Priority Research Paper Volume 1, Issue 8 pp 699—713

Figure 4. Electrophysiological, Ca ²⁺ influx, and expression evidence of functional K_ATP channels in adult IPCs. (A1-C) Membrane depolarization of adult IPCs in response to glucose and glibenclamide. (A1) Trace of membrane potential from an adult IPC in the whole brain preparation shows that exposure to high glucose (80 mM) evoked a reversible membrane depolarization. (A2) Trace of membrane potential from an adult IPC shows that exposure to a commonly known K_ATP channel blocker, glibenclamide (glib, 20 μM) also evoked a reversible membrane depolarization. (B1 and B2) traces of membrane potential from adult non-IPCs show that these cells do not respond to glucose or glibenclamide. (C) Average membrane potential response to glucose and glibenclamide of IPCs (N=5) and non-IPCs (N=3). Glucose (*) and glibenclamide (**) significantly increased membrane potential of IPCs as compared to non-IPCs, *p and **p <0.05 (Student's t test). Each bar represents mean + S.E.M. (D1-E) Glucose-dependent Ca²⁺ influx measured in adult IPCs. (D1) Normalized fluorescence trace (∆F/F) (see Materials and Methods) recorded from an acutely dissociated adult IPC shows that exposure to glucose (80 mM) increased fluorescence intensity, thus indicating an increase in intracellular Ca²⁺. (D2) Normalized fluorescence trace (∆F/F) recorded from an acutely dissociated non-IPC shows that glucose does not increase intracellular Ca²⁺ in these cells. (E). An average of normalized fluorescence intensity in response to glucose demonstrates a significant increase in Ca²⁺ influx recorded from IPCs (N=6) as compared to non-IPCs (N=3). *P= 0.007 (Student's t test). Each bar represents mean + S.E.M. (FI) In situ hybridization of whole mount adult brains demonstrates dSur expression in IPCs (F-G) when probed with anti-sense dSur probes and dilp2 expression (HI) when probed with anti-sense dilp2 probes. The IPCs marked in squares are shown in panel G for dSur signals and panel I for dilp2 signals. (J) Quantitative real-time PCR analysis reveals an average of 33% reduction in dSur transcripts when the IPCs are partially ablated using an IPC-specific driver, dilp3-Gal4 to drive the expression of a pro-apoptotic gene, reaper. The housekeeping gene GAPDH was used as the reference gene. Each bar represents mean + S.E.M. N=5, *p<0.001 (Student's t test). Control: dilp3-Gal4/w1118.