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.     

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.     

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.     

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 hypoxia on the respiratory activity generated by semi-isolated medullo-spinal preparations of early postnatal rats

The effects of short- and long-lasting (2-min-long and up to 30-min-long) hypoxia episodes on the inspiratory activity (IA) recorded from then. phrenicus were tested in experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of newborn (the lst day of life) and 4- to 5-day-old rats. Hypoxia was provided by superfusion of the preparations with low-O₂ solution. Short-lasting hypoxia evoked no significant modulation of the IA in preparations of newborn animals, while two-phase responses (an initial, up to 30 sec, increase in the frequency of inspiratory discharges, followed by their longer, up to 4 min, suppression) were observed in 4- to 5-day-old preparations. Long-lasting hypoxia suppressed activity in then. phrenicus of 1-day-old preparations, and this effect was replaced in five cases by the development of sporadic low-amplitude and short-lasting inspiratory discharges. These shortened discharges were qualified as gasps. The responses of 4- to 5-day-old SIMSP to long-lasting hypoxia episodes were more complex. An initial increase in the IA frequency lasted up to 30 sec, and in 4–6 min it was followed by complete suppression of the activity. In some of the SIMSP, permanent tonic activity appeared in then. phrenicus within the period of total absence of inspiratory discharges, which could be followed by generation of short low-amplitude gasping discharges. Against the background of gasping pattern, eupnoe-like discharges appeared in four preparations. Under control conditions, transerve section of the ventrolateral medullary regions (VLMR) at a middle level of then. hypoglossus root abolished respiratory activity in all studied SIMSP. Yet, in some of the SIMSP of both newborn and 4- to 5-day-old animals long-lasting hypoxia testing evoked weak tonic activity in then. phrenicus followed by the appearance of gasping discharges. After the transection of the VLMR at the caudal edge of then. hypoglossus root, long-lasting hypoxia evoked only weak tonic responses in some SIMSP of both age groups, and there were no phasic discharges in this case. The results of our experiments, first, show that the respiratory activity in newborn animals is more resistant to hypoxia than that in 4-to 5-day-old rats, and, second, they allow us to suppose that the gasp-generating medullary structures are localized in more caudal medullary regions. We discuss the questions of how the eupnoe-generating and gasp-generating medullary structures are formed in rats during their initial four to five postnatal days, and what specific features are typical of hypoxia-related respiratory responses in these animals.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 121–131, March–June, 1996.     

Parameters of discharges of respiratory neurons of the ventrolateral medullary regions in early postnatal rats

In experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of newborn (1st day of life) and 4- to 5-day-old rats, we studied the parameters of extracellularly recorded spike activity of respiratory neurons of the ventrolateral medullary regions (VLMR). In SIMSP of 4- to 5-day-old rats, the frequency of discharges of pre-inspiratory, inspiratory, and expiratory neurons is shown to be significantly higher, while the dispersion of its values is considerably lower, as compared with the corresponding values for newborn animals. In the majority of pre-inspiratory and inspiratory neurons of SIMSP of newborn rats, irregular low-frequency discharges are usually generated within the interinspiration phase. The relative intensity of suppression of discharges of pre-inspiratory and expiratory neurons within an inspiration phase is much lower in SIMSP of newborn rats, as compared with that in 4- to 5-day-old preparations. The activity of most pre-inspiratory neurons manifests a trend toward transformation from a two-phase pattern in newborn rats (with two frequency peaks, pre- and post-inspiratory) to a monophasic pattern (with one pre-inspiratory frequency peak) typical of 4- to 5-day-old animals. The effects of electrical stimulation of the site of localization of pre-inspiratory neurons showed that in SIMSP of both age groups of rats an inspiratory response could be evoked in then. phrenicus only in the case when stimulation was applied within the second half of an interinspiratory phase. Therefore, it can be supposed that the respiratory network in newborn animals is to a considerable extent immature in the morphofunctional aspect. It seems probable that in early postnatal rats pre-inspiratory neurons are involved in the medullary mechanisms foron-off switching of the inspiratory and expiratory phases.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 207–217, July–October, 1996.     

Role of Glutamate in the Mechanisms of Adaptation of the System of Respiratory Control in Rats to Intermittent Hypoxia

In experiments on Wistar rats, we studied the role of changes in the state of glutamatergic transmission in the course of adaptation of the system of respiratory control to intermittent hypoxia. The volume/temporal parameters of respiration were estimated according to characteristics of EMG activity (amplitude, integral intensity of EMG discharges) recorded from the diaphragmatic muscle. Changes in EMG activity of the diaphragm induced by acute hypoxia (breathing a 12% О₂-containing gas mixture) were estimated before and after of a 14-day-long course of intermittent hypoxia trainings and before and after inductions of a blocker of NMDA receptors, МK-801. The results prove that the glutamatergic transmitter system is significantly involved in the reaction of the respiratory system to presentation of a hypoxic stimulus within all stages of formation of the ventilatory response, both before and after the action of intermittent hypoxia. Blocking of NMDA receptors under conditions of adaptation to intermittent hypoxia exerted a more intense influence on the amplitude of respiratory EMG discharges of the diaphragm than on their frequency.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

Formation and maintenance of ventilatory long-term facilitation require NMDA but not non-NMDA receptors in awake rats

N-methyl-d-aspartate (NMDA) receptor antagonism in the phrenic motonucleus area eliminates phrenic long-term facilitation (pLTF; a persistent augmentation of phrenic nerve activity after episodic hypoxia) in anesthetized rats. However, whether NMDA antagonism can eliminate ventilatory LTF (vLTF) in awake rats is unclear. The role of non-NMDA receptors in LTF is also unknown. Serotonin receptor antagonism before, but not after, episodic hypoxia eliminates pLTF, suggesting that serotonin receptors are required for induction, but not maintenance, of pLTF. However, because NMDA and non-NMDA ionotropic glutamate receptors are directly involved in mediating the inspiratory drive to phrenic, hypoglossal, and intercostal motoneurons, we hypothesized that these receptors are required for both formation and maintenance of vLTF. vLTF, induced by five episodes of 5-min poikilocapnic hypoxia (10% O(2)) with 5-min normoxia intervals, was measured with plethysmography in conscious adult male Sprague-Dawley rats. Either (+/-)-2-amino-5-phosphonovaleric acid (APV; NMDA antagonist, 1.5 mg/kg) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; non-NMDA antagonist, 10 mg/kg) was systemically (ip) injected approximately 30 min before hypoxia. APV was also injected immediately after or 20 min after episodic hypoxia in additional groups. As control, vehicle was similarly injected in each rat 1-2 days before. Regardless of being injected before or after episodic hypoxia, vehicle did not alter vLTF ( approximately 23%), whereas APV eliminated vLTF while having little effect on baseline ventilation or hypoxic ventilatory response. In contrast, CNQX enhanced vLTF ( approximately 34%) while decreasing baseline ventilation. Collectively, these results suggest that activation of NMDA but not non-NMDA receptors is necessary for formation and maintenance of vLTF in awake rats.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.     

Effects of MK-801 and CNQX on various neurotoxic responses induced by kainic acid in mice

Effects of MK-801 (a NMDA receptor blocker) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker) on several neurotoxic responses induced by kainic acid (KA) were examined in ICR mice. In a lethality test, intracerebroventricular (i.c.v.) pretreatment of MK-801 (1 microg), but not CNQX (0.5 microg), attenuated the time to lethality induced by KA (0.5 microg) administered i.c.v. In the memory test (a passive avoidance test), MK-801, but not CNQX, prevented the memory loss induced by KA (0.1 microg). The damage induced by KA (0.1 microg) administered i.c.v. in the hippocampus was markedly concentrated in the CA3 pyramidal neurons. Both MK-801 and CNQX blocked the pyramidal cell death in CA3 hippocampal region induced by KA. In the immunocytochemical study, KA dramatically increased the phosphorylated ERK (p-ERK) and decreased the phosphorylated CREB (p-CREB) in the hippocmapus. Both MK-801 and CNQX attenuated, in part, the increased p-ERK and the decreased p-CREB induced by KA. In addition, both MK-801 and CNQX partially reduced the increased c-Fos and c-Jun protein expression in hippocampus induced by KA. Our results suggest that both NMDA and non-NMDA receptors are involved in supraspinally administered KA-induced pyramidal cell death in CA3 region of hippocampus in the mouse and the p-ERK and the dephosphorylation of CREB protein may play an important role in CA3 region cell death of the hippocampus induced by KA administered supraspinally. Furthermore, c-Fos and c-Jun proteins may serve as third messengers responsible for CA3 pyramidal cell death induced by supraspinally administered KA.     

Excitatory synaptic currents in Purkinje cells

The N-methyl-D-aspartate (NMDA) and non-NMDA classes of glutamate receptor combine in many regions of the central nervous system to form a dual-component excitatory postsynaptic current. Non-NMDA receptors mediate synaptic transmission at the resting potential, whereas NMDA receptors contribute during periods of postsynaptic depolarization and play a role in the generation of long-term synaptic potentiation. To investigate the receptor types underlying excitatory synaptic transmission in the cerebellum, we have recorded excitatory postsynaptic currents (EPSCS), by using whole-cell techniques, from Purkinje cells in adult rat cerebellar slices. Stimulation in the white matter or granule-cell layer resulted in an all-or-none synaptic current as a result of climbing-fibre activation. Stimulation in the molecular layer caused a graded synaptic current, as expected for activation of parallel fibres. When the parallel fibres were stimulated twice at an interval of 40 ms, the second EPSC was facilitated; similar paired-pulse stimulation of the climbing fibre resulted in a depression of the second EPSC. Both parallel-fibre and climbing-fibre responses exhibited linear current-voltage relations. At a holding potential of -40 mV or in the nominal absence of Mg2+ these synaptic responses were unaffected by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). NMDA applied to the bath failed to evoke an inward current, whereas aspartate or glutamate induced a substantial current; this current was, however, largely reduced by CNQX, indicating that non-NMDA receptors mediate this response. These results indicate that both types of excitatory input to adult Purkinje cells are mediated exclusively by glutamate receptors of the non-NMDA type, and that these cells entirely lack NMDA receptors.     

Direct Evidence That Excitotoxicity in Cultured Neurons Is Mediated via N-Methyl-D-Aspartate (NMDA) as well as Non-NMDA Receptors

Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L-glutamate, kainate (KA), N-methyl-D-aspartate (NMDA), or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L-aspartic acid beta-hydroxamate was added at 500 microM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate-induced cytotoxicity by blocking only one of the glutamate receptor subtypes with either the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) or with one of the specific non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non-NMDA glutamate agonists with no effect on NMDA-induced cell death. Likewise, APV prevented NMDA-induced cell death without affecting the KA- or AMPA-induced neurotoxicity. It is concluded that EAA-dependent neurotoxicity is induced by NMDA as well as non-NMDA receptors.     

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 modulation of cholinergic transmission of activity of the medullary respiratory generator of newborn rats:in vitro study

Periodic phasic activity (respiratory discharges) was recorded from the spinal ventral rootsC ₄−C ₅ in the experiments on isolated medullo-spinal preparations of 1- to 3-day-old rats. Applications of acetylcholine (ACh) in different concentrations and modifications of pH were tested under normal conditions and after treatment with cholinoblockers, atropine and benzohexonium, and an acetylcholinesterase blocker, physostigmine. ACh intensified periodic respiratory activity in a concentration-dependent manner, and the duration of postactivatory depression showed similar dependence. ACh-induced effects were facilitated after pH of the superfusing solution had been decreased. Atropine decreased the frequency of respiratory discharges and suppressed responses to ACh applications and pH modification. Contrastingly, benzohexonium and, especially, physostigmine in concentrations, exerting no significant independent influence on the frequency of respiratory activity, increased sensitivity of preparations and facilitated responses to ACh applications and pH decrease. Involvement of cholinergic neuronal mechanisms, localized in the chemosensitive region of the ventral surface of the medulla, in generation of periodic activity by the medullary respiratory generator is 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.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

A glutamate receptor channel with high affinity for domoate and kainate.

The non-NMDA family of glutamate receptors comprises a growing number of structurally related subunits (GluR-A to -D or -1 to -4; GluR-5, -6; KA-1). GluR-A to -D appear to constitute the major AMPA receptor subtypes but the functional and pharmacological characteristics of the other subunits are unresolved. Using a mammalian expression system we demonstrate here that homomeric GluR-5 receptors exhibit properties of a high affinity domoate (KD approximately 2 nM) and kainate (KD approximately 70 nM) binding site. For these receptors, the rank order of ligands competing with [3H]kainate binding was domoate much greater than quisqualate approximately glutamate much greater than AMPA approximately CNQX. The respective receptor channels were gated in decreasing order of sensitivity by domoate, kainate, glutamate and AMPA. In contrast to recombinantly expressed GluR-A to -D channels, currents elicited at GluR-5 receptor desensitize channels to all agonists. This property is characteristic of currents in peripheral neurons on sensory ganglia. These findings suggest the existence of at least two distinct types of non-NMDA receptor channels, both gated by AMPA and kainate, but differing in pharmacology and current properties.     

羟基马桑毒素所致的大鼠海马锥体细胞痫样放电活动

本研究采用离体海马脑片电生理研究技术,细胞外记录海马锥体细胞群体锋电位(population spike,PS),观察羟基马桑毒素(tutin)对大鼠海马脑片CA1区锥体细胞电活动的影响,探讨tutin是否具有致痛作用及其致痫机制。结果如下:(1)用40、30和20μg/ml浓度的tutin灌流海马脑片,可显著增高由顺向刺激Schaffer侧支所诱发的PS的幅度,灌流tutin 30min时,PS第一个波的幅度分别为对照的(388.7±20.1)%、(317.2±19.1)%和(180.9±11.6)%(各组n=5,P<0.05)。(2)伴随PS波幅的增高,可出现成串痫样放电波,波数4～11个不等。(3)灌流tutin后的部分脑片(n=9/34),在未刺激Schaffer侧支时也出现自发的成串、高幅痫样放电。(4)灌流CNQX阻断非NMDA受体后,再灌流tutin,PS幅度和放电波数均无显著性变化,即CNQX可完全抑制tutin所致的痫样放电;灌流AP-5阻断NMDA受体后,tutin仍可使PS幅度增高但放电波数无显著性增加,即AP-5可部分抑制tutin所致的痫样放电。上述结果表明,tutin可使海马脑片锥体细胞兴奋活动增强,具有致痫作用;兴奋性谷氨酸受体尤其是非NMDA受体可能介导tutin的致痫作用。