Enteric glial S100B controls spontaneous activity in enteric motor neurocircuits

Enteric glia regulate gut motor function through interactions with enteric neurons but specific glial mechanisms remain poorly understood. Patterns of gut motility such as colonic motor complexes (CMC) are driven by central pattern generators in the enteric nervous system (ENS). These enteric circuits share similarities with central pattern generators in the brain in which astrocytes play key roles through mechanisms that involve the protein S100B. We hypothesized that enteric glial S100B controls gut motor function by modulating ENS excitability and coordinated activity. Glial S100B release was studied by ELISA in whole-mount preparations while CMC motor activity was studied in organ bath experiments. Cellular activity in the ENS was studied using Ca 2+ imaging in whole-mount preparations of myenteric plexus from Wnt1 CreGCaMP5g-tdTom mice expressing genetically encoded calcium indicators (GCaMP5g) in Wnt1+ cells (glia and neurons) or ChAT CreGCaMP5g-tdTom mice in which GCaMP5g expression is in ChAT+ neurons. S100B was manipulated using arundic acid (AA, 50 μM) pentamidine (300 μM), and S100B antibodies (1:1000) or recombinant S100B protein (rS100B, 50ug/ml). Spontaneous S100B release by myenteric plexus whole-mounts was reduced by 51% in the presence of AA. Reducing S100B release or blocking its extracellular buffering activity with pentamidine and AA diminished CMCs (p<0.0001) in a reversible manner. Live cell imaging studies showed that changes in neuron and glial excitability underlie the effects of S100B on CMCs. Inhibiting S100B production with AA reduced spontaneous glial and neuronal activity in the myenteric plexus (p<0.0001). Pentamidine also reduced the amplitude of spontaneous enteric neuron responses (p<0.01) and this effect was most prominent in males. Opposite effects were observed in females where inhibiting S100B with pentamidine increased spontaneous neuron peak amplitudes (p<0.0001). Because rhythmic activity among cholinergic neurons in the proximal colon is thought to underlie CMC generation, we evaluated Ca 2+ activity in proximal ChAT+ myenteric neurons. Inhibiting S100B release in those samples increased spontaneous cholinergic neuron activity (p<0.05) in a reversible manner and adding exogenous S100B (rS100B) decreased response amplitude in ChAT+ neurons. These data show that the glial mediator S100B controls cellular activity in gut neurocircuits that underlie motor behaviors of the intestine. Changes to S100B production and secretion by glia have the potential to impact both neuron and glial activity and neuron subtypes that control gut motor patterns, which may contribute to digestive diseases. Disclosure of funding sources: The authors have no conflicts of interest to declare. NIH grants R01DK120862 and R01DK103723 to Dr. Gulbransen. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.