The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K + currents in cultured neostriatal neurones, was investigated in medium‐spiny neurones and cholinergic interneurones contained in neostriatal brain slices from 5–26‐day‐old rats. In these cells, adenosine‐5′‐triphosphate (ATP) (100–1000 μ M ), 2‐methylthioadenosine‐5′‐triphosphate (2MeSATP), α,β ‐methyleneadenosine‐5′‐triphosphate ( α,β meATP, 30–300 μ M , each) and adenosine‐5′‐ O ‐(3‐thiotriphosphate (ATP γ S) (100 μ M ) failed to evoke P2X receptor currents even when 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 0.1 μ M ), apyrase (10 U ml −1 ) or intracellular Cs + was used to prevent occluding effects of the ATP breakdown product adenosine, desensitisation of P2X receptors by endogenous ATP and an interference with the activation of K + channels, respectively. P2X receptor agonists were also ineffective in outside‐out patches withdrawn from the brain slice tissue. Muscimol (10 μ M ) evoked GABA A receptor‐mediated currents under all these conditions. When used as a control, locus coeruleus neurones responded with P2X receptor‐mediated currents to ATP (300 μ M ), 2MeSATP and α,β meATP (100 μ M , each). ATP and adenosine‐5′‐diphosphate (ADP) (100 μ M , each) did not activate K + currents in the neostriatal neurones. Despite the observed lack of function, P2X 2 and P2Y 1 immunofluorescence was found in roughly 50% of the medium‐spiny neurones and cholinergic interneurones. A role of ATP in synaptic transmission to striatal medium‐spiny neurones and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under certain yet unknown conditions. British Journal of Pharmacology (2004) 143 , 119–131. doi: 10.1038/sj.bjp.0705916

Immunocytochemical and Co(2+) uptake studies revealed that in primary cultures of rat cortical neurones, the majority of neurones are gamma-aminobutyric acid (GABA) immunopositive and can express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. By fura-2 microfluorimetry, it was shown that the stimulation with the selective agonist (S)-AMPA (0.3-300 microM) induced a concentration-dependent but cell-variable increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (EC(50) value 7.4 microM) in more than 80% of the medium-sized multipolar neurones studied. The AMPA-induced rise in [Ca(2+)](i) seems to be due to Ca(2+) entry through AMPA receptor channels, because the response was abolished in Ca(2+)-free solution and by AMPA receptor selective antagonists, but was not significantly influenced by cyclopiazonic acid, an inhibitor of the endoplasmatic Ca(2+)-ATPase, by selective N-methyl-D-aspartic acid (NMDA) receptor antagonists, as well as the Na(+) channel blocker tetrodotoxin and the majority of tested Ca(2+) channel blockers. In conclusion, the results indicate that the cerebral cortical neurones in culture represent mostly GABAergic interneurone-like cells and the majority of them possess Ca(2+)-permeable AMPA receptors, important for intracellular signal transduction and neuronal plasticity.

Activation of adenosine A(1) receptors by endogenous adenosine plays a neuroprotective role under various pathophysiological conditions including hypoxia. Intracellular recordings were made in rat pyramidal cells of the somatosensory cortex. Hypoxia (5 min) induced a membrane depolarization and a decrease of input resistance. The A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 100 microM) reversibly inhibited the hypoxic depolarization. The inhibition was also present after blockade of the A(2A), A(2B) and A(3) receptor subtypes by selective antagonists. CPA had no effect on the hypoxic decrease of input resistance. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A(1) receptor antagonist, which did not alter hypoxic depolarization when given alone abolished the inhibitory effect of CPA. Neither CPA nor DPCPX influenced membrane potential or apparent input resistance under normoxic conditions. The novel pyrimidoindole (R)-9-(1-methylbenzyl)-2-(4'-pyridyl)-9H-pyrimido[4,5-b]indole-4-amine (APPPI, 1 and 10 microM) reversibly diminished hypoxic depolarization but had no significant effect on input resistance. The effect of APPPI at a concentration of 1 microM, but not at 10 microM, was blocked by DPCPX (0.1 microM). CPA (100 microM) inhibited [(3)H]-noradrenaline ([(3)H]-NA) release from rat hippocampal brain slices significantly only in the presence of rauwolscine (0.1 microM), an alpha(2)-adrenoceptor antagonist. APPPI (1 and 10 microM) exhibited an inhibitory effect similar to that observed with CPA. The effects of both CPA and APPPI were antagonized by DPCPX (0.1 microM). The present data suggest that mainly presynaptic mechanisms prevent neurons from hypoxic changes by an inhibition of transmitter release. However, in contrast to CPA, APPPI exhibited additional effects, which require further investigation.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Kinya Kuriyama. The presentations were (1) Modulation of neuroreceptors and ion channels by alcohol, by T. Narahashi; (2) Inhibition by ethanol of NMDA and AMPA receptor-channels, by P. Illes, K. Wirkner, W. Fischer, K. Mühlberg, P. Scheibler, and C. Allgaier; (3) Effects of ethanol on metabotropic glutamate receptors, by K. Minami; (4) Acute alcohol actions on the 5-HT3 ligand-gated ion channel, by D. Lovinger; (5) Inhibition of NMDA receptors by MK801 attenuates ethanol-induced taurine release from the hippocampus, by F. Lallemand, R.J. Ward, and P. DeWitte; and (6) Effect of ethanol on voltage-operated Ca2+ channels in hepatic stellate cells, by T. Itatsu, Y. Takei, H. Oide, M. Hirose, X. E. Wang, S. Watanabe, M. Tateyama, R. Ochi, and N. Sato.

Acamprosate has recently been introduced in relapse prophylaxis in weaned alcoholics. Using fura-2 microfluorimetry, the present study investigates whether acamprosate affects N-methyl-d-aspartate (NMDA) or K+-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) in rat cultured mesencephalic neurones. Both application of NMDA (plus glycine) and elevation of extracellular K+ induced rapid increases in [Ca2+]i which respectively were insensitive and sensitive to ω-conotoxin (ω-CTX) MVIIC, a blocker of voltage-dependent Ca2+ channels (VDCCs). Acamprosate (100 µM and 300 µM) significantly attenuated the response induced by NMDA as well as that induced by K+ in a concentration-dependent manner. Concurrent application of ω-CTX MVIIC and acamprosate impaired the K+-induced increase in [Ca2+]i to the same extent as ω-CTX MVIIC alone. The present data suggest that acamprosate inhibits Ca2+ influx through both NMDA receptors and VDCCs.

1 NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+](i)) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT-PCR.2 NMDA (1-100 mu M, plus 10 mu M glycine) induced a concentration-dependent increase in [Ca2+](i) (EC50 = 5.7 mu M). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 mu M) and nitrendipine (1 mu M), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 mu M), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response.3 Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 mu M, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively.4 Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3' splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable.5 In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.

1 NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+](i)) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT-PCR.2 NMDA (1-100 mu M, plus 10 mu M glycine) induced a concentration-dependent increase in [Ca2+](i) (EC50 = 5.7 mu M). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 mu M) and nitrendipine (1 mu M), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 mu M), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response.3 Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 mu M, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively.4 Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3' splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable.5 In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.

The effects of 2,2,2-trichloroethanol, the active compound of the sedative-hypnotic chloral hydrate, were investigated on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) in cultured mesencephalic and cortical neurones by means of the fura-2 method. Trichloroethanol inhibited the NMDA response in a concentration-dependent manner in cortical (IC50 = 2.76 mM) and mesencephalic neurones (IC50 = 1.12 mM), with a maximum effect of approximately 85 and 94%, respectively. Ethanol was considerably less potent than trichloroethanol. In conclusion, the trichloroethanol-induced impairment of NMDA receptor function may contribute to the sedative-hypnotic properties of chloral hydrate.

Coloured and odoriferous lamp oils available as household articles repeatedly lead to ingestion intoxications in children in some cases resulting in a bad course. Labels on containers of lamp oils are misleading, e.g. ''pure liquid paraffin'' or ''without noxious substances'', causing considerable misconceptions during therapeutic use by treating doctors.Analyses of four arbitrary chosen lamp oils by GC and GC/MS show that the main components (approx. 98%) are the unbranched saturated hydrocarbons n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane. Tests according to the German Pharmacopoeia 10th ed. prove also that ''paraffin''-labelled products are not at all identical with medical/pharmaceutical paraffinum perliquidum or liquidum. Lamp oils are low viscous and high toxic petroleum distillates and should be labelled ''toxic to humans''

Coloured and odoriferous lamp oils available as household articles repeatedly lead to ingestion intoxications in children in some cases resulting in a bad course. Labels on containers of lamp oils are misleading, e.g. pure liquid paraffin or without noxious substances, causin...