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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.043729

Journal of Cell Science 122, 1211-1219 (2009)
Published by The Company of Biologists 2009
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Activity of the AMPA receptor regulates drebrin stabilization in dendritic spine morphogenesis

Hideto Takahashi1,2,*, Hiroyuki Yamazaki1, Kenji Hanamura1, Yuko Sekino1,3,4 and Tomoaki Shirao1,{ddagger}

1 Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan
2 ERCGSM, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan
3 CREST, JST, Kawaguchi, Saitama, 332-0012, Japan
4 Division of Neuronal Network, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo, 1108-8639, Japan


Figure 1
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  		Fig. 1. Synaptic activity regulates drebrin clustering. (A) Drebrin immunolabeling of cultured hippocampal neurons treated with vehicle (control), TTX or picrotoxin from 8 DIV for 7 days. Upper panels show the representative low-magnification images of each treated neuron. Lower panels shows the boxed region in the respective upper panel. Scale bars: 20 µm (upper), 10 µm (lower). (B) Quantitative analysis of the densities of drebrin clusters. TTX treatment suppressed drebrin clustering, whereas picrotoxin treatment enhanced it (>14 neurons for each condition). **P<0.01, *P<0.05 versus control.

 

Figure 2
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  		Fig. 2. AMPAR activity is involved in drebrin clustering. (A) Drebrin immunolabeling of neurons treated with vehicle (control) or the following agents from 8 DIV for 7 days: the AMPAR and kainite-receptor blocker CNQX (40 µM), the specific AMPAR blocker GYKI (10 µM), the Ca2+-permeable (GluR2-subunit-lacking) AMPAR blocker PhTX (10 µM), the NMDAR blocker AP5 (50 µM), GYKI (10 µM) plus AP5 (50 µM), or the metabotropic glutamate receptor blocker MCPG (100 µM). Scale bar: 5 µm. (B) Quantitative analysis of the densities of drebrin clusters after these treatments. CNQX, GYKI and GYKI plus AP5 treatments suppressed drebrin clustering (> ten neurons for each treatment). *P<0.01 versus control. (C) Western blots showing the representative effect of GYKI and CNQX treatment on the expression of total drebrin (drebrin E and A) and β-actin. According to the densitometric analysis, GYKI and CNQX treatments do not change the expression level of drebrin protein (n=3 separate cultures; GYKI, 109.8±7.7% of control, P=0.33; CNQX, 89.1±8.3% of control, P=0.32).

 

Figure 3
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  		Fig. 3. Chronic AMPAR blockade suppresses drebrin clustering without affecting synapsin-I clustering. (A) Double labeling of neurons at 7 DIV for drebrin (green) and synapsin I (red). (B,C) Double labeling for drebrin and synapsin I of neurons treated with vehicle (control), GYKI or AP5 from 8 to 15 DIV (B) or to 22 DIV (C). (D,E) Quantitative analysis of the densities of drebrin clusters (D) and synapsin-I clusters (E) after these treatments. GYKI treatment reduced the developmental increase of drebrin cluster density without any significant change in synapsin-I cluster density (>15 neurons for each condition). *P<0.01 versus control at same DIV. Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. AMPAR activity enhances drebrin clustering in dendritic spines. (A) Merged drebrin (green), synapsin-I (red) and F-actin (blue) images in neurons treated from 8 to 15 DIV with vehicle (control), GYKI (10 µM), AP5 (50 µM), or CTZ (100 µM) plus AP5. GYKI-treated neurons have few drebrin clusters along their dendrites. By contrast, CTZ plus AP5-treated neurons have more drebrin clusters on dendritic protrusions than do controls. Scale bar: 5 µm. (B) Densities of total or synaptic drebrin clusters on dendritic protrusions and on dendritic shafts. GYKI treatment significantly decreases drebrin cluster density on dendritic protrusions, especially synaptic ones, without affecting that on dendritic shafts. CTZ plus AP5 treatment increases drebrin cluster density on dendritic protrusions (16 neurons examined for each treatment). *P<0.01, #P<0.02 versus control. (C) Densities of total synapsin-I clusters along the same dendrites. None of treatments change synapsin-I cluster density.

 

Figure 5
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  		Fig. 5. AMPAR activity stabilizes drebrin clusters in dendritic spines by increasing the size of the fraction of stable drebrin. (A) Time-lapse sequence during FRAP of a neuron containing eGFP–drebrin-A. The fluorescence of the drebrin cluster in the green circle is almost completely lost at the first image after bleaching (0 minutes), whereas a neighboring cluster in the red square is unaffected. The subsequent images show gradual recovery of fluorescence in the bleached cluster. Scale bar: 3 µm. (B) Fluorescence intensities were normalized in the green circle (green plots) and red square (red plots) shown in A. The green line is derived from the equation described in the Materials and Methods section. The fluorescent recovery was up to about 70% of the pre-bleach level. The intensity of the unbleached neighboring spine (red outlined square) moderately fluctuated but was not systematically altered. (C) The representative time-lapse sequences during FRAP of drebrin of the vehicle control neurons and those in the presence of CNQX (40 µM), AP5 (100 µM), AMPA (1 µM) plus AP5 (100 µM), or CTZ (100 µM) plus AP5 (100 µM). Scale bar: 1 µm. (D,E) FRAP curves of drebrin in vehicle control neurons and in those in the presence of CNQX, AMPA plus AP5, or CTZ plus AP5 (D), or AP5 (E). Fluorescence recovery is an index of drebrin turnover. (F,G) The stable fraction (F) and time constant (G) of drebrin under each condition. AMPAR blockade by CNQX decreases the size of the stable drebrin fraction. By contrast, AMPAR activation by AMPA, or CTZ plus AP5, increases the stable drebrin fraction. NMDAR blockade by AP5 shortens the time constant of dynamic drebrin without affecting the stable drebrin fraction (>29 protrusions were examined for each condition). *P<0.01 versus control.

 

Figure 6
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  		Fig. 6. An increase in the stable drebrin fraction is accompanied by increased drebrin clustering. (A) The representative time sequences during FRAP of drebrin of the vehicle control neurons and those in the presence of cytoD (10 µM). Scale bar: 1 µm. (B) FRAP curves of drebrin in neurons treated with vehicle (control) or cytoD. (C) Stable drebrin fraction under each condition (32 protrusions for each condition). (D) Double labeling of neurons treated with vehicle (control) or cytoD for 1 hour for drebrin (green) and F-actin (red). Scale bar: 10 µm. (E,F) Densities of drebrin (E) and F-actin (F) clusters in neurons with and without cytoD treatment. The short-term cytoD treatment enhances clustering of drebrin and F-actin along dendrites (ten neurons for each condition). *P<0.01 versus control.

 

Figure 7
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  		Fig. 7. Chronic AMPAR blockade induces filopodia-like thin spines. (A) Fluorescent images of mGFP-expressing cultured hippocampal neurons treated from 8 to 15 DIV with vehicle (control), GYKI (10 µM), AP5 (50 µM) or MCPG (100 µM). GYKI-treated neurons have many thin headless dendritic protrusions. Scale bar: 5 µm. (B,C) Cumulative frequency plot (B) and average (C) width of dendritic protrusions after each treatment. GYKI treatment significantly decreases the width of dendritic protrusions, whereas neither AP5 treatment nor MCPG treatment affects its width (Kolmogorov-Smirnov test and Student's t-test; >1000 protrusions and >14 neurons examined for each treatment). **P<0.00001 versus control.

 

Figure 8
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  		Fig. 8. Chronic AMPAR blockade inhibits synaptic targeting of PSD-95 during spine morphogenesis. (A) Merged PSD-95 (green), synapsin-I (red) and F-actin (blue) images in neurons treated with vehicle (control) or GYKI (10 µM) from 8 to 15 DIV. The upper panels in A represent PSD-95 immunolabeling as gray-scale images. Scale bar: 5 µm. (B,C) Quantification of synaptic PSD-95 clusters on dendritic protrusions (B), or total PSD-95 clusters on dendritic shafts (C) in control and GYKI-treated neurons. GYKI treatment significantly decreases the density of synaptic PSD-95 clusters on dendritic protrusions (14 neurons examined for each condition). *P<0.01 versus control.

 

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© The Company of Biologists Ltd 2009