Assessing Neuron-Astrocyte Spatial Interactions Using the Neuron-Astrocyte Proximity Assay

Abstract

Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno-associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Förster resonance energy transfer imaging in cultured cells Basic Protocol 2: Förster resonance energy transfer imaging with the neuron-astrocyte proximity assay in acute brain slices.

Keywords: astrocyte; imaging; neuron; optical; synapse.

Figure 1.

Outline of the experimental paradigm for NAPA experiments in situ. On the far left are listed the time considerations for the corresponding steps. In the middle we report the stepwise protocol from project inception culminating in data interpretation. On the right are listed the related figures, and relevant sections of the paper which correspond to steps in the protocol. We estimate the full protocol will take approximately 6 weeks to complete.

Figure 2.

Neuron-astrocyte proximity assay (NAPA) experimental setup in vitro and in situ. A. Experimental workflow to measure FRET in cultured HEK293T cells. B. Experimental workflow to measure FRET ex vivo in acute brain slices following viral transduction in vivo.

Figure 3.

Bleed through and cross-talk (SBT) calculations to measure sensitized emission FRET (SE-FRET). A. Diagram of the two channels’ imaging conditions for the GFP alone, i.e. donor bleed through SBT model. B. Diagram of the two channels’ imaging conditions for the mCherry alone, i.e. acceptor cross-talk SBT model. C. Diagram of the three channels’ imaging conditions for the sensitized emission FRET experiment between GFP and mCherry. D. Förster equation used to calculate FRET efficiency, E.

Figure 4.

Donor bleed through and acceptor cross-talk calculations in HEK293T cells. A. The GFP channel images (ex. 488 nm / em. 505 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm/ em. 615 nm). B. These stacks can then be used to create FOV donor montages of the GFP and FRET channels, these montages can then be stacked in a single file, the donor montage stack. C. From the montage stack we can graph the raw donor bleed through model and by thresholding the PixFRET viewer, we can determine the final donor model. D. For the acceptor controls, the mCherry channel images (ex. 543 nm/ em. 615 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm/ em. 615 nm). These stacks can then be used to create multiple FOV acceptor montages of the mCherry and FRET channels. E. The mCherry and FRET channel montages can then be stacked in a single file, the acceptor montage stack. F. From the montage stack we can graph the raw acceptor cross-talk model and by thresholding the PixFRET viewing window, we can determine the final acceptor model.

Figure 5.

Calculation of sensitized emission FRET efficiency in HEK293T cells. A. Example of the image order that should be used in creating the three channel FRET stacks before quantification in PixFRET. B. Representative image of FRET efficiency from the GFP-mCherry fusion experiment. C. Masked image of FRET from panel B, showing the calculated FRET efficiency from individual cells.

Figure 6.

FRET efficiency based on tandem GFP-mCherry in HEK293T cells. A. Representative images of the HEK293T cells expressing, GFP alone (top panels), mCherry alone (middle panels) and tandem GFP-mCherry (bottom panels). B. Quantification of FRET efficiency from 20 cells in these three conditions (Mean ± SEM).

Figure 7.

Calculation of donor bleed through and acceptor cross talk in brain slices. A. The GFP channel images (ex. 488 nm/ em. 505 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm / em. 615 nm). B. These stacks can then be used to create FOV donor montages of the GFP and FRET channels. The channels can then be stacked in a single file, the donor montage stack. C. From the montage stack we can graph the raw donor bleed through model and by thresholding the PixFRET viewer, we can determine the final donor model. D. For the acceptor controls, the mCherry channel images (ex. 543 nm/ em. 615 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm/ em. 615 nm). These stacks can then be used to create multiple FOV acceptor montages of the mCherry and FRET channels. E. The mCherry and FRET channel montages can then be stacked in a single file, the acceptor montage stack. F. From the montage stack we can graph the raw acceptor cross-talk model and by thresholding the PixFRET viewing window, we can determine the final acceptor model.

Figure 8.

Calculation of sensitized emission FRET efficiency in brain slices. A. Diagram of astrocyte-synapse proximity assay on corticostriatal projections. B. Example of the image order that should be used in creating the three channel FRET stacks before quantification in PixFRET. C. Representative image of all imaging channels and FRET efficiency from the NAPA experiment. The white outline represents estimated edge of astrocyte territory based on NAPA-a expression. D. Quantification of FRET efficiency from 11 astrocytes (Mean ± SEM).