Nonuniform distribution of cell proliferation in the adult teleost retina

Teleost fish continue to grow throughout life, and their eyes enlarge correspondingly. Within the eye, the retina grows by stretching existing tissue and adding new cells. Cell addition occurs in two ways: First, all cell types except rod photoreceptors are added circumferentially at the edge of the eye where the retina meets the iris; second, rod photoreceptors are generated from a population of rod progenitor cells which divide throughout the outer nuclear layer (ONL). To determine the spatial distribution of rod progenitor cells across the teleost retina, we labeled dividing cells with an antibody to proliferating cell nuclear antigen (PCNA) throughout a 24 h period. We found a significantly higher density of dividing rod precursor cells at the nasal and temporal margins than in the central retina throughout the 24 h cycle. At night, the density of dividing cells is significantly greater at the nasal pole of the eye. The difference between cell division at the center and the margin was reduced at night when the density of cell division in the central retina increased significantly. Taken together, these data suggest that the eye grows asymmetrically, with more cells added at the nasal pole. Possible developmental causes and functional consequences of the reported distribution of cell divisions in time and location are presented.     

Timing and location of rhodopsin expression in newly born rod photoreceptors in the adult teleost retina

Labeling of newly divided retinal cells with bromodeoxyuridine (BrdU) and a rhodopsin mRNA probe revealed that rhodopsin is first expressed by new rod photoreceptors 2 days after cell birth in an adult cichlid fish. Most new cells that expressed rhodopsin had nuclei located in the vitreal half of the outer nuclear layer (ONL), lending further support to the hypothesis that movement from scleral to vitreal ONL is associated with rod differentiation.     

成人脑神经干细胞体内外增殖、分化和迁移研究

目的探索从成人脑组织获取的神经干细胞（成人-hNSCs）在体外的增殖能力、分化特性、以及在裸鼠颅内的存活、迁移及分化情况。方法分别留取癫痫患者手术切除的颞叶脑组织和10W左右人类自然流产胎儿纹状体组织,体外分离成单细胞悬液,无血清培养基培养、传代并诱导分化。软琼脂糖集落形成实验检测NSCs的增殖能力。免疫荧光法检测NSCs标志物神经上皮巢蛋白（Nestin）和诱导分化后神经元标志物13．tubllin以及神经胶质细胞标志物胶质纤维酸性蛋白（GFAP）的表达;利用动物立体定向仪将体外悬浮培养2W的人NSCs移植入裸鼠颅内,检测NSCs在裸鼠脑组织局部的存活、迁移和分化状况。结果成人-hNSCs集落形成能力较胚胎脑组织来源的NSCs（胎儿-hNSCs）明显减弱,免疫荧光染色显示分离的NSCs呈Nestin阳性,诱导分化后可见（β-tubllin和GFAP阳性的神经细胞,其中80％的细胞为GFAP阳性的星形胶质细胞,20％左右为β-tubllin阳性细胞。分别将成人-hNSCs和胎儿．hNSCs移植入裸鼠纹状体,1个月后,冰冻切片,荧光显微镜观察到来源于成人脑组织来源的NSCs仅见沿针道的近距离迁移,免疫荧光染色在成人-hNSCs移植裸鼠颅内只检测到GFAP阳性的星形胶质细胞。而胎儿-hNSCs可穿过针道,沿大脑廉向脑实质广泛迁移,免疫荧光染色能检测到GFAP和少量（β-tubllin阳性细胞。结论成人脑组织和胚胎纹状体组织中均能成功分离到神经前体细胞,而与胎儿．hNSCs相比,成人-hNSCs体外增殖能力、多向分化潜能和体内迁移能力都明显减弱。     

Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain

In contrast to mammals, fish exhibit an enormous potential to produce new cells in the adult brain. By labeling mitotically dividing cells with 5-bromo-2'-deoxyuridine (BrdU), we have characterized the development of these cells in the zebrafish (Danio rerio). Proliferation zones were located in specific regions of the olfactory bulb, dorsal telencephalon (including a region presumably homologous to the mammalian hippocampus), preoptic area, dorsal zone of the periventricular hypothalamus, optic tectum, torus longitudinalis, vagal lobe, parenchyma near the rhombencephalic ventricle, and in a region of the medulla oblongata lateral to the vagal motor nucleus, as well as in all three subdivisions of the cerebellum, the valvula cerebelli, the corpus cerebelli, and the lobus caudalis cerebelli. In the valvula cerebelli and the corpus cerebelli, the young cells migrated from their site of origin in the molecular layers to the corresponding granule cell layers. By contrast, in the lobus caudalis cerebelli and optic tectum, no indication of a migration of the newly generated cells over wider distances could be obtained. BrdU-labeled cells remained present in the brain over at least 292 days post-BrdU administration, indicating a long-term survival of a significant portion of the newly generated cells. The combination of BrdU immunohistochemistry with immunolabeling against the neural marker protein Hu, or with retrograde tracing, suggested a neuronal differentiation in a large portion of the young cells.     

Characterization of cell proliferation throughout the brain of the African cichlid fish Astatotilapia burtoni and its regulation by social status

New cells are added in the brains of all adult vertebrates, but fishes have some of the greatest potential for neurogenesis and gliogenesis among all taxa, partly due to their indeterminate growth. Little is known, however, about how social interactions influence cell proliferation in the brain of these fishes that comprise the largest group of vertebrates. We used 5-bromo-2'-deoxyuridine (BrdU) to identify and localize proliferation zones in the telencephalon, diencephalon, mesencephalon, and rhombencephalon that were primarily associated with ventricular surfaces in the brain of the African cichlid fish Astatotilapia burtoni. Cell migration was evident in some regions by 1 day post injection, and many newborn cells coexpressed the neuronal marker HuC/D at 30 days, suggesting they had differentiated into neurons. To test the hypothesis that social status and perception of an opportunity to rise in rank influenced cell proliferation, we compared numbers of BrdU-labeled cells in multiple brain nuclei among fish of different social status. Socially suppressed subordinate males had the lowest numbers of proliferating cells in all brain regions examined, but males that were given an opportunity to rise in status had higher cell proliferation rates within 1 day, suggesting rapid upregulation of brain mitotic activity associated with this social transition. Furthermore, socially isolated dominant males had similar numbers of BrdU-labeled cells compared with dominant males that were housed in a socially rich environment, suggesting that isolation has little effect on proliferation and that reduced proliferation in subordinates is a result of the social subordination. These results suggest that A. burtoni will be a useful model to analyze the mechanisms of socially induced neurogenesis in vertebrates.     

Regular mosaics of large displaced and non-displaced ganglion cells in the retina of a cichlid fish

Large retinal ganglion cells in the tilapid cichlid fish Oreochromis spilurus (standard length 15-54 mm) were filled with horseradish peroxidase and studied in flatmounts. Three types, with distinct patterns of dendritic stratification, formed spatially independent, nonrandom mosaics. One type (about 0.3% of all ganglion cells) resembled the outer (off) alpha cells of mammals. They were very large, with thick primary dendrites and large, sparsely branched planar trees in the outer part of the inner plexiform layer (IPL). About 300 were arrayed regularly across each retina, their exact number and spacing depending on its size. Their somata were often displaced into the IPL, even where neighbours in the mosaic were orthotopic. Another type (0.8%) resembled the inner (on) alpha cells of mammals. These had slightly smaller somata that were never displaced and smaller trees in the middle layers of the IPL. About 800 were arrayed uniformly and regularly across each retina. A rarer type (0.06-0.08%) had two planar trees: one forming a coarse mosaic in the outer part of the inner plexiform layer (co-planar with the trees of outer alpha-like cells) and another in the outer plexiform layer. These "biplexiform" cells were smaller and rounder than alpha-like cells and always displaced. The dendrites were finer and less tapered. Cells in which we could identify an outer plexiform tree failed to cover the retina completely, but were nonrandomly distributed. We draw three main conclusions: (1) some nonmammalian vertebrates have separate inner and outer mosaics of large ganglion cells like those of mammals, (2) the vertical displacement of ganglion cell somata can vary widely within a single mosaic and may thus be functionally irrelevant, and (3) biplexiform ganglion cells exist in fish but differ in morphology from the biplexiform types described in some other vertebrates.     

Interphotoreceptor matrix in the human retina: cone-like domains surround a small population of rod photoreceptors.

The interphotoreceptor matrix (IPM) in mammalian retinas is subdivided into rod and cone specific compartments: peanut agglutinin (PNA) binding glycoconjugates are associated with cones, whereas wheat germ agglutinin (WGA) binding glycoconjugates are associated with rods. To establish the identity of a photoreceptor cell type in the human retina with rod dimensions but with a matrix domain which stains with PNA, double label studies, using PNA-ferritin to decorate the extracellular domains and immunocytochemical techniques using a rod specific anti-opsin antibody were conducted. The PNA-binding domains were observed in the cone-associated IPM as well as in the IPM surrounding a small population of rod-shaped photoreceptors. The outer segments of these rod-shaped photoreceptors showed intense labeling with a rod specific anti-opsin antibody as did all other rods which were free of PNA-labeling. A quantitative analysis of all retinal quadrants indicates that this novel rod represents approximately 0.3% of the total rod population in the human retina.     

The organization of retinal projections to the diencephalon and pretectum in the cichlid fish, Haplochromis burtoni

The organization of retinofugal projections was studied in a cichlid fish by labelling small groups of retinal ganglion cell axons with either horseradish peroxidase or cobaltous lysine. Two major findings resulted from these experiments. First, optic tract axons show a greater degree of pathway diversity than was previously appreciated, and this pathway diversity is related to the target nuclei of groups of axons. The most striking example is the formation of the medial optic tract. Fibers that will become the medial optic tract move abruptly away from their neighbors, at about the level of the optic chiasm, and coalesce at the dorsomedial edge of the marginal optic tract. The medial optic tract projects to the thalamus, the dorsal pretectum, and the deep layer of the optic tectum. The axial optic tract is a group of fibers which segregates from the most medial portion of the marginal optic tract, at about the level of the optic chiasm. The axial tract stays medial to the marginal optic tract for a few hundred microns and then curves laterally to rejoin the marginal optic tract. At least some axial trat axons terminate in the suprachiasmatic nucleus. Within the marginal optic tract, retinal ganglion cell axons from a given retinal quadrant are always segregated into at least two groups. The smaller group projects to the superficial pretectal nucleus. The larger group projects to the superficial layer of the optic tectum. Second, each nontectal retinal termination site receives a unique pattern of retinal input. Within the pretectum the parvocellular superficial pretectal nucleus receives a highly retinotopically organized input from all retinal regions; the basal optic nucleus receives a roughly retinotopically organized input from all retinal regions; the dorsal pretectum receives an input from all retinal regions; and the central pretectal nucleus receives input only from the ventral hemiretina. Within the diencephalon the thalamus receives an input from all retinal regions, but this input is not retinotopically organized; the suprachiasmatic nucleus receives input from the region of central retina that lies just dorsal to the optic nerve head, via the axial optic tract. The accessory optic nucleus receives input from the dorsal hemiretina.     

Drug-induced changes in catecholaminergic cells of the fish retina

The changes in fluorescence intensity and number of visible catecholaminergic cells (CA-cells), as revealed by means of a histofluorescence technique, were used as indicators of the effects of various pharmacological agents upon CA-cells in the retina of fishes (Cyprinus carpio and Eugerres plumieri). The study includes in vivo and in vitro experiments. In the in vivo experiments, intravitreal injection, two or three hours before eye enucleation, of 10 microgram L-DOPA, dopamine, or noradrenaline accentuated CA-cell fluorescence and increased the number of visible cells, whereas 10 microgram of tyramine, octopamine, synephrine, or adrenaline reduced the endogenous fluorescence. Intramuscular injection of reserpine (3 mg/kg) abolished CA-cell fluorescence. In the in vitro experiments, pieces of isolated retinas were incubated for three or 30 minutes in media containing different drugs. Only minor changes in fluorescence were detected after three minutes of incubation, but after 30 minutes, dopamine (20 microM) markedly enhanced CA-cell fluorescence. Carbachol (20 mM), acetylcholine (10 mM) plus BW-anticholinesterase (1 mM) or substance P(1.6 x 10(-2) mM), all reduced CA-cell fluorescence. Kainic acid (20 mM) abolished fluorescence from CA-cell somata, while fluorescent fiber networks remain unchanged. L-aspartate (5 mM) and GABA (10 mM) in the incubation medium did not influence fluorescence intensity. The results are relevant to, and consistent with, electrophysiological observations of dopamine-mediated spatial effects on horizontal cell potentials.     

Identification of a cone bipolar cell in cat retina which has input from both rod and cone photoreceptors

It has been generally accepted that rod photoreceptor cells in the mammalian retina make synaptic contact with only a single population of rod bipolar cells, whereas cone photoreceptors contact a variety of cone bipolar cells. This assumption has been challenged in rodents by reports of a type of cone bipolar cell which receives input from both rods and cones. Questions remained as to whether similar pathways are present in other mammals. We have used an antiserum against the glutamate transporter GLT1-B to visualize a population of cone bipolar cells in the cat retina which make flat contacts with axon terminals of both rod and cone photoreceptor cells. These cells are identified as OFF-cone bipolar cells and correspond morphologically to type cb1 (CBa2) cone bipolar cells which are a major source of input to OFF-beta ganglion cells in the cat retina. The GLT1-B transporter was also localized to processes making flat contacts with photoreceptor terminals in rat and rabbit retinas. Examination of tissue processed for the GluR1 glutamate receptor subunit showed that cb1 cone bipolar cells, like their rodent counterparts, express this alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-selective receptor at their contacts with rod spherules. Thus, a direct excitatory pathway from rod photoreceptors to OFF-cone bipolar cells appears to be a common feature of mammalian retinas.     

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

In teleost fish, unlike other vertebrates, the retina continues to grow throughout the animal's life both by stretching of the mature tissue and by the addition of new cells. Following larval development, new retinal cell birth is known to occur in a rim at the periphery of the mature retina and in the outer nuclear layer (ONL). We have now found that cell birth and proliferation also occurs in the inner nuclear layer (INL) of the mature fish retina. In rainbow trout (Onchoryncus mykiss), proliferative cells exist in the INL of fish of all ages, at least up to 2 years posthatching. The proliferative cells form clusters in the INL that align in radial columns, reaching from the inner to the outer plexiform layers. The density of proliferative cell clusters changes along the equatorial plane of the retina and is highest near both the nasal and temporal poles. Our data suggest that, after birth, the proliferative cells migrate away from the INL and into the ONL, with a half-time of about 3 days, and their cell bodies can be seen in the outer plexiform layer. Once they are in the ONL, the proliferative cells continue to divide and likely give rise to the precursor cells that differentiate into new rod photoreceptors. J. Comp. Neurol. 394:271–282, 1998. © 1998 Wiley-Liss, Inc.     

Lithium enhances proliferation and neuronal differentiation of neural progenitor cells in vitro and after transplantation into the adult rat spinal cord

Transplantation of neural progenitor cells (NPCs) holds great potential for the treatment of spinal cord injuries. The survival and differential fates of transplanted NPCs in the cord are key factors contributing to the success of the therapy. In this study, we investigate the effects of lithium, a widely used antidepressant drug, on the survival, proliferation and differentiation of spinal cord-derived NPCs in cultures and after transplantation into the spinal cord. Our results show that clinically relevant doses of lithium increase the proliferation of grafted NPCs at 2 weeks post-grafting and neuronal generation by grafted NPCs at 2 weeks and 4 weeks post-grafting. However, lithium does not cause preferential differentiation of NPCs into astrocytes or oligodendrocytes both in vitro and after transplantation. Our results also show that chronic treatment with lithium (up to 4 weeks) reduces microglia and macrophage activation, indicating that lithium treatment can affect the host immune response. The results of the present study provide evidence that lithium may have therapeutic potential in cell replacement strategies for CNS injury due to its ability to promote proliferation and neuronal generation of grafted NPCs and reduce the host immune reaction.     

Cyclic changes in dopamine and DOPAC content, and tyrosine hydroxylase activity in the retina of a cichlid fish

Using high-performance liquid chromatography combined with electrochemical detection (HPLC-ED) we measured the content of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) as well as the activity of tyrosine hydroxylase in the retina of a teleost fish during a normal 12 h dark-light cycle. All 3 parameters showed a distinct diurnal pattern with oscillations during phases of changing illumination and more stable levels throughout the light and dark phase. While tyrosine hydroxylase activity levels roughly paralleled dopamine values, the time course of DOPAC content was a mirror image of dopamine levels. The results suggest that changing dopamine levels provide an adaptational signal for the retinal neural network; its metabolism seems at least in part to be controlled by endogenous factors.     

Ultrastructural study of the pituicytes in the pituitary gland of the teleost Diplodus sargus

An electron microscopic study was performed on the pituitary gland of the Mediterranean teleost fish Diplodus sargus to analyse the morphological characteristics of the pituicytes. In this class of vertebrates, the pituicytes have, like other astroglial cells, a trophic and support function, but they may also play an active role in the release of neurohormones. Most of the pituicytes were of the Dark type. Their shape appeared irregular with long, thin processes protruding from the cellular body. The pituicytes protruded from the neurohypophysis as far as the adenohypophysis. Their cellular bodies were mainly located in the posterior neurohypophysis. In the adenohypophysis, pituicytic processes were intermingled with cells of the pars intermedia and pars distalis, though being more numerous in the former. These processes sometimes surrounded the whole adenohypophyseal cell. This provides further evidence for the possible role of the pituicytes in controlling the release of the pituitary hormones given that, in teleost fishes, there is no distinct portal system or true median eminence.     

Identification and localization of myosin superfamily members in fish retina and retinal pigmented epithelium

Myosins are cytoskeletal motors critical for generating the forces necessary for establishing cell structure and mediating actin‐dependent cell motility. In each cell type a multitude of myosins are expressed, each myosin contributing to aspects of morphogenesis, transport, or motility occurring in that cell type. To examine the roles of myosins in individual retinal cell types, we first used polymerase chain reaction (PCR) screening to identify myosins expressed in retina and retinal pigmented epithelium (RPE), followed by immunohistochemistry to examine the cellular and subcellular localizations of seven of these expressed myosins. In the myosin PCR screen of cDNA from striped bass retina and striped bass RPE, we amplified 17 distinct myosins from eight myosin classes from retinal cDNA and 11 distinct myosins from seven myosin classes from RPE cDNA. By using antibodies specific for myosins IIA, IIB, IIIA, IIIB, VI, VIIA, and IXB, we examined the localization patterns of these myosins in retinas and RPE of fish, and in isolated inner/outer segment fragments of green sunfish photoreceptors. Each of the myosins exhibited unique expression patterns in fish retina. Individual cell types expressed multiple myosin family members, some of which colocalized within a particular cell type. Because much is known about the functions and properties of these myosins from studies in other systems, their cellular and subcellular localization patterns in the retina help us understand which roles they might play in the vertebrate retina and RPE. J. Comp. Neurol. 513:209–223, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of horizontal cells generating different spectral responses in the retina of a teleost fish (Eugerres plumieri)

Six different types of spectral responses were recorded from horizontal cells under mesopic conditions in perfused retina, isolated from the dark-adapted mojarra (Eugerres plumieri). They were tentatively termed photopic Lr-, Lg1-, Lg2-, Lb-, and C-type, and scotopic L-type. The Lr-, Lg-, and Lb-type responses showed a maximum peak at 605, 550, and 516 nm respectively, while the C-type was composed of hyperpolarizing potentials in response to shorter wavelengths and depolarizing potentials in response to longer wavelengths (so-called R/G-type). The scotopic L-type has a peak at 516 nm in the spectral response and a slow decay phase in the waveform response. Following a brief period of diffuse illumination, it was found that the Lg1-type response is altered to the Lr-type, while both Lg2- and Lb-type responses change to the C-type. Intracellular marking with Lucifer or Procion yellow identified the cellular origins of different response types: external (He) and medial horizontal (Hm) cells for the Lr-type, internal horizontal (Hi) cells for the C-type, and rod-horizontal (Hr) cells for the scotopic L-type. Only He cells were found to possess an axon, while dye coupling was seen between axonless Hm, Hi, or Hr cells but not between He cells. The morphology of these fluorescent dye-marked cells was the same as that of the respective cells observed in Golgi-stained materials.     

成年大鼠脑创伤后神经前体细胞的增殖及迁移

目的 研究液压冲击性脑损伤后成年大鼠神经前体细胞的增殖及迁移。方法 制作液压冲击性脑损伤模型，免疫组织化学方法动态检测巢蛋白（Nestin）和5溴脱氧尿苷（BrdU）的表达。BrdU标记方法确定增列殖的前体细胞；Nestin的表达用于确定神经前体细胞。结果 同正常对照组相比较，伤侧皮层、海马及室下区的Nestin阳性细胞数于伤后1d明显增多，7d达高峰,30d消失；BrdU阳性细胞数于作后3d达高峰，而7d以后逐渐减小，室下区BrdU阳性细胞及Nestin阳性细胞经胼胝体向对侧迁移。结论 液压冲击性脑损伤可激发成年大鼠神经前体细胞增殖及迁移。     

Topography of ganglion cells and photoreceptors in the sheep retina

Retinal topographies of some cell types and distribution of the tapetum lucidum in the sheep's eye were investigated in this study. The tapetum was observed macroscopically in the fundus. The topographical distributions of retinal ganglion cells (RGCs), cones, and rods were simultaneously analyzed in retinal whole mounts stained with cresyl violet. Short‐wavelength‐sensitive (S) cones were immunocytochemically identified in retinal whole mounts. The tapetum was located dorsal to the optic disc, with the nasal part elongated horizontally and the temporal part expanded dorsally. RGCs were distributed densely in the area centralis, horizontal visual streak, and anakatabatic area. The highest density in the area centralis was approximately 18,000 RGCs/mm². Cones showed high density in the horizontal area crossing the optic disc and dorsotemporal area, whereas rods showed high density in the horizontal area, which was greater in height than the horizontal area of high cone density. S cones showed high density in the dorsotemporal retina. The rod/cone ratios were high horizontally in the dorsal retina to the optic disc, with a mean value of 11:1. The cone/RGC and rod/RGC ratios were lower in the horizontal and dorsotemporal retina, and the rod/cone/RGC ratio was lowest in the area centralis (9:1:1). The retinal topographies and distribution of the tapetum were specialized in the horizontal and dorsotemporal fundus. This suggests that sheep have better visual acuity in horizontal and anteroinferior visual fields and that this specialization is related to the visual ecology of sheep. J. Comp. Neurol. 518:2305–2315, 2010. © 2010 Wiley‐Liss, Inc.     

GDNF和NO对局灶性脑缺血大鼠皮质和尾壳核神经干细胞增殖和分化的影响

目的观察胶质细胞源性神经营养因子（GDNF）对局灶性脑缺血大鼠皮质和尾壳核神经干细胞（NSCs）增殖分化以及nNOS阳性神经元分布的影响，探讨GDNF和NO对内源性NSCs增殖分化影响的作用机制。方法制作右侧局灶性脑缺血模型，左侧脑室注射GDNF，5-溴脱氧尿嘧啶（BrdU）标记DNA合成期（S期）细胞，免疫组化观察正常组、假手术组、脑缺血组、生理盐水组和GDNF组大鼠局灶性脑缺血90min后再灌注时间分别为3d、7d、14d、21d、28d的BrdU／nNOS、BrdU／NeuN、BrdU／GFAP双标阳性细胞。结果GDNF组较脑缺血组和生理盐水组行为学恢复明显；脑缺血后皮质和尾壳核BrdU和nNOS阳性细胞增多，BrdU／nNOS（38．23％±6．87％）、Br—dU／NeuN（52．38％±13．29％）、BrdU／GFAP（13．51％±8．78％）双标细胞阳性率明显增加，与其它组比较均有显著性差异（P〈0．05）。结论局灶性脑缺血激活内源性NSCs，GDNF可调节NO释放，促进NSCs增殖、分化。