The Enigma of the Dichotomic Pressure Response of GluN1-4a/b Splice Variants of NMDA Receptor: Experimental and Statistical Analyses

Abstract

Professional deep-water divers, exposed to hyperbaric pressure (HP) above 1.1 MPa, develop High Pressure Neurological Syndrome (HPNS), which is associated with central nervous system (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 analysis of variance (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.

Keywords: CNS hyperexcitability; HPNS; diving physiology; oocyte; pressure.

Figure 1

Different hyperbaric pressure (HP) effects on GluN1-4a/b splice variants. GluN1-4a (A) and GluN1-4b (B) splice variants were co-expressed with the GluN2A subunit. Currents for each of the splice variants were either augmented (lower traces) or decreased (upper traces) at HP. For all experiments the applied co-agonists were glutamate (100 μM) and glycine (10 μM) with no [Mg²⁺]_o added. The 20 s agonists application time is indicated by horizontal bars. The HP effects are reversed after full decompression for all subtypes.

Figure 2

(A) cRNA-quality check. Single bands indicate stable cRNA (no fragmentation) (B) Correlations between amplitude and I/V slope changes at HP. Percent changes (%Δ) between HP and controls. Each point represents one subunit combination (either GluN1-4a or -4b co-expressed with GluN2A) with its SEM for amplitude change and corresponding I/V slope change that exhibited increased (2 points) or corresponding decreased (2 points) response. The slope of about 0.95 suggests that the change in input conductance is sufficient to explain the current amplitude change.

Figure 3

HP effects on glutamate dose-response. GluN1-4a/b were co-expressed with GluN2A. There were no significant changes in the dose-response curve and the EC₅₀ at HP in both N-methyl-D-aspartate receptor (NMDAR) subtypes. The result for each condition were averaged (n = 6) and dose-response curve fitting was performed. The GluN1-4b graph has been published earlier in Bliznyuk et al. (2015).

Figure 4

Clustering analysis of all data (n = 73) when k = 2. Histograms are presented on the left (A) and the corresponding silhouette values on the right (B). Silhouette values higher than 0.5 are considered significant.

Figure 5

Alpha function distribution analysis. (A) Distribution of the negative (decreased) response group. (B) Distribution of the positive (increased) response group. Note the values of the Alpha function at the bottom of each histogram.