Journal: Cell reports

Article Title: Spatially non-overlapping Ca 2+ signals drive distinct forms of neurotransmission

doi: 10.1016/j.celrep.2023.113201

Figure Lengend Snippet: (A) Experimental design for calcium phosphate transfection of GCaMP8s-Syb2 in cultured hippocampal neurons. (B) Cartoon representation of GCaMP8s-Syb2 on presynaptic vesicles. (C) Representative image of neuronal synapses before and after high-frequency stimulation. (D) Representative spontaneous presynaptic Ca 2+ transient (sPreCT) trace. (E) Histogram of detected spontaneous event amplitudes compared to the noise of the trace, from individual synapses (ROIs). (F) Histogram of sPreCT frequencies of all synapses (green) and active synapses only (black). n = 1,744 synapses. (G) Bar graph comparison of the sPreCT frequencies between all synapses versus active synapses only. n = 1,744 synapses. (H) Representative evoked presynaptic Ca 2+ transient (ePreCT) trace. (I) Histogram of detected evoked event amplitudes compared to the noise of the trace, from individual synapses (ROIs). (J) Histogram distribution of the likelihood of detecting an ePreCT with each stimulation. n = 1,115 synapses. Note that the majority of synapses (~70%) respond to every stimulation with high fidelity (≥90%). (K) Averaged detected sPreCT and ePreCT events. (L–N) Comparison of the kinetics of ePreCT (N = 12 coverslips) versus sPreCT (N = 10 coverslips) in terms of Ca 2+ amplitudes (L), rise time (M), and decay time (N). Welch’s t test. Graphs are mean ± SEM. Significance reported as ***p < 0.001. NS, non-significance.

Article Snippet: Neuronal transfections were performed on DIV 7 using a Ca 2+ phosphate kit (ProFection Mammalian Transfection System, Cat #E1200, Promega), based on a previously described method.

Techniques: Transfection, Cell Culture, Comparison

Journal: Cell reports

Article Title: Spatially non-overlapping Ca 2+ signals drive distinct forms of neurotransmission

doi: 10.1016/j.celrep.2023.113201

Figure Lengend Snippet: (A) Experimental design and representative traces of VGCC blockade on ePreCTs. (B and C) Effects of VGCC blockade on ePreCT likelihood to stimulation (B) and amplitudes (C). N = 9 coverslips. One-way ANOVA. (D) Experimental design and representative traces of VGCC blockade on sPreCTs. (E and F) Effects of VGCC blockade on sPreCT frequencies (E) and amplitudes (F). Normalized values for GCaMP8s-Syb2 imaging were calculated by dividing each synaptic value after treatment by the average of the coverslip during before conditions. sPreCT frequencies were so infrequent that we could not normalize synapse by synapse without losing information; thus, we had to normalize this way, and we kept it consistent across other measurements in this experiment. N = 9 coverslips. One-way ANOVA was performed for these experiments. (G) Representative traces of baseline Ca 2+ before and after VGCC blockade. (H) Changes in baseline fluorescence after VGCC blockade compared with controls. N = 5 coverslips for treatment groups; N = 3 coverslips for control groups. Two-way ANOVA. (I) Frequency distribution of the noise of the trace before and after treatment, with the standard deviation demarcated by dotted lines. (J) Comparison of the standard deviation of the trace after VGCC blockade. One-way ANOVA. (K) Comparison of the standard deviation of the trace after vehicle control addition. One-way ANOVA. (L) Comparison of standard deviation after vehicle control compared with after VGCC blockade. Two-way ANOVA. (M) Representative iGluSnFR traces before and after VGCC blockade. (N–P) Effect of VGCC blockade on glutamate evoked release probability (N), spontaneous glutamate frequency (O), and amplitudes (P). Normalized values for iGluSnFR were made by normalizing synapse by synapse such that all normalized “before” values are 1. N = 10 coverslips for evoked glutamate events; N =7 coverslips for spontaneous glutamate events. One-way ANOVA was performed on these experiments. Graphs are mean ± SEM. Significance reported as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS, non-significance.

Article Snippet: Neuronal transfections were performed on DIV 7 using a Ca 2+ phosphate kit (ProFection Mammalian Transfection System, Cat #E1200, Promega), based on a previously described method.

Techniques: Imaging, Fluorescence, Control, Standard Deviation, Comparison

Journal: Cell reports

Article Title: Spatially non-overlapping Ca 2+ signals drive distinct forms of neurotransmission

doi: 10.1016/j.celrep.2023.113201

Figure Lengend Snippet: (A) Experimental design of ryanodine on spontaneous PreCTs with representative traces. (B and C) Effect of ryanodine inhibition on sPreCT frequencies (B) and amplitudes (C). N = 7 coverslips. Welch’s t test. (D) Experimental design of ryanodine on evoked PreCTs with representative traces. (E and F) Effect of ryanodine receptor inhibition on ePreCT likelihood to stimulation (E) and ePreCT amplitudes (F). N = 7 coverslips. Welch’s t test. Note that DMSO treatment by itself reduces the likelihood of Ca 2+ responses to stimulation. (G) Effect of ryanodine inhibition on baseline Ca 2+ fluorescence. N = 7 coverslips for treatment group; N = 6 coverslips for control group. Two-way ANOVA. (H) Frequency distribution of the noise of the trace before and after ryanodine inhibition, with the standard deviation demarcated by dotted lines. (I) Comparison of the standard deviation of the fluorescent trace before and after ryanodine inhibition. N = 7 coverslips. Welch’s t test. (J) Experimental paradigm of ryanodine on glutamate release via iGluSnFR recordings, with representative traces. (K–N) Effect of ryanodine inhibition on spontaneous glutamate event frequencies (K), spontaneous glutamate event amplitudes (L), evoked release probability (M), and evoked glutamate event amplitudes (N). N = 7 coverslips for treatment group; N = 6 coverslips for control group. Welch’s t test. (O) Comparison of spontaneous presynaptic Ca 2+ event frequencies with spontaneous glutamate event frequencies. N = 1,844 synapses for sPreCTs; n = 2,896 synapses for spontaneous glutamate events. Welch’s t test. Graphs are mean ± SEM. Significance reported as *p < 0.05. ****p < 0.0001. NS, non-significance.

Article Snippet: Neuronal transfections were performed on DIV 7 using a Ca 2+ phosphate kit (ProFection Mammalian Transfection System, Cat #E1200, Promega), based on a previously described method.

Techniques: Inhibition, Fluorescence, Control, Standard Deviation, Comparison

Journal: Cell reports

Article Title: Spatially non-overlapping Ca 2+ signals drive distinct forms of neurotransmission

doi: 10.1016/j.celrep.2023.113201

Figure Lengend Snippet: (A) Photobleaching schematic on its use-dependent property. (B) Experimental paradigm of photobleaching presynaptic Ca 2+ transients at rest or with stimulation. (C and D) Effect of photobleaching at rest on ePreCT event likelihood to stimulation (C) and ePreCT amplitudes (D). N = 4 coverslips per group. One-way ANOVA. (E and F) Effect of photobleaching at rest on sPreCT frequencies (E) or sPreCT amplitudes (F). N = 6 coverslips for 10 min of photobleaching; N = 7 coverslips at 30 min of photobleaching. One-way ANOVA. (G and H) Effect of photobleaching with stimulation on ePreCT event likelihood to stimulation (G) and ePreCT amplitudes (H). N = 5 coverslips for all groups. One-way ANOVA. (I and J) Effect of photobleaching with stimulation on sPreCT frequencies (I) or sPreCT amplitudes (J). N = 4 coverslips for all groups. One-way ANOVA. Graphs are mean ± SEM. Significance reported as *p < 0.05, **p < 0.01, and ***p < 0.001. NS, non-significance.

Article Snippet: Neuronal transfections were performed on DIV 7 using a Ca 2+ phosphate kit (ProFection Mammalian Transfection System, Cat #E1200, Promega), based on a previously described method.

Techniques:

Journal: Cell reports

Article Title: Spatially non-overlapping Ca 2+ signals drive distinct forms of neurotransmission

doi: 10.1016/j.celrep.2023.113201

Figure Lengend Snippet: (A) Experimental paradigm. (B and C) Effect of perfusing high K + while photobleaching on sPreCT frequencies (B) and sPreCT amplitudes (C). N = 8 coverslips. Welch’s t test for (B) and Kolmogorov-Smirnov test for (C). (D–F) Effect of perfusing high K + while photobleaching on ePreCT likelihood to stimulation (D), ePreCT amplitudes (E), and baseline Ca 2+ signal (F). N = 9 coverslips. Welch’s t test. Graphs are mean ± SEM. Significance reported as *p < 0.05, **p < 0.01, and ****p < 0.0001. NS, non-significance.

Article Snippet: Neuronal transfections were performed on DIV 7 using a Ca 2+ phosphate kit (ProFection Mammalian Transfection System, Cat #E1200, Promega), based on a previously described method.

Techniques:

Journal: Cell reports

Article Title: Spatially non-overlapping Ca 2+ signals drive distinct forms of neurotransmission

doi: 10.1016/j.celrep.2023.113201

Figure Lengend Snippet: (A) Experimental paradigm of photobleaching at rest on baseline Ca 2+ signal. (B) Photobleaching at rest on baseline Ca 2+ . N = 6 coverslips for 10 min; N = 7 for 30 min of photobleaching. Two-way ANOVA. (C) Experimental paradigm of photobleaching with stimulation on baseline Ca 2+ . (D) Photobleaching with stimulation on baseline Ca 2+ . N = 6 coverslips for 10 min; N = 7 for 30 min of photobleaching. The same control baseline group was used as for (B). Two-way ANOVA. (E) Comparison of the effect of photobleaching at rest versus with stimulation on baseline Ca 2+ signal. N = 6 coverslips for 10 min. N = 7 for 30 min of photobleaching per group. Two-way ANOVA. (F) Comparison of baseline Ca 2+ signal in 2 versus 8 mM Ca 2+ . N = 23 coverslips for 2 mM and N = 27 coverslips for 8 mM Ca 2+ . Welch’s t test. (G) Comparison of photobleaching on baseline Ca 2+ signal in 2mM Ca 2+ (N = 6 coverslips for 10 min, N = 7 for 30 min of photobleaching) versus 8 mM Ca 2+ (N = 7 coverslips for 10 min, N = 6 for 30 min of photobleaching). Two-way ANOVA. (H) Representative baseline Ca 2+ signals before and after photobleaching. (I) Frequency distribution of the noise of the trace before and after photobleaching. (J and K) Standard deviation of the fluorescent trace before and after photobleaching at rest (N = 5 coverslips for 10 min, N = 6 for 30 min of photobleaching) (J) and with stimulation (N = 6 coverslips for 10 min, N = 7 for 30 min of photobleaching) (K). One-way ANOVA. (L) Comparison of the standard deviations between photobleaching for 30 min at rest (N = 6) and with stimulation (N = 7). Two-way ANOVA. Graphs are mean ± SEM. Significance reported as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS, non-significance.

Article Snippet: Neuronal transfections were performed on DIV 7 using a Ca 2+ phosphate kit (ProFection Mammalian Transfection System, Cat #E1200, Promega), based on a previously described method.

Techniques: Control, Comparison, Standard Deviation

Journal: Cell reports

Article Title: Spatially non-overlapping Ca 2+ signals drive distinct forms of neurotransmission

doi: 10.1016/j.celrep.2023.113201

Figure Lengend Snippet: (A) Experimental paradigm of photobleaching in 8 mM Ca 2+ . (B) Comparison of photobleaching with stimulation on ePreCT likelihood to stimulation in 2 (N = 4 coverslips for 10 min, N = 5 for 30 min of photobleaching) versus 8 mM Ca 2+ (N = 7 coverslips for 10 min, N = 6 for 30 min of photobleaching). Two-way ANOVA. (C) Comparison of photobleaching with stimulation on ePreCT amplitudes in 2 (N = 4 coverslips for 10 min, N = 4 for 30 min of photobleaching) versus 8 mM Ca 2+ (N = 7 coverslips for 10 min, N = 6 for 30 min of photobleaching). Two-way ANOVA. (D) Experimental paradigm of fluorescence recovery over time. (E–G) Effect of fluorescence recovery over time on ePreCT likelihood to stimulation (E), ePreCT amplitudes (F), and baseline Ca 2+ signal (G). N = 5 coverslips at 1 h of photobleaching; N = 6 coverslips for 5 h; N = 5 coverslips for 14 h. One-way ANOVA. (H) Cartoon schematic of local and non-local vesicle turnover and subsequent fluorescence recovery over time after photobleaching. Graphs are mean ± SEM. Significance reported as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS, non-significance.

Article Snippet: Neuronal transfections were performed on DIV 7 using a Ca 2+ phosphate kit (ProFection Mammalian Transfection System, Cat #E1200, Promega), based on a previously described method.

Techniques: Comparison, Fluorescence