Size Effects in Internal Friction of Nanocrystalline Aluminum Films.

Al thin film is extensively used in micro-electromechanical systems (MEMS) and electronic interconnections; however, most previous research has concentrated on their quasi-static properties and applied their designs on larger scales. The present study designed a paddle-like cantilever specimen with metal films deposited on the upper surface to investigate the quasi-static properties of Al thin film at room temperature under high vacuum conditions at microscopic scales. Energy loss was determined using a decay technique in the oscillation amplitude of a vibrating structure following resonant excitation. Grain size and film thickness size were strictly controlled considering the quasi-static properties of the films. This study found that the internal friction of ultra-thin and thin Al films was more dependent on the grain boundaries than film thickness.

Using Digital Image Correlation on SEM Images of Strain Field after Ion Beam Milling for the Residual Stress Measurement of Thin Films.

The residual stress of thin films during the deposition process can cause the components to have unpredictable deformation and damage, which could affect the service life and reliability of the microsystems. Developing an accurate and reliable method for measuring the residual stress of thin films at the micrometer and nanometer scale is a great challenge. To analyze the residual stress regarding factors such as the mechanical anisotropy and preferred orientation of the materials, information related to the in-depth lattice strain function is required when calculating the depth profiles of the residual strain. For depth-resolved measurements of residual stress, it is strategically advantageous to develop a measurement procedure that is microstructurally independent. Here, by performing an incremental focused ion beam (FIB) ring-core drilling experiment with various depth steps, the digital image correlation (DIC) of the specimen images was obtained. The feasibility of DIC to FIB images was evaluated after the translation test, and an appropriate procedure for reliable results was established. Furthermore, the condition of the film in the function of residual stress was assessed and compared to elucidate the applicability of this technology.

Foxp2 controls synaptic wiring of corticostriatal circuits and vocal communication by opposing Mef2c.

Cortico-basal ganglia circuits are critical for speech and language and are implicated in autism spectrum disorder, in which language function can be severely affected. We demonstrate that in the mouse striatum, the gene Foxp2 negatively interacts with the synapse suppressor gene Mef2c. We present causal evidence that Mef2c inhibition by Foxp2 in neonatal mouse striatum controls synaptogenesis of corticostriatal inputs and vocalization in neonates. Mef2c suppresses corticostriatal synapse formation and striatal spinogenesis, but can itself be repressed by Foxp2 through direct DNA binding. Foxp2 deletion de-represses Mef2c, and both intrastriatal and global decrease of Mef2c rescue vocalization and striatal spinogenesis defects of Foxp2-deletion mutants. These findings suggest that Foxp2-Mef2C signaling is critical to corticostriatal circuit formation. If found in humans, such signaling defects could contribute to a range of neurologic and neuropsychiatric disorders.

P2X7 R-mediated Ca(2+) -independent d-serine release via pannexin-1 of the P2X7 R-pannexin-1 complex in astrocytes.

D-serine is a coagonist of N-methyl-d-aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity-dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d-serine from astrocytes and discovered a novel Ca(2+) -independent release mechanism mediated by P2X7 receptors (P2X7 R). Using [(3) H] d-serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X7 R agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), stimulated [(3) H]D-serine release and that were abolished by P2X7 R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca(2+) . The P2X7 R-mediated d-serine release was inhibited by pannexin-1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and (10) Panx-1 peptide, and shRNAs, and stimulation of P2X7 R induced P2X7 R-pannexin-1 complex formation. Simply incubating astrocytes in Ca(2+) /Mg(2+) -free buffer also induced the complex formation, and that enhanced basal d-serine release through pannexin-1. The P2X7 R-mediated d-serine release assayed in Ca(2+) /Mg(2+) -free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca(2+) -dependent PKC inhibitor, Gö6976, inhibited the P2X7 R-mediated d-serine release. Thus, we conclude that in astrocytes, P2X7 R-pannexin-1 complex formation is crucial for P2X7 R-mediated d-serine release through pannexin-1 hemichannel. The release is Ca(2+) -independent and regulates by a Ca(2+) -independent PKC. The activated P2X7 R per se is also functioned as a permeation channel to release d-serine in part. This P2X7 R-mediated d-serine release represents an important mechanism for activity-dependent neuron-glia interaction.

DDA3 stabilizes microtubules and suppresses neurite formation.

We have previously shown that DDA3 - also known as proline/serine-rich coiled-coil protein 1 (PSRC1) - is a microtubule-associated protein that promotes cell growth by stimulating the ß-catenin pathway. Here, we report that DDA3 can bundle and stabilize microtubules in vivo and in vitro. We found that overexpression of DDA3 increased the abundance of acetylated and tyrosinated microtubules. We employed PC12 and N2a cell lines, as well as cultured hippocampal neurons, and demonstrated that overexpression of DDA3 suppressed neurite/axon outgrowth, whereas its depletion accelerated neurite/axon formation and elongation. Knockdown of DDA3 reduced ß3-tubulin levels in N2a cells, which contributed to the spontaneous neurite formation caused by DDA3 depletion. Consistent with its role in suppressing neuritogenesis, DDA3 was downregulated during induced neuronal differentiation. Moreover, expression of DDA3 was detected in the rat brain at embryonic (E) day E15 and in the cortical region at E17, the period of active neurogenesis. Levels of cortical DDA3 decreased at the beginning of E19, when active neuritogenesis is completed. Overall our results demonstrate that DDA3 is a so-far-unknown microtubule-stabilizing protein that is involved in regulating neurite formation and elongation.

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms.

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.

Dendritic spines of developing rat cortical neurons in culture.

The formation of spines and their association with synapses were examined in developing cultured rat cortical neurons using fluorescence labeling techniques. Small protrusions were found on the processes of cultured cortical neurons after seven days in vitro (DIV), and the density of protrusions almost halved during the second week in vitro, after which it remained unchanged throughout the third week in vitro. The proportion of protrusions associated with the accumulation of the presynaptic marker, synaptophysin, increased steadily from <5% at 7 DIV to approximately 50% at 21 DIV. Based on the absence or presence of an enlargement at the end, protrusions on processes were further divided into filopodia and spines, respectively. The percentage of protrusions that were classified as spines increased steadily from approximately 5% at 3-4 DIV to approximately 80% at 18-20 DIV. The percentage of spines associated with synaptophysin accumulation increased gradually as the cortical neurons developed in vitro, reaching a plateau of approximately 40% after two weeks. However, the percentage of filopodia associated with synaptophysin accumulation never exceeded 5% during the first three weeks in vitro. Double-label staining the microfilaments and beta-tubulin or phosphorylated neurofilament H of cultured neurons further revealed many spines without any nearby axon-like processes. These findings suggest that spines are the dominant form of protrusion on the processes of more mature cortical neurons, that spines are the preferential sites where synapses reside, and that maintaining constant contact with axons is not essential for the formation of spines in cultured cortical neurons.

Cholesterol deficiency perturbs actin signaling and glutamate homeostasis in hippocampal astrocytes.

This study was undertaken to explore the possibility that cholesterol deficiency may perturb the physiological functions of astrocytes, thus rendering cells vulnerable to the cytotoxicity induced by glutamate (Glu). Cholesterol deprivation induces astrocyte stellation, which is accompanied by disruption of cortical actin, and phosphorylation of extracellular signal-regulated kinase (ERK) in an astrocyte-specific manner. Moreover, cholesterol reduction decreases the activity of glutamine synthetase (GS) while enhancing the capacity of Glu transporter. Using [(3)H]d-aspartate as a tracer, we found a marked efflux of [(3)H]d-aspartate from cholesterol-deficient astrocytes after Glu stimulation. Changes in the actin cytoskeleton, cell morphology, ERK phosphorylation and GS level gradually recovered in astrocytes after the withdrawal of cholesterol depletion. Moreover, withdrawal of cholesterol deprivation attenuated cell loss in cholesterol-deficient astrocytes during Glu exposure. Taken together, our data suggest that, upon Glu exposure, there would be an increase in intracellular Glu as a consequence of enhanced Glu uptake and reduced degradation of Glu by GS in cholesterol-deficient astrocytes. This in turn leads to a concentration gradient favoring Glu release, thereby causing the accumulation of cytotoxic levels of Glu extracellularly. It is thus concluded that the detrimental effect of cholesterol deprivation may, in part, arise from the impairment in Glu homeostasis.

Cholesterol deficiency increases the vulnerability of hippocampal glia in primary culture to glutamate-induced excitotoxicity.

Cholesterol, a molecule critical for cellular function, is found in particular high concentration in the brain and has been implicated to synaptic plasticity and neuronal regeneration. This study was undertaken to investigate the mechanism by which cholesterol shortage modulates glutamate (Glu)-induced excitotoxicity in hippocampal cell cultures. A combined treatment of lovastatin and beta-cyclodextrin reduced cellular content of cholesterol while having no significant effect on cell viability in neuron/glia mixed cultures. The experimental manipulation, nonetheless, exacerbated Glu-induced membrane damage and loss of mitochondrial activity in mixed cultures. Analysis of [3H]thymidine incorporation revealed cholesterol deficiency impaired cell proliferation in mixed cultures after Glu exposure, indicating considerable loss of glia. Indeed, it was found that cholesterol deprivation potentiated the release of lactate dehydrogenase (LDH) and the impairment in mitochondrial reduction of WST-1 reagent in astrocyte-enriched cultures subjected to Glu exposure. The detrimental effect of cholesterol shortage, nevertheless, was not observed in cultured neurons. Notably, the pretreatment of lovastatin and beta-cyclodextrin caused a decrease in the content of cellular LDH while having no effect on cell cycle profile and cellular activity of WST-1 reduction in astrocyte-enriched cultures. In contrast, removal of cholesterol had no effect on LDH content in neuron-enriched cultures. It is concluded that the differential vulnerability of cholesterol-depleted neural cells to excitotoxic damage may, in part, be ascribed to cholesterol shortage destabilizing the plasma membrane of astrocytes, thus rendering them less capable of withstanding Glu insult.

Development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos and rat pups at birth.

We studied the development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos at the 18th day of gestation (E18) and rat pups at birth (P0). Between 7 and 14 days in vitro (DIV), large increases in the amplitudes and frequencies of the spontaneous excitatory postsynaptic currents (EPSCs) of both cultured E18 and P0 neurons were observed. The EPSCs of E18 neurons were mediated primarily by alpha-amino-3-hydroxy-5-methyl-4-iso-xazole-propionic acid (AMPA) receptors at 7 DIV and by both N-methyl-D-aspartate (NMDA) and AMPA receptors at 14 DIV. Consistently, immunostaining indicated significant increases in the proportion of the clusters of NR1, an NMDA receptor subunit, which were associated with the accumulation of synaptophysin, a presynaptic marker, in cultured E18 neurons between 7 and 14 DIV. The proportion of NR1 clusters residing in synaptic regions and the proportion of synapses that colocalized with NR1 clusters in 7-day-old P0 neurons were not different statistically from those found in 7-day-old E18 neurons. However, cultured P0 neurons at 7 DIV displayed clear EPSCs mediated by NMDA receptors. Our results suggest that the targeting of NMDA receptors to synaptic regions lag behind the synaptic clustering of AMPA receptors during the in vitro development of cultured rat E18 cortical neurons. The results further suggest that the cortical neurons at P0 differ from those at E19 in certain cellular properties; as a result, the currents mediated by the synaptic NMDA receptors in 7-day-old P0 neurons are larger than those mediated by the synaptic NMDA receptors in 7-day-old E18 neurons.