Age-related changes in glutamate release in the CA3 and dentate gyrus of the rat hippocampus

The present studies employed a novel microelectrode array recording technology to study glutamate release and uptake in the dentate gyrus, CA3 and CA1 hippocampal subregions in anesthetized young, late-middle aged and aged male Fischer 344 rats. The mossy fiber terminals in CA3 showed a significantly decreased amount of KCl-evoked glutamate release in aged rats compared to both young and late-middle-aged rats. Significantly more KCl-evoked glutamate release was seen from perforant path terminals in the DG of late-middle-aged rats compared young and aged rats. The DG of aged rats developed an increased glutamate uptake rate compared to the DG of young animals, indicating a possible age-related change in glutamate regulation to deal with increased glutamate release that occurred in late-middle age. No age-related changes in resting levels of glutamate were observed in the DG, CA3 and CA1. Taken together, these data support dynamic changes to glutamate regulation during aging in subregions of the mammalian hippocampus that are critical for learning and memory.     

GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.     

Aged F344 rats exhibit altered electrophysiological activity in locomotor-unrelated but not locomotor-related striatal neurons

Multi-wire electrode arrays were chronically implanted and striatal electrophysiological activity was recorded in young (4-9 months) versus aged (24-29 months) Fischer 344 (F344) rats in order to determine whether locomotor-related striatal neurons exhibit age-related changes in electrophysiological activity during freely-moving conditions. Individual neurons were classified as locomotor-related if they exhibited significant differences in their firing rates between periods of locomotion versus periods of non-movement. While the activity of locomotor-related striatal neurons did not differ between young and aged rats, neurons that were not related to locomotion exhibited significantly greater activity in the aged rats during both periods of non-movement and bouts of locomotion. These results suggest that in the aged striatum, increased activity of nonlocomotor-related neurons may contribute to hypokinesia through their influence on basal ganglia output nuclei. Such studies may aid in the understanding of movement disorders seen in aging and Parkinson's disease.     

Glutamate transporters GLAST and EAAT4 regulate postischemic Purkinje cell death: an in vivo study using a cardiac arrest model in mice lacking GLAST or EAAT4

Cerebellar Purkinje cells represent a group of neurons highly vulnerable to ischemia. Excitotoxicity is thought to be an important pathophysiological mechanism in Purkinje cell death following ischemia. The glutamate transporter is the only mechanism for the removal of glutamate from the extracellular fluid in the brain. Therefore, glutamate transporters are believed to play a critical role in protecting Purkinje cells from ischemia-induced damage. Two distinct glutamate transporters, GLAST and EAAT4, are expressed most abundantly in the cerebellar cortex. GLAST is expressed in Bergmann glia, whereas EAAT4 is concentrated in the perisynaptic regions of Purkinje cell spines. However, the in vivo functional significance of these glial and neuronal glutamate transporters in postischemic Purkinje cell death is largely unknown. To clarify the role of these glutamate transporters in the protection of Purkinje cells after global brain ischemia, we evaluated Purkinje cell loss after cardiac arrest in mice lacking GLAST or EAAT4. We found that Purkinje cells with low EAAT4 expression were selectively lost after cardiac arrest in GLAST mutant mice. This result demonstrates that GLAST plays a role in preventing excitotoxic cerebellar damage after ischemia in concert with EAAT4.     

Downregulation of glutamate transporters is associated with elevation in extracellular glutamate concentration following rat microsphere embolism

Sodium-dependent glutamate transporters expressed in astroglial cells and neurons are essential for clearance of extracellular glutamate. In the present study, we found elevation of extracellular glutamate concentration associated with concomitant downregulation of glutamate transporters following rat microsphere embolism (ME). A marked increase in extracellular glutamate in the rat striatum was observed by microdialysis immediately after ME induction, and glutamate remained elevated at least 12 h after ischemia. Concomitantly, impairment of high KCl (146 mM)-induced glutamate release was observed in the striatum 12 h after ME. Consistent with the persistent increase in extracellular glutamate, expression of the glutamate transporters EAAC1 and GLT-1 significantly decreased 6 h after insult without a change in GLAST levels. GLT-1 expression was restored to basal levels within 48 h, whereas EAAC1 expression remained decreased up to at least 72 h after ME. Restoration of GLT-1 was associated with increased expression of the astroglial marker GFAP, whereas markedly reduced EACC1 levels were correlated with reduced levels of the neuronal marker MAP2, likely due to loss of vulnerable neurons. Taken together, downregulation of glutamate transporters after ME is associated with dysregulation of basal glutamate concentrations and KCl-induced glutamate release in the brain.     

Spinal glial glutamate transporters downregulate in rats with taxol-induced hyperalgesia

Changes in the expression of glial glutamate transporters (GLAST and GLT-1) were examined in the spinal cord of rats with chemotherapy (taxol)-induced mechanical hyperalgesia. Immunohistochemical studies show that the expression of both GLAST and GLT-1 in the L4-L5 spinal dorsal horn is decreased by 24% (P<0.001) and 23% (P<0.001), respectively, in rats with taxol-induced hyperalgesia as compared with those in control rats. These changes were further confirmed using an enzyme-linked immunosorbent assay that confirmed downregulation of GLAST by 36% (P<0.05) and GLT-1 by 18% (P<0.05) in the L4-L5 spinal cord of taxol-treated rats. These data indicate that downregulation of glutamate transporters may contribute to the development of hyperalgesia induced by taxol and suggest that glutamate transporters may be a new target for treatment of pain.     

Aged Fischer 344 rats exhibit altered orolingual motor function: relationships with nigrostriatal neurochemical measures

The present study utilized a novel behavioral preparation to measure differences in orolingual motor function between young (6 months) and aged (24 months) Fischer 344 (F344) rats. Rats were trained to lick an isometric force-sensing operandum for water reinforcement so that the number of licks per session, licking rhythm and lick force could be compared between the two groups. The aged rats exhibited a greater number of licks per session, but a slowed licking rhythm, compared to the young rats. Lick force did not differ significantly between the groups. The dopamine (DA) uptake inhibitor nomifensine decreased all three measures in both groups. Analyses of whole brain tissue content of DA, 3,4 dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the substantia nigra and dorsal striatum revealed no significant differences between the two age groups. Differences were observed between the two groups with respect to relationships between behavioral and neurochemical tissue measures. Striatal DA content and the number of licks per session were positively correlated for the young rats but not for the aged rats. In the aged rats, but not the young rats, positive correlations were also observed between licking rhythm and the DOPAC+HVA/DA ratio in the substantia nigra. These findings suggest that age-related alterations in orolingual motor function may relate in part to functional changes in DA neuronal circuits.     

Decline in striatal dopamine D1 and D2 receptor activation in aged F344 rats

Aging differentially affects receptor function. In the present electrophysiological study we compared neuronal responsiveness to locally applied dopamine D₁ and D₂ receptor agonist in the striatum of female Fischer 344 rats aged 3 and 26–27 months. In a subgroup of the old rats, the nigrostriatal dopamine bundle was destroyed unilaterally with 6-hydroxydopamine (6-OHDA) to assess receptor plasticity in response to denervation. Spontaneous firing rate of striatal neurons was higher in aged compared to young rats. Higher doses of the D₁ agonist SKF 38393 or the D₂ agonist quinpirole were required to elicit a 50% change in firing rate in aged compared to young rats. No difference with SKF 38393 or quinpirole was detected between 6-OHDA denervated and control (nonlesioned) striatum in aged rats. Supersensitivity to D₂ agonists has been reported following 6-OHDA lesions in young rats. These observations suggest that D₂ receptors in aged rat striatum might not be as plastic as in younger rats.     

Nomifensine reveals age-related changes in K(+)-evoked striatal DA overflow in F344 rats

To investigate the influence of age-associated changes in DA uptake on measures of potassium-stimulated DA overflow in the striatum, microdialysis was conducted in anesthetized young (6-month-old) versus aged (24-month-old) F344 rats. Extracellular levels of DA, DOPAC, and HVA were measured under basal and potassium-stimulated (10, 25, 50, & 100 mM) conditions. Basal levels of DA and metabolites did not differ significantly between the two age groups. At the 50 and 100 mM concentrations, potassium stimuli significantly increased DA overflow and decreased DOPAC and HVA--effects that did not differ with age. The addition of the DA uptake inhibitor nomifensine (100 microM) to the perfusion solutions revealed differences between the two age groups. Nomifensine augmented potassium-evoked DA overflow at the 50 mM concentration in both groups, but only amplified the effect of the 100 mM concentration in the young animals. The results demonstrate that decreased DA transporter function in aged rats masks age-related differences in K(+)-evoked striatal DA release when microdialysis methods are used, resulting in net equalization of K(+)-evoked striatal DA overflow in young versus aged F344 rats.     

Up-regulation of spinal glutamate transporters contributes to anti-hypersensitive effects of valproate in rats after peripheral nerve injury

Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of repeated administration of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in the spinal dorsal horn in rats after L5-L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn. Repeated oral administration of valproate reduced hypersensitivity, restored down-regulated expression of GLT-1 and GLAST in the spinal dorsal horn, and enhanced analgesia from the glutamate transporter activator riluzole. This analgesia from valproate was blocked by the selective GLT-1 blocker dihydrokainic acid (DHK). These data suggest that valproate restores down-regulated expression of glutamate transporters in the spinal cord to presumably reduce glutamate signaling and to reduce hypersensitivity after nerve injury, and that combination of valproate with riluzole produces enhanced analgesia which relies on the spinal glutamate transporters.     

The ionic stoichiometry of the GLAST glutamate transporter in salamander retinal glia

Maintaining a low extracellular glutamate concentration in the central nervous system is important for terminating synaptic transmission and preventing excitotoxic cell death. The stoichiometry of the most abundant glutamate transporter, GLT-1, predicts that a very low glutamate concentration, ∼2 n m , should be reached in the absence of glutamate release, yet microdialysis measurements give a value of ∼1 μ m . If other glutamate transporters had a different stoichiometry, the predicted minimum glutamate concentration could be higher, for example if those transporters were driven by the cotransport of 2 Na⁺ (rather than of 3 Na⁺ as for GLT-1). Here we investigated the ionic stoichiometry of the glutamate transporter GLAST, which is the major glutamate transporter expressed in the retina and cerebellum, is expressed in other adult brain areas at a lower level than GLT-1, and is present throughout the brain early in development when expression of GLT-1 is low. Glutamate transport by GLAST was found to be driven, as for GLT-1, by the cotransport of 3 Na⁺ and 1 H⁺ and the counter-transport of 1 K⁺, suggesting that the minimum extracellular glutamate concentration should be similar during development and in the adult brain. A less powerful accumulation of glutamate by GLAST than by GLT-1 cannot be used to explain the high glutamate concentration measured by microdialysis.     

Endothelins negatively regulate glial glutamate transporter expression

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system which at high extracellular levels leads to neuronal over-stimulation and subsequent excitotoxic neuronal cell death. Both the termination of glutamatergic neurotransmission and the prevention of neurotoxic extracellular glutamate concentrations are predominantly achieved by the uptake of extracellular glutamate into astroglia through the high-affinity glutamate transporters, excitatory amino acid transporter-2/glutamate transporter-1 (EAAT-2/GLT-1) and EAAT-1/glutamate aspartate transporter (GLAST). Although several injury-induced growth factors such as epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) potently stimulate the expression of glutamate transporters in cultured astroglia, GLT-1 and/or GLAST expression temporarily decreases during acute brain injuries eventually contributing to secondary neuronal cell death. We now demonstrate that the stimulatory influences of these injury-regulated growth factors are overridden by endothelins (ETs), a family of peptides also upregulated in the injured brain. Exposure of cultured cortical astroglia to ET-1, ET-2, and ET-3 resulted in a major loss of basal glutamate transporter expression after 72 hours and the complete prevention of the known stimulatory influences of dibutyryl cyclic (dbc)AMP, pituitary adenylate cyclase-activating polypeptide (PACAP), EGF, and TGFalpha on both GLT-1 and GLAST expression. With all ET isoforms, the inhibitory effects were detectable with similar low nanomolar concentrations and persisted in endothelin B-receptor deficient astroglia, suggesting that the inhibitory action is equally induced by endothelin A and B receptors. In astroglial cultures maintained with endothelins alone or in combination with PACAP, the inhibitory action was remarkably long-lasting and was still detectable after 7 days. In apparent contrast, glutamate transporter expression partially recovered between days 5 and 7 in cultures maintained with a combination of ETs and the injury-regulated growth factors EGF or TGFalpha. These findings point to ETs as major mediators of injury-dependent down-regulation of glial glutamate transporters and subsequent glutamate-induced brain damage.     

Blood-brain transport of triiodothyronine is reduced in aged rats

An age-related decline in blood-brain barrier transport of thyroid hormones may contribute to the central nervous system changes with aging. To test this hypothesis, the brain uptake index (BUI) of levo (L) and dextro (D) triiodothyronine (T3) was determined in male Fischer 344 rats at 6 months of age (young) and 26 months of age (aged). Young rats pair fed with aged were included to control for reduced food intake in aged rats. The L-T3 BUI of aged rats (22.4 +/- 2.1%) was significantly reduced compared to young rats (29.5 +/- 2.0%) or young rats pair fed with aged rats (28.5 +/- 2.5%) (p less than 0.05). This could not be attributed to age-related changes in BBB permeability or to reduced cerebral blood flow. At steady state conditions, the brain uptake of either L-T3 or D-T3 was not altered with aging. There were no significant changes in plasma or brain binding of T3. These results indicate that the reduced BBB transport of T3 in aged rats is counterbalanced by a reduction in T3 clearance from the brain.     

Spatial reference and working memory across the lifespan of male Fischer 344 rats

Loss of mnemonic function is among the earliest and most disconcerting consequences of the aging process. This study was designed to provide a comprehensive profile of spatial mnemonic abilities in male Fischer 344 (F344) rats across the lifespan. Young, middle-aged, and aged F344 rats were trained in spatial reference and working memory versions of the water maze task. There was a progressive age-related decline in spatial reference memory across the lifespan. Reliable individual differences were observed among aged rats, with some aged rats performing as well as young cohorts and others performing outside this range. An age-related delay-dependent decline was observed on a working memory version of the water maze task although no relationship between performance on reference and working memory tasks was present. Notably, middle-aged rats were impaired relative to young on both tasks. Together these data demonstrate that individual differences in spatial reference memory exist among aged F344 rats and provide novel data demonstrating an unrelated decline in working memory across the lifespan, suggesting that age-related mnemonic dysfunction may occur across multiple brain systems.     

Contribution of glutamate transporter GLT-1 to removal of synaptically released glutamate at climbing fiber-Purkinje cell synapses

Rapid removal of synaptically released glutamate from the extracellular space ensures a high signal-to-noise ratio in excitatory neurotransmission. In the cerebellum, glial glutamate transporters, GLAST and GLT-1, are co-localized in the processes of Bergmann glia wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, the decay kinetics of climbing fiber-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(−/−) mice are not different from those in wild-type (WT) mice. This raises a possibility that GLT-1 plays a significant role in clearing glutamate at CF-PC synapses despite its smaller amount of expression. Here, we studied the functions of GLT-1 and GLAST in the clearance of glutamate using GLAST(−/−) mice and GLT-1(−/−) mice. In the presence of cyclothiazide (CTZ) that attenuates the desensitization of AMPA receptors, the decay time constant of CF-EPSCs (τ_w) in GLT-1(−/−) mice was slower than that in WT mice. However, the degree of this prolongation of τ_w was less prominent compared to that in GLAST(−/−) mice. The values of τ_w in GLT-1(−/−) mice and GLAST(−/−) mice were comparable to those estimated in WT mice in the presence of a potent blocker of glial glutamate transporters (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA) at 10 and 100 nM, which reduced the amplitudes of glutamate transporter currents elicited by CF stimulation in Bergmann glia to ∼81 and ∼28%, respectively. We conclude that GLT-1 plays a minor role compared to GLAST in clearing synaptically released glutamate at CF-PC synapses.     

Age-related alteration in excitatory amino acid neurotransmission in rat brain

The excitatory amino acids as neurotransmitters in the neocortex, hippocampus, striatum, thalamus, amygdala, nucleus basalis of Meynert and cerebellum from rats aged 4 months, 12 months and 24 months have been examined by measuring sodium-dependent high affinity uptake of D-[3H]-aspartate into preparations containing synaptosomes. Calcium-dependent K(+)-stimulated release of endogenous glutamate from the nucleus basalis was also measured. The hippocampus and cerebellum failed to show significant age-related changes in uptake of D-[3H]-aspartate. D-[3H]-aspartate uptake decreased significantly in the neocortex (29%), striatum (29%), nucleus basalis (26%), amygdala (19%) and thalamus (16%) in the middle-aged rats as compared to the young rats, but the changes were not progressive with age. The release of glutamate from the nucleus basalis was unaltered during the aging process.     

Localisation of novel forms of glutamate transporters and the cystine-glutamate antiporter in the choroid plexus: Implications for CSF glutamate homeostasis

The choroid plexus is a structure within each ventricle of the brain that is composed of fenestrated vessels surrounded by secretory epithelial cells. The epithelial cells are linked by tight junctions to create a permeability barrier. The epithelial cells are derived from neuroectoderm, and are thus defined by some authors as a subtype of macroglia. Glutamate is a tightly regulated substance in the CSF, as it is in the rest of the brain. In the brain macroglia express multiple sodium dependent and independent glutamate transporters and are the main regulators of extracellular glutamate. However, the identities of the transporters in the choroid plexus and their localisations have remained poorly defined. In this study we examined the expression and distribution of multiple splice variants of classical sodium-dependent glutamate transporters, as well as the cystine-glutamate antiporter, and the PDZ protein NHERF1, (which acts as a molecular anchor for proteins such as the glutamate transporter GLAST). We identified three forms of sodium-dependent transporters (GLAST1a, GLAST1c and GLT1b) that are expressed at the apical surface of the epithelial cells, a location that matches the distribution of NHERF1 and the cystine-glutamate antiporter. We propose that this coincident localisation of GLAST1a/GLAST1c/GLT1b and the cystine-glutamate antiporter would permit the cyclical trafficking of glutamate and thus optimise the accumulation of cystine for the formation of glutathione in the choroid plexus.     

Expression and distribution of ‘high affinity’ glutamate transporters GLT1, GLAST,EAAC1 and of GCPII in the rat peripheral nervous system

l ‐Glutamate is one of the major excitatory neurotransmitters in the mammalian central nervous system, but recently it has been shown to have a role also in the transduction of sensory input at the periphery, and in particular in the nociceptive pathway. An excess of glutamate is implicated in cases of peripheral neuropathies as well. Conventional therapeutic approaches for treating these diseases have focused on blocking glutamate receptors with small molecules or on reducing its synthesis of the receptors through the inhibition of glutamate carboxypeptidase II (GCPII), the enzyme that generates glutamate. In vivo studies have demonstrated that the pharmacological inhibition of GCPII can either prevent or treat the peripheral nerve changes in both BB/Wor and chemically induced diabetes in rats. In this study, we characterized the expression and distribution of glutamate transporters GLT1, GLAST, EAAC1 and of the enzyme GCPII in the peripheral nervous system of female Wistar rats. Immunoblotting results demonstrated that all glutamate transporters and GCPII are present in dorsal root ganglia (DRG) and the sciatic nerve. Immunofluorescence localization studies revealed that both DRG and sciatic nerves were immunopositive for all glutamate transporters and for GCPII. In DRG, satellite cells were positive for GLT1 and GCPII, whereas sensory neurons were positive for EAAC1. GLAST was localized in both neurons and satellite cells. In the sciatic nerve, GLT1 and GCPII were expressed in the cytoplasm of Schwann cells, whereas GLAST and EAAC1 stained the myelin layer. Our results give for the first time a complete characterization of the glutamate transporter system in the peripheral nervous system. Therefore, they are important both for understanding glutamatergic signalling in the PNS and for establishing new strategies to treat peripheral neuropathies.     

Molecular regulation of glutamate and GABA transporter proteins by valproic acid in rat hippocampus during epileptogenesis

Epileptiform discharges and behavioral seizures may be the consequences of the presence of either excessive excitation associated with the neurotransmitter glutamate or from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). Synaptic effects of these neurotransmitters are terminated by the action of transporter proteins that remove these amino acids from the synaptic cleft. The glial transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), and the neuronal transporter excitatory amino acids carrier-1 (EAAC-1) limit excitation initiated by synaptic release of glutamate. Transporter proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3) remove GABA from synaptic regions. To assess the molecular effects of the antiepileptic drug valproate, albino rats with chronic, spontaneous, recurrent seizures induced by amygdalar injection of FeCl3 were treated for 14 days with either valproic acid or with saline as an injection control. Regions of the hippocampus were assayed for glutamate and GABA transporters by western blot. While epileptogenesis is thought to correlate with the downregulation of GLAST and upregulation of EAAC-1, valproate caused an increase in the quantity of GLAST protein measured in the hippocampus. Valproate treatment decreased GLT-1 in both control and experimental animals in both hippocampi. EAAC-1 was unchanged by valproate treatment. GABA transporters GAT-1 and GAT-3 in the hippocampus were upregulated by FeCl3 injection into the amygdala. However, valproate caused the downregulation of these GABA transporters in both control and experimental animals. Altered molecular regulation of glutamate appears to be critical in the development of sustained, spontaneous limbic seizures. Our data suggest that valproate may have unique mechanisms of action; specifically, it may affect the removal of glutamate by upregulating GLAST and decreasing GABA transport, which could result in increased tissue concentrations of GABA.