LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

GLS1 Mutant Mice Display Moderate Alterations of Hippocampal Glutamatergic Neurotransmission Associated with Specific Adaptive Behavioral Changes.

Significant alterations in glutamatergic neurotransmission have been reported in major depressive disorder (MDD) that could underlie psychiatric traits. Studies were mainly interested in synaptic dysfunction in the prefrontal cortex, a key structure involved in depressive-like behavior, however hippocampus has been shown to be important in MDD. As cognitive deficits such as hippocampus-memory process were observed in MDD, we investigated in a mild hypoglutamatergic model behaviors related to depression and memory, synaptic transmission parameters and glutamatergic state specifically in the hippocampus. We thus characterized these phenotypes in adult male mice partially depleted in glutaminase type 1 or GLS1 (GLS1 HET), the enzyme responsible for glutamate synthesis in neurons, that we previously characterized as displaying moderate lower levels of glutamate in brain. We showed that GLS1 mutant mice display AMPA-R-mediated response deficits after prolonged repetitive stimulation with electrophysiological recording and inability to sustain glutamate release by microdialysis experiments with no consequences on behavioral spatial learning performances. However, their ability to escape from unpleasant but repeated escapable condition was attenuated whereas they were more immobile in the unescapable situation in the FST during re-test. These results show that GLS1 mutant mice display moderate impairments of hippocampal glutamatergic neurotransmission and moderate changes in adaptive behaviors that have been shown to participate to the development of depressive-like state.

Increase of KCC2 in hippocampal synaptic plasticity disturbances after perinatal ethanol exposure.

Low to moderate perinatal ethanol exposure (PEE) may have disastrous consequences for the central nervous system resulting notably in permanent cognitive deficits. Learning and memory are mediated in the hippocampus by long-term potentiation (LTP) and long term depression (LTD), two forms of synaptic plasticity. PEE decreases LTP but also abnormally facilitates LTD (Kervern et al. ) through a presently unknown mechanism. We studied in rat hippocampus slice, the involvement of the chloride co-transporters NKCC1 and KCC2, in the role of GABAA inhibitions in facilitated LTD after moderate PEE. After PEE and in contrast to control slices, facilitated LTD in CA1 field was reduced by the GABAA receptor antagonist bicuculline with no changes in sensitivity to bicuculline and in GABA and benzodiazepine binding sites. Also, sensitivity to diazepam was unaltered, whereas aberrant LTD was blocked. Immunohistochemistry and protein analysis demonstrated an increase in KCC2 protein level at cell membrane in CA1 after PEE with no change in NKCC1 expression. Specifically, both monomeric and dimeric forms of KCC2 were increased in CA1. Bumetanide (10-100 µM), a dose-dependent blocker of NKCC1 and KCC2, or VU0240551 (10 µM) a specific antagonist of KCC2, corrected the enhanced LTD and interestingly bumetanide also restored the lower LTP after PEE. These results demonstrate for the first time an upregulation of the KCC2 co-transporter expression after moderate PEE associated with disturbances in GABAergic neurotransmission modulating bidirectional synaptic plasticity in the hippocampus. Importantly, bumetanide compensated deficits in both LTP and LTD, revealing its potential therapeutic properties.

Two Binges of Ethanol a Day Keep the Memory Away in Adolescent Rats: Key Role for GLUN2B Subunit.

BACKGROUND: Binge drinking is common in adolescents, but the impact of only a few binges on learning and memory appears underestimated. Many studies have tested the effects of long and intermittent ethanol exposure on long-term synaptic potentiation, and whether long-term synaptic depression is affected remains unknown. METHODS: We studied the effects of one (3 g/kg, i.p.; blood ethanol content of 197.5±19 mg/dL) or 2 alcohol intoxications (given 9 hours apart) on adolescent rat's memory and synaptic plasticity in hippocampus slice after different delay. RESULTS: Animals treated with 2 ethanol intoxications 48 hours before training phase in the novel object recognition task failed during test phase. As learning is related to NMDA-dependent mechanisms, we tested ketamine and found the same effect as ethanol, whereas D-serine prevented learning deficit. In hippocampus slice, NMDA-dependent long-term synaptic depression was abolished 48 hours after ethanol or ketamine but prevented after D-serine or in a low-Mg(2+) recording medium. Long-term synaptic depression abolition was not observed 8 days after treatment. An i.p. treatment with MK-801, tetrahydroisoxazolopyridine, or muscimol was ineffective, and long-term synaptic potentiation, intrinsic excitability, and glutamate release remained unaffected. The input/ouput curve for NMDA-fEPSPs was shifted to the left 48 hours after the binges with a stronger contribution of GluN2B subunit, leading to a leftward shift of the Bienenstock-Cooper-Munro relationship. Interestingly, there were no cellular effects after only one ethanol injection. CONCLUSION: Two ethanol "binges" in adolescent rats are sufficient to reversibly abolish long-term synaptic depression and to evoke cognitive deficits via a short-lasting, repeated blockade of NMDA receptors only, inducing a change in the receptor subunit composition. Furthermore, ethanol effects developed over a 48-hour period of abstinence, indicating an important role of intermittence during a repeated long-duration binge behavior.

Aberrant NMDA-dependent LTD after perinatal ethanol exposure in young adult rat hippocampus.

Irreversible cognitive deficits induced by ethanol exposure during fetal life have been ascribed to a lower NMDA-dependent synaptic long-term potentiation (LTP) in the hippocampus. Whether NMDA-dependent long-term depression (LTD) may also play a critical role in those deficits remains unknown. Here, we show that in vitro LTD induced with paired-pulse low frequency stimulation is enhanced in CA1 hippocampus field of young adult rats exposed to ethanol during brain development. Furthermore, single pulse low frequency stimulation, ineffective at this age (LFS600), induced LTD after ethanol exposure accompanied with a stronger response than controls during LFS600, thus revealing an aberrant form of activity-dependent plasticity at this age. Blocking NMDA receptor or GluN2B containing NMDA receptor prevented both the stronger response during LFS600 and LTD whereas Zinc, an antagonist of GluN2A containing NMDA receptor, was ineffective on both responses. In addition, LFS600-induced LTD was revealed in controls only with a reduced-Mg(2+) medium. In whole dissected hippocampus CA1 field, perinatal ethanol exposure increased GluN2B subunit expression in the synaptic compartment whereas GluN2A was unaltered. Using pharmacological tools, we suggest that LFS600 LTD was of synaptic origin. Altogether, we describe a new mechanism by which ethanol exposure during fetal life induces a long-term alteration of synaptic plasticity involving NMDA receptors, leading to an aberrant LTD. We suggest this effect of ethanol may reflect a delayed maturation of the synapse and that aberrant LTD may also participates to long-lasting cognitive deficits in fetal alcohol spectrum disorder.

Selective impairment of some forms of synaptic plasticity by oligomeric amyloid-ß peptide in the mouse hippocampus: implication of extrasynaptic NMDA receptors.

Alzheimer's disease is characterized by the loss of memory and synaptic damage. Evidence is accumulating for a causal role of soluble oligomeric species of amyloid-ß peptide (Aßo) in the impairment of synaptic plasticity and cognition but the precise mechanisms underlying these effects are still not clear. Synaptic plasticity such as long-term potentiation is thought to underlie learning and memory. While the effect of Aß on long-term potentiation is well documented, a more general understanding of Aß action on various aspects of plasticity involving synaptic and extrasynaptic receptors and the nature of the mechanisms involved in its effects are lacking. Using a combination of electrophysiological and biochemical techniques in mouse hippocampal slices, we show here that Aßo drastically affects synaptic plasticities induced by high stimulation frequencies through the involvement of extrasynaptic glutamate receptors. Experiments on hippocampal slices as well as on cultured cortical neurons show that Aßo potentiates extrasynaptic NMDA receptors-mediated responses. Pharmacological characterization indicates that GluN2B-containing NMDARs are involved in these responses. When synaptic and extrasynaptic glutamate receptor-mediated effects are dissociated using cortical neurons in culture, it appears that Aßo has differential effects on these two receptors types. We conclude that the pool of extrasynaptic GluN2B-containing NMDARs is a major target of Aßo in the hippocampus. During high frequency stimulation, Aßo dramatically impairs long-term neuronal responses.

Reversal of age-related oxidative stress prevents hippocampal synaptic plasticity deficits by protecting D-serine-dependent NMDA receptor activation.

Oxidative stress (OS) resulting from an imbalance between antioxidant defenses and the intracellular accumulation of reactive oxygen species (ROS) contributes to age-related memory deficits. While impaired synaptic plasticity in neuronal networks is thought to underlie cognitive deficits during aging, whether this process is targeted by OS and what the mechanisms involved are still remain open questions. In this study, we investigated the age-related effects of the reducing agent N-acetyl-L-cysteine (L-NAC) on the activation of the N-methyl-D-aspartate receptor (NMDA-R) by its co-agonist D-serine, because alterations in this mechanism contribute greatly to synaptic plasticity deficits in aged animals. Long-term dietary supplementation with L-NAC prevented oxidative damage in the hippocampus of aged rats. Electrophysiological recordings in the CA1 of hippocampal slices indicated that NMDA-R-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) were depressed in aged animals, deficits that could be reversed by exogenous D-serine. Chronic treatment with L-NAC, but not acute application of the reducing agent, restored potent D-serine-dependent NMDA-R activation and LTP induction in aged rats. In addition, it is also revealed that the age-related decrease in D-serine levels and in the expression of the synthesizing enzyme serine racemase, which underlies the decrease in NMDA-R activation by the amino acid, was rescued by long-term dietary treatment with L-NAC. Our results indicate that protecting redox status in aged animals could prevent injury to the cellular mechanisms underlying cognitive aging, in part by maintaining potent NMDA-R activation through the D-serine-dependent pathway.

Perinatal alcohol exposure in rat induces long-term depression of respiration after episodic hypoxia.

RATIONALE: Little is known about the effects of alcohol exposure during pregnancy, which is responsible for fetal alcohol syndrome and the respiratory network functions, especially respiratory network plasticity (e.g., long-term facilitation) elicited after repeated short-lasting hypoxic episodes. The mechanism of induction of respiratory long-term facilitation involves 5-HT(2A/2C) receptors, which also participate in the response to hypoxia. Because fetal alcohol exposure is known to reduce serotonin centrally, and synaptic plasticity in the hippocampus, we hypothesized that alcohol exposure during gestation might impair respiratory long-term facilitation after hypoxic episodes. OBJECTIVES: To analyze the effects of prenatal and postnatal alcohol exposure on respiratory long-term facilitation in 5- to 7-day-old rats. METHODS: Respiratory frequency and amplitude were measured in vivo and in an in vitro rhythmic medullary slice before and after three hypoxia episodes or three applications of a 5-HT(2A/2C) receptor agonist in vitro. 5-HT(2A/2C) receptor mRNA was measured from the slice. MEASUREMENTS AND MAIN RESULTS: Alcohol exposure impaired respiratory long-term facilitation and induced long-term depression of respiration in both in vivo and in vitro models. Alcohol altered 5-HT(2A/2C) mRNA expression, although 5-HT(2A/2C) agonist efficacy was not altered in increasing rhythmic activity in slices. However, a higher concentration of 5-HT(2A/2C) agonist was necessary to induce transient facilitation in slices from ethanol-exposed animals, suggesting disturbances in induction and maintenance mechanisms of respiratory long-term facilitation. CONCLUSIONS: Respiratory facilitation after repeated hypoxia was converted to long-term depression in rats treated with alcohol in utero. Alcohol exposure during pregnancy may therefore induce long-term maladaptive behavior of the respiratory system in neonates.