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Article

Journal of Cerebral Blood Flow & Metabolism (1998) 18, 803–807; doi:10.1097/00004647-199807000-00010

Low Extracellular Dopamine Levels Are Maintained in the AnoxicTurtle (Trachemys scripta) Striatum

Supported by the National Science Foundation grant IBN:9507961.

Sarah L Milton and Peter L Lutz

Department of Biological Sciences, Florida Atlantic University, Boca Raton, Florida, U.S.A.

Correspondence: Sarah L Milton, Department of Biological Sciences, Bldg 12, Florida Atlantic University, 777 Glades Rd,Boca Raton, FL 33431, U.S.A.

Received 26 August 1997; Revised 17 November 1997; Accepted 18 November 1997.

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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.

Keywords:

Anoxia, Dopamine, GBR 12909, Microdialysis, Striatum, Trachemys scripta

Abbreviations:

ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; DA, dopamine; EEAs, excitatory amino acids

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). Hypoxicconditions deplete the high-energy phosphate stores needed to maintain cellular integrity,leading to decreased energy availability, neuron depolarization, and the release ofneurotoxic excitatory amino acids (EAAs).

Extracellular levels of the monoamine dopamine (DA) also have been implicated as animportant cause of pathogenesis in the hypoxic/ischemic brain, particularly in the striatum(Globus et al., 1988). Unlike the EAAs, however, where uncontrolled release occurs onlyafter depolarization (Katayama et al., 1991), excessive increases in extracellular DA areseen 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 oxygenpressure in the newborn piglet to 11% oxygen resulted in a 200% increase in extracellularDA (Huang et al., 1994). Dopamine may contribute to neuronal damage by modulating therelease of EAAs, through the production of oxygen-free radicals, by inhibitingsodium/potassium adenosine triphosphatase (Na+/K+ ATPase), and by uncoupling glucosemetabolism 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) increasedrelease 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 ofextra 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 ofincreased 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 tolow oxygen, a few species, including the freshwater turtle Trachemys scripta, demonstrateextended tolerance to brain anoxia (Lutz and Nilsson, 1997). The turtle brain can maintainadenosine triphosphate (ATP) levels and ion gradients for at least 48 hours of anoxia atroom temperature (Chih et al., 1989), lowering metabolic rates to equal the energy suppliedby glycolysis alone (Lutz and Nilsson, 1997).

The neurotransmitter system plays an important role in this process. Reducedneuroexcitability 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 avoidingdepolarization (Nilsson and Lutz, 1991).

However, it is not yet known how extracellular DA fits into this model. This neurotransmitteris of special interest because increases in extracellular DA and neuronal damage are seen inmammals even in mild hypoxia (Huang et al., 1994), well before oxygen levels are lowenough to cause depolarization and the catastrophic release of EAAs. It may be that theturtle brain is similar to that of mammals and that extracellular DA increases duringhypoxia, in which case one would expect the brain to have defense mechanisms against anyelevation in extracellular DA. Conversely, the turtle may not respond like the mammal andmay be able to maintain low concentrations of extracellular DA during hypoxia and evenover prolonged anoxia.

The purpose of this study was to distinguish between these two possibilities, and in thelatter case, to determine whether low extracellular DA is maintained by decreasing DArelease during hypoxia/anoxia or whether instead DA uptake mechanisms are functioning inthe face of continued release.

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MATERIALS AND METHODS

MaterialsThe 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 purchasedfrom Tocris Cookson (St. Louis, MO, U.S.A.). All other chemicals and reagents werepurchased from Sigma Chemicals (St. Louis, MO, U.S.A.).

MethodsExperiments were performed at 25°C on 24 freshwater turtles (T. scripta elegans). Animalswere divided into four groups: 1) controls (6 hours air); 2) anoxic controls (3 hours air, 3hours 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 mmolouabain 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 mixturepumped from a 1.5-L rebreathing bag. Animals were maintained on 1.7% isoflurane once asurgical plane was achieved (Shaw et al., 1992).

After exposing the skull, a 1-cm diameter hole was trephined and the skull cap removed. Asmall incision through the dura mater exposed the cerebral hemispheres. A stereotaxicinstrument and guide were used to insert a CMA/12 microdialysis probe (3 mm membranelength, Bioanalytical Systems, Inc., Acton, MA, U.S.A.) into the striatum (5 mm depth fromthe cerebral surface). After a 2-hour stabilization period in which no sampling occurred, theprobe was perfused with unbuffered sodium turtle Ringer's solution at 1.5 L/minute with aCMA/100 microdialysis pump (Carnegie Medecin, Solna, Sweden). Anoxia was induced bychanging the breathing mixture to certified 99.99% nitrogen (County Welding, PompanoBeach, Florida, U.S.A.) and isoflurane. For experimental animals, the specific DA transportblocker GBR 12909 (2 mol) in turtle Ringer's solution was delivered through themicrodialysis probe. Normoxic and anoxic brains were depolarized by the addition of 2 mmolouabain in turtle Ringer's solution through the microdialysis probe. Dialysate was collectedfor 30-minute intervals and analyzed immediately. Probe recovery was determined fromknown standards in vitro (Huang et al., 1994); DA recovery averaged 14.2 3.7% (meanstandard error of the mean).

Samples were analyzed for monoamine content using reversed-phase high-performanceliquid chromatography with electrochemical detection as adapted from Nilsson (1990). A 20-

L aliquot of dialysate was injected into a Waters 510 high-performance liquidchromatography pump (flow rate 1.3 mL/min) (Waters, Milford, MA, U.S.A.). The mobilephase consisted of 100 mmol l-1 sodium phosphate4, 9% (v/v) methanol, 0.63 mmol l-1

sodium octyl sulfate, and 0.2 mmol l-1 ethylenediamine tetraacetic acid. pH 3.6. Sampleswere separated on a catecholamine C18 column (3 m, 100 4.6 mm; Alltech, Deerfield,IL, U.S.A.) and detected by an LC-3 electrochemical detector with a glassy carbon workingelectrode set at +750 mV. Concentrations were determined by integrating the area underthe peak compared with known standards. Integrations were performed using the DynamaxMacIntegrator II integrator and software (Rainin Instrument, Woburn, MA, U.S.A.).

Methylene blue was injected through the microdialysis probe at the end of each experimentto identify probe location. Data were used only from those turtles in which probe location inthe 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 ofchanges was determined nonparametrically (Kruskal-Wallis test for unequal variances) usingthe 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 consideredstatistically significant. Although statistical analysis was performed on data from all timepoints (control versus experimental groups), figure (1 and figure 3) show peak values onlyas the time course of changes in DAec varied between animals.

Figure 1.

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).

Full figure and legend (117K)

Figure 3.

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).

Full figure and legend (128K)

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RESULTS

NormoxiaBasal DA levels in the turtle striatum (80 10 pmol/mL) were similar to those reported byothers in the mammalian brain (Baker et al., 1991; Goiny et al., 1991). (Normoxic andpreanoxic animals were pooled because there was no significant difference in basalextracellular DA between groups.) Basal concentrations increased nearly threefold (286196%) on perfusion of the striatum over a 3-hour period with the DA uptake blocker GBR12909 (Fig. 1); at such low concentrations of DA blocker, this increase most likely is due todecreased reuptake rather than increased release (Heikkila and Manzino, 1984). Inhibitionof Na+/K+ ATPase with ouabain (normoxic controls) caused nearly a fivefold increase inextracellular 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 iongradients within the brain.

AnoxiaThe effect of anoxia on extracellular striatal dopamine is shown in Figure 2; extracellulardopamine levels did not change significantly from control values even after 3 hours ofanoxia. That this lack of increase in striatal extracellular dopamine in the anoxic turtle brainis due at least in part to the continued activity of Na+/K+ ATPase and/or continuouslymaintained ion gradients, rather than to an overall depletion of DA within the brain, isindicated by the near threefold increase in extracellular levels on ouabain depolarization(Fig. 3). By contrast, extracellular dopamine levels still averaged only 109 63% of basallevels over 3 hours of anoxia (Fig. 2). Other work performed in this laboratory shows thatthe Na+/K+ ATPase, although working at reduced levels, still is active in the telencephalonof the anoxic T. scripta (Hylland et al., 1997). Previous work indicates that DA transport issodium dependent (Horn, 1990); thus, the continued maintenance of ATPase activity andion gradients would be crucial to DA reuptake. Addition of DA transport blocker to theanoxic 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 normoxicanimals treated with GBR 12909; this could indicate that rates of DA turnover continue atnear 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 duringanoxia and normoxia.

Figure 2.

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.

Full figure and legend (59K)

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DISCUSSION

This study shows that the turtle brain is unlike that of both the mammalian adult and thecomparatively hypoxia-tolerant neonate because turtle striatal extracellular DA levels didnot increase significantly even after 3 hours of anoxia, whereas mammals experience largeincreases in extracellular DA, even under mild hypoxia (Huang et al., 1994). Although 3hours of anoxia resulted in only an average 9% increase in extracellular DA in the turtlestriatum, severe hypoxia or hypoxia/ischemia causes as much as a 175- to 500-foldincrease in the rat (Globus et al., 1987; Globus et al., 1988; Pastuszko et al., 1996). Themaintenance of low extracellular DA probably is an important adaptation to anoxiatolerance, protecting the turtle brain from the neuronal damage that is associated withmassive DA release seen in hypoxic/ischemic mammalian brains.

Extracellular DA levels are determined by three distinct processes in the brain: rate ofrelease into the extracellular space, rate of reuptake, and washout rate. Although hypoxiaincreases 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 theturtle) and temporary (lasting only 1–2 hours) (Hylland et al., 1994), and therefore unlikelyto have any major effect over a 3-hour anoxic period. Although it has not beendemonstrated directly that DA is able to cross the blood-brain barrier of the turtle brain, themuch greater increases in striatal extracellular levels in the mammalian brain duringischemia (decreased washout) versus hypoxia indicate that at least some DA is able toescape (Globus et al., 1988; Baker et al., 1991). Therefore, it would appear unlikely thatsignificant differences in washout rates are responsible for the major increases in striatalextracellular DA reported in mammals versus the relatively stable levels observed in theanoxic turtle brain.

Extracellular DA levels thus would be affected primarily either by changing monoaminerelease, decreasing (or increasing) reuptake, or both. However, it is unlikely that the lowlevels of extracellular DA observed in this study during anoxia were due to decreasedintracellular DA supplies and thus decreased release. Because monoamine synthesisrequires oxygen, whole-brain DA levels do decrease over time during anoxia, but thesedecreases are only 10% over the first 4 hours (Nilsson et al., 1990). In addition, theouabain experiments described show that in both air and anoxia, depolarization of the turtlebrain results in massive DA release, as occurs in the mammal. The turtle striatum thereforeis clearly capable of releasing large quantities of DA. The difference in dopamine releasealso is not due to differences in intracellular DA levels because basal whole-brain levels arereported to be similar in the mammalian and turtle brains (Nilsson et al., 1990). Normoxicextracellular DA concentrations in this study also were similar to those previously reportedfor 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 DArelease 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 thereis evidence that K+

ATP channels are activated during the first hour of anoxia in the turtlebrain and may therefore have a similar effect to decrease DA release during that period, thechannels do not remain activated if anoxia continues (Pek and Lutz, unpublished data) andthus would not have an effect over the 3 hours of anoxic exposure used in theseexperiments.

Dopamine also may decrease release, and reduced rates of uptake may be sufficient tomaintain extracellular DA at basal levels (or even that both release and reuptake increaseduring anoxia) such that the difference between release and reuptake remains the same inboth anoxia and normoxia. However, there was no significant difference in DA releasebetween normoxic and anoxic turtles treated with GBR 12909, and in fact, the rate ofrelease in the anoxic brain was even slightly higher than in the normoxic brain (2 hours topeak DAec values vs. 3 hours in the normoxic brain, data not shown), indicating that rates ofDA release during anoxia are similar to normoxic rates.

Dopamine uptake is known to be the primary route of DA removal from the extracellularspace during normoxia (Iversen, 1971); this study demonstrated that reuptake mechanismscontinue to function during anoxia even though inward cellular transport occurs via anenergy-dependent process (Akiyama et al., 1991). The main route of DA uptake occursthrough 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 transportsite during hypoxia in the rat brain and suggest that this is an adaptation to speed removalof excess extracellular DA, this transporter is not quantitatively significant enough forneuroprotection 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 inboth the normoxic and anoxic turtle brain, this uptake mechanism is important inmaintaining 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 lowto prevent DA increases in the extracellular space when the high-affinity transportmechanism is blocked. It would be interesting to investigate which mechanisms allow theDA transporter to remain active in the anoxic turtle brain because its functioning uses ATPand the transporter must then be one of the basal metabolic costs to be paid even duringanoxia.

Thus, the question is raised of what function, if any, the continuous release of DA plays inthe electrically and metabolically suppressed brain. The turtle's ability to prevent the releaseof other potentially neurotoxic compounds, such as the EAAs, implies that DA release isfunctional rather than accidental. Because no known oxygen-independent pathway formonoamine synthesis is known in vertebrates, however, the nouveau synthesis of DAcannot replace vesicular losses and the continued functioning of DA reuptake would then berequired to replenish intracellular stores.

Thus, the anoxia-tolerant turtle brain is unlike that of the mammal. Whereas in themammalian brain, extracellular DA levels increase significantly even during mild hypoxia,low DA concentrations are maintained in the turtle striatum, even during 3 hours ofcomplete anoxia. This probable adaptation to anoxia allows the turtle to escape theneurotoxic effects of DA release. One key mechanism of this adaptation is the continuedfunction of the DA uptake mechanism during anoxia. It would be of interest to furtherinvestigate what role DA uptake and release play in the anoxic turtle brain.

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