6-cyano-7-nitroquinoxaline-2-3-dione and Hypoxia

Both orexin neurons in the lateral hypothalamus and spinally-projecting pre-sympathetic neurons (PSNs) in the paraventricular nucleus of the hypothalamus (PVN) play an important role in the regulation of cardiovascular function under normal conditions and during cardiovascular challenges such as hypoxia and/or hypercapnia. We have previously established, using selective optogenetic excitation of orexin neurons and pathways, there is a heterogeneous neurotransmission from orexin neurons to PSNs in the PVN. This study was undertaken to test whether this pathway is altered by acute exposure to hypoxia alone and/or combined hypoxia and hypercapnia (H/H). To test this hypothesis, we selectively expressed channelrhodopsin-2 (ChR2) in orexin neurons in the lateral hypothalamus and photoactivated ChR2-expressing fibers to evoke postsynaptic currents in spinally-projecting PSNs in an in vitro slice preparation in rats. In accordance with previously published data, two subpopulations of spinally-projecting PSNs were established, including those with glutamatergic or GABAergic inputs from orexin neurons. Hypoxia alone did not alter the peak amplitude of either glutamatergic or GABAergic neurotransmission, however, H/H significantly inhibited both glutamatergic and GABAergic neurotransmission from orexin neurons to SPNs. In conclusion, H/H may modulate cardiovascular function by affecting heterogeneous pathways from orexin neurons to spinally-projecting PSNs in the PVN.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; GABA Antagonists; Green Fluorescent Proteins; Hypercapnia; Hypoxia; In Vitro Techniques; Male; Neurons; Optogenetics; Orexins; Paraventricular Hypothalamic Nucleus; Pyridazines; Rats; Rats, Transgenic; Rhodopsin; Synaptic Transmission; Time Factors

During the perinatal period, the brain is highly vulnerable to hypoxia and inflammation, which often cause white matter injury and long-term neuronal dysfunction such as motor and cognitive deficits or epileptic seizures. We studied the effects of moderate hypoxia (HYPO), mild systemic inflammation (INFL), or the combination of both (HYPO+INFL) in mouse somatosensory cortex induced during the first postnatal week on network activity and compared it to activity in SHAM control animals. By performing in vitro electrophysiological recordings with multi-electrode arrays from slices prepared directly after injury (P8-10), one week after injury (P13-16), or in young adults (P28-30), we investigated how the neocortical network developed following these insults. No significant difference was observed between the four groups in an extracellular solution close to physiological conditions. In extracellular 8mM potassium solution, slices from the HYPO, INFL, and HYPO+INFL group were more excitable than SHAM at P8-10 and P13-16. In these two age groups, the number and frequency of spontaneous epileptiform events were significantly increased compared to SHAM. The frequency of epileptiform events was significantly reduced by the NMDA antagonist D-APV in HYPO, INFL, and HYPO+INFL, but not in SHAM, indicating a contribution of NMDA receptors to this pathophysiological activity. In addition, the AMPA/kainate receptor antagonist CNQX suppressed the remaining epileptiform activity. Electrical stimulation evoked prominent epileptiform activity in slices from HYPO, INFL and HYPO+INFL animals. Stimulation threshold to elicit epileptiform events was lower in these groups than in SHAM. Evoked events spread over larger areas and lasted longer in treated animals than in SHAM. In addition, the evoked epileptiform activity was reduced in the older (P28-30) group indicating that cortical dysfunction induced by hypoxia and inflammation was transient and compensated during early development.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Age Factors; Animals; Bicuculline; Disease Models, Animal; Excitatory Amino Acid Antagonists; Exploratory Behavior; Female; GABA-A Receptor Antagonists; Gene Expression Regulation; Hypoxia; In Vitro Techniques; Inflammation; Male; Mice; Mice, Inbred C57BL; Neurons; Potassium Chloride; Somatosensory Cortex

Protein tyrosine kinase (PTK) mediated the tyrosine phosphorylation modification of neuronal receptors and ion channels. Whether such modification resulted in changes of physiological functions was not sufficiently studied. In this study we examined whether the hypoxic respiratory response-which is the enhancement of breathing in hypoxic environment could be affected by the inhibition of PTK at brainstem ventral respiratory neuron column (VRC). Experiments were performed on urethane anesthetized adult rabbits. Phrenic nerve discharge was recorded as the central respiratory motor output. Hypoxic respiratory response was produced by ventilating the rabbit with 10% O2-balance 90% N2 for 5 minutes. The responses of phrenic nerve discharge to hypoxia were observed before and after microinjecting PTK inhibitor genistein, AMPA receptor antagonist CNQX, or inactive PTK inhibitor analogue daidzein at the region of ambiguus nucleus (NA) at levels 0-2 mm rostral to obex where the inspiratory subgroup of VRC were recorded. Results were as follows: 1. the hypoxic respiratory response was significantly attenuated after microinjection of genistein and/or CNQX, and no additive effect (i.e., further attenuation of hypoxic respiratory response) was observed when genistein and CNQX were microinjected one after another at the same injection site. Microinjection of daidzein had no effect on hypoxic respiratory response. 2. Fluorescent immunostaining showed that hypoxia significantly increased the number of phosphotyrosine immunopositive neurons in areas surrounding NA and most of these neurons were also immunopositive to glutamate AMPA receptor subunit GluR1. These results suggested that PTK played an important role in regulating the hypoxic respiratory response, possibly through the tyrosine phosphorylation modification of glutamate AMPA receptors on the respiratory neurons of ventral respiratory neuron column.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Stem; Female; Hypoxia; Male; Microinjections; Neurons; Phosphotyrosine; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Rabbits; Receptors, AMPA; Respiration

Recent studies have indicated that dynamic alterations in the structure of postsynaptic density (PSD) are involved in the pathogenesis of many central nervous system disorders, including ischemic stroke. Homer is the newly identified scaffolding protein located at PSD and regulates synaptic function. Homer1a, an immediate early gene, has been shown to be induced by several stimulations, such as glutamate, brain-derived neurotrophic factor, and trauma. However, whether acidosis mediated by acid-sensing ion channels (ASICs) and hypoxia during cerebral ischemia can change Homer1a expression remains to be determined.. We investigated that acidosis and hypoxia selectively and rapidly upregulated Homer1a expression, but not Homer1b/c in cultured cortical neurons. We also found that Homer1a exhibited induction expression in brain cortex of the middle cerebral artery occlusion (MCAO) rats. Additionally, acid-evoked Homer1a mRNA induction depended on extracellular signal-regulated kinase1/2 (ERK1/2) and Akt activity, and ASIC1a-mediated calcium influx whereas hypoxia depended only on ERK1/2 activity. Also, we demonstrated that continuous acidosis and hypoxia resulted in pronounced cell injury and Homer1a knockdown with small interfering RNA aggravated this damage induced by 3 h acid and hypoxia incubation in neuro-2a cells.. Homer1a might act as an activity-dependent regulator responding to extracellular stimuli during cerebral ischemia.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; Amiloride; Animals; Carrier Proteins; Cell Survival; Cells, Cultured; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Homer Scaffolding Proteins; Hypoxia; Infarction, Middle Cerebral Artery; Neurons; Peptides; Rats; Rats, Sprague-Dawley; Signal Transduction; Spider Venoms; Time Factors; Up-Regulation

NMDA-type glutamate receptors mediate both trophic and excitotoxic signalling in CNS neurons. We have previously shown that blocking NMDAR- post-synaptic density-95 (PSD95) interactions provides significant protection from excitotoxicity and in vivo ischaemia; however, the mechanism of neuroprotection is unclear. Here, we report that blocking PSD-95 interactions with the Tat-NR2B9c peptide enhances a Ca²⁺-dependent protective pathway converging on cAMP Response Element binding protein (CREB) activation. We provide evidence that Tat-NR2B9c neuroprotection from oxygen glucose deprivation and NMDA toxicity occurs in parallel with the activation of calmodulin kinase signalling and is dependent on a sustained phosphorylation of the CREB transcription factor and its activator CaMKIV. Tat-NR2B9c-dependent neuroprotection and CREB phosphorylation are blocked by coapplication of CaM kinase (KN93 and STO-609) or CREB (KG-501) inhibitors, and by siRNA knockdown of CaMKIV. These results are mirrored in vivo in a rat model of permanent focal ischaemia. Tat-NR2B9c application significantly reduces infarct size and causes a selective and sustained elevation in CaMKIV phosphorylation; effects which are blocked by coadministration of KN93. Thus, calcium-dependent nuclear signalling via CaMKIV and CREB is critical for neuroprotection via NMDAR-PSD95 blockade, both in vitro and in vivo. This study highlights the importance of maintaining neuronal function following ischaemic injury. Future stroke research should target neurotrophic and pro-survival signal pathways in the development of novel neuroprotective strategies.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Infarction; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Cells, Cultured; Cerebral Cortex; CREB-Binding Protein; Disease Models, Animal; Disks Large Homolog 4 Protein; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glucose; Hypoxia; In Vitro Techniques; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Neurons; Nimodipine; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors

In response to low ambient oxygen levels the western painted turtle brain undergoes a large depression in metabolic rate which includes a decrease in neuronal action potential frequency. This involves the arrest of N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) currents and paradoxically an increase in γ-aminobutyric acid receptor (GABAR) currents in turtle cortical neurons. In a search for other oxygen-sensitive channels we discovered a Ca(2+)-activated K(+) channel (K(Ca)) that exhibited a decrease in open time in response to anoxia. Single-channel recordings of K(Ca) activity were obtained in cell-attached and excised inside-out patch configurations from neurons in cortical brain sheets bathed in either normoxic or anoxic artificial cerebrospinal fluid (aCSF). The channel has a slope conductance of 223pS, is activated in response to membrane depolarization, and is controlled in a reversible manner by free [Ca(2+)] at the intracellular membrane surface. In the excised patch configuration anoxia had no effect on K(Ca) channel open probability (P(open)); however, in cell-attached mode, there was a reversible fivefold reduction in P(open) (from 0.5 ± 0.05 to 0.1 ± 0.03) in response to 30-min anoxia. The inclusion of the potent protein kinase C (PKC) inhibitor chelerythrine prevented the anoxia-mediated decrease in P(open) while drip application of a phorbol ester PKC activator decreased P(open) during normoxia (from normoxic 0.4 ± 0.05 to phorbol-12-myristate-13-acetate (PMA) 0.1 ± 0.02). Anoxia results in a slight depolarization of turtle pyramidal neurons (∼8 mV) and an increase in cytosolic [Ca(2+)]; therefore, K(Ca) arrest is likely important to prevent Ca(2+) activation during anoxia and to reduce the energetic cost of maintaining ion gradients. We conclude that turtle pyramidal cell Ca(2+)-activated K(+) channels are oxygen-sensitive channels regulated by cytosolic factors and are likely the reptilian analog of the mammalian large conductance Ca(2+)-activated K(+) channels (BK channels).

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Biophysics; Calcium; Cerebral Cortex; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Female; Hypoxia; In Vitro Techniques; Ion Channel Gating; Male; Membrane Potentials; Oxygen; Patch-Clamp Techniques; Phorbol Esters; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Probability; Pyramidal Cells; Sodium Channel Blockers; Tetraethylammonium; Tetrodotoxin; Turtles; Valine

Excessive presynaptic glutamate release after cerebral ischaemia leads to neuronal death mainly through excessive calcium entry of N-methyl-D-aspartate receptors (NMDARs). Our recent study reported that cerebroside can open large-conductance Ca²⁺-activated K⁺ (BKCa) channels. The present study evaluated the effects of cerebroside-A (CS-A), a single molecule isolated from an edible mushroom, on brain injury after focal or global ischaemia in adult male mice and rats. We herein report that treatment with CS-A after 60-min middle cerebral artery occlusion dose-dependently reduced the cerebral infarction with at least a 6-h efficacious time-window, which was partially blocked by the BKCa channel blocker charybdotoxin (CTX). Treatment with CS-A after 20 min global cerebral ischaemia (four-vessel occlusion) significantly attenuated the death of pyramidal cells in hippocampal CA1 area, which was also sensitive to CTX. CS-A, by opening the BKCa channel, could prevent excessive glutamate release after oxygen-glucose deprivation (OGD). In addition, CS-A could inhibit NMDAR Ca²⁺ influx, which did not require the activation of the BKCa channel. Furthermore, CS-A blocked the OGD-induced NMDAR-dependent long-term potentiation in hippocampal CA1 region. These findings indicate that treatment with CS-A after stroke exerts potent neuroprotection through prevention of excessive glutamate release and reduction of Ca²⁺ influx through NMDARs.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Brain Ischemia; Calcium; Cerebral Infarction; Cerebrosides; Charybdotoxin; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glucose; Glutamic Acid; Hippocampus; Hypoxia; In Vitro Techniques; Infarction, Middle Cerebral Artery; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; N-Methylaspartate; Neuroprotective Agents; Neurotoxins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Valine

Cardioinhibitory cardiac vagal neurons (CVNs) do not receive inspiratory-related excitatory inputs under normal conditions. However, excitatory purinergic and serotonergic pathways are recruited during inspiratory activity after episodes of hypoxia and hypercapnia (H/H). Prenatal nicotine (PNN) exposure is known to dramatically change cardiorespiratory responses and decrease the ability to resuscitate from H/H. This study tested whether PNN exposure alters excitatory neurotransmission to CVNs in the nucleus ambiguus during and after H/H. Spontaneous and inspiratory evoked excitatory postsynaptic currents were recorded in CVNs from rats that were exposed to nicotine (6 mg x kg(-1) x d(-1)) throughout the prenatal period. In contrast to unexposed animals, in PNN animals H/H recruited excitatory neurotransmission to CVNs during inspiratory-related activity that was blocked by the alpha3beta4 nicotinic acetylcholine receptor (nAChR) blocker alpha-conotoxin AuIB (alpha-CTX AuIB, 100 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 microM) and d(-)-2-amino-5-phosphonopentanoic acid (AP5, 50 microM), selective AMPA/kainate and N-methyl-d-aspartate receptor blockers, respectively. Following H/H, there was a significant increase in inspiratory-related excitatory postsynaptic currents that were unaltered by alpha-CTX AuIB or ondansetron, a 5-HT3 receptor blocker, but were subsequently inhibited by pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (100 microM), a purinergic receptor blocker and CNQX and AP5. The results from this study demonstrate that with PNN exposure, an excitatory neurotransmission to CVNs is recruited during H/H that is glutamatergic and dependent on activation of alpha3beta4-containing nAChRs. Furthermore, exposure to PNN abolishes a serotonergic long-lasting inspiratory-related excitation of CVNs that is replaced by recruitment of a glutamatergic pathway to CVNs post H/H.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Animals, Newborn; Conotoxins; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agents; Female; Heart; Hypercapnia; Hypoxia; In Vitro Techniques; Male; Neurons; Nicotine; Nicotinic Antagonists; Ondansetron; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Serotonin; Serotonin Antagonists; Synaptic Transmission; Vagus Nerve; Valine

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.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Disease Models, Animal; Excitatory Amino Acid Antagonists; Heart Rate; Hypoxia; Long-Term Potentiation; Male; Motor Neurons; Neural Pathways; Pulmonary Ventilation; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Respiratory Mechanics; Respiratory Muscles; Tidal Volume; Time Factors; Valine; Wakefulness

Inhibitory GABAergic and glycinergic neurotransmission to cardioinhibitory cardiac vagal neurons (CVNs) increase during inspiratory activity and likely mediate respiratory sinus arrhythmia, while the frequency of excitatory postsynaptic currents (EPSCs) in CVNs are unaltered during the different phases of respiration. However, following hypoxia and hypercapnia (H/H), the parasympathetic activity to the heart increases and thus far, identification of the pathways and neurotransmitters that are responsible for exciting CVNs post H/H are unclear. This study identifies different excitatory pathways to CVNs recruited post H/H. Spontaneous and inspiratory-related EPSCs were recorded in CVNs before, during, and after 10 min of H/H in an in vitro slice preparation that retains rhythmic respiratory activity. Before and during H/H, EPSCs in CVNs were completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and d(-)-2-amino-5-phosphonopentanoic acid (AP5), selective AMPA/kainate and N-methyl-d-apartate (NMDA) receptor blockers, respectively. However, after H/H, there was a significant increase in EPSCs during each inspiratory burst. While some of the inspiratory-related EPSCs were blocked by the broad purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and the specific P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate monolithium trisodium salt (TNP-ATP) a P2X receptor blocker, most of the recruited excitatory neurotransmission to CVNs is serotonergic because odansetron, a selective 5-HT3 antagonist, abolished the majority of the spontaneous and inspiratory-related EPSCs evoked during recovery from H/H. The results from this study suggest that following episodes of H/H, two nonglutamatergic excitatory pathways, purinergic and serotonergic, activating P2X and 5-HT3 receptors, respectively, are recruited to excite CVNs in the post H/H recovery period.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Animals; Animals, Newborn; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hypercapnia; Hypoxia; In Vitro Techniques; Neurons; Nucleus Accumbens; Ondansetron; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Respiration; Serotonin; Serotonin Antagonists; Vagus Nerve; Valine

Physiological activity-dependent long-term changes in synaptic transmission, as long-term potentiation (LTP) are thought to be the substrate of learning and memory. However, a form of postsynaptic pathological LTP at the CA3-CA1 synapses has been demonstrated following few minutes of anoxia and aglycemia in vitro. The ischemia LTP shared many molecular mechanisms with the physiological LTP, and was believed to be involved in the delayed neuronal death following ischemia. However, the role of the presynaptic component in this regard is not known. Here we show that a short period of oxygen-glucose deprivation can induce a form of LTP (lasting for hours) of the presynaptic response at the CA3-CA1 synapses. This form of LTP is independent of postsynaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, but Ca(2+) dependent. This presynaptic LTP may represent a presynaptic hyperexcitability of the afferent fibers following ischemia, and responsible for the excitotoxicity to the CA1 neurons (ischemia-induced increases of glutamate release that kills neurons) and the postsynaptic pathological ischemic LTP.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Glucose; Hippocampus; Hypoxia; In Vitro Techniques; Long-Term Potentiation; Phosphorus Compounds; Rats; Rats, Sprague-Dawley; Synapses; Tetrodotoxin; Time Factors

The respiratory role of excitatory amino acid (EAA) receptors within the Bötzinger complex (BötC) and the pre-Bötzinger complex (pre-BötC) was investigated in alpha-chloralose-urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30-50 nL) of EAA receptor antagonists. Blockade of both N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors by 50 mM kynurenic acid (KYN) within the BötC induced a pattern of breathing characterized by low-amplitude, high-frequency irregular oscillations superimposed on tonic phrenic activity and successively the disappearance of respiratory rhythmicity in the presence of intense tonic inspiratory discharges (tonic apnea). KYN microinjections into the pre-BötC caused similar respiratory responses that, however, never led to tonic apnea. Blockade of NMDA receptors by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1, 10 and 20 mM) within the BötC induced increases in respiratory frequency and decreases in peak phrenic amplitude; the highest concentrations caused tonic apnea insensitive to chemical stimuli. Blockade of non-NMDA receptors by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1, 10 and 20 mM) within the BötC produced only less pronounced increases in respiratory frequency. Responses to D-AP5 in the pre-BötC were similar, although less pronounced than those elicited in the BötC and never characterized by tonic apnea. In the same region, CNQX provoked increases in respiratory frequency similar to those elicited in the BötC, associated with slight reductions in peak phrenic activity. The results show that EAA receptors within the investigated medullary subregions mediate a potent control on both the intensity and frequency of inspiratory activity, with a major role played by NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Blood Pressure; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Hypercapnia; Hypoxia; Kynurenic Acid; Male; Medulla Oblongata; Microinjections; Neurons; Phrenic Nerve; Rabbits; Respiration; Time Factors; Vagotomy

Hypoxia is the most common cause of perinatal seizures and can be refractory to conventional anticonvulsant drugs, suggesting an age-specific form of epileptogenesis. A model of hypoxia-induced seizures in immature rats reveals that seizures result in immediate activation of the phosphatase calcineurin (CaN) in area CA1 of hippocampus. After seizures, CA1 pyramidal neurons exhibit a downregulation of GABA(A) receptor (GABA(A)R)-mediated inhibition that was reversed by CaN inhibitors. CaN activation appears to be dependent on seizure-induced activation of Ca2+-permeable AMPA receptors (AMPARs), because the upregulation of CaN activation and GABA(A)R inhibition were attenuated by GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] or Joro spider toxin. GABA(A)R beta2/3 subunit protein was dephosphorylated at 1 h after seizures, suggesting this subunit as a possible substrate of CaN in this model. Finally, in vivo administration of the CaN inhibitor FK-506 significantly suppressed hypoxic seizures, and posttreatment with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) or FK-506 blocked the hypoxic seizure-induced increase in CaN expression. These data suggest that Ca2+-permeable AMPARs and CaN regulate inhibitory synaptic transmission in a novel plasticity pathway that may play a role in epileptogenesis in the immature brain.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Blotting, Western; Calcineurin; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Gene Expression Regulation, Developmental; Hippocampus; Hypoxia; Immunoprecipitation; In Vitro Techniques; Neural Inhibition; Patch-Clamp Techniques; Rats; Receptors, AMPA; Receptors, GABA-A; Seizures; Synapses; Synaptic Transmission; Tacrolimus; Time Factors

The cytosolic redox status modulates ion channels and receptors by oxidizing/reducing their sulfhydryl (SH) groups. We therefore analyzed to what degree SH modulation affects hippocampal susceptibility to hypoxia. In rat hippocampal slices, severe hypoxia caused a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). Oxidizing SH groups by 5,5'-dithiobis 2-nitrobenzoic acid (DTNB, 2 mM) postponed HSD by 30%, whereas their reduction by 1,4-dithio-dl-threitol (DTT, 2 mM) or alkylation by N-ethylmaleimide (500 microM) hastened HSD onset. The DTNB-induced postponement of HSD was not affected by tolbutamide (200 microM), dl-2-amino-5-phosphonovaleric acid (150 microM), or 6-cyano-7-nitroquinoxaline-2,3-dione (25 microM). It was abolished, however, by Ni2+ (2 mM), withdrawal of extracellular Ca2+, charybdotoxin (25 nM), and iberiotoxin (50 nM). In CA1 neurons DTNB induced a moderate hyperpolarization, blocked spontaneous spike discharges and postponed the massive hypoxic depolarization. DTT induced burst firing, depolarized glial cells, and hastened the onset of the massive hypoxic depolarization. Schaffer-collateral/CA1 synapses were blocked by DTT but not by DTNB; axonal conduction remained intact. Mitochondria did not markedly respond to DTNB or DTT. While the targets of DTT are less clear, the postponement of HSD by DTNB indicates that sulfhydryl oxidation increases the tolerance of hippocampal tissue slices against hypoxia. We identified as the underlying mechanism the activation of BK channels in a Ca(2+)-sensitive manner. Accordingly, ionic disregulation and the loss of membrane potential occur later or might even be prevented during short-term insults. Therefore well-directed oxidation of SH groups could mediate neuroprotection.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aniline Compounds; Animals; Calcium; Charybdotoxin; Cortical Spreading Depression; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamine; Hippocampus; Hypoxia; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Neurons; Nickel; Oxidation-Reduction; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Sulfhydryl Reagents; Tolbutamide; Valine; Xanthenes

The most ubiquitous form of arrhythmia is respiratory sinus arrhythmia in which the heart beat slows during expiration and heart rate increases during inspiration. Whereas respiratory sinus arrhythmia benefits pulmonary gas exchange respiratory dysfunction presents a major challenge to the cardiorespiratory system. Hypoxia evokes a pronounced bradycardia mediated by increases in parasympathetic cardiac activity. It has been hypothesized that the fatal events in sudden infant death syndrome (SIDS) are exaggerated cardiorespiratory responses to hypoxia. This study tests whether premotor cardiac vagal neurons receive rhythmic respiratory-related excitatory synaptic inputs during normoxia and hypoxia, and if animals exposed to nicotine in the prenatal period have exaggerated responses to hypoxia. Premotor cardiac vagal neurons in the nucleus ambiguus were identified in rats by the presence of a fluorescent tracer in medullary slices that generate rhythmic inspiratory-related motor discharge. Respiratory activity was recorded from the hypoglossal nerve and excitatory synaptic events in cardiac vagal neurons were isolated using patch clamp techniques. Adult female rats were implanted with osmotic minipumps that delivered nicotine at a level approximately equivalent to those that occur in moderate to heavy smokers. During normal eupneic respiration, as well as during hypoxia, premotor cardiac vagal neurons from control animals did not receive any rhythmic respiratory-related excitatory inputs. However in animals exposed to nicotine throughout the prenatal period respiratory bursts during hypoxia dramatically increased the frequency of excitatory synaptic events in cardiac vagal neurons. In summary, in animals exposed to nicotine throughout the prenatal period, but not in unexposed animals, respiratory bursts that occur during hypoxia dramatically increase the frequency of excitatory synaptic events in cardiac vagal neurons. This study establishes a likely neurochemical mechanism for the heart rate responses to hypoxia and a link between prenatal nicotine exposure and exaggerated bradycardia responses during hypoxia that may contribute to sudden infant death syndrome.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Animals, Newborn; Brain Stem; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glycine Agents; Hypoxia; Neural Pathways; Neurons; Nicotine; Nicotinic Agonists; Picrotoxin; Rats; Rats, Sprague-Dawley; Respiration; Strychnine; Valine

Intracellular pH (pH(i)) is an important factor for understanding cellular processes associated with the response of central neurons to metabolic disturbances such as anoxia or ischemia. In the present study, pH(i) was fluorometrically measured in 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescin (BCECF)-filled, voltage-clamped dorsal vagal neurons (DVN) of brainstem slices from rats during metabolic disturbances activating ATP-sensitive K(+) (K(ATP)) channels. Chemical anoxia induced by cyanide, rotenone or p-trifluoromethoxy-phenylhydrazone (FCCP) decreased pH(i) by >0.4 pH units. Untreated neurons with normal pH(i) baseline (7.2) responded to glucose-free superfusate after a delay of 7-16 min with a progressive fall of pH(i). In contrast, pH(i) increased by >0.2 pH units after approximately 10 min in cells that had a mean pH(i) of 6.8 due to incomplete recovery from a CN(-)induced acid load prior to glucose depletion. Metabolic arrest, induced by cyanide in glucose-free solution after 30 min preincubation in glucose-free saline, caused a progressive glutamate-mediated inward current with no change of pH(i). Upon metabolic arrest, depolarization-evoked pH(i) decreases ( approximately 0.2 pH units) were abolished, whereas glucose-free superfusate slightly delayed their recovery without major effects on amplitude. The glucose-dependent pH(i) fall coincided with activation of the K(ATP) channel-mediated outward current, while K(ATP) currents due to anoxia or metabolic arrest could reach their maximum in the absence of a major pH(i) change. The results indicate that the anoxic pH(i) decrease is due to enhanced glycolysis and lactate formation with often no obvious effect on K(ATP) channel activity. The origin of glucose-dependent acidosis and its relation to K(ATP) channel activity remain to be determined.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Stem; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cyanides; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Fluoresceins; Glucose; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Intracellular Space; Male; Membrane Potentials; Membrane Proteins; Neurons; Patch-Clamp Techniques; Potassium Channels; Rats; Rotenone; Time Factors; Vagus Nerve; Valine

Pressor and sympathoexcitatory responses induced by microinjection of glutamate (Glu) into the dorsomedial medulla (DM) were depressed after hypoxia in anesthetized cats. This study was undertaken to investigate which Glu receptor subtypes would be involved in the post-hypoxic depression. Hypoxia was induced by inhalation of a 5% O(2) and 95% N(2) gas mixture. The pressor and sympathoexcitatory responses to microinjections of N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) into the DM were significantly depressed after hypoxia, suggesting the involvement of both NMDA and AMPA receptors. However, pretreatment with kynurenic acid or DL-2-amino-5-phosphonopentanoic acid, but not 6-cyano-7-nitroquinoxaline-2,3-dione, 5 min before hypoxia could effectively prevent the post-hypoxic depression of Glu-induced responses. These results further suggest that post-hypoxic depression of Glu-induced responses in the DM was predominately mediated by NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Blood Gas Analysis; Blood Pressure; Cats; Depression, Chemical; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Heart Rate; Hypoxia; Kynurenic Acid; Male; Medulla Oblongata; Microinjections; N-Methylaspartate; Receptors, N-Methyl-D-Aspartate; Spinal Nerves

An important but poorly understood event associated with ischemia is anoxic depolarization (AD), a sudden and profound depolarization of neurons and glia in cortical and subcortical gray matter. Leao first measured the AD as a wave of electrical silence moving across the cerebral cortex in 1947 and noted its similarity to spreading depression (SD). SD is harmless when coursing through normoxic cortical tissue as during migraine aura. However for 3-4 h following focal ischemia, the additional metabolic stress arising from recurring SD in the penumbra expands the ischemic core, so SD blockade is potentially beneficial therapeutically. In the present study, we measured intrinsic optical signals (IOSs) to monitor anoxic depolarization in submerged rat neocortical slices during O2/glucose deprivation (OGD). After approximately 6 min of OGD, the AD was imaged as a focal increase in light transmittance which then propagated across neocortical gray at approximately 2 mm/min. Although the slice was globally stressed, the AD always initiated focally, sometimes at multiple sites. Its propagation was coincident with a transient negative shift in the extracellular potential, the electrical signature of AD. Acute damage to neocortex (measured as a delayed decrease in LT and as a loss of the evoked field potential) followed only where the AD had propagated, so it is the combined metabolic demands of AD and OGD that acutely damages all layers of the neocortex. Glutamate receptor antagonists (2 mM kynurenate or 25 microM AP-5/10 microM CNQX) did not block AD initiation, slow its propagation or prevent post-AD damage. This study shows that acute ischemic damage is greatly exacerberated by AD during metabolic stress and that glutamate receptor antagonists are not protective. Using this slice model, therapeutically tolerable drugs that block the AD and SD can be investigated.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Brain Ischemia; Cortical Spreading Depression; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hypoxia; Kynurenic Acid; Male; N-Methylaspartate; Neocortex; Neurons; Rats; Rats, Sprague-Dawley; Receptors, Glutamate

In the rhythmic brain stem slice preparation, spontaneous respiratory activity is generated endogenously and can be recorded as output activity from hypoglossal XII rootlets. Here we combine these recordings with measurements of the intrinsic optical signal (IOS) of cells in the regions of the periambigual region and nucleus hypoglossus of the rhythmic slice preparation. The IOS, which reflects changes of infrared light transmittance and scattering, has been previously employed as an indirect sensor for activity-related changes in cell metabolism. The IOS is believed to be primarily caused by cell volume changes, but it has also been associated with other morphological changes such as dendritic beading during prolonged neuronal excitation or mitochondrial swelling. An increase of the extracellular K(+) concentration from 3 to 9 mM, as well as superfusion with hypotonic solution induced a marked increase of the IOS, whereas a decrease in extracellular K(+) or superfusion with hypertonic solution had the opposite effect. During tissue anoxia, elicited by superfusion of N(2)-gassed solution, the biphasic response of the respiratory activity was accompanied by a continuous rise in the IOS. On reoxygenation, the IOS returned to control levels. Cells located at the surface of the slice were observed to swell during periods of anoxia. The region of the nucleus hypoglossus exhibited faster and larger IOS changes than the periambigual region, which presumably reflects differences in sensitivities of these neurons to metabolic stress. To analyze the components of the hypoxic IOS response, we investigated the IOS after application of neurotransmitters known to be released in increasing amounts during hypoxia. Indeed, glutamate application induced an IOS increase, whereas adenosine slightly reduced the IOS. The IOS response to hypoxia was diminished after application of glutamate uptake blockers, indicating that glutamate contributes to the hypoxic IOS. Blockade of the Na(+)/K(+)-ATPase by ouabain did not provoke a hypoxia-like IOS change. The influences of K(ATP) channels were analyzed, because they contribute significantly to the modulation of neuronal excitability during hypoxia. IOS responses obtained during manipulation of K(ATP) channel activity could be explained only by implicating mitochondrial volume changes mediated by mitochondrial K(ATP) channels. In conclusion, the hypoxic IOS response can be interpreted as a result of cell and mitochondrial swelling. C

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine; Adenosine Triphosphate; Animals; Animals, Newborn; Anti-Bacterial Agents; Antihypertensive Agents; Diazoxide; Energy Metabolism; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glyburide; Hypoglossal Nerve; Hypoglycemic Agents; Hypoxia; Kainic Acid; Macrolides; Mice; Mitochondrial Swelling; N-Methylaspartate; Optics and Photonics; Organ Culture Techniques; Ouabain; Potassium Channels; Respiratory Center; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin

In severe hypoxia the breathing frequency is modulated in a biphasic manner: an initial increase (augmentation) is followed by a depression and cessation of breathing (apnea). Using a mouse slice preparation that contains the functional respiratory network, we aimed at identifying the neurons responsible for this frequency modulation. Whole-cell patch recordings revealed that expiratory neurons become tonically active during anoxia, indicating that these neurons cannot be responsible for the respiratory frequency modulation. Inspiratory neurons tended to depolarize (by 6.9 mV; n = 9), and the frequency of rhythmic activity was significantly increased during anoxia (from 0.16 to 0.4 Hz; n = 9). After the blockade of network activity with 6-cyano-7-nitroquinoxaline-2, 3-dione, most inspiratory neurons became tonically active (72%; n = 25, non-pacemaker). In anoxia, the membrane potential of these non-pacemaker neurons did not change (-0.26 mV; n = 6), and their tonic activity ceased. Only a subpopulation of inspiratory neurons remained rhythmically active in the absence of network activity (pacemaker neurons, 28%, 7 of 25 inspiratory neurons). In anoxia two subgroups of pacemaker neurons were differentiated; one group showed a transient increase in the bursting activity, followed by a decrease and cessation of rhythmic activity. These neurons tended to depolarize (by 10.3 mV) during anoxia. The second group remained rhythmic during the entire anoxic exposure and exhibited no depolarization. The time course of the frequency modulation in all pacemaker neurons resembled that of the intact network. We conclude that pacemaker neurons are primarily responsible for the frequency modulation in anoxia and that in the respiratory network there is a strict separation between rhythm- and pattern-generating mechanisms.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Biological Clocks; Excitatory Amino Acid Antagonists; Female; Hypoglossal Nerve; Hypoxia; Male; Mice; Neurons; Patch-Clamp Techniques; Respiratory Mechanics; Solitary Nucleus

Studies in slices suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by D(-)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-D-aspartate (NMDA) and GABAA (gamma-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Aging; Animals; Animals, Newborn; Benzodiazepines; Bicuculline; Cations, Divalent; Electric Stimulation; Embryonic and Fetal Development; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Hippocampus; Hypoxia; Lysine; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Synapses; Tetrodotoxin

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca2+ concentration ([Ca2+]i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen-glucose deprivation) elicited a gradual increase in [Ca2+]i in normal Krebs solution. At high extracellular sodium concentrations ([Na+]o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na+]o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na+]o was reversed by pre-incubation of the slice at a low [Na+]o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na+]o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na+-Ca2+ exchanger only slightly influenced the effects of high [Na+]o. These observations suggest that disturbance of the transmembrane gradient of Na+ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na+-dependent glutamate transporter rather than an exocytotic process.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acid Transport System X-AG; Animals; ATP-Binding Cassette Transporters; Bepridil; Biological Transport; Calcium; Calcium Channel Blockers; Chelating Agents; Dicarboxylic Acids; Excitatory Amino Acid Antagonists; Fura-2; Glutamic Acid; Hippocampus; Hypoxia; Neurotransmitter Uptake Inhibitors; Organ Culture Techniques; Pyrrolidines; Rats; Rats, Wistar; Sodium; Sodium-Calcium Exchanger

It has been shown previously that hypoxia reduced presumed presynaptic Ca2+ entry in area CA1 of rat hippocampal slices, perhaps due to energy depletion in presynaptic endings. This would imply that hypoglycaemia also affects presynaptic Ca2+ uptake. We therefore studied effects of glucose withdrawal on stimulus induced changes in [Ca2+]o in area CA1 of rat hippocampal slices. After blockade of synaptic transmission by application of the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 microM) and L-(+)-2-amino-5-phosphonovaleric acid (2APV, 30 microM) stimulation of Schaffer collaterals resulted in small but significant reductions in [Ca2+]o which could be attributed to presynaptic Ca2+ entry. Stimulation of the alveus resulted in somewhat reduced Ca2+ signals mediated to a large extent by Ca2+ entry into dendrites and somatas of pyramidal cells. Removal of glucose results in an augmentation of the presumed presynaptic Ca2+ entry by a factor of 2-3. Decreases in [Ca2+]o induced by repetitive stimulation of the alveus were initially augmented and then disappeared presumably due to irreversible damage of pyramidal neurones. This finding suggests that depletion of ATP alone does not account for the depressive effect of hypoxia on presynaptic Ca2+ entry.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Evoked Potentials; Glucose; Hippocampus; Hypoxia; Rats; Rats, Wistar; Synaptic Transmission

EPSPs/IPSPs were recorded with intracellular electrodes from CA1 neurons close to site of stimulation. Brief anoxia (3 min) abolished EPSPs but reduced IPSPs by 64.8 +/- 4.0% (n = 10); the remaining IPSP was presumed to be monosynaptic. The effects of anoxia on purely monosynaptic IPSPs were examined after pharmacological blockade of excitatory synaptic transmission with 2 mM kynurenate or 20 microM CNQX + 20 microM APV. In these tests, after 3 min of anoxia the slopes of IPSPs vs. membrane potential were reduced by only 38.2 +/- 4.3% (n = 12). The present study demonstrates that, contrary to previous reports, inhibitory synaptic transmission is quite resistant to anoxia.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Evoked Potentials; Hippocampus; Hypoxia; In Vitro Techniques; Kynurenic Acid; Membrane Potentials; Quinoxalines; Rats; Rats, Sprague-Dawley; Synapses; Synaptic Transmission

Rat embryonic hippocampal neurons cultured on astrocyte feeder-layers were sensitive to different excitotoxic stimuli after 10-12 DIV. Almost all neurons (approximately 95%) died within 20 h after a transient exposure for 10 min to 50 microM glutamate, a continuous exposure to either 25 microM NMDA or 250 microM kainate or after a 15-min deprivation of glucose and oxygen. Dizocilpine at 10 microM protected neurons against the glutamate- and NMDA-mediated toxicity as well as against 30 min glucose and oxygen deprivation. However, it failed to protect against kainate toxicity and prolonged glucose/oxygen deprivation (60 min). An additional treatment with CNQX (100 microM) protected neurons even under the latter two conditions. This indicates that the vast majority of neurons was sensitive to different excitotoxic stimuli acting through different types of glutamate receptors leading to calcium overload of the cells which might be the common denominator of triggering cell death under these conditions. Expression of calcium-binding proteins, such as calbindin D28K or calretinin, might increase the intracellular calcium buffer capacity of neurons, thus, rendering them more resistant to calcium overload. Therefore, we analysed whether neurons expressing these calcium-binding proteins would survive these toxic stimuli. Indeed, a small population of the neurons (3-5%) survived, including a subpopulation of calretinin-positive but not calbindin D28K-positive neurons. This implies that the expression of calcium-binding proteins per se does not render neurons more resistant towards these excitotoxic stimuli. Moreover, most of the surviving calretinin-positive neurons showed morphological damage as indicated by loss of neurites. When cytotoxicity due to calcium overload was induced by an exposure of the cells to the calcium ionophore 4-bromo-A23187 rather than by activation of glutamate receptors, calretinin-positive cells were found not to be significantly more resistant than the vast majority of neurons. This may indicate that the lower sensitivity of a subpopulation of calretinin-positive neurons to excitotoxic stimuli may be due to a lower expression of glutamate receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calbindin 1; Calbindin 2; Calbindins; Calcimycin; Cell Death; Cells, Cultured; Dizocilpine Maleate; Glucose; Glutamic Acid; Hippocampus; Hypoxia; Immunohistochemistry; Kainic Acid; Mice; Nerve Tissue Proteins; Neurons; Oxygen Consumption; Rats; S100 Calcium Binding Protein G

Calcium plays a prominent role in the neuronal degeneration which accompanies stroke and there has been much conjecture about the possible source of this Ca2+. The transmembrane Ca2+ transporting processes are considered likely candidates for the ischemia-induced rise in intracellular Ca2+. In the present paper we have monitored metabolism in the cerebral cortex in vitro before, during and after aglycaemic hypoxia using 31P and 1H NMR spectroscopy. We used the recovery of cellular metabolites phosphocreatine, ATP, lactate, glutamate and N-acetyl aspartate determined by NMR as an indicator of cell damage caused by hypoxia. Phosphocreatine concentration recovered to only approximately 58% of its control level following 15 min of aglycaemic hypoxia in the presence of 1.2 mM Ca2+. The ratios of phosphocreatine/ATP, lactate/N-acetyl aspartate and glutamate/N-acetyl aspartate did not differ at 1 h of recovery from the prehypoxia levels showing that the hypoxia resistant cells were metabolically viable. In the absence of external Ca2+, phosphocreatine recovery improved to approximately 80%. Ten mM Mg2+ or 25 microM diltiazem in the presence of 1.2 mM Ca2+ improved recovery of phosphocreatine to approximately 85%. Two other antagonists of L-type voltage-gated Ca(2+)-channels, verapamil and nifedipine, did not protect the cerebral cortex from hypoxic damage. N-methyl-D-aspartate (100 microM) applied during hypoxia with 1.2 mM Ca2+ did not augment the loss of phosphocreatine indicating that the cellular damage was not potentiated by the drug, even when 30 mM K+ was present. The presence of N-methyl-D-aspartate did not weaken the protective effect of diltiazem. Blockade of N-methyl-D-aspartate or non-N-methyl-D-aspartate channels did not alleviate cellular damage caused by hypoxic insult. The present results suggest that the immediate, Ca(2+)-mediated neuronal damage in the cerebral cortex may be mediated by Ca2+ influx through L-type voltage-gated Ca(2+)-channels.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cerebral Cortex; Diltiazem; Excitatory Amino Acid Antagonists; Hydrogen; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Magnesium; Magnetic Resonance Spectroscopy; N-Methylaspartate; Neurons; Phosphocreatine; Phosphorus; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Chickens; Dizocilpine Maleate; Edema; Electron Transport; Glycolysis; Hypoglycemia; Hypoxia; Iodoacetates; Iodoacetic Acid; Ischemia; N-Methylaspartate; Potassium Cyanide; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Retina; Tetrodotoxin

Neurotoxicity of glutamate in conjunction with subcritical hypoxia was determined in vitro using hippocampal neurons obtained from 18-day-old rat fetuses. Neurons were plated at a low density and maintained for 3 days in a chemically defined medium without glutamate. When glutamate + was added after subcritical hypoxic stress, a low dose of glutamate, even at 10 microM, could cause significant neuronal loss in the following 24 h. The observed neurotoxicity to low glutamate dose (10-100 microM) could completely be prevented by 5 microM of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This protective effect of CNQX was more potent than that of MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). The mechanism by which glutamate is transformed from a neurotransmitter to a neurotoxin is discussed.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Glutamates; Glutamic Acid; Hippocampus; Hypoxia; Nervous System Diseases; Neurons; Pregnancy; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, AMPA; Receptors, Kainic Acid; Receptors, Neurotransmitter