The Increased Activity of TRPV4 Channel in the Astrocytes of the Adult Rat Hippocampus after Cerebral Hypoxia/Ischemia

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult

Distinct Expression/Function of Potassium and Chloride Channels Contributes to the Diverse Volume Regulation in Cortical Astrocytes of GFAP/EGFP Mice

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green–fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K+ and Cl− channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K2P) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K2P channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K+ effluxes. Additionally, the inhibition of Na+-K+-Cl− co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K+ channels (Kir4.1), K2P channels (TREK-1 and TWIK-1) and Cl− channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K2P channels

List of used inhibitors.

<p>KCC – K⁺-Cl⁻ co-transporter, NKCC – Na⁺-K⁺-Cl⁻ co-transporter, EAAT – excitatory amino acid transporter, VRAC – volume-regulated anion channel, K_2P – two-pore domain potassium channel, Kir - inwardly rectifying potassium channel.</p><p>Stock solutions were dissolved at 1,000× final concentration in DMSO (*); ethanol (**) or methanol (***). BaCl₂ was dissolved at 1 mM concentration in double-distilled H₂O (ddH₂O).</p

Experiment II: gene expression profiling of distinct astrocytic subpopulations.

<p><b>A:</b> Bar plot with SEM for all the expressed genes; significant differences are indicated with asterisks (<i>p</i><0.05 (*), <i>p</i><0.01 (**), <i>p</i><0.001 (***). <b>B:</b> Principal component analysis. The identification of 2 astrocytic subpopulations is along the first principal component, which accounts for most of the variation in the measured data. <b>C:</b> Clustering of astrocytes using Kohonen SOMs. The expression levels of all genes were mean-centered. Each dot represents one cell. <b>D:</b> Dendrogram based on all astrocytic genes. The y-axis shows the distance between groups.</p

Currents evoked by 4αPDD in hippocampal astrocytes <i>in situ</i> are reduced by calcium-free extracellular solution, Ruthenium Red or RN1734.

<p>(<b>A–C, left</b>) Time course of 4αPDD-evoked currents measured from the ramp protocol in astrocytes 7D after H/I (for the voltage protocol see the inset) prior to and during 4αPDD (10 µM) application and after removing extracellular Ca²⁺ (Ext2_ØCa, <b>A</b>) or after the application of TRPV4 inhibitors, such as Ruthenium Red (RR, 10 µM, <b>B</b>) or RN1734 (10 µM, <b>C</b>). (<b>A-C, right</b>) The traces of the steady state currents (same cells as in left) obtained in Ext2 solution (black lines), during 4αPDD application (red lines) and after removing extracellular Ca²⁺ (Ext2_ØCa, <b>A</b>) or after the application of TRPV4 inhibitors, such as Ruthenium Red (RR, 10 µM, <b>B</b>) or RN1734 (10 µM, <b>C</b>), are indicated by blue lines. Representative traces of steady state currents were obtained at the times indicated by asterisks of the corresponding colors.</p

Inhibitors of Cl⁻ channels differently affect OGD-induced swelling in HR-and LR-astrocytes.

<p>The effect of 100 µM NPPB, a non-specific inhibitor of chloride channels (<b>A</b>), 30 µM Tamoxifen (<b>B</b>) and 30 µM DCPIB (<b>C</b>), inhibitors of volume regulated anion channels. <b>A–C top:</b> Time course of volume changes in HR-astrocytes (filled circles) and LR-astrocytes (empty circles) during 40-minute OGD (control) and during 20-minute OGD followed by 20-minute co-application of ACSF_OGD plus an inhibitor in HR-/LR-astrocytes (filled/empty triangles). <b>A–C bottom:</b> The effect of the inhibitors was evaluated in each individual cell and expressed as the percent cell volume increase/decrease related to the maximal volume after 20-minute OGD, which was set as 0%. Note that in HR-astrocytes the application of VRAC inhibitors (Tamoxifen and DCPIB) reduced the swelling induced by OGD, while in LR-astrocytes the application of DCPIB resulted in an additional swelling. Asterisks indicate significant differences from controls (<i>p</i><0.05 (*, significant), <i>p</i><0.01 (**, very significant), <i>p</i><0.001 (***, extremely significant)).</p