INTRODUCTION

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GLUTAMATE HAS LONG BEEN IDENTIFIED as the major excitatory neurotransmitter in the vertebrate nervous system (8), with intracellular concentrations ranging from 50 µM in astrocytes to as high as 10 mM in glutamatergic neurons (9). Basal extracellular glutamate levels, however, are maintained at only 1-3 µM, which provides a high signal-to-noise ratio at the synapse while simultaneously preventing excitotoxicity (9). Under conditions of oxygen deprivation, however, such as in hypoxia or ischemia, the extracellular compartment of the mammalian brain is flooded with high levels of excitatory compounds such as glutamate, aspartate, and dopamine. Pathological increases in extracellular glutamate are thought to play a central role in hypoxic/ischemic neuronal degeneration and reperfusion injuries (15).

Under basal conditions, glutamate levels in the extracellular compartment are determined by both synaptic release and continuous leakage from nonvesicular stores, balanced by high-affinity reuptake mechanisms (1). These energy- and sodium-dependent transporters include both neuronal and astroglial subtypes, with the majority of glutamate uptake occurring through glial transporters such as GLT-1 (9). With a net movement of 3 Na⁺ cotransported into the cell and 1 K⁺ countertransported for each glutamate anion, energy costs are estimated to be at least 1.5 ATP per glutamate anion (35). Glutamate reuptake may thus constitute a large fraction of the total energy requirement of neurotransmission (35) and is likely to be severely compromised during conditions of energy deficit such as hypoxia or ischemia. Low ATP inhibits these transport processes, leading to glutamate increases in the extracellular space both by continuous glutamate leakage and by membrane depolarization as the sodium pump slows. The breakdown in transmembrane Na⁺ and K⁺ ion gradients may also cause transporters to run in reverse, releasing additional glutamate into the extracellular space (36). Excess glutamate then activates ionotropic and metabotropic receptors (GluR) that trigger a cascade of events resulting in neuronal death. The majority of hypoxic/ischemic glutamate release to the extracellular space is thought to occur by this reversal of glutamate reuptake transporters (25).

The freshwater turtle Trachemys scripta, however, is able to survive days to months of anoxia (15); the brain maintains ion gradients and avoids anoxic depolarization for at least 48 h. Basal levels of extracellular glutamate (20) and dopamine (19) are also maintained during periods of oxygen deprivation. It has been assumed until now that low extracellular glutamate levels were maintained during anoxia simply by preventing release into the extracellular space. However, recent work on dopamine homeostasis indicates that neurotransmitter balance in the anoxic turtle brain is a much more dynamic process that may use blockage of release as well as continued release and reuptake (19; Milton and Lutz, unpublished observations). The turtle's ability to prevent pathological increases in extracellular glutamate may then be a combination of 1) decreased synaptic release, 2) decreased glutamate leakage, 3) maintained or increased glutamate reuptake, and/or 4) prevention of glutamate transporter reversal. The purpose of this study was to investigate the relative roles of these mechanisms in maintaining low extracellular glutamate levels in the early anoxic (1-2 h) turtle brain, when the animal experiences a temporary energy crisis.

The particular roles of adenosine and ATP-sensitive potassium (K_ATP) channel activation on glutamate release were also examined, as these mechanisms are known to be neuroprotective in the hypoxic/ischemic mammal as well as in the anoxia-tolerant turtle. Activated K_ATP channels play an important role in decreasing membrane permeability (channel arrest) in the anoxic turtle (23) and are thought to ameliorate neuronal injury in the hypoxic mammalian brain by hyperpolarizing the neuronal membrane (7) and by inhibiting glutamate and dopamine (DA) release (37, 38). K_ATP channels, in turn, may be modulated by adenosine (27). Adenosine A₁ receptor activation in the mammal inhibits many neurons both pre- and postsynaptically by modulating ionic currents and by decreasing neurotransmitter release (8, 28). Adenosine plays an important role in the anoxic turtle brain as well, acting as a "retaliatory metabolite" to balance energy supply and demand (15, 21). Perfusing the anoxic isolated turtle cerebellum with adenosine receptor blockers causes a rapid depolarization (15), whereas superfusion of the anoxic brain in situ with the adenosine receptor blocker aminophylline prevents the normal anoxia-induced increase in cerebral blood flow (12).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. The studies described were approved by the institutional animal care and use committee. Freshwater turtles were purchased from a commercial supplier (Lemberger, Oshkosh, WI). The general glutamate transport blocker L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and the specific blocker of the astrocytic GLT-1 glutamate transporter dihydrokinate (DHK; originally a gift from B. Hassel) were purchased from Tocris Cookson (St. Louis, MO). The specific K_ATP channel blocker 2,3-butanedione monoxime (BDM) and K_ATP channel opener 7-chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide (Diazoxide) were purchased from Research Biochemicals International (Natick, MA). All other chemicals and reagents were purchased from Sigma Chemicals (St. Louis, MO).

Methods. Experiments were performed at 25°C on the freshwater turtle Trachemys scripta elegans. Turtles were anesthetized with AErrane (Isoflurane, USP, Anaquest) in air. Anesthesia was induced using a mixture of 4% isoflurane in air pumped from a 1.5-liter rebreathing bag. Animals were maintained on 1.7% isoflurane once a surgical plane was achieved (31).

Normoxic treatments. Normoxic treatment groups were as follows: 1) normoxic controls (4-h respiration on air, no drugs); 2) non-PDC drug controls: 1-h baseline sampling followed by 1-h individual drug administration with one of the following: 500 µM K_ATP channel opener Diazoxide (16), 200 µM adenosine (18), 100 µM general adenosine receptor blocker theophylline (24), and 500 µM K_ATP blocker BDM (Milton and Lutz, unpublished observations); and 3) normoxic PDC or DHK animals: 1-h baseline sampling followed by perfusion of experimental drugs (above) coadministered with glutamate uptake blocker PDC [2.5 mM (22)] or DHK [1 mM (29)]. All normoxic treatment groups were respired on air; sampling occurred following a 1-h recovery period after insertion of the microdialysis probe.

Anoxic treatments. Drug concentrations for anoxic treatments were as per normoxic treatments above. Experimental anoxic treatments were as follows.

Anoxic controls. One-hour air respiration was followed by 2-h N₂ respiration.

Administration of the K_ATP blocker BDM. After the 1-h normoxic recovery period, animals were respirated on air for 30 min followed by 30-min N₂ respiration to obtain baseline glutamate values. The K_ATP blocker BDM was infused through the microdialysis probe beginning at 30-min N₂ respiration; sampling followed for an additional 1.5 h.

L-Trans-PDC administration. Glutamate transport blockade by L-trans-PDC was used to determine rates of glutamate release in anoxic vs. normoxic turtles. After the 1-h normoxic recovery period, animals were respirated on nitrogen for 30 min in which no sampling occurred. Samples were collected to determine anoxic baseline glutamate levels for the following hour, after which the general glutamate uptake blocker PDC was administered through the microdialysis probe for three additional 20-min sampling periods.

Coadministration of adenosine and K_ATP antagonists with PDC. To assess the respective roles of adenosine and K_ATP channels in preventing glutamate release, the general adenosine receptor antagonist theophylline and/or K_ATP blocker BDM were coadministered with PDC in the anoxic turtle as described above (1-h baseline sampling followed by 1-h drug exposure in anoxic animals). Because of rapid glutamate uptake by excitatory amino acid (EAA) transporters, changes in extracellular glutamate can be highly variable and difficult to detect (26); therefore all anoxic experiments other than basal normoxic and anoxic controls were performed by the coperfusion (reverse dialysis) of each drug in the presence of the general glutamate uptake inhibitor L-trans-PDC to provide background glutamate levels sufficiently high to detect changes. PDC possesses strong glutamate transporter affinity and negligible EAA-receptor affinity (2) and reliably increases extracellular glutamate in the mammalian brain without causing neurohistological damage (17) when applied through the microdialysis probe in concentrations such as were used in this study (22, 26). The seemingly high concentrations of drugs typically used in microdialysis studies are necessary because of the incomplete permeability of the dialysis membrane as well as the torturous diffusion pathways through tissue (32), such that only a fraction of the drug infused through a microdialysis probe actually reaches the tissue.

	RESULTS

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Glutamate homeostasis. Basal levels of extracellular glutamate averaged 3.4 ± 0.8 µM in the turtle striatum; there was no significant difference between normoxic and anoxic baseline levels. As has been previously described (20), baseline levels of excitatory neurotransmitters (glutamate and aspartate) did not increase over 4 h of anoxia, whereas levels of the inhibitory neurotransmitter GABA increased significantly (Fig. 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effects of anoxia on extracellular levels of inhibitory and excitatory amino acids measured by intracerebral microdialysis in the striatum of freshwater turtles exposed to 4-h anoxia at 25°C. Time 0 indicates the start of ventilation on 99.99% nitrogen. Values are means ± SE from 5 animals. *Significantly different from normoxia, P < 0.05. Asp, aspartate; Glu, glutamate.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Perfusion of the anoxic turtle striatum with the general glutamate uptake blocker L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC; 2.5 mM) or the specific astrocytic GLT-1 uptake blocker dihydrokinate (DHK; 1 mM) decreases extracellular glutamate levels compared with normoxic animals. Values are means ± SE, n = 5 animals/group. Anoxic refers to the mean of samples collected between 1- and 2-h nitrogen respiration. O2, normoxic groups; N2, anoxic experimental groups. *Anoxic group significantly different from normoxic group, P < 0.05.

Factors modulating glutamate release. As activation of both adenosine A₁ receptors and K_ATP channels is thought to play a role in decreasing hypoxic/ischemic glutamate release by reducing neurotransmitter release (8, 38), these two key factors were manipulated to determine their relative potential roles in the observed decrease in glutamate release in the anoxic turtle brain.

Adenosine. As shown in Fig. 3, glutamate release in the normoxic turtle can be modulated by adenosine. When the normoxic turtle brains were perfused with adenosine in the presence of PDC, extracellular glutamate levels were significantly reduced below PDC perfusion alone to levels nearly equal to anoxic animals perfused with PDC (290 ± 27 vs. 300 ± 27%; Fig. 2). Adenosine receptor activation, then, reduces glutamate release in the normoxic turtle.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Adenosine (AD) and ATP-sensitive potassium channels (KATP) modulate glutamate release in the turtle striatum: coperfusion of AD or the KATP opener diazoxide (DZ) with the glutamate uptake blocker PDC decreased extracellular glutamate to levels comparable to those seen in the anoxic PDC-treated animal. However, blocking either AD receptors [theophylline (Theo)] or KATP [2,3-butanedione monoxime (BDM)] in the anoxic animal did not significantly increase extracellular glutamate above levels observed in anoxic PDC turtles. Simultaneously blocking both KATP and AD receptors in the anoxic animal does prevent the anoxia-induced decrease in glutamate release. Values are means ± SE, n = 5 animals/group. *Experimental group significantly different from normoxic (PDC) group, P < 0.05.

K_ATP channels. We next investigated the role of K_ATP channels, which have been shown to play a protective role in the hypoxic/ischemic mammalian brain, in part, by decreasing glutamate release (38) and have also been demonstrated to inhibit DA release in the 1-h anoxic turtle brain (Milton and Lutz, unpublished observations). As with adenosine, opening K_ATP channels in the normoxic brain in the presence of PDC decreased extracellular glutamate levels by 49% compared with normoxic glutamate increases in the presence of PDC alone (normoxic/PDC; Fig. 3). K_ATP activation in normoxia thus decreases glutamate release to levels observed in baseline anoxic PDC animals.

K_ATP channels and adenosine. Despite the fact that neither blocking adenosine receptors nor K_ATP channels individually significantly elevated anoxic glutamate release, the two mechanisms together exhibit a powerful synergistic effect (Fig. 3). Blocking both K_ATP channels and adenosine receptors simultaneously in the anoxic brain increased extracellular glutamate levels to 480 ± 63% of basal, significantly higher than anoxic/PDC animals and not statistically different from levels seen in normoxic/PDC animals (570 ± 27%). The inhibition of adenosine receptors and K_ATP channels at the same time then prevents the anoxia-induced decrease in glutamate release.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The basal extracellular glutamate levels reported here are in line with previous reports for both the normoxic mammalian brain (6, 9) and the normoxic and anoxic turtle (20). As in the mammalian brain, low extracellular glutamate levels are maintained during normoxia by rapid reuptake into cells surrounding the synaptic cleft; the results reported here indicate that ~50% of this reuptake is astrocytic. Unlike the anoxia-sensitive mammal, however, where extracellular glutamate levels increase dramatically in response to ischemic injury (13), glutamate levels remain low in the anoxic turtle striatum due to a combination of continued glutamate reuptake and a reduction in glutamate release. As in the normoxic brain, glial cell transporters account for approximately one-half (or more) of anoxic glutamate uptake. Other transport mechanisms most probably remain active in anoxia as well, accounting for a similar proportion of glutamate uptake as in normoxia such that the overall pattern of glutamate uptake into neurons and glial cells is maintained.

Our normoxic studies also indicate that activating either adenosine receptors or K_ATP channels independently is sufficient to decrease glutamate release. It should be noted, however, that when adenosine receptors were pharmacologically activated by us in the normoxic brain, K_ATP channels would likely have remained unstimulated due to high intracellular ATP levels, although a direct link between adenosine and K_ATP channels has been suggested in ischemic mammalian brain (10, 27). Conversely, when K_ATP channels were stimulated pharmacologically, adenosine receptors would be inactive due to the high ATP and low adenosine levels of the normoxic brain.

But while adenosine receptor activation decreased glutamate release in the normoxic animal, blocking adenosine receptors alone in the anoxic turtle brain was not sufficient to prevent the observed anoxia-induced decrease in glutamate release. This is in direct contrast to the mammalian brain, where adenosine receptor blockade augments ischemic glutamate release (28), and it suggests that other factors are activated in the anoxic turtle that are able to decrease glutamate release and compensate for adenosine receptor blockade. One such potential mechanism to suppress glutamate release is the stimulation of K_ATP channels, which are activated during hypoxia and ischemia in response to decreased intracellular ATP. Although the anoxia-tolerant turtle is able to maintain basal ATP levels over long-term anoxia, there is a temporary drop in ATP during the initial hour of anoxia while the turtle is in transition to a hypometabolic state (15). Decreased ATP levels stimulate ATP-dependent potassium channels, an effect reinforced by adenosine A₁ receptor stimulation (30); activation has been shown to modulate DA homeostasis and ion channel downregulation in the early anoxic turtles (Milton and Lutz, unpublished observations; 23). Activated K_ATP channels have also been shown to hyperpolarize the mammalian neuronal membrane and consequently inhibit neuronal excitability and neurotransmitter release, including the release of glutamate (38). However, this mechanism is probably not operational in the turtle as there is no evidence of hyperpolarization in the anoxic turtle brain (15).

Although stimulating K_ATP channels in the normoxic brain decreased glutamate release nearly 50%, as was the case with adenosine, blocking K_ATP channel activation alone in the early anoxic brain did not prevent the decrease in glutamate release. As with adenosine, it would appear that other factors in the anoxic turtle are able to compensate for the pharmacological blockade of K_ATP channels. The increase in extracellular adenosine as intracellular ATP breaks down remains a possible important factor. Although blocking adenosine receptors in the early anoxic turtle did not affect glutamate release, increases in adenosine are known to play an important role in anoxic survival. Striatal extracellular adenosine levels in the anoxic turtle increase significantly from the onset of anoxia as ATP levels fall, peaking at 12 times basal levels by 90 min (21). Adenosine is critical for survival in both the anoxic turtle, where blocking A₁ receptors in the anoxic cerebellum induces depolarization (15), and in the ischemic mammal, where adenosine receptor blockade increases excitotoxin release (28). Our results indicate that activation of either adenosine receptors or K_ATP channels in the normoxic or anoxic turtle brain is sufficient to block glutamate release. This may be the result of a general neuronal inhibition of the cell brought about by decreased ATP levels (activating K_ATP channels) and/or the release of adenosine and activation of A₁ receptors. Blocking adenosine receptors as we did would enhance the activation of K_ATP channels by increasing energy demand and lowering ATP levels. Blocking K_ATP channels could similarly enhance the effectiveness of A₁ receptor activation by increasing adenosine release. Thus, when either mechanisms is blocked in the early anoxic turtle, the other is sufficiently stimulated by high adenosine or decreased ATP to compensate and induce the ~50% decrease in glutamate release.

This hypothesis is supported by our results, in that blocking adenosine receptors and K_ATP channels simultaneously in the presence of PDC greatly increased glutamate release in the anoxic turtle to levels not significantly different from normoxic/PDC turtles, i.e., the anoxia-induced decrease in glutamate release was almost completely abolished when both potential pathways were blocked. Similar synergistic interactions with adenosine have been reported for both GABA and acetylcholine, while de Mendonca and Ribiero (4) reported that the contribution of group 3 metabotropic glutamate receptors to hypoxia-induced synaptic depression could only be detected when adenosine A₁ receptors were blocked. Lucchi et al. (14) suggested that adenosine and GABA may act in a redundant or perhaps overprotective manner, with GABA acting as a substitute for some of adenosine's actions in hypoxia when adenosine was not operative. Only when adenosine receptors were blocked was the cosuppression by GABA revealed, which accounted for ~20% of the hypoxic decrease in synaptic transmission. Similarly, Coelho et al. (3) reported a muscarinic receptor-induced depression of synaptic transmission during hypoxia that was only apparent when adenosine A₁ receptors were blocked, suggesting a link through a common transduction signal. Although a direct link between the A₁ receptor and K_ATP channels has also been suggested for the mammalian brain (10, 27), the present experiments suggest that these mechanisms are functioning independently in the early anoxic turtle, in that blocking either mechanism alone did not alter glutamate release.

The continued release and reuptake of glutamate in the anoxic turtle, albeit at a significantly reduced rate, suggest that the anoxic animal may be attempting to retain neuronal function while simultaneously cutting energy expenditures. As glutamate uptake occurs via a Na⁺/glutamate cotransport mechanism maintained by Na⁺/K⁺-ATPase, decreasing glutamate release conserves energy by decreasing uptake requirements while continuing to maintain basal extracellular glutamate levels. A similar process has been demonstrated for DA homeostasis in the anoxic turtle brain, where K_ATP stimulation decreases release during early anoxia (Milton and Lutz, unpublished observations) while DA release and reuptake continue over long-term anoxia such that basal extracellular DA levels are maintained (19). The dependence of glutamate transport on the Na⁺/K⁺ gradient also implies that while membrane permeability has been shown to decrease in the anoxic turtle brain (5), the near-complete channel arrest reported in the anoxia-tolerant turtle hepatocytes (13) will not be an option for turtle neurons. If the turtle must maintain a degree of ion flux to support glutamate reuptake, energetic costs as high as 1.5 ATP per glutamate may represent a significant fraction of the energy expenditure required under anoxic conditions and thus put a limit on the degree of metabolic reduction that can be achieved. We speculate that the maintenance of basal extracellular glutamate levels, as with the stable DA levels reported previously, acts to maintain the functional integrity of the turtle neuronal network, preserving neural tone and allowing the full recovery of operational activity after hours or even months of anoxia. The maintenance of neural tone by low levels of neurotransmitters has recently been described in the mammalian brain (34). Further studies will determine the pathways of glutamate release (vesicular vs. nonvesicular stores) as well as investigate the mechanisms that control glutamate homeostasis over long-term anoxic exposure.