Switching hypoxia tolerance strategies in a brainstem network of bullfrogs, Lithobates catesbeianus

Abstract

Strategies to survive brain anoxia vary widely among vertebrates. Along a spectrum, some animals arrest neuronal activity to conserve ATP, while others can sustain neuronal functions during energetic stress. The neural circuit that produces breathing in the adult American bullfrog, Lithobates catesbeianus, is one example of a network that arrests activity during anoxia. This response is considered an energy saving arrest because 1) a hypoxia sensor that inhibits the network through noradrenergic signaling appears to contribute, and 2) activity recovers after anoxia. Unexpectedly, we found that this network becomes remarkably functionally tolerant to hypoxia after frogs emerge from 3–5 weeks of cold‐acclimation. Normally, control networks fail to produce rhythmic activity after 10.1±6.4 minutes of hypoxia (0% O2) at 22°C (n=9); however, networks at 22°C from cold‐acclimated frogs continue to produce coordinated respiratory bursts for about 16 times longer (159.1 ±42.2 minutes; n=5; p=0.001; Mann‐Whitney U test), representing a shift from an arrest strategy to functional tolerance. Thus, the transition may reflect a reduction in the hypoxia sensor (noradrenergic signaling) and/or a shift in energy use/metabolism. To begin to address these possibilities, we sought to identify if noradrenergic signaling is the sole cause of the arrest in control networks. To test this, we exposed in vitro brainstem preparations to norepinephrine and then hypoxia. We expected that pretreatment with norepinephrine would occlude the arrest during hypoxia if noradrenergic signaling was part of the oxygen sensor. However, this did not occur; preparations stopped activity over a similar time course during hypoxia when bathed with (n=6) or without (n=9) norepinephrine, implying the arrest does not involve noradrenergic signaling. We then hypothesized that metabolic deficits may contribute. To test this, we applied inhibitors of glycolysis [2‐deoxy‐D‐glucose substituted for D‐glucose (n=5) or 1 mM iodoacetate (n=4)] or mitochondrial respiration [1 mM sodium cyanide (n=5)] in baseline O2 tensions. Each inhibitor recapitulated the actions of hypoxia, demonstrating that deficits in ATP synthesis, regardless of oxygen tensions, produce an arrest. Cellular energy sensors, such as KATP channels and AMP‐dependent protein kinase, are known to reduce neuronal activity when ATP falls; however, activation of these mechanisms pharmacologically failed to arrest activity [KATP activator; 600 μM Diazoxide (n=5], AMPK activator; 1 mM AICAR (n=4)]. In sum, we hypothesize that deficits in ATP synthesis stop network output during hypoxia and that improvements in metabolic function or energy use contribute to the striking shift to functional hypoxia tolerance after cold‐acclimation.Support or Funding InformationThis work was supported by start‐up funds to JS provided by UNC‐Greensboro.