Abstract
The uncontrolled increase of extracellular dopamine (DA) has been implicated in the pathogenesis of hypoxic/ischemic damage in the mammalian brain. But unlike the harmful release of excitatory neurotransmitters such as glutamate and aspartate, which occurs on brain depolarization, excessive extracellular DA levels occur even with mild hypoxia in the mammalian brain. The purpose of this study was to determine whether hypoxia/anoxia provokes a similar increase in the anoxic tolerant turtle brain. Extracellular DA was measured in the striatum of the turtle using microdialysis followed by high-performance liquid chromatography analysis. Results show that extracellular DA was held to normoxic levels over 4 hours of anoxia. Treatment with the specific DA transport blocker GBR 12909 during anoxia resulted in a significant increase in DA to 236% over basal levels. The ability to maintain low striatal extracellular DA may be an important adaptation for anoxic survival in the turtle brain; a contributing factor is the continued functioning of DA uptake mechanisms during anoxia.
Many factors have been identified in the pathophysiology of hypoxic/ischemic brain damage; one major factor appears to be the uncontrolled release of excitatory neurotransmitters, such as glutamate and aspartate, after brain depolarization (Huang et al., 1994). Hypoxic conditions deplete the high-energy phosphate stores needed to maintain cellular integrity, leading to decreased energy availability, neuron depolarization, and the release of neurotoxic excitatory amino acids (EAAs) (Rothman and Olney, 1986; Baker et al., 1991; Siesjo and Katsura, 1992).
Extracellular levels of the monoamine dopamine (DA) also have been implicated as an important cause of pathogenesis in the hypoxic/ischemic brain, particularly in the striatum (Globus et al., 1988). Unlike the EAAs, however, where uncontrolled release occurs only after depolarization (Katayama et al., 1991), excessive increases in extracellular DA are seen in mild hypoxia and even in the normally hypoxia-tolerant mammalian neonates (Binienda et al., 1994; Huang et al., 1994). For example, a decrease in cortical oxygen pressure in the newborn piglet to 11% oxygen resulted in a 200% increase in extracellular DA (Huang et al., 1994). Dopamine may contribute to neuronal damage by modulating the release of EAAs, through the production of oxygen-free radicals, by inhibiting sodium/potassium adenosine triphosphatase (Na+/K+ ATPase), and by uncoupling glucose metabolism from cerebral blood flow (Lutz and Nilsson, 1997).
Increases in extracellular DA may be caused by several possible mechanisms, including 1) decreased reuptake during hypoxia (Akiyama et al., 1991; Huang et al., 1994); 2) increased release from intracellular stores (Globus et al., 1988; Gordon et al., 1990); and/or 3) decreased cerebral blood flow (decreased washout). Evidence for the decreased reuptake of extra cellular dopamine (DAec) comes from studies of striatal synaptosomes in the rat (Pastuszko et al., 1982; Akiyama et al., 1991), although there have been many reports of increased DA release from the hypoxic mammalian brain (Gordon et al., 1990; Huang et al., 1994).
Although most vertebrates share the mammalian central nervous system vulnerability to low oxygen, a few species, including the freshwater turtle Trachemys scripta, demonstrate extended tolerance to brain anoxia (Lutz and Nilsson, 1997). The turtle brain can maintain adenosine triphosphate (ATP) levels and ion gradients for at least 48 hours of anoxia at room temperature (Chih et al., 1989), lowering metabolic rates to equal the energy supplied by glycolysis alone (Lutz and Nilsson, 1997).
The neurotransmitter system plays an important role in this process. Reduced neuroexcitability may be mediated by the early release of adenosine in the turtle brain, followed by increases in the inhibitory neurotransmitter gamma aminobutyric acid (GABA); thus, the uncontrolled release of excitatory amino acids is prevented by avoiding depolarization (Nilsson and Lutz, 1991).
However, it is not yet known how extracellular DA fits into this model. This neurotransmitter is of special interest because increases in extracellular DA and neuronal damage are seen in mammals even in mild hypoxia (Huang et al., 1994), well before oxygen levels are low enough to cause depolarization and the catastrophic release of EAAs. It may be that the turtle brain is similar to that of mammals and that extracellular DA increases during hypoxia, in which case one would expect the brain to have defense mechanisms against any elevation in extracellular DA. Conversely, the turtle may not respond like the mammal and may be able to maintain low concentrations of extracellular DA during hypoxia and even over prolonged anoxia.
The purpose of this study was to distinguish between these two possibilities, and in the latter case, to determine whether low extracellular DA is maintained by decreasing DA release during hypoxia/anoxia or whether instead DA uptake mechanisms are functioning in the face of continued release.
MATERIALS AND METHODS
Materials
The studies described were approved by the institutional animal care and use committee. Freshwater turtles (Trachemys scripta) were purchased from a commercial supplier (Lemberger, Oshkosh, WI, U.S.A.). The DA blocker GBR 12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl]piperazine dihydrochloride) was purchased from Tocris Cookson (St. Louis, MO, U.S.A.). All other chemicals and reagents were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.).
Methods
Experiments were performed at 25°C on 24 freshwater turtles (T. scripta elegans). Animals were divided into four groups: 1) controls (6 hours air); 2) anoxic controls (3 hours air, 3 hours nitrogen [N2]); 3) an experimental air group (3 hours air, 3 hours air and DA blocker); and 4) an experimental anoxic group (3 hours air, 3 hours N2, 3 hours N2 and DA blocker). Normoxic and anoxic controls were depolarized after the 6-hour control period with 2 mmol ouabain in turtle Ringer's solution.
Turtles were anesthetized with AErrane (isoflurane USP, Fort Dodge Animal Health, Ft. Dodge, Iowa, U.S.A.) in air. Anesthesia was induced using a 4% isoflurane-in-air mixture pumped from a 1.5-L rebreathing bag. Animals were maintained on 1.7% isoflurane once a surgical plane was achieved (Shaw et al., 1992).
After exposing the skull, a 1-cm diameter hole was trephined and the skull cap removed. A small incision through the dura mater exposed the cerebral hemispheres. A stereotaxic instrument and guide were used to insert a CMA/12 microdialysis probe (3 mm membrane length, Bioanalytical Systems, Inc., Acton, MA, U.S.A.) into the striatum (5 mm depth from the cerebral surface). After a 2-hour stabilization period in which no sampling occurred, the probe was perfused with unbuffered sodium turtle Ringer's solution at 1.5 μL/minute with a CMA/100 microdialysis pump (Carnegie Medecin, Solna, Sweden). Anoxia was induced by changing the breathing mixture to certified 99.99% nitrogen (County Welding, Pompano Beach, Florida, U.S.A.) and isoflurane. For experimental animals, the specific DA transport blocker GBR 12909 (2 μmol) in turtle Ringer's solution was delivered through the microdialysis probe. Normoxic and anoxic brains were depolarized by the addition of 2 mmol ouabain in turtle Ringer's solution through the microdialysis probe. Dialysate was collected for 30-minute intervals and analyzed immediately. Probe recovery was determined from known standards in vitro (Huang et al., 1994); DA recovery averaged 14.2 ± 3.7% (mean ± standard error of the mean).
Samples were analyzed for monoamine content using reversed-phase high-performance liquid chromatography with electrochemical detection as adapted from Nilsson (1990). A 20-μL aliquot of dialysate was injected into a Waters 510 high-performance liquid chromatography pump (flow rate 1.3 mL/min) (Waters, Milford, MA, U.S.A.). The mobile phase consisted of 100 mmol 1−1 sodium phosphate4, 9% (v/v) methanol, 0.63 mmol 1−1 sodium octyl sulfate, and 0.2 mmol 1−1 ethylenediamine tetraacetic acid. pH 3.6. Samples were separated on a catecholamine C18 column (3 μm, 100 × 4.6 mm; Alltech, Deerfield, IL, U.S.A.) and detected by an LC-3 electrochemical detector with a glassy carbon working electrode set at +750 mV. Concentrations were determined by integrating the area under the peak compared with known standards. Integrations were performed using the Dynamax MacIntegrator II integrator and software (Rainin Instrument, Woburn, MA, U.S.A.).
Methylene blue was injected through the microdialysis probe at the end of each experiment to identify probe location. Data were used only from those turtles in which probe location in the striatum was verified (N = 6 per group).
Baseline levels were defined as the average of the first four normoxic samples after a 1-hour presampling period (Huang et al., 1994). All values are expressed as percent of control ± standard deviation because of between-animal variability. Statistical significance of changes was determined nonparametrically (Kruskal—Wallis test for unequal variances) using the SAS/JMP statistical package (Cary, North Carolina, U.S.A.). In cases of equal variances, one-way analysis of variance (ANOVA)/Student's t-test was used; P < 0.05 was considered statistically significant. Although statistical analysis was performed on data from all time points (control versus experimental groups), figures (1 and 3) show peak values only as the time course of changes in DAec varied between animals.
RESULTS
Normoxia
Basal DA levels in the turtle striatum (80 ± 10 pmol/mL) were similar to those reported by others in the mammalian brain (Baker et al., 1991; Goiny et al., 1991). (Normoxic and preanoxic animals were pooled because there was no significant difference in basal extracellular DA between groups.) Basal concentrations increased nearly threefold (286 ± 196%) on perfusion of the striatum over a 3-hour period with the DA uptake blocker GBR 12909 (Fig. 1); at such low concentrations of DA blocker, this increase most likely is due to decreased reuptake rather than increased release (Heikkila and Manzino, 1984). Inhibition of Na+/K+ ATPase with ouabain (normoxic controls) caused nearly a fivefold increase in extracellular DA to a mean peak of 510 ± 381% above basal levels. As in the mammal, keeping extracellular DA levels low apparently depends on the maintenance of ATP and ion gradients within the brain.
Changes in striatal extracellular dopamine (DA) in the normoxic turtle on addition of the specific DA transport blocker GBR 12909 or the sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) blocker ouabain. Mean percent change ± standard deviation, N = 12 controls, N = 6 per experimental group. Pairs of letters indicate significantly different means (P < 0.05).
Anoxia
The effect of anoxia on extracellular striatal dopamine is shown in Figure 2; extracellular dopamine levels did not change significantly from control values even after 3 hours of anoxia. That this lack of increase in striatal extracellular dopamine in the anoxic turtle brain is due at least in part to the continued activity of Na+/K+ ATPase and/or continuously maintained ion gradients, rather than to an overall depletion of DA within the brain, is indicated by the near threefold increase in extracellular levels on ouabain depolarization (Fig. 3). By contrast, extracellular dopamine levels still averaged only 109 ± 63% of basal levels over 3 hours of anoxia (Fig. 2). Other work performed in this laboratory shows that the Na+/K+ ATPase, although working at reduced levels, still is active in the telencephalon of the anoxic T. scripta (Hylland et al., 1997). Previous work indicates that DA transport is sodium dependent (Horn, 1990); thus, the continued maintenance of ATPase activity and ion gradients would be crucial to DA reuptake. Addition of DA transport blocker to the anoxic brain resulted in a peak increase in striatal extracellular dopamine of 236 ± 98% within 2 hours, indicating that DA release and reuptake mechanisms continue to function (Fig. 3). This increase is not significantly different from the increase observed in normoxic animals treated with GBR 12909; this could indicate that rates of DA turnover continue at near normoxic levels, although it also may mean that release and reuptake rates are lower (or higher) than basal rates, with the difference between the two remaining constant during anoxia and normoxia.
Anoxia causes no significant elevation in striatal extracellular dopamine (DA) over a 3-hour anoxic insult. Depolarization causes a significant increase in both the normoxic and anoxic brains within 1 to 3 hours. Mean percent change from basal levels ± standard deviation, N = 6.
Changes in striatal extracellular dopamine (DA) in the anoxic turtle on addition of the specific DA transport blocker GBR 12909 or the sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) blocker ouabain. Mean percent change ± standard deviation, N = 6 per group. Pairs of letters indicate significantly different means (P < 0.05).
DISCUSSION
This study shows that the turtle brain is unlike that of both the mammalian adult and the comparatively hypoxia-tolerant neonate because turtle striatal extracellular DA levels did not increase significantly even after 3 hours of anoxia, whereas mammals experience large increases in extracellular DA, even under mild hypoxia (Huang et al., 1994). Although 3 hours of anoxia resulted in only an average 9% increase in extracellular DA in the turtle striatum, severe hypoxia or hypoxia/ischemia causes as much as a 175- to 500-fold increase in the rat (Globus et al., 1987; Globus et al., 1988; Pastuszko et al., 1996). The maintenance of low extracellular DA probably is an important adaptation to anoxia tolerance, protecting the turtle brain from the neuronal damage that is associated with massive DA release seen in hypoxic/ischemic mammalian brains.
Extracellular DA levels are determined by three distinct processes in the brain: rate of release into the extracellular space, rate of reuptake, and washout rate. Although hypoxia increases cerebral blood flow in both the mammalian (Morii et al., 1987) and turtle brains (Davies, 1991), in the turtle brain this hyperemia is modest (mean increase of 50% in the turtle) and temporary (lasting only 1–2 hours) (Hylland et al., 1994), and therefore unlikely to have any major effect over a 3-hour anoxic period. Although it has not been demonstrated directly that DA is able to cross the blood—brain barrier of the turtle brain, the much greater increases in striatal extracellular levels in the mammalian brain during ischemia (decreased washout) versus hypoxia indicate that at least some DA is able to escape (Globus et al., 1988; Baker et al., 1991). Therefore, it would appear unlikely that significant differences in washout rates are responsible for the major increases in striatal extracellular DA reported in mammals versus the relatively stable levels observed in the anoxic turtle brain.
Extracellular DA levels thus would be affected primarily either by changing monoamine release, decreasing (or increasing) reuptake, or both. However, it is unlikely that the low levels of extracellular DA observed in this study during anoxia were due to decreased intracellular DA supplies and thus decreased release. Because monoamine synthesis requires oxygen, whole-brain DA levels do decrease over time during anoxia, but these decreases are only 10% over the first 4 hours (Nilsson et al., 1990). In addition, the ouabain experiments described show that in both air and anoxia, depolarization of the turtle brain results in massive DA release, as occurs in the mammal. The turtle striatum therefore is clearly capable of releasing large quantities of DA. The difference in dopamine release also is not due to differences in intracellular DA levels because basal whole-brain levels are reported to be similar in the mammalian and turtle brains (Nilsson et al., 1990). Normoxic extracellular DA concentrations in this study also were similar to those previously reported for the mammalian brain (Baker et al., 1991; Wood et al., 1992; Huang et al., 1994).
However, in the electrically quiescent turtle brain (Fernandes et al., 1997), the rate of DA release may be diminished. In the mammal, it has been shown that K+ATP channels activated during periods of energy challenge reduce DA release (Tanaka et al., 1995). Although there is evidence that K+ATP channels are activated during the first hour of anoxia in the turtle brain and may therefore have a similar effect to decrease DA release during that period, the channels do not remain activated if anoxia continues (Pek and Lutz, unpublished data) and thus would not have an effect over the 3 hours of anoxic exposure used in these experiments.
Dopamine also may decrease release, and reduced rates of uptake may be sufficient to maintain extracellular DA at basal levels (or even that both release and reuptake increase during anoxia) such that the difference between release and reuptake remains the same in both anoxia and normoxia. However, there was no significant difference in DA release between normoxic and anoxic turtles treated with GBR 12909, and in fact, the rate of release in the anoxic brain was even slightly higher than in the normoxic brain (2 hours to peak DAec values vs. 3 hours in the normoxic brain, data not shown), indicating that rates of DA release during anoxia are similar to normoxic rates.
Dopamine uptake is known to be the primary route of DA removal from the extracellular space during normoxia (Iversen, 1971); this study demonstrated that reuptake mechanisms continue to function during anoxia even though inward cellular transport occurs via an energy-dependent process (Akiyama et al., 1991). The main route of DA uptake occurs through both a sodium-dependent high-affinity uptake mechanism (Horn, 1990); a sodium-independent low-affinity mechanism also has been reported (Mireylees et al., 1986). Although Berndt et al. (1993) report a 50% increase in the Vmax of the low affinity transport site during hypoxia in the rat brain and suggest that this is an adaptation to speed removal of excess extracellular DA, this transporter is not quantitatively significant enough for neuroprotection in the mammalian brain.
Because GBR 12909 specifically blocks the high-affinity DA transporter (van der Zee et al., 1980; Heikkila and Manzino, 1984; Andersen, 1989), causing a rise in extracellular DA in both the normoxic and anoxic turtle brain, this uptake mechanism is important in maintaining low extracellular DA in the turtle under both conditions. If a low-affinity, sodium-independent transporter exists in the turtle, as in the mammal, its activity is too low to prevent DA increases in the extracellular space when the high-affinity transport mechanism is blocked. It would be interesting to investigate which mechanisms allow the DA transporter to remain active in the anoxic turtle brain because its functioning uses ATP and the transporter must then be one of the basal metabolic costs to be paid even during anoxia.
Thus, the question is raised of what function, if any, the continuous release of DA plays in the electrically and metabolically suppressed brain. The turtle's ability to prevent the release of other potentially neurotoxic compounds, such as the EAAs, implies that DA release is functional rather than accidental. Because no known oxygen-independent pathway for monoamine synthesis is known in vertebrates, however, the nouveau synthesis of DA cannot replace vesicular losses and the continued functioning of DA reuptake would then be required to replenish intracellular stores.
Thus, the anoxia-tolerant turtle brain is unlike that of the mammal. Whereas in the mammalian brain, extracellular DA levels increase significantly even during mild hypoxia, low DA concentrations are maintained in the turtle striatum, even during 3 hours of complete anoxia. This probable adaptation to anoxia allows the turtle to escape the neurotoxic effects of DA release. One key mechanism of this adaptation is the continued function of the DA uptake mechanism during anoxia. It would be of interest to further investigate what role DA uptake and release play in the anoxic turtle brain.