Mate choice is an evolutionarily critical decision that requires the detection of multiple sex-specific signals followed by central integration of these signals to direct appropriate behavior. The mechanisms controlling mate choice remain poorly understood. This study shows that the glial amino-acid transporter Genderblind controls whether Drosophila melanogaster males will attempt to mate with other males. genderblind (gb) mutant males showed no alteration in heterosexual courtship or copulation, but are attracted to normally unappealing male species-specific chemosensory cues. As a result, genderblind mutant males courted and attempted to copulate with other Drosophila males. This homosexual behavior could be induced within hours using inducible RNAi, suggesting that genderblind controls nervous system function rather than its development. Consistent with this, and indicating that glial genderblind regulates ambient extracellular glutamate to suppress glutamatergic synapse strength in vivo, homosexual behavior could be turned on and off by altering glutamatergic transmission pharmacologically and/or genetically (Grosjean, 2008).

Mate selection is an important decision that relies on proper detection and integration of multiple sensory cues. To aid the process, many animals perform elaborate courtship rituals that are designed to attract and differentiate between potential sexual partners. In the fruit fly Drosophila melanogaster, courtship typically begins when a male fly identifies and approaches a suspected conspecific female. To confirm his suspicions and to test whether she is sexually receptive, he will tap her with his foreleg (to evaluate nonvolatile pheromones via chemoreceptors on his leg), sing a species-specific courtship song (by extending and vibrating a wing) and lick her genitalia (to sample pheromones). If she is acceptable and does not reject him (by extending her ovipositor, striking him with her wings or legs, or simply running away), he will mount her, curl his abdomen and attempt copulation (Grosjean, 2008).

Much of the 'wiring' required for Drosophila courtship develops under the control of well-studied sex-specific transcription factors, including those encoded by the genes transformer, fruitless, doublesex and dissatisfaction, which also determine whether brains develop as 'male' or 'female'. As expected, flies with genetically male brains carry out typical male behaviors and flies with genetically female brains show typical female behaviors (Grosjean, 2008).

Atypical behavior includes homosexual courtship. Homosexual (male-male or female-female) courtship, regardless of whether heterosexual (male-female) courtship is also altered, represents an inability to distinguish sex-specific cues or an inability to respond appropriately to these cues. In Drosophila melanogaster, the ability to discriminate between males and females depends on visual, acoustic and chemical cues, including 7-tricosene and cis-vaccenyl acetate (cVA), which are perceived by taste and olfaction, respectively (van der Goes van Naters, 2007; Lacaille, 2007). Flies that do not produce 7-tricosene and/or cVA are courted by males, and male flies that cannot sense these pheromones inappropriately court other males (Grosjean, 2008).

But what controls whether cues such as 7-tricosene and cVA are attractive or repulsive? The central mechanisms controlling sexual behavior remain unknown. This study shows that homosexual behavior in Drosophila is controlled by glutamatergic synapse strength, which in turn is regulated by a glial amino-acid transporter that has been named Genderblind on the basis of the mutant phenotype. Consistent with this conclusion, it was found that homosexual behavior could be turned on and off in a period of hours by genetic alteration of Genderblind abundance and/or by pharmaceutical manipulation of glutamatergic synapse strength. Genderblind represents a previously unknown form of neural circuit modulation and an unexpected means of regulating an evolutionarily critical behavior (Grosjean, 2008).

Male flies carrying the KG07905 P{SUPor-P} transposon insertion in the gb (CG6070) gene showed frequent homosexual interactions, including singing to other males, genital licking and attempted copulation. In contrast, wild-type and control flies (including those carrying P{SUPor-P} transposon insertions in other genes) rarely showed these homosexual behaviors (Grosjean, 2008).

The P{SUPor-P}CG6070[KG07905] insertion lies in the predicted 5' UTR of the gb gene, and therefore might disrupt gb transcription, mRNA trafficking and/or mRNA stability. To determine whether gb mRNA was reduced in gb[KG07905] mutants, real-time RT-PCR was performed. Quantitative real-time RT-PCR using mRNA extracted from adult male flies showed a significant reduction of gb mRNA in gb[KG07905] mutants compared with wild type, demonstrating that the KG07905 insertion does indeed cause a loss of gb mRNA and that gb[KG07905] is a mRNA hypomorph (Grosjean, 2008).

Loss of gb mRNA should lead to loss of Genderblind protein. To confirm this, and to also determine whether incidence of male-male courtship might be directly proportional to Genderblind protein loss, Genderblind protein was measured from five different genotypes using immunoblots probed with antibody to Genderblind. The total amount of Genderblind protein in gb[KG07905] mutants was 35 +/- 12% of that found in wild type, consistent with the reductions in gb mRNA that was measured in the same genotypes by real-time RT-PCR. Furthermore, there was a strong inverse correlation between total Genderblind protein quantity and homosexual courtship (Grosjean, 2008).

Three other experiments confirmed that the homosexual behavior observed in gb[KG07905] mutant male flies was caused by loss of gb function. First, precise excision of the transposon inserted in gb (P{SUPor-P}CG6070[KG07905]) completely rescued the courtship phenotype. Second, gb mutant homosexual courtship was phenocopied by expression of gb RNAi. Third, a chromosomal deletion of gb, Df(3R)Exel6206, was unable to complement the defect induced by the mutation; double heterozygote (Df/gb) males showed high levels of homosexual courtship behavior, equal to that observed in gb mutant homozygotes (Grosjean, 2008).

Although gb[KG07905] mutants showed prominent homosexual behavior, they also showed heterosexual behavior. Therefore, they were presumably bisexual. To confirm this, gb[KG07905] and wild-type male flies were presented simultaneously with a wild-type passive (decapitated) male and a wild-type passive (decapitated) virgin female, either of which could be chosen as a sexual partner. Wild-type males always chose to court the female. In contrast, gb mutant males courted wild-type males and females with equal intensity and probability. Detailed examination of gb mutant heterosexual courtship and copulation revealed no alterations in copulation frequency, latency or duration. gb mutant males also showed normal locomotor activity. Thus, the gb courtship phenotype appears to be specific to male-male interactions (Grosjean, 2008).

To rule out possible group effects that might have arisen in these assays, single-pair courtship assays were carried out using passive (decapitated) partners. These assays confirmed that individual gb[KG07905] mutant males court both males and females with equal likelihood, unlike wild-type males. Notably, precise excision males courted decapitated wild-type males more often than did wild-type males. However, precise excision males are white-eyed, and thus are effectively blind. Wild-type males assayed under dim red light, where they are also blind, show similar levels of homosexual courtship. Therefore, the level of courtship in precise excision males is equivalent to that of wild type under similar sensory constraints. Precise excision males engaged in heterosexual courtship with decapitated wild-type females 49.7% +/- 5.0 of the time, which was also indistinguishable from wild type (Grosjean, 2008).

Altered sexual discrimination in gb mutant males could be a result of a misinterpretation of sex-specific sensory cues. To test this hypothesis and to identify these cues, homosexual courtship was measured under dim red light, in which Drosophila are virtually blind. In this condition, wild-type and precise-excision control males showed slightly higher than normal homosexual courtship, confirming the importance of visual cues for sexual discrimination. However, gb mutant males still showed much higher homosexual courtship, indicating that misinterpretation of nonvisual cues is the primary cause of the gb mutant phenotype. To confirm this, homosexual courtship directed toward desat1 mutant males was measured. desat1 mutants are genetically deficient for the production of several pheromones, including 7-tricosene (Marcillac, 2005). Homosexual courtship was reduced to wild-type levels when gb mutant males were partnered with desat1 mutant males. However, homosexual courtship was restored to the high levels typical of gb mutants when synthetic 7-tricosene was topically applied to the cuticles of the desat1 mutant male partners. Thus, gb mutant homosexual behavior represents an altered response to chemosensory cues, including 7-tricosene. Consistent with the idea that gb mutant males misinterpret chemical signals, gb mutant males also showed abnormally high courtship to mated wild-type females, which acquire inhibitory male chemical signals (including cVA) during copulation. The chemical signals misinterpreted by gb mutant males appear to be species-specific, as gb mutant males reacted normally to potential partners from other Drosophila species (Grosjean, 2008).

To determine whether gb mutant males might overreact to other chemosensory stimuli, olfactory trap assays were carried out using standard Drosophila food as bait. Significantly more gb mutant males were trapped in these assays, compared with wild type or precise excision controls. This difference was confirmed in single-fly trap assays, where 60% of gb mutants were trapped after 34 h, compared with 33% of precise excision controls (precise excision, n = 15; gb, n = 10). These results support the idea that gb mutants have fundamental defects in chemosensory processing that cause them to overreact to certain chemical signals. Therefore the attention of this study turned toward determining the mechanism by which Genderblind might alter chemosensory processing (Grosjean, 2008).

Genderblind has been shown to be a highly conserved glial amino-acid transporter subunit and a critical regulator of ambient extracellular glutamate (Augustin, 2007). In gb[KG07905] mutants, ambient extracellular glutamate is reduced to approximately 50% of normal (Augustin, 2007). Ambient extracellular glutamate bathes the nervous system and generally suppresses glutamatergic synapse strength via constitutive desensitization of glutamate receptors (Augustin, 2007; Featherstone, 2008). To test whether the homosexual behavior of gb mutant males might be attributable to increased glutamatergic synapse strength in chemosensory circuits, the following series of experiments were carried out. First, a Genderblind-specific antibody was used to examine Genderblind expression in the adult male brain. In particular, whether Genderblind protein might be expressed in the adult male nervous system near brain centers that are known to be involved in chemical sensation and integration, was examined. As expected, Genderblind was distributed throughout adult male Drosophila brain, including areas associated with olfactory and gustatory sensation. More precisely, Genderblind was detected in the subesophagial ganglia that receive inputs from gustatory neurons (some of which process 7-tricosene), in the antennal lobe and in the calyces that are involved in the higher integration of pheromonal inputs (including olfactory inputs for cVA sensation). In contrast, no expression was detected in the central complex region or in the different lobes of the mushroom bodies, which are involved in locomotion and olfactory learning, respectively. Genderblind immunoreactivity was reduced to background levels after expression of gb RNAi, indicating that the antibody is specific. Genderblind presence in glia was also examined. In larvae, Genderblind is exclusively expressed in glia. Consistent with this, Genderblind immunoreactivity in adult brains is excluded from neurons and was partially associated with cells expressing the glial transcription factor Repo. Genderblind is also abundant in areas of the brain containing glutamatergic neurons. Thus, immunohistochemical data support the possibility that Genderblind could modulate glutamatergic neurotransmission in pathways that control processing and/or integration of chemical stimuli (Grosjean, 2008).

To further explore the mechanism by which Genderblind regulates homosexual behavior, RNAi was used. As expected, gb mutant homosexual behavior could be phenocopied by constitutive expression of gb RNAi using the Gal4/UAS system (UASgb.RNAi;TubGal4) (Grosjean, 2008).

Genderblind appeared to be expressed exclusively in glia. To confirm that the gb mutant homosexual phenotype was a result of the loss of glial Genderblind, out cell type-specific knockdown of gb was performed. Duplication of the gb mutant phenotype by RNAi was maximal when gb RNAi was expressed in all brain tissues, but was only partial when gb RNAi was expressed under control of RepoGal4, consistent with the fact that some Genderblind protein is expressed in glia that do not express Repo. There is no available Gal4 driver that is specific for Genderblind glia (Grosjean, 2008).

To test whether the gb mutant courtship phenotype could be a result of a developmental alteration rather than acute modulation of neural circuit function, inducible RNAi was used. The TubGal80ts transgene is a ubiquitously expressed conditional repressor of Gal4 that is active at low temperatures (25°C), but not at high temperatures (30°C). In UASgb.RNAi;TubGal4,TubGal80ts males at 25°C, all genetic components for gb RNAi expression are present, but RNAi expression is actively repressed by TubGal80ts. UASgb.RNAi;TubGal4,TubGal80ts males showed low levels of homosexuality (similar to wild type) when reared continuously at 25°C. However, when UASgb.RNAi;TubGal4,TubGal80ts adult males reared at 25°C were moved to 30°C 24 h before testing, the gb mutant homosexual phenotype was completely restored. Homosexual behavior in these conditions could not have been an artifact of high temperature or the presence of TubGal80ts, as UASgb.RNAi;TubGal80ts males at 30°C did not show homosexual behavior. This ability to switch on homosexual behavior in adult males suggests that Genderblind regulates brain function rather than development, which is consistent with the hypothesis that Genderblind indirectly regulates glutamatergic synapse strength (Grosjean, 2008).

If homosexual courtship in gb mutants was a result of increased glutamatergic synapse strength in the CNS, then increasing CNS glutamatergic synapse strength independent of Genderblind should also cause high levels of homosexual behavior. To test this, the strength of glutamatergic synapses was tested in adult male brains by overexpressing the Drosophila vesicular glutamate transporter (DVGluT). Overexpression of DVGluT has previously been shown to overload synaptic vesicles with glutamate and lead to increased glutamate secretion at synapses (Daniels, 2004). As predicted, overexpression of DVGluT (UASDVGluT;TubGal4) caused high levels of homosexual courtship. UASDVGluT;TubGal4-induced homosexual courtship, as in gb mutants, included all aspects of sexual behavior, including singing, genital licking and attempted copulation. Occasionally, UASDVGluT;TubGal4 males even attempted copulation with inappropriate body regions, suggesting that increased glutamatergic synapse strength was a strong proximate cause of homosexual courtship and that homosexual courtship might represent a restricted example of general ectopic courtship. Overexpression of DVGluT in mushroom body neurons (UASDVGluT;MB247Gal4) had no effect, consistent with the lack of genderblind expression in mushroom bodies. But the gb mutant homosexual phenotype was partially duplicated by DVGluT overexpression specifically in adult brain chemosensory centers, consistent with the idea that gb mutant homosexuality is a result of increased glutamatergic synapse strength in circuits associated with processing of chemical stimuli (Grosjean, 2008).

As a further test of the hypothesis that the gb mutant phenotype is a result of increased glutamatergic synapse strength, glutamate receptor function was pharmacologically and genetically altered . Gamma-D-glutamylglycine (γ-DGG) is a competitive glutamate-receptor antagonist. If gb mutant homosexuality is a result of increased glutamatergic neurotransmission, then γ-DGG should eliminate gb mutant homosexuality. As predicted, adult gb mutant male flies reverted to low (wild type) levels of homosexual courtship when fed apple juice containing 25 mM γ-DGG for 21 h. This dose of γ-DGG did not seem to disrupt coordination, and had no significant effect on locomotory activity (Grosjean, 2008).

Glutamatergic neurotransmission is mediated by two different types of receptors: ionotropic (pore-forming) glutamate receptors and metabotropic (G protein-coupled) glutamate receptors. The increased glutamatergic neurotransmission underlying the gb mutant homosexual phenotype could occur via either receptor type or even both. Concanavalin A (ConA) is a glutamate-receptor agonist that inhibits ionotropic receptor desensitization. If Drosophila homosexual behavior is caused by increased glutamatergic neurotransmission via ionotropic glutamate receptors, then ingestion of ConA should induce homosexual behavior. Consistent with this, adult wild-type flies that were fed apple juice containing 40 mM ConA for 21 h before testing showed increased homosexual courtship. As with γ-DGG, the dose of ConA that was used did not seem to disrupt coordination and had no significant effect on locomotory activity (Grosjean, 2008).

However, ConA (which disrupts transmission via ionotropic glutamate receptors) did not induce as high a level of homosexual courtship as was measured in either gb mutants or after ingestion of γ-DGG (which disrupts transmission via both ionotropic and metabotropic glutamate receptors). This suggests that the enhanced glutamatergic transmission causing gb mutant homosexual behavior is only partially attributable to overactivation of ionotropic glutamate receptors. To test whether some of the gb mutant homosexual behavior might also be a result of overactivation of metabotropic glutamate receptors, homosexual courtship was measured in gb; mGluRA[112b] double mutant males, in which loss of gb function was combined with a small deletion that specifically removes mGluRA, the only functional metabotropic glutamate receptor encoded by the Drosophila genome. Deletion of mGluRA partially rescued the gb mutant homosexual phenotype, which is consistent with the idea that gb mutant homosexual courtship is a result of increased neurotransmission via both ionotropic and metabotropic glutamate receptors (Grosjean, 2008).

Taken together, these data suggest that Drosophila homosexual behavior is controlled by glutamatergic synapse strength and that Genderblind normally suppresses homosexual behavior by suppressing glutamatergic synapse strength (Grosjean, 2008).

This study was prompted by the observation that gb[KG07905] mutant males showed strong homosexual courtship. Similar homosexual courtship has been observed in flies with other transposon insertions (Zhang, 1995; Hing, 1996). In those cases, homosexual courtship was attributed to misexpression of white, an eye color gene that is commonly engineered into Drosophila transposons as a transgenic marker. Because gb[KG07905] mutants also contain a transgenic white gene, the possibility that homosexual courtship in these experiments might simply be caused by misexpression of white was considered. However, no evidence was seen that homosexual courtship can be triggered by the presence of white-expressing transposons that did not otherwise disrupt specific genes. Other studies (An, 2000; Svetec, 2005) have also cast doubt on the conclusion that white misexpression invariably causes male-male courtship (Grosjean, 2008).

The fraction of time spent in homosexual courtship by gb[KG07905] and gb[KG07905]/Df mutants was statistically identical, implying that gb[KG07905] is a null allele by traditional genetic criteria. However, real-time PCR and Genderblind immunoblot data clearly demonstrate that gb[KG07905] is not a null, and readers are cautioned not to over-interpret courtship index values. The maximum obtainable courtship is never 100%, even between wild-type male and female flies . Male flies spend substantial amounts of time in search and grooming behaviors. Neither searching nor grooming counts as courtship behavior, and the maximal obtainable courtship values are therefore limited to 60%-80%. Indeed, qualitatively far more vigorous courtship was measured after overexpression of DVGluT, but this did not lead to a higher courtship index when compared with gb[KG07905] or gb[KG07905]/Df, as noncourtship behavior was not substantially altered (Grosjean, 2008).

The fact that homosexual behavior in Drosophila seems to be controlled by glutamatergic circuits is notable, since the Drosophila CNS is generally thought to rely primarily on acetylcholine for neurotransmission. However, there are increasing indications that glutamatergic transmission is also important, despite being overlooked, in the fly CNS, including evidence that (1) large portions of the Drosophila CNS are glutamatergic, (2) in situ data show that many different ionotropic glutamate receptor subunits are expressed in the CNS, (3) the ionotropic glutamate receptor subunit GluRIID has been shown to be important in central pattern generation and (5) both NMDA receptor homologs in the Drosophila genome are expressed in CNS memory centers and are required for proper olfactory memory formation (Grosjean, 2008).

Genderblind has high homology to mammalian xCT proteins, which together with 4F2hc subunits, form heteromeric cystine/glutamate transporters that secrete glutamate in exchange for extracellular cystine (Sato, 1999). Most of the focus on cystine/glutamate transporters to date has been on their ability to import cystine. However, cystine/glutamate transporters are also potentially important regulators of ambient extracellular glutamate bathing the nervous system. Pharmacological studies support the idea that cystine-glutamate transporters regulate ambient extracellular glutamate in rat brain (Baker, 2002), and it has been shown that ambient extracellular glutamate in gb mutant flies is halved when compared with controls. Ambient extracellular glutamate, in both mammals and flies, can regulate glutamatergic transmission via steady-state glutamate receptor desensitization. Consistent with this idea, it was possible to both phenocopy and rescue the gb mutant homosexual phenotype by pharmacological manipulation of glutamatergic transmission, including the use of the desensitization inhibitor ConA (Grosjean, 2008).

The findings stated in this study recent description of gb mutant synaptic phenotypes (Augustin, 2007) all support the idea that Genderblind regulates ambient extracellular glutamate, and that this in turn regulates glutamatergic signaling in Drosophila chemosensory processing centers. Similar regulation, although perhaps not in chemosensory centers, may occur in mammals. In healthy mammalian brains, ambient extracellular glutamate concentration varies spatially and temporally, and these changes in ambient extracellular glutamate may contribute to behavioral states or mood. For example, melatonin alters glial glutamate uptake and this triggers circadian changes in ambient extracellular glutamate. Pharmacological manipulation of cystine/glutamate exchange in rats alters ambient extracellular glutamate, cocaine withdrawal and effects of phencyclidine. However, the idea that Genderblind-type transporters might volumetrically regulate glutamatergic signaling in vivo remains controversial. As a first step toward resolving this controversy, a gb cDNA was cloned using primers designed to amplify the gb cDNA that is predicted by Flybase. It was hoped that gb could be misexpressed and overexpressed to test whether specific glutamatergic circuits might be altered in a Genderblind dose-dependent manner. However, pan-cellular expression of this cDNA failed to rescue the cellular phenotypes or the behavioral changes. Transgenic cDNA rescue in Drosophila does not always work, or can be misleading, and there are several reasons why the gb cDNA might have failed to rescue the mutant phenotypes. One possibility is that the gb locus encodes multiple protein isoforms and that these isoforms must be expressed in a specific spatiotemporal pattern to recapitulate normal synaptic circuit modulation. This conclusion is supported by quantitative RT-PCR data. Genderblind-type transporters are also multi-subunit complexes (Lim, 2005; Burdo, 2006; Shih, 2006), and expression of each subunit might need to be carefully coordinated for proper function (Grosjean, 2008).

In addition to demonstrating a behavioral role for Genderblind, these results also suggest a physiological model for Drosophila sexual preference that parallels a model recently proposed for mice. In this model, wild-type flies are 'pre-wired' for both heterosexual and homosexual behavior, but Genderblind-based transporters suppress the glutamatergic circuits that promote homosexual behavior. In gb mutants, the repression of homosexual behavior does not occur and flies become bisexual. Heterosexual courtship is not altered in gb mutants, indicating that circuits driving heterosexual courtship are not regulated by Genderblind. This could be because circuits promoting heterosexual courtship are not glutamatergic, or because they are perfused by a different ambient extracellular glutamate pool than the one that is regulated by genderblind-based transporters (Grosjean, 2008).

Glutamate transport is highly regulated as glutamate directly acts as a neurotransmitter and indirectly regulates the synthesis of antioxidants. Although glutamate deregulation has been repeatedly linked to serious human diseases such as HIV infection and Alzheimer's, glutamate's role in the immune system is still poorly understood. A putative glutamate transporter in Drosophila melanogaster, polyphemus (polyph), was found to play an integral part in the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles. When infected with S. aureus, polyph flies show an increase in both susceptibility and bacterial growth. Additionally, the expression of two known glutamate transporters, genderblind and excitatory amino acid transporter 1, in blood cells affects the flies' ability to phagocytose and survive after an infection. Consistent with previous data showing a regulatory role for glutamate transport in the synthesis of the major antioxidant glutathione, polyph flies produce more reactive oxygen species (ROS) as compared to wild-type flies when exposed to S. aureus. In conclusion, this study has demonstrated that a polyph-dependent redox system in blood cells is necessary to maintain the cells' immune-related functions. Furthermore, the model provides insight into how deregulation of glutamate transport may play a role in disease (Gonzales, 2013).

It was hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, a novel Drosophila xCT gene was identified and functionally characterized, that has been named 'genderblind' (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200%-300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, these results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses (Augustin, 2007).

The primary physiological role of xCT transporters remains controversial. Although xCT transporters mediate 1:1 exchange between extracellular cystine and intracellular glutamate, glutamate excretion is generally ignored, and xCT transporters are often assumed to function primarily as a cystine-uptake mechanism for glutathione synthesis and protection from oxidative stress (see for example Shih, 2006). However, this bias ignores several important facts: (1) xCT transporters also export glutamate. (2) Mammalian brain xCT appears most abundant in 'border areas between the brain proper and periphery' (Burdo, 2006), specifically 'several regions facing the CSF,' including ventricle walls and meninges (Sato, 2002), consistent with the idea that xCT transporters are important for regulation of free glutamate content of CSF but not for cystine uptake in all brain cells. (3) Mammalian xCT transporters appear to be dispensable for cystine uptake and glutathione synthesis (Chung, 2005). Instead, glutathione synthesis in neurons and glia may be regulated by excitatory amino acid transport (EAAT) family proteins. EAATs are best known as sodium-dependent transporters for glutamate uptake, but EAATs also efficiently import cysteine, the reduced form of cystine used in glutathione synthesis (see Chung, 2005). In agreement, overexpression of Drosophila gb (Tub-Gal4;UAS-gb) causes shortened lifespan and neurodegeneration, consistent with increased glutamate secretion but the exact opposite phenotype that one would expect if the role of GB were cystine uptake for neuroprotection. (4) Microdialysis of rat brains with inhibitors of xCT function (Baker, 2002) leads to a decrease in nonvesicular glutamate secretion (Augustin, 2007).

Accordingly, it is argued that glutamate export by xCT transporters is at least as important as cystine import, particularly in the nervous system. Full acceptance of this idea, however, requires one to accept the idea that xCT transporters maintain ambient extracellular glutamate in the nervous system for good reasons and that extracellular glutamate in the brain is not merely a potentially pathological byproduct of glutamatergic transmission. The data suggest that ambient extracellular glutamate regulates constitutive receptor desensitization for control of synaptic glutamate receptor abundance (Augustin, 2007).

A link between glutamate receptor desensitization and clustering has not previously been demonstrated. It is well known that desensitization functionally eliminates glutamate receptors on a short time scale (tens to hundreds of milliseconds). The data suggest that constitutive desensitization is, on a longer time scale (hours), also associated with removal of receptors from the synapse. The EC₅₀ for activation of Drosophila larval muscle glutamate receptors is ~2 mM, and significant numbers of receptors can be desensitized at considerably lower concentrations. Because 2 mM is near the concentration of glutamate bathing NMJ receptors in vivo, it must be concluded that one-half or more of Drosophila larval muscle glutamate receptors are constitutively desensitized, and therefore delocalized, in vivo. This conclusion is consistent with the 200%-300% increase in postsynaptic glutamate receptor abundance that was observed after switching NMJs to culture media containing 0 mM glutamate (Augustin, 2007).

At first, the idea that many glutamate receptors should be desensitized (and subsequently delocalized) in vivo seems surprising. However, constitutive desensitization (and subsequent delocalization) of ligand-gated ion channels by ambient ligand is analogous to constitutive inactivation of voltage-gated ion channels by resting membrane potential. Constitutive inactivation of voltage-gated channels is a common and important regulator of membrane excitability. For example, at a typical rat skeletal muscle resting potential of -90 mV, approximately two-thirds of rat skm-1 skeletal muscle sodium channels are inactivated. As a result, only one-third of channels in the membrane are normally available for generation of action potentials. However, if resting membrane potential is modified or the voltage dependence of sodium channel inactivation is slightly shifted by (for example) channel phosphorylation, then the number of functionally available sodium channels in the membrane can change quickly and dramatically, with consequent large effects on cell excitability. In the case of glutamate receptors, the number of functionally available receptors at a synapse, and therefore synaptic strength, could similarly be quickly and effectively altered by relatively minor changes in ambient glutamate levels (perhaps because of regulation of xCT-mediated transport) or changes in the concentration dependence of receptor desensitization as a result of (for example) receptor phosphorylation. These possibilities have not been explored (Augustin, 2007).

A physiological role for ambient extracellular glutamate also has medical implications. Abnormal levels of CSF glutamate have been linked to a variety of human neurodevelopmental and neurodegenerative disorders, including anxiety/stress-related disorders, Rett syndrome, autism, and all forms (both familial and sporadic) of amyotrophic lateral sclerosis. Furthermore, xCT and 4F2hc have specifically been implicated in development, behavior, and disease. For example, lysinuric protein intolerance, a recessive disorder characterized by severe mental retardation, is caused by mutations in the human xCT gene SLC7A7 [solute carrier family 7 (cationic amino acid transporter, y+ system), member 7]. Similarly, 4F2hc is required for tumor transformation in human cancers. Finally, human xCT protein was recently identified as the fusion-entry receptor for Kaposi's sarcoma-associated herpes virus. Not surprisingly, therefore, extracellular glutamate and xCT transporters are beginning to be targeted for pharmacological inhibition. These results suggest that pharmacological inhibition of xCT transport could considerably ameliorate neuropathologies exacerbated by extracellular glutamate but raise the caveat that tampering with extracellular glutamate could have unexpected developmental and/or psychotropic effects (Augustin, 2007 and references therein).