During pre-hearing stages of development in mice, periodic transient elevations of cytosolic free Ca2+ concentration occur spontaneously in the greater epithelial ridge (GER) and propagate as intercellular Ca2+ waves invading variable portions of the GER. Prior work by our and other groups indicates that intercellular Ca2+ waves in the GER rely on the interplay between IP3, generated intracellularly, and ATP, released at the apical surface of cochlear non-sensory cells. A vast body of data supports the hypothesis that, in the developing cochlea, ATP is released through connexin hemichannels [1], however a direct proof is lacking. To test this hypothesis, we designed and built a closed microfluidic chamber (10 ?l max. volume) in which the transparent roof was covered with plated HEK23T cells (facing the fluid interior of the chamber), stably expressing P2Y2 purinergic receptors and sensitive to ATP in the nM range. These biosensor cells sitted at <100 ?m from the surface of an organotypic cochlear culture plated on the chamber bottom. After loading both biosensor cells and cochlea with Ca2+-selective dyes, or by using genetically encoded GCaMP6s Ca2+ sensors, this architecture allowed us to monitor Ca2+ responses in HEK293T biosensor during propagation of Ca2+ waves in the GER of the cochlea underneath. To image Ca2+ dynamics while discriminating optical signals originating from the two focal planes, we stepped up and down, rapidly and repeatedly, the objective of a custom-made multi-photon microscope. For these experiments, the saline solution trapped within the chamber contained an endolymphatic Ca2+ concentration (20 mM) and ARL67156 (100 mM), an inhibitor of ectonucleotidases. Ca2+ signals disappeared upon replacing ARL67156 with apyrase (40 U/ml, an enzyme that catalyzes the sequential hydrolysis of ATP) in the extracellular medium, confirming that ATP mediated both Ca2+ wave propagation in the GER and the ensuing Ca2+ responses in the HEK293T biosensors. To determine the source of the released ATP, we tested cochlear organotypic cultures from mutant mice with global deletion of pannexin 1 (Panx1-/-), and another strain with targeted deletion of connexin 26 in the sensory epithelium of the cochlea (Gjb2-/-). Using the microfluidic chamber, we determined that Ca2+ signals in the ATP biosensor cells were the same irrespective of whether they faced Panx1-/- or age-matched Panx1+/+ or Cx26+/+ cochlear cultures. In contrast, Ca2+ signals strongly depressed in the presence of Gjb2-/- cultures. Furthermore, Ca2+ waves in the GER were reversibly inhibited by flufenamic acid (50 mM) and the anti-connexin antibody abEC1.1 (952 nM) [2], both of which are not effective on pannexin 1 channels [3]. Together, these results validate our working hypothesis and confirm that pannexin 1 channels are not involved in the ATP release process that mediates Ca2+ wave propagation in the GER.
Simultaneous detection of Ca2+ signaling and ATP release in the developing cochlea
2019
Abstract
During pre-hearing stages of development in mice, periodic transient elevations of cytosolic free Ca2+ concentration occur spontaneously in the greater epithelial ridge (GER) and propagate as intercellular Ca2+ waves invading variable portions of the GER. Prior work by our and other groups indicates that intercellular Ca2+ waves in the GER rely on the interplay between IP3, generated intracellularly, and ATP, released at the apical surface of cochlear non-sensory cells. A vast body of data supports the hypothesis that, in the developing cochlea, ATP is released through connexin hemichannels [1], however a direct proof is lacking. To test this hypothesis, we designed and built a closed microfluidic chamber (10 ?l max. volume) in which the transparent roof was covered with plated HEK23T cells (facing the fluid interior of the chamber), stably expressing P2Y2 purinergic receptors and sensitive to ATP in the nM range. These biosensor cells sitted at <100 ?m from the surface of an organotypic cochlear culture plated on the chamber bottom. After loading both biosensor cells and cochlea with Ca2+-selective dyes, or by using genetically encoded GCaMP6s Ca2+ sensors, this architecture allowed us to monitor Ca2+ responses in HEK293T biosensor during propagation of Ca2+ waves in the GER of the cochlea underneath. To image Ca2+ dynamics while discriminating optical signals originating from the two focal planes, we stepped up and down, rapidly and repeatedly, the objective of a custom-made multi-photon microscope. For these experiments, the saline solution trapped within the chamber contained an endolymphatic Ca2+ concentration (20 mM) and ARL67156 (100 mM), an inhibitor of ectonucleotidases. Ca2+ signals disappeared upon replacing ARL67156 with apyrase (40 U/ml, an enzyme that catalyzes the sequential hydrolysis of ATP) in the extracellular medium, confirming that ATP mediated both Ca2+ wave propagation in the GER and the ensuing Ca2+ responses in the HEK293T biosensors. To determine the source of the released ATP, we tested cochlear organotypic cultures from mutant mice with global deletion of pannexin 1 (Panx1-/-), and another strain with targeted deletion of connexin 26 in the sensory epithelium of the cochlea (Gjb2-/-). Using the microfluidic chamber, we determined that Ca2+ signals in the ATP biosensor cells were the same irrespective of whether they faced Panx1-/- or age-matched Panx1+/+ or Cx26+/+ cochlear cultures. In contrast, Ca2+ signals strongly depressed in the presence of Gjb2-/- cultures. Furthermore, Ca2+ waves in the GER were reversibly inhibited by flufenamic acid (50 mM) and the anti-connexin antibody abEC1.1 (952 nM) [2], both of which are not effective on pannexin 1 channels [3]. Together, these results validate our working hypothesis and confirm that pannexin 1 channels are not involved in the ATP release process that mediates Ca2+ wave propagation in the GER.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
