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.

Ahn, A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. 1998, Pubmed

Ahn, A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. 1998, Pubmed
Alessi, PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. 1995, Pubmed
Alsina, Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF. 2001, Pubmed , Xenbase
Bailey, Toward a molecular definition of long-term memory storage. 1996, Pubmed
Bailey, Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons. 1997, Pubmed
Balschun, Does cAMP response element-binding protein have a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent memory? 2003, Pubmed
Barco, Gene expression profiling of facilitated L-LTP in VP16-CREB mice reveals that BDNF is critical for the maintenance of LTP and its synaptic capture. 2005, Pubmed
Barco, Expression of constitutively active CREB protein facilitates the late phase of long-term potentiation by enhancing synaptic capture. 2002, Pubmed
Bartsch, Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change. 1995, Pubmed
Bito, CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression. 1996, Pubmed
Bourtchuladze, Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein. 1994, Pubmed
Christopherson, Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. 2005, Pubmed
Corsi, Up-regulation of NR2B subunit of NMDA receptors in cerebellar granule neurons by Ca2+/calmodulin kinase inhibitor KN93. 1998, Pubmed
Davis, Protein synthesis and memory: a review. 1984, Pubmed
Davis, Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity. 1996, Pubmed
Deisseroth, Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. 1998, Pubmed
Deisseroth, Signaling from synapse to nucleus: the logic behind the mechanisms. 2003, Pubmed
DeZazzo, nalyot, a mutation of the Drosophila myb-related Adf1 transcription factor, disrupts synapse formation and olfactory memory. 2000, Pubmed
English, Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. 1996, Pubmed
Finkbeiner, CREB: a major mediator of neuronal neurotrophin responses. 1997, Pubmed
Gass, Deficits in memory tasks of mice with CREB mutations depend on gene dosage. 1998, Pubmed
Goelet, The long and the short of long-term memory--a molecular framework. , Pubmed
Hardingham, Control of recruitment and transcription-activating function of CBP determines gene regulation by NMDA receptors and L-type calcium channels. 1999, Pubmed
He, Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3. 2000, Pubmed , Xenbase
Ho, Impaired synaptic plasticity and cAMP response element-binding protein activation in Ca2+/calmodulin-dependent protein kinase type IV/Gr-deficient mice. 2000, Pubmed
Huang, Trk receptors: roles in neuronal signal transduction. 2003, Pubmed
Impey, Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation. 1998, Pubmed
Je, Distinct mechanisms for neurotrophin-3-induced acute and long-term synaptic potentiation. 2005, Pubmed , Xenbase
Kandel, Genes, synapses, and long-term memory. 1997, Pubmed
Kandel, The molecular biology of memory storage: a dialogue between genes and synapses. 2001, Pubmed
Kang, An important role of neural activity-dependent CaMKIV signaling in the consolidation of long-term memory. 2001, Pubmed
Kaplan, Neurotrophin signal transduction in the nervous system. 2000, Pubmed
Kelleher, Translational control by MAPK signaling in long-term synaptic plasticity and memory. 2004, Pubmed
Lee, Estrogen induces phosphorylation of cyclic AMP response element binding (pCREB) in primary hippocampal cells in a time-dependent manner. 2004, Pubmed
Li, Dynamic redistribution of calmodulin in HeLa cells during cell division as revealed by a GFP-calmodulin fusion protein technique. 1999, Pubmed
Liou, Regulation of quantal secretion by neurotrophic factors at developing motoneurons in Xenopus cell cultures. 1997, Pubmed , Xenbase
Liou, Regulation of quantal secretion from developing motoneurons by postsynaptic activity-dependent release of NT-3. 1997, Pubmed , Xenbase
Lonze, Function and regulation of CREB family transcription factors in the nervous system. 2002, Pubmed
Lu, Acute and long-term synaptic modulation by neurotrophins. 2004, Pubmed
Lu, Neurotrophic regulation of the development and function of the neuromuscular synapses. 2003, Pubmed
Lu, BDNF and activity-dependent synaptic modulation. 2003, Pubmed
Markus, Raf and akt mediate distinct aspects of sensory axon growth. 2002, Pubmed
Marshall, Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. 1995, Pubmed
Martin, MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. 1997, Pubmed
Matthies, Protein kinase A inhibitors prevent the maintenance of hippocampal long-term potentiation. 1993, Pubmed
Mayford, Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia. 1992, Pubmed
Mochizuki, Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. 2001, Pubmed
Murphy, Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons. 1998, Pubmed
Murphy, Morphological plasticity of dendritic spines in central neurons is mediated by activation of cAMP response element binding protein. 1997, Pubmed
O'Connell, CREB phosphorylation coincides with transient synapse formation in the rat hippocampal dentate gyrus following avoidance learning. 2000, Pubmed
Pang, Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. 1995, Pubmed
Perron, Distinct neurite outgrowth signaling pathways converge on ERK activation. 1999, Pubmed
Picciotto, Immunochemical localization of calcium/calmodulin-dependent protein kinase I. 1995, Pubmed , Xenbase
Pittenger, Reversible inhibition of CREB/ATF transcription factors in region CA1 of the dorsal hippocampus disrupts hippocampus-dependent spatial memory. 2002, Pubmed
Poo, Neurotrophins as synaptic modulators. 2001, Pubmed
Ramanan, SRF mediates activity-induced gene expression and synaptic plasticity but not neuronal viability. 2005, Pubmed
Rammes, Synaptic plasticity in the basolateral amygdala in transgenic mice expressing dominant-negative cAMP response element-binding protein (CREB) in forebrain. 2000, Pubmed
Ribar, Cerebellar defects in Ca2+/calmodulin kinase IV-deficient mice. 2000, Pubmed
Rosenblum, The role of extracellular regulated kinases I/II in late-phase long-term potentiation. 2002, Pubmed
Ruthazer, Stabilization of axon branch dynamics by synaptic maturation. 2006, Pubmed , Xenbase
Schuster, Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. 1996, Pubmed
Schuster, Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity. 1996, Pubmed
Silva, CREB and memory. 1998, Pubmed
Tartaglia, Protein synthesis-dependent and -independent regulation of hippocampal synapses by brain-derived neurotrophic factor. 2001, Pubmed
Tokumitsu, Activation mechanisms for Ca2+/calmodulin-dependent protein kinase IV. Identification of a brain CaM-kinase IV kinase. 1994, Pubmed
Tyler, BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses. 2001, Pubmed
Verhage, Synaptic assembly of the brain in the absence of neurotransmitter secretion. 2000, Pubmed
Walton, A dominant repressor of cyclic adenosine 3',5'-monophosphate (cAMP)-regulated enhancer-binding protein activity inhibits the cAMP-mediated induction of the somatostatin promoter in vivo. 1992, Pubmed
Wang, Neurotrophins promote maturation of developing neuromuscular synapses. 1995, Pubmed , Xenbase
West, Regulation of transcription factors by neuronal activity. 2002, Pubmed
Wu, Activity-dependent CREB phosphorylation: convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. 2001, Pubmed
Wu, Nerve growth factor activates persistent Rap1 signaling in endosomes. 2001, Pubmed
Yang, PI-3 kinase and IP3 are both necessary and sufficient to mediate NT3-induced synaptic potentiation. 2001, Pubmed , Xenbase
Yin, CREB and the formation of long-term memory. 1996, Pubmed
York, Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1. 2000, Pubmed
York, Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. 1998, Pubmed