Optic fiber development between dual transplants of retina and superior colliculus placed in the occipital cortex

Summary Fetal rat retina was excised from donor rats at 15 days of gestation and transplanted to the occipital cortex of neonatal host rats in combination with and adjacent to: 1) the appropriate portion of the superior colliculus to serve as a specific target tissue in an attempt to stimulate an outgrowth of optic fibers from the isolated retinal transplant; 2) a sample of the medial thalamus to provide a alternate tissue not normally recipient to the retinofugal projection, thereby serving as a control to further test for the target specificity of the axonal outgrowth from the retinal transplant. After 5–30 days of development samples of transplant and surrounding host cortex were removed and subjected to light and electron microscopic study.The fetal retina has been shown to develop successfully while located in the occipital cortex but this tissue does not form a significant number of optic fibers nor does it demonstrate the normal quantity of ganglion cells, (Matthews et al. 1981). A dual transplant of fetal retina and medial thalamus demonstrates the major types of neurons and sensory elements in the retina as well as concentrations of large neurons in the thalamus. Examination of these transplants with a silver stain, as well as electron microscopy, revealed a network of axons coursing within the transplanted thalamic neuropil but few axons traversing the interface between the retinal and thalamic transplant. Additionally, no axons coursed out of the retina into the surrounding host cortex.Similar studies of dual transplants of retina and superior colliculus revealed marked concentrations of fibers projecting from the periphery of the retinal tissue, extending deep into the adjacent superior colliculus and merging with the tangle of axons found within this tissue. It should be emphasized, however, that such outgrowths from the retina were clearly restricted to those portions in apposition to the transplanted tectum. No axons were found extending from the opposite surface of the retinal transplant in contact with the host cortex.A quantitative analysis of ganglion cell populations in the transplant demonstrated that these were somewhat reduced in all retinal transplants with the exception of those regions located adjacent to a transplant of superimental enlargement of a peripheral target organ during early stages of development reduces the amount of spontaneous neuronal degeneration resulting from a failure to establish functional connections with the available postsynaptic sites in the target.Our experiments indicate that the use of multiple transplants may provide a useful model system for further exploration of the relationship between developing CNS neurons and tissues within the CNS or the periphery which receive their input.     

神经干细胞玻璃体内移植后的分化及对视网膜节细胞的影响

目的： 观察视神经(ON)微挤压断后玻璃体内移植神经干细胞（NSCs）的分化情况及其对视网膜节细胞(RGCs)轴突再生的促进作用。方法: 在成年大鼠球后1 mm处微挤压断ON，在玻璃体内注入Hoechst33342标记的NSCs 2×10⁴个，实验动物分对照组(MC组、MC+PBS组)、实验组(MC+NSCs组)，各组动物分别存活3、4、5周。用粒蓝逆行标记再生的RGCs，在荧光镜下观察视网膜平铺片再生RGCs的数量变化。另取实验组5只动物存活4周后取眼球切片观察NSCs分化情况。结果: 存活3、4 、5周，再生的RGCs 数目实验组与对照组有显著差异（P<0.01）。4周后移植的NSCs表达NF、CNP、GFAP；未见NSCs迁移至视网膜内。结论: 玻璃体内注入NSCs可显著促进ON微挤压断后RGCs轴突的再生，并在玻璃体内分化为神经元、星形胶质细胞和少突胶质样细胞。     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

The human optic nerve: fascicular organisation and connective tissue types along the extra-fascicular matrix

Fibres in the mammalian optic nerve are arranged into fascicles between which there is an extra-fascicular matrix containing connective tissue, a feature similar to that found in association with fibres in peripheral nerves, but not otherwise found in the CNS. The relationship between these major features of the nerve architecture are not known. We have addressed this question by examining the fascicular organisation of the optic nerve and the distribution of connective tissue and specific collagen types in the human. We have also examined the spatial development of connective tissue in the human nerve to determine when and from where it originates. Fibres are grouped into fascicles at all locations along the nerve, except intracranially, close to the chiasm where this pattern is lost. Relatively large fascicular numbers are found directly behind the eye and in the region of the optic canal, but decline in the mid-orbital segment of the nerve. Connective tissue is present in the extra-fascicular matrix throughout the fasciculated segment, but in many cases it does not fully encircle fascicles. The proportion of matrix occupied by connective tissue is similar along the length of the nerve (approximately 60%). Within the matrix, collagen types I, III, IV, V and VI are present throughout fasciculated regions. Staining for types V and VI appeared relatively weak compared with that for the other types. Although the collagen types in the nerve are similar to those at the lamina cribrosa and in peripheral nerves, they did not appear to be differentially distributed as in regions of the PNS. Connective tissue enters the nerve at a number of wide-spread locations early in development, consistent with the notion that it enters the nerve with the blood supply. It is present within the matrix before it is established at the lamina cribrosa.     

Voltage-gated sodium channel organization in neurons: Protein interactions and trafficking pathways

In neurons, voltage-gated sodium (Nav) channels underlie the generation and propagation of the action potential. The proper targeting and concentration of Nav channels at the axon initial segment (AIS) and at the nodes of Ranvier are therefore vital for neuronal function. In AIS and nodes, Nav channels are part of specific supra-molecular complexes that include accessory proteins, adhesion proteins and cytoskeletal adaptors. Multiple approaches, from biochemical characterization of protein–protein interactions to functional studies using mutant mice, have addressed the mechanisms of Nav channel targeting to AIS and nodes. This review summarizes our current knowledge of both the intrinsic determinants and the role of partner proteins in Nav targeting. A few fundamental trafficking mechanisms, such as selective endocytosis and diffusion/retention, have been characterized. However, a lot of exciting questions are still open, such as the mechanism of differentiated Nav subtype localization and targeting, and the possible interplay between electrogenesis properties and Nav concentration at the AIS and the nodes.     

Immunohistochemical localization of protein kinase C-alpha in the retina of pigs during postnatal development

The cellular localization and protein expression level of protein kinase C (PKC)-alpha was examined in pig retina at different ages. Western blot analysis detected PKC-alpha in the retinas of 3-day-old piglets and indicated significantly increased expression in 6-month-old young adult and 2-year-old adult pigs. Immunohistochemistry of 3-day-old retinas revealed intense PKC-alpha reactivity in the inner plexiform and inner nuclear cell layers, weak reactivity in the ganglion cell layer, and few positive cells in the outer nuclear cell layer. The cellular localization of PKC-alpha in the adult retina was similar, with staining more intense than that in neonates. PKC-alpha was co-localized in some glial fibrillary acidic protein-positive cells and glutamine synthetase-positive cells in the retina. This study demonstrates that the protein level of retinal PKC-alpha is increased with maturation and suggests that PKC-alpha plays a role in signal transduction pathways for postnatal development in porcine retina.     

Extracellular space in the developing retina assessed by electron microscopy: laminar and topographic distribution

The extent of extracellular space (ECS) in the developing retina of the cat has been measured by electron microscopy in material fixed using techniques developed by others to preserve ECS. ECS is generally greater in foetal than in adult material. It is particularly marked in the plexiform layers of retina at the time of synaptogenesis and in the axon layer at the time of axon growth. The changes in ECS occur first in the central retina, and spread to the periphery. These observations suggest that the high volumes of ECS found in the foetus are not artefactual, but accompany and may play a role in developmental processes.     

Amiloride-sensitive sodium transport of the rat distal colon during early postnatal development

To evaluate developmental changes in colonic sodium transport, the sensitivity of the transepithelial potential and short-circuit current to amiloride was investigated. The amiloride-sensitive short-circuit current (I _sc ^Na ), which represents the electrogenic sodium transport through Na⁺ channels, rose significantly from day 5, reached a peak on day 10, and entirely disappeared after weaning. The maximum rate of electrogenic, amiloride-sensitive sodium transport was 12.0 Eq/cm² · h. TheI _sc ^Na was suppressed by adrenalectomy and/or premature weaning but not by a mineralocorticoid antagonist, spironolactone. On the contrary, treatments which increase aldosterone levels in vivo (low-sodium diet, furosemide-induced natriuresis, high dietary intake of potassium) stimulated theI _sc ^Na . The effect of adrenalectomy increased during postnatal development. The sensitivity ofI _sc ^Na to aldosterone was highest at the end of the weaning period. High-sodium diet, which causes a decrease in circulating aldosterone, was associated with a partial inhibition ofI _sc ^Na (P<0.016). These data suggest that the distal colon of neonatal rats can transport sodium via an electrogenic, amiloride-sensitive mechanism and that adrenocortical hormones exert the main regulatory control of this pathway.Parts of this study have been presented at the 6th Symposium on Developmental Pharmacology, Schloss Reinhardsbrunn 1986     

Expression of preproenkephalin mRNA in rat superior cervical ganglion during postnatal development

The number of principal neurons in the rat superior cervical ganglion (SCG) exhibiting enkephalin-peptide immunoreactivity is reported to be limited. To better determine the degree of enkephalinergic phenotype in sympathetic neurons, sections of SCGs from rats aged newborn to adult were processed for in situ hybridization histochemistry, using a [³⁵S]cRNA probe directed against preproenkephalin (PPENK). > 50% of principal ganglion neurons express mRNA for PPENK in adults. Striking variability in labeling intensity is observed. PPENK mRNA is detected in developing ganglia beginning at postnatal days 4–7. Both the number of cells and intensity of labeling increases with postnatal development. These results indicate that expression of PPENK mRNA is more widespread than expression of enkephalin peptides and develops postnatally.     

Reduced expression of Na(v)1.6 sodium channels and compensation by Na(v)1.2 channels in mice heterozygous for a null mutation in Scn8a

The voltage-gated sodium channel alpha subunit Na(v)1.6, encoded by the Scn8a gene, accumulates at high density at mature nodes of Ranvier of myelinated axons, replacing the Na(v)1.2 channels found at nodes earlier in development. To investigate this preferential expression of Na(v)1.6 at adult nodes, we examined isoform-specific expression of sodium channels in mice heterozygous for a null mutation in Scn8a. Immunoblots from these +/- mice had 50% of the wild-type level of Na(v)1.6 protein, and their optic-nerve nodes of Ranvier had correspondingly less anti-Na(v)1.6 immunofluorescence. Protein level and nodal immunofluorescence of the Na(v)1.2 alpha subunit increased in Scn8a(+/-) mice, keeping total sodium channel expression approximately constant despite partial loss of Na(v)1.6 channels. The results are consistent with a model in which Na(v)1.6 and Na(v)1.2 compete for binding partners at sites of high channel density, such as nodes of Ranvier. We suggest that Na(v)1.6 channels normally occupy most of the molecular machinery responsible for channel clustering because they have higher binding affinity, and not because they are exclusively recognized by mechanisms for transport and insertion of sodium channels in myelinated axons. The reduced amount of Na(v)1.6 protein in Scn8a(+/-) mice is apparently insufficient to saturate the nodal binding sites, allowing Na(v)1.2 channels to compete more successfully.     

Quantitative analysis of proliferation and cell cycle length during development of the rat retina

The rat retina has been a useful model system for the study of the development of the central nervous system (CNS). In order to facilitate future studies on the mechanisms that control retinal growth, we have quantified the proliferation of retinal cells and the length of the cell cycle throughout development. For each day during development, the number of mitotic and postmitotic cells per retina, the proportion of cycling cells, S phase length, and cell cycle length were determined through quantification of cell numbers and ³H-thymidine labeling. As retinal development proceeds, the proportion of cycling cells decreases, and cell cycle length increases, in part due to an increase in S phase length. © 1996 Wiley-Liss, Inc.     

Pharmacological properties of neuronal TTX-resistant sodium channels and the role of a critical serine pore residue

Voltage-gated sodium channels can be characterized by their sensitivity to inhibitors. Na_v1.5 is sensitive to block by cadmium and extracellular QX-314, but relatively insensitive to tetrodotoxin and saxitoxin. Na_v1.4 is tetrodotoxin- and saxitoxin-sensitive but resistant to cadmium and extracellular QX-314. Na_v1.8 and Na_v1.9 generate slowly inactivating (I_TTXr-Slow) and persistent (I_TTXr-Per) currents in sensory neurons that are tetrodotoxin-resistant. Tetrodotoxin sensitivity is largely determined by the identity of a single residue; tyrosine 401 in Na_v1.4, cysteine 374 in Na_v1.5 and serine 356 and 355 in Na_v1.8 and Na_v1.9. We asked whether Na_v1.8 and Na_v1.9 share other pharmacological properties as a result of this serine residue. I_TTXr-Slow and I_TTXr-Per were saxitoxin-resistant and resistant to internal QX-314. I_TTXr-Slow was also resistant to external QX-314 and displayed a approximately fourfold higher sensitivity than I_TTXr-Per to cadmium. The impact of the serine residue was investigated by replacing tyrosine 401 in Na_v1.4 with serine (Y401S) or cysteine (Y401C). Both mutants were resistant to tetrodotoxin and saxitoxin. Whereas Na_v1.4-Y401C displayed an increased sensitivity to cadmium and extracellular QX-314, the serine substitution did not alter the sensitivity of Na_v1.4 to cadmium or QX-314. Our data indicates that while the serine residue determines the sensitivity of I_TTXr-Slow and I_TTXr-Per to tetrodotoxin and saxitoxin, it does not determine their insensitivity to QX-314 or their differential sensitivities to cadmium.This work was supported in part by grants from the Medical Research Service and the Rehabilitation Research Service, Department of Veterans Affairs, and by grants from The Eastern Paralyzed Veterans Association and the Paralyzed Veterans of America.     

小鼠肝生后发育的形态学

目的:探讨小鼠肝生后发育的形态学变化。方法:H-E染色观察小鼠生后肝的形态学变化、Giemsa染色观察造血细胞的变化、免疫组织化学检测血小板内皮细胞黏附分子(CD31)和基质细胞衍生因子(SDF-1)在肝中的表达及变化。结果:肝小叶生后1周开始出现,2周末分化完成。肝板间可见圆形或椭圆形的巨核细胞,胞质嗜酸性、多核;血窦内幼稚血细胞成簇,形态似小淋巴细胞,核呈深蓝色。肝内造血细胞数量自P1~P7逐渐增加,P7达到顶峰,与P1、P5、P13、P15相比差异具有统计学意义,P15几乎消失。巨核细胞在P1数量最多,随发育逐渐减少,至P15消失;棕黄色SDF-1颗粒分布于肝细胞细胞质,P1~P7平均光密度值(AOD值)逐渐降低,与P14~P28相比差异有统计学意义。CD31阳性信号位于血窦内皮细胞胞质,P1~P14AOD值逐渐增大,P28最高,与P1~P7相比明显增高。结论:小鼠肝生后2周内还有造血能力,肝小叶和血窦则在2周后发育完善。     

Ganglion cell survival in embryonic rabbit retina transplanted to the midbrain of neonatal rats

Summary Fetal rabbit retinae can grow and differentiate when transplanted to the collicular region of neonatal rats. In addition, the observed survival of retinal ganglion cells within grafts is associated with the extension of axons into the superior colliculus of the host brain, suggesting that the factors influencing the guidance of axons and the survival of ganglion cells may be homologous across different mammalian orders.     

Immunohistochemical localization of protein kinase C (PKC) beta I in the pig retina during postnatal development

In order to investigate the expression of protein kinase C (PKC) beta I in the retinas of pigs during postnatal development, we analyzed retinas sampled from 3-day-old and 6-month-old pigs by Western blotting and immunohistochemistry. Western blot analysis detected the expression of PKC beta I in the retinas of 3-day-old piglets and it was increased significantly in the retinas of 6-month-old adult pigs. Immunohistochemical staining showed PKC beta I in the retinas of both groups. Immunohistochemistry of 3-day-old retinas revealed weak PKC beta I reactivity in the ganglion cell layer, inner plexiform layer, inner nuclear cell layer, outer plexiform layer and rod and cone cell layer. In the 6-month-old pig retina, the cellular localization of PKC beta I immunostaining was similar to that of the 3-day-old retina, where PKC beta I was localized in some glial fibrillary acidic protein-positive cells, glutamine synthetase-positive cells, parvalbumin-positive cells, and PKC alpha-positive cells in the retina. This is the first study to show the expression and cellular localization of PKC beta I in the retina of pigs with development, and these results suggest that PKC beta I, in accordance with PKC alpha, plays important roles in signal transduction pathways in the pig retina with development.     

Adaptation and dynamics in X-cells and Y-cells of the cat retina

Summary On the basis of the spatial summation properties of their receptive fields, cat retinal ganglion cells were classified as either X-cells (linear) or Y-cells (non-linear). Responses were then obtained to a small, centered spot, square-wave modulated in time and superimposed on various levels of diffuse, steady background illumination. When fully dark-adapted, both X-cells and Y-cells produced responses that were entirely sustained. When well lightadapted but still in the scotopic range, both cell types produced largely transient responses with only a very small sustained component. The sustained or transient nature of responses is, therefore, not an invariant characteristic of X-cells and Y-cells in the scotopic range. We also conclude that the mechanism which controls the center's sensitivity in the scotopic range is similar though not identical in the two types of cells.     

大鼠海马γ-氨基丁酸-A受体在生后发育中的表达

目的:研究大鼠生后发育过程中海马组织γ-氨基丁酸-A(GABA-A)受体的表达规律。方法:用免疫组织化学和PCR技术,检测不同年龄大鼠海马组织GABA-A受体及编码该受体的mRNA表达,并用图像分析方法进行定量研究。结果:大鼠海马组织在生后3 d已经出现GABA-A受体免疫反应,以后逐渐增强,到生后30 d达到最高值,海马各区GABA-A受体免疫反应强度没有显著的差别;编码GABA-A受体的mRNA表达也有类似的增龄性增多,但其表达高峰值提前到14 d。结论:生后大鼠海马GABA-A受体表达在一定时期内呈增龄性表达增强的趋势。     

大鼠生后视网膜发育过程中Nurr1与TH的相关关系研究

目的:观察核受体相关因子1(nuclear receptor-related factor 1,Nurr1)与多巴胺能神经细胞特异性标记物酪氨酸羟化酶(TH)在生后大鼠视网膜发育过程中的表达变化,探明Nurr1与视网膜多巴胺能神经元的相关关系。方法:石蜡包埋组织切片,免疫组织化学双重标记。结果:Nurr1在视网膜发育过程中的表达出现了显著的动态变化,Nurr1阳性产物主要表达在内核层细胞,生后3～7 d达到高峰,之后随着细胞的成熟阳性表达又逐渐减少,成熟的视网膜组织内仅见少量Nurr1阳性细胞,在视网膜神经细胞从幼稚到成熟的分化过程中仅见个别TH阳性细胞。Nurr1与TH的免疫组化双标结果显示两蛋白可以共表达在同一细胞中,但众多的Nurr1阳性细胞不表达TH。结论:Nurr1在大鼠视网膜多巴胺能神经细胞及非多巴胺能神经细胞从幼稚到成熟的分化过程中可能具有重要的调控作用。