RNASeq‐derived transcriptome comparisons reveal neuromodulatory deficiency in the CO₂ insensitive brown Norway rat

Abstract

Key pointsr Increases in carbon dioxide (CO 2 ) provide a major chemical stimulus to breathe through activation of the ventilatory CO 2 chemoreflex, which is heavily influenced by the brainstem serotonergic (5-HT) system. r Brown Norway (BN) rats have an inherent and extremely low ventilatory sensitivity to hypercapnia, which can be augmented with selective serotonin reuptake inhibition.r Using mRNA sequencing, we show that BN rats have reduced medullary raphé expression of multiple 5-HT neuron-specific genes, predictive of lower monoamine levels by informatics pathway analyses and confirmed by high-performance liquid chromatography measurements.r BN rats also showed reduced thyrotropin-releasing hormone (TRH) expression, where injections of the TRH analogue Taltirelin caused greater increases in baseline ventilation, body temperature and the ventilatory CO 2 chemoreflex in BN rats compared to control Sprague-Dawley rats.r These data establish a molecular basis of a neuromodulatory deficiency in BN rats, and further suggest an important functional role for TRH signalling in the mammalian CO 2 chemoreflex.Abstract Raphé-derived serotonin (5-HT) and thyrotropin-releasing hormone (TRH) play important roles in fundamental, homeostatic control systems such as breathing and specifically the ventilatory CO 2 chemoreflex. Brown Norway (BN) rats exhibit an inherent and severe ventilatory insensitivity to hypercapnia but also exhibit relatively normal ventilation at rest and during other conditions, similar to multiple genetic models of 5-HT system dysfunction in mice. Herein, we tested the hypothesis that the ventilatory insensitivity to hypercapnia in BN rats is due to altered raphé gene expression and the consequent deficiencies in raphé-derived neuromodulators such as TRH. Medullary raphé transcriptome comparisons revealed lower expression of multiple 5-HT neuron-specific genes in BN compared to control Dahl salt-sensitive rats, predictive of reduced central nervous system monoamines by bioinformatics analyses and confirmed by high-performance liquid chromatography measurements. In particular, raphé Trh mRNA and peptide levels were significantly reduced in BN rats, and injections of the stable TRH analogue Taltirelin (TAL) stimulated breathing dose-dependently, with greater effects in BN versus control Sprague-Dawley rats. Importantly, TAL also effectively normalized the ventilatory CO 2 chemoreflex in BN rats, but TAL did not affect CO 2 sensitivity in control Sprague-Dawley rats. These data establish a molecular basis of the neuromodulatory deficiency in BN rats, and further suggest an important functional role for TRH signalling in the mammalian CO 2 chemoreflex.

Raphe gene expression changes implicate immune-related functions in ventilatory plasticity following carotid body denervation in rats

“…Upon completion of physiological protocols, the rats were deeply anesthetized (20% isoflurane in propylene glycol) and upon respiratory arrest the brain extracted, embedded with embedding media and flash frozen and stored (−80 °C) until being placed into a chilled stainless steel matrix for ~1 mm serial sectioning (Puissant et al, 2015). Frozen tissue punches (~0.85 mm in diameter) were collected from the ventral midline from 2 serial sections beginning with the one containing Obex as previously described (Puissant et al, 2015), and stored in Eppendorf tubes (−80 °C) until RNA extraction.…”

“…Frozen tissue punches (~0.85 mm in diameter) were collected from the ventral midline from 2 serial sections beginning with the one containing Obex as previously described (Puissant et al, 2015), and stored in Eppendorf tubes (−80 °C) until RNA extraction.…”

“…Total RNA samples were processed and made into cDNA libraries per manufacturer instruction (Illumina) for mRNA sequencing (HiSeq 2000) as described previously. Sequence data were aligned to the rat reference genome for identification and quantification of transcripts, and differential expression determined using an in-house pipeline described previously (Puissant et al, 2015; Liu et al, 2014). …”

“…Herein we utilized mRNA Sequencing (Puissant et al, 2015) (RNASeq) to measure gene expression changes in the 5-HT neuron-rich medullary raphe nuclei at two time points after CBD in rats to gain insights into mechanisms driving the restoration of breathing following CBD. Here we tested the hypothesis that the restoration of eupneic breathing acutely or chronically after CBD depends on 5-HT-specific and/or glutamate receptor-dependent changes in medullary raphe gene expression.…”

Going to WAR: using a rat model of audiogenic seizure to uncover potential links to ventilatory dysfunction in epilepsy

“…It appears that WAR rats have fewer (presumptive) chemosensitive neurons along with general reductions in serotonergic and other excitatory neuromodulatory signalling within the respiratory network, which distinguishes the WAR model from other inbred rat strains with inherently reduced CO 2 sensitivity and reduced serotoninergic function (Puissant et al . ).…”