Developmental expression of glutamate transporters and glutamate dehydrogenase in astrocytes of the postnatal rat hippocampus

Glutamate is the major excitatory transmitter in the CNS and plays distinct roles in a number of developmental events. Its extracellular concentration, which mediates these activities, is regulated by glutamate transporters in glial cells and neurons. In the present study, we have used nonradioactive in situ hybridization, immunocytochemistry, and immunoblotting to show the cellular and regional expression of the high-affinity glutamate transporters GLAST (EAAT1) and generic GLT1 (EAAT2; glial form of GLT1) in the rat hippocampus during postnatal development (P1-60). The results of transporter expression were compared with the localization and activity pattern of glutamate dehydrogenase (GDH), an important glutamate-metabolizing enzyme. The study showed that both transporters and GDH were demonstrable at P1 (day of birth). The expression of GLAST (detected by in situ hybridization and immunocytochemistry) in the early postnatal development was higher than GLT1. Thereafter, the expression of both transporters increased, showing adult levels at between P20 and P30 (detected by in situ hybridization and immunoblotting). At these time points, the expression of GLT1 appeared to be significantly higher than the GLAST expression. GLT1 and GLAST proteins were demonstrable only in astrocytes. The increase of GDH activities (steepest increase from P5-P8), which were localized preferentially in astrocytes, was in agreement with the increase of transporter expression, preferentially with that of GLT1. These observations suggest that the extent of glutamate transporter expression and of glutamate-metabolizing GDH activity in astrocytes is intimately correlated with the formation of glutamatergic synapses in the developing hippocampus.     

Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood–brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate‐induced endfeet sodium transients were diminished by TFB‐TBOA, suggesting that they were generated by sodium‐dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT‐1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ～120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double‐deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long‐lived and inter‐cellularly acting indicator of synaptic activity at the blood–brain barrier, which likely constitutes an important component of neuro‐metabolic coupling in the brain. GLIA 2017;65:293–308     

Adenosine A(2A) receptors modulate glutamate uptake in cultured astrocytes and gliosomes

Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to synaptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular glutamate. We now report that adenosine A(2A) receptors (A(2A) R) control the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalemmal vesicles (gliosomes) from adult rats, whereas A(1) R and A(3) R were devoid of effects. Thus, the acute exposure to the A(2A) R agonist, CGS 21680, inhibited glutamate uptake, an effect prevented by the A(2A) R antagonist, SCH 58261, and abbrogated in cultured astrocytes from A(2A) R knockout mice. Furthermore, the prolonged activation of A(2A) R lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and protein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate transporters by astrocytic A(2A) R provides a novel candidate mechanism to understand the ability of A(2) (A) R to control synaptic plasticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters.     

Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning-induced ischemic tolerance of neurons in neuron/astrocyte co-cultures

Sublethal ischemia leads to increased tolerance against subsequent prolonged cerebral ischemia in vivo. In the present study, we investigated the roles of the astrocytic glutamate (Glu) transporter GLT-1 in preconditioning (PC)-induced neuronal ischemic tolerance in cortical neuron/astrocyte co-cultures. Ischemia in vitro was simulated by subjecting cultures to both oxygen and glucose deprivation (OGD). A sublethal OGD (PC) increased the survival rate of neurons significantly when cultures were exposed to a lethal OGD 24 h later. The extracellular concentration of Glu increased significantly during PC, and treatment with an inhibitor of N-methyl-D-actetate (NMDA) receptors significantly reversed the PC-induced ischemic tolerance of neurons, suggesting that the increase in extracellular concentration of Glu during PC was critical to the development of PC-induced neuronal ischemic tolerance via the activation of NMDA receptors. Treatment with a GLT-1 blocker during PC suppressed this increase in Glu significantly, and antagonized the PC-induced neuronal ischemic tolerance. This study suggested that the reversed operation of GLT-1 was crucial to the development of neuronal ischemic tolerance.     

Alcohol consumption increases basal extracellular glutamate in the nucleus accumbens core of Sprague–Dawley rats without increasing spontaneous glutamate release

Glutamate neurotransmission in the nucleus accumbens core (NAc) mediates ethanol consumption. Previous studies using non‐contingent and voluntary alcohol administration in inbred rodents have reported increased basal extracellular glutamate levels in the NAc. Here, we assessed basal glutamate levels in the NAc following intermittent alcohol consumption in male Sprague‐Dawley rats that had access to ethanol for 7 weeks on alternating days. We found increased basal NAc glutamate at 24 h withdrawal from ethanol and thus sought to identify the source of this glutamate. To do so, we employed a combination of microdialysis, slice electrophysiology and western blotting. Reverse dialysis of the voltage‐gated sodium channel blocker tetrodotoxin did not affect glutamate levels in either group. Electrophysiological recordings in slices made after 24 h withdrawal revealed a decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency relative to controls, with no change in sEPSC amplitude. No change in metabotropic glutamate receptor 2/3 (mGlu2/3) function was detected as bath application of the mGlu2/3 agonist LY379268 decreased spontaneous and miniature EPSC frequency in slices from both control and ethanol‐consuming rats. The increase in basal glutamate was not associated with changes in the surface expression of GLT‐1, however, a decrease in slope of the no‐net‐flux dialysis function was observed following ethanol consumption, indicating a potential decrease in glutamate reuptake. Taken together, these findings indicate that the increase in basal extracellular glutamate occurring after chronic ethanol consumption is not mediated by an increase in action potential‐dependent glutamate release or a failure of mGlu2/3 autoreceptors to regulate such release.     

Astrocytic glutamate uptake is slow and does not limit neuronal NMDA receptor activation in the neonatal neocortex

Glutamate uptake by astrocytes controls the time course of glutamate in the extracellular space and affects neurotransmission, synaptogenesis, and circuit development. Astrocytic glutamate uptake has been shown to undergo post‐natal maturation in the hippocampus, but has been largely unexplored in other brain regions. Notably, glutamate uptake has never been examined in the developing neocortex. In these studies, we investigated the development of astrocytic glutamate transport, intrinsic membrane properties, and control of neuronal NMDA receptor activation in the developing neocortex. Using astrocytic and neuronal electrophysiology, immunofluorescence, and Western blot analysis we show that: (1) glutamate uptake in the neonatal neocortex is slow relative to neonatal hippocampus; (2) astrocytes in the neonatal neocortex undergo a significant maturation of intrinsic membrane properties; (3) slow glutamate uptake is accompanied by lower expression of both GLT‐1 and GLAST; (4) glutamate uptake is less dependent on GLT‐1 in neonatal neocortex than in neonatal hippocampus; and (5) the slow glutamate uptake we report in the neonatal neocortex corresponds to minimal astrocytic control of neuronal NMDA receptor activation. Taken together, our results clearly show fundamental differences between astrocytic maturation in the developing neocortex and hippocampus, and corresponding changes in how astrocytes control glutamate signaling. GLIA 2015;63:1784–1796     

Neuronal activity regulates glutamate transporter dynamics in developing astrocytes

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT-1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. Green fluorescent protein (GFP)-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse three-dimensional confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses.     

Glial glutamate transporter expression patterns in brains from multiple mammalian species

It is generally assumed that rodent brains can be used as representative models of neurochemical function in other species, such as humans. We have compared the distributions of the predominant glial glutamate transporters in rodents, rabbits, cats, pigs, monkeys, and humans. We identify similarities but also significant differences between species. GLT-1v, which is abundantly expressed by rodent astrocytes, is expressed only in a rare subset of astrocytes of cats and humans, and appears to be absent from brains of rabbits and monkeys. Conversely, in the pig brain GLT-1v is expressed only by oligodendrocytes. GLAST and GLT-1alpha expression differed significantly between species; while rodents and rabbits exhibited uniform expression patterns in cortex, higher species, including cats, pigs, monkeys, and humans, exhibited heterogeneities in cortical and hippocampal expression. Patches devoid of labeling intermingling with patches of strong labeling were evident in areas such as temporal cortex and frontal cortex. In addition, we noted that in human motor cortex, there were inconsistencies in labeling for the C-terminal of GLT-1alpha and common domains of GLT-1, suggesting that the C-terminal region may be missing or that an unidentified splicing is present in many human astrocytes. Collectively our data suggest that assumptions as to the roles of glutamate transporters in any species may need to be tested empirically.     

Rapid increase of glial glutamate uptake via blockade of the protein kinase A pathway

Glutamate is the main excitatory neurotransmitter in the vertebrate central nervous system. Removal of this transmitter from the synaptic cleft by glial and neuronal transporter systems plays an important role in terminating glutamatergic neurotransmission. The effects of different activators and blockers of PKA and PKC on glutamate uptake were studied in primary glial cells cultivated from the rat cortex using the patch-clamp recording technique and immunocytochemical methods. GF 109203X enhances glutamate-induced membrane currents in a concentration- and time-dependent manner. After pre-application for 40 s the maximal transport capacity was increased by 30-80%. The estimated Km-value of the transport system did not change after drug application and the enhanced glutamate uptake was reversible within a few minutes upon washout. Activators and blockers of the PKC pathway did not affect glutamate uptake, whereas H89, a selective blocker of PKA, mimicked the effects of GF 109203X, indicating involvement of the protein kinase A pathway. The GF 109203X-induced increase in transport capacity is likely to be mediated by GLAST since the GLT-1 selective blocker dihydrokainate was unable to block basal or stimulated glutamate uptake. Furthermore, the increase in transport activity may well be based on an increase in cell surface expression of the transporter protein since preincubation with cytochalasin-B, a protein that blocks actin polymerization, almost completely abolished the effect of GF 109203X and H89. These results indicate that GF 109203X and H89 enhance glial glutamate uptake via blockade of the PKA. The described effect may affect glutamatergic neurotransmission by reducing the glutamate concentration in the synaptic cleft.     

Endothelin downregulates the glutamate transporter GLAST in cAMP-differentiated astrocytes in vitro.

Endothelin (ET) is a putative pathogenetic mediator associated with brain trauma and ischemia. Because a link between neuronal damage after these injuries and glial Na(+)-dependent L-glutamate transporter activity has been suggested, we investigated the effect of ET on the glutamate clearance ability of astrocytes. Dibutyryl cyclic adenosine monophosphate (dBcAMP), which is widely used to induce differentiation of cultured astrocytes, markedly increased [(3)H]glutamate transport activity in a concentration- and time-dependent manner. In the presence of ET, however, dBcAMP decreased the glutamate uptake. This effect was efficiently prevented by an antagonist of ET(B) receptor, but not of ET(A) receptor. ET per se was virtually ineffective. Eadie-Hofstee analysis demonstrated that dBcAMP increased the V(max) value of glutamate uptake activity by 43.4% in the absence of ET, but decreased it by 41.4% in the presence of ET, without apparent changes in the K(m) value. Accordingly, Western blot analysis indicated that the change in transport activity correlated closely with that in expression level of the glial glutamate transporter GLAST. These results may represent the mechanisms by which ET aggravates trauma- and ischemia-elicited neuronal damage.     

Curcumin protects against staurosporine toxicity in rat neurons

Objective Curcumin is extracted from the turmeric plant (Curcuma longa Linn.) and is widely used as a food additive and traditional medicine. The present study investigated the activity of curcumin against staurosporine (STS) toxicity in cell culture. Methods Rat hippocampal neurons in primary culture were exposed to STS (20 μmol/L) and treated with curcumin (20 μmol/L). Cell viability was tested by MTT assay and reactive oxygen species (ROS) were measured using the MitoSOX red mitochondrial superoxide indicator. Western blot was used to assess changes in the levels of caspase-3 (Csp3), heat shock protein 70 (Hsp70) and Akt. Results The results showed that curcumin protects against STS-induced cytotoxicity in rat hippocampal neurons. Csp3, Hsp70, Akt and ROS activation may be involved in this protection. Conclusion Curcumin could be a potential drug for combination with STS in cancer treatment to reduce the unwanted cytotoxicity of STS.     

Inhibition of glutamine transport into mitochondria protects astrocytes from ammonia toxicity

Hepatic encephalopathy (HE) is a major neurological complication that occurs in the setting of severe liver failure. Ammonia is a key neurotoxin implicated in this condition, and astrocytes are the principal neural cells histopathologically and functionally affected. Although the mechanism by which ammonia causes astrocyte dysfunction is incompletely understood, glutamine, a by-product of ammonia metabolism, has been strongly implicated in many of the deleterious effects of ammonia on astrocytes. Inhibiting mitochondrial glutamine hydrolysis in astrocytes mitigates many of the toxic effects of ammonia, suggesting the involvement of mitochondrial glutamine metabolism in the mechanism of ammonia neurotoxicity. To determine whether mitochondriaare indeed the organelle where glutamine exerts its toxic effects, we examined the effect of L-histidine, an inhibitor of mitochondrial glutamine transport, on ammonia-mediated astrocyte defects. Treatment of cultured astrocytes with L-histidine completely blocked or significantly attenuated ammonia-induced reactive oxygen species production, cell swelling, mitochondrial permeability transition, and loss of ATP. These findings implicate mitochondrial glutamine transport in the mechanism of ammonia neurotoxicity.     

Norepinephrine protects cortical neurons against microglial-induced cell death

Interleukin-1 beta (IL-1beta) is one of the main cytokines involved in the inflammatory response; it has multiple effects that can contribute to cell damage, one of which is the upregulation of the inducible form of nitric oxide (NO) synthase (NOS2) in certain cell types. We demonstrated previously that in vivo, cortical microglial inflammatory responses were increased when noradrenaline (NE) levels were depleted, suggesting that NE can reduce microglial activation. In the present report, we examined the role of IL-1beta in neurotoxicity induced by microglial-conditioned media, and possible neuroprotective effects of NE. Incubation of cortical neurons with conditioned media (CM) obtained from lipopolysaccharide (LPS)-treated microglia induced neuronal NOS2 expression and increased neuronal cell death, and these responses were reduced if the neurons were coincubated with interleukin-1 receptor antagonist. Cotreatment of microglial cells with LPS plus NE potently blocked IL-1beta production and reduced the ability of the CM to induce neuronal NOS2 and cell death. These results suggest that microglial release of IL-1beta is an important activator of neuronal inflammatory responses, and that protective effects of NE upon neurons involve a reduction of microglial-derived IL-1beta.     

Acetoacetate protects neuronal cells from oxidative glutamate toxicity

Glutamate cytotoxicity contributes to neuronal degeneration in many central nervous system (CNS) diseases, such as epilepsy and ischemia. We previously reported that a high-fat and low-carbohydrate diet, the ketogenic diet (KD), protects against kainic acid-induced hippocampal cell death in mice. We hypothesized based on these findings that ketosis resulting from KD might inhibit glutamate cytotoxicity, resulting in inhibition of hippocampal neuronal cell death. Therefore, we investigated the role of ketone bodies [acetoacetate (AA) and beta-hydroxybutyrate (beta-OHB)] both in a mouse hippocampal cell line (HT22) and in rat primary hippocampal neurons. As a result, we found that pretreatment with 5 mM lithium AA and 4 mM Na beta-OHB protected the HT22 hippocampal cell line and primary hippocampal neuronal culture against 5 mM glutamate toxicity and that up to 2 hr of pretreatment with 5 mM AA had a protective effect against 5 mM glutamate toxicity in the HT22 cell line. Pretreatment with 5 mM AA decreased ROS production of HT22 cell line at 2 and 8 hr exposure of glutamate, and it decreased the appearance of annexin V-positive HT22 cells, which are indicative of an early stage of apoptosis, and propidium iodide-positive HT22 cells, which are indicative of necrosis.     

Alpha-cyano-4-hydroxycinnamate decreases both glucose and lactate metabolism in neurons and astrocytes: implications for lactate as an energy substrate for neurons.

The rates of uptake and oxidation of [U-(14)C]lactate and [U-(14)C]glucose were determined in primary cultures of astrocytes and neurons from rat brain, in the presence and absence of the monocarboxylic acid transport inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN). The rates of uptake for 1 mM lactate and glucose were 7.45 +/- 1.35 and 8.80 +/- 1.0 nmol/30 sec/mg protein in astrocytes and 2.36 +/- 0.19 and 1.93 +/- 0.16 nmol/30 sec/mg protein in neuron cultures, respectively. Lactate transport into both astrocytes and neurons was significantly decreased by 0.25-1.0 mM 4-CIN; however, glucose uptake was not affected. The rates of (14)CO(2) formation from 1 mM lactate and glucose were 12.49 +/- 0.77 and 3.42 +/- 0.67 nmol/hr/mg protein in astrocytes and 29.32 +/- 2.81 and 10.04 +/- 1.79 nmol/hr/mg protein in neurons, respectively. Incubation with 0.25 mM 4-CIN decreased the oxidation of lactate and glucose to 57.1% and 54.1% of control values in astrocytes and to 13.2% and 41.6% of the control rates in neurons, respectively. Preincubation with 4-CIN further decreased the oxidation of both glucose and lactate. Studies with glucose specifically labeled in the one and six positions demonstrated that 4-CIN decreased mitochondrial glucose oxidation but did not impair the metabolism of glucose via the pentose phosphate pathway in the cytosol. The lack of effect of 4-CIN on glutamate oxidation demonstrated that overall mitochondrial metabolism was not impaired. These findings suggest that the impaired neuronal function and tissue damage in the presence of 4-CIN observed in other studies may be due in part to decreased uptake of lactate; however, the effects of 4-CIN on mitochondrial transport would significantly decrease the oxidative metabolism of pyruvate derived from both glucose and lactate.     

Differential expression of glutamate dehydrogenase in cultured neurons and astrocytes from mouse cerebellum and cerebral cortex.

Glutamate dehydrogenase (GDH) specific activities, kinetic properties and allosteric regulation were studied in extracts from cultured neurons and astrocytes prepared from mouse cerebral cortex and cerebellum. Considerable differences were observed in the specific activity of the enzyme among the different cell types with astrocytes expressing the highest GDH activity. This may reflect the functional importance of these cells in glutamate uptake and metabolism. Among the neurons, the glutamatergic cerebellar granule cells showed a GDH specific activity that was 60% higher (P < 0.01) than that of the GABAergic cerebral cortical neurons. Also, the K(m) for ammonia was 1.7-fold higher in the cortical neurons than in the other cell types. These findings may reflect a particular need for the glutamatergic granule cells to synthesize glutamate via the GDH pathway. No differences were observed among the different cell types with regard to the allosteric properties of GDH expressed by these cells.     

Lipopolysaccharide increases microglial GLT-1 expression and glutamate uptake capacity in vitro by a mechanism dependent on TNF-alpha

This study investigates the effect of microglial activation on microglial glutamate transporters in vitro. Stimuli known to activate microglia and/or to be associated with pathological conditions with an impaired astroglial glutamate uptake were compared. Morphological changes and marked increases in ED1 antigen expression were found after 8-h incubation of rat microglia in 56 mM KCl, 1 ng/ml lipopolysaccharide (LPS), and 100 microM glutamate, as well as in acidic and basic conditions, showing that the cells were activated. Of the stimuli used, only LPS induced a significant release of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), and was the only stimulus that increased microglial GLT-1 expression and glutamate uptake capacity after 12-h incubation. This effect was probably mediated by TNF-alpha, since this cytokine mimicked the effect of LPS. Furthermore, the effect of LPS was blocked by thalidomide, an inhibitor of TNF-alpha synthesis. Additionally, neutralizing antibodies against TNF-alpha also blocked the increase, indicating TNF-alpha as an inducer of GLT-1 expression in microglia. It was also found that preincubation with glutamate dose-dependently inhibited the microglial glutamate uptake. This could suggest different physiological functions for microglial and astroglial glutamate uptake and might indicate a reciprocal control of GLT-1 expression between microglia and astrocytes.     

High extracellular gamma-aminobutyric acid protects cultured neurons against damage induced by the accumulation of endogenous extracellular glutamate

The glutamate uptake inhibitor L-trans-2,4-pyrrolidine-dicarboxylate (PDC) induces glutamate accumulation and neuronal damage in cultured cells. We have used dissociated cortical cells in culture to study whether the toxicity induced by inhibiting glutamate uptake with PDC could be blocked by the simultaneous inhibition of gamma-aminobutyric acid (GABA) uptake, because both types of transporters are affected during an ischemic event. After 6 hr of exposure to 100 microM GABA or to four different GABA uptake inhibitors, the concentration of extracellular GABA was augmented from the basal 2 microM value to about 25 microM and 5 microM, respectively. These increases, however, did not result in protection against the neuronal damage induced by the accumulation of extracellular glutamate because of the simultaneous exposure to PDC. In contrast, when 100 microM GABA and an inhibitor of GABA uptake were added, after 6 hr the concentrations of GABA reached 50 microM, and neurons were protected from PDC-induced toxicity. The addition of the GABA(A) and GABA(B) receptor agonists muscimol and baclofen also partially protected against PDC-induced damage. The results suggest that the excitotoxic damage resulting from chronic gradual elevation of extracellular glutamate may be prevented by high concentrations of extracellular GABA, an effect mediated by activation of GABA(A) and GABA(B) receptors.     

Evidence for estrogenic regulation of gonadotropin-releasing hormone neurons by glutamatergic neurons in the ewe brain: An immunohistochemical study using an antibody against vesicular glutamate transporter-2

Gonadotropin-releasing hormone (GnRH) secretion is controlled by various factors, including the excitatory neurotransmitter glutamate. Estrogen (E) regulates GnRH secretion by means of E-responsive cells in the brain that relay the feedback effects to the preoptic area (POA). We used an antibody to vesicular glutamate transporter 2 (VGluT2) to label glutamatergic neurons in the areas of the ewe brain that control GnRH secretion. VGluT2-immunoreactive cells were observed in the arcuate nucleus (ARC)/ventromedial hypothalamic nucleus (VMH) complex, POA, bed nucleus of stria terminalis (BnST), and A1 and A2 cell groups in the brainstem. In three ewes, E receptor-alpha was detected in 52-61% of glutamatergic neurons in ARC/VMH, 37-52% of neurons in the POA, and 37-58% of neurons in the BnST. E injection (i.m. or i.v.) increased the percentage of glutamatergic cells that expressed Fos protein in the ARC (P < 0.01 and P < 0.001, respectively). In six ewes, injection of the retrograde tracer Fluoro-Gold into the POA labeled cells in the ARC and 6-29% of these were also VGluT2-immunoreactive. Double-labeling of varicosities in the POA showed colocalization of VGluT2 in 12.5 +/- 3% of dopamine beta-hydroxylase-immunoreactive terminals, indicating that a subset of glutamatergic inputs could arise from brainstem noradrenergic neurons cells. In the POA, 60% of GnRH neurons had close appositions that were VGluT2-immunoreactive. We conclude that E-responsive glutamatergic neurons arising from the brainstem, the BnST, and ARC/VMH provide input to the POA and may be involved in the regulation of GnRH secretion.