Oxygen Sensing Neurons and Neuropeptides Regulate Survival after Anoxia in Developing C. elegans

Abstract

Hypoxic brain injury remains a major source of neurodevelopmental impairment for both term and preterm infants. The perinatal period is a time of rapid transition in oxygen environments and developmental resetting of oxygen sensing. The relationship between neural oxygen sensing ability and hypoxic injury has not been studied. The oxygen sensing circuitry in the model organism C. elegans is well understood. We leveraged this information to investigate the effects of impairments in oxygen sensing on survival after anoxia. There was a significant survival advantage in developing worms specifically unable to sense oxygen shifts below their preferred physiologic range via genetic ablation of BAG neurons, which appear important for conferring sensitivity to anoxia. Oxygen sensing that is mediated through guanylate cyclases (gcy-31, 33, 35) is unlikely to be involved in conferring this sensitivity. Additionally, animals unable to process or elaborate neuropeptides displayed a survival advantage after anoxia. Based on these data, we hypothesized that elaboration of neuropeptides by BAG neurons sensitized animals to anoxia, but further experiments indicate that this is unlikely to be true. Instead, it seems that neuropeptides and signaling from oxygen sensing neurons operate through independent mechanisms, each conferring sensitivity to anoxia in wild type animals.

Preconditioning ofCaenorhabditis elegansto Anoxic Insult by Inactivation of Cholinergic, GABAergic, and Muscle Activity

“…Since the preconditioning phenomena has been described throughout the animal kingdom, insight into this physiological response in a genetically tractable organism may have broad application. [39][40][41] One salient feature of our investigations here and in prior publications (Flibotte et al, 2014 andDoshi et al,2019) is variability of survival after a 48-hour anoxic insult.…”

“…Two days later L4 stage animals were collected and assayed for survival after anoxic exposure. Protocol and details described by Theresa Stiernagle in Wormbook chapter entitled Maintenance of C. elegans and used in previous studies to synchronize C. elegans for hypoxia assays in Flibotte et al ., 2014 and Doshi et al ., 2019. 13,18,24…”

“…For anoxic insult, 30 mid L4 stage animals per genotype were picked to an NGM plate, which was placed in a Bio-Bag (Type A anaerobic environmental system, Becton-Dickinson Company, Franklin Lakes, New Jersey), anoxic conditions were induced and maintained for 48 hours at 20°C as described previously. 13,25 Bags were then opened, and animals were allowed to recover in ambient oxygen for 24 hours before being scored for survival. Surviving animals were identified as those that moved spontaneously or after gentle prodding with a platinum wire.…”

“…elegans prefers oxygen between 5 and 12% 11 and has the ability to sense and respond to shifts in oxygen that fall outside the preferred range. 12,13 C. elegans are capable of surviving low oxygen stress and use a variety of pathways to achieve this depending on the degree and duration of oxygen deprivation. [14][15][16][17] We found that ablation of the oxygen-sensing BAG (but not the URX) neurons rendered animals resistant to an anoxic insult.…”

“…[14][15][16][17] We found that ablation of the oxygen-sensing BAG (but not the URX) neurons rendered animals resistant to an anoxic insult. 13 We postulated the neural circuit in which the BAG neuron was embedded secreted a factor(s) that heightened peripheral tissue sensitivity to anoxia. This hypothesis was derived from the observations that: 1) inhibition of neuropeptide processing (e.g., C. elegans with egl-3 or egl-21 mutation) and secretion (e.g., C. elegans with unc-31 mutation) protected against anoxia, 2) nervous systemspecific rescue of egl-3 expression in egl-3 mutant animals restored sensitivity to anoxia, and 3) we identified one neuropeptide nlp-40 and its receptor aex-2 that are likely to be involved in this process.…”