Unmet Need:
AMPA-type glutamate receptors (AMPARs) are crucial molecules to study to understand the function the nervous system. These receptors mediate the majority of fast excitatory activities in the mammalian brain, and their regulation is regarded as a key mechanism underlying long-lasting changes that give rise to learning and memory. Elucidating how synaptic molecules such as AMPA receptors mediate neuronal communication and tracking them during behavior is crucial to understand cognition and disease, but current technological barriers exist that limit the ability to visualize such proteins in mouse models. Modern proteomic and transcriptomic methods provide biologists with myriad candidate proteins, but in many cases, there are no tools (e.g. monoclonal or polyclonal antibodies) available to effectively study these targets. One alternative approach that addresses this challenge is to fluorescently tag proteins to visualize them in living tissue. There is a strong need for a new genetic labeling strategy that allows direct visualization of endogenous AMPAR expression in a manner that does not impair synaptic function, plasticity, or behavior and enables further understanding of cognition and disease.
Technology Overview:
Johns Hopkins researchers have developed a SEP-GluA1 knockin mouse model that enables the robust visualization of excitatory synapses throughout the brain. This knockin mouse line contains an AMPAR GluA1 subunit tagged with super ecliptic pHluorin (SEP), a pH-sensitive variant of GFP that fluoresces at neutral pH and is quenched at acidic pH. When coupled to the extracellular N-terminal domain of the AMPAR, this SEP tag reports the concentration of functional receptors at the cell surface, as the fluorescence of receptors localized in acidic, internal compartments such as endosomes and Golgi is quenched. This genetic labeling strategy also avoids confounders arising from manipulation of the AMPAR C-terminus, a region important for proper function and trafficking to the postsynaptic membrane. These SEP-GluA1 knockin mouse models used in conjunction with in vivo 2 photon microscopy, enables longitudinal tracking of synaptic plasticity underlying behavior at brain-wide scale with single-synapse resolution.
Stage of Development:
Mouse model is developed.
Publication:
Graves, Roth, et al. eLife, 10:e66809, 2021
