Abstract
Ventilation is continuously adjusted by a neural network to maintain blood gases and pH. Acute CO2 and/or pH regulation requires neural feedback from brainstem cells that encode CO2/pH to modulate ventilation, including but not limited to brainstem serotonin (5-HT) neurons. Brainstem 5-HT neurons modulate ventilation and are stimulated by hypercapnic acidosis, the sensitivity of which increases with increasing postnatal age. The proper function of brainstem 5-HT neurons, particularly during post-natal development is critical given that multiple abnormalities in the 5-HT system have been identified in victims of Sudden Infant Death Syndrome. Here, we tested the hypothesis that there are age-dependent increases in expression of pH-sensitive ion channels in brainstem 5-HT neurons, which may underlie their cellular CO2/pH sensitivity. Midline raphe neurons were acutely dissociated from neonatal and mature transgenic SSePet-eGFP rats [which have enhanced green fluorescent protein (eGFP) expression in all 5-HT neurons] and sorted with fluorescence-activated cell sorting (FACS) into 5-HT-enriched and non-5-HT cell pools for subsequent RNA extraction, cDNA library preparation and RNA sequencing. Overlapping differential expression analyses pointed to age-dependent shifts in multiple ion channels, including but not limited to the pH-sensitive potassium ion (K+) channel genes kcnj10 (Kir4.1), kcnj16 (Kir5.1), kcnk1 (TWIK-1), kcnk3 (TASK-1) and kcnk9 (TASK-3). Intracellular contents isolated from single adult eGFP+ 5-HT neurons confirmed gene expression of Kir4.1, Kir5.1 and other K+ channels, but also showed heterogeneity in the expression of multiple genes. 5-HT neuron-enriched cell pools from selected post-natal ages showed increases in Kir4.1, Kir5.1, and TWIK-1, fitting with age-dependent increases in Kir4.1 and Kir5.1 protein expression in raphe tissue samples. Immunofluorescence imaging confirmed Kir5.1 protein was co-localized to brainstem neurons and glia including 5-HT neurons as expected. However, Kir4.1 protein expression was restricted to glia, suggesting that it may not contribute to 5-HT neuron pH sensitivity. Although there are caveats to this approach, the data suggest that pH-sensitive Kir5.1 channels may underlie cellular CO2/pH chemosensitivity in brainstem 5-HT neurons.
Highlights
Chronic physiological regulation of arterial pH is controlled by the kidney, but arterial oxygen, carbon dioxide (CO2), and pH levels are acutely regulated by chemoreflex-mediated changes in breathing
Total RNA extracted from the FAC-sorted enhanced green fluorescent protein (eGFP)+ and eGFP− cell pools was of high quality and sufficient quantity (Nanodrop 2000), and RT-qPCR experiments validated the specificity of the sort
We tested the hypothesis that gene transcripts encoding pH sensitive proteins, including known pH-sensitive ion channels are developmentally regulated in 5-HT neurons by applying fluorescent cell sorting and subsequent RNA sequencing to determine age-related and region-specific differential gene expression profiles
Summary
Chronic physiological regulation of arterial pH is controlled by the kidney, but arterial oxygen, carbon dioxide (CO2), and pH levels are acutely regulated by chemoreflex-mediated changes in breathing. Central (brain) respiratory chemoreceptors are resident CNS cells (neurons/glia) whose pH- or CO2-dependent activity can affect ventilation (Richerson et al, 2005). Some brainstem cells have been identified as CO2/pH-sensitive and involved in the control of breathing, and in some cases the location, neurochemical identity, and molecular mechanism(s) of pH sensitivity have been determined. The activity of Phox2b+ RTN neurons is modulated by raphe-derived neuromodulators (serotonin and substance P), and via ATP released by local pH-sensitive glia (Mulkey et al, 2007a), which appear to rely on heteromeric, inwardly rectifying, pH-sensitive K+ (Kir4.1/Kir5.1) channels for their chemosensitivity (Wenker et al, 2010). Other populations of CO2-sensitive neurons in the Locus Coeruleus (LC; catecholaminergic neurons) and solitary complex are thought to rely on yet to be identified pHsensitive K+ channels (Martino and Putnam, 2007; Li and Putnam, 2013)
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