Teruhiko Matsumiya

Rosmarinic acid and caffeic acid produce antidepressive-like effect in the forced swimming test in mice.

We previously showed that rosmarinic acid from the leaves of Perilla frutescens Britton var. acuta Kudo (Perillae Herba) has antidepressive-like activity. The aim of the present study was to examine (i) whether caffeic acid, a major metabolite of rosmarinic acid, also has antidepressive-like activity, and (ii) whether these substances inhibit either the uptake of monoamines to synaptosomes or mitochondrial monoamine oxidase activity. Rosmarinic acid (2 mg/kg, i.p.) and caffeic acid (4 mg/kg, i.p.) each significantly reduced the duration of immobility in the forced swimming test in mice. In contrast, neither substance, at doses that produced a significant reduction in the immobile response in the forced swimming test, affected spontaneous motor activity. These results indicate that, like rosmarinic acid, caffeic acid also possesses antidepressive-like activity. In neuropharmacological studies, neither rosmarinic acid (10 x (-9)-10 x (-3) M) nor caffeic acid (10 x (-9)-10 x (-3) M) affected either the uptake of monoamines to synaptosomes or mitochondrial monoamine oxidase activity in the mouse brain. These results suggest that both caffeic acid and rosmarinic acid may produce antidepressive-like activity via some mechanism(s) other than the inhibition of monoamine transporters and monoamine oxidase.

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 35.7u2003±u20034.1u2003µm and a maximal velocity (Vmax) of 49.1u2003±u20032.0u2003pmol/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 THAu2003>u2003TBAu2003>u2003TEA. 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 17β‐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.

Adrenocortical suppression blocks the enhancement of memory storage produced by exposure to psychological stress in rats

Several reports have indicated that various stress stimuli modulate learning and memory processes. In the present study, the effects of adrenocortical suppression with the 11beta-hydroxylase inhibitor metyrapone on the psychological stress-induced changes in memory storage in inhibitory avoidance training and in serotonin turnover in various brain regions were investigated in rats. Retention of one-trial inhibitory avoidance and the plasma corticosterone level were significantly enhanced by post-training exposure to psychological stress for 1 h. Pretreatment with metyrapone (12.5 or 25 mg/kg, s.c.) 90 min beforehand dose-dependently blocked the enhancement of memory storage and of the plasma corticosterone level produced by psychological stress. These results suggest that the adrenocortical system may contribute to the memory-enhancing effect of psychological stress. In a neurochemical study, a significant increase in serotonin turnover in the hippocampus and limbic forebrain, including the nucleus accumbens, were observed in rats that were exposed to psychological stress. In contrast to the behavioral experiments, these changes in serotonin turnover produced by exposure to psychological stress were not antagonized by pretreatment with metyrapone; instead, a further increase in serotonin turnover was observed only in the hippocampus. These results suggest that the serotonergic system in the hippocampus might be selectively regulated by adrenal steroids in response to stress, and imply the existence of negative feedback mechanisms via a hippocampal serotonergic system in the memory enhancement associated with corticosterone and psychological stress.

Pharmacological characterization and visualization of the glial serotonin transporter

Astrocytes contain transport systems that are capable of removing various neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Glial serotonin transporter (SERT) plays an important role in the re-uptake of 5-hydroxytryptamine (5-HT). We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins using specific site-directed monoclonal antibodies (MoAb). Furthermore, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method, we addressed the expression of SERT mRNA in cultured rat astrocytes. We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [3H]5-HT into cultured astrocytes and cortical synaptosomes. Tricyclic antidepressants (clomipramine and imipramine) as well as selective serotonin re-uptake inhibitors (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [3H]5-HT uptake in both preparations. In contrast, the inhibitory effects of NE uptake inhibitors (nisoxetine and desipramine) and cocaine were weaker than those of 5-HT uptake inhibitors. In addition, dopamine (DA) uptake inhibitors (nomifensine and GBR-12935) exhibited a Ki value in the low micromolar range. The inhibitory potencies were in the order 5-HT uptake inhibitors (clomipramine, fluvoxamine, fluoxetine, imipramine and zimelidine) > NE uptake inhibitors (nisoxetine and desipramine) = cocaine > DA uptake inhibitors (nomifensine and GBR-12935). There was no difference in the order of the inhibitory effects of various monoamine uptake inhibitors between the two preparations. A correlation analysis of the potencies of various monoamine uptake inhibitors in the inhibition of [3H]5-HT into cultured astrocytes and cortical synaptosomes produced a highly significant correlation coefficient of 0.9893 (P < 0.0001). Immunocytochemical staining using anti-SERT MoAb in cultured astrocytes revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or golgi membranes, showed a considerable level of immunoreactivity. Extracts of astrocytes and synaptosomes from the cortex were immunoblotted with anti-SERT MoAb. SDS-PAGE/Western blots indicate that anti-SERT MoAb recognized two bands of 120 and 73 kDa in both preparations. RT-PCR demonstrated that astrocytes in cultured expressed mRNA for the cloned SERT protein, which has been characterized as the neuronal SERT. These pharmacological experiments indicate that this uptake process takes place through glial SERT that is very similar to neuronal SERT. Furthermore, the present data also indicate that the presence of the mRNA and protein for the neuronal SERT were established in cultured rat astrocytes, and the polypeptide portion of SERT in astrocytes and frontal cortex could be the same gene product.

Astroglial dopamine transport is mediated by norepinephrine transporter.

Abstract. The aim of this study was to clarify the characteristics of the dopamine (DA) transport mechanism in cultured rat cortical astrocytes. Reverse transcription-polymerase chain reaction (RT-PCR) with DA transporter (DAT)-, norepinephrine (NE) transporter (NET)- and organic cation transporter 3 (OCT3)-specific primers demonstrated that rat cortical astrocytes and frontal cortex expressed DAT, NET and OCT3 mRNA. Specific [3H]DA and [3H]NE uptake were each partly inhibited by 1xa0µM decynium 22, an extraneuronal monoamine transporter (EMT) and OCT inhibitor. The selective NE uptake inhibitor nisoxetine (0.1xa0µM) and the tricyclic antidepressant desipramine (1xa0µM) very potently inhibited the specific uptake of both [3H]DA and [3H]NE in astrocytes. In contrast, 0.1xa0µM GBR-12935, a selective and potent DA uptake inhibitor, had no inhibitory activity on the uptake of either compound. These results suggest that cortical astrocytes regulate extracellular DA and NE concentrations through the uptake of DA and NE by the glial NET but not by DAT. Furthermore, an uptake2 mechanism contributes to DA uptake in cortical astrocytes.

Expression and functional characterization of the extraneuronal monoamine transporter in normal human astrocytes

In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT‐PCR with EMT‐specific primers demonstrated the presence of EMT mRNA in NHA. The RT‐PCR products were subjected to restriction‐site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT‐PCR products from NHA. Sequence analysis demonstrated that the sequences of RT‐PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti‐EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti‐EMT polyclonal antibody recognized a band of 63u2003kDa. Immunocytochemical staining using anti‐EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature‐dependent [3H]MPP+ uptake in NHA for 60u2003min. Temperature‐dependent [3H]MPP+ uptake increased in a time‐dependent manner for the initial 45u2003min and almost reached a plateau level (8.70u2003±u20030.59u2003pmol/mg protein) at 60u2003min. In the presence of 3u2003µm decynium22 (D22) (the most potent EMT inhibitor), temperature‐dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39u2003±u20030.76u2003pmol/mg protein at 60u2003min). D22‐sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25–200u2003µm. Km for this process was 78.01u2003±u20037.64u2003µm and Vmax was 295.4u2003±u200312.8u2003pmol/mg protein/min. D22‐sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential‐sensitive. Moreover, D22‐sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22‐sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.

Different effects of 5-HT1A receptor agonists and benzodiazepine anxiolytics on the emotional state of naive and stressed mice: a study using the hole-board test

Abstract. Objectives: The effects of 5-HT1A receptor agonists on the emotional behavior of naive or stressed mice were examined and compared with those of benzodiazepine anxiolytics. Methods: Changes in the emotional state of mice were evaluated in terms of changes in exploratory activity, i.e. total locomotor activity, numbers and duration of rearing and head-dipping and latency to the first head-dipping, using an automatic hole-board apparatus. Results: The 5-HT1A receptor full agonists flesinoxan (0.03–1xa0mg/kg, IP) and 8-OH-DPAT (0.03–1xa0mg/kg, IP), and the partial agonist buspirone (0.3–10xa0mg/kg, IP) dose-dependently decreased all of the exploratory behaviors. Significant decreases in both the number and duration of head-dips, and an increase in the latency to head-dipping were observed at 30xa0min after exposure to acute restraint stress (60xa0min). These emotional changes were scarcely improved by post-stress treatment with 5-HT1A receptor agonists, at doses that alone did not produce a significant behavioral effect. In contrast, pretreatment with flesinoxan (0.1–1xa0mg/kg, IP) or 8-OH-DPAT (0.1–1xa0mg/kg, IP) 24xa0h prior to exposure to stress dose-dependently suppressed the decrease in various exploratory behaviors that was observed immediately after the exposure to acute restraint stress. Moreover, pretreatment with buspirone (1–10xa0mg/kg, IP) 24xa0h prior to exposure to stress also significantly suppressed the decrease in rearing behavior and the increase in head-dip latency. However, changes in the emotional response to stress stimuli were not observed in mice that had been pretreated with the benzodiazepine anxiolytics diazepam (0.1–1xa0mg/kg, IP) and chlordiazepoxide (2–8xa0mg/kg, IP). Conclusions: The present study clearly demonstrates that the behavioral effects of 5-HT1A receptor agonists in both naive and stressed mice were quite different from those of benzodiazepine anxiolytics, as previously reported by us. Notably, 5-HT1A receptor agonists but not benzodiazepine anxiolytics protect against various emotional changes produced by stress stimuli, and the results suggest that activation of 5-HT1A receptors may facilitate some mechanism(s) involved in the recognition of and/or ability to cope with stressful situation.

Functional expression of the norepinephrine transporter in cultured rat astrocytes.

We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time‐dependent manner, and this uptake involves temperature‐ and Na+‐sensitive mechanisms. The Na+‐dependent [3H]NE uptake was saturable, and the Km for the process was 539.3u2003±u200355.4u2003nm and the Vmax was 1.41u2003±u20030.03u2003pmol/mg protein/min. Ouabain, a Na+‐K+ ATPase inhibitor, significantly inhibited Na+‐dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+‐dependent [3H]NE uptake. On the other hand, GBR‐12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT‐PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti‐NET polyclonal antibody recognized a major band of 80u2003kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re‐uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.

Rosmarinic acid and caffeic acid reduce the defensive freezing behavior of mice exposed to conditioned fear stress.

AbstractnRationale. We previously showed that rosmarinic acid from the leaves of Perilla frutescens Britton var. acuta Kudo (Perillae Herba) and its major metabolite caffeic acid have antidepressive-like activity in the forced swimming test.nObjective. The present study was designed to examine whether rosmarinic acid and caffeic acid might also be effective in other types of stress model. Methods: The conditioned fear stress paradigm was used as a stress model for assessing the effects of rosmarinic acid and caffeic acid.nResults. Rosmarinic acid (0.25–4 mg/kg, IP) induced a dose-dependent, U-shaped reduction in the duration of the defensive freezing behavior of mice exposed to conditioned fear stress. Caffeic acid (1–8xa0mg/kg, IP) also dose-dependently reduced this freezing behavior. However, neither substance, at doses that produced a significant reduction in the freezing behavior, affected spontaneous motor activity.nConclusions. These results confirm that rosmarinic acid and caffeic acid may inhibit the emotional abnormality produced by stress.

The novel κ-opioid receptor agonist TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine in mice

The effects of the novel kappa-opioid receptor agonist TRK-820 on the rewarding and locomotor-enhancing effects of morphine were investigated in mice. Morphine (1-5 mg/kg, s.c.) caused a dose-related preference for the drug-associated place. In contrast, TRK-820 (0.003-0.03 mg/kg, s.c.) did not produce a significant preference for either compartment of the test box. In combination studies, co-injection of TRK-820 (0.01 and 0.03 mg/kg, s.c.) with morphine significantly suppressed the morphine (5 mg/kg, s.c.)-induced place preference, and this effect of TRK-820 was antagonized by pretreatment with nor-BN1 (3 mg/kg, s.c.), a selective kappa-opioid receptor antagonist. TRK-820 also suppressed morphine-induced hyperlocomotion, and this suppression was also blocked by nor-BNI. These results suggest that TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine through the activation of kappa-opioid receptors. Thus, we propose that TRK-820 may be useful for controlling pain while reducing undesirable side-effects.