Protective effect of Oren-gedoku-to (Huang-Lian-Jie-Du-Tang) against impairment of learning and memory induced by transient cerebral ischemia in mice.

The protective effect of Oren-gedoku-to (OGT; Huang-Lian-Jie-Du-Tang), a traditional Chinese medicine, against impairment of learning and memory induced by transient cerebral ischemia was investigated in mice. The cerebral ischemia caused a reduction of step-down latency and an increase of step-down errors in the passive avoidance task. Pretreatment with oral administration of OGT (2, 4 or 8 g of herbs per kg) once daily for 5 days prolonged the step-down latency significantly and decreased the step-down errors as compared with those of sham-operated controls. In the Morris water maze test, the cerebral ischemia caused an increase in the latency until finding the platform in the training trial and a decrease in the percentage of swimming in the quadrant of the former platform in the probe trial. Oren-gedoku-to (OGT; 2, 4 and 8 g/kg, p. o.) shortened the latency of escaping markedly onto the platform in the training trial and increased the percentage of crossing the former platform quadrant in the probe trial. A reference drug, tacrine (0.5 and 1.0 mg/kg, p.o.), prevented the reduction of step-down latency in the passive avoidance task and shortened the escape latency in the Morris water maze task. Furthermore, OGT significantly protected against cerebral ischemia-induced reduction in the acetylcholine (ACh) content of the cerebral cortex, hippocampus and striatum. These results indicate that the protective effects of OGT against the impairment of learning and memory induced by transient cerebral ischemia may be associated with preventing the decrease in the ACh content of the mouse brain.

Delivery of antisense oligonucleotides to neuroblastoma cells.

pH-Sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesterol hemisuccinate (3:2 mol/mol) were applied in delivery of antisense oligodeoxynucleotides (asODN) into NG 108-15 neuroblastoma and glioma cells. Fluorescently labelled asODN were entrapped in liposomes by a modified freeze-thawing method (20% encapsulation efficiency). The uptake of asODN (free or entrapped in liposomes) by NG 108-15 cells was monitored by fluorescence-activated cell sorting and confocal microscopy. Delivery of asODN was significantly improved when antisense were entrapped in liposomes as compared to free (nonliposomal) asODN. The uptake was dose-dependent and optimum was achieved after 2 h incubation.

Involvement of diazepam binding inhibitor and its fragment octadecaneuropeptide in social isolation stress-induced decrease in pentobarbital sleep in mice.

Diazepam binding inhibitor (DBI) and its fragment, octadecaneuropeptide (ODN), are putative endogenous ligands for benzodiazepine (BZD) receptors and have been shown to act as an inverse BZD receptor agonist in the brain. A previous study suggested that the social isolation stress-induced decrease in pentobarbital sleep in mice was partly due to endogenous substances with an inverse BZD receptor agonist-like property. In this study, we examined the effects of DBI and ODN on pentobarbital sleep in group-housed and socially isolated mice to test the possible involvement of DBI and ODN in a social isolation-induced decrease in pentobarbital sleep. The socially isolated mice showed significantly shorter durations of pentobarbital (50 mg/kg, intraperitoneally, i. p.) sleep compared to the group-housed animals. When injected intracerebroventricularly (i.c.v.), DBI and ODN (3 and 10 nmol) dose-dependently shortened the pentobarbital-induced sleeping time in group-housed mice at the same dose range, but these peptides had no effect on the sleeping time in socially isolated animals. In contrast, flumazenil (16.5-33 nmol, i.c.v.), a BZD receptor antagonist, reversed the pentobarbital sleeping time in socially isolated mice to the level of group-housed animals without affecting the sleeping time in group-housed animals. The effects of DBI and ODN in group-housed mice were significantly blocked by flumazenil (33 nmol, i.c.v.). Moreover, the effect of flumazenil in socially isolated mice was significantly attenuated by DBI and ODN (10 nmol, i.c.v.). These results suggest that the changes in the activity of DBI and/or ODN are partly involved in the social isolation-induced decrease in the hypnotic action of pentobarbital in mice.

Diazepam binding inhibitor (DBI) gene expression in the brains of socially isolated and group-housed mice.

Diazepam binding inhibitor (DBI), a putative endogenous polypeptide ligand for benzodiazepine (BZD) receptors, has been shown to act as an inverse BZD receptor agonist in the brain. We previously suggested that the social isolation stress-induced decrease in pentobarbital sleeping time in mice was partly due to an increase in the activity of endogenous substances with an inverse BZD receptor agonist-like property such as DBI. In this study, we examined whether the DBI gene expression is affected by socially isolated stress. Consistent with the previous findings, the in situ hybridization result showed very strong signals of DBI mRNA around the regions of the third ventricle, especially the lining cells, the arcuate nucleus of the hypothalamus and the cerebellum, in both socially isolated and group-housed animals. Unexpectedly, however, semi-quantitative experiments with reverse transcription polymerase chain reaction technique revealed that socially isolated mice had significantly less expression of DBI mRNA in the hypothalamus than group-housed animals, and no difference in the expression in the other brain areas was observed between two animal groups. We discuss the relationship between the decrease of DBI mRNA expression in the hypothalamus and the decrease of GABA(A) receptor function following long-term social isolation in mice.

Influence of chronic treatment with imipramine on mRNA levels in rat brain: elevation of glyceraldehyde-3-phosphate dehydrogenase levels.

The differential display method was used to identify the intrinsic factor that changes its mRNA expression level in rat brain after a 14-day oral administration of 20 mg/kg imipramine. The expression of a 180-bp band was markedly enhanced by imipramine. The results of sequencing and a data base search revealed that the isolated clone was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with a one-base difference. Enhancement of the expression by imipramine was observed in the amygdala. Quantitative PCR showed that imipramine treatment significantly elevated the GAPDH/beta-actin ratio in the cortex. These findings suggest that long-term treatment with imipramine stimulates GAPDH mRNA expression.

Brain serotonin neurotransmission: an overview and update with an emphasis on serotonin subsystem heterogeneity, multiple receptors, interactions with other neurotransmitter systems, and consequent implications for understanding the actions of serotonergic drugs.

Knowledge about serotonergic neurotransmission has been expanding rapidly. Recent research has delineated 15 molecularly different serotonin receptors and multiple, discrete neuronal and nonneuronal (including endocrine) pathways and mechanisms that mediate the many functions of serotonin. Nonetheless, gaps remain regarding aspects of the anatomy and physiology of serotonin in its roles as a neurotransmitter, a neuromodulator, and a hormone. Few serotonin receptor-selective drugs are available for clinical use. A group of selective serotonin reuptake inhibitors (SSRIs) remain the agents with greatest therapeutic utility, although the mechanisms underlying their delayed efficacy, which clearly result from adaptive consequences following repeated administration rather than early uptake inhibition of serotonin by itself, are incompletely understood and appear to involve changes in signal transduction and gene expression in serotonergic and other neurotransmitter systems.

Inhibitory effects of corymine-related compounds on glycine receptors expressed in Xenopus oocytes.

We examined the effects of 4 corymine-related compounds on glycine-induced chloride current in Xenopus oocytes. Dihydrocorymine, N-demethyl-3-epi-dihydrocorymine and deformylcorymine dose-dependently decreased the glycine current with IC50 values of 34, 37 and 55 microM, respectively. The effect of these compounds on the glycine current was more potent than that of pleiocarpamine (IC50 > 1 mM). N-demethyl-3-epi-dihydrocorymine and dihydrocorymine, at 100 microM, also decreased the gamma-aminobutyric acid-induced current by 65% and 22%, respectively, whereas deformylcorymine and pleiocarpamine failed. The inhibitory action of deformylcorymine on the glycine current was noncompetitive. These results suggest that deformylcorymine is a novel specific noncompetitive glycine receptor antagonist. The structure-activity relationship of these compounds was discussed.

A possible mechanism underlying corymine inhibition of glycine-induced Cl- current in Xenopus oocytes.

We previously reported that corymine, an alkaloid extracted from the leaves of Hunteria zeylanica native to Thailand, inhibited glycine-induced chloride current using a receptor expression model of Xenopus oocytes. In this study, we investigated the mechanism underlying the inhibitory action of this alkaloid on glycine current using the same model. Corymine inhibited glycine current in a noncompetitive fashion. Co-application with strychnine, a competitive glycine receptor antagonist, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, corymine decreased the ED50 value of strychnine, but did not change that of DIDS. Moreover, the inhibitory effects of corymine and either strychnine or DIDS were additive. The desensitization phase of glycine current showed two exponentials and corymine preferentially inhibited the fast component, whereas strychnine affected both of them to the same extent and DIDS preferentially inhibited the slow component. When these drugs were applied repeatedly, the inhibitory effects of corymine and strychnine were not use-dependent and reversible, while the effect of DIDS was use-dependent and irreversible. The inhibitory effect of corymine on gamma-aminobutyric acid (GABA) current was less potent than the effect on glycine current, while this alkaloid failed to affect acetylcholine and serotonin currents. These results demonstrate that corymine inhibits glycine-gated CI- channels by interacting with the site different from that of DIDS.

Identification of opioid receptor subtypes in antinociceptive actions of supraspinally-administered mitragynine in mice.

Mitragynine (MG), a major alkaloidal constituent extracted from the plant Mitragyna speciosa Korth, is known to exert an opioid-like activity. Our previous study showed the involvement of opioid systems in the antinociceptive activity of MG in the tail-pinch and hot-plate tests in mice. In the present study, to clarify the opioid receptor subtypes involved in the antinociceptive action of MG, we investigated the effects of selective antagonists for mu-, delta- and kappa- opioid receptors on antinociception caused by the intracerebroventricular (i.c.v.) injection of MG in the tail-pinch and hot-plate tests in mice. The coadministration of a selective mu-opioid antagonist, cyprodime (1-10 microg, i.c.v.) and the pretreatment with a selective mu1-opioid antagonist naloxonazine (1-3 microg, i.c.v.) significantly antagonized the antinociceptive activities of MG (10 microg, i.c.v.) and morphine (MOR, 3 microg, i.c.v.) in the tail-pinch and hot-plate tests. Naltrindole (1-5 ng, i.c.v.), a selective delta-opioid antagonist, also blocked the effects of MG (10 microg, i.c.v.) without affecting MOR (3 microg, i.c.v.) antinociception. Nor-binaltorphimine, a selective kappa-opioid antagonist, significantly attenuated MG (10 microg, i.c.v.) antinociception in the tail-pinch test but not in the hot-plate test at the dose (1 microg, i.c.v.) that antagonized the antinociceptive effects of the selective kappa-opioid agonist U50,488H in both tests, while it had no effect on MOR antinociception in either tests. These results suggest that antinociception caused by i.c.v. MG is dominantly mediated by mu- and delta-opioid receptor subtypes, and that the selectivity of MG for the supraspinal opioid receptor subtypes differs from that of MOR in mice.

Enhancement of serotonin 2C receptor mRNA expression by antidepressants possessing the receptor-blocking activity in the rat brain.

The effects of repeated oral administration of antidepressants on the serotonin 2C receptor subtype (5-HT2CR) mRNA level in the rat brain were examined. Imipramine (20 mg/kg, p.o.) enhanced the hybridization signal in a time (days)-dependent manner, reaching a maximum at day 4 and maintaining a high level until day 14. Desipramine and mianserin, which have 5-HT2CR antagonistic activity, also stimulated the mRNA expression to about same extents as imipramine, but nomifensine, which has no effect on 5-HT2CR, was ineffective. These results suggest that long-term treatment with antidepressants, which act as 5-HT2CR antagonists, stimulates 5-HT2CR mRNA expression.

Inhibitory effects of corymine, an alkaloidal component from the leaves of Hunteria zeylanica, on glycine receptors expressed in Xenopus oocytes.

We previously reported that corymine, an alkaloidal compound extracted from the leaves of Hunteria zeylanica native to Thailand, potentiated convulsions induced by either picrotoxin or strychnine. Therefore, to clarify the mechanism of action of corymine, the effects of corymine on gamma-aminobutyric acid (GABA) and glycine receptors were examined. We used Xenopus oocytes expressing these receptors and the two-electrode voltage-clamp method. The receptors expressed in oocytes injected with rat brain and spinal cord RNA showed the pharmacological properties of GABAA and glycine receptors, respectively. Corymine (1-100 microM) partially (20-30%) reduced the GABA responses in oocytes injected with rat brain RNA, while marked (up to 80%) dose-dependent reductions were observed in the glycine responses in oocytes injected with rat spinal cord RNA. These observations suggest that corymine was more effective against the glycine receptors than the GABA receptors. The ED50 of corymine on the glycine response was 10.8 microM. Corymine, at 30 microM, caused a shift to the right, with a lower maximal response, of the glycine concentration-response curve. This indicated that the action of corymine on glycine receptors is neither competitive nor purely non-competitive. These observations suggest that a binding site other than the glycine recognition site of the glycine receptors is the site of action of corymine.

Effects of mitragynine on cAMP formation mediated by delta-opiate receptors in NG108-15 cells.

Mitragynine, a major constituent of the young leaves of Mitragyna speciosa KORTH., has been reported to exert antinociceptive activity in mice. To determine the mechanism the influence of mitragynine on cAMP content was measured in NG108-15 cells which possess delta opioid receptors and alpha 2B-adrenoceptors. Mitragynine inhibited the forskolin-stimulated cAMP content in a concentration dependent manner as well as morphine and noradrenaline. Mitragynine- and morphine-induced inhibition of cAMP content were blocked by naloxone. Although idazoxane inhibited noradrenaline-induced inhibition of the cAMP content, idazoxane had no effect on mitragynine-induced inhibition. These results suggest that mitragynine acts directly on opioid receptors, but not on alpha 2-adrenoceptors, to show antinociceptive activity.

NMDA antagonists potentiate scopolamine-induced amnesic effect.

The effects of N-methyl-D-aspartate NMDA receptor antagonists on scopolamine-induced amnesia and on delay-interposed short-term memory performance were investigated using an 8-arm radial maze in rats. Scopolamine, a muscarinic antagonist, deteriorated the radial maze performance, while MK-801, an NMDA receptor channel blocker and CGS-19755, a competitive NMDA receptor antagonist, showed no obstruction to the spatial cognition in the non-delayed maze task. MK-801 (0.01-0.03 mg/kg, i.v.) and CGS-19755 (1-10 mg/kg, i.v.) significantly augmented scopolamine-induced deficit in the non-delayed maze task and impaired the short-term memory in the 5-min delay-interposed task. These results suggest that NMDA antagonists have a negative action on short-term memory and that the interaction between the NMDA and the central muscarinic system plays a role in modulating the cognitive function.

NMDA-but not AMPA-receptor antagonists augment scopolamine-induced spatial cognitive deficit of rats in a radial maze task.

The effect of NMDA and AMPA receptor antagonists on a scopolamine-induced spatial cognitive deficit was investigated in rats using an 8-arm radial maze. The NMDA antagonists, MK801 and CGS19755, robustly augmented scopolamine-induced deficits but had no effect on spatial cognition when administered alone. In contrast, augmentation of the scopolamine-induced deficits was not observed when the selective AMPA antagonist, YM90K, was administered with scopolamine. These results suggest that the NMDA but not AMPA subtypes of the ionotropic glutamate receptors play important roles in regulation of the central cholinergic function related to the spatial learning and memory processes.

Neuropharmacological actions of Pluchea indica Less root extract in socially isolated mice.

The effects of Pluchea indica Less root extract (PI-E) on locomotor activity and pentobarbital-induced sleep, social isolation-induced aggressive behavior, motor coordination in the rotarod test, pentylenetetrazole-induced convulsion and nociceptive responses in the tail-pinch test were examined in mice. Socially isolated mice showed higher locomotor activity and shorter duration of pentobarbital sleep than group-housed mice. PI-E (50-100 mg/kg, p.o.) significantly decreased locomotor activity and prolonged pentobarbital sleep in a dose-dependent manner in isolated mice but not in group-housed mice. At a large dose (400 mg/kg, p.o.), PI-E not only decreased locomotor activity but also prolonged pentobarbital sleep in group-housed mice. The reference drug diazepam, at 0.5 mg/kg, also suppressed the locomotor activity in isolated mice but not in group-housed mice. Moreover, diazepam, at 0.1 and 0.5 mg/kg, significantly prolonged pentobarbital sleep in both isolated mice and group-housed mice. The effects of PI-E and diazepam on pentobarbital sleep in isolated mice were significantly attenuated by flumazenil (1 mg/kg, i.v.). PI-E (50-100 mg/kg), as well as diazepam (0.5-5 mg/kg, p.o.), dose-dependently suppressed social isolation-induced aggressive behavior, but it had no effect on pentylenetetrazole-induced convulsion, motor coordination in the rotarod test, or nociceptive response in the tail pinch test in group-housed mice. These results suggest that PI-E attenuates pathophysiological changes caused by social isolation stress in mice, and that the GABAergic system is partly involved in the action of PI-E on a social isolation-induced decrease in pentobarbital sleep.

Behavioral studies on alkaloids extracted from the leaves of Hunteria zeylanica.

The effects of a crude methanol extract, butanol- and chloroform-fractions, and a pure compound, corymine, extracted from the leaves of H. zeylanica on locomotor activity and rearing, pentobarbital-induced sleep, and drug-induced convulsions were studied in mice. The methanol extract dose-dependently decreased rearing without a significant effect on locomotor activity at doses of 15, 60 and 120 mg/kg. It did not significantly prolong the sleeping time but potentiated the convulsions induced by strychnine, but not that by either picrotoxin or pentylenetetrazole, at a dose of 120 mg/kg. The butanol-fraction significantly prolonged sleeping time at a dose of 125 mg/kg but did not affect either of the convulsive drugs. The chloroform fraction prolonged sleeping time at doses of 62.5 and 125 mg/kg and potentiated the convulsions induced by either strychnine or picrotoxin, but not that by pentylenetetrazole, at doses of 15, 30, 60 and 120 mg/kg. Corymine did not significantly prolong sleeping time, but potentiated the convulsions induced by either strychnine or picrotoxin, not by pentylenetetrazole, at doses of 2, 8 and 15 mg/kg. These results suggest that crude alkaloidal extracts of H. zeylanica leaves produce biphasic effects on the central nervous system (CNS), depression and stimulation, while the pure compound, corymine, has a unique central stimulatory effect in mice.

Influence of aging on rolipram-sensitive phosphodiesterase activity and [3H]rolipram binding in the rat brain.

To clarify the quantitative and qualitative changes in type IV phosphodiesterase (PDE IV) with aging, phosphodiesterase (PDE) activity and [3H]rolipram binding in the cytosolic fraction from the brains of young and aged rats were examined. In all areas of the aged (100-week-old) rat brain except for hippocampus, the PDE activity was decreased by about half that in the young (10-week-old) animals. However, inhibition % by 100 microM rolipram and by 100 microM isobutylmethylxanthine (IBMX) was not significantly different between the aged and young rats. On the other hand, [3H]rolipram binding and rolipram-sensitive PDE activity did not change with aging in the hippocampus, although both were decreased in other brain regions. These results suggest that PDE IV does not decrease with aging and maintains its cAMP degrading activity in the hippocampus. It may be involved in the dysfunction of the hippocampus with aging.

Permanent occlusion of bilateral internal carotid arteries produces cognitive deficits in two learning behavior tasks.

We investigated the effects of permanent bilateral occlusion of the internal carotid arteries (2ICAO) on the learning and memory performances in rats to evaluate the permanent 2ICAO rats as a model for vascular dementia. The learning and memory performance was tested by a step-through passive avoidance task and an 8-arm radial maze task. Permanent 2ICAO decreased cerebral blood flows in the cortex and hippocampus by 46.3 +/- 3.3 and 21.1 +/- 4.6%, respectively, when measured at 15 min after occlusion. In the passive avoidance task, the 2ICAO rats showed no impairment of learning or of memory retention when tested 1 h after learning trial, while they showed a shorter latency than sham-operated rats when tested 24 h after learning trial. In the radial maze learning task, the non-pretrained 2ICAO rats showed impairment. The pretrained 2ICAO rats had no deficit in the radial maze retention task but they showed impaired performance when a 3-min delay was interposed in the task. These results suggest that permanent 2ICAO is a useful animal model for studying vascular dementia.

Imipramine-induced increase in 5-HT2C receptor mRNA level in the rat brain.

Repeated oral administration of 20 mg/kg imipramine elevated the level of 5-HT2C mRNA in the rat brain. Hybridization signals in nearly all regions stained by digoxigenin-labeled antisense cRNA probe, such as the hippocampus, choroid plexus, habenular nucleus, and dorsomedial hypothalamic nucleus, were more intense following imipramine treatment. These results suggest that long-term treatment with imipramine stimulates 5-HT2C receptor gene expression.

Studies on the activation mechanisms of guanylyl cyclase by serotonin, probably through a novel subtype of serotonin receptor (5-HTGC).

Characterization of the serotonin-induced increase in guanosine 3',5'-cyclic monophosphate (cyclic GMP) was investigated and compared with that induced by atrial natriuretic peptide (ANP) in NG108-15 cells. The cyclic GMP formed by serotonin or ANP was transported in a similar manner to the extracellular medium, although the cyclic GMP formed by bradykinin was not. Serotonin and ANP raised cyclic GMP additively. Serotonin-induced cyclic GMP formation was completely inhibited by pretreatment with 100 nM 12-o-tetradecanoylphorbol 13-acetate (TPA), although that induced by ANP was only partially inhibited and the effects were blocked by pretreatment with staurosporin. In membrane preparations, ANP stimulated cyclic GMP formation in the presence of ATP, but serotonin did not. Serotonin-stimulated cyclic GMP formation was found to occur in neuroblastoma N18TG-2, but not in glioma C6Bu-1. These results suggest that a novel subtype of serotonin receptors (5-HTGC) which stimulates membrane-bound guanylyl cyclase, different from that stimulated by natriuretic peptide, may exist especially in neurons.