The parafacial respiratory group (pFRG) is a conditional oscillator known to drive active expiration and enhance ventilation during high metabolic and respiratory demands, through the recruitment of abdominal (ABD) expiratory muscles. At rest and under anesthesia, pFRG neurons appear to be silent, but when disinhibited or activated, they trigger active expiration. Furthermore pFRG modulation appears to be critical for recruitment of expiratory ABD activity during REM sleep. There is extensive evidence for functional modulation of pFRG and its role in active expiration, although no systematic analysis of neuroanatomical projections to the pFRG is currently available. Thus, the objectives of this study are: 1) to identify brainstem regions that directly project to pFRG; and 2) define phenotypes of these projections, mainly excitatory and/or inhibitory.To test the objective 1, we used a retrograde virus (herpes simplex virus) injected unilaterally in the pFRG area in adult male rats that expressed the reporter protein eYFP (enhanced yellow fluorescent protein) driven by the neuronal promoter synapsin in order to track neurons projecting to the pFRG. To determine the phenotype of these projections (objective 2), we combined immunofluorescent labeling for eYFP with different neuronal markers: somatostatin (SST), choline acetyltransferase (CHAT), tyrosine hydroxylase (TH), paired Like Homeobox 2 (PHOX2B) and 5‐hydroxytryptamine (5HT); and we used RNAScope assay to target mRNA for Vesicular Glutamate Transporter (Vglut2); Glutamate Decarboxylase 1 (Gad1) and Glycine Transporter 2 (Glyt2).We identified several brainstem structures that directly project to pFRG ipsilaterally and contralaterally: in the medulla, projecting neurons were located in the ventral respiratory column (Bötzinger Complex, preBötzinger Complex, rostral ventral respiratory group, retrotrapezoid nucleus ‐ RTN and pFRG), the nucleus of the solitary tract (NTS), the reticular formation (RF), the vestibular nuclei (extended to the pons and midbrain), the medullary raphe and the nucleus of Roller. Several projecting neurons were also located rostrally in the pons and midbrain within the Kölliker‐Fuse nucleus, the parabrachial nucleus, the periaqueductal grey, the pedunculopontine nucleus (PPT), the laterodorsal tegmental (LDT) and the A5 area. Overall, the ipsilateral distribution was more extensive than in the contralateral side of the virus injection. Phenotype analysis demonstrated the presence of both excitatory (Vglut2+) and inhibitory (Gad1/Glyt2+) cells projecting to pFRG deriving from all the above regions, in addition to PHOX2B+ cells located in the RTN, NTS and part of the RF. Moreover, we observed lack of direct projections to the pFRG area from TH+ cells in the NTS, A5 and Locus coeruleus; 5HT+ cells in the raphe nuclei; SST+ cells in the preBötzinger Complex and CHAT+ cells in the PPT and LDT.The present study provides evidence that pFRG neurons receive excitatory and inhibitory inputs from several breathing‐ and non‐breathing related brainstem regions that could be involved with their conditional activity and the modulation of active expiration in various physiological conditions.Support or Funding InformationCIHR 156338, NSERC 435843; WCHRI Fellowship (VB)
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