Binucleated and Multinucleated Neurons are Formed by Fusion.

In the era of molecular biology and atomic force microscopy, some important macroscopic issues such as simultaneous bidirectional axonal flow or neuronal multinucleosis remain unaddressed. However, these issues have to be addressed, because they distort the results of our current achievements. Using videorecording technique, we studied adhesive contacts between neurons and their processes and kinetics of anastomosis retraction between the cell bodies up to their complete fusion with introduction of neurites into the cell cytoplasm and formation of binuclear cells. Three proofs refuting the mechanism of binuclearity formation by amitosis are presented. Live trinuclear neurons without signs of amitotic division were identified. Electron microscopy showed that fusion of many living neurons into one simplest during centrifugation of isolated cells.

Lubinska Phenomenon: Simultaneous Bidirectional Axoplasmic Flow in Nerve Fibers.

Experiments on live mollusk neurons isolated with a neurite fragmentsat its various levels demonstrated that axoplasm is characterized by mechanical strain realized in the form of retraction up to complete invagination of the axoplasm into the soma. Changes in axon geometry were attributed to neuroplasm movement. It was found that the direction of axoplasm movement depends on the location of adhesion points. It was always simultaneous and oppositely directed, as is the case with contractile myofibrils. The formation of distant paired adhesion sites can promote moving away of the axoplasm mass and organelles carried by it. The velocity and activity of axoplasm movement depend on the quantity and intensity of adhesion points along the axon.

[Contractile Tone and Contraction as Important Physiological Properties of Terminals on the Processes of Living Neurons].

An attempt to summarize some static morphological renderings of reversible structural alterations of nervous processes, as well as receptor and synaptic terminals, to compare them with the mechanisms of actual transformation of living neurons and to find a common kinetic characteristic for these phenomena has been made. The contractile tone and contraction of processes of living isolated neurons are reported. The dependence of the direction of retraction on the localization of the adhesion site of the isolated cell has been detected. The retraction bulb has been identified as an indicator of all contractions of motor and sensory terminals, both alive and fixed. The process of transformation of growth cones into retraction bulbs has been investigated. The presence of mechanical tension in preterminals and interneuronal contacts has been demonstrated in vitro. Similarity of the kinetics of tissue receptor sensory terminals and growth cones has been detected during in vivo experiments. The kinetics of asynaptic dendrite contraction has been compared to the well-characterized structural variability of dendritic spines. The hypothesis of a common origin of the contractile tone of all nervous elements as one of the principal nonelectrophysiological properties of a neuron has been put forward.

[ISOMETRIC RETRACTION AND THE INVISIBLE PROCESSES OF NERVE CELLS].

Recently, a large number of physiological studies on stress and hibernation had described an unusual morphological phenomenon of the rapid disappearance and reapperance of apical dendrites of pyramidal neurons of the hippocampus, prefrontal cortex and other parts of the brain. In this article an attempt is maid to explain this phenomenon on the basis of morphological analysis of natural elastic properties of neuroplasm and structural kinetics of partially preserved processes of the living isolated neurons. The neuroplasm displacement with its bidirectional flow was identified in the processes. A new physiological phenomenon is described--the isometric retraction of nerve cell processes, during which the neuroplasm fluxes were directed to the opposite sides, leading to abrupt thinning of middle parts of the processes and to a thickening of both ends. It is suggested that the extremely attenuated processes can reach the submicroscopic sizes, becoming invisible in the light microscope. The repeated reversible "disappearance" and "appearance" of the processes was demonstrated supravitally in the culture of neurons and of C-1300 neuroblastoma cells. Reduction of the diameter of the processes to a limit of their visibility was demonstrated by the example of their natural stretching. The same effect was observed in the areas between the reversible varicosities of the processes. These areas became extremely thin, and then invisible. Becoming thinner, the processes were capable of sharp extension. A review of the available literature and our own data allow to conclude that the phenomenon of the disappearance of the apical dendrites was due to their isometric retraction, which lead to the emergence of "invisible processes".

[Simultaneous opposite axonal currents in neural process. Retraction hypothesis].

Bidirectional axonal current of organelles and molecules in the nerve fibers was demonstrated using radioautography, the horseradish peroxidase and in virology. However, the mechanism of this phenomenon and regulation of the currents direction in axoplasm still remain not entirely understood. In this article we used the model of living single neurons of mollusk isolated with fragment of neural process at its different levels. It was proved that the axoplasm has a mechanical tone, which is realized in the form of retraction up to complete axoplasm invagination in the neuron soma. The geometry changing of the living axon was treated as its transport neuroplasm mass. It turned out that the direction of axoplasm mass depends on the location of its adhesion sites. It is always simultaneous and bidirectional opposite, as it is the case with contractile muscle fibers.

Contractile activity of living isolated neurons and its inhibition by cytochalasin B.

Contractile activity of damaged neuronal axons of Lymnaea stagnalis and Planorbis corneus vulgaris mollusks and the possibility of inhibiting their retraction by cytochalasin B were studied. In experimental series I (control), the neuronal axons contracted in Ringer's fluid in 90% cases. In series II and III (cytochalasin B in concentrations of 0.02 and 0.2 mM), the percentage of non-contracting neurons was 50 and 70%, respectively. Presumably, the fiber retraction mechanism was involved in the formation of diastasis after nerve cutting and damage to conduction tracts. The nerve diastasis formed at the expense of not only elastic characteristics of the nerve sheath and glia, but also due to nerve fiber retraction. Experiments with cytochalasin B demonstrated that F-actin filaments were involved in the retraction of myelin-free nerve fibers.

[Interdependent changes of the axon and Schwann cell in the process of reactive remodeling of a myelinated nerve fiber].

Using the inverted phase-contrast microscope, the living undamaged frog sciatic nerve fibers and the fibers mechanically injured to varying degrees, were studied. It was found that the swelling of myelin incisures (MI) (of Schmidt-Lanterman) occured according to the principles similar to those controlling the changes of the myelin gap (node of Ranvier) and depended on the swelling of a Schwann cell (SC) perikaryon. It was detected that this was a single process, which which could be united in a complex of nonspecific changes of a myelinated nerve fiber. It was also demonstrated that under the action of mechanical injury and hypotonic solution, swelling of MI, nodes of Ranvier and SC perikaryon occurred without modifications of outer fiber diameter, due to the pronounced local axon thinning. Electron microscopic study of the cytoskeletal axonal structures showed that there was not a simple local contraction of an axon, but a significant local increase in the density of cytoskeletal components of the axoplasm (by 200-275%). Reactive reversible remodeling of a myelinated fiber suggests a new type of interaction between the axon and SC, the mechanism of reversible translocation of liquid axoplasmic fraction to the glial cell cytoplasm.

[Enucleation, formation of cyto- and karyoplasts, and their fusion with neuronal body].

In this research that was performed on isolated neurons of mollusk Lymnaea stagnalis, using neuron enucleation, the cytoplast was obtained which was then fused with another neuron resulting in cybrid formation. The experiments performed have shown that the isolated neurons are able to fuse with each other, forming binuclear neurons; also, like all other cells, they could be enucleated with the formation of cyto- and karyoplasts and, after fusion, they can form cell body-cytoplast, cytoplasts-karyoplast, and other complexes. This is associated with the appearance of all doubtless indicators of fusion described for fusion of nerve cell bodies. This work demonstrates the possibility to artificially fuse the amputated neuroplasm fragment with neuronal cell body--the metabolic center of another cell. Theoretically, this means that in vivo amputated neuronal process also can be fused with a novel cell.

[Neuron contractile and electrical activities as affected by colchicine].

The purpose of this investigation was to analyze the contractile activity of traumatized nerve cell processes and to try to inhibit their retraction by colchicine solution. Isolated living neurons of mollusks (Lymnaea stagnalis and Planorbis comeus vulgaris) were studied using phase contrast and time-lapse microvideorecording. In the control group, contractile activity of nerve cell processes in Ringers solution was detected in 92% of cases. Application of colchicine resulted in the inhibition of retraction of nerve fibers in 86% of neurons. In the experiments designed to study neuron electrical activity, leech Retzius neurons were used. It was found that ganglion incubation in colchicine solution of increased the frequency of spontaneous pulse activity from 0.22 to 0.75 imp/s. The amplitude of spontaneous potentials decreased from 46.9 to 37 mV, the threshold was reduced by 18%, spontaneous spike duration increased from 4.3 ms to 7.1 ms, while the latent period of the response to irritating stimulus increased from 25.0 to 37.9 ms. During the irritation with a frequency of 7-10 Hz, neuron generated higher frequency of pulse activity, than in norm. Thus, it was possible to show, that cochicine can inhibit the contractive activity of the traumatized nerve cell processes, preserving an electroexcitable membrane in a satisfactory state. These results suggest that it is possible to partially inhibit the nerve fiber retraction in vivo, thus preventing the diastasis increase in the nerves that impedes their contact surgical approximation and promotes the development of a massive scar in severed area.

[Two-nuclear neurons: sincitial fusion or amitotic division].

In the review the history of research two-nuclear neurons is stated and two hypotheses about mechanisms of their formation are analysed: by sincitial fusion or amytotic divisions. The facts of discrepancy of the former orthodox cellular theory categorically denying possibility sincitial of communications in nervous system and of sincitial fusion neurons are mentioned. As an example results of ultrastructural researches of occurrence sincitium in a cortex of the big brain of rats, in autonomic ganglions, in hypocampus and a cerebellum of adult animals are presented. The video data of the sincitial fusion of live neurons and the mechanism of formation multinuclear neurons in tissue culture are analyzed. Existing data about amytotic a way of formation two-nuclear neurons are critically considered. The conclusion becomes, that the mechanism of formation two-nuclear neurons is cellular fusion. Simultaneously the review confirms our representations about existence in nervous system sincitial interneural communications.

[Early reactive changes of myelin sheath in the area of myelin sheath gaps (nodes of Ranvier) in nerve fibers (a supravital study)].

Using the inverted phase contrast microscope, the supravital study of structural dynamics of single myelin sheath gaps (nodes of Ranvier) of isolated frog myelin nerve fibers was performed after mechanical injury and in the medium with the decreased ion force under the conditions which induce, in electrophysiological experiments, the expression of the axolemmal K+-channels in the paranodal area. Videorecording has shown that within this area the myelin sheath stratification appeared that was associated with the swelling of Schwann cell cytoplasm enclosed in the terminal membranous loops of myelin. An increase of the degree of stratification of the lamellar myelin complexes make them invisible in the light microscope; therefore, it is not the translocation of the myelin sheath from the node cleft that is recorded, as many authors believed, but a shift of only the visible border of the compact, yet unstratified myelin sheath. Hence, the removal of myelin (demyelination) was absent, and the electrophysiological effect can be accounted for by a significant fall of electrical resistance in paranodal area as a result of swelling of terminal loops and stratification of the myelin sheath. Preparations examination also revealed a decrease of the axonal diameter in, which is proportional to swelling of the myelin sheath terminal parts. Since the outer fiber diameter did not change, it can be concluded that the process observed is the result of swelling of the Schwann cell cytoplasm due to the axoplasm water fraction which may be a peculiar process of axo-glial interactions.

[Fusion of brain neurons in rat embryos].

Syncytial interneuronal connections were studied in the sensomotor cortex and caudate nucleus of twenty 14-22 day rat embryos. It was shown that with the extremely weak development of glial processes, many neuronal bodies and their processes were in the direct contact with each other. The contacting membranes in these areas formed oblong and dot-like contacts resembling gap and tight junctions. As a result, the intercellular cleft experienced varicose-like deformations. In the area of contacts, barely visible membrane pores were formed that broadened to form large perforations. The perforation margins presented the rounded shape of fused plasma membranes of adjacent neurons. Inside the perforations, residual vesicular membranous bodies were formed. The areas of the paired membranes between perforations were fragmented, thus increasing the number of residual vesicles, until the neurons fused with each other completely by unifying the neuroplasm of contacting cells. The results of these studies suggest that that the fusion of neurons in vertebrate brain cortex and brainstem nuclei could occur not only in pathology, but also in normal animals at the stage of embryonic development.

[Contraction of the injured neuronal processes].

The investigation was performed on the isolated living neurons of a mollusk (Lymnaea stagnalis). The purpose of this study was to examine the contractile activity of the injured neuronal processes. Retraction of latter in Ringer's solution was found in 90% of the cases. The specific club-shaped structure (retraction bulb) served as a marker of contraction. The speed of process contraction fluctuated in different neurons from 0.03 to 9 microm/ min. As a result of usual linear contraction, the process diameter was increased on the average by 35%, while the cell body volume was increased on the average by 30%. The three forms of contractile activity were distinguished: linear contraction, isometric contraction (reduction of a process thickness with no change in its length) and mixed form of contraction. It is suggested that the mechanism of retraction takes part in the formation of diastasis after nerve sectioning and injury of the brain conducting pathways. Diastasis in the nerve is formed not only due to the elastic properties of its fibrous sheath and glia, but also as a result of nerve fiber retraction.

[Syncytial perforations of human neuronal embryo membranes].

An electron microscopy study of the anlage of cerebral cortex of human embryo has been carried with the aim of determining the presence of syncytial interneuronal connections in embryogenesis. It has been determined that, in part of the neurons, the glial embryo is absent and their external cell membranes are directly attached to each other by forming elongated or dotted tight junctions. Sometimes these junctions are perforated and, on their basis, the true syncytial interneuronal connections are formed. Natural structural properties of these connections are the following: formation of the base of tight membrane contacts, obligatory rounding of perforation edges, and the presence of residual particles in the form of spherical vesicles in the lumen of perforations. Results obtained allowed us to conclude that, in the anlage of cerebral cortex of embryos obtained during surgical abortion of pregnancy, apart from the formation of synaptic contacts, or until their formation, there is the possibility of syncytial interneuronal connections appearing. This should be considered during the transplantation of the developing brain.

Water translocation from the axial cylinder to myelin sheath structures of the nerve fiber.

We studied isolated myelinated nerve fibers from frog sciatic nerve surviving in Ringer solution or in water-free liquid perfluorodecalin immiscible with water or mineral oil. Swelling of incisures and perikaryon, loosening of myelin in the node, and formation of the axial cylinder varicosities were found in the fibers surviving in Ringer solution after 5-7 h. The same process, swelling of Schmidt-Lantermann myelin incisures, Schwann cell perikaryon, and loosening of myelin lamellae in the Ranvier nodes was found in water-free perfluorodecalin medium. However, swelling of the perikaryon and incisures spread along the axial cylinder and the reaction of the fiber developed in perfluorodecalin much later and unfolded slower than in the control. These changes developed much sooner and progressed much more rapidly than in perfluorodecalin in fibers surviving in mineral oil. Swelling of the myelin sheath structures in water-free medium indicated an uncommon new form of the neuron-glia relationships: water translocation from the axial cylinder to Schwann cell under unfavorable conditions.

Interneuronal membrane contacts and syncytial perforations in CA2 hippocampal area after brain trauma.

Membranes of pyramid neuron bodies located in CA2 hippocampal area were studied by electron microscopy after gunshot craniocerebral injury. In control group, asynaptic contacts and interneuronal syncytial perforations forming from tight junctions were observed. Contacts and perforations increased in size after trauma. Their number was maximum after severe gunshot injury. They reached their maximal size on days 5-7 after the injury.

Inhibition of contractions of nerve processes in calcium-free medium.

The possibility of blocking contractile activity of damaged neurites was studied on viable isolated neurons from Lymnaea stagnalis mollusks. Retraction was blocked in more than 80% cells plunged in Ringer's solution free from Ca(2+) or containing 20 mM CoCl(2). Nimodipine and nitrendipine significantly inhibited neurite contractions. Inhibition of neurite contractile activity can be useful for reducing diastasis after nerve crossing.

[Glio-neuronal and glio-glial syncytial cytoplasmic connections in peripheral nerve trunks of the crayfish Astacus leptodactylus].

The paper considers various aspects of glial sheaths of neuritis in the crayfish peripheral nerve trunks and roots. There are revealed dotted glio-neurite tight junctions and a varicose deformation of the intercellular glio-neurite cleft. Rupture of membranes in the area of contact leads to formation of the glio-neurite pore (less than 10 nm) that is enlarged and forms wide (up to 240 nm) syncytial perforations. At the edge of perforation, either remnants of tight junctions are present or damaged membranes that fuse and are rounding. The lumen of perforations always contains residual membranous bodies in the form of vesicles. Their deviation from the median line can indicate a mutual translocation of substances of the glio- and neuroplasm. In the adjacent layers of the multilayer glial sheath there is noted a similar phenomenon of formation of the glio-glial syncytial connection terminating by fusion of neighbor glial layers, which is terminated by fusion of neighbor glial layers into the single lamina. The process begins from the varicose deformation of interglial clefts, which appears as a result of massive formation of dotted and expanded tight membranous contacts. As a result of transformation of ellipsoid varicose deformations into the spherical ones, syncytial pores (less than 10 nm) between them are formed, which are enlarged and break the paired gliolemmas into fragments. As a result, the adjacent glial layers are united. Since this process in intact animals occurs on the background of undamaged nerve structures, a suggestion is put forward about its reversibility and the functional nature.

[Contribution to the discussion on syncytial connections in the nervous system].

The paper discusses data on the existence of the syncytial interneuronal connections. Firstly, the discovery of synapses in principle is not a proof of the absence of syncytial connections in the nervous system. Secondly, there is a light microscopical evidence of the existence of the syncytial connections. These are found in giant axons of crustaceans, polychaetes, and other invertebrates, as well as during similar fusion of 2-3 processes into one fiber covered with myelin sheath in vertebrate neurons. In tissue culture, after the destruction of the neuronal body, its process connected to another neuron does not die, as it has syncytial connection with the latter. Thirdly, under the electron microscope, interneuronal syncytial connections were demonstrated in piglet intramural nervous system, in cat caudal mesenteric ganglion, in rabbit and frog hippocampus and cerebellum, and in cat cerebral cortex. Structural regularities of such connections have been described. By observing changes of contacting membranes in pathology, it was established that the essence of the process consists in a modification of tight junctions which are not refuted by anyone. The syncytial pores and perforations are also observed in intact animals in neurons without obvious lesions.

Neuron division or enucleation.

The classical Bielschowsky-Gross neurohistological method was used to reproduce all the morphological phenomena interpreted by many authors as signs of neuron division, budding, and fission. It is suggested that these signs are associated with the effects of enucleation, which occurs in many cells of other tissue types in response to a variety of chemical and physical treatments. Studies were performed using neurons isolated from the mollusk Lymnaea stagnalis and exposed in tissue culture to the actin microfilament inhibitor cytochalasin B. Phase contrast time-lapse video recording over periods of 4-8 h demonstrated nuclear displacement, ectopization, and budding, to the level of almost complete fission of the neuron body. This repeats the pattern seen in static fixed preparations in "normal" conditions and after different experimental treatments. Budding of the cytoplasm was also sometimes seen at the early stages of the experiments. Control experiments in which cultured neurons were exposed to the solvent for cytochalasin B, i.e., dimethylsulfoxide (DMSO), did not reveal any changes in neurons over a period of 8 h. We take the view that the picture previously interpreted as neuron division and fission can be explained in terms of the inhibition of actin microfilaments, sometimes developing spontaneously in cells undergoing individual metabolic changes preventing the maintenance of cytoskeleton stability.