[Cryopreserved neurons of a mollusc are capable of morphological differentiation in culture in vitro]. / Kriokonservirovannye neirony molluska sposobny k morfoligicheskoi diffentsirovke v kul'ture in vitro.

It was shown in culture in vitro that neurons isolated from the cryopreserved brain of adult molluscs Lymnaea stagnalis L. retain viability. Isolated brains were frozen in liquid nitrogen vapors at a rate of 400-500 degrees C/min in the presence of 2 M dimethylsulfoxide. The samples were then plunged into liquid nitrogen and stored from 1 month to 2 years. Upon thawing and removing the cryoprotectant, the neurons were isolated from the brain and then introduced into a cellular culture in vitro. It was shown that the thawed neurons were capable of regenerating new nerve processes similar to those formed by unfrozen neurons in the control.

[Proliferation and morphological differentiation of neurblastoma cells in cultured under the effect of avermectins]. / Proliferatsiia i morfologicheskaia differentsirovka kletok neiroblastomy v kul'ture pod vliianiem avermektinov.

The effect of natural avermectin complex (Aversectin C) and Abamectin on the processes of proliferation and morphological differentiation of the neural cells was studied using N1E-115 murine neuroblastoma cells (clone C-1300) as a model. Aversectin C in concentrations 10(-7)-10(-8) was shown to induce morphological differentiation of cultured nervous cells. Treatment with Abamectin resulted in the changes of proliferation pattern of the cells. Morphological differentiation of the cultured nervous cells treated with Aversectin C was associated with electrophysiological one.

[The morphological differentiation of murine neuroblastoma NIE-115 cells]. / Morfologicheskaia differentsirovka kletok myshinoi neiroblastomy NIE-115.

Characters and kinesis of mouse neuroblastoma (MN) cells morphological differentiation affected by non-serum, hypo- and hyperosmotic media and salt solution were studied. The nature of morphological differentiation was found not to depend on the inductors, while kinesis varies significantly. Morphological differentiation speed is directly proportional to the extent of non-specific action and probability of the following cell death. on the other hand, the number of irreversibly differentiated cells is inversely proportional to the action strength. For the induction of morphological differentiation a minimal nutrition media do not containing serum is sufficient and a common growth media changed once in 3-5 days according to how it acidifies is better to use for the prolonged maintenance of it. Universality of neuronal morphological differentiation is under discussion according tot he data obtained from MN cells and cultured mollusk isolated neurons.

Differentiation and apoptosis of murine neuroblastoma cells N1E115.

The mode and the kinetics of differentiation and death of murine N1E115 neuroblastoma cells induced by dimethyl sulfoxide and other nonspecific factors in vitro were investigated. After morphological differentiation neuroblastoma cells die by apoptosis which is indicated by characteristic morphological features and by internucleosomal DNA fragmentation. Durations of both differentiation and apoptosis are dependent on the nature of stimuli used. Protein synthesis inhibitor cycloheximide does not prevent differentiation and apoptosis of neuroblastoma cells induced by dimethyl sulfoxide and even accelerates both processes. The relationship between cell death and differentiation is discussed.

Influence of biologically active substances isolated from Galleria mellonella on neurons of Lymnaea stagnalis in culture.

A procedure for isolating biologically active substances from Galleria mellonella using a culture of isolated giant neurons of mollusc Lymnaea stagnalis as a test-system is described. Fractions capable of activating neurites and inhibiting aggregation of neuronal cells within a range of concentrations from 1 to 30 micrograms/ml were isolated. The fractions obtained have in their chemical composition about 10.5% N, also contain P and S. They have a carbohydrate component.

Ca2+ and pH affect the neurite formation in cultured mollusc isolated neurones.

Neurite formation in neurones isolated from adult molluscs in culture has been shown to depend on the total content of Ca in the cells, intracellular Ca2+ concentration, intracellular acid-alkaline balance, extracellular pH, and the capacity and composition of buffers. The neurones with a low total Ca content prior to cultivating (1.2 mmol/kg) and low buffer capacity of cytoplasm (pH artificially shifted to the acidic level) possess the most pronounced capability of neurite regeneration. Optimal media for neurite regeneration appear to contain sodium bicarbonate as a buffer either alone or with small additions of organic buffers (1.5-3 mM) at pHs increasing from 7.6 to 8.2 under equilibrium with air. In the absence of sodium bicarbonate, when only organic buffers are used (Tris-HCl; HEPES-Na2CO3), at constant pH values ranging from 7.5 to 8.2, no neurites are formed. Artificial enhancement of intracellular Ca2+ concentration at the beginning of culture completely inhibits neurite outgrowth, and when applied on the third to fifth days of culture, it causes retraction of the neurites already formed. Neurones loaded with calcium (10 mmol/kg) form no neurites regardless of medium composition and concentrations of buffers used at pHs ranging from 7.5 to 8.2. The results obtained allow to suggest that neurite regeneration is controlled by Ca2+- and pH-regulating intracellular systems.

The effect of elevated potassium on the adult mollusc giant neurone survival and neurite formation in culture.

The number of viable neurones calculated as soon as adult mollusc ganglia tissue was digested and over different periods of cultivation, show that elevated K+ prevents degradation of intercellular contacts, preserves neurones against lysis, prolongs the time course of neurone survival in culture and inhibits neurite formation. K+ depolarizing effect is maintained throughout the whole course of cultivation; when elevated K+ is substituted by normal K+ the resting potential of neurones recovers. The effect of elevated K+ on neurone survival and the capability for neurite formation is not due to membrane depolarization, but seems to be related to an influence on intracellular ionic homeostasis.

[Reactive changes in living nerve endings in cultures of isolated neurons devoid of glia]. / Reaktivnye izmeneniia zhivykh nervnykh okonchanii v kul'ture izolirovannykh neironov, lishennykh glii.

Reactive changes in terminal apparatus of mollusc mature neurons treated with pronase and cultivated in 20% medium with saline solutions added have been studied by means of modern methods of differential and coloured interferential contrast. The culture used can serve to study mechanisms of an urgent nonspecific reaction of varicose rearrangement of the terminal apparatus, as well as delayed reactive changes of an "excessive" growth type, autapses formation, self amputation etc. In the glia-deprived culture, glia is not an initial organizing factor of varicose deformity of the processes. Mechanisms of varicose rearrangement of thick and thin fibres are discussed.

[Effect of ATP on protein synthesis in cultured nerve tissue of the edible snail]. / Vliianie ATF na sintez belka v kul'tiviruemoi nervnoi tkani vinogradnoi ulitki.

External ATP in concentrations of 10(-6)--10(-3) M is shown to stimulate the label incorporation from intracellular labeled pool of 14C-leucine into proteins of mollusc nervous tissue. The maximum effect (by 45% higher than in control) is observed at the 10(-5) ATP concentration. In solutions with high concentration of bivalent ions, ATP action increases by 10--15%. Being incubated for an hour in physiological solutions without energic substrates nervous tissue loses 30--50% of labeled amino acids. Outwashing of 14C-leucine depends only a little on the bivalent ion concentration in the external solution and on the presence of helating agents. Addition of 10(-4) M ATP into the solution, completely inhibits the washing of amino acids out of tissue. At low bivalent ion concentrations 14C-leucine incorporation into nervous tissue in the presence of ATP changes inversely to the ATP concentration: low ATP concentrations (10(-5)--10(-6) M) activate label incorporation by 60--40%, whereas high concentrations lead to the corresponding inhibition. This inhibition is due to helating action of ATP.

[Use of a histological fixative for extraction of the RNA precursor pool from mollusk nerve tissue]. / Ispol'zovanie gistologicheskogo fiksatora dlia ékstraktsii pula predshestvennika RNK iz nervnoi tkani molliuskov.

The incorporation of 3H-uridine into the nucleotide pool of the molluscan nerve tissue was calculated in a routine fixative solution (formalin--alcohol--acetic acid, 9:3:1). The optimal conditions for the precursor output from the tissue being studied, the results obtained well compare with those obtained by common biochemical techniques. Our method gives a possibility to use the same sample of tissue for historadiographical investigation of labeled RNA and for cytochemical determination of nuclear acid amounts.

Completely isolated molluscan neurons. An ultrastructural study.

The neurons of the molluscs Lymnaea and Helix isolated by fermentative digestion followed by mechanical treatment do not differ ultrastructurally from intact ones. These cells have sufficient metabolic reserves and incorporate into RNA 8% of the total radioactive pool, even more than neurons in ganglia under equal conditions. Neuronal damage can occur, mainly during the pipetting, and this is usually expressed in vacuolization of the cytoplasm. It is important to note that alterations in cell ultrastructure develop earlier than changes in the membrane electrical properties. The surface of the isolated neurons is enlarged two-fold due to the infoldings of the cell membrane. So, the specific resistance of soma membrane of these neurons was calculated as 78 +/- 13 komega-sq. cm. On the surface of isolated neurons scraps of glial and neuronal processes not connected with their own cell bodies, and as a consequence not powerful, are sometimes found. Some endings of the neuronal processes on the surface of isolated neurons are ultrastructurally similar to the axo-somatic synapses.