Hypoxia-induced silencing of NMDA receptors in turtle neurons

Abstract

Hypoxia-induced suppression of NMDA receptors (NMDARs) in western painted turtle (Chrysemys picta) cortical neurons may be critical for surviving months of anoxic dormancy. We report that NMDARs are silenced by at least three different mechanisms operating at different times during anoxia. In pyramidal neurons from cerebrocortex, 1-8 min anoxia suppressed NMDAR activity (Ca(2+) influx and open probability) by 50-60%. This rapid decrease in receptor activity was controlled by activation of phosphatase 1 or 2A but was not associated with an increase in [Ca(2+)](i). However, during 2 hr of anoxia, [Ca(2+)](i) in cerebrocortical neurons increased by 35%, and suppression of NMDARs was predicted by the increase of [Ca(2+)](i) and controlled by calmodulin. An additional mechanism of NMDAR silencing, reversible removal of receptors from the cell membrane, was found in cerebrocortex of turtles remaining anoxic at 3 degrees C for 3-21 d. When suppression of NMDARs was prevented with phosphatase inhibitors, tolerance of anoxia was lost. Silencing of NMDARs is thus critical to the remarkable ability of C. picta to tolerate life without oxygen.

Fig. 1.

Comparison of Chrysemyscerebrocortical neurons and rat CA1 neurons. A, Differences in viability of turtle and rat neurons 5 hr after bouts of anoxia. *p < 0.05 denotes significant difference between turtle and rat groups (ANOVA). B, Anoxia protects turtle neurons from NMDA neurotoxicity, but not when NMDA receptor activity is stimulated with 0.1 nm forskolin or 0.5 μm okadaic acid. *p < 0.05 denotes significant difference from control (Dunnett's test).Numbers above bars indicate number of slices studied, and error bars show 1 SEM.

Fig. 2.

Decrease in NMDA receptor activity during anoxia.A, Decrease in mean NMDA receptor open probability in cell-attached patches during 90 min of anoxia. Patch pipettes contained 10 μm NMDA. *p < 0.05 denotes significant decrease compared with normoxic control (Dunnett's test). Error bars show 1 SEM; n = 6–8 for each data point. B, Examples of [Ca²⁺]_i changes produced by 100 μm NMDA (arrows) in cortical sheets in normoxic and anoxic conditions and after recovery from anoxia.C, Mean NMDA receptor activity (NMDA Δ Ca²⁺) in cortical sheets during anoxia and recovery.D, Example of the decrease in NMDA receptor activity observed during anoxia in dissociated cortical neurons. NMDA (100 μm) was applied for 10 sec periods.

Fig. 3.

Changing abundance of NMDA receptors during anoxia and recovery. Top portion shows representative Western immunoblots of NR1 NMDA subunits. Bottom graph shows changes in receptor abundance during 3 hr to 3 weeks of anoxia (submergence in 3°C anoxic water) and recovery.

Fig. 4.

Role of phosphatases in the regulation of NMDA receptors during anoxia. A, Example showing that the nonspecific phosphatase inhibitor okadaic acid prevents NMDA receptor inactivation in a dissociated pyramidal neuron during anoxia.B, Phosphatase inhibitors prevent receptor silencing in cortical sheets during anoxia. Bars show mean NMDA Δ Ca²⁺ responses during normoxia, anoxia, and anoxia with 1 μm calyculin or 120 nm okadaic acid.Numbers above bars indicate n values. *p < 0.05 compared with oxic control (Dunnett's test). C, Decreases in NMDA receptor open probability during anoxia are prevented with calyculin A (1 μm). *p < 0.05 denotes significant difference from control (Dunnett's test); n = 9, both groups.D, Calyculin does not affect NMDA receptor open probability with oxygen available; n = 5.

Fig. 5.

[Ca²⁺]_i predicts NMDA receptor activity in both oxic and anoxic neurons in cortical sheets. A, Two hours of in vitro anoxia increased [Ca²⁺]_i by 32% (70 nm). B, Significant correlation of [Ca²⁺]_i to NMDA receptor activity in both oxic or anoxic neurons. Lines show least-squares linear regression for each group of neurons.

Fig. 6.

Regulation of NMDA receptor activity during anoxia by Ca²⁺-dependent processes. A, Inhibition of the Ca²⁺ binding protein calmodulin reduced NMDA receptor silencing during anoxia. Cortical sheets were treated with 10 nm calmidazolium 30 min before and during 2 hr anoxia or with O₂ present. NMDA Δ Ca²⁺ was then measured. B, The calcineurin inhibitor cypermethrin, at two concentrations, did not prevent NMDA receptor silencing during anoxia. C, Effects of increasing PKA activity with forskolin or inhibiting it with H8 on NMDA receptor activity during normoxia or anoxia. Test compounds were present during the 2 hr period before assay of receptor activity. For all graphs, bars show means ± SEM, and numbersare number of observations; and *p < 0.05 denotes significant difference compared with control condition within each group (ANOVA).