N-methyl-D-aspartate effect on spontaneous activity of 16–20-day-old chick embryo spinal cord

The effects of N-methyl-D-aspartate (NMDA) and the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (2-APV), on spontaneous activity of dorsal and ventral roots (DR and VR, respectively) generated by isolated spinal cord from 16–20-day-old chick embryo were studied. This activity was synchronous oscillations of electrotonic potentials in DR and VR. There was no impulse activity in the VR. When NMDA was applied at 2–25 µM, the amplitudes of the oscillations increased, the impulse activities in VR and DR developed, and the tonic component of electrotonic potentials appeared. At 20 µM, 2-APV decreased both, the spontaneous and NMDA induced activity. After sectioning of the spinal cord, the neuronal network of the isolated dorsal arm conserved the capacity to generate spontaneous activity in the DR which increased after NMDA application. There was no rhythm in the ventral part of the spinal cord. The localization of the NMDA-sensitive neuronal network, generator of the rhythmic (motor) activity, in the spinal cord is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 2, pp. 205–213, March–April, 1991.     

Modification of cyclical activity in the spinal cord of the 16- to 20-day-old chick embryo due to changes in Ca2+ and Mg2+ concentrations

In isolated segments of the 16- to 20-day old chick embryo, increasing the Mg²⁺ concentration from 1.3 to 5.0 mmoles/liter in the superfusate caused complete suppression of spontaneous rhythmic activity that appeared as synchronous cyclical oscillations of electrotonic potentials in the dorsal and ventral roots. A similar change was recorded when the Ca²⁺ concentration was decreased from 2.6 to 1.0 mmole/liter, but in this case tonic discharges of impulses (spikes) could occur. Further, during the disappearance of the spontaneous activity due to changing the concentration of Ca²⁺ or Mg²⁺, in six out of eight experiments another type of rhythmic activity was seen, appearing as oscillations of electrotonic potentials in the ventral roots that were independent of oscillations in the dorsal roots. The amplitude of these oscillations of potential in the ventral roots was up to 200 µV, and their duration was up to 400 msec. The highest frequency of this activity was 0.4 sec^–1. The possible functional significance of the observed patterns of activity is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 333–338, May–June, 1991.     

Effects of vasopressin and oxytocin on evoked dorsal horn cell activity in an isolated segment of spinal cord

During experiments on an isolated segment of the spinal cord of 2- to 3-week-old rats, a study was made of the effects of vasopressin and oxytocin on the activity of dorsal horn cells produced by stimulating the afferent root. Both field and action potentials were recorded in single cells. It was established that vasopressin and oxytocin produced reversible inhibition of the postsynaptic component of field potentials. The amplitude of potentials was reduced by 33–39% by vasopressin and by 12–34% using oxytocin. The effect of the test substances depended on the concentration used and the duration of their action on the brain. Both vasopressin and oxytocin reversibly depressed discharges of single dorsal horn cells evoked by stimulating the dorsal root. These two neuropeptides prolonged latency, and reduced the number of evoked potentials or completely suppressed response. A facilitatory effect was recorded in a small number of cells. We deduced from our findings that their hypothalamospinal neurohormonal system inhibits transmission of afferent impulses at the level of interneurons of the dorsal horn.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 634–640, September–October, 1985.     

Effects of barium on isolated frog spinal cord

The effects of Ba2+ were studied in vitro on the isolated frog spinal cord. Ba2+ (25 microM-5 mM) caused a concentration-dependent depolarization of ventral (VR) and dorsal (DR) roots. TTX and Mg2+ substantially reduced the depolarization suggesting that interneuronal effects were involved. Ba2+ (25-500 microM) markedly increased the frequency and duration of spontaneous VR and DR potentials and substantially enhanced the duration (and frequently the amplitude) of VR and DR potentials evoked by DR stimulation. Higher concentrations of Ba2+ (1-5 mM) reduced both spontaneous and evoked potentials. Ba2+ (25-500 microM) enhanced the amount of K+ released by a DR volley and by application of L-glutamate and L-aspartate. The cation reduced VR and DR root depolarizations produced by elevated [K+]0. VR potentials induced by L-glutamate, L-aspartate, GABA and glycine and DR depolarizations caused by GABA were reduced by Ba2+. These results show that Ba2+ has complex actions on reflex transmission, interneuronal activity, the postsynaptic actions of excitatory and inhibitory amino acids and the evoked release of K+.     

Acetylcholinesterase distribution in chick spinal cord cultures

Summary Explants of 10–12 day chick embryo spinal cord were cultured by coverslip-roller tube method for 3–80 days. The cellular and subcellular localization of acetylcholinesterase activity in cultured neurons was studied by the thiocholine techniques of Karnovsky and Roots and Lewis and Shute.At the light microscopic level, acetylcholinesterase was demonstrated in the neurons of both ventral and dorsal horn regions. Occasionally neurons migrated in the outgrowth zone exhibited strong intracellular activity.At the electron microscopic level, acetylcholinesterase activity was found in the nuclear envelope, granular endoplasmic reticulum and the Golgi apparatus of the neurons. No enzyme reaction was detected in the glial cell cytoplasm.     

Nature and kinetic characteristics of L-DOPA uptake in rat renal proximal tubules

Summary The present study was performed with the aim to determine the kinetics and the caracteristics of cellular uptake of L-3,4-dihydroxyphenylalanine (L-DOPA) in rat renal proximal tubules. Incubation of renal tubules at 4°C in the presence of increasing concentrations of L-DOPA results in a linear and concentration-dependent accumulation of the substrate. In experiments carried out at 37°C, the accumulation of L-DOPA in renal tubules was found to be greater than that occurring at 4°C and showed a trend for saturation. The saturable component of L-DOPA uptake was derived from the total amount of L-DOPA accumulated in renal tubules at 37°C subtracted with the values obtained in experiments conducted at 4°C. The V_max and K_m values for the saturable component of L-DOPA uptake in renal tubules were, respectively, 241 ± 32 fmol µg protein^–1min^–1 and 567 ± 63 µM. Cyanine 863 (5 and 10 µM) was found to decrease the tubular uptake of L-DOPA, whereas probenecid (50 µM) did not change the rate of uptake of L-DOPA into renal tubules. The V_max and K_m values for the saturable component of L-DOPA uptake in renal tubules incubated in the presence of 10 µM cyanine 863 were, respectively, 97 ± 11 fmol µg protein^–1min^–1 and 160 ± 22 µM. It is suggested that the anionic L-DOPA may behave as an amphoteric substance, both hydroxyl groups in the aromatic ring determining the binding of the molecule to the organic cation transporter.     

Effect of vasopressin and oxytocin on spontaneous unit activity of dorsal horn cells in the isolated spinal cord of young rats

The effect of vasopressin and oxytocin, added to the perfusion solution, on the spontaneous firing rate of single dorsal horn cells was studied in experiments on an isolated segment of spinal cord from rats aged 2–3 weeks. Both neuropeptides were found to have a mainly inhibitory action. Under the influence of vasopressin the spontaneous firing rate of 74% of cells (29 of 39 cells responding to vasopressin) fell, and only in 26% (10 of 39 cells) did it rise. Oxytocin inhibited spontaneous activity of 67% of cells (14 of 21 which responded) and excited 33% of neurons (seven of 21). The effects were dose-dependent and reversible. The cells either responded in the same way to vasopressin and oxytocin or they responded to application of one peptide but not of the other.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 3, pp. 314–320, May–June, 1985.     

Effect of corticosterone on noradrenergic nuclei in the pons-medulla and [3H]NA release from terminals in hippocampal slices

The aim of the present study was to investigate possible membrane and genomic effects of corticosterone on the noradrenergic system of the rat brain. Corticosterone effects were studied in vivo by treating rats s.c. with 10 mg/kg corticosterone for 7 or 14 days. In the first two experiments corticosterone significantly decreased th noradrenaline (NA) and dopamine (DA) levels in the pons-medulla, an area which contains the A1-A7 noradrenergic cell groups, while the NA and DA levels in the dorsal hippocampus remained unchanged. In a third experiment where the locus coeruleus (LC) and the A1 and A2 nuclei (A1,A2) were analysed separately, NA levels were unchanged but total MHPG levels and the total MHPG/NA ratio were decreased in the A1,A2 area. Chronic corticosterone treatment (14 days) did not alter the ₂-adrenoceptor-mediated modulation of [³H]NA release from dorsal hippocampal slices. Neither the spontaneous outflow nor the electrically stimulated release of [³H]NA from dorsal hippocampal slices of untreated rats was affected by exposure of the slices to corticosterone (10^–7 M–10^–4 M) in the superfusion buffer. Thus, chronic corticosterone treatment of rats altered the noradrenergic system of the pons-medulla, but did not change the ₂-adrenoceptor-mediated modulation of NA release in the dorsal hippocampus, a major terminal area of the LC neurons. Corticosterone also did not appear to have a direct membrane effect on the NA terminals in the dorsal hippocampus of the rat.     

Effect of serotonin on neuronal background activity in a hemisegment isolated from the infant rat spinal cord

Neuronal background activity was investigated in a hemisegment of the lumbar section of the spinal cord before and after addition of serotonin (5-HT — 1 × 10^–8–10^–4 M) in 14- to 22-day-old rats. Reversible changes in background firing rate were recorded in 50% and 70.6% of dorsal and ventral horn interneurons respectively. Excitatory response predominated; in the dorsal horn, 62.4% of all cells responding to 5-HT showed an excitatory response, 8.4% an inhibitory reaction, and 29.2% a two-stage response. In the ventral horn, an excitatory and two-stage response were recorded in 91.6% and 8.4% of cells respectively. Application of 5-HT induced an increase in firing rate and depolarization in the ventral horn. Findings from this study would point to a primarily excitatory effect of 5-HT on background in segmental neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 335–343, May–June, 1989.     

Action of precursors of noradrenaline synthesis on membrane receptors of chick embryo spinal neurons

Effects of noradrenaline precursors on glycine and N-methyl-D-aspartate (NMDA) receptors in spinal cord neurons recently isolated from chick embryo were investigated using whole cell patch-clamp and concentration clamp techniques. Both L-alanine and L-DOPA were found to be glycine agonists capable of potentiating NMDA response, while L-tyrosine does not activate glycine but can potentiate NMDA response. Lastly, L-phenylalanine and dopamine do not interact with either glycine or NMDA receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 665–670, September–October, 1990.     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Catechol-O-methyltransferase inhibition protects against 3,4-dihydroxyphenylalanine (DOPA) toxicity in primary mesencephalic cultures: new insights into levodopa toxicity

Inhibition of catechol-O-methyltransferase (COMT) has protective effects on levodopa (L-DOPA), but not D-DOPA toxicity towards dopamine (DA) neurons in rat primary mesencephalic cultures [Mol. Pharmacol. 57 (2000) 589]. Here, we extend our recent studies to elucidate the mechanisms of these protective effects. Thus, we investigated the effects of all main L-DOPA/DA metabolites on survival of tyrosine hydroxylase immunoreactive (THir) neurons in primary rat mesencephalic cultures. 3-O-Methyldopa, homovanillic acid, dihydroxyphenyl acetate and 3-methoxytyramine had no effects at concentrations up to 300 micro M after 24h, whereas DA was more toxic than L-DOPA with toxicity at concentrations of >or=1 micro M. The coenzyme of COMT, S-adenosyl-L-methionine (SAM), and its demethylated product S-adenosylhomocystein caused no relevant alteration of THir neuron survival or L-DOPA toxicity. In contrast, inhibition of SAM synthesis by selenomethionine showed time- and dose-dependent increase of THir neuron survival, but did not affect L-DOPA toxicity. L-DOPA-induced lipid peroxidation in mesencephalic cultures was not modified by the COMT inhibitor Ro 41-0960 (1 micro M). Increased contamination of the cultures with glial cells attenuated L- and D-DOPA toxicity, but caused significant enhancement of protection by COMT inhibitors against L-DOPA toxicity only. Investigations of L-DOPA uptake in rat striatal cultures using HPLC revealed a significant reduction of extracellular L-DOPA concentrations by Ro 41-0960. Our data confirm that L-DOPA toxicity towards DA neurons is mediated by an autooxidative process, which is attenuated by glial cells. In addition, we demonstrate a second mechanism of L-DOPA toxicity in vitro mediated by a COMT- and glia-dependent pathway, which is blocked by COMT inhibitors, most likely due to enhanced glial uptake of L-DOPA.     

Posttetanic changes in hippocampal minimal evoked potentials

Changes in the EEG induced by a single spike were recorded in the hippocampus of an unanesthetized rabbit. Summation of focal electrical activity synchronous with spontaneous single unit discharges at the symmetrical point of contralateral hemisphere revealed no stable potentials which could reflect these changes. In two cases discharges identified as activity of Shaffer's collaterals were recorded in area CA₁. Summation of post-spike changes in evoked activity recorded by the same microelectrode showed stable negative waves with an amplitute of 40–60 µV, which could have been evoked by single spikes. The curve of amplitude of the averaged evoked potentials versus near-threshold current strength stimulating the intrahippocampal pathways was not smooth in most experiments but stepwise in character. It is suggested that the minimal evoked potential corresponding to the first step (amplitude 40–80 µV) reflects a response to stimulation of one fiber. After above-threshold tetanization prolonged posttetanic potentiation of the minimal evoked potentials did not arise in CA₁ in response to stimulation of Shaffer's collaterals. Minimal evoked potentials recorded in area CA₃ in response to stimulation of the dentate fascia showed clear potentiation. The results are in agreement with the hypothesis of the synaptic localization of the mechanisms responsible for prolonged posttetanic potentiation.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 2, pp. 124–134, March–April, 1977.     

Comparison of spontaneous cortical unit activity in several brain areas of unanesthetized cats

Spontaneous unit activity in association area 5 and some projection areas of the cortex (first somatosensory, first and second auditory areas) were studied in cats immobilized with D-tubocurarine in which the index of specific spontaneous activity, the mean frequency, types of spontaneous activity, and statistical parameters — distribution of interspike intervals and autocorrelation function — were determined. The results showed that spontaneous unit activity in the association area differs from that in the projection areas in both intensity and character. A special feature of the spontaneous activity of the auditory areas was a well-marked volley distribution of activity. In the somatosensory area the level of spontaneous activity as reflected in all indices was the lowest. In the association cortex the largest number of neurons with spontaneous activity lay at a depth of 500–1000 µ corresponding to cortical layers III–IV. In the first auditory area neurons with spontaneous activity were concentrated at a depth of 1400 µ (layer V) and in the somatosensory area at a depth of 1000–1400 µ (alyers IV–V). The possible functional significance of these differences is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 1, pp. 13–21, January–February, 1976.     

Uptake and release ofmeta-tyramine,para-tyramine,and dopamine in rat striatal slices

The uptakes of high-affinity concentrations (10^–8 M) ofmeta-tyramine (m-TA),para-tyramine (p-TA), and dopamine (DA) into rat striatal slices have been shown to be inhibited by DNP and ouabain. We now demonstrate that cocaine (5×10^–6 M) and low concentrations of sodium ion (26×10^–3 M) also reduced these uptakes. The spontaneous efflux and the release [induced by an elevated concentration of potassium ion (5×10^–2 M)] of each of the previously accumulated amines were studied in the presence and absence of added calcium ions. The spontaneous efflux of each amine (especially the tyramines) was enhanced by the absence of calcium ions. Part of this enhancement seemed to be due to an inhibition of a calcium-dependent reuptake. The elevated concentration of potassium ion proved to be an effective releaser of each amine; and for DA, such release was decreased by the removal of calcium. Form- andp-TA, however, the removal of calcium either did not reduce or completely abolished the releases depending upon the duration of the calcium removal. The significance of these findings is discussed.     

Depolarizing effects of dopamine on motoneurons from a segment of spinal cord isolated from newborn rats

The effects on dorsal root potentials of applying dopamine to the perfusing fluid were investigated in experiments on a segment isolated from the spinal cord of 13- to 18-day-old rats. Dopamine induced slow, dose-dependent depolarization in motoneurons in 28 trials out of 32, retained in the solution blocking synaptic transmission. Threshold concentration of dopamine in the normal perfusing fluid measured 1·10^–6 M and 1·10^–5 M in a calcium-free perfusate containing magnesium or manganese ions. Depolarization was accompanied by an increased rate of motor discharges recorded from the ventral root. Segmental reflex response produced by dorsal root stimulation was depressed following depolarization. Hyperpolarization in response to dopamine was observed in 4 out of 32 experiments. Dopamine-induced electrotonic dorsal root potentials were suppressed by prior haloperidol application to the brain.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 735–741, November–December, 1987.     

Mass Fragmentographic Analysis of Monoamine Metabolites in the Spinal Cord of Rat After the Administration of Morphine

Abstract: A mass fragmentographic method was used in which homovanillic acid (HVA), methoxyhydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) were measured from a single sample. The results describe the effect of morphine on the metabolism of the major monoamines, dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in the spinal cord. Morphine has very little effect on the metabolism of DA and NA in the spinal cord. However, morphine causes a significant increase in the metabolism of spinal 5-HT. The increase in 5-HIAA induced by morphine is not restricted to the dorsal horn. The three main functional regions of the cord—dorsal horn (sensory), zona intermedia (autonomic), and ventral horn (somatic motor)—are affected to the same degree. The results indicate that morphine causes a generalized activation of serotonin neurons in the spinal cord. There appears to be little or no selectivity for those serotonergic neurons that innervate the dorsal horn. The results are discussed with reference to current data which indicate a fairly strong link between descending serotonergic nerves and the mechanism of action of morphine-induced analgesia.     

Possible use of an analgesic (fentanyl) during electrophysiological investigations of the cerebellar cortex

The effect of the central analgesic fentanyl on evoked potentials and responses of single Purkinje cells in the cerebellar cortex to stimulation of the sensomotor cortex and of somatic nerves was investigated in unanesthetized cats. Injection of fentanyl in an analgesic dose (10–30 µg/kg, intravenously) had no appreciable effect on evoked potentials and led only to very slight changes in spontaneous activity and responses of Purkinje cells. As a rule fentanyl, in a dose of 30 µg/kg, depressed, but in a dose of 10 µg/kg, facilitated responses and spontaneous activity of Purkinje cells. This effect of fentanyl is negligible compared with the action of barbiturate anesthetics on responses of cerebellar cortical neurons. Consequently, fentanyl can be used for analgesia during electrophysiological investigations of the cerebellar cortex in unanesthetized cats.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 585–592, November–December, 1979.     

Inhibitory effect of dopamine on the transmission of excitation in isolated rat spinal cord

The effects of dopamine on ventral root potential produced by a single supramaximal dorsal root stimulation of the dorsal root was investigated during experiments on isolated superfused spinal cord segments from 10–16-day old rats. A reciprocal dose-dependent inhibition of the mono- and polysynaptic components of reflex response was also observed. Minimum effective concentration was 1×10^–8 M dopamine. Extent of reflex response increased in step with dopamine concentration, so that the amplitude of the monosynaptic component of ventral root potential was decreased by 20% and 87% of baseline level by the action of 10^–4 and 10^–3 M dopamine respectively on the cord. The amplitude of the polysynaptic component was thereby decreased by an average of 18% and 87%. Findings indicate that dopaminergic brainstem-spinal pathways contribute to the governing of impulse transmission in the segmental reflex arcs. Inhibition of dopaminergic synaptic transmission probably underlies the increase in latency already described in the literature, as well as the increase observed in the threshold of reflex motor response to nociceptive action following either stimulation of the dopaminergic brainstem structures or intravenous administration of dopamine agonists.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 616–621, September–October, 1986.     

Inhibitory effect of calcium-binding protein regucalcin on Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca²⁺/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca²⁺ (50 µM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 µM); the inhibitory effect was complete at 1.0 µM. Regucalcin (1.0 µM) did not have an appreciable effect on basal activity without Ca²⁺ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160–480 U/ml). However, regucalcin (1.0 µM) did not inhibit Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 µM Ca²⁺ added. Meanwhile, Cd² (25–100 µM)-induced decrease in Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 µM). The present results suggest that regucalcin can regulate Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca²⁺ in liver cells.