Yoram Grossman

Professional divers who are exposed to high pressure (HP) above 1.1 MPa suffer from high pressure neurological syndrome (HPNS), which is characterized by reversible CNS hyperexcitability and cognitive and motor deficits. HPNS remains the final major constraints on deep diving at HP. Prolonged and repetitive exposure to HP during deep sea saturation dives may result in permanent memory and motor impairment. Previous studies revealed that CNS hyperexcitability associated with HPNS is largely induced by N-methyl-D-aspartate receptors (NMDARs). NMDARs that contain the GluN2A subunit are the only ones that show a large (∼60%) current increase at He HP. NMDAR subtypes that contain other GluN2 members show minor decrease or no change of the current. Immunoprecipitation was used in order to test the hypothesis that current augmentation may result from inserting additional NMDARs into the membrane during the 20–25 min compression. The results indicated that there is no increase in surface expression of NMDARs in the oocyte membrane under HP conditions. In contrast, consistent increase in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin was discovered. GAPDH and β-actin are cytosolic proteins which involve in various cellular control processes, increase of their expression suggests the presence of a general cellular stress response to HP. Understanding the precise hyperexcitation mechanism(s) of specific NMDAR subtypes and other possible neurotoxic processes during HP exposure could provide the key for eliminating the adverse, yet reversible, short-term effects of HPNS and hopefully the deleterious long-term ones.

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.

Divers that are exposed to high pressure (HP) above 1.1 MPa suffer from High Pressure Neurological Syndrome (HPNS), which is implicated with central nervous system (CNS) malfunction. Marine mammals performing extended and deep breath-hold dives are exposed to almost 20 MPa without apparent HPNS symptoms. N-methyl-D-aspartate receptor (NMDAR) has repeatedly been implicated as one of the major factors in CNS hyperexcitability as part of HPNS. Electrophysiological studies in rat brain slices at He HP showed a significant increase in the synaptic NMDAR response, followed by postsynaptic excitability changes. Molecular studies of Rattus norvegicus NMDARs have revealed that different subunit combinations of the NMDAR exhibit different, increased or decreased, current responses under He HP conditions. The purpose of the present research was to disclose if the breath-hold deep diving mammals exhibit NMDAR structural modifications related to HP. We used sequence alignment and homology structure modeling in order to compare deep diving marine mammals’ NMDARs to those of terrestrial mammals. We discovered that deep diving mammals have a special tertiary TMD structure of the GluN2A subunit that differs from that of the terrestrial mammals. In addition, the GluN2A subunit has a group of four conserved a.a. substitutions: V68L (N-terminal domain, NTD) and V440I (agonist-binding domain, ABD) are cetacean specific, E308D (N-terminal domain, NTD) and I816V (transmembrane domain, TMD) were also singularly found in some terrestrial mammals. Since I816V is localized in M4 α-helix region, which is critical for NMDAR activation and desensitization, we hypothesize that the presence of all 4 substitutions rather than a single one, is the combination that may enable HP tolerance. Furthermore, additional special substitutions that were found in the marine mammals’ NTD may affect the Zn²⁺ binding site, suggesting less or no voltage-independent inhibition by this ion. Our molecular studies of NMDARs containing the GluN2A subunit showed that HP removal of the Zn²⁺ voltage-independent inhibition could be the mechanism explaining its current increase at HP. Thus, this mechanism could play a crucial role in the CNS hyperexcitability at HP. Less or no voltage-independent Zn²⁺ inhibition, different conformations of the TMD, and special mutation in the M4 α-helix region of cetaceans’ NMDAR, may give them the advantage they need in order to perform such deep dives without CNS malfunction.

Professional deep-water divers, exposed to hyperbaric pressure (HP) above 1.1 MPa, develop High Pressure Neurological Syndrome (HPNS), which is associated with CNS hyperexcitability. It was previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic response, increases neuronal excitability and potentially causes irreversible neuronal damage. Our laboratory has reported differential current responses under HP conditions in NMDAR subtypes that contain either GluN1-1a or GluN1-1b splice variants co-expressed in Xenopus laevis oocytes with all four GluN2 subunits. Recently, we reported that the increase in ionic currents measured under HP conditions is also dependent on which of the eight splice variants of GluN1 is co-expressed with the GluN2 subunit. We now report that the NMDAR subtype that contains GluN1-4a/b splice variants exhibited 'dichotomic' (either increased or decreased) responses at HP. The distribution of the results is not Normal thus ANOVA test and clustering analysis were employed for statistical verification of the grouping. Furthermore, the calculated constants of alpha function distribution analysis for the two groups were similar, suggesting that the mechanism underlying the switch between an increase or a decrease of the current at HP is a single process, the nature of which is still unknown. This dichotomic response of the GluN1-4a/b splice variant may be used as a model for studying reduced response in NMDAR at HP. Successful reversal of other NMDAR subtypes response (i.e. current reduction) may allow the elimination of the reversible malfunctioning short term effects (HPNS), or even deleterious long term effects induced by increased NMDAR function during HP exposure.

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 Ca2+ currents based on synaptic activity modified by HP. We have recently demonstrated that currents in genetically identified cellular voltage-dependent Ca2+ channels (VDCCs), Ca(V)1.2 and Ca(V)3.2 are selectively affected by HP. This work further elucidates the HPNS mechanism by examining HP effect on Ca2+ currents in neuronal VDCCs, Ca(V)2.2 and Ca(V)2.1, which are prevalent in presynaptic terminals, expressed in Xenopus oocytes. HP augmented the Ca(V)2.2 current amplitude, much less so in a channel variation containing an additional modulatory subunit, and had almost no effect on the Ca(V)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.

Professional deep‐water divers exposed to hyperbaric pressure (HP) above 1.1MPa develop high‐pressure neurological syndrome, which is associated with central nervous system hyperexcitability. It was previously reported thatHPaugmentsN‐methyl‐D‐aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. In addition, we have reported thatHP(10.1MPa) differentially affects ionic currents, measured by the two‐electrode voltage‐clamp technique, of eight specificNMDARsubtypes generated by the co‐expression of GluN1‐1a or GluN1‐1b with one of the four GluN2(A–D) subunits inXenopus laevisoocytes. We now report that eight GluN1 splice variants, when co‐expressed with GluN2A, mediate different ionic currents at normal andHP(5.1MPa). These data, in conjunction with our previous results, indicate that both GluN1 and GluN2 subunits play a critical role in determiningNMDARcurrents under normal andHPconditions. These data, given the differential spatial distribution of the differentNMDARsubtypes 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‐termHPhealth decrement due to repetitive deep dives by professional divers.

Professional deep sea divers experience motor and cognitive impairment, known as High Pressure Neurological Syndrome (HPNS), when exposed to pressures of 100 msw (1.1MPa) and above, considered to be the result of synaptic transmission alteration. Previous studies have indicated modulation of presynaptic Ca2+ currents at high pressure. We directly measured for the first time pressure effects on the currents of voltage dependent Ca2+ channels (VDCCs) expressed in Xenopus oocytes. Pressure selectivity augmented the current in CaV1.2 and depressed it in CaV3.2 channels. Pressure application also affected the channels' kinetics, such as ƮRise, ƮDecay. Pressure modulation of VDCCs seems to play an important role in generation of HPNS signs and symptoms.

Presynaptic Ca2+-dependent mechanisms have already been implicated in depression of evoked synaptic transmission by high pressure (HP). Therefore, pressure effects on terminal Ca2+ currents were studied in Rana pipiens peripheral motor nerves. The terminal currents, evoked by nerve or direct stimulation, were recorded under the nerve perineurial sheath with a loose macropatch clamp technique. The combined use of Na+ and K+ channel blockers, [Ca2+](o) changes, voltage-dependent Ca2+ channel (VDCC) blocker treatments and HP perturbations revealed two components of presynaptic Ca2+ currents: an early fast Ca2+ current (I-CaF), possibly carried by N-type (Ca(V)2.2) Ca2+ channels, and a late slow Ca2+ current (I-CaS), possibly mediated by L-type (Ca(V)1) Ca2+ channels. HP reduced the amplitude and decreased the maximum (saturation level) of the Ca2+ currents, I-CaF being more sensitive to pressure, and may have slightly shifted the voltage dependence. HP also moderately diminished the Na+ action current, which contributed to the depression of VDCC currents. Computer-based modeling was used to verify the interpretation of the currents and investigate the influence of HP on the presynaptic currents. The direct HP reduction of the VDCC currents and the indirect effect of the action potential decrease are probably the major cause of pressure depression of synaptic release.

We simulate the inhibition of Ia-glutamatergic excitatory postsynaptic potential (EPSP) by preceding it with glycinergic recurrent (REN) and reciprocal (REC) inhibitory postsynaptic potentials (IPSPs). The inhibition is evaluated in the presence of voltage-dependent conductances of sodium, delayed rectifier potassium, and slow potassium in five [Formula: see text]-motoneurons (MNs). We distribute the channels along the neuronal dendrites using, alternatively, a density function of exponential rise (ER), exponential decay (ED), or a step function (ST). We examine the change in EPSP amplitude, the rate of rise (RR), and the time integral (TI) due to inhibition. The results yield six major conclusions. First, the EPSP peak and the kinetics depending on the time interval are either amplified or depressed by the REC and REN shunting inhibitions. Second, the mean EPSP peak, its TI, and RR inhibition of ST, ER, and ED distributions turn out to be similar for analogous ranges of G. Third, for identical G, the large variations in the parameters’ values can be attributed to the sodium conductance step ([Formula: see text]) and the active dendritic area. We find that small [Formula: see text] on a few dendrites maintains the EPSP peak, its TI, and RR inhibition similar to the passive state, but high [Formula: see text] on many dendrites decrease the inhibition and sometimes generates even an excitatory effect. Fourth, the MN's input resistance does not alter the efficacy of EPSP inhibition. Fifth, the REC and REN inhibitions slightly change the EPSP peak and its RR. However, EPSP TI is depressed by the REN inhibition more than the REC inhibition. Finally, only an inhibitory effect shows up during the EPSP TI inhibition, while there are both inhibitory and excitatory impacts on the EPSP peak and its RR.