Transcriptome of neonatal preBötzinger complex neurones in Dbx1 reporter mice

Abstract

We sequenced the transcriptome of brainstem interneurons in the specialized respiratory rhythmogenic site dubbed preBötzinger Complex (preBötC) from newborn mice. To distinguish molecular characteristics of the core oscillator we compared preBötC neurons derived from Dbx1-expressing progenitors that are respiratory rhythmogenic to neighbouring non-Dbx1-derived neurons, which support other respiratory and non-respiratory functions. Results in three categories are particularly salient. First, Dbx1 preBötC neurons express κ-opioid receptors in addition to μ-opioid receptors that heretofore have been associated with opiate respiratory depression, which may have clinical applications. Second, Dbx1 preBötC neurons express the hypoxia-inducible transcription factor Hif1a at levels three-times higher than non-Dbx1 neurons, which links core rhythmogenic microcircuits to O2-related chemosensation for the first time. Third, we detected a suite of transcription factors including Hoxa4 whose expression pattern may define the rostral preBötC border, Pbx3 that may influence ipsilateral connectivity, and Pax8 that may pertain to a ventrally-derived subset of Dbx1 preBötC neurons. These data establish the transcriptomic signature of the core respiratory oscillator at a perinatal stage of development.

Inspiratory rhythmogenic activity is burst-independent and opioid-sensitive

“…Which preBötC neurons does DAMGO target to depress inspiratory rhythm? We hypothesized that DAMGO was acting on preBötC Dbx1 + neurons, which are essential for respiratory rhythm generation (Bouvier et al, 2010; Gray et al, 2010; Wang et al, 2014) and contain μOR mRNA (Hayes et al, 2017). We first sought to determine whether these neurons express μOR protein using immunohistochemistry.…”

“…We therefore localized preBötC using tachykinin precursor peptide (TAC1) expression, which colocalizes with NK1R, ventral to nucleus ambiguus (NA) as a marker for preBötC (Fig. S1; Hayes et al, 2017). To measure μOR and TAC1 expression, we performed fluorescence in situ hybridization, which avoids the non-specific labeling that may occur with some μOR antibodies (Schmidt et al, 2013) and allows localization of TAC1 expression to somata (Fig.…”

Genetic Variation Regulates Opioid-Induced Respiratory Depression in Mice

“…None of the coding SNPs are the type with the most deleterious effects, such as a stop loss, stop gain or coding region insertion (frameshift). All four of these protein-coding genes are expressed in the mouse pre-Bötzinger complex, a group of brainstem interneurons that control regulation of respiratory rythmogenicity 32 , and are thus mechanistically plausible. Three of these four pre-Bötzinger complex-expressed protein-coding genes contain polymorphisms that cause non-synonymous (Cn) amino acid changes.…”

“…GALTN11 has been shown to uniquely glycosylate at least 313 glycoproteins in HEK cells 33 . Using the Jaccard similarity tool in the GeneWeaver 34 system for integrative functional genomics, we determined that of the 313 human glycopeptides, 287 mouse orthologs are expressed in the mouse pre-Bötzinger complex 32 (Table S2). Four of those genes Hs6st2 35 , Fn1 36 , Lrp1 35 , and Sdc4 37 were identified by the Comparative Toxicogenomic Database 38 as morphine-associated genes, and one additional pre-Bötzinger-expressed glycoprotein, Cacna2d1 , has been shown to have differential brain expression in mice following morphine treatment 39 .…”

“…Differential coding sequence non-synonymous amino acid substitution SNPs (Cn) that differed between the high and low allele groups were identified. GeneWeaver’s database (RRID:SCR_003009) was searched to identify the overlap among tissue-specific expression profiles from Allen Brain Atlas as well as datasets derived from Entrez GEO profiles (RRID:SCR_004584) for pre-Bötzinger neurons 32 (GS 273275), diaphragm (GS273269) 68 and lung 69 with the QTL positional candidates,. The gene sets were overlapped using the Jaccard similarity and GeneSet graph tools 34 .…”