An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

The vasculature of the rat embryo: an electron microscope study

The ultrastructure of portions of the arterial and venous systems of the 11.5 day old Wistar rat embryos has been studied by scanning and transmission electron microscopy. The vessels at this stage of development are in the form of capillaries, and the arterial and venous types can be distinguished by the morphology of the endothelial cells by SEM. The endothelial cells of the arterial vessels gave prominent nuclear bulges and numerous microvilli apart from their spindle shape, whilst those of the veins appear flattened, are polygonal in shape, and have few microvilli. Transmission electron microscopy shows that the endothelial cells of the arteries and veins are identical in structure. The ultrastructure of these cells resembles that of endothelial cells at later stages of development including the adult type in that mature forms of cytoplasmic organelles are obtained. In studies on the intercellular junctions and fenestrations with lanthanum nitrate, the impression is formed that the vessels at this stage are impermeable to small molecular size particles, compared with adult capillaries. This suggests that cytoplasmic vesicles must play a major role in the transport of macromolecules in the 11.5 day embryonic vessels.     

An electron microscope study of myofibril formation in embryonic rabbit skeletal muscle

Trunk and limb muscles from fetal and newborn rabbits were investigated by means of light and electron microscopes. At 14 days gestation, the presumptive myoblasts migrate away from the myotome to form the anlage of the muscle of the trunk and limb. Among the population of undifferentiated cells, the myoblasts were recognized due to the presence of actin and myosin filaments. The aggregates of thin and thick filaments appear at the periphery of the cells. There is a great variety of filament assembly. The presence of Z band material appears to be essential for sarcomere formation. At 14 days of gestation the myotubes are more numerous in the limb than in the trunk. The presence of unmaturated fibrils with absence of the M line in the sarcomeres was observed. By day 18 of gestation the myotubes are wider and aggregate to form small bundles. The myofibrils were more numerous and the vesicles of the SR precursor, partly incrustated with ribosomes were dispersed among them. At day 22 of gestation the myotubes are thicker because of the myofibrils which are far more numberous. The sarcomeres were more fully developed, with the M line present. At day 28 of gestation and 3 days after delivery the already developed myofibers were present with a well organized SR system and fully developed sarcomeres.     

Troponin in embryonic chick skeletal muscle cells in vitro. An immunoelectron microscope study

The fine structurel distribution of troponin on thin filaments in developing myofibrils was investigated by the use of immunoelectron microscopy. Embryonic chick skeletal muscle cells grown in vitro were treated with antibodies against each of the troponin components (troponin T, I, and C) from adult chicken muscles. Each antibody was distributed along the thin filaments with a period of 38 nm. It is concluded that these newly synthesized regulatory proteins are assembled at their characteristic position from the initial phases of myofibrillogenesis.     

The rat-tail artery maintained in culture: an experimental model

The rat-tail artery was maintained in vitro for 2 weeks to investigate its suitability as an experimental model. The criteria were that (a) it should retain the overall histological organization with normal ultrastructural appearance of the smooth-muscle cells; (b) stored neurotransmitter which could be activated by experimental treatment should be absent; and (c) smooth-muscle ion transport mechanisms should fall within normal range. Vessels were maintained in Falcon tissue-culture dishes in Dulbecco's modified Eagle's medium. Either 2% or no serum supplement was found to be more suitable than 10% serum due to the high rate of cell proliferation induced by the latter. Light and electron microscopy of cross sections of the vessels indicated that the overall normal vessel architecture was retained, and the ultrastructural features predicted normal function. There were no discernible differences dependent on the length (up to 8- to 10-cm lengths) of the cultured vessel. Preliminary experiments with fluorescent microscopy showed that stored neurotransmitter in the nerves of the vessel wall was no longer present after 48 hr. Ultrastructural examination revealed that storage vesicles in vitro lost their dense cores, representing noradrenalin, between 41 and 48 hr in culture. Normal ion transport mechanisms were retained in the smooth-muscle cells of the arteries in vitro for up to 2 weeks when tested with ion-specific electrodes. Morphological and physiological evidence support the suitability of the rat-tail artery as a model for experimental testing of vascular tissues.     

Hymenolepis diminuta: an electron microscope study of ion absorption

Ten-day-old Hymenolepis diminuta were incubated in media containing either the divalent cations barium or lead, or the divalent anions sulfate or ferrocyanide. Mitochondria of the tegument, but of no other tissue with the time scale used, accumulated all these ions in conspicuous amounts.By visual inspection mitochondria appeared to accumulate ions in the following order of preference, lead > sulfate > barium > ferrocyanide. These results have been interpreted with reference to Lehninger's theory of ion accumulation in vertebrate mitochondria, in that it is suggested the results depend on the presence or absence of permeant cations. From the viewpoint of ion accumulation, the mitochondria of Hymenolepis diminuta appear to be similar to vertebrate mitochondria.Ion deposits also occurred attached to apical and basal plasma membranes of the tegument and to the membranes of the multitubular complex. Reactions at these sites appear to be due to the properties of their associated glycocalyx. The presence of deposits in and around multitubular complexes also supports the previous suggestion that this structure may play a role in ion or water transport in this tapeworm. The deposits which occurred in the interstitial material appear to be the result of a reaction between acidic groups present in the matrix material of this connective tissue and cations in the incubating and fixing solutions.     

Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion

The effect of intercellular interactions on the determination and differentiation of early embryonic brain cells was tested by immunomagnetic cell separation techniques. Using the A2B5 monoclonal antibody, which in chick brain reacts with a neuron-specific surface ganglioside, we produced initially pure populations of optic tectum cells devoid of the antigen. A coincident depletion of neurofilament(+) cells (95%) and nonneuronal growth characteristics of the separated A2B5(-) cells indicated that the vast majority of neurons had been removed initially. Surprisingly, A2B5(+) cells rapidly appeared in separated A2B5(-) cell cultures. After 1 day, the percentage of A2B5(+) cells in separated cell cultures equalled those in unseparated cultures (approximately 50%). By a week in culture, A2B5(+) cells developed neuronal morphology and contained neurofilaments. A2B5(-) to (+) conversion was a regulated phenomenon in that removal of different proportions of the (+) cells resulted in different numbers of (-) to (+) conversions. New DNA synthesis was not required for the acquisition of cell surface A2B5 antigen or for differentiation of cells into definitive A2B5(+) neurons. Our results demonstrate that postmitotic embryonic brain contains cells which are capable of replacing depleted neurons in vitro.     

Spermiogenesis in Lumbricus herculeus; an electron microscope study

Small pieces of the sperm sacs of Lumbricus herculeus were fixed for 4 hours in chrome-osmium, embedded in methacrylate, sectioned with a Porter-Blum microtome, and studied with a R.C.A. EMU-2C electron microscope. Each spermatid of a group developing synchronously is attached by a cytoplasmic strand to a common nutrient protoplasmic mass. This mass contains mitochondria and yolk bodies but is anucleate. The proximal centriole, that is, the centriole nearer the nucleus, is at first associated with a small peg which becomes firmly attached to the nuclear membrane. Later these two bodies become separated during the development of the middle-piece which is differentiated in the usual manner from a nebenkern formed by the fusion of 6 or 7 mitochondria. The acrosome develops in relation to the dictyosome (Golgi body), itself composed of 8 or more individual flattened sacs and situated in the cytoplasm opposite the point of attachment of the spermatid to the nutrient mass. Soon after its formation, the acrosome becomes incorporated into a cytoplasmic appendage or acrosome carrier. The carrier moves from its original position, along the lateral border of the elongating nucleus, to the distal margin of the nucleus where the acrosome is deposited. No evidence was found of a centriole located at the point of junction between nucleus and acrosome as suggested by earlier workers.     

Light and electron microscope studies of effects of 50 Hz electromagnetic fields on preincubated chick embryo

We investigated the effects of an electromagnetic field (EMF) of 50 Hz, 1.33-7.32 mT on sections of preincubated white leghorn chicken embryos using light, SEM and TEM microscopes. Five hundred healthy, fresh, and fertilized eggs (55-65 g) were divided into three groups of experimental (n = 18-20), control (n = 60), and sham (n = 50). Experimental eggs (inside the coil) were exposed to 15 different intensities (1.33-7.32 mT) for morphological surveys and to the known most effective intensities for light, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) studies. Sham groups were located inside the same coil with no exposure for 24 h before incubation. Control, sham, and experimental groups were then incubated in an incubator (38 +/- 0.5 degrees C, 60% humidity) for 4 days. At the end of this period, embryos were removed from their shells, prepared for morphometric, light, and SEM/TEM studies. Results of light microscopic studies (serial sections, 6mu) and morphometric data showed significant differences between different groups (P < 0.005). Larger and abnormal brain cavities, spina bifida, monophthalmia, microphthalmia, anophthalmia, and growth retardation were shown on SEM. TEM sections demonstrated that the nucleus was condensed, the nuclear envelope disappeared, and mitochondria degenerated. Golgi apparatus and endoplasmic reticulum were the least affected organelles. The Telencephlon was the most affected region, and the retina was altered more than the lens. We conclude that EMFs affect the brain, especially the Telencephalon and eye of preincubated-exposed chick embryo at the morphological and cellular level, nuclei are the most affected part, and our data agrees with "Ubeda's windows effects" of EMFs on preincubated chick embryos.     

The nucleus of the basal optic root in the pigeon: an electron microscope study

The ultrastructure of the nucleus of the basal optic root in an avian species (Columba livia) was investigated. The ectomamillary nucleus (EMN) in which terminates the basal optic tract reveals three types of neurons: 1) small round neurons bearing a scanty cytoplasm in organelles, 2) medium-sized neurons, spindle-shaped with a dense population of organelles and 3) large multipolar neurons with well developed perikaryal elements. Some of these neurons have their inner plasma-membrane which fuse to make junctional zones alternating between attachment plates and gap junctions. The analysis of the neuropil displays four types of vesicle-containing profiles (VCP), Type I VCP, identified as optic terminals, are numerous (49%), contain round vesicles (500-550 A) and establish Gray type I contacts principally with dendrites. They also participate in serial and triadic arrangements. Type II VCP have lighter hyaloplasm and are less numerous (6,7%). Rounded vesicles (450-500 A) with a clear content synapse also with Gray type I active zones on dendrites. Some of these profiles have the peculiarity of both a chemical and electrical transmission known as mixed synapses. Type III VCP are larger and contain a mixed population of rounded and flattened vesicles which synapse according to Gray type II. Type IV VCP are characterized by a light hyaloplasm where the microtubules are a predominant organelle. Their active zones are also of Gray type II.     

Paraneuronal pseudobranchial neurosecretory cells in scorpion catfish Heteropneustes fossilis: an environment scanning electron microscope and transmission electron microscope study

Yadav, L., Sengar, M., Zaccone, D. and Gopesh, A. 2011. Paraneuronal pseudobranchial neurosecretory cells in scorpion catfish Heteropneustes fossilis: an environment scanning electron microscope and transmission electron microscope study. —Acta Zoologica (Stockholm) 00 : 1–8. Pseudobranchial neurosecretory system (PNS), found in the gill region of certain groups of teleosts, falls under the category of the ‘diffuse neuroendocrine system’ (DNES). The cells belonging to the system share morpho‐functional features with the paraneuronal cells observed in respiratory tract and airway surfaces of higher vertebrates. On the basis of the experimental observations, a role in condition of hypoxia has been recorded for this system. In an attempt to elucidate the ultrastructure of pseudobranchial neurosecretory cells, present investigation was undertaken using environment scanning electron microscope (ESEM) and TEM in an air‐breathing catfish, Heteropneustes fossilis. The external morphology of PNS under ESEM appeared as a mass of cells supplied with nerves and blood capillaries. Each cell mass is made up of numerous pear‐shaped neurosecretory cells, confirmed by neurosecretion‐specific acid violet stain. The TEM investigation of the cells revealed the presence of different sizes of dense‐cored vesicles in the cytoplasm, which was observed as granular cytoplasm under light microscope. Presence of large number of mitochondria in the cytoplasm confirmed active involvement of these cells in the physiology of fishes. Although lacuna prevails regarding the exact function of this system of fish, its probable role in hypoxic condition and surfacing behavior are speculated.     

The development of the tongue in the chick embryo: observation under a scanning electron microscope

The morphological features of the chick embryo tongue from the 8th day of incubation till hatching and during the early post incubation period have been investigated by means of Scanning Electron Microscope. At the SEM, it is possible to observe that already at the 8th day of incubation, the body and the root are separated by a low smooth-surfaced ridge. In the following days this ridge develops, giving rise to the so-called lingual spines, whose significance is still uncertain. As concerns the evolutive pattern of the superficial layer of the epithelium, in the first days of the considered incubation period the cells appear dome-shaped and have microvilli on their apical surface; afterwards they tend to become more flattened, and the microvilli are replaced by a thick net of microplicae. In the last days of incubation and after hatching desquamative phenomena become evident. The above described evolutive process can be regarded as a common feature of the whole dorsal lingual surface; only few regional differences are to be noted, such as the earlier development of the microplicae on the apex and borders of the tongue. In particular, the microplicae observed at the apex of the tongue show a typical aspect and arrangement; they run regularly parallel to each other. On the lingual dorsal surface taste bud-like structures have never been observed.     

Incorporation and stabilization of 3H-tetracycline in embryonic chick bone: an autoradiographic study

Tibiae from 11-day-old chick embryos, injected with 3H-tetracycline, were autoradiographically analyzed at different stages to localize and study the dynamics of the initial phases of bone mineralization. 3H-tetracycline was localized within newly formed trabeculae, but only at the surfaces of older trabeculae, indicating that the incorporation of tetracycline into bone occurs at active sites of calcification. It takes between 24 and 36 h for injected tetracycline to become stabilized and incorporated into the mineralized matrix. Absence of 3H-tetracycline grains over the osteoblasts suggests a paracellular pathway for incorporation of tetracycline into the mineralizing bone matrix.