Activity-driven local ATP synthesis is required for synaptic function

Abstract

Cognitive function is tightly related to metabolic state, but the locus of this control is not well understood. Synapses are thought to present large ATP demands; however, it is unclear how fuel availability and electrical activity impact synaptic ATP levels and how ATP availability controls synaptic function. We developed a quantitative genetically encoded optical reporter of presynaptic ATP, Syn-ATP, and find that electrical activity imposes large metabolic demands that are met via activity-driven control of both glycolysis and mitochondrial function. We discovered that the primary source of activity-driven metabolic demand is the synaptic vesicle cycle. In metabolically intact synapses, activity-driven ATP synthesis is well matched to the energetic needs of synaptic function, which, at steady state, results in ∼10(6) free ATPs per nerve terminal. Despite this large reservoir of ATP, we find that several key aspects of presynaptic function are severely impaired following even brief interruptions in activity-stimulated ATP synthesis.

Figure 1. Syn-ATP: a genetically-encoded ATP reporter

(A) Fluorescence (red) and luminescence (green) images of Syn-ATP in the presence of 2 mM luciferin expressed in hippocampal neurons. Scale bar 10 μm. (B) Correlation between individual bouton luminescence and fluorescence intensities from a sample experiment (left). Average correlation coefficient measured across cells = 0.83 (n = 50) (right). (C) Fluorescence (red) and luminescence (green) images acquired before (top) and after (middle) permeabilization in the presence of luciferin, followed by addition of ATP (bottom). Scale bar = 10 μm. (D) ATP titration curve of Syn-ATP fit with Michaelis-Menten relationship yielding K_m (ATP) = 2.3 ± 0.6 mM, n = 10. Error bars are SEM. See also Figures S1 & S2.

Figure 2. Activity drives large ATP consumption

(A) Average ATP measured in neurons in the absence and presence of TTX. The Box whisker plots represent median (line), mean (point), 25 – 75 percentile (box), 10 – 90 percentile (whisker), 1 – 99 percentile (X) and min - max (−) ranges. (B) Fluorescence and luminescence images acquired before and after 3 min incubation in deoxyglucose and oligomycin (dGlu+Oligo). Scale bar 5 μm. Pseudocolor intensity scales is in arbitrary units. (C) Corresponding fluorescence, luminescence intensities (top) and L/F normalized to the initial resting L/F value measured during the time course of the experiment in comparison with dGlu+Oligo treatment in the presence or absence of TTX (bottom). (D) Average normalized L/F measured after 3 min incubation in dGlu+Oligo in the presence or absence of TTX (n = 4). See also Figure S3.

Figure 3. Stimulation drives ATP synthesis

(A) Representative L/F time course from a collection of 30 boutons from one neuron during a 60 s period of 10 Hz AP firing and separate cytoplasmic pHluorin based measurements of pH measured under the same condition, averaged across experiments (n = 12) (top), which was used to obtain a calibrated ATP time course (middle). ATP time course averaged across experiments (n = 30 cells) (bottom). Grey dotted line indicates baseline average of first 5 min during the pre-stimulation period; Error bars are SEM. (B) Average ATP time course measured pairwise in the absence and presence of Oligo (n = 9, top) or dGlu (n = 21, middle) during a 60s period of 10 Hz AP firing. (Bottom) Average ΔATP_presyn measured over the stimulus period (During stimulation) or averaged over 6 min after the stimulation (Post-stimulation) showed that ATP levels dropped significantly compared to control in dGlu (p < 0.000001) or in Oligo (p = 0.0003) during the stimulus period. (C) Single neuron example of resting presynaptic ATP levels (ATP_presyn) in TTX in the absence or presence of Oligo (top) or dGlu (middle). Grey arrows indicate 10 and 40 min time points. Average drop in ATP (ΔATP_presyn) measured after 10 min (n = 5 cells; p = 0.64, Oligo; p = 0.27, dGlu; p < 0.000001, Oligo+dGlu) or 40 min (n = 5 cells; p = 0.35, Oligo; p = 0.0004, dGlu; p < 0.000001, Oligo+dGlu) in Oligo, dGlu or a combination of both compared to the initial resting ATP level (bottom). Error bars are SEM. Student’s t-test was used to determine all the statistics. See also Figure S3.

Figure 4. The vesicle cycle presents a large presynaptic ATP burden

Average [ATP]_presyn dynamics in response to 600 AP (10 Hz) in the presence of dGlu or Oligo with (A) zero external Ca²⁺, n = 9 (dGlu), n = 9 (Oligo) and (C) in Munc13 KD neurons, n = 8 (dGlu), n = 13 (Oligo). Black dotted lines represent extrapolated baseline in the absence of stimulation and grey dotted lines represent baseline average of first 5 min during the pre-stimulation period (see Experimental procedures). (B) Resting ATP_presyn was elevated in Munc13 KD compared to WT neurons (p < 0.000001). Chronic TTX treatment of Munc13 KD and WT however eliminated any differences (p = 0.94), while WT treated with TTX showed a modest increase in ATP_presyn values compared to WT neurons (p = 0.04), same as in Figure 2A. (D) Average drop in ATP_presyn (ΔATP_presyn) during the stimulus period (During stimulation) or subsequent 6 min post-stimulus period (Post-stimulation) in the presence of dGlu (left) or Oligo (right) for the conditions shown in (A, C and Figure 3B) show that ATP depletion monitored during stimulation is smaller in the absence of extracellular Ca²⁺ (p = 0.03, dGlu, n = 9 and p = 0.008, Oligo, n = 9) while the ΔATP_presyn measured in the post-stimulation period was not statistically significant from its respective baselines either in the absence of external Ca²⁺ (p = 0.7, dGlu, n = 9 and p = 0.1, Oligo, n = 9) or upon removal of Munc13 (p = 0.7, dGlu, n = 24 and p = 0.06, Oligo, n = 13). Error bars are SEM. Student’s t-test was used to determine statistical significances. See also Figure S4.

Figure 5. Presynaptic function relies on activity-driven ATP synthesis

Sample vGlut-pHluorin (vG-pH) traces showing (A) 100 AP responses before and after 5 min incubation in dGlu and (E) 600 AP responses before and after 5 min incubation in Oligo. Average ratio of the stimulus response in (B) dGlu compared to control (ΔF_dGlu/ΔF_control) at 100 AP (n = 9) and (F) Oligo compared to control (ΔF_Oligo/ΔF_control) at 600 and 100 AP (n = 4). Average endocytic block measured as the fraction of vG-pH fluorescence remaining at (C) 5 endocytic time constants (5τ) of the control at the end of 100 AP in control and dGlu (ΔF_5τ/ΔF_100AP), n = 9 and (G) at 2 endocytic time constants (2τ) at the end of 600 AP in control and Oligo (ΔF_2τ/ΔF_600AP), n = 4. Average vesicular pH determined from the vG-pH responses to NH₄Cl and acid quenching in (D) dGlu (n = 6) and (H) Oligo (n = 4). Error bars are SEM. See also Figure S5.