Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons

Abstract

Despite being the most abundant synaptic vesicle membrane protein, the function of synaptophysin remains enigmatic. For example, synaptic transmission was reported to be completely normal in synaptophysin knockout mice; however, direct experiments to monitor the synaptic vesicle cycle have not been carried out. Here, using optical imaging and electrophysiological experiments, we demonstrate that synaptophysin is required for kinetically efficient endocytosis of synaptic vesicles in cultured hippocampal neurons. Truncation analysis revealed that distinct structural elements of synaptophysin differentially regulate vesicle retrieval during and after stimulation. Thus, synaptophysin regulates at least two phases of endocytosis to ensure vesicle availability during and after sustained neuronal activity.

Figure 1. Syp regulates endocytosis of SVs following cessation of persistent neuronal activity

(A) Average traces of wild-type (black) and syp −/− (white) neurons expressing synaptotagmin 1-pHluorin (syt1-pH) in response to 300 stimuli at 10 Hz. Average from 3 coverslips, 20 boutons each. (B) Average traces of wild-type (black) and syp −/− (white) neurons expressing SV2A-pHluorin (SV2A-pH) in response to 300 stimuli at 10 Hz. Average from 6 coverslips, 30 boutons each. (C) Fluorescence images showing pre-synaptic boutons expressing SV2A-pH in wt and syp −/− neurons, before stimulation (t = 0) and following cessation of stimulation (t = 41s or 80 s) at 10 Hz for 30 s. ‘t’ indicates a time-point on the horizontal axis of the plot shown in (B). Scale bars are 2 μm. (D) Average SV2A-pH traces of syp −/− (white, 6 coverslips, 30 boutons each) and wt-syp rescue (grey, 5 coverslips, 30 boutons each) neurons in response to 300 stimuli at 10 Hz. (E) Average SV2A-pH traces of wild-type (black) and syp −/− (white) neurons in response to 50 stimuli at 10 Hz. Average from 4 coverslips, 40 boutons each. (F) Comparison of average post-stimulus endocytic time constants between wild-type, syp −/− and wt-syp rescue neurons. All ΔF values were normalized to the maximal fluorescence intensity change. The decay phases of ΔF traces from syt1-pH or SV2A-pH were fitted with single exponential functions before normalization, and the time constants were calculated from the fits. (G) Protocol for FM 1-43 pulse-chase experiments. Neurons were stimulated at 10 Hz for 30 s. After a short delay (30 s), neurons were exposed to FM 1-43 for 3 min and then washed in Ca²⁺ solution for 10 min. Two trains of 900 pulses (10 Hz) were delivered to evoke unloading of FM 1-43. After a 10 min rest, neurons were stimulated at 10 Hz for 30 s in the presence of FM 1-43. This was followed by washing and unloading in the same manner as the first round. (H) Sample images showing boutons labeled with FM 1-43 during the delayed loading (Left panels) and the maximal loading (Right panels). Scale bars are 1.5 μm. (I) Average ΔF₁/ΔF₂ ratio of wt and syp −/− neurons. Average from 4 coverslips, 40 boutons each. All error bars are SEM. **, p < 0.001 (two-tailed, unpaired t-test throughout).

Figure 2. Endocytosis during sustained synaptic transmission is reduced in syp −/− neurons

(A) Protocol for measuring time-course and extent of endocytosis during neuronal activity using bafilomycin (Baf) to prevent vesicle re-acidification. Neurons expressing SV2A-pH were stimulated at 10 Hz for 30 s in the absence of Baf. After a 10 min rest, neurons were stimulated at 10 Hz for 120 s in the presence of Baf. Images were acquired during each phase of stimulation. (B) Average SV2A-pH traces from wt neurons in response to 300 stimuli at 10 Hz. Traces represent averages from 6 coverslips, 30 boutons each. Values were normalized to the maximum fluorescence change at the end of 1200 stimuli in presence of Baf. The short and long arrows (also in (C) and (D)) indicate the extent of endocytosis and exocytosis at the end of the 300 stimuli (vertical dashed line) at 10 Hz respectively. (C) The same experiment was repeated in syp −/− neurons (6 coverslips, 30 boutons each) (D) The same experiment was repeated in wt-syp rescue samples (5 coverslips, 30 boutons each). (E) Time-courses of exocytosis (Baf) and endocytosis for wt, syp −/− and wt-syp rescue neurons during stimulation (30 s, 10 Hz). Each endocytosis time-course was calculated by subtracting the SV2A-pH trace that was acquired in the absence of Baf, from the ‘Baf’ trace in panels (B–D). Endocytosis traces were fitted with linear functions (solid line shown as an example for ‘wt’). Endocytic rates were determined empirically by calculating slope of the fitted lines. (F) Endocytic rates (in arbitrary units per second (AU/s)) as determined in (E). (G) Average magnitude of endocytosis as a fraction of exocytosis after delivery of 300 pulses. (H) Average exocytic time constants estimated by fitting the rising phase of Baf traces in panels (B–D) with single exponential functions. All error bars are SEM. **, p < 0.001.

Figure 3. The C-terminal cytoplasmic tail of synaptophysin regulates endocytosis during persistent neuronal activity, but is not important for post-stimulus endocytosis

(A) (Left) Average SV2A-pH traces of syp −/− (white, 6 coverslips, 30 boutons each), wt-syp rescue (black, 5 coverslips, 30 boutons each) and ΔC-syp neurons (grey, 5 coverslips, 30 boutons each) in response to 300 stimuli at 10 Hz. (Right) Comparison of average post-stimulus endocytic time constants between syp −/−, wt-syp rescue and ΔC-syp neurons. (B) Endocytosis during neuronal activity was assayed using the same protocol as shown in Fig. 2(A). Each trace is an average of the same number of samples as in (A). Time-courses of exocytosis (Baf) and endocytosis for syp −/− (Left), wt-syp rescue (Middle) and ΔC-syp neurons (Right) during the 30 s stimulation protocol. (C) Time-courses of exocytosis (Baf) and endocytosis for syp −/−, wt-syp rescue ΔC-syp neurons during the 30 s stimulation at 10 Hz. Each endocytosis time-course was calculated by subtracting the SV2A-pH trace that was acquired in the absence of Baf, from the ‘Baf’ trace in each of the three panels in (B). Endocytic rates were determined by fitting the slope with linear functions as in Fig. 2(E). (D) Endocytic rates (in AU/s) as determined in (C). (E) Average magnitude of endocytosis as a fraction of exocytosis after delivery of 300 stimuli. All error bars are SEM. *, p < 0.05, **, p < 0.001.

Figure 4. Time courses of synaptic depression and recovery in syp −/− neurons

(A) Representative traces of evoked inhibitory post-synaptic currents (IPSCs) in wt (top) and syp −/− (bottom) during 100 stimuli at 10 Hz in 3 mM Ca²⁺. (B) (Top) Normalized IPSC amplitudes in wt (black) and syp −/− (white) neurons during sustained 10 Hz stimulation. Values are averages from 7 wt and 7 syp −/− neurons. Amplitude values were normalized to the first response in the train. (Bottom) Averages of the first twenty responses during 10 Hz stimulation are shown for clarity. (C) Representative traces of IPSCs in wt-syp (Top) and ΔC-syp (Bottom) neurons during 100 stimuli at 10 Hz in 3 mM Ca²⁺. (D) Normalized IPSC amplitudes from wt-syp rescue (grey) and ΔC-syp neurons (grey triangle) are overlaid with wt and syp −/− data from (B). Values are averages from 8 wt-syp and 6 ΔC-syp samples. (E) Representative traces of IPSCs from vesicle pool recovery experiments. Neurons were subjected to two separate phases of stimulation: 200 stimuli at 10 Hz in 4 mM Ca²⁺ for maximal depletion of the recycling SV pool, and then a recovery phase with mild stimulation at 0.5 Hz. (F) Normalized IPSC amplitudes from wt (black) and syp −/− (white) neurons during the recovery phase. Amplitude values were normalized to the first response in the depletion phase and fitted with single exponential functions. Values are averages from 8 wt and 8 syp −/− neurons. All error bars are SEM.