The structure and development of the rat retina: an immunofluorescence microscopical study using antibodies specific for intermediate filament proteins

Rat retina structure was studied between embryonic day 14 and adult with antibodies specific for vimentin, glial fibrillary acidic protein (GFA) and the proteins of the neurofilament triplet. Vimentin could be detected in radial processes throughout the retina at all stages studied. These processes are believed to correspond, in the developing retina, to ventriculocytes, and in the mature retina to Müller cells. They could not normally be stained with any of the other intermediate filament antibodies employed here. We did find, however, that some older albino rats possessed GFA staining in addition to vimentin in these processes. Since we never saw such staining in the retinae of mature non-albino rats, and the retinae of older albino rats often showed signs of degeneration, we concluded that such GFA expression was most likely pathological. Neurofilament protein-positive processes were first detectable at embryonic day 15 1/2 in the inner regions of the retina, and corresponded to the axons of retinal ganglion cells. Such processes were equivalently displayed with antibodies to 68 K and 145 K protein, but were negative with 200 K protein. Some 68 K and 145 K positive fibers could also be decorated with vimentin antibody at this stage, though at later stages this was not the case. At later development stages more 68 K and 145 K neurofilament positive processes appeared, and after the first post-natal week progressively more of such processes became in addition 200 K positive, so that almost all neurofilament positive fibers in the adult stained for all three proteins. Such fibers, in the mature retina corresponded to 68 K and 145 K positive optic nerve fibers, and the processes of neurones in the inner plexiform layer. All fibers in the mature optic nerve fiber layer, but not all of those in the inner plexiform layer were stainable with 200 K antibodies. At 4 days post-natal we were able to detect 68 K and 145 K protein positive profiles in the outer regions of the developing retina, the prospective outer plexiform layer. Such profiles were always in addition vimentin positive, but negative for 200 K protein. During further development such profiles became ordered into a well defined layer and from about post-natal day 13 all of them began to acquire 200 K protein. They could be identified as the processes of horizontal cells. They continued to express vimentin in addition to the three triplet proteins in the adult, a so far unprecedented situation. We were able to detect neurofilament staining in the mature retina only in the above described regions, the inner and outer nuclear layer and the photoreceptor processes being completely free of staining. GFA was first detected in short processes adjacent to the inner limiting membrane which penetrated the optic nerve fiber layer. Such profiles were first detectable in the eye of the newborn animal, and were invariably identically stainable with vimentin at this age. These profiles could be stained with both vimentin and GFA at all later stages examined, although GFA staining became very much stronger than vimentin staining in some profiles in the adult. The results presented here are discussed in terms of development of the different retinal cell types.     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

An interspecies comparison of gangliosides and neutral glycolipids in adrenal glands

Gangliosides and neutral glycolipids of adrenal glands of mouse, rat, guinea pig, rabbit, cat, pig, cow, monkey, and chicken were analyzed by thin layer chromatography (TLC). The major gangliosides common to all species had lactosylceramide in their core structure. GM3 containing N-acetylneuraminic acid (NeuAc) was the major ganglioside in rat, guinea pig, rabbit, and cat, whereas GM3 containing N-glycolylneuraminic acid (NeuGc) was the major one in mouse, cow, and monkey. GD3 was also detected in all species except mouse and GD3(NeuAc)2 was the major in pig adrenal gland. GM4(NeuAc) was detected in the adrenal glands of guinea pig and chicken but not in those of the other species. In the neutral glycolipid fractions, galactosylceramide, glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide were detected and the proportions of these glycolipids varied among the species. Guinea pig and chicken adrenal glands contained large amounts of galactosylceramide, this being consistent with the presence of GM4 in these two species. Globopentaosylceramide was detected in mouse, guinea pig, cat, and chicken by the TLC-immunostaining procedure.     

The immunological relatedness of neurofilament proteins of higher vertebrates

We prepared intermediate filaments from the nervous system of several different species, representing mammals, birds and reptiles. These were examined using a panel of polyclonal and monoclonal antibodies originally raised against pig or rat neurofilament proteins. All species studied possessed a single major protein of apparent molecular weight between 68 K and 75 K immunologically related to the lowest molecular weight rat and pig neurofilament protein. All birds and mammals possessed two proteins immunologically related respectively to the pig and rat middle and high molecular weight neurofilament proteins. These data show that the neurofilament triplet proteins represent an evolutionarily conserved three member protein family in birds and mammals, and allow us to suggest a new nomenclature for these three homologous proteins: "H" for the heaviest subunit, "M" for the middle subunit and "L" for the lightest subunit. We found that many monoclonal antibodies stained both the H- and M-proteins of all mammalian and avian species examined, suggesting a close immunological relatedness between these two proteins. The reptiles examined appeared to have only one high molecular weight protein, which was immunologically related to both of the high molecular weight mammalian and avian neurofilament proteins. We also noted a curious situation in neurofilament preparations derived from cows. Both the highest and the middle cow neurofilament proteins were stained by all antibodies which were specific solely for the high molecular weight protein in other species.     

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species

Summary The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in para-benzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues. Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.     

The distribution of the neurofilament triplet proteins within individual neurones

The three proteins of the mammalian neurofilament ‘triplet’ were purified from rat sciatic nerve as individual polypeptides. Antibodies were raised in rabbits and in guinea pigs. When tested by the very sensitive immune-blotting technique some of the antibodies proved to be completely specific for the peptide to which they had been raised. Others, however, exhibited weak cross-reactivity with other proteins of the triplet. Cross-reacting IgGs could be removed by appropriate antigen affinity chromatography. Thus a series of rabbit and guinea pig antibodies specific for each of the triplet proteins was obtained. The antibodies were used in immunofluorescence microscopy on cultured rat dorsal root ganglion cells. Only cells with a neuronal morphology were stained by these antibodies, some very strongly and some extremely weakly. When double immunofluorescence was performed it was found that cells stained in an equivalent manner with any combination of antibodies. Neurones which stained strongly with any one antibody could be stained strongly with any other and the converse was true for weakly staining cells. When fine profiles in the growth cones of positive cells were examined it was found that these profiles, representing single or small numbers of neurofilaments, were stained in an identical manner in double immunofluorescence. The results show that the distribution of the three proteins is identical at the level of resolution of the light microscope in rat dorsal root ganglion neurones in tissue culture, and lend support to the supposition that all three triplet polypeptides are contained within each individual neurofilament.     

Hexokinase A from mammalian brain: comparative peptide mapping and immunological studies with monoclonal antibodies

Immunological reactivity of partially purified hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from brain of several vertebrate species has been compared by using enzyme-linked immunosorbent assay and seven monoclonal antibodies raised against the rat brain enzyme. The epitopes recognized by three of these antibodies have been rather widely conserved among the species examined (rat, mouse, guinea pig, rabbit, cat, dog, sheep, cow, pig, chicken), while this was not the case for the epitopes recognized by the other antibodies, which differed markedly in their distribution among these species. The domain structure of these enzymes has been examined by peptide mapping (after limited tryptic digestion) in conjunction with immunoblotting techniques employing monoclonal antibodies. The results indicate that the overall domain structure of these enzymes is similar to that previously described for rat brain hexokinase A, but that there are significant differences in the size of these domains in enzymes from different species.     

眼科常用实验动物视网膜血管的比较

目的比较眼科常用实验动物视网膜血管尤其是视网膜毛细血管的情况,为实验时正确选择动物模型提供基础。方法取猕猴、家猪、新西兰大白兔、犬、猫、SD大鼠、C57小鼠以及豚鼠的正常眼球数个,完整剥离整个视网膜,用ADPase法进行血管染色,对视网膜血管进行形态学的比较。结果猕猴视网膜大血管从视盘穿出,分成四支分别供应视网膜四个象限,每条血管逐级分支最后成为毛细血管,其毛细血管呈网状分布,在赤道处分成两层,至周边变成一层,且有发育良好的黄斑区毛细血管拱环结构。家猪视网膜大血管由视盘发出后放射状走行,毛细血管也呈网状分布,无黄斑拱环结构。兔仅视盘两侧部分视网膜可见血管,毛细血管网状不明显。犬的视网膜血管也放射状走行,但迂曲明显,毛细血管不成网状。猫、大鼠、小鼠的视网膜大血管均由视盘发出,猫的分成上、鼻下、颞下三支,大鼠、小鼠的各方向均有,区域性不明显,三者的毛细血管网均发育良好,至周边部仍很密集,呈两层分布。豚鼠视网膜无可见的血管。结论用于研究人视网膜血管尤其是毛细血管时,可选用猕猴、家猪、猫、大鼠和小鼠作为动物模型;但要研究人黄斑区血管时,仅可选用猕猴等灵长类动物。     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Summary Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in parabenzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues.Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.Abbreviations GFAP glial fibrillary acidic protein - NSE neuron-specific enolase - PBS phosphate-buffered saline - PAP peroxidase anti-peroxidase - FITC fluorescein-isothiocyanate     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.     

An immunofluorescence microscopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein within the adult rat brain

A collection of antibodies specific to different intermediate filament proteins were applied to frozen sections of adult rat brains. The relative distribution of these proteins was then studied using double label immunofluorescence microscopy. Antibodies specific to each of the neurofilament "triplet" proteins (of approximate molecular weight 68 K, 145 K and 200 K) stained exclusively neuronal structures. The distribution of these three antigens was in general identical, except that certain neurofilament populations such as those in the dendrites and cell bodies of pyramidal cells of the hippocampus and cerebral cortex, contained relatively little if any 200 K protein. Some neurone populations, such as the granule cells of the cerebellar cortex, could not be visualized by neurofilament antibodies, indicating that neurofilaments may not be essential for function of all neurones in vitro. Antibodies to GFA and vimentin stained an entirely different population of processes, none of which stained with any of the neurofilament antibodies. Vimentin antibody stained sheath material around the brain, a monolayer of ependymal cell bodies lining the ventricles, fibrous material associated within the choroid plexus, the walls of blood vessels and capillaries, and the processes of cells in certain regions. GFA antibody stained a second layer of sheath material under the vimentin layer, and numerous processes visible throughout the brain. Some specific populations of GFA-positive processes proved to stain also with vimentin. These included the processes of Golgi "epithelial" cells (Bergmann glial fibres), those of certain astrocytes in bundles of myelinated fibers. In addition, some processes apparently derived from ependymal cells proved to stain for both vimentin and GFA, whilst other could only be reliably visualized by vimentin alone. These results are discussed in terms of the previously described morphological characteristics of the various cell types of the brain.     

Species identification of animal cells by nested PCR targeted to mitochondrial DNA

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418     

Intermediate Filaments of Schwann Cells

Abstract: Intermediate filaments were prepared from distal stumps of rabbit sciatic nerve 5 weeks after nerve section, at which time Schwann cells account for 85–90% of the cell area. A polypeptide of molecular weight 58,000 was the main component of this fraction. An antiserum raised in guinea pig against this polypeptide stained all cells present in the distal stump, as well as Schwann cells and 3T3 cells in culture. The identity of the molecular weight 58,000 polypeptide obtained from distal stumps with vimentin was proved with one and two-dimensional sodium dodecyl sulfate pol yacrylamide gel electrophoresis and with immunoautoradiography. It is concluded that the intermediate filament subunit of undifferentiated Schwann cells is vimentin. The possibility that Schwann cells in normal nerve may have another type of intermediate filament besides vimentin cannot be ruled out.     

TNAP activity is localized at critical sites of retinal neurotransmission across various vertebrate species

Evidence is emerging with regard to the role of tissue non-specific alkaline phosphatase (TNAP) in neural functions. As an ectophosphatase, this enzyme might influence neural activity and synaptic transmission in diverse ways. The localization of the enzyme in known neural circuits, such as the retina, might significantly advance an understanding of its role in normal and pathological functioning. However, the presence of TNAP in the retina is scarcely investigated. Our multispecies comparative study (zebrafish, cichlid, frog, chicken, mouse, rat, golden hamster, guinea pig, rabbit, sheep, cat, dog, ferret, squirrel monkey, human) using enzyme histochemistry and Western blots has shown the presence of TNAP activity in the retina of several mammalian species, including humans. Although the TNAP activity pattern varies across species, we have observed the following trends: (1) in all investigated species (except golden hamster), retinal vessels display TNAP activity; (2) TNAP activity consistently occurs in the photoreceptor layer; (3) in majority of the investigated species, marked TNAP activity is present in the outer and inner plexiform layers. In zebrafish, frog, chicken, guinea pig, and rat, TNAP histochemistry has revealed several sublayers of the inner plexiform layer. Frog, golden hamster, guinea pig, mouse, and human retinas possess a subpopulation of amacrine cells positively staining for TNAP activity. The expression of TNAP in critical sites of retinal signal transmission across a wide range of species suggests its fundamental, evolutionally conserved role in vision.     

Rat pancreas adenylate cyclase. VI. Role of the enzyme in secretin stimulated enzyme secretion.

1. The responsiveness of adenylate cyclase and enzyme secretin for secretin and the C-terminal octapeptide of pancreozymin has been investigated in particulate fractions of the pancreas of five different species. 2. The adenylate cyclase is sensitive to the C-terminal octapeptide of pancreozymin in all species investigated. 3. The enzyme is much more sensitive to secretin in rat and cat than in mouse and rabbit, whereas with guinea pig intermediate values are obtained. 4. The enzyme secretion is stimulated by secretin in pancreatic fragments of rat and cat, but not in those of mouse and rabbit. 5. These results suggest that in species where secretin stimulated enzyme secretion, it does so by stimulating the adenylate cyclase system.     

Self-digestion of erythrocyte membranes of various mammalian species

1. The rate of self-digestion of main membrane proteins, spectrin and band 3 protein, was studied for erythrocyte membranes of eight mammalian species: man, cow, pig, cat, rabbit, hamster, mouse and rat. 2. Spectrin and band 3 protein were most rapidly degraded in human and pig ghosts. The rates of proteolysis for other species were similar. 3. The rates of self-digestion were correlated neither with lifetime of red cells nor lifespan of animals of various species.