Dissipation of transmembrane potassium gradient is the main cause of cerebral ischemia-induced depolarization in astrocytes and neurons

Abstract

Membrane potential (V_M) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K⁺ and glutamate has been shown to underlie an ischemia-induced V_M depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K⁺ gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K⁺ gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K⁺ contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K⁺ loss and depolarization should facilitate the development of future stroke therapy.

Keywords: Astrocytes; Brain ischemia; Ion channel; NG2 glia; Neurons.

Figure 1. Electrophysiological responses of hippocampal astrocyte, NG2 glia and pyramidal neuron to OGD

(a-c) An astrocyte identified by SR101 staining, an NG2 glia devoid of SR101 staining, and a pyramidal neuron in CA1 region. (d-f) Representative whole-cell current profiles of an astrocyte, an NG2 glia, and a pyramidal neuron. Inset in (d), voltage steps (V_COM) were from -180 mV to +20 mV in 10 mV increments. The bottom panel in (f), action potentials induced by 100–200 pA/50 ms current pulses (I_COM). (g-i) A 30 min OGD-induced V_M response of three cell types. Inset in (i), the neuronal action potentials occurred and vanished during OGD-induced depolarization.

Figure 2. OGD induces no detectable damage to astrocytic and NG2 glial membrane ion channels

(a) A single freshly dissociated astrocyte stained with SR101. (b) Schematic illustration of OGD-induced K⁺ gradient dissipation (left, “[K⁺]_i non-clamped”), and masked this [K⁺]_i loss due to dialysis of the cell by electrode solution (right, “[K⁺]_i clamped”). (c-d) Under [K⁺]_i clamped condition, OGD-induced depolarization was eliminated. NS: no significant difference (P>0.05, ANOVA). (e) Unchanged R_in in astrocyte during recording (P>0.05, paired sample t-test). (f-g) Dual patch single astrocyte recording for analysis of membrane conductance (G_M). In VC mode (left electrode), the cell was held at -80 mV. The delivered command voltages (V_COM) were ±50 mV/25 ms separated by 30 ms intervals. The V_COM-induced membrane current (ΔI_M) was recorded using the same electrode. The CC mode (right electrode) was set in I=0 mode to record the V_COM-induced change in membrane voltage (ΔV_M). (g) Shows the recorded ΔV_M and ΔI_M. (h) The summary shows no OGD-induced change in astrocyte G_M (P>0.05, paired sample t-test). (i-j) Unaltered current profile of NG2 glia in control and OGD. Inset in (i), V_COM ranging from -180 mV to +40 mV in 10 mV increments. (k-m) The I-V plots of NG2 glial IKa, IKd, and IK_ir before and 30 min in OGD. Current at each step was compared between control and OGD and no significant change was found in these ion channels (P>0.05, paired sample t-test).

Figure 3. A short-term OGD does not alter the activity of voltage-gated ion channels or the excitability of cultured hippocampal neurons

(a-b) DIC image and a representative whole-cell current profile of a cultured hippocampal neuron. The cultured hippocampal neuron expressed voltage-gated Na⁺ and K⁺ current conductance as neurons in hippocampal slices. Inset in (b), V_COM ranging from -180 mV to +40 mV in 10 mV increments. (c) Three different patterns of neuronal spontaneous firing response to OGD. (d) In 10/23 cultured neurons, OGD induced a small depolarization compared to a large neuronal depolarization in slice (#, P<0.01, t-test). (e) In 13/23 cultured neurons, OGD induced no detectable depolarization (NS, P>0.05, ANOVA). (f-i) Representative induced action potentials in control and OGD. I_COM in insets: 100 pA/500 ms in (f); 200 pA/500 ms in (h). (j-k) The number and amplitude of induced action potentials were not altered after 30 min OGD (P>0.05, paired sample t-test).

Figure 4. OGD does not alter the activity of neuronal membrane ion channels

(a-b) Representative current profiles of a cultured hippocampal neuron in control and OGD. Inset in (a), V_COM ranging from -180 mV to +40 mV in 10 mV increments. (c) A 30 min OGD induced no change in the current density of voltage-gated ion channels: INa at V_COM -30~-40 mV; IKa and IKd both at V_COM +40 mV (P>0.05, paired sample t-test). (d-f) The I-V plots of INa, IKa, and IKd. Current at each step was compared between control and OGD and no significant change was found in these ion channels (P>0.05, paired sample t-test).

Figure 5. OGD-induced depolarization increases with more astrocytes coupling into a syncytium

(a) SR101 staining of a single freshly dissociated astrocyte, and three “miniature syncytia” with a varied number of astrocytes as indicated. AS: astrocyte. (b) OGD-induced V_M depolarization increases with the number of coupled astrocytes. (c) Quantification of OGD-induced ΔV_M in (b) (NS, P>0.05; *, P<0.05, ANOVA). (d) Representative astrocyte whole-cell currents recorded before and at the end of 30 min OGD under uncoupled and varied coupling syncytial sizes. (e) Normalized I-V plots show an OGD-induced positive shift in I-V plot (depolarization), but no suppression of passive conductance change in astrocyte in situ. (f) OGD induced neither a shift in I-V plot nor reduction in passive conductance in single astrocytes.

Figure 6. K⁺ gradient dissipation contributes significantly to OGD-induced astrocyte depolarization

(a) Schematic diagram of conventional whole-cell mode (left), and cell-attached mode (right) for V_M recording in syncytial-coupled astrocytes. (b) SR101 fluorescence in recording electrode shows cytoplasm-electrode exchange in whole-cell mode (blue arrow), but not in cell-attached mode. (c-d) Representative V_M recording in whole-cell mode (c) and in cell-attached mode (d) of astrocytes in situ. Insets show unchanged R_in. (e) Quantification of V_M shows no significant difference of the V_M recorded in whole-cell and cell-attached modes (P>0.05, t-test). (f) The OGD-induced astrocytes ΔV_M is significantly greater in cell-attached mode than in whole-cell mode (P<0.01, t-test). (g) Representative V_M recording of NG2 glia in cell-attached mode. Insets show unchanged R_in. (h) The OGD-induced NG2 glia ΔV_M is comparable in cell-attached mode and in whole-cell mode (P>0.05, t-test).

Figure 7. K⁺ gradient dissipation contributes significantly to OGD-induced neuronal depolarization

(a) Cell-attached V_M recording from a hippocampal pyramidal neuron in situ. Two types of V_M responses to OGD are shown in (b) (fully reversible) and (c) (partially reversible). Inset in (b), the neuronal action potentials occurred and vanished during OGD-induced depolarization. (d) The resting V_M recorded in cell-attached (a) and conventional whole-cell modes are comparable in pyramidal neurons (P>0.05, t-test). (e) For the neurons with fully reversible V_M, OGD-induced depolarization (ΔV_M) was significantly greater in cell-attached mode than that of whole-cell mode (P<0.01, t-test). The OGD-induced depolarization from neurons with partially reversible V_M is shown in a separate column in (e) but was not included in the statistical comparison.