Potassium-Dependent Coupling of Retinal Astrocyte Light Response to Müller Glia

Abstract

Astrocytes throughout the central nervous system mediate a variety of functions to support proper tissue physiology, including the regulation of blood flow and providing metabolic support to neurons. There is also growing appreciation for their role in directly modulating neuronal excitability and information transfer. Recently, we reported that astrocytes in the retina exhibit an array of neuronal-associated microstructural motifs whose structure and placement suggest roles in monitoring neuronal electrical activity or direct modulation of excitability. In this study, we record whole-cell patch clamp responses of astrocytes in intact retina to both light and voltage step as a precursor to studying the detailed physiology of individual microstructural motifs. Retinal astrocytes exhibit small amplitude, graded depolarization to both light ON and OFF stimuli with waveforms that closely resemble those of Müller glial endfeet, from which we also recorded. Depolarization is due to potassium influx, with the major source likely being focal release from Müller endfeet onto astrocyte soma. Both macroglia additionally share current-voltage relationships and exhibit stimulus-dependent changes in ionic permeability. The results suggest a pathway of communication from Müller cells to astrocytes that could support broader retinal modulation beyond potassium spatial buffering.

Keywords: Müller glia; astrocyte; electrophysiology; light; potassium; retina.

FIGURE 1

Astrocytes demonstrate small amplitude, graded depolarization to light stimulus. (A) Example astrocyte recorded from in current clamp mode and filled with lucifer yellow dye, scale 25 μm. (B–D) Astrocytes depolarize to light onset and offset with variability in voltage stability during light presentation. A 3‐s 525‐nm light stimulus is indicated by the green bar. (E) An example astrocyte trace lacking a distinct response to light offset (black arrow). (F) An example astrocyte trace where brief hyperpolarization precedes depolarization to both ON and OFF stimuli. *marks hyperpolarization.

FIGURE 2

Astrocyte light response is dependent on extracellular K+ but not voltage‐gated sodium channels. (A) An astrocyte response to 525‐nm light is plotted in conjunction with the voltage change from picospritzing an Ames' solution with an additional supplemental 1.4 mM of KCl for 20 ms from 112 μm near the same cell. Dashed lines show the onset and offset of light and Picospritz pulses. (B) Light response is disrupted and eventually abolished when potassium channels are blocked by 1 mM of barium chloride. Each curve is a light response trace from the same astrocyte at a specific timepoint following bath application of BaCl₂. In the legend, the time at which the recording begins is noted along with the starting voltage for that trace. Each plotted trace is baseline corrected to 0 mV starting voltage, as BaCl₂ depolarizes the cell. (C) Injection of current to bring the cell back to its pre‐BaCl₂ RMP does not restore the light response. Traces in B and C are from the same astrocyte. Three cells from different mice were recorded. (D–F) 500 nM of tetrodotoxin rapidly abolishes retinal ganglion cell spiking (< 5 min, 99.95% ± 0.05% spike reduction), but at a 20‐min incubation, most astrocytes retain a light response (9/10 cells, ON response amplitude % of baseline: 60.8% ± 10.9%, AVG ± SEM).

FIGURE 3

The astrocyte light response resembles that of Müller glia. (A) Three representative Müller glia endfoot current clamp recordings in response to 525‐nm light. Cell identity was verified through dye filling. Scale is 25 μm. (B–E) The kinetics of the astrocyte and Müller glia light responses are similar. (B) Müller glia and astrocytes differ in RMP (p < 0.0001, Mann–Whitney). (C) The magnitude of depolarization differed both between cell types (A‐ON vs. MG‐ON, p = 0.0131) and also between light onset and offset within a given cell type (A‐ON vs. A‐OFF p < 0.0001, MG‐ON vs. MG‐OFF p < 0.0001). 1‐way ANOVA + Tukey post hoc. (D) Time to reach depolarization peak was only different between MG ON and OFF responses (p = 0.0023, Kruskal–Wallis + Dunn's multiple comparisons). (E) Depolarization rate was not significantly different between groups. However, both macroglia have a slower depolarization rate to light offset than onset (astrocytes p < 0.0001; MG p = 0.0136). Kruskal–Wallis + Dunn's multiple comparisons. (F) Averaged light response curves for α‐ON RGCs, Müller glia, and astrocytes with semi‐manual determination of response latency (glial voltage signal increases 1.5 standard deviations above baseline; peak location for first RGC action potential). Response latencies for ON RGCs, Müller glia, and astrocytes were 72.3 ± 3.5, 88.4 ± 8.1, and 100.0 ± 4.1 ms, respectively (trace count: RGC 124, Müller glia 25, astrocyte 63. p < 0.0001 A‐RGC; p = 0.0170 MG‐RGC, Kruskal–Wallis + Dunn's multiple comparisons). Bars are mean ± SEM. (G) Area density of RGC soma and linear density at eccentric rings for axons crossing on a path to the ONH. (H) Amplitude of astrocyte depolarization to light is not dependent on eccentricity (n = 13). (I) Example patched astrocyte near multiple RGC axon bundles which has a light‐induced depolarization amplitude of 0.62 mV. Magenta (Lucifer yellow) shows an astrocyte, and white shows axons (beta iii tubulin). Scale is 20 μm. (J) Müller glia density as a function of eccentricity is relatively constant.

FIGURE 4

Astrocytes and Müller glia share diverse rectification patterns which adapt to repeated stimulation. (A) Astrocytes and Müller glia can be found in varying states of ionic permeability. Whole‐cell voltage clamp recordings in response to voltage step from a holding potential of −80 mV show current–voltage relationships exhibiting outward, low, and inward rectification polarity (Number of astrocytes: Outward 30, low flux 6, inward 9. Number of Müller glia endfeet: Outward 8, low flux 4, inward 11). Points indicate mean and bars indicate SEM. Both cell types have similar linearly interpolated reversal potentials for both outward and inward current (astrocyte: Outward −71.2 ± 0.6 mV, inward −65.2 ± 1.6 mV. Müller glia: Outward −69.6 ± 0.8 mV, inward −67.9 ± 0.7 mV). Astrocytes (B) and Müller glia (C) can switch between states of ionic permeability in a stimulus‐dependent manner. Repeated acquisition of the voltage step protocol changes the current–voltage relationship. Astrocyte rectification curves are well fit by potassium and calcium currents observed in salamander Müller glia. (D) Re‐created data from Newman 1985 showing isolated potassium and calcium currents from enzymatically dissociated salamander Müller glia. (E–H) Least‐squares regression between weighted curves in D and example individual astrocyte IV curves. Traces in E–H are outward rectification, shifted outward rectification, complex, and low flux respectively. Many traces can be well approximated. Inward rectification curves in A, B could not be well approximated due to a lack of inward current in the positive voltage region of the Newman curves. R ² values for outward fit, outward fit +50 pA correction, shifted outward fit, complex fit, and low flux fit are as follows: 0.71, 0.99, 0.92, 0.85, 0.06 (noise @ ~0pA).

FIGURE 5

Astrocyte light response is not due to calcium. (A–C) Astrocytes show an increase in intracellular calcium in response to ATP but not light. Example fluorescence time course in A. Scale 10 μm. (B) 0/170 astrocytes in 73 total frames of view (frame area: 224 × 168 μm², 3 mice/4 retinae) depolarized to light. (C) Fluorescence profile of representative astrocyte. (D) Müller glia endfeet show calcium response to both light and ATP. (E) A light response was observed in Müller cells in 8/73 total frames of view (224 × 168 μm², 3 mice/4 retinae). Scale 5 μm. (F) Fluorescence profile of representative Müller cell. For each cell, fluorescence intensity was determined for pixels in an ROI within the cell body or endfoot, respectively. 490‐nm light served as both the excitation and recording light, and it was verified that astrocytes and Müller glia depolarize to 490‐nm light as they do for 525 nm (not shown). In A and D, the second frame differs for the example astrocyte and Muller endfoot. Because the astrocytes lack a light response, we show the point of ATP addition (Noted by cyan line in C). For the Muller endfoot, we show a frame “Light Max” demonstrating the light response which is more informative than the timepoint where ATP is released (simply a darker frame). For both cells, the Max ΔF/F₀ is the maximum of the time course after ATP addition.

FIGURE 6

Potassium diffusion modeling supports a dual origin of the astrocyte light response. (A) Estimate of free and restricted diffusion of potassium using Fick's law. Using the Δlatency in light‐induced depolarization for astrocyte‐RGC (27.7 ms) and astrocyte‐Müller glia (11.6 ms), the free diffusion distance of potassium in this time is 14.7 and 9.5 μm, respectively. Restricted diffusion distances are 9.5 and 6.2 μm. (B, C) Simulated change in astrocyte voltage as a function of the number of axons within a distance bounded by restricted potassium diffusion from RGCs. The potassium released per unit surface area of an axon was estimated from the literature and voltage change estimated using the Goldman–Hodgkin–Katz (GHK) equation. (D) Random‐walk simulation of potassium which reaches the surface over time for an 18 × 18 bundle of axons. (E) An astrocyte voltage response to 525‐nm light is shown with an estimate of how the extracellular concentration of K+ would have to change to fit that curve (GHK equation). (F) Plot of how RMP changes with changing extracellular potassium (GHK model). (G) Confocal image of an astrocyte illustrating the restricted diffusion distance of potassium for RGC and Müller cell release. The green region shows the distance from the edge of the cell body that RGC‐originating potassium could diffuse to reach the edge of the astrocyte cell body within the Δlatency period. The cyan region illustrates this distance for Müller‐derived potassium. Scale for full size image is 20 μm and for the zoomed image is 5 μm. (H) Patched astrocyte with gap junction‐mediated spread of dye to Müller glia. Lucifer yellow primarily labeled the astrocyte (magenta) while neurobiotin primarily labeled the Müller glia. Circled region indicates the astrocyte cell body. Scale is 10 μm.

FIGURE 7

Astrocyte light response is sensitive to gap junction blockade. 200 μM of meclofenamate, 200 μM of carbenoxolone, and 500 μM of octanol all disrupt the astrocyte light response. Plots show current clamp whole‐cell recordings of astrocytes in response to 525‐nm light (green bar) at baseline (black) and following drug application (cyan). Effects of meclofenamate are seen within 5 min, whereas the effects of carbenoxolone and octanol occur between 20 and 30 min (data plot times).

FIGURE 8

Model of astrocyte light response. Retinal cross section (A) and zoomed in (B) view of a potassium‐based model of astrocyte depolarization to light. Most of the potassium arises from Müller glia siphoning of potassium to the inner retina. This potassium is released extracellularly and through gap junction connections with astrocytes. Extracellular potassium released at focal sites of endfoot association with astrocyte cell bodies depolarizes the cells. A small amount of potassium released from RGC axons also contributes to astrocyte depolarization.