Respiratory responses to external ammonia in zebrafish (Danio rerio)

Comp Biochem Physiol A Mol Integr Physiol. 2021 Jan:251:110822. doi: 10.1016/j.cbpa.2020.110822. Epub 2020 Oct 13.

Abstract

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 μM (NH4)2SO4 caused increases in ventilation amplitude (AVENT) without affecting frequency (fVENT), resembling the ventilatory response to hypercapnia rather than hypoxia, during which fVENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca2+] in response to 1 mM NH4Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in fVENT (AVENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the fVENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels.

Keywords: CRISPR/Cas9 knockout; Calcium imaging; Chemoreception; Immunohistochemistry; Morpholino knockdown; Ventilation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ammonia / metabolism
  • Ammonia / pharmacology*
  • Animals
  • Blood Proteins / metabolism
  • Calcium / metabolism
  • Cation Transport Proteins / metabolism
  • Gills / cytology
  • Gills / drug effects
  • Gills / metabolism
  • Hyperventilation / physiopathology*
  • Immunohistochemistry
  • Larva / cytology
  • Larva / drug effects
  • Larva / metabolism
  • Membrane Glycoproteins / metabolism
  • Neuroepithelial Cells / drug effects
  • Neuroepithelial Cells / metabolism
  • Respiratory Physiological Phenomena / drug effects*
  • Zebrafish / genetics
  • Zebrafish / metabolism
  • Zebrafish / physiology*
  • Zebrafish Proteins / metabolism

Substances

  • Blood Proteins
  • Cation Transport Proteins
  • Membrane Glycoproteins
  • Rhag protein, zebrafish
  • Rhbg protein, zebrafish
  • Zebrafish Proteins
  • rhcgb protein, zebrafish
  • Ammonia
  • Calcium