Fura-2 Calcium Imaging Is a Valid Method of Investigating Signalling & Integrative Properties of the Subfornical Organ

Abstract

Research into the integration of signalling molecules at the subfornical organ (SFO) is typically conducted through in vitro electrophysiology methods on dissociated SFO neurons. We performed Fura-2 calcium imaging on dissociated SFO neurons and compared our observations to past electrophysiology data to determine if our imaging protocol can be used as an alternative, high-throughput method to investigate the integrative properties of the SFO. Sequential bath-application of 10-8 M cholecystokinin (CCK), a signalling molecule involved in energy balance, and 10-8 M Angiotensin-II (ANG), an excitatory signalling molecule involved in fluid balance, was performed on dissociated SFO neurons in hypoglycemic (1 mM glucose), normoglycemic (5 mM glucose), and hyperglycemic (10 mM glucose) conditions. ANG caused an increase in intracellular calcium ([Ca2+]i) in 32.8% (n = 20/61), 53.7% (n = 29/54) and 69.8% (n = 30/43) of SFO neurons at 1, 5 and 10 mM glucose, respectively, which is similar to the proportion of SFO neurons which depolarized to ANG in electrophysiology experiments [33.3% (n = 4/12) at 1 mM, 50.0% (n = 16/32) at 5mM, and 72.7% (n = 8/11) at 10 mM glucose]. These results suggest that ANG-related depolarizations and detectable [Ca2+]i increases are associated with each other. CCK caused an increase in [Ca2+]i in 31.1% (n = 19/61), 35.2% (n = 19/54), and 44.2% (n = 19/43) of SFO neurons at 1, 5 and 10 mM glucose, respectively, which is similar to the overall proportion of CCK-responsive neurons in electrophysiology experiments [41.2% (n = 7/17) at 1mM, 48.9% (n = 22/45) at 5 mM, and 47.6% (n = 10/21) at 10 mM glucose]. CCK elicits both excitatory and inhibitory responses, so our results suggest that increases in [Ca2+]i may be associated with both types of events. Lastly, we observed four subpopulations of SFO neurons: those who respond exclusively to ANG (n = 48/158), those who respond exclusively to CCK (n = 26/158), those who respond to both peptides (n = 31/158), and those who respond to neither peptide (n = 53/158). The observation of four subpopulations aligns with past electrophysiological data. Overall, our results suggest that SFO neurons respond to ANG and CCK similarly in imaging and electrophysiology trials and that Fura-2 calcium imaging is a reliable high-throughput method for researching signal transduction and the integrative properties of the SFO.