Article Figures & Data
Figures
- Figure 1.
Arc/Arg3.1 mRNA localizes in the same dendritic lamina marked by the band of polymerized actin. A , Selective localization of Arc/Arg3.1 mRNA in the middle molecular layer of the dentate gyrus in a rat that received repeated 400 Hz trains for 90 min. B , Phalloidin staining reveals the band of polymerized actin in the middle molecular layer of the dentate gyrus from the same experiment. C , D , Arc/Arg3.1 mRNA expression ( C ) and phalloidin staining ( D ) for polymerized actin in the dentate gyrus contralateral to the HFS. The stimulated and control figures are from the same sections. E , The graph illustrates OD measurements of Arc/Arg3.1 mRNA expression across the molecular layer in the areas illustrated in A (Arc/Arg3.1mRNA HFS) and C (Arc/Arg3.1mRNA Con). F , Optical density measurements of phalloidin staining across the molecular layer in B and D . G–I , Colocalization of Arc/Arg3.1 protein and F-actin in the activated dendritic lamina. G , Fluorescence immunocytochemistry for Arc/Arg3.1 protein in an animal that received repeated HFS. H , Phalloidin staining for F-actin in the same section. I , Overlay of Arc/Arg3.1 and F-actin. Arrows indicate the band of Arc protein ( G ) and polymerized actin ( H ) in the middle molecular layer. GCL, granule cell layer. Arrows point out the middle molecular layer, which has been activated. Scale bars, 100 μm.
- Figure 2.
Actin polymerization depends on NMDA receptor and Rho kinase activation, and inhibition of actin polymerization blocks Arc/Arg3.1 mRNA localization. A and B illustrate phalloidin staining from an experiment in which a micropipette filled with MK801 was positioned in the dentate gyrus during the period of HFS. A , Phalloidin staining on the control (unstimulated) side. B , Phalloidin staining on the side that received HFS. The micropipette containing MK801 was located between the arrows. Note the band of polymerized actin in areas distant from the micropipette (arrowheads), and the absence of the band in the area surrounding the micropipette (arrows). C–J , Phalloidin staining and In situ hybridization for Arc/Arg3.1 mRNA in experiments in which micropipettes filled with the Rho kinase inhibitor were positioned in the dentate gyrus during the period of HFS. In these experiments, three bouts of 10 trains of 400 Hz were delivered to induce LTP, and then 400 Hz were delivered at a rate of 1/10 s for an additional 30 min. The animal was killed ∼50 min after the first high-frequency train. C , Phalloidin staining reveals a blockade of actin polymerization in the area surrounding the micropipette filled with the Rho kinase inhibitor (arrows). Note the prominent band of polymerized actin in areas distant from the micropipette (arrowheads). D , Phalloidin staining of a section showing a band of phalloidin staining surrounding the track of a saline filled electrode. E , Distribution of Arc/Arg3.1 mRNA as revealed by in situ hybridization in the control dentate gyrus contralateral to the side that received HFS. F–H , Arc/Arg3.1 mRNA targeting to the activated dendritic lamina is blocked by inhibition of Rho kinase. F , Pattern of labeling for Arc/Arg3.1 mRNA in the area surrounding the micropipette with the Rho kinase inhibitor. Arrowheads indicate the band of Arc/Arg3.1 mRNA in the activated dendritic lamina in areas distant from the micropipette. In the area immediately surrounding the micropipette, Arc/Arg3.1 mRNA is distributed diffusely across the dendritic lamina rather than being localized in a band in the activated segment (arrows), which indicates the blockade of Arc/Arg3.1 mRNA targeting as a result of inhibition of Rho kinase. G , High-magnification photomicrograph illustrating the localization of Arc/Arg3.1 mRNA in sections posterior to the drug application site. Note the band of Arc/Arg3.1 mRNA in the middle molecular layer of dentate gyrus (arrowheads). H , High-magnification photomicrograph illustrating Arc/Arg3.1 mRNA distribution in the area surrounding the micropipette with the Rho kinase inhibitor. Arrows indicate the areas in which Arc/Arg3.1 mRNA targeting is blocked. Graphs in I and J illustrate the optical density measurement across the molecular layer in areas surrounding the micropipette filled with the Rho kinase inhibitor (ROCK Inh) and areas distant from the micropipette (Distant site). I) Optical-density measurements of phalloidin staining for F-actin are as shown in C. J , Optical-density measurements of Arc/Arg3.1 mRNA distribution as shown in F . GCL, granule cell layer. Scale bars, 100 μm.
- Figure 3.
Inhibition of Rho kinase attenuates LTP. A , Example traces of responses during baseline recording with a saline-filled electrode, after placement of the micropipette filled with the Rho kinase inhibitor, after the delivery of 3× 10 400 Hz trains (S1, S2, and S3), and after 30 min of repeated delivery of 400 Hz trains at 1/10 s intervals. B , The graph plots the average EPSP slope expressed as a percentage of the baseline across three separate experiments. Twenty-two test responses were collected with the saline-filled electrode; and forty test responses were collected after replacement of the Rho kinase inhibitor-filled electrode. After the baseline recording, three bouts of 400 Hz stimulation were applied and 10 test responses were taken after each bout. Then, continuous HFS was delivered for 30 min followed by 90 min of test stimulation to assess the extent of LTP. The graph shows the average EPSP slope of the first 50 responses after 30 min HFS and the last 50 responses before the end of the experiment. Note the slight drop of EPSP slope during the replacement of the saline-filled electrode with the Rho kinase inhibitor-filled electrode. EPSP slope increased by an average of 16% after three bouts of HFS, and by 23% after 30 min of continuous HFS, declining to 17% by the end of the testing period. In similar experiments with saline-filled micropipettes, the average percentage increase in field EPSP slope was 63 ± 6.4% (Steward and Worley, 2001b).
- Figure 4.
Latrunculin B blocks actin polymerization and Arc/Arg3.1 mRNA induction. A and B show an experiment in which micropipettes filled with latrunculin B (Latr) were positioned in the dentate gyrus during the period of HFS. In these experiments, HFS was delivered for 30 min. A , Phalloidin staining reveals a blockade of actin polymerization in the area surrounding the micropipette filled with the latrunculin B (arrows). Note the prominent band of polymerized actin in areas distant from the micropipette (arrowheads). B , High-magnification pictures of the pattern of phalloidin staining in the site of latrunculin B application. C–E , Distribution of Arc/Arg3.1 mRNA in experiments in which micropipettes filled with latrunculin B were positioned in the dentate gyrus during the period of HFS. In these experiments, HFS was delivered for 90 min. C , Arc/Arg3.1 mRNA expression in the dentate gyrus contralateral to HFS. D , Arc/Arg3.1 mRNA expression in the area surrounding the latrunculin B-filled micropipette. Note the blockade of Arc/Arg3.1 mRNA induction in the area of latrunculin B application (arrow) and strong expression in areas distant from the site of drug application (arrowheads). E , Plot of the average OD across the molecular layer from the case shown in A and B . Pink represents the OD in the stimulated dentate gyrus, red illustrates OD values in the dentate gyrus on the control side contralateral to the stimulation, and blue represents the OD in the latrunculin B application site. Error bars indicate the SD of the five measurements at each level. Sti, Stimulation; DG, dentate gyrus; GCL, granule cell layer. Scale bar, 100 μm.
- Figure 5.
Latrunculin B blocks the targeting of Arc/Arg3.1 mRNA to active synaptic sites. A–D show an experiment in which the rat was given an ECS, followed by HFS of the perforant path on one side with a latrunculin B-filled recording micropipette positioned in the dentate gyrus. A , Phalloidin staining in the dentate gyrus on the control side contralateral to HFS. B , Phalloidin staining in the dentate gyrus on the side that received HFS of the perforant path with latrunculin B in the micropipette. Arrows point out the latrunculin B application site. Note the blockade of the band of phalloidin staining. C , Arc/Arg3.1 mRNA expression in the dentate gyrus that received ECS only. Note that Arc/Arg3.1 mRNA is distributed uniformly across the molecular layer after induction by an ECS. D , Arc/Arg3.1 mRNA induced by an ECS selectively localizes in the activated dendritic lamina after HFS except in areas of latrunculin B application. Arrows point out the latrunculin B application sites. Note the diffusive distribution of Arc/Arg3.1 mRNA in the drug sites and the absence of a band of labeling in the middle molecular layer. In areas distant from the drug site, Arc/Arg3.1 mRNA is selectively localized (arrowheads). E , F , High-magnification pictures of Arc/Arg3.1 mRNA localization in the site distant from the latrunculin B application and the site with drug application. Latr, Latrunculin B; GCL; granule cell layer; DG, dentate gyrus. Scale bars, 100 μm.
- Figure 6.
Actin polymerization alone is not sufficient for Arc/Arg3.1 mRNA targeting. A–D illustrate an experiment in which actin polymerization was induced by delivering HFS for 30 min to induce the band of polymerized actin. The rat was allowed to recover from the anesthesia, and then received an ECS 2 h after the cessation of HFS, which strongly activates Arc/Arg3.1 transcription. The rat was perfused 2 h after the ECS. A , Control pattern of phalloidin staining in the dentate gyrus contralateral to the HFS. B , Phalloidin staining in the dentate gyrus on the side of the stimulation. Note that the band of phalloidin staining is still evident 4 h after the cessation of HFS. C , Arc/Arg3.1 mRNA distribution on the side of dentate gyrus contralateral to the HFS. Arc mRNA expression is strongly induced as a result of the ECS and Arc/Arg3.1 mRNA is distributed diffusely across the molecular layer. D , Similarly, Arc/Arg3.1 mRNA induced by an ECS is also distributed diffusely across the molecular despite the presence of the band of polymerized actin. E , Optical-density measurements of phalloidin staining in the dentate gyrus on the side that received HFS (red line) and Arc/Arg3.1 mRNA distribution in the dentate gyrus on both the side that received HFS (blue line) and on the control side with ECS only. Note the peak in the middle molecular layer for F-actin and the absence of any indication of higher levels of labeling for Arc/Arg3.1 mRNA in the middle layer on the HFS side compared with the side that received ECS only. Thus, the band of polymerized actin from previous synaptic activation is not sufficient to capture Arc/Arg3.1 mRNA induced by a seizure. Scale bars, 100 μm.
- Figure 7.
Blockade of ERK phosphorylation blocks the targeting of Arc/Arg3.1 mRNA to the activated dendritic lamina. A , p-ERK immunostaining in the control dentate gyrus contralateral to the stimulated side. B , p-ERK immunostaining in the dentate gyrus that received HFS. Note the massive ERK phosphorylation in dendrites and also the band of increased immunostaining in the middle molecular layer (arrow), which is in the same location as the band of polymerized actin ( C ). D–F show the colocalization of p-ERK and Arc/Arg3.1 protein in the activated dendritic lamina. D , Fluorescence immunocytochemistry for p-ERK (red). E , Fluorescence immunocytochemistry for Arc/Arg3.1 protein. F , Overlay of Arc/Arg3.1 protein and p-ERK. G–L show an experiment in which Arc/Arg3.1 expression was induced by an ECS, and HFS was then delivered beginning 2 h after the ECS to induce localization in the activated dendritic lamina (Steward and Worley, 2001b). In this experiment, a micropipette filled with U0126 was present at the site indicated by the arrow. G and H illustrate p-ERK staining contralateral and ipsilateral to the stimulation. Note the blockade of ERK phosphorylation in the area surrounding the micropipette (arrows). I , Higher-magnification view of the area of blockade. J , Arc/Arg3.1 mRNA distribution on the side contralateral to the HFS. Arc/Arg3.1 mRNA that was induced by the ECS is distributed uniformly across the dendritic lamina. K , On the side of the HFS, Arc/Arg3.1 mRNA is selectively localized in the activated dendritic lamina except in the area in which ERK phosphorylation is blocked (arrows). L , Higher-magnification view of the area of blockade. Note that in the area of blockade, Arc/Arg3.1 mRNA is distributed uniformly across the dendritic lamina as on the contralateral side that did not receive HFS. Con, Control; GCL, granule cell layer. Scale bars, 100 μm.
- Figure 8.
ERK phosphorylation blockade by U0126 has no effect on actin polymerization, but inhibition of actin polymerization inhibition by either the Rho kinase inhibitor or latrunculin B (Latr) blocks ERK phosphorylation. A , ERK phosphorylation is blocked by local application of the MEK inhibitor U0126, as pointed out by arrows. Arrowheads point out site distant from drug application. B , Actin polymerization is not affected by the ERK phosphorylation inhibition. C , ERK phosphorylation is blocked by the Rho kinase inhibitor. D , ERK phosphorylation is blocked by latrunculin B. Arrows point out the drug application site and ERK phosphorylation blockade. Scale bars, 100 μm. The section shown in C comes from the same case illustrated in Figure 2, F and H (for the Rho kinase inhibitor).
- Figure 9.
A possible model illustrating the roles of actin polymerization and ERK activation in Arc/Arg3.1 mRNA localization. Under resting conditions (top), NMDAR, Ca2+ channels, Rho, and ERK are in an inactive state. RNA granules are being transported along microtubules. After HFS (bottom), NMDA receptor activation triggers actin polymerization in spines, which is mediated by Rho GTPase activation and causes a dramatic alteration in spine morphology. Specifically, the spine neck becomes shorter and wider; the spine head becomes larger and often concave (Fifkova and Anderson, 1981; Desmond and Levy, 1990; Bonhoeffer and Yuste, 2002). More prolonged HFS, as delivered here, accentuates these changes (Steward and Worley, 2001a). HFS activates transcription of Arc/Arg3.1 mRNA, which is then transported into dendrites along microtubules. Actin polymerization in the dendritic spines, when accompanied by ERK activation and maybe other signaling molecules, causes Arc/Arg3.1 mRNA to dissociate from the microtubule-based transport machinery in the dendritic shaft and associate with the modified actin network at the active synapse.
