Elisa Landucci

Neuroprotection by group I mGlu receptors in a rat hippocampal slice model of cerebral ischemia is associated with the PI3K-Akt signaling pathway: a novel postconditioning strategy?

Ischemic postconditioning is defined as a repetitive series of brief interruptions of reperfusion applied immediately after ischemia. In this study, postconditioning was investigated by first exposing rat organotypic hippocampal slices to 30min oxygen-glucose deprivation (OGD), which promotes selective CA1 pyramidal cell death, and 5min later to either a brief period (3min) of OGD or to a low dose (10microM) of 3,5-dihydroxyphenylglycine (DHPG) for 30min. Both protocols attenuated CA1 neuronal injury, as revealed 24h later by measuring the intensity of propidium iodide fluorescence in this region. The beneficial effects were observed when DHPG postconditioning was applied up to 15min after OGD, but not at later time points, and was not additive with the neuroprotective effects of a preconditioning DHPG treatment. The attenuation of the OGD-induced CA1 injury evoked by postconditioning was prevented when mGlu1 and mGlu5 receptor antagonists and inhibitors of phosphatidylinositol 3-kinase and Akt activity were present in the incubation medium during the 5min recovery period after OGD and the 30min exposure to DHPG. The PI3K inhibitor was also able to prevent the reduction of NMDA toxicity induced by the DHPG treatment. Finally, DHPG increased the phosphorylation of Akt in a transient and mGlu1/mGlu5-dependent manner. Our results show that activation of the mGlu1/mGlu5-PI3K-Akt signaling pathway plays a crucial role in the mechanisms of postconditioning evoked by DHPG and point to this strategy as a possible novel therapeutic tool for stroke and cerebral ischemia.

Pharmacological effects of 3‐iodothyronamine (T1AM) in mice include facilitation of memory acquisition and retention and reduction of pain threshold

3‐Iodothyronamine (T1AM), an endogenous derivative of thyroid hormones, is regarded as a rapid modulator of behaviour and metabolism. To determine whether brain thyroid hormone levels contribute to these effects, we investigated the effect of central administration of T1AM on learning and pain threshold of mice either untreated or pretreated with clorgyline (2.5 mg·kg−1, i.p.), an inhibitor of amine oxidative metabolism.

Differential role of mGlu1 and mGlu5 receptors in rat hippocampal slice models of ischemic tolerance

Activation of glutamate receptors has been proposed as a key factor in the induction of ischemic tolerance. We used organotypic rat hippocampal slices exposed to 30 min oxygen–glucose deprivation (OGD) to evaluate postischemic pyramidal cell death in the CA1 subregion. In this model, 10 min exposure to OGD 24 h before the exposure to toxic OGD was not lethal and reduced the subsequent OGD neurotoxicity by ∼ 53% (ischemic preconditioning). Similarly, a 30 min exposure to the group I mGlu receptor agonist DHPG (10 µM) significantly reduced OGD neurotoxicity 24 h later (pharmacological preconditioning). Ischemic tolerance did not develop when either the selective mGlu1 antagonists LY367385 and 3‐MATIDA or the AMPA/KA antagonist CNQX were present in the incubation medium during exposure to sublethal OGD. Neither the NMDA antagonist MK801 nor the mGlu5 antagonist MPEP affected the preconditioning process. On the other hand, pharmacological preconditioning was prevented not only by LY367385 or CNQX, but also by MPEP. In preconditioned slices, the toxic responses to AMPA or NMDA were reduced. The neurotoxicty of 100 µM DHPG in slices simultaneously exposed to a mild (20 min) OGD was differentially altered in the two preconditioning paradigms. After ischemic preconditioning, DHPG neurotoxicity was reduced in a manner that was sensitive to LY367385 but not to MPEP, whereas after pharmacological preconditioning it was enhanced in a manner that was sensitive to MPEP but not to LY367385. Our results show that mGlu1 and mGlu5 receptors are differentially involved in the induction and expression of ischemic tolerance following two diverse preconditioning stimuli.

Histamine mediates behavioural and metabolic effects of 3-iodothyroacetic acid, an endogenous end product of thyroid hormone metabolism

3‐Iodothyroacetic acid (TA1) is an end product of thyroid hormone metabolism. So far, it is not known if TA1 is present in mouse brain and if it has any pharmacological effects.

In the brain of mice, 3-iodothyronamine (T1AM) is converted into 3-iodothyroacetic acid (TA1) and it is included within the signaling network connecting thyroid hormone metabolites with histamine.

3-iodothyronamine (T1AM) and its oxidative product, 3-iodotyhyroacetic acid (TTA1A), are known to stimulate learning and induce hyperalgesia in mice. We investigated whether i)TA1 may be generated in vivo from T1AM, ii) T1AM shares with TA1 the ability to activate the histaminergic system. Tandem mass spectrometry was used to measure TA1 and T1AM levels in i) the brain of mice following intracerebroventricular (i.c.v.) injection of T1AM (11μgkg(-1)), with or without pretreatment with clorgyline, (2.5mgkg(-1) i.p.), a monoamine oxidase inhibitor; ii) the medium of organotypic hippocampal slices exposed to T1AM (50nM). In addition, learning and pain threshold were evaluated by the light-dark box task and the hot plate test, respectively, in mice pre-treated subcutaneously with pyrilamine (10mgkg(-1)) or zolantidine (5mgkg(-1)), 20min before i.c.v. injection of T1AM (1.32 and 11μgkg(-1)). T1AM-induced hyperalgesia (1.32 and 11μgkg(-1)) was also evaluated in histidine decarboxylase (HDC(-/-)) mice. T1AM and TA1 brain levels increased in parallel in mice injected with T1AM with the TA1/T1AM averaging 1.7%. Clorgyline pre-treatment reduced the increase in both T1AM and TA1. TA1 was the main T1AM metabolite detected in the hippocampal preparations. Pretreatment with pyrilamine or zolantidine prevented the pro-learning effect of 1.32 and 4μgkg(-1) T1AM while hyperalgesia was conserved at the dose of 11μgkg(-1) T1AM. T1AM failed to induce hyperalgesia in HDC(-/-) mice at all the doses. In conclusion, TA1 generated from T1AM, but also T1AM, appears to act by modulating the histaminergic system.

Neuroprotective Effects of the Novel Glutamate Transporter Inhibitor (–)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic Acid, Which Preferentially Inhibits Reverse Transport (Glutamate Release) Compared with Glutamate Reuptake

(±)-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-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 IC50 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 EAATs 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.

Mild activation of poly(ADP‐ribose) polymerase (PARP) is neuroprotective in rat hippocampal slice models of ischemic tolerance

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP‐ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen‐glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N‐methyl‐d‐aspartate (NMDA) or (S)‐3,5‐dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD‐induced CA1 damage. Exposure of slices to the PARP‐1 and ‐2 inhibitors TIQ‐A, PJ‐34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration‐dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP‐ribose) polymer product, but was not associated with consumption of cellular NAD+ or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z‐VAD‐FMK. The modest but significant increase in caspase‐3/7 induced by preconditioning, however, was not associated with PARP‐1 cleavage, as occurred with staurosporine. Finally, TIQ‐A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase‐3/7.

MPP+‐dependent inhibition of Ih reduces spontaneous activity and enhances EPSP summation in nigral dopamine neurons

1‐Methyl‐4‐phenylpyridinium (MPP+), a potent parkinsonizing agent in primates and rodents, is a blocker of mitochondrial complex I, therefore MPP+‐induced parkinsonism is believed to depend largely on mitochondrial impairment. However, it has recently been proposed that other mechanisms may participate in MPP+‐induced toxicity. We tackled this issue by probing the effects of an acute application of MPP+ on substantia nigra pars compacta (SNc) dopamine (DA) neurons.

Novel 3-Carboxy- and 3-Phosphonopyrazoline Amino Acids as Potent and Selective NMDA Receptor Antagonists: Design, Synthesis, and Pharmacological Characterization

The design and synthesis of new N1‐substituted 3‐carboxy‐ and 3‐phosphonopyrazoline and pyrazole amino acids that target the glutamate binding site of NMDA receptors are described. An analysis of the stereochemical requirements for high‐affinity interaction with these receptors was performed. We identified two highly potent and selective competitive NMDA receptor antagonists, (5S,αR)‐1 and (5S,αR)‐4, which exhibit good in vitro neuroprotective activity and in vivo anticonvulsant activity by i.p. administration, suggesting that these molecules may have potential use as therapeutic agents.

Rat Hippocampal Slice Culture Models for the Evaluation of Neuroprotective Agents

Organotypic slices cultured for weeks in vitro represent an extremely valuable strategy for the investigation of the long-term properties of neuronal circuits under physiological and pathological conditions. Here, we describe how to prepare rat organotypic hippocampal slice cultures and how to expose them for appropriate periods of time to excitotoxic agents or to oxygen and glucose deprivation conditions, in order to mimic the pattern of pyramidal cell damage which is observed in vivo and in other in vitro models. This preparation is very useful not only to study synaptic plasticity or the pathways and mechanisms of neurodegeneration but also to evaluate the effects of neuroprotective agents.