O. S. Sotnikov

Syncytial coupling of neurons in tissue culture and early ontogenesis

Computerized time-lapse video recording was used to detect the process of formation of syncytial couplings between the processes of different neurons in dissociated neuron cultures. These studies showed that once the processes of one neuron had formed connections with another neuron, death of the cell body (its trophic center) was not followed by Wallerian degeneration. Translocation of cytoplasmic varicosities along the branches of one neuron to another was observed over periods of several hours. Electron microscopic studies of the nerve processes of cells in the intramural intestinal plexus in the early postnatal period demonstrated all the transitional states from fusion and perforation of the membranes of contacting dendrites to complete fusion of the neuroplasm of processes with formation of residual membranous structures at the location of the former intercellular contact.

Primary sensory neurons in the central nervous system

Published data and our own results relating to exteroceptor and a variety of interceptor neurons in the brain and spinal cord, such as intraspinal Hesse ocelli and light-sensitive epiphyseal and ependymal neurons, are presented. Light-sensitive ganglion neurons in invertebrates are also described, along with intrinsic spinal cord bipolar sensory neurons within the spinal cord, primary chemo-and thermosensitive neurons, and sensory unipolar neurons associated with the three fine “central nerves” of Motavkin, which perforate the sheath of the spinal cord and ending with bush-like receptors close to vessels or near the ependyma of the central canal. Data on all known intracortical interoceptors in vertebrates are generalized into a single scheme. It is hypothesized that the brains of animals and humans have an intrinsic sensory innervation comparable with the innervation of other organs and containing local primary sensory neurons and their asynaptic dendrites, which can be divided into two groups: interceptor and exteroceptor.

Axon reactions precede demyelination in experimental models of multiple sclerosis.

nating disease are currently known. Among these, multiple sclerosis is a widespread disease in Europe and the US. Despite the fact that the etiology of multiple sclerosis remains unknown, it has been demonstrated that an autoimmune mechanism plays an important role in the pathogenesis of demyelination [25]. Chronic recurrent experimental allergic encephalomyelitis (crEAE) is a valuable model for studies of the pathogenesis of multiple sclerosis in animals, as the clinical and pathological changes in this condition correspond to those seen in human patients with multiple sclerosis [4, 12, 13]. Multiple sclerosis is regarded as a primary demyelinating disease in the early stages of which the myelin sheath is damaged while the axon is preserved. However, data have been obtained indicating axon damage also occurs at the early stages of multiple sclerosis. On the other hand, it has been demonstrated that formation of the myelin sheath (myelination) involves various complex interactions between neurons and oligodendrocytes in the CNS, these interactions in the peripheral nervous system occurring between neurons and Schwann cells during several phases of myelinogenesis [7, 9, 11, 14, 18]. Recent data [18–20] show that during periods of active myelination, some of the myelin phospholipids are synthesized in neuron bodies. These are transported with the axon flow and only then enter the myelin sheath of the fiber [10]. We thus suggested that demyelination, which occurs in multiple sclerosis, is not the result of primary damage to the myelin sheath but is associated with damage to the neuron [21, 22]. It is therefore of interest to use electron microscopy to obtain detailed data on the structural responses of axons and glio-axonal interactions during demyelinating processes in experimental models of multiple sclerosis, which was the aim of the present work.

Cytoplasmic syncytial interneuronal connection in molluscs

The absolute criteria developed by the authors have been presented; they allow revealing cytoplasmic syncytial connections between processes of nerve cells in vivo and in vitro at the light microscopy level by using classical methods and time lapse videoshooting in the phase contrast. With aid of electron microscopy, metastable membrane contacts and their perforations, cytoplasmic syncytial interneuronal pores, and fusion of nerve processes are demonstrated. In the culture of isolated molluscan neurons, the process of formation of syncytial connections between processes of the same neuron or of different neurons is reproduced. Processes of one neuron, which have syncytial connection with another neuron, are shown to remain viable after death of its neuronal soma. The cytoplasmic varicosities formed on processes of one neuron are able to overcome the place of syncytial contact with processes of another neuron and to move to the body of the latter. A hypothesis is put forward that the cytoplasmic syncytial connection between nerve processes is formed under the conditions of the absence of their glial sheaths.

Syncytial Cytoplasmic Anastomoses between Neurites in Caudal Mesenteric Ganglion Cells in Adult Cats

The fact that most published data on syncytial cytoplasmic anastomoses are based on the autonomic nervous system in the early postnatal period of development, when many nerve fibers are poorly ensheathed by glia or have no glial sheaths at all, has led to the assumption that these anastomoses do not exist in adults because of the significant development of the glia and glial insulation of individual neurites from each other. We tested this assumption using electron microscopic studies of the caudal mesenteric ganglion in adult cats. A high level of glial ensheathing of neurites was observed. However, syncytial pores were seen between contacting neurites lacking glial sheaths in almost every specimen. This is the first report describing axodendritic synapses with perforations in the presynaptic zone outside the synaptic specializations in the autonomic nervous system. It is suggested that although syncytial cytoplasmic connections are seen in adult animals, they do not contradict the neuron theory.

Cytoplasmic Syncytial Connections Between Neuron Bodies in the CNS of Adult Animals

Studies of neurons in the dentate gyrus and hippocampal fields CA1 and CA2 and cerebellar granule cells were performed to test the hypothesis that there are syncytial connections between the bodies of neurons in adult higher vertebrates. Electron microscopic investigations showed that these cells were densely packed and had incomplete glial coatings. The outer cell membranes of these cells were found to be in contact, and membrane contacts in the form of tight junctions and gap junctions were seen. These areas showed membrane perforation and the establishment of syncytial connections between neurons, with all the expected ultrastructural characteristics. These connections could form between several contacting neurons, resulting in a unified functional cell cluster. These studies support the hypothesis that cytoplasmic syncytial interneuronal connections, along with synaptic and contact-type electrical connections, form not only in tissue cultures and the autonomic nervous system during early postnatal ontogenesis, but also in the CNS in adult vertebrates.

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.

The question of the fusion of neuron processes.

The authors, whilst accepting the neuron theory, present data indicating the possibility that neuronal syncytia exist when myelin-coated ring-like structures form in the dendritic field, in nerve arcades close to neuron bodies, and on formation of thick, straight anastomoses between neuron bodies. Studies using computerized time-lapse videomicroscopy in cultures of isolated neurons demonstrated the mechanism by which these structures form. This report provides the first evidence of the time parameters of the fusion of the processes of a single live neuron; the fusion of fragments of an isolated glial-free fiber was demonstrated.

Analysis of the effects of antibodies to gangliosides on the electrical activity of Retzius neurons in the leech and on the functional activity of influx sodium current channels.

The effects of anti-ganglioside antibodies on the functional states of two types of influx Na+ current channels were studied. Experiments used 20% anti-ganglioside antiserum prepared by standard methods by immunizing rabbits with total bovine brain gangliosides. These experiments showed that incubation of neurons in physiological saline containing antiserum induced discordance in the operation of the two types of influx current Na+ channels responsible for spike generation. This reaction was found to be associated with the slowed rate of activation of TTX-sensitive Na+ channels. Synaptic stimulation of cells in the presence of antiserum induced blockade of TTX-insensitive influx Na+ current channels. High-frequency synaptic activation of cells (10 Hz) showed that apart from blockade of TTX-insensitive Na+ channels, anti-ganglioside antibodies prevented plastic rearrangements in the gate system of TTX-sensitive Na+ channels. This resulted in impairment of the development of the acclimation process – the response of the neuron to high-frequency stimulation seen in normal conditions.

Non-electrical functions of neurons.

Classical histological preparations of metasympathetic nervous system impregnated with silver salts were compared with the dynamics of the development of plexuses in cultures of isolated neurons and electron microscopic studies to investigate the non-electrical properties of neurons. Two modes of neurite contraction were demonstrated, along with a means of translocation of the neuron soma within neurites, and formation of tail processes and autotomy. It is hypothesized that nerve receptor endings have tissue trophic effects, with autoamputation of terminals with subsequent activation of their lysosomal proteases, which appear to be growth factors for surrounding tissues. The morphogenetic functions of neurocytes are analyzed and evidence is presented for the occurrence of constant reconstruction of previously formed plexuses in healthy adult animals.