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In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca2+ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.

Limited information is available on the brain expression and role of GPR35, a Gi/o coupled receptor activated by kynurenic acid (KYNA). In mouse cultured astrocytes, we detected GPR35 transcript using RT-PCR and we found that KYNA (0.1 to 100 µM) decreased forskolin (FRSK)-induced cAMP production (p<0.05). Both CID2745687 (3 µM, CID), a recently described GPR35 antagonist, and GPR35 gene silencing significantly prevented the action of KYNA on FRSK-induced cAMP production. In these cultures, we then evaluated whether GPR35 activation was able to modulate intracellular Ca(2+) concentration ([Ca(2+)]i ) and [Ca(2+)]i fluxes. We found that both KYNA and zaprinast, a phosphodiesterase (PDE) inhibitor and GPR35 agonist, did not modify either basal or peaks of [Ca(2+)]i induced by challenging the cells with ATP (30 µM). However, the [Ca(2+)]i plateau phase following peak was significantly attenuated by these compounds in a store-operated Ca(2+) channel (SOC)-independent manner. The activation of GPR35 by KYNA and zaprinast was also studied at the CA3-CA1 synapse in the rat hippocampus. Evoked excitatory post synaptic currents (eEPSCs) were recorded from CA1 pyramidal neurons in acute brain slices. The action of KYNA on GPR35 was pharmacologically isolated by using NMDA and α7 nicotinic receptor blockers and resulted in a significant reduction of eEPSC amplitude. This effect was prevented in the presence of CID. Moreover, zaprinast reduced eEPSC amplitude in a PDE5- and cGMP-independent mechanism, thus suggesting that glutamatergic transmission in this area is modulated by GPR35. In conclusion, GPR35 is expressed in cultured astrocytes and its activation modulates cAMP production and [Ca(2+)]i. GPR35 activation may contribute to KYNA effects on the previously reported decrease of brain extracellular glutamate levels and reduction of excitatory transmission.

Infants born to epileptic women treated with antiepileptic drugs (AEDs) have an increased risk of major congenital malformations (MCMs). In order to determine the role of maternal epilepsy we conducted a prospective cohort study on three cohorts of pregnant women: (i) 385 epileptic women treated with AEDs, (ii) 310 non-epileptic women treated with AEDs, (iii) 867 healthy women not exposed to AEDs (control group). The rate of MCMs in the epileptic group (7.7%) was not statistically higher than in the non-epileptic one (3.9%) (p=0.068). The rate in the first group was higher compared to the control group (p=0.001), while the rate in the second one was not (p=0.534). Our data confirm that AEDs therapy is the main cause of the increased risk of malformations in the offspring of epileptic women; however a teratogenic role of the maternal epilepsy itself cannot be excluded., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This review is intended to offer updated information on the involvement of cannabinoids in the process of inflammation, focusing on immune/allergic reactions, inflammatory pain and neuroinflammation and discussing the interactions among endocannabinoid metabolism, prostanoids and nitric oxide. Two types of cannabinoid receptors, CB1 and CB2, which belong to the G protein-coupled receptor family, have been identified and are targeted by numerous exogenous and endogenous ligands. The activation of CB2 receptors on mast cells has direct antiinflammatory effects, causing decreased release of pro-inflammatory mediators by these cells. The activation of CB1 receptors on bronchial nerve endings has bronchodilator effects by acting on the airway smooth muscle and may be beneficial in airway hyperreactivity and asthma. Moreover, pharmacologic interference with endocannabinoid metabolism has been demonstrated to result in anti-nociceptive activity, mediated by CB1 and CB2 receptors, in animal models of inflammatory pain. The presence of endocannabinoid machinery in the central nervous system, together with high levels of CB1 expression, suggests that the endocannabinoid system is an important modulator of neuroinflammation and a possible drug target. In selected conditions, the activation of CB1 receptors in cerebral blood vessels can have beneficial antiischemic effects. However, as endocannabinoids can also bind to vanilloid receptors, they may also mediate neurotoxic effects.

It is usually assumed that kynurenic acid (KYNA) modifies neuronal function because it antagonizes the glycine site of the NMDA receptors and/or the neuronal cholinergic α7 nicotine receptors. It is not clear, however, whether the basal levels of KYNA found in brain extracellular spaces are sufficient to interact with these targets. Another reported target for KYNA is GPR35, an orphan receptor negatively coupled to G(i) proteins. GPR35 is expressed both in neurons and other cells (including glia, macrophages and monocytes). KYNA affinity for GPR35 in native systems has not been clarified and the low-affinity data widely reported in the literature for the interaction between KYNA and human or rat GPR35 have been obtained in modified expression systems. Possibly by interacting with GPR35, KYNA may also reduce glutamate release in brain and pro-inflammatory cytokines release in cell lines. The inhibition of inflammatory mediator release from both glia and macrophages may explain why KYNA has analgesic effects in inflammatory models. Furthermore, it may also explain why, KYNA administration (200 mg/kg ip × 3 times) to mice treated with lethal doses of LPS, significantly reduces the number of deaths. Finally, KYNA has been reported as an agonist of aryl hydrocarbon receptor (AHR), a nuclear protein involved in the regulation of gene transcription and able to cause immunosuppression after binding with dioxin. Thus, KYNA has receptors in the nervous and the immune systems and may play interesting regulatory roles in cell function.

G-protein coupled receptor 35 (GPR35) is a former "orphan receptor" expressed in brain and activated by either kynurenic acid or zaprinast. While zaprinast has been studied as a phosphodiesterase inhibitor, kynurenic acid (KYNA) is a tryptophan metabolite and has been proposed as the endogenous ligand for this receptor. In the present work, we showed that GPR35 is present in the dorsal root ganglia and in the spinal cord and in order to test the hypothesis that GPR35 activation could cause analgesia, we administered suitable doses of zaprinast or we increased the local concentration of KYNA by administering a precursor (kynurenine) or by inhibiting its disposal from the CNS (with probenecid). We used the "writhing test" induced by acetic acid i.p. injection in mice. KYNA and kynurenine plasma and spinal cord levels were measured with HPLC techniques. Kynurenine (30, 100, 300 mg/kg s.c.) increased plasma and spinal cord levels of KYNA and decreased the number of writhes in a dose dependent manner. Similarly, probenecid was able to increase KYNA levels in plasma and spinal cord, to reduce the number of writes and to amplify kynurenine effects. Furthermore, zaprinast had antinociceptive effects in the writhing test without affecting KYNA levels. In agreement with its affinity for GPR35 receptor (approximately 10 times higher than that of KYNA), zaprinast action occurred at relatively low doses. No additive actions were obtained when kynurenine and zaprinast were administered at maximally active doses. Our results suggest that GPR35 could be an interesting target for innovative pharmacological agents designed to reduce inflammatory pain. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Unlabelled: The diagnosis of hepatic encephalopathy (HE) relies on clinical, neurophysiological, psychometric and laboratory variables. The relationships between such tests remain debated. The aim of this study was to determine the laboratory correlates/prognostic value of neurophysiological/psychometric abnormalities in patients with cirrhosis. Seventy-two patients and 14 healthy volunteers underwent EEG and paper-and-pencil psychometry (PHES). Blood was obtained for C reactive protein (CRP), interleukin 6 (IL6), tumor necrosis factor (TNF)α, ammonia and indole/oxindole. Patients were followed prospectively for a median of 22 months in relation to the occurrence of death, transplantation and HE-related hospitalizations. Thirty-three patients had normal PHES and EEG, 6 had abnormal PHES, 18 abnormal EEG and 13 abnormal PHES and EEG. Patients with abnormal PHES had higher CRP (17 ± 22 vs 7 ± 6, P < 0.01), IL6 (32 ± 54 vs 12 ± 13, P < 0.05) and TNFα (17 ± 8 vs 11 ± 7, P < 0.001) levels than those with normal PHES. Patients with abnormal EEG had higher indole (430 ± 270 vs 258 ± 255, P < 0.01) and ammonia (66 ± 35 vs 45 ± 27, P < 0.05) levels than those with normal EEG. Psychometric test scores showed significant correlations with CRP, TNFα and IL6; EEG indices with ammonia and IL6. CRP and TNFα concentrations were independent predictors of abnormal PHES, ammonia and indole of abnormal EEG on multivariate analysis. Seven patients were lost to follow-up; of the remaining 65, 20 died and 14 underwent transplantation; 15 developed HE requiring hospitalization. PHES and EEG performance were independent predictors of HE and death (P < 0.05)., Conclusion: PHES and EEG abnormalities in patients with cirrhosis have partially different biochemical correlates and independently predict outcome., (Copyright © 2010 American Association for the Study of Liver Diseases.)

Objectives: Intestinal bacteria metabolize tryptophan into indole, which is then further metabolized into oxindole, a sedative compound putatively involved in the pathophysiology of hepatic encephalopathy (HE). The aim of this study was to measure indole and oxindole levels in patients with cirrhosis with or without HE and to establish whether an intestinal production and a hepatic metabolism of these substances exist., Methods: We studied 10 healthy subjects (controls) and 51 cirrhotic patients: 17 without HE, 14 with a minimal HE, 8 with overt HE, and 12 who had undergone a transjugular intrahepatic portosystemic shunt (TIPS) procedure. In the last group, blood was collected from the artery, and the portal and hepatic veins during TIPS construction and from the peripheral veins before, immediately after, and at weekly intervals during the first month after TIPS., Results: Plasma indole levels were significantly higher in patients with overt HE. Oxindole levels were higher in cirrhotics than in controls. Indole and ammonia were significantly correlated (r=0.66). Peripheral and splanchnic determinations showed that indole was produced in the intestine and cleared by the liver, similar to ammonia. TIPS implantation increased both indole and ammonia levels. After TIPS, the psychometric performance worsened in 4 of the 12 patients. The increase in indole plasma concentrations in these four patients was higher than in those who remained stable after undergoing TIPS., Conclusions: Indole correlates with HE and has a significant intestinal production and hepatic extraction; its level increases after TIPS and is related to psychometric performance. These data suggest that indole may be involved in the pathophysiology of HE.

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor that is expressed throughout the central nervous system. PAR1 activation by brain-derived as well as blood-derived proteases has been shown to have variable and complex effects in a variety of animal models of neuronal injury and inflammation. In this study, we have evaluated the effects of PAR1 on lesion volume in wild-type or PAR1-/- C57Bl/6 mice subjected to transient occlusion of the middle cerebral artery or injected with NMDA in the striatum. We found that removal of PAR1 reduced infarct volume following transient focal ischemia to 57% of control. Removal of PAR1 or application of a PAR1 antagonist also reduced the neuronal injury associated with intrastriatal injection of NMDA to 60% of control. To explore whether NMDA receptor potentiation by PAR1 activation contributes to the harmful effects of PAR1, we investigated the effect of NMDA receptor antagonists on the neuroprotective phenotype of PAR1-/- mice. We found that MK801 reduced penumbral but not core neuronal injury in mice subjected to transient middle cerebral artery occlusion or intrastriatal NMDA injection. Lesion volumes in both models were not significantly different between PAR1-/- mice treated with and without MK801. Use of the NMDA receptor antagonist and dissociative anesthetic ketamine also renders NMDA-induced lesion volumes identical in PAR1-/- mice and wild-type mice. These data suggest that the ability of PAR1 activation to potentiate NMDA receptor function may underlie its harmful actions during injury.

An emerging body of evidence supports a key role for the endocannabinoid system in numerous physiological and pathological mechanisms of the central nervous system. In the recent past, many experimental studies have examined the putative protective or toxic effects of drugs interacting with cannabinoid receptors or have measured the brain levels of endocannabinoids in in vitro and in vivo models of cerebral ischemia. The results of these studies have been rather conflicting in supporting either a beneficial or a detrimental role for the endocannabinoid system in post-ischemic neuronal death, in that cannabinoid receptor agonists and antagonists have both been demonstrated to produce either protective or toxic responses in ischemia, depending on a number of factors. Among these, the dose of the administered drug and the specific endocannabinoid that accumulates in each particular model appear to be of particular importance. Other mechanisms that have been put forward to explain these discrepant results are the effects of cannabinoid receptor activation on the modulation of excitatory and inhibitory transmission, the vasodilatory and hypothermic effects of cannabinoids, and their activation of cytoprotective signaling pathways. Alternative mechanisms that appear to be independent from cannabinoid receptor activation have also been suggested. Endocannabinoids probably participate in the mechanisms that are triggered by the initial ischemic stimulus and lead to delayed neuronal death. However, further information is needed before pharmacological modulation of the endocannabinoid system may prove useful for therapeutic intervention in stroke and related ischemic syndromes.

Experimental evidence indicates that metabotropic glutamate (mGlu) receptors of the mGlu1 and mGlu5 subtypes play a differential role in models of cerebral ischemia and that only mGlu1 receptors are implicated in the pathways leading to postischemic neuronal injury. The localization of mGlu1 receptors in GABA-containing interneurons rather than in hippocampal CA1 pyramidal cells that are vulnerable to ischemia has prompted experimental studies that have demonstrated mGlu1 receptor antagonist agents attenuate postischemic injury by enhancing GABA-mediated neurotransmission, thus providing a new viewpoint on the neuroprotective mechanism of these pharmacological agents. In view of the recent discovery of a functional interaction between group I mGlu receptors and the cannabinoid system in the modulation of synaptic transmission, we propose a novel mechanism that predicts that the neuroprotective effects of mGlu1 receptor antagonists on CA1 pyramidal cells are mediated by a mechanism that overcomes the "synaptic circuit break" operated by endocannabinoids on GABAergic transmission.

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.

Astrocytes express a wide range of G-protein coupled receptors that trigger release of intracellular Ca2+, including P2Y, bradykinin and protease activated receptors (PARs). By using the highly sensitive sniffer-patch technique, we demonstrate that the activation of P2Y receptors, bradykinin receptors and protease activated receptors all stimulate glutamate release from cultured or acutely dissociated astrocytes. Of these receptors, we have utilized PAR1 as a model system because of favourable pharmacological and molecular tools, its prominent expression in astrocytes and its high relevance to neuropathological processes. Astrocytic PAR1-mediated glutamate release in vitro is Ca2+ dependent and activates NMDA receptors on adjacent neurones in culture. Activation of astrocytic PAR1 in hippocampal slices induces an APV-sensitive inward current in CA1 neurones and causes APV-sensitive neuronal depolarization in CA1 neurones, consistent with release of glutamate from astrocytes. PAR1 activation enhances the NMDA receptor-mediated component of synaptic miniature EPSCs, evoked EPSCs and evoked EPSPs in a Mg2+-dependent manner, which may reflect spine head depolarization and consequent reduction of NMDA receptor Mg2+ block during subsequent synaptic currents. The release of glutamate from astrocytes following PAR1 activation may also lead to glutamate occupancy of some perisynaptic NMDA receptors, which pass current following relief of tonic Mg2+ block during synaptic depolarization. These results suggest that astrocytic G-protein coupled receptors that increase intracellular Ca2+ can tune synaptic NMDA receptor responses.

Recent studies implicated involvement of the 5-hydroxytryptamine4 (5-HT4) receptor in cognitive and emotional processes. The highest 5-HT4 receptor densities in the brain are found in the limbic system including the hippocampus. Here we used the selective 5-HT4 receptor full agonist, N-pentyl-N'-aminoguanidine carbazimidamide (SDZ-216454) to characterize effects of 5-HT4 receptor activation in whole-cell and field recordings in the area CA1 in hippocampal slices prepared from 3 to 4- and 6 to 9-week-old rats, respectively. Extracellular recordings showed that transient 5-HT4 receptor activation by 10-20 min application of SDZ-216454 induces field excitatory postsynaptic potential (fEPSP)-population spike potentiation (ESP(5-HT4)), which persisted for as long as we held the recordings (> 2 h). ESP(5-HT4) displayed characteristics different from EPSP-spike potentiation that accompanies long-term potentiation; it developed without an associated increase in synaptic transmission, was independent on afferent input, activity of postsynaptic neurons and N-methyl-d-aspartate receptor activation; and was expressed in the presence of GABA receptor antagonists. ESP(5-HT4) was also induced by transient application of the natural neurotransmitter, 5-HT. The increase in the evoked population spike (PS) induced by SDZ-216454 was not prevented by blockers of hyperpolarization-activated cation current (Ih), Cs+ and ZD-7288, but was mimicked and occluded by 150 microm Ba2+. Whole-cell voltage-clamp recordings from pyramidal neurons demonstrated that SDZ-216454 application increases membrane resistance with a concomitant decrease in a Ba2+-sensitive inwardly rectifying K+ current and the Ba2+-insensitive K+ current underlying slow afterhyperpolarization (I(sAHP)). We conclude that 5-HT4 receptor activation may cause a long-lasting excitability increase in CA1 pyramidal neurons by inhibition of a Ba2+-sensitive inwardly rectifying K+ current.

Background: Neuroleptic malignant syndrome (NMS) is a rare side effect of antipsychotic therapy characterized by fever, muscular rigidity, altered mental status, increased level of serum creatinine phosphokinase, and increased number of white blood cells. The mortality rate of patients with NMS remains elevated., Methods: We examined the clinical records of patients diagnosed with severe NMS admitted to the Clinical Toxicology Unit, Florence University Hospital, between 1990 and 2004., Results: Eight patients presented with this neurological disorder. All were treated with supportive therapy, which included dantrolene, levodopa/benserazide, benzodiazepines, metamizole and/or paracetamol, and antibiotics. Five survived and three died. Of the three deceased, two had large hemorrhages in the gastrointestinal tract, and one had massive liver damage and diffuse hemorrhages throughout the body., Conclusion: Our results suggest that gastrointestinal bleeding is a frequent cause of death in NMS patients. Bleeding may occur as a consequence of commonly accepted medical treatments (especially the use of cyclooxygenase inhibitors as antipyretic agents) and NMS-induced changes in blood coagulation status. To increase the survival rate of these patients, it is necessary to avoid using drugs that may facilitate gastrointestinal lesions and to utilize procedures known to decrease the risk of bleeding.

Glutamate plays an important role in the regulation of dopamine neuron activity. In particular, the glutamatergic input from the subthalamic nucleus is thought to provide control over dopamine neuron firing patterns. The degeneration of dopamine neurons in the substantia nigra pars compacta (SNc) observed in Parkinson's disease (PD) is believed to be due to a complex interplay of factors, including oxidative stress and mitochondrial dysfunction. Although glutamate is not the primary cause of cell death in PD, there is evidence suggesting excessive glutamate release onto dopamine neurons may play a role in continued degeneration. Although many studies have focused on the role of glutamate in the SNc, little work has been directed at exploring the modulatory control of glutamate release in this region. Previous studies have found a high-potency inhibitory effect of nonselective group III mGluR agonist on glutamatergic transmission in the SNc. Using whole-cell patch-clamp methods and novel pharmacological tools, we have determined that mGluR4 mediates the group III mGluR modulation of excitatory transmission in the rat SNc. The group III mGluR-selective agonist l-(+)-2-amino-4-phosphonobutyric acid inhibits excitatory transmission in the SNc at low micromolar concentrations with a maximal inhibition occurring at 3 muM. This effect was potentiated by the mGluR4-selective allosteric modulator N-phenyl-7-(hydroxymino)cyclopropa[b]chromen-1a-carboxamide and was not mimicked by the mGluR8-selective agonist (S)-3,4-dicarboxyphenylglycine. Interestingly, in an attempt to employ knockout mice to confirm the role of mGluR4, we discovered an apparent species difference suggesting that in mice, both mGluR4 and mGluR8 modulate excitatory transmission in the SNc.

The serine proteases tissue plasminogen activator, plasmin, and thrombin and their receptors have previously been suggested to contribute to neuronal damage in certain pathological situations. Here we demonstrate that mice lacking protease-activated receptor 1 (PAR1) have a 3.1-fold reduction in infarct volume after transient focal cerebral ischemia. Intracerebroventricular injection of PAR1 antagonist BMS-200261 reduced infarct volume 2.7-fold. There are no detectable differences between PAR1-/- and WT mice in cerebrovascular anatomy, capillary density, or capillary diameter, demonstrating that the neuroprotective phenotype is not likely related to congenital abnormalities in vascular development. We also show that the exogenously applied serine proteases thrombin, plasmin, and tissue plasminogen activator can activate PAR1 signaling in brain tissue. These data together suggest that if blood-derived serine proteases that enter brain tissue in ischemic situations can activate PAR1, this sequence of events may contribute to the harmful effects observed. Furthermore, PAR1 immunoreactivity is present in human brain, suggesting that inhibition of PAR1 may provide a novel potential therapeutic strategy for decreasing neuronal damage associated with ischemia and blood-brain barrier breakdown.

1 5-hydroxyindole (5-OHi) is a proposed tryptophan metabolite able to cause convulsions when systemically injected into rodents. We studied its effects using microdialysis in vivo and electrophysiological approaches in vitro. 2 Local administration of 5-OHi into the CA1 region of the rat hippocampus, via a microdialysis probe, significantly increased glutamate concentrations in the dialysates. 3 In rat hippocampal slices, using extracellular recordings in the CA1 region, 5-OHi (30-300 microM) increased the amplitude of population spikes and fEPSPs. 4 In the same preparation, using intracellular recordings in CA1 pyramidal neurons, 5-OHi reduced the latency of firing induced by direct depolarization and increased both evoked excitatory and slow inhibitory postsynaptic potential amplitudes, without affecting the resting membrane potential, the after-hyperpolarization or the neuronal input resistance. It also altered GABA(A)-mediated neurotransmission by increasing the frequency and the amplitude of pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSC). 5 In separate experiments, performed by measuring AMPA or NMDA-induced depolarization in cortical wedges, 5-OHi did not modify glutamate receptor agonist responses. 6 Our results show that 5-OHi causes convulsions, modifies the properties and the function of the hippocampal circuitry, and facilitates the output of both excitatory and inhibitory transmitters.

Group I metabotropic glutamate receptors (mGluRs) 1 and 5 frequently colocalize in the same neurons throughout the CNS. Because both receptors can couple to the same effector systems, the purpose of their cellular coexpression remains unclear. Here, we report that group I mGluR1 and mGluR5 have distinct functional roles in type II neurons of the rat globus pallidus (GP). Type II GP neurons form a large population of GABAergic projection neurons that are characterized by the presence of inwardly rectifying current I(h), low-threshold voltage-activated calcium current I(t), and activity at rest. Although immunocytochemical analysis reveals a high degree of neuronal colocalization of the two group I mGluRs in the GP, activation of mGluR1 only directly depolarizes type II GP neurons. Interestingly, blockade of mGluR5 by a highly selective antagonist, methylphenylethynylpyridine, leads to the potentiation of the mGluR1-mediated depolarization in this neuronal subpopulation. Metabotropic GluR1 desensitizes during repeated activation with the agonist in type II GP neurons, and blocking mGluR5 prevents the desensitization of the mGluR1-mediated depolarization. Elimination of the activity of protein kinase C (PKC) by an application of 1 microm bisendolylmaleimide or 1 microm chelerythrine, both protein kinase C inhibitors, potentiates the mGluR1-mediated response and prevents the desensitization of mGluR1 in type II GP neurons, suggesting that the effect of mGluR5 on mGluR1 signaling may involve PKC. Together, these data illustrate a novel mechanism by which mGluR1 and mGluR5, members of the same family of G-protein-coupled receptors, can interact to modulate neuronal activity in the rat GP.

An 80-year-old diabetic patient was admitted to the hospital because of sudden unconsciousness and severe metabolic acidosis. His son reported the possibility of cyanide poisoning. Clinical data and the detection of cyanide in blood and gastric material confirmed this possibility. Supportive therapy and the following antidotes--sodium nitrite two doses 300 mg i.v., sodium thiosulfate 3 g i.v., and hydroxocobalamin 4 g in 24 hours--were administered immediately and the patient completely recovered in 48 hours. Our observations suggest that timely and appropriate use of antidotes for cyanide intoxication may prevent death, even in aged diabetic patients.

The activation of group I metabotropic glutamate receptors (mGluRs) produces a variety of actions that lead to alterations in excitability and synaptic transmission in the CA1 region of the hippocampus. The group I mGluRs, mGluR1 and mGluR5, are activated selectively by (S)-3,5-dihydroxyphenylglycine (DHPG). To identify which of these mGluR subtypes are responsible for the various actions of DHPG in area CA1, we took advantage of two novel subtype-selective antagonists. (S)-(+)-alpha-amino-a-methylbenzeneacetic acid (LY367385) is a potent competitive antagonist that is selective for mGluR1, whereas 2-methyl-6-(phenylethynyl)-pyridine (MPEP) is a potent noncompetitive antagonist that is selective for mGluR5. The use of these compounds in experiments with whole-cell patch-clamp recording and Ca(2+)-imaging techniques revealed that each group I mGluR subtype plays distinct roles in regulating the function of CA1 pyramidal neurons. The block of mGluR1 by LY367385 suppressed the DHPG-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the direct depolarization of CA1 hippocampal neurons. In addition, the increase in the frequency of spontaneous IPSCs (sIPSCs) caused by the DHPG-induced depolarization of inhibitory interneurons also was blocked by LY367385, as was the DHPG-induced inhibition of transmission at the Schaffer collateral-->CA1 synapse. On the other hand, the block of mGluR5 by MPEP antagonized the DHPG-induced suppression of the Ca(2+)-activated potassium current (I(AHP)) and potentiation of the NMDA receptor. Finally, antagonism of the DHPG-induced suppression of evoked IPSCs required the blockade of both mGluR1 and mGluR5. These data suggest that mGluR1 and mGluR5 play distinct roles in the regulation of the excitability of hippocampal CA1 pyramidal neurons.

Oxindole administration (1-100 mg/kg i.p.) to mammals decreases locomotor activity, reduces muscular tone and blood pressure and at larger doses causes coma and death. Utilizing both HPLC and GC/MS, we showed that oxindole is present in the blood, brain and other organs of several animal species, including humans. We demonstrated that oxindole is a tryptophan metabolite able to significantly decrease neuronal excitability by modifying the function of voltage-operated sodium channels. Its synthesis requires the availability of indole, which is formed in the gut. When liver function is impaired, a sufficient amount of indole reaches systemic circulation and is oxidized into oxindole, which seems to be one of the responsible agents for the neurological symptoms found in the course of liver impairment.

1. The aim of the present work was to investigate the electrophysiological effects of oxindole, a tryptophan metabolite present in rat blood and brain, and recently proposed as a contributing factor in the pathogenesis of hepatic encephalopathy. 2. Using rat hippocampal slices in vitro and extra- or intracellular recordings, we evaluated oxindole effects on the neurotransmission of the CA1 region following orthodromic stimulation of the Schaffer collaterals. 3. Oxindole (0.3-3 mM) decreased the amplitude of population spikes extracellularly recorded at the somatic level and of the fEPSPs recorded at the dendritic level. In intracellular recordings, oxindole (0.1-3 mM) did not affect the resting membrane potential or the neuronal input resistance, but reduced the probability of firing action potentials upon either synaptic or direct activation of the pyramidal cells. 4. Oxindole (0.3-3 mM) increased the threshold and the latency of firing action potentials elicited by depolarizing steps without changing the duration or the peak amplitude of the spikes. It also significantly increased the spike frequency adaptation induced by long lasting (400 ms) depolarizing stimuli. 5. In separate experiments, performed by measuring AMPA or NMDA-induced responses in cortical slices, oxindole (1-3 mM) did not modify glutamate receptor agonist responses. 6. Our results show that concentrations of oxindole which may be reached in pathological conditions, significantly decrease neuronal excitability by modifying the threshold of action potential generation.

Rats treated with oxindole (10-100 mg/kg i.p.), a putative tryptophan metabolite, showed decreased spontaneous locomotor activity, loss of the righting reflex, hypotension, and reversible coma. Brain oxindole levels were 0.05 +/- 0.01 nmol/g in controls and increased to 8.1 +/- 1.7 or 103 +/- 15 nmol/g after its administration at doses of 10 or 100 mg/kg i.p., respectively. To study the role that oxindole plays in the neurological symptoms associated with acute liver failure, we measured the changes of its concentration in the brain after massive liver damage, and we investigated the possible metabolic pathways leading to its synthesis. Rats treated with either thioacetamide (0.2 and 0.4 g/kg i.p., twice) or galactosamine (1 and 2 g/kg i.p.) showed acute liver failure and a large increase in blood or brain oxindole concentrations (from 0.05 +/- 0.01 nmol/g in brains of controls to 1.8 +/- 0.3 nmol/g in brains of thioacetamide-treated animals). Administration of tryptophan (300-1,000 mg/kg p.o.) caused a twofold increase, whereas administration of indole (10-100 mg/kg p.o.) caused a 200-fold increase, of oxindole content in liver, blood, and brain, thus suggesting that indole formation from tryptophan is a limiting step in oxindole synthesis. Oral administration of neomycin, a broad-spectrum, locally acting antibiotic agent able to reduce intestinal flora, significantly decreased brain oxindole content. Taken together, our data show that oxindole is a neurodepressant tryptophan metabolite and suggest that it may play a significant role in the neurological symptoms associated with acute liver impairment.

1. Mouse cortical wedge preparations were used in order to study the effects of metabotropic glutamate receptor (mGluR) agonists and antagonists on the depolarization induced by N-methyl-D-aspartate (NMDA) or by (S)-alpha-amino-4-bromo-3-hydroxy-5-isoxazolepropionic acid (AMPA). 2. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (30-300 microM) significantly potentiated the depolarizations induced by NMDA, leaving unchanged those mediated by AMPA. This potentiation developed slowly and lasted for up to 60 min provided that the slices were continuously perfused with the mGluR agonist. 3. Concentration-response curves to NMDA in the absence and in the presence of 1S,3R-ACPD (100 microM) indicated that the potentiation was due to increased affinity of the NMDA receptor complex for its agonist. The maximal responses to NMDA were not potentiated. 4. Selective agonists of group 1 mGluR such as quisqualate (Quis) (30 microM) or (RS)-3,5-dihydroxyphenylglycine (DHPG) (300 microM) did not potentiate NMDA responses. Similarly, selective agonists of group 2 mGluRs, such as (2S,3S,4S)-alpha-carboxycyclopropyl-glycine (L-CCG-I) (3-30 microM), and of group 3, such as L-2-amino-4-phosphonobutyric acid (L-AP4) (100 microM) were inactive in our test. A number of other putative mGluR agents having partial agonist activity on mGluRs in brain slices and in expression systems, such as 1R,3S-ACPD (500 microM), DL-2-amino-3-phosphonopropionic acid (DL-AP3) (300 microM) and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG; 500 microM), when placed in the experimental protocol we used, did not change NMDA responses. 5. Available mGluR antagonists, such as DL-AP3 (1 mM), (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) (500 microM), S-4-carboxyphenylglycine (4CPG; 500 microM) and S-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG; 500 microM), did not reduce 1S,3R-ACPD potentiation of NMDA responses. 6. It is concluded that the potentiation of NMDA currents induced by the mGluR agonist 1S,3R-ACPD, in mouse cortical wedges, has a pharmacological profile which is different from that of the three mGluR groups so far described in expression systems.

Several potent and selective agonists of the glutamate (L-GLU) receptors of N-methyl-D-aspartate (NMDA) type have been tested on the L-[3H]GLU binding to rat cortical membranes, on the depolarization of mouse cortical wedges and on the contraction of guinea pig longitudinal muscle myenteric plexus preparations with the aim of comparing the NMDA receptors present in the cortex and those present in the gut. When the depolarization of the cortical wedges was evaluated, the EC50 values of the agonists were (microM): (R,S)-(tetrazol-5-yl)-glycine (TG) 0.3; trans-4-hydroxy-(S)-pipecolic acid-4-sulfate (t-HPIS) 0.7; 1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACBD) 0.8; NMDA 8; (2S,3R,4S) cyclopropylglutamate (L-CGA C) 12; quinolinic acid (QUIN) 400. When the contraction of the longitudinal muscle myenteric plexus was evaluated, the EC50 values were (microM): L-CGA C 1; TG 8; ACBD 50; t-HPIS 100; QUIN 500 and NMDA 680. When the displacement of NMDA specific L-[3H]GLU binding from rat cortical membranes was evaluated, the IC50 values were (microM): L-CGA C 0.003; TG 0.005; ACBD 0.044; t-HPIS 0.062; NMDA 0.31 and QUIN 15. No significant correlation was found when the EC50 values obtained in the ileum were plotted against the EC50 values obtained in the cortex (r = 0.47). In particular it was noted that L-CGA C was approximately three orders of magnitude more potent than NMDA when tested in the ileum but had a potency not significantly different from that of NMDA when tested in the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)