Interareal Synaptic Inputs Underlying Whisking-Related Activity in the Primary Somatosensory Barrel Cortex.

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 (Vm ) 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 Gi/o -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 subset of wS1 neurons, which were accompanied by depolarization and substantial reduction of slow-wave oscillatory fluctuations of Vm Despite an extensive innervation, inhibition of inputs from the whisker primary motor cortex (wM1) to wS1 did not alter the spike rates and Vm dynamics 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 Vm dynamics of wS1 neurons. Notably, silencing inputs from wTLM markedly decreased the modulation depth of whisking phase-tuned neurons in wS1, 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.

Iris-derived induced pluripotent stem cells that express GFP in all somatic cells of mice and differentiate into functional retinal neurons.

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.

A subset of cone bipolar cells expresses the Na+ channel SCN2A in the human retina.

Some bipolar cells in the human retina are known to express voltage-gated Na+ channels. However, it is unclear which types of channels are expressed, and whether Na+ channel expression is limited to specific types of bipolar cells. In the present study, we examined the types of voltage-gated Na+ channels expressed in human bipolar cells and the morphology of bipolar cells with voltage-gated Na+ currents. To investigate the expression of voltage-gated Na+ channels in human bipolar cells, we examined whether Na+ channel transcripts could be detected in single bipolar cells using the reverse transcription polymerase chain reaction (RT-PCR) technique. The voltage-gated Na+ current was recorded from isolated bipolar cells using the patch-clamp recording technique. Types of bipolar cells that have the Na+ currents were investigated by analyzing their morphology after staining with Lucifer yellow. Using RT-PCR, the SCN2A Na+ channel was detected in 5 of 6 isolated bipolar cells. This suggests that a subset of human bipolar cells expresses the SCN2A Na+ channel. Under voltage-clamp conditions, depolarizing voltage steps induced a fast transient inward current in cone bipolar cells with axon terminal boutons that stratified at the ON layer, which includes the stratum 3, 4, and 5 of the inner plexiform layer (IPL, n = 2/11 cells). The fast transient inward current of isolated bipolar cells was blocked by 1 µM of tetrodotoxin (TTX), a voltage-gated Na+ channel blocker. No fast transient inward current was recorded with axon terminals that stratify at the OFF layer, which includes stratum 1 and 2 of the IPL (n = 4). Thus, a subset of ON cone bipolar cells at least expresses the putative voltage-gated Na+ channel SCN2A in the human retina. The Na+ channels in the bipolar cells may serve to amplify the release of neurotransmitter, glutamate, when membrane potential is rapidly depolarized and thereby selectively accelerating light responses.

The Effect of Histamine on Inward and Outward Currents in Mouse Retinal Amacrine Cells.

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.

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.

Effects of Progesterone and Other Gonadal Hormones on Glutamatergic Circuits in the Retina.

BACKGROUND: Gonadal hormones function in the retina; however, their targets have not yet been identified. Therefore, the present study examined the effects of progesterone and other gonadal hormones on glutamatergic circuits in the retina. METHODS: Extracellular glutamate concentrations, which correspond to the amount of glutamate released, were examined using an enzyme-linked fluorescent assay system. The activity of glutamatergic synapses between bipolar cells and ganglion cells was investigated using a patch clamp technique. Changes in retinal thickness during pregnancy were assessed using optical coherence tomography (OCT) images. RESULTS: Progesterone and pregnenolone sulfate increased extracellular glutamate concentrations, whereas estrogen and testosterone did not. Progesterone increased the activity of glutamatergic synapses between bipolar cells and ganglion cells. A temporal decrease in the thickness of the peripheral retina was observed in the 1st trimester. CONCLUSIONS: Progesterone, but not estrogen or testosterone, activated glutamate release in the mouse retina. Increases in the concentration of progesterone during pregnancy did not induce any detectable change in retinal thickness.

Long-range axonal projections of transplanted mouse embryonic stem cell-derived hypothalamic neurons into adult mouse brain.

The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

Novel Technique for Retinal Nerve Cell Regeneration with Electrophysiological Functions Using Human Iris-Derived iPS Cells.

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.

Isolation of multipotent stem cells from mouse adipose tissue.

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 multipotent 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, osteoblasts, 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, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells. The rate of differentiation into adipocytes, osteoblasts 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).

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., 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 microM 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.

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.

Suppression by an h current of spontaneous Na+ action potentials in human cone and rod photoreceptors.

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 approximately 10 to 15 mV, and surprisingly generated spontaneous action potentials. The spontaneous spikes were blocked by 1 microM 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.

A novel G protein alpha subunit in embryo of the ascidian, Halocynthia roretzi.

A cDNA clone encoding a novel G protein alpha subunit, HrGalpha(n) was isolated from the larvae of ascidian, Halocynthia roretzi. In contrast with overall amino acid identity (63%) with G protein alpha subunit of G(i) or G(o) subclass, HrGalpha(n) 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 HrGalpha(n) defines a novel subclass within G(i) class of G protein alpha subunits. The zygotic expression of HrGalpha(n) 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 HrGalpha(n) became restricted and was observed in the muscle, mesenchyme and a part of trunk lateral cells in tailbud embryos. With HrGalpha(n)-GFP fusion-gene construct it was showed that the genomic fragment containing 2674 bp upstream of the putative translation start site of HrGalpha(n) 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 HrGalpha(n) in the signaling system regulates the cell fate during the embryogenesis of the ascidian.

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.