An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales

Abstract

Astrocytes are complex bushy cells that serve important functions through close contacts between their processes and synapses. However, the spatial interactions and dynamics of astrocyte processes relative to synapses have proven problematic to study in adult living brain tissue. Here, we report a genetically targeted neuron-astrocyte proximity assay (NAPA) to measure astrocyte-synapse spatial interactions within intact brain preparations and at synaptic distance scales. The method exploits resonance energy transfer between extracellularly displayed fluorescent proteins targeted to synapses and astrocyte processes. We validated the method in the striatal microcircuitry following in vivo expression. We determined the proximity of striatal astrocyte processes to distinct neuronal input pathways, to D1 and D2 medium spiny neuron synapses, and we evaluated how astrocyte-to-excitatory synapse proximity changed following cortical afferent stimulation, during ischemia and in a model of Huntington's disease. NAPA provides a simple approach to measure astrocyte-synapse spatial interactions in a variety of experimental scenarios. VIDEO ABSTRACT.

Keywords: FRET; astrocyte; connectome; striatum; synapses; wiring.

Figure 1. Neuron astrocyte proximity assay to assess astrocyte-synapse interactions

A. Diagram of the NAPA assay. Presynaptic terminals are labeled by extracellular, synapse-targeted membrane tethered mCherry. Fine astrocyte processes opposing these terminals are labeled by extracellular membrane-tethered eGFP. Astrocyte-synapse interactions can be seen at three distance scales. B. Graph of intermolecular FRET distances for GFP and mCherry or the intercellular FRET distance for the NAPA-a and NAPA-n probes that contain extended linkers. The arrows above indicate the distance scales over which we can resolve astrocyte synapse interactions with NAPA, corresponding to the inset cartoons in panel A. C. NAPA-n was targeted to synapses by the Neurexin-1 transmembrane and intracellular c-terminal domains. NAPA-a was targeted to astrocyte processes by the PDGFR transmembrane domain. The lower panels show NAPA-a and NAPA-n cDNA constructs. D. Images of colocalization and FRET in adjoining HEK-293 cells that express either the NAPA-a or NAPA-n. Colocalization and FRET occurred at the junctions of cells. E. Quantification of colocalization and FRET for NAPA-n and NAPA-a, NAPA-n and Lck-GFP or NAPA-a and cytosolic GFP as assessed by membrane colocalization as a percent of the overlap area (left), FRET area as a percent of colocalization area (middle) and FRET efficiency within the FRET ROIs (right).

Figure 2. AAVs selectively target NAPA components to entire striatal astrocytes and presynaptic terminals

A. Schematic illustrating the approach. B. AAV vectors generated for NAPA-a and NAPA-n, each employing cell-specific promoters. C. Representative image showing the distribution of NAPA-a following AAV2/5 microinjections into the dorsolateral striatum. D. Representative images of the cellular expression and distribution of NAPA-a in green and the overlap with S100β in red. E. Quantification of the percentage of S100β positive cells that colocalized with NAPA-a. F. Representative image of NAPA-a and NeuN. G. Quantification of the percentage of NeuN positive cells that colocalized with NAPA-a. H. Size of the astrocyte territory as assessed by IHC, labeled with NAPA-a, Lck-GFP, or S100β. I. Representative image of the brain distribution of NAPA-n when microinjected into the rostral intralamminar nucleus of the thalamus. J. Representative image of the cellular distribution and overlap of NAPA-a and NeuN. K. Quantification of the percentage of NeuN positive cells that colocalized with NAPA-n. L. Representative images and quantification of the thalamostriatal projection labeled by NAPA-n and imaged within the striatum.

Figure 3. Astrocyte-synapse proximity was pathway specific

A. Representative images of MSN collateral projections expressing NAPA-n and their contacts with astrocyte processes expressing NAPA-a assessed by colocalization and FRET. B–D. As in A, but for corticostriatal, thalamostriatal and nigrostriatal inputs. In A–D, the areas of colocalization are shown in yellow, but the areas of FRET are shown on a color scale that reflects FRET efficiency, as shown in the figure. E. Diagram of striatal inputs and their plausible contacts with astrocyte processes. F. Quantification of striatal input colocalization and FRET with dorsolateral striatal astrocyte processes expressed as a percentage of the astrocyte territory area. G. Quantification of the astrocyte area displaying the closest detected astrocyte-synapse interactions as assessed by the area of FRET expressed as a percentage of the total colocalization area per cell. H. Quantification of the relative proximity between astrocyte processes and various striatal inputs as assessed by their FRET efficiency. I. Example FRET image of an astrocyte with superimposed circles of increasing diameter. Such circles were used to count the number of FRET ROIs as a function of distance in J. K. Summary plot for the distance of FRET ROIs to the center of the soma.

Figure 4. SBF-SEM of striatal astrocyte processes with presynaptic terminals and MSN dendritic spines

A. Representative image of three manually traced MSN dendrites within a ~2180 µm³ block of the striatum that was scanned by SBF-SEM. B. Representative image and three-dimensional reconstruction of a striatal astrocyte process that was closely opposed to the pre- and postsynaptic membranes of an excitatory synapse. C. Representative image and threedimensional reconstruction of a striatal astrocyte process that was more distal to the pre- and postsynaptic membranes of an excitatory synapse. D. Quantification of the distance between the nearest astrocyte process and the pre- and post-synaptic membranes of 116 synapses from 3 mice. In the box and whisker plots shown in D, the mean is shown with a circle, the s.e.m. with the box, the median with a horizontal line and the 10–90% range with the whiskers. E. Representation of the distances between the nearest astrocyte process and the pre- and post-synaptic membranes, binned to <10 nm, 10–100 nm and distances that are greater than 100 nm. The data show that striatal astrocytes interact with excitatory synapses at separable distance scales, with around half of the distances being the most proximate at <10 nm. F–J. As in A–E, but for TH-positive terminals in relation to astrocyte processes (see Table 1).

Figure 5. Striatal astrocytes contact D1 and D2 MSNs equivalently

A. Representative image of a striatal astrocyte that expresses Lck-GFP under the control of the GfaABC₁D promoter in BAC D1-tdTomato mice. B. Representative image of a striatal astrocyte that expresses tdTomato under the control of the GfaABC₁D promoter in BAC D2-GFP mice. C. Quantification of the number of neurons within the territory of individual striatal astrocytes as assessed by NeuN staining, D2-GFP or D1-tdTomato labeling, from images such as in A and B. D. Quantification of the density of neurons in the dorsolateral striatum as assessed by NeuN staining, D2-GFP or D1-tdTomato labeling. E. Image of the brain distribution of GFP when AAV2/1 FLEX CAG GFP was microinjected in the striatum of BAC D1-Cre mice. F. Image of the brain distribution of GFP when AAV2/1 FLEX CAG GFP was microinjected in the striatum of BAC A2a-Cre mice. In E and F, the expected projection patterns of D1 and D2 MSNs are seen within the basal ganglia circuit. G. Images of the dorsolateral striatum when AAV2/1 FLEX CAG GFP was microinjected in the striatum of either BAC D1-Cre or BAC A2a-Cre mice, as in E and F. H. Representative image of a μ-crystallin positive dorsolateral striatal astrocyte in BAC D1-Cre mouse that had been microinjected with AAV2/1 FLEX NAPA-n. I. Image of a μ-crystallin positive dorsolateral striatal astrocyte in BAC A2a-Cre mouse that had been microinjected with AAV2/1 FLEX NAPA-n. J. Quantification of the number of neurons per μ-crystallin positive striatal astrocyte in BAC D1-Cre and BAC A2a-Cre mice, as in H and I. K. Schematic depicting the somata of both D1 or D2 MSNs within the territory of an individual striatal astrocyte, which our data indicate interact equivalently with both cell types. L. Image showing biocytin labeling of coupled astrocytes in the striatum, when a single striatal astrocyte was dialyzed with biocytin for 30 min. M. Quantification of the percentage of S100β positive cells that were also biocytin positive in the striatum following intracellular dialysis of a single astrocyte: these are shown as the coupled cells. N. Representative current waveforms for a striatal astrocyte in response to stepwise changes membrane potential. O. Average current-voltage relationship (I/V curve) of dorsolateral striatal astrocytes.

Figure 6. NAPA analyses of the contacts between D1 and D2 MSN collaterals and striatal astrocyte processes

A. Representative images of NAPA-n expression within D1 MSN collaterals and their contacts with astrocyte processes expressing NAPA-a. B. Quantification of the area of the striatal astrocyte territory occupied by colocalized (yellow bar) and FRET ROIs (red bar) for D1 MSNs. C. Representative images of NAPA-n expression within D2 MSN collaterals and their contacts with astrocyte processes expressing NAPA-a. D. Quantification of the area of the striatal astrocyte territory occupied by colocalized (yellow bar) and FRET ROIs (red bar) for D2 MSNs. E. The number of colocalized ROIs for D1 and D2 collaterals in the territory of striatal astrocytes. F. The number of FRET ROIs for D1 and D2 collaterals in the territory of striatal astrocytes. G. The FRET efficiency of ROIs for either D1 projections or D2 collaterals within the territory of striatal astrocytes. H. Quantification of the areas displaying FRET as a percent of total area of colocalization for either D1 or D2 MSNs.

Figure 7. Electrical field stimulation (EFS) of cortical axons and effects on astrocyte spatial interactions with corticostriatal synapses

A. Photograph of a parasagittal brain slice to show the location of the imaging site in relation to EFS. B. Input-output curve for evoked EPSCs onto MSNs. C. Representative trace for evoked EPSCs recorded from MSNs during a train of EFS. D. Representative traces for iGluSnFR signals measured from striatal astrocytes during cortical stimulation. E. Average data for experiments such as those in panel D. F. Average data for experiments such as those in in panel D, but with calcium imaging using Lck-GCaMP6f. G. Representative images showing astrocyte territories and FRET ROIs for the indicated conditions. H–J. Averaged data for experiments such those shown in panel G, reporting FRET efficiencies, FRET ROI areas and the areas of colocalization. K–M. Individual and average data for FRET efficiency over time before and after EFS under control conditions and in the presence of TTX.

Figure 8. Altered proximity during OGD and in R6/2 Huntington’s disease model mice

A. Representative images for astrocyte territory areas, colocalization and FRET under control conditions and in OGD. The insets (1 to 4) are blown up to the right. B. Time course for how astrocyte territory area increased in OGD (data normalized to –OGD). C. Summary bar graphs for colocalization area, FRET area and FRET efficiency under control conditions. D. Representative images of dorsolateral striatal astrocytes expressing NAPA-a in either WT or R6/2 mice. E. Area of dorsolateral striatal astrocytes as assessed by a flattened projection of the soma or of the entire territory. F. Representative images of lucifer yellow filled striatal astrocytes in either WT or R6/2 mice. G. Volume of dorsolateral striatal astrocytes as assessed by 3D reconstructions of the astrocyte somata or territory in WT or R6/2 mice from images such as in F. H. Cartoon showing that we microinjected AAVs for NAPA-n into either the motor cortex or thalamus, with NAPA-a microinjected into the striatum. Using this approach, we studied spatial interactions of corticostriatal and thalamostriatal inputs with astrocytes in WT and R6/2 mice. I–J. The scatter graphs plot areas of colocalization and FRET in WT and R6/2 mice.