Extended Data Fig. 6: Control experiments for AMPK requirement in cortex glia to sustain K-AM formation in MB neurons.
From: Glia fuel neurons with locally synthesized ketone bodies to sustain memory under starvation
(a) In fed flies, AMPKα expression in cortex glia is not required for LTM (F2,52 = 0.33, P = 0.717). When AMPKα RNAi expression was not induced, K-AM was normal (F2,21 = 1.02, P = 0.378). (b) Inhibition of AMPKα expression in adult cortex glia using a second non-overlapping RNAi impaired K-AM in starved flies (F2,33 = 9.95, P = 0.0004), whereas memory formed in fed flies after either massed or spaced training was not affected (ARM: F2,27 = 0.23, P = 0.799; LTM: F2,27 = 0.187, P = 0.831). Non-induced flies showed no K-AM defect (F2,27 = 0.16, P = 0.855). (c) In wild-type Canton S fly heads, starvation did not change the mRNA level of either AMPK, HMGS, Chk, ACAT1 or Sln (AMPK: t4 = 0.382, p = 0.722, HMGS: t4 = 1.40, p = 0.24; Chk t6 = 0.033, p = 0.98; ACAT1 t10 = 0.439, p = 0.669; Sln: t6 = 0.389, p = 0.711). (d) In starved flies expressing the Laconic FRET sensor in cortex glia, application of 5 mM NaAz induced a strong increase in the Laconic FRET ratio, reaching the saturation level of the sensor (mean trace ± s.e.m.). The last 100 s of recording (red bar), when the Laconic FRET sensor had reached saturation, were used to normalize; the initial Laconic ratio was measured during the 2-min baseline recording before NaAz application. In starved flies, the lactate basal concentration in cortex glia expressing AMPKα RNAi was similar to the genotypic control (AMPK: t15 = 0.279, P = 0.784). (e) Application of 10 mM acetoacetate (red dashed line) results in a decreased Laconic ratio followed by a plateau in cortex glia of starved control flies, showing lactate efflux from cortex glia after acetoacetate bath application (mean trace ± s.e.m.). Inhibition of HMGS expression did not change the lactate efflux evoked by acetoacetate application (t15 = 0.004, P = 0.99). Quantification of the mean Laconic ratio at the plateau was performed during the last 100 s of recording (red line). (f) In starved flies, the lactate basal concentration in cortex glia expressing HMGS RNAi was similar to the genotypic control (HMGS: t12 = 0.112, P = 0.913). n represents either a group of 40–50 flies analyzed together in a behavioral assay (a and b), mRNA extracted from a group of 50 flies (c) or the response of a single recorded fly (d-f). Data are expressed as mean ± s.e.m. with dots as individual values, and analyzed by one-way ANOVA with post hoc testing by Newman-Keuls pairwise comparisons test (a and b) or by unpaired two-sided t-test (c-f). Asterisks refer to the least significant P-value of post hoc comparison between the genotype of interest and the genotypic controls (a and b) or to the P-value of the unpaired t-test comparison (c-f). **P<0.01, ns: not significant.
