Ezia Guatteo

Resistance to NMDA toxicity correlates with appearance of nuclear inclusions, behavioural deficits and changes in calcium homeostasis in mice transgenic for exon 1 of the huntington gene

Transgenic Huntingtons disease (HD) mice, expressing exon 1 of the human HD gene (lines R6/1 and R6/2), are totally resistant to striatal lesions caused by the NMDA receptor agonist quinolinic acid (QA). Here we show that this resistance develops gradually over time in both R6/1 and R6/2 mice, and that it occurred earlier in R6/2 (CAG‐155) than in R6/1 (CAG‐115) mice. The development of the resistance coincided with the appearance of nuclear inclusions and with the onset of motor deficits. In the HD mice, hippocampal neurons were also resistant to QA, especially in the CA1 region. Importantly, there was no change in susceptibility to QA in transgenic mice with a normal CAG repeat (CAG‐18). R6/1 mice were also resistant to NMDA‐, but not to AMPA‐induced striatal damage. Interestingly, QA‐induced current and calcium influx in striatal R6/2 neurons were not decreased. However, R6/2 neurons had a better capacity to handle cytoplasmic calcium ([Ca2+]c) overload following QA and could avoid [Ca2+]c deregulation and cell lysis. In addition, basal [Ca2+]c levels were increased five‐fold in striatal R6/2 neurons. This might cause an adaptation of R6 neurons to excitotoxic stress resulting in an up‐regulation of defense mechanisms, including an increased capacity to handle [Ca2+]c overload. However, the increased level of basal [Ca2+]c in the HD mice might also disturb intracellular signalling in striatal neurons and thereby cause neuronal dysfunction and behavioural deficits.

Acute action of rotenone on nigral dopaminergic neurons – involvement of reactive oxygen species and disruption of Ca2+ homeostasis

Rotenone is a toxin used to generate animal models of Parkinson’s disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05–1 μm) effects on SNc neurons in acute rat midbrain slices, using whole‐cell patch‐clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide‐sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca2+] ([Ca2+]i) (73.8 ± 7.7 nm) and intracellular [Na+] (3.1 ± 0.6 mm) (all with 1 μm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca2+]i rise was abolished by removing extracellular Ca2+, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N‐(p‐amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine‐123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca2+]i rise resulted in a large (46.6 ± 25.3 nm) Ca2+ response when baseline [Ca2+]i was increased by a ‘priming’ protocol that activated voltage‐gated Ca2+ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca2+]i and the rotenone‐induced [Ca2+]i rise. This correlation was not seen in non‐dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP‐gated K+ channels and TRPM2‐like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone‐induced [Ca2+]i rise by a small increase in baseline [Ca2+]i.

Metabotropic Glutamate Receptor 1 Mediates the Electrophysiological and Toxic Actions of the Cycad Derivative β-N-Methylamino-l-Alanine on Substantia Nigra Pars Compacta DAergic Neurons

Amyotrophic lateral sclerosis–Parkinson dementia complex (ALS-PDC) is a neurodegenerative disease with ALS, parkinsonism, and Alzheimers symptoms that is prevalent in the Guam population. β-N-Methylamino alanine (BMAA) has been proposed as the toxic agent damaging several neuronal types in ALS-PDC, including substantia nigra pars compacta dopaminergic (SNpc DAergic) neurons. BMAA is a mixed glutamate receptor agonist, but the specific pathways activated in DAergic neurons are not yet known. We combined electrophysiology, microfluorometry, and confocal microscopy analysis to monitor membrane potential/current, cytosolic calcium concentration ([Ca2+]i) changes, cytochrome-c (cyt-c) immunoreactivity, and reactive oxygen species (ROS) production induced by BMAA. Rapid toxin applications caused reversible membrane depolarization/inward current and increase of firing rate and [Ca2+]i in DAergic neurons. The inward current (IBMAA) was mainly mediated by activation of metabotropic glutamate receptor 1 (mGluR1), coupled to transient receptor potential (TRP) channels, and to a lesser extent, AMPA receptors. Indeed, mGluR1 (CPCCOEt) and TRP channels (SKF 96365; Ruthenium Red) antagonists reduced IBMAA, and a small component of IBMAA was reduced by the AMPA receptor antagonist CNQX. Calcium accumulation was mediated by mGluR1 but not by AMPA receptors. Application of a low concentration of NMDA potentiated the BMAA-mediated calcium increase. Prolonged exposure to BMAA caused significant modifications of membrane properties, calcium overload, cell shrinkage, massive cyt-c release into the cytosol and ROS production. In SNpc GABAergic neurons, BMAA activated only AMPA receptors. Our study identifies the mGluR1-activated mechanism induced by BMAA that may cause the neuronal degeneration and parkinsonian symptoms seen in ALS-PDC. Moreover, environmental exposure to BMAA might possibly also contribute to idiopathic PD.

Neuroprotective effect of hydrogen peroxide on an in vitro model of brain ischaemia

Reactive oxygen species (ROS) have been postulated to play a crucial role in the pathogenesis of ischaemia‐reperfusion injury. Among these, hydrogen peroxide (H2O2) is known to be a toxic compound responsible for free‐radical‐dependent neuronal damage. In recent years, however, the ‘bad reputation’ of H2O2 and other ROS molecules has changed. The aim of this study was to assess the protective role of H2O2 and modification in its endogenous production on the electrophysiological and morphological changes induced by oxygen/glucose deprivation (OGD) on CA1 hippocampal neurons.

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta.

We investigated the effect of changes in membrane‐voltage on intracellular sodium concentration ([Na+]i) of dopamine‐sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole‐cell patch‐clamp techniques were combined with microfluorometric measurements of [Na+]i using the Na+‐sensitive probe, sodium‐binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously

Protective role of hydrogen peroxide in oxygen‐deprived dopaminergic neurones of the rat substantia nigra

Hydrogen peroxide (H2O2) is a reactive oxygen species, responsible for cytotoxic damage through the formation of hydroxyl radicals. Dopamine (DA) neurones of the substantia nigra pars compacta (SNc) are highly sensitive to metabolic stress, and they typically respond to energy deprivation with membrane hyperpolarization, mainly through opening of ATP‐dependent K+ channels. Accordingly, H2O2 (3 mm) induced a tolbutamide‐sensitive outward current in DA neurones. Conversely, in a hypoxic medium, H2O2 reverted membrane hyperpolarization, which is associated with oxygen deprivation in DA neurones, restored their action potential firing, and reduced the hypoxia‐mediated outward current in a concentration‐dependent manner, between 0.1 and 3 mm (IC50 0.6 ± 0.1 mm). Notably, H2O2 did not counteract membrane hyperpolarization associated with hypoglycaemia, moreover, when catalase was inhibited with 3‐amino‐1,2,4‐triazole (3‐AT; 30 mm), H2O2 did not reduce hypoxia‐mediated outward current. The counteracting action of H2O2 on hypoxia‐mediated effects was further confirmed by single‐unit extracellular recordings of presumed DA neurones in acute midbrain slices preparations, using a planar multi‐electrode array device. Whilst a prolonged period of hypoxia (40 min) caused firing suppression, which did not recover after perfusion in normoxic conditions, the presence of H2O2 (3 mm) during this prolonged hypoxic period rescued most of the neurones from irreversible firing inhibition. Accordingly, morphological studies showed that H2O2 counteracts the cytochrome c release provoked by prolonged hypoxic treatment. Taken together, our data suggest that H2O2 prevents the metabolic stress of DA neurones induced by hypoxia by serving as a supplementary source of molecular oxygen, through its degradation by catalase.

Memantine Inhibits ATP-Dependent K+ Conductances in Dopamine Neurons of the Rat Substantia Nigra Pars Compacta

1-Amino-3,5-dimethyl-adamantane (memantine) is a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist used in clinical practice to treat neurodegenerative disorders that could be associated with excitotoxic cell death. Because memantine reduces the loss of dopamine neurons of the substantia nigra pars compacta (SNc) in animal models of Parkinsons disease, we examined the effects of this drug on dopamine cells of the SNc. Besides inhibition of NMDA receptor-mediated currents, memantine (30 and 100 μM) increased the spontaneous firing rate of whole-cell recorded dopamine neurons in a midbrain slice preparation. Occasionally, a bursting activity was observed. These effects were independent from the block of NMDA receptors and were prevented in neurons dialyzed with a high concentration of ATP (10 mM). An increase in firing rate was also induced by the ATP-sensitive potassium (KATP) channel antagonist tolbutamide (300 μM), and this increase occluded further effects of memantine. In addition, KATP channel-mediated outward currents, induced by hypoxia, were inhibited by memantine (30 and 100 μM) in the presence of the NMDA receptor antagonist (5S, 10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (10 μM). An increase in the spontaneous firing rate by memantine was observed in dopamine neurons recorded with extracellular planar 8 × 8 multielectrodes in conditions of hypoglycemia. These results highlight KATP channels as possible relevant targets of memantine effects in the brain. Moreover, in view of a proposed role of KATP conductances in dopamine neuron degeneration, they suggest another mechanism of action underlying the protective role of memantine in Parkinsons disease.

An electrophysiological analysis of the protective effects of felbamate, lamotrigine, and lidocaine on the functional recovery from in vitro ischemia in rat neocortical slices

We used field potential recording techniques to examine whether felbamate (FBM), lamotrigine (LTG), and lidocaine (LID) protect against the irreversible functional damage induced by transient ischemia. Five minutes of ischemia caused a depression of the field potential in rat cortical slices, which did not recover even after more than 1 h of washout. The N‐methyl‐D‐aspartate (NMDA) antagonist ketamine (50 μM) protected against depression of the field caused by ischemia. On the other hand, the non‐NMDA antagonist 6‐cyano‐7‐nitroquinoxaline‐2.3‐dione (CNQX) (10 μM) had protective effects only if co‐applied with ketamine. We found that either FBM (30–300 μM), which did not modify the amplitude of the field EPSP, or LTG (10–300 μM), which reversibly depressed the excitatory synaptic transmission, had a marked protective effect when superfused before and during the ischemic insult. After FBM (100 μM) and LTG (100 μM), the field EPSP recovered by 84 ± 1% and 73 ± 2.7% of control, respectively. Furthermore, LID (30–300 μM) was less effective than FBM and LTG in inducing a functional recovery from the damage caused by ischemia (58 ± 1.8%). The rank order of potency, based on the maximal protection caused by the three drugs, was FBM > LTG > LID. Our results suggest that a noticeable neuroprotection can be obtained during glucose and O2 deprivation by preventive therapeutic regimens which use the two recently marketed anticonvulsant drugs, FBM and LTG. Synapse 30:371–379, 1998.

Over-expression of N-type calcium channels in cortical neurons from a mouse model of Amyotrophic Lateral Sclerosis.

Voltage-gated Ca(2+) channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca(2+) channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca(2+) channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K(+) concentration (25 mM, 5h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 μM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium. These data provide robust evidence for an excess of N-type Ca(2+) expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.

Neuregulin 1 signalling modulates mGluR1 function in mesencephalic dopaminergic neurons.

Neuregulin 1 (NRG1) is a trophic factor that has an essential role in the nervous system by modulating neurodevelopment, neurotransmission and synaptic plasticity. Despite the evidence that NRG1 and its receptors, ErbB tyrosine kinases, are expressed in mesencephalic dopaminergic nuclei and their functional alterations are reported in schizophrenia and Parkinson’s disease, the role of NRG1/ErbB signalling in dopaminergic neurons remains unclear. Here we found that NRG1 selectively increases the metabotropic glutamate receptor 1 (mGluR1)-activated currents by inducing synthesis and trafficking to membrane of functional receptors and stimulates phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway, which is required for mGluR1 function. Notably, an endogenous NRG1/ErbB tone is necessary to maintain mGluR1 function, by preserving its surface membrane expression in dopaminergic neurons. Consequently, it enables striatal mGluR1-induced dopamine outflow in in vivo conditions. Our results identify a novel role of NRG1 in the dopaminergic neurons, whose functional alteration might contribute to devastating diseases, such as schizophrenia and Parkinson’s disease.