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.2021 May 4;118(18):e2012137118.
doi: 10.1073/pnas.2012137118.

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Affiliations

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Rhodé van Westen et al. Proc Natl Acad Sci U S A..

Abstract

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

Keywords: dense core vesicles; hippocampal neurons; neuromodulators; synaptotagmin-1; synaptotagmin-7.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Synaptotagmin-1 and synaptotagmin-7 are required for neuropeptide vesicle fusion. (A) Stimulation paradigm with a single vesicle fluorescence intensity trace. (B,Left) Representative image of NPY-SEP–infected neuron during Tyrode’s NH4 application. (Scale bar: 20 µm.) (Right) Neurite before (baseline) and during (16 × 50 AP at 50 Hz) stimulation and during NH4 application (NH4). (Scale bar: 10 µm.) (C) Median number of neuropeptide vesicle fusion events per neuron in a cumulative plot in Syt1-KD (green), Syt7-KO (red), and Syt1-KD/Syt7-KO (blue) neurons, respectively. (D) Fusing fraction is reduced in Syt1-KD, Syt7-KO, and Syt1-KD/Syt7-KO neurons [X2 (4) = 14.05,P = 0.0028]. *P < 0.05, **P < 0.01, nonsignificantP > 0.05. (E) Deficiency of Syt1 or Syt7 does not affect the total number of DCVs per neuron [X2 (4) = 1.233,P = 0.75]. The numbers before and after the dash represent the number of independent experiments/animals and the number of neurons, respectively.
Fig. 2.
Fig. 2.
Synaptotagmin-1 and synaptotagmin-7 cross-rescue neuropeptide vesicle fusion. (A) Median number of neuropeptide vesicle fusion events in WT, and Syt1-KO neurons with and without Syt1-OE in a cumulative plot. (B) Fusing fraction in Syt1-KO neurons is rescued by Syt1-OE [X2 (3) = 19.28,P < 0.0001]. For lentiviral infection, 2 µL lentivirus was used. For the effects of different volumes of lentivirus, seeSI Appendix, Fig. S4. (C andD) Same asA andB but with Syt7-OE. The fusing fraction in Syt1-KO neurons is rescued by Syt7-OE [X2 (3) = 20.96,P < 0.0001]. Neurons with a fusing fraction above 0.9 were plotted at 0.9 for visualization purposes. (E andF) Same asA andB but with Syt7-KO neurons with Syt7-OE and Syt1-OE. The fusing fraction in Syt7-KO neurons is rescued by Syt7-OE and Syt1-OE [X2 (4) = 15.96,P = 0.0012]. *P < 0.05, **P < 0.01, ***P < 0.001, nonsignificantP > 0.05. The numbers before and after the dash represent the number of independent experiments/animals and the number of neurons, respectively.
Fig. 3.
Fig. 3.
Overexpression of Syt1-OE and Syt7-OE in WT neurons increases neuropeptide vesicle fusion. (A) Median number of neuropeptide vesicle fusion events in WT neurons with and without Syt1-OE in a cumulative plot. (B) Fusing fraction is increased in WT neurons with Syt1-OE (Mann–WhitneyU = 193,P < 0.05). Neurons with a fusing fraction above 0.9 were plotted at 0.9 for visualization purposes. (C andD) Same asA andB but with Syt7-OE (Mann–WhitneyU = 174,P < 0.05). *P < 0.05, nonsignificantP > 0.05. The numbers before and after the dash represent the number of independent experiments/animals and the number of neurons, respectively.
Fig. 4.
Fig. 4.
Syt1, and to lesser extent Syt7, colocalizes and co-travels with NPY-mCherry.(A) Representative image of neuron immunostained for Syt1, NPY-SEP, and VGluT1. (Scale bars: large image: 20 µm; zoomed image: 5 µm.) To detect extrasynaptic Syt1, higher laser powers were required than for synaptic Syt1 detection (SI Appendix, Fig. S6A andB). (B) Quantification of colocalization between Syt1, NPY-SEP, and VGluT1 [X2 (5) = 31.74,P < 0.0001]. (C) Representative image of neuron immunostained for Syt7, NPY-SEP, and VGluT1. (Scale bars: large image: 20 µm; zoomed image: 5 µm.) (D) Quantification of colocalization between Syt7, NPY-SEP, and VGluT1 [X2 (5) = 25.25,P < 0.0001]. (E) Cartoon of Syt-SEP, localized at the plasma membrane or at acidic intracellular compartments, at a neutral pH (7.4) and during bath application of acidic pH (pH 5.5) or NH4-contaning imaging solution. (F andG) Average Syt1-SEP (n = 7 cells) (F) and Syt7-SEP (n = 7 cells) (G) fluorescence response during bath application of pH 5.5 or 50 mM NH4 Tyrode’s solution. (H) Quantification of plasma membrane/intracellular fraction of Syt1-SEP and Syt7-SEP [X2 (4) = 24.56,P < 0.0001]. (I) Cartoon of co-traveling of NPY-mCherry and Syt-SEP vesicles. (J) mCherry-Syt1 and mCherry-Syt7 partially co-travel with NPY-SEP vesicles. (Scale bars: 5 µm.) (K) Quantification of co-traveling of NPY with Syt1-SEP or Syt7-SEP and vice versa [X2 (4) = 19.47,P < 0.0002]. The numbers before and after the dash represent the number of neurites and the number of trafficking vesicles, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, nonsignificantP > 0.05.
Fig. 5.
Fig. 5.
Fusing NPY-mCherry vesicles colocalize with Syt1-SEP and to lesser extent with Syt7-SEP events. (A) Schematic representation of NPY-mCherry events co-fusing with Syt-SEP. (B) Representative images of fusing NPY-mCherry with and without increase of Syt1-SEP. (C) Percentage of colocalization of fusing NPY-mCherry events (red lines of typical example traces) with sudden appearance of Syt1-SEP (green lines of typical example traces).n represents the number of cells and the number of extrasynaptic NPY fusion events. Synapses were defined by an accumulation of VGluT1 vesicles/above threshold VGluT1 fluorescence. Fusion events that do not overlap with these VGluT1 accumulations were considered extrasynaptic. (D) Representative images of fusing NPY-mCherry with and without increase of Syt7-SEP. (E) Same asC but with Syt7-SEP.n represents the number of cells and the number of NPY fusion events. The numbers before and after the dash represent the number of neurons and the number of DCVs, respectively. Scale bars represent 10 s (x-axis) and 0.2 ΔF/F (y-axis).
Fig. 6.
Fig. 6.
Synaptotagmin-1, but not Syt7, is required for event duration. (A) Representative images of NPY-SEP events. (B) Representative fluorescence traces of NPY-SEP event ROIs. The duration was defined as the time between the onset of NPY-SEP (baseline fluorescence + 2 SD) and return to baseline levels. Scale bar represents 2 s (x-axis) and 1 ΔF/F (y-axis). (C) Normalized cumulative median event duration per neuron in WT, Syt1-KO, and Syt1-KO neurons with Syt1-OE. For lentiviral infection, 2 µL lentivirus was used. For the effects of different volumes of lentivirus, seeSI Appendix, Fig. S4. (D) Event duration in Syt1-KO neurons is rescued by Syt1-OE [X2 (3) = 13.27,P = 0.0013]. (E) Normalized cumulative median event duration per neuron in WT, Syt1-KO, and Syt1-KO neurons with Syt7-OE. (F) Event duration in Syt1-KO neurons is not rescued by Syt7-OE [X2 (3) = 13.28,P = 0.0013]. (G) Normalized cumulative median event duration per neuron in WT neurons, Syt7-KO, and Syt7-KO neurons with Syt7-OE. (H) Event duration is unaltered in Syt7-KO neurons with Syt1-OE and Syt7-OE [X2 (4) = 1.475,P = 0.67]. All data presented in this figure corresponds to data presented in Fig. 2. The numbers before and after the dash represent the number of independent experiments/animals and the number of neurons, respectively. **P < 0.01, ***P < 0.001, nonsignificantP > 0.05. The text refers to a WT event duration of 12.0 ± 1.3 s, computed over all three experiments, for representative purposes.
Fig. 7.
Fig. 7.
Syt1-OE, but not Syt7-OE, increases DCV event duration. (A andB) Fusion duration is prolonged in WT neurons with Syt1-OE (Mann–WhitneyU = 218,P < 0.05). (C andD) Fusion event duration is not affected in WT neurons with Syt7-OE (Mann–WhitneyU = 258,P = 0.89). The numbers before and after the dash represent the number of neurites and the number of trafficking vesicles, respectively. All data presented in this figure corresponds to data presented in Fig. 3. *P < 0.05, nonsignificantP > 0.05.
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