Journal: Scientific Reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

doi: 10.1038/srep31997

Figure Lengend Snippet: ( a ) Representative images of exogenous vGlut1 (vG-pH) and Epsin1 (Epsin1-mKate2) [top] or endogenous vGlut1 (green) and Epsin1 (red) [bottom] in primary cultured hippocampal neurons. Neurons were fixed at 14–18 days in vitro (DIV) and stained with anti-vGlut1 (green) and anti-Epsin1 (red) for endogenous vGlut1 and Epsin1, respectively. Scale bar, 5 μm. ( b ) Distribution of Epsin1 corresponds to each nerve terminal in terms of endogenous (left) and exogenous (right) levels. Higher levels of Epsin1 localize at synapse as a function of synaptic size.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Cell Culture, In Vitro, Staining

Journal: Scientific Reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

doi: 10.1038/srep31997

Figure Lengend Snippet: ( a ) Representative snapshots of vGlut1-pHluorin at the resting state and after applying NH 4 Cl in control and Epsin1-KD1 or -KD2 neurons, (top) NH 4 Cl−, absence of NH 4 Cl indicates steady-state under resting conditions and (bottom) NH 4 Cl+, presence of NH 4 Cl indicates application of NH 4 Cl to reveal synaptic bouton with the total vesicle population. Scale bar, 5 μm. ( b ) Mean values of surface accumulation at rest in control, Epsin1-KD1, and Epsin1-KD2 neurons. Surface accumulation of vG-pH was measured by calculating the fold change in response to NH 4 Cl application. The surface fraction in control neurons was 5.35 ± 0.97% (n = 9), while that measured in Epsin1-KD1 and Epsin1-KD2 neurons was 15.12 ± 1.36% (n = 9) and 17.12 ± 3.53% (n = 7), respectively. ( c ) Total vesicle pool size (TVS) was determined by measuring absolute changes in fluorescence intensity in response to application of NH 4 Cl under the same imaging conditions. In Epsin1-KD1 neurons, TVS was reduced to ~58%, compared to that in control neurons. (Con) = 2346 ± 298 a.u. (n = 7), (Epsin1-KD1) = 1368 ± 120 a.u (n = 9), (Epsin1-KD2) = 1226 ± 194 (n = 7), **p < 0.01, ***p < 0.001. One-way ANOVA.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Control, Fluorescence, Imaging

Journal: Scientific Reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

doi: 10.1038/srep31997

Figure Lengend Snippet: ( a ) Representative traces of the vG-pH response to 100 action potential (AP) firing from control (black), Epsin1-KD1 (red) and Epsin1-KD2 (green) neurons. Neurons transfected with vG-pH and Epsin1-KD1 or Epsin1-KD2 were stimulated with 100 AP at 10 Hz. ( b ) Mean values of post-stimulus endocytic time constants from control, Epsin1-KD1, and Epsin1-KD2 neurons (τ endo con = 12.69 ± 0.98 s, n = 9; τ endo Epsin1-KD1 = 29.02 ± 3.78 s, n = 9; τ endo Epsin1-KD2 = 24.41 ± 1.80 s, n = 9). ( c,d ) Representative traces of vG-pH response to 300 AP at 10 Hz with (red)/without (black) bafilomycin A1 (BAF) from control ( c ) and Epsin1-KD1 ( d ) neurons. ( e,f ) Endocytosis during neural activity is strongly suppressed in Epsin1-KD1 neurons. Endocytosis during stimulation in control neurons (black). Endocytosis during stimulation was analyzed as ΔF 300Baf+ -ΔF 300Baf− . Con endo = 26.4 ± 0.2% (n = 9), Epsin1KD-1 endo = 15.7 ± 0.3% (n = 9), * p < 0.05, ** p < 0.01; One-way ANOVA.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Control, Transfection, Activity Assay

Journal: Scientific Reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

doi: 10.1038/srep31997

Figure Lengend Snippet: ( a ) Representative vG-pH trace responses to 100 AP from control (black), Epsin1-KD1 (red), and rescue (green) neurons. For rescue experiments, neurons were transfected with shRNA1-Epsin1, shRNA1-insensitive Epsin1, and vG-pH. ( b ) Mean values of post-stimulus endocytic time constants from control, Epsin1-KD1, and rescue neurons. ( c ) Endocytosis during stimulation is restored upon shRNA-insensitive Epsin1 expression in Epsin1-KD1 neurons, calculated as for The amount of endocytosis during stimulation (22.83 ± 2.9%) is similar to that of the control. ( e,f ) Surface accumulation of vG-pH and total vesicle pool size (TVS) are restored to control levels in rescue neurons. Surface fraction rescue 5.0 ± 1.2% (n = 7), TVS rescue = 2056 ± 292 a.u (n = 7). ( g ) Representative images of rescue neurons. Neurons expressing shRNA1-Epsin1, shRNA-insensitive Epsin1 and vG-pH were fixed and stained with anti-Epsin1 (red). ( h ) Expression of Epsin1 is almost completely restored (99.2 ± 5.7%) to levels in non-transfected neurons by re-expressing shRNA-insensitive Epsin1 in Epsin1-KD1 neurons. * p < 0.05, ** p < 0.01. One-way ANOVA; ns means non-significant.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Control, Transfection, shRNA, Expressing, Staining

Journal: Scientific Reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

doi: 10.1038/srep31997

Figure Lengend Snippet: ( a ) Schematic diagram of each domain deletion mutant of Epsin1 (ΔENTH, ΔUIMs, ΔCLAP and ΔNPFs). ( b ) All deletion mutants are located placed at nerve termini of cultured hippocampal neurons. Neurons co-transfected with each deletion mutant, vG-pH and shRNA1-Epsin1 were fixed after 14~21 DIV. Scale bar, 5 μm. ( c ) Mean values of endocytic time constants from control, ΔENTH, ΔUIM, ΔCLAP, and ΔNPFs rescue neurons. In ΔENTH, ΔCLAP and ΔNPFs rescue neurons, kinetics of endocytosis is impaired, but not in ΔUIMs rescue cells. τ endo con = 12.69 ± 0.99 s, n = 9; τ endo ΔENTH = 17.90 ± 1.44 s, n = 10; τ endo ΔUIMs = 12.53 ± 0.88 s, n = 15; τ endo ΔCLAP = 23.57 ± 2.25 s, n = 8; τ endo ΔNPFs = 17.85 ± 0.82 s, n = 8. *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA; ns means non-significant.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Mutagenesis, Cell Culture, Transfection, Control

Journal: Scientific Reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

doi: 10.1038/srep31997

Figure Lengend Snippet: ( a,b ) Representative post-stimulus traces of vG-pH response to 25 (black), 50 (red), 100 (blue), and 300 (green) AP, respectively, from control ( a ) and Epsin1-KD1 ( b ) neurons. The kinetics of post-stimulus endocytosis was measured in neurons subjected to a range of stimuli (25, 50, 100, and 300 AP). ( c ) Mean values of endocytic time constants at 25, 50, 100 and 300 AP in control and Epsin1-KD1 neurons. The time constant in Epsin1-KD1 neurons is altered from 100AP but in control neurons from 300 AP. τ endo con 25AP = 12.23 ± 0.53 s, τ endo con 50AP = 12.38 ± 0.71 s, τ endo con 100AP = 12.07 ± 0.85 s, τ endo con 300AP = 21.44 ± 1.63 s, n = 8 ; τ endo Epsin1-KD1 25AP = 21.87 ± 2.09 s, τ endo Epsin1-KD1 50AP = 20.03 ± 1.29 s, τ endo Epsin1-KD1 100AP = 28.83 ± 2.45 s, τ endo Epsin1-KD1 300AP = 50.50 ± 8.63 s, n = 10. ( d ) Normalized time constants in control and Epsin1-KD1 neurons under exposure to various stimuli. The time constant is normalized by the average value of 25–50 AP under each condition. Overlay of normalized time constants under various stimulation conditions in control (black) and Epsin1-KD1 (red) neurons. Endocytic capacity is significantly decreased in Epsin1-KD1 neurons. ** p < 0.01, One-way ANOVA.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Control

Journal: Scientific Reports

Article Title: Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

doi: 10.1038/srep31997

Figure Lengend Snippet: ( a,c ) Mean values of time constants of post-stimulus endocytosis of VAMP2-pHluorin ( a ; VAMP2-pH) or synaptophysin-pHluorin ( c ; physin-pH) in response to various stimuli (25, 50, 100, and 300 AP, respectively) in control and Epsin1-KD neurons. τ endo VAMP2-pH Con 25AP = 16.55 ± 1.07 s, τ endo VAMP2-pH Con 50AP = 17.76 ± 0.88 s, τ endo VAMP2-pH Con 100AP = 17.30 ± 0.57 s, τ endo VAMP2-pH Con 300AP = 24.49 ± 1.08 s, n = 6 ; τ endo VAMP2-pH-Epsin1-KD1 25AP = 25.05 ± 3.10 s, τ endo VAMP2-pH-Epsin1-KD1 50AP = 28.39 ± 2.18 s, τ endo VAMP2-pH-Epsin1-KD1 100AP = 42.13 ± 3.72 s, τ endo VAMP2-pH Epsin1-KD1 300AP = 59.02 ± 7.80 s, n = 9. τ endo physin-pH-con 25AP = 10.71 ± 1.36 s, τ endo physin -pH-con 50AP = 11.80 ± 1.42 s, τ endo physin -pH-con 100AP = 10.77 ± 1.03 s, τ endo physin -pH-con 300AP = 15.29 ± 1.57 s, n = 6 ; τ endo physin-pH-Epsin1-KD1 25AP = 18.64 ± 1.14 s, τ endo physin-pH-Epsin1-KD1 50AP = 20.26 ± 1.08 s, τ endo physin-pH-Epsin1-KD1 100AP = 29.43 ± 2.54 s, τ endo physin-pH-Epsin1-KD1 300AP = 41.28 ± 5.037 s, n = 8. **p < 0.01. One-way ANOVA. ( b,d ) Normalized mean values of endocytic time constants of VAMP2-pH or physin-pH in control and Epsin1-KD neurons. **p < 0.01, One-way ANOVA.

Article Snippet: Rat Epsin1 cDNA was obtained from Addgene (ID#21066).

Techniques: Control

Journal: eLife

Article Title: The ion channel ppk301 controls freshwater egg-laying in the mosquito Aedes aegypti

doi: 10.7554/eLife.43963

Figure Lengend Snippet:

Article Snippet: 15x-QUAS-dTomato-T2A-GCaMP6s was generated by Gibson assembly of the following PCR-amplified fragments: Plasmid backbone and Mos arms from PUb-mCD8:GFP-T2A-dsRed:NLS-SV40 (described above); 15x-QUAS from pBAC-ECFP-15xQUAS_TATA-SV40 (Addgene #104875, gift of Chris Potter); dTomato-T2A-GCaMP6s PCR-amplified from a vector synthesized (Genscript).

Techniques: Generated, Knock-In, Expressing, Membrane, Produced, Plasmid Preparation