Circadian Clock Gene Expression in Regions of Interest to Respiratory Control

Abstract

Circadian rhythms are endogenous, ~24‐hour cycles that synchronize biological functions to external cues known as zeitgebers. Circadian rhythms emerge from self‐sustaining oscillations in a transcription‐translation feedback loop of core clock genes (Bmal1, Clock, Per1 and Per2). The central clock (suprachiasmatic nucleus) and peripheral tissues throughout the body utilize this same circadian machinery. Core clock components also exert tissue‐specific effects, including tissue‐appropriate gene regulation. Although clock gene networks have been established throughout the body, little is known concerning clock genes in the neuromuscular system regulating breathing. We tested the hypothesis that clock genes oscillate in key regions of the respiratory circuit by characterizing mRNA expression patterns of core clock genes in the medulla, ventral spinal cord and diaphragm throughout the day via qPCR. Samples were collected from Sprague Dawley rats entrained to a 12‐hour light/dark cycle at 4 zeitgeber times (ZT), including (n = 8 per group): ZT5 (11am), ZT11 (5pm), ZT17 (11pm) and ZT23 (5am); ZT0 corresponds to beginning of light cycle. Cosinor analysis revealed that Bmal1, Clock, Per1 and Per2 clock genes were rhythmic in all 3 regions (all p<0.01). The expression pattern of Per1 and Per2 was antiphase to Bmal1 and Clock in all regions. We hypothesize that clock genes may influence the potential to elicit serotonin‐dependent respiratory motor plasticity, such as intermittent‐hypoxia‐induced phrenic long‐term facilitation. Thus, we tested the specific hypotheses that BMAL1 protein is expressed within phrenic motor neurons and molecules necessary for phrenic long‐term facilitation (5‐HT2A receptors, BDNF and TrkB) exhibit rhythmic gene expression in brainstem and spinal regions involved in this form of plasticity. Using immunofluorescence, we demonstrate that the clock protein BMAL1 is expressed within cholera toxin B labelled phrenic motor neurons. The plasticity‐related gene Bdnf was expressed rhythmically in medulla and in ventral spinal (C3–C5) homogenates (p<0.001), peaking in the active phase. TrkB mRNA (Ntrk2) was expressed rhythmically within the medulla (p<0.01), but not the ventral spinal cord. 5‐HT2A receptor mRNA (Htr2a) did not exhibit rhythmic expression in either region. In conclusion, daily rhythms are observed in transcripts from clock genes and molecules necessary and sufficient for phrenic long‐term facilitation (e.g. BDNF). Although we did not uncouple cellular clocks from zeitgebers (light/dark) in this initial study, we confirm the potential for local circadian regulation in regions of interest to respiratory motor control.Support or Funding InformationUniversity of Florida Graduate School Preeminence Award (MNK), NIH T32 HD043730 (MDS) and the McKnight Brain Institute (GSM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.