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-D-aspartate (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 96 h median lethal concentration (LC(50)) values of 199 μmol l(-1) and 4132 μmol l(-1) for NH(3) and total ammonia ([T(Amm)]=[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(4)Cl (3-5 mmol l(-1)) for 5 days, despite 6-fold increases in plasma [T] to ~1300 μmol l(-1) and 3-fold increases in brain [T(Amm)] to 6700 μmol l(-1). Muscle [T(Amm)] increased by almost 8-fold from ~900 μmol kg(-1) wet mass (WM) to greater than 7000 μmol kg(-1) WM by 48 h, 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 μmol kg(-1) WM to 15,000 μmol kg(-1) WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5-8 mg kg(-1)) or ethanol (1-8 mg kg(-1)) increased trout survival time by 1.5-2.0-fold during exposure to 2 mmol l(-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 (8-9.5 mmol l(-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 5 mmol l(-1) or 10 mmol l(-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 3-5 mmol l(-1) T(Amm) for 5 days, which was followed by a restoration of NR1 subunit abundance after 3 days 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.