Investigation of spinal neuronal mechanisms of movement control systems

Morphological and electrophysiological investigations of the means whereby the principal descending motor systems (the cortico-, rubro-, reticulo-, and vestibulo-spinal tracts) are connected with the segmental interneuronal apparatus and motoneurons show that these connections can be based on two different principles. Descending systems either activate motoneurons directly (monosynaptically) or are connected primarily with various interneuron systems, exerting their influence in that case by regulating the activity of simpler or more complex spinal mechanisms. The older descending system (reticulo- and vestibulo-spinal) possess a monosynaptic excitatory action of motoneurons; the evolutionarily newer descending systems, which transmit the most complex motor signals from the cerebral and cerebellar cortex to the spinal cord (cortico- and rubro-spinal), terminate synaptically in every case on interneurons. It is only in primates that a few cortico-spinal fibers form monosynaptic connections with motoneurons. The chief ways of action of the descending systems on interneurons are: control of the afferent inflow into the interneuron system by presynaptic inhibition of the corresponding synapses; control of the interneuron system by postsynaptic interaction with afferent influences; control of motoneurons through the specialized interneuron apparatus. The investigation shows that the last of these mechanisms functions in the cortico- and rubro-spinal, and possibly also in the reticulo- and vestibulo-spinal systems. The functional role of the various means of connection of the descending systems with the spinal neurons in the system of movement control is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 2, pp. 189–202, March–April, 1970.     

Calcium Signaling in <Emphasis Type="Italic">Carassius</Emphasis> Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca²⁺ ([Ca²⁺] _і ) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca²⁺, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.     

Capsaicin-induced death of cultured neurons of the rat hippocampus

We studied the effects of an agonist of vanilloid receptors (VRs), capsaicin, and of an antagonist of VRs of type 1, capsazepin, on cultured neurons of the rat hippocampus. In cultures incubated for 1 day in a medium containing 10 μM capsaicin, the numbers of cytologically normal cells and those with manifestations of necrosis and apoptosis were, on average, 46.4 ± 3.3, 30.7 ± 2.4, and 22.9 ± 5.4%. The latter two values were more than three times greater than the respective indices under control conditions (P < 0.05). Coincubation of the cells with 10 μM capsaicin and 25 μM capsazepin decreased the normalized number of apoptotic units by about one-third, while the number of cells with necrotic changes showed nearly no changes. Using confocal microscopy and staining the cells with a fluorescent dye, JC-1, we found that incubation with capsaicin resulted in a dramatic drop in the mitochondrial potential in the great majority of cultured cells, while capsazepin somewhat smoothed this effect. Thus, our data show that the cytotoxic effect of capsaicin is related to changes in the mitochondrial potential and is at least partially mediated by activation of type-1 VRs. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 13–20, January–February, 2008.     

Roles of Nicotinic and Muscarinic Receptors in Calcium Signaling and Transmitter Release

Activation of acetylcholine receptors (AChR) triggers catecholamine release from adrenal chromaffin cells and release of neurotransmitters in neuron-to-neuron and neuromuscular junctions, including those on smooth muscle cells. Calcium ions play the role of the main intracellular messenger, which mediates these processes. In our study, we explored the properties of Ca²⁺ signaling triggered by activation of AChR by analyzing the characteristics of Ca²⁺ transients induced by selective activation of nicotinic (nAChR) and muscarinic (mAChR) cholinoreceptors using Fura-2 fluorescent measurements in experiments on rat chromaffin cells. Two populations of chromaffin cells, which in a different manner responded to AChR stimulation, were classified. We found that the mean frequency of quantum release induced by ACh is considerably higher than that during hyperpotassium cell depolarization. Comparative analysis of single secretory events showed that, in the case of stimulation by ACh, single secretory spikes demonstrate faster kinetic characteristics than those induced by depolarization. Statistical analysis of the integral magnitude (area) of single secretory spikes evoked by both types of stimulation showed no significant difference despite amplitude and kinetic dissimilarities between such secretory events. Mathematical modeling of the dynamics of the exocytotic processes led to the conclusion that the reason for the specific kinetic characteristics of single secretory responses may be different diameters of the secretory pores formed during fusion of secretory vesicles with the plasma membrane.     

Targeted mutagenesis identifies Asp-569 as a catalytically critical residue in T7 RNA polymerase

In order to look more closely at a well-conserved region in T7 RNA polymerase (T7 RNAP) containing, as shown earlier, the functionally essential residues Pro-563 and Tyr-571, we used targeted mutagenesis to change those residues within this region that are invariant in all single-subunit RNA polymerases, and characterized the mutant enzymes in vitro. The most interesting finding of this study was the crucial importance of the acidic group of Asp-569. In addition, we have shown that the phenolic ring is the most significant functional group of Tyr-571, with the hydroxy group also contributing to promoter binding.     

Pyramidal influences on interneurons of spinal segmentary reflector arcs in cat

Microelectrode discharges of potentials have been realized from segmentary interneurons of the dorsal horn and intermediate nucleus of the spinal cord in cat at the L₆–L₇ level by electrical stimulation of the sensorimotor region of the brain cortex. It has been established that corticifugal influences on segmentary interneurons of the system of the flexor reflex and on neurons activated by high threshold muscle afferents (groups Ib, II, and III), or high threshold cutaneous afferents are predominantly excitatory. Interneurons activated by muscle afferents of group Ia or by the lowest threshold cutaneous fibers are weakly subjected to pyramidal influences. The mean latencies of excitatory postsynaptic potentials (EPSP's) and discharges evoked under the influence of pyramidal volley, for the neurons under study in the system of afferents of the flexor reflex are equal to 11.8±2.6 and 20.1±1.8 msec, respectively; for interneurons, excited only by high threshold muscle afferents, they are equal to 15.5±3.6 and 16.3±2.2 msec, respectively; and for interneurons, excited by high threshold cutaneous fibers they are equal to 11.8±2.6 and 18.3±1.4 msec, respectively. Possible pathways of activating segmentary interneurons from the lateral sensorimotor region of the brain cortex have been discussed.The A. A. Bogomolets Institute of Physiology, Academy of Sciences, Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 17–25, January–February, 1970.     

Changes in the ionic mechanisms of electrical excitability in the somatic membrane of rat dorsal root ganglion neurons during ontogenesis. Correlations between inward current densities

Correlations between densities of various types of inward currents in the somatic membrane of dorsal root ganglion neurons were studied in three different rat age groups: 5–9 days, 45 days, and 90 days. A linear relationship was found in neurons with "slow" tetrodotoxin-sensitive sodium current between the densities of high-threshold calcium current and "slow" sodium current (Bravias-Pearson's correlation coefficient: r=0.84 and 0.70 for n₁=16 and n₂=28, respectively). No such correlation was observed in neurons with low-threshold calcium inward current. Cells with only two types of channel — "fast" sodium and high-threshold calcium — present in their somatic membrane manifested an inverse correlation (r=–0.48, where n₄=95) between the densities of transmembrane currents passing through these channels. No inverse relationship was observed in the density of "fast" sodium and high-threshold calcium currents in neurons with tetradotoxinresistant "slow" sodium and/or low threshold calcium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 820–827, November–December, 1986.     

Structural characteristics of connections between medial descending systems and spinal neurons

Structural and ultrastructural changes in the medial part of the ventral horn were studied in segments of the cat spinal cord following destruction of the ventral column at the level C₁–C₂. Analysis of results obtained by the Fink — Heimer method showed that degenerating preterminals occur mainly in Rexed's lamina VIII and also in ventromedial zones of lamina VII. Preterminals of descending pathways of the ventral column are also found in the intermediate nucleus of Cajal (central part of lamina VI) and in the ventromedial motor nucleus. Fewer of these preterminals are present in the thoracic and, in particular, in the lumbar segments. Staining by the Holländer — Vaaland method revealed degenerating myelinated axons of small diameter (3–5 µ), evidently collaterals of descending fibers entering the gray matter, in lamina VIII. Degenerative changes in myelinated axons may be manifested either as marked condensation and shrinking or as the appearance of numerous neurofilaments, polymembraneous structures, and cytolysomes. Degeneration also affects axon terminals (axo-dendritic, axo-somatic, and axo-axonal) with spherical or flattened synaptic vesicles. Counting the relative numbers of intact terminals of the various types and their comparison with the corresponding figures for normal animals shows that most connections of descending fibers with spinal neurons are axo-dendritic in character. No degenerating terminals were found on the soma of the "dark" neurons or their processes.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 6, pp. 579–586, November–December, 1972.     

Voltage-gated ion channels of inflowing current expressed in Xenopus oocytes after injection of rat brain mRNA

The expression of two types of voltage-gated ion channels of the inflowing current ("fast" sodium channels, sensitive to tetrodotoxin, and high-threshold calcium channels) was detected by electrophysiological methods in the membrane ofXenopus oocytes, after injection of poly(A)⁺-mRNA from the brains of 18- to 20-day-old rats. When Cd²⁺ (200 µmoles/liter) was added to the extracellular solution, the barium current through the expressed calcium channels was completely suppressed, but no sensitivity to D-600 (20 µmoles/liter) and nitrendipine (50 µmoles/liter) was exhibited. A peptide blocker of the high-threshold calcium channels of the neuron membrane, -conotoxin GVIA, in a concentration of 1 µmole/liter led to 20–40 min suppression of the barium current expressed in the oocyte. Steady-state inactivation of this current could be described by the Boltzman formula, using the values of the half-inactivation potential V_1/2=–50 mV and the steepness factor k=14 mV. It is concluded that in potential-dependent and pharmacological properties, the calcium channels expressed in the oocyte, despite the absence of any appreciable time-dependent inactivation, most resemble the high-threshold inactivatable (HTI- or N-type) calcium channels of the neuron membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 344–353, May–June, 1991.