Mahito Ohkuma

ACTION SPECTRUM FOR THE PHOTOPHOBIC RESPONSE OF CIONA INTESTINALIS (ASCIDIEACEA, UROCHORDATA) LARVAE IMPLICATES RETINAL PROTEIN

Abstract Ascidian tadpole larvae change swimming behavior during the course of development. The photic behavior of the larvae of Ciona intestinalis was monitored by a computerized cell‐tracking system with a time resolution of 0.1 s. Newly hatched larvae swim at an average speed of 1.4 mm/s but show no response to light stimuli. The swimming speed of the larvae became slower (0.4 mm/s) 3 h after hatching and they were induced to swim more rapidly by a sudden decrease in light intensity 4 h after hatching. During the course of development, the maximal speed of swimming behavior increased with time until 8 h after hatching and then plateaued. The action spectrum for the step‐down photophobic response of the larvae was determined at around 8 h after hatching and was fitted to Dartnalls nomogram with the absorbance maximum of the pigment located at 505 nm. These results suggest retinal proteins in the ocellus of the larvae are the photoreceptors for the photobehavior.

Patch-clamp recording of human retinal photoreceptors and bipolar cells.

Photoreceptors and retinal bipolar cells are considered as nonspiking neurons; however, we recently showed that human rod photoreceptors can generate sodium action potentials in response to membrane depolarization from membrane potentials of −60 or −70 mV ( Kawai et al., Neuron30 [2001] 451). We performed patch‐clamp recording of human cone photoreceptors and retinal bipolar cells to examine whether functional voltage‐gated sodium channels are expressed in these cells as well as rod photoreceptors. Under current‐clamp conditions, the injection of depolarizing current steps into a cone photoreceptor‐induced marked action potentials. These action potentials were blocked by 1 µM tetrodotoxin, a voltage‐gated sodium channel blocker. Under voltage‐clamp conditions, depolarizing voltage steps‐induced a fast transient inward current in several bipolar cells (n = 4/78). This current was activated from −70 to +20 mV (maximal at −10 mV) and inactivated within 5 ms. The 10–90% rise time of this current was shorter than another inward current (less than one‐hundredth). These results indicate that human cones and bipolar cells express voltage‐gated sodium channels as rod photoreceptors. Sodium channels may serve to amplify the release of a neurotransmitter and to accelerate the light–dark change in photosignals.

Requirement of the tumour suppressor APC for the clustering of PSD‐95 and AMPA receptors in hippocampal neurons

Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C‐terminal region with postsynaptic density (PSD)‐95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant‐negative construct that disrupts the APC–PSD‐95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD‐95 and a glutamate receptor subunit, and reduced alpha‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate (AMPA)‐induced activity of AMPA receptors; however, the clustering of an N‐methyl‐d‐aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses.

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

Summary. 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 μM 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 100 μM NMDA and 50 μM 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 [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.

Acetylcholine enhances excitability by lowering the threshold of spike generation in olfactory receptor 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 μM 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.

Heterotrimeric G Protein α and β Subunit Genes of the Ascidian, Halocynthia roretzi

In order to investigate G protein-coupling signal transduction system of ascidians in developmental stages and in embryonic neural system, we have cloned G protein α and β subunit genes and studied their spatial and temporal expression patterns in the ascidian, Halocynthia roretzi. Five different cDNA clones of G protein α subunit were isolated from a cDNA library of ascidian larvae. The deduced amino acid sequences of three of them (HrGiα, HrGqα, and HrGsα) showed high homology to those of α subunits of human Gi Gq, and Gs, respectively. The other two (HrGnα and HrGxα) was supposed to define a novel subfamily within the Gj and Gq families, respectively. Northern blot analysis revealed that messages of all these G protein α subunits were found during embryogenesis and in adult organs. Whole-mount in situ hybridization of tadpole larvae showed a distinct spatial expression pattern of the each α subunit gene.

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

A novel G protein α subunit in embryo of the Ascidian, Halocynthia roretzi

Abstract A cDNA clone encoding a novel G protein α subunit, HrGαn was isolated from the larvae of ascidian, Halocynthia roretzi. In contrast with overall amino acid identity (63%) with G protein α subunit of Gi or Go subclass, HrGαn has a unique amino acid sequence, which lacks a residue for pertussis toxin substrate, but retains for cholera toxin substrate for ADP-ribosylation. The sequence characteristics and molecular phylogenetic analysis suggest that HrGαn defines a novel subclass within Gi class of G protein α subunits. The zygotic expression of HrGαn was first detected at the 64-cell stage and observed in all blastomeres except for B7.4, B7.5 and B7.6 cells till the 110-cell stage. As progress of the developmental stages, the expression of HrGαn became restricted and was observed in the muscle, mesenchyme and a part of trunk lateral cells in tailbud embryos. With HrGαn-GFP fusion-gene construct it was showed that the genomic fragment containing 2674 bp upstream of the putative translation start site of HrGαn contained the regulatory sequence responsible for the expression in the muscle and mesenchyme cells, and that the regulatory sequence functioned also in Ciona intestinalis. Our results suggest a possible involvement of HrGαn in the signaling system regulates the cell fate during the embryogenesis of the ascidian.

Light Regulated GnRH Neurons in Biological Clock for Reproduction in the Ascidian, Halocynthia roretzi

Since ascidians spawn at a fixed latency after sunrise, light must regulate a biological clock for reproduction. The photoreceptor might drives the change in gonadal activity via the gonadotropin-releasing hormone (GnRH) system. Retinal proteins in the cerebral ganglion of the ascidian, Halocynthia roretzi, were visualized by the time-resolved difference fluorescence imaging and immunohistochemical method which showed the retinal protein bearing cells located close to the GnRH bearing cells.

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-β-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.