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Neuronal activity drives pathway-specific depolarization of astrocyte distal processes

Moritz Armbruster, Saptarnab Naskar, Jacqueline Garcia, Mary Sommer, Elliot Kim, Yoav Adam, Philip G Haydon, Edward S Boyden, Adam E Cohen, Chris G Dulla

Preprint posted on July 04, 2021 https://www.biorxiv.org/content/10.1101/2021.07.03.450922v1

Eavesdropping neuron-astrocyte conversations: the Dulla lab unravels a novel form of neuron-astrocyte crosstalk using genetically encoded voltage imaging

Selected by Kristina Kuhbandner

Categories: neuroscience

Background

Electrical signaling plays a central role in the transmission of information between neurons. How neurons generate and propagate these signals has been extensively studied in the past. Importantly, to fulfil their duties, neuronal cells also need the support of other cells such as oligodendrocytes and astrocytes. Astrocytes physically and functionally interact with neuronal synapses, and play a role in regulating extracellular levels of potassium (K+) and the neurotransmitter glutamate, thereby controlling excitatory neurotransmission. For example, the clearance of glutamate is facilitated by excitatory amino acid transporters (EAATs) located on distal astrocyte processes (DAPs).  These transporters are known to be voltage-dependent and can be inhibited upon neuronal activity. Importantly, until now it has been assumed that astrocytes themselves are not electrically active, because following neuronal activity only minimal membrane potential changes were observed at the soma of astrocytes (1). To explain this discrepancy, Armbruster and colleagues hypothesized that local depolarization at distal processes might be responsible for the activity-induced inhibition of EAATs.

To investigate whether DAPs undergo electrical changes that can’t be detected in the soma, they specifically expressed the genetically encoded voltage indicators (GEVIs) Archon1 or Arclight in mouse cortical astrocytes (2, 3). These membrane-bound fluorescent sensors report membrane voltage dynamics as changes in fluorescent signal intensities.

Main findings

1. Astrocytes show fast, activity-dependent depolarization

To specifically express the GEVIs Archon1 or Arclight in astrocytes, the authors employed AAV-mediated transduction under the control of a modified GFAP promoter. After electrical stimulation of ascending axons in acute brain slices, confocal images were taken to measure changes in fluorescence intensities to investigate activity-induced membrane potential changes in astrocytes. Both, Archon1 and Arclight exhibited stimulus-evoked changes in fluorescence (F/F0) consistent with astrocyte depolarization (Fig. 1A, B). Of note, depolarization time derived from analysis of GEVI F/F0 was approximately five times faster than depolarization time measured in the soma by whole cell patch clamping.

2. Activity-induced astrocyte depolarization is locally restricted, stable and pathway- specific

To specifically determine depolarized regions in astrocytes, principal component analysis/independent component analysis was performed. In the identified “hotspots” of astrocyte depolarization, stimulus-evoked fluorescent changes were significantly enhanced and stable over repeated trials. Then, Armbruster et al. compared fluorescence changes in hotspots after either stimulating ascending cortical or Layer II/III intracortical axons (Fig. 1C). Interestingly, there was only minimal spatial overlap of depolarized hotspots evoked by stimulation of either ascending or intracortical axons, indicating that stimulus-dependent depolarization of astrocytes is pathway-specific (Fig. 1 D).

Figure 1: Fast, activity-dependent and pathway-specific depolarization of astrocytes. (A) Archon1 and (B) Arclight exhibit progressive depolarization with increasing stimuli number (x-axis). (C) Illustration of the experimental setting to study pathway-specific depolarization using ascending (red) or intracortical (blue) stimulators. (D) Example ROI maps of Arclight-expressing astrocytes after stimulating either ascending or intracortical axons (modified after Armbruster et al., 2021, Fig.1 and Fig.3)

3. Astrocyte depolarization is regulated by EAAT activity and presynaptic K+ release

Next, the authors wanted to investigate how neuronal activity causes depolarization of DAPs. Therefore, they manipulated different factors known to be involved in neuron-astrocyte crosstalk. Fluorescent signals were abrogated after using Tetrodotoxin to block voltage-gated sodium channels, verifying that neuronal activity is required for astrocyte depolarization. Inhibition of glutamate uptake by EAATs only caused a partial reduction of GEVI fluorescent changes, indicating that additional mechanisms may contribute to the regulation of astrocyte depolarization (Fig. 2A). Subsequently, the expression of the astrocytic voltage-dependent K+ channel Kir4.1, which buffers increased extracellular K+ concentrations following neuronal activity, was manipulated. While Kir4.1 overexpression significantly reduced depolarization, its inhibition resulted in increased depolarization (Fig. 2 B). These findings suggest that Kir4.1-mediated K+ uptake helps to regulate activity-dependent depolarization and that the increase in extracellular K+ levels is the main trigger for DAP depolarization.

Figure 2. EAAT activity and increased extracellular K+ levels contribute to astrocyte depolarization. Reduced depolarization following (A) application of the EAAT-inhibitor TFB-TBOA or (B) overexpression (OE) of Kir4.1 (modified after Armbruster et al., Fig. 5).

4. DAP depolarization affects the clearance of glutamate

Finally, the authors investigated whether activity-induced depolarization of DAPs modulates astrocyte function, e.g. glutamate uptake. It has been shown that glutamate clearance by EAATs is inhibited by depolarization and slowed down by neuronal activity.

To visualize glutamate clearing after manipulation of activity-dependent depolarization, the fluorescent glutamate reporter iGluSnFr was used. When depolarization was decreased by Kir4.1-overexpression, the slowing of glutamate clearance was reduced. Contrarily, enhancing depolarization by blocking Kir4.1 increased the activity-dependent slowing of glutamate clearance. This demonstrates that EAAT function is inhibited by activity-dependent depolarization of DAPs.

Why I like this preprint

For a long time, neuroscience research mainly focused on neurons. Only in the last decades, other cells in the central nervous system have gained more attention and their critical role in neurological functions has been unraveled. However, their exact contributions remain a mystery. In this study, Armbruster et al. use an elegant approach to investigate the electrical response of astrocytes to neuronal activity.  Their findings challenge the prior assumption that astrocytes are not electrically active and provide a method to further decipher the language of neuron-astrocyte communication. Ultimately, this will foster our knowledge about astrocyte-neuron interactions and their implications in health and disease.

Questions to the authors

  1. The present experiments are performed under “normal” conditions. However, during neuroinflammatory processes and reactive astrocytosis, DAP properties might be altered. Are you planning to investigate DAP depolarization also in reactive astrocytes, for example after application of LPS?
  2. Do you think a disruption in this form of astrocyte-neuron communication might be involved in the pathophysiology of neurological diseases such as schizophrenia, which is associated with glutamatergic dysregulation?

References

  1. Zhou et al., Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive. J Neurophysiol 95,134-43 (2006).
  2. D. Piatkevich et al., A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat Chem Biol 14, 352-360 (2018).
  3. Jin et al., Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron 75, 779-785 (2012).

Tags: astrocytes, electrophysiology, neuron-astrocyte crosstalk

Posted on: 20th July 2021 , updated on: 21st July 2021

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Author's response

Chris Dulla shared

1) The present experiments are performed under “normal” conditions. However, during
neuroinflammatory processes and reactive astrocytosis, DAP properties might be
altered. Are you planning to investigate DAP depolarization also in reactive astrocytes,
for example after application of LPS?

This is an excellent question! These are some of the next experiments on the horizon for us. There is absolutely reason to believe that neuronal activity may have very different effects on astrocyte membrane potential (Vm) in the injured or inflamed brain. First, many of the proteins that we think regulate astrocyte membrane potential (Kir4.1, ATPases, other potassium channels) are known to be altered in these pathological states where astrocytes are reactive. Second, we propose that astrocyte depolarization affects the function of glutamate transporters. Those proteins (GLT-1 and GLAST) can also be changed when astrocytes are reactive. Finally, reactive astrocytes have dramatically different morphologies from normal astrocytes. Because the spatial compartmentalization of electrical signals is a key aspect of how we believe astrocyte voltage is regulated, altering astrocyte morphology and their physical interactions with neurons could have big effects on how neuronal activity affects astrocyte Vm. If these changes occur in reactive astrocytes, another interesting downstream effect could be the disruption of the spatial and synapse-level specificity of activity-induced astrocyte depolarization. This could compound other synaptic dysfunctions that occur during neuroinflammation and injury.

2) Do you think a disruption in this form of astrocyte-neuron communication might be
involved in the pathophysiology of neurological diseases such as schizophrenia, which is
associated with glutamatergic dysregulation?

Again – an excellent question. There are links between astrocyte function and schizophrenia, including the expression and function of glutamate transporters and associated proteins. If neuronal activity alters astrocyte Vm, or the spatial coupling of Vm to glutamate transporters, this novel form of neuron-astrocyte communication could be disrupted in schizophrenia. This also brings up an interesting point that we have not yet explored: How does this system function in the immature or adolescent brain? This could be critical to understanding its implications in schizophrenia and other neurodevelopmental disorders. We believe other diseases where glutamate signaling is known to be disrupted (epilepsy, ASD, Alzheimer’s Disease) may also alter the coupling between neuronal activity, astrocyte Vm, and modulation of glutamate uptake. We are excited to use new tools and imaging modalities to explore these possibilities!

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