The ferroptosis inducer RSL3 triggers interictal epileptiform activity in mice cortical neurons.

Epilepsy is a neurological disorder characterized by recurrent seizures, which result from excessive, synchronous discharges of neurons in different brain areas. In about 30% of cases, epileptic discharges, which vary in their etiology and symptomatology, are difficult to treat with conventional drugs. Ferroptosis is a newly defined iron-dependent programmed cell death, characterized by excessive accumulation of lipid peroxides and reactive oxygen species. Evidence has been provided that ferroptosis is involved in epilepsy, and in particular in those forms resistant to drugs. Here, whole cell patch clamp recordings, in current and voltage clamp configurations, were performed from layer IV principal neurons in cortical slices obtained from adult mouse brain. Application of the ferroptosis inducer RAS-selective lethal 3 (RSL3) induced interictal epileptiform discharges which started at RSL3 concentrations of 2 µM and reached a plateau at 10 µM. This effect was not due to changes in active or passive membrane properties of the cells, but relied on alterations in synaptic transmission. In particular, interictal discharges were dependent on the excessive excitatory drive to layer IV principal cells, as suggested by the increase in frequency and amplitude of spontaneously occurring excitatory glutamatergic currents, possibly dependent on the reduction of inhibitory GABAergic ones. This led to an excitatory/inhibitory unbalance in cortical circuits. Interictal bursts could be prevented or reduced in frequency by the lipophilic antioxidant Vitamin E (30 µM). This study allows identifying new targets of ferroptosis-mediated epileptic discharges opening new avenues for the treatment of drug-resistant forms of epilepsy.

Coincident Pre- and Post-Synaptic Cortical Remodelling Disengages Episodic Memory from Its Original Context.

The view that the neocortex is remotely recruited for long-term episodic memory recall is challenged by data showing that an intense transcriptional and synaptic activity is detected in this region immediately after training. By measuring markers of synaptic activity at recent and remote time points from contextual fear conditioning (CFC), we could show that pre-synaptic changes are selectively detected 1 day post-training when the memory is anchored to the training context. Differently, pre- and post-synaptic changes are detected 14 days post-training when the memory generalizes to other contexts. Confirming that coincident pre- and post-synaptic remodelling mediates the disengagement of memory from its original context, DREADDs-mediated enhancement of cortical neuron activity during CFC training anticipates expression of a schematic memory and observation of bilateral synaptic remodelling. Together, our data show that the plastic properties of cortical synapses vary over time and specialise in relation to the quality of memory.

NGF steers microglia toward a neuroprotective phenotype.

Microglia are the sentinels of the brain but a clear understanding of the factors that modulate their activation in physiological and pathological conditions is still lacking. Here we demonstrate that Nerve Growth Factor (NGF) acts on microglia by steering them toward a neuroprotective and anti-inflammatory phenotype. We show that microglial cells express functional NGF receptors in vitro and ex vivo. Our transcriptomic analysis reveals how, in primary microglia, NGF treatment leads to a modulation of motility, phagocytosis and degradation pathways. At the functional level, NGF induces an increase in membrane dynamics and macropinocytosis and, in vivo, it activates an outward rectifying current that appears to modulate glutamatergic neurotransmission in nearby neurons. Since microglia are supposed to be a major player in Aß peptide clearance in the brain, we tested the effects of NGF on its phagocytosis. NGF was shown to promote TrkA-mediated engulfment of Aß by microglia, and to enhance its degradation. Additionally, the proinflammatory activation induced by Aß treatment is counteracted by the concomitant administration of NGF. Moreover, by acting specifically on microglia, NGF protects neurons from the Aß-induced loss of dendritic spines and inhibition of long term potentiation. Finally, in an ex-vivo setup of acute brain slices, we observed a similar increase in Aß engulfment by microglial cells under the influence of NGF. Our work substantiates a role for NGF in the regulation of microglial homeostatic activities and points toward this neurotrophin as a neuroprotective agent in Aß accumulation pathologies, via its anti-inflammatory activity on microglia.

TRPV1 channels are critical brain inflammation detectors and neuropathic pain biomarkers in mice.

The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication.

Increased D-aspartate brain content rescues hippocampal age-related synaptic plasticity deterioration of mice.

Until recently, free d-amino acids were thought to be involved only in bacterial physiology. Nevertheless, today there is evidence that D-serine, by acting as co-agonist at NMDARs, plays a role in controlling neuronal functions in mammals. Besides D-serine, another D-amino acid, D-aspartate (D-Asp), is found in the mammalian brain with a temporal gradient of occurrence: high in embryo and low in adult. In this study, we demonstrate that D-Asp acts as an endogenous NMDAR agonist, since it triggers currents via interaction with each of NR2A-D receptor subunits. According to its pharmacological features, we showed that oral administration of D-Asp strongly enhances NMDAR-dependent LTP in adulthood and, in turn, completely rescues the synaptic plasticity decay observed in the hippocampus of aged animals. Therefore, our findings suggest a tantalizing hypothesis for which this in-embryo-occurring D-amino acid, when "forced" over its physiological content, may disclose plasticity windows inside which it counteracts the age-related reduction of NMDAR signaling.

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP.

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.

Trace amines depress D(2)-autoreceptor-mediated responses on midbrain dopaminergic cells.

BACKGROUND AND PURPOSE: Although trace amines (TAs) are historically considered 'false neurotransmitters' on the basis of their ability to induce catecholamine release, there is evidence that they directly affect neuronal activity via TA receptors, ligand-gated receptor channels and/or sigma receptors. Here, we have investigated the effects of two TAs, tyramine (TYR) and beta-phenylethylamine (beta-PEA), on electrophysiological responses of substantia nigra pars compacta (SNpc) dopaminergic cells to the D(2) receptor agonist, quinpirole. EXPERIMENTAL APPROACH: Electrophysiological recordings of D(2) receptor-activated G-protein-gated inward rectifier K(+) channel (GIRK) currents were performed on dopaminergic cells from midbrain slices of mice and on Xenopus oocytes expressing D(2) receptors and GIRK channels. KEY RESULTS: TYR and beta-PEA reversibly reduced D(2) receptor-activated GIRK currents in a concentration-dependent manner on SNpc neurones. The inhibitory effect of TAs was still present in transgenic mice with genetically deleted TA(1) receptors and they could not be reproduced by the selective TA(1) agonist, o-phenyl-3-iodotyramine (O-PIT). Pretreatment with antagonists of sigma1 and sigma2 receptors did not block TA-induced effects. In GTPgammaS-loaded neurones, the irreversibly-activated GIRK-current was still reversibly reduced by beta-PEA. Moreover, beta-PEA did not affect basal or dopamine-evoked GIRK-currents in Xenopus oocytes. CONCLUSIONS AND IMPLICATIONS: TAs reduced dopamine-induced responses on SNpc neurones by acting at sites different from TA(1), sigma-receptors, D(2) receptors or GIRK channels. Although their precise mechanism of action remains to be identified, TAs, by antagonizing the inhibitory effects of dopamine, may render dopaminergic neurones less sensitive to autoreceptor feedback inhibition and hence enhance their sensitivity to stimulation.

Connexin 26 (GJB2) mutations, causing KID Syndrome, are associated with cell death due to calcium gating deregulation.

The autosomic dominant KID Syndrome (MIM 148210), due to mutations in GJB2 (connexin 26, Cx26), is an ectodermal dysplasia with erythematous scaly skin lesions, keratitis and severe bilateral sensorineural deafness. The Cx26 protein is a component of gap junction channels in epithelia, including the cochlea, which coordinates the exchange of molecules and ions. Here, we demonstrate that different Cx26 mutants (Cx26D50N and Cx26G11E) cause cell death in vitro by the alteration of intra-cellular calcium concentrations. These results help to explain the pathogenesis of both the hearing and skin phenotypes, since calcium is also a potent regulator of the epidermal differentiation process.

N-ethyl lidocaine (QX-314) protects striatal neurons against ischemia: an in vitro electrophysiological study.

In this study, we have investigated the neuroprotective actions of the membrane impermeable, lidocaine analog, N-ethyl lidocaine (QX-314) in the striatum. The effects of this drug were compared with those caused by the strictly-related-compound and sodium channel blocker lidocaine. To address this issue, electrophysiological recordings were performed in striatal slices, in control condition (normoxia) and during combined oxygen and glucose deprivation (in vitro ischemia). Either QX-314 or lidocaine induced, to some extent, a protection of the permanent electrophysiological alteration (field potential loss) caused by a period (12 min) of ischemia. Thus, both compounds permitted a partial recovery of the ischemic depression of the corticostriatal transmission and reduced the amplitude of the ischemic depolarization in medium spiny neurons. However, while QX-314, at the effective concentration of 100 microM, slightly reduced the amplitude of the excitatory field potential and did not affect the current-evoked spikes discharge of medium spiny striatal neurons, equimolar lidocaine depressed the field potential and eliminated repetitive spikes on a depolarizing step. On the basis of these observations, our results suggest the use of QX-314 as a neuroprotective agent in ischemic brain disorders.

Rapid constitutive and ligand-activated endocytic trafficking of P2X receptor.

P2X receptors mediate a variety of physiological actions, including smooth muscle contraction, neuro-endocrine secretion and synaptic transmission. Among P2X receptors, the P2X(3) subtype is expressed in sensory neurons of dorsal root- and trigeminal-ganglia, where it performs a well-recognized role in sensory and pain transmission. Recent evidence indicates that the strength of P2X(3)-mediated responses is modulated in vivo by altering the number of receptors at the plasma membrane. In the present study, we investigate the trafficking properties of P2X(3) receptor in transfected HEK293 cells and in primary cultures of dorsal root ganglion neurons, finding that P2X(3) receptor undergoes rapid constitutive and cholesterol-dependent endocytosis. We also show that endocytosis is accompanied by preferential targeting of the receptor to late endosomes/lysosomes, with subsequent degradation. Furthermore, we observe that at steady state the receptor localizes predominantly in lamp1-positive intracellular structures, with a minor fraction present at the plasma membrane. Finally, the level of functional receptor expressed on the cell surface is rapidly up-regulated in response to agonist stimulation, which also augments receptor endocytosis. The findings presented in this work underscore a very dynamic trafficking behavior of P2X(3) receptor and disclose a possible mechanism for the rapid modulation of ATP-mediated responses potentially relevant during physiological and pathological conditions.

Ethanol enhances GABAB-mediated inhibitory postsynaptic transmission on rat midbrain dopaminergic neurons by facilitating GIRK currents.

It is largely accepted that an activation of the dopaminergic system underlies the recreational and convivial effects of ethanol. However, the mechanisms of action of this drug on the dopaminergic neurons are not fully understood yet. In the present study, we have used intracellular electrophysiological techniques (current and single-electrode voltage-clamp) to investigate the actions of ethanol on the gamma-aminobutyric acid (GABA)(B)-mediated inhibitory postsynaptic potentials (IPSPs) in rat midbrain dopaminergic neurons. Ethanol (10-200 mM) augmented, in a concentration-dependent and reversible manner, the amplitude of the GABA(B)-IPSP. In addition, the GABA(B) agonist baclofen generated G-protein-gated inward rectifying K(+) channels (GIRK)-related membrane hyperpolarizations/outward currents that were potentiated by ethanol. The potentiating effect of ethanol persisted in tetrodotoxin (TTX)-treated neurons, suggesting a postsynaptic site of action. These effects of ethanol were not changed by manipulating adenyl cyclase, protein kinases and phospholipase C activity, or by chelating intracellular Ca(2+) with EGTA. Interestingly, the outward current caused by the intracytoplasmatic diffusion of the irreversible G-protein activator GTPgammaS was transiently enhanced by ethanol. Our observations suggest that the action of ethanol occurs on activated GIRK channels downstream of the GABA(B) receptors. These enhancing effects of ethanol on GABA(B)-induced synaptic responses could modulate alcohol intake and the altered mental and motor performance of individuals in an acute intoxicative phase.

Differential effect of carbamazepine and oxcarbazepine on excitatory synaptic transmission in rat hippocampus.

In this study, we have compared the effects of two structurally related compounds carbamazepine (CBZ) and oxcarbazepine (OXC), both in current use for the treatment of epilepsy and bipolar disorder, on fast excitatory transmission in rat hippocampal slices. Using electrophysiological recordings, we have investigated the effects of CBZ and OXC on repetitive action potential discharge of CA1 pyramidal neurons demonstrating that both compounds produced firing inhibition with similar IC(50) values. Moreover, we show that bath applied CBZ (0.01-1 mM) exerted a concentration-dependent decrease in the amplitude of the field excitatory postsynaptic potentials with an IC(50) of approximately 194.3 microM. When OXC was used at the same concentrations, the concentration-response curve was shifted to the right (IC(50) of approximately 711.07 microM). In addition, we demonstrated that CBZ and OXC reduced, to a different extent, both evoked excitatory postsynaptic currents and NMDA-, AMPA-, and KA-mediated inward currents, CBZ being more potent than OXC. These data highlight distinct presynaptic and postsynaptic sites of action for both compounds and suggest that CBZ, by markedly depressing postsynaptic ionotropic glutamate receptors-mediated responses, may produce more severe cognitive and memory impairment. Thus, we assume that relatively high doses of OXC could be better tolerated than therapeutically equivalent doses of CBZ, justifying the preferential use of OXC as first-line treatment in the therapy of neurological and psychiatric disorders, particularly when compared with CBZ.

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 Parkinson's 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 microM) 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 (K(ATP)) channel antagonist tolbutamide (300 microM), and this increase occluded further effects of memantine. In addition, K(ATP) channel-mediated outward currents, induced by hypoxia, were inhibited by memantine (30 and 100 microM) 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 microM). An increase in the spontaneous firing rate by memantine was observed in dopamine neurons recorded with extracellular planar 8 x 8 multielectrodes in conditions of hypoglycemia. These results highlight K(ATP) channels as possible relevant targets of memantine effects in the brain. Moreover, in view of a proposed role of K(ATP) conductances in dopamine neuron degeneration, they suggest another mechanism of action underlying the protective role of memantine in Parkinson's disease.

Trace amines reduce GABA(B) receptor-mediated presynaptic inhibition at GABAergic synapses of the rat substantia nigra pars compacta.

Trace amines (TAs) act in the mammalian brain through amphetamine-like effects and as endogenous agonists of specific receptors. We now show that tyramine and beta-phenylethylamine, in the presence of specific dopamine (DA) receptor antagonists, inhibit the GABA(B)-dependent presynaptic inhibition of GABAergic inputs to midbrain DA neurons. Our results further extend the role of TAs as neuromodulators and propose a novel mechanism by which they modulate DA neurons.

Distinct mechanisms of presynaptic inhibition at GABAergic synapses of the rat substantia nigra pars compacta.

We investigated the mechanisms of presynaptic inhibition of GABAergic neurotransmission by group III metabotropic glutamate receptors (mGluRs) and GABA(B) receptors, in dopamine (DA) neurons of the substantia nigra pars compacta (SNc). Both the group III mGluRs agonist L-(+)-2-amino-4-phosphonobutyric acid (AP4, 100 microM) and the GABA(B) receptor agonist baclofen (10 microM) reversibly depressed the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) to 48.5 +/- 2.7 and 79.3 +/- 1.6% (means +/- SE) of control, respectively. On the contrary, the frequency of action potential-independent miniature IPSCs (mIPSCs), recorded in tetrodotoxin (TTX, 1 microM) and cadmium (100 microM) were insensitive to AP4 but were reduced by baclofen to 49.7 +/- 8.6% of control. When the contribution of voltage-dependent calcium channels (VDCCs) to synaptic transmission was boosted with external barium (1 mM), AP4 became effective in reducing TTX-resistant mIPSCs to 65.4 +/- 3.9% of control, thus confirming a mechanism of presynaptic inhibition involving modulation of VDCCs. Differently from AP4, baclofen inhibited to 58.5 +/- 6.7% of control the frequency mIPSCs recorded in TTX and the calcium ionophore ionomycin (2 microM), which promotes Ca2+-dependent, but VDCC-independent, transmitter release. Moreover, in the presence of alpha-latrotoxin (0.3 nM), to promote a Ca2+-independent vesicular release of GABA, baclofen reduced mIPSC frequency to 48.1 +/- 3.2% of control, while AP4 was ineffective. These results indicate that group III mGluRs depress GABA release to DA neurons of the SNc through inhibition of presynaptic VDCCs, while presynaptic GABA(B) receptors directly impair transmitter exocytosis.