Changes in the morphometric parameters of neuroblastoma cells cultured in a high pH medium

The development of a population of morphologically differentiated mouse neuroblastoma cells was investigated during culture in a medium with a pH of 8.0–8.2. The original population split into two subpopulations — one of proliferating and the other differentiating cells — on the third day of culture in modified medium. Changes in the morphometric parameters of cells differentiating with time was investigated in vivo. A significant correlation between somatic dimensions and neurite length was found in differentiating cells. This implies that degree of morphological differentiation may be determined by size of the soma when using this technique for inducing differentiation. The patterns noted may serve for further research into morphofunctional changes produced by induced differentiation of neuroblastoma cells.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 213–219, March–April, 1988.     

Morphological differentiation of neuroblastoma cells induced by dimethylsulfoxide

Morphological features of neuroblastoma cells grown in culture in the presence of dimethylsulfoxode (DMSO) were studied. Morphological differentiation, expressed as the appearance of long axon-like processes (neurites), an increase in size of the cells, and inhibition of cell division, was observed in neuroblastoma cells of line C 1300, subline N-18-TG₂A₁, incubated in medium containing 1% DMSO. In the early stages of culture in normal growth medium the cells possess primary features of morphological differentiation. Quantitative criteria for the development of these features depending on duration of culture in modified medium were worked out. An increase in the total length of the neurites of cells differentiating under the influence of DMSO is a linear function of time. The rate of growth of the neurites is 20.0±3.0 µ/h. The area of cross-section of the soma of the differentiated cells is 6–7 times greater than the corresponding parameter in the control. An increase in the DMSO concentration in the culture medium (1.5 and 2.0%) does not induce rapid growth of the neurites or an increase in size of the cell soma, but it does block mitosis. Characteristics of morphological differentiation of neuroblastoma cells are compared with probable functional changes in these cells.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 519–527, July–August, 1984.     

Sodium and calcium channels in the somatic membrane of artificially differentiated neuroblastoma cells

Using an intracellular dialysis technique a study was made on calcium and sodium inward currents at the neuroblastoma somatic cell membrane in suspension and during the course of artificial morphological differentiation produced by raising the pH of the culture medium to 8.0–8.2. The density of sodium currents in the somata of cells cultured in the suspension averaged 7.3±0.8 µA/µF, while this value varied from 37±5.2 to 54.7±3.6 µA/µF at various stages of culture. These values equalled 1.4±0.2 and 1.1±0.2 to 2.8±0.4 µA/µF in the case of calcium currents. Reciprocal changes were produced in the density of sodium and calcium channels by altering the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 207–214, March–April, 1986.     

Murine neuroblastoma cells absorb anti-cytoplasmic tetrodotoxin-sensitive protein antibodies

The properties were investigated of polyclonal antibodies obtained by immunizing with a fraction of cytoplasmic glycoproteins forming sodium channels in liposomes. It was shown that these antibodies can be absorbed by intact murine neuroblastoma cells. Graphs plotting intensity of absorption against numbers of absorbant cells follow a characteristic course dependent on life of the cells in culture and serum concentration in the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 98–105, January–February, 1988.     

Excitation of accessory motoneurons by stimulation of Deiters' nucleus in the cat

Acute experiments on cats under chloralose-pentobarbital anesthesia showed that application of single stimuli to Deiters' nucleus evoked monosynaptic EPSPs in motoneurons of the accessory nucleus. Latent periods of EPSPs ranged from 1.3 to 2.3 msec (mean 1.8±0.3 msec), their rise time was 0.5–1.0 msec, and their duration 7–10 msec. During repetitive stimulation the EPSPs were weakly potentiated, but with an increase in the strength of stimulation applied to Deiters' nucleus they readily changed into action potentials. In some motoneurons polysynaptic EPSPs with latent periods of the order of 6.0 msec appeared on the descending phase of these EPSPs.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 5, pp. 515–519, September–October, 1981.     

Common features of antigenic determinants present on mammalian nerve cell membrane structures and in cytoplasmic tetrodotoxin-sensitive proteins

Possible antigenic features common to nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins were investigated by means of an immobilized immunoenzyme test. It was found that antiserum obtained by immunizing rabbits with a purified preparation of cytoplasmic tetrodotoxin-sensitive proteins can react with antigenic determinants present on the membrane fraction of bovine brain cells, on rat synaptosomes and on cells of a clonal line of mouse neuroblastoma. It was shown by means of an inhibition assay test that antibodies of the same specificity contribute to the observed response. Findings would indicate the presence of antigenic determinants common to nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins. This is consistent with the hypothesis that cytoplasmic tetrodotoxin-sensitive proteins have certain features in common with membrane sodium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 369–372, May–June, 1987.     

Calcium currents of differentiated mouse neuroblastoma cells

Differentiated mouse neuroblastoma cells (line C 1300, clone N-18-TG₂A₁) were investigated by intracellular dialysis. A slow component was found in the potential-dependent inward current of these cells. The results of investigation of changes in amplitude of this component during variation of the ionic composition of the external and internal solutions showed that this component is due to transport of calcium ions. A calcium current was observed in all cells tested. The region of its activation was between –70 and –65 mV; maximal values of this current were reached when the membrane potential was between –30 and –40 mV. The kinetic characteristics of this current were examined. In its kinetics and potential-dependence, this calcium current of the mouse neuroblastoma cell membrane is analogous to the fast component of the calcium current in normal neurons of rat spinal ganglia.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 527–531, July–August, 1984.     

Monoclonal antibodies to cytoplasmic tetrodotoxin-sensitive brain protein. Effects of veratrine on binding between antibodies and neuroblastoma cells

The effects were studied of neurotoxins, which modulate the activity of voltagedependent sodium channels, on binding between neuroblastoma cells and monoclonal antibodies to cytoplasmic tetrodotoxin-sensitive protein displaying the properties of tetrodotoxin-sensitive sodium channles when incorporated into the liposomes. Binding between antibodies and the monolayer of viable (unfixed) cells recorded by immunoenzyme testing was found to break down in the presence of veratrine (veratridine). It is postulated that the antibodies obtained bind with an antigenic determinant located at or near the veratrine binding site.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 794–800, November–December, 1988.     

Action of neurotoxin from the sea anemoneHomostichanthus duerdemi on inward sodium current in mammalian neurons

The action of purified toxin from the sea anemoneHomostichanthus duerdemi (HTX-1) on the inward sodium current was studied in experiments on isolated neurons from rat spinal ganglia and neuroblastoma cells of clone N-18F1, by an intracellular perfusion and voltage clamp method. HTX-1 was found to delay inactivation of the tetrodotoxin-(TTX-)sensitive inward sodium current and to make it incomplete, but virtually without affecting its activation. The relationship between the fraction of sodium channels modified by the toxin and the HTX-1 concentration is described by a Langmuir isotherm with association constant of (1.1 ± 0.1)·10^–7 M (holding potential –100 mV). Under the influence of the toxin the peak inward sodium current was increased by about 80%. Binding of HTX-1 with TTX-sensitive sodium channels is distinguished by strong potential-dependence: at a holding membrane potential of 0 mV the binding constant was an order of magnitude less than at a potential of –100 mV. In the case of brief action of HTX-1 on the nerve cell membrane (under 5 min) the effect of the toxin was completely reversible, but if the time of action of HTX-1 exceeded 30 min, subsequent washing with normal solution for 90 min did not abolish the effect completely.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Pacific Institute of Bioorganic Chemistry, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 14, No. 4, pp. 402–409, July–August, 1982.     

Properties of the slow excitatory postsynaptic potential in mammalian sympathetic ganglion cells

Two types of slow excitatory postsynaptic potentials (EPSPs) with different properties were found in neurons of the rabbit superior cervical sympathetic ganglion. In our group of neurons slow EPSPs increased during artificial hyperpolarization and decreased during depolarization of the membrane. The input resistance of the cells fell or remained unchanged during the development of slow EPSPs. In the second group of cells slow EPSPs increased during depolarization and decreased during hyperpolarization. The reversal potential of these responses, determined by extrapolation, was –78.9±3.6 mV. Depolarization responses to activation of muscarinic cholinergic receptors by acetylcholine or carbachol developed in 53% of neurons with an increase in input resistance and had a reversal potential of –83.2±6.7 mV. It is suggested that in cells of the first group the ionic mechanism of the slow EPSPs is similar to that of the fast EPSPs, whereas in cells of the second group its main component is a decrease in the potassium conductance of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 371–379, July–August, 1981.     

Effects of stimulating the superior colliculus on motoneurons of the neck muscle in the cat

Postsynaptic potentials produced by stimulating three sites of the midbrain superior colliculus were examined in motoneurons innervating the sternocleidomastoid, the trapezius, and the platysma cervical muscles in anesthetized cats. Stimulating the ipsilateral colliculus produced EPSP in the motoneurons as well as action potentials with a latency of 1.5–3.5 msec, averaging 2.6 ± 0.1 msec. Stimulation of the contralateral colliculus evoked EPSP with a latency of 1.5–3.2 msec and averaging 2.1 ± 0.1 msec together with IPSP with latency ranging from 2.6 to 5.0 msec. It is postulated that these postsynaptic responses are both monosynpatic and bisynaptic in nature. This type of synaptic action is assumed to be one of the mechanisms responsible for coordinated head movements produced by tectofugal impulses.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 197–202, March–April, 1986.     

Modulation by polyamines of the phosphorylation of the channel-forming cytosol protein of bovine brain

It was shown that the protein from the cytoplasm of bovine brain cells, which possesses functional similarity to the protein of membrane potential-dependent sodium channels, is the substrate of cAMP-dependent phosphorylation. Polyamines appreciably inhibit the phosphorylation of cytosolic protein. Spermine proves to be the most active here; in a concentration of 5 mM the inhibition reaches 40%. A possible modulation by polyamines of the functioning of sodium channels in the cell is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. R. E. Kavetskii Institute of Problems of Oncology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 603–607, September–October, 1991.     

Integration of soluble tetrodotoxin-sensitive brain proteins with liposomal membranes

Interaction of tetrodotoxin-sensitive proteins of the soluble cytoplasmic fraction of mammalian brain with liposomes was studied. Tetrodotoxin-sensitive proteins were shown to be incorporated in an orderly transmembrane manner into liposomes of a particular chemical composition (phosphatidylcholine-phosphatidylserine-cholesterol, brain phospholipids-cholesterol).A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 716–725, September–October, 1984.     

Action of thiamine on auditory evoked potentials in the guinea pig

A technique is proposed for quantifying the effects of physiologically active substances at the periphery of the auditory analyzer. It was found that applying 1×10^–11 to 1×10^–3 M thiamine to the membrane of guinea pig cochlear round window (fenestra rotunda) produces a rise in the amplitude and a reduction in the latency of the N₁ and N₂ components of auditory nerve action potentials, waves I and II of brainstem auditory evoked potentials occurring in response to an acoustic stimulus. It is suggested that this effect is produced by facilitated synaptic transmission at synapses between hair cells and spiral ganglia neurons under the action of thiamine penetrating into the cochlea.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. A. I. Kolomiichenko Research Institute of Otolaryngology, Ministry of Public Health of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 654–660, September–October, 1986.     

Phenylalanine intraperitoneal injection effect on high-voltage calcium channels in rat hippocampal neurons

Neurons from the hippocampus of one-day-old (controls) rats intraperitoneally injected with phenylalanine were isolated and cultured [3, 8]. The potential-dependent calcium input channel (I_Ca) in hippocampal neurons of controls after five-seven days of culture was made up of two components, low and high voltage, when the membrane potential was fixed under intracellular perfusion. The high-voltage channel was 69±13% of the total calcium channel at Vt = –10 mV. After rats were injected with phenylalanine, the high-voltage I_Ca significantly decreased to 32±14% of the total channel in the neurons at the same Vt. The low-voltage I_Ca did not change significantly.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 98–104, January–February, 1991.     

Inhibition in hippocampal pyramidal neurons during peripheral stimulation

Responses of hippocampal pyramidal neurons were investigated intracellularly in unanesthetized rabbits immobilized with tubocurarine. A single stimulus, applied to the sciatic nerve, evoked prolonged (up to 2.5 sec) hyperpolarization of the cell membrane, accompanied by inhibition of action potentials. The latent period of the evoked hyperpolarization was 48±16.4 msec, and its amplitude 2.5±1.9 mV. In some neurons the development of hyperpolarization potentials was preceded by excitation. The suggestion is made that hyperpolarization of the membrane of pyramidal cells during peripheral stimulation is manifested as an inhibitory postsynaptic potential (IPSP), generated with the participation of hippocampal interneurons. The possibility of prolonged tonic action of interneurons from outside as a cause of prolonged inhibition of the pyramidal neurons is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 278–284, November–December, 1969.     

Ionic mechanisms of depolarization responses in Helix pomatia neurons to glutamate application

Reversal potentials of transmembrane ionic currents induced by glutamate were determined in various D neurons ofHelix pomatia. Two types of neurons were found with mean reversal potentials of –10.6±1.2 and –40.0±0.6 mV. Neurons of the first group responded under ordinary conditions to glutamate application by a volley of action potentials. Neurons of the second group did not generate action potentials under the same conditions during glutamate application. With an increase in the dose of mediator the amplitude of D responses in these neurons increased only up to a certain limit, without reaching the critical depolarization level of the cell; a fall in the external chloride ion concentration led to a decrease in their reversal potential. The possible ionic mechanisms of glutamate-dependent depolarization responses of these groups of neurons are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 572–577, November–December, 1982.     

Ongoing activity in identified neurons of the rat superior cervical ganglion before and after partial denervation of the submandibular gland

Ongoing activity was investigated in rat superior certical ganglion neurons innervating the submandibular gland using intracellular recording techniques. These cells had previously been labelled by fluorescent marker. Ongoing activity was found in 11% of test cells, with a firing rate of 0.1±0.01 Hz. No ongoing activity occurred in the remainder (89% out of 95). Following an experimentally induced reduction in the number of neurons innervating the gland (which had previously been partially denervated), numbers of cells manifesting ongoing activity rose significantly (from 11 to 42%) and ongoing spike rate rose, at 0.3±0.03 Hz. These changes in neurons innervating the partially denervated gland are thought to result from increased convergence on these neurons of influences from preganglionic fibers.Studies carried out at the Anatomy and Neurobiology Department of Washington University Medical School (St. Louis, USA).A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 826–832, November–December, 1989.     

Inhibitory and excitatory processes in thalamic motor nuclei neurons in the normal nigrostriatal dopaminergic system and following injury to it produced by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Excitatory and inhibitory processes in neurons of the thalamic anteroventral and ventrolateral (VA–VL) motor nuclei were investigated in two sets of experiments on cats anesthetized with Ketalar and immobilized by myorelaxant during the course of stimulation of the red nucleus region and following chronic administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 5 mg/kg i.m. over a 5-day period): It was found 48 h after the last MPTP injection that up to 48% neurons belonging to the substantia nigra pars compacta had been destroyed and that dopamine level had declined to 30% of that found in intact animals. In cats injected with MPTP inhibitory processes declined significantly in both duration and efficacy in VA–VL relay and non-relay neurons, while latency of orthodromic excitatory response to red nucleus stimulation declined. It is suggested that attenuation of dopamine-modulated GABA-ergic nigrothalamic influences underlies the low level of inhibition occurring.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Endocrinology and Metabolism, Ukrainian Ministry of Public Health, Kiev. Institute of Organic Chemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 5, pp. 620–629, September–October, 1989.     

Comparative analysis of vestibulospinal neuron sizes in the guinea pig

A comparison was made between dimensions of vestibular neurons labeled with horseradish peroxidase projecting to the spinal cord and cells stained with neutral red not differentiated into vestibulospinal and not forming descending projections. The cells in nondifferentiated areas of descending, medial and lateral vestibular nuclei include neurons of all sizes. In the caudorostral direction of the vestibular complex, the number of small and average neurons decreased and the number of large and gigantic neurons increased. The vestibulospinal populations included cells of average, large and gigantic size, and large and gigantic neurons were dominant. In the caudorostral direction, neurons of various sizes were distributed relatively evenly without forming differentiated groups.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 616–624, September–October, 1991.