The Role of Chloride in the Breathing‐related Response to Central Acidosis in Developing Birds

Abstract

Rhythmic spontaneous neural activity (rSNA) is thought to contribute to the organization and development of motor circuits. In spinal motor networks, rSNA has a role in synapse refinement, axon trajectory, and the formation of neuromuscular junctions. In the zebra finch embryo, extracellular recordings of hindbrain breathing‐related motor activity also show that rSNA burst pattern, shape, and neurotransmitter signaling change in an age‐dependent manner. Further, the role of chloride‐mediated neurotransmission exhibits a switch in polarity such that GABAergic and glycinergic signaling transitions from excitatory to inhibitory over the course of 24–48 hours. Despite recognizing these ontogenetic changes in hindbrain rSNA, we are just beginning to explore whether the changing physiochemical environment within the eggshell shapes rhythmic motor activity. As a bird embryo grows within the egg, it experiences progressive alterations in respiratory gases (↓PO2, ↑PCO2), an emerging acidosis, as well as modifications to the anatomical structures that facilitate gas exchange. Data show that lowering and raising the pH of the superfusate bathing the isolated avian hindbrain influences breathing‐linked rSNA as a function of age. Embryos employing diffusive gas exchange within the egg show a decreased rate of rSNA, while freely breathing birds employing pulmonary gas exchange show an increased rate of cranial nerve motor output. Given these findings, our objective here was to test mechanisms contributing to this pH response. We speculate that chloride is an important player as a result of three observations: 1) chloride‐mediated neurotransmission shifts in polarity at nearly the same embryonic age as the switch in the rSNA response to low pH; 2) chloride transport is involved in pH homeostasis; 3) GABAA receptors conduct both chloride and hydrogen carbonate, which are tied to pH regulation. To explore the first point, we recorded rSNA and breathing‐related motor rhythms while lowering pH under chloride free conditions. Results show that the removal of chloride eliminates rSNA in embryonic days (E) 4–6 embryos. On E7‐10, chloride removal decreases the rate of motor output compared with low pH alone. Related to the second point, we treated the hindbrain with low pH and bumetanide, an antagonist for the transmembrane sodium‐potassium‐chloride cotransporter (NKCC1). Data show that, compared to controls, the instantaneous frequency increases in E7‐10 birds. To address the third point, we applied the GABAA receptor antagonist picrotoxin (PTX) during low pH treatments. Results show that PTX and low pH treatment decreases rSNA frequency to 49 ± 11% of control in <E11 birds and increases frequency to 129 ± 20% of control at >E11. Together, our results suggest that interfering with chloride gradients and blocking GABAA receptor activity during low pH treatment has consequences for rSNA that are similar to lowering pH alone. In contrast, the inhibition of NKCC1 with bumetanide seems to disrupt the rhythmic response to central acidosis in the zebra finch hindbrain, at least prior to the onset of breathing.Support or Funding InformationIdaho IDeA Network of Biomedical Research and Excellence Program Grant # P20 GM103408 and the National Institute of Neurological Disorders and Stroke via the AREA Program Grant # 1R15NS087521‐01A1.