Synaptotagmin-7 Is an Asynchronous Calcium Sensor for Synaptic Transmission in Neurons Expressing SNAP-23

Synchronization of neurotransmitter release with the presynaptic action potential is essential for maintaining fidelity of information transfer in the central nervous system. However, synchronous release is frequently accompanied by an asynchronous release component that builds up during repetitive stimulation, and can even play a dominant role in some synapses. Here, we show that substitution of SNAP-23 for SNAP-25 in mouse autaptic glutamatergic hippocampal neurons results in asynchronous release and a higher frequency of spontaneous release events (mEPSCs). Use of neurons from double-knock-out (SNAP-25, synaptotagmin-7) mice in combination with viral transduction showed that SNAP-23-driven release is triggered by endogenous synaptotagmin-7. In the absence of synaptotagmin-7 release became even more asynchronous, and the spontaneous release rate increased even more, indicating that synaptotagmin-7 acts to synchronize release and suppress spontaneous release. However, compared to synaptotagmin-1, synaptotagmin-7 is a both leaky and asynchronous calcium sensor. In the presence of SNAP-25, consequences of the elimination of synaptotagmin-7 were small or absent, indicating that the protein pairs SNAP-25/synaptotagmin-1 and SNAP-23/synaptotagmin-7 might act as mutually exclusive calcium sensors. Expression of fusion proteins between pHluorin (pH-sensitive GFP) and synaptotagmin-1 or -7 showed that vesicles that fuse using the SNAP-23/synaptotagmin-7 combination contained synaptotagmin-1, while synaptotagmin-7 barely displayed activity-dependent trafficking between vesicle and plasma membrane, implying that it acts as a plasma membrane calcium sensor. Overall, these findings support the idea of alternative syt∶SNARE combinations driving release with different kinetics and fidelity

Regulation of Ca2+ channels by SNAP-25 via recruitment of syntaxin-1 from plasma membrane clusters

SNAP-25 regulates Ca(2+) channels, with potentially important consequences for diseases involving an aberrant SNAP-25 expression level. How this regulation is executed mechanistically remains unknown. We investigated this question in mouse adrenal chromaffin cells and found that SNAP-25 inhibits Ca(2+) currents, with the B-isoform being more potent than the A-isoform, but not when syntaxin-1 is cleaved by botulinum neurotoxin C. In contrast, syntaxin-1 inhibits Ca(2+) currents independently of SNAP-25. Further experiments using immunostaining showed that endogenous or exogenous SNAP-25 expression recruits syntaxin-1 from clusters on the plasma membrane, thereby increasing the immunoavailability of syntaxin-1 and leading indirectly to Ca(2+) current inhibition. Expression of Munc18-1, which recruits syntaxin-1 within the exocytotic pathway, does not modulate Ca(2+) channels, whereas overexpression of the syntaxin-binding protein Doc2B or ubMunc13-2 increases syntaxin-1 immunoavailability and concomitantly down-regulates Ca(2+) currents. Similar findings were obtained upon chemical cholesterol depletion, leading directly to syntaxin-1 cluster dispersal and Ca(2+) current inhibition. We conclude that clustering of syntaxin-1 allows the cell to maintain a high syntaxin-1 expression level without compromising Ca(2+) influx, and recruitment of syntaxin-1 from clusters by SNAP-25 expression makes it available for regulating Ca(2+) channels. This mechanism potentially allows the cell to regulate Ca(2+) influx by expanding or contracting syntaxin-1 clusters

Trends in Antibiotic Use by Birth Season and Birth Year

OBJECTIVES: We examined 2 birth cohort effects on antibiotic prescribing during the first year of life (henceforth, infancy) in Denmark: (1) the birth season effect on timing and overall occurrence of antibiotic prescribing, and (2) the birth year effect amid emerging nationwide pneumococcal vaccination programs and changing prescribing guidelines. METHODS: We linked data for all live births in Denmark from 2004 to 2012 (N = 561 729) across the National Health Service Prescription Database, Medical Birth Registry, and Civil Registration System. Across birth season and birth year cohorts, we estimated 1-year risk, rate, and burden of redeemed antibiotic prescriptions during infancy. We used interrupted time series methods to assess prescribing trends across birth year cohorts. Graphical displays of all birth cohort effect data are included. RESULTS: The 1-year risk of having at least 1 redeemed antibiotic prescription during infancy was 39.5% (99% confidence interval [CI]: 39.3% to 39.6%). The hazard of a first prescription increased with age throughout infancy and varied by season; subsequently, Kaplan-Meier-derived risk functions varied by birth season cohort. After rollout of a first vaccination program and new antibiotic prescribing guidelines, 1-year risk decreased by 4.4% over 14 months (99% CI: 3.4% to 5.5%); it decreased again after rollout of a second vaccination program by 6.9% over 3 years (99% CI: 4.4% to 9.3%). CONCLUSIONS: In Denmark, birth season and birth year cohort effects influenced timing and risk of antibiotic prescribing during infancy. Future studies of antibiotic stewardship, effectiveness, and safety in children should consider these cohort effects, which may render some children inherently more susceptible than others to downstream antibiotic effects

Synaptotagmin-7 Is an Asynchronous Calcium Sensor for Synaptic Transmission in Neurons Expressing SNAP-23

Synchronization of neurotransmitter release with the presynaptic action potential is essential for maintaining fidelity of information transfer in the central nervous system. However, synchronous release is frequently accompanied by an asynchronous release component that builds up during repetitive stimulation, and can even play a dominant role in some synapses. Here, we show that substitution of SNAP-23 for SNAP-25 in mouse autaptic glutamatergic hippocampal neurons results in asynchronous release and a higher frequency of spontaneous release events (mEPSCs). Use of neurons from double-knock-out (SNAP-25, synaptotagmin-7) mice in combination with viral transduction showed that SNAP-23-driven release is triggered by endogenous synaptotagmin-7. In the absence of synaptotagmin-7 release became even more asynchronous, and the spontaneous release rate increased even more, indicating that synaptotagmin-7 acts to synchronize release and suppress spontaneous release. However, compared to synaptotagmin-1, synaptotagmin-7 is a both leaky and asynchronous calcium sensor. In the presence of SNAP-25, consequences of the elimination of synaptotagmin-7 were small or absent, indicating that the protein pairs SNAP-25/synaptotagmin-1 and SNAP-23/synaptotagmin-7 might act as mutually exclusive calcium sensors. Expression of fusion proteins between pHluorin (pH-sensitive GFP) and synaptotagmin-1 or -7 showed that vesicles that fuse using the SNAP-23/synaptotagmin-7 combination contained synaptotagmin-1, while synaptotagmin-7 barely displayed activity-dependent trafficking between vesicle and plasma membrane, implying that it acts as a plasma membrane calcium sensor. Overall, these findings support the idea of alternative syt∶SNARE combinations driving release with different kinetics and fidelity

Syt-7 limits the build-up of release during prolonged stimulation in the presence of SNAP-23.

<p>A: Examples of 40 Hz, 100 AP trains in the four groups. Color coding as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114033#pone-0114033-g002" target="_blank">Figure 2</a>. B: Cumulative release during and after a 40 Hz, 100 AP train. Train stimulation is indicated by a horizontal bar. Shown are SNAP-25 KO neurons rescued with SNAP-25 (black), or SNAP-23 (blue), compared to SNAP-25/Syt-7 DKO neurons rescued with SNAP-25 (red) and SNAP-23 (green). The thick lines are means of all experiments; thin lines indicate the mean ± SEM (number of cells: SNAP-25+syt7, n = 25 cells; SNAP-25−syt7, n = 31 cells; SNAP-23+syt7, n = 19 cells; SNAP-23−syt7, 26 cells). C: Zoom-in of the first 0.5 s of (B), showing that in the presence of SNAP-23, but in the absence of syt-7, release becomes stronger than in the presence of syt-7 at ∼0.4 s (train starts at 0.1 s). **: p<0.01.</p

SNAP-23 expressing neurons display asynchronous release, which is shifted to later times by the lack of synaptotagmin-7.

<p>A: Example traces (stimulus artifacts and action potential associated currents have been blanked); arrows indicate the five stimuli. Color coding as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114033#pone-0114033-g002" target="_blank">Figure 2</a>. B: Charge transfer plotted versus stimuli at 50 Hz. The expression of SNAP-23 reduced the charge, which was further exacerbated by the deletion of syt-7. C: Charge transferred as a result of 50 Hz stimulation (5 stimuli). “During STP”: Integrated release during the interval from the beginning of the train until 20 ms after the train. “Post STP”: Release after the 5th EPSC (+20 ms) until current has relaxed to baseline. “Cumulative”: “During STP”+“Post STP”. Deletion of syt-7 in the presence of SNAP-23 shifted release to after the short train of action potentials. D: As (B), 5 Hz instead of 50 Hz. E: As (C), 5 Hz instead of 50 Hz. *: p<0.05; **: p<0.01; ***:p<0.001.</p