Extended Data Fig. 8: Palmitic acid (PA) supports synaptic vesicle recycling in hypometabolic condition.

a, Fluorescence intensity traces (normalized to first peak) of vGlut1-pHluorin at synaptic boutons of dissociated hippocampal neurons following periodic action potential firing (as in Fig. 4a) under three conditions: absence of external fuel (N = 9), presence of 5 mM glucose (N = 5), or pre-fed and maintained with PA (N = 5). Lower panels show representative kymographs of synaptic boutons under these conditions. b, Fluorescence intensity traces of vGlut-pHluorin at synaptic boutons in PA pre-fed or following oligomycin treatment in PA-fed hippocampal neurons. Sample sizes were N = 5 for both PA and PA + Oligo. treated conditions. c, Fluorescence intensity traces of vGlut1-pHluorin at synaptic boutons of dissociated hippocampal neurons maintained with 0, 0.16, 1.2, and 5 mM glucose, in presence of 2 µM oligomycin. Sample sizes were N = 6 for 0 mM Glucose + Oligo, N = 5 for 0.16 mM Glucose + Oligo, N = 4 for 1.2 mM Glucose + Oligo and N = 5 for 5 mM Glucose + Oligo conditions. d, Fluorescence intensity traces of vGlut1-pHluorin at synaptic boutons of hippocampal neurons maintained with following media: subphysiological 0.16 mM glucose + oligomycin (N = 5), pre-fed and maintained with PA + 0.16 mM glucose (N = 6), or pre-fed and maintained with PA + 0.16 mM glucose + etomoxir (N = 6). e, Synaptic vGlut1-pHluorin traces in lactate and pyruvate in presence of etomoxir, or lactate and pyruvate with etomoxir and oligomycin. Sample size for each condition was N = 5. f, Fluorescence intensity traces of vGlut-pHluorin at synaptic boutons after periodic AP firing in acutely PA-fed neurons (5 min.) compared to no fuel conditions. Sample size for each condition was N = 5. Data (in a-f) are presented as mean ± SE. p-values (ns, #p = 0.063; ##p = 0.01; ††p = 0.01; ns, p = 0.25; *p = 0.04; **p = 0.01) were determined using unpaired samples two-tailed t-test.

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