Background/Objectives: The esophagus connects the oral cavity to the stomach and terminates at the gastroesophageal junction (GEJ) where the lower esophageal sphincter (LES), a thickening of the circular smooth muscle is located. The mouse esophagus has been described to consist entirely of skeletal muscle, despite this, phasic contractile activity is present in the distal portion. In other gastrointestinal (GI) muscles phasic activity is associated with slow waves generated by interstitial cells of Cajal (ICC). Smooth muscle cells (SMCs) form an electrical syncytium with ICC and a second type of interstitial cell, PDGFRa⁺ cells (SIP syncytium; Sanders et al., 2014). ICC are present throughout the mouse esophagus (Rumessen et al., 2001) while PDGFRa⁺ cells have been observed in the human esophagus (Chen et al., 2013). The aims of our study were to characterize the distribution of SIP cells, and evaluate the functional role of ICC within the mouse esophagus and LES.

Methods: Dual labeling immunohistochemistry was performed on tissues from wildtype (C57Bl/6) and smooth muscle eGFP (smMHC^-eGFP/+) mice. ICC were labeled with antibodies against Kit or ANO1. PDGFRa⁺ cells were labeled with antibodies against PDGFRa or SK3. Enteric motor neurons and glia were labeled with antibodies against nNOS, VIP (inhibitory), vChAT, TH (excitatory) and GFAP (glia) respectively. Images were acquired with a Leica Stellaris 5 confocal microscope and processed using LasX software. Contractile activity was recorded from LES clasp and distal esophagus muscle strips. ICC Ca²⁺ transients were imaged from the Kit-GCaMP6f mouse GEJ using confocal spinning disc microscopy. The contributions of ANO1 and Cav_L channels were determined with Ani9 and nifedipine or pinacidil respectively.

Results: SMCs were present throughout the esophagus though their density declined proximally. In contrast to other GI regions, only intramuscular ICC (ICC-IM) were present; these cells expressed ANO1 and declined in density proximally. Submucosal and intramuscular PDGFRa⁺ cells were distributed throughout the esophagus and LES. Only intramuscular PDGFRa⁺ cells expressed SK3 though this expression declined proximally. SIP cells were closely associated with one another as well as with enteric neurons and glia. The LES clasp generated tone whereas the distal esophagus exhibited phasic contractions. Contractile activity in both muscles was abolished by inhibiting ANO1 or Cav_L. As observed previously, ICC-IM in the LES exhibited asynchronous Ca²⁺ activity (Type I ICC-IM) (Drumm et al., 2022). In contrast, in the distal esophagus, two Ca²⁺ signaling behaviors were present with a second ICC-IM population exhibiting whole-cell Ca²⁺ transients (Type II ICC-IM). Whole-cell Ca²⁺ transients in Type II ICC-IM were abolished by inhibiting ANO1 or Cav_L revealing underlying Type I-like asynchronous Ca²⁺ transients. This suggests that these cells likely regulate motility within this region.

Discussion/Conclusions: Our studies revealed that SIP cells are distributed throughout the mouse GEJ and that they are closely associated with one another as well as with enteric neurons and glia. Functional studies suggest that ICC-IM play an important role in regulating motility within the GEJ. Future studies will utilize mice to evaluate the role of SIP cells in neuromuscular transmission within the GEJ.