Effect of nitric oxide on the respiratory activity generated under hypoxic conditions by medullo-spinal preparations from early postnatal rats

Experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats were carried out to study the effects of a blocker of nitric oxide synthase (NO synthase), methyl ester of N^G-nitro-L-arginine (MENA), and an exogenic NO donor, nitroglycerin, on the respiratory activity. Inspiratory discharges (ID) were recorded from the phrenic nerve under superfusion of SIMSP with a standard saline and a solution saturated with anoxic isocapnic gas mixture. Under normal conditions, 3-min-long applications of 1.0 μM MENA evoked no significant changes in the parameters of inspiratory activity; yet 10.0 μM of this blocker evoked a significant drop in the amplitude and an increase in the ID frequency. Three-min-long applications of 1.0 μM nitroglycerin significantly decreased the ID frequency and somewhat increased their amplitude and integral intensity. Higher doses of nitroglycerin (10.0 μM) significantly increased the amplitude and integral intensity of ID and in a lesser extent lowered their frequency. Under conditions of 3-min-long hypoxia, 10-min-long preliminary superfusion of SIMSP with the 1.0 μM MENA-containing saline resulted in no significant changes of respiratory activity, as compared with the hypoxia effect in the norm. Applied before the hypoxic test, 10 μM MENA resulted in significant decreases in the amplitude and integral intensity of ID; concurrently their frequency became higher, as compared with the respective parameters measured at hypoxic testing of the intact preparations. Ten-min-long superfusion with 1.0 μM nitroglycerin-containing solution at subsequent hypoxic testing significantly increased the amplitude and integral intensity of ID and decreased their frequency; these shifts developed during the first half of exposure to the hypoxic solution. Increased (to 10 μM) nitroglycerin concentration resulted in less intensive shifts in the ID frequency within the first half of a hypoxic episode. In a part of the tests, the second half of exposure of SIMSP to the hypoxic solution was characterized by the appearance of low-amplitude short ID against the background of suppressed eupnea-like respiratory activity; we qualified such discharges as gasping discharges. The experimental data confirm the involvement, of NO in the central regulation of the frequency and amplitude parameters of inspiratory activity generated by SIMSP of early postnatal rats both under normoxic and hypoxic conditions. The role NO plays under hypoxic conditions in modifications of parameters of respiratory activity and in modulation of the functional, levels of the bulbar respiratory generator is discussed.     

Involvement of nitric oxide in realization of the NMDA influence on the respiratory rhythm generated by medullo-spinal preparations of early postnatal rats

The effects of blocking of NO synthase on the dynamics of NMDA-induced changes in the respiratory activity were studied in experiments on semi-isolatedin situ medullo-spinal preparations of 3- to 4-day-old rats. The experiments were carried out with the ventrolateral regions of the medulla (VLMR) left intact or when the rostral portion of this region (corresponding to the chemosensitive zoneM) had been separated by a transversial transection. Three-min-long application of 5.0 μM NMDA increased the frequency of inspiratory discharges (ID); the shifts were more intensive after separation of the VLM rostral portion. Superfusion of preparations with a solution containing 10.0 μM of an NO synthase inhibitor, methyl ester of N^G-nitro-L-arginine (MENA), increased the frequency and decreased the amplitude and integral intensity of ID generated by the preparations with the intact VLM, whereas after separation of the rostral VLM portion this inhibitor decreased the ID frequency. Application of 5 μM NMDA against the background of 10-min-long superfusion with the 10 μM MENA-containing solution resulted in no significant increase in the ID frequency. After the rostral VLM portion had been removed, NMDA application after superfusion with the MENA-containing solution led to frequency shifts which did not significantly differ from those in the absence of the blockade of NO synthase. Against the MENA influence, NMDA-induced depression of the ID amplitude became significantly more intensive. The experiments showed that during the early postnatal period endogenous NO is involved in realization of the NMDA influence on the parameters of respiratory activity of rats. Possible mechanisms of the influences exerted by activation of NMDA receptors and by the associated NO synthesis on regulation of the respiratory rhythmogenesis and their specificity within the early postnatal period are discussed.     

Impact of NMDA Receptors and Nitric Oxide on pH Sensitivity of Medullary Mechanisms Controlling Respiratory Rhythm

We studied the dynamics of modifications of the respiratory activity generated by semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats related to a drop in the pH of superfusing solution from 7.4 to 7.0. Reactions were recorded in the norm and under conditions of preliminary applications of a noncompetitive blocker of NMDA receptors, ketamine; an inhibitor of nitric oxide synthase (NOS), N^G-nitro-L-arginine methyl ester (L-NAME); a substrate for NO synthesis, L-arginine; or an exogenous NO donor, sodium nitroprusside (SN). Under control conditions, test applications of the solution with pH 7.0 resulted in a significant increase in the frequency of inspiratory discharges (ID) recorded from the phrenic nerve and drops in their amplitude and integral intensity. Such SIMSP extracellular acidification-induced responses were inhibited in a dose-dependent manner by ketamine and L-NAME (the effect of the latter was more intensive). The effects of agents increasing the NO level in the tissues were not uniform: L-arginine potentiated an increase in the ID frequency related to application of the acidified solution, while SN inhibited such a reaction. Our findings allow us to suppose that the stimulating influences of the pH-sensitive chemoreceptor structures of the ventrolateral medulla (VLM) on the activity of the medullary respiratory generator of early postnatal rats are realized with the involvement of NMDA receptors of excitatory amino acids and the process of enzyme-mediated NO production. It seems probable that endogenous synthesis of NO in VLM structures mediates and potentiates the effect of activation of the NMDA receptors on the medullary generator of the respiratory rhythm.     

Involvement of NMDA receptors in the control of respiratory rhythm generated by medullo-spinal preparations of early postnatal rats

Effects of a non-competetive blocker of glutamate NMDA receptors, ketamine, on respiratory activity recorded from the phrenic nerve were studied in experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3− to 4-day-old rats. The experiments were carried out under conditions where the ventrolateral medullary region (VLM) was left intact, or its rostral portion (projectionally corresponding to the chemosensitiveM zone) was separated by transection. Three-min-long application of 1.0 μM ketamine evoked a slight increase in the duration of inspiratory discharges (ID) and a statistically significant increase in their frequency. After the rostral VLM had been separated, similar ketamine application resulted in significant increases in the duration, amplitude, and integral intensity of ID and some drop in their frequency. An increase to 10 μM ketamine concentration in the superfusing solution determined a significant rise of the ID duration, which indicates the possibility of inhibition of the mechanisms switching inspiration to expiration. Concurrently, the ID frequency significantly dropped, while their amplitude and integral intensity increased. After separation of the rostral VLM, the latter ketamine concentration ceased to increase the ID duration, and their frequency and amplitude significantly dropped. Application of ketamine in the concentration of 100 μM resulted in rather profound decreases of all measured ID parameters, and separation of the rostral VLM exerted no influence on the direction of the above modifications. Thus, we obtained evidence of the involvement of NMDA receptors of the VLM in the control of temporal and frequency-amplitude parameters of respiratory activity of early postnatal rats. Possible localization of NMDA receptors and mechanisms of their involvement in inspiration-expiration switching and tonic inhibitory control of respiratory rhythms are discussed.     

Blocking of glutamate ionotropic receptors: Influence on the respiratory activity generated by medullo-spinal preparations of rats in hypoxia

We studied the influences of a non-competitive blocker of glutamate NMDA-receptors ketamune and of a competitive blocker of AMPA-kainate non-NMDA receptors, CNQX, on the respiratory activity generelated by superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats. We compared the ampes recorded under conditions of superfusion, a standard solution and the solution saturated with an anoxic isocapine gas mixture were compared; pO₂ in these solutions were 440±22 and 41±8 mm Hg, respectively. The experments were carried out with the ventrolateral medullary region (VLMR) left intact or after separation of its rostral part, which propertchonally corresponded to the chemosensitiveM zone. A 3-min-long hypoxic test initially evoked an increase in the frequency of inspiratory discharges (IR) in the phrenic nerve followed by a frequency drop within the final half of the test. After the rostral VLMR had been separated, the hypoxic test did not elicit a significant decrease in the IR frequency. After preliminary application of 1.0 or 10.0 μM ketamine or CNQX on intact preparations, the IR frequency under hypoxic conditions dropped within the first half of the test and increased in the second half, while the amplitude and integral intensity of these discharges were depressed more intensively than in hypoxia with no applications. Using ketamme and CNQX in the same concentrations resulted in significant drops in the amplitude, frequency, and integral intensity of IR recorde din the hypoxic test. Our experiments showed that in the early postnatal period glutamate ionotropic receptors of rostral VLMR neurons are involved in the control of IR frequency under hypoxic conditions. The possible role of glutamatergic control of the respiratory rhythm and mechanisms of the influences resulting from blocking of NMDA and non-NMDA receptors on the parameters of respiratory activity are discussed.     

Neurochemical mechanisms responsible for depotentiation of synaptic transmission

It was established in experiments on murine hippocampal slices that low-frequency (1 sec⁻¹, 15 min) stimulation of the Schaffer collaterals applied 45 to 60 min after their high-frequency repetitive stimulation (60 sec⁻¹, 0.5 sec) results, in 2/3 of the slices, in reduction of the amplitude of population EPSP recorded from pyramidal neurons of theCA1 area, almost to its level before high-frequency stimulation. Depotentiation was practically completely prevented by application of a non-competitive blocker of NMDA glutamate receptors (GR), ketamine (100 μM), was weakened by a blocker of voltage-dependent L-type Ca²⁺ channels, nifedipine (10 μM), and remained significant after a competitive blocker of the AMPA/kainate receptors, CNQX (10 μM), had been applied to the slices. Depotentiation was significantly reduced by 10 μM of a calmodulin inhibitor, trifluoroperazine, by an increase in the intracellular cAMP concentration caused by activation of A2-adenosine receptors and D5-dopamine receptors, but was resistant to the action of 50 μM of a protein kinase C (PKC) inhibitor, polymixin B. Nootropic compounds possessing anti-amnestic activity enhanced the depotentiation. It is suggested that depotentiation is due to an increase in the intracellular Ca²⁺ concentration, activation of protein phosphatases, and dephosphorylation of pre- and post-synaptic substrates involved in the expression of long-term post-tetanic potentiation of synaptic transmission, which result from cooperative activation of NMDA GR and metabotropic GR.     

Effects of Chronic Introduction of Antidepressants on NMDA-Induced Damage to Neurons of the Rat Hippocampus and Cerebral Cortex

In in vitro studies on superfused slices obtained from the rat hippocampus and cortex, we found that 50 μM N-methyl-D-aspartate (NMDA) applied to the slices in the presence of 10 μM glycine for 15 min exerts a significant damaging action to neurons of these structures. One hour after termination of the action of NMDA, this was manifested in more than a twofold decrease in the synaptic reactivity of pyramidal neurons of the hippocampal СА1 area and layers II/III of the cerebral cortex. The excitotoxic effect of NMDA was prevented by application of competitive (D-2-amino-5-phosphonovaleric acid, 50 μM) and noncompetitive (ketamine, 100 μM) blockers of NMDA receptors. A blocker of glycine-binding sites of NMDA receptors (compound ТСВ 24.15, 10 μM) weakened NMDA-induced damage to the neurons. A competitive blocker of glutamate АМРА receptors, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μM), and a local anesthetic, lidocaine hydrochloride (50 μM), did not modify the excitotoxic effect of NMDA. A blocker of voltagedependent L-type calcium channels, verapamil (20 μM), demonstrated some trend to intensification of NMDA excitotoxic action. An inhibitor of tyrosine-protein phosphatases, sodium vanadate, when i.p. injected into rats in a dose of 15 mg/kg 6 h prior to the electrophysiological experiment, decreased the damaging action of NMDA. Two-hour-long treatment of cerebral slices with 1 μM genistein, an inhibitor of tyrosine kinases, weakened the neuroprotective effect of sodium vanadate. Chronic injections (14 days in daily doses of 20 mg/kg) of antidepressants belonging to different functional classes (imipramine, fluoxetine, and pyrazidol) into rats decreased (similarly to blockers of NMDA receptors) the excitotoxic action of NMDA receptors. Neuroprotective effects of antidepressants were weakened upon the action of genistein. We conclude that the neuroprotective activity of antidepressants under conditions of excitotoxic action of NMDA is mainly determined by an increase in the activity of tyrosine kinases in the cytoplasm and/or neuronal nucleus.     

Effects of low-frequency tetanic stimulation of the synaptic inputs on different forms of long-term potentiation in the rat hippocampus

In experiments on transversal slices of the dorsal hippocampus of rats, we found that low-frequency stimulation of the mossy fibers (MF) against the background of pre-settled long-term post-tetanic potentiation in the MF-CA3 pyramidal neuron (PN) dendrites synaptic system evoked depotentiation in all studied slices. Depotentiation was considerably decreased by a non-competitive blocker of the NMDA glutamate receptors, ketamine (100 μM), as well as by an inhibitor of calmodulin, trifluoroperazine (10 μM), and an inhibitor of calcineurin, cyclosporin A (250 μM). At the same time, depontentiation was not changed by 50 μM polymixin B, an inhibitor of protein kinase C. Long-term potentiation of synaptic transmission in the Schaffer collaterals (SchC)-CA1 PN dendrites system, which was evoked by 2.5-min-long anoxia/aglycemia episodes, resulted exclusively from enhancement of the NMDA component of population EPSP, while their AMPA component was not modified, i.e., in this case potentiation was of a postsynaptic nature. Under these conditions, low-frequency stimulation of SchC resulted in a further increase in the intensity of synaptic transmission due to increases in both the NMDA and AMPA components of population EPSP. The above form of potentiation could be suppressed by 100 μM ketamine, 10 μM trifuoroperazine, 250 μM cyclosporin A, or 10 μM N-nitro-L-arginine. Weak (near-threshold) high-frequency stimulation of SchC induced long-lasting potentiation of synaptic transmission due to an isolated increase in the AMPA component of population EPSP, i.e., this potentiation was of a postsynaptic nature. In the latter case, low-frequency SchC stimulation resulted in further facilitation of synaptic transmission. Intensive tetanic high-frequency stimulation of the above fibers induced long-term potentiation of a presynaptic nature, while their low-frequency stimulation depotentiated synaptic transmission.     

Homocysteine Induces Hypophosphorylation of Intermediate Filaments and Reorganization of Actin Cytoskeleton in C6 Glioma Cells

In this study, we investigated the actions of high homocysteine (Hcy) levels (100 and 500 μM) on the cytoskeleton of C6 glioma cells. Results showed that the predominant cytoskeletal response was massive formation of actin-containing filopodia at the cell surface that could be related with Cdc42 activation and increased vinculin immunocontent. In cells treated with 100 μM Hcy, folic acid, trolox, and ascorbic acid, totally prevented filopodia formation, while filopodia induced by 500 μM Hcy were prevented by ascorbic acid and attenuated by folic acid and trolox. Moreover, competitive NMDA ionotropic antagonist DL-AP5 totally prevented the formation of filopodia in both 100 and 500 μM Hcy treated cells, while the metabotropic non-selective group I/II antagonist MCPG prevented the effect of 100 μM Hcy but only slightly attenuated the effect induced by of 500 μM Hcy on actin cytoskeleton. The competitive non-NMDA ionotropic antagonist CNQX was not able to prevent the effects of Hcy on the reorganization of actin cytoskeleton in the two concentrations used. Also, Hcy-induced hypophosphorylation of vimentin and glial fibrillary acidic protein (GFAP) and this effect was prevented by DL-AP5, MCPG, and CNQX. In conclusion, our results show that Hcy target the cytoskeleton of C6 cells probably by excitoxicity and/or oxidative stress mechanisms. Therefore, we could propose that the dynamic restructuring of the actin cytoskeleton of glial cells might contribute to the response to the injury provoked by elevated Hcy levels in brain.     

一氧化氮对呼吸节律性放电的调节作用

实验旨在探讨一氧化氮(nitric oxide,NO)在基本呼吸节律产生和调节中可能的作用。制作新生大鼠离体延髓脑片标本,主要包含面神经后核内侧区,前包钦格复合体、腹侧呼吸组以及背侧呼吸组的一部分。同时保留舌下神经根,用改良Kreb′s液灌流脑片并记录与之相连的舌下神经根呼吸节律性放电(respiratory rhythmical discharge activity,RRDA),在灌流液中分别给予不同浓度的NO供体硝普钠(sodium nitroprusside,SNP),NO合成前体L—精氨酸(L—Arginine,L-Arg)以及神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)特异性抑制剂7-nitro indazole (7-NI),观察其对RRDA的影响。结果显示,nNOS的特异性抑制剂7-NI对吸气时程和放电强度有明显抑制,而NO合成前体L—Arg,以及NO供体SNP对呼吸放电活动没有明显的影响。这提示,在哺乳动物基本呼吸节律的产生和调节中,NO可能对吸气中止和呼吸幅度具有调节作用。     

Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin

NADH-dependent NO scavenging in barley extracts is linked to hemoglobin (Hb) expression and is inhibited by SH-reagents. Barley Hb has a single cysteine residue. To determine whether this cysteine was critical for NO scavenging, barley Hb and a mutated version, in which the single Cys⁷⁹ was replaced by Ser, were over-expressed in Escherichia coli and purified to near homogeneity. The purified proteins exhibited very low NO-scavenging activity (12–14 nmol min⁻¹ mg⁻¹ protein) in the presence of NADH or NADPH. This activity was insensitive to SH-reagents. Addition of an extract from barley roots to either of the purified proteins resulted in high NADH-dependent NO turnover in a reaction that was sensitive to SH-reagents. A protein was purified from barley roots and identified by mass-spectrometry analysis as a cytosolic monodehydroascorbate reductase. It efficiently supported NADH-dependent NO scavenging in the presence of either native or mutated barley Hb. Ascorbate strongly facilitated the rate of metHb reduction. The K _m for Hb was 0.3 μM, for ascorbate 0.6 mM and for NADH 4 μM. The reaction in the presence of monodehydroascorbate reductase was sensitive to SH-reagents with either form of the Hb. We conclude that metHb reduction and NO turnover do not involve direct participation of the Cys⁷⁹ residue of barley Hb. NO scavenging is facilitated by monodehydroascorbate reductase mediating a coupled reaction involving ferric Hb reduction in the presence of ascorbate and NADH.     

Neurochemical mechanisms underlying NMDA-independent long-term post-tetanic potentiation of synaptic transmission

In experiments performed on rat transversial slices of the rat dorsal hippocampus, we found that high-frequency tetanic stimulation of the mossy fibers (MF) and short-term action of 1 μM kainic acid on the slices resulted in long-term potentiation of the population spikes evoked inCA3 pyramidal neurons by single stimuli applied to the MF. The tetanus-and kainate-induced potentiations of synaptic transmission were accompanied by a decrease in the degree of paired facilitation at a 50-msec-long interstimulus interval; they were additive, prevented by 10 μM CNQX, a competitive antagonist of AMPA/kainate receptors, and insensitive to 100 μM ketamine, a noncompetitive antagonist of NMDA-glutamate receptors. Both types of potentiation were enhanced by 10 μM (1S, 3R)-ACPD, an agonist of metabotropic glutamate receptors, as well as by 1 μM pyracetam or 50 μM dichlothiazide, substances weakening AMPA/kainate receptor desensitization. The effects produced by high-frequency tetanic stimulation of the MF and by kainic acid were prevented by 50 μM polymixin B, a protein kinase C blocker, and weakened by 10 μM trifluoroperazine, a calmodulin inhibitor, or 1 μM pirenzepine, an M1 acetylcholine receptor blocking agent. In total, the above data suggest that the tetanus- and kainate-induced potentiations of transmission in the synapses formed by the MF and dendrites ofCA3 pyramidal neurons are due to the combined activation of pre-synaptic high-affinity kainate-preferring receptors, located in the membranes of the MF varicosities, and post-synaptic phosphoinositide metabolism-coupled metabotropic glutamate receptors and 1 and M1 acetylcholine receptors. This activation results in a significant increase in the activity of epsilon-form protein kinase C, phosphorylation of protein substrates involved in vesicular glutamate release from the MF varicosities, and long-term enhancement of presynaptic glutamate release.     

Lung respiratory rhythm and pattern generation in the bullfrog: role of neurokinin-1 and μ-opioid receptors

Location of the lung respiratory rhythm generator (RRG) in the bullfrog brainstem was investigated by examining neurokinin-1 and μ-opioid receptor (NK1R, μOR) colocalization by immunohistochemistry and characterizing the role of these receptors in lung rhythm and episodic pattern generation. NK1R and μOR occurred in brainstems from all developmental stages. In juvenile bullfrogs a distinct area of colocalization was coincident with high-intensity fluorescent labeling of μOR; high-intensity labeling of μOR was not distinctly and consistently localized in tadpole brainstems. NK1R labeling intensity did not change with development. Similarity in colocalization is consistent with similarity in responses to substance P (SP, NK1R agonist) and DAMGO (μOR agonist) when bath applied to bullfrog brainstems of different developmental stages. In early stage tadpoles and juvenile bullfrogs, SP increased and DAMGO decreased lung burst frequency. In juvenile bullfrogs, SP increased lung burst frequency, episode frequency, but decreased number of lung bursts per episode and lung burst duration. In contrast, DAMGO decreased lung burst frequency and burst cycle frequency, episode frequency, and number of lung bursts per episode but increased all other lung burst parameters. Based on these results, we hypothesize that NK1R and μOR colocalization together with a metamorphosis-related increase in μOR intensity marks the location of the lung RRG but not necessarily the lung episodic pattern generator.     

In Vitro Dose-Dependent Inhibition of the Intracellular Spontaneous Calcium Oscillations in Developing Hippocampal Neurons by Ketamine

Spatial and temporal abnormalities in the frequency and amplitude of the cytosolic calcium oscillations can impact the normal physiological functions of neuronal cells. Recent studies have shown that ketamine can affect the growth and development and even induce the apoptotic death of neurons. This study used isolated developing hippocampal neurons as its study subjects to observe the effect of ketamine on the intracellular calcium oscillations in developing hippocampal neurons and to further explore its underlying mechanism using Fluo-4-loaded laser scanning confocal microscopy. Using a semi-quantitative method to analyze the spontaneous calcium oscillatory activities, a typical type of calcium oscillation was observed in developing hippocampal neurons. In addition, the administration of NMDA (N-Methyl-D-aspartate) at a concentration of 100 µM increased the calcium oscillation amplitude. The administration of MK801 at a concentration of 40 µM inhibited the amplitude and frequency of the calcium oscillations. Our results demonstrated that an increase in the ketamine concentration, starting from 30 µM, gradually decreased the neuronal calcium oscillation amplitude. The inhibition of the calcium oscillation frequency by 300 µM ketamine was statistically significant, and the neuronal calcium oscillations were completely eliminated with the administration of 3,000 µM Ketamine. The administration of 100, 300, and 1,000 µM NMDA to the 1 mM ketamine-pretreated hippocampal neurons restored the frequency and amplitude of the calcium oscillations in a dose-dependent manner. In fact, a concentration of 1,000 µM NMDA completely reversed the decrease in the calcium oscillation frequency and amplitude that was induced by 1 mM ketamine. This study revealed that ketamine can inhibit the frequency and amplitude of the calcium oscillations in developing hippocampal neurons though the NMDAR (NMDA receptor) in a dose-dependent manner, which might highlight a possible underlying mechanism of ketamine toxicity on the rat hippocampal neurons during development.     

Early Anoxic Damage to the Hippocampus and Its Modifications Resulting From Chronic Influences of Antidepressants

We examined the actions of 5- or 7.5-min-long episodes of oxygen/glucose deprivation, OGD (temperature, 37°C), on pyramidal neurons of the CA1 hippocampal area and granular neurons of the gyrus dentatus. The respective damage to these neurons was manifested as an irreversible decrease in the amplitude of field EPSPs developing in such neuronal populations. Antagonists of NMDA receptors, D-2-amino-5 phosphonovaleric acid (50 μM), ketamine (50 μM), and compound TSB 24.15 (10 μM), demonstrated neuroprotective activities under these conditions, but only in the case of the 5-min-long exposure to OGD. A blocker of AMPAreceptors, DNQX (10 μM), combined with a local anesthetic, lidocaine hydrochloride (50 μM), induced comparable effects at the 7.5-min-long exposure. A blocker of calcium channels, verapamil (20 μM), exerted no effect on the level of injury of the neurons induced by the OGD influence. An inhibitor of tyrosine phosphoprotein phosphatases, sodium orthovanadate (15 mg/ml), demonstrated protective activities at both exposures, 5 and 7.5 min long. Chronic (during two weeks) preliminary injections of imipramine, fluoxetine, and pyridazol (everyday doses 20 mg/kg) into experimental animals resulted in noticeable weakening of the OGD-induced impairments of hippocapmal slices in the case of both exposures used, 5 and 7.5 min. The neuroprotective effects of chronically introduced antidepressants were augmented under the action of sodium orthovanadate. It is supposed that neuroprotective actions of preliminarily chronically introduced antidepressants with respect to the anoxic damage to hippocampal neurons is determined (at least to a considerable extent) by intensification of expression of neurotrophins. Under the influence of the latter, the functional activity of NMDA receptors decreases, and consequences of OGD-induced increase in the intracellular Ca²⁺ concentration are weakened.     

Glycine and serine as tentative agonists for metabotropic glutamate receptors

In experiments on slices of the rat hippocampus, glycine (Gly) and serine (Ser) in concentrations of 100 μM to I mM were found to reversible increase the amplitudes of population EPSP (pEPSP) in pyramidal neurons of theCA1 hippocampal area, evoked by single electrical stimuli applied to Schaffer collaterals (SchC). This potentiation was not affected by 100 μM of a non-competetive antagonist of NMDA glutamate receptors (GR), ketamine, but was considerably weakened by 500 μM of a competitive antagonist of metabotropic GR (mGR), (±)-4-carboxyphenylglycine (CFG). The effects of Gly and Ser were not observed in the presence of 50 μM of a blocker of protein kinase C (PKC) catalytic subunit, polymixin B, but were not modified by preliminary action on the slices of 10 μM of a calmodulin inhibitor, substance W-7. Gly and Ser also enhanced long-term post-tetanic potentiation (LTPP) of synaptic transmission caused by high-frequency rhythmic stimulation of SchC. Low-frequency (1/sec, 15 min) SchC stimulation abolished the potentiation of synaptic transmission evoked either by high-frequency SchC stimulation or by the actions of Gly and Ser. The data allow us to suggest that Gly and Ser in millimolar concentrations activate mGR, enhance relay functions of the synapses of pyramidal neurons in theCA1 hippocampal region, and facilitate plastic modifications in these synapses.     

Pacemaker properties of respiratory neurons of the ventrolateral medullary regions in early postnatal rats

Spike activity of respiratory neurons of the ventrolateral medullary regions was studied under conditions of blocking of synaptic transmission. The experiments were carried out on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of newborn (1st day of life) and 4- to 5-day-old rats. Part of the pre-inspiratory and (to a somewhat lesser extent) expiratory neurons of newborn rats appeared most resistive to superfusion of preparations with a low-Ca²⁺ (0.2 mM) and Mg²⁺-rich (5.0 mM) solution. Spike activity in some neurons of these groups was preserved up to 40 and 25 min, respectively, after mass inspiratory discharges in then. phrenicus had disappeared. Similar neurons in 4- to 5-day-old SIMSP were less resistive. Inspiratory neurons in animals of both age groups demonstrated no pacemaker properties. Coagulation of the regions where pre-inspiratory neurons are localized (the retrofacial zone) did not evoke irreversible blockade of respiratory rhythm in all SIMSP of 4- to-5-day-old rats and in most SIMSP of newborn animals. At the same time, coagulation of the zone where inspiratory neurons are concentrated (the pre-Bötzinger complex) resulted in the blockade of respiratory rhythm in all SIMSP, with no exceptions.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 273–284, November–December, 1996.     

Different Mechanisms of NMDA-Mediated Protection Against Neuronal Apoptosis: A Stimuli-Dependent Effect

The mechanisms of protective effect of N-methyl-D-aspartate (NMDA) receptor stimulation on apoptosis of neurons at their early stage of development are poorly understood. In the present study, we investigated the effects of NMDA on staurosporine (St)- and low-potassium (LP)-evoked apoptotic cell death in primary cerebellar granule cell (CGC) cultures at 7 days in vitro (DIV). We found that NMDA (200 μM) attenuated the St (0.5 μM)- and LP (5 mM KCl)-induced neuronal cell death in 7 but not 12 DIV CGC as confirmed by LDH release and MTT reduction assays. Moreover, NMDA attenuated St-and LP-evoked DNA fragmentation and cytosolic apoptosis inducing factor (AIF) protein level but not caspase-3 activation induced by both pro-apoptotic factors. Neuroprotective effects of NMDA on St-induced apoptosis in CGC were attenuated by inhibitors of ERK/MAPK-signaling, PD 98059 and U0126 but not by NMDA receptor antagonists, AP-5 (100 μM) and MK-801 (1 μM) or by inhibitors of PI3-K/Akt pathway (LY 294002 and wortmannin). In contrast to staurosporine model of apoptosis, AP-5 and MK-801 but not inhibitors of PI3-K/Akt and MAPK/ERK1/2 prevented the NMDA-mediated neuroprotection in LP-induced apoptosis of CGC. In separate experiments, we observed also the anti-apoptotic action of NMDA on St (0.5 μM)- and salsolinol (250 μM)-evoked cell death in human neuroblastoma SH-SY5Y cells without its influence on caspase-3 activity, induced by these pro-apoptotic factors. These data indicate that neuroprotection evoked by NMDA in CGC strongly depends on used pro-apoptotic agent and could engage NMDA channel function or be connected with the activation of pro-survival MAPK/ERK1/2 pathway. It is also suggested that anti-apoptotic effects of NMDA is connected with inhibition of fragmentation of DNA via caspase-3-independent mechanism.     

Ammonium and nitrate uptake by Laminaria saccharina and Nereocystis luetkeana originating from a salmon sea cage farm

In the laboratory, ammonium and nitrate uptakes were measured for juvenile Laminaria saccharina (L.) Lamour. and Nereocystis luetkeana (Mert.) Post. et Rupr. originating from a salmon sea cage farm in northwestern British Columbia, Canada. The effect of various concentrations of NH4+ and NO3-, which are typical of salmon farming environments, on uptakes values were examined. Both L. saccharina and Nereocystis revealed simultaneous uptake of NH4+ and NO3- when both NH4+ and NO3- were present in the medium. During a 3-h incubation, mean uptake rates of NH4+ and NO3- by L. saccharina ranged from 6.0–8.9 and 4.6–10.6 μmol gdw-1 h-1, respectively, and by Nereocystis, they ranged from 6.6–9.3 μmol gdw-1 h-1 and 6.1–17.0 μmol gdw-1 h-1, respectively. The highest uptake rates (14.8 μmol NH4+ gdw-1 h-1by L. saccharina and 27.2 μmol NO3- gdw-1 h-1 by Nereocystis) occurred at the highest concentration (40 μM NH4+ plus 30 μM NO3-) during a 1 h incubation. Nitrate uptake by both L. saccharina and Nereocystis increased linearly up to the highest nitrate level tested (30 μM), whereas uptake rates of ammonium were stable beyond 10 μM NH4+ to reach approximately 10 and 13 μmol gdw-1 h-1, respectively, for L. saccharina and Nereocystis. Unlike L. saccharina, Nereocystis showed a significant preference for NO3- when more than 20 μM NO3- was present in the medium ( p <0.05). Both L. saccharina and Nereocystis would be suitable for integrated cultivation of salmon/kelp. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla

Functional evidence suggests that nitric oxide (NO) signalling in the rostral ventrolateral medulla (RVLM) is cGMP-dependent and that this pathway is impaired in hypertension. We examined cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, comparing adult (>18 weeks) Wistar–Kyoto (WKY, n = 4) and spontaneously hypertensive rats (SHR, n = 4). Double label immunohistochemistry for cGMP-immunoreactivity (IR) and C1 neurons [as identified by phenylethanolamine N-methyltransferase (PNMT-IR) or tyrosine hydroxylase TH-IR)], or neuronal NO synthase (nNOS) neurones, failed to reveal cGMP-IR neurons in the RVLM of either strain, despite consistent detection of cGMP-IR in the nucleus ambiguus (NA). This was unchanged in the presence of isobutylmethylxanthine (IBMX; 0.5 mM, WKY, n = 4, SHR n  = 2) and in young animals (WKY, 10-weeks, n = 3). Incubation of RVLM-slices (WKY, 10-weeks, n = 9) in DETA-NO (100 μm; 10 min) or NMDA (10 μM; 2 min) did not uncover cGMP-IR. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase (sGC)-IR was found throughout neurones of the RVLM, but did not co-localise with PNMT, TH or nNOS-IR neurons (WKY, 10-weeks, n = 6). Results indicate that within the RVLM, cGMP is not detectable using immunohistochemistry in the basal state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application. Kellysan Powers-Martin and Anna M. Barron contributed equally.