Hideto Takahashi

Down‐regulation of drebrin A expression suppresses synaptic targeting of NMDA receptors in developing hippocampal neurones

Drebrin is a major F‐actin‐binding protein in the brain. We have recently demonstrated that drebrin A (neurone‐specific isoform) clusters at synapses and governs targeting of the post‐synaptic density 95 protein to synapses during development. To determine the role of drebrin A on excitatory synapse formation, we analysed whether the suppression of drebrin A expression affects filopodia‐spine morphology and synaptic targeting of NMDA receptors in cultured hippocampal neurones. Suppression of developmentally programmed up‐regulation of drebrin A by antisense treatment significantly decreased the density and width of filopodia‐spines. Immunocytochemistry showed that the antisense treatment did not attenuate synaptic clustering of NMDA receptors under conditions that permitted spontaneous activities but inhibited the accelerated targeting of NMDA receptors into synapses by its antagonist d‐(–)‐2‐amino‐5‐phosphonopentanoic acid. These results indicate that drebrin A up‐regulation plays a pivotal role in spine morphogenesis and activity‐dependent synaptic targeting of NMDA receptors.

Tomographic features of a lamellar macular hole formation and a lamellar hole that progressed to a full-thickness macular hole

PURPOSE To report optical coherence tomography of a lamellar macular hole and a lamellar macular hole that progressed to a full-thickness macular hole. METHODS Case Reports. RESULTS Case 1. In the right eye of a 66-year old man, a lenticular-shaped split was present in the inner neurosensory retina corresponding to the fovea. Three months later, an operculum formed anterior to the fovea where the retina was attenuated. Case 2. In the left eye of a 58-year old woman, a foveal cyst was seen in the inner neurosensory retina and the inner wall of the cyst was elevated, where a slightly detached posterior vitreous cortex was attached. One month later, the inner wall was operculated, leaving a thin foveal bottom. Four months later, the hole progressed to a full-thickness macular hole. CONCLUSION Lamellar macular hole appears to form when the inner wall of a split or cyst in the neurosensory retina at the fovea is avulsed by vitreous traction.

Three-dimensional observations of developing macular holes

PURPOSE To describe the morphologic features of idiopathic macular holes and vitreous traction during macular hole evolution. METHODS We prospectively examined 89 eyes of 82 patients with idiopathic macular holes (stage 1, 15 eyes; stage 2, 16 eyes; stage 3, 50 eyes; stage 4, eight eyes) using optical coherence tomography. In addition to optical coherence tomography, scanning laser ophthalmoscopy was performed in all 15 eyes with stage 1 hole, six of 16 eyes with stage 2, and 19 of 50 eyes with stage 3. RESULTS In stage 1 eyes, optical coherence tomography revealed retinal split or cystic changes at the fovea in 11 of 15 eyes (73%) and foveal retinal detachment in four eyes (27%). Two eyes with foveal cysts progressed to stage 2, and one developed a stage 3 hole. In one eye with retinal detachment, the detached retina thinned and developed dehiscence. Optical coherence tomography showed a vitreous cortex that was detached in the perifoveal area but attached on the fovea in 11 of the 15 stage 1 eyes. In stage 2 macular holes, retinal tissue extending from the perifoveal retina formed a flap. Scanning laser ophthalmoscopy demonstrated intraretinal radiating striae, which corresponded to a foveal cyst or perifoveal cystic changes. CONCLUSIONS Macular holes start as retinal splits or foveal cysts in most cases. The anterior wall of the cyst serves as a flap in stage 2 and an operculum in stage 3 holes. Radiating striae correspond to retinal splits or cysts and presumably represent an elevation of Henle fiber. In a few macular holes, foveal detachment is the initial change. The detached retina thins and eventually develops a hole. In both courses, anterior traction of the slightly detached vitreous cortex appears to be a major contributing factor to macular hole formation.

Optical coherence tomography images of spontaneous macular hole closure

PURPOSE To investigate optical coherence tomography images of spontaneous macular hole closure. METHOD Case report. In a 60-year-old woman with full-thickness macular hole, posterior vitreous detachment, and previous branch retinal vein occlusion, we observed the entire course of spontaneous macular hole closure by use of optical coherence tomography. RESULTS Spontaneous macular hole closure began as the inward protrusion of the tissue around the margin of the macular hole. The protruding tissue then connected to bridge the macular hole, which mimicked a foveal retinal detachment. The bridged tissue gradually thickened, and the foveal detachment and perifoveal cysts resolved. The fovea eventually regained its normal configuration. CONCLUSIONS The bridging of the protruding retinal tissue over the macular hole plays a key role in spontaneous macular hole closure.

Activity of the AMPA receptor regulates drebrin stabilization in dendritic spine morphogenesis

Spine morphogenesis mainly occurs during development as a morphological shift from filopodia-like thin protrusions to bulbous ones. We have previously reported that synaptic clustering of the actin-binding protein drebrin in dendritic filopodia governs spine morphogenesis and synaptic PSD-95 clustering. Here, we report the activity-dependent cellular mechanisms for spine morphogenesis, in which the activity of AMPA receptors (AMPARs) regulates drebrin clustering in spines by promoting drebrin stabilization. In cultured developing hippocampal neurons, pharmacological blockade of AMPARs, but not of other glutamate receptors, suppressed postsynaptic drebrin clustering without affecting presynaptic clustering of synapsin I (synapsin-1). Conversely, the enhancement of the action of AMPARs promoted drebrin clustering in spines. When we explored drebrin dynamics by photobleaching individual spines, we found that AMPAR activity increased the fraction of stable drebrin without affecting the time constant of drebrin turnover. An increase in the fraction of stable drebrin corresponded with increased drebrin clustering. AMPAR blockade also suppressed normal morphological maturation of spines and synaptic PSD-95 clustering in spines. Together, these data suggest that AMPAR-mediated stabilization of drebrin in spines is an activity-dependent cellular mechanism for spine morphogenesis.

Myosin II ATPase Activity Mediates the Long-Term Potentiation-Induced Exodus of Stable F-Actin Bound by Drebrin A from Dendritic Spines

The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca2+ influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

Tomographic features of early macular hole closure after vitreous surgery.

PURPOSE To report the optical coherence tomographic features of macular hole closure in the first months after vitreous surgery. METHODS We studied prospectively the tomographic features of 28 eyes (28 patients) with idiopathic macular holes before and after vitreous surgery by optical coherence tomography. We compared the best-corrected visual acuity levels with the postoperative tomographic features. RESULTS The 25 eyes with successfully sealed macular holes had one of two tomographic features within 1 month postoperatively: simple closure (normal foveal configuration) in 14 eyes (56%) or a bridge formation at the fovea that mimicked a foveal retinal detachment in 11 eyes (44%). It took an average of 2.0 months (range, 0.8 to 3.5 months) for the bridge tissue to attach to the retinal pigment epithelium. Best-corrected visual acuity quickly improved in the former group; visual improvement began 1 month after attachment of the bridge tissue in the latter group. A closed hole reopened 4 months postoperatively in one eye with a bridge formation. CONCLUSIONS Idiopathic macular holes have one of two patterns early after surgical closure, simple closure or a bridge formation. Visual improvement starts after the fovea assumes a normal configuration. The bridge formation appears to reflect an early phase and fragile condition in the anatomic closure of macular holes.

Spikar, a novel drebrin-binding protein, regulates the formation and stabilization of dendritic spines.

Dendritic spines are small, actin‐rich protrusions on dendrites, the development of which is fundamental for the formation of neural circuits. The actin cytoskeleton is central to dendritic spine morphogenesis. Drebrin is an actin‐binding protein that is thought to initiate spine formation through a unique drebrin‐actin complex at postsynaptic sites. However drebrin overexpression in neurons does not increase the final density of dendritic spines. In this study, we have identified and characterized a novel drebrin‐binding protein, spikar. Spikar is localized in cell nuclei and dendritic spines, and accumulation of spikar in dendritic spines directly correlates with spine density. A reporter gene assay demonstrated that spikar acts as a transcriptional co‐activator for nuclear receptors. We found that dendritic spine, but not nuclear, localization of spikar requires drebrin. RNA‐interference knockdown and overexpression experiments demonstrated that extranuclear spikar regulates dendritic spine density by modulating de novo spine formation and retraction of existing spines. Unlike drebrin, spikar does not affect either the morphology or function of dendritic spines. These findings indicate that drebrin‐mediated postsynaptic accumulation of spikar regulates spine density, but is not involved in regulation of spine morphology.

Molecular cloning and dendritic localization of rat SH3P7

SH3P7 was originally isolated by cloning SH3 domain ligand targets from a mouse embryo cDNA library. SH3P7 is an actin‐binding protein implicated in antigen reception, JNK1 signalling, and Rac activation. It contains a drebrin homology sequence in its N‐terminal region and a cortactin homology sequence (SH3 domain) in its C‐terminal region. Both drebrin and cortactin are actin‐binding proteins, and both have been suggested as possible regulators of the actin cytoskeleton in neurons. In the present study, we performed cDNA cloning of rat SH3P7, performed RT‐PCR analysis, generated polyclonal antibodies against the recombinant rat SH3P7 protein, and examined the distribution of SH3P7 in the rat brain using immunohistochemistry. Sequence analysis revealed that there were at least four isoforms of the SH3P7 protein: SH3P7r1–SH3P7r4. RT‐PCR analysis revealed that the predominant isoforms expressed in the brain were SH3P7r1 and SH3P7r3. The relative levels of isoform expression were similar among regions. Immunohistochemistry revealed that the most intense immunolabelling for SH3P7 was observed in the hippocampus and cerebellar cortex. Double‐labelling studies with anti‐SH3P7 antibody and other neuronal marker proteins revealed that SH3P7 was located primarily in dendrites, and in moderate amounts in cell bodies. Immunoreactivity was absent in the presynaptic terminals. In cultured astrocytes, SH3P7 was localized at protrusive structures of the cell periphery and in the cell body. We concluded that SH3P7 is ubiquitous in the rat brain, and occurs as several isoforms. Also, its dendritic localization suggests that SH3P7 is functionally linked to actin cytoskeleton organization in dendrites.

Increase in AMPA receptor-mediated miniature EPSC amplitude after chronic NMDA receptor blockade in cultured hippocampal neurons

Synaptic scaling has been reported as scaling up of AMPA receptors (AMPAR)-mediated miniature excitatory postsynaptic currents (mEPSCs) induced by blockade of action potentials or AMPAR. Here, we show a novel type of synaptic scaling induced by N-methyl-D-aspartate receptors (NMDAR) blockade. In the present study, we analyzed AMPAR-mediated mEPSCs of D-(-)-2-amino-5-phosphonopentanoic acid (AP5)-treated hippocampal neurons (16 days in vitro) for 48 h in low-density cultures, using a whole-cell patch-clamp technique. The mEPSC amplitudes recorded from chronic AP5-treated neurons (25.5+/-0.3 pA; n=30 neurons) were significantly larger than that recorded from control neurons (21.6+/-0.2 pA; n=30 neurons, p<0.05), whereas the frequency of mEPSCs was not changed. Immunocytochemistry showed that the number of synapsin I clusters of AP5-treated neurons was not different from that of control neurons. Cumulative amplitude histograms revealed that the amplitude of mEPSCs was scaled multiplicatively after AP5 treatment. GluR2-lacking AMPAR were not involved in the scaling observed here. Together, our data indicate that NMDAR activity, as well as AMPAR activity, is involved in the negative feedback plasticity of AMPAR-mediated synaptic activity.