Tsukasa Gotow

Excitatory and inhibitory effects of histamine on molluscan neurons

Histamine elicited depolarization (excitation) in some neurons and hyperpolarization (inhibition) in other neurons of the central nervous system of the marine mollusc, Onchidium verruculatum. The histamine sensitive region was along the axon at some distance from the soma. H1-receptor blockers (SA-97 and mepyramine) suppressed the excitatory (H1) response without affecting the inhibitory (H2) response, while H2-receptor blockers (burimamide and metiamide) suppressed the H2-response without affecting the H1-response. The H1-response was associated with a marked increase in membrane conductance and was blocked by removal of the external Na. The H2-response consisted of a hyperpolarization without much change in conductance, compared with the hyperpolarization of same amplitude produced by glutamate in the same neuron. Passive polarization of the membrane and reduction of Cl concentrations to 1/5-1/25 caused no significant change in H2-response. The H2-response was slightly suppressed in K-free saline. Thus, it seems difficult to account for the hyperpolarization only by an increase in K or Cl conductance. Complete removal of Na and addition of ouabain blocked the H2-response, suggesting a contribution of an electrogenic Na-pump to the hyperpolarization. However, in 20 mM Na saline with or without K, histamine still caused clear hyperpolarization. In this solution, the histamine response was not affected by ouabain. Although it is difficult to exclude the possibility that an increase in K conductance may be responsible for the hyperpolarization, it is tentatively proposed as a hypothesis that the H2-response involved some active transport mechanism, different from a ouabain-sensitive electrogenic Na-pump.

Single K+ channels closed by light and opened by cyclic GMP in molluscan extra-ocular photoreceptor cells ☆

We report the first recordings of the light-sensitive channel which is active during dark and is closed by light in the Onchidium extra-ocular photoreceptor cells. This light-sensitive channel was K-selective and was not blocked by extracellular Ca2+ and Mg2+. Application of cyclic GMP to excised inside-out patches activated (opened) a channel that appeared to be the same as the light-sensitive channel recorded from the same membrane in the intact cell.

Light-increased cGMP and K+ conductance in the hyperpolarizing receptor potential of Onchidium extra-ocular photoreceptors

The phototransduction mechanism of the extra-ocular photoreceptor cells Ip-2 and Ip-1 in the mollusc Onchidium ganglion was examined. Previous work showed that the depolarizing receptor potential of another extra-ocular photoreceptor cell, A-P-1 is produced by a decrease of the light-sensitive K+ conductance activated by a second messenger, cGMP and is inactivated by the hydrolysis of cGMP. Here, a hyperpolarizing receptor potential of Ip-2 or Ip-1 was associated with an increase in membrane conductance. When Ip-2 or Ip-1 was voltage-clamped near the resting membrane potential, light induced an outward photocurrent corresponding to the above hyperpolarization. The spectral sensitivity had a peak at 510 nm. The shift of reversal potentials of the photocurrent depended on the Nernst equation of K(+)-selective conductance. The photocurrent was blocked by 4-AP and L-DIL, which are effective blockers of the A-P-1 light-sensitive K+ conductance. These results suggested that the hyperpolarization is mediated by increasing a similar light-sensitive K+ conductance to that of A-P-1. The injection of cGMP or Ca2+ into a cell produced a K+ current that mimicked the photocurrent. 4-AP and L-DIL both abolished the cGMP-activated K+ current, while TEA suppressed only the Ca(2+)-activated K+ current. These results indicated that cGMP is also a second messenger that regulates the light-sensitive K+ conductance. The photocurrent was blocked by LY-83583, a guanylate cyclase (GC) inhibitor, but was unaltered by zaprinast, a phosphodiesterase (PDE) inhibitor. Together, the present results suggest that increasing the internal cGMP in Ip-2 or Ip-1 cells light-activates GC rather than inhibits PDE, thereby leading to an increase of the light-sensitive K+ conductance and the hyperpolarization.

Light-dependent K+ Channels in the Mollusc Onchidium Simple Photoreceptors Are Opened by cGMP

Light-dependent K+ channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268–272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K+ channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K+ and 200 mM Mg2+ to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K+ concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants τo1, τo2 and τc1, τc2, respectively, similar to those of the light-dependent channel. In both the channels, τo1 and τo2 in ms ranges were similar to each other, although τc2 over tens of millisecond ranges was different. τo1, τo2, and the mean open time τo were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of τo2 or τo. In both, the reversal potentials using 200- and 450-mM K+-filled pipettes were close to the K+ equilibrium potentials, suggesting that both the channels are primarily K+ selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K+ pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K+ channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.

A light-induced decrease of cyclic GMP is involved in the photoresponse of molluscan extraocular photoreceptors

The depolarizing photoreceptor potential in the molluscan extraocular photoreceptor, A-P-1, results from the light-induced suppression of specific K+ currents. An application of cGMP or IBMX (inhibitor of phosphodiesterase) to A-P-1-evoked light-suppressed currents, similar to the above specific K+ currents. Thus, the light-induced decrease of cGMP levels in A-P-1 may be responsible for the photoreceptor potential, like vertebrate phototransductions.

Simple photoreceptors in some invertebrates: Physiological properties of a new photosensory modality

Simple photoreceptors, namely photoresponsive neurons without microvilli and/or cilia have long been known in the central ganglion of crayfish, Aplysia, Onchidium and Helix. Recently, similar simple photoreceptors, ipRGCs were discovered in the mammalian retinas. A characteristic common to all of their photoreceptor potentials shows a slow kinetics and little adaptation, contrasting with the fast and adaptive photoresponses in eye photoreceptors. Furthermore, these simple photoreceptors are not only first-order photosensory cells, but also second-order interneurons. Such characteristics suggested that simple photoreceptors function as a new sensory modality, non-image-forming vision, which is different from the image-forming vision of eye photoreceptors. The Onchidium simple photoreceptors A-P-1 and Es-1 respond to light with a depolarizing receptor potential, caused by closing of light-dependent, cGMP-gated K+ channels, as in vertebrate cGMP cascade mediated by Gt-type G-protein. The same simple photoreceptors Ip-2 and Ip-1 are hyperpolarized by light, owing to opening of the same K+ channels. This shows the first demonstration of a new type of cGMP cascade, in which Ip-2/Ip-1 are hyperpolarized when light activates guanylate cyclase (GC) through a Go-type G-protein. The ipRGCs, as involved in non-imaging function of ipRGCs, contribute to pupillary light reflex and circadian clocks. However, their function as interneurons has not been ascertained. In Onchidium simple photoreceptors, A-P-1/Es-1 and Ip-2/Ip-1 cells the photoreceptor potentials play a role in LTP-like long-lasting potentiation (LLP) of the non-imaging functions, e.g., excitatory tactile or inhibitory pressure synaptic transmission and the subsequent behavioral responses. It was also shown that this LLP is effective, even if their photoresponse is subthreshold.

Involvement of a Go-type G-protein coupled to guanylate cyclase in the phototransduction cGMP cascade of molluscan simple photoreceptors

Simple photoreceptors, namely photoresponsive neurons, designated as A-P-1, Es-1, Ip-2 and Ip-1, exist in the sea slug Onchidium ganglion. Previous works has shown that, of these, Ip-2 and Ip-1 respond to light with a hyperpolarizing receptor potential, caused by the opening of light-dependent, cGMP-gated K+ channels, whereas A-P-1 and Es-1 are depolarized by light, owing to the closing of the same K+ channels. The present study of Ip-2 or Ip-1 cells was undertaken to identify the G-proteins that couple light to the activation of guanylate cyclase (GC), thereby leading to the opening of K+ channels and the consequent hyperpolarizing photocurrents. The specific channel blocker, 4-aminopyridine (4-AP), and a GC inhibitor, LY-83583, both suppressed this hyperpolarizing photocurrent. N-ethylmaleimide and GDP-beta-S also inhibited this photocurrent, consistent with the involvement of G-proteins. Mastoparan an activator of both Go- and Gi-type G-proteins, induced an outward current. Furthermore, benzalkonium chloride (C(16)BAC), a selective activator of Go, dose-dependently generated an outward current similar to that induced by mastoparan. Both of these outward currents were susceptible to 4-AP, LY-83583 and N-ethylmaleimide. Taken together, these results suggest that phototransduction in Ip-2 or Ip-1 cells is triggered by a Go-type G-protein coupled to GC. Thus, this new cGMP cascade contrasts with the conventional phototransduction cGMP cascade mediated by the Gt-type G-protein coupled to phosphodiesterase, seen in the vertebrate photoreceptors and the above A-P-1 or Es-1 cells.

4-Aminopyridine and l-cis-diltiazem block the cGMP-activated K+ channels closed by light in the molluscan extra-ocular photoreceptors

The cGMP-activated K+ channels closed by light lead to the depolarizing photocurrent of photoreceptors in the Onchidium ganglion. Whole-cell current records showed that external application of 100-200 microM 4-aminopyridine or 200-400 microM l-cis-diltiazem completely blocked the macroscopic photocurrent at any depolarizing and hyperpolarizing potentials. Single-channel current recordings suggested that both 4-aminopyridine and l-cis-diltiazem act to block the cGMP-activated K+ channels in their open state from inside the cell.

An analysis of histamine-induced inhibitory response in molluscan neurons

Mechanisms of the histamine-induced inhibitory response (the H2-response) in neurons of the marine mollusc Onchidium, were further investigated following the preceding paper. The H2-response in normal saline was blocked by ouabain, but the response recovered after a short exposure to Na-free solution containing ouabain. The recovery was only transient in the continuous presence of ouabain. When external Na was reduced to about 1/8 normal concentration (60 mM), the H2-response became sensitive to removal of external Ca, but insensitive to ouabain. The suppressing effect of Ca removal and the recovery by Ca readmission appeared very slowly. However, in about 1/3 normal Na concentration (150 mM) the H2-response was suppressed by removal of the Ca, only in the presence of ouabain. The Na-gradient may be regulated by the ouabain-insensitive transportk, such as a Na-Ca exchange in addition to the ouabain-sensitive Na-pump. The Na-Ca exchange probably dominates over the ouabain-sensitive Na-pump only when passive Na-influx is reduced in a low external Na concentration. The H2-response was markedly inhibited by DNP (5 X 10(-4)M) and cyanide (2 X 10(-3)M), while the hyperpolarization produced by glutamate, which was accompanied by a large reduction of membrane resistance, was not affected by these metabolic inhibitors. Over a wide range of external Na concentrations, the membrane potential was lower in presence than in the absence of external Ca. This may be explained by the hypothesis that there is an electrogenic Na-Ca exchange in which Ca-influx is coupled with Na-efflux. According to a similar hypothesis, the H2-response is produced by the transport system in which Ca-efflux is coupled with Na-influx and the system is controlled by the transmembrane Na gradient.

Characterization of long-lasting histaminergic inhibition in a beating pacemaker neuron of Onchidium

A single BPSP (excitatory-inhibitory postsynaptic potential) was monosynaptically produced in an identified Onchidium neuron, Be-1, with a beating rhythm upon stimulation of the cardiac nerve. The BPSPs summated to produce an inhibition of long duration (ILD) upon blockage of the beating rhythm after repeated stimulation, so that the BPSPs seemed to be functionally inhibitory. Ten stimuli (1-2 Hz) applied to the cardiac nerve usually evoked an ILD (0.5-1 min) of about 10 mV. The early and middle phases of this ILD reversed near -80 to -85 mV, but the late phase did not reverse at more negative potentials. None of the phases was significantly affected by low Cl or Na solutions or by high Ca solutions. However, by changing the external K, the shift of the reversal potentials for the early and middle phases reached about 65% of that predicted for the K electrode, although the late phase was insensitive to the external K. Intracellular tetraethylammonium (TEA) attenuated the amplitude of the ILD but did not shorten the duration. These suggest that the ILD has another conductance-independent mechanism simultaneously with the increase in K conductance. Several lines of evidence suggested that a ouabain-sensitive Na pump does not contribute to the ILD. Inhibitors of energy supply, 2,4-dinitrophenol sodium salt (DNP) and cyanide, selectively and reversibly reduced the ILD. Simultaneous applications of intracellular TEA and DNP completely abolished the ILD. As for the ionic basis, the histamine-induced inhibitory response in Be-1 was closely related to the ILD. Cimetidine specifically blocked the ILD and histamine-induced inhibitory response, which were mimicked by 2-methylhistamine, but not by dimaprit. It is concluded that the ILD, mediated by some histamine receptor other than the H1 or H2 type, results from an increase in K conductance and a hyperpolarizing ion pump insensitive to ouabain.