A Pipeline for the Registration of Calcium Transient Data to Structural Networks of the Interstitial Cells of Cajal

Interstitial cells of Cajal (ICC) generate electrical pacemaker activity in the gastrointestinal (GI) tract known as slow waves, which regulate GI motility. ICC express both the Kit receptor tyrosine kinase protein and a Ca²⁺-activated Cl^--channel, encoded by the anoctamin1 (Ano1) protein, which is an essential contributor to the Ca²⁺ cycling of ICC and slow wave pacemaking. Recent dye-loading imaging studies have demonstrated Ca²⁺ transients in ICC in isolated tissue preparations. The main aim of this study was to develop a method that allows Ca²⁺ transients to be registered to structural ICC network data. Confocal image stacks of ICC labeled for Kit or Ano1 and Ca²⁺ recording data were processed using a thresholding protocol. The Ca²⁺ transients were then registered to the ICC structural network. First, a general idea of the placement was found by mapping the field-of-view of the Ca²⁺ transient data to the distorted tissue that contained the ICC network image. The errors in the registration were then corrected for by warping the internal Ca²⁺ transient field according to the structural network. In data sets from tissues with induced, targeted knockdown of Ano1 expression in a subset of ICC, agreement between the Ca²⁺ transient data and structural network was 68 ± 10%. This level of agreement allowed selective extraction of Ca²⁺ data from Ano1-positive (Ano1+) and Ano1-negative (Ano1-) ICC. In the future, this technique will allow investigation into the functional properties of ICC in relation to the level of knockdown of specific ICC associated proteins.