Voltage-gated Ca 2ϩ channels in presynaptic terminals initiate the Ca 2ϩ inflow necessary for transmitter release. At a variety of synapses, multiple Ca 2ϩ channel subtypes are involved in synaptic transmission and plasticity. However, it is unknown whether presynaptic Ca 2ϩ channels differ in gating properties and whether they are differentially activated by action potentials or subthreshold voltage signals. We examined Ca 2ϩ channels in hippocampal mossy fiber boutons (MFBs) by presynaptic recording, using the selective blockers -agatoxin IVa, -conotoxin GVIa, and SNX-482 to separate P/Q-, N-, and R-type components. Nonstationary fluctuation analysis combined with blocker application revealed a single MFB contained on average ϳ2000 channels, with 66% P/Q-, 26% N-, and 8% R-type channels. Whereas both P/Q-type and N-type Ca 2ϩ channels showed high activation threshold and rapid activation and deactivation, R-type Ca 2ϩ channels had a lower activation threshold and slower gating kinetics. To determine the efficacy of activation of different Ca 2ϩ channel subtypes by physiologically relevant voltage waveforms, a six-state gating model reproducing the experimental observations was developed. Action potentials activated P/Q-type Ca 2ϩ channels with high efficacy, whereas N-and R-type channels were activated less efficiently. Action potential broadening selectively recruited N-and R-type channels, leading to an equalization of the efficacy of channel activation. In contrast, subthreshold presynaptic events activated R-type channels more efficiently than P/Q-or N-type channels. In conclusion, single MFBs coexpress multiple types of Ca 2ϩ channels, which are activated differentially by subthreshold and suprathreshold presynaptic voltage signals.

Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.

Ca 2؉-dependent phospholipid binding to the C 2 A and C 2 B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca 2؉ binding to the C 2 B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca The synaptic vesicle protein synaptotagmin 1 acts as a major Ca 2ϩ sensor in neurotransmitter release at excitatory and inhibitory synapses (1, 2). This function can be attributed to Ca 2ϩ binding to the two C 2 domains of synaptotagmin 1 (referred to as the C 2 A and C 2 B domain; Ref.3). The C 2 A and C 2 B domains bind three and two Ca 2ϩ ions, respectively, through loops located at the tips of similar ␤-sandwich structures (4 -7). Both C 2 domains bind to negatively charged phospholipids, including phosphoinositides, as a function of Ca 2ϩ , and exhibit comparable apparent Ca 2ϩ affinities (7-9). Furthermore, in the absence of Ca 2ϩ , the C 2 B domain, but not the C 2 A domain, of synaptotagmin 1 avidly binds to inositolpolyphosphates (such as inositol 1,3,4,5-tetrakisphosphate) and to phosphoinositides (such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 2 ) via a polybasic sequence that is located in a ␤-strand on the side of the domain (10, 11). Moreover, the C 2 domains interact Ca 2ϩ -dependently and -independently with individual SNARE proteins such as syntaxin1 and SNAP-25 and with SNARE complexes (12-17). Finally, the synaptotagmin C 2 domains engage in additional interactions in vitro, including binding of the clathrin adaptor protein complex and Ca 2ϩ channels (21-23). Although the biochemical properties of synaptotagmin 1 have been studied in detail, the functional importance of individual properties has remained unclear. Ca 2ϩ -dependent phospholipid binding by synaptotagmin 1 in vitro correlates with its functional role in Ca 2ϩ triggering of release in vivo, as demonstrated with both loss-of-function and gain-offunction mutations (1,24). This correlation suggests that Ca 2ϩ -dependent phospholipid binding represents a crucial step in synaptotagmin 1 function. However, mutational studies revealed that although both C 2 domains of synaptotagmin 1 are involved in Ca 2ϩ -triggered release, Ca 2ϩ binding to the C 2 A domain only boosts release, whereas Ca 2ϩ binding to the C 2 B domain is essential for synchronous release (1, 25-29). Thus, it is puzzling that the two C 2 domains of synaptotagmin 1 appear to exhibit similar Ca 2ϩ -dependent phospholipid binding properties in vitro but a striking functional asymmetry in vivo. The differential requirements of the C 2 A versus C 2 B domain for the Ca 2ϩ triggering of release could potentially arise from the unique ability of the C 2 B domain (but not the C 2 A domain) to bind to phosphoinositides in a Ca 2ϩ -independent manner (10, 11). Indeed, consistent with this idea, microinjection of soluble inositol polyphosphates into nerve terminals potently inhibits release (30). Two observations, however, argue against this interpretation. First, the C 2 B domai...

Efficient removal of heat via thermal interface materials has become one of the most critical challenges in the development of modern microelectronic devices. However, traditional polymer composites present limited thermal conductivity even when highly loaded with highly thermally conductive fillers due to the lack of efficient heat transfer channels. In this work, vertically aligned and interconnected graphene networks are first used as the filler, which is prepared by a controlled three-step procedure: formation of graphene oxide liquid crystals, oriented freeze casting, and high-temperature annealing reduction under Ar. The obtained composite, at an ultralow graphene loading of 0.92 vol %, exhibits a high thermal conductivity (2.13 W m–1 K–1) that is equivalent to a dramatic enhancement of 1231% compared to the pure matrix. Furthermore, the composite also presents a much reduced coefficient of thermal expansion (∼37.4 ppm K–1) and increased glass transition temperature (135.4 °C). This strategy provides an insight for the design of high-performance composites with potential to be used in advanced electronic packaging.

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease.

Recently, metal-assisted chemical etching (MaCE) has been proposed as a promising wet-etching method for the fabrication of micro- and nanostructures on silicon with low cost. However, uniform vertical trench etching with high aspect ratio is still of great challenge for traditional MaCE. Here we report an innovated MaCE method, which combined the use of a nanoporous gold thin film as the catalyst and a hydrofluoric acid (HF)-hydrogen peroxide (H2O2) mixture solution with a low HF-to-H2O2 concentration ratio (ρ) as the etchant. The reported method successfully fabricated vertical trenches on silicon with a width down to 2 μm and an aspect ratio of 16. The geometry of the trenches was highly uniform throughout the 3D space. The vertical etching direction was favored on both (100)- and (111)-oriented silicon substrates. The reported method was also capable of producing multiple trenches on the same substrate with individually-tunable lateral geometry. An etching mechanism including a through-catalyst mass-transport process and an electropolishing-favored charge-transport process was identified by a comparative study. The novel method fundamentally solves the problems of distortion and random movement of isolated catalysts in MaCE. The results mark a breakthrough in high-quality silicon trench-etching technology with a cost of more than 2 orders of magnitude lower than that of the currently available methods.

In adult neurogenesis young neurons connect to the existing network via formation of thousands of new synapses. At early developmental stages, glutamatergic synapses are sparse, immature and functionally 'silent', expressing mainly NMDA receptors. Here we show in 2- to 3-week-old young neurons of adult mice, that brief-burst activity in glutamatergic fibers is sufficient to induce postsynaptic AP firing in the absence of AMPA receptors. The enhanced excitability of the young neurons lead to efficient temporal summation of small NMDA currents, dynamic unblocking of silent synapses and NMDA-receptor-dependent AP firing. Therefore, early synaptic inputs are powerfully converted into reliable spiking output. Furthermore, due to high synaptic gain, small dendritic trees and sparse connectivity, neighboring young neurons are activated by different distinct subsets of afferent fibers with minimal overlap. Taken together, synaptic recruitment of young neurons generates sparse and orthogonal AP firing, which may support sparse coding during hippocampal information processing.

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