Protection of Malonate-Induced GABA But Not Dopamine Loss by GABA Transporter Blockade in Rat Striatum

Experimental Neurology 176, 193–202 (2002) doi:10.1006/exnr.2002.7917

Protection of Malonate-Induced GABA But Not Dopamine Loss by GABA Transporter Blockade in Rat Striatum Gail D. Zeevalk, Lawrence Manzino, and Patricia K. Sonsalla Department of Neurology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Received October 12, 2001; accepted February 11, 2002

INTRODUCTION Previous work has shown that overstimulation of GABA A receptors can potentiate neuronal cell damage during excitotoxic or metabolic stress in vitro and that GABA A antagonists or GABA transport blockers are neuroprotective under these situations. Malonate, a reversible succinate dehydrogenase/mitochondrial complex II inhibitor, is frequently used in animals to model cell loss in neurodegenerative diseases such as Parkinson’s and Huntington’s diseases. To determine if GABA transporter blockade during mitochondrial impairment can protect neurons in vivo as compared with in vitro studies, rats received a stereotaxic infusion of malonate (2 ␮mol) into the left striatum to induce a metabolic stress. The nonsubstrate GABA transport blocker, NO711 (20 nmol) was infused in some rats 30 min before and 3 h following malonate infusion. After 1 week, dopamine and GABA levels in the striata were measured. Malonate caused a significant loss of striatal dopamine and GABA. Blockade of the GABA transporter significantly attenuated GABA, but not dopamine loss. In contrast with several in vitro reports, GABA A receptors were not a downstream mediator of protection by NO711. Intrastriatal infusion of malonate (2 ␮mol) plus or minus the GABA A receptor agonist muscimol (1 ␮mol), the GABA A Cl ⴚ binding site antagonist picrotoxin (50 nmol) or the GABA B receptor antagonist saclofen (33 nmol) did not modify loss of striatal dopamine or GABA when examined 1 week following infusion. These data show that GABA transporter blockade during mitochondrial impairment in the striatum provides protection to GABAergic neurons. GABA transporter blockade, which is currently a pharmacological strategy for the treatment of epilepsy, may thus also be beneficial in the treatment of acute and chronic conditions involving energy inhibition such as stroke/ischemia or Huntington’s disease. These findings also point to fundamental differences between immature and adult neurons in the downstream involvement of GABA receptors during metabolic insult. © 2002 Elsevier Science (USA) Key Words: GABA transporter; malonate; Parkinson; Huntington; ischemia.

GABA serves as the major inhibitory neurotransmitter in the CNS. Exposure of neurons to an excitotoxic or metabolic challenge causes an early release of GABA into the extracellular space (12, 55) prior to release of other amino acids such as glutamate or aspartate (55). The majority of the GABA that is released is the result of reversal of the high affinity GABA transporter (56, 57). In some neuronal systems, the increase in extracellular GABA during excitotoxic or metabolic stress has been implicated in the pathological sequelae of events. Blockade of the GABA transporter with subsequent attenuation of GABA release prevented acute cell swelling due to glutamate (56) or metabolic impairment (57) in the isolated embryonic chick retina. Consistent with protection against acute cell swelling, GABA transport blockers have been shown to protect against irreversible neuronal damage in primary cortical cultures exposed to NMDA (36) or mesencephalic cultures exposed to the succinate dehydrogenase/mitochondrial complex II inhibitor, malonate (48). Evidence from several laboratories suggest that downstream mediators of GABA transporter involvement in excitotoxic or metabolic stress involves Cl ⫺ flux and/or GABA A receptor activation. Low Cl ⫺ or Cl ⫺ channel blockers were neuroprotective against acute (53) and delayed (12, 13, 16) excitotoxicity. Kainate dramatically increased Cl ⫺ influx and was significantly attenuated by GABA A receptor antagonists (12). Activation of GABA A receptors with muscimol or GABA potentiated excitotoxin-mediated damage in cortical cultures (16), and in cerebellar granule cell cultures (12), whereas blockade of GABA A receptors was found to be protective. It should be noted, however, that GABA A receptor activation during excitotoxicity in some studies was found to be protective (36). Mitochondrial inhibition with malonate produces damage to mesencephalic dopamine and GABA neurons that is mediated in part by NMDA receptors (52). In this system, GABA A receptors also appear to be downstream mediators of the protective effects of GABA transporter

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blockade in that GABA A receptor activation potentiated malonate-induced damage, while GABA A receptor antagonists were protective (48). Consistent with these findings, GABA A receptor activation during oxygen/ glucose deprivation potentiated damage to cultured cortical neurons (36). There is also evidence to suggest that under certain circumstances, overstimulation of GABA receptors can be detrimental. A subpopulation of hippocampal neurons in culture (29, 51) or fetal forebrain GABA neurons (22) were killed by exposure to muscimol, GABA or ␥-vinyl-GABA. The GABA transport blocker, tiagibine, is used clinically in the treatment of epilepsy and is well tolerated in humans. The efficacy of GABA transport blockers versus other insults such as excitotoxicity or metabolic inhibition in vitro suggests that GABA transport blockers may have utility in the treatment of conditions such as hypoxia/ischemia or in neurodegenerative conditions that may involve a metabolic or secondary excitotoxic component such as Parkinson’s or Huntington’s diseases (4, 43). To date, the protective effects of GABA transporter blockade or the involvement of GABA A receptors in potentiating damage during excitotoxicity or metabolic inhibition has focused mainly on studies using cultured or embryonic neuronal preparations. Immature neurons, however, may use GABA as an excitatory neurotransmitter; see (14) for review. Thus, the involvement of the GABAergic system during mitochondrial disturbances or subsequently, a secondary excitotoxicity in the mature nervous system may not duplicate what is observed in vitro. The current studies were, therefore, undertaken to examine the ability of the GABA uptake blocker NO711 to protect against striatal dopamine or GABA loss due to intrastriatal infusion of malonate in the adult rat and to determine if activation or antagonism of GABA A receptors during malonate exposure modulated toxicity. MATERIAL AND METHODS

Intrastriatal infusion. Male Sprague–Dawley rats (300 –350 g), approximately 12 weeks of age; Harlan Farms, Indianapolis, IN) were used in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Studies were approved by the local Institutional Animal Care Committee. Rats were grouped two per cage in a room maintained at 20 –22°C on a 12-h light/dark cycle with food and water available ad libitum. Animals were anesthetized intramuscularly with a mixture of ketamine (50 mg/ kg), xylazine (2.5 mg/kg), and acetyl promazine (0.625 mg/kg) prior to cannula placement. The left striatum was then cannulated such that the tip of the cannula resided just above the region to be infused. Coordinates for needle placement corresponded to those in the rat

atlas of Konig and Klippel (25) (AP 7.2; L, 2.6; and DV, 0.4). Treatment protocol and tissue preparation. All solutions were pH adjusted to 7.6. NO711 (20 nmol), picrotoxin (50 nmol), saclofen (33 nmol), or saline were directly infused into the left striatum using an adapted microsyringe pump 30 min prior to infusion of malonate (2 ␮mol). In rats receiving muscimol (1 ␮mol), the muscimol was coinfused with malonate. Infusion volume was 1 ␮l with a delivery rate of 0.5 ␮l/min. The needle was left in place for an additional min prior to its removal. All animals received a second infusion, 3 h later that consisted of either NO711, muscimol, picrotoxin, saclofen, or saline at the above indicated doses. Animals were allowed to recover for 7 days. Two min prior to sacrifice, rats were given a tail vein injection of 3-mercaptopropionic acid to inhibit glutamic acid decarboxylase activity and prevent GABA synthesis (26). Striata were removed, laid flat, and the nucleus accumbens ventral to the anterior commissure was dissected away. The remaining striatum was subdivided into three regions of equal mass that were designated ventral, middle, and dorsal striatum. Each region was homogenized in 0.2 N perchloric acid and analyzed for dopamine and GABA. Detection of dopamine. Dopamine was measured by high-performance liquid chromatography (HPLC) with electrochemical detection as described in detail previously (47). Briefly, homogenates were centrifuged and a 15-␮l aliquot of the supernatant was injected onto a HPLC (BAS, West Lafayette, IN) equipped with a Hewlett-Packard integrator. The mobile phase was composed of 26 ml of acetonitrile, 21 ml of tetrahydrofuran, and 960 ml of 0.15 M monochloroacetate, pH 3.0, containing 50 mg/l of EDTA and 200 mg/l sodium octyl sulfate. Concentrations of dopamine and metabolites were determined by comparison to peak heights of known standards. Detection of GABA. GABA in the extract was quantified by fluorescent detection of the o-phthalaldehyde adduct as described previously (37). Prior to derivitization, extracts were neutralized to pH 5 with K 2CO 3. A Beckman HPLC system Gold Model 338 fitted with a Beckman 175 fluorometer was used in the separation and detection. The derivatives were separated on a Beckman, 5 ␮m, C 18 Ultrasphere ODS column. Samples were reacted with an equal volume of o-phthalaldehyde reagent and injected at 1.25 min. The programmed gradient elution was formed from 2 buffers: (A) 0.1M sodium acetate, pH 5.9, in 10% methanol, and (B) 80% methanol. Quantitation was by comparison to known standards of amino acids. Statistics. For comparison of drug treatments against malonate, differences in the left infused striata versus control contralateral noninfused striata were analyzed using ANOVA (Instat 2, GraphPad) followed

STRIATAL GABA TRANSPORT BLOCKADE AND MALONATE

by post hoc Tukey comparison. Comparison between saline and NO711, muscimol, picrotoxin, or saclofen controls were done by comparing mean values from left infused striata using ANOVA plus Tukey post hoc comparison. Statistical probability of P ⬍ 0.05 or better was considered significant. RESULTS

In order to examine if the protective effects of GABA transporter blockade during mitochondrial inhibition with malonate was protective in vivo, similar to what our laboratory reported in vitro in mesencephalic cultures (48), Sprague–Dawley rats were stereotaxically infused with 2 ␮mol malonate in 1 ␮l into the left striatum. The right noninfused striatum served as each animal’s own control. Some animals received an infusion of NO711 (20 nmol) or saline 30 min prior to malonate infusion followed by a second infusion of NO711 or saline 3 h later. Animals were allowed to recover for 1 week and then striatal levels of dopamine and GABA were measured. To circumvent confounding factors such as nonspecific damage at the site of infusion, the striatum was subdivided into 3 regions of equal mass termed ventral, middle, and dorsal striata. In some animals, the infusion site was denoted prior to measurement of dopamine and GABA and determined to be at the interface between the dorsal and middle third of the tissue (Fig. 1a). Striatal dopamine levels in left and right striatal regions in NO 711 control animals one week after infusion were, ventral: 12.2 ⫾ 1.3 and 12.9 ⫾ 0.6; middle 10.8 ⫾ 1.5 and 11.5 ⫾ 1.2; dorsal 6.2 ⫾ 1.5 and 6.6 ⫾ 1.0 ␮g/gm tissue ⫾ SD, respectively, and did not differ significantly from saline infused animals (see Table 1). Striatal GABA levels in left and right striata in NO711 control rats were ventral, 2.44 ⫾ 0.49 and 2.02 ⫾ 0.37; middle, 2.55 ⫾ 0.51 and 2.24 ⫾ 0.51; and dorsal, 2.93 ⫾ 0.91 and 2.19 ⫾ 0.59 ␮mol/g tissue ⫾ SD, respectively, and were statistically similar to saline infused animals (Table 1). Malonate caused a 50 and 60% loss of dopamine in the middle and dorsal striata, respectively (Figs. 1c and 1d). No loss of dopamine was observed in the ventral third of the tissue (Fig. 1b). The nonsubstrate transport blocker NO711 showed no protective effect against dopamine terminal loss produced by malonate. In contrast, NO711 protected GABA neurons from damage caused by malonate. Malonate reduced GABA content 23 and 45% in the middle and dorsal striata, respectively. In the presence of malonate plus NO711, GABA loss was significantly attenuated in the middle and dorsal regions (Figs. 2b and 2c) and statistically different from animals treated with malonate alone. Similar to findings with dopamine, GABA levels were not reduced in the ventral third of the striatum. In previous work, our laboratory has shown that the bulk of GABA release during energy stress is caused by

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reversal of the GABA transporter and blocked by the nonsubstrate GABA transport inhibitors, SKF 89976A or NO711 (56, 57). In mesencephalic cultures, protection by NO711 was observed to be due to a decrease in extracellular GABA, which in turn attenuated activity at GABA A receptors (48). To determine if the protection afforded by NO711 against striatal GABA loss 1 week following mitochondrial inhibition with malonate was mediated via a secondary action on GABA A receptors as observed in vitro, rats were infused with 1 ␮l of either saline or 2 ␮mol malonate into the left striatum. Some rats received a coinfusion of 1 ␮mol muscimol along with malonate. A second infusion of muscimol or saline was given 3 h later. To insure that there was sufficient muscimol to activate GABA A receptors, the concentration chosen for infusion (1 ␮mol in 1 ␮l) was in excess of that needed to elicit GABA A receptor activation in the basal ganglia of rats receiving centrally administered muscimol (0.8 nmol/␮l) (11). Animals recovered for 1 week and striatal dopamine and GABA levels were measured. Infusion of muscimol into the left striatum did not alter dopamine or GABA levels when examined at 1 week postinfusion (Table 1). Malonate caused a significant reduction in dopamine in the middle (54% loss) and dorsal (71% loss) thirds of the striatum and no loss in the ventral third (Fig. 3). Similarly, GABA levels were reduced in the middle striatum by 24% and dorsal striatum by 44% with no loss observed in the ventral tissue. Activation of GABA A receptors with the GABA A agonist muscimol did not significantly alter malonate toxicity in the dopamine or GABA populations (Fig. 3). In addition to examining the effects of GABA A receptor activation during energy deprivation in the striatum in vivo, the effects of GABA A receptor antagonism of the GABA A Cl ⫺ site with picrotoxin were studied. Rats were infused into the left striatum with either saline or malonate (2 ␮mol). Some rats received an infusion of picrotoxin (50 nmol) 30 min prior to malonate. Three hours later, a second infusion of picrotoxin and/or saline was administered. The concentration of picrotoxin chosen for this study was based on a pilot dose–response study and represented one half the maximal dose that could be administered that resulted in minimal seizure activity. Animals recovered for 1 week and then striatal dopamine and GABA levels were measured. Consistent with the lack of effect of GABA A receptor activation on malonate-induced damage in the striatum, antagonism of GABA A receptors during malonate exposure did not modify toxicity to either the dopamine or GABA populations (Fig. 4). Saclofen, a GABA B receptor antagonist was also tested against malonate and consistent with in vitro studies was found to be without effect in any region of the striatum. Combined total striatal infused versus noninfused ratios for dopamine were 0.91 ⫾ 0.026, 0.66 ⫾ 0.11, and 0.66 ⫾ 0.095 for left and right side ⫾ SEM for

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FIG. 1. (a) Schematic of rat striatum showing the infusion site (arrow) and location of subdivisions for dopamine and GABA measurement. Dopamine levels were measured separately in the ventral (b), middle (c), and dorsal (d) thirds of the tissue one week following infusion of malonate (2 ␮mol) or malonate (2 ␮mol) plus the GABA uptake blocker NO711 (20 nmol) as described under Materials and Methods. Results are expressed as dopamine levels in the infused left striatum divided by levels in the contralateral noninfused right striatum ⫾ SEM. Absolute tissue levels in control NO711 left and right striata are given in the text and did not differ significantly from saline infused animals (Table 1). Malonate caused a significant decrease in tissue dopamine in the middle and dorsal thirds of the striatum. NO711 did not attenuate dopamine loss. The n is from 6 to 7 animals per group. a Different from control.

saclofen (33 nmol), malonate (2 ␮mol), or saclofen plus malonate, respectively. Combined GABA values were 1.15 ⫾ 0.07, 0.88 ⫾ 0.09, and 0.92 ⫾ 0.11, left and right striata ⫾ SEM for saclofen, malonate, and saclofen plus malonate, respectively. The n was derived from five animals per group. DISCUSSION

Two major observations arise from the current study. The first is that the intrastriatal administration of the GABA uptake blocker NO711 can protect striatal

GABA neurons, but not dopamine terminals, from damage due to mitochondrial inhibition with the competitive, reversible succinate dehydrogenase/mitochondrial complex II inhibitor, malonate. Second, in contrast with in vitro studies, protection by GABA transporter blockade in the adult rat brain does not involve GABA A receptors. Our finding of protection of striatal GABA neurons in vivo by GABA transporter blockade during energy impairment is consistent with the protection of GABA neurons observed in vitro in rat mesencephalic cultures (48). In animal models of ischemia, protection of hippocampal CA1 neurons has been ob-

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TABLE 1 Striatal Dopamine and GABA Tissue Levels Ventral Left

Middle

Dorsal

Right

Left

Right

Left

Right

14.41 ⫾ 0.43 14.19 ⫾ 0.76 13.69 ⫾ 0.84

12.12 ⫾ 1.2 12.13 ⫾ 0.81 11.08 ⫾ 0.76

12.65 ⫾ 0.34 12.73 ⫾ 0.33 12.09 ⫾ 0.93

7.80 ⫾ 1.80 7.65 ⫾ 1.01 6.95 ⫾ 1.40

8.88 ⫾ 1.49 8.01 ⫾ 0.62 7.05 ⫾ 0.50

1.64 ⫾ 0.49 1.91 ⫾ 0.20 1.66 ⫾ 0.28

2.21 ⫾ 0.50 2.70 ⫾ 0.73 2.20 ⫾ 0.60

2.06 ⫾ 0.41 2.21 ⫾ 0.48 2.08 ⫾ 0.40

1.82 ⫾ 0.53 2.28 ⫾ 0.66 2.06 ⫾ 0.59

2.07 ⫾ 0.91 2.06 ⫾ 0.46 2.03 ⫾ 0.57

Dopamine (␮g/g tissue ⫾ SD) Saline Musc Picro

13.62 ⫾ 0.73 13.70 ⫾ 1.41 12.78 ⫾ 0.63

GABA (␮mol/g tissue ⫾ SD) Saline Musc Picro

2.02 ⫾ 0.45 2.45 ⫾ 0.76 2.00 ⫾ 0.64

Note. Sprague–Dawley rats received an infusion of either saline, muscimol (musc, 1 ␮mol) or picrotoxin (picro, 0.05 ␮mol) in 1 ␮l into the left striatum followed 3 h later by a 2nd infusion. After 1 week of recovery, the striata were removed and subdivided into 3 regions of equal mass (ventral, middle, and dorsal) striatum and dopamine and GABA levels were measured separately in each region. N is from 6 animals per group.

served with GABA transporter inhibition (23, 24), although the intraperitoneal administration of GABA transport blockers produces hypothermia and there is some question as to its contribution in this paradigm (23). Intrastriatal infusion of NO711, as done in the present study, eliminates this confounding factor and demonstrates the ability of GABA transporter blockade to protect against mitochondrial impairment in striatum in vivo. In addition to acute metabolic insults such as hypoxia/ischemia, a number of neurodegenerative diseases have been associated with metabolic defects including Parkinson’s and Huntington’s diseases (4, 43). In sporadic Parkinson’s disease, defects in complex I have been most consistently observed, see (52) for discussion. In addition, defects in combined complex II/III activity, in ␣-ketoglutarate dehydrogenase and in Coenzyme Q10 have been reported (34, 46, and see 52 for discussion). Mesencephalic cultures contain midbrain dopamine neurons, the major neurotransmitter population lost in Parkinson’s disease. Our previous observation that GABA transporter blockers protected mesencephalic dopamine and GABA neurons from damage due to the mitochondrial inhibitor malonate (48), suggested the possibility that modulation of the GABAergic system may protect the dopaminergic neurons from mitochondrial inhibition in vivo. An intrastriatal infusion of malonate produces loss of both striatal GABA neurons and dopamine terminals (54) when examined 1 week following infusion. As observed previously (35, 54) and in the present study (Figs. 1 and 2) a greater loss of striatal dopamine as compared with GABA is observed in both the rat and mouse brain with malonate. In contrast with the protection found against malonate-induced striatal GABA loss with NO711, loss of dopamine 1 week following malonate exposure was not attenuated by the GABA

transport inhibitor. The lack of protection against dopamine loss is counter to our in vitro findings. In mesencephalic cultures, NO711 provided dose dependent protection of the dopamine population from malonate. Other nonsubstrate GABA transport blockers, such as SKF 89976A, that inhibit the carrier but are not themselves transported, or substrate blockers such as nipecotic acid, that compete for transport, were also protective (48). The difference in findings for GABA transporter inhibition and protection against toxicity in the dopamine population in vitro, but not in vivo, may be related to the mechanism of protection in different age neurons. Protection by GABA transport inhibitors in cultured mesencephalic neurons was consistent with a secondary action at GABA A receptors (48). The GABA agonist, muscimol, potentiated malonate-induced damage. Antagonism of GABA A receptors with bicuculline or picrotoxin provided protection. This mechanism of action supports several in vitro studies from other laboratories to show that excitotoxin-induced damage was exacerbated by GABA A receptor activation and protected by GABA A and/or glycine receptor antagonists (12, 16, 20). Malonate toxicity in mesencephalic cultures produces a secondary excitotoxicity (52) and can be blocked by NMDA antagonists, similar to what has been reported in vivo (18). Thus, in cultured mesencephalic neurons, it is speculated that GABA transporter blockade prevents GABA transporter reversal and release of GABA during energy impairment. The increase in extracellular GABA may then activate GABA A receptors to exacerbate a secondary excitotoxicity. Potentiation of excitotoxic damage by GABA A receptor activation has mainly been observed in cultured neurons which are known to be immature. During development, activation of GABA A receptors can elicit depolarizing responses in some neurons (39); see (14)

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FIG. 2. GABA levels in the ventral (a), middle (b), and dorsal (c) striatum from the same animals as described in the legend of Fig. 1 were measured 1 week following infusion of malonate (2 ␮mol) or malonate (2 ␮mol) plus NO711 (20 nmol). GABA levels were significantly decreased in the middle and dorsal striata. NO711 significantly attenuated GABA loss. N is from 6 to 7 animals per group. a Different from control; b different from malonate alone.

for review. During early postnatal life, GABA A receptor activation provides a major excitatory drive to pyramidal cells and it has been speculated that GABA A receptor activation during development may provide the excitatory drive for growth and differentiation (14). The excitatory action at GABA A receptors in immature neurons may thus underlie the potentiation of excitotoxicity or metabolic stress observed in in vitro studies. In the mature nervous system, on the other hand, enhancement of GABA A receptor activity can attenuate excitotoxicity due to direct exposure to kainate (30) or as a secondary consequence to traumatic brain injury (38). There is also evidence as to the protective effect of GABA potentiation during ischemia (see (17) for review). The lack of potentiation of malonate-induced damage and the trend towards protection in rat striatum with GABA A receptor activation in the present study would be in keeping with these observations. In striatum, GABA can activate presynaptic GABA A receptors on dopamine terminals (40, 42) as well as GABA A-like autoreceptors on GABAergic neurons (15). GABA B receptors are also present in high concentration in rat striatum (21, 50) and consistent with in vivo (41) or in vitro (36) studies, modulation of these receptors did not alter toxicity due to energy inhibition. GABA C receptors are structurally related to GABA A receptors, but their distribution in mammals appears to be limited to retina (8) and were, therefore, not examined. The mechanism underlying protection of striatal GABA neurons against malonate by NO711 in the adult rat brain is uncertain at present, although the selective effect on GABA neurons suggests that it might be due to a direct effect of transporter blockade. NO711, also known as NNC711, has a high affinity for the GABA transporter, GAT1 (7), that is present on striatal GABA neurons (3), consistent with an action on GABA, but not other neuronal elements in striatum. Metabolic impairment is known to release GABA via reversal of the GABA transporter (57). In vivo, intrastriatal infusion of malonate increases extracellular GABA as well as glutamate (33). Prevention of GABA transporter reversal may have two effects on GABA neurons during energy inhibition. It may help to maintain energy status by conserving ATP usage by ATPase pumps that are indirectly needed for GABA removal from the extracellular space and second, it may partially reduce Na ⫹ and Cl ⫺ movement during excitotoxicity or metabolic stress that could contribute to ionic imbalance and subsequent swelling. The GABA transporter requires the presence of Na ⫹ and Cl ⫺ and is electrogenic (9, 10). Ion movement through the transporter can occur in the absence of GABA (9, 10). Nonsubstrate transport blockers such as NO711 suppress GABA-induced currents through the transporter (49) consistent with the interpretation that inhibitors can protect by blocking ionic movements. This could also

STRIATAL GABA TRANSPORT BLOCKADE AND MALONATE

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FIG. 3. GABA (a, b, c) and dopamine (d, e, f) levels in the striata of animals one week following infusion with malonate (2 ␮mol) or malonate (2 ␮mol) plus muscimol (1 ␮mol) as described under Materials and Methods. Malonate significantly decreased dopamine and GABA levels in the middle and dorsal striata. The GABA A agonist, muscimol, did not significantly alter dopamine or GABA loss caused by malonate, although there was a trend towards protection in both populations. N is from 6 animals per group. aDifferent from control.

help explain the prevention of acute cell swelling in ex vivo retina exposed to either glutamate or metabolic inhibitors (56, 57). It should be noted that in the ex vivo retinal model of metabolic impairment, only nonsubstrate transport blockers prevented acute cell swelling (57) further supporting that ion and/or GABA movement through the transporter during conditions of stress contributed to the acute cellular edema. In mesencephalic cultures, both nonsubstrate and substrate blockers were protective. However, under the long exposure condition in this paradigm, i.e., 24 h, it was found that substrate inhibitors such as nipecotic acid

caused long term irreversible inhibition of GABA transport (48). Damage to GABA neurons caused by intrastriatal infusion of malonate resembles that found in Huntington’s disease in that there is sparing of NADPH diaphorase positive neurons and somatostatin (6). Deficiencies in energy metabolism have been hypothesized as contributing to the pathology in Huntington’s disease. Deficient oxidative metabolism in muscle from symptomatic Huntington patients (28), reduced glucose metabolism (2), increased lactate concentration and lactate to pyruvate ratios (27), and mitochondrial complex

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FIG. 4. GABA (a, b, c) and dopamine (d, e, f) levels in rat striata one week following infusion with malonate (2 ␮mol) or malonate (2 ␮mol) plus picrotoxin (0.05 ␮mol) as described under Materials and Methods. Both GABA and dopamine levels were significantly reduced in the middle and dorsal striata. The presence of picrotoxin was without effect on malonate-induced toxicity. The n if from 6 animals per group. a Different from control.

defects in the caudate nucleus (19) have been described. Animal models of Huntington’s disease also implicate a secondary excitotoxicity downstream of energy impairment as contributing to GABA cell loss (5). A number of approaches have been used to attenuate striatal GABA loss in animal models of Huntington’s disease including: NMDA receptor antagonism (5), free radical scavengers (32), nitric oxide synthase (NOS) inhibitors (31) or neuronal NOS knock out animals (44), and modulation of apoptotic signaling proteins (1, 45). To our knowledge, this is the first report to show that inhibition of the GABA transporter with a nonsubstrate blocker can protect striatal GABA neurons

during energy inhibition with malonate and suggests that this approach may be useful in the treatment of Huntington’s disease. Of interest, tiagabine, (R(-)-N[4,4-bis(3-methylthein-2-yl)but-3-enyl]nipecotic acid, hydrochloride), a lipophilic derivative of nipecotic acid is currently in use in the treatment of epilepsy and similar to its close structural analog, NO711, has a high affinity for GAT1 transporters (7). In summary, our findings demonstrate that blocking GABA transport with a nonsubstrate GAT1 inhibitor provided protection of striatal GABA neurons during impairment in energy metabolism. Unlike immature cultured neurons, in vivo protection did not involve

STRIATAL GABA TRANSPORT BLOCKADE AND MALONATE

GABA A receptors. The involvement of metabolic defects in Huntington’s disease and the histologically similar pattern of striatal cell loss observed with malonate in striatum in Huntington’s disease suggests that GABA transporter blockade may have therapeutic relevance to this disease. Acute conditions such as hypoxia/ischemia may also benefit from GABA transporter blockers, however, given the differences in outcome with GABA A receptor activation during excitotoxicity or energy inhibition in the adult and immature nervous system, caution needs to be observed when developing approaches in treatment strategies. ACKNOWLEDGMENTS The authors thank Cindy Song for her skillful technical assistance. This work was supported by Public Health Service Grant NS36157 and by a grant from The American Parkinson Disease Association, Inc.

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