大熊 真人

Body movements influence brain-wide neuronal activities. In the sensory cortex, thalamocortical bottom-up inputs and motor-sensory top-down inputs are thought to affect the dynamics of membrane potentials (V_m) of neurons and change their processing of sensory information during movements. However, direct perturbation of the axons projecting to the sensory cortex from other remote areas during movements has remained unassessed, and therefore the interareal circuits generating motor-related signals in sensory cortices remain unclear. Using a G_i-coupled opsin, eOPN3, we here inhibited interareal signals incoming to the whisker primary somatosensory barrel cortex (wS1) of awake male mice and tested their effects on whisking-related changes in neuronal activities in wS1. Spontaneous whisking in air induced the changes in spike rates of a fraction of wS1 neurons, which were accompanied by depolarization and substantial reduction of slow-wave oscillatory fluctuations of V_m. Despite an extensive innervation, inhibition of inputs from the whisker primary motor cortex (wM1) to wS1 did not alter the spike rates and V_mdynamics of wS1 neurons during whisking. In contrast, inhibition of axons from the whisker-related thalamus (wTLM) and the whisker secondary somatosensory cortex (wS2) to wS1 largely attenuated the whisking-related supra- and sub-threshold V_mdynamics of wS1 neurons. Notably, silencing inputs from wTLM markedly decreased the modulation depth of whisking phase-tuned neurons, while inhibiting wS2 inputs did not impact the whisking variable tuning of wS1 neurons. Thus, sensorimotor integration in wS1 during spontaneous whisking is predominantly facilitated by direct synaptic inputs from wTLM and wS2 rather than from wM1. Significance statementThe traditional viewpoint underscores the importance of motor-sensory projections in shaping movement-induced neuronal activity within sensory cortices. However, this study challenges such established views. We reveal that the synaptic inputs from the whisker primary motor cortex do not alter the activity patterns and membrane potential dynamics of neurons in the whisker primary somatosensory cortex (wS1) during spontaneous whisker movements. Furthermore, we make a novel observation that inhibiting inputs from the whisker secondary somatosensory cortex (wS2) substantially curtails movement-related activities in wS1, leaving the tuning to whisking variables unaffected. These findings provoke a reconsideration of the role of motor-sensory projections in sensorimotor integration and bring to light a new function for wS2-to-wS1 projections.

When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.

Regenerative medicine in ophthalmology that uses induced pluripotent stem cells (iPS) cells has been described, but those studies used iPS cells derived from fibroblasts. Here, we generated iPS cells derived from iris cells that develop from the same inner layer of the optic cup as the retina, to regenerate retinal nerves. We first identified cells positive for p75NTR, a marker of retinal tissue stem and progenitor cells, in human iris tissue. We then reprogrammed the cultured p75NTR-positive iris tissue stem/progenitor (H-iris stem/progenitor) cells to create iris-derived iPS (H-iris iPS) cells for the first time. These cells were positive for iPS cell markers and showed pluripotency to differentiate into three germ layers. When H-iris iPS cells were pre-differentiated into neural stem/progenitor cells, not all cells became positive for neural stem/progenitor and nerve cell markers. When these cells were pre-differentiated into neural stem/progenitor cells, sorted with p75NTR, and used as a medium for differentiating into retinal nerve cells, the cells differentiated into Recoverin-positive cells with electrophysiological functions. In a different medium, H-iris iPS cells differentiated into retinal ganglion cell marker-positive cells with electrophysiological functions. This is the first demonstration of H-iris iPS cells differentiating into retinal neurons that function physiologically as neurons.

The expression of H1 receptor has been reported in amacrine cells of mouse and rat retinae. However, we assumed that other types of histamine receptors also function in amacrine cells. In order to confirm that histamine modulates the membrane potential in mouse amacrine cells, we measured voltage-gated currents using whole-cell configuration. Under voltage-clamp conditions, the amplitude of voltage-gated outward currents was enhanced by the application of 100 µM histamine in 65% of amacrine cells. Histamine also increased the amplitudes of voltage-gated inward currents in 72% of amacrine cells. When antagonists of the histamine H1, H2, or H3 receptors were applied to histamine-sensitive amacrine cells, all three types of these inhibitors reduced the effect of histamine. Moreover, we classified recorded cells into seven types based on their morphological characteristics. Two of the seven types, diffuse multistratified cells and AII amacrine cells, responded significantly to histamine. These results indicate that histamine affected the membrane potential via three types of histamine receptors. Furthermore, there were differences in the responses to histamine among types of amacrine cells. Histamine may be one of the important neurotransmitters and/or neuromodulators in the visual processing.

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.

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 iHCENY1 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.

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.

Background: Embryonic stem (ES) cells, bone marrow, adipose tissue or other genetically modified stem cells are being widely used in basic research in the field of regenerative medicine. However, there is no specific surface antigen that can be used as a marker of muttipotent stem cells. Objective: We tried to isolate and collect putative multipotent stem cells from mouse subcutaneous adipose tissue using the p75 neurotrophin receptor (p75NTR) as a marker. Methods: Adipose tissue was processed for immunostaining using antibodies anti-CD90, anti-CD105 and anti-Sca-1 as general mesenchymal stem cell (MSC) markers, and anti-p75NTR, an epithelial stem cell and MSC marker. Subsequently, the expression of cell surface markers in adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells) was examined by flow cytometry (fluorescence-activated cell sorting: FACS). Finally, ADSVF cells positive for p75NTR were sorted and cultured to induce their differentiation into adipocytes, osteobtasts, chondrocytes, smooth muscle cells and neuronal cells. Results: Cells positive for several of these markers were found in the deep layers of adipose tissue. Among them, those positive for p75NTR differentiated into adipocytes, osteobtasts, chondrocytes, smooth muscle cells and neuronal cells. The rate of differentiation into adipocytes, osteobtasts and neuronal cells was higher for p75NTR-positive cells than for p75NTR-negative cells. Conclusions: p75NTR proved to be a useful marker to isolate adipose tissue-derived stem cells (ASCs). (c) 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

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.

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., Neuron 30 [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 mu M 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.

Bone marrow stroma cells (MSCs) have been shown to differentiate into multiple lineages and have great potential for regenerative therapy. We have obtained hMSCs from 6 human adults. They were all positive for CD13, CD44, and CD90 and weakly positive for CD49, while negative for CD45, suggesting that they were different from hematopoetic stem cells. hMSCs were induced to become neuronal cells and maintained as long as three weeks in the serum free medium supplemented with N2. An increase in the amount of mRNA was observed for the NeuroD1, neurofilament M and H, MAP2, neuron-specific enolase, tryptophan hydroxylase, Nurr1, and neuron specific Na⁺ channel genes, and the existence of voltage-gated Na⁺ channels that were sensitive to tetrodotoxin was confirmed electro physiologically. These results suggested that hMSCs differentiated into serotonergic neural cells. However, the expression of genes specific for stroma cells, the Big-h3 and vimentin-genes, was observed equally during the induction process, indicating that the expression pattern was not the completely same as in genuine neural cells. hMSCs cultured with serial passages showed aging phenomena at the cellular level under the various conditions examined in this study. Trials to isolate cellular clones proliferating indefinitely have not succeeded.

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.

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.

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 alpha and beta subunit genes and studied their spatial and temporal expression patterns in the ascidian, Halocynthia roretzi. Five different cDNA clones of G protein alpha subunit were isolated from a cDNA library of ascidian larvae. The deduced amino acid sequences of three of them (HrG(i)alpha, HrG(q)alpha, and HrG(s)alpha) showed high homology to those of alpha subunits of human G(i), G(q), and G(s), respectively. The other two (HrG(n)alpha and HrG(x)alpha) was supposed to define a novel subfamily within the G(i) and G(q) families, respectively. Northern blot analysis revealed that messages of all these G protein a 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 alpha subunit gene. A cDNA clone of G protein beta subunit (HrG beta) was isolated from a cDNA library of ascidian larvae. The expression of HrG beta overlapped with the expression of all alpha subunit genes in tadpole larvae, suggesting that HrG beta couples with multiple alpha subunits in ascidian tadpole larvae.

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. Photoresponses of the cerebral ganglion of ascidian, Halocynthia roretzi, were examined and two light-evoked responses recorded extracellularly, a light-evoked slow potential and light inhibition of high frequency spontaneous discharges. These results suggest that pacemaker signals of GnRH neurons might be regulated by photoreceptor activation. Thus, the photosignal might proceed from photoreceptor cell to GnRH neuron intercellularly.

Since ascidians, a primitive chordate, spawn at a fixed latency after sunrise, light must regulate a biological clock for reproduction in the ascidians. A retinal protein found in the cerebral ganglion of the ascidian is a candidate for the photoreceptor that might drives the change in gonadal activity via the gonadotropin-releasing hormone (GnRH) system. Photoresponses of the cerebral ganglion of ascidian, Halocynthia roretzi, were examined and two light-evoked responses recorded extracellularly, a light-evoked slow potential and light inhibition of high frequency spontaneous discharges. These results suggest that pacemaker signals of GnRH neurons might be regulated by photoreceptor activation. Immunohistochemical studies showed photoreceptor cells located close to the GnRH neurons and thus the photosignal might proceed from photoreceptor cell to GnRH neuron intercellularly. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

Retinal proteins in the cerebral ganglion of the ascidian, Halocynthia roretzi, were successfully visualized and their localization was determined by the time-resolved fluorescence difference imaging method. This visualizes retinal proteins in the tissue even though the concentration of the retinal proteins is low and there is considerable endogenous fluorescence. Heterogeneous retinal proteins in the same tissues could be distinguished by differential sensitivities to photobleaching, standardized by rhodopsin and retinochrome in octopus retina. Retinal proteins in the ocellus of ascidian larva, which is composed of only about 20 photoreceptor cells, were successfully visualized. Retinal proteins in the cerebral ganglion of adult Halocynthia roretzi, localized mainly at the surface of the anterodorsal root and the posterodorsal root. In the cross sections along the anteroposterio axis of the cerebral ganglion, the cells bearing retinal proteins were found in the peripheral cellular cortex mainly at the dorsal surface. Close localization of retinal protein and GnRH bearing cells suggests that retinal protein may trigger the biological clock for spawning in this ascidian.

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 Dartnall's 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.