Persistent elevation of D-Aspartate enhances NMDA receptor-mediated responses in mouse substantia nigra pars compacta dopamine neurons.

Dopamine neurons in the substantia nigra pars compacta regulate not only motor but also cognitive functions. NMDA receptors play a crucial role in modulating the activity of these cells. Considering that the amino-acid D-Aspartate has been recently shown to be an endogenous NMDA receptor agonist, the aim of the present study was to examine the effects of D-Aspartate on the functional properties of nigral dopamine neurons. We compared the electrophysiological actions of D-Aspartate in control and D-aspartate oxidase gene (Ddo(-/-)) knock-out mice that show a concomitant increase in brain D-Aspartate levels, improved synaptic plasticity and cognition. Finally, we analyzed the effects of L-Aspartate, a known dopamine neuron endogenous agonist in control and Ddo(-/-) mice. We show that D- and L-Aspartate excite dopamine neurons by activating NMDA, AMPA and metabotropic glutamate receptors. Ddo deletion did not alter the intrinsic properties or dopamine sensitivity of dopamine neurons. However, NMDA-induced currents were enhanced and membrane levels of the NMDA receptor GluN1 and GluN2A subunits were increased. Inhibition of excitatory amino-acid transporters caused a marked potentiation of D-Aspartate, but not L-Aspartate currents, in Ddo(-/-) neurons. This is the first study to show the actions of D-Aspartate on midbrain dopamine neurons, activating not only NMDA but also non-NMDA receptors. Our data suggest that dopamine neurons, under conditions of high D-Aspartate levels, build a protective uptake mechanism to compensate for increased NMDA receptor numbers and cell hyper-excitation, which could prevent the consequent hyper-dopaminergia in target zones that can lead to neuronal degeneration, motor and cognitive alterations.

Increased levels of p70S6 phosphorylation in the G93A mouse model of Amyotrophic Lateral Sclerosis and in valine-exposed cortical neurons in culture.

The higher risk factor for Amyotrophic Lateral Sclerosis (ALS) among Italian soccer players is a question that is still debated. One of the hypotheses that have been formulated to explain a possible link between ALS and soccer players is related to the abuse of dietary supplements and drugs for enhancing sporting performance. In particular, it has been reported that branched-chain amino acids (BCAAs) are widely used among athletes as nutritional supplements. To observe the possible effect of BCAAs on neuronal electrical properties, we performed electrophysiological experiments on Control cultured cortical neurons and on neurons after BCAA treatment. BCAA-treated neurons showed hyperexcitability and rapamycin was able to suppress it and significantly reduce the level of mTOR, Akt and p70S6 phosphorylation. Interestingly, the hyperexcitability previously reported in cortical neurons from a genetic mouse model of ALS (G93A) was also reversed by rapamycin treatment. Moreover, both G93A and valine-treated neurons presented significantly higher levels of Pp70S6 when compared to control neurons, strongly indicating the involvement of this substrate in ALS pathology. Finally, we performed electrophysiological experiments on motor cortex slices from Control and G93A mice and those fed with a BCAA-enriched diet. We observed that neuron excitability was comparable between G93A and BCAA-enriched diet mice, but was significantly higher than in Control mice. These findings, besides strongly indicating that BCAAs specifically induce hyperexcitability, seem to suggest the involvement of p70S6 substrate in ALS pathology.

Brivaracetam (ucb 34714) inhibits Na(+) current in rat cortical neurons in culture.

Brivaracetam (ucb 34714; BRV), a new antiepileptic drug (AED) candidate, is a pyrrolidone derivative displaying a markedly higher affinity than levetiracetam (LEV; Keppra) to the synaptic vesicle protein SV2A, shown to be the brain-specific binding site of LEV. The higher affinity for SV2A correlates significant antiepileptic activity in animal epilepsy models in vitro and in vivo. Since many AEDs act upon inhibiting neuronal Na(+) currents, this study explored putative activity of BRV on the properties of these currents. Voltage-activated Na(+) currents were recorded by whole-cell patch-clamp on neuronal somas of rat neocortical neurons, grown in dissociated cell culture for up to 12 days. BRV, dissolved at the desired final concentration (between 0.2microM and 1mM) was applied by a multi-barrel pipette system near the soma of the recorded neuron. BRV produced a concentration-dependent inhibition of voltage-dependent Na(+) currents with IC(50) values of 41microM at the holding potential of -100mV, and of 6.5microM at the holding potential of -60mV. The voltage-dependence of activation and the kinetics of fast inactivation were not modified in the presence of BRV (30microM). Conversely, the recovery from fast inactivation was significantly slower and the voltage of half-maximal inactivation was shifted toward hyperpolarized value after BRV perfusion in a concentration-dependent manner. Furthermore, BRV (30microM) induced a significant use-dependent block at 50Hz stimulation frequency. These results indicate that BRV is able to modulate the voltage-activated Na(+) inflow in cortical neurons, which conceivably might contribute to the antiepileptic activity of this drug.

Gene expression profiles of APP and BACE1 in Tg SOD1G93A cortical cells.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Abeta peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer's disease, as APP, beta- and gamma-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of gamma-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.

Increased persistent sodium current determines cortical hyperexcitability in a genetic model of amyotrophic lateral sclerosis.

Cortical hyperexcitability has been observed in Amyotrophic Lateral Sclerosis (ALS) patients. Familial ALS accounts for 10% of all cases and mutations of the Cu,Zn superoxide dismutase (SOD1) gene have been identified in about 20% of the familial cases. The aim of this study was to investigate whether in a mouse model of ALS the cortical neurons developed hyperexcitability due to intrinsic properties of the single cell. We first examined the passive membrane properties and the pattern of repetitive firing in cultured cortical neurons from Control mice and transgenic mice expressing high levels of the human mutated protein (Gly(93)-->Ala, G93A). The former did not display significantly differing values between Control and G93A cortical neurons. However, the threshold potential and time of the first action potential decreased significantly and the firing frequency increased significantly in the G93A compared to Control neurons. The analysis of the voltage-dependent sodium currents revealed that the fast transient sodium current was unaffected by the SOD1 mutation whereas the persistent sodium current was significantly higher in the mutated neurons. Finally, Riluzole, a selective blocker of the persistent sodium current at low concentrations, decreased the firing frequency in G93A neurons, strongly indicating an involvement of this current in the observed hyperexcitability. These are the first data that demonstrate an intrinsic hyperexcitability in the G93A cortical neurons due to a higher current density of the persistent sodium current in the mutated neurons and open up new prospects of understanding ALS disease etiopathology.

GAB(A) receptors present higher affinity and modified subunit composition in spinal motor neurons from a genetic model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the selective degeneration of motor neurons in the spinal cord, brainstem and cerebral cortex. In this study we have analysed the electrophysiological properties of GABA(A) receptors and GABA(A) alpha1 and alpha2 subunits expression in spinal motor neurons in culture obtained from a genetic model of ALS (G93A) and compared with transgenic wild type SOD1 (SOD1) and their corresponding non transgenic litter mates (Control). Although excitotoxic motor neuron death has been extensively studied in relation to Ca(2+)-dependent processes, strong evidence indicates that excitotoxic cell death is also remarkably dependent on Cl(-) ions and on GABA(A) receptor activation. In this study we have analysed the electrophysiological properties of GABA(A) receptors and the expression of GABA(A)alpha(1) and alpha(2) subunits in cultured motor neurons obtained from a genetic model of amyotrophic lateral sclerosis (G93A) and compared them with transgenic wild-type Cu,Zn superoxide dismutase and their corresponding non-transgenic littermates (Control). In all tested motor neurons, the application of gamma-aminobutyric acid (GABA) (0.5-100 mum) evoked an inward current that was reversibly blocked by bicuculline (100 mum), thus indicating that it was mediated by the activation of GABA(A) receptors. Our results indicate that the current density at high GABA concentrations is similar in control, Cu,Zn superoxide dismutase and G93A motor neurons. However, the dose-response curve significantly shifted toward lower concentration values in G93A motor neurons and the extent of desensitization also increased in these neurons. Finally, multiplex single-cell real-time polymerase chain reaction and immunofluorescence revealed that the amount of GABA(A)alpha(1) subunit was significantly increased in G93A motor neurons, whereas the levels of alpha(2) subunit were unchanged. These data show that the functionality and expression of GABA(A) receptors are altered in G93A motor neurons inducing a higher Cl(-) influx into the cell with a possible consequent neuronal excitotoxicity acceleration.

Altered calcium homeostasis in motor neurons following AMPA receptor but not voltage-dependent calcium channels' activation in a genetic model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brain stem and motor cortex. By combining electrophysiological recordings with imaging techniques, clearance/buffering capacity of cultured spinal cord motor neurons after a calcium accumulation has been analyzed in response to AMPA receptors' (AMPARs') activation and to depolarizing stimuli in a genetic mouse model of ALS (G93A). Our studies demonstrate that the amplitude of the calcium signal in response to AMPARs' or voltage-dependent calcium channels' activation is not significantly different in controls and G93A motor neurons. On the contrary, in G93A motor neurons, the [Ca(2+)](i) recovery to basal level is significantly slower compared to control neurons following AMPARs but not voltage-dependent calcium channels' activation. This difference was not observed in G93A cultured cortical neurons. This observation is the first to indicate a specific alteration of the calcium clearance linked to AMPA receptors' activation in G93A motor neurons and the involvement of AMPA receptor regulatory proteins controlling both AMPA receptor functionality and the sequence of events connected to them.

Modulation of AMPA receptors in cultured cortical neurons induced by the antiepileptic drug levetiracetam.

PURPOSE: The present study explores the hypothesis that the antiepileptic mechanism of action of levetiracetam (LEV) is related to effects on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels in mouse cortical neurons in culture. METHODS: The neurons were subjected to the whole-cell configuration of the patch-clamp recording technique and were 8-12 days old in culture. RESULTS: Kainate elicited concentration-dependent (EC(50)= 80 microM) inward currents in all the patched cells. LEV (5-200 microM) itself did not induce inward or outward currents on all patched neurons, whereas it was effective on the kainate- and AMPA-induced current because it significantly decreased the amplitude of these currents. LEV was also able to significantly decrease the total membrane conductance during kainate perfusion, indicating that its effect was not dependent on the cellular voltage membrane potential. Further evidence that LEV modulated the ionotropic non-NMDA receptors came from the analysis of miniature excitatory postsynaptic currents (mEPSCs). In fact, LEV significantly decreased both the amplitude and the frequency of mEPSCs, as shown by the relative cumulative distributions. CONCLUSIONS: These results reveal that AMPA receptors are modulated by LEV because a significant decrease in the kainate- and AMPA-induced currents and a decrease in amplitude and in frequency of mEPSCs have been observed in cortical neurons in culture. The described effect of LEV on AMPA receptors in cortical neurons is probably due to the etheromeric composition of the receptors and may be considered as a possible new antiepileptic mechanism of action.

Voltage-dependent sodium channels in spinal cord motor neurons display rapid recovery from fast inactivation in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brain stem, and motor cortex. Previous evidence showed that in a mouse model of a familial form of ALS expressing high levels of the human mutated protein Cu,Zn superoxide dismutase (Gly(93)-->Ala, G93A), the firing properties of single motor neurons are altered to induce neuronal hyperexcitability. To determine whether the functionality of the macroscopic voltage-dependent Na(+) currents is modified in G93A motor neurons, in the present work their physiological properties were examined. The voltage-dependent sodium channels were studied in dissociated motor neurons in culture from nontransgenic mice (Control), from transgenic mice expressing high levels of the human wild-type protein [superoxide dismutase 1 (SOD1)], and from G93A mice, using the whole cell configuration of the patch-clamp recording technique. The voltage dependency of activation and of steady-state inactivation, the kinetics of fast inactivation and slow inactivation of the voltage-dependent Na(+) channels were not modified in the mutated mice. Conversely, the recovery from fast inactivation was significantly faster in G93A motor neurons than that in Control and SOD1. The recovery from fast inactivation was still significantly faster in G93A motor neurons exposed for different times (3-48 h) and concentrations (5-500 microM) to edaravone, a free-radical scavenger. Clarification of the importance of these changes in membrane ion channel functionality may have diagnostic and therapeutic implications in the pathogenesis of ALS.

AMPA receptors are modulated by tachykinins in rat cerebellum neurons.

The peptides of the tachykinin family are widely distributed within the mammalian peripheral and central nervous systems and play a well-recognized role as neuromodulators, although their direct action on cerebellum granule cells have not yet been demonstrated. We have examined the effect of the best known members of the family, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors from rat cerebellar granule cells in culture to assess the ability of these peptides to regulate the glutamatergic input. Both NKA and NKB, but not SP, produce a significant enhancement of ionic current through AMPA receptors activated by the agonist kainate in 53.5 and 46% of patched neurons, respectively. This effect was not observable in the presence of MEN 10,627 and Trp(7)betaAla(8), NKA and NKB competitive antagonist receptors, respectively, indicating that the current modulations were mediated by the respective receptors. NKB also produces a significant enhancement of ionic current through the AMPA receptors activated directly by its agonist AMPA and cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA receptors. The presence of NK3 receptors was demonstrated in these neurons by RT-PCR amplification of total RNA extracted from cerebellar granule cells, using NK3-specific primer pairs. Immunocytochemistry experiments, using a specific polyclonal antibody directed against NK3, also confirmed the presence of NK3 receptors and their co-localization with the GLUR2 AMPA subunit in about 54% of cerebellar granule neurons. This study adds the tachykinins to the list of neuromodulators capable of exerting a excitatory action on cerebellar granule cells.