Intracellular biogenesis of collagen fibrils in 'activated fibroblasts' of tendo Achillis. An ultrastructural study in the New Zealand rabbit.

We have studied the formation of collagen fibrils in 'activated fibroblasts' of tendo Achillis of rabbits. The tendon was in the process of regeneration after experimental partial tenotomy. Samples were taken from the peri-incisional region and analysed by transmission electron microscopy. Ultrastructural examination showed the presence of a 'fine dense granular substance' inside the rough endoplasmic reticulum and procollagen filaments. These come together to form collagen fibrils in the dilated vacuoles of the rough endoplasmic reticulum. The possible intra- and extracellular origin of collagen fibrils is suggested. Within the cell biosynthesis of collagen fibrils take place with the formation of collagen substance which gives rise to procollagen filaments. These make contact in parallel apposition to produce striated 'spindle-shaped bodies' which elongate by the longitudinal attachment of more procollagen filaments and form intracellular nascent collagen fibrils.

The use of different fixatives and hydrophilic embedding media (Historesin and Unicryl) for the study of embryonic tissues.

The effects of different fixatives, dehydration procedures, and embedding media on the structural and ultrastructural preservation of young chick embryos (Hamburger and Hamilton stages 18-24) have been studied by means of light and electron microscopy techniques. Under the light microscope, the results obtained with the use of Bouin, glutaraldehyde, or glutaraldehyde-paraformaldehyde mixtures, followed by partial dehydration of the samples and the embedding with two different polar resins (Historesin and Unicryl), were compared with the results obtained using conventional paraffin-embedding methods. Cell and tissue shrinkage was determined by comparing blood cells from those embryos embedded in either of resins with those embedded in paraffin. Samples were also compared with blood smears, either methanol-fixed or unfixed, obtained from embryos at the same Hamburger and Hamilton stages. The results obtained when Unicryl and Araldite were used for electron microscopy have also been compared. When ultrastructural images from glutaraldehyde-tannic acid/osmium tetroxide fixed, Unicryl embedded samples were compared with those from araldite embedded samples, the same good results were observed with either of the resins. Araldite embedding requires a complete dehydration of the samples, while Unicryl allows the embedding of partially dehydrated embryos with optimal ultrastructural results. We suggest that these polar resins can be considered as complementary tools for embedding delicate embryonic tissues, allowing partial dehydration of the specimens with an excellent cell and tissue preservation.

Demonstration of atrial natriuretic peptide immunoreactivity and ultrastructurally related secretory granules in myoblasts of the chick myotome.

In this study, an ultrastructural and immunohistochemical examination was carried out on the presence of an 'atrial-natriuretic-peptide (ANP)-like factor' in the myoblasts of the brachial myotome of chicken embryos at Hamburger and Hamilton stages 17 +/- to 19. The results of the immunohistochemical study indicate that, at these stages of their development, the myoblasts of the brachial myotome show ANP immunoreactivity. The ultrastructural analysis of the myoblasts shows the presence of 'granules' or 'secretory vesicles', similar to the secretory granules which contain natriuretic peptides. The demonstration of immunoreactivity to ANP in the myotome and the specific localization of secretory vesicles in myoblasts, as the first evidence relating ANP with skeletal muscle development, reveal a new site at which an ANP-like factor may be synthesized. The hypothesis of a paracrine and/or autocrine function(s) during myogenesis is suggested.

Fusion mechanism of the myoblasts in the myotome of the chick embryo.

We have studied the fusion process of myoblasts in the mytome corresponding to H.H. stages 22, 23 and 24 from calcitonin-treated chick embryos and their untreated controls. The micrograph images of this process were studied to detect the ultrastructural changes in myoblast morphology that could be associated with the known hormonal and biochemical changes that take place in preparation of fusion. Once actin and myosin myofilament differentiation and sarcomerogenesis had begun, the myotome myoblasts fused in bundles of 10-14 myoblasts, and the following was observed during this process: plasmatic membrane interdigitation and invagination; the appearance of cytoplasmic flaps covering other myoblasts and fading at the point of contact; plasmatic membranes that fade and disintegrate; membrane rupturing; double closed lamina; small ringed lamina; large disperse vesicles; small vesicles (liposomes), and semi-dense amorphous material. Seven stages were established: 1--Membrane rupture; 2--Double closed and elongated lamina; 3--Small ringed lamina aligned lengthwise; 4--Large dispersed vesicles; 5--Imprecise boundaries with amorphous material in diffuse areas; 6--Cytoplasm fusion; and 7--Prefunctional syncitium. Answers to the questions: "how", "when", "where", "why" and "for what purpose" the fusion of myoblasts takes place are suggested.

Hematoencephalic barrier. Ultrastructure and histophysiology of the endothelium capillary of the neuronal nuclei of the mesencephalon.

The ultrastructure of the dorsal periaqueductal nucleus capillaries of the mesencephalon in the cat was studied under the electron microscope in relation to the hematoencephalic barrier, and its four structural levels: 1. Endothelium; 2. Basal membrane; 3, Pericytes; and 4. Glial prolongations. An analysis was performed of what occurs in these four components (in a non-experimental histophysiological state, and without manipulation by markers) in the thinnest capillaries of the centre of the mesencephalic neuronal nucleus. Special attention was placed on the first diffusion barrier formed by the endothelium capillary as the intimate guardian of the Central Nervous System (C.N.S.) neurons. The C.N.S. capillaries are formed from the continuous endothelium, with no fenestrations, and hermetic joining complexes, without pinocytosis vesicles on both sides of the plasmatic membrane (adluminal and external), and surrounded by a continuous basal membrane. The non-fenestrated capillaries of the C.N.S. are less permeable than those with similar characteristics located in other areas. In the C.N.S. these capillaries form a selective physiological barrier which determines the size of the molecules that are permitted to cross the capillary wall. It is suggested that the electron-dense globules found in the endothelium cytoplasm may be molecules assimilated from the blood, which might represent the first level or step to the selective diffusion entrusted to the hematoencephalic barrier. It is also suggested that the elongated electron-dense particles found in the endothelium cytoplasm and basal membrane may be macromolecules which are normally retained for an active defensive function. They would represent the first and second level or steps of the retention performed by the hematoencephalic barrier which blocks their passage to the confined space of the perivascular capillary.

Biogenesis of the ciliary roots: participation of the dictyosomes of Golgi's complex.

In this study, the hypothesis of a possible biogenesis of the ciliary roots is suggested, after observing the cilia neurons under the electron microscope, which were found as an exception in the periaqueductal nucleus of the mesencephalon in the domestic cat, conserving the potential to differentiate the cilia, basal bodies and ciliary roots. The dictyosomes of Golgi's complex and Golgi's vesicles participated in this biogenesis. Vesicles of approximately 71.6 nm in diameter had become separated from the periphery of the flattened discoid cisterns of the dictyosome and were aligned normally, in tangential contact with each other, forming rows of vesicles or 'ringed chains', whose points of contact formed the beginning of the 'periodic striation' of a thin ciliary root. Later, the lateral walls of the vesicles and the molecules of the intracisternal proteins gave rise to the interperiodic microfilaments, when the carrier proteins were transformed into structural proteins of the ciliary roots. The parallel apposition of several ringed chains or thin ciliary roots, with their rings joined at the same level (or transversal striations), gave rise to thicker striated roots. This hypothesis of an ultrastructural biogenesis of the striated ciliary roots involves the following six stages: stage I = separation of Golgi's vesicles from the periphery of the flattened disk of dictyosomes near the basal body, with a diameter of over 71.6 nm; stage II = reinforcement of the membrane of the vesicles at the two opposite polar ends of its larger diameter; stage III = alignment of vesicles to form ringed chains, due to the tangential contact between their reinforced points; initiation of the 71.6-nm striation period, roots ringed linearly; stage IV = formation of joining microfilaments between periods (69.2 nm) with the lateral walls of the vesicles and the molecules of the proteins in their content; stage V = lengthening of the thin ciliary roots due to the coupling of new Golgi's vesicles at their ends so that their length increases as a result of the addition of terminal vesicles; stage VI = increase in thickness of the thin ciliary roots, due to the parallel apposition of several ringed chains or thin ringed ciliary roots, at the point where their transversal striation points coincide.

Communicating synapses: types and functional interpretation. Exceptions to Cajal's neuron theory.

The neurons of the dorsal periaqueductal nucleus of the mesencephalon and their synaptic contacts were observed under a transmission electron microscope. We found various types of synapses which constituted an exception to Cajal's neuron theory (law of neuron independence). Some of these synapses had an open communicating or continuity 'passage' between the presynaptic bouton of a neuron (first neuron) and the postsynaptic portion of another neuron (second neuron). The 'communicating' passage (located in the synaptosome) is formed by the continuity of the presynaptic and postsynaptic membrane, and its limits or rims are the reflexion points of the membranes. When only two neurons intervene they could be termed 'simple communicating synapses'. We found three types: I = communicating axosomatic synapses; II = communicating axodendritic synapses, and III = communicating axoaxonic synapses'. When three neurons intervene in the synaptic contact, they could be termed 'complex communicating synapses'. In these, the first and second neurons form a normal synapse, but the lateral portion of the presynaptic bouton of the first neuron also enters into contact with a third neuron, with which it establishes an open communicating or continuity passage. The points of these passages are collateral to the synapse, and may be in the presynaptic or pre-postsynaptic portions simultaneously, communicating collaterally with the third neuron. We found a further three types: IV = complex communicating axosomatic and dendritic synapses; V = complex communicating axoaxonic and somatic synapses, and VI = complex communicating axodendritic and double-somatic synapses. It is suggested that communicating synapses may constitute an exception to Cajal's neuron theory, representing functional states for the acceleration, retardation or modulation of the synaptic function. The neurotransmitters would pass en masse through the communicating passage and the depolarization wave would pass through the rims without being retarded. In the simple communicating synapses, their action would be intensifying. In the complex communicating synapses, their action would be modulating or retarding, since the collateral communicating passage would function as an 'escape valve' through which part of the impulse reaching the presynaptic bouton would escape.

First ultrastructural differentiation of myoblasts of chicken embryos: appearance of the initial filaments.

A study is made of the structure and ultrastructure of myoblasts located in the myotome in chicken embryos, between stages 18 and 19, for electron microscope observation of the occurrence of the first myogenetic filamentous molecules. We suggest a hypothesis for the formation of initial filaments consisting of an initial synthesis of actin globular molecules, carried out in the centre of the myoblast, near the nucleus, with the participation of RNA and the ribosomes. These molecules accumulate peripherically in areas below the plasmatic membrane where they polymerize into actin filamentous molecules which form the initial filaments. These move towards the sharp ends of the myoblast, under the plasmatic membrane, and thence to the interior of the cytoplasm, where they are evenly distributed. This genesis of initial filaments is independent of the influence of the nervous system. We postulate the existence of a single type of myoblast, of fibrillar form, with a dark central area containing the nucleus and the cell organelles, and two sharp, light end zones which contain only the initial filaments in a very light cytoplasmic matrix.

Exceptions to Cajal's neuron theory: communicating synapses.

Ultrastructural images of some neurons and their synaptic connections, belonging to the nucleus of the periaqueductal grey substance in the domestic cat mesencephalon, are shown. The finding that some axosomatic synapses showed an open communication between the pre- and postsynaptic portion attracted our attention. In this way a continuity is made between the presynaptic bouton of one neuron (axon) and the postsynaptic portion of the other (neuronal soma). Synapses having these interneuronal communications could be denominated communicating synapses. Accepting Cajal's neuron theory and his law of neuronal independence, it is very difficult to interpret these images. We wonder if this type of communicating synapses could be the exception that proves the rule of the neuron independence.

High-energy adhering junctional complexes or with mitochondrial coupling.

A variety of adhering junction is found in the ependyma of the domestic cat with a coupling of mitochondria. These are symmetrically situated (in mirror form) at both sides of the intercellular cleft, which always maintain the same separating distance, thereby leaving a limiting cellular space of a constant amplitude. The hypothesis is put forward that the energy (ATP), provided by the mitochondria over adhering junctional complexes, would produce separate fields of force which would position in a lengthwise direction the molecules which give rise to the anchoring filaments. The mitochondrial energy provided and the electrostatic forces generated would produce an adhering, intercellular junction which is functionally very strong and which could be called: high-energy adhering junctional complexes or with mitochondrial coupling.

Ultrastructural basis of the coordination of ciliary movement. Contractile function of the interconnecting cross-striated roots in ependymal epithelium.

A hypothesis for contraction was suggested (molecular basis of the transmission of ciliary coordination) by means of the slipping and interdigitation of the microfilaments of one period with those of the adjacent periods, occurring at the level of the periodic band. The amplitude of the period is greater in the relaxed than in the contracted state, while the reverse is true with the periodic band amplitude. The microfilament length is constant, probably varying with the species. This hypothesis of the slipping and interdigitation of the microfilaments would be the basis of the ciliary coordination from cilium to cilium and from cell to cell, because of the anchoring of the bunches in the peribasal complexes and in the adherent connection complexes, respectively.

Ultrastructure of the basal body and the ciliary roots of the ependymal epithelium of the third ventricle in the cat.

A study was made of thee ultrastructure of the elements surrounding the basal bodies and their cross-striated ciliay roots in the ependymal epithelium of the third ventricle of domestic cats, perfused with glutaraldehyde and postfixed with 3% osmium tetroxide. The structures observed were compared with those described by other authors in several ciliated epithelia and in different animal species. A description is given of a complex anchoring system of the basal bodies of the cilia which are formed of (1) a peribasal complex consisting of (a) sail-shaped fibres, (b) transversal arched filaments, (c) longitudinal arched filaments and (d) a peribasal space, and (2) of striated ciliary roots consisting of (a) anchoring ciliary roots and (b) interconnecting of interbasal ciliay roots. The structure of the cross-striate is also described; they exhibit of a period of 640 A, with periodical bands and intraperiodical bands. The authors suggests an essential participation of the striated ciliary roots with their peribasal complex in the activation and coordination of the movements of the cilia.

Ultrastructure of the neural canal closure in the chicken embryo.

We have studied the neuroectoderm of the chicken embryo, from the beginning of somitic segmentation up to the state at which it has seven somities, i.e. the period covering the passage from the 'neural canal' into the 'neural tube'. This paper was devoted mainly to the study of the ultrastructural cytodifferentiation which takes place during the stages in which the neural canal closes up, and at the level of the first area of contact between the 'neural crests'-roughly at the level of the third somite. We used eggs from a hen of the White Leghorn breed, incubated at 38 degrees C, from which we extracted chicken embryos after 24-30 h of incubation, corresponding to Hamburger-Hamilton's stages 7, 8 and 9. Thus we were able to obtain several series of embryos with three, six and seven somites. The neural canal, or tube, at the level of the third somite, was fixed in glutaraldehyde at 6.25% for 30 min and postfixed in 1% osmium tetroxide for 2 h embedded in Araldite, and the sections were then stained with lead citrate. We observed that the vacuoles in the free edge of the neural canal gradually disappear as the canal closes up, while we gradually witness the appearance of the 'closure apparatus' (or the safety or occlusion apparatus) of what is beginning to form the ependymal epithelium, and the first rudimentary outlines of the cilia. All these changes begin to be observed at the seven-somite stage, i.e. when the neural canal is beginning to close up. The 'closure apparatus' consists of a number of intercellular joint complexes, of the 'close-join't type, between which we observe a number of fine filaments, like a terminal velum', or veil, which we call 'interconnecting filaments'. In the 'raphe', whereby contact is established between the neural crests, we observe the initial stages of fusion between the vacuolated edges, with the plasmatic membrane of these cells forming very fine cytoplasmic 'tongues' which interdigitate with cells from the opposite neural crest and finally constitute the so-called close joints.