Development of negative ion extractor in the high-power and long-pulse negative ion source for fusion application.

High power and long-pulse negative ion extractor, which is composed of the plasma grid (PG) and the extraction grid (EXG), is newly developed toward the neutral beam injector for heating and current drive of future fusion machines such as ITER, JT-60 Super Advanced and DEMO reactor. The PG is designed to enhance surface production of negative ions efficiently by applying the chamfered aperture. The efficiency of the negative ion production for the discharge power increased by a factor of 1.3 against that of the conventional PG. The EXG is also designed with the thermal analysis to upgrade the cooling capability for the long pulse operation of >1000 s required in ITER. Though the magnetic field for electron suppression is reduced to 0.75 of that in the conventional EXG due to this upgrade, it was experimentally confirmed that the extracted electron current can be suppressed to the allowable level for the long pulse operation. These results show that newly developed extractor has the high potential for the long pulse extraction of the negative ions.

Endocannabinoids contribute to metabotropic glutamate receptor-mediated inhibition of GABA release onto hippocampal CA3 pyramidal neurons in an isolated neuron/bouton preparation.

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in various brain regions. In hippocampus, it is well known that endocannabinoids suppress presynaptic inhibitory neurotransmitter release in CA1 region. However, endocannabinoid signaling in CA3 region remains to be examined. Here we investigated whether presynaptic inhibition can be caused by activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and following presynaptic cannabinoid receptor type 1 (CB1 receptor) using mechanically dissociated rat hippocampal CA3 pyramidal neurons with adherent functional synaptic boutons. Application of group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) reversibly suppressed spontaneous inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX), frequency of miniature IPSCs was significantly reduced by DHPG, while there were no significant changes in minimum quantal size and sensitivity of postsynaptic GABA(A) receptors to the GABA(A) receptor agonist muscimol, indicating that this suppression was caused by a decrease in GABA release from presynaptic nerve terminals. Application of CB1 synthetic agonist WIN55212-2 (mesylate(R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone) or endocannabinoid 2-arachidonoylglycerol also suppressed the spontaneous IPSC. The inhibitory effect of DHPG on spontaneous IPSCs was abolished by SR-141716 (5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), a CB1 receptor antagonist. Furthermore, postsynaptic application of GDP-betaS blocked the DHPG-induced inhibition of spontaneous IPSCs, indicating the involvement of endcannabinoid-mediated retrograde synaptic signaling. These results provide solid evidence for retrograde signaling from postsynaptic group I mGluRs to presynaptic CB1 receptors, which induces presynaptic inhibition of GABA release in rat hippocampal CA3 region.

Presynaptic inhibition caused by retrograde signal from metabotropic glutamate to cannabinoid receptors.

We report a type of synaptic modulation that involves retrograde signaling from postsynaptic metabotropic glutamate receptors (mGluRs) to presynaptic cannabinoid receptors. Activation of mGluR subtype 1 (mGluR1) expressed in cerebellar Purkinje cells (PCs) reduced neurotransmitter release from excitatory climbing fibers. This required activation of G proteins but not Ca2+ elevation in postsynaptic PCs. This effect was occluded by a cannabinoid agonist and totally abolished by cannabinoid antagonists. Depolarization-induced Ca2+ transients in PCs also caused cannabinoid receptor-mediated presynaptic inhibition. Thus, endocannabinoid production in PCs can be initiated by two distinct stimuli. Activation of mGluR1 by repetitive stimulation of parallel fibers, the other excitatory input to PCs, caused transient cannabinoid receptor-mediated depression of climbing fiber input. Our data highlight a signaling mechanism whereby activation of postsynaptic mGluR retrogradely influences presynaptic functions via endocannabinoid system.

Endogenous cannabinoid as a retrograde messenger from depolarized postsynaptic neurons to presynaptic terminals.

Cannabinoid receptors are the molecular targets for the active component Delta(9)-tetrahydrocannabinol of marijuana and hashish, and constitute a major family of G protein-coupled seven-transmembrane-domain receptors. They consist of type 1 (CB1) and type 2 (CB2) receptors of which the CB1 is rich in various regions of the CNS. Accumulated evidence suggests that endogenous cannabinoids function as diffusible and short-lived intercellular messengers that modulate synaptic transmission. Recent studies have provided strong experimental evidence that endogenous cannabinoids mediate signals retrogradely from depolarized postsynaptic neurons to presynaptic terminals to suppress subsequent neurotransmitter release, driving the synapse into an altered state. In hippocampal neurons, depolarization of postsynaptic neurons and resultant elevation of [Ca(2+)](i) lead to transient suppression of inhibitory transmitter release (depolarization-induced suppression of inhibition, DSI). In cerebellar Purkinje cells, on the other hand, depolarization-induced elevation of [Ca(2+)](i) causes transient suppression of excitatory transmitter release (depolarization-induced suppression of excitation, DSE). DSI and DSE appear to share the same properties and may be a general and important mechanism by which the postsynaptic neuronal activity can influence the amount of transmitter release.

Influence of film additives on stabilizing drug release rates from pellets coated with acrylic polymers.

The objective of this study was to investigate the influence of talc and triethyl citrate (TEC) on stabilizing the drug release rates following curing and storage at elevated temperature of pellets coated with an aqueous acrylic polymeric dispersion. Core pellets containing anhydrous theophylline (20%), microcrystalline cellulose, and polyvinylpyrrolidone were prepared by extrusion-spheronization. The aqueous dispersions were prepared by adding up to 30% TEC as a plasticizer and talc up to 200% as an antiadherent to a mixture of Eudragit RS 30D/RL 30D (95:5). The theophylline pellets were coated in a fluidized-bed coating unit and then cured at elevated temperatures. Theophylline pellets were successfully coated with the Eudragit dispersions that contained up to 200% talc, based on the dry polymer weight, and the coating efficiency was greater than 93%. Our results demonstrated that the polymer, which was plasticized by TEC, was able to function as a film-forming agent for dispersions containing high levels of talc. No sticking of the coated pellets was observed during the coating process or during the curing or equilibrating phase, even with high levels of TEC in the film. The dissolution rate of theophylline from the coated pellets was delayed when the film coating dispersion contained high levels of talc. Additionally, the stability of the drug release profiles from the coated pellets after storage was significantly improved. Furthermore, a modified dissolution testing used to simulate mechanical stresses that may be encountered in vivo showed the film coated pellets would have sufficient strength. The results of this study demonstrated that high levels of film additives in the acrylic dispersion contributed to the stabilization of the drug release rates as well as the reproducibility of the coating process.

Endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals.

Endogenous cannabinoids are considered to function as diffusible and short-lived modulators that may transmit signals retrogradely from postsynaptic to presynaptic neurons. To evaluate this possibility, we have made a paired whole-cell recording from cultured hippocampal neurons with inhibitory synaptic connections. In about 60% of pairs, a cannabinoid agonist greatly reduced the release of the inhibitory neurotransmitter GABA from presynaptic terminals. In most of such pairs but not in those insensitive to the agonist, depolarization of postsynaptic neurons and the resultant elevation of intracellular Ca2+ concentration caused transient suppression of inhibitory synaptic currents, which is mainly due to reduction of GABA release. This depolarization-induced suppression was completely blocked by selective cannabinoid antagonists. Our results reveal that endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals to cause the reduction of transmitter release.

Advantages of immunostaining over DNA analysis using PCR amplification to detect p53 abnormality in long-term formalin-fixed tissues of human colorectal carcinomas.

To study the appropriate period for formalin fixation in order to detect p53 abnormalities in formalin-fixed tissue, we used seven surgically resected human colorectal cancer specimens. The immunohistochemical reactivity of p53 immunostaining and amplification of DNA by polymerase chain reaction (PCR) of the p53 gene were compared after various periods of 10% formalin fixation (1 day, and 1, 2, 4, and 8 weeks). For comparative immunostaining, we used the monoclonal antibody Ki-67 (MIB-1), and for comparative polymerase chain reaction (PCR), K-ras at codon 12 was amplified. Immunostaining was performed by the streptavidin-biotin method with microwave retrieval, and PCR amplifications were performed by the nested PCR method. p53 and Ki-67 immunoreactivity did not change essentially for up to 2 weeks and 1 week, respectively, of formalin fixation. PCR amplification for p53 at exon 8 and K-ras at codon 12 was successful until 1 day and 2 weeks, respectively, of formalin-fixation for the specimens of all seven cases. Thereafter, the amplification tended to worsen as the fixation time lengthened. Further, the DNA was more successfully amplified in the second PCR than in the first. These results suggest that to detect p53 abnormality in specimens that have been formalin-fixed for long periods, immunohistochemical staining may have advantages over DNA analysis with PCR amplification.

Application of tumbling melt granulation method to prepare controlled-release beads by coating with mixture of functional non-meltable and meltable materials.

A new coating method for use in preparing controlled-release beads was developed by modifying the tumbling melt granulation technique. The dissolution rate of the drug from the beads was controlled by coating the mixture of meltable and non-meltable materials by heating in a centrifugal fluidizing granulator without using any solvent. In experiments using talc as the non-meltable material, the resultant beads showed the sufficient ability to suppress the dissolution of the drug and no change in the dissolution characteristics by wetting agent and in a stability test at high temperature. Using functional polymer as the non-meltable material, the controlled-release beads with various dissolution characteristics could be prepared: entero-soluble-release beads using entero-soluble polymer, and zero-order-release beads using hydrophilic gel-forming agent and talc.