The effect of high pressure on the NMDA receptor: molecular dynamics simulations.

Professional divers exposed to ambient pressures above 11 bar develop the high pressure neurological syndrome (HPNS), manifesting as central nervous system (CNS) hyperexcitability, motor disturbances, sensory impairment, and cognitive deficits. The glutamate-type N-methyl-D-aspartate receptor (NMDAR) has been implicated in the CNS hyperexcitability of HPNS. NMDARs containing different subunits exhibited varying degrees of increased/decreased current at high pressure. The mechanisms underlying this phenomenon remain unclear. We performed 100 ns molecular dynamics (MD) simulations of the NMDAR structure embedded in a dioleoylphosphatidylcholine (DOPC) lipid bilayer solvated in water at 1 bar, hydrostatic 25 bar, and in helium at 25 bar. MD simulations showed that in contrast to hydrostatic pressure, high pressure helium causes substantial distortion of the DOPC membrane due to its accumulation between the two monolayers: reduction of the Sn-1 and Sn-2 DOPC chains and helium-dependent dehydration of the NMDAR pore. Further analysis of important regions of the NMDAR protein such as pore surface (M2 α-helix), Mg2+ binding site, and TMD-M4 α-helix revealed significant effects of helium. In contrast with previous models, these and our earlier results suggest that high pressure helium, not hydrostatic pressure per se, alters the receptor tertiary structure via protein-lipid interactions. Helium in divers' breathing mixtures may partially contribute to HPNS symptoms.

Selective pressure modulation of synaptic voltage-dependent calcium channels-involvement in HPNS mechanism.

Exposure to hyperbaric pressure (HP) exceeding 100 msw (1.1 MPa) is known to cause a constellation of motor and cognitive impairments named high-pressure neurological syndrome (HPNS), considered to be the result of synaptic transmission alteration. Long periods of repetitive HP exposure could be an occupational risk for professional deep-sea divers. Previous studies have indicated the modulation of presynaptic Ca(2+) currents based on synaptic activity modified by HP. We have recently demonstrated that currents in genetically identified cellular voltage-dependent Ca(2+) channels (VDCCs), CaV 1.2 and CaV 3.2 are selectively affected by HP. This work further elucidates the HPNS mechanism by examining HP effect on Ca(2+) currents in neuronal VDCCs, CaV 2.2 and CaV 2.1, which are prevalent in presynaptic terminals, expressed in Xenopus oocytes. HP augmented the CaV 2.2 current amplitude, much less so in a channel variation containing an additional modulatory subunit, and had almost no effect on the CaV 2.1 currents. HP differentially affected the channels' kinetics. It is, therefore, suggested that HPNS signs and symptoms arise, at least in part, from pressure modulation of various VDCCs.

The N-methyl-D-aspartate receptor's neglected subunit - GluN1 matters under normal and hyperbaric conditions.

Professional deep-water divers exposed to hyperbaric pressure (HP) above 1.1 MPa develop high-pressure neurological syndrome, which is associated with central nervous system hyperexcitability. It was previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. In addition, we have reported that HP (10.1 MPa) differentially affects ionic currents, measured by the two-electrode voltage-clamp technique, of eight specific NMDAR subtypes generated by the co-expression of GluN1-1a or GluN1-1b with one of the four GluN2(A-D) subunits in Xenopus laevis oocytes. We now report that eight GluN1 splice variants, when co-expressed with GluN2A, mediate different ionic currents at normal and HP (5.1 MPa). These data, in conjunction with our previous results, indicate that both GluN1 and GluN2 subunits play a critical role in determining NMDAR currents under normal and HP conditions. These data, given the differential spatial distribution of the different NMDAR subtypes in the central nervous system, may offer a partial explanation for the mechanism governing the complex signs and symptoms of high-pressure neurological syndrome, and an explanation for the suspected long-term HP health decrement due to repetitive deep dives by professional divers.

Is glucose-6-phosphate dehydrogenase deficiency a risk factor for hyperbaric oxygen exposure?

Divers and patients lacking glucose-6-phosphate dehydrogenase (G6PD) may face a serious threat of central nervous system oxygen toxicity (CNS-OT) during exposure to hyperbaric oxygen (HBO), due to the important part played by G6PD in cellular redox balance. Our objective was to investigate G6PD deficiency as a risk factor for CNS-OT. We exposed G6PD-deficient (G6PDdef) and wild type (WT) mice to HBO at 405 kPa. Latency to CNS-OT was measured by observing the animal and monitoring the time to appearance of convulsions. Changes in glutathione peroxidase (GPx) and catalase activity were measured in red blood cells, and levels of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) and 3-nitrotyrosine (NT) were measured in extracts of whole brain tissue by Western blot analysis. Unexpectedly, latency to CNS-OT was more than twice as long in G6PDdef mice compared with WT (36.9 ± 15.4 and 15.6 ± 13.2 min, respectively, P < 0.005). No significant differences were found in GPx and catalase activity or in protein levels of eNOS. However, nNOS and NT levels were lower in G6PDdef mice compared with WT (50.6%, P < 0.01 and 52.8%, P < 0.05, respectively). Our results suggest that the enhanced resistance of G6PDdef mice to HBO is due in part to a reduction in nNOS and NT levels in the brain. We conclude that G6PD deficiency at the level of the animals in the present study may not be a risk factor for developing CSN-OT, but this remains to be verified for human subjects.

Low susceptibility to inert gases and pressure symptoms in TREK-1-deficient mice.

Nervous disorders may occur after an organism is saturated with inert gases, which may alter the lipid bilayer structure, according to their liposolubility coefficient. Increase in the nitrogen partial pressure induces a neurological syndrome called 'nitrogen narcosis'. By contrast, high pressures of helium induce epilepsy, an high-pressure nervous syndrome symptom. On the basis of an analogy with anaesthetic mechanisms, we used TREK-1 knockout mice, earlier described to volatile the anaesthetics resistance. These mice had a higher threshold of resistance to the narcotic effects of nitrogen and to the death after recurrent epileptic seizure induced by high pressure. TREK-1 channels seem to play a key role in modulating the anaesthetic potential of inert gases and in neuroprotection.

High pressure modulation of NMDA receptor dependent excitability.

Pressure above 1.1 MPa induces in mammals and humans the high pressure neurological syndrome (HPNS). HPNS is characterized by cognitive and motor decrements associated with sleep disorders, EEG changes, tremor, and convulsions that ultimately may lead to death. Previous theories proposed that augmented response of the glutamatergic N-methyl-D-aspartate receptor (NMDAR) or reduced GABAergic inhibition may be involved. Recently, we have reported that isolated NMDAR response was augmented at high pressure. We now test whether this augmentation induces neuronal hyperexcitability. We studied high pressure effects on pharmacologically isolated NMDAR field excitatory postsynaptic potentials (fEPSPs) and on their efficacy in generating population spikes (PSs). Sprague-Dawley male rats were used. Hippocampal coronal brain slices were prepared, constantly superfused with physiological solutions, gas-saturated at normobaric pressure, and compressed up to 10.1 MPa with helium. fEPSPs and PSs were recorded from the dendritic and the somatic layers of CA1 pyramidal neurons in response to Schaefer collaterals stimulation with trains of five stimuli at 25 Hz. Pressure caused PSs to appear earlier in the train. However, PS delay, rise time and decay time were increased and PS amplitude, frequency, and number were decreased in the last responses in the train. The decrease in late fEPSPs was associated with a reduction of the total number of PSs in the train, apparently without a change in the synaptic efficacy. These results may partially explain the neuronal hyperexcitability observed at pressure. Therefore, it is postulated that significant hyperexcitability is attained at pressure only when the normal fast fEPSP is intact.

Recent neurochemical basis of inert gas narcosis and pressure effects.

Compressed air or a nitrogen-oxygen mixture produces from 0.3 MPa nitrogen narcosis. The traditional view was that anaesthesia or narcosis occurs when the volume of a hydrophobic site is caused to expand beyond a critical amount by the absorption of molecules of a narcotic gas. The observation of the pressure reversal effect on general anaesthesia has for a long time supported the lipid theory. However, recently, protein theories are in increasing consideration since results have been interpreted as evidence for a direct anaesthetic-protein interaction. The question is to know whether inert gases act by binding processes on proteins of neurotransmitter receptors. Compression with breathing mixtures where nitrogen is replaced by helium which has a low narcotic potency induces from 1 MPa, the high pressure nervous syndrome which is related to neurochemical disturbances including changes of the amino-acid and monoamine neurotransmissions. The use of narcotic gas (nitrogen or hydrogen) added to a helium-oxygen mixture, reduced some symptoms of the HPNS but also had some effects due to an additional effect of the narcotic potency of the gas. The researches performed at the level of basal ganglia of the rat brain and particularly the nigro-striatal pathway involved in the control of the motor, locomotor and cognitive functions, disrupted by narcosis or pressure, have indicated that GABAergic neurotransmission is implicated via GABAa receptors.

Helium-oxygen pressure induces striatal glutamate increase: a microdialysis study in freely-moving rats.

In rat, helium pressures induce locomotor and motor activity which requires dopaminergic and N-methyl-D-aspartate (NMDA) receptor activities at striatal level. However, biochemical studies have suggested that pressure exposure may increase striatal glutamate level. We used microdialysis technique to study the effects of pressure on glutamate level in the striatum and the effects of local administration of D1 (SCH23390) or D2 (sulpiride) on these changes. Pressures increase both glutamate and glutamine levels in striatal microdialysates. Administration of sulpiride (1 microM) or SCH23390 (1 microM) by reverse microdialysis did not affect significantly pressure induced glutamate increase. So, protective effects of D1 and D2 antagonists against locomotor and motor hyperactivity (LMA) are probably independent of the processes involved in the striatal glutamate increase evoked by pressure.

Crucial role of the 5-HT2C receptor, but not of the 5-HT2A receptor, in the down regulation of stimulated dopamine release produced by pressure exposure in freely moving rats.

Helium pressure of more than 2 MPa is a well known factor underlying pressure-dependent central neuroexcitatory disorders, referred to as the high-pressure neurological syndrome. This includes an increase in both serotonin (5-HT) and dopamine (DA) release. The relationship between the increase in 5-HT transmission produced by helium pressure and its effect on DA release has been clarified in a recent study, which have first demonstrated that the helium pressure-induced increase in DA release was dependent on some 5-HT receptor activation. In the present study, we examined in freely moving rats the role of 5-HT2A and 5-HT2C receptors in the increase in DA release induced by 8 MPa helium pressure. We used the 5-HT2A receptor antagonist ketanserin and the 5-HT2C receptor agonist m-CPP which have been demonstrated to reduce DA function. Because neither ketanserin is an ideal 5-HT2A receptor antagonist nor m-CPP an ideal 5-HT2C receptor agonist, additional experiments were made at normal pressure to check up on the selectivity of ketanserin and m-CPP for 5-HT2A and 5-HT2C receptors, respectively. Administration of m-CPP reduced both DA basal level and the helium pressure-induced increase in DA release, whereas administration of ketanserin only showed a little effect on the increase in DA release produced by high helium pressure. These results suggest that the 5-HT2C receptor, but not the 5-HT2A receptor, would play a crucial role in the helium pressure-induced increase in DA release. This further suggests that helium pressure may simultaneously induce an increase in 5-HT transmission at the level of 5-HT2A receptors and a decrease in 5-HT transmission at the level of 5-HT2C receptors.

[Effect of calcium channel blockers on developing nervous syndrome of high pressure and nitrogen narcosis in mice]. / Vliianie blokatorov kal'tsievykh kanalov na razvitie azotnogo narkoza i nervnogo sindroma vysokikh davlenií u mysheí.

In the experiments conducted on mice which prior to compression in a heliox environment have been injected the blockers of various types of calcium channels (flunarezine, verapramil and nifedipine) as well as bemethyl (actoprotector) and oxymethacye (antioxidant) there escaped detection of noticeable effect of these drugs on developing the high pressure nervous syndrome (HPNS). On exposure to the hyperbaric nitrogen-oxygen environment verapromil (phenylalkulamine blocker of L-type calcium channels) had a protection effect with respect to a convulsive component of the nitrogen narcosis.

Glycine activation of human homomeric alpha 1 glycine receptors is sensitive to pressure in the range of the high pressure nervous syndrome.

The effect of hyperbaric pressure on the inhibitory glycine receptor has been investigated in voltage-clamped Xenopus oocytes microinjected with cRNA encoding the human alpha-1 glycine receptor subunit. Heterologous expression of the human alpha-1 subunit generated functional glycine-gated channels with properties typical of native receptors. Glycine elicited a concentration-dependent inward current which reversed polarity at -25 mV and was antagonised by nanomolar concentrations of strychnine. Concentration-response curves established for the homomeric alpha-1 glycine receptor at 5, 10 and 15 MPa were progressively shifted to the right with respect to the concentration response curve established at atmospheric pressure (0.1 MPa). Pressure had no effect on the maximal response. The EC(50) values at 0.1, 5, 10 and 15 MPa were 190 mu M, 222 mu M, 338 mu M and 482 mu M, respectively. The results demonstrate that a receptor comprised solely of the human alpha-subunit is sensitive to pressure in the range that affects divers and at which the native rat spinal cord receptor is affected. This finding is discussed in the context of the postulated binding sites for glycine and the implications for the design of drugs to protect divers from the effects of pressure.

Involvement of 5-HT3 receptor in the pressure-induced increase in striatal and accumbens dopamine release and the occurrence of behavioral disorders in free-moving rats.

Rats exposed to high pressure developed locomotor and motor activity (LMA) that correlated with an increase of DA release in both the nucleus accumbens and the caudate-putamen. We investigated the effects of the 5-HT3 receptor antagonist MDL 72222 on these pressure-induced neurochemical and behavioral disorders. MDL 72222 totally blocked the pressure-induced increase in accumbens DA release and the development of LMA, whereas it only reduced the increase in striatal DA release. This suggest that both LMA and the increase of DA release in the nucleus accumbens, but not in the caudate-putamen, could specifically result from a 5-HT3 receptor activation in rats exposed to high pressure.

Effects of elevated pressures of inert gases on cytosolic free Ca2+ of cultured human neuroblastoma cells stimulated with carbachol: relevance to high pressure neurological syndrome.

Suspended cells of the human neuroblastoma line SK-N-SH were exposed to elevated pressures of non-narcotic helium (He) and the narcotic gases nitrogen (N2), and argon (Ar) and stimulated with carbachol. He, 18 and 36 atmospheres absolute (ATA), equivalent to 544 and 1120 feet of seawater, potentiated the increase in [Ca2+]i induced by carbachol, as measured by Fura-2. Carbachol-stimulated increases in [Ca2+]i were not significantly altered from values in 1 ATA air by either N2 or Ar at the same pressures. The response to carbachol of cells exposed to 36 ATA of He and slowly decompressed to 1 ATA was indistinguishable from that of cells never exposed to pressure. Thus this pressure-potentiated increase in [Ca2+]i is compatible with excitation, is reversible and is not elicited by narcotic gases. It was observed, moreover, at pressures encountered by commercial deep-sea divers. The High Pressure Neurological Syndrome (HPNS) encountered by divers breathing He/O2 mixtures at high pressures, and its known antagonism by N2, may be due in part to effects on neuronal [Ca2+]i levels since an increase in these would most likely result in an excitatory response.

Effects of high pressure on striatal dopamine release in freely moving rats: a microdialysis study.

When human divers and experimental animals are exposed to high pressure of helium-oxygen mixture, they develop the high pressure neurological syndrome, characterized by nausea, vertigo, tremor, myoclonus, EEG modifications and convulsions. Free-moving rats were stereotaxically implanted in the anterior caudate nucleus with a microdialysis probe to measure dopamine, dihydroxyphenylacetic acid and homovanillic acid levels during different phases of a simulated dive up to 5.1 MPa. Compression was found to cause an increase in extracellular dopamine and dihydroxyphenylacetic acid concentrations, but not in homovanillic acid. This represents a specific effect of high pressure on the dopaminergic pathway. Recent findings on D2 autoreceptors, showing a decrease in receptor affinity under pressure, allow us to conclude that pressure increases dopamine synthesis through a direct action on D2 autoreceptors.

Effect of pressure on the release of endogenous dopamine from rat striatum and the role of sodium-calcium exchange.

Exposure to environmental pressures in excess of 20 atm abs can precipitate a hyperexcitability state known as high pressure neurologic syndrome (HPNS). Little is known about the underlying neurochemical basis of this syndrome. An in vitro model of the synthesis and release of endogenous dopamine (DA) from rat striatal slices has been used to examine the mechanism underlying the effects of high pressures of He. He at 100 atm abs produced changes in DA release which were strikingly similar to those of the cardiac glycoside, ouabain. Neither pressure nor ouabain (1-10 microM) had any significant effects on the spontaneous (nonevoked) release of DA or its metabolite 3,4-dihydroxyphenylacetic acid, but both pressure and ouabain significantly enhanced the stimulated release of DA which was evoked by a 6-min exposure to 35 mM KCl (P less than 0.05 and P less than 0.001). In both cases, this effect was dependent on the presence of extracellular Ca2+. Augmentation of evoked DA release by both ouabain and He pressure was reversed (P less than 0.05) by 3,4-dichlorobenzamil, a selective antagonist of the membrane Na+/Ca2+ exchange mechanism. The results suggest that pressure exerts its effects on DA release by increasing intracellular-free Ca2+ exchange after pressure-inhibition of the activity of the membrane Na,K-ATPase.

Pressure-induced striatal dopamine release correlates hyperlocomotor activity in rats exposed to high pressure.

Free-moving rats chronically implanted in the striatum with multifiber carbon electrodes selective to dopamine were compressed in a helium-oxygen mixture to 80 bars. Extracellular dopamine level and behavioral symptoms of high-pressure neurological syndrome were simultaneously recorded. Under these conditions, the extracellular level of dopamine monitored by differential pulse voltammetry was found to be pressure dependent, and hyperlocomotor activity, a behavioral symptom of high-pressure neurological syndrome, was found to be linked to these pressure-induced changes in dopamine release.

The effects of the competitive NMDA receptor antagonist CPP on the high pressure neurological syndrome in a primate model.

Neurophysiological interactions between the competitive N-methyl-D-aspartate (NMDA) preferring receptor antagonist, CPP (3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonate) and the high pressure neurological syndrome (HPNS) have been investigated in the non-human primate Papio anubis. Eight animals were exposed on two occasions to environmental pressures of 81 atmospheres absolute (ATA) in a hyperbaric chamber, using helium and oxygen. One exposure followed pretreatment with CPP (either 5 or 10 mg/kg i.v. plus 5 mg/kg/hr infusion), the other a saline control. Pretreatment with CPP delayed moderate signs of face tremor and myoclonus and abolished severe signs of whole body tremor and seizure activity. By 81 ATA, scores representing severity of HPNS were significantly reduced by CPP to a mean score, reflecting a level of just mild to moderate limb tremoring (P less than 0.001). Changes in the EEG were observed in channels associated with the frontal, parietal and occipital regions of the left cortex. Amplitude and frequency spectra were calculated and changes with pressure in the 4 conventional wavebands were analysed. The most striking change was the complete prevention by CPP of the 100% increase in the amplitude of alpha waves at 81 ATA in the frontal region (P less than 0.001). It is concluded that NMDA transmission has a major role in the expression of HPNS.

A 23187-stimulated calcium uptake and GABA release by cerebrocortical synaptosomes: effects of high pressure.

Guinea pig cerebrocortical synaptosome preparations were used to study the effect of compression to 62 ATA on 45Ca2+ uptake and [3H]GABA release using a calcium ionophore A 23187, which bypasses the voltage-sensitive calcium channel. Pressure was found to exert a suppressive effect on the A 23187-induced release of [3H]GABA, while having no significant effect on A 23187-stimulated 45Ca2+ uptake. On the other hand, both depolarization-induced 45Ca2+ uptake and [3H]GABA release were inhibited by pressure exposure. These results suggest that pressure may suppress GABA release by affecting pre-synaptic events subsequent to calcium influx.

Effects of high pressure on the release of excitatory amino acids by brain synaptosomes.

Excitatory amino acid antagonists have been shown to protect against the hyperexcitability associated with exposure to high pressure. This suggests that these excitatory neurotransmitter substances may play a role in the development of the symptoms of high pressure nervous syndrome (HPNS). Using a superfusion technique, we investigated the effect of exposure to 67.7 ATA of pressure on the release of aspartate and glutamic acid by isolated presynaptic nerve terminals from the guinea pig cerebral cortex. Pressure exposure was found to significantly increase the depolarization-induced release of aspartate by these synaptosomes. On the other hand, compression to 67.7 ATA had no effect on glutamic acid release. These findings suggest that increased aspartate release may be a contributing factor in the etiology of HPNS.

Regional amino acid concentration in the brains of rats exposed to high pressures.

Regional amino acid concentrations were measured in rat brain fixed by microwave irradiation at three levels of elevated atmospheric pressure corresponding to different phases of the high-pressure neurological syndrome [20 atmospheres absolute (ATA), no clinical signs; 60 ATA, tremor; 85 ATA, severe tremor and myoclonic jerks]. No changes in amino acid content occurred at 20 or 60 ATA. At 85 ATA glutamine content increased in hippocampus, striatum, cerebellum, and substantia nigra, and gamma-aminobutyric acid content increased in hippocampus. It is suggested that enhanced glutamate release in various subcortical structures contributes to the myoclonic activity observed at 85 ATA.