Functional expression of choline transporter-like protein 1 (CTL1) in human neuroblastoma cells and its link to acetylcholine synthesis.

We examined the molecular and functional characterization of choline uptake into human neuroblastoma cell lines (SH-SY5Y: non-cholinergic and LA-N-2: cholinergic neuroblastoma), and the association between choline transport and acetylcholine (ACh) synthesis in these cells. Choline uptake was saturable and mediated by a single transport system. Removal of Na(+) from the uptake buffer strongly enhanced choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium. The increase in choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger (NHE) inhibitor. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), NHE1 and NHE5 mRNA are mainly expressed. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. ChAT mRNA was expressed at a much higher level in LA-N-2 cells than in SH-SY5Y cells. The conversion of choline to ACh was confirmed in both cells, and was enhanced in Na(+)-free conditions. These findings suggest that CTL1 is functionally expressed in both SH-SY5Y and LA-N-2 cells and is responsible for choline uptake that relies on a directed H(+) gradient as a driving force, and this transport functions in co-operation with NHE1 and NHE5. Furthermore, choline uptake through CTL1 is associated with ACh synthesis in cholinergic neuroblastoma cells.

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells.

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K(m)) of 16.5 microM and a maximal velocity (V(max)) of 133.9 pmol/mg protein/min. The V(max) value of choline uptake was strongly enhanced in the absence of Na(+) without any change in K(m) values. The increase in choline uptake under Na(+)-free conditions was inhibited by Na(+)/H(+) exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H(+) electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.

Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells.

We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H(+) electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.

Expression and functional characterization of choline transporter in human keratinocytes.

Choline is essential for synthesis of the major membrane phospholipid phosphatidylcholine. Moreover, it serves as a precursor for synthesis of the neurotransmitter acetylcholine (ACh). Keratinocytes of the epidermis synthesize and release ACh. The uptake of choline is the rate-limiting step in both ACh synthesis and choline phospholipid metabolism, and it is a prerequisite for keratinocyte proliferation. However, the nature of the choline transport system in keratinocytes is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake into cultured human keratinocytes. Choline uptake into keratinocytes was independent of extracellular Na(+), saturable, and mediated by a single transport system with an apparent Michaelis-Menten constant of 12.3 muM. Choline uptake was reduced when the keratinocyte membrane potential was depolarized by high K(+). These results provide evidence that the choline transport activity is potential-sensitive. Various organic cations inhibit the choline transport system. RT-PCR demonstrated that keratinocytes expressed mRNA for choline transporter-like protein 1 (CTL1), mainly the CTL1a subtype. The present biochemical and pharmacological data suggest that CTL1a is functionally expressed in human keratinocytes and is responsible for the uptake of choline and organic cations in these cells.

[Physiological functions of carnitine and carnitine transporters in the central nervous system].

L-Carnitine is an essential co-factor in the metabolism of lipids and consequently in the production of cellular energy. This molecule has important physiological roles, including its involvement in the beta-oxidation of fatty acids by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane as acylcarnitine esters. In the brain, L-carnitine and acetyl-L-carnitine have important roles in cerebral bioenergetics and in neuroprotection through a variety of mechanisms including their antioxidant properties and in the modulation and promotion of synaptic neurotransmission, most notably cholinergic neurotransmission. Acetyl-L-carnitine was successfully applied as pharmacological agents for treatment of chronic degenerative diseases of the senile brain and for slowing down the progression of mental deterioration in Alzheimer's disease, and they may involve both the cholinergic neuronal transmission activity of acetyl-L-carnitine and its ability to enhance neuronal metabolism in mitochondria. Astrocytes are able to produce large amounts of ketone bodies, which are thought to supply adjacent neurons with easily transferable substrates for generation of energy. Thus, the L-carnitine uptake mechanism becomes the rate-limiting step for astrocyte ketogenesis. Several carnitine transporters have been known to be present in peripheral tissues. In this review, the functional expression and physiological role of carnitine transporters in central nervous system is further discussed.

Antidepressants enhance the antinociceptive effects of carbamazepine in the acetic acid-induced writhing test in mice.

Some antidepressants, as well as antiepileptics, are effective for treating pain of varying etiology. The present study was designed to characterize the antinociceptive effects of imipramine, a tricyclic antidepressant, fluvoxamine, a selective serotonin reuptake inhibitor, milnacipran, a serotonin noradrenaline reuptake inhibitor, and carbamazepine, an antiepileptic drug, using the acetic acid-induced writhing test in mice. Imipramine (1.25-10 mg/kg, i.p.), fluvoxamine (5-40 mg/kg, i.p.) and milnacipran (2.5-20 mg/kg, i.p.) all dose-dependently and significantly reduced the number of writhes induced by the injection of acetic acid (0.8% (v/v)), although the maximal effect of milnacipran was weaker than those of imipramine and fluvoxamine. Similarly, carbamazepine (5-20 mg/kg, i.p.) also showed a dose-dependent and significant antinociceptive effect. In combination studies, the co-administration of a sub-effective dose of carbamazepine (5 mg/kg, i.p.) with imipramine (1.25 and 2.5 mg/kg, i.p.), fluvoxamine (10 mg/kg, i.p.) or milnacipran (1.25 and 2.5 mg/kg, i.p.) significantly reduced the number of writhes. Additionally, the hole-board test revealed that the medications with significant antinociceptive effects barely produced changes in motor activity that could possibly affect writhing behavior. Thus, the present study demonstrated that the antinociceptive effect of carbamazepine is enhanced by combination with imipramine, fluvoxamine and milnacipran. Therefore, the combined therapy using antidepressants and carbamazepine may be useful clinically for the control of pain.

Zeta-sarcoglycan is a functional homologue of gamma-sarcoglycan in the formation of the sarcoglycan complex.

The sarcoglycans (SGs), transmembrane components of the dystrophin-associated glycoprotein complex, are stable and functional only when they assemble into a tetrameric complex in muscle cells. A defect in any one of the four SG members disrupts the entire SG complex (SGC) and causes limb-girdle muscular dystrophy. zeta-SG has been recently found as a transmembrane protein homologous to gamma-SG and delta-SG. To characterize zeta-SG in complex formation, we co-transfected expression vectors encoding all six SGs (alpha-, beta-, gamma-, delta-, epsilon- and zeta-SG) and dystroglycan into Chinese hamster ovary cells. Immunoprecipitation analysis showed that zeta-SG or gamma-SG formed a SGC with beta-SG and delta-SG plus alpha-SG or epsilon-SG, revealing that zeta-SG can form two types of SGCs (alpha-beta-zeta-delta or epsilon-beta-zeta-delta). This result indicates the functional resemblance of zeta-SG to gamma-SG rather than delta-SG, although phylogenetic analysis suggests that zeta-SG is evolutionally closer to delta-SG than to gamma-SG. Reverse transcription (RT)-PCR showed that the expression pattern of the transcript was almost the reciprocal of that of gamma-SG in various mouse tissues and that the zeta-SG transcript was especially abundant in the brain, suggesting that zeta-SG might play a particular role in the central nervous system.

Functional expression of the organic cation/carnitine transporter 2 in rat astrocytes.

In this study, we sought to identify the transporters that mediate the uptake of L-carnitine and acetyl-L-carnitine in cultured rat cortical astrocytes. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake were both saturable, and mediated by a single Na(+)-dependent transport system. Uptake of both was inhibited by L-carnitine, D-carnitine, acetyl-L-carnitine and various organic cations. Acylcarnitines (acetyl-, butyryl-, hexanoyl-, octanoyl- and palmitoyl-L-carnitine) also interacted with L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine transport. 2-Amino-2-norbornane carboxylic acid, a known inhibitor of amino acid transporter B(0,+) (ATB(0,+)), did not cause any significant inhibition. A highly significant correlation was found between the potencies of acylcarnitines in the inhibition of L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake and the acyl chain length of acylcarnitines. The expression of mRNA for organic cation/carnitine transporters (OCTNs), carnitine transporter 2 (CT2) and ATB(0,+) in astrocytes was investigated by reverse transcription (RT)-PCR. OCTN2 mRNA was expressed in astrocytes, whereas the expression of OCTN1, OCTN3 and CT2 mRNA could not be detected. ATB(0,+) mRNA was expressed at very low levels in astrocytes. Western blotting analysis indicated that anti-OCTN2 polyclonal antibody recognized a band of 70 kDa in both kidney and astrocyte preparations. OCTN2 immunoreactivity was detected in rat astrocytes by immunocytochemical staining. Inhibition of OCTN2 expression by RNA interference significantly inhibited L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake into astrocytes. These results suggest that OCTN2 is functionally expressed in rat astrocytes, and is responsible for L-carnitine and acetyl-L-carnitine uptake in these cells.

Caffeic acid attenuates the decrease in cortical BDNF mRNA expression induced by exposure to forced swimming stress in mice.

We previously reported that caffeic acid produces antidepressive-like effects in the forced swimming test in mice, an animal model of depression. Increased evidence suggests that brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family that has high affinity for the tyrosine kinase receptor B (TrkB), plays an important role in the pathophysiology and treatment of depression. The present study examined whether caffeic acid affects the expression levels of BDNF and TrkB mRNA in brain regions of mice subjected to a forced swimming test. Caffeic acid (4 mg/kg, i.p.) reduced the duration of immobility of mice in the forced swimming test. The levels of BDNF mRNA in the frontal cortex as well as TrkB mRNA in the amygdala were significantly decreased after the forced swimming test, and the former reduction was significantly inhibited by caffeic acid (4 mg/kg, i.p.). Caffeic acid (4 mg/kg, i.p.) did not modify the levels of BDNF and TrkB mRNA in brain regions of naive mice. These results suggest that caffeic acid can attenuate the down-regulation of BDNF transcription that results from stressful conditions.

Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes.

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.

Effects of a 5-HT7 receptor antagonist DR4004 on the exploratory behavior in a novel environment and on brain monoamine dynamics in mice.

The present study examined whether serotonin (5-hydroxytryptamine; 5-HT)7 receptors play a role in the modulation of emotionality in mice using the selective 5-HT7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydrobenzo (c,d)indol-2-(1H)-one (DR4004). The emotionality of mice was evaluated in terms of exploratory activity in the hole-board test. The mice treated with DR4004 (2.5-10 mg/kg, i.p.) displayed a dose-dependent decrease in locomotor activity by moving less distance in the hole-board, and statistically significant decreases were observed at 5 and 10 mg/kg. On the other hand, DR4004 (10 mg/kg, i.p.) did not affect spontaneous motor activity. In a neurochemical study, decreases in amygdaloid dopamine and 5-HT turnover were observed in mice in which locomotor activity in the hole-board test was attenuated following the administration of DR4004 (10 mg/kg, i.p.). Also, a simple linear regression analysis revealed that locomotor activity on the hole-board was significantly correlated with dopamine and 5-HT turnover in amygdala. Furthermore, co-injection of the selective dopamine reuptake inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR12909; 1.25-5 mg/kg, i.p.) or the selective 5-HT reuptake inhibitor fluvoxamine (20 mg/kg, i.p.) significantly reversed the DR4004 (10 mg/kg, i.p.)-induced decrease in locomotor activity in the hole-board test. These findings constitute the behavioral evidence that 5-HT7 receptors may play a role in the modulation of emotionality. Furthermore, it is also suggested that amygdaloid dopamine and 5-HT neuronal systems may be involved in this modulation.

Characterization of the anxiolytic-like effects of fluvoxamine, milnacipran and risperidone in mice using the conditioned fear stress paradigm.

It has been known that rodents exhibit the immobility when tested in the same environment in which they had been previously exposed to aversive stimuli. This behavior is called conditioned fear stress-induced freezing behavior, and has been used as a model of anxiety. Using this animal model, the present study tried to characterize the anxiolytic-like effects of fluvoxamine, a selective serotonin reuptake inhibitor, milnacipran, a serotonin noradrenaline reuptake inhibitor and risperidone, an atypical antipsychotic in mice. Fluvoxamine (1.25-10 mg/kg, intraperitoneally (i.p.)) and milnacipran (0.5-4 mg/kg, i.p.) each dose-dependently and significantly suppressed the conditioned fear stress-induced freezing behavior in mice, an indicator of anxiety, and milnacipran had a weaker effect than fluvoxamine. While risperidone also significantly suppressed freezing behavior at a low dose (0.01 mg/kg, i.p.), a high dose (0.04 mg/kg, i.p.) decreased spontaneous motor activity. On the contrary, sulpiride, a typical antipsychotic (2-8 mg/kg, i.p.), did not affect freezing behavior. In a combination study, the suppressive effect of a low dose of risperidone (0.01 mg/kg, i.p.) on freezing behavior was significantly antagonized by the co-administration of low/middle doses of fluvoxamine (1.25 and 2.5 mg/kg, i.p.), whereas a high dose of fluvoxamine (10 mg/kg, i.p.) was unaffected. Additionally, the co-administration of milnacipran (0.5-2 mg/kg, i.p.) also tended to inhibit the suppressive effect of risperidone (0.01 mg/kg, i.p.). These findings indicate that fluvoxamine, milnacipran and risperidone may each be clinically effective at treating anxiety disorders, but their effects may be attenuated in combination with other medications.

Evaluation of Rhubarb using antioxidative activity as an index of pharmacological usefulness.

We attempted to estimate the pharmacological activity by measuring the concentrations of a composition ingredient using a multivariate statistical analysis method. Medicinal herb of Rhubarb has been many largely unrecognized biochemical and pharmacological effect components. Therefore, we attempted to estimate the antioxidative activity of Rhubarb on low-density lipoprotein (LDL) of its components. Thirty specimens of Rhubarb from various origins were used, chose nine components of anthraquinones, two components of anthrones, two components of flavan-3-ols, one component of procyanidin, one component of naphthalene, two components of phenylbutanones and one component of stilbene. Quantitative analysis of 18 components was performed with high-performance liquid chromatography (HPLC) and antioxidative activities were measured with plasma taken from spontaneous familial hypercholesterolemia model rabbits. There was considerable variation among the specimens in the concentration of components and antioxidative activities on LDL. As a result of multiple regression analysis, significant multiple correlation coefficient for antioxidative activities on LDL (R=0.914, P<0.01) was found in relation to the concentrations of five components: aloe-emodin, chrysophanol, emodin 1-O-beta-D-glucoside, lindleyin and 6-hydroxymusizin 8-O-beta-D-glucoside. Three of the five components were not active in promoting antioxidative activity and there was no significant correlation between the concentrations of the most active component and the activity. We consider this a useful method for selecting of Rhubarb and propose a new scientific approach for the selection of natural medicines.

[The role of glial monoamine transporters in the central nervous system].

Monoamine transport systems play a very important role in determining the concentrations of monoamines in the synaptic cleft, and therefore the magnitude and duration of the effects of transmitters. Several transport systems for monoamines have been described. The first to be recognized were uptake, a Na(+)-dependent, high-affinity, cocaine-sensitive neuronal transporter, which includes dopamine transporter, norepinephrine transporter and serotonin transporter, and uptake1, a Na(+)-independent, low-affinity, high-capacity, steroid-sensitive extraneuronal transporter. Recently, molecular identification of the uptake2 transporter has been reported, and this has been called extraneuronal monoamine transporter in humans, and organic cation transporter3 in rats. Astrocytes contain these two transport systems that can remove monoamine neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Since monoamine oxidase and catechol-O-methyl-transferase are present in astroglial cells, their glial uptake systems are likely to play an important role in regulating extracellular monoamine concentrations. This uptake system may be characterized as a second line of defense that inactivates monoamines that have escaped neuronal re-uptake, and thus prevents uncontrolled spreading of the signal. In this review, the identification of monoamine transporters in astrocytes is described and the physiological role of glial monoamine transporters in monoaminergic neurotransmission is discussed.

Caffeic acid produces antidepressive- and/or anxiolytic-like effects through indirect modulation of the alpha 1A-adrenoceptor system in mice.

We previously reported that caffeic acid produce antidepressive- and/or anxiolytic-like effects in two different types of stress models. It has recently been reported that caffeic acid affects the alpha1A-adrenoceptor system. The present study examined whether the alpha1A-adrenoceptor system is involved in the antidepressive- and/or anxiolytic-like effects of caffeic acid. Caffeic acid reduced the duration of immobility and freezing of mice produced by forced swimming and conditioned fear stress, respectively. These effects of caffeic acid were suppressed by the alpha1- and alpha1A-adrenoceptor antagonists. However, caffeic acid did not alter the binding of [3H]prazosin to alpha1A-adrenoceptor in mouse cortical membranes. These results suggest that indirect modulation of the alpha1A-adrenoceptor system may be involved in the antidepressive- and/or anxiolytic-like effects of caffeic acid.

Pathophysiologic characteristics of the activity-stress paradigm in animal models: inhibitory effect of glucose on these responses.

This review provides a discussion of the pathophysiologic significance of animal models of the activity-stress paradigm and the role of plasma glucose level in the appearance of physical stress responses of those models. Many research reports have demonstrated that animal models exposed to activity-stress are useful as a "symptomatic model" of anorexia nervosa and obsessive-compulsive disorder as well as peptic ulcer. Our findings show that a decrease in plasma glucose concentration is an important factor in determining the activity-stress-induced physical responses. Further investigation of the pathophysiology of activity-stressed animal models may contribute to the development of new therapeutics for diseases such as anorexia nervosa and obsessive-compulsive disorder.

Effect of a selective inducible nitric oxide synthase inhibitor on intraocular nitric oxide production in endotoxin-induced uveitis rabbits: in vivo intraocular microdialysis study.

Inducible nitric oxide (NO) synthase (iNOS) is believed to contribute to the pathogenesis of endotoxin-induced uveitis (EIU). In the present study, we investigated the inhibitory effects of N(G)-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, and S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBITU), a potent and selective iNOS inhibitor, on intraocular NO production in EIU rabbits using an in vivo intraocular microdialysis technique. The flare level in the anterior chamber increased from 1h after the injection of 100 micro g/kg lipopolysaccharide (LPS), and continued to increase for 24h. Aqueous humor protein concentrations were significantly increased at 24h after LPS-injection. These changes were significantly reduced by L-NAME (10mg/kg) and PBITU (1mg/kg), but not by D-NAME (10mg/kg). The increase in NO(2)(-) and NO(3)(-) levels in the dialysate induced by LPS was significantly inhibited by L-NAME (10mg/kg) and PBITU (1mg/kg), but not by D-NAME (10mg/kg). These results suggest that activation of iNOS may play a key role in the development of EIU, and selective inhibitors of iNOS may have therapeutic applications in the treatment of EIU.

Modulation of passive avoidance in mice by the 5-HT1A receptor agonist flesinoxan: comparison with the benzodiazepine receptor agonist diazepam.

The effects of the 5-HT(1A) receptor agonist flesinoxan on passive avoidance in mice were compared with those of the benzodiazepine receptor agonist diazepam. In preliminary experiments, the retention latency to enter a dark compartment in mice subjected to single-training sessions with 0.6-mA electric foot shocks for 4, 8, or 16 s slightly increased in all of the test sessions (immediately, 24 h, and 1 week after the training sessions), but none of these changes were significant. In contrast, mice subjected to double-training sessions with 0.6-mA electric foot shocks for 16 s showed a significant increase in retention latency in all of the test sessions. Pretreatment with either flesinoxan or diazepam 30 min before the double-training sessions with 0.6-mA electric foot shocks for 16 s significantly decreased the retention latency in test sessions 24 h and 1 week later. In contrast, mice pretreated with flesinoxan 24 h before the single-training sessions with 0.6-mA electric foot shocks for 4, 8, or 16 s showed a significant increase in retention latency in the test sessions 24 h and/or 1 week later. Similar enhancements of retention latency in the test sessions 24 h and/or 1 week later were observed also in mice pretreated with flesinoxan 24 h before the double-training sessions. However, in this time interval following injection, pretreatment with diazepam did not affect the retention latency of mice in any of the test sessions. Neither flesinoxan nor diazepam, at the same doses and time intervals used in the passive avoidance study, modified the thresholds for flinching and jumping elicited by electrical stimuli. These results suggest that the activation of 5-HT(1A) receptors, but not benzodiazepine receptors, has a dual effect on the formation of learning and memory for an aversive event that depends on the time interval following receptor activation.