Thyroid hormone supplementation dampens GABAergic inhibition of the brainstem respiratory network in newborn rat

Abstract

We previously showed that thyroid hormones (THs) deficiency potentiates GABAergic inhibition in the core elements of the respiratory control circuit. To understand the underlying mechanisms, we tested the hypotheses that 1) cellular mechanisms including K+/Cl− co‐transporters contribute to the delay of GABAergic system development in TH deficient newborn rats and 2) TH supplementation following birth can reverse this enhanced inhibition. Thyroid hormone deficiency in the foetus was recreated by administrating an antithyroid susbstance (Methimazole; MMI) in drinking water of the pregnant dam at concentration of 0,02% (weight per volume; w/v) from the first day of pregnancy (gestational day 1; GD1) to the day of experiment with the newborn. TH supplementation was performed by injecting newborn rats (1 day old) intraperitoneally with Levothyroxine (L‐T4). Experiments were done on Sprague Dawley rats aged of 4 days. Protein levels of newborn co‐transporters were evaluated by western blot analysis. Each brainstem lysate samples were migrated on SDS acrylamide gel under denaturing conditions. Gel was then transferred on nitrocellulose membrane and blocked in 5% non‐fat dry milk in TBS tween. Membranes were incubated overnight with primary antibody for NKCC1 (Cell signaling) or KCC2 (Cell signaling) followed by 1h exposure to secondary antibody (Licor IRDye 800CW). Fictive breathing was measured by extracting the brainstem with a part of the spinal cord and placing it in a chamber where it was superfused with an artificial cerebrospinal fluid (aCSF). The output signal from the respiratory network was recorded by a suction electrode placed on the fourth ventral root representing the inspiratory signal sent to the diaphragm via the phrenic nerve. Brainstems were then exposed to GABAA receptor agonist (muscimol) or antagonist (bicuculline) for 20 minutes and respiratory frequency response was observed. Data show that NKCC1 and KCC2 protein expressions were similar between MMI‐treated and control groups (MMI vs Control: NKCC1 p=0.5260; KCC2 p=0.3309), but L‐T4 supplementation in TH deficient pups increased both co‐transporters protein expression (MMI/L‐T4 vs MMI: NKCC1 p=0.0044; KCC2 p<0.0001). In agreement with these results, L‐T4 supplementation decreased respiratory frequency response to muscimol in MMI‐treated pups (MMI/L‐T4 vs MMI: p<0.0001). This suggests the involvement of different mechanisms in the potentiation of GABAergic inhibition by TH deficiency with a specific action of TH supplementation on GABAergic system development. These results provide new hypotheses regarding the origins of respiratory disorders of the newborn involving reduced respiratory drive such as apnea of prematurity.Support or Funding InformationSupported by NSERCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.