Journal: Journal of Neurophysiology

Article Title: Determinants of synaptic strength vary across an axon arbor

doi: 10.1152/jn.00615.2011

Figure Lengend Snippet: Presynaptic heterogeneity is correlated with the amount of postsynaptic NMDA receptors and PSD-95. Local axon segments were stimulated with 70 pulses delivered at 20 Hz to measure evoked release, followed by NH4Cl saline perfusion to measure total vesicle pool sizes. Post hoc immunostaining was performed with PSD-95, NR1, GluR1, or MAP2 antibodies to reveal postsynaptic specializations or dendrites. We examined 6–8 neurons for each marker. A: representative images of evoked ΔF (1st panel), total vesicle pool size (2nd panel), post hoc immunostaining of NR1 clusters (3rd panel; green channel is pHluorin, red channel is NR1), and colocalized pixels between NR1 and synaptophysin-pHluorin (4th panel). Arrowheads indicate the terminals plotted in B and C. Scale bar, 5 μm. B: correlation plot of total vesicle pool size and evoked ΔF of terminals shown in A (correlation coefficient r = 0.98, Pearson correlation test, P = 0.02). C: correlation plot of NR1 intensity and evoked ΔF of terminals shown in A (r = 0.99, Pearson correlation test, P = 0.006). D: correlation plot of NR1 intensity and total vesicle pool size of terminals shown in A (r = 0.97, Pearson correlation test, P = 0.03). E: the average correlation coefficient between evoked release and NR1 (9 segments; r = 0.8 ± 0.04) or PSD-95 (11 segments; r = 0.80 ± 0.04) was significantly different from the MAP2 correlation coefficient (Mann-Whitney U-test: NR1, *P = 0.003; PSD-95, *P = 0.003) but not that of GluR1 (r = 0.6 ± 0.1, P = 0.27). Evoked release was not significantly correlated with the amount of MAP2 for terminals in 7/8 axon segments measured (r = 0.4 ± 0.1). F: the average correlation coefficients between total vesicle pool size and NR1 (r = 0.8 ± 0.03) or PSD-95 (r = 0.80 ± 0.04) were significantly different from the MAP2 correlation coefficient (Mann-Whitney U-test: NR1, *P = 0.008; PSD-95, *P = 0.01) but not that of GluR1 (r = 0.7 ± 0.1, P = 0.17). Total vesicle pool size was not significantly correlated with the amount of MAP2 for terminals in 6/8 segments measured (r = 0.5 ± 0.1). G: cumulative distribution of total vesicle pool size in presynaptic terminals colocalized with PSD-95 increased significantly from 10 to 21 DIV (Kruskal-Wallis test, *P < 0.02). H: cumulative distribution of total vesicle pool size in presynaptic terminals not colocalized with PSD-95 was not significantly different from 10 to 21 DIV (Kruskal-Wallis test, P = 0.13). Cc, correlation coefficient.

Article Snippet: Student's t -test was used for parametrically distributed values; the Mann-Whitney U -test or Wilcoxon matched-pairs test was used for nonparametrically distributed values; the Kruskal-Wallis ANOVA test was used for multiple group data comparison; the Pearson correlation test was used to compare correlated values; and the F -test was used to compare linear regressions (GraphPad Prism).

Techniques: Saline, Immunostaining, Marker, MANN-WHITNEY

Journal: Journal of Neurophysiology

Article Title: Determinants of synaptic strength vary across an axon arbor

doi: 10.1152/jn.00615.2011

Figure Lengend Snippet: Spatial distribution of evoked release and total vesicle pool size across single axon arbors. A: Neurolucida tracing of a neuron transfected with synaptophysin-pHluorin and mCherry (not shown) to enable long-distance axon tracking. Colors indicate axon segments that were stimulated and imaged. Images taken after stimulation show the distribution of releasing presynaptic terminals (arrows). Scale bar, 100 μm. B: evoked release of individual presynaptic terminals, color coded by segment. C: the average random pair ratios of evoked release for terminals in color-coded axon segments shown in A (range: 2.9 ± 0.5 to 4.4 ± 0.5; average of all 5 segments from this neuron, white bar), as well as 22/24 axon segments from 6 neurons (average of terminals in all segments: 3.4 ± 0.2), are significantly higher than that for the two-repeats control (indicated by dashed line, 1.9 ± 0.2; Mann-Whitney U-test, P < 0.05). The random pair ratios of evoked release for terminals in color-coded segments and all segments from all axons are not significantly different (1-way ANOVA, P = 0.66). D: total vesicle pool size of individual presynaptic terminals, color coded by segment. E: the average random pair ratios of total vesicle pool size for terminals in short axon segments shown in A (range: 3.3 ± 0.6 to 5.0 ± 0.9; average of all 5 segments from this neuron, white bar), as well as 18/24 axon segments from 6 neurons (average of terminals in all segments: 3.2 ± 0.2), are significantly higher than the two-repeats control (indicated by dashed line, 1.9 ± 0.1; Mann-Whitney U-test, P < 0.05). The random pair ratios of total vesicle pool size for terminals in color-coded segments and all segments from all axons are not significantly different (1-way ANOVA, P = 0.92). F: relationship between coefficient of variance (CV) of evoked release and total vesicle pool size of presynaptic terminals from the most proximal (white circles; r = 0.65, Pearson correlation test, P < 0.05) and the most distal segments (black circles; r = 0.82; Pearson correlation test, P < 0.05) from 13 neurons (slope = 0.96 ± 0.05; Pearson correlation test, P < 0.0001).

Article Snippet: Student's t -test was used for parametrically distributed values; the Mann-Whitney U -test or Wilcoxon matched-pairs test was used for nonparametrically distributed values; the Kruskal-Wallis ANOVA test was used for multiple group data comparison; the Pearson correlation test was used to compare correlated values; and the F -test was used to compare linear regressions (GraphPad Prism).

Techniques: Transfection, Control, MANN-WHITNEY

Journal: Journal of Neurophysiology

Article Title: Determinants of synaptic strength vary across an axon arbor

doi: 10.1152/jn.00615.2011

Figure Lengend Snippet: Evoked release and release fraction are higher in distal compared with proximal segments of individual axon arbors. A: average evoked release in proximal axon segments (black symbols; 1.3 ± 0.2 × 104 AFU) is slightly but significantly smaller than distal segments (1.9 ± 0.3 × 104 AFU; 16 neurons; Wilcoxon matched-pairs test, P < 0.001). The average evoked release in proximal compared with distal segments is indicated by red symbols. B: average total vesicle pool size in proximal axon segments (black symbols; 15.2 ± 3.9 × 104 AFU) of individual neurons is not significantly different from that in distal segments (10.1 ± 2.0 × 104 AFU; 16 neurons; Wilcoxon matched-pairs test, P = 0.17). The average of total vesicle pool size in proximal compared with distal segments is indicated by red symbols. C: average release fraction in proximal axon segments (black symbols; 0.15 ± 0.02) is significantly smaller than that in distal segments (0.30 ± 0.04; 16 neurons; Wilcoxon matched-pairs test, P = 0.003). The average release fraction of proximal compared with distal segments is indicated by red symbols. D: relationship between evoked release and total vesicle pool size of presynaptic terminals from axon segments I-V from the representative neuron shown and color coded in Fig. 3A. Correlation coefficient and Pearson correlation test results for each segment: segment I, r = 0.67, P = 0.15; segment II, r = 0.86, P = 0.006; segment III, r = 0.81, P = 0.002; segment IV, not available; segment V, r = 0.88, P = 0.004. In this example, as for all axons measured (24 segments, 6 neurons), some segments have a similar regression slope, indicative of similar release fraction (segments I and II, segments III and IV), whereas release fraction is significantly different among other segments (1-way ANOVA, P = 0.007). E: relationship between evoked release and total vesicle pool size of all terminals in the most proximal segments (gray) and the most distal segments (black) from 13 neurons (evoked release: r = 0.90, Spearman correlation test, P < 0.0001; total vesicle pool size: r = 0.85, Spearman correlation test, P < 0.0001). The linear regression lines are significantly different (average proximal slope = 0.05 ± 0.005, distal slope = 0.14 ± 0.009; F-test, P < 0.001), indicating that distal terminals have a significantly higher release fraction compared with proximal terminals.

Article Snippet: Student's t -test was used for parametrically distributed values; the Mann-Whitney U -test or Wilcoxon matched-pairs test was used for nonparametrically distributed values; the Kruskal-Wallis ANOVA test was used for multiple group data comparison; the Pearson correlation test was used to compare correlated values; and the F -test was used to compare linear regressions (GraphPad Prism).

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