Journal: The Journal of Physiology

Article Title: Involvement of Ca_V2.2 channels and α₂δ‐1 in homeostatic synaptic plasticity in cultured hippocampal neurons

doi: 10.1113/jp283600

Figure Lengend Snippet: Figure 2. Monitoring presynaptic Ca2+ transients in hippocampal neurons with Sy-GCaMP6f and VAMP-mOr2 A, images showing the expression of Sy-GCaMP6f in putative boutons during stimulation with one AP (arrowheads pointing at exemplary boutons). B, changes in Sy-GCaMP6f fluorescence after stimulation with 10 APs were used to identify responding boutons (arrowheads). C, VAMP-mOr2 fluorescence after stimulation with 200 APs at 10 Hz allowed identification of functional vesicle-releasing synapses based on an increase in fluorescence following the increase in pH to which it is exposed during vesicle fusion. D, up to 75 ROIs were selected per field of view to show changes in fluorescence over baseline (F/F0) of averages of 5−8 repeats of one AP stimulation. Responses from releasing (blue open circles) and non-releasing (blue filled circles) boutons were distinguished based on VAMP-mOr2 responses. n for releasing boutons = 57 fields of view, n for non-releasing boutons = 34 fields of view. E, responses from releasing (blue open circles) and non-releasing (blue filled circles) boutons after stimulation with 10 Aps. n for releasing boutons = 52 fields of view, n for non-releasing boutons = 32 fields of view. F, increase in VAMP-mOr2 fluorescence (orange line) after stimulation with 200 APs was used to identify releasing boutons (orange line) and non-releasing boutons (black line). n for releasing boutons = 23, n for non-releasing boutons = 11. Based on this, responses to 1 and 10 APs were categorised into releasing and non-releasing boutons. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Plated cells on 22 mm2 coverslips were transferred to a laminar-flow perfusion and stimulation chamber (imaging chamber with field stimulation series 20, Warner Instruments, Holliston, MA, USA) and mounted on an epifluorescence microscope (Zeiss Axiovert 200M) under continuous perfusion at 23°C at 0.5 ml/min with Ca2+ perfusion buffer containing (in mm) 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 Hepes (buffered to pH 7.4) and 30 glucose.

Techniques: Expressing, Fluorescence, Functional Assay

Journal: The Journal of Physiology

Article Title: Involvement of Ca_V2.2 channels and α₂δ‐1 in homeostatic synaptic plasticity in cultured hippocampal neurons

doi: 10.1113/jp283600

Figure Lengend Snippet: Figure 3. TTX treatment increases presynaptic Ca2+ transient amplitudes in more mature mouse hippocampal boutons A, Sy-GCaMP6f fluorescence changes in functionally releasing presynaptic boutons after stimulation with one AP in control (grey) and TTX (magenta) conditions at DIV 14−15 (left panel) shown as averaged traces with SEM. At DIV 14−15, no significant differences were observed between presynaptic Ca2+ transient amplitudes in control (grey) and TTX-treated (magenta) boutons (right panel). n = 7 biological replicates, two-tailed paired t test P = 0.15; n corresponds to independent experiments and data are shown as means ± SD (black). B, Sy-GCaMP6f fluorescence changes at DIV 18−22 in TTX (blue)-treated presynaptic terminals and control (grey) untreated terminals during one AP stimulation. Traces are averaged values and are shown as means ± SEM (left panel). At DIV 18−22, TTX treatment (blue) induced an increase in presynaptic Ca2+ transient amplitudes compared to control boutons (grey; right panel). n = 11 biological replicates, two-tailed paired t test, P = 0.007; n corresponds to independent experiments and data are shown as means ± SD (black). C, Sy-GCaMP6f fluorescence at DIV 14−15 in control (grey) and TTX-treated (magenta) presynaptic boutons during stimulation with 10 APs. Traces are averaged values and are shown as means ± SEM (left panel). Comparison of the Ca2+ transient amplitudes reveal similar levels for control and TTX-treated neurons (right panel). n = 7 biological replicates, two-tailed paired t test, P = 0.38; n corresponds to independent experiments and data are shown as mean ± SD (black). Averaged Sy-GCaMP6f traces of control (grey) and TTX-treated (blue) boutons (left panel) shown ± SEM (left panel). D, in more mature cultures at DIV 18−22, Sy-GCaMP6f fluorescence increased after TTX treatment (blue) compared to control boutons (grey). n = 11 biological replicates, two-tailed paired t test, P = 0.01; n corresponds to independent experiments and data are shown as means ± SD (black). [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Plated cells on 22 mm2 coverslips were transferred to a laminar-flow perfusion and stimulation chamber (imaging chamber with field stimulation series 20, Warner Instruments, Holliston, MA, USA) and mounted on an epifluorescence microscope (Zeiss Axiovert 200M) under continuous perfusion at 23°C at 0.5 ml/min with Ca2+ perfusion buffer containing (in mm) 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 Hepes (buffered to pH 7.4) and 30 glucose.

Techniques: Fluorescence, Control, Two Tailed Test, Comparison

Journal: The Journal of Physiology

Article Title: Involvement of Ca_V2.2 channels and α₂δ‐1 in homeostatic synaptic plasticity in cultured hippocampal neurons

doi: 10.1113/jp283600

Figure Lengend Snippet: Figure 4. CaV2.2 channels mediate increased Ca2+ transients during HSP at DIV 18−22 A, averaged Ca2+ transients from control (grey) and TTX-treated (magenta) boutons at DIV 14−15 and post-ConTx application (red traces for control + ConTx and orange traces for TTX + ConTx). Values are normalised to control neurons. n for control = 9, n for TTX = 9. B, at DIV 14−15, contribution of CaV2.2 channels to Ca2+ transient amplitudes after electrical stimulation with one AP was not significantly different from untreated neurons (red data points) and after TTX incubation (orange data points). Two-tailed unpaired t test, P = 0.083, n for control = 9, n for TTX = 9; n corresponds to fields of view from three independent experiments and data are shown as means ± SD; values are normalised to their respective control pre-toxin incubation. C, averaged Sy-GCaMP6f traces for control and TTX-treated presynaptic boutons in grey and blue, respectively, and decreased traces after application of ConTx in control neurons (red) and TTX-treated neurons (orange). Values are normalised to control neurons; n for control = 9, n for TTX = 12. D, at DIV 18−22, CaV2.2 channels contribute more to Ca2+ flux after the induction of HSP with TTX. Two-tailed unpaired t test, P = 0.007; n for control = 9, n for TTX = 12; n corresponds to fields of view from three independent experiments and data are shown as means ± SD; values are normalised to their respective

Article Snippet: Plated cells on 22 mm2 coverslips were transferred to a laminar-flow perfusion and stimulation chamber (imaging chamber with field stimulation series 20, Warner Instruments, Holliston, MA, USA) and mounted on an epifluorescence microscope (Zeiss Axiovert 200M) under continuous perfusion at 23°C at 0.5 ml/min with Ca2+ perfusion buffer containing (in mm) 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 Hepes (buffered to pH 7.4) and 30 glucose.

Techniques: Control, Incubation, Two Tailed Test

Journal: The Journal of Physiology

Article Title: Involvement of Ca_V2.2 channels and α₂δ‐1 in homeostatic synaptic plasticity in cultured hippocampal neurons

doi: 10.1113/jp283600

Figure Lengend Snippet: Figure 5. Overexpression of α2δ-1 does not change Ca2+ transients resulting from one AP but decreases the contribution of CaV2.2 channels A, confocal images of immunostaining for α2δ-1_HA in α2δ-1-overexpressing neurons (top row) and control, empty vector (EV)-transfected neurons (bottom row). α2δ-1_HA is shown in green and transfection marker mCherry in magenta. Maximum intensity projection of z-stacks and tile scan, optical section, 0.279 μm; confocal mode ×40; scale bar, 50 μm. B, similar Sy-GCaMP6f fluorescence changes after stimulation with one AP of EV (grey traces) and α2δ-1-overexpressing (OE; green traces) neurons shown as means ± SEM. Averaged fluorescence traces of 5−8 repeats of stimulation with one AP; for each field of view, 10−75 ROIs were chosen for analysis of change in fluorescence over baseline fluorescence (F/F0). C, Ca2+ transient amplitudes after stimulation with one AP were similar in EV (grey) and α2δ-1-overexpressing (OE; green) neurons. n = 12 biological replicates; paired t test, P = 0.18; n corresponds to independent experiments and data are also shown as means ± SD (black). D, contribution of CaV2.2 channels to Ca2+ transients after stimulation with one AP was 47.1 ± 20.65% (grey) in EV neurons and 28.1 ± 16.38% (green) in neurons overexpressing α2δ-1. n for EV control = 10 fields of view, n for α2δ-1 = 8 fields of view; n corresponds to fields of view from five independent experiments and data are shown as means ± SD; two-tailed unpaired t test, P = 0.05. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Plated cells on 22 mm2 coverslips were transferred to a laminar-flow perfusion and stimulation chamber (imaging chamber with field stimulation series 20, Warner Instruments, Holliston, MA, USA) and mounted on an epifluorescence microscope (Zeiss Axiovert 200M) under continuous perfusion at 23°C at 0.5 ml/min with Ca2+ perfusion buffer containing (in mm) 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 Hepes (buffered to pH 7.4) and 30 glucose.

Techniques: Over Expression, Immunostaining, Control, Plasmid Preparation, Transfection, Marker, Fluorescence, Two Tailed Test

Journal: The Journal of Physiology

Article Title: Involvement of Ca_V2.2 channels and α₂δ‐1 in homeostatic synaptic plasticity in cultured hippocampal neurons

doi: 10.1113/jp283600

Figure Lengend Snippet: Figure 6. During HSP, surface and total levels of endogenous α2δ-1 decrease while overexpression of α2δ-1 prevents HSP A, biotinylation experiments of control and TTX-treated hippocampal neurons showing the total endogenous amount of α2δ-1 (WCL) and surface fractions, respectively (top gel). Bands for GAPDH are shown in the bottom gel. B, quantification of the intensity of endogenous α2δ-1 protein bands reveals decreased levels of total and surface α2δ-1 after the induction of HSP with TTX (green data points) compared to control, untreated neurons (grey data points). Data have been normalised to controls in each experiment, and analysed by one-sample two-tailed t test compared to a theoretical value of 1. WCL P = 0.0072 and surface P = 0.042; n for WCL = 4 independent experiments, n for surface fraction = 3 independent experiments, averaged duplicates for each experiment. C, Ca2+ transient amplitudes after stimulation with one AP are larger in TTX-treated EV than in control EV conditions (grey data points) but similar in α2δ-1-overexpressing neurons with (green) and without TTX (dark grey data points). One-way ANOVA, F(3,40) = 6.484, P = 0.001, Tukey’s multiple comparisons test, EV vs. EV + TTX, P = 0.0008; α2δ-1 vs. α2δ-1 + TTX, P = 0.99; n corresponds to independent experiments and data are shown as means ± SD; n for EV and EV + TTX = 13, n for α2δ-1 and α2δ-1 + TTX = 9. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Plated cells on 22 mm2 coverslips were transferred to a laminar-flow perfusion and stimulation chamber (imaging chamber with field stimulation series 20, Warner Instruments, Holliston, MA, USA) and mounted on an epifluorescence microscope (Zeiss Axiovert 200M) under continuous perfusion at 23°C at 0.5 ml/min with Ca2+ perfusion buffer containing (in mm) 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 Hepes (buffered to pH 7.4) and 30 glucose.

Techniques: Over Expression, Control, Two Tailed Test