Honey, I shrunk the extracellular space: Measurements and mechanisms of astrocyte swelling

Abstract

Astrocyte volume fluctuation is a physiological phenomenon tied closely to the activation of neural circuits. Identification of underlying mechanisms has been challenging due in part to use of a wide range of experimental approaches that vary between research groups. Here, we first review the many methods that have been used to measure astrocyte volume changes directly or indirectly. While the field has recently shifted towards volume analysis using fluorescence microscopy to record cell volume changes directly, established metrics corresponding to extracellular space dynamics have also yielded valuable insights. We then turn to analysis of mechanisms of astrocyte swelling derived from many studies, with a focus on volume changes tied to increases in extracellular potassium concentration ([K⁺ ]_o ). The diverse methods that have been utilized to generate the external [K⁺ ]_o environment highlight multiple scenarios of astrocyte swelling mediated by different mechanisms. Classical potassium buffering theories are tempered by many recent studies that point to different swelling pathways optimized at particular [K⁺ ]_o and that depend on local/transient versus more sustained increases in [K⁺ ]_o .

Keywords: AQP4; ATPase; ECS; buffering; potassium; pump; volume; water.

FIGURE 1

Summary of techniques to measure or estimate astrocyte swelling. (1) Increases in extracellular tissue resistance indicate reduction in the volume of the ECS, which is inversely correlated to cell volume changes. (2) Recovery of fluorescent indicator in the ECS after photobleaching. Slowed diffusion rates are indicative of constricted ECS. (3) Dimming of cytosolic fluorescent indicator is an indicator of cell swelling due to dilution by water, while increased signal intensity results from cell shrinking. (4) Real‐time volume measurements in fluorescently labeled cells through edge detection of binarized images overlaid over the baseline image (see Figure 2). (5) Example of real‐time volume measurement in an astrocyte. An image taken after 3 min in 26 mM K⁺ is overlaid onto the baseline image, with expansion of the cell border marked by the arrows (4), followed by recovery to baseline volume after washout of high K⁺ (5) (from Risher et al., 2009). (6) 3D confocal morphometry is similar to real‐time volume imaging in its use of z‐stacks and edge detection with thresholding, but with some notable differences. Rather than thresholding after z‐stack compression, in 3D morphometry unit areas are calculated for each thresholded image individually and converted to square microns, summed, and multiplied by the distance between neighboring sections in the stack. A number of additional image processing steps are also applied, including correction for photobleaching due to the larger z‐stacks (50–60 individual sections) and duration of recordings (up to 80 min). For details, see Chvatal et al., . 3D morphometry offers improved spatial resolution at the expense of temporal resolution, enabling imaging in bulk astrocyte processes. (7) Luminescent single‐walled carbon nanotubes can be tracked over long timescales to measure variances in the local architecture of the ECS (from Godin et al., 2017). Presumably, movement of the nanotubes would be hindered by cell swelling‐induced compression of the ECS. (8) Super‐resolution shadow imaging (SUSHI) combined with fluorescent labeling of astrocytes enables simultaneous measurement of local pools of ECS and individual astrocytic perisynaptic processes. SUSHI offers data collection at rapid timescales and with unprecedented spatial resolution (from Arizono et al., 2021).

FIGURE 2

Real‐time volume measurements. Morphometric analysis of fluorescently labeled cells begins with the collection of a z‐stack of images at one point in time (a). Image processing allows for compression of the z‐stack into a single optical section (b), where all of the z‐axis pixels are flattened onto a single plane. Compressed z‐stacks are collected across individual timepoints (c), and methods such as edge detection after thresholding of average or maximum intensity projections are used to estimate cellular volume changes in the overlaid images (d). The temporal resolution is limited by the time needed to acquire each individual z‐stack, but 1 min intervals are achievable at sufficient signal‐to‐noise to permit accurate edge detection of the soma and major processes despite dimming of cytosolic indicator (Lauderdale et al., ; Murphy, Davila, et al., 2017).

FIGURE 3

Transmembrane proteins involved in astrocyte swelling in elevated [K⁺]_o, along with their substrates. All have the potential to move substrates outward as well under certain conditions. Aquaporin‐4 has been tested in many in vivo models in comparison to the other proteins shown. Potassium inwardly rectifying channels are supported by the classical potassium siphoning hypothesis, while the sodium‐potassium ATPase has received renewed attention in recent studies due to the uniquely K⁺‐sensitive isoform expressed by astrocytes. The sodium‐potassium‐chloride and potassium‐chloride cotransporters are strongly supported by primary culture experiments performed using pathological potassium concentrations, but may be less physiologically relevant than the other proteins. Other evidence points to a role for the sodium bicarbonate cotransporter NBCe1 and monocarboxylate cotransporters (MCT1 and 4) at [K⁺]_o levels closer to basal [K⁺]_o. Evidence supports the astrocyte sodium‐potassium pump as the main K⁺ entry pathway in more elevated and sustained extracellular K⁺ conditions. Key: Aquaporin‐4: (AQP4); sodium potassium chloride cotransporter: (NKCC1); inwardly rectifying potassium channel: (Kir); monocarboxylate transporters: (MCT); sodium‐potassium ATPase: (NKA); sodium bicarbonate cotransporter: (NBCe1)

FIGURE 4

Local/transient versus sustained models of astrocyte swelling. (a) In many studies, low‐level stimulation of neuronal afferents is used to generate local increases in [K⁺]_o. Potassium is released presynaptically from voltage‐gated potassium channels and postsynaptically from neurotransmitter‐gated ion channels (receptors in dark blue, neurotransmitter pink) resulting in transient and local increases in [K⁺]_o. Extruded potassium ions will enter the perisynaptic astrocyte processes and either released back into the ECS at areas of lower [K⁺]_o or siphoned into the vasculature at astrocyte endfeet. This local uptake and redistribution of potassium fits more closely with classical K⁺‐buffering/siphoning models. A Newton's cradle analogy could be used to model this type of K⁺ movement (inset). Because the siphoning of K⁺ out of the cell occurs almost concurrently with K⁺ influx (Newman et al., 1984), it is not clear whether such an instantaneous electromotive (rather than diffusional) redistribution of K⁺ would result in any appreciable or detectable swelling. (b) High frequency stimulation or models using bath application of potassium will produce more sustained, non‐directional K⁺ increases that will engulf the cell. In these instances, other K⁺ handling mechanisms are activated resulting in increased intracellular K⁺ concentration and readily detectable cell swelling using standard confocal and 2PLSM fluorescent imaging techniques. Active synapses are depicted in green. Specific swelling mechanisms are summarized in Figure 5.

FIGURE 5

Summary of proposed mechanisms underlying astrocyte swelling and volume maintenance/recovery at different [K⁺]_o. Conditions are shown along a range from: (a) Local/transient rises in [K⁺]_o near baseline levels through (d) pathological levels of [K⁺]_o. (a) Transient elevations in [K⁺]_o due to local synaptic activity may enter astrocyte processes via Kirs along with water through AQP4 or other pathways (see text). K⁺ and water is redistributed locally to areas of lower activity or siphoned via astrocyte endfeet into the vasculature on a very rapid timescale. (b) Increases in [K⁺]_o up to 5–6 mM by bath application or more sustained stimulation of neuronal afferents likely affects a larger area of the astrocyte domain resulting in more widespread and observable changes in astrocyte and ECS volume. Monocarboxylate transporters, NBCe1, and the Na⁺/K⁺ ATPase may all contribute to increased intracellular K⁺ concentrations coupled to astrocyte volume increases in these conditions. With the inward ion movements under the auspices of transporters and pumps, the role of Kir4.1 and AQP4 is likely to ease intracellular K⁺ and water burden through efflux to areas of lower activity or at endfeet into the vasculature. (c) At more moderate K⁺ approaching ceiling levels during seizure or high frequency stimulation, the Na⁺/K⁺ ATPase plays a primary role in sequestering [K⁺]_o into astrocytes. (d) Strong cellular depolarization due to application of very high levels of [K⁺]_o (up to 100 mM) likely activates NKCC1 and reverses the direction of KCC, resulting in rapid ion influx and associated cell swelling. It is estimated that the astrocyte isoform of the Na⁺/K⁺ ATPase is effective at [K⁺]_o ≤ ~12 mM and therefore would be rapidly overwhelmed in these conditions. In (b), (c), and (d), data from several labs suggest that AQP4 is not required for water entry into astrocytes, which may instead occur by diffusing directly across the lipid bilayer, by passing through other dedicated water pore proteins such as the glucose or glutamate transporters, or in the case of (d), directly through NKCC/KCC co‐transporters.