4107
INTRODUCTION
Stress and routine activities such as feeding or vigorous swimming
can lead to increased blood and tissue ammonia concentrations in
fish (Ortega et al., 2005; Wicks and Randall, 2002; Wang et al.,
1994b), in addition to exposure to high ambient concentrations of
ammonia arising from anthropogenic or natural sources (Randall
and Tsui, 2002). Typical ammonia concentrations are usually less
than 10 mol l
–1
in natural freshwaters (Environment Canada and
Health Canada, 2001), but concentrations may approach 0.5 mmol l
–1
or more in highly eutrophic waters or waters receiving ammonia-
contaminated municipal, industrial or agricultural effluents
(Environment Canada and Health Canada, 2001; Eddy, 2005).
Increased internal ammonia, or hyperammonemia, can also result
from the degradation of food or crowding in aquaculture facilities.
Although fish are more tolerant to ammonia than mammals, their
susceptibility to ammonia’s neurotoxic effects varies widely among
different species (Ip et al., 2001; Randall and Tsui, 2002; Eddy,
2005). In mammals, toxic increases in ammonia, often caused by
acute liver failure or in-born errors of urea metabolism, lead to
seizures, coma and eventually death (Felipo and Butterworth,
2002). Fishes exhibit similar symptoms to acutely toxic levels of
ammonia including hyperventilation and hyper-excitability, followed
by convulsions, coma and death (Eddy, 2005).
The injurious cascade of neurophysiological responses that
characterize ammonia toxicity are similar in many respects to those
associated with anoxic/ischemic injury. It is well known that anoxia
The Journal of Experimental Biology 214, 4107-4120
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jeb.057513
RESEARCH ARTICLE
The relationship between NMDA receptor function and the high ammonia tolerance
of anoxia-tolerant goldfish
Michael P. Wilkie
1,
*, Matthew E. Pamenter
2,†
, Stephanie Duquette
1
, Hadi Dhiyebi
1
, Navjeet Sangha
1
,
Geoffrey Skelton
1
, Matthew D. Smith
1
and Leslie T. Buck
2
1
Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada, N2L 3C5 and
2
Department of Cell and Systems Biology,
University of Toronto, Toronto, ON, Canada, M5S 3G5
*Author for correspondence (mwilkie@wlu.ca)
†
Present address: Department of Pediatrics, University of California San Diego, La Jolla, CA 92093-0735, USA
Accepted 30 August 2011
SUMMARY
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 mol l
–1
and 4132 mol 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
Amm
] to ~1300 mol l
–1
and 3-fold increases in brain [T
Amm
] to 6700 mol l
–1
. Muscle [T
Amm
] increased by
almost 8-fold from ~900 mol kg
–1
wet mass (WM) to greater than 7000 mol 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 mol kg
–1
WM to 15,000 mol 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.
Key words: ammonia toxicity, excitotoxicity, glutamate NR1 receptor, glutamine, methionine sulfoximine, MK801, urea.
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