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The developing brain is particularly vulnerable to alcohol: Drinking during pregnancy can lead to a number of physical, learning, and behavioral disorders in the newborn. It has been demonstrated that immature and mature brain tissues display a differential sensitivity to ethanol (EtOH) toxicity and that cerebral structure and function are diversely impaired according to the stage of synaptic maturation. Rat organotypic hippocampal slice cultures were exposed for 7 days to EtOH (100 to 300 mM) after 2 days (immature) or 10 days (mature) of culture in vitro; EtOH was then removed from the medium, and 24 hours later, slices were analyzed by fluorescence microscopy, Western blotting, electrophysiology, and electron microscopy to explore the molecular mechanisms of EtOH toxicity in the developing hippocampus. EtOH withdrawal elicited a selective CA1 pyramidal cell injury in mature slices, but not in immature slices. A significant increase in the expression of pre‐ and postsynaptic proteins in mature slices revealed that slice maturation is presumably associated with the development of new synapses. Incubation with chronic EtOH for 7 days and its removal from the medium induced a significant decrease in GluA1 and GluA2 expression levels; a significant reduction in the expression of synaptophysin and GluN2A was observed only after EtOH withdrawal. Whole‐cell patch‐clamp recordings showed that incubation with EtOH for 7 days induced a significant decrease in spontaneous excitatory postsynaptic current (sEPSC)frequency in CA1 pyramidal cells of immature slices and a trend toward a decrease in sEPSCamplitude. Electron microscopy revealed a disorganization of neurotubuli in immature slices after chronic exposure to EtOH. These results indicate that prolonged incubation with EtOH and its subsequent withdrawal from the medium induce an impairment of excitatory synaptic transmission and possibly an incorrect formation of neuronal circuits in developing hippocampus in vitro, which is suggestive of mechanisms that may lead to mental retardation in fetal alcohol spectrum disorders. Prolonged incubation with ethanol and its subsequent removal from the medium produce, in developing brain tissue, alterations in the expression of pre‐ and post‐synaptic proteins, an impairment of excitatory synaptic transmission and a dramatic disorganization of dendritic microtubules and neuronal processes. An incorrect formation of neuronal circuits and a compromised synaptic transmission in the developing hippocampal neurons are suggestive of mechanisms that may lead to mental retardation, which may be of relevance for the alterations observed in newborns affected by FASD.

(±)-3-Hydroxy-4,5,6,6 a-tetrahydro-3 aH-pyrrolo [3,4 -d]-isoxazole-4-carboxylic acid (HIP-A) and (±)-3-hydroxy-4,5,6, 6 a-tetrahydro-3 aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) are selective inhibitors of excitatory amino acid transporters (EAATs), as potent as dl-threo-β-benzyloxyaspartic acid (TBOA). We report here that the active isomers are (–)-HIP-A and (+)-HIP-B, being approximately 150- and 10-fold more potent than the corresponding enantiomers as inhibitors of [3H]aspartate uptake in rat brain synaptosomes and hEAAT1–3-expressing cells. Comparable IC50values were found on the three hEAAT subtypes. (–)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]d-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]l-glutamate uptake. New data suggest that the noncompetitive-like interaction described previously is probably the consequence of an insurmountable, long-lasting impairment of EAAT’s function. Some minutes of preincubation are required to induce this impairment, the duration of preincubation having more effect on inhibition of glutamate-induced release than of glutamate uptake. In organotypic rat hippocampal slices and mixed mouse brain cortical cultures, TBOA, but not (–)-HIP-A, had toxic effects. Under ischemic conditions, a neuroprotective effect was found with 10 to 30 μM (–)-HIP-A, but not with 10 to 30 μM TBOA or 100 μM (–)-HIP-A. The effect of (–)-HIP-A suggests that, under ischemia, EAATs mediate both release (reverse transport) and uptake of glutamate. The neuroprotection with the lower (–)-HIP-A concentrations may indicate a selective inhibition of the reverse transport confirming the data obtained in synaptosomes. The selective interference with glutamate-induced glutamate release might offer a new strategy for neuroprotective action.

(+/-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo [3,4 -d]-isoxazole-4-carboxylic acid (HIP-A) and (+/-)-3-hydroxy-4,5,6, 6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) are selective inhibitors of excitatory amino acid transporters (EAATs), as potent as DL-threo-beta-benzyloxyaspartic acid (TBOA). We report here that the active isomers are (-)-HIP-A and (+)-HIP-B, being approximately 150- and 10-fold more potent than the corresponding enantiomers as inhibitors of [3H]aspartate uptake in rat brain synaptosomes and hEAAT1-3-expressing cells. Comparable IC(50) values were found on the three hEAAT subtypes. (-)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]L-glutamate uptake. New data suggest that the noncompetitive-like interaction described previously is probably the consequence of an insurmountable, long-lasting impairment of EAAT's function. Some minutes of preincubation are required to induce this impairment, the duration of preincubation having more effect on inhibition of glutamate-induced release than of glutamate uptake. In organotypic rat hippocampal slices and mixed mouse brain cortical cultures, TBOA, but not (-)-HIP-A, had toxic effects. Under ischemic conditions, a neuroprotective effect was found with 10 to 30 microM (-)-HIP-A, but not with 10 to 30 microM TBOA or 100 microM (-)-HIP-A. The effect of (-)-HIP-A suggests that, under ischemia, EAATs mediate both release (reverse transport) and uptake of glutamate. The neuroprotection with the lower (-)-HIP-A concentrations may indicate a selective inhibition of the reverse transport confirming the data obtained in synaptosomes. The selective interference with glutamate-induced glutamate release might offer a new strategy for neuroprotective action.

Recent experimental evidence indicates that erythropoietin (Epo), in addition to its hormonal role in regulating red cell production, operates as a neuroprotective agent. So far, the neuroprotective effect of human recombinant Epo (rhEpo) has been mainly demonstrated in models of cerebral ischemia/hypoxia and in selected in vivo studies of traumatic neuronal injury. To further investigate the potential role of this multifunctional trophic factor in post-traumatic cell death, we examined the protective effects of rhEpo in a newly developed model of mechanical trauma in organotypic hippocampal slices. Organotypic rat hippocampal slices were subjected to traumatic injury by allowing a stylus to impact on the CA1 area with an energy of 6 μJ. Hippocampal damage was identified and measured 24 and 48 h later with the fluorescent dye propidium iodide (PI). In untreated slices, the impact induced a significant increase in the mean hippocampal PI fluorescence, co-localized with the area of impact at 24 h (primary post-traumatic injury) and progressively spread to the whole slice between 24 and 48 h (secondary post-traumatic injury). Addition of rhEpo (1–100 UI/mL) or of the NMDA antagonist MK-801 (30 μM) immediately after the traumatic injury reduced hippocampal damage by approximately 30% when observed 24 h later. At 48 h after trauma, the protective effect of rhEpo was greater (by about 47%) and significantly more pronounced than that of MK-801 (28%). Our results suggest that the neuroprotective activity of rhEpo is particularly effective against delayed, secondary post-traumatic damage. This well tolerated agent could provide a therapeutic benefit in pathologies involving post-traumatic neurodegeneration.

Excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme catalyzing the transfer of ADP-ribose units from NAD to acceptor proteins, induces cellular energy failure by NAD and ATP depletion and has been proposed to play a causative role in a number of pathological conditions, including ischemia/reperfusion injury. In this study, we used an in vitro enzyme activity assay to characterize a series of newly synthesized isoquinolinone derivatives as potential PARP-1 inhibitors. Several compounds displayed powerful inhibitory activity: thieno[2,3-c]isoquinolin-5-one (TIQ-A) displayed a submicromolar IC50of 0.45 ± 0.1 μM, whereas the 5-hydroxy and 5-methoxy TIQ-A derivatives had IC50values of 0.39 ± 0.19 and 0.21 ± 0.12 μM, respectively. We then examined the neuroprotective effects of the newly characterized compounds in cultured mouse cortical cells exposed to 60 min of oxygen and glucose deprivation (OGD). When PARP-1 inhibitors were present in the incubation medium during OGD and the subsequent 24-h recovery period, they significantly attenuated neuronal injury. TIQ-A provided neuroprotection even when added to the culture 30 min after OGD and was able to reduce the early activation of PARP induced by OGD as detected by flow cytometry. When the IC50values observed in the PARP-1 activity assay for selected compounds were compared with their IC50values for the neuroprotective activity, a significant correlation (r= 0.93, P< 0.01) was observed. Our results suggest that TIQ-A and its derivatives are a new class of neuroprotectants that may be helpful in studies aimed at understanding the involvement of PARP-1 in physiology and pathology.

Metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) appear to be distinct from any known mGlu receptor subtype linked to phospholipase C or adenylyl cyclase. The availability of antagonists is necessary for understanding the role of these receptors in the central nervous system, but selective ligands have not yet been identified. In a previous report, we observed that 3,5-dihydroxyphenylglycine (3,5-DHPG) inhibits the PLD response induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate in adult rat hippocampal slices. We now show that the antagonist action of 3,5-DHPG (IC50= 70 μM) was noncompetitive in nature and nonselective, because the drug was also able to reduce PLD activation elicited by 100 μM norepinephrine and 1 mM histamine. In the search for a selective and more potent antagonist, we examined the effects of sixteen stereoisomers of 2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCG) on the PLD-specific transphosphatidylation reaction resulting in the formation of [3H]phosphatidylethanol. The (2R,1′S,2′R,3′S)-PCCG stereoisomer (PCCG-13) antagonized the formation of [3H]phosphatidylethanol induced by 100 μM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate in a dose-dependent manner and with a much lower IC50value (25 nM) compared with 3,5-DHPG. In addition, increasing concentrations of PCCG-13 were able to shift to the right the agonist dose-response curve but had no effect when tested on other receptors coupled to PLD. The potent, selective, and competitive antagonist PCCG-13 may represent an important tool for elucidating the role of PLD-coupled mGlu receptors in adult hippocampus.

We examined the pharmacological profile of 1-aminoindan-1,5-dicarboxylic acid (AIDA), a rigid (carboxyphenyl)glycine derivative acting on metabotropic glutamate receptors (mGluRs). In cells transfected with mGluR1a, AIDA competitively antagonized the stimulatory responses of glutamate and (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S,3 R)-ACPD] on phosphoinositide hydrolysis (p A2= 4.21). In cells transfected with mGluR5a, AIDA displayed a much weaker antagonist effect. In transfected cells expressing mGluR2, AIDA (≤1 mM) did not affect the inhibition of forskolin-stimulated adenylate cyclase activity induced by (1 S,3 R)-ACPD, but at large concentrations, it displayed a modest agonist activity. In rat hippocampal or striatal slices, AIDA (0.1–1 mM) reduced the effects of (1 S,3 R)-ACPD on phospholipase C but not on adenylate cyclase responses, whereas (+)-α-methyl-4-carboxyphenylglycine (0.3–1 mM) was an antagonist on both transduction systems. In addition, AIDA (0.3–1 mM) had no effect on mGluRs coupled to phospholipase D, whereas (+)-α-methyl-4-carboxyphenylglycine (0.5–1 mM) acted as an agonist with low intrinsic activity. In rat cortical slices, AIDA antagonized the stimulatory (mGluR1-mediated) effect of (1 S,3 R)-ACPD on the depolarization-induced outflow ofd-[3H]aspartate, disclosing an inhibitory effect ascribable to (1 S,3 R)-ACPD activating mGluR2 and/or mGluR4. Finally, mice treated with AIDA (0.1–10 nmol i.c.v.) had an increased pain threshold and difficulties in initiating a normal ambulatory behavior. Taken together, these data suggest that AIDA is a potent, selective and competitive mGluR1a antagonist.