Synaptotagmin-1 enables frequency coding by suppressing asynchronous release in a temperature dependent manner

Abstract

To support frequency-coded information transfer, mammalian synapses tightly synchronize neurotransmitter release to action potentials (APs). However, release desynchronizes during AP trains, especially at room temperature. Here we show that suppression of asynchronous release by Synaptotagmin-1 (Syt1), but not release triggering, is highly temperature sensitive, and enhances synchronous release during high-frequency stimulation. In Syt1-deficient synapses, asynchronous release increased with temperature, opposite to wildtype synapses. Mutations in Syt1 C2B-domain polybasic stretch (Syt1 K326Q,K327Q,K331Q) did not affect synchronization during sustained activity, while the previously observed reduced synchronous response to a single AP was confirmed. However, an inflexible linker between the C2-domains (Syt1 9Pro) reduced suppression, without affecting synchronous release upon a single AP. Syt1 9Pro expressing synapses showed impaired synchronization during AP trains, which was rescued by buffering global Ca²⁺ to prevent asynchronous release. Hence, frequency coding relies on Syt1's temperature sensitive suppression of asynchronous release, an aspect distinct from its known vesicle recruitment and triggering functions.

Figure 1

Raising temperature enables frequency-coded signalling during sustained transmission. (a) Typical EPSCs evoked by a 150-AP train with variable frequencies of 40, 20 and 10 Hz in autaptic hippocampal neurons (wild type) at room temperature (22 ± 1 °C) and (c) near-physiological temperature (32 ± 1 °C). (b) Raster plot of cells firing above 30% amplitude of first evoked response at 22 °C and (d) 32 °C.

Figure 2

Raising temperature synchronizes neurotransmission during sustained stimulation and suppresses asynchronous release. (a) Representative traces of mEPSC recordings at 22 °C (top) and 32 °C (bottom). Boxplots showing (b) mEPSC frequency (22 °C: 9.655 ± 5.34 Hz, n = 9; 32 °C: 22.27 ± 14.1 Hz, n = 9), (c) mEPSC amplitude (22 °C: 24.25 ± 3.20 pA, n = 9; 32 °C: 33.50 ± 5.05 pA, n = 9), and (d) mEPSC charge at 22 °C and 32 °C (22 °C: 0.1010 ± 0.00363 pC, n = 9; 32 °C: 0.1099 ± 0.0139 pC, n = 9). (e) Single EPSC representative traces and (f) boxplot of charge transferred per EPSC at 22 °C and 32 °C (22 °C: 70.34 ± 19.0 pC, n = 6; 32 °C: 78.15 ± 12.0 pC, n = 6). (g) Representative traces of 40 Hz train stimulation (100 pulses, 2.5 s) at 22 °C and 32 °C. The expanded traces illustrate steady state release dynamics, and single pulse zooms display division synchronous and asynchronous release. (h) Cumulative plots (mean ± S.E.M.) of charge transferred synchronously and asynchronously at 22 °C and (i) 32 °C. (j) Cumulative total charge (mean ± S.E.M.) at 22 °C and 32 °C. (k) Boxplots with late-train charge averaged over the final 20 pulses of the 40 Hz train, for paired 22 °C and 32 °C recordings. Displayed for total charge (22 °C: 15.53 ± 8.21 pC, n = 9; 32 °C: 28.54 ± 6.83 pC, n = 9), and synchronous (22 °C: 1.135 ± 0.293 pC, n = 9; 32 °C: 13.27 ± 5.134 Hz, n = 9) and asynchronous charge (22 °C: 14.69 ± 8.50 Hz, n = 9; 32 °C: 10.29 ± 5.92 Hz, n = 9) separately. (l) Boxplot with fraction of late-train charge transfer during the final 20 pulses released synchronously (22 °C: 0.07162 ± 0.0239, n = 9; 32 °C: 0.4872 ± 0.0658, n = 9). (m) Typical traces of asynchronous tail release, normalized to peak release. (n) Boxplot with decay tau from single exponential fits of asynchronous tail release at 22 °C and 32 °C (22 °C: τ = 0.5694 ± 0.129 s, n = 9; 32 °C: τ = 0.1843 ± 0.0854 s, n = 9). (*p < 0.05, Wilcoxon signed-rank test).

Figure 3

Raising temperature in Synaptotagmin-1 deficient synapses enhances asynchronous release instead of suppressing it. (a) Representative traces of mEPSC recordings at 22 °C (top) and 32 °C (bottom), and (b) boxplot showing mEPSC frequency (22 °C: 9.002 ± 3.09 Hz, n = 7; 32 °C: 56.38 ± 35.7 Hz, n = 7). (c) Single EPSC representative traces and (d) boxplot of charge transferred per EPSC at 22 °C and 32 °C (22 °C: 12.19 ± 4.57 pC, n = 6; 32 °C: 4.037 ± 1.59 pC, n = 6). (e) Representative traces of 40 Hz train stimulation (100 pulses, 2.5 s) at 22 °C and 32 °C; single pulse zooms display division synchronous and asynchronous release. (f) Cumulative plots (mean ± S.E.M.) of charge transferred synchronously and asynchronously at 22 °C and (g) 32 °C. (h) Cumulative total charge (mean ± S.E.M.) at 22 °C and 32 °C. (i) Boxplots with late-train charge averaged over the final 20 pulses of the 40 Hz train, for paired 22 °C and 32 °C recordings. Displayed for total charge (22 °C: 8.711 ± 0.905 pC, n = 7; 32 °C: 17.79 ±  ± 4.03 pC, n = 7), and synchronous (22 °C: 0.5791 ± 0.157 pC, n = 7; 32 °C: 1.594 ± 0.353 pC, n = 7) and asynchronous charge (22 °C: 8.132 ± 1.17 pC, n = 7; 32 °C: 15.84 ± 3.57 pC, n = 7) separately. (j) Boxplot with fraction of late-train charge transfer during the final 20 pulses released synchronously (22 °C: 0.07414 ± 0.00782, n = 7; 32 °C: 0.1104 ± 0.00939, n = 7). (k) Typical traces of asynchronous tail release, normalized to peak release. (l) Boxplot with decay tau from single exponential fits of asynchronous tail release at 22 °C and 32 °C (22 °C: τ = 1.120 ± 0.186 s, n = 6; 32 °C: τ = 0.5694 ± 0.174 s, n = 6). (*p < 0.05, Wilcoxon signed-rank test).

Figure 4

Deficient Ca²⁺ binding in Syt1 D363N mutant suppresses all forms of release and delays the onset of the asynchronous component. (a) Representative traces of mEPSC recordings from Syt1 KO neurons rescued with WT (top) or D363N mutant (bottom) constructs, and (b) boxplot showing mEPSC frequency (Syt1 WT: 11.11 ± 7.85 Hz, n = 19; Syt1 D363N: 2.585 ± 1.82 Hz, n = 26). (c) Representative traces of 40 Hz train stimulation (100 pulses, 2.5 s) in Syt1 WT or D363N expressing synapses. The expanded traces illustrate steady state release dynamics, and single pulse zooms display division synchronous and asynchronous release. (d) Cumulative plots (mean ± S.E.M.) of charge transferred synchronously and asynchronously in Syt1 WT, or (E) Syt1 D363N expressing synapses. (f) Cumulative total charge (mean ± S.E.M.) in Syt1 WT and D363N expressing synapses. (g) Boxplots with late-train charge averaged over the final 20 pulses of the 40 Hz train, for Syt1 WT and D363N expressing synapses. Displayed for total charge (Syt1 WT: 41.30 ± 18.3 pC, n = 19; Syt1 D363N: 17.51 ± 14.5 pC, n = 25), and synchronous (Syt1 WT: 1.703 ± 0.676 pC, n = 19; Syt1 D363N: 2.651 ± 1.404 pC, n = 25) and asynchronous charge (Syt1 WT: 39.64 ± 19.9 pC, n = 19; Syt1 D363N: 15.61 ± 13.0 pC, n = 25) separately. (h) Representative traces of 500 mM hypertonic sucrose responses, and (i) boxplot of RRP charge estimates for Syt1 WT and D363N expressing synapses (Syt1 WT: 3.039 ± 0.219 nC, n = 3; Syt1 D363N: 1.105 ± 0.923 nC, n = 7). (j) Representative traces of 40 Hz train stimulation in Syt1 D363N rescue and Syt1 KD synapses, scaled to the peak asynchronous current. (k) Asynchronous charge plot (mean ± S.E.M.), normalized to peak mean charge for Syt1 D363N rescue and Syt1 KD synapses. (l) Boxplot of pulse number containing the peak asynchronous charge for Syt1 D363N rescue and Syt1 KD synapses (Syt1 D363N: 31 ± 7, n = 25; Syt1 KD: 11 ± 1, n = 7). All recordings at room temperature (RT), ~22 °C unmonitored. (*p < 0.05, Wilcoxon rank sum test).

Figure 5

Syt1 3K expressing synapses show impaired first evoked response but have normal release inhibition and late train synchronous release. (a) Representative traces of mEPSC recordings from Syt1 KO neurons rescued with WT (top) or 3K mutant (bottom) constructs, and (b) boxplot showing mEPSC frequency (Syt1 WT: 10.97 ± 8.72 Hz, n = 70; Syt1 3 K: 9.388 ± 6.42 Hz, n = 72). (c) Single EPSC representative traces and (d) boxplot of charge transferred per EPSC in Syt1 WT and 3 K expressing synapses (Syt1 WT: 68.81 ± 28.6 pC, n = 33; Syt1 3 K: 40.53 ± 19.5 pC, n = 24). (e) Representative traces of 10 Hz train stimulation (20 pulses, 2 s) in Syt1 WT or 3K expressing synapses; single pulse zooms display division synchronous and asynchronous release. (f) Cumulative plots (mean ± S.E.M.) of charge transferred synchronously and asynchronously in Syt1 WT, or (g) Syt1 3K expressing synapses. (h) Cumulative total charge (mean ± S.E.M.) in Syt1 WT and 3K expressing synapses. (i) Boxplots with late-train charge averaged over the final 3 pulses of the 10 Hz train, for Syt1 WT and 3K expressing synapses. Displayed for total charge (Syt1 WT: 65.09 ± 29.1 pC, n = 33; Syt1 3K: 96.07 ± 34.9 pC, n = 24), and synchronous (Syt1 WT: 19.41 ± 36.3 pC, n = 33; Syt1 3K: 36.28 ± 17.6 pC, n = 24) and asynchronous charge (Syt1 WT: 39.07 ± 19.5 pC, n = 33; Syt1 3K: 55.80 ± 21.2 pC, n = 24) separately. (j) Boxplot with fraction of late-train charge transfer during the final 3 pulses released synchronously (Syt1 WT: 0.3834 ± 0.0616, n = 33; Syt1 3K: 0.4038 ± 0.0522, n = 24). All recordings at room temperature (RT), ~22 °C unmonitored. (*p < 0.05, Wilcoxon rank sum test).

Figure 6

Impaired release inhibition in Syt1 9Pro expressing synapses decreases late-train synchronous release and increases asynchronous release. (a) Representative traces of mEPSC recordings from Syt1 KO neurons rescued with WT (top) or 9Pro mutant (bottom) constructs, and (b) boxplot showing mEPSC frequency (Syt1 WT: 12.33 ± 6.33 Hz, n = 28; Syt1 9Pro: 32.76 ± 20.4 Hz, n = 23). (c) Single EPSC representative traces and (d) boxplot of charge transferred per EPSC in Syt1 WT and 9Pro expressing synapses (Syt1 WT: 163.5 ± 112 pC, n = 37; Syt1 9Pro: 241.5 ± 152 pC, n = 37). (e) Representative traces of 10 Hz train stimulation (20 pulses, 2 s) in Syt1 WT or 9Pro expressing synapses; single pulse zooms display division synchronous and asynchronous release. (f) Cumulative plots (mean ± S.E.M.) of charge transferred synchronously and asynchronously in Syt1 WT, or (G) Syt1 9Pro expressing synapses. (h) Cumulative total charge (mean ± S.E.M.) in Syt1 WT and 9Pro expressing synapses. (i) Boxplots with late-train charge averaged over the final 3 pulses of the 10 Hz train, for Syt1 WT and 9Pro expressing synapses. Displayed for total charge (Syt1 WT: 133.8 ± 63.8 pC, n = 27; Syt1 9Pro: 152.8 ± 61.6 pC, n = 28), and synchronous (Syt1 WT: 67.66 ± 32.9 pC, n = 27; Syt1 9Pro: 45.16 ± 22.4 pC, n = 28) and asynchronous charge (Syt1 WT: 60.76 ± 21.0 pC, n = 27; Syt1 9Pro: 92.49 ± 39.10 pC, n = 28) separately. (j) Boxplot with fraction of late-train charge transfer during the final 3 pulses released synchronously (Syt1 WT: 0.5409 ± 0.0360, n = 27; Syt1 9Pro: 0.3654 ± 0.0755, n = 28). All recordings at room temperature (RT), ~22 °C unmonitored. (*p < 0.05, Wilcoxon rank sum test).

Figure 7

Impaired synchronization in Syt1 9Pro is rescued when global Ca²⁺ is buffered by EGTA-AM. (a) Representative traces of 20 Hz train stimulation (20 pulses, 1 s) in Syt1 WT or 9Pro expressing synapses in the absence (Naive) or (g) presence of EGTA-AM; single pulse zooms display division synchronous and asynchronous release. (b) Cumulative plots (mean ± S.E.M.) of charge transferred synchronously and asynchronously in naive Syt1 WT and (h) in the presence of EGTA-AM, or (C) naive Syt1 9Pro expressing synapses and (i) in the presence of EGTA-AM. (d) Cumulative total charge (mean ± S.E.M.) in naive and (j) EGTA-AM Syt1 WT and 9Pro conditions. (e) Boxplots with late-train charge averaged over the final 3 pulses of the 20 Hz train, for naive and (k) EGTA-AM Syt1 WT and 9Pro conditions. Displayed for total charge (WT Naive: 118.8 ± 46.3 pC, n = 40; 9Pro Naive: 108.2 ± 44.4 pC, n = 41; WT EGTA-AM: 47.93 ± 17.5 pC, n = 11; 9Pro EGTA-AM: 36.53 ± 15.39 pC, n = 10), and synchronous (WT Naive: 31.47 ± 13.5 pC, n = 40; 9Pro Naive: 16.28 ± 6.84 pC, n = 41; WT EGTA-AM: 38.70 ± 14.7 pC, n = 11; 9Pro EGTA-AM: 31.59 ± 14.6 pC, n = 10)and asynchronous charge (WT Naive: 73.23 ± 39.2 pC, n = 40; 9Pro Naive: 87.43 ± 39.2 pC, n = 41; WT EGTA-AM: 6.145 ± 3.08 pC, n = 11; 9Pro EGTA-AM: 4.383 ± 2.16 pC, n = 10) separately. (f,l) Boxplot with fraction of late-train charge transfer during the final 3 pulses released synchronously for naive and EGTA-AM conditions respectively (WT Naive: 0.2728 ± 0.0592, n = 40; 9Pro Naive: 0.1426 ± 0.0195, n = 41; WT EGTA-AM: 0.8634 ± 0.0243, n = 11; 9Pro EGTA-AM: 0.8718 ± 0.0245, n = 10). All recordings at room temperature (RT), ~22 °C unmonitored. (*p < 0.05, Wilcoxon rank sum test).

Figure 8

Impaired release inhibition Syt1 9Pro reduces the temperature dependent increase in synchronization. (a) Representative traces of 40 Hz train stimulation (100 pulses, 2.5 s) at 22 °C and 32 °C, for Syt1 WT and 9Pro expressing synapses. The expanded traces illustrate steady state release dynamics, and single pulse zooms display division synchronous and asynchronous release. (b,f) Cumulative plots (mean ± S.E.M.) of charge transferred synchronously and asynchronously in Syt1 WT and 9Pro respectively at 22 °C and (c,g) 32 °C. (d,h) Cumulative total charge (mean ± S.E.M.) at 22 °C and 32 °C in Syt1 WT and 9Pro expressing synapses respectively. (e,i) Boxplots with late-train charge averaged over the final 20 pulses of the 40 Hz train, for paired 22 °C and 32 °C recordings in Syt1 WT and 9Pro respectively. Displayed for total charge (WT 22 °C: 14.75 ± 2.79 pC; WT 32 °C: 22.73 ± 5.41 pC, n = 17; 9Pro 22 °C: 21.19 ± 13.1 pC; 9Pro 32 °C: 23.19 ± 5.90 pC, n = 22), and synchronous (WT 22 °C: 0.8044 ± 0.246 pC; WT 32 °C: 10.89 ± 3.92 pC, n = 17; 9Pro 22 °C: 0.9444 ± 0.364 pC; 9Pro 32 °C: 8.504 ± 5.00 pC, n = 22) and asynchronous charge (WT 22 °C: 14.02 ± 2.41 pC; WT 32 °C: 10.40 ± 5.16 pC, n = 17; 9Pro 22 °C: 19.83 ± 12.6 pC; 9Pro 32 °C: 14.17 ± 4.42 pC, n = 22) separately. (j) Boxplot with fraction of late-train charge transfer during the final 20 pulses released synchronously (WT 22 °C: 0.05344 ± 0.00995, n = 19; 9Pro 22 °C: 0.07128 ± 0.0256, n = 22; WT 32 °C: 0.5444 ± 0.1122, n = 17; 9Pro 32 °C: 0.3707 ± 0.0648, n = 22). (k,l) Typical traces of asynchronous tail release, normalized to peak release at 22 °C and 32 °C respectively for Syt1 WT and 9Pro expressing synapses. (m) Boxplot with decay tau from single exponential fits of asynchronous tail release in Syt1 WT and 9Pro neurons at 22 °C and 32 °C (WT 22 °C: τ = 0.4956 ± 0.127 s, n = 19; 9Pro 22 °C: τ = 0.6869 ± 0.160 s, n = 21; WT 32 °C: τ = 0.1534 ± 0.0879 s, n = 16; 9Pro 32 °C: τ = 0.1762 ± 0.0743 s, n = 21). (*p < 0.05, Wilcoxon signed-rank test for paired and Wilcoxon rank sum test for independent samples).

Figure 9

Syt1’s release inhibitory function synchronizes release during high-frequency stimulation. Working model for the effect of Syt1’s release inhibitory and vesicle recruitment functions on release coming from rest (First evoked release; left column) and during high-frequency stimulation (right column), split between global Ca²⁺ induced release in the interpulse-interval (i.e. 75 ms), and peak Ca²⁺ induced release right after the AP (i.e. 25 ms). With vesicles not subject to Syt1’s release inhibitory function in green (unclamped), and clamped vesicles in red. (a,c,e,g) First evoked release after a period of rest during peak Ca²⁺ in WT synapses at 32 °C (a), in WT synapses at 22 °C (c), in Syt1 9Pro synapses at 32 °C with a shift towards unclamped vesicles due to impaired Syt1 release inhibition (e), and in Syt1 3K synapses at 32 °C with a decreased number of available vesicles due to impaired constitutive vesicle recruitment (g). (b,d,f,h) Late-train release during high-frequency stimulation, due to global Ca²⁺ (left), and peak Ca²⁺ (right), in WT synapses at 32 °C (b) where rapid transition of newly primed vesicles to the clamped (red) state suppresses asynchronous release due to global Ca²⁺ (b, left), preserving vesicles for synchronous release during peak Ca²⁺ (b, right). In WT synapses at 22 °C (d), where a slower on-rate of release inhibition leaves vesicles unclamped causing asynchronous release (d, left), and leaving insufficient vesicles for a synchronous response (d, right). In Syt1 9Pro synapses at 32 °C (f) where impaired release inhibition leaves vesicles unclamped (f, left), leaving too few vesicles for synchronous release (f, right). And in Syt1 3 K synapses at 32 °C (h) where global Ca²⁺ drives increased Ca²⁺-dependent recruitment, increasing the available pool for synchronous release (h, right). In all panels insets show representations of the post-synaptic response based on the depicted fusion events, with synchronous responses in dark grey, asynchronous responses in light grey, and the baseline represented by the dashed line.