INTRODUCTION

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Hypoglycemia is a common occurrence in diabetic patients and can damage both white matter and gray matter brain tissue (Auer 1986; Siesjö 1988), and in vivo evidence suggests that aglycemia can occur in the brain as a consequence of severe systemic hypoglycemia (Silver and Erecinska 1994). Prior studies from our laboratory have indicated that aglycemic injury to the adult rat optic nerve (RON), a representative central white matter tract, requires the presence of extracellular Ca²⁺ (Brown et al. 2001a). The degree of injury, as determined by the area under the evoked compound action potential (CAP), increased with bath [Ca²⁺] (Brown et al. 2001a) as was the case for anoxia-mediated white matter injury (Brown et al. 2001b; Stys et al. 1990). Evidence indicates that the extracellular Ca²⁺ acts as a source for toxic Ca²⁺ influx into intracellular compartments mediated by reverse Na⁺-Ca²⁺ exchange (Brown et al. 2001b; Stys et al. 1992) and voltage-gated Ca²⁺ channels (Brown et al. 2001b). The intracellular Ca²⁺ overload leads to axonal death and loss of nerve function.

In this report, we studied the effects of bath [Ca²⁺] on aglycemic injury in the adult RON. Based on previous studies (Brown et al. 2001b; Stys et al. 1990), we expected that increasing [Ca²⁺]_o during aglycemia would worsen subsequent recovery. Our results indicated, however, that increasing bath [Ca²⁺] above 2 mM resulted in increased CAP recovery after aglycemia, the first recorded report of increased extracellular [Ca²⁺] being neuroprotective. Analysis of this finding has led us to conclude that Ca²⁺ effects on white matter injury are complex and include both direct mediation of intracellular damage as well as biophysical effects on transmembrane voltage gradients affecting ion channel gating.

	METHODS

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Preparation

All experiments were carried out in accordance with the guidelines for Animal Care of the University of Washington. Long Evans rats aged 45+ days old were deeply anesthetized with CO₂ then decapitated. The optic nerves (5-10 mm long) were cut at the optic chiasm and behind the orbit and placed in an interface perfusion chamber (Medical Systems, Greenvale, NY) (see also Stys et al. 1990). RONs were maintained at 37°C and perfused with artificial cerebrospinal fluid (ACSF) bubbled with 95% O₂-5% CO₂ which contained (in mM): 153 Na⁺, 3 K⁺, 2 Mg²⁺, 2 Ca²⁺, 143 Cl, 26 HCO, 1.25 HPO4^2-, and 10 glucose, pH 7.45. We used this hyperglycemic concentration of glucose as the diffusion distance for glucose from the tissue-bath interface to cells and axons is up to 250 µm. Given the high rate of glucose consumption in mammalian brain at 37°C, glucose would be exhausted within the first 100 µm if the bath concentration were the same as in plasma. A suction electrode back filled with ACSF was attached to the distal end of the nerve and stimulated every 30 s (WPI, Sarasota, FL: Isostim 320). A recording electrode filled with ACSF was attached to the proximal end of the nerve to record the CAP, which was evoked by a 125% supramaximal stimulus (50 µs duration). Nerves were allowed to equilibrate for 60 min before recording commenced. Aglycemia was initiated by perfusion with glucose-free ACSF, and in these instances, the solution in the stimulating and recording electrodes was switched to glucose-free ACSF. No osmotic compensation was made but the change in osmolarity was only about 3%. Ca²⁺-free ACSF was made by omitting CaCl₂ and adding 5 mM ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Introduction of test ACSF containing test divalent cation solution occurred 20 min prior to the 1-h period of aglycemia to allow the extracellular space (ECS) and bath solution to equilibrate (Brown et al. 2001a,b).

Electrodes

Ion-sensitive microelectrodes were made with double-barreled piggyback glass (WPI, PB150F-6) according to the method of Borrelli et al. (1985) with slight modifications. Electrodes were pulled on an upright puller producing tips of about 1 µm in diameter (Narashige, East Meadow, NY: PP83). These were subsequently beveled to a tip diameter of 2-5 µm (Sutter, Novato, CA: BV-10). The tip of the ion-sensitive barrel was filled with hexamethyldisilazine (Fluka, Ronkonkoma, NY: 52619) and baked at 160°C for 1 h. The indifferent barrel was filled with 150 mM NaCl, and the ion-sensitive barrel was back-filled with (in mM) 120 NaCl, 3 KCl, 20 HEPES, and mM CaCl₂ adjusted to pH 7.2 with 1 M HCl. The ion-sensitive barrel was filled at the tip by back injection with a short (100-400 µm) column of Ca²⁺-sensitive liquid ion sensor (Fluka, Cocktail A 21098). Electrodes were calibrated in a solution containing 120 mM NaCl, 3 mM KCl, 20 mM HEPES, and Ca²⁺ concentrations of 20 µM, 200 µM, and 2 mM. All electrodes were individually calibrated and only those showing stable, near Nernstian responses (i.e., 25-30 mV) to decade changes in [Ca²⁺] were used for experimental measurements. Electrodes were recalibrated after each experiment, and data from electrodes with greater than a 5 mV deviation in response to decade changes in [Ca²⁺] were discarded. The average between the initial and final calibration responses was used to evaluate the experimental measurement. The latency to drop of [Ca²⁺]_o was measured by defining baseline [Ca²⁺]_o as the average value during the first 40 min of the experiment. Threshold for recording a "[Ca²⁺]_o decline" was set at a drop of 0.05 mM [Ca²⁺]_o below this baseline. The ion-sensitive barrel was connected to an Axoclamp 2A amplifier (Axon Instruments) via a high-impedance headstage (HS-2 × 0.0001 M), and the indifferent signal was subtracted from the ion-sensitive signal using a differential amplifier (Stanford Research Systems, Sunnyvale, CA: SRS 560). The signal was amplified 100 times, filtered at 1 Hz, and acquired at 1 Hz. CAPs were recorded from a suction electrode connected to a separate Axoclamp 2A amplifier, and the signal was amplified 500 times (SRS 560), filtered at 30 kHz, and acquired at 20 kHz.

Data analysis

Data were acquired on-line (Axon Instruments, Digidata 1200A) using proprietary software (Axon Instruments, Axotape). CAP area was calculated using pClamp (Axon Instruments), and the ion-sensitive signal was converted to [Ca²⁺]_o using a template created in Excel (Microsoft, Redmond, WA) based on the Nernst equation. Data are presented as means ± SE. Significance was determined by ANOVA using Tukey's posttest.

	RESULTS

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Bath [Ca²⁺] during aglycemia determines latency to onset of CAP failure and CAP recovery

The effects of 60 min of aglycemia on CAP area in adult RONs are shown in Fig. 1. Latency to the onset of CAP failure was defined as the time from aglycemia onset to the point when CAP area dropped below 95% of its normalized baseline value (Wender et al. 2000). Latency to the start of CAP failure in aglycemia depended on bath [Ca²⁺] (Fig. 1A). In control ACSF containing 2 mM Ca²⁺ and 2 mM Mg²⁺, the latency to CAP failure was 30.5 ± 1.3 min (n = 8). As the baseline [Ca²⁺] in the ACSF was decreased to 1, 0.5, or 0 mM Ca²⁺ (Mg²⁺ was constant at 2 mM), the latency to CAP failure decreased to 20.0 ± 2.1 min (n = 6, P < 0.001 compared with 30.5 min), 10.3 ± 0.7 min (n = 6, P < 0.001 compared with 20.0 min), or 6.0 ± 1.7 min (n = 6, P < 0.05 compared with 10.3 min), respectively. Thus in these experiments, decreasing Ca²⁺ correspondingly reduces the total divalent ion concentration [compare with Fig. 3C in Brown et al. (2001a), where decreases in Ca²⁺ were compensated by equimolar substitution with Mg²⁺ to ensure the total divalent ion concentration stayed constant]. Although the latency to CAP failure changed with [Ca²⁺], it did not appear that the rate of CAP decline varied, once initiated. CAP recovery after aglycemia was also dependent on bath [Ca²⁺] (Fig. 1). In 2 mM Ca²⁺ CAP recovered to 48.8 ± 3.9% (n = 6). The degree of CAP recovery increased as bath Ca²⁺ was decreased. In 1, 0.5, or 0 mM Ca²⁺, CAP recovery was 62.2 ± 6.0, 69.5 ± 9.7, or 99.1 ± 3.8%, respectively. Specimen records of CAPs before, during, and after 60 min of aglycemia at different test concentrations of Ca²⁺ are shown in Fig. 1B. Control CAPs in each condition exhibit the standard three peaks (Foster et al. 1982). These data indicated that aglycemia-mediated irreversible injury to the adult RON required extracellular Ca²⁺ and that the degree of injury was related to the bath [Ca²⁺], strongly suggesting that during aglycemia Ca²⁺ moves from the ECS to intracellular compartments.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Latency of compound action potential (CAP) decline during aglycemia and subsequent levels of CAP recovery depend on bath [Ca2+]. A: the long bar indicates the period of superfusion with artificial cerebrospinal fluid (ACSF) containing variable [Ca2+]. The short bar indicates the period of aglycemia. In 2 mM Ca2+, CAP failure has the longest latency, while CAP recovery is the smallest. Lower concentrations of Ca2+ shortened latency but increased CAP recovery. Each trace is the average of 6 separate nerves. B: individual CAPs recorded at the times indicated in A as letters above traces. Scale bars are 1 mV and 1 ms for all traces.

Increased bath Ca²⁺ above 2 mM improves CAP recovery

We explored the effects of bath [Ca²⁺] above 2 mM during aglycemia on latency to CAP failure and CAP recovery (Fig. 2). Solutions containing the test concentration of Ca²⁺ were introduced 20 min prior to the 1-h period of aglycemia, after which nerves recovered for 1 h in control ACSF containing 2 mM Ca²⁺. In these experiments, [Mg²⁺] was held constant at 2 mM. Because aglycemia-induced white matter injury depends on Ca²⁺ influx, we expected greater injury with higher ambient [Ca²⁺]. However, as the [Ca²⁺] of the bath was increased from 2 to 5 or 10 mM, the degree of CAP recovery from aglycemia increased from 48.8 ± 3.9% (n = 6) to 74.3 ± 7.5% (n = 7) or 91.1 ± 5.2% (n = 6), respectively (Fig. 2A). In fact, in 5 or 10 mM [Ca²⁺], CAP area declined during aglycemia but never reached zero. The protective effect of increasing Ca²⁺ on aglycemia-induced injury may be related to the effect of bath Ca²⁺ on latency to CAP decline, i.e., charge screening. Latency to the start of CAP failure during aglycemia was dependent on bath [Ca²⁺]. Increasing the bath Ca²⁺ from 2 to 5 or 10 mM, respectively, increased the latency to CAP decline to 29.9 ± 1.5 min (n = 6, P < 0.00001, vs. 8.8 min), 41.2 ± 1.7 (n = 6, P < 0.001, vs. 29.9 min), or 51.8 ± 1.2 (n = 6, P < 0.001, vs. 41.2 min), respectively. The relationship between the latency to the start of CAP failure and bath Ca²⁺ during aglycemia is illustrated in Fig. 5A.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Latency of CAP decline during aglycemia and subsequent levels of CAP recovery depend on bath [Ca2+]. A: higher [Ca2+]s increase latency of CAP decline during aglycemia and increase the magnitude of CAP recovery. Traces are averages of 6 separate nerves. B: individual CAPs recorded at the times indicated in A as letters above traces. Scale bars are 1 mV and 1 ms for all traces.

Increasing bath Mg²⁺ increases latency to CAP failure during aglycemia

The effect of [Ca²⁺] on latency to onset of CAP failure during aglycemia was unexpected. We hypothesized that Ca²⁺ might be exerting this action due to "charge screening effects" (Hille 2001). As charge-screening effects are not solely exhibited by Ca²⁺ but are shared by all divalent cations (Hille 2001), we predicted that increasing the concentration of a divalent cation other than Ca²⁺ should also increase latency to CAP failure. We tested this idea by altering ACSF [Mg²⁺]. Charge screening effects are well documented for Mg²⁺ (Hille et al. 1975), and Ca²⁺ channels are not permeable to Mg²⁺ nor are they blocked by this ion. In this series of experiments, bath Ca²⁺ was kept constant at 0.2 mM to minimize the contribution of Ca²⁺ to charge screening. As Mg²⁺ was increased from 1 to 2 or 5 mM the latency to CAP failure increased significantly from 1.3 ± 1.0 min (n = 2) to 12.9 ± 1.1 min (n = 4) or 25.7 ± 1.3 min (n = 6), respectively (Fig. 3). These results are consistent with a charge screening effect (see DISCUSSION).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Increasing [Mg2+] from 1 to 2 or 5 mM progressively increased the latency to CAP failure. Bath [Ca2+] was held constant at 0.2 mM. Data are averaged from 6 nerves in each condition.

Effects of increasing bath Mg²⁺ on CAP recovery

Increasing Mg²⁺ from 2 to 5 or 10 mM, keeping Ca²⁺ constant at 2 mM, resulted in increased latency to CAP failure and increased CAP recovery (Fig. 4). In 2 mM Mg²⁺ ACSF, the latency to CAP failure was 29.9 ± 1.5 min and CAP recovery was 48.8 ± 3.9% (n = 6) of control. Increasing Mg²⁺ to 5 or 10 mM increased latency to CAP failure to 43.8 ± 2.8 or 47.9 ± 7.8 min, respectively, and increased CAP recovery to 97.7 ± 7.1 or 102.0 ± 7.1%, respectively (Fig. 4A). Thus increasing [Mg²⁺], like increasing [Ca²⁺], enhances CAP recovery from 60 min of aglycemia. In fact, CAP recovery in both 5 and 10 mM Mg²⁺ was greater than in 5 mM Ca²⁺ ACSF (94.9 ± 8.4 vs. 78.9 ± 9.2%, P < 0.05) or 10 mM Ca²⁺ (102.0 ± 7.0 vs. 91.1 ± 5.2%, not significant: see Fig. 5B).

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effect of bath [Mg2+] on CAP responses to aglycemic injury. A: the long bar indicates the period of superfusion with ACSF containing variable [Mg2+]. The short bar indicates 1 h of aglycemia. Higher [Mg2+] caused increased latency to CAP failure during aglycemia and improved CAP recovery. B: individual CAPs recorded at the times indicated in A as letters above traces. Scale bars are 1 mV and 1 ms for all traces.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Aglycemia effects on the CAP are dependent on bath divalent cation concentration. A: latency to CAP failure during aglycemia increased as a function of [Ca2+] () or [Mg2+] (). B: CAP recovery after 1 h of aglycemia was a function of [Ca2+] () or [Mg2+] ().

[Ca²⁺]_o changes during aglycemia in nerves bathed in various bath [Ca²⁺]

The pattern of changes in [Ca²⁺]_o before, during, and after a 1-h period of aglycemia was studied while varying bath [Ca²⁺]. The level to which [Ca²⁺]_o fell during aglycemia was determined by bath [Ca²⁺] (Fig. 6). In all these experiments, Mg²⁺ was constant at 2 mM. In 2 mM Ca²⁺, the baseline [Ca²⁺]_o of 1.55 ± 0.02 mM fell to 0.45 ± 0.04 mM at the end of 1 h of aglycemia and recovered to 1.44 ± 0.05 mM (n = 6). In 5 or 10 mM Ca²⁺, baseline [Ca²⁺]_o was 3.36 ± 0.15 mM (n = 4) or 7.90 ± 0.11 mM (n = 4), respectively, and fell to 2.93 ± 0.08 mM or 7.74 ± 0.21 mM, respectively, during 1 h of aglycemia (Fig. 6A). The lower the bathing [Ca²⁺], the shorter the latency to [Ca²⁺]_o decrease and the greater the degree of [Ca²⁺]_o decrease.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Effects of aglycemia on change in [Ca2+]o as a function of baseline [Ca2+]o in various bath [Ca2+]. [Ca2+]o was recorded with Ca2+-sensitive microelectrodes in bath [Ca2+] of 2, 5, or 10 mM. A: the integral of [Ca2+]o decrease below baseline was used to estimate the total amount of Ca2+ influx during aglycemia. , the integral used in calculations. B: plot of CAP recovery vs. integral of [Ca2+]o decrease (2 mM, ; 5 mM, ; or 10 mM, ). Aglycemia-induced injury increased in a linear fashion as a function of the integral of [Ca2+]o decrease.

The latency to the onset of [Ca²⁺]_o decline following application of aglycemia progressively increased as bath [Ca²⁺]_o increased. In 2 mM Ca²⁺, the latency to [Ca²⁺]_o decrease below baseline was 40.7 ± 1.5 min (n = 6), which rose to 44.0 ± 0.6 min (n = 4, not significantly different from 40.7 min) in 5 mM and 49.7 ± 2.2 min (n = 4, not significantly different from 44.0 min) in 10 mM [Ca²⁺].

The relationship between the total amount of Ca²⁺ entering the intracellular compartments and injury was examined. We have used the integral of [Ca²⁺]_o below baseline during aglycemia as a crude qualitative measure of net amount of Ca²⁺ influx (Brown et al. 2001a). Fig. 6A illustrates () how the integral of [Ca²⁺]_o decrease during aglycemia was measured. Data shown are the averages of nerves exposed to aglycemia at 2 mM (n = 6), 5 mM (n = 4), or 10 mM (n = 4) bath [Ca²⁺]. The relationship between integral of Ca²⁺ decrease during aglycemia and CAP recovery is illustrated in Fig. 6B, demonstrating that there is a linear relationship between injury and the amount of Ca²⁺ leaving the ECS, presumably entering intracellular compartments.

	DISCUSSION

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The brain requires both glucose as an energy source and oxygen to oxidize the glucose to maintain the transmembrane ion gradients on which excitability depends (Clarke and Sokoloff 1999). Only recently have the effects of energy deprivation on CNS axons [i.e., white matter (WM)], independent of the cell bodies been assessed. The importance of a continual supply of glucose and oxygen to WM has been demonstrated by the irreversible injury that occurs to the adult RON, when either glucose (Brown et al. 2001a) or oxygen (Brown et al. 2001b; Stys et al. 1990) is withdrawn.

Prior studies have indicated that central axonal injury from energy deprivation is dependent on the presence of extracellular Ca²⁺ (Brown et al. 2001a). Our results show that the effects of [Ca²⁺] on WM injury during aglycemia are complex. While lowering bath [Ca²⁺] from 2 mM is progressively protective, so too is raising [Ca²⁺] from 2 mM. These contrary findings are best explained by two interesting actions of extracellular Ca²⁺. On the one hand, Ca²⁺ acts as an intracellular agent of destruction, and in this context, lowering its concentration in the extracellular space is highly protective (Brown et al. 2001a). On the other hand, Ca²⁺ and other divalent cations have powerful biophysical effects on membrane potential and ion channel gating (Hille 2001) such that higher extracellular concentrations have the net effect of decreasing excitability (Frankenhauser 1957) and blocking the toxic Ca²⁺ influx previously mentioned. These antagonistic actions are strikingly diverse either side of 2 mM; the toxic role of this ion predominates from near 0 to 2 mM, while the protective role dominates the picture at [Ca²⁺]_o's above 2 mM. These data indicate the importance of maintaining constant extracellular divalent cation concentration to avoid such effects.

Thus if Ca²⁺ enters intracellular compartments, its toxic actions result in cell death. However, the extracellular actions of Ca²⁺ on ion channel gating render cells less excitable with increasing extracellular [Ca²⁺]. In this way, extracellular Ca²⁺ can be viewed as a regulator of voltage-gated Ca²⁺ channel gating. In addition to the effects on Ca²⁺ channels, extracellular Ca²⁺ has two other effects relevant to this study. First, increasing Ca²⁺ results in a depolarization of resting membrane potential. Indeed, pioneering studies on squid axons showed that decreasing Ca²⁺ in the perfusate resulted in spontaneous firing of action potentials, whereas the squid axons perfused in Ca²⁺ containing perfusate was quiescent (Frankenhauser 1957; Frankenhauser and Hodgkin 1957). Second, in a study examining the relationship between membrane potential and energy deprivation in adult RON, inhibition of glycolysis or oxidative phosphorylation led to delayed membrane depolarization. Omitting Ca²⁺ from the perfusate resulted in both and accelerated membrane depolarization and an increased rate of depolarization increased (Leppanen and Stys 1997).

These effects of divalent cations have been incorporated into our model illustrated in Fig. 7. The resting membrane potential of the adult RON has been calculated at 80 mV (Stys et al. 1997) in ACSF containing 2 mM Ca²⁺ (black trace), and under similar conditions the threshold for activation of Na⁺ channels is 60 mV (Campbell and Hille 1976). On removal of glucose from the ACSF, the membrane potential is maintained at rest, probably due to the nerves' ability to utilize energy stores (Wender et al. 2000). When these stores are depleted, the membrane potential depolarizes. Once the depolarization reaches threshold the Na⁺ channels inactivate resulting in CAP failure (at the time indicated by the arrow). In our recording conditions and using the grease gap method (Leppanen and Stys 1997), this latency period was about 30 min. In 0 Ca²⁺ ACSF (red trace) the membrane potential is depolarized slightly (Leppanen and Stys 1997), and threshold for Na⁺ channel activation is hyperpolarized compared with 2 mM Ca²⁺ (Campbell and Hille 1976). Removal of glucose results in accelerated depolarization (Leppanen and Stys 1997). This coupled to the relatively depolarized resting membrane potential and hyperpolarized threshold results in more rapid CAP failure. Conversely, in 5 mM Ca²⁺ ACSF (blue trace), the resting membrane potential will be slightly hyperpolarized compared with control (Leppanen and Stys 1997), and threshold for Na⁺ channel activation will be depolarized compared with 2 mM Ca²⁺ (Campbell and Hille 1976). Thus CAP failure will be delayed compared with control conditions.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Schematic effect of bath [Ca2+] on the electric field within the axon membrane during aglycemia. The effects of aglycemia on membrane potential in nerves bathed in [Ca2+] of 0, 2, or 5 mM are shown. According to this model, higher [Ca2+]o neutralized the negative charge on fixed surfaces particles causing lower field strength within the membrane. Conversely low [Ca2+]o increased the field within the membrane. Transmembrane voltage would be the same in both conditions. Because channel activation depends on the field within the membrane, high [Ca2+]o "functionally" hyperpolarizes the membrane (- - -). At a given rate of depolarization, it takes longer to reach Na+ channels threshold in high [Ca2+]o compared with low [Ca2+]o.

The consequences of these effects of divalent cations to injury are as follows. In Fig. 5, we showed that the drop in [Ca²⁺]_o is intimately related to CAP failure. Thus delaying the latency to CAP failure by increasing extracellular divalent cation concentration resulted in an increased latency after the onset of aglycemia before Ca²⁺ entered intracellular compartments. We predict that the more Ca²⁺ that leaves the ECS, the more injury should occur. This is seen if CAP recovery is compared between experiments where 5 mM Mg²⁺:2 mM Ca²⁺ ACSF or 5 mM Ca²⁺:2 mM Mg²⁺ were employed (97.7 ± 7.1 vs. 74.3 ± 7.5%). There is an increased latency to CAP failure in both conditions that is not significantly different. However, the CAP recovery is significantly greater in 5 mM Ca²⁺:2 mM Mg²⁺. This is because there is a greater gradient for Ca²⁺ influx than in 5 mM Mg²⁺:2 mM Ca²⁺ and hence more injury occurs. This difference is not as pronounced in 10 mM Ca²⁺ ACSF as the 51.8 min latency to CAP failure limits the duration of Ca²⁺ influx.

Experiments investigating the effects of divalent cations on anoxic injury also support this theory. Increasing Mg²⁺ in ACSF to 10 mM during a 60-min anoxic insult increased CAP recovery from 29.7 to 54.9% (Stys et al. 1990). Although the effect of Mg²⁺ on latency to CAP failure was not described, we would expect that it would delay the failure of CAP and thus lead to a decreased amount of toxic Ca²⁺ influx leading to improved CAP recovery. Additional supportive evidence from the author states CAP area falls to near zero within a minute of anoxia onset in 0 mM Ca²⁺/5 mM EGTA versus 5-6 min in normal Ca²⁺ (P. Stys, personal communication).