Journal: PLoS Biology

Article Title: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons

doi: 10.1371/journal.pbio.1002516

Figure Lengend Snippet: (A) Mitochondria-occupied and -free presynaptic Ca 2+ dynamics were monitored using vGlut1-GCaMP5G and mito-mTagBFP in axons of cultured cortical neurons following ex utero electroporation at E15.5 and imaged at 15–17 DIV. (B) Representative image of vGlut1-GCaMP5G peak signal during 20 AP at 10 Hz and mito-mTagBFP. vGlut1-GCaMP5G image is converted to ratio view normalized by Fmax signal followed by ionomycin (5 μM) incubation. White arrows point to mitochondria-associated presynaptic site and orange arrows point to mitochondria-free presynaptic site. (C–H) vGlut1-GCaMP5G signals in mitochondria-free boutons show significantly increased normalized peak values and total charge transfer (area under curve) during repetitive stimulation (20 AP and 100 AP at 10 Hz). n = 62 for mito-associated boutons, and 40 for mito-free boutons from 14 neurons in 20 AP condition. n = 34 for mitochondria-associated boutons and 23 for mitochondria-free boutons from 9 neurons in 100 AP condition. * p < 0.05 and ** p < 0.01, Mann-Whitney test. Scale bar = 3 μm. Individual values are available in .

Article Snippet: We conjugated mitochondrial targeting sequences from human cytochrome c oxidase subunit VIII to mTagBFP, mCherry and GCaMP5G (from pCMV-GCaMP5G; Douglas Kim and Loren Looger, Addgene #31788) by swapping DsRED1 in pCAG-mitoDsRED1 [ ]. pCAG:vGlut1-mCherry and pCAG:vGlut1-GCaMP5G were created by fusion of rat vGlut1 and mCherry or GCaMP5G.

Techniques: Cell Culture, Electroporation, Incubation, MANN-WHITNEY

Journal: PLoS Biology

Article Title: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons

doi: 10.1371/journal.pbio.1002516

Figure Lengend Snippet: (A–C) MCU inhibitor Ru360 reduced mitochondrial calcium influx during repetitive stimulation (100 AP at 10 Hz). Mito-GCaMP5G signals at the same presynaptic boutons were imaged before and after Ru360 (10 μM) incubation. Peak value and total charge transfer (area under curve) were significantly reduced by Ru360 application. n = 40 for water, 40 for Ru360 from 11 neurons. **** p < 0.0001, *** p < 0.001, Paired t test. (D and E) Acute inhibition of MCU by application of MCU inhibitor Ru360 (10 μM applied 3 min before and during imaging) increased presynaptic calcium (vGlut1-GCaMP5G) accumulation during repetitive stimulation with 100 AP at 10 Hz. n = 22 for water, 21 for Ru360 from 6 neurons. ** p < 0.01, Mann-Whitney test. (F–K) MCU expression level was significantly reduced in LKB1 knockout (Nex-Cre;LKB1 F/F ) whole-cell lysate from hippocampus, whereas other mitochondrial components, cytochrome c oxidase III (COXIII) and voltage-dependent anion channel (VDAC), were not significantly changed. (G) MCU intensity normalized by COXIII. (H) COXIII intensity normalized by actin. ( I ) Western blot for VDAC and Actin. (J) VDAC intensity normalized by actin. n = 5 for wild type (WT), 5 for knockout (KO). ( K ) Quantification of MCU expression in WT and LKB1 KO normalized to actin. * p < 0.05, Mann-Whitney test. Individual values are available in .

Article Snippet: We conjugated mitochondrial targeting sequences from human cytochrome c oxidase subunit VIII to mTagBFP, mCherry and GCaMP5G (from pCMV-GCaMP5G; Douglas Kim and Loren Looger, Addgene #31788) by swapping DsRED1 in pCAG-mitoDsRED1 [ ]. pCAG:vGlut1-mCherry and pCAG:vGlut1-GCaMP5G were created by fusion of rat vGlut1 and mCherry or GCaMP5G.

Techniques: Incubation, Inhibition, Imaging, MANN-WHITNEY, Expressing, Knock-Out, Western Blot

Journal: PLoS Biology

Article Title: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons

doi: 10.1371/journal.pbio.1002516

Figure Lengend Snippet: (A–D) Loss of LKB1 reduced intra-mitochondrial calcium influx during evoked release imposed by 100 AP at 10 Hz and MCU overexpression rescued the defect. (A) Captured images from mito-GCaMP5G signals at vGlut1-mCherry presynaptic sites (not shown). (B–D) LKB1 deletion decreased maximum intensity and total charge transfer (area under the curve) of mito-GCaMP5G signals and MCU overexpression on LKB1-deficient neurons rescued intra-mitochondrial calcium import. n = 72 for control from 21 neurons, 75 for LKB1-null from 22 neurons, and 51 for LKB1-deletion+MCU from 12 neurons. **** p < 0.0001, * p < 0.05, Mann-Whitney test. Scale bar = 10 μm. (E–G) Conditional LKB1 deletion in cortical pyramidal neurons increased presynaptic Ca 2+ during evoked release imposed by 100 AP at 10 Hz and MCU overexpression restored the defect. (E) Captured images from mitochondria-associated vGlut1-GCaMP5G signal at presynaptic boutons at specific times before and after stimulation. (F–G) LKB1-null neurons show significant increase of total intensity of vGlut1-GCaMP5G (area under the curve) and the increased intensity returned to control level following MCU overexpression. n = 38 for control from 11 neurons, 37 for LKB1-deletion from 10 neurons, 35 for LKB1-deletion+MCU from 12 neurons. * p < 0.05, *** p < 0.001. Mann-Whitney test. Individual values are available in .

Article Snippet: We conjugated mitochondrial targeting sequences from human cytochrome c oxidase subunit VIII to mTagBFP, mCherry and GCaMP5G (from pCMV-GCaMP5G; Douglas Kim and Loren Looger, Addgene #31788) by swapping DsRED1 in pCAG-mitoDsRED1 [ ]. pCAG:vGlut1-mCherry and pCAG:vGlut1-GCaMP5G were created by fusion of rat vGlut1 and mCherry or GCaMP5G.

Techniques: Over Expression, Control, MANN-WHITNEY

Journal: PLoS Biology

Article Title: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons

doi: 10.1371/journal.pbio.1002516

Figure Lengend Snippet: (A) WT neurons display significantly increased vGlut1-GCaMP5G peak values in mitochondria-free boutons compared to mitochondria-associated boutons during repetitive stimulation (20 AP at 10 Hz). (B) LKB1-deficient neurons display no significant difference in presynaptic calcium accumulation (vGlut1-GCaMP5G) between mitochondria-associated and mitochondria–free boutons. (C) Following 20 AP at 10 Hz stimulation, mitochondria-associated presynaptic boutons in LKB1-null axons show significantly increased presynaptic Ca 2+ (vGlut1-GCaMP5G) compared to WT boutons. (D) Histogram of peak values for each condition. n = 34 for mito-associated boutons, and 36 for mito-free boutons from 16 neurons in WT. n = 51 for mitochondria-associated boutons and 19 for mitochondria-free boutons from 18 neurons in LKB1-null. ** p < 0.01, Mann-Whitney test. Individual values are available in .

Article Snippet: We conjugated mitochondrial targeting sequences from human cytochrome c oxidase subunit VIII to mTagBFP, mCherry and GCaMP5G (from pCMV-GCaMP5G; Douglas Kim and Loren Looger, Addgene #31788) by swapping DsRED1 in pCAG-mitoDsRED1 [ ]. pCAG:vGlut1-mCherry and pCAG:vGlut1-GCaMP5G were created by fusion of rat vGlut1 and mCherry or GCaMP5G.

Techniques: MANN-WHITNEY

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Inhibitor toxicity measured in HEK293T cells using a standard dead cell apoptosis assay. Healthy cells are not fluorescent (green), dead cells are propidium iodide (PI) and annexin V (AV) positive (red). Apoptotic cells are only AV positive (grey), while necrotic cells are only PI positive (black). Hoescht nuclear counterstain was to determine the total number of cells. Values are mean +/- SEM, n = 7–8 for each condition from three independent trials. ( b ) Inhibitor toxicity measured in primary cultures of rat cortical neurons using PI (Dead) and Hoechst (Total) stains. Values are mean +/- SEM, n = 10 fro each condition from two independent trials. ( c ) Representative anti-GFP immunoblots showing time-course of cleavage for DNV and PRV washout with the modified EDVVCC/QMSY model. PRV has the fastest apparent dissociation from the protease, as the model is almost completely cleaved within 1 hr following washout. ( d ) Representative, individual ROI (black) and average (green) iGluSnFR traces showing the Ca 2+ -dependence of release in WT rat hippocampal neurons transduced with iGluSnFR. The examples shown were from a single FOV for each condition. ( e ) Quantification of miniature glutamate transients (mGT) in WT rat hippocampal neurons at 15 DIV using iGluSnFR in 2 mM extracellular Ca 2+ . One µM TTX was used to block action potentials where indicated; in some experiments, forskolin (10 µM) was also included to increase mGT frequency. Addition of PRV drastically increased the rate of spontaneous glutamate release events; TTX blocked this increase. A low dose of PRV (0.5 µM) did not influence spontaneous release. Two separate trials were conducted; bars represent means and individual dots represent events/s from a FOV (n) imaged for one minute, n = 4 to 10. ( f ) Normalized, average traces (n = 5 to 6) of evoked glutamate release from a single action potential in WT neurons, and WT neurons treated with 0.5, 1, or 3 µM PRV, in 1 mM extracellular Ca 2+ . ( g ) Representative confocal images of 20 DIV rat hippocampal neurons stained with anti-PSD95, anti-vGLUT1, and anti-MAP2. Neurons grown in vehicle, 0.5 µM, or 5.0 µM PRV. Quantitation of synapse number to the right, control (grey) 0.5 µM (green), or 5.0 µM PRV (blue).

Article Snippet: The glutamate sensor, iGluSnFR, was PCR amplified using the iGluSnFR template (with super-folding mutations) (pAAV.hSynapsin.SF-iGluSnFR.A184V was a gift from Loren Looger [Addgene plasmid # 106175; http://n2t.net/addgene :106175; RRID: Addgene_106175 ]) , and subcloned into our lentivirus transfer plasmid (CamKII promoter) after the addition of a Golgi export sequence , and an ER exit motif , to the carboxy terminus.

Techniques: Apoptosis Assay, Western Blot, Modification, Transduction, Blocking Assay, Staining, Quantitation Assay, Control

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Representative, individual ROI (black) and average (green) iGluSnFR traces from a single-field stimulus, recorded optically at 100 Hz. The examples shown are from a single FOV for each condition. ( b ) Histograms of iGluSnFR (ΔF/F 0 ) peaks plotted using 10 ms bins. Peaks were binned over the entire 1.5 s of recording; a 0.5 s epoch, just before and after the stimulus, is shown. Samples were color-coded as follows: WT (black), Syt1 KO (+CRE) (red), S1KO + S1-SELF + 0.5 µM PRV (green); the S1-SELF PRV washout samples were: 2 (purple), 4 (blue), 6 (orange), and 8 hr (grey). The histograms include all combined data from four independent trials, with 10 to 16 FOVs for each group. Comparisons between all conditions, and statistical analysis, are provided in . ( c ) Representative images showing temporally color-coded max projections (time projection) of iGluSnFR ΔF/F 0 peaks 100 ms after a single stimulus. Temporal color code is from red (time of stimulation) to purple (100 ms after the stimulus). Scale bar, 5 µm. Figure 5—source data 1. Loss of SYT1 via knockoff disrupts synchronous release. Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for the histograms shown in .

Article Snippet: The glutamate sensor, iGluSnFR, was PCR amplified using the iGluSnFR template (with super-folding mutations) (pAAV.hSynapsin.SF-iGluSnFR.A184V was a gift from Loren Looger [Addgene plasmid # 106175; http://n2t.net/addgene :106175; RRID: Addgene_106175 ]) , and subcloned into our lentivirus transfer plasmid (CamKII promoter) after the addition of a Golgi export sequence , and an ER exit motif , to the carboxy terminus.

Techniques: Comparison

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Average, normalized, traces of iGluSnFR ΔF/F 0 from . Samples are color coded as follows: WT (black), Syt1 KO (+CRE) (red), S1KO + S1-SELF + 0.5 µM PRV (green), S1-SELF 2 hr washout (purple), S1-SELF 4 hr washout (blue), S1-SELF 6 hr washout (orange), and S1-SELF 8 hr washout (grey). Values from four independent trials, with 10 to 16 field of views for each group.

Article Snippet: The glutamate sensor, iGluSnFR, was PCR amplified using the iGluSnFR template (with super-folding mutations) (pAAV.hSynapsin.SF-iGluSnFR.A184V was a gift from Loren Looger [Addgene plasmid # 106175; http://n2t.net/addgene :106175; RRID: Addgene_106175 ]) , and subcloned into our lentivirus transfer plasmid (CamKII promoter) after the addition of a Golgi export sequence , and an ER exit motif , to the carboxy terminus.

Techniques:

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) mEPSC rates in WT (black), S1KO (red), and neurons expressing S1-SELF (+ PRV green and −4 hr PRV blue). Values are mean +/- SEM, 12 to 18 neurons per condition (n). Comparisons between conditions and statistical analysis, are provided in . ( b ) Representative traces from ( a ). ( c ) Average mEPSC amplitude. ( d ) Average mEPSC 10–90% half-width. No statistical differences between groups in ( c ) or ( d ) were observed. ( e ) Representative images showing temporally color-coded max projections (time projection) of iGluSnFR ΔF peaks. mGT denotes miniature glutamate release events. Temporal color code is from red (start of image acquisition) to purple (60 s after image acquisition start). Scale bar, 5 µm. ( f ) mGT rates in WT, S1KO, and neurons expressing S1-SELF, recorded optically with iGluSnFR. Values are mean +/- SEM from three independent trials, with 13 to 21 field of views for each group. Comparisons between conditions and statistical analysis, are provided in . ( g ) Percentage of iGluSnFR ΔF/F 0 peaks within 10 ms following a single stimulus (data are from ). Values are mean +/- SEM from four independent trials, with 10 to 16 field of views for each group. Comparisons between conditions and statistical analysis, are provided in . ( h ) Inverse correlation between synchronous release (≤10 ms) and spontaneous (mGT) release in WT, S1KO, and S1-SELF neurons. Data were fitted using a linear regression; r 2 = 0.7671, p-value=0.0097. Figure 6—source data 1. Acute knockoff of SYT1 unclamps spontaneous release as determined electrophysiologically. Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for data in . Figure 6—source data 2. Acute knockoff of SYT1 unclamps spontaneous release via iGluSnFR. Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for data in . Figure 6—source data 3. Acute knockoff of SYT1 decreases synchronous release (increases asynchronous release). Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for histogram data in .

Article Snippet: The glutamate sensor, iGluSnFR, was PCR amplified using the iGluSnFR template (with super-folding mutations) (pAAV.hSynapsin.SF-iGluSnFR.A184V was a gift from Loren Looger [Addgene plasmid # 106175; http://n2t.net/addgene :106175; RRID: Addgene_106175 ]) , and subcloned into our lentivirus transfer plasmid (CamKII promoter) after the addition of a Golgi export sequence , and an ER exit motif , to the carboxy terminus.

Techniques: Expressing, Comparison

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Example trace of background-subtracted, iGluSnFR ΔF spontaneous events, from cultured hippocampal neurons expressing S1-SELF, 6 hr after washout of PRV; four spontaneous events were detected (green arrows). ( b ) Histogram of background ROI peak ΔF (grey) compared to manually identified, ROI ΔF peaks (green) of spontaneous events. Background peaks are centered at 3 a.u., while mGT events are centered at 9 a.u., n = 141 for each group. ( c ) The peak amplitude of mGT events (green, panel C) and random ROIs (grey), plotted against the peak signal-to-noise ratio at each ROI. This graph indicates that low-intensity events were identified because the background noise, for those specific events, was also low, n = 141 for each group. ( d ) Representative anti-CDK5 immunoblot of WT, Syt1 KO (generated using a CRE virus), and KO neurons expressing S1-SELF. (Ø) PRV means that the culture has never seen inhibitor (PRV). (Ø) denotes a condition in which cultures have never been exposed to PRV.

Article Snippet: The glutamate sensor, iGluSnFR, was PCR amplified using the iGluSnFR template (with super-folding mutations) (pAAV.hSynapsin.SF-iGluSnFR.A184V was a gift from Loren Looger [Addgene plasmid # 106175; http://n2t.net/addgene :106175; RRID: Addgene_106175 ]) , and subcloned into our lentivirus transfer plasmid (CamKII promoter) after the addition of a Golgi export sequence , and an ER exit motif , to the carboxy terminus.

Techniques: Cell Culture, Expressing, Western Blot, Generated, Virus