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J Neurophysiol 81: 174-183, 1999;
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The Journal of Neurophysiology Vol. 81 No. 1 January 1999, pp. 174-183
Copyright ©1999 by the American Physiological Society

Intraneuronal [Ca2+] Changes Induced by 2-Deoxy-D-Glucose in Rat Hippocampal Slices

S. Tekkök1, I. Medina2, and K. Krnjevic'1

1 Department of Anaesthesia Research and Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada; and 2 Institut National de la Santé et de la Recherche Médicale Unité 29, 75014 Paris, France

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Tekkök, S., I. Medina, and K. Krnjevic'. Intraneuronal [Ca2+] changes induced by 2-deoxy-D-glucose in rat hippocampal slices. J. Neurophysiol. 81: 174-183, 1999. Temporary replacement of glucose by 2-deoxyglucose (2-DG; but not sucrose) is followed by long-term potentiation of CA1 synaptic transmission (2-DG LTP), which is Ca2+-dependent and is prevented by dantrolene or N-methyl-D-aspartate (NMDA) antagonists. To clarify the mechanism of action of 2-DG, we monitored [Ca2+]i while replacing glucose with 2-DG or sucrose. In slices (from Wistar rats) kept submerged at 30°C, pyramidal neurons were loaded with [Ca2+]-sensitive fluo-3 or Fura Red. The fluorescence was measured with a confocal microscope. Bath applications of 10 mM 2-DG (replacing glucose for 15 ± 0.38 min, means ± SE) led to a rapid but reversible rise in fluo-3 fluorescence (or drop of Fura Red fluorescence); the peak increase of fluo-3 fluorescence (Delta F/F0), measured near the end of 2-DG applications, was by 245 ± 50% (n = 32). Isosmolar sucrose (for 15-40 min) had a smaller but significant effect (Delta F/F0 = 94 ± 14%, n = 10). The 2-DG-induced Delta F/F0 was greatly reduced (to 35 ± 15%, n = 16) by D,L-aminophosphono-valerate (50-100 µM) and abolished by 10 µM dantrolene (-4.0 ± 2.9%, n = 11). A substantial, although smaller effect, of 2-DG persisted in Ca2+-free 1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) medium. Two adenosine antagonists, which do not prevent 2-DG LTP, were also tested; 2-DG-induced Delta F/F0 (fluo-3) was not affected by the A1 antagonist 8-cyclopentyl-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX 50 nM; 287 ± 38%; n = 20), but it was abolished by the A1/A2 antagonist 8-SPT; 25 ± 29%, n = 19). These observations suggest that 2-DG releases glutamate and adenosine and that the rise in [Ca2+] may be triggered by a synergistic action of glutamate (acting via NMDA receptors) and adenosine (acting via A2b receptors) resulting in Ca2+ release from a dantrolene-sensitive store. The discrepant effects of sucrose and 8-SPT on Delta F/F0, on the one hand, and 2-DG LTP, on the other, support other evidence that increases in postsynaptic [Ca2+]i are not essential for 2-DG LTP.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

2-Deoxyglucose (2-DG) competes with glucose for uptake into brain cells and phosphorylation by hexokinase (Pirolo and Allen 1986; Sols and Crane 1954). Because 2-DG-6-phosphate is not further metabolized, 2-DG suppresses glycolysis and ATP is rapidly depleted (Tower 1958). As a result, cellular mechanisms are depressed, especially those that are very dependent on glycolytically generated ATP, such as the Na/K pump (Paul et al. 1979), ATP-sensitive K channels (Weiss and Lamp 1987), the control of cell volume by Na-K-Cl cotransport (Flatman 1991), and Na/H exchange (Wu and Vaughan-Jones 1994).

When applied to hippocampal slices, 2-DG reversibly blocks synaptic transmission (Bachelard et al. 1984). In the CA1 region, the recovery of transmission is regularly followed by sustained potentiation (2-DG LTP) (Tekkök and Krnjevic' 1995). Like other types of long-term potentiation (LTP) (Bliss and Collingridge 1993; Hammond et al. 1994; Larkman and Jack 1995; Lynch et al. 1983), 2-DG LTP is Ca2+-dependent (Tekkök and Krnjevic' 1996); a unique feature, however, is its association with postsynaptic hyperpolarization (Zhao et al. 1997) rather than the usual depolarization. We therefore wanted to know whether 2-DG produces detectable changes in [Ca2+]i of postsynaptic CA1 neurons.

We soon found that 2-DG consistently raises [Ca2+]i in the pyramidal cells. Albeit in keeping with the idea that increases in postsynaptic [Ca2+]i are essential for LTP, the changes in [Ca2+]i could be incidental, as suggested by separate experiments (Zhao and Krnjevic' 1997) in which intracellular ethylene glycol-bis(beta -aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), which suppressed tetanic LTP, failed to prevent 2-DG LTP. Nevertheless, if postsynaptic [Ca2+]i does play an essential role, agents that prevent or modify 2-DG LTP can be expected to have similar effects on the 2-DG-induced rise in [Ca2+]i.

For example, like tetanic LTP (Bliss and Collingridge 1993; Malenka and Nicoll 1993), 2-DG LTP is prevented by N-methyl-D-aspartate (NMDA) antagonists, such as 2-amino-5-phosphonovalerate (APV) (Tekkök and Krnjevic' 1995). Unlike tetanic LTP, 2-DG LTP is suppressed by dantrolene (O'Mara et al. 1995; Tekkök and Krnjevic' 1996), a blocker of Ca2+ release from internal stores (Henzi and McDermott 1992; van Winkle 1976). On the other hand, adenosine antagonists that prevent the 2-DG-induced block of synaptic transmission do not abolish 2-DG LTP (Krnjevic' and Tekkök 1996). Because these agents are known to influence [Ca2+]i, they were also tested in the present experiments. Preliminary reports of the results were published in two abstracts (Krnjevic' et al. 1996; Tekkök et al. 1997).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Preparation of slices

Brains were removed from male Wistar rats aged 15 or 30 postnatal days (P15-P30) that had been killed by decapitation. Transverse hippocampal slices (450-µm thick) were cut in ice-cold artificial cerebrospinal fluid (ACSF) with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK). The slices were allowed to stabilize for 1-2 h in ACSF at room temperature. Standard ACSF contained (in mM) 126 NaCl, 3.5 KCl, 2 CaCl2, 1.3 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 10 glucose; it was saturated with 95% O2-CO2 (pH of 7.3). Osmolality (~295 mOsm) was checked with a Wescor vapor pressure osmometer (Logan, UT).

Dye loading in slices

Hippocampal slices were transferred to a recording chamber located on the stage of an Axioscope Karl Zeiss microscope (×40 water-immersion objective) and superfused with standard ACSF (2 ml/min) at 30°C. The pyramidal stratum and cells were readily identified under the microscope; a group of such cells was loaded with the Ca2+-sensitive dye fluo-3 (Kao et al. 1989) by a prolonged (15-20 min) but very localized application of fluo-3 acetoxymethyl ester (AM). The dye was ejected slowly from a micropipette by 0.3- to 1-s pressure pulses applied at 0.2 s-1 (from a Picospritzer, General Valve, Fairfield, NJ). A stock solution of fluo-3 AM (0.5 mM) was prepared by adding 4.6 µl dimethyl sulfoxide (DMSO) and 110 µl H2O to 50 µg of fluo-3 AM. Just before use a 7 µl aliquot of stock solution was diluted to 1-10 µM by adding the required amount of ACSF. The final concentration of DMSO was <0.1%. Further details of this method were recorded in Leinekugel et al. (1995).

[Ca2+]i measurements

The fluorescence measurements were done with a confocal laser scanning microscope (MRC BIORAD 600) equipped with an argon-krypton laser and a photomultiplier. Excitation was delivered at 488 nm and the emission intensity was measured at >500 nm. In two experiments Fura Red fluorescence (>800 nm), which is reduced by Ca2+ (Lipp and Niggli 1993), was used to confirm that the fluo-3 fluorescence changes were indeed caused by corresponding increases in [Ca2+].

The changes in fluorescence were analyzed off-line on a computer with the program Fluo (IMSTAR, Paris, France). The fluorescence was measured within the limits of a given cell (mainly the soma) drawn on the initial control image, as illustrated in Fig. 1A; data were thus obtained within the same cell (or within several labeled cells) in successive images at regular intervals of 20-30 s, or 2-5 s when strong depolarizing agents were applied. The difference (Delta F) between the mean fluorescence measured in a given image and the corresponding control value for each cell (F0; mean of 10 baseline images) was expressed as a percentage or fraction of the control (Delta F/F0). During prolonged recordings small adjustments of focus or cell position were occasionally necessary, especially when superfusing slices with Ca2+-free medium.


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  		FIG. 1. 2-Deoxy-D-glucose (2-DG) causes marked, but reversible increase in fluorescence of Ca2+-sensitive fluo-3 in CA1 neurons, in slice from 30-postnatal-day-old (P30) rat. A: pyramidal layer at high magnification after very localized prolonged release of fluo-3 acetoxymethyl ester (AM; 20 µM) from micropipette. Left: initial control image. Right: much increased fluorescence elicited by 2-DG [10 mM 2-DG replaced glucose in artificial cerebrospinal fluid (ACSF) for 15 min]. Different intensities of fluorescence in control image reflect variable efficacy of fluo-3 loading in 10 different neurons (labeled in image at right). Fluorescence in each of these 10 cells was measured at regular 20-s intervals throughout experiment. B: time course of 2-DG-induced increase in fluorescence (Delta F/F0; where F0 is mean fluorescence during initial control recording) in 3 cells (areas outlined in both images in A) showing the smallest (4), largest (5), and intermediate (10) Delta F/F0, as well as effects of subsequent brief application of mixture of kainate (KA; 50 µM) and N-methyl-D-aspartate (NMDA; 100 µM) from a micropipette. C: mean ± SE for all 10 neurons.

2-DG or other drug applications

Most agents were applied in the bath. For control tests of a cell's viability and responsiveness, brief pulses of glutamate (0.1-1 mM with 100 µM glycine) or a mixture of NMDA (10-100 µM) and kainate (KA; 50 µM) were applied by pressure from a micropipette. Such near-maximal responses were also useful for comparison with changes elicited in the same cell by 2-DG. In some experiments hypertonic saline (3 Osm), which stimulates glutamate release (e.g., Malgaroli and Tsien 1992), was applied instead of the excitatory aminoacids.

Fluo-3, fluo-3 AM, and Fura Red AM were obtained from Molecular Probes (Eugene, OR); 8-cyclopentyl-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX; made up in a 0.5 mM DMSO stock solution) and 8-(p-sulfophenyl)-theophylline (8-SPT) from Research Biochemicals (Natick, MA), and 2-DG, D,L-APV and dantrolene were obtained from Sigma Chemical (St. Louis, MO; a 20 mM dantrolene stock solution was prepared in DMSO, avoiding exposure to strong light).

As a check for any direct interference between dantrolene and fluo-3 fluorescence, NMDA and dantrolene were applied to cultured hippocampal neurons (prepared as described by Medina et al. 1996). Increases in [Ca2+]i evoked by brief microejections of NMDA, together with 1 µM tetrodotoxin (TTX) and 100 µM Cd2+ to block voltage-gated Na+ and Ca2+ channels, were recorded at 5-min intervals in the absence or presence of 10 µM dantrolene; in five control runs (no dantrolene), the second response was virtually unchanged (98 ± 4.9% of first, means ± SE); in six test runs, the second NMDA response (after applying 10 µM dantrolene for 4 min) was a little smaller (89 ± 3.9% of first), but the paired difference (9 ± 6.2%) was not significant.

All values given are means ± SE and the significance of differences was assessed by Student's t-test or, when more appropriate, the Fisher-Behrens d-test, for populations with unequal variances (Campbell 1989).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Bath applications of 2-DG (10 mM, replacing glucose in ACSF for 10-20 min) regularly elicited a change in fluo-3 fluorescence (indicative of a rise in [Ca2+]i) in various hippocampal pyramidal neurons, including CA1 neurons in slices from both mature (P30) and immature (P15) rats, CA3 neurons in P5 slices, and cultured neurons. Because they were the most relevant for the electrophysiological studies on 2-DG LTP (Tekkök and Krnjevic' 1995, 1996), only the observations on the CA1 region of slices from mature rats were described in detail.

Effects of 2-DG on fluorescence in slices from mature rats (P30)

We made a detailed examination of 32 CA1 pyramidal layer neurons, loaded with fluo-3, in 8 slices kept at 30°C. Representative results are illustrated in Fig. 1. In 10 labeled cells, identified by visual inspection (Fig. 1A, left, control image), the fluorescence rose when ACSF glucose (10 mM) was substituted by equimolar 2-DG (Fig. 1A, image at right). The three plots in the graph below (Fig. 1B) illustrate the full range of changes observed in these cells, the smallest and largest relative increases (cells 4 and 5, respectively), as well as an intermediate response (cell 10). For each cell, the relative fluorescence change (Delta F; from its mean control value F0) was plotted as Delta F/F0 in the graph in Fig. 1B. The bottom graph (Fig. 1C) summarizes the mean data (±SE) obtained from all 10 cells.

Typically, the fluorescence started rising within 2-3 min and reached a peak near the end of the applications of 2-DG. For the 10 cells of Fig. 1, the mean value of peak Delta F/F0 was 75 ± 18.0%. The fluorescence returned slowly toward the initial baseline over the next ~20 min (Delta F/F0 was 3.1 ± 0.92% by 25 min). A brief local ejection of a mixture of KA (50 µM) and NMDA (10 µM; from a micropipette) produced an even sharper, but much more quickly reversible, fluorescence peak in the same 10 cells (80 ± 16%), indicating that the cells remained viable after such applications of 2-DG, confirming previous electrophysiological studies (Bachelard et al. 1984; Tekkök and Krnjevic' 1995, 1996). The 2-DG-induced Delta F/F0 averaged 92 ± 9.5% (n = 10) of the peak responses evoked in the same cells by KA/NMDA.

The data obtained from 32 neurons in P30 slices are summarized by histograms in Fig. 2A: 2-DG labels the peak values of Delta F/F0 (245 ± 44.6%) recorded near the end of the applications of 2-DG (15 ± 0.30 min). In 25 cells, half-decay was observed after washing for 9.1 ± 0.79 min. In a P30 slice kept at room temperature (25°C), two CA1 neurons showed comparable changes of fluorescence, with increases by 530 and 250%, respectively.


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  		FIG. 2. 2-DG elicits larger increases in fluo-3 fluorescence in slices from mature (P30) than in slices from immature (P15) rats, as well as reductions in Fura Red fluorescence, which is depressed by Ca2+. Mean data (+SE) from 32 CA1 neurons in P30 slices (A) and 11 CA1 neurons in P15 slices (B). Mean fluorescence during 5-min period preceding onset of 2-DG application (Control) is reference level for 2-DG-evoked changes: 2-DG is mean value recorded at end of application, and Wash is mean level during final 5 min of wash. C: data from 2 Fura Red-labeled neurons in P30 slice (open circle  and black-square). There is a marked 2-DG-induced, at least partly reversible, drop in fluorescence; larger effect of KA + NMDA (applied as in Fig. 1) was only partly reversible.

For control, 2-DG was applied to five cells (in 2 P30 slices) loaded with Fura Red AM; the fluorescence of Fura Red diminishes when [Ca2+] rises (Lipp and Niggli 1993). As illustrated by data from two cells in Fig. 2C, there was a temporary reduction in fluorescence, comparable in its time course to increases in fluo-3 fluorescence.

Changes in fluorescence in slices from immature rats or in cultured hippocampal neurons

In P15 slices, reversible increases in fluo-3 signal were seen in all 11 CA1 neurons studied (Fig. 2B). The average increase (165 ± 39%) was one-third less than in P30 slices, but this difference was not statistically significant. The rise in fluorescence began relatively late (after 7.4 ± 1.3 vs. 3.8 ± 0.33 min in P30 slices; P < 0.01) and the halfdecay time was nearly twice as long (16 ± 2.6 min vs. 9.1 ± 0.79 min, P < 0.01). In P15 slices, hypertonic tests also evoked nonsignificantly smaller increases in fluorescence (87 ± 19%, n = 12; cf. 150 ± 46%, n = 8 for P30 slices under comparable conditions). A marked Delta F/F0 was also seen when 2-DG was applied to three cultured hippocampal neurons kept at room temperature (not shown).

Sucrose-containing glucose-free ACSF

In several experiments glucose was replaced with sucrose instead of 2-DG, for periods of 15-40 min. Such glucose-free perfusions resulted in smaller, more variable and typically delayed increases in fluo-3 fluorescence, as illustrated by data from three cells in Fig. 3A. The overall peak changes recorded in 10 CA1 neurons (all in P30 slices kept at 30°C) are summarized by the histograms in Fig. 3B. The average increase (by 94 ± 14%) was approximately one-third of the mean increase elicited by 2-DG (down by 62 ± 11%; d-test, P < 0.01). The mean Delta F/F0 induced by sucrose was 64 ± 3.8% of that produced by KA/NMDA in the same 10 cells; this was significantly smaller than in experiments in which glucose was replaced by 2-DG (d-test, P < 0.01).


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  		FIG. 3. Equimolar replacement of glucose with sucrose can also enhance fluo-3 fluorescence. A: different symbols indicate changes recorded in 3 CA1 neurons in P30 slice. After long control recording, glucose-free ACSF (containing 10 mM sucrose) was superfused for 38 min. B: mean response (+SE) to such glucose-free ACSF (data from 10 CA1 neurons in 3 slices from P30 rats).

Effects of D,L-APV

To ensure full block of NMDA receptors, 100 µM D,L-APV was superfused for >= 10-15 min before testing 2-DG on 16 cells in eight P30 slices. As illustrated in Fig. 4, A and B, in the presence of APV the responses to either 2-DG or KA/NMDA were much diminished. The 2-DG-induced increases in fluo-3 fluorescence were smaller by nearly 90%, peak Delta F/F0 (35 ± 15%) being only just significant (P < 0.05). In the seven cells in which KA/NMDA was also tested, peak Delta F/F0 was reduced to 69 ± 3.5% (d-test, P < 0.05). A similar or even more complete suppression of 2-DG's effect was observed in P15 slices, both at 30°C (8 cells; Fig. 4) and at room temperature (9 cells).


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  		FIG. 4. NMDA antagonist D,L-aminophosphonovalerate (APV) depresses 2-DG-induced increases in fluo-3 signal in CA1 neurons in slices from adult (P30) and immature (P15) rats. A: after recording a stable baseline signal from 3 cells, APV (100 µM) and 2-DG (10 mM; replacing glucose) were bath-applied as indicated. Note relatively small responses to 2-DG and KA + NMDA (cf. Fig. 1). B: mean data (+SE) obtained from 16 P30 cells and 8 P15 cells, also at 30°C. C: APV, effect of APV alone (during 5 min preceding addition of 2-DG); APV + 2-DG, largest rise evoked by 2-DG (in presence of APV); Wash, during last 5 min of wash.

Ca2+-free superfusion

In similar experiments on submerged slices, Ca2+-free medium containing 100 µM EGTA fully suppressed synaptic transmission within 2-3 min; but 2-DG LTP was prevented only by very prolonged Ca2+-free treatment (90 min) (Tekkök and Krnjevic' 1996). In the present experiments, such long recordings were not feasible; even shorter Ca2+-free perfusions led to changes in cell size or shape, which hindered accurate monitoring of cellular fluorescence. Nevertheless, it was clear that even in Ca2+-free medium, 2-DG evoked significant increases in fluorescence in most cells.

Thus after preperfusion with nominally Ca2+-free medium (11.5 ± 1.7 min, n = 10), the usual applications of 2-DG (in Ca2+-free medium) to 10 cells in P15 slices resulted in a peak increase by 109 ± 31%, less than one-half that observed in standard ACSF (d-test, P < 0.05). Moreover, the fluorescence peaked early (9.3 ± 1.4 min before the end of the 14-min applications of 2-DG) and decayed rapidly. In P30 slices, 2-DG elicited comparable increases in fluo-3 fluorescence in 8 of 12 cells.

Corresponding reductions in Fura Red fluorescence were evoked in four P30 cells by 2-DG in Ca2+-free medium containing 1 mM EGTA (Fig. 5). As mentioned previously, even such brief pretreatment with Ca2+-free ACSF and EGTA abolishes synaptic transmission, which requires that [Ca2+]o drop by >90% from its standard 2-mM level (Dingledine and Somjen 1981). Judging by the substantial remaining mean Delta F/F0, the 2-DG-induced [Ca2+]i rise is not determined solely by Ca2+ influx.


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  		FIG. 5. Ca2+-free medium does not suppress 2-DG-induced change in fluorescence. Fura-Red fluorescence was measured in 4 cells (P30 slice). Superfusion with Ca2+-free, 1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ACSF began 2 min before start of 15-min 2-DG application, as indicated. Subsequent test of KA + NMDA was less effective than usual, probably owing to residual EGTA.

Dantrolene

At a concentration of 10 µM, dantrolene suppresses 2-DG LTP, but not tetanic LTP (Tekkök and Krnjevic' 1996). By itself it produced no significant change in fluo-3 fluorescence in P30 slices (4.0 ± 7.0%) or in NMDA responses of cultured neurons (see METHODS). However, in the presence of dantrolene, 2-DG failed to elicit a detectable Delta F/F0 in 11 neurons in three P30 slices (as in the 3 cells illustrated in Fig. 6). Overall, there was no significant change (-4.3 ± 2.9%). These cells were fully responsive to KA/NMDA (107 ± 15%, n = 11; Fig. 6)


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  		FIG. 6. Dantrolene fully suppresses 2-DG-evoked increase in fluorescence in P30 slices. Three CA1 neurons in slice superfused with 10 µM dantrolene show no effect of 2-DG, although all 3 responded vigorously to brief pulse of KA + NMDA (KA).

Adenosine antagonists

The following two agents were tested: DPCPX, a high affinity selective antagonist of A1 receptors (Fredholm et al. 1994; Lohse et al. 1987), and 8-SPT, a less selective drug that blocks A1 and A2 receptors (Fredholm et al. 1994; Lewis et al. 1994). Both prevent the 2-DG-induced suppression of synaptic transmission, but not the subsequent LTP (Krnjevic' and Tekkök 1996).

TESTS WITH DPCPX. Superfusion with 50 nM DPCPX led to a small but significant rise in baseline fluorescence (by 34 ± 15%, P < 0.05, in 20 P30 cells in 4 slices). After further adding 2-DG while recording from 19 of these cells, Delta F/F0 rose much further (Fig. 7A), to a mean of 287 ± 38%, not different from the change produced by 2-DG in ACSF (245 ± 45%). When normalized by the hypertonic effect on each cell, the mean response to 2-DG was 127 ± 29% (paired data from 15 cells). DPCPX evidently does not interfere with the action of 2-DG.


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  		FIG. 7. In presence of adenosine A1 antagonist 8-cyclopentyl-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX), 2-DG elicits large increases in fluo-3 signal (in P30 slices). A: time course of fluorescence increase in 3 cells in slice treated with DPCPX (50 nM). B: summary of data obtained from 20 cells in presence of DPCPX. Labels are as described in legend to Fig. 4.

TESTS WITH 8-SPT. The results were quite different in experiments with 2-DG and 8-SPT (on 19 cells; also in 4 slices). By itself 8-SPT (10 µM) produced either no change or only a slight reduction in baseline fluorescence. However, it suppressed the responses to 2-DG cells (Fig. 8); the overall Delta F/F0 was -25 ± 29%. The contrast between the effects of DPCPX and 8-SPT was just as clear when the 2-DG-induced changes in fluorescence were normalized by the responses to hypertonic tests, which were particularly large (Fig. 8A), averaging 200 ± 48% (n = 10); in the presence of 8-SPT, the 2-DG-induced Delta F/F0 was only 22 ± 4.9% (n = 10). This is equivalent to a reduction by 83 ± 5.5% when compared with the Delta F/F0 observed in the presence of DPCPX.


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  		FIG. 8. A1/A2 antagonist 8-(p-sulfophenyl)-theophylline (8-SPT) abolishes 2-DG-induced increase in fluo-3 signal (CA1 neurons in P30 slices). A: different symbols are data from 3 cells showing no effect of 2-DG in presence of 8-SPT (10 µM). Note sharp responses to hypertonic test (HYP). B: summary of data from 19 cells. Labels are as described in legend to Fig. 4.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

2-DG raises neuronal [Ca2+]i

Applications of 2-DG (10-20 min) result in a major, but reversible, increase in neuronal fluo-3 fluorescence. Fluo-3's sensitivity to Ca2+ is well established (Kao et al. 1989). It was used to monitor [Ca2+]i in hippocampal and neocortical neurons in cultures (Burgard and Hablitz 1995; Segal and Manor 1992) and slices (Leinekugel et al. 1995; Markram et al. 1995). Together with corroborating reductions in Fura Red fluorescence (Lipp and Niggli 1993), the fluo-3 data indicate that 2-DG raises neuronal [Ca2+]i, which is in agreement with mostly incidental observations on various cultured cells, such as cardiac myocytes (Eisner et al. 1989; Wang et al. 1995), Ehrlich ascites cells (Rakowska and Wojtczak 1995; Teplova et al. 1993), vascular epithelium (Ziegelstein et al. 1992), and hippocampal neurons (Wang et al. 1994).

Even with the confocal microscope, we could not exclude a possible contribution of fluo-3 signal from nerve endings forming synapses on the observed cell body, but this is unlikely to be a major factor. In some initial experiments, in which four pyramidal neurons were loaded directly with fluo-3 from patch electrodes, 2-DG evoked Delta F/F0's that were quite comparable in magnitude and time course to those reported here.

The reversible rise in [Ca2+]i induced in P30 slices had the following characteristics. It began after 1-2 min and reached a peak near the end of the applications. On washing, it decayed with a half-time of ~9 min. The 2-DG-induced Delta F/F0 being 92 ± 9.5% of that produced in the same cells by KA/NMDA, peak [Ca2+]i was probably near 0.5-1 µM, the ceiling level observed with various high-affinity calcium probes during applications of excitotoxic agents (Burgard and Hablitz 1995; Dubinsky and Rothman 1991; Lei et al. 1992; Segal and Mannor 1993), or even higher in view of the micromolar levels recently observed by Hyrc et al. (1997) with the use of a lower affinity Ca2+ probe.

Mechanism of [Ca2+]i rise induced by 2-DG

2-DG could raise cytoplasmic Ca2+ by emptying Ca2+ stores after the depletion of ATP needed for Ca2+ transport (Teplova et al. 1993; Ziegelstein et al. 1994) or by activating special Ca2+ leak channels (Wang et al. 1995). If a second messenger triggers Ca2+ release, it cannot be NADH (the reduced form of nicotinamide-adenine dinucleotide), which stimulates Ca2+ release from inositol-1,4,5-trisphosphate-3 (IP3)-sensitive stores during hypoxia (Kaplin et al. 1996), because 2-DG suppresses glycolysis and thus would lower NADH levels. Adenosine released after ATP breakdown is another possible mediator of the [Ca2+]i rise, as discussed in the following section.

Effects of various agents on [Ca2+]i rise

BLOCK OF NMDA RECEPTORS. The marked reduction in Delta F/F0 by APV is surprising. Although both 2-DG (Tower 1958) and glucose lack (Gibson et al. 1989; Ogata et al. 1995; Takata et al. 1995) induce glutamate leakage, which could activate Ca2+ influx via NMDA receptors, they also consistently hyperpolarize hippocampal neurons (Knöpfel et al. 1990; Spuler et al. 1989; Zhao et al. 1997). Such hyperpolarization might be expected to prevent activation of NMDA receptors. This result, however, is in keeping with several reports that, even in the presence of Mg2+, glutamate can elicit Ca2+ influx into cells having a quite negative resting potential (Burgard and Hablitz 1995; Garaschuk et al. 1996; Yuste and Katz 1991).

CA-FREE MEDIA. Although more difficult to measure accurately in Ca-free ACSF because cells were not stable, 2-DG-induced Delta F/F0's appeared to be much smaller and their durations especially shorter than in standard ACSF. Nevertheless, the Delta F/F0's were relatively large considering that such a Ca-free medium causes an early block of transmission (Tekkök and Krnjevic' 1996), indicating a major depletion of [Ca2+]o (Dingledine and Somjen 1981). Thus Ca2+ influx probably accounts for only part of the rise in [Ca2+]i.

DANTROLENE. The idea that Ca2+ influx is responsible for only part of the rise in [Ca2+]i is strongly supported by dantrolene's action. Dantrolene neither binds to glutamate receptors (Frandsen and Schousboe 1991) nor depresses NMDA-evoked currents (Lei et al. 1992). Its only known effect is to block Ca2+ release from internal stores (Henzi and MacDermott 1992; Mody and MacDonald 1995; van Winkle 1976). In its presence, NMDA elicits a much smaller rise of [Ca2+]i owing to depression of Ca2+-induced Ca2+ release (Frandsen and Schousboe 1991; Lei et al. 1992; Mody and MacDonald 1995; Segal and Manor 1992). Therefore NMDA receptor-mediated Ca2+ influx triggers internal Ca2+ release (from a dantrolene-sensitive store), which greatly enhances the observed rise in [Ca2+]i (as emphasized by Segal and Manor 1992 and Lei et al. 1992).

ADENOSINE ANTAGONISTS. Hypoglycemia results in the extracellular accumulation of adenosine (Fowler 1993; Lipton and Whittingham 1984; Zhu and Krnjevic' 1993). Judging by the effects of DPCPX and 8-SPT, adenosine mediates two major actions of 2-DG, early suppression of excitatory postsynaptic potentials (EPSPs) (Krnjevic' and Tekkök 1996) and neuronal hyperpolarization (Zhao et al. 1997).

In a variety of cells, adenosine raises [Ca2+]i by activating phospholipase C and IP3 formation (Glowinski et al. 1994; Iredale et al. 1994; Kohl et al. 1990; Porter and McCarthy 1995; Spielman et al. 1992; White et al. 1992). Adenosine may also potentiate IP3 formation initiated by glutamate via NMDA receptors (Sladeczek et al. 1988) by a mechanism akin to the marked synergism between adenosine and several ligands that produce IP3 in striatal neurons (Glowinski et al. 1994; Ogata et al. 1995).

Against expectations, these adenosine antagonists had quite dissimilar effects in the present experiments; apart from causing a minor rise in baseline fluorescence, DPCPX was essentially ineffective, whereas 8-SPT virtually abolished the 2-DG-induced increases in [Ca2+]i. Because it is less selective than DPCPX, 8-SPT would block both A2 and A1 receptors (Fredholm et al. 1994). The present results therefore suggest that the adenosine receptors involved in the 2-DG-induced [Ca2+]i increase are of the A2 and not the A1 type. A variety of evidence suggests that the relevant A2 receptors are likely to be of the A2b subtype. Binding studies have demonstrated their presence in the hippocampus (Collis and Hourani 1993; Lee and Reddington 1986). In hippocampal slices, adenosine raises astrocytic [Ca2+]i by an A2b receptor-triggered mechanism (Porter and McCarthy 1995), most likely involving activation of phospholipase C (Yakel et al. 1992); the rise in [Ca2+]i is suppressed by 8-SPT but not by 8-cyclopentyltheophylline (CPT; a blocker of both A1 and A2a receptors according to Fredholm et al. 1994). That A2b receptors can affect neuronal responses is indicated by the following two previous findings: 1) when applied together with CPT ,adenosine does not depress but rather enhances hippocampal EPSPs (Garaschuk et al. 1992) and 2) the induction of hippocampal LTP is prevented by an A2 antagonist (Sekino et al. 1991). Thus the 2-DG-evoked [Ca2+]i rise may depend on adenosine release and the selective activation of A2b receptors.

An important question is whether 2-DG's effects on neurons could be mediated indirectly by adenosine acting on glia. This intriguing possibility is supported by some experiments on striatal neurons; adenosine stimulates neuronal IP3 formation in slices but not in pure cultures, except when neurons were cocultured with glia (El-Etr et al. 1989). If glia are the primary target for the action of adenosine, neurons may be affected secondarily by the release of a glial messenger, such as arachidonic acid (Delumeau et al. 1991).

Our results are thus consistent with the following sequence of events (summarized in Fig. 9). By depleting cellular ATP, 2-DG causes an early leakage of both glutamate and adenosine; glutamate generates an influx of Ca2+ via NMDA receptors, and adenosine releases Ca2+ from dantrolene-sensitive internal stores by activating A2b receptors and stimulating phospholipase C. Their combined, possibly synergistic, action on internal Ca2+ stores markedly enhances the rise in [Ca2+]i.


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  		FIG. 9. Schematic model of supposed mechanism of 2-DG-induced [Ca2+]i rise in CA1 neurons. 2-DG causes release of both glutamate and adenosine from another (or possibly the same) nerve cell or from a glia. NMDAR, NMDA receptor; PLPC, phospholipase C; A2bR, A2b-type adenosine receptor.

How does the [Ca2+]i rise relate to the 2-DG-induced block of transmission?

Typically, population spikes begin to diminish ~5 min after the start of 2-DG application, drop to 50% after 10 min, and vanish after 13 min. They recover fully only ~12 min after the return to standard ACSF (Tekkök and Krnjevic' 1995). EPSPs begin to fail somewhat later; they always reach a minimum 2-3 min after the end of 2-DG application, but then recover quickly (within another 2-3 min). Thus there is broad agreement in time course of spike depression, increase in fluorescence, and hyperpolarization of CA1 neurons (Zhao et al. 1997).

Could the loss of spikes be due to a Ca2+-evoked hyperpolarization (via GK(Ca))? Probably not because both dantrolene and intracellular EGTA should prevent any rise in somatic [Ca2+]i, yet neither prevents 2-DG-induced transmission block (Zhao and Krnjevic' 1997) or indeed the hyperpolarizations (Zhao et al. 1997). By contrast, DPCPX suppresses hyperpolarizations and greatly delays and reduces transmission block, but not the rise in [Ca]i. Clearly, adenosine is the agent responsible for these effects of 2-DG.

Correlation between the observed increases in [Ca2+]i and 2-DG LTP

The results of some of the tests showed good agreement. The [Ca2+]i rise and 2-DG LTP were both blocked by APV and dantrolene, quite resistant to DPCPX, and relatively insensitive to Ca2+-free medium. These findings could suggest that the observed rise in postsynaptic [Ca2+]i is the trigger for 2-DG LTP at CA1 synapses.

The results of other tests, on the other hand, argue against a simple correlation. The fact that 8-SPT suppresses the [Ca2+]i rise but tends to enhance 2-DG LTP (Krnjevic' and Tekkök 1996) is an obvious major discrepancy. Another is the effect of sucrose. Even prolonged replacement of glucose with sucrose consistently failed to induce LTP (Krnjevic' and Tekkök 1996), whereas sucrose elicited increases in fluorescence in most slices, albeit smaller and more variable than those produced by 2-DG. Such variable effects are in general agreement with the results of previous tests of glucose removal on neuronal [Ca2+]i: either no change, in cultured neurons from striatum (Williams et al. 1995) or hippocampus (except after >12 h) (Cheng et al. 1993), or clear increases in dissociated sensory neurons (Duchen et al. 1990) and CA3 neurons in slices (Knöpfel et al. 1990; Takata et al. 1995).

The somatic [Ca2+]i increases observed in CA1 neurons are thus unlikely to be the trigger for the induction of 2-DG LTP. This conclusion is strongly supported by the recent finding that injections of EGTA into CA1 neurons do not prevent 2-DG LTP (Zhao and Krnjevic' 1997), although in the same cells they suppress tetanic LTP (as originally reported by Lynch et al. 1983). The crucial Ca2+ signal may not be readily visible because it occurs at a distant site (if postsynaptic, in dendritic spines; if presynaptic, in nerve terminals; if extraneuronal, in glia, which then act on neighboring synapses via another mediator).

    ACKNOWLEDGEMENTS

  We are grateful to Dr. Jean-Marie Godfraind for help in the preparation of Fig. 6 and to M. Sweet for photographic skills.

  This research was financially supported by Institut National de la Santé et de la Recherche Médicale and the Medical Research Council of Canada. S. Tekkök had a scholarship from Hacettepe University, Ankara, Turkey.

  Present addresses: I. Medina, Enders 13, Children's Hospital, 320 Longwood Ave., Boston, MA 02115; S. Tekkök, Dept. of Neurology, Washington University School of Medicine, Box 8111, 600 South Euclid Ave., St Louis, MO 63110.

    FOOTNOTES

  Address for reprint requests: K. Krnjevic', McIntyre Centre, Rm. 1208, 3655 Drummond St., Montreal, Quebec H3G 1Y6, Canada.

  Received 24 December 1997; accepted in final form 23 September 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References