Sabrina Siliquini

Decreased NR2B Subunit Synaptic Levels Cause Impaired Long-Term Potentiation But Not Long-Term Depression

The discovery of the molecular mechanisms regulating the abundance of synaptic NMDA receptors is essential for understanding how synaptic plasticity, as well as excitotoxic events, are regulated. However, a complete understanding of the precise molecular mechanisms regulating the composition of the NMDA receptor complex at hippocampal synapse is still missing. Here, we show that 2 h of CaMKII inhibition leads to a specific reduction of synaptic NR2B-containing NMDA receptors without affecting localization of the NR2A subunit; this molecular event is accompanied by a dramatic reduction in the induction of long-term potentiation (LTP), while long-term depression induction is unaffected. The same molecular and functional results were obtained by disrupting NR2B/PSD-95 complex with NR2B C-tail cell permeable peptide (TAT-2B). These data indicate that NR2B redistribution between synaptic and extrasynaptic membranes represents an important molecular disturbance of the glutamatergic synapse and affects the correct induction of LTP.

Mechanisms underlying the impairment of hippocampal long-term potentiation and memory in experimental Parkinson's disease.

Although patients with Parkinsons disease show impairments in cognitive performance even at the early stage of the disease, the synaptic mechanisms underlying cognitive impairment in this pathology are unknown. Hippocampal long-term potentiation represents the major experimental model for the synaptic changes underlying learning and memory and is controlled by endogenous dopamine. We found that hippocampal long-term potentiation is altered in both a neurotoxic and transgenic model of Parkinsons disease and this plastic alteration is associated with an impaired dopaminergic transmission and a decrease of NR2A/NR2B subunit ratio in synaptic N-methyl-d-aspartic acid receptors. Deficits in hippocampal-dependent learning were also found in hemiparkinsonian and mutant animals. Interestingly, the dopamine precursor l-DOPA was able to restore hippocampal synaptic potentiation via D1/D5 receptors and to ameliorate the cognitive deficit in parkinsonian animals suggesting that dopamine-dependent impairment of hippocampal long-term potentiation may contribute to cognitive deficits in patients with Parkinsons disease.

Effects of central and peripheral inflammation on hippocampal synaptic plasticity.

The central nervous system (CNS) and the immune system are known to be engaged in an intense bidirectional crosstalk. In particular, the immune system has the potential to influence the induction of brain plastic phenomena and neuronal networks functioning. During direct CNS inflammation, as well as during systemic, peripheral, inflammation, the modulation exerted by neuroinflammatory mediators on synaptic plasticity might negatively influence brain neuronal networks functioning. The aim of the present study was to investigate, by using electrophysiological techniques, the ability of hippocampal excitatory synapses to undergo synaptic plasticity during the initial clinical phase of an experimental model of CNS (experimental autoimmune encephalomyelitis, EAE) as well as following a systemic inflammatory trigger. Moreover, we compared the morphologic, synaptic and molecular consequences of central neuroinflammation with those accompanying peripheral inflammation. Hippocampal long-term potentiation (LTP) has been studied by extracellular field potential recordings in the CA1 region. Immunohistochemistry was performed to investigate microglia activation. Western blot and ELISA assays have been performed to assess changes in the subunit composition of the synaptic glutamate NMDA receptor and the concentration of pro-inflammatory cytokines in the hippocampus. Significant microglial activation together with an impairment of CA1 LTP was present in the hippocampus of mice with central as well as peripheral inflammation. Interestingly, exclusively during EAE but not during systemic inflammation, the impairment of hippocampal LTP was paralleled by a selective reduction of the NMDA receptor NR2B subunit levels and a selective increase of interleukin-1β (IL1β) levels. Both central and peripheral inflammation-triggered mechanisms can activate CNS microglia and influence the function of CNS synapses. During direct CNS inflammation these events are accompanied by detectable changes in synaptic glutamate receptors subunit composition and in the levels of the pro-inflammatory cytokine IL1β.

Hippocampal synaptic plasticity, memory, and epilepsy: effects of long-term valproic acid treatment.

BACKGROUND Memory impairment is commonly associated with epilepsy, and the use of antiepileptic drugs (AEDs) causes additional neuropsychologic deficits that are of particular concern in learning-age children and elderly patients. The aim of this study was to investigate hippocampal synaptic plasticity and morphology as well as hippocampal-dependent memory in physiologic conditions and in a genetic model of epilepsy following chronic treatment with the widely used AED valproic acid (VPA). METHODS Mice lacking the presynaptic scaffolding protein Bassoon were used as a model of epilepsy. Electrophysiologic recordings were used to analyze basal glutamatergic synaptic transmission, paired-pulse facilitation, and activity-dependent long-term potentiation (LTP) in the CA1 area. Dendritic morphology and spine density were analyzed, and glutamate-related signaling was investigated by Western blot analysis. Social transmission of food preference test was used to investigate nonspatial hippocampal memory. RESULTS VPA treatment significantly reduced seizures frequency and mortality in epileptic mice. Long-term potentiation was absent at CA1 synapses of untreated epileptic mutant mice that also showed significant dendritic abnormalities. Treatment with VPA rescued physiologic LTP but did not reverse morphological abnormalities and deficits in nonspatial hippocampal memory observed in mutant epileptic mice. Moreover, VPA was found to induce per se dendritic abnormalities and memory dysfunction in normal animals. CONCLUSIONS The impairment of hippocampal synaptic plasticity in epileptic mice, rescued by VPA treatment, might represent the mechanism underlying epilepsy-induced memory deficits. Moreover, the demonstration that VPA induces morphologic alterations and impairment in specific hippocampal-dependent memory task might explain the detrimental effects of antiepileptic treatment on cognition in human subjects.

Electrophysiology and Pharmacology of Striatal Neuronal Dysfunction Induced by Mitochondrial Complex I Inhibition

Reduced activity of the mitochondrial respiratory chain and in particular of complex I is implicated not only in the etiology of Parkinsons disease but also in other forms of parkinsonism in which striatal neurodegeneration occurs, such as progressive supranuclear palsy. The pesticide rotenone inhibits mitochondrial complex I and reproduces features of these basal ganglia neurological disorders in animal models. We have characterized the electrophysiological effects of rotenone in the striatum as well as potential neuroprotective strategies to counteract the detrimental effects of this neurotoxin. We found that rotenone causes a dose-dependent and irreversible loss of the corticostriatal field potential amplitude, which was related to the development of a membrane depolarization/inward current in striatal spiny neurons, coupled to an increased release of both excitatory amino acids and dopamine (DA). In particular, we have investigated whether glutamate, DA, and GABA systems might represent possible targets for neuroprotection against rotenone-induced striatal neuronal dysfunction. Interestingly, whereas modulation of glutamatergic transmission was not neuroprotective, blockade of D2-like but not D1-like DA receptors significantly reduced the rotenone-induced effects via a GABA-mediated mechanism. In addition, because antiepileptic drugs (AEDs) modulate multiple transmitter systems, we have analyzed the possible neuroprotective effects of some AEDs against rotenone. We found that carbamazepine, unlike other tested AEDs, exerts a potent neuroprotective action against rotenone-induced striatal neuronal dysfunction. This neuroprotection was observed at therapeutically relevant concentrations requiring endogenous GABA. Differential targeting of GABAergic transmission may represent a possible therapeutic strategy against basal ganglia neurodegenerative disorders involving mitochondrial complex I dysfunction.

Memantine reduces neuronal dysfunctions triggered by in vitro ischemia and 3-nitropropionic acid

Memantine, a low-affinity uncompetitive NMDA receptor antagonist, has been widely utilized for the treatment of Alzheimers disease. A possible neuroprotective role of this drug in pathophysiological conditions involving an altered energetic metabolism of the basal ganglia has never been addressed. Thus, we have characterized the electrophysiological effect of memantine on striatal spiny neurons recorded under control conditions and after in vitro ischemia (oxygen and glucose deprivation). Memantine reduced in a dose-dependent manner (EC(50)=5 microM) the irreversible loss of field potential amplitude induced by in vitro ischemia. The neuroprotective effect of memantine against in vitro ischemia was even more potent (EC(50)=3.2 microM) in the absence of external magnesium, a condition enhancing NMDA-mediated glutamatergic transmission. Memantine was also able to block long-term potentiation recorded from spiny neurons following a brief ischemic episode. Moreover, memantine showed protection against irreversible field potential loss induced by 3-nitropropionic acid (3-NP), an inhibitor of the mitochondrial complex II, without influencing toxicity induced by rotenone, a complex I inhibitor. Memantine could represent a potential neuroprotective agent in pathophysiological conditions involving an altered energy metabolism of basal ganglia.

Mechanisms underlying altered striatal synaptic plasticity in old A53T-α synuclein overexpressing mice

The interactions between certain α-synuclein (SNCA) conformations and dopamine (DA) metabolism cause selective DA neuron degeneration in Parkinsons disease (PD). Preclinical research on PD took advantage of increasing studies involving different animal models which express different forms of mutated SNCA. Transgenic animals expressing mutant α-synucleins such as mice transgenic for A53T-SNCA (TG) are considered valuable models to assess specific aspects of the pathogenesis of synucleinopathies and PD. In this study we performed electrophysiological recordings in corticostriatal slice preparations from young TG overexpressing mice, in which extracellular striatal DA levels appeared to be normal, and in old TG mice, characterized by abnormalities in striatal DA signaling and impaired long-term depression (LTD). We report no difference in TG mice from the two groups of age of either the basal membrane properties and synaptic striatal excitability in respect to age-matched wild-type mice. Furthermore, in old TG mice, showing plastic abnormalities and motor symptoms, we investigated the mechanisms at the basis of the altered LTD. In old TG mice LTD could not be restored by treatments with acute application of DA or by subchronic treatment with L-3,4-dihydroxyphenylalanine (L-DOPA). Conversely, the application of the phosphodiesterase inhibitor zaprinast fully restored LTD to normal conditions via the stimulation of a cyclic guanosine monophosphate (GMP)-protein kinase G-dependent intracellular signaling pathway. These results suggest that, in addition to the dopaminergic alterations reported in this genetic model of PD, other signal transduction pathways linked to striatal synaptic plasticity are altered in an age-dependent manner.

Electrophysiological actions of zonisamide on striatal neurons: Selective neuroprotection against complex I mitochondrial dysfunction.

Since the anti-epileptic drug Zonisamide (ZNS) seems to exert beneficial effects in Parkinsons (PD) disease, we have investigated the electrophysiological effects of ZNS in a rat corticostriatal slice preparation. ZNS affected neither the resting membrane potential nor the input resistance of the putative striatal spiny neurons. In contrast, this drug depressed in a dose-dependent manner the current-evoked repetitive firing discharge with a EC(50) value of 16.38 microM. ZNS also reduced the amplitude of glutamatergic excitatory postsynaptic potentials (EPSPs) with a EC(50) value of 32.5 microM. Reduced activity of the mitochondrial respiratory chain, particularly complex I and II, is implicated in the pathophysiology of PD and Huntingtons (HD) diseases, respectively. Thus, ZNS was also tested in two different in vitro neurotoxic models obtained by acutely exposing corticostriatal slices either to rotenone, a selective inhibitor of mitochondrial complex I, or to 3-nitropropionic acid (3-NP), an inhibitor of complex II. Additionally, we also investigated the effect of ZNS in an in vitro model of brain ischemia. Interestingly, low concentrations of ZNS (0.3, 1, 3 and 10 microM) significantly reduced the rotenone-induced toxicity protecting striatal slices from the irreversible loss of corticostriatal field potential (FP) amplitude via a GABA-mediated mechanism. Conversely, this drug showed no protection against 3-NP and ischemia-induced toxicity. Our data indicate that relatively high doses of ZNS are required to decrease striatal neuronal excitability while low concentrations of this drug are sufficient to protect striatum against mitochondrial impairment suggesting its possible use in the therapy of basal ganglia neurodegenerative diseases.

A critical role of NO/cGMP/PKG dependent pathway in hippocampal post-ischemic LTP: modulation by zonisamide.

Abstract Nitric oxide (NO) is an intercellular retrograde messenger involved in several physiological processes such as synaptic plasticity, hippocampal long-term potentiation (LTP), and learning and memory. Moreover NO signaling is implicated in the pathophysiology of brain ischemia. In this study, we have characterized the role of NO/cGMP signaling cascade in the induction and maintenance of post-ischemic LTP (iLTP) in rat brain slices. Moreover, we have investigated the possible inhibitory action of zonisamide (ZNS) on this pathological form of synaptic plasticity as well as the effects of this antiepileptic drug (AED) on physiological activity-dependent LTP. Finally, we have characterized the possible interaction between ZNS and the NO/cGMP/PKG-dependent pathway involved in iLTP. Here, we provided the first evidence that an oxygen and glucose deprivation episode can induce, in CA1 hippocampal slices, iLTP by modulation of the NO/cGMP/PKG pathway. Additionally, we found that while ZNS application did not affect short-term synaptic plasticity and LTP induced by high-frequency stimulation, it significantly reduced iLTP. This reduction was mimicked by bath application of NO synthase inhibitors and a soluble guanyl cyclase inhibitor. The effect of ZNS was prevented by either the application of a NO donor or drugs increasing intracellular levels of cGMP and activating PKG. These findings are in line with the possible use of AEDs, such as ZNS, as a possible neuroprotective strategy in brain ischemia. Moreover, these findings strongly suggest that NO/cGMP/PKG intracellular cascade might represent a physiological target for neuroprotection in pathological forms of synaptic plasticity such as hippocampal iLTP.

A2A adenosine receptor antagonists protect the striatum against rotenone-induced neurotoxicity

Adenosine A2A receptor has emerged as an attractive non-dopaminergic target in the experimental pharmacological therapy for Parkinsons disease (PD). Moreover, it has been postulated that A2A adenosine receptor antagonists exert neuroprotective effects in experimental models of PD and progressive supranuclear palsy (PSP). Interestingly, in both these pathological conditions a deficit of mitochondrial complex I has been found. Thus, utilizing extracellular and intracellular recordings from corticostriatal brain slices, we have tested the possible neuroprotective action of two A2A receptor antagonists, ST1535 and ZM241385, on the irreversible electrophysiological effects induced by the acute application of rotenone, a pesticide acting as a selective inhibitor of mitochondrial complex I activity. Both these antagonists reduced the rotenone-induced loss of corticostriatal field potential amplitude as well as the membrane depolarization caused by this toxin on striatal spiny neurons. The use of A2A receptor antagonists might represent a promising neuroprotective strategy in basal ganglia disorders involving a deficit of mitochondrial complex I activity.