Potassium Uptake Mechanisms of Cultured Oligodendrocytes Studied with lon-sensitive Electrodes

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1 Potassium Uptake Mechanisms of Cultured Oligodendrocytes Studied with lon-sensitive Electrodes H. Kettenmann, R. K. Orkand, and M. Schachner Introduction Physiological studies over the past two decades of the functional roles of glial cells in the operation of the nervous system have focused attention on the abilities of these cells to regulate ions and take up amino acids in the neuronal microenvironment [17]. Because glial membranes are selectively permeable to K+ [9,10] and the regulation of this ion is important in the control of nerve transmission [15], special attention has been paid to a possible role of glial cells in K+ homeostasis [5, 8, 18]. Following the observation in the bee retina, made with the use of ion-sensitive electrodes, that during photoreceptor stimulation the K + activity [K +1 of glial cells increased [3], the question arose as to the mechanisms producing this increase. Two types of processes have been considered: (a) space-independent net uptake; and (b) space-dependent uptake via spatial buffer currents [1, 3-6, 9, 11]. With the first mechanism, K + is taken up by a transport mechanism as a consequence of a rise in [K +]0 which may be uniform over the cell surface. For the second, an uneven distribution of K + produces a current which results from the difference between the membrane potential Vm and the potassium equilibrium potential E k This current drives K + into the glial cell in regions where [K +]0 is elevated and out of the cells where [K +]0 is low. Such a mechanism depends on a high relative K + permeability. In cell culture, one has the advantage of having independent control over both the ionic environment and membrane currents of glial cells. We have used oligodendrocytes in culture to study K + uptake and membrane permeability. Methods Oligodendrocytes were studied in 4- to 6-week-old explant cultures of embryonic (day 13) mouse spinal cord obtained as described previously [9]. They were identified with morphological criteria established through the use of cell-type-specific monoclonal antibodies [12]. Recordings were made on the stage of an inverted microscope at about 30 C in a CO2 atmosphere sufficient to maintain ph 7.3. For recording of membrane potential and current injection, single- or double-barrelled electrodes were filled with 1 or 2 mmolll potassium acetate, KCI, or NaCI (20-60 Mil). Double-barrelled K + -sensitive.electrodes using Corning as the exchanger in the silanized barrel were made as described by Sonnhof et al. [13]. The bathing solution was the culture medium and the cells were grown in Eagle's basal medium with Earle's salts supplemented with 10% calf serum. It contained (in Ion Measurements in Physiology and Medicine Edited by M. Kessler et al. Springer Yerlag Berlin Heidelberg 1985

2 Potassium Uptake Mechanisms 195 mmolll): NaCI116; KCI5.3; CaC}z1.8; NaHC0 3, 26; NaH2P04, 1; MgS04, 0,8; and glucose 5.5. For perfusion, the normal Ringer's solution contained only the listed salts and glucose. To raise [K +] in the bath, NaCI was partially replaced by equimolar amounts of KCl. Cells were penetrated under visual control with the aid of two-step motor-driven micromanipulators [14]. Results Net Uptake of Potassium When the culture is superfused with a solution containing increased K +, the cells are uniformly depolarized and any increase in [K +]j must result from net uptake. Figure 1 is a diagram of the setup and Fig. 2 illustrates the results of an experiment in which it was demonstrated that an increase in [K +]0 leads to an increase in [K +1 in cultured oligodendrocytes. In these experiments, K + -sensitive electrodes are positioned both inside and outside the glial cell so that the K + gradient can be monitored continuously. Under the conditions of these experiments, the effect could be quite dramatic; a doubling of [K+]o leading to a 20mmolll increase in [K+1. The K + uptake increased as [K +]0 increased and reached a plateau after 2-10 min. The result was the same whether the solution containing the increased K + was superfused over the cell from a blunt-tipped pipette or if the entire bathing solution was exchanged. As [K +1 increased, the membrane hyperpolarized. By comparing the glial membrane potential with the potential expected from the Nernst equation for K + (as measured by the internal and external K + -sensitive electrodes), it was found that the membrane potential is simply determined by the K + gradient [9]. These experiments provide clear evidence that glial cells in culture can respond to a uniform increase in [K +]0 by taking up K +. K+ Permeability Revealed by Na+ Intracellular Iontophoresis The theory of spatial buffering demands a high K + permeability. Movements of K + across the membrane are a result of a difference between Vm and E k We therefore tried to generate such a discrepancy by injection of Na + and monitoring Vm and [K +]j. Oligodendrocytes were penetrated with both a double-barrelled K + -sensitive electrode and a Na + -filled electrode for current injection [7]. The experimental arrangement used is shown in Fig.3. In Fig. 4, the results of an experiment are illustrated. In the absence of current Vm = Ek and [K +1 is steady. When the cell is depolarized by only a few m V by the injection of Na +, the outward membrane current is carried by K + and [K +1 falls. At the end of the current pulse, the potential returns to a slightly depolarized level as expected from the fall in [K +]j. With inward current the opposite occurs and K + enters the cell, leading to an increase in [K +1 and hyperpolarization. The changes are not symmetrical, possibly because the water movements depend on the direction of current flow. However, the results are clear in suggesting that ionic current across the glial membrane is carried predominantly by K+ ions and that only a few mv driving force is sufficient to make a significant change in [K +]j.

3 196 H. Kettenmann et at. Discussion The main results of these studies demonstrate that oligodendrocytes in culture are capable of buffering changes in [K +]0. When they are surrounded by a homogeneous increase in [K +]0' there is a net uptake of K + into the cell [1, 8,9]. The mecha- Fig. 1. Diagram of experimental setup for determining K + uptake. [K +10 was raised either by pressure superfusion of the cell Cvia a blunt electrode positioned over the cell E 2, or by exchanging the bath solution. A double-barrelled K + -sensitive electrode, Elo was positioned in the vicinity of the cell to monitor [K +)0. A second double-barrelled K + -sensitive electrode E3 was inserted into the cell to record membrane potential and [K +)i :t [K+) i Vm :t mmo" I 15f 7 mv ~ ~ r ,..., 10 rt 5 ~ L ~ "- [Kl o Ve 10 [ Smin Fig. 2. Increase in [K +)i with raised [K+)o. Top trace is [K +)i, second trace Vrn, third trace [K+Io, bottom trace the voltage of the reference barrel of the extracellular electrode. K + was raised for 1-3 min to four increasing levels. Note the progressive increase in [K +)i while [K +10 is maintained constant

4 Potassium Uptake Mechanisms 197 Fig. 3. Diagram of experimental setup for injecting current while measuring Vrn and [K+li. While current is passed through 2, Vrn and [K +li are recorded by 1 [ K+} I mmol; : ~ " ' ~ Vm mv I na 2 [ ~ ~ _ - t / ~ Noel Fig. 4. Effect of displacing Vrn on [K +li' The top trace is [K +li, the middle trace Vrn, and the bottom trace displays the output of the current monitor. For a given potential change, depolarization causes a greater fall in [K +li than the increase resulting from hyperpolarization nisms involved possibly include Na + I K + active transport, transport of K + with an anion (Cl- or HC0 3 -), exchange of K + for another cation [16], or passive KCl uptake. When [K +]0 is not homogeneous the passive spread of potential along the cell should cause the membrane potential to deviate from Ek and a deviation of just a few m V, given the high relative K + permeability, should be sufficient to drive K + into the cell in regions where the cell is relatively hyperpolarized and out of the cell where Vrn exceeds Ek [6]. In additon, the results raise questions as to how [K+]i is regulated in these cells so that, when conditions are restored, [K +]i returns to control levels, and how water movements mask or accentuate the observed changes in [K +]i [3, 4]. In that the recovery of [K +]i following cation injection occurs similarly

5 198 H. Kettenmann et al.: Potassium Uptake Mechanisms whether the injected ion is Na +, Li +, or tetramethylammonium ([7] and Kettenmann et al. in preparation), it would appear that recovery is not primarily dependent on the activity of the N a + IK + pump. The further use of ion-sensitive electrodes offers a promising approach to study these mechanisms. At this point, it appears that glial membranes are well suited to play an important role in K + homeostasis in the nervous system. Acknowledgments. We thank B. Berger for skillful technical assistance. Supported by Hermann and Lilly Schilling-Stiftung, Alexander von Humboldt Stiftung (RKO) and Deutsche Forschungsgemeinschaft (Ke 3291/1). References 1. Coles JA, Orkand RK (1983) Modification of potassium movement through the retina of the drone (apis melifera d) by glial uptake. J Physiol (Lond) 340: Coles JA, Tsacopoulos M (1979) K+ activity in photoreceptors, glial cells, and extracellular space in the drone retina: changes during photostimulation. J Physiol (Lond) 290: Dietzel I, Heinemann U, Hofmeier G, Lux HD (1980) Transient changes in the size of the extracellular space in the sensorimotor cortex of cats in relation to stimulus induced changes in potassium concentration. Exp Brain Res 40: Dietzel I, Heinemann U, Hofmeier G, Lux HD (1982) Stimulus-induced changes in extracellular Na+ and Cl- concentration in relation to changes in the size of the extracellular space. Exp Brain Res 46: Gardner-Medwin AR (1980) Membrane transport and solute migration affecting the brain cell microenvironment. In: Nicholson C (ed) Dynamics of the brain cell microenvironment. Neurosci Res Program Bull 18: Gardner-Medwin AR (1983) Analysis of potassium dynamics in brain tissue. J Physiol (Lond) 335: Grossman RG, Seregin A (1977) Glial-neural interaction demonstrated by the injection of Na + and Li + into cortical glia. Science 195: Hertz L (1978) An intense potassium uptake into astrocytes its further enhancement by high concentrations of potassium and its possible involvement in potassium homeostasis at the cellular level. Brain Res 145: Kettenmann H, Sonnhof U, Schachner M (1983) Exclusive potassium dependence of the membrane potential in cultured mouse oligodendrocytes. J Neurosci 3: Kuffler SW, Nicholls JG, Orkand RK (1966) Physiological properties of glial cells in the central nervous system of Amphibia. J Neurophysiol 29: Orkand RK, Nicholls JG, Kuffler SW (1966) Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of Amphibia. J Neurophysiol29: Sommer I, Schachner M (1981) Monoclonal antibodies (01 to 04) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev BioI 83: Sonnhof U, Richter DW, Taugner R (1977) Electrotonic coupling between frog spinal motoneurons. An electrophysiological and morphological study. Brain Res 138: Sonnhof U, Foerderer R, Schneider W, Kettenmann H (1982) Cell puncturing with a step motor driven manipulator with simultaneous measurement of displacement. Pflugers Arch 392: Sykova E, Orkand RK (1980) Extracellular potassium accumulation and transmission in frog spinal cord. Neuroscience 5: Thomas RC (1982) Snail neuron intracellular ph regulation. In: Nuccitelli R, Deamer DW (eds) Intracellular ph: Its measurement, regulation, utilization in cellular functions. Liss, New York, pp Treherne JE (1981) Glial-neurone interactions. J Exp Bioi Varon SS, Somjen GG (1979) Neuron-glia interactions. Neurosci Res Program Bull 17: 1-239