Wafer-scale epitaxial modulation of quantum dot density.

Precise control of the properties of semiconductor quantum dots (QDs) is vital for creating novel devices for quantum photonics and advanced opto-electronics. Suitable low QD-densities for single QD devices and experiments are challenging to control during epitaxy and are typically found only in limited regions of the wafer. Here, we demonstrate how conventional molecular beam epitaxy (MBE) can be used to modulate the density of optically active QDs in one- and two- dimensional patterns, while still retaining excellent quality. We find that material thickness gradients during layer-by-layer growth result in surface roughness modulations across the whole wafer. Growth on such templates strongly influences the QD nucleation probability. We obtain density modulations between 1 and 10 QDs/µm2 and periods ranging from several millimeters down to at least a few hundred microns. This method is universal and expected to be applicable to a wide variety of different semiconductor material systems. We apply the method to enable growth of ultra-low noise QDs across an entire 3-inch semiconductor wafer.

Modeling non-syndromic autism and the impact of TRPC6 disruption in human neurons.

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.

Localization and quantification of 5-hydroxytryptophan and serotonin in the central nervous systems of Tritonia and Aplysia.

Serotonin (5-hydroxytryptamine, 5-HT) plays a central role in several behaviors in marine molluscs and other species. In an effort to better understand the regulation of 5-HT synthesis, we used high performance liquid chromatography (HPLC) with electrochemical detection and immunohistochemistry to measure and map the distribution of the immediate precursor of 5-HT, 5-hydroxytryptophan (5-HTP), in two model opisthobranch molluscs, the nudibranch Tritonia diomedea and the anaspid Aplysia californica. HPLC measurements showed that 5-HTP is present at approximately the same level as the 5-HT metabolite, 5-hydroxyindolacetic acid (5-HIAA) but is more than 100 times lower in concentration than either 5-HT or dopamine in the same tissue. Specific 5-HTP immunoreactivity was colocalized with serotonin in both species. The overall intensity of 5-HTP immunoreactivity in individual ganglia agreed with HPLC measurements for those ganglia. The intensity of 5-HTP immunolabeling varied between cell types and was correlated with the intensity of 5-HT immunolabeling. In particular, differences in staining intensity were consistently seen among the three dorsal swim interneurons of the Tritonia swim central pattern generator circuit. Some nonserotonergic neurons also displayed low levels of 5-HTP immunolabeling that were above background levels. Together, these results support the notion that production of 5-HTP is a rate-limiting step in serotonin synthesis and suggest that there may be additional regulation that allows 5-HTP to accumulate to varying levels.

Filopodial calcium transients promote substrate-dependent growth cone turning.

Filopodia that extend from neuronal growth cones sample the environment for extracellular guidance cues, but the signals they transmit to growth cones are unknown. Filopodia were observed generating localized transient elevations of intracellular calcium ([Ca2+]i) that propagate back to the growth cone and stimulate global Ca2+ elevations. The frequency of filopodial Ca2+ transients was substrate-dependent and may be due in part to influx of Ca2+ through channels activated by integrin receptors. These transients slowed neurite outgrowth by reducing filopodial motility and promoted turning when stimulated differentially within filopodia on one side of the growth cone. These rapid signals appear to serve both as autonomous regulators of filopodial movement and as frequency-coded signals integrated within the growth cone and could be a common signaling process for many motile cells.

Specific frequencies of spontaneous Ca2+ transients upregulate GAD 67 transcripts in embryonic spinal neurons.

Spontaneous Ca2+ transients expressed prior to synaptogenesis regulate the developmental appearance of GABA in cultured Xenopus spinal neurons. We find that glutamic acid decarboxylase (GAD) immunoreactivity is also Ca(2+)-dependent and parallels the appearance of GABA. We show that xGAD 67 transcripts first appear in the embryonic spinal cord during the period in which these Ca2+ spikes are generated, in a pattern that is temporally and spatially appropriate to account for differentiation of GABAergic interneurons. RNase protection and competitive quantitative RT-PCR demonstrate that transcript levels are approximately threefold greater when neurons are cultured in the presence of extracellular Ca2+ that permits generation of transients than when cultured in its absence. The frequency of spontaneous Ca2+ spikes plays a crucial role in the regulation of transcripts, since reimposition of Ca2+ transients at the frequency generated in cultured neurons rescues normal expression. We conclude that naturally occurring low frequencies of these Ca2+ transients regulate levels of xGAD 67 mRNA in differentiating neurons.

Coding of neuronal differentiation by calcium transients.

Excitability has long been recognized as the basis for rapid signaling in the mature nervous system, but roles of channels and receptors in controlling slower processes of differentiation have been identified only more recently. Voltage-dependent and transmitter-activated channels are often expressed at early stages of development prior to synaptogenesis, and allow influx of Ca(2+). Here we examine the functions of spontaneous transient elevations of intracellular Ca(2+) in embryonic neurons. These Ca(2+) transients abruptly raise levels of Ca(2+) as much as tenfold, for brief periods, repeatedly, and can be highly localized. Like cloudbursts on the developing landscape, Ca(2+) transients modulate growth and stimulate differentiation, in a frequency-dependent manner, probably by changes in phosphorylation or proteolysis of regulatory and structural proteins in local regions. We review the mechanisms by which Ca(2+) transients are generated and their effects in regulating motility via the cytoskeleton and differentiation via transcription.

Antisense suppression of potassium channel expression demonstrates its role in maturation of the action potential.

A developmental increase in delayed rectifier potassium current (I(Kv)) in embryonic Xenopus spinal neurons is critical for the maturation of excitability and action potential waveform. Identifying potassium channel genes that generate I(Kv) is essential to understanding the mechanisms by which they are controlled. Several Kv genes are upregulated during embryogenesis in parallel with increases in I(Kv) and produce delayed rectifier current when heterologously expressed, indicating that they could encode channels underlying this current. We used antisense (AS) cRNA to test the contribution of xKv3.1 to the maturation of I(Kv), because xKv3.1 AS appears to suppress specifically heterologous expression of potassium current by xKv3.1 mRNA. The injection of xKv3.1 AS into embryos reduces endogenous levels of xKv3.1 mRNA in the developing spinal cord and reduces the amplitude and rate of activation of I(Kv) in 40% of cultured neurons, similar to the percentage of neurons in which endogenous xKv3.1 transcripts are detected. The current in these mature neurons resembles that at an earlier stage of differentiation before the appearance of xKv3.1 mRNA. Furthermore, AS expression increases the duration of the action potential in 40% of the neurons. No change in voltage-dependent calcium current is observed, suggesting that the decrease in I(Kv) is sufficient to account for lengthening of the action potential. Computer-simulated action potentials incorporating observed reductions in amplitude and rate of activation of I(Kv) exhibit an increase in duration similar to that observed experimentally. Thus xKv3.1 contributes to the maturation of I(Kv) in a substantial percentage of these developing spinal neurons.

Sustained upregulation in embryonic spinal neurons of a Kv3.1 potassium channel gene encoding a delayed rectifier current.

Differentiation of electrical excitability entails changes in the currents that generate action potentials in spinal neurons of Xenopus embryos, resulting in reduced calcium entry during impulses generated at later stages of development. A dramatic increase in delayed rectifier current (I(Kv)) during the first day of development plays the major role in this process. Identification of potassium channel genes responsible for the increase in I(Kv) is critical to understanding the molecular mechanisms involved. Several members of the Shaw Kv3 gene subfamily encode delayed rectifier currents, indicating that they could contribute to the upregulation of I(Kv) that reduces the duration of action potentials. We isolated a Xenopus (x) Kv3.1 gene whose expression is restricted to the central nervous system, which is upregulated throughout the period during which I(Kv) develops in vivo. The fraction of neurons in which transcripts of this gene are detected by single-cell RT-PCR increases to 40% with time in culture, paralleling the development of I(Kv) in neurons in vitro. Expression of xKv3.1 mRNA generates a delayed rectifier potassium current in oocytes, suggesting that xKv3. 1 contributes to the maturation of I(Kv) and shortening of the action potential.

Regulation of calcineurin by growth cone calcium waves controls neurite extension.

Growth cones generate spontaneous transient elevations of intracellular Ca(2+) that regulate the rate of neurite outgrowth. Here we report that these Ca(2+) waves inhibit neurite extension via the Ca(2+)-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca(2+)-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca(2+) transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca(2+)-dependent neurite extension in the spinal cord. Ca(2+) wave frequency and Ca(2+)-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca(2+) waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca(2+)-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca(2+) waves act to regulate neurite extension.

Differentiation of electrical excitability in motoneurons.

Investigation of the differentiation of electrical properties of motoneurons has been stimulated by the importance of these neurons for embryonic behavior and facilitated by their experimental accessibility. In this review, we examine the development of different patterns of excitability and their functions, and discuss the emergence of repetitive firing and localization of ion channels in axons and dendrites. Finally, we summarize studies of the role of extrinsic factors in differentiation. These changes associated with differentiation of young motoneurons may presage those occurring later in the context of plasticity in the mature nervous system.

Ca(2+)-permeable AMPA receptors and spontaneous presynaptic transmitter release at developing excitatory spinal synapses.

At many mature vertebrate glutamatergic synapses, excitatory transmission strength and plasticity are regulated by AMPA and NMDA receptor (AMPA-R and NMDA-R) activation and by patterns of presynaptic transmitter release. Both receptors potentially direct neuronal differentiation by mediating postsynaptic Ca(2+) influx during early development. However, the development of synaptic receptor expression and colocalization has been examined developmentally in only a few systems, and changes in release properties at neuronal synapses have not been characterized extensively. We recorded miniature EPSCs (mEPSCs) from spinal interneurons in Xenopus embryos and larvae. In mature 5-8 d larvae, approximately 70% of mEPSCs in Mg(2+)-free saline are composed of both a fast AMPA-R-mediated component and a slower NMDA-R-mediated decay, indicating receptor colocalization at most synapses. By contrast, in 39-40 hr embryos approximately 65% of mEPSCs are exclusively fast, suggesting that these synapses initially express predominantly AMPA-R. In a physiological Mg(2+) concentration (1 mM), mEPSCs throughout development are mainly AMPA-R-mediated at negative potentials. Embryonic synaptic AMPA-R are highly Ca(2+)-permeable, mEPSC amplitude is over twofold larger than at mature synapses, and mEPSCs frequently occur in bursts consistent with asynchronous multiquantal release. AMPA-R function in this motor pathway thus appears to be independent of previous NMDA-R activation, unlike other regions of the developing nervous system, ensuring a greater reliability for embryonic excitatory transmission. Early spontaneous excitatory activity is specialized to promote AMPA-R-mediated synaptic Ca(2+) influx, which likely has significant roles in neuronal development.

Calcium signaling in the developing Xenopus myotome.

Embryonic Xenopus myocytes generate spontaneous calcium (Ca(2+)) transients during differentiation in culture. Suppression of these transients disrupts myofibril organization and the formation of sarcomeres through an identified signal transduction cascade. Since transients often occur during myocyte polarization and migration in culture, we hypothesized they might play additional roles in vivo during tissue formation. We have tested this hypothesis by examining Ca(2+) dynamics in the intact Xenopus paraxial mesoderm as it differentiates into the mature myotome. We find that Ca(2+) transients occur in cells of the developing myotome with characteristics remarkably similar to those in cultured myocytes. Transients produced within the myotome are correlated with somitogenesis as well as myocyte maturation. Since transients arise from intracellular stores in cultured myocytes, we examined the functional distribution of both IP(3) and ryanodine receptors in the intact myotome by eliciting Ca(2+) elevations in response to photorelease of caged IP(3) and superfusion of caffeine, respectively. As in culture, transients in vivo depend on Ca(2+) release from ryanodine receptor (RyR) stores, and blocking RyR during development interferes with somite maturation.

Long-term protection of mice against Leishmania major with a synthetic peptide vaccine.

A single synthetic T cell epitope (PT3), obtained from the histidine zinc-binding region of the metalloprotease gp63, was employed in a vaccine trial using two virulent strains of L. major. When a single subcutaneous injection of PT3 was delivered with the Thl stimulating adjuvant poloxamer 407, BALB/c mice were protected for at least 10 months against the disease. Vaccinated mice were largely free of lesions on termination of the experiment. Protection was similar for both L. major MRHP/SU/59 Neals and L. major WHOM/IR/-/173 strains which manifest different disease sequelae. Thus, these data provide evidence that a single subcutaneous injection of a single synthetic T cell epitope is sufficient to provide long-lasting protection against two highly virulent strains of L. major in BALB/c mice.

In vivo regulation of axon extension and pathfinding by growth-cone calcium transients.

Growth cones at the tips of extending neurites migrate through complex environments in the developing nervous system and guide axons to appropriate target regions using local cues. The intracellular calcium concentration ([Ca2+]i) of growth cones correlates with motility in vitro, but the physiological links between environmental cues and axon growth in vivo are unknown. Here we report that growth cones generate transient elevations of [Ca2+]i as they migrate within the embryonic spinal cord and that the rate of axon outgrowth is inversely proportional to the frequency of transients. Suppressing Ca2+ transients by photorelease of a Ca2+ chelator accelerates axon extension, whereas mimicking transients with photorelease of Ca2+ slows otherwise rapid axonal growth. The frequency of Ca2+ transients is cell-type specific and depends on the position of growth cones along their pathway. Furthermore, growth-cone stalling and axon retraction, which are two important aspects of pathfinding, are associated with high frequencies of Ca2+ transients. Our results indicate that environmentally regulated growth-cone Ca2+ transients control axon growth in the developing spinal cord.

Properties of ectopic neurons induced by Xenopus neurogenin1 misexpression.

We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3beta, which inhibits NeuroD function in vivo, blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.

Development of electrical excitability in embryonic neurons: mechanisms and roles.

Xenopus spinal neurons serve as a nearly ideal population of excitable cells for study of developmental regulation of electrical excitability. On the one hand, the firing properties of these neurons can be directly examined at early stages of differentiation and membrane excitability changes as neurons mature. Underlying changes in voltage-dependent ion channels have been characterized and the mechanisms that bring about these changes are being defined. On the other hand, these neurons have been shown to be spontaneously active at stages when action potentials provide significant calcium entry. Calcium entry provokes further elevation of intracellular calcium via release from intracellular stores. The resultant transient elevations of intracellular calcium encode differentiation in their frequency. Recent studies have shown that different neuronal subpopulations enlist distinct mechanisms for regulation of excitability and recruit specific programs of differentiation by particular patterns of activity.