Extended Data Fig. 5: Control experiments for KB export by cortex glia to sustain K-AM formation in MB neurons.

(a) In fed flies, when Chk expression was downregulated in adult cortex glia, LTM was normal (F_2,37 = 0.96, P = 0.393). When Chk RNAi expression was not induced, K-AM was normal (F_2,32 = 0.40, P = 0.677). (b) Since starvation induced strong lethality in Chk^MB04207 homozygous flies, we used heterozygous Chk^MB04207/+ flies to confirm Chk function in K-AM. Furthermore, since both Chk^MB04207 flies (which are in a w¹¹¹⁸ background) and w¹¹¹⁸ flies have lower memory scores, we only counted heterozygous females (w¹¹¹⁸/+; Chk^MB04207/+) and not hemizygous males (w¹¹¹⁸/Y; Chk^MB04207/+), and counted only heterozygous w¹¹¹⁸/+ females as control flies. Heterozygous w¹¹¹⁸/+; Chk^MB04207/+ female flies had a strong K-AM defect as compared to control w¹¹¹⁸/+;+ female flies (t₂₇ = 3.24, P = 0.0032). Neither ARM (t₂₃ = 0.89, P = 0.379) nor LTM (t₂₆ = 0.10, P = 0.918) was affected in these heterozygous w¹¹¹⁸/+;Chk^MB04207/+ female flies. (c) Inhibition of Sln expression in adult cortex glia did not impair K-AM (F_2,36 = 0.23, P = 0.794). (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 Chk RNAi was similar to the genotypic control (t₁₄ = 0.743, P = 0.469). n represents either a group of 40–50 flies analyzed together in a behavioral assay (a–c) or the response of a single recorded fly (d). 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 c) or by unpaired two-sided t-test (b and d). Asterisks refer to the least significant P-value of post hoc comparison between the genotype of interest and the genotypic controls (a and c) or to the P-value of the unpaired t-test comparison (b and d). **P<0.01, ns: not significant.