Yuko Kaneko

Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt.

SummaryElectrical membrane properties of solitary spiking cells during newt (Cynops pyrrhogaster) retinal regeneration were studied with whole-cell patch-clamp methods in comparison with those in the normal retina.The membrane currents of normal spiking cells consisted of 5 components: inward Na+ and Ca++ currents and 3 outward K+ currents of tetraethylammonium (TEA)-sensitive, 4-aminopyridine (4-AP)-sensitive, and Ca++-activated varieties. The resting potential was about -40mV. The activation voltage for Na+ and Ca++ currents was about -30 and -17 mV, respectively. The maximum Na+ and Ca++ currents were about 1057 and 179 pA, respectively.In regenerating retinae after 19–20 days of surgery, solitary cells with depigmented cytoplasm showed slowrising action potentials of long duration. The ionic dependence of this activity displayed two voltage-dependent components: slow inward Na+ and TEA-sensitive outward K+ currents. The maximum inward current (about 156 pA) was much smaller than that of the control. There was no indication of an inward Ca++ current.During subsequent regeneration, the inward Ca++ current appeared in most spiking cells, and the magnitude of the inward Na+, Ca++, and outward K+ currents all increased. By 30 days of regeneration, the electrical activities of spiking cells became identical to those in the normal retina. No significant difference in the resting potential and the activation voltage for Na+ and Ca++ currents was found during the regenerating period examined.

Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of adult newt

Adult newts can regenerate their entire retina through transdifferentiation of the retinal pigment epithelium (RPE). The objective of this study was to redescribe the retina regeneration process by means of modern biological techniques. We report two different antibodies (RPE‐No.112 and MAB5428) that recognize the newt homolog of RPE65, which is involved in the visual cycle and exclusively label the RPE cell‐layer in the adult newt eye. We analyzed the process of retinal regeneration by immunohistochemistry and immunoblotting and propose that this process should be divided into nine stages. We found that the RPE65 protein is present in the RPE‐derived new retinal rudiment at 14 days postoperative (po) and in the regenerating retinas at the 3–4 cell stage (19 days po). These observations suggest that certain characteristics of RPE cells overlap with those of retinal stem/progenitor cells during the period of transdifferentiation. However, RPE65 protein was not detected in either retinal stem/progenitor cells in the ciliary marginal zone (CMZ) of adult eyes or in neuroepithelium present during retina development, where it was first detected in differentiated RPE. Moreover, the gene expression of RPE65 was drastically downregulated in the early phase of transdifferentiation (by 10 days po), while those of Connexin43 and Pax‐6, both expressed in regenerating retinas, were differently upregulated. These observations suggest that the RPE65 protein in the RPE‐derived retinal rudiment may represent the remainder after protein degradation or discharge rather than newly synthesized protein. J. Comp. Neurol. 40:391–407, 2006.

An immunohistochemical study of regenerating newt retinas

Light-microscopical examination was carried out to investigate the emergence and development of several classes of immunoreactive cells in regenerating retinas of the adult newt (Triturus pyrrhogaster) after total retinal ablation. Immunoreactive proliferating cell nuclear antigen (ir-PCNA, a marker for replicating cells) was present in nuclei of all neuroblasts in the early mono-layered to several-layered stages (15-20 days after retinal ablation; days 15-20), but was lost progressively in an intermediate-to-central/peripheral order as cells and layers increased (days 20-25). Cells, which had lost ir-PCNA, began to separate to form the outer nuclear, inner nuclear and ganglion cell layers around days 25-30 (the cell separation stage). Finally, the location of ir-PCNA was restricted to a band of neuroblast cells at the retinal margin (days 30-35) as seen in intact adult retinas. Visinin-immunoreactive (ir) cells, mainly destined to be cones, appeared first singly or as clusters at the most distal layer in the intermediate region of retinas multi-layered with PCNA-ir neuroblasts, which was followed by appearance of opsin-ir rod outer segments and tyrosine hydroxylase-ir amacrine cells around the cell separation stage. Shortly later, cells respectively immunoreactive to glutamic acid decarboxylase, neuropeptide Y, serotonin, glucagon, glutamine synthetase, glial fibrillary acidic protein, substance P and protein kinase C were found to emerge also in an intermediate-to-central/peripheral sequence. Some of the glucagon-ir cells appeared to be of an interplexiform type.

Pax‐6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax‐6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax‐6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax‐6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax‐6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax‐6 became similar to that of a normal retina. It was found that Pax‐6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax‐6 changed as cell differentiation proceeded during retinal regeneration.

Expression of Nav1.1 in rat retinal AII amacrine cells

In retinal ganglion cells (RGCs), the expression of various types of voltage-gated sodium channel (Nav) alpha-subunits (Nav1.1, Nav1.2, Nav1.3, and Nav1.6) has been reported. Like RGCs, certain subsets of retinal amacrine cells, including AII amacrine cells, generate tetrodotoxin (TTX)-sensitive action potentials in response to light; however, the Nav subtypes expressed in these cells have not been identified. We examined the Nav subtypes expressed in rat retinal amacrine cells by in situ hybridization (ISH) using RNA probes specific for TTX-sensitive Na(v)s (Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7). Our results confirmed that Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are localized in the ganglion cell layer (GCL). Interestingly, Nav1.1 was expressed not only in the GCL, but also in the inner nuclear layer (INL). The cell bodies of the Nav1.1-positive cells in the INL were located at the INL/inner plexiform layer (IPL) border. The cell bodies of AII amacrine cells are located close to the INL/IPL border, and these cells can be labeled with antibodies against parvalbumin (PV). Therefore, we combined ISH with immunohistochemistry and discovered that most of the PV-immunoreactive cells located at the INL/IPL border express Nav1.1. Our results show that AII amacrine cells express Nav1.1.

Expression pattern of a newt Notch homologue in regenerating newt retina.

We isolated part of a newt Notch homologue, N-Notch, from regenerating newt retina. The spatio-temporal pattern of N-Notch expression was studied by in situ hybridization at different stages of newt retinal regeneration. Proliferating cells were confirmed by the injection of bromodeoxyuridine (BrdU). In the early stage of regeneration, when the retina was one to two cells thick, all proliferating retinal progenitors expressed N-Notch. As the thickness of the retina increased with regeneration, N-Notch expression decreased in BrdU-positive cells on the vitreal side of the retina. Subsequently, presumptive retinal ganglion cells that were BrdU-negative cells appeared at the vitreal edge of the regenerating retina. These differentiating cells did not express N-Notch. Later, N-Notch expression decreased in the BrdU-positive cells on the scleral surface of the retina. Subsequently, presumptive photoreceptor cells that were BrdU-negative cells appeared in this region. These differentiating cells also did not express N-Notch. The proliferating retinal progenitors ceased expressing N-Notch and then stopped dividing during the differentiation of ganglion cells and photoreceptor cells. It was found that retinal regeneration involves the expression of an important developmental signaling molecule, Notch, in retinal progenitors and the expression of Notch ceased as cell differentiation proceeded during retinal regeneration.

The occurrence of apoptosis during retinal regeneration in adult newts

The present study examined the occurrence of apoptosis, identified by an in situ technique for detecting DNA fragmentation, in the regenerating retina of adult newts following ablation of the retina. Apoptosis occurs in the initial phase of regeneration when retinal precursor cells are actively proliferating. In the late stage of regeneration, when two synaptic layers are forming, apoptosis occurs mainly in the ganglion cell layer and inner nuclear layer. We found that apoptosis occurred with proliferation, differentiation, formation of retinal layers and retinotectal projections during retinal regeneration. Our findings suggest that apoptosis is closely related to these phenomena.

CLONING AND DISTRIBUTION OF A PUTATIVE TETRODOTOXIN-RESISTANT NA+ CHANNEL IN NEWT RETINA

Abstract Overlapping cDNA clones spanning the entire coding region of a Na+ channel were isolated from newt retina. The coding region predicts a 2,007 amino acid protein, designated nRNaCh (newt retina sodium channel), which is homologous to other Na+ channels. In situ hybridization indicated that nRNaCh is expressed exclusively in spiking neurons, where a tetrodotoxin (TTX)-resistant Na+ current has been recorded. Therefore, nRNaCh cDNA is sure to encode the TTX-resistant Na+ channel of newt retina. Sequence comparisons show that nRNaCh is more homologous to TTX-sensitive Na+ channels than to TTX-resistant Na+ channels. The length of the S5-S6 loop of repeat I of nRNaCh is similar to that of TTX-sensitive channels, whereas TTX-resistant Na+ channels have a deletion. The 3rd position in the SS2 region of repeat I of nRNaCh is a non-aromatic amino acid (Ala), which is a common feature of TTX-resistant channels. These findings suggest that whether the amino acid at the 3rd position in the SS2 region of repeat I is aromatic or non-aromatic determines the TTX sensitivity of Na+ channels, not the overall structure of the channel.

Development of voltage-gated currents in newt retinal neuroblasts in culture

Neuroblasts dissociated from early regenerating newt retina (18-20 days after surgery) survived in culture and extended neurites. They were classified into three groups on the basis of neurite outgrowth patterns; monopolar, bipolar, and multipolar varieties. Freshly dissociated neuroblasts (3-15 h in culture) were characterized by slow-rising action potentials with long duration, mediated by inward Na+ currents. After a few days in culture, these cells produced much briefer action potentials mediated by the characteristic Na+, Ca2+ and K+ currents of ganglion cells dissociated from normal retina. The results suggest that neuroblasts in early regenerating retina differentiate in vitro into spiking cells, possibly ganglion cells, characterized by their morphology and voltage-dependent ionic currents.

Cadherin Expression during Retinal Regeneration in the Adult Newt

Abstract The present study used an immunohistochemical technique to examine the expression of cadherins in the regenerating retina of adult newts. After surgical removal of the neural retina, retinal pigment epithelial (RPE) cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, retinal cells originating from RPE cells are undifferentiated precursor cells. Both E-cadherin and R-cadherin are expressed at the surface of these precursor cells. As regeneration proceeds, precursor cells differentiate into retinal neurons. R-cadherin is expressed at the surface of the differentiated neurons, but E-cadherin is lost to the differentiated neurons. The difference in expression pattern suggests that each cadherin plays distinctive roles in retinal regeneration. And our finding that Ecadherin is expressed transiently by undifferentiated precursor cells implies the importance of cell-cell adhesions by E-cadherin for differentiation.