Purinergic and Cholinergic Drugs Mediate Hyperventilation in Zebrafish: Evidence from a Novel Chemical Screen

Abstract

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O2 chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (fV) maximally at 100 μM (EC50 = 24.5 μM). Hypoxia elevated fV from 93.8 to 145.3 breaths min-1. Atropine reduced the hypoxic response only at 100 μM. Suramin and A-317491 maximally reduced fV at 50 μM (EC50 = 30.4 and 10.8 μM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O2-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O2 sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.

Hif-1α paralogs play a role in the hypoxic ventilatory response of larval and adult zebrafish (Danio rerio)

“…Consistent with other studies on larval zebrafish (Rahbar et al, 2016;Coe et al, 2017), older larvae (10-15 dpf ) and adults rapidly returned f V to normoxic levels after a bout of hypoxia. In contrast, f V remained significantly elevated at 4 and 7 dpf during recovery in wild-type zebrafish, whereas in the Hif-1A −/− and Hif-1B −/− larvae at 4 and 7 dpf, f V returned to normoxic levels rapidly following hypoxia.…”

Automated Non-invasive Video-Microscopy of Oyster Spat Heart Rate during Acute Temperature Change: Impact of Acclimation Temperature

“…Indeed a recent report in the adult oyster suggests that biosensors could monitor HR and health in “sentinel” animals (Andrewartha et al, 2016 ). This concept is similar to those proposed for other species, such as zebrafish, as biological models or pharmacologic screens (Liu et al, 2016 ; Rahbar et al, 2016 ). HRV has been used to explore cardiac regulatory mechanisms, which could be employed to assess cardiac phenotype between triploid and diploid spat or other marine invertebrates.…”

A role for dopamine in control of the hypoxic ventilatory response via D2 receptors in the zebrafish gill

“…Curves for dose versus response were prepared to display concentration-dependent trends and to estimate the effective concentration of each drug that gave a half-maximal f v response (EC 50 ). Percent maximum f v for domperidone (at 300 μM) and percent maximum inhibition for quinpirole (at 150 μM) were calculated to normalize the data following procedures described in Rahbar et al (2016) and Coe et al (2017). For estimation of EC 50 , a line constrained from the origin at zero to the maximal response was fit to the data using a nonlinear ligand binding model with least squares following the equation: y = b max ⋅x h /(k d h + x h ) (Prism v5.0, RRID: SCR_002798; GraphPad Software Inc.).…”