Astrocytes are glial cells that interact with neuronal synapses via their distal processes, where they remove glutamate and potassium (K+) from the extracellular space following neuronal activity. Astrocyte clearance of both glutamate and K+ is voltage dependent, but astrocyte membrane potential (Vm) is thought to be largely invariant. As a result, these voltage dependencies have not been considered relevant to astrocyte function. Using genetically encoded voltage indicators to enable the measurement of Vm at peripheral astrocyte processes (PAPs) in mice, we report large, rapid, focal and pathway-specific depolarizations in PAPs during neuronal activity. These activity-dependent astrocyte depolarizations are driven by action potential-mediated presynaptic K+ efflux and electrogenic glutamate transporters. We find that PAP depolarization inhibits astrocyte glutamate clearance during neuronal activity, enhancing neuronal activation by glutamate. This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyte–neuron interaction.
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Asymmetric dysregulation of glutamate dynamics across the synaptic cleft in a mouse model of Alzheimer’s disease
Acta Neuropathologica Communications Open Access 14 February 2023
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Source data are provided with this paper. The datasets generated during and analyzed during the current study are available from the corresponding author on request.
All computer code used to collect and analyze data are available from the corresponding author on request.
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We thank members of the Dulla, Haydon and Rios laboratories, and J. Raimondo and J. Diamond for helpful comments on the manuscript. We thank Y. Yang (Tufts) for EAAT2-tdTomato mice. We thank L. Looger (UCSD), V. Pieribone (Yale), B. Khakh (UCLA) and S. Grinstein (University of Toronto) for making available plasmids and constructs. This work was supported by the NIH (nos. NS113499, NS104478 and NS100796 to C.G.D.; MH117042 to AEC).
The authors declare no competing interests.
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Extended Data Fig. 1 GEVI expression does not induce reactive astrocytosis.
Representative confocal IHC images stained for GFAP in Arclight infected and Archon1-EGFP (EGFP fluorescence shown) infected cortices. Additionally, staining in matched uninfected contralateral cortex. Neither GEVI infected or uninfected contralateral cortex shows high GFAP levels, indicative of a lack of reactive astrocytosis. In order to verify the sensitivity of our GFAP antibody, we stained slices 3 days following controlled cortical impact, a robust model of traumatic brain injury. In this positive control, astrocytes are labeled with the astrocyte specific marker Sox9 and shows high levels of GFAP staining and astrocytosis. Scalebar = 50 µm.
Extended Data Fig. 2 GEVI expression does not change astrocyte morphology.
Example confocal sections and reconstruction of the astrocyte cell-fill reporter tdTomato either uninfected controls (left column), Arclight infected (middle column), or Archon infected (right column). No significant changes were observed in total astrocyte volume or soma volume between control or GEVI infected astrocytes. N= 8 control, 5 Arclight, 6 Archon astrocytes. One way ANOVA Control v Arclight p = 0.23, Control v Archon p = 0.058, Arclight v Archon p = 0.81. All panels: Error bars = Standard error of the mean.
Extended Data Fig. 3 Correcting pH transients.
Average pH transients (Lyn-pHluorin) and Arclight GEVI responses to A) 1 Stim, B) 5 Stimuli at 100Hz, C) 10 Stimuli at 100Hz. D) The GEVI decays are corrected for the pH changes using the difference in the Arclight and pHluorin traces. n = 9 Slices/ 3 mice (pHluorin). n = 17 slices/6 mice (Arclight). All panels: Error bars = Standard error of the mean.
Extended Data Fig. 4 Astrocyte membrane probes primarily localize to astrocyte process rather than soma.
Example confocal images from a 3D Z-stack of astrocyte targeted Arclight (a membrane targeted GEVI) and an astrocyte cell fill (GFAP-tdTomato). We subsequently quantified the Arclight fluorescence originating from the soma membrane compared to the total astrocyte Arclight fluorescence in all Z-sections. Soma fluorescence represents 1.0 ± 0.0005% of the total Arclight fluorescence N = 6 astrocytes. Scale bar = 10 μm.
Extended Data Fig. 5 Confocal Laser Scanning Microscopy shows ROI hotspots have skewed distributions.
Half of individual ROI hotspots from Fig. 3g,h, are fit with 2D gaussians to determine one-sided standard deviations for X and Y- axis. Both Arclight and Archon show significantly more skewed fluorescence distribution along the Y-axis. Box-Whisker plot, Box = 25, 50, 75th percentile, whiskers = 5-95th percentile, square = mean. *** = p < 0.001. N= 1109 ROIs (Arclight, p = 1.1E-30) and N=104 ROIs (Archon p = 3.3E-11).
Extended Data Fig. 6 K+ wash-on calibration induces uniform depolarization.
Example image of Arclight (scale bar = 30 μm) and response image to +5mM K+ wash-on, shows largely uniform voltage response across the field. Outliers, such as those highlighted with white arrows, tend to be areas excluding the Arclight sensor such as somas, or blood vessels. Distribution of pixel responses to +5mM K+ shows a uniform distribution of depolarization responses.
Extended Data Fig. 7 Kir4.1 Overexpression.
A) Confocal example image of immunofluorescence staining of Kir4.1 in Kir4.1 overexpression (Kir4.1-OE), (AAV5-GFAP-Kir4.1-EGFP) or control (AAV5-GFAP-GFP) infected cortex. B) Quantification of widefield Kir4.1 IHC staining shows significantly enhanced Kir4.1 staining. Scale bar = 50 µm. Two-sample t-test, n=3, 4 mice, p = 0.038. C, D) Astrocyte whole-cell voltage clamp shows enhanced Ba2+ (Kir4.1 inhibitor) sensitive currents in Kir4.1-OE compared to control-infected cortex. 9 cells/3 mice each. E) Ba2+- sensitive currents are significantly increase in Kir4.1-OE astrocytes, p = 0.028. F) Western blot quantification of Kir4.1-OE (AAV5-GFAP=Kir4.1-mCherry), shows significantly increased Kir4.1 protein compared to control virus (AAV5-GFAP-tdTomato). p = 0.049 * = p < 0.05 All panels: Error bars = Standard error of the mean.
Extended Data Fig. 8 Kir4.1 depolarizes astrocyte soma during neuronal activity.
Astrocyte-whole cell current clamp recordings were made in the cortex to measure somatic Vm. In order to isolate the effects of Kir4.1 on astrocyte Vm during neuronal activity, glutamate transporter activity was blocked with TFB-TBOA and responses to 10 stimuli at 100Hz were recorded before and after blockade of Kir4.1 with Ba2+. A) Average paired traces before (black) and after (red) inhibition of Kir4.1 with Ba2+, and B) the Ba2+-sensitive ΔVm. These recordings show that Kir4.1 depolarizes astrocyte soma during neuronal activity. C and D) Expanded time scale (dashed boxes in A & B) to show Vm during stimulus. N = 5 cells. All panels: Error bars = Standard error of the mean.
Extended Data Fig. 9 The effects of low Ca2+ on presynaptic release do not correlate with the effects on glutamate clearance and astrocyte depolarization.
Utilizing data from Figs. 6, 7, and Extended Data Fig. 10 we plotted the effects of Low Ca2+ aCSF on presynaptic release (x-axis) and glutamate clearance/astrocyte depolarization (y-axis) for 1, 5, and 10 stimuli at 100Hz. In each condition, Low Ca2+ is normalized to control. Dashed lines represent no change from control. Left of the dashed line on the x-axis represents a reduction in presynaptic release (as assayed by GTC, iGluSnFr, or NMDA peak amplitude). Beneath the dashed line represents an enhanced glutamate clearance/reduced depolarization (as assayed by GTC/iGluSnFr/NMDA decays and Arclight peaks). 1 Stim responses (grey) shows the largest change in presynaptic release, with the smallest effect on glutamate clearance/depolarization. 10 Stimuli at 100Hz (blue) shows the smallest presynaptic release effect with the largest glutamate clearance/depolarization effect.
Extended Data Fig. 10 Low Ca2+ Glutamate Transporter Currents.
Glutamate transporter currents were recorded from astrocytes with Control or Low Ca2+ aCSF, showing enhanced glutamate clearance following trains of stimulation. Two way repeated measures ANOVA * = p<0.05. n = 10 cells/3 mice, p = 0.046. All panels: Error bars = Standard error of the mean.
Supplementary Figs. 1 and 2 (with legends) and Table 1.
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Armbruster, M., Naskar, S., Garcia, J.P. et al. Neuronal activity drives pathway-specific depolarization of peripheral astrocyte processes. Nat Neurosci 25, 607–616 (2022). https://doi.org/10.1038/s41593-022-01049-x
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