Fig. 5
Axonal FFN270 release reveals two populations of NE synapses. a Change in FFN270 puncta fluorescence in the barrel cortex of acute slice during the course of a locally applied electrical stimulation (10 Hz, 3000 pulses, starts at t: 0 min). In red are destaining puncta identified during electrical stimulation (t-1/2: 34.6 s), and in blue are puncta during no stimulation (n = 2–3 slices per animal, 3 animals for each condition). b A representative FFN 270 labeled noradrenergic axon during electrical stimulation. FFN270 (d) strongly colocalized with the ChR2-EYFP signal (c 72.4%, 92/127 axons, 6 animals) in TH-Cre animals injected with floxed-ChR2-EYFP in the LC. The ChR2 was then locally stimulated with 470 nm light (10 Hz, 2400 pulses), and the change in FFN270 signal after stimulation (e) was measured. f When averaged across each slice, there was a significant decrease in remaining FFN270 signal that colocalized with ChR2-EYFP (ChR+, 42.6 ± 5.9%) compared to those that did not (ChR−, 72.4 ± 3.3%, P < 0.0001, two-tailed unpaired t-test, n = 2–3 slices per animal from 5 animals, data presented with min/max whiskers). g We also compared fluorescence remaining of the individual axons across all trials, and observed a similar trend (ChR+: 37.1 ± 3.3%, ChR−: 72.0 ± 2.6%, P < 0.0001, Mann–Whitney). The individual axons that comprise the ChR− population follow a normal distribution (D'Agostino & Pearson test, P = 0.3), while the ChR+ population does not (P = 0.003). The ChR+ population closely follows a double Gaussian distribution (right panel histogram, R2 = 0.91) with high (64% with 15.1% F remaining) and low releasing (36% with 75.4% F remaining) populations. All data listed as mean ± SEM. Scale bar: 10 µm
