We measured activity‐dependent changes in [K+]o with K+‐selective microelectrodes in adult rat optic nerve, a CNS white matter tract, to investigate the factors responsible for post‐stimulus recovery of [K+]o. Post‐stimulus recovery of [K+]o followed a double‐exponential time course with an initial, fast time constant, τfast, of 0.9 ± 0.2 s (mean ±s.d.) and a later, slow time constant, τslow, of 4.2 ± 1 s following a 1 s, 100 Hz stimulus. τfast, but not τslow, decreased with increasing activity‐dependent rises in [K+]o. τslow, but not τfast, increased with increasing stimulus duration. Post‐stimulus recovery of [K+]o was temperature sensitive. The apparent temperature coefficients (Q10, 27–37°C) for the fast and slow components following a 1 s, 100 Hz stimulus were 1.7 and 2.6, respectively. Post‐stimulus recovery of [K+]o was sensitive to Na+ pump inhibition with 50 μM strophanthidin. Following a 1 s, 100 Hz stimulus, 50 μM strophanthidin increased τfast and τslow by 81 and 464%, respectively. Strophanthidin reduced the temperature sensitivity of post‐stimulus recovery of [K+]o. Post‐stimulus recovery of [K+]o was minimally affected by the K+ channel blocker Ba2+ (0.2 mm). Following a 10 s, 100 Hz stimulus, 0.2 mm Ba2+ increased τfast and τslow by 24 and 18%, respectively. Stimulated increases in [K+]o were followed by undershoots of [K+]o. Post‐stimulus undershoot amplitude increased with stimulus duration but was independent of the peak preceding [K+]o increase. These observations imply that two distinct processes contribute to post‐stimulus recovery of [K+]o in central white matter. The results are compatible with a model of K+ removal that attributes the fast, initial phase of K+ removal to K+ uptake by glial Na+ pumps and the slower, sustained decline to K+ uptake via axonal Na+ pumps.

1. Whole cell and cell-attached patch-clamp recordings were obtained from rat spinal cord astrocytes maintained in culture for 6-14 days. It was found that the resting conductance in these astrocytes is primarily due to inwardly rectifying K+ (Kir) channels. 2. Two types of astrocytic Kir channels were identified with single-channel conductances of approximately 28 and approximately 80 pS, respectively. Channels displayed some voltage dependence in their open probability, which was largest (0.8-0.9) near the K+ equilibrium potential (Ek) and decreased at more negative potentials. The resting potential closely followed Ek, so it can be assumed that Kir channels have a high open probability at the resting potential. 3. The conductance of inwardly rectifying K+ currents (Kir) depended strongly on [K+]o and was approximately proportional to the square-root of [K+]o. 4. Kir currents inactivated in a time- and voltage-dependent manner. The Na+ dependence of inactivation was studied with ion substitution experiments. Replacement of [Na+]o with choline or Li+ removed inactivation. This dependence of current inactivation on [Na+]o resembles the previously described block of Kir channels in other systems by [Na+]o. 5. Kir currents were also blocked in a dose-dependent manner by Cs+ (Kd = 189 microM at -140 mV), Ba2+ (Kd = 3.5 microM), and tetraethylammonium (TEA; 90% block at 10 mM) but were insensitive to 4-aminopyridine (4-AP; 5 mM). In the current-clamp mode, Ba2+ and TEA inhibition of Kir currents was associated with a marked depolarization, suggesting that Kir channel activity played a role in the establishment of the negative resting potential typical of astrocytes. 6. These biophysical features of astrocyte inwardly rectifying K+ channels are consistent with those properties required for their proposed involvement in [K+]o clearance: 1) high open probability at the resting potential, 2) increasing conductance with increasing [K+]o, and 3) rectification, e.g., channel closure, at positive potentials. It is proposed, therefore, that the dissipation of [K+]o following neuronal activity is mediated primarily by the activity of astrocytic Kir channels.

We used an in vitro model for glioma cell invasion (transwell migration assay) and patch-clamp techniques to investigate the role of volume-activated Cl(-) currents (I(Cl,Vol)) in glioma cell invasion. Hypotonic solutions ( approximately 230 mOsm) activated outwardly rectifying currents that reversed near the equilibrium potential for Cl(-) ions (E(Cl)). These currents (I(Cl,Vol)) were sensitive to several known Cl(-) channel inhibitors, including DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The IC(50) for NPPB inhibition of I(Cl,Vol) was 21 microm. Under isotonic conditions, NPPB (165 microm) blocked inward currents (at -40 mV) and increased input resistance in both standard whole-cell recordings and amphotericin perforated-patch recordings. Reducing [Cl(-)](o) under isotonic conditions positively shifted the reversal potential of whole-cell currents. These findings suggest a significant resting Cl(-) conductance in glioma cells. Under isotonic and hypotonic conditions, Cl(-) channels displayed voltage- and time-dependent inactivation and had an I(-) > Cl(-) permeability. To assess the potential role of these channels in cell migration, we studied the chemotactic migration of glioma cells toward laminin or vitronectin in a Boyden chamber containing transwell filters with 8 microm pores. Inhibition of I(Cl,Vol) with NPPB reduced chemotactic migration in a dose-dependent fashion with an IC(50) of 27 microm. Time-lapse video microscopy during patch-clamp recordings revealed visible changes in cell shape and/or movement that accompanied spontaneous activation of I(Cl,Vol), suggesting that I(Cl,Vol) is activated during cell movement, consistent with the effects of NPPB in migration assays. We propose that I(Cl,Vol) contributes to cell shape and volume changes required for glioma cell migration through brain tissue.

Ion channels in inexcitable cells are involved in proliferation and volume regulation. Glioma cells robustly proliferate and undergo shape and volume changes during invasive migration. We investigated ion channel expression in two human glioma cell lines (D54MG and STTG-1). With low [Ca2+]i, both cell types displayed voltage-dependent currents that activated at positive voltages (more than +50 mV). Current density was sensitive to intracellular cation replacement with the following rank order; K+ > Cs+ approximately = Li+ > Na+. Currents were >80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine (1 mM), tetrandrine (30 microM), and tetraethylammonium ion (TEA; 1 mM). Extracellular phloretin (100 microM), an activator of BK(Ca2+) channels, and elevated intracellular Ca2+ negatively shifted the I-V curve of whole cell currents. With 0, 0.1, and 1 microM [Ca2+]i, the half-maximal voltages, V(0.5), for whole cell current activation were +150, +65, and +12 mV, respectively. Elevating [K+]o potentiated whole cell currents in a fashion proportional to the square-root of [K+]o. Recording from cell-attached patches revealed large conductance channels (150-200 pS) with similar voltage dependence and activation kinetics as whole cell currents. These data indicate that human glioma cells express large-conductance, Ca2+ activated K+ (BK) channels. In amphotericin-perforated patches bradykinin (1 microM) activated TEA-sensitive currents that were abolished by preincubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the responses of glioma cells to stimuli that increase [Ca2+]i.

Astrocytes contribute to virtually every aspect of brain function, including ionic homeostasis, energy metabolism, and synaptic signaling. The varied and important roles of astrocytes have evolved to allow increasingly complex nervous systems to operate efficiently and with high fidelity. For example, astrocytes figure prominently in glutamatergic synaptic transmission, an elemental event of brain function: high-affinity glutamate uptake into astrocytes improves the temporal and spatial fidelity of glutamatergic signaling and astrocytes subsequently shuttle glutamine back to neurons for the synthesis of more glutamate. The important and dynamic contributions of astrocytes to normal brain function demand that the interactions between neurons and astrocytes be viewed as a "partnership," a harmonious collaboration to produce a desired function. The historical view of astrocytes as simple "support cells" is no longer valid and should be discarded. It is more accurate to view astrocytes as "partner cells." Future investigations of the intimate neuron-astrocyte partnership will require stringent and novel methodologies. This timely book on methodological approaches for studying astrocytes will provide modern neuroscientists with indispensable technical advice to help unravel the mysteries of the beautiful and successful marriage between astrocytes and neurons.