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 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.     

Roles of NMDA receptors and nitric oxide in responses of the medullary respiratory generator of early postnatal rats to hypoxia and acidosis

The dynamics of changes in the frequency of the respiratory activity recorded from the n. phrenicus under conditions of 3-min-long applications of 5 μM N-methyl-D-aspartate (NMDA), an anoxic gas mixture-saturated saline, or an acidified (pH 7.0) solution were studied in the experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats. Test applications were performed on the intact SIMSP or on those preliminarily influenced by the following substances: a non-competitive NMDA receptor blocker, ketamine (10 μM); an inhibitor of NO synthase, methyl ester of N^G-nitro-L-arginine (MENA, l0 μM); hemoglobin, which binds NO (Hb, 0.3 μM); an NO donor, sodium nitroprusside (SNP, 10 μM); or/and a competitive blocker of non-NMDA receptors, CNQX (1.0 μM). Application of NMDA increased the frequency of the respiratory discharges, and the effect was blocked by MENA, Hb, and SNP. Addition of Hb to the SNP-containing solution neutralized the effect of the latter. In hypoxia, ketamine blocked an increase in the respiratory frequency within the initial 90-sec segment of the test and decreased the rhythm suppression within the second test half. MENA increased the respiration discharge frequency throughout the test. CNQX exerted no Influence on the frequency in the initial period and decreased its suppression within the second test half. Preliminary ketamine and MENA applications made smaller the increment of the discharge frequency at application of the solution with pH 7.0; the MENA effect was stronger. In addition, using a histochemical technique, we studied spatial distribution of the neurons containing an NO synthase marker, NADPH-diaphorase (NADPH-d), in frontal sections of the medulla of 4-day-old rats. NADPH-d-positive cells were observed within the limits of the dorsal and ventral respiratory neuronal groups (DRG and VRG, respectively). Their density was the highest in the rostral VRG part (in the region of the lateral paragigantocellular nucleus). Our results show that in early postnatal rats NMDA receptors and endogenous NO are actively involved in the control of respiratory rhythm generated by SIMSP under hypoxic and acidotic conditions. The results of morphohistochemical study can be considered a neuroanatomical support for the active NO role in the control of medullary respiratory rhythm in the early postnatal period.     

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.     

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.     

Respiratory activity generated by semi-isolated medullo-spinal preparation and recorded from the phrenic nerve of newborn rats

Inspiratory activity generated by superfusedin situ semi-isolated medullo-spinal preparations of newborn (one-day-old) and four- to five-day-old rats was recorded from then. phrenicus before and after transverse sectioning of the ventrolateral part of the medulla (VLPM) at different levels. Under similar experimental conditions, the frequency of inspiratory discharges (ID) and their integral intensity, reflecting the volume and temporal parameters of inspiration, are much lower in one-day-old rats, as compared with those in four- to five-day-old animals. Specific roles of different VLPM levels in respiration control in young rats are demonstrated. Transection of the VLPM below the most rostral VLPM portion, corresponding to theM chemosensitive zone, caused a significant increase in the ID frequency and a decrease in the ID integral intensity. Transection performed below the intermediate VLPM region, corresponding to theS chemosensitive zone, resulted in a significant decrease in both ID frequency and ID integral intensity, up to total ID blockade in 5 of 12 1-day-old preparations. This finding can be interpreted as an indication of morphofunctional immaturity of the respiratory network in the caudal VLPM regions in newborn animals. Comparative analysis of ID pattern showed that this activity in one-day-old rats is more or less gasping-like, while that in four- to five-day-old animals is eupnoe-like. The results allow us to conclude that the level of maturity of morphofunctional organization of medullary respiratory networks considerably differs in newborn and older animals. The mechanisms responsible for formation and control of respiratory activity in early postnatal period of rats are discussed.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 387–395, September–December, 1995.     

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 bilobalide on amino acid release and electrophysiology of cortical slices

Summary.  This study investigated the effects of bilobalide, a constituent of Ginkgo biloba, on potassium and veratridine-induced release of glutamate and aspartate from mouse cortical slices. We also studied its effects on spontaneous and N-methyl-D-aspartate (NMDA)-induced depolarizations elicited in magnesium-free artificial cerebrospinal fluid (aCSF) as well as its effect on NO-711 (a γ-aminobutyric acid (GABA) uptake inhibitor)-induced depolarizations. Bilobalide, 100 μM significantly reduced both glutamate and aspartate release elicited by potassium or veratridine. Bilobalide (5–100 μM) also significantly reduced the frequency of NO-711 induced depolarizations, however, it had no effect on spontaneous or on NMDA-induced depolarizations at 5–200 μM. These results suggest that the neuroactive properties of bilobalide may be mediated by a reduction in excitatory amino acid neurotransmitter release. Received June 25, 2001 Accepted October 4, 2001     

Nitric Oxide Synthase Activity Endogenously Modulates NMDA Receptors

Abstract: We tested the possibility that endogenous nitric oxide synthase activity regulated NMDA receptors in primary cultured striatal neurons. We monitored NMDA-induced increase in intra-cellular Ca²⁺ levels with fura-2 ratio imaging, while nitric oxide synthase activity was either increased with l -arginihe (the natural substrate of nitric oxide synthase) or inhibited using nitro- l -arginine (a specific inhibitor of nitric oxide synthase). We found that the NMDA receptor effect was slowly but strongly diminished after an l -arginine (1 m M , 15 min) treatment ( l -arginine preincubation reduced the 100 μM NMDA-induced maximal effect by 30–50%). The l -arginine blockade of NMDA receptors was long-lasting but could be partially reversed by hemoglobin (100 μM , 10 min), which binds nitric oxide. This was not observed when the neurons were treated with l -arginine together with nitro- l -arginine. Our data strongly suggest that physiological nitric oxide synthase activity could regulate NMDA receptors.     

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.     

The nNos/cGMP mediation of the depressor response to NMDA receptor stimulation in the caudal ventrolateral medulla

Using in vivo voltammetry to directly measure extracellular nitric oxide (NO) levels, our previous studies suggested that the neuronal NO synthase (nNOS) and cyclic guanosine monophosphate (cGMP) signal transducing systems are involved in the cardiovascular responses elicited by activation of N-methyl-D-aspartate (NMDA) receptors in the rostral ventrolateral medulla. In this study, we examined if the depressor responses elicited by activation of NMDA receptors in the caudal ventrolateral medulla (CVLM) also depend on the actions of nNOS and soluble guanylyl cyclase. In anesthetized cats, microinjection of NMDA into the CVLM produced hypotension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 2-amino-5-phosphonopentanoate (a NMDA receptor competitive antagonist), 7-nitroindazole (a nNOS inhibitor) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). These findings suggest that NO is also involved in the NMDA-induced depressor responses of the CVLM.     

Participation of the opioid- and serotonergic systems of the ventrolateral medullary regions in the control of respiratory activity in early postnatal rats

The effects of blockers of opioid and serotonin receptors (naloxone and methisergide, respectively) on the respiratory activity recorded from then. phrenicus of semi-isolated medullo-spinal preparations (SIMSP) were studied in newborn (1-day-old) and 4- to 5-day-old rats. The preparations were superfusedin situ, and inspiratory discharges (ID) were recorded before and after transverse sections of the ventrolateral medullary regions (VLMR) at different levels. Naloxone evoked an increase in the ID frequency in then. phrenicus both at the initial configuration of preparations and after successive transections of the VLMR between theM andS chemosensitive zones and between theS andL zones. The relative intensity of this effect was significantly higher in newborn rats than in 4- to 5-day-old ones. In contrast, methisergide made the ID frequency lower at all configurations of the SIMSP, and this effect was more intensive in 4- to 5-day-old animals. In the course of progressive separations of more rostral VLMR zones, the effects of naloxone and methisergide applications on the ID frequency became weaker. This allows us to suppose that in newborn rats the rostral VLMR portions provide more intensive opioidergic inhibitory control influences on the activity of respiratory networks than those in 4- to 5-day-old animals. At the same time, tonic activating influences from serotonergic VLMR neurons on the mechanisms generating respiratory rhythm are more intensive in 4- to 5-day-old animals than those in newborn ones. Thus, it can be supposed that the levels of maturation of the opioid- and serotonergic neurotransmitter systems, which provide tonic control of respiratory activity generated in the VLMR, are different in newborn and 4- to 5-day-old animals. The problems of how the opioid- and serotonergic mechanisms controlling respiratory rhythm generation are formed in the course of early stages of ontogenetic development are discussed.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 62–73, January–February, 1996.     

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.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (I_gaba and I_Mus), respectively in the Mg²⁺-free external solution containing 1 μmol/L glycine at a holding potential (V _H ) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of I_gaba; (ii) when the neurons were incubated in a Ca²⁺-free bath or pre-loaded with a membrane-permeable Ca²⁺ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on I_GAba disappeared. Cd²⁺ (10 μmol/L) or La³⁺ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of I_gaba by NMDA application; (iii) the suppression of I_GAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca²⁺-dependent and CaMKII is involved in the process of the Ca²⁺-dependent inhibition.     

Cerebral Taurine Release Mechanisms In Vivo: Pharmacological Investigations in Rats Using Microdialysis for Proof of Principle

Cerebral taurine acts as neurotransmitter, as neuromodulator, or as osmoregulator. To investigate its release mechanisms in vivo, we combined the microdialysis technique with a variety of experimental paradigms. Taurine release was stimulated by either NMDA, NO or a hypotonic solution locally with or without the addition of the NMDA antagonists APV or Ketamine, or the NO synthase inhibitor L-NAME. Alternatively, the neuroprotective drug lubeluzole was applied i.v. NMDA, NO or the hypotonic solution stimulated the release of taurine. NMDA-mediated taurine release was inhibited by either APV, Ketamine or the NO synthase inhibitor L-NAME. Lubeluzole had no effect. Under the hypotonic conditions only lubeluzole was effective. These data confirm in vivo that the NMDA-induced taurine release is mediated via the NO cascade. By contrast, the release after a hypotonic stimulus is not related to the NO cascade. Instead, Na⁺- and/or Ca²⁺- mediated events might have been attenuated by lubeluzole.     

NADPH-Diaphorase-containing neurons in the medullary structures involved in generation of the respiratory activity in neonatal rats

Using a histochemical technique, we examined distribution of the neurons containing a marker of nitric oxide synthase (NOS), NADPH-diaphorase (NADPH-d), on frontal slices of the medulla and upper cervical spinal segments of 4-day-old rats. It was demonstrated that NADPH-d-positive cells are present within the dorsal and ventral medullary respiratory groups. The highest density of the labeled middle-size multipolar neurons (27.9±2.6 cells per 0.1 mm² of the slice) was observed in the rostral part of the ventral respiratory group, within the reticular lateral paragigantocellular nucleus. Similar NADPH-d-positive neurons were also observed in other reticular formation structures: rostroventrolateral reticular, gigantocellular, and ventral medullary nuclei, and in the ventral part of the paramedial nucleus. There were no labeled neurons in the lateral reticular nucleus. Single small and medium-size labeled neurons were found at all rostro-caudal levels of thenucl. ambiguous (nuclei retrofacialis, ambiguous, andretroam-biguous). Groups of NADPH-d-positive neurons were also revealed within the dorsal respiratory group, along the whole length of thenucl. tractus solitarii (mostly in its ventrolateral parts). Single labeled neurons were also observed in thenucl. n. hypoglossi, and their groups were observed in the dorsal motor part of thenucl. n. vagus. Involvement of the structures containing NADPH-d-positive neurons in the processes related to generation of the respiratory activity is discussed. Our neuroanatomical experiments prove that in early postnatal mammals NO is actively involved in generation and regulation of the medullary respiratory rhythm. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 128–136, March–April, 2000.     

Neuroprotective Potential of Biphalin,Multireceptor Opioid Peptide,Against Excitotoxic Injury in Hippocampal Organotypic Culture

Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. Findings: (1) 0.025–0.1 μM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.     

NGP1-01 is a Brain-permeable Dual Blocker of Neuronal Voltage- and Ligand-operated Calcium Channels

Calcium overload of neurons leads to cell death and is a key feature in neurodegenerative diseases. The polycyclic amine NGP1-01 blocks L-type voltage operated calcium channels in cardiomyocytes. Here, we tested whether NGP1-01 blocks neuronal calcium channels. NGP1-01 (1 μM) inhibited depolarization-induced calcium influx by 78% in cortical neurons preloaded with fura-2 AM, with a potency similar to nimodipine. NGP1-01 (1 μM) also inhibited N-methyl-d-aspartate (NMDA)-induced (1 mM) calcium influx by 52%, only slightly less potent than memantine. Using in vivo-microdialysis, we monitored choline release during NMDA infusion as a measure of excitotoxic membrane breakdown. Intraperitoneal injection of NGP1-01 (40 mg/kg) reduced NMDA-induced membrane breakdown by 31% (P<0.01) while memantine (10 mg/kg) reduced choline release by 40%. Our results demonstrate that NGP1-01 simultaneously blocks both major neuronal calcium channels and is sufficiently brain-permeable. We conclude that NGP1-01 is a promising lead structure for a new class of dual-mechanism neuroprotective agents.     

Delta-9-tetrahydrocannabinol protects cardiac cells from hypoxia via CB2 receptor activation and nitric oxide production

Delta-9-tetrahydrocannabinol (THC), the major active component of marijuana, has a beneficial effect on the cardiovascular system during stress conditions, but the defence mechanism is still unclear. The present study was designed to investigate the central (CB1) and the peripheral (CB2) cannabinoid receptor expression in neonatal cardiomyoctes and possible function in the cardioprotection of THC from hypoxia. Pre-treatment of cardiomyocytes that were grown in vitro with 0.1 – 10 μM THC for 24 h prevented hypoxia-induced lactate dehydrogenase (LDH) leakage and preserved the morphological distribution of α-sarcomeric actin. The antagonist for the CB2 (10 μM), but not CB1 receptor antagonist (10 μM) abolished the protective effect of THC. In agreement with these results using RT-PCR, it was shown that neonatal cardiac cells express CB2, but not CB1 receptors. Involvement of NO in the signal transduction pathway activated by THC through CB2 was examined. It was found that THC induces nitric oxide (NO) production by induction of NO synthase (iNOS) via CB2 receptors. L-NAME (NOS inhibitor, 100 μM) prevented the cardioprotection provided by THC. Taken together, our findings suggest that THC protects cardiac cells against hypoxia via CB2 receptor activation by induction of NO production. An NO mechanism occurs also in the classical pre-conditioning process; therefore, THC probably pre-trains the cardiomyocytes to hypoxic conditions.     

Acute tolerance to ethanol inhibition of NMDA-induced responses in rat rostral ventrolateral medulla neurons

The present study was performed to examine the effects of acute ethanol exposure on N-methyl-D-aspartate (NMDA)-induced responses and the development of acute tolerance in rat rostral ventrolateral medulla (RVLM) in vivo and in vitro. Repeated microinjections of NMDA (0.14 nmol) into the RVLM every 30 min caused reproducible increases in mean arterial pressure in urethane-anesthetized rats weighing 325–350 g. Intravenous injections of ethanol (0.16 or 0.32 g, 1 ml) inhibited NMDA-induced pressor effects in a blood-concentration-dependent and reversible manner. The inhibitory effect of ethanol was reduced over time during continuous infusion of ethanol or on the second injection 3.5 h after prior injection of a higher dose of ethanol (0.32 g). A high dose of ethanol (0.32 g) had no significant effects on-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid,-aminobutyric acid and glycine-induced changes in blood pressure. In vitro studies showed that ethanol (10–100 mM) dose-dependently inhibited inward currents elicited by pressure ejection of NMDA (10 mM) in RVLM neurons of neonatal brainstem slice preparations. When the superfusion time of ethanol (100 mM) was increased to 50 min, its inhibitory effect decreased gradually after 30–40 min in 60% of RVLM neurons examined. These data suggested that ethanol inhibition and subsequent tolerance development is associated with changed sensitivity to NMDA in the RVLM, which may play important roles in the ethanol regulation of cardiovascular function.