Abstract
Neural systems encode information in the frequency of action potentials, which is then decoded by synaptic transmission. However, the rapid, synchronous release of neurotransmitters depletes synaptic vesicles (SVs), limiting release at high firing rates. How then do synapses convey information about frequency? Here, we show in mouse hippocampal neurons and slices that the adaptor protein AP-3 makes a subset of SVs that respond specifically to high-frequency stimulation. Neurotransmitter transporters slot onto these SVs in different proportions, contributing to the distinct properties of release observed at different excitatory synapses. Proteomics reveals that AP-3 targets the phospholipid flippase ATP8A1 to SVs; loss of ATP8A1 recapitulates the defect in SV mobilization at high frequency observed with loss of AP-3. The mechanism involves recruitment of synapsin by the cytoplasmically oriented phosphatidylserine translocated by ATP8A1. Thus, ATP8A1 enables the subset of SVs made by AP-3 to release at high frequency.
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Data availability
The original mass spectra for the SV proteomics are shown in Supplementary Table 1. The sequence of primers are shown in Supplementary Table 2. Source data are provided with this paper.
Code availability
The code used for the analysis of the pHluorin imaging is provided with this paper as Supplementary Code 1.
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Acknowledgements
We thank J. Maas and other members of the Edwards laboratory for helpful discussion, J. Maas, X. Chen and K. Bender for help with electrophysiology, and R. Nicoll for help with the experimental design and evaluating the data. All confocal images were acquired at the UCSF Center for Advanced Light Microscopy-Nikon Imaging Center supported by an NIH S10 Shared Instrumentation grant (no. 1S10OD017993-01A1). MS was provided by the MS Resource at UCSF (A.L. Burlingame, Director) supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the UCSF Program for Breakthrough Biomedical Research. This work was supported by NIH grant nos. R01 MH50712 and R01 NS103938 to R.H.E. The funders had no role in study design, data collection, analysis, decision to publish or preparation of the paper.
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H.X., S.J. and R.H.E. conceptualized the work. M.K. performed the initial SV preparations. J.A.O.-P. performed the proteomics analysis with support from A.B. J.L. prepared several of the primary neuronal cultures and ATP8A1-related constructs. H.X. and R.H.E. wrote the paper.
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Extended data
Extended Data Fig. 1 VAMP7+ and VGLUT2+ SVs differ in the frequency dependence of exocytosis.
a, Hippocampal neurons from WT mice were immunostained for endogenous synaptophysin (green), VAMP7 (red) and MAP2 (blue). The area inside the white rectangle is shown below at higher magnification. Arrowheads indicate punctae double labeling for synaptophysin and VAMP7. Scale bars, 20 μm. Representative images were from three independent experiments. b, Hippocampal neurons expressing VAMP7-, VAMP2- or VGLUT2-pHluorin (pH) were stimulated at 5, 10, 25 and 50 Hz for 20 s and the fluorescence normalized to total pHluorin as shown in Fig. 1. Graphs show the initial rate of fluorescence increase per action potential (P = 2.3 × 0−14, 5 and 10 Hz; 3.7 × 10−14, 25 Hz; 1.44 × 10−4, 50 Hz). n = 20/3 c, Graph shows the time constant for endocytosis of VAMP7- and VGLUT2-pH (P = 1.15 × 10−13, 5 Hz; 9.3 × 10−14, 10 Hz; 2.19 × 10−10, 25 Hz; 1.85 × 10−10, 50 Hz). n = 18/3 d-f, Hippocampal neurons expressing VAMP7- or VGLUT2-pH were stimulated at 5, 10, 25 and 50 Hz for 60-120 s in the presence of bafilomycin (d). The initial rate of fluorescence increase per action potential (e) and the proportion of pHluorin reporters (f) as a function of stimulation frequency. e, P = 3.9 × 10−14, 5 Hz; 4.1 × 10−14, 10 Hz; 1.11 × 10−13, 25 Hz; 0.27, 50 Hz (n = 18/3). f, P = 3.4 × 10−14, 5 Hz; 3.41 × 10−14, 10 Hz; 3.4 × 10−14, 25 Hz; 0.007, 50 Hz (n = 23/3). g, The initial rate/AP at 50 Hz normalized to the rate at 5 Hz in the presence or absence of bafilomycin: without bafilomycin (n = 20/3), P = 8.66 × 10−6, VGLUT2; 1.75 × 10−7, VAMP7; with bafilomycin (n = 18/3), P = 9.86 × 10−6, VGLUT2; 4.86 × 10−8, VAMP7. n = x/y where x is the number of fields examined and y the number of independent experiments. The data indicate mean ± s.e.m. for individual coverslips containing 50–100 boutons each. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-way (b,c,e,f) or one-way ANOVA (g) with Tukey’s multiple comparisons test.
Extended Data Fig. 2 VAMP7+ SVs differ from canonical SVs in pH, Ca++ sensitivity and coupling to presynaptic Ca++ channels.
a, Hippocampal neurons expressing VGLUT1-, VGLUT2-, VAMP2- or VAMP7-pH were imaged in Tyrode’s solution and 2-methanesulfonic acid (MES, pH 5.5) was added to quench cell surface pHluorin, then 50 mM NH4Cl to alkalinize the intracellular pool and reveal the total pHluorin fluorescence. Left, imaging; middle, fluorescence traces; right, surface expression of pHluorin reporters (P = 1.0 × 10−15, n = 19/3). Scale bar, 20 µm. b, The baseline intracellular fluorescence determined by subtraction of the cell surface protein and normalization to total in NH4Cl was used to determine the lumenal pH for each SV reporter (P = 1.92 × 10−10, n = 10/3). c-f, Hippocampal neurons from WT mice expressing VAMP7- (c), VGLUT2- (e) or VGLUT1-pH (f) were stimulated at 5, 10, 25 and 50 Hz in 0, 0.5, 2 and 4 mM Ca++, and the fluorescence response normalized as above (n = 15/3 for c and e, 20/3 for f and 17/3 for d). Graph shows the time constant for endocytosis at 50 Hz (d) (P = 0.027). g Hippocampal neurons expressing VAMP7- or VGLUT2-pH were incubated with 100 μM EGTA-AM for 15 min in Tyrode’s buffer and then stimulated at 10 Hz for 20 s. Scatterplot shows the peak response in EGTA-AM relative to controls (P = 2 × 10−15, n = 23/3). h, Hippocampal neurons expressing GLUT4-, VAMP7- or VGLUT1-pH were incubated with 1 mM AICAR for 30 min and the fluorescence response normalized as above. The scatter plot indicates the pH of GLUT4+ vesicles, determined from the response to acid quenching and neutralization with NH4Cl (n = 33/3). n = x/y where x is the number of fields examined and y the number of independent experiments. The data indicate mean ± s.e.m. for individual coverslips containing 50-100 boutons each. ****, P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test (a,b,d) or two-tailed unpaired t test (g).
Extended Data Fig. 3 VAMP7-pH and VGLUT2-pH exocytosis differ in dependence on VAMP2.
a, Hippocampal neurons from WT mice expressing VAMP7-, VGLUT1-, VMAT2- or VGLUT2-pH were treated with tetanus toxin (TeNT) for 16-18 hours and then stimulated at 5, 10, 25 and 50 Hz, with the fluorescence normalized as in Fig. 1. Graphs indicate fluorescence at the end-stimulation. VAMP7, P = 1.73 × 10−9, 10 Hz; 3.56 × 10−10, 25 Hz; 3.56 × 10−10, 50 Hz (n = 12/3). VGLUT1, P = 1.008 × 10−9,10 Hz; 4.0 × 10−14, 25 Hz; 4.0 × 10−14, 50 Hz (n = 15/3). VMAT2, P = 0.0012, 10 Hz; 7.22 × 10−9, 25 Hz; 3.56 × 10−10, 50 Hz (n = 12/3). VGLUT2, P = 0.02, 5 Hz; 6.57 × 10−8, 10 Hz; 3.56 × 10−10, 25 Hz and 50 Hz (n = 12/3). b, Hippocampal neurons from WT or VAMP2 KO mice expressing VAMP7- or VGLUT2-pH were stimulated at 5, 10, 25 and 50 Hz and the fluorescence response normalized as above. Graphs indicate the response at the end-stimulation. VAMP7, P = 5.12 × 10−9, 10 Hz; 1.08 × 10−7, 25 Hz; 3.56 × 10−10, 50 Hz (n = 12/3). VGLUT2, P = 3.98 × 10−5, 5 Hz; 3.56 × 10−10, 10 Hz; 3.56 × 10−10, 25 Hz; 3.56 × 10−10, 50 Hz (n = 12/3). c, Hippocampal neurons from WT or VAMP7 KO mice expressing VGLUT1-, VMAT2- or VGLUT2-pH were treated with TeNT for 16-18 hours and then stimulated at 50 Hz, with the fluorescence normalized as above. Graphs indicate fluorescence at the end-stimulation. VGLUT1, P = 1.05 × 10−7 (n = 15/3). VMAT2, P = 6.40 × 10−13 (n = 13/3). VGLUT2, P = 0.39 (n = 15/3). d, Hippocampal neurons from embryonic synaptotagmin 1 (Syt1) KO and littermates expressing VAMP7- or VGLUT2-pH were stimulated at 5, 10, 25 and 50 Hz. Graphs indicate the peak response. VAMP7, P = 1.14 × 10−4, 25 Hz; 2.61 × 10−7, 50 Hz (n = 11/3). VGLUT2, P = 0.0015, 5 Hz; 1.13 × 10−6, 10 Hz; 3.79 × 10−12, 25 Hz; 8.44 × 10−7, 50 Hz (n = 11/3). n = x/y where x is the number of fields examined and y the number of independent experiments. The data indicate mean ± s.e.m. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by two-way ANOVA (a,b,d) or one-way ANOVA (c) with Tukey’s multiple comparisons test.
Extended Data Fig. 4 Loss of AP-3 specifically impairs release at high frequency.
a, Hippocampal neurons from WT and mocha mice expressing VAMP7-pH were stimulated at 5, 10, 25 and 50 Hz for 60-150 s in the presence of 60 nM bafilomycin and the fluorescence response normalized as shown in Fig. 1 (above). Statistical analysis of the response at 4 s is shown below. 25 Hz: P = 6 × 10−15, 2 s; 1.22 × 10−12, 3 s; 9 × 10−14, 4 s. 50 Hz: P = 2.16 × 10−7, 1 s; 5.71 × 10−11, 2 s; 2.01 × 10−12, 3 s; 2.9 × 10−14, 4 s). n = 16/3. b, The initial rate of fluorescence increase per action potential in WT and mocha neurons: P = 0.029,10 Hz; 1.133 × 10−6, 25 Hz; 3.56 × 10−10, 50 Hz (n = 12/3). c, The proportion of VAMP7-pH that responds to stimulation (recycling pool size) in WT and mocha neurons: P = 0.0084, 10 Hz; 1.12 × 10−13, 25 Hz; 5.2 × 10−14, 50 Hz (n = 16/3). n = x/y where x is the number of fields examined and y the number of independent experiments. The data indicate mean ± s.e.m. for individual coverslips containing 50-100 boutons each. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test.
Extended Data Fig. 5 Effect of the mocha mutation on short-term plasticity in CA1 strata radiatum and lacunosum-moleculare.
a, Hippocampal region CA1 with whole cell recording electrode in the pyramidal cell layer. For VGLUT1+ inputs, stimulating electrodes were placed in stratum radiatum between regions CA3 and CA1. For VGLUT2+ inputs, stimulating electrodes were placed in stratum lacunosum-moleculare between regions CA3 and CA1. b, Representative traces from three independent experiments in response to stimulation at 1 and 25 Hz from stratum radiatum of WT and mocha slices. c, Representative traces from three independent experiments in response to stimulation at 5, 25 and 50 Hz from stratum lacunosum-moleculare of WT and mocha slices. d, Analysis of the normalized stratum lacunosum-moleculare EPSCs at 10, 30 and 90 action potentials as a function of stimulation frequency (n = 11 cells from three WT mice; n = 14 cell for 5 and 50 Hz, 9 cells for 25 Hz from three mocha mice). There is no significant difference between the genotypes by two-way ANOVA with Tukey’s multiple comparisons test. The data indicate mean ± s.e.m.
Extended Data Fig. 6 Proteomic comparison of SVs from WT and mocha mice.
a, Diagram of SV purification. After velocity sedimentation in glycerol, fractions 5-9 (pink) were collected, sedimented and analyzed by LC-MS/MS. b, Enrichment of SV proteins. Equal amounts of protein (0.5 µg) from the different stages of SV purification (H, homogenate; P1, tissue debris, nuclei, and large myelin fragments; P2, synaptosomes; LP1, synaptic plasma membrane and associated organelles; LS2, synaptosomal cytoplasm; LP2, synaptic vesicles) were analyzed by immunoblotting. Representative blots were from three independent experiments. c, Fractionation of synaptic vesicles (LP2) by glycerol velocity sedimentation showing the migration of VAMP7, VGLUT2 and synaptophysin. Representative blots were from three independent experiments. d, Fold enrichment of proteins in mocha SVs relative to WT. e, Quantitative western analysis of SV proteins in WT and mocha mice. Scatterplots show the fraction relative to WT. P = 4.24 × 10−5, VAMP7; 0.04, Syt1, 0.0019, VGLUT1; 8.05 × 10−6, ZnT-3; 3.24 × 10−3, ATP8A1; 0.0052, Scamp1 (n = 3 independent experiments). The data indicate mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by unpaired two-tailed t-test.
Extended Data Fig. 7 Loss of AP-3 redistributes ATP8A1 away from axon terminals in hippocampus and cerebellum.
a, Slices from adult WT and mocha mice were immunostained for ATP8A1 (red), synaptophysin (green), Hoechst (blue) and MAP2 (purple). Scale bar, 400 μm. Representative images were from three independent experiments. b,c, Cerebellar slices from adult WT and mocha mice were immunostained for ATP8A1 (red), synaptophysin (green) and Hoechst (blue). Images were acquired at low (b) and high magnification (c). Scale bars, 400 μm (b) and 100 μm (c). Scatterplot indicates the fluorescence intensity of ATP8A1 from the molecular layer of WT and mocha slices: b, P = 3.36 × 10−6; c, P = 2.69 × 10−8, ATP8A1; c, 0.9809, synaptophysin (15 images from 3 independent experiments). The data indicate mean ± s.e.m. ****, P < 0.0001 by unpaired two-tailed t-test.
Extended Data Fig. 8 Loss of ATP8A1 selectively impairs the response to high frequency stimulation.
a, Quantitative western analysis of the LP2 (SV) fraction from WT and ATP8A1 KO mice (n = 3 independent experiments). b, Hippocampal neurons from WT and ATP8A1 KO mice expressing VAMP7-pH were stimulated at 5, 10, 25 and 50 Hz for 60-150 s in the presence of bafilomycin and the response normalized to NH4Cl (n = 11/3). c, The initial rate of fluorescence increase per action potential in WT and ATP8A1 KO neurons (P = 0.99, 10 Hz; 0.0057, 25 Hz; 2.22 × 10−4, 50 Hz). n = 11/3. d, The recycling pool size in WT and ATP8A1 KO neurons (P = 0.99, 10 Hz; 1.82 × 10−6, 25 Hz; 7.20 × 10−9, 50 Hz). n = 11/3. e, Hippocampal neurons from ATP8A1 KO mice infected with virus encoding WT or E191Q ATP8A1 (with IRES-mCherry) were lysed with SDS-sample buffer and blotted with ATP8A1 antibody. The expression level (right) was normalized to β-action (n = 3 independent experiments. P = 0.3982). n = x/y where x is the number of fields examined and y the number of independent experiments. The data indicate mean ± s.e.m. for individual coverslips containing 50-100 boutons each. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA (c,d) with Tukey’s multiple comparisons test or unpaired two-tailed t-test (e).
Extended Data Fig. 9 High salt releases endogenous synapsins from SVs.
a, WT and mocha neurons expressing GFP-synapsin 1 were stimulated at 5 and 25 Hz and the fluorescence response over boutons normalized to the baseline before stimulation. Analysis (right) shows the time constants of dispersion during stimulation and of reclustering after stimulation (n = 15 coverslips for 5 Hz and 12 coverslips for 25 Hz from three independent experiments. P = 6 × 10−5). b, LP2 (SVs) were treated with high salt (0.3 M glycine, 0.2 M NaCl) for 10-90 min, the membranes sedimented and then immunoblotted for synapsins and VGLUT2 (n = 3 independent experiments). c, Quantitative western analysis shows that LS2 does not contain the membrane protein VGLUT2 (n = 3 independent experiments). d, LS2 fraction contains synapsins. Scatterplot shows the amount of synapsin 1 and 2 in WT and ATP8A1 KO mice (P = 0.16, synapsin 1; 0.016, synapsin 2). n = 3 independent experiments. e, SV fractions (LP2) from WT and ATP8A1 KO mice were treated with high salt buffer to remove endogenous synapsins and then incubated with presynaptic cytosol (LS2) from WT animals in the presence or absence of 0.2 mM ATP. After incubation, the SVs were sedimented and SV-bound synapsin quantified by western analysis. ATP increases synapsin binding to WT but not ATP8A1 KO SVs: synapsin 1 WT (P = 0.0025), ATP8A1 KO (P = 0.1217); synapsin 2 WT (P = 0.0230), ATP8A1 KO (P = 0.9325) and loss of ATP8A1 reduces binding to SVs: synapsin 1, without ATP (P = 4.67 × 10−7); synapsin 1 with ATP (P = 1.41 × 10−8). n = 4 independent experiments The data indicate mean ± s.e.m. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by unpaired two-tailed t-test (a,d) or one-way ANOVA (e) with Tukey’s multiple comparison s test.
Extended Data Fig. 10 The ATP8A1 KO has no effect on mEPSC frequency or amplitude of CA1 pyramidal neurons.
a,b, Representative mEPSCs of CA1 pyramidal neurons from WT and ATP8A1 KO mice (left). Frequency (middle) and amplitude (right) of mEPSCs from WT and ATP8A1 KOs. ns, P = 0.39 for mEPSC frequency; P = 0.84 for mEPSC amplitude by unpaired two-tailed t-test. The data indicate mean ± s.e.m. n = 6 slices from three animals.
Supplementary information
Supplementary Table 1
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Sequences of primers
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Code used to analyze the pHluorin imaging
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Xu, H., Oses-Prieto, J.A., Khvotchev, M. et al. Adaptor protein AP-3 produces synaptic vesicles that release at high frequency by recruiting phospholipid flippase ATP8A1. Nat Neurosci 26, 1685–1700 (2023). https://doi.org/10.1038/s41593-023-01434-0
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DOI: https://doi.org/10.1038/s41593-023-01434-0
