Neuroscience Letters, 117 (1990) 295-299

295

Elsevier Scientific Publishers Ireland Ltd. NSL 07153

Cytosolic calcium during glucose deprivation in hippocampal pyramidal cells of rats T h o m a s K n 6 p f e l 1, A n d r e a s S p u l e r 2, P e t e r G r a f e 2 a n d B e a t H . G / i h w i l e r l 1Brain Research Institute, Zurich (Switzerland) and :Department of Physiology, University of Munich, Munich (F.R.G.)

(Received 17 May 199(I;Accepted 1 June 1990) Key words: Hypoglycemia; Hippocampus; Intracellular recording; Intracellular calcium; Fura-2; Sulfo-

nylurea Glucose deprivation (GD) results in a hyperpolarization by turning on a potassium conductance (gK,GD) in hippocampal CA3 pyramidal cells. We used combined intracellular and microfluorometric recording techniques to evaluate whether gK.GDis activated by a rise in the concentration of intracellular calcium ([Ca2+]i). We found that the activation of gK.GDis only followed, but not preceded by a rise in [Ca2+]i. Furthermore, gK,ODis not blocked by the sulfonylurea glibenclamide, a blocker of ATP-regulated potassium conductance. We conclude that activation of gK,ODdoes not simply reflect breakdown of the calcium of ATP homeostasis, but on the contrary might represent an active restoring mechanism which delays the pathological consequences of sustained glucose deficiency.

The m a m m a l i a n brain relies on a persistent supply o f glucose. Lack o f this energydelivering substrate (glucose deprivation, G D ) results in a loss o f consciousness and an isoelectric electroencephalogram [3]. Hypoglycemic conditions also reduce the intracellular concentration o f A T P ([ATP]i) and raise the intracellular concentration o f Ca 2+ [3, 6]. Both increases in [Ca2+]i and decreases in [ATP]i have been shown to activate neuronal potassium conductances and are therefore possible activators o f gr~,GD. C o m b i n e d intracellular and microfluorometric recordings o f m e m b r a n e properties and [Ca2+]i respectively, were used to investigate these possibilities. Slices o f h i p p o c a m p u s were obtained from 5 to 6-day-old rats and cultured on glass coverslips for 3-5 weeks [4, 5]. F o r electrophysiological experiments, cultures were transferred to a temperature-controlled (32°C) perfused c h a m b e r m o u n t e d on the stage o f an inverted microscope and superfused at 0.5 ml.min-1 with T y r o d e solution at 32°C containing (in mM): N a + 148.9, K + 2.7, C1- 152.2, Ca 2+ 3.8, M g 2+ 2.5, H C O 3 - 11.6, H2PO4- 0.4, D-glucose 5.6. CA3 pyramidal cells were impaled with a microelectrode filled with 2 M K-methyl-SO4 (tip resistance 45-55 Mr2) and singleCorrespondence: T. Kn6pfel, Institut fiir Hirnforschung der Universitfit Ziirich, August-Forel-Strasse 1, CH-8029, Zurich, Switzerland.

0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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r n e r n b r ~ potential (rnV) Fig. 1. Effect of glucose deprivation on membrane properties of CA3 pyramidal cells in hippocampal slicecultures. A pyramidal cell was voltage-damped at about - 6 0 mV and superfused with a solution containing 1 /~M TTX. The current-voltage relationship of the cell membrane was assessed by 150 ms voltage pulses to between - 130 and - 5 0 mV. The current induced by glucose deprivation (loo) was evaluated by digitally subtracting the current flowing through resting conductances from the current measured during glucose deprivation. IOD was assessed first with 2.7 mM (open symbols) and then with 8.1 mM K + (filled circles) in the superfusate. The current reversed at values close to the predicted potassium reversal potentials of - 104 mV and - 7 5 mV calculated for 2.7 mM and 8.1 mM external, respectively, and 140 mM internal K +.

electrode current- or voltage-clamped using a Axoclamp-2 amplifier (Axon Instruments Inc.; switching frequency 2 kHz, clamp-gain 2.5-5 nA/mV). GD was achieved by switching from the control superfasate containing 5.6 mM glucose and 1 ~tM TTX to a glucose-free solution and resulted after a variable time period (15.5+3.6 min, n = 38) in a hyperpolarization of the neuronal membrane, as seen in acute hippocampal slices [11]. Accordingly, in voltage-clamped cells, GD induced an outward current (Ioo). The dependency of this current on membrane potential as well as extracellular concentration of potassium suggests that /GD substantially represents a potassium conductance [11] (Fig, 1). Following return to a glucose containing solution, lad rapidly declined. This decline was sometimes preceded by a brief further hyperpolarization which probably reflects the activation of an electrogenic transport (cf. Fig. 2). To investigate the temporal relation between [Ca2+]i and gK,GD we combined intracellular recordings with microfluorometric measurements of [Ca2+]i using the fluorescent Ca 2+ indicator fura-2 which was intracellularly injected through the recording microelectrode [8] (n = 8). These experiments clearly established that the onset of the GD-induced hyperpolarizations preceded by several minutes any rise in [Ca2+]i (Fig. 2A). Changes in [Ca2+]i were more striking when the cells were depolarized by constant current injection. In these depolarized cells, the steady state [Ca2+]i was elevated, most likely because of an activation of voltage-gated Ca2+-conductances [7]. Under these conditions, GD repolarized the membrane and this hyperpolarization was initially accompanied by a drop in [Ca2+]i (Fig. 2B). Only later did [Ca2+]i rise.

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Fig. 2. Effect of glucose deprivation on cytosolic free Ca 2+ ([Ca2+]i) of a CA3 pyramidal cell. A: under current-clamp conditions (membrane potential - 65 mV), glucose deprivation (GD) induced a hyperpolarization which was followed only after about 200 s by a significant rise in [Ca2+]i. Reperfusion with glucosecontaining solution restored membrane potential as well as cytosolic calcium. B: depolarization of the same neuron to about - 5 0 mV (by constant injection of 0.2 nA current through the recording electrode) resulted in a rise of [Ca2+]i. Under these conditions, glucose deprivation induced a hyperpolarization which was first accompanied by a fall in [Ca2+]i. Only subsequently did [Ca2+]i rise. C: the amplifier was then switched to voltage-clamp mode (only a partial voltage-clamp was achieved due to the high resistance of the fura-2 electrode). Under these conditions, glucose deprivation resulted in an outward current followed by a delayed rise of [Ca2+]i. After return to glucose containing solution the membrane of the cell was clamped to a more negative potential which resulted in a fall of [Ca2+]~. All recordings were from the same cell. [Ca2+]i was measured every 20 s during the recording. Onset of IGD indicated by vertical broken lines. To measure [Ca2+]~ and membrane potential simultaneously, neurons of the hippocampal CA3 region were impaled with thin-walled microelectrodes the very tip of which contained the calcium indicator fura-2 (1 mM) and 150 mM K-methyl-SO4 and backfilled with 2 M K-methyl-SO4. After intracellular injection of the dye ( - 0 . 3 to - 0 . 6 nA constant current for 20--40 min) epifluorescence was excited either at 366 or 405 nm using a mercury arc lamp. To measure the fluorescence intensity, an image of the cell body was projected through a 470 nm barrier filter onto a photodiode. To prevent bleaching of the dye, illumination was restricted to 100 ms intervals repeated at 0.05 Hz. Calculation of [Ca2+]~ from fura-2 fluorescence intensities was based on a modified ratio method [8] using calibration constants evaluated from calibration solutions (pH 7.4) containing 131 mM K +, 10 mM Na ÷, 1 mM Mg 2+, 123.5 mM CI-, 5 mM HEPES, 10 pm fura-2, 5 mM of the Ca 2÷ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and varying concentrations of free Ca ~+ (free [Ca 2÷] was calculated using a Kd of 107 nM for BAPTA).

T h e initial fall in [Ca2+]i c a n be e x p l a i n e d by a c l o s u r e o f t h e v o l t a g e - g a t e d c o n d u c t a n c e s d u e to t h e r e p o l a r i z a t i o n o f the m e m b r a n e . I n a g r e e m e n t w i t h this h y p o t h e s i s t h e initial d e c r e a s e in [Ca2+]i w a s p r e v e n t e d by c l a m p i n g the p o t e n t i a l at the s a m e r e l a t i v e l y d e p o l a r i z e d level (Fig. 2C). V o l t a g e j u m p s to m o r e n e g a t i v e m e m b r a n e p o t e n t i a l s w e r e , h o w e v e r , f o l l o w e d b y a d r o p in [Ca2+]i (see Fig. 2C). H y p o g l y c e m i a (in situ) i n d u c e s a rise in the e x t r a c e l l u l a r c o n c e n t r a t i o n o f K ÷ w h i c h is f o l l o w e d , b u t n o t p r e c e d e d , by a fall in t h e e x t r a c e l l u l a r c o n c e n t r a t i o n o f C a 2÷ [6]. T o g e t h e r w i t h o u r m e a s u r e m e n t s ofgK,GD a n d [Ca2+]i this s t r o n g l y s u g g e s t s t h a t gK,GD is n o t a c t i v a t e d by a rise in [Ca2+]i. H o w e v e r , o u r d a t a d o n o t d i f f e r e n t i a t e

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between an extra- and/or intracellular origin of the late rise in [Ca2+]i. Hypoxia, an other model for energy deprivation, has been proposed to increase a potassium conductance resulting from breakdown of Ca 2+ homeostasis [9]. We presume that gK,GD and the hypoxia-induced g~: are either produced by distinct ion channels, or that the latter conductance is also not activated by a rise in [Ca 2+]i. An alternative activator of gK,GD could be ATP. An ATP-dependent potassium conductance was first described in heart [10], and more recently shown to exist in cortical neurones [2]. ATP-sensitive potassium channels have been demonstrated to underlie increased hypothalamic firing following an increase in extracellular glucose [1] and are typically blocked by sulphonylureas such as glibenclamide [12]. Glibenclamide (1-10 gM) failed to prevent an increase in gK,GD in hippocampal pyramidal cells (Fig. 3, n = 15). It has previously been observed that the ATP content of cortical tissue can be unchanged during severe hypoglycemic depression of the EEG [3] and is reduced only after hypogiycemic changes in extracellular ion concentrations [6]. Together with our observation this latter finding suggests that gK,GD is not activated by a fall in [ATP]i. At present, the direct link between GD and IGD is unknown. It is unlikely that IGD is turned on by extraceUular accumulation of ions, given the low diffusion barriers in these thin cultures. More conceivable is a shift in the phosphorylation/dephosphorylation equilibrium of a potassium channel. Such a conductance might be inactive in a phosphorylated state at rest and activated following dephosphorylation under hypoglycemic conditions. The functional implications of an activation ofgK,GD and the resulting hyperpolarization of the neuronal membrane might be two-fold. First, hyperpolarization will reduce energy consumption by reducing the probability of discharges of action potentials. This block of activity might be involved in the loss of consciousness during hypoglycemia (in coma diabeticum). Second, [Ca2+]i will be maintained at low levels due to closure of voltage-gated Ca2+-conductances. ThuS, gK,GD might serve as an emergency measure which delays the onset of pathological conditions leading to Ca 2+ accumulation and eventually cell death. We thank L. Rietsehin, R. Emch and R. Schoeb for their excellent technical help and Dr. S.M. Thompson for critical reading of the manuscript. This study was supported by the Swiss National Science Foundation (Grant N641).

299 1 Ashford, M.L.J., Boden, P.R. and Treherne, J.M., Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive K + channels, Pfliigers Arch., 415 (1990) 479-483. 2 Ashford, M.L.J., Sturgess, C.N., Trout, N.J., Gardner, N.J. and Hales, C.N., Adenosine-5'-triphosphate-sensitive ion channels in neonatal rat cultured central neurones, Pflfiger's Arch., 412 (1988) 297 304. 3 Feise, G., Kogure, K., Busto, R., Scheinberg, P. and Reinmuth, O.M., Effect of insulin hypoglycemia upon cerebral energy metabolism and EEG activity in the rat, Brain Res., 126 (1976) 263- 280. 4 Gfihwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329 342. 5 Gfihwiler, B.H. and Kn6pfel, T., Cultures of brain slices. In H. Jahnsen (Ed.) Preparations of Vertebrate Central Nervous System in Vitro, Wiley, 1990, pp. 77. 6 Harris, R.J., Wieloch, T., Symon, L., and Siesj6, B.K., Cerebral extracellular calcium activity in severe hypoglycemia: Relation to extracellular potassium and energy state, J. Cereb. Blood Flow Metab., 4 (1984) 187 193. 7 Kn6pfel, Y., Charpak, S., Brown, D.A. and G/ihwiler, B.H., Cytosolic free calcium in hippocampal CA3 pyramidal cells. In J. Storm-Mathisen, J. Zimmer and O.P. Ottersen (Eds.), Progress in Brain Research, Vol. 83, Elsevier, Amsterdam, 1990, pp. 189 195. 8 Kn6pfel, T., Vranesic, l., Gfihwiler, B.H. and Brown, D.A., Muscarinic and beta-adrenergic depression of the slow Ca 2+ activated potassium conductance in hippocampal CA3 pyramidal cells is not mediated by a reduction of depolarization-induced cytosolic Ca 2~ transients, Proc. Natl. Acad. Sci. U.S.A., in press. 9 Leblond, J. and Krnjevic, K., Hypoxic changes in hippocampal neurons, J. Neurophysiol., 62 (1989) 113. 10 Noma, A., AYP-regulated K + channels in cardiac muscle, Nature, 305 (1983) 147 148. 11 Spuler, A., Endres, W. and Grafe, P., Glucose depletion hyperpolarizes guinea pig hippocampal neurons by an increase in potassium conductance, Exp. Neurol., 100 (1988) 248-252. 12 Surges, N.C., Ashford, M.L.J., Cook, D.L. and Hales, C.N., The sutfonylurea receptor may be an ATP-sensitive potassium channel, Lancet, 2 (1985) 474-475.

Cytosolic calcium during glucose deprivation in hippocampal pyramidal cells of rats.

Glucose deprivation (GD) results in a hyperpolarization by turning on a potassium conductance (gK,GD) in hippocampal CA3 pyramidal cells. We used comb...
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