Non-Excitatory Amino Acids, Melatonin, and Free Radicals: Examining the Role in Stroke and Aging.

The aim of this review is to explore the relationship between melatonin, free radicals, and non-excitatory amino acids, and their role in stroke and aging. Melatonin has garnered significant attention in recent years due to its diverse physiological functions and potential therapeutic benefits by reducing oxidative stress, inflammation, and apoptosis. Melatonin has been found to mitigate ischemic brain damage caused by stroke. By scavenging free radicals and reducing oxidative damage, melatonin may help slow down the aging process and protect against age-related cognitive decline. Additionally, non-excitatory amino acids have been shown to possess neuroprotective properties, including antioxidant and anti-inflammatory in stroke and aging-related conditions. They can attenuate oxidative stress, modulate calcium homeostasis, and inhibit apoptosis, thereby safeguarding neurons against damage induced by stroke and aging processes. The intracellular accumulation of certain non-excitatory amino acids could promote harmful effects during hypoxia-ischemia episodes and thus, the blockade of the amino acid transporters involved in the process could be an alternative therapeutic strategy to reduce ischemic damage. On the other hand, the accumulation of free radicals, specifically mitochondrial reactive oxygen and nitrogen species, accelerates cellular senescence and contributes to age-related decline. Recent research suggests a complex interplay between melatonin, free radicals, and non-excitatory amino acids in stroke and aging. The neuroprotective actions of melatonin and non-excitatory amino acids converge on multiple pathways, including the regulation of calcium homeostasis, modulation of apoptosis, and reduction of inflammation. These mechanisms collectively contribute to the preservation of neuronal integrity and functions, making them promising targets for therapeutic interventions in stroke and age-related disorders.

Identification of Non-excitatory Amino Acids and Transporters Mediating the Irreversible Synaptic Silencing After Hypoxia.

The contribution of excitatory amino acids (AA) to ischemic brain injury has been widely described. In addition, we reported that a mixture of non-excitatory AA at plasmatic concentrations turns irreversible the depression of synaptic transmission caused by hypoxia. Here, we describe that the presence of seven non-excitatory AA (L-alanine, L-glutamine, glycine, L-histidine, L-serine, taurine, and L-threonine) during hypoxia provokes an irreversible neuronal membrane depolarization, after an initial phase of hyperpolarization. The collapse of the membrane potential correlates with a great increase in fiber volley amplitude. Nevertheless, we show that the presence of all seven AA is not necessary to cause the irreversible loss of fEPSP after hypoxia and that the minimal combination of AA able to provoke a solid, replicable effect is the mixture of L-alanine, glycine, L-glutamine, and L-serine. Additionally, L-glutamine seems necessary but insufficient to induce these harmful effects. We also prove that the deleterious effects of the AA mixtures on field potentials during hypoxia depend on both the identity and concentration of the individual AA in the mixture. Furthermore, we find that the accumulation of AA in the whole slice does not determine the outcome caused by the AA mixtures on the synaptic transmission during hypoxia. Finally, results obtained using pharmacological inhibitors and specific substrates of AA transporters suggest that system N and the alanine-serine-cysteine transporter 2 (ASCT2) participate in the non-excitatory AA-mediated deleterious effects during hypoxia. Thus, these AA transporters might represent therapeutical targets for the treatment of brain ischemia.

"MiR-7 controls cholesterol biosynthesis through posttranscriptional regulation of DHCR24 expression".

Dysregulation of cholesterol homeostasis is associated with several pathologies including cardiovascular diseases and neurological disorders such as Alzheimer's disease (AD). MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of cholesterol metabolism. We previously established the role of miR-7 in regulating insulin resistance and amyloidosis, which represents a common pathological feature between type 2 diabetes and AD. We show here an additional metabolic function of miR-7 in cholesterol biosynthesis. We found that miR-7 blocks the last steps of the cholesterol biosynthetic pathway in vitro by targeting relevant genes including DHCR24 and SC5D posttranscriptionally. Intracranial infusion of miR-7 on an adeno-associated viral vector reduced the expression of DHCR24 in the brain of wild-type mice, supporting in vivo miR-7 targeting. We also found that cholesterol regulates endogenous levels of miR-7 in vitro, correlating with transcriptional regulation through SREBP2 binding to its promoter region. In parallel to SREBP2 inhibition, the levels of miR-7 and hnRNPK (the host gene of miR-7) were concomitantly reduced in brain in a mouse model of Niemann Pick type C1 disease and in murine fatty liver, which are both characterized by intracellular cholesterol accumulation. Taken together, the results establish a novel regulatory feedback loop by which miR-7 modulates cholesterol homeostasis at the posttranscriptional level, an effect that could be exploited for therapeutic interventions against prevalent human diseases.

Novel mechanism of hypoxic neuronal injury mediated by non-excitatory amino acids and astroglial swelling.

In ischemic stroke and post-traumatic brain injury (TBI), blood-brain barrier disruption leads to leaking plasma amino acids (AA) into cerebral parenchyma. Bleeding in hemorrhagic stroke and TBI also release plasma AA. Although excitotoxic AA were extensively studied, little is known about non-excitatory AA during hypoxic injury. Hypoxia-induced synaptic depression in hippocampal slices becomes irreversible with non-excitatory AA, alongside their intracellular accumulation and increased tissue electrical resistance. Four non-excitatory AA (l-alanine, glycine, l-glutamine, l-serine: AGQS) at plasmatic concentrations were applied to slices from mice expressing EGFP in pyramidal neurons or astrocytes during normoxia or hypoxia. Two-photon imaging, light transmittance (LT) changes, and electrophysiological field recordings followed by electron microscopy in hippocampal CA1 st. radiatum were used to monitor synaptic function concurrently with cellular swelling and injury. During normoxia, AGQS-induced increase in LT was due to astroglial but not neuronal swelling. LT raise during hypoxia and AGQS manifested astroglial and neuronal swelling accompanied by a permanent loss of synaptic transmission and irreversible dendritic beading, signifying acute damage. Neuronal injury was not triggered by spreading depolarization which did not occur in our experiments. Hypoxia without AGQS did not cause cell swelling, leaving dendrites intact. Inhibition of NMDA receptors prevented neuronal damage and irreversible loss of synaptic function. Deleterious effects of AGQS during hypoxia were prevented by alanine-serine-cysteine transporters (ASCT2) and volume-regulated anion channels (VRAC) blockers. Our findings suggest that astroglial swelling induced by accumulation of non-excitatory AA and release of excitotoxins through antiporters and VRAC may exacerbate the hypoxia-induced neuronal injury.

Hypoxia-induced depression of synaptic transmission becomes irreversible by intracellular accumulation of non-excitatory amino acids.

The intracellular accumulation of some amino acids (AAs), mainly glutamine, can contribute to brain edema observed during liver failure. We recently demonstrated that individual applications of high concentrations (10 mM) of some non-excitatory AAs increase the electrical resistance of hippocampal slices, indicating cell swelling. Therefore, we pondered whether an AA mixture's application might cause cell swelling at a physiological concentration range. In rat hippocampal slices, we carried out extra- and intracellular electrophysiological recordings and AAs analysis to address this question. We applied a mixture of 19 AAs at their plasmatic concentrations (Plasma solution: Ala, Gly, Gln, His, Ser, Tau, Thr, Arg, Leu, Met, Pro, Val, Asn, Cys, Phe, Ile, Lys, Tyr, and Trp). This solution was afterward divided into two according to the individual AAs at 10 mM concentration inducing synaptic potentiation (Plasma1, containing the first seven AAs of Plasma) or not (Plasma2, with the remaining AAs). Plasma application increased evoked field potentials requiring extracellular chloride. This effect was mimicked by the Plasma1 but not the Plasma2 solution. Plasma1-induced potentiation was independent of changes in release probability, basic electrophysiological membrane properties, and NMDAR activation. AAs in Plasma1 act cooperatively to accumulate intracellularly and to induce synaptic potentiation. In the presence of Plasma1, the reversible synaptic depression caused by a 40-min hypoxia period turned into an irreversible disappearance of synaptic potentials through an NMDAR-dependent mechanism. The presence of a system A transport inhibitor did not block Plasma1-mediated effects. These results indicate that cell swelling, induced by the accumulation of non-excitotoxic AAs through unidentified transporters, might foster deleterious effects produced by hypoxia-ischemia episodes.

Intracellular accumulation of amino acids increases synaptic potentials in rat hippocampal slices.

The application of high concentrations of taurine induces long-lasting potentiation of synaptic responses and axon excitability. This phenomenon seems to require the contribution of a transport system with a low affinity for taurine. The prototypic taurine transporter TauT (SLC6A6) was discarded by experimental evidence obtained in TauT-KO mice. The purpose of the present study was to determine whether the proton-coupled amino acid transporter 1 (PAT1; SLC36A1) which is a transport system with low affinity and high capacity for a great variety of amino acids including taurine, contributes to the taurine-induced synaptic potentiation. In rat hippocampal slices, the application of several amino acids (L- and D-alanine, L-glutamine, ß-guanidinopropionic acid, glycine, L-histidine, L- and D-serine, sarcosine, L- and D-threonine) imitated the synaptic potentiation induced by taurine. The magnitude of the potentiation caused by some of these amino acids was even greater than that induced by taurine. By contrast, the application of other amino acids (L-arginine, betaine, L-leucine, L-methionine, L- and D-proline, and L-valine) did not induce potentiation. The behaviour of these different amino acids on synaptic potentiation is not compatible with a role of PAT1 in synaptic potentiation. There was a positive correlation between the accumulation of the different amino acids in the slice and the magnitude of synaptic potentiation induced by them. Some of the amino acids inducing synaptic potentiation, like taurine and L-threonine, also increased electrical resistance of the slice, whereas L-leucine did not modify this parameter. Modifications induced by either taurine or L-threonine in synaptic potentiation, slice resistance and amino acid accumulation were dependent on extracellular chloride concentration. These findings support the idea that the accumulation of amino acids throughout the action of transporters causes cell swelling enhancing the electrical resistance of the slice, which by itself could be sufficient to increase field synaptic potentials.

Characterisation of the mechanisms underlying the special sensitivity of the CA2 hippocampal area to adenosine receptor antagonists.

Recent studies have underscored the importance of the CA2 area in social memory formation. This area, a narrow transition zone between hippocampal CA3 and CA1 areas, is endowed with special connectivity and a distinctive molecular composition. In particular, adenosine A1 receptors (A1R) are enriched in CA2, and based on the prominent synaptic potentiation induced by A1R antagonists (e.g., caffeine) in this area, it has been proposed that CA2 is under the strong tonic control of A1R activation. It is unclear whether this special sensitivity of CA2 to A1R antagonists is due to an elevated extracellular concentration of adenosine or to a different A1R function. Here, using the recording of field potentials evoked simultaneously in CA2 and CA1 by Schaffer collateral stimulation, we confirm that the application of A1R antagonists, caffeine and DPCPX has a stronger effect on synaptic responses in CA2 than in those evoked in CA1. This difference was, at least partially, explained by the action of A1R antagonists on presynaptic A1Rs. We found that caffeine-induced potentiation in CA2 was restricted to Schaffer collateral synapses, but not to those formed by temporoammonic inputs. We also observed that the apparent affinity of an A1R agonist is similar for A1R in both CA2 and CA1 areas, which indicates that the tonic activation of A1R in both areas is comparable. Furthermore, we show that the direct activation of adenylyl cyclase with forskolin in the presence of rolipram, a phosphodiesterase inhibitor, greatly enhances the synaptic potentials in CA2 compared to CA1. The forskolin-induced potentiation was exacerbated in the presence of caffeine or DPCPX, accentuating the differences between the two areas. These results indicate that the tonic activation of A1Rs in area CA2 is not different to that of other hippocampal areas, but it is more efficiently coupled to the downstream effectors.

The taurine transporter substrate guanidinoethyl sulfonate mimics the action of taurine on long-term synaptic potentiation.

Taurine is especially abundant in rodent brain where it appears to be involved in osmoregulation and synaptic plasticity mechanisms. The demonstration of a physiological role for taurine has been hampered by the difficulty in modifying taurine levels in most tissues, including the brain. We used an experimental strategy to reduce taurine levels, involving treatment with guanidinoethyl sulfonate (GES), a structural analogue of taurine that, among other properties, acts as a competitive inhibitor of taurine transport. GES delivered in the drinking water of rats for 1 month effectively reduced taurine levels in brain structures (hippocampus, cerebellum and cortex) and outside the brain (heart, muscle, kidney, liver and plasma) by between 50 and 80 %, depending on the tissue. This partial taurine depletion did not affect either basal synaptic transmission or the late phase of long-term potentiation (late-LTP) in hippocampal slices. In vivo microdialysis studies in the hippocampus revealed that GES treatment reduced extracellular taurine levels and the magnitude of taurine released in response to the application of either N-methyl-D-aspartate (NMDA) or a hypoosmotic solution, without affecting release mechanisms. Finally, we demonstrated in hippocampal slices that a brief GES application can mimic taurine action on the conversion of a decremental LTP into a perdurable late-LTP, concluding that GES might replace taurine function in some mechanisms such as those implicated in synaptic plasticity.

Taurine content in different brain structures during ageing: effect on hippocampal synaptic plasticity.

A reduction in taurine content accompanies the ageing process in many tissues. In fact, the decline of brain taurine levels has been associated with cognitive deficits whereas chronic administration of taurine seems to ameliorate age-related deficits such as memory acquisition and retention. In the present study, using rats of three age groups (young, adult and aged) we determined whether the content of taurine and other amino acids (glutamate, serine, glutamine, glycine, alanine and GABA) was altered during ageing in different brain areas (cerebellum, cortex and hippocampus) as well non-brain tissues (heart, kidney, liver and plasma). Moreover, using hippocampal slices we tested whether ageing affects synaptic function and plasticity. These parameters were also determined in aged rats fed with either taurine-devoid or taurine-supplemented diets. With age, we found heterogeneous changes in amino acid content depending on the amino acid type and the tissue. In the case of taurine, its content was reduced in the cerebellum of adult and aged rats, but it remained unchanged in the hippocampus, cortex, heart and liver. The synaptic response amplitude decreased in aged rats, although the late phase of long-term synaptic potentiation (late-LTP), a taurine-dependent process, was not altered. Our study highlights the stability of taurine content in the hippocampus during ageing regardless of whether taurine was present in the diet, which is consistent with the lack of changes detected in late-LTP. These results indicate that the beneficial effects of taurine supplementation might be independent of the replenishment of taurine stores.

Cooperation of taurine uptake and dopamine D1 receptor activation facilitates the induction of protein synthesis-dependent late LTP.

Co-activation of NMDA and dopamine receptors is required for the induction of the late phase of LTP (L-LTP) that is dependent on new protein synthesis. Other neuromodulatory substances may also contribute to this process. Here, we examined whether taurine is one of the neuromodulators contributing to L-LTP induction, since it is known that taurine uptake induces a long-lasting synaptic potentiation dependent on protein synthesis, and taurine uptake inhibition blocks L-LTP induced by tetanization. Experiments were conducted using rat hippocampal slices where field synaptic potentials were evoked and recorded in CA3-CA1 synapses. Taurine (1 mM) applied 10 min before a high frequency stimulation (HFS) train converted a transitory early-LTP (E-LTP) into an L-LTP dependent on protein synthesis. This taurine effect was blocked by a taurine uptake inhibitor. A facilitation of L-LTP induction was also obtained by pre-application of SKF38393, a D1/D5 dopamine receptor (D1R) agonist. In this case, LTP facilitation was not affected by the taurine uptake inhibitor. Nevertheless, when taurine and SKF38393 were simultaneously pre-applied at a concentration that individually did not modify E-LTP, they produced a synergistic mechanism that facilitated the induction of L-LTP with a sole HFS train. This facilitation of L-LTP was blocked by inhibiting either taurine uptake or D1R activation. Taurine and SKF38393 activated different signaling pathways to transform E-LTP into L-LTP. Taurine-induced L-LTP facilitation required MAPK activation, while D1R-agonist-induced facilitation depended mainly on PKA activation and partially on MAPK activation. On the other hand, the synergistic mechanisms induced by the cooperative action of taurine and SKF38393 were impaired by inhibitors against MAPK, PKA and PI3-K. This pharmacological profile resembles that displayed by L-LTP induced by three HFS trains at 10-min intervals. These results indicate that taurine uptake is necessary and cooperates with other neurotransmitter systems in the induction of L-LTP.

L-DOPA treatment selectively restores spine density in dopamine receptor D2-expressing projection neurons in dyskinetic mice.

BACKGROUND: L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia is an incapacitating complication of L-DOPA therapy that affects most patients with Parkinson's disease. Previous work indicating that molecular sensitization to dopamine receptor D1 (D1R) stimulation is involved in dyskinesias prompted us to perform electrophysiological recordings of striatal projection "medium spiny neurons" (MSN). Moreover, because enhanced D1R signaling in drug abuse induces changes in spine density in striatum, we investigated whether the dyskinesia is related to morphological changes in MSNs. METHODS: Wild-type and bacterial artificial chromosome transgenic mice (D1R-tomato and D2R-green fluorescent protein) mice were lesioned with 6-hydroxydopamine and subsequently treated with L-DOPA to induce dyskinesia. Functional, molecular, and structural changes were assessed in corticostriatal slices. Individual MSNs injected with Lucifer-Yellow were detected by immunohistochemistry for three-dimensional reconstructions with Neurolucida software. Intracellular current-clamp recordings with high-resistance micropipettes were used to characterize electrophysiological parameters. RESULTS: Both D1R-MSNs and D2R-MSNs showed diminished spine density in totally denervated striatal regions in parkinsonian mice. Chronic L-DOPA treatment, which induced dyskinesia and aberrant FosB expression, restored spine density in D2R-MSNs but not in D1R-MSNs. In basal conditions, MSNs are more excitable in parkinsonian than in sham mice, and excitability decreases toward normal values after L-DOPA treatment. Despite this normalization of basal excitability, in dyskinetic mice, the selective D1R agonist SKF38393 increased the number of evoked action potentials in MSNs, compared with sham animals. CONCLUSIONS: Chronic L-DOPA induces abnormal spine re-growth exclusively in D2R-MSNs and robust supersensitization to D1R-activated excitability in denervated striatal MSNs. These changes might constitute the anatomical and electrophysiological substrates of dyskinesia.

Systemic injection of kainic acid differently affects LTP magnitude depending on its epileptogenic efficiency.

Seizures have profound impact on synaptic function and plasticity. While kainic acid is a popular method to induce seizures and to potentially affect synaptic plasticity, it can also produce physiological-like oscillations and trigger some forms of long-term potentiation (LTP). Here, we examine whether induction of LTP is altered in hippocampal slices prepared from rats with different sensitivity to develop status epilepticus (SE) by systemic injection of kainic acid. Rats were treated with multiple low doses of kainic acid (5 mg/kg; i.p.) to develop SE in a majority of animals (72-85% rats). A group of rats were resistant to develop SE (15-28%) after several accumulated doses. Animals were subsequently tested using chronic recordings and object recognition tasks before brain slices were prepared for histological studies and to examine basic features of hippocampal synaptic function and plasticity, including input/output curves, paired-pulse facilitation and theta-burst induced LTP. Consistent with previous reports in kindling and pilocapine models, LTP was reduced in rats that developed SE after kainic acid injection. These animals exhibited signs of hippocampal sclerosis and developed spontaneous seizures. In contrast, resistant rats did not become epileptic and had no signs of cell loss and mossy fiber sprouting. In slices from resistant rats, theta-burst stimulation induced LTP of higher magnitude when compared with control and epileptic rats. Variations on LTP magnitude correlate with animals' performance in a hippocampal-dependent spatial memory task. Our results suggest dissociable long-term effects of treatment with kainic acid on synaptic function and plasticity depending on its epileptogenic efficiency.

Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement.

Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.

High potassium induces taurine release by osmosensitive and osmoresistant mechanisms in the rat hippocampus in vivo.

The high potassium-evoked taurine efflux in the nervous tissue has been entirely considered to be the result of the cell swelling produced by KCl influx via passive Donnan forces. However, the extracellular taurine increase evoked in the hippocampus by applying 6-100 mM KCl through microdialysis probes, which saturates at a concentration of 25 mM KCl, is not congruent with the mentioned osmosensitive release of taurine stimulated by high potassium. Therefore, we studied whether the taurine release elicited by different high KCl concentrations (25, 50, 75, or 100 mM) was blocked under hypertonic conditions (+100 mM sucrose). Taurine release stimulated by 25 mM KCl was totally osmosensitive, but that released by higher KCl concentrations became progressively osmoresistant, achieving more than the 60% of the extracellular taurine enhancement during 100 mM KCl perfusion. The osmoresistant taurine release evoked by 100 mM KCl perfusion was partially reduced by a solution without Ca(2+) and with high Mg(2+), or by D,L-2-amino-5-phosphopentanoic acid, an N-methyl-D-aspartic acid (NMDA) receptor antagonist. Moreover, the release of taurine induced by a hypoosmotic solution was reduced by the presence of either high K(+) (75 mM) or NMDA (100 microM). These results indicate that although moderately high [K(+)] evoke the osmosensitive release of taurine, higher [K(+)] inhibit it and trigger the release of taurine by an osmoresistant mechanism. This last component is partially mediated by NMDA receptors activated by the glutamate released during potassium-induced depolarization.

D1 but not D5 dopamine receptors are critical for LTP, spatial learning, and LTP-Induced arc and zif268 expression in the hippocampus.

Recent evidence suggests that glutamatergic and dopaminergic afferents must be activated to induce persistent long-term potentiation (LTP) in the hippocampus. Whereas extensive evidence supports the role of glutamate receptors in long-lasting synaptic plasticity and spatial learning and memory, there is less evidence regarding the role of dopamine receptors in these processes. Here, we used dopamine D(1) receptor knockout (D(1)R(-/-)) mice to explore the role of D(1)R in hippocampal LTP and its associated gene expression. We show that the magnitude of early and late phases of LTP (E-LTP and L-LTP) was markedly reduced in hippocampal slices from D(1)R(-/-) mice compared with wild-type mice. SCH23390, a D(1)/D(5)R antagonist, did not further reduce L-LTP in D(1)R(-/-) mice, suggesting that D(5)Rs are not involved. D(1)R(-/-) mice also showed a significant reduction of D(1)R-induced potentiation of N-Methyl-D-aspartic acid-mediated currents, via protein kinase activated by cyclic adenosine 3',5'-monophosphate activation. Finally, LTP-induced expression of the immediate early genes zif268 and arc in the hippocampal CA1 area was abolished in D(1)R(-/-) mice, and these mice showed impaired learning. These results indicate that D(1)R but not D(5)R are critical for hippocampal LTP and for the induction of Zif268 and Arc, proteins required for the transition from E-LTP to L-LTP and for memory consolidation in mammals.

Glycine-induced long-term synaptic potentiation is mediated by the glycine transporter GLYT1.

The negative symptoms of schizophrenia are reverted by treatment with glycine or other agonists of the glycine-B site which facilitate NMDA receptor function. On the other hand, there are experimental observations showing that exogenous application of glycine (0.5-10mM) results in a long-lasting potentiation of glutamatergic synaptic transmission (LTP-GLY). The characterization of the mechanisms underlying LTP-GLY could be useful to develop new therapies for schizophrenia. The main goal of this work is to deepen the understanding of this potentiation phenomenon. The present study demonstrates in rat hippocampal slices that superfusion of glycine 1mM during 30 min produces a potentiation of excitatory postsynaptic potentials in CA3-CA1 pathway lasting at least 1h. Glycine application does not modify neither presynaptic fiber volley nor paired-pulse facilitation of synaptic potentials. This LTP-GLY is independent of both strychnine-sensitive glycine receptors and nifedipine-sensitive calcium channels. Interestingly, LTP-GLY is not inhibited but strengthened by NMDA receptors antagonists such as AP-5 or MK-801. In contrast, LTP-GLY is partially or totally blocked with the antagonists of glycine transporter GLYT1, sarcosine or ALX-5407, respectively. These results indicate that LTP-GLY requires the activation of GLYT1, a glycine transporter co-localized and associated to NMDA receptors. In addition, the fact that NMDA receptor inhibition increases LTP-GLY magnitude, opens the possibility that these receptors could have a negative control on GLYT1 activity.

Taurine potentiates presynaptic NMDA receptors in hippocampal Schaffer collateral axons.

We have previously shown that activation of presynaptic N-methyl-d-aspartate (NMDA) receptors (NMDAR) enhances the amplitude of the presynaptic fibre volley (FV) evoked in Schaffer collateral axons of rat hippocampal slices, by a mechanism independent of extracellular Ca(2+). Here we compared the pharmacological characteristics of presynaptic NMDARs affecting axon excitability (activated by 10-300 microM NMDA for 10 min), with those mediating field excitatory postsynaptic potentials (NMDA-fEPSP). We found that NMDA-induced potentiation was completely inhibited by NVP-AAM077, an antagonist of NR2A-containing NMDAR, but not by ifenprodil, an NR2B-selective antagonist. The inhibitor of the glycine-binding site in NMDARs, 7-clorokynurenic acid (7-CK), was more potent against NMDA-fEPSP (IC(50) = 6.3 +/- 1.3 microM) than against the NMDA-induced FV potentiation (IC(50) = 26.5 +/- 1.3 microM). Moreover, both post- and presynaptic NMDAR-mediated phenomena were enhanced by glycine and d-serine, but taurine, an endogenous analogue of glycine, only enhanced the latter (EC(50) = 19 microM). Taurine was able to block the inhibitory effect of low doses of 7-CK on NMDA-induced FV potentiation, while glycine and d-serine only reduced the effects of higher concentrations of this drug. Surprisingly, the enhancing effect of taurine on NMDA-induced FV potentiation was blocked when it was co-applied with glycine. Furthermore, the glutamate released synaptically with a train of stimuli also increased FV amplitude by a mechanism dependent on NMDARs; this was potentiated by taurine but not by co-application of taurine and glycine. These results reveal that presynaptic NMDARs have unique properties that mediate the facilitation of axon excitability.

Presynaptic NMDA autoreceptors facilitate axon excitability: a new molecular target for the anticonvulsant gabapentin.

Gabapentin is a drug with anticonvulsant and analgesic properties causing the reduction of neurotransmitter release. We show that one of the mechanisms implicated in this effect of gabapentin is the reduction of the axon excitability measured as an amplitude change of the presynaptic fibre volley (FV) in the CA1 area of rat hippocampal slices. Interestingly, we found that gabapentin-induced depression of FV is mimicked and occluded by NMDA receptor (NMDA-R) antagonists, indicating that these receptors are located presynaptically and are activated by ambient levels of glutamate. Conversely, NMDA application (20 microM, 10 min) elicits a reversible FV potentiation which is reduced by gabapentin. Both NMDA- and gabapentin-induced FV changes are partially explained by modifications in the firing threshold of individual fibres. Increasing [K(+)](o) does not mimic or occlude (at a concentration of 6.5 mM) the effect of NMDA on FV amplitude, which makes it unlikely that a rise in [K(+)](o) induced by NMDA receptor activation could indirectly participate in the potentiation of the FV. The NMDA-induced FV potentiation is independent of extracellular calcium presence but is completely inhibited in a low-Na(+) solution (50% reduction) or under NMDA channel block (high Mg(2+) or MK 801). These findings suggest that sodium entry through presynaptic NMDA-R channels facilitates axon excitability. The interaction of gabapentin with this newly described mechanism might contribute to its therapeutic benefits.

Role of taurine uptake on the induction of long-term synaptic potentiation.

Taurine application in the CA1 area of rat hippocampal slices induces a long-lasting potentiation of excitatory synaptic transmission that has some mechanistic similitude with the late phase of long-term potentiation (L-LTP). Previous indirect evidence such as temperature and sodium dependence indicated that taurine uptake is one of the primary steps leading to the taurine-induced synaptic potentiation. We show that taurine-induced potentiation is not related to the intracellular accumulation of taurine and is not impaired by 2-guanidinoethanesulphonic acid, a taurine transport inhibitor that is a substrate of taurine transporter. We have found that taurine uptake in hippocampal synaptosomes was inhibited by SKF 89976A, a GABA uptake blocker that is not transportable by GABA transporters. SKF 89976A prevents the induction of synaptic potentiation by taurine application. This effect is neither mimicked by nipecotic acid, a broad inhibitor of GABA transporters that does not affect taurine uptake, nor by NO-711, a specific and potent inhibitor of GABA transporter GAT-1. In addition, L-LTP induced by trains of high-frequency stimulation is also inhibited by SKF 89976A, and taurine, at a concentration that does not change basal synaptic transmission, overcomes such inhibition. We conclude that taurine induces synaptic potentiation through the activation of a system transporting taurine and that taurine uptake is required for the induction of synaptic plasticity phenomena such as L-LTP.

Evaluation of vapor recovery systems efficiency and personal exposure in service stations in Mexico City.

Results of a field study on the efficiency of vapor recovery systems currently used in gasoline service stations in Mexico City are presented. Nine gasoline stations were studied, representing the several technologies available in Mexico City. The test was applied to a fixed vehicular fleet of approximately 10 private and public service vehicles. Each one of the gasoline service stations tested reported efficiencies above 80% in the recovery of vapor losses from gasoline which is the minimum permissible value by Mexican regulations. Implications to the emissions inventory are discussed. A second goal of this study was to measure the potential exposure of service attendants to three important components of gasoline: benzene; toluene; and xylenes. The influence of spatial location of personnel within the service station was also evaluated by measuring levels of the three compounds both at the refueling area and in the service station office. Results are discussed and compared to a previous study.