Je HS , Yang F , Zhou J , Lu B .

Intramural NIH HHS

	Figure 1. NT-3 activates CREB via CaMKs. X. laevis nerve-muscle cultures were treated with or without inhibitors for 1 h, followed by application of NT-3. The cultures were fixed 30 min later and processed for immunocytochemistry using anti-pCREB antibody. (A) pCREB staining images of cultures treated with or without 5 ng/ml NT-3. The yellow arrow indicates dark nuclear staining by pCREB. N, neuron; M, muscle cell. Bar, 20 μm. (B) Fluorescence intensity profiles of pCREB staining in the cell body of spinal neurons. Higher magnification of neuronal cell body is shown in the top right corner. The intensity of pCREB fluorescence was monitored along the axis drawn on the cell body of spinal neurons. Bar, 10 μm. n = 8 for untreated neurons; n = 10 for NT-3–treated neurons. (C) Quantification of peak intensity of pCREB fluorescence under various conditions. The number associated with each column represents the number of cells analyzed. *, significantly higher than the respective NT-3(–) group, by ANOVA, followed by post hoc tests; P < 0.01. (D) Time course of CREB activation upon NT-3 treatment with or without 10 μM MAPK inhibitor (PD098059) or 1 μM CaMK inhibitor (KN93). The peak intensities of pCREB fluorescence at various time points were monitored after NT-3 treatment. Data are presented as the mean ± the SEM.
	Figure 2. Presynaptic expression of DnCREB prevents the long-term effect of NT-3 on synaptic transmission. In this and all other figures, presynaptic expression of a specific gene was achieved by embryo mRNA injection. The acute effect of NT-3 was studied by directly applying 25 ng/ml NT-3 to 1-d-old cultures, and the long-term effect of NT-3 was studied by treating cultures with 5 ng/ml NT-3 for 48 h. The frequency of SSCs was used to monitor changes in synaptic efficacy. (A) Inhibition of CREB activation did not prevent acute potentiation of synaptic transmission by NT-3. (top) A sample recording showing that acute application of NT-3 still elicits a rapid increase in the frequency of SSCs recorded from a synapse in which K-CREB is expressed presynaptically (N+). (bottom) Summary of effect of acute application of NT-3 on control synapses (N–) without K-CREB, and synapses with presynaptic (N+) expression of K-CREB. Each data point represents normalized SSCs frequency (averaged from 10 min of recording) from a single synapse before and after NT-3 application. (B and D) Presynaptic, but not postsynaptic, expression of K-CREB prevents NT-3–induced long-term synaptic potentiation on SSC frequency (B) and ESC amplitude (D). M+, K-CREB expressed postsynaptically. (C) Presynaptic expression (N+) of A-CREB prevents NT-3–induced long-term synaptic potentiation. The number associated with each column represents the number of cells analyzed. Data are presented as the mean ± the SEM.
	Figure 3. NT-3–induced CREB activation and long-term physiological change are mediated by CaMKIV. (A) Inhibition of CaMKs prevents NT-3–induced long-term synaptic potentiation. Cultures were treated with or without 1 μM KN93, which is a specific inhibitor for CaMKs. (B and C) Expression of Dn-CaMKIV abolishes CREB activation induced by NT-3. (B) Bright-field (top left), pCREB (top right, red), Dn-CaMKIV (bottom left, green), and merged fluorescence images (bottom right) of three spinal neurons, two of which express Dn-CaMKIV, as indicated by coexpression of GFP. After application of NT-3, the Dn-CaMKIV–negative neuron exhibit marked CREB phosphorylation (red). In contrast, the Dn-CaMKIV–expressing neuron (green) shows no CREB phosphorylation. Note that the neuron in the middle expresses a lower level of Dn-CaMKIV and shows a reduced CREB phosphorylation. Bar, 10 μm. White dashed lines indicate the outline of neuronal cell bodies. (C) Quantitative analysis of pCREB levels in Dn-CaMK (+) and (–) neurons before and after NT-3 application. (D and E) Inhibition of NT-3–induced long-term synaptic potentiation by presynaptic expression of Dn-CaMKIV. Dn-CaMKIV was expressed in spinal neurons. SSC frequency and ESC amplitude were measured in synapses treated with or without NT-3 for 2 d. (D) Effects on SSC frequency. (E) Effects on ESC amplitude. The number associated with each column represents the number of cells analyzed. Data are presented as the mean ± the SEM.
	Figure 4. NT-3 induces the translocation of CaM into the nucleus of spinal neurons. (A) Expression of the GFP-CaM fusion gene in X. laevis spinal neurons. Hoffman and fluorescent images (left and middle) show a spinal neuron expressing GFP-CaM. Bars (A and B), 20 μm. (right) Western blots showing the expression of the GFP-CaM in 1-d-old neural tubes injected with or without (Control) CaM mRNA. (B) Redistribution of CaM in spinal neurons induced by NT-3. (top) Color-coded images of a CaM-GFP–expressing spinal neuron before and after NT-3 application. Two ROIs for monitoring fluorescence changes (free-hand ROI traces over nucleus and axon) are shown. At the peak of the response (30 min after NT-3 application), the nuclear area is bright red, indicating a substantial accumulation of GFP-CaM in the region. (bottom) Application of NT-3 evoked a slow, but sustained, increase (from 10 min after NT-3 application) in the fluorescence signal from the nucleus region (blue trace), accompanied by a corresponding decrease in the fluorescence intensity from the axonal area (pink trace). Averaged florescence signals at 30 min are shown on the right. The number associated with each column represents the number of cells analyzed. Data are presented as the mean ± the SEM.
	Figure 5. Synaptic sites are revealed by double labeling with SYP-GFP and Cy5-α-BTX. (A) Phase-contrast image of nerve-muscle coculture. M, myocytes. (B) Labeling of axons of a spinal neuron by rhodamine dextran. (C) Labeling of synaptic varicosities by SYP-GFP. Note the discrete SYP puncta located in presynaptic terminals innervating the myocytes. (D) Labeling of postsynaptic AChRs with Cy5-α-BTX. (E) Merge of images in C and D. (F) Enlargement of the areas boxed in E. Note that SYP-GFP (green) and Cy5-α-BTX (red) puncta are juxtaposed against each other at the synapses. Bar, 10 μm.
	Figure 6. SYP-GFP–labeled synaptic varicosities are colocalized with FM dye–labeled puncta. Neurons transiently transfected with SYP-GFP were loaded with FM 4–64 dye by rapid depolarization (2 min) with high K+ and neurons, followed by extensive washing.(A) Phase-contrast images of nerve-muscle coculture. (B) Labeling of synaptic varicosities by SYP-GFP. (C) Labeling of synaptic varicosities by FM 4–64. Yellow arrows indicate synaptic varicosities in which SYP-GFP and FM dye are colocalized. (D) Merge of images in B and C. More than 20 neurons were examined, and consistent colocalization of SYP-GFP and FM dye were observed in puncta located in the synaptic regions. Bar, 10 μm.
	Figure 7. Long-term NT-3 treatment increases the number of synaptic sites via MAPK, but not CREB. (A) Effect of NT-3 on the number of synaptic sites. Synaptic sites (yellow spots, indicated by yellow arrows) are revealed by synaptic boutons (labeled by SYP-GFP, green) directly juxtaposed to postsynaptic AChR (labeled by Cy5-α-BTX; red). (B) Failure of K-CREB to block a NT-3–induced increase on synaptic sites. (C) Selective blockade of NT-3–induced long-term changes in synaptic sites by inhibition of MAPK pathway. The MEK inhibitor U0126, but not the PKA inhibitor Rp-cAMP or the CaMK inhibitor KN93, prevented the increase in synaptic sites induced by NT-3. The number associated with each column represents the number of cells analyzed. Data are presented as the mean ± the SEM.
	Figure 8. Rap1, not Ras, is required for long-term morphological changes by NT-3. (A) Activation of Ras and Rap1 in NT-3–stimulated X. laevis spinal neurons. Intensity-modulated display images of spinal neurons expressing Raichu–Ras or –Rap1 and stimulated by NT-3. Bar, 10 μm. Yellow arrows indicate spots with increased level of Ras or Rap1 activity (B) Quantitative analysis of Ras and Rap1 activities in neuronal soma. The relative changes in fluorescence intensity were monitored over time. n = 4. (C) Lack of effects of presynaptic expression of Dn-Ras or -Rap1 on NT-3–induced changes in SSC frequency. (D) Inhibition of NT-3–induced increase in synaptic site by presynaptic expression of Dn-Rap1, but not Dn-Ras. The number associated with each column represents the number of cells analyzed. Data are presented as the mean ± the SEM.
	Figure 9. A model for mechanisms underlying long-term modulation of structures and functions of synapses by NT-3.

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