The relationship between NMDA receptor function and the high ammonia tolerance of anoxia-tolerant goldfish

Abstract

Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-Daspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96h median lethal concentration (LC 50) values of 199 μmoll -1 and 4132 μmoll -1 for NH 3 and total ammonia ([TAmm] [NH 3]+[NH 4+]), respectively. These values were ∼5-6 times greater than corresponding NH 3 and T Amm LC 50 values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH 4Cl (3-5mmoll -1) for 5days, despite 6-fold increases in plasma [T Amm] to ∼1300 μmoll -1 and 3-fold increases in brain [T Amm] to 6700 μmoll -1. Muscle [T Amm] increased by almost 8-fold from ∼900 μmolkg -1 wet mass (WM) to greater than 7000 μmolkg -1 WM by 48h, and stabilized. Although urea excretion rates (J Urea) increased by 2-3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ∼3000 μmolkg -1 WM to 15,000 μmolkg -1 WM after 48h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5-8mgkg -1) or ethanol (1-8mgkg -1) increased trout survival time by 1.5-2.0-fold during exposure to 2mmoll -1 ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8-9.5mmoll -1 T Amm) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro exposure to 5mmoll -1 or 10mmoll -1 T Amm reversibly potentiated NMDA receptor currents. This observation suggested that goldfish neurons may not be completely resistant to ammonia-induced excitotoxicity. Subsequent western blot and densitometric analyses revealed that NMDA receptor NR1 subunit abundance was 40-60% lower in goldfish exposed to 3-5mmoll -1 T Amm for 5days, which was followed by a restoration of NR1 subunit abundance after 3days recovery in ammonia-free water. We conclude that the goldfish brain may be protected from excitotoxicity by downregulating the abundance of functional NMDA receptors during periods when it experiences increased internal ammonia. © 2011. Published by The Company of Biologists Ltd.