Imaging Synaptic Protein Dynamics Using Photoactivatable Green Fluorescent Protein

Abstract

Considerable evidence has accumulated that structural changes in dendritic spines and their synapses are associated with adaptive functional changes in cortical circuits, such as during circuit refinement in young animals and in learning and memory in adults. Understanding the mechanisms of circuit plasticity requires detailed investigation of the structural dynamics of dendritic spines and how they are regulated by neural activity and sensory experience. Studying the dynamic localization of synaptic proteins in dendritic spines and how their stabilization and exchange rates influence spine structural plasticity is also important. This protocol describes imaging approaches to study synaptic protein dynamics in dendritic spines of the rodent cerebral cortex. It gives a strategy for generating photoactivatable green fluorescent protein (PA-GFP)-tagged synaptic proteins and in vitro and in vivo transfection methods for coexpression of these proteins with a spectrally separable cell-filling marker (DsRed-Express). Methods for tracking synaptic protein localization using photoactivation and time-lapse imaging of PA-GFP in spiny pyramidal neuron dendrites are given. A discussion of imaging hardware and software preferences is also included. The methods described here can be used to study the dynamic processes underlying spine synapse development during the formation and plasticity of neural circuits in the mammalian brain. Alternatively, use pregnant female mice if in vivo imaging will be performed.Reagents for testing PA-GFP-tagged synaptic proteins for their ability to retain native protein function (see Step 2)

Molecular mechanisms of dendrite morphogenesis

“…These include tools that allow for genetic manipulation of murine models, control of neuron specific activation or inhibition in vivo , for example using optogenetic tools (Tye and Deisseroth, 2012), advanced imaging molecules (Weimer et al, 2012) and techniques and high efficiency gene sequencing techniques. These tools should allow us to develop new and critical insights into how the dendritic arbor develops and is maintained through life, how dendritic arborization contributes to neural circuit formation and function and how aberrations in dendritic arbors contribute to the pathology of various neurological disorders.…”

Smart fluorescent proteins: Innovation for barrier‐free superresolution imaging in living cells

“…PA-GFP can be photoactivated (emission max 520 nm) from its dark equilibrium state by ultraviolet (UV) irradiation. (Patterson & Lippincott-Schwartz 2002Stark & Kulesa 2005;Gray et al 2006;Murray & Saint 2007;Weimer et al 2012).…”