Mutsuaki Ueda

Dual effect of serotonin on formalin-induced nociception in the rat spinal cord.

To examine the role of the descending serotonergic system in the regulation of spinal nociceptive processing, the effects of serotonin (5-HT) and selective ligands for 5-HT receptor subtypes on persistent nociception were investigated. Formalin (5% formaldehyde) injected into the plantar region of the rat hindpaw induced two phases of aversive responses such as licking and biting. Intrathecal administration of selective 5-HT3 receptor antagonists, granisetron (0.1-100 pmol/rat) and ondansetron (1-1000 pmol/rat), reduced the second phase of the formalin-induced aversive responses without affecting the first one. The antinociceptive effect of granisetron (100 pmol/rat) was abolished when 5-HT was depleted from the lumbar cord by pretreatment with 5,7-dihydroxytryptamine (5,7-DHT). In the 5,7-DHT-treated rats, intrathecal administration of 1-(m-chlorophenyl)-biguanide, a selective 5-HT3 receptor agonist, facilitated the aversive responses in the second phase whereas that of 8-OH-DPAT, a selective 5-HT1A receptor agonist, suppressed them. Intrathecal administration of 5-HT showed a dual effect on the second phase of the aversive responses in the 5,7-DHT-treated rats; 5-HT inhibited the aversive responses when administered at a low dose (0.1 nmol/rat) but facilitated them at a high dose (1 nmol/rat). In addition, the inhibitory and facilitatory effects of intrathecal 5-HT were blocked by its co-administration with NAN190, a 5-HT1A receptor antagonist, and granisetron, respectively. These results suggest that 5-HT suppresses formalin-induced nociception in the spinal cord via the 5-HT1A receptor and facilitates it via the 5-HT3 receptor.

Mechanism of the pathogenesis of glutamate neurotoxicity in retinal ischemia

Abstract · Purpose: This study was carried out to examine the involvement of glutamate and nitric oxide neurotoxicity in ischemia/reperfusion-induced retinal injury in vivo. · Methods: We monitored glutamate release from in vivo cat retina during and after pressure-induced ischemia using a microdialysis technique. Morphometric studies were performed to study the effects of MK-801 (dizocilpine), L-NAME (Nω-nitro-l-arginine methyl ester), and D-NAME (Nω-nitro-d-arginine methyl ester) on the histological changes in the rat retina induced by ischemia or intravitreal injection of NMDA (N-methyl-d-aspartate; 200 nmol). · Results: A large release of glutamate occurred during ischemia, followed by a marked release after reperfusion. Histological changes occurred selectively in the inner part of the retina after ischemia as well as intravitreal injection of NMDA. Pretreatment with intravenous injection of MK-801 or L-NAME significantly inhibited the ischemic injury of the inner retina. Intravitreal injection of L-NAME inhibited NMDA-induced neurotoxicity in the retina. · Conclusion: These findings indicate that nitric oxide mediates neurotoxic actions of glutamate which are responsible for ischemic injury in the retina.

Evidence that glutamate is released from capsaicin-sensitive primary afferent fibers in rats: study with on-line continuous monitoring of glutamate

The aim of this study is to elucidate whether the excitatory amino acid glutamate is released from capsaicin-sensitive primary afferent fibers, and to compare the releasing effect of capsaicin on glutamate with that on substance P. The release of glutamate was measured using a fluorometric on-line continuous monitoring system, in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, 2-min application of capsaicin produced an increased outflow of glutamate, as well as an increase in the release of immunoreactive substance P from dorsal horn slices of the rat. The release of glutamate was concentration-dependently increased by capsaicin at concentrations in the range of 0.1-3 microM, and the release evoked by 10 microM capsaicin was not higher than that evoked by 3 microM. On the other hand, capsaicin at concentrations of 1-10 microM produced a concentration-dependent increase in the release of immunoreactive substance P, without effect at 0.1 microM. The amount of glutamate release evoked by 3 microM capsaicin was about 42.8 pmol.mg-1 protein, and 290 times that of immunoreactive substance P. The release of glutamate by 3 microM capsaicin was suppressed by the depletion of calcium from the superfusate. Capsaicin at 3 microM failed to increase the release of glutamate from the dorsal horn slices of the rats made an L4-L6 dorsal rhizotomy. These results suggest that capsaicin evoked the release of glutamate from primary afferent fibers in the dorsal horn and that glutamate may play an important role in pain transmission between primary afferent fibers and dorsal horn neurons.

Inhibition of NMDA receptors and nitric oxide synthase reduces ischemic injury of the retina.

This study was performed to examine the roles of body temperature, NMDA receptors and nitric oxide (NO) synthase in post-ischemic retinal injury in rats. Cell loss in the ganglion cell layer and thinning of the inner plexiform layer were observed 7 days after ischemia. Cell loss in the ganglion cell layer but not thinning of the inner plexiform layer was reduced by hypothermia during ischemia. Intravenous injection of dizocilpine (MK-801) or Nomega-nitro-L-arginine methyl ester (L-NAME) prior to ischemia ameliorated retinal injury. These results suggest that activation of NO synthase following NMDA receptor stimulation is involved in ischemia-induced retinal injury.

Opioidergic inhibition of capsaicin-evoked release of glutamate from rat spinal dorsal horn slices

We investigated the effects of opioid agonists on the capsaicin-evoked release of glutamate from nociceptive primary afferent fibers of the rat (6-8 weeks) using a fluorometric on-line continuous monitoring system for glutamate. In the presence of 0.3 microM tetrodotoxin, the application of 3 microM capsaicin to spinal dorsal horn slices produced an evoked glutamate release (55.9 +/- 4.02 pmol.mg-1 protein, n = 15). DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin; 0.3-10 microM) and morphine (1-30 microM), mu-opioid agonists, produced a concentration-dependent reduction (approximately 85 and approximately 77% reduction, respectively) in the capsaicin (3 microM)-evoked release of glutamate. These inhibitory effects were significantly antagonized by naloxone (1 microM). DPDPE ([D-Pen2,5]enkephalin; 1-10 microM), a delta-opioid agonist, also reduced the capsaicin-evoked release in a concentration-dependent manner (approximately 59% reduction). Naltrindole (1 microM), a selective delta-antagonist, significantly antagonized the inhibitory effect of DPDPE (10 microM). In contrast, neither U-50,488H (1-10 microM) nor U-69,593 (10 microM), kappa-opioid agonists, had any effects on the evoked release of glutamate. These results suggest that mu-, and delta-opioid agonists modulate pain transmission in the spinal dorsal horn, at least in part, by inhibiting the release of glutamate from capsaicin-sensitive primary afferents.

Detection of capsaicin-evoked release of glutamate from spinal dorsal horn slices of rat with on-line monitoring system

We examined whether capsaicin evokes the release of glutamate from unmyelinated primary afferent fibers, using a fluorometric on-line monitoring system, in which immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In this system, an application of capsaicin (1 microM) resulted in an increased outflow of glutamate from dorsal horn slices in a tetrodotoxin-resistant manner, while capsaicin (3 microM) was without effect on ventral horn slices. This effect of capsaicin on the dorsal horn slices was in a concentration-dependent manner at 0.1-3 microM and the effect at 10 microM was not larger than that at 3 microM. The competitive capsaicin antagonist capsazepine significantly reduced the release of glutamate evoked by 1 microM capsaicin. Twice exposures of dorsal horn slices to capsaicin led a significant decrease in the release of glutamate evoked by the second capsaicin stimulation, indicating desensitization to capsaicin. Thus, using on-line monitoring system, we actually demonstrated that capsaicin induced the release of glutamate from the dorsal horn, probably from capsaicin-sensitive primary afferent terminals.

Alpha2-adrenoceptor-mediated inhibition of capsaicin-evoked release of glutamate from rat spinal dorsal horn slices

It is known that the descending noradrenergic system suppresses nociceptive transmission in the spinal dorsal horn. To determine whether noradrenaline-alpha-adrenoceptor systems exert inhibitory influence on the release of glutamate from the nociceptive primary afferents, the effects of alpha-adrenoceptor agonists on the capsaicin (3 microM)-evoked release of glutamate from rat spinal dorsal horn slices were examined using an on-line continuous monitoring system for glutamate. The application of alpha 2-agonists clonidine (0.1-10 microM) and ST-91 (1 and 10 microM), respectively, decreased the capsaicin-evoked release of glutamate in a concentration-dependent manner. The partial alpha 2-agonist oxymetazoline (1 and 10 microM) produced a slight inhibition in the evoked release of glutamate. In contrast, the alpha 1-agonist phenylephrine (1 and 10 microM) did not show any significant effects on the evoked release of glutamate. The inhibitory action of 10 microM clonidine on the evoked glutamate release was antagonized by the alpha 2-antagonist yohimbine (1 microM) but not by the opioid antagonist naloxone (10 microM). These findings suggest that noradrenaline, probably released from the descending inhibitory system, reduces the release of glutamate from the capsaicin-sensitive primary afferent terminals through alpha 2-adrenoceptors.

L-DOPA neurotoxicity is mediated by glutamate release in cultured rat striatal neurons.

The exposure of cultured rat striatal neurons to L-DOPA caused marked cell death. The L-DOPA cytotoxicity was inhibited by the addition of Mg2+ to and by the removal of Ca2+ from the culture medium, and also by the application of tetrodotoxin. Moreover, prolonged application of L-DOPA increased the glutamate content in the culture medium. These results indicate that L-DOPA produces neurotoxicity by facilitating glutamate release.

EFFECT OF REPEATED COLD STRESS ON CAPSAICIN-EVOKED RELEASE OF GLUTAMATE FROM RAT SPINAL DORSAL HORN SLICES

We investigated the effect of repeated cold stress (RCS) on the capsaicin-evoked release of glutamate from the primary afferent fibers of the rat, and compared this with the effect of inoculation of complete Freunds adjuvant (adjuvant inoculation). The release of glutamate was measured using a fluorometric on-line continuous monitoring system in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, the application of 1 microM capsaicin to spinal dorsal horn slices evoked glutamate release (18.6 +/- 1.2 pmol mg(-1) protein, n = 11). In rats subjected to RCS (RCS rats), the release of glutamate evoked by 1 microM capsaicin was markedly increased to 272% (n = 6, P < 0.05) of the value for the control group, although the basal release was not significantly altered (n = 6, P > 0.05). Adjuvant inoculation produced a significant increase in the basal and capsaicin (1 microM) evoked release of glutamate to 141 and 344% (n = 6, P < 0.05) of the value for the control group, respectively. The present results suggest that the facilitated release of glutamate from capsaicin-sensitive primary afferent terminals in the spinal dorsal horn is, at least in part, involved in the hyperalgesia of RCS rats as well as the complete Freunds adjuvant-induced hyperalgesia.

Specific binding sites for bifemelane in the hippocampus of the guinea pig, relevant to its pharmacological actions

Bifemelane has an anti-amnesic effect, produces the translocation of protein kinase C in the hippocampal CA3 region but not in CA1 and enhances long-term potentiation in the mossy fibre-CA3 system but not in the Schaffer collateral-CA1 system. The present study examined the specific binding of [3H]bifemelane in membrane preparations of guinea pig hippocampus and regional differences in such a binding. The binding of [3H]bifemelane was reversible and greater when incubated at 4 degrees C than at 25 or 37 degrees C. The binding of [3H]bifemelane appeared to be composed of at least 2 different affinity components. Imipramine significantly suppressed the binding of [3H]bifemelane at 1 microM and, in the presence of 1 microM imipramine, the low-affinity component of the binding of [3H]bifemelane was eliminated. The density of specific binding sites for 1 nM [3H]bifemelane was significantly higher in the hippocampal CA3 region than in the CA1. The specific binding of 1 nM [3H]bifemelane was not inhibited by other nootropic drugs, such as idebenone, calcium hopantenate, vinpocetine, indeloxazine and piracetam. The present results suggest that there are specific binding sites for bifemelane in hippocampus, which are different from those for other nootropic drugs tested and that the regional differences in the pharmacological susceptibilities to bifemelane are at least, in part, attributed to those in the density of binding sites for bifemelane.