Relationship between the fluorescence intensity of rhodamine-labeled orexin A and the calcium responses in cortical neurons: An in vivo two-photon calcium imaging study

Neural responses to a ligand vary widely between neurons; however, the mechanisms underlying this variation remain unclear. One possible mechanism is a variation in the number of receptors expressed in each neural membrane. Here, we synthesized a rhodamine-labeled orexin A compound, enabling us to quantify the amount of orexin binding to its receptors, OX1 and OX2, which principally couple to the G(q/11) in protein. The rhodamine intensity and calcium response were measured under tetrodotoxin application from insular cortical glutamatergic neurons in Thy1-GCaMP6s transgenic mice using an in vivo two-photon microscope. Applying rhodamine-labeled orexin A (10 mu M) to the cortical surface gradually and heterogeneously increased both the intensity of the rhodamine fluorescence and [Ca2+](i). Calcium responses started simultaneously with the increase in rhodamine-labeled orexin fluorescence and reached a plateau within several minutes. We classified neurons as high- and low-responding neurons based on the peak amplitude of the [Ca2+](i) increase. The rhodamine fluorescence intensity was larger in the high-responding neurons than the low-responding neurons. Preapplication of SB334867 and TCS-OX2-29, OX1 and OX2 antagonists, respectively, decreased the proportion of high-responding neurons. These results suggest that the diverse receptor expression level in neural membranes is involved in mechanisms underlying varied neural responses, including [Ca2+](i) increases. (C) 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.