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. 2017 Feb 21:11:34.
doi: 10.3389/fncel.2017.00034. eCollection 2017.

Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

Madeleine M Puissant  1 , Gary C Mouradian Jr  2 , Pengyuan Liu  3 , Matthew R Hodges  4
Affiliations

Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

Madeleine M Puissant et al. Front Cell Neurosci. .

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.

Keywords: RNA sequencing; chemoreception; control of breathing; potassium channels; serotonin.

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Figures

FIGURE 1
FIGURE 1
Methods for FACS collection of dissociated eGFP-expressing (eGFP+) and non-eGFP (eGFP-) medullary raphe neurons. (A) Representative data plotting forward-scatter (FSC) versus side-scatter (SSC) of all recorded sort events (cells). An area (P1) was identified as inclusive of raphe neurons, confirmed in (B) by sorting and plotting the FSC versus NeuN fluorescence of NeuN-labeled (Alexa Fluor 546-tagged) cells (Green events in A and B). (C) Gate establishment for the identification of eGFP+ (5-HT neurons) from ePet-eGFP:SS brainstem tissues by plotting FSC and the fluorescence intensity of Alexa Fluor 488 from collected P1/NeuN+ events, designated eGFP+ (red) or eGFP- (purple). (C’) The distribution of Alexa Fluor 488 intensity versus event counts. (D) qPCR data showing fold-enrichment of 5-HT neuron-specific genes slc6a4 (SERT) and tph2 (tryptophan hydroxylase 2) in eGFP+ (5-HT-enriched) versus non-5-HT neuron-enriched cell pools (P < 0.05; One-way ANOVA).
FIGURE 2
FIGURE 2
All plasma membrane-associated genes that were upregulated with increasing age in 5-HT neuron-enriched cell pools. Plasma Membrane molecules identified in IPA were significantly increased (q < 0.01) with increasing age in 5-HT neuron-enriched cell pools by FACS. (A) Gene name, description, protein type and relative change in expression (greatest increase (red) to smallest increase (yellow) for log fold-change with increasing age) is shown for identified plasma membrane-associated molecules. (B) Representation of the type of plasma membrane molecules categorized by transporters, ion channel, G-protein coupled receptors (GPCR), enzymes, or other.
FIGURE 3
FIGURE 3
Assumption-based identification of candidate genes through unique and overlapping differential expression patterns highlight the potential involvement of potassium ion (K+) channels. (A) Upregulated plasma membrane molecules [>0.5 log-fold increase) from age-specific (young vs adult) rostral 5-HT RNA sequencing comparison; (blue)] and region-specific [caudal versus rostral adult 5-HT neurons; (yellow)] with common genes in middle (tan). Note that the genes listed in red encode proteins with known pH sensitivity, and those with asterisks bind to and/or associate with pH-sensitive proteins. (B) Fold change in expression (young vs. old) of all K+ channel genes plotted against overall abundance (fragments per kilobase of transcript per million reads; FPKM), where those in red are downregulated, green are upregulated, and those in blue are not differentially regulated with age.
FIGURE 4
FIGURE 4
Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 & Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific (Tph2 and Ddc), neuronal specific (Map2 and Nefl), and glial specific (Gfap, Aqp4, and Sox10) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR (n = 7; ∗ vs. Gfap, Aqp4, and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR Ct-values demonstrating 5-HT specific expression of K+ channel genes, including Kcnj10 and Kcnj16 (n = 7).
FIGURE 5
FIGURE 5
Age-related changes in the expression of select K+ channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9, and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe (∗P < 0.05 vs. P0 by one-Way ANOVA). Western blots from raphe tissues collected from different ages showed increases in both a high molecular weight band (∼250 kDa) Kir4.1HMW and expected (42.5 kDa) Kir4.1 bands (B), Kir5.1HMW (∼250 kDa) and expected (48 kDa) Kir5.1 bands (C), and TASK-1 (45–65 kDa) and TASK-3 (45 kDa) (D) with respective quantification normalized to β-actin (E; ∗P < 0.05 vs. P0 by one-way ANOVA). Note that the quantification for Kir5.1 protein in (E) was from two blots from P0 (n = 3), P7 (n = 4), P18 (n = 6), and adult (n = 3), but only one is shown in (C).
FIGURE 6
FIGURE 6
Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C), but not neurons (D–F). Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L). The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.
FIGURE 7
FIGURE 7
Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A) and TPH (red; B) or Kir5.1 (green; D) and TPH (red; E). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F) but not Kir4.1 (correlation coefficient = 0.109; C) is co-localized to TPH+ 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I). Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G) and Kir5.1 (green; H) also show intense co-localization (yellow; I) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I).
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