Fusao Kawai

Retinal horizontal cells form a broad receptive field, which contributes to generating antagonistic surround responses in retinal bipolar cells. Here, I found that depolarizing responses of yellow/red, blue-type horizontal cells (Y/RB HCs) exhibit a larger receptive field than hyperpolarizing responses at monochromatic lights between 480 nm and 520 nm. Because bipolar cells play a key role in the detection of visual contrast, depolarization or hyperpolarization of Y/RB HCs may regulate the size of the surround receptive field in the bipolar cells.

Olfactory perception is influenced by behavioral states, presumably via efferent regulation. Using the whole cell version of patch-clamp recording technique, we discovered that acetylcholine, which is released from efferent fibers in the olfactory mucosa, can directly affect the signal encoding in newt olfactory receptor cells (ORCs). Under current-clamp conditions, application of carbachol, an acetylcholine receptor agonist, increased the spike frequency of ORCs and lowered their spike threshold. When a 3-pA current to induce near-threshold depolarization was injected into ORCs, 0.0 spikes/s were generated in control solution and 0.5 spikes/s in the presence of carbachol. By strong stimuli of injection of a 13-pA current into ORCs, 9.1 and 11.0 spikes/s were generated in control and carbachol solutions, respectively. A similar result was observed by bath application of 50 xM acetylcholine. Under voltage-clamp conditions, carbachol increased the peak amplitude of a voltage-gated sodium current by 32% and T-type calcium current by 39%. Atropine, the specific muscarinic receptor antagonist, blocked the enhancement by carbachol of the voltage-gated sodium current and T-type calcium current, suggesting that carbachol increases those currents via the muscarinic receptor rather than via the nicotinic receptor. In contrast, carbachol did not significantly change the amplitude of the L-type calcium current or the delayed rectifier potassium current in the ORCs. Because T-type calcium current is known to lower the threshold in ORCs, we suggest that acetylcholine enhance excitability by lowering the threshold of spike generation in ORCs via the muscarinic receptor. © 2013 the American Physiological Society.

PURPOSE. The h current (I(h)) is a hyperpolarization-activated current that plays important roles in the physiological functions of different types of cells. In the retina of lower vertebrates, I(h) contributes to the rod responses to light stimuli by bringing the membrane potential back to the dark level in the presence of continuous light. The purpose of this study was to determine how dopamine modulates I(h) in human rods and regulates voltage responses. METHODS. A patch-clamp recording technique was used on surgically excised human retinas to investigate the effects of dopamine on the I(h) of isolated rods. Dopamine was applied in the superfusate. RESULTS. Dopamine reversibly decreased the amplitude of the I(h) induced by hyperpolarizing voltage steps from a holding potential of -60 mV. At a voltage step of -100 mV, 20 mu M dopamine decreased the amplitude of I(h). The D2 dopamine agonist quinpirole inhibited I(h), but the D1 agonist SKF-38393 had no effect. Dopamine-induced reduction of I(h) amplitude was blocked by the D2 dopamine antagonist sulpiride. Under current-clamp conditions, an injection of hyperpolarizing current steps to rods produced voltage responses that exhibited a gradual decay. Adding dopamine to the superfusate inhibited the decay in the voltage responses. Quinpirole also inhibited the voltage decay, whereas SKF-38393 was ineffective. CONCLUSIONS. Dopamine reduced I(h) through a D2 receptor and inhibited the gradual decay in the voltage response through a D2 receptor, indicating that dopamine slows the recovery phase of responses to light stimuli by inhibiting Ih in human rods. (Invest Ophthalmol Vis Sci. 2011;52:4113-4117) DOI: 10.1167/iovs.10-6983

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 Ca2+ 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 [Ca2+](i) response of ORCs, suggesting that linalool suppresses Ca2+ currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells.

PURPOSE. The sense of vision in humans is robust, and visual flickering is rarely experienced. To investigate this mechanism, electrophysiological and molecular biological techniques were used on human cone and rod photoreceptors. METHODS. Voltage-gated currents were recorded using the patch-clamp technique on isolated human cones, and especially their voltage-gated Na+ currents were analyzed in detail. Whether Na+ channel transcripts could be detected in single photoreceptors using RT-PCR was also examined, to test the expression of voltage-gated Na+ channels in cones and/or rods. RESULTS. Under current-clamp conditions, blocking h currents (hyperpolarization-activated cationic currents) with Cs+, Tl+, or ZD7288 hyperpolarized the resting potentials of cones and rods by similar to10 to 15 mV, and surprisingly generated spontaneous action potentials. The spontaneous spikes were blocked by 1 muM tetrodotoxin, but not by 1 mM Co2+, suggesting that they were Na+ spikes rather than Ca2+ spikes. Under voltage-clamp conditions, application of Cs+ and ZD7288 markedly decreased the steady inward current through the h channel. This is consistent with Cs+-induced hyperpolarization under a current-clamp condition. SCN2 Na+ channel was observed in both cones and rods by single-cell RT-PCR analysis, suggesting that human photoreceptors express the SCN2 Na+ channel. CONCLUSIONS. The data confirmed that voltage-gated Na+ channels were expressed not only in human rods but also in cones by electrophysiological and molecular biological experiments. These results suggest that the h current may contribute to preventing visual flickering by inhibiting the generation of spontaneous Na+ spikes in human photoreceptors.

Olfaction beams with the transduction of the information carried by odorants into electrical signals in olfactory receptor cells (ORCs). The binding of odor molecules to specific receptor proteins on the ciliary surface of ORCs induces the receptor potentials. This initial excitation causes a slow and graded depolarizing voltage change, which is encoded into a train of action potentials. Action potentials of ORCs are generated by voltage-gated Na+ currents and T-type Ca2+ currents in the somatic membrane. Isolated ORCs. which have lost their cilia during the dissociation procedure, are known to exhibit spike frequency accommodation by injecting the steady current. This raises the possibility that somatic ionic channels in ORCs may serve for odor adaptation at the level of spike encoding. although odor adaptation is mainly accomplished by the ciliary transduction machinery. This review discusses current knowledge concerning the mechanisms of spike generation in ORCs. It also reviews how neurotransmitters and hormones modulate ionic currents and action potentials in ORCs. (C) 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

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 Ca2+ imaging technique. Bath application of 100 muM glutamate rose [Ca2+](i) under application of the voltage-gated Ca2+ channel blocker. Thus, [Ca2+](i) rises in the neurons were most likely attributable to Ca2+ influx via Ca2+-permeable glutamate-gated channels rather than voltage-gated Ca2+ channels. A similar increase of [Ca2+](i) was observed by application of 100muM NMDA and 50muM kainate, suggesting that both NMDA and AMPA/kainate receptors were expressed in newt retinal neurons. Application of odorants, 1mM amyl acetate and acetophenone, reversibly reduced [Ca2+](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.

We used the whole-cell patch-clamp recording technique on surgically excised human retina to examine whether human rod photoreceptors express hyperpolarization-activated cationic currents (I-h) and to analyze the effects of I-h on rod's voltage responses. Hyperpolarizing voltage steps from a holding potential of -60 mV evoked a slow inward-rectifying current in both rods in retinal slices and isolated rods. The slow inward-rectifying currents induced by hyperpolarization were markedly reduced by 3 mM Cs+ (a blocker of I-h) in the bath, but not by 3 mM Ba2+ (an anomalous rectifier K+ current blocker) or 1 mM SITS (a Cl- current blocker). A concentration-response curve for block by Cs+ of the inward currents could be fitted by the Hill equation with a half-blocking concentration (IC50) of 41 muM and a Hill coefficient of 0.91. The time course of the inward current activation was well described at all recorded voltages by the sum of two exponentials. Under current-clamp conditions, injection of steps of current, either hyperpolarizing or depolarizing, elicited an initial rapid voltage change that was followed by a gradual decay in the voltage response. The decay in the voltage responses was eliminated by bath application of 3 mM Cs+. The voltage dependence, pharmacology, and kinetics of the slow inward-rectifying currents described above suggest that human rods express I-h. We suggest that I-h becomes activated in the course of large hyperpolarizations generated by bright-light illumination and may modify the waveform of the photovoltage in human rods. (C) 2002 Elsevier Science B.V. All rights reserved.

Certain ganglion cells in the mammalian retina are known to express a cGMP-gated cation channel. We found that a cGMP-gated current modulates spike responses of the ganglion cells in mammalian retinal slice preparation. In such cells under current clamp, bath application of the membrane-permeant cGMP analog (8-bromo-cGMP, 8-p-chlorophenylthio-cGMP) or a nitric oxide donor (sodium nitroprusside, S-nitroso-N-acetyl-penicillamine) depolarized the membrane potential by 5-15 mV, and reduced the amount of current needed to evoke action potentials. Similar effects were observed when the membrane potential was simply depolarized by steady current. The responses to cGMP are unaffected by inhibitors of cGMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase. The response to cGMP persisted in Ca2+-free bath solution with Ca2+ buffers in the pipette. Under voltage clamp, cGMP analogs did not affect the response kinetics of voltage-gated currents. We conclude that cGMP modulates ganglion cell spiking simply by depolarizing the membrane potential via the inward current through the cGMP-gated channel. Modulation of this channel via the long-range NO-synthase amacrine cell may contribute to control of contrast gain by peripheral mechanisms.

The olfactory system is thought to accomplish odor adaptation through the ciliary transduction machinery in olfactory receptor cells (ORCs). However, ORCs that have lost their cilia can exhibit spike frequency accommodation in which the action potential frequency decreases with time despite a steady depolarizing stimulus. This raises the possibility that somatic ionic channels in ORCs might serve for odor adaptation at the level of spike encoding, because spiking responses in ORCs encode the odor information. Here I investigate the adaptational mechanism at the somatic membrane using conventional and dynamic patch-clamp recording techniques, which enable the ciliary mechanism to be bypassed. A conditioning stimulus with an odorant-induced current markedly shifted the response range of action potentials induced by the same test stimulus to higher concentrations of the odorant, indicating odor adaptation. This effect was inhibited by charybdotoxin and iberiotoxin, Ca2+-activated K+ channel blockers, suggesting that somatic Ca2+-activated K+ currents regulate odor adaptation by modulating spike encoding. I conclude that not only the ciliary machinery but also the somatic membrane currents are crucial to odor adaptation.

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.

Mechanisms underlying action potential initiation in olfactory receptor cells (ORCs) during odor stimulation were investigated using conventional and dynamic patch-clamp recording techniques. Under current-clamp conditions, action potentials generated by a least effective odor-induced depolarization were almost completely blocked by 0.1 mM Ni2+, a T-type Ca2+ channel blocker, but not by 0.1 mM Cd2+, a high voltage-activated Ca2+ channel blocker. Under voltage-clamp conditions, depolarizing voltage steps induced a fast transient inward current, which consisted of Na+ (I-Na) and T-type Ca2+ (I-Ca, (T)) currents. The amplitude of I-Ca,I-T was approximately one- fourth of that of I-Na (0.23 +/- 0.03, mean +/- SEM). Because both I-Na and I-Ca,(T) are known to show rapid inactivation, we examined how much I-Na and I-Ca,(T) are activated during the gradually depolarizing initial phase of receptor potentials. The ratio of I-Ca,(T) /I-Na during a ramp depolarization at the slope of 0.5 mV/msec was 0.56 +/- 0.03. Using the dynamic patch-clamp recording technique, we also recorded I-Ca,I-T and I-Na during the generation of odor-induced action potentials. This ratio of I-Ca,I-T /I-Na was 0.54 +/- 0.04. These ratios were more than twice as large as that (0.23) obtained from the experiment using voltage steps, suggesting that I-Ca,I-T carries significant amount of current to generate the action potentials. We conclude that I-Ca,I-T contributes to enhance odor sensitivity by lowering the threshold of spike generation in ORCs.

The kinetics of glutamate concentration in the synaptic cleft is an important determinant of synaptic function. To elucidate peak concentration of glutamate released from a single vesicle in the cleft, spontaneous excitatory postsynaptic currents (sEPSCs) in Off-bipolar cells from the sliced newt retina were analyzed using whole-cell patch clamp recording and the computer simulation. The sEPSCs were blocked by an AMPA/kainate (KA) antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and prolonged by cyclothiazide. However, an N-methyl-D-aspartate (NMDA) antagonist, D-2-amino-5-phosphonopentanoic acid (D-AP5), was ineffective. These suggest that sEPSCs in Off-bipolar cells are mediated exclusively by AMPA/KA receptors. sEPSCs simulated by a detailed kinetic model of AMPA receptor best approximated the data, when peak glutamate concentration was 10 mu M. Therefore, it was concluded that peak concentration of glutamate released from a single vesicle would be elevated to approximately 10 mu M at the newt Off-bipolar dendrite. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

The AMPA receptor, ubiquitous in brain, is termed "ionotropic" because it gates an ion channel directly. We found that an AMPA receptor can also modulate a G-protein to gate an ion channel indirectly. Glutamate applied to a retinal ganglion cell briefly suppresses the inward current through a cGMP-gated channel. AMPA and kainate also suppress the current, an effect that is blocked both by their general antagonist CNQX and also by the relatively specific AMPA receptor antagonist GYKI-52466. Neither NMDA nor agonists of metabotropic glutamate receptors are effective. The AMPA-induced suppression of the cGMP-gated current is blocked when the patch pipette includes GDP-beta-S, whereas the suppression is irreversible when the pipette contains GTP-gamma-S. This suggests a G-protein mediator, and, consistent with this, pertussis toxin blocks the current suppression. Nitric oxide (NO) donors induce the current suppressed by AMPA, and phosphodiesterase inhibitors prevent the suppression. Apparently, the AMPA receptor can exhibit a "metabotropic" activity that allows it to antagonize excitation evoked by NO.

Olfactory perception is influenced by hormones. Here we report that adrenaline can directly affect the signal encoding of olfactory receptor cells. Application of adrenaline suppressed action potentials near threshold and increased their frequency in response to strong stimuli, resulting in a narrower dynamic range. Under voltage-clamp conditions, adrenaline enhanced sodium current and reduced T-type calcium current. Because sodium current is the major component of spike generation and T-type calcium current lowers the threshold in olfactory receptor cells, the effects of adrenaline on these currents are consistent with the results obtained under current-clamp conditions. Both effects involved a common cytoplasmic pathway, cAMP-dependent phosphorylation. We suggest that adrenaline may enhance contrast in olfactory perception by this mechanism.

Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch-clamp technique. Under voltage clamp, membrane depolarization to voltages between -90 mV and +40 mV from a holding potential (Vh) Of -100 mV generated time- and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate for individual current components (Na+ current: I-Na, T-type Ca2+ current: I-Ca,I-T, L-type Ca2+ current: I-Ca,I-L, delayed rectifier K+ current: I-Kv and Ca2+-activated K+ current: I-K(Ca)) could be fitted by the Hill equation. Half-blocking concentrations for each current were 0.11 mM (I-Na), 0.15 mM (I-Ca,I-T), 0.14 mM (I-CaL), 1.7 mM (I-Kv), and 0.17 mM (I-K(Ca)), and Hill coefficient was 1.4 (I-Na), 1.0 (I-Ca,I-T), 1.1 (I-Ca,I-L), 1.0 (I-Kv), and 1.1 (I-K(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of I-Na was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were -53 mV at control, -66 mV at 0.01 mM, and -84 mV at 0.1 mM. These phenomena are similar to the suppressive effects of local anesthetics (lidocaine and benzocaine) on I,, in various preparations, suggesting that both types of suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels.