Tritium Efflux Research Articles

Li-rod structure in high-temperature gas-cooled reactor as a tritium production device for fusion reactors

Production of tritium using a high-temperature gas-cooled reactor (HTGR) has been studied for a prior engineering test with tritium handling and for the startup operation of a demonstration fusion reactor. For this purpose, the hydrogen absorption speed of Zr in a Li-loading rod for the reactor operation is experimentally measured, and an analysis model is presented to evaluate the tritium outflow from the Li rod in a high-temperature engineering test reactor (HTTR). On the basis of the presented model, the structure of the Li-loading rod for the demonstration test using the HTTR is proposed.

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [3H]D-Aspartate Release from Bovine Isolated Retinae.

We investigated the pharmacological actions of a slow-releasing H2S donor, GYY 4137; a substrate for the biosynthesis of H2S, L-cysteine and its precursor, N-acetylcysteine on potassium (K+; 50mM)-evoked [3H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10nM-10µM), L-cysteine (100nM-10µM) and N-acetylcysteine (10µM-1mM) elicited a concentration-dependent decrease in K+-evoked [3H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10µM, the rank order of activity was as follows: L-cysteine > GYY 4137 > N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H2S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3mM) reversed the inhibitory responses caused by GYY 4137, L-cysteine and N-acetylcysteine on K+-evoked [3H]D-aspartate release. Glibenclamide (300µM), an inhibitor of KATP channels blocked the inhibitory action of GYY 4137 and L-cysteine but not that elicited by N-acetylcysteine on K+-induced [3H]D-aspartate release. The inhibitory effect of GYY 4137 and L-cysteine onK+-evoked [3H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), L-NAME (300µM). Furthermore, a specific inhibitor of inducible NOS(iNOS), aminoguanidine (10µM) blocked the inhibitory action of L-cysteine on K+-evoked [3H]D-aspartate release. We conclude that both donors and substrates for H2S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of KATP channels and NO synthase.

Study on Tritium Production Using a High-Temperature Gas-Cooled Reactor for Fusion Reactors: Evaluation of Tritium Outflow by Non-Equilibrium Diffusion Simulations

Performance of tritium production for fusion reactors, using a high-temperature gas-cooled reactor (HTGR) is examined. From the viewpoints of tritium recovery and environmental safety, tritium outflow from Li rods should be suppressed to the same level as the liquid radioactive waste from the pressurized water reactors (PWRs) in Japan. Methods for suppressing tritium leakage from Li rods are studied. The tritium outflow is reevaluated accurately on the basis of non-equilibrium simulations and the influence of coolant temperature on tritium leakage is clarified. The approach using Zr in the Li rod to reduce the tritium pressure and the resulting suppression of tritium leakage are also investigated.The results of the non-equilibrium simulation show that the tritium outflow is approximately 40% lower than the outflow reported in a previous study. Although the electric power generation efficiency is reduced, lowering the coolant temperature to 600 K results in a reduction of the tritium outflow to ~1/30 compared to the outflow in the case of a coolant temperature of 800 K. The incorporation of Zr into the Li rod can suppress tritium outflow (to ~1/200 compared to the case without Zr) to below the outflow level in PWRs in Japan.

Synergy between 5-HT4 receptor stimulation and phosphodiesterase 4 inhibition in facilitating acetylcholine release in human large intestinal circular muscle.

Gastroprokinetic properties of 5-HT4 receptor agonists, such as prucalopride, are attributed to activation of 5-HT4 receptors on cholinergic nerves innervating smooth muscle in the gastrointestinal smooth muscle layer, increasing acetylcholine release and muscle contraction. In porcine stomach and colon, phosphodiesterase (PDE) 4 has been shown to control the signaling pathway of these 5-HT4 receptors. The aim of this study was to investigate the PDE-mediated control of these 5-HT4 receptors in human large intestine. Circular smooth muscle strips were prepared from human large intestine; after incubation with [³H]-choline, electrically induced tritium outflow was determined as a measure for acetylcholine release. The influence of PDE inhibition on the facilitating effect of prucalopride on electrically induced acetylcholine release was studied. The non-selective PDE inhibitor IBMX enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence, while rolipram and roflumilast (PDE4) enhanced the prucalopride-induced facilitation to the same extent as IBMX. In human large intestinal circular muscle, the intracellular pathway of 5-HT4 receptors facilitating cholinergic neurotransmission to large intestinal circular smooth muscle is controlled by PDE4. If the synergy between 5-HT4 receptor agonism and PDE4 inhibition is confirmed in a functional assay with electrically induced cholinergic contractions of human large intestinal circular smooth muscle strips, combination of a selective 5-HT4 receptor agonist with a selective PDE4 inhibitor might enhance the in vivo prokinetic effect of the 5-HT4 receptor agonist in the large intestine.

Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro

5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [3H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1–1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01–0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect.

Study on a method for loading a Li compound to produce tritium using high-temperature gas-cooled reactor

Tritium production using high-temperature gas-cooled reactors and its outflow from the region loading Li compound into the helium coolant are estimated when considering the suppression of tritium outflow. A Li rod containing a cylindrical Li compound placed in an Al2O3 cladding tube is assumed as a method for loading Li compound. A gas turbine high-temperature reactor of 300MW electrical nominal capacity (GTHTR300) with 600MW thermal output power is considered and modeled using the continuous-energy Monte Carlo transport code MVP-BURN, where burn-up simulations are carried out. Tritium outflow is estimated from equilibrium solution for the tritium diffusion equation in the cladding tube. A GTHTR300 can produce 400–600g of tritium over a 180-day operation using the chosen method of loading the Li compound while minimizing tritium outflow from the cladding tube. Optimizing tritium production while suppressing tritium outflow is discussed.

Altered acetylcholine release in the hippocampus of dystrophin-deficient mice

Mild cognitive impairments have been described in one-third of patients with Duchenne muscle dystrophy (DMD). DMD is characterized by progressive and irreversible muscle degeneration caused by mutations in the dystrophin gene and lack of the protein expression. Previously, we have reported altered concentrations of α7- and β2-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal membranes of dystrophic (mdx) mice. This suggests that alterations in the central cholinergic synapses are associated with dystrophin deficiency. In this study, we examined the release of acetylcholine (ACh) and the level of the vesicular ACh transporter (VAChT) using synaptosomes isolated from brain regions that normally have a high density of dystrophin (cortex, hippocampus and cerebellum), in control and mdx mice at 4 and 12months of age. ACh release evoked by nicotinic stimulation or K+ depolarization was measured as the tritium outflow from superfused synaptosomes preloaded with [3H]-choline. The results showed that the evoked tritium release was Ca2+-dependent and mostly formed by [3H]-ACh. β2-containing nAChRs were involved in agonist-evoked [3H]-ACh release in control and mdx preparations. In hippocampal synaptosomes from 12-month-old mdx mice, nAChR-evoked [3H]-ACh release increased by 57% compared to age-matched controls. Moreover, there was a 98% increase in [3H]-ACh release compared to 4-month-old mdx mice. [3H]-ACh release evoked by K+ depolarization was not altered, while the VAChT protein level was decreased (19%) compared to that of age-matched controls. In cortical and cerebellar preparations, there was no difference in nAChR-evoked [3H]-ACh release and VAChT levels between mdx and age-matched control groups. Our previous findings and the presynaptic alterations observed in the hippocampi of 12-month-old mdx mice indicate possible dysfunction of nicotinic cholinergic synapses associated with dystrophin deficiency. These changes may contribute to the cognitive and behavioral abnormalities described in dystrophic mice and patients with DMD.

The facilitating effect of prucalopride on cholinergic neurotransmission in pig gastric circular muscle is regulated by phosphodiesterase 4

The influence of the selective 5-HT4 receptor agonist prucalopride on acetylcholine release from cholinergic nerve endings innervating pig gastric circular muscle and the possible regulation of this effect by phosphodiesterases (PDEs) was investigated, as PDEs have been shown to control the response to 5-HT4 receptor activation in pig left atrium. Circular muscle strips were prepared from pig proximal stomach and either submaximal cholinergic contractions or tritium outflow after incubation with [3H]-choline, induced by electrical field stimulation, were studied. Prucalopride concentration-dependently increased the amplitude of submaximal cholinergic contractions and of acetylcholine release induced by electrical field stimulation. The effect of the highest concentration tested (0.3 μM) on cholinergic contractions was antagonized by the selective 5-HT4 receptor antagonist GR113808 but not by granisetron or methysergide; the antagonism of prucalopride by GR113808 was confirmed in the release assay. The non-selective PDE-inhibitor 3-isobutyl-methyl-xanthine (IBMX) concentration-dependently reduced the amplitude of the cholinergic contractions; 3 μM IBMX reduced the cholinergic contractions maximally by 16% but it enhanced the facilitating effect of prucalopride from 51 to 83%. IBMX (10 μM) induced and enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence the effect of prucalopride on acetylcholine release but the PDE4-inhibitor rolipram (1 μM) enhanced the facilitating effect of prucalopride to the same extent as IBMX. These results demonstrate that 5-HT4 receptors are present on the cholinergic nerves towards the pig gastric circular muscle, facilitating acetylcholine release; the intracellular transduction pathway of this facilitation is regulated by PDE4. Combination of a 5-HT4 receptor agonist with selective inhibition of the PDE involved in this regulation of transmitter release might enhance the prokinetic effect of the 5-HT4 receptor agonist.

P.1.g.020 Effect of reversal of light–dark cycle on dentate gyrus of hippocampal formation in vivo

We have previously described a population of 5-hydroxytryptamine neurons which repetitively fires bursts of usually two (but occasionally three or four) action potentials, with a short (<20 ms) interspike interval within a regular low-frequency firing pattern. Here we used a paradigm of electrical stimulation comprising twin pulses (with 7- or 10-ms inter-pulse intervals) to mimic this burst firing pattern, and compared the effects of single- and twin-pulse electrical stimulations in models of pre- and postsynaptic 5-hydroxytryptamine function. Firstly, we measured the effect of direct electrical stimulation (2 Hz for 2 min) of rat brain slices on efflux of preloaded [3H]5-hydroxytryptamine. In this in vitro model, twin-pulse stimulation increased the efflux of tritium by about twice as much as did single-pulse stimulation. This effect was evident in the medial prefrontal cortex (area under the curve: 2.59±0.34 vs 1.28±0.22% relative fractional release), as well as in the caudate–putamen (3.93±0.65 vs 2.17±0.51%) and midbrain raphe nuclei (5.42±1.05 vs 2.51±0.75%). Secondly, we used in vivo microdialysis to monitor changes in endogenous extracellular 5-hydroxytryptamine in rat medial prefrontal cortex in response to electrical stimulation (3 Hz for 10 min) of the dorsal raphe nucleus. In this model, twin-pulse stimulation of the dorsal raphe nucleus increased 5-hydroxytryptamine by approximately twice as much as did single-pulse stimulation at the same frequency (area under the curve: 50.4±9.0 vs 24.2±4.4 fmol). Finally, we used in vivo extracellular recording to follow the response of postsynaptic neurons in the rat medial prefrontal cortex to 5-hydroxytryptamine released by dorsal raphe stimulation. Electrical stimulation of the dorsal raphe nucleus (1 Hz) induced a clear-cut poststimulus inhibition in the majority of cortical neurons tested. In these experiments, the duration of poststimulus inhibition following twin-pulse stimulation was markedly longer than that induced by single-pulse stimulation (200±21 vs 77±18.5 ms).Taken together, the present in vitro and in vivo data suggest that in 5-hydroxytryptamine neurons, short bursts of action potentials will propagate along the axon to the nerve terminal and will enhance both the release of 5-hydroxytryptamine and its postsynaptic effect.

Facilitatory effect of nicotine on adrenergic neuroeffector transmission in the isolated ear artery of the rabbit.

The effects of nicotine were studied on perfusion pressure and vasoconstrictor responses to sympathetic nerve stimulation in the isolated ear artery of the rabbit. Infusions of nicotine (50 micro M) produced a transient increase in perfusion pressure and potentiated responses to nerve stimulation; these effects of nicotine were unaffected by atropine (0.3 micro M) and abolished or significantly reduced respectively by hexamethonium (300 micro M) or mecamylamine (1 micro M). In experiments with ear arteries previously labelled with [3H] noradrenaline an infusion of nicotine (50 micro M) produced a transient increase in tritium efflux and the potentiation of responses to nerve stimulation in the presence of nicotine was accompanied by a statistically significant increase in stimulation-induced tritium efflux; these effects of nicotine were abolished by hexamethonium (300 micro M) or mecamylamine (1 micro M).

Mechanism of the indirect adrenergic effect of histamine in cat cerebral arteries.

KCl (50 mM), tyramine (10(-7) M), and histamine (10(-4) M) induced an increase in tritium release from cat cerebral arteries preincubated with [3H]noradrenaline, this increase being due in part to noradrenaline. When calcium was absent from the superfusion medium, only tyramine (10(-7) M) enhanced the tritium outflow. Colchicine (10(-3) M) partially inhibited the increase in radioactivity brought about by 10(-4) M histamine. KCl (50 mM) also evoked release of radioactivity from cerebral arteries preloaded with [3H]histamine; this release was unaffected by reserpine pretreatment or removal of both superior cervical sympathetic ganglia. Neither tyramine (10(-7) M) nor compound 48/80 (300 micrograms ml-1) altered the spontaneous tritium outflow from cerebral blood vessels preincubated with [3H]histamine. These results suggest that histamine is not accumulated by sympathetic nerve endings and elicits its noradrenaline-releasing effect by means of an exocytotic process.

Release of [3H]noradrenaline induced by 5-hydroxytryptamine from cat pial arteries.

Pial arteries of cats were used to analyse the effects of 5-hydroxytryptamine (5-HT) on the release of [3H]noradrenaline. To achieve this the vessels were preincubated with [3H]noradrenaline and the effect of different concentrations of 5-HT (10(-6), 10(-5), 10(-4) M) on the release of tritium was studied. 5-HT elicited release of radioactivity in a dose-dependent manner. Removal of both superior cervical sympathetic ganglia 15 days before the experiment of pretreatment of the animals with reserpine (3 mg kg-1, total dose) produced a significant decrease in the outflow of tritium induced by 5-HT. In these arteries, the amount of radioactivity retained at the end of the experiment was much diminished. Cocaine (10(-6) M) caused a significant decrease in the tritium efflux induced by 5-HT (1"0(-5) M). These results show that 5-HT has an indirect adrenergic effect in the pial arteries of the cat only at high doses of 5-HT, and confirm that sympathetic innervation of these vessels mainly comes from the superior cervical ganglia.

Lack of circadian variation in the sensitivity of rat terminal 5-HT1B autoreceptors.

The sensitivity of terminal 5-HT1B autoreceptors in the cerebral cortex and hippocampus to both agonist and antagonist was determined at four different time points in the light:dark cycle of the rat to evaluate whether changes in their responsiveness underlie the circadian rhythm in 5-hydroxytryptamine (5-HT) release. No significant circadian differences were evident in the apparent pIC50 values calculated for 5-HT to inhibit K(+)-evoked tritium efflux or in the apparent pA2 values calculated for methiothepin to antagonize the effect of 5-HT, at the different time points, in either brain region. These findings suggest that the sensitivity of terminal 5-HT1B autoreceptors in rat cerebral cortex and hippocampus does not change in a circadian pattern, during the light:dark cycle, and argues against such a change influencing the circadian variation in 5-HT release.

Role of Presynaptic Purinoceptors and Cyclic AMP on the Noradrenaline Release in Cat Cerebral Arteries

Field electrical stimulation (ES), K+ (50 mM) or ionophore X-537A (0.01 mM) induced tritium release from cat cerebral arteries preincubated with [3H]noradrenaline (NA). Adenosine and AMP (0.5 mM) did not modify tritium release caused by ionophore X-537A, but these agents and ATP (0.5 mM) significantly reduced that elicited by ES and K+; this reduction was antagonized by 1-methyl-3-isobutylxanthine (MIX; 0.05 mM). Inosine (0.5 mM) and the agonist of purinergic A2-receptors, 5'N-ethyl-carboxamide adenosine (NECA; 0.5 mM) had no effect, but the agonist of purinergic A2-receptors L-N6-phenylisopropyl adenosine (L-PIA; 0.1 mM) diminished tritium efflux caused by ES and K+. The adenosine inhibition of ES-induced radioactivity release was not affected by indomethacin (0.05 mM). MIX (0.05 mM) increased tritium release evoked by ES and K+. Agents that increase intracellular cyclic (c)AMP levels, such as dibutyryl cAMP (0.5 mM), the phosphodiesterase inhibitor Ro 20-1724 (0.1 mM), and the activators of adenylate cyclase, forskolin (0.005 mM) and NaF (2 mM) reduced tritium secretion elicited by ES and K+. However, the intracellular increase of cyclic GMP (cGMP) caused by 8-Br-cGMP did not affect this secretion. Dipyridamole (0.05 mM) and the adenosine deaminase inhibitor erythro-9-2-hydroxy-3 nonyl adenosine (EHNA; 0.1 mM) also produced inhibition of tritium secretion elicited by ES and K+. Dipyridamole reduced both the uptake of [3H]NA and [3H]adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cocaine-like action of diphenhydramine in cat cerebral arteries

Diphenhydramine (5.3 X 10(-7) M) significantly reduced the tritium efflux evoked by 10(-7) M tyramine from cat cerebral arteries preloaded with [3H]noradrenaline but not that brought about by 50 mM KCl. These results indicate the ability of diphenhydramine to block the amine neuronal uptake.

Differential Effects of Potassium Channel Blockers on Dopamine Release from Rat Striatal Slices

The effects of different potassium channel blockers on tritiated dopamine [( 3H]DA) release were investigated in rat striatal slices in the presence of pargyline and nomifensine (10 microM each). 4-Aminopyridine (4-AP; 10 and 30 microM) and 3,4-diaminopyridine (3,4-DAP; 30 microM) markedly increased the basal tritium outflow, whereas tetraethylammonium (TEA; 100-1000 microM) was without effect. The facilitating effect of 4-AP (10 microM) on spontaneous release was Ca(2+)- and K(+)-dependent. Moreover, the 4-AP-induced increase in spontaneous release was abolished in the presence of tetrodotoxin, indicating that voltage-dependent Na+ channels were involved in the release mechanism. 4-AP (10 and 30 microM) induced a dose-dependent decrease in K(+)-evoked [3H]DA release. This effect was confirmed with 3,4-DAP (30 microM). When striatal slices were depolarized with veratridine (5 microM), these two aminopyridines increased the evoked release of [3H]DA. TEA increased both K(+)- and veratridine-evoked [3H]DA release. These biochemical results are consistent with electrophysiological differences between the mechanism of action of aminopyridines and that of TEA.

Indirect adrenergic effect of histamine in human cerebral arteries: influence of post-mortem period.

Histamine (10(-4) M) increased the radioactivity released from human cerebral arteries obtained within 6 h of death and preincubated with [3H]noradrenaline. In the presence of 10(-6) M cocaine or if 7 or more hours had elapsed since death, 10(-4) M histamine was unable to change basal levels of tritium outflow. The radioactivity retained by the tissue was higher when cerebral blood vessels were obtained within a post-mortem period of 6 h. These results suggest that histamine may release noradrenaline from the sympathetic innervation of human cerebral arteries and that the function of this innervation lasts only 6 h after death.

Effects of Cannabinor, a Novel Selective Cannabinoid 2 Receptor Agonist, on Bladder Function in Normal Rats

BackgroundCannabinoid (CB) receptors may be involved in the control of bladder function; the role of CB receptor subtypes in micturition has not been established. ObjectivesOur aim was to evaluate the effects of cannabinor, a novel CB2 receptor agonist, on rat bladder function. Design, setting, and participantsSprague Dawley rats were used. Distribution of CB2 receptors in sensory and cholinergic nerves of the detrusor was studied. Selectivity of cannabinor for human and rat CB receptors was evaluated. Effects of cannabinor on rat detrusor and micturition were investigated. MeasurementsImmunohistochemistry, radioligand binding, tritium outflow assays, organ bath studies of isolated bladder tissue, and cystometry in awake rats were used. Results and limitationsCB2 receptor immunoreactivity was expressed in the urothelium and in sensory and cholinergic bladder nerves. Cannabinor exhibited similar binding at human and rat CB2 receptors and a 321-fold functional selectivity for the CB2 receptor versus the CB1 receptor. Cannabinor had no effect on isolated detrusor muscle function. In vivo, cannabinor 3.0mg/kg increased micturition intervals and volumes by 52% (p<0.05) and 96% (p<0.01), respectively, and increased threshold and flow pressures by 73% (p<0.01) and 49% (p<0.001), respectively. Cannabinor 0.3 or 1.0mg/kg or vehicle did not affect urodynamic parameters. ConclusionsConsidering that CB2 receptors are localized on sensory nerves and on the urothelium and that cannabinor had effects on “afferent” urodynamic parameters, peripheral CB2 receptors may be involved in sensory functions of rat micturition. Effects of cannabinor on cholinergic nerve activity in normal bladder tissue appear to be limited.

Neurochemical evidence that stimulation of CB1 cannabinoid receptors on GABAergic nerve terminals activates the dopaminergic reward system by increasing dopamine release in the rat nucleus accumbens

We examined the effect of cannabinoid receptor activation on basal and electrical field simulation-evoked (25 V, 2 Hz, 240 shocks) [ 3H]dopamine efflux in the isolated rat nucleus accumbens in a preparation, in which any effect on the dendrites or somata of ventral tegmental projection neurons was excluded. The cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212-2, 100 nM) significantly enhanced stimulation-evoked [ 3H]dopamine release in the presence of the selective dopamine transporter inhibitor 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine dihydrochloride (GBR12909, 100 nM). GBR12909 (100 nM–1 μM), when added alone, increased the evoked [ 3H]dopamine efflux in a concentration-dependent manner. The stimulatory effect of WIN55,212-2 on the evoked tritium efflux was inhibited by the selective CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 100 nM) and by the GABA A receptor antagonist bicuculline (10 μM). Repeated application of N-methyl- d aspartate (1 mM) under Mg 2+-free conditions, which directly acts on dopaminergic terminals, reversibly increased the tritium efflux, but WIN55,212-2 did not affect N-methyl- d aspartate-evoked [ 3H]dopamine efflux, indicating that WIN55,212-2 has no direct action on dopaminergic nerve terminals. AM251 (100 nM) alone also did not have an effect on electrical stimulation-evoked [ 3H]dopamine efflux. Likewise, the selective CB2 receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630, 0.3 μM) and the anandamide transport inhibitor (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11, 10 μM) had no significant effect on electrically evoked [ 3H]dopamine release. This is the first neurochemical evidence that the activation of CB1 cannabinoid receptors leads to the augmentation of [ 3H]dopamine efflux via a local GABA A receptor-mediated disinhibitory mechanism in the rat nucleus accumbens.

GABAA and strychnine-sensitive glycine receptors modulate N-methyl-d-aspartate–evoked acetylcholine release from rat spinal motoneurons: A possible role in neuroprotection

Increasing experimental and clinical evidence suggests that abnormal glutamate transmission might play a major role in a vast number of neurological disorders.As a measure of glutamatergic excitation, we have studied the acetylcholine (ACh) release induced by N-methyl-d-aspartate (NMDA) receptor stimulation in primary cultured rat ventral horn spinal neurons and we have evaluated the possibility to limit the consequences of the hyperactivation of glutamatergic receptors, by recruiting the inhibitory transmission mediated by GABA and glycine. For this purpose, we have exposed cell cultures, previously loaded with [3H]choline, to NMDA, which increased the spontaneous tritium efflux in a concentration-dependent manner. Tritium release is dependent upon external Ca2+, tetrodotoxin, Cd2+ ions and ω-conotoxin GVIA, but not on ω-conotoxin MVIIC nor nifedipine, suggesting the involvement of N-type voltage-sensitive calcium channels. NMDA-mediated [3H]ACh release was completely prevented by MK-801, 5,7-diclorokynurenic acid and ifenprodil, while it was strongly inhibited by a lower external pH, suggesting that the involved NMDA receptors contain NR1 and NR2B subunits. Muscimol inhibited NMDA-evoked [3H]ACh release and its effect was antagonized by SR95531 and potentiated by diazepam, indicating the involvement of benzodiazepine-sensitive GABAA receptors. Also glycine, via strychnine-sensitive receptors, inhibited the effect of NMDA.It is concluded that glutamate acts on the NMDA receptors situated on spinal motoneurons to evoke ACh release, which can be inhibited through the activation of GABAA and glycine receptors present on the same neurons. These data suggest that glutamatergic overload of receptors located onto spinal cord motoneurons might be decreased by activating GABAA and glycine receptors.