1. Intracellular recordings were made from layer V pyramidal neurons in slices of rat cingulate cortex. Electrodes contained potassium methylsulphate and biocytin for subsequent histology. 2. Synaptic potentials were separated pharmacologically with DL-2-amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), bicuculline, and 2-hydroxysaclofen into components mediated, respectively, by N-methyl-D-aspartate (NMDA) type excitatory amino acid, non-NMDA type excitatory amino acid, gamma-aminobutyric acid (GABAA), and GABAB receptors. Components mediated at excitatory amino acid, GABAA and GABAB receptors reversed polarity at -11, -76, and -108 mV, respectively. 3. When synaptic potentials were evoked by stimulation to the subcortical white matter, 5-hydroxytryptamine (5-HT; 1-100 microM) reversibly reduced the amplitude of NMDA, non-NMDA, GABAA, and GABAB components. Selective agonists and antagonists were used to show that this resulted from activation of 5-HT1B receptors. 4. When synaptic potentials were evoked by stimulation within layer V, 5-HT reduced the amplitude only of the NMDA and non-NMDA components but did not affect the GABAA and GABAB components. 5-HT did not change the amplitude of depolarizations evoked by direct application of glutamate. 5. It is concluded that 5-HT presynaptically inhibits the release of excitatory amino acids at synapses onto prefrontal pyramidal neurons and at synapses onto local feed-forward inhibitory interneurons. 6. 5-HT also hyperpolarized, depolarized, or did not change the membrane potential. The hyperpolarization involved 5-HT1A receptors and resulted from potassium conductance increase. The depolarization involved 5-HT2 receptors and resulted from potassium conductance decrease.

Edaravone was originally developed as a potent free radical scavenger, and has been widely used to treat acute ischemic stroke in Japan since 2001. Free radicals play an important role in the pathogenesis of a variety of diseases, such as cardiovascular diseases and stroke. Therefore, free radicals may be targets for therapeutic intervention in these diseases. Edaravone shows protective effects on ischemic insults and inflammation in the heart, vessel, and brain in experimental studies. As well as scavenging free radicals, edaravone has anti-apoptotic, anti-necrotic, and anti-cytokine effects in cardiovascular diseases and stroke. Edaravone has preventive effects on myocardial injury following ischemia and reperfusion in patients with acute myocardial infarction. Edaravone may represent a new therapeutic intervention for endothelial dysfunction in the setting of atherosclerosis, heart failure, diabetes, or hypertension, because these diseases result from oxidative stress and/or cytokine-induced apoptosis. This review evaluates the potential of edaravone for treatment of cardiovascular disease, and covers clinical and experimental studies conducted between 1984 and 2013. We propose that edaravone, which scavenges free radicals, may offer a novel option for treatment of cardiovascular diseases. However, additional clinical studies are necessary to verify the efficacy of edaravone.

Neurons in the nucleus accumbens septi in brain slices from adult male rats were studied with patch clamp recording in the whole-cell conformation. Cells filled with Lucifer Yellow were identified as medium spiny neurons. Electrical stimulation close to the recorded cell evoked excitatory and inhibitory synaptic currents. In the presence of picrotoxin or bicuculline, stimulation at a holding potential of -90 mV evoked an inward excitatory current that was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), identifying it as an excitatory postsynaptic current (EPSC) mediated by glutamate acting at AMPA/kainate receptors. Serotonin (5-hydroxytryptamine, 5-HT; 3-100 microM in the bath) decreased the EPSC in about 90% of the cells. The action of 5-HT was mimicked by N-(3-trifluoromethylphenyl)-piperazine HCl (TFMPP), but not by (+/-)-8-hydroxydipropylaminotetralin (8-OH-DPAT) or (+/-)-2,5-dimethoxy-4-iodoamphetamine HCl (DOI). The 5-HT effect was antagonized by pindolol or cyanopindolol, but not by spiperone, ketanserin or tropisetron. Taken together, these results indicate that 5-HT acts at 5-HT1B receptors. The effect of 5-HT was potentiated by cocaine (0.3-3 microM) or the selective serotonin reuptake inhibitor citalopram. Miniature synaptic currents recorded in the presence of tetrodotoxin were inhibited by CNQX, identifying them as spontaneous miniature EPSCs. 5-HT reduced the frequency of these miniature EPSCs without affecting their amplitude, which indicates a presynaptic site of action. This presynaptic inhibition by 5-HT might be involved in the behavioural effects of cocaine.

Rett syndrome (RTT) is a neurodevelopmetal disorder associated with mutations in the methyl-CpG–binding protein 2 (MeCP2) gene. MeCP2-deficient mice recapitulate the neurological degeneration observed in RTT patients. Recent studies indicated a role of not only neurons but also glial cells in neuronal dysfunction in RTT. We cultured astrocytes from MeCP2-null mouse brain and examined astroglial gene expression, growth rate, cytotoxic effects, and glutamate (Glu) clearance. Semi-quantitative RT-PCR analysis revealed that expression of astroglial marker genes, including GFAP and S100β, was significantly higher in MeCP2-null astrocytes than in control astrocytes. Loss of MeCP2 did not affect astroglial cell morphology, growth, or cytotoxic effects, but did alter Glu clearance in astrocytes. When high extracellular Glu was added to the astrocyte cultures and incubated, a time-dependent decrease of extracellular Glu concentration occurred due to Glu clearance by astrocytes. Although the shapes of the profiles of Glu concentration versus time for each strain of astrocytes were grossly similar, Glu concentration in the medium of MeCP2-null astrocytes were lower than those of control astrocytes at 12 and 18 h. In addition, MeCP2 deficiency impaired downregulation of excitatory amino acid transporter 1 and 2 (EAAT1/2) transcripts, but not induction of glutamine synthetase (GS) transcripts, upon high Glu exposure. In contrast, GS protein was significantly higher in MeCP2-null astrocytes than in control astrocytes. These findings suggest that MeCP2 affects astroglial genes expression in cultured astrocytes, and that abnormal Glu clearance in MeCP2-deficient astrocytes may influence the onset and progression of RTT.

In CA1 pyramidal neurons in rat hippocampal tissue slices, superfusion with ischemia-simulating medium produced a rapid depolarization after 6 min of exposure. The membrane potential eventually reached 0 after 5 min (a persistent depolarization), even when oxygen and glucose were reintroduced. The role of various ions in the reversal of this persistent depolarization after reintroduction of oxygen and glucose was investigated. The peak of the persistent depolarization was decreased in solutions containing reduced Na+ or Ca2+ and in solutions containing Co2+ or Ni2+. In contrast, the depolarization was not affected by reduction of external K+ or Cl- or by addition of tetrodotoxin (TTX), flunarizine, or nifedipine. These results suggest that sustained Na+ and Ca2+ influxes produce the persistent depolarization. The membrane potential recovered after reintroduction of oxygen and glucose in low Ca2+, low Cl-, or K+-rich medium and in TTX- or tetraethylammonium-containing medium, but not in low Na+ or low K+ medium and in flunarizine- or nifedipine-containing medium. Either reduction in extracellular Ca2+ or addition of Co2+ was the most effective in promoting recovery from the persistent depolarization, suggesting that Ca2+ influx has a key role in causing the membrane dysfunction. The peak of the persistent depolarization was reduced by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-5-phosphonopentanoic acid (AP5), DL-amino-3-phosphonopropionic acid (AP3), or DL-amino-4-phosphonobutyric acid, suggesting that activation of non-N-methyl-D-aspartate (non-NMDA), NMDA, and metabotropic glutamate (Glu) receptors is involved in the generation and maintenance of the persistent depolarization. Among these Glu receptor antagonists, only CNQX or AP5 was able to reduce dose dependently the level of depolarization, suggesting that Ca2+ influx via both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate type II receptors and NMDA receptors contributes to the membrane dysfunction. trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) did not affect the peak potential of the persistent depolarization, but it dose-dependently restored the membrane potential. AP3 antagonized the protective action of t-ACPD. The membrane potential also recovered after reintroduction when the slice was pretreated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, ryanodol 3-(1H-pyrrole-2-carboxylate), 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride, and procaine, suggesting that raised [Ca2+]i from Ca2+-induced Ca2+ release pool contributes to the membrane dysfunction. It, therefore, is concluded that raised [Ca2+]i has a dominant role in causing irreversible changes. The increase in [Ca2+]i during the persistent depolarization may be the result of Ca2+ entry via both a leaky membrane and Glu-activated receptor channels as well as Ca2+ released from internal stores.

Intracellular recordings were made from layer V pyramidal neurons in slices of rat anterior cingulate cortex, using electrodes that contained potassium methylsulfate and biocytin. [Met5]enkephalin (300 nM to 30 microM) reversibly reduced the amplitude of EPSPs evoked by stimulation of the subcortical white matter; the half-maximal concentration was about 800 nM. These EPSPs were blocked by (+/-)-2- amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. [Met5]enkephalin also reduced the amplitude of bicuculline-sensitive IPSPs evoked by stimulation within layer V; the half-maximal concentration was about 60 nM. Both these actions of [Met5]enkephalin were mimicked by the delta-selective agonist DPDPE (Tyr-D-Pen-Gly-Phe-D- Pen) but not by the mu-selective agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly- ol); they were blocked by the delta-selective antagonist naltrindole (apparent dissociation constant of about 0.3 nM) but not by the mu- selective antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). [Met5]enkephalin did not change the amplitudes of depolarizations evoked by direct application of glutamate or hyperpolarizations evoked by direct application of muscimol (at -55 mV). Fifty percent (22 of 45) of pyramidal cells were hyperpolarized by [Met5]enkephalin; this resulted from an increase in potassium conductance, and it was mimicked by DPDPE and blocked by naltrindole. Five of seven nonpyramidal cells were hyperpolarized by [Met5]enkephalin; this was mimicked by DAMGO and blocked by CTOP.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute ischemic stroke (AIS) is a major cause of mortality and disability and remains a serious and significant global health problem. The development of neurovascular protectants to treat AIS successfully has been beset by disappointments and setbacks. Many promising candidates have lacked significant pleiotropic protective activity for brain tissue and cerebral blood vessels in clinical trials, while those with protective activity have had poor bioavailability or high toxicity. Moreover, the majority of agents did not confer significant neurovascular protection or clinical efficacy, as measured by standard behavioral endpoints in clinical trials of heterogeneous populations of patients with AIS. The recombinant tissue plasminogen activator alteplase is approved in many countries for the treatment of AIS in the first 3 h after symptom onset. Many drug candidates have been subject to clinical trials, including those with anti-excitotoxic, anti-inflammatory, antioxidant, antiapoptotic/regenerative, calcium/adrenergic-modulating/antihypertensive, thrombolytic, nootropic/stimulant, fluid regulatory, or oxygen-delivering mechanisms of action. Some agents, such as tenecteplase, edaravone and minocycline, may be approved for global use in the future. This review evaluates almost all neurovascular protectants subject to clinical trial evaluation for the treatment of AIS, and includes 241 studies conducted between 1978 and 2014. The development of agents that reduce brain injury after AIS will require new and different approaches based on a deeper understanding of the pathophysiology of AIS. Moreover, the future treatment for AIS is likely to lie in combination therapy rather than monotherapy. Additional approaches to the testing and use of neurovascular protectants should be considered.

In response to oxygen deprivation, CA1 pyramidal neurons show a hyperpolarization (hypoxic hyperpolarization), which is associated with a reduction in neuronal input resistance. The role of extra- and intracellular Ca2+ ions in hypoxic hyperpolarization was investigated. The hypoxic hyperpolarization was significantly depressed by tolbutamide (100 microM); moreover, the response was reversed in its polarity in medium containing tolbutamide (100 microM), low Ca2+ (0.25 mM), and Co2+ (2 mM), suggesting that the hypoxic hyperpolarization is mediated by activation of both ATP-sensitive K+ (KATP) channels and Ca(2+)-dependent K+ channels. The hypoxic depolarization in medium containing tolbutamide, low Ca2+, and Co2+ is probably due to inhibition of the electrogenic Na(+)-K+ pump and concomitant accumulation of interstitial K+. Hypoxic hyperpolarizations were depressed in either low Ca2+ (0.25 or 1.25 mM) or high Ca2+ (5 or 7.5 mM) medium (control: 2.5 mM), indicating that there is an optimal extracellular Ca2+ concentration required to produce the hypoxic hyperpolarization. Bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM (50-100 microM), procaine (300 microM), or ryanodine (10 microM) significantly depressed the hypoxic hyperpolarization, suggesting that Ca2+ released from intracellular Ca+ stores may have an important role in the generation of hypoxic hyperpolarization. The high-affinity calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonomide hydrochloride (W-7) (5 microM) completely blocked, whereas the low-affinity calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonomide hydrochloride (W-5) (50 microM) did not affect, the hypoxic hyperpolarization. The calmodulin inhibitor trifluoperazine (50 microM) also suppressed the hypoxic hyperpolarization. In addition, calcium/ calmodulin kinase II inhibitor 1-[N,O-bis (1,5-isoquinol-inesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-pip erazine (KN-62) (10 microM) markedly depressed the amplitude and net outward current of the hypoxic hyperpolarization without affecting the reversal potential. In contrast, neither the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexa-hydro-1,4-diazepin hydrochloride (ML-7) (10 microM) nor the protein kinase A inhibitor N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinolinesulfonamide (H-89) (1 microM) significantly altered the hypoxic hyperpolarization. These results suggest that calmodulin kinase II, which is activated by calmodulin, may contribute to the generation of the hypoxic hyperpolarization. In conclusion, the present study indicates that, in the majority of hippocampal CA1 neurons, the hypoxic hyperpolarization is due to activation of both KATP channels and Ca(2+)-dependent K+ channels.