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Conditional knockout of astrocytic Kir4.1 channels impairs central respiratory drive despite maintaining a subset of Cx26 + astrocytes within the RTN
Author(s) -
Hawkins Virginia E,
Kolling Fred W,
Nelson Craig E,
Mulkey Daniel K
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.772.13
Subject(s) - astrocyte , purinergic receptor , neuroscience , patch clamp , conditional gene knockout , biology , brainstem , chemoreceptor , neuron , microbiology and biotechnology , electrophysiology , gap junction , membrane potential , connexin , chemistry , central nervous system , extracellular , intracellular , biochemistry , receptor , gene , phenotype
Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO 2 /H + . The retrotrapezoid nucleus (RTN) has been identified as a key locus for central respiratory control. Neurons in this region are intrinsically sensitive to changes in CO 2 /H + (i.e. are chemosensitive). RTN astrocytes also function as chemoreceptors by sensing CO 2 /H + through inhibition of an inwardly rectifying (Kir4.1‐like) K + channel, and respond by releasing ATP via connexin 26 (Cx26) hemichannels. This CO 2 /H + ‐dependent purinergic drive from RTN astrocytes has been shown to increase the chemosensitive RTN neuron response by ~30%. However, it is not known whether Kir4.1 in astrocytes is required for RTN chemoreceptor function, and little is known regarding the molecular identity of the chemosensitive astrocyte cell subset. To investigate this we generated an inducible astrocyte specific Kir4.1 channel knockout (Kir4.1 cKO) using GFAP‐CreERT2 and Kir4.1 floxed mouse lines, along with a TdTomato reporter line. Whole‐cell voltage clamp recordings of RTN astrocytes in juvenile brainstem slices from Kir4.1 cKO mice showed the absence of a Kir4.1‐like, CO 2 /H + ‐sensitive current. RTN neurons from Kir4.1 cKO brainstem slices show a reduced purinergic component to their CO 2 /H + response, suggesting deletion of Kir4.1 from astrocytes may disrupt the ability of these cells to respond normally to changes in CO 2 /H + . Respiratory activity was measured in adult mice by whole body plethysmography and showed that Kir4.1 cKO mice hypoventilate in 100% O 2 and have a reduced tidal volume response to hypercapnia, compared to controls. For examination of gene expression, RTN GFAP‐TdT + astrocytes from both control and Kir4.1 cKO brainstems were acutely dissociated and single cells were isolated by FACS into 96‐well plates. Single cell qPCR revealed that the majority of astrocytes within the RTN express glial markers Kcnj10 (Kir4.1), Aqp4 , Aldh1L1 and Gfap, and NeuN was probed for to exclude neuronal transcript contamination. A subset of RTN astrocytes expressed Gjb2 (Cx26), which unlike Kcnj10 expression, was not greatly reduced in astrocytes from the Kir4.1 cKO mouse RTN. These results demonstrate that Kir4.1 channels in astrocytes contribute to the central drive to breathe, and suggest that RTN astrocytes are a heterogeneous population, with a discrete subset that expresses Cx26. These findings provide insight into the specialized nature of astrocyte chemoreceptors and may identify new targets important for the central control of breathing. Support or Funding Information NIH HL104101 (DKM) and F32HL126381 (VEH)