Fig. 2.

NT-3 in synaptosomes: K⁺-induced-release of NT-3 and other proteins from axon terminals and binding of neurotrophins and neuropeptides to synaptosomes. A, SDS gel shows that the large majority of NT-3 recovered from synaptosomes (Synapt.) migrates identically to native NT-3 at ∼14 kDa. The arrow indicates minor degradation products. Three month exposure. B, Plot of anterogradely transported ¹²⁵I-NT-3 in the contralateral tectum (cpm) as a function of the amount of ¹²⁵I-NT-3 remaining in the injected eye. C, Plot of the¹²⁵I-NT-3 in the synaptosomal fraction from the contralateral tectum (filled circles) as a function of the amount of ¹²⁵I-NT-3 remaining in the injected eye. When the¹²⁵I-NT-3 in the eye was competed with 20–50-fold excess cold NT-3, only background cpm were found in the synaptosomes (open circles). Note that the amounts of NT-3 in the tectum and in the synaptosomal peak saturate at ∼10 × 10⁶-15 × 10⁶ cpm in the eye.D, Representative examples show decreased size of synaptosomal peaks (SY) from tectum depolarized in high K⁺ buffer (right panel) compared with low K⁺ buffer (left panel). E, Quantification of D reveals a 15–20% decrease in NT-3 content in depolarized terminals (HiK, high K⁺ buffer; ***p ≤ 0.001, ANOVA, error bars indicate SEM). The average of the values for low K⁺ buffer (LoK, 60 min) was taken as 100%. Comparison of incubation for 60 min versus 0 min in low K⁺ shows lack of basal (constitutive) release. Numbers of experiments are indicated on bars. F, Release of radiolabeled tetanus toxin C fragment (Tet tox) was not statistically significant in high K⁺ buffer.G, Radiolabeled cytochrome c(Cyt.C) was not released in high K⁺ buffer compared with low K⁺buffer. H, Depolarized tecta showed a 25–50% release of substance P (SP) from tectal terminals, measured by radioimmunoassay in 500 μl samples (1:6 dilution). *p ≤ 0.05, t test, HiK versus LoK, 60 min, error bars indicate SEM. I, When homogenized tecta were incubated with radiolabeled NT-3, NGF, or cytochromec (Cyt C), NT-3 bound to tectal terminals which was competed by excess cold NT-3, whereas NGF and cytochromec showed very little binding. SY, Synaptosomal peak; GM, general membrane fraction.