the projects conducted by lab alumni on the alumni page, and our previous publications on the publications page.
Project 1: Explore the mechanism of cleft acidification during PHP
Details forthcoming!
Project 2: Explore morphology of the synapse at the NMJ using immunofluorescence
This project aims to expand our knowledge on the morphology and structure of the synapse at the vertebrate neuromuscular junction. Immunofluorescent dyes and antibodies are applied on stained and fixed mouse muscle tissue, and imaged with a confocal microscope to test its efficacy in labeling structural components of the synapse, and correlate their locations relative to each other.
The synapse with immunofluorescence labels are 3D-rendered to visualize its structural features.
The nerve terminal is marked in blue fluorescence, the ACh receptors in red, and the Schwann cells in green.
We aim to test a combination of fluorophores and antibodies to establish protocols for clean imaging outcomes.
This is an important project toward understanding the spatial relationship between the structures that play role in PHP, which will also be important for when measuring pH in the cleft (see Project 4).
Project 3: Computationally simulate pH response to neurotransmitter release
pH is an important indicator that could support our proposal that there is acidification (pH drop) in the synaptic cleft during PHP, as more vesicles with acidic contents are released into the cleft.
Many factors, including the width of the cleft, entering and exiting ion species, function of the PMCA (plasma membrane calcium ATPase; removes H+ from the cleft) can influence cleft pH during neurotransmitter release.
In their 2021 paper, Feghhi and colleagues built a MATLAB computational model to simulate the concentration of ion species as they react and diffuse through the cleft of a Drosophila neuromuscular junction. One of the ion species in the model is H+, whose concentration was used to plot the pH as reactions and diffusion progress over time.
With permission and support from Dr. Feghhi and collaborators, we aim to modify the model to simulate the pH in the mouse NMJ cleft during the release cycle of the ACh neurotransmitter. The simulation model could help provide more insights into the effect of ACh receptor (nAChR) block and external conditions on the cleft pH.
Project 4: Measure pH in the synaptic cleft during PHP
We have successfully tested for expression of fluorescent pH indicator in the postsynaptic NMJ membrane of AAV-injected mice. We will use the same fluorophore to measure the pH in the NMJ stimulated at different frequencies, and observe the pH change upon application of dTC (d-tubocurarine; blocks ACh receptors). By inserting different types of pH probes, we hope to measure and examine pH surrounding various synaptic proteins, including the PMCA.
Project 5: Observe the response to transient acidification in the synaptic cleft
We previously observed that the drop in extracellular pH by ~0.2 units via sustained changes to pH result in ACh release. We aim to extend this finding by transiently altering pH by using two approaches: First, applying acidific buffer from a pipette near NMJ through pressure pulse. Second, photolytically uncaging NPE-caged proton in the synaptic cleft.
We will observe the effect of transient acidification on neurotransmitter release (measured in QC, quantal content).
Project 6: Explore PHP in the presence of postsynaptic action potentials
In our typical electrophysiology experiments, we apply blockers to inhibit the voltage-gated Na2+ channels, which causes action potentials (AP) and preclude the EPPs that we are interested in measuring. However, preculduing AP prevents us from addressing the Ca2+ release triggered by muscle AP, and how it may impact neurotransmitter release.
In this project, we will perform electrophysiology without inhibiting voltage-gated Na2+ channels while inhibiting muscle contraction with a myosin inhibitor – meaning AP will be present in the muscle, while contraction is avoided.
Using same methods from Project 4, we will measure pH without and with the voltage-gated Na2+ channels blocked. We will then monitor muscle APs to assess presence of PHP while ASICs (a structure which we proposed is activated by extracellular acidification to induce PHP, in Zhu et al., 2021) is blocked or when the buffer pH is set to constant.