Optical measurement of glutamate in slice preparations of the mouse retina.

Signaling by glutamatergic synapses plays an important role in visual processing in the retina. In this study, we used an enzyme-linked fluorescence assay system to monitor the dynamics of extracellular glutamate in a slice preparation from the mouse retina. High K stimulation induced an elevation of fluorescence in the inner plexiform layer (IPL) of the retina when glutamate transporters were inhibited by dl-threo-ß-benzyloxyaspartic acid (TBOA). The high K-induced fluorescence signals in the IPL were inhibited by the calcium channel blocker Cd2+. Blockade of GABAergic and glycinergic circuits by picrotoxin and strychnine also elevated the fluorescence signals in the IPL. Thus, the enzyme-linked fluorescence assay system might be useful for monitoring the bulk concentration of extracellular glutamate released by synapses in the inner retina.

Mechanism of the formation of dural arteriovenous fistula: the role of the emissary vein.

Dural arteriovenous fistula (DAVF) can be separated into two types: DAVF which drains through an affected sinus (sinus type) and DAVF with direct reflux to the cortical vein (non-sinus type). The present report attempted to clarify the mechanism of formation and development of DAVF focusing on the emissary vein (EV) hypothesis.First, inflammation occurs at the penetrating point of the EV on the dura due to idiopathic or secondary causes. Local inflammatory reactions induce vessel dilatation and neovascularization, and subsequently create arteriovenous (AV) connections on the arteriole level. Although EV communicating with dural arteries might play a role as draining routes at first, they start to degrade due to compression of enlarged emissary arteries or to a hemodynamic shift to the drainage pathway of least resistance. Following the occlusion of drainage pathway through EV into the sinus or cortical veins may form, resulting in clinically detectable DAVF. The AV shunt then expands to the surrounding dura associated with recruitment of feeders from distant sites induced by expression of angiogenetic factors and a shift in the hemodynamic balance. In sinus type DAVF, the sinus is progressively compartmentalized and finally occludes due to thrombogenesis with activated coagulopathy or to hemodynamic hypertrophy of the sinus wall. This progression results in the mature, aggressive DAVF with drainage impairments. Previous mechanistic hypotheses focusing on sinus hypertension and sinus thromboses cannot explain the pathogenesis of non-sinus type of DAVF. Although the etiology of DAVF may be concerned by the thrombo-occlusive change of sinus, the unique theory presented in this report may enable an understanding of the common etiology of both types of DAVF.

Protective effect against 17beta-estradiol on neuronal apoptosis in hippocampus tissue following transient ischemia/recirculation in mongolian gerbils via down-regulation of tissue transglutaminase activity.

We analyzed the protective effect of 17beta-estradiol (17beta-ED) injection against delayed neuronal death in the hippocampus tissue of the brain in Mongolian gerbils after transient ischemia/recirculation treatment, especially in relation with bcl-2 gene expression and enzymatic activity changes of caspase-3 and tissue transglutaminase (tTGase). Daily intraperitoneal injection of 17beta-ED to the animal after the ischemia stimulated the expression of an apoptosis suppressor gene, bcl-2, in the hippocampal tissue for a week. The gradually increasing apoptotic enzyme activity of caspase-3 and increased number of TUNEL positive fragmented neuronal nuclei caused by ischemic attack in the gerbil brain were clearly suppressed by 17beta-ED administration. The reduced activity and enzyme protein of tTGase, a neurodegenerative marker of apoptosis in the hippocampus after ischemia, were also restored to nearly normal levels by 17beta-ED injection. These results suggest that daily 17beta-ED administration to the gerbil after transient ischemic insult with progressing neuronal deteriorative changes in hippocampus tissue can effectively prevent apoptotic changes through a molecular cascade involving gene expression regulation.

Linalool suppresses voltage-gated currents in sensory neurons and cerebellar Purkinje cells.

Linalool is a major component of essential oils and possesses various biological effects in sensory or central nervous systems. To investigate the pharmacological and biophysical effects of linalool on voltage-gated currents in sensory neurons, we used the whole-cell patch clamp and the Ca(2+) imaging techniques. Under the voltage clamp, membrane depolarization generated time- and voltage-dependent current responses in newt olfactory receptor cells (ORCs). Linalool significantly and reversibly suppressed the voltage-gated currents in ORCs. The dose-suppression relation of linalool for the voltage-gated Na(+) current could be fitted by the Hill equation with a half-blocking concentration of 0.56 mM and a Hill coefficient of 1.2. To test whether linalool suppresses voltage-gated currents in ORCs specifically or suppresses currents in other neurons generally, we next examined the effects of linalool on voltage-gated currents in newt retinal neurons and rat cerebellar Purkinje cells. Linalool suppressed the voltage-gated currents not only in retinal horizontal cells and ganglion cells but also in Purkinje cells. Furthermore, bath application of linalool inhibited the KCl-induced [Ca(2+)](i) response of ORCs, suggesting that linalool suppresses Ca(2+) currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells.

Direct suppression by odorants of ionotropic glutamate receptors in newt retinal neurons.

Odorants are known to suppress voltage-gated channels not only in olfactory receptor cells but also in neurons of outside of the olfactory system. Here we found that odorants suppress glutamate-gated channels in newt retinal neurons using the Ca(2+) imaging technique. Bath application of 100 microM glutamate rose [Ca(2+)](i) under application of the voltage-gated Ca(2+) channel blocker. Thus, [Ca(2+)](i) rises in the neurons were most likely attributable to Ca(2+) influx via Ca(2+)-permeable glutamate-gated channels rather than voltage-gated Ca(2+) channels. A similar increase of [Ca(2+)](i) was observed by application of 100 microM NMDA and 50 microM kainate, suggesting that both NMDA and AMPA/kainate receptors were expressed in newt retinal neurons. Application of odorants, 1 mM amyl acetate and acetophenone, reversibly reduced [Ca(2+)](i) increased by glutamate, NMDA and kainate. This suggests that odorants can suppress not only voltage-gated channels but also ligand-gated channels such as NMDA and AMPA/kainate receptors.

Na(+) action potentials in human photoreceptors.

Mammalian photoreceptors are hyperpolarized by a light stimulus and are commonly thought to be nonspiking neurons. We used the whole-cell patch-clamp technique on surgically excised human retina to examine whether human photoreceptors can elicit action potentials. We discovered that human rod photoreceptors express voltage-gated Na(+) channels, and generate Na(+) action potentials, in response to membrane depolarization from membrane potentials of -60 or -70 mV. Na(+) spikes in human rods were elicited at the termination of a light response that hyperpolarized the potential well below -50 mV. This served to amplify the release of a neurotransmitter when a bright light is turned off, and thus selectively amplify the off response to the light signal.

Modulation by cGMP of the voltage-gated currents in newt olfactory receptor cells.

Effects of cGMP on voltage-gated currents in the somatic membrane of isolated newt olfactory receptor cells were investigated using the whole-cell mode of the patch-clamp technique. Under voltage clamp, membrane depolarization generated time- and voltage-dependent current responses, a transient inward current and a sustained outward current. When cGMP or a membrane permeant analog of cGMP, 8-p-chlorophenylthio-cGMP (CPT-cGMP), was applied to the recorded cell, the amplitude of the transient inward current increased markedly, but that of the sustained outward current did not change significantly. When each current was isolated by pharmacological agents, 0.1 mM CPT-cGMP increased the peak amplitude of a Na(+) current (I(Na)) by approximately 40%, a T-type Ca(2+) current (I(Ca,T)) by approximately 40%, and an L-type Ca(2+)current (I(Ca,L)) by approximately 10%; however it did not change significantly the amplitude of a delayed rectifier K(+) current (I(K)). A selective cGMP-dependent protein kinase inhibitor, KT5823, blocked the enhancement by cGMP of I(Na) and I(Ca,T), suggesting that cGMP increases these currents via cGMP-dependent phosphorylation. Under current-clamp conditions, application of CPT-cGMP lowered the current threshold of action potentials induced by current injection, and increased the maximum spike frequency in response to strong stimuli. We suggest that cGMP may lower the threshold in olfactory perception by decreasing the current threshold to generate spikes, and also prevent the saturation of odor signals by increasing the maximum spike frequency.

Direct suppression by odorants of cyclic nucleotide-gated currents in the newt photoreceptors.

Odorants are known to suppress the cyclic nucleotide-gated (CNG) current in olfactory receptor cells. It is unclear, however, whether odorants suppress the olfactory CNG current directly or whether they suppress the current by decreasing the second messenger (cAMP) through the activation of phosphodiesterase. We found that odorants also suppress CNG currents in photoreceptor cells. Under voltage clamp, an odorant puff immediately suppressed the currents induced by the intracellular cGMP in isolated newt rods and cones. Odorants also suppressed the currents induced by another cGMP analog (8-p-chlorophenylthio-cGMP, which strongly resists hydrolysis by phosphodiesterase), suggesting that the second messenger metabolism via phosphodiesterase is not involved in the suppression by odorants. This suggests that odorants suppress the CNG currents directly rather than via the second messenger system in photoreceptors, and also likely in olfactory receptor cells.

Odorants suppress voltage-gated currents in retinal horizontal cells in goldfish.

Odorants are known to suppress non-selectively voltage-gated currents in olfactory receptor cells. We found that odorants also suppress voltage-gated currents in neurons of outside of the olfactory system. Under voltage clamp, odorants such as amyl acetate, limonene, and acetophenone suppressed non-selectively voltage-gated currents (a Ca(2+) current, a delayed rectifier K(+) current, a fast transient K(+) current, and an anomalous rectifier K(+) current) in horizontal cells from the goldfish retina. An amyl acetate puff completely and immediately suppressed the Ca(2+) current (I(Ca)) and the delayed rectifier K(+) current induced by repetitive depolarizations, suggesting that amyl acetate is a closed-channel blocker. Odorants did not change significantly the activation curve of I(Ca), but made the slope of inactivation curve of I(Ca) gentler and shifted its half-inactivation voltage toward a negative voltage. These results are similar to the effects of odorants on voltage-gated currents in olfactory receptor cells. This suggests that odorants may suppress the voltage-gated currents in retinal horizontal cells by the same mechanism described in olfactory receptor cells.

Is cyclic AMP involved in the defibrillating effect of sotalol?

Ventricular fibrillation induced in animals pretreated with sotalol, a class III antiarrhythmic agent, would spontaneously terminate and revert into a sinus rhythm. This phenomenon has been attributed to the class III action of this drug, i.e., prolongation of myocardial action potential duration and effective refractory period. Since various observations suggested that these alone cannot explain the defibrillating phenomenon, we hypothesised that sotalol affected ventricular intercellular synchronization by increasing intercellular coupling. Our recent experimental studies have shown that sotalol antagonized the cellular decoupling to guinea pig ventricular muscle strip caused by perfusion with either a hypoxic normal Tyrode's solution or an oxygenated high Ca2+ Tyrode's solution. We assumed that the most likely mechanism for the restoration of intercellular coupling would be increasing intracellular cAMP concentration. In order to test this hypothesis, we studied the modification of this sotalol-induced recoupling by a cAMP dependent protein kinase inhibitor. The results clearly supported our assumption since the addition of Arg-Gly-Tyr-Ala-Leu- Gly (pure A- kinase inhibitor) prevented the aforementioned cellular recoupling action of sotalol in a dose-dependent manner. It can thus be concluded that changes in intracellular cAMP level are involved in the synchronizing / defibrillating effect of sotalol.

Sotalol facilitates spontaneous ventricular defibrillation by enhancing intercellular coupling. An entirely new mechanism for its antiarrhythmic action.

We have previously shown that sotalol, a class III antiarrhythmic agent, helps spontaneous ventricular defibrillation in various mammalian species. Since we hypothesized that self ventricular defibrillation depends on a high degree of intercellular synchronization, and since the major electrophysiological action of sotalol causing prolongation of action potential duration (APD), cannot fully explain its defibrillating property, we carried out a series of studies to examine the effect of sotalol on intercellular myocardial coupling. Guinea pig right ventricular muscle preparations were superfused in a tissue bath and the spread of intracellularly injected fluorescent dye (Lucifer yellow CH) to the neighboring cells was studied under various conditions. When either the Ca2+ concentration of Tyrode's solution was elevated to 6 mM or the solution was made hypoxic by not bubbling O2 (n = 3 each), no spread of the injected dye was observed. The addition of 1 microM sotalol to the high Ca2+ solution or 0.5 microM to the hypoxic superfusate (n = 3 each) caused a wide spreading of the dye, thus strongly suggesting a marked improvement in the intercellular coupling. These results show an entirely new property of sotalol, i.e., enhancement of cellular synchronization, which may better explain its ability to cause spontaneous ventricular defibrillation than its class III action. Our previous demonstration of successful spontaneous ventricular defibrillation by several other agents that are known to enhance intercellular coupling but have contrasting actions on APD further substantiates our hypothesis.

Arachidonic acid blocks gap junctions between retinal horizontal cells.

Horizontal cells in turtle retinae are electrically coupled via gap junctions, and the input resistances of the cells are too low to be measured. However, intracellular injection of arachidonic acid into horizontal cells caused great increases in the input resistances of the cells, and the cells could be easily polarized by intracellular current injection. The injection of arachidonic acid also caused decrease in light responses of horizontal cells to surround illumination, and blocked dye-couplings with Lucifer Yellow CH. On the other hand, injection of a lipoxygenase inhibitor or a guanylate cyclase inhibitor into horizontal cells suppressed the decoupling effect of arachidonic acid. These findings suggest that lipoxygenase metabolites of arachidonic acid block gap junctions by activating guanylate cyclase.

[Effect of nitroprusside on gap junctions of single tracheal smooth muscle cells excised from rats].

To investigate the effect of nitroprusside on gap junctions of tracheal smooth muscle cells, we ionophoretically injected sodium nitroprusside into single tracheal smooth muscle cells excised from rats. The input resistance of the tracheal smooth muscle cells increased after the injection of nitroprusside. This results suggests that nitroprusside blocks gap junctions of tracheal smooth muscle cells.

[Intracellular messengers and their roles in retinal gap junctions].

Intracellular injection of cAMP or cGMP into retinal horizontal cells blocked the gap junctions between the cells. Similar results were obtained when L-arginine was injected into the cells. L-Arginine is a substrate of nitric oxide (NO) which is believed to activate soluble guanylate cyclase to produce cGMP. The endothelium-derived relaxing factor (EDRF) in the blood vessels has been identified as NO. With respect to the nervous systems, production of NO and its synthase have been found in the brain, and NO has been discussed in relation to such phenomena as synaptic plasticity, long-term potentiation, and development. The decoupling effect of L-arginine suggests the presence of the L-arginine: NO: cGMP pathway in the retina as well. Before injection of cAMP, cGMP or L-arginine, the applied current leaked through the gap junctions. After the injection, the horizontal cells could be easily polarized by intracellular current injection, and the synaptic mechanisms were analyzed by measuring I-V curves. In luminosity-type (H1) horizontal cells, the reversal potential of light responses was estimated at about 0 mV. In addition, conductance decreases were detected during illumination. These findings support the widely accepted hypothesis that glutamate is released from the photoreceptors in darkness. In chromaticity-type cells (H2 and H3 cells), the reversal potentials of light responses were about 0 mV, suggesting that the ionic mechanisms of synaptic transmission are common among horizontal cell types.

Synaptic inputs to turtle horizontal cells analyzed after blocking of gap junctions by intracellular injection of cyclic nucleotides.

Intracellular injection of cAMP or cGMP into turtle horizontal cells significantly increased the input resistances, and the cells could thus be easily polarized by current injection, suggesting that the cyclic nucleotides blocked gap junctions between cells. Then, synaptic inputs onto triphasic chromaticity-type cells were analyzed. Hyperpolarizing and depolarizing light responses were all reduced with depolarizing current, and their polarities were reversed by further depolarization. Their reversal potentials coincided at around 0 mV. This level was the same as observed in luminosity-type and biphasic chromaticity-type cells, suggesting that the ionic mechanisms of synaptic transmission are common among horizontal cell types.

Arginine blocks gap junctions between retinal horizontal cells.

Functions of nitric oxide are of common interest among a variety of tissues, since it activates soluble guanylate cyclase to produce cGMP. Here we report that intracellular application of L-arginine, the precursor of nitric oxide, blocked gap junctions between horizontal cells of the turtle retina. The input resistances of the cells were greatly increased and the cells were thereby easily polarized by current injections through microelectrodes. This procedure enables us to plot precise I-V curves and the reversal potential of light responses was estimated at around 0 mV. These results were quite similar to those obtained by intracellular application of cGMP, suggesting that the L-arginine:nitric oxide:cGMP pathway is present in retinal horizontal cells.

Decoupling of horizontal cells in carp and turtle retinae by intracellular injection of cyclic AMP.

1. Horizontal cells are electrically coupled through gap junctions. This is a disadvantage in elucidating the membrane properties of the cells. In order to block gap junctions, adenosine 3',5'-cyclic monophosphate (cyclic AMP) or its analogues, dibutyryl cyclic AMP and 8-bromo cyclic AMP, were ionophoretically injected into horizontal cells of the carp or turtle retina. 2. Before injection of the chemicals the input resistance of the cell was so low as to be unmeasurable, because the applied current leaked through gap junctions. After injection, however, the input resistance was significantly increased. 3. After the injection dye-coupling between horizontal cells was not observed when examined by intracellular injection of Lucifer Yellow dye, supporting the idea that high concentrations of intracellular cyclic AMP block gap junctions. 4. In this situation responses to light delivered to the receptive field centre were increased in amplitude, while responses to light delivered to the receptive field surround were greatly diminished. 5. After injection horizontal cells were readily polarized by conventional intracellular current injection. The hyperpolarizing light responses in carp and turtle luminosity-type cells (H1 cells) could be reversed by depolarizing the horizontal cells, and the reversal potentials were estimated to be about 0 mV. In addition, the resistance increase which accompanied the hyperpolarizing light responses could be detected. 6. In turtle biphasic chromaticity-type horizontal cells (H2 cells), hyperpolarizing light responses to shorter wavelengths and depolarizing ones to longer wavelengths could be reversed by depolarizing the horizontal cells. Both responses have almost the same reversal potential at about 0 mV. The membrane resistance changes associated with light responses were also detected; the resistance increased during the hyperpolarizing response, while it decreased during the depolarizing response. These observations suggest that the ionic mechanisms of both responses are probably the same, irrespective of their polarities.

Coexistence of NMDA and non-NMDA receptors on turtle horizontal cells revealed using isolated retina preparations.

Effects of glutamate and its agonists on horizontal cells appeared diversely when studied in turtle eyecup preparations. Consistent results were obtained when the isolated retina preparations were used. Not only kainate and quisqualate but also N-methyl-D-aspartate (NMDA) caused a sustained depolarization and light-evoked responses were suppressed for as long as these agonists were superfused. A selective antagonist of NMDA, 2-amino-5-phosphonovalerate (APV), hyperpolarized horizontal cells and reduced their light-evoked responses. These results indicate the coexistence of NMDA and non-NMDA receptors on turtle horizontal cells.