The mammalian target of rapamycin (mTOR) signaling pathway is a powerful regulator of cell proliferation, growth, synapse maintenance and cell fate. While intensely studied for its role in cancer, the role of mTOR signaling is just beginning to be uncovered in specific cell types that are implicated in neurodevelopmental disorders. Previously, loss of the Tsc1 gene, which results in hyperactive mTOR, was shown to affect the function and molecular properties of GABAergic cortical interneurons (CINs) derived from the medial ganglionic eminence. To assess if other important classes of CINs could be impacted by mTOR dysfunction, we deleted Tsc1 in a caudal ganglionic eminence-derived interneuron group, the vasoactive intestinal peptide (VIP)+ subtype, whose activity disinhibits local circuits. Tsc1 mutant VIP+ CINs reduced their pattern of apoptosis from postnatal days 15-20, resulting in increased VIP+ CINs. The mutant CINs exhibited synaptic and electrophysiological properties that could contribute to the high rate of seizure activity in humans that harbor Tsc1 mutations.

Ecosystem level ozone (O₃) fluxes during four different years were examined at a subalpine forest site in the Colorado Rocky Mountains. The local mountain-valley wind system and the proximity of the Denver Metropolitan area leads to high summertime ozone episodes on many afternoons. The timing between these episodes and the ecosystem processes controlling photosynthesis during the growing season plays a critical role in determining the amount of ozone deposition. Light and vapor pressure deficit (VPD) were the most dominant environmental drivers controlling the deposition of O₃ at this site through their influence on stomatal conductance. 81% of the daytime O₃ uptake was predicted to occur through the stomata. Stomatal uptake decreased at high VPD and temperatures leading to an overall decrease in O₃ flux; however, we did observe a non-stomatal conductance for O₃ that increased slightly with temperature before leveling off at higher values. During the growing season, O₃ deposition fluxes were enhanced after midday precipitation events and continued at elevated levels throughout the following night, implying a role for surface wetness. From nighttime data, evidence for both the presence of water films on the needles and non-closure of the plant stomata were observed. During the winter (non-growing) season, the ozone deposition velocity showed a consistent dependency on the latent heat flux. Although the mechanism is unclear, it is apparent that precipitation events play a role here through their influence on latent heat flux.

Background

Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories.

Results

We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient.

Conclusion

This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols.

Herein, a novel method, magnetic effervescent tablet-assisted ionic liquid-based dispersive liquid-liquid microextraction (META-IL-DLLME), was pioneered for extraction and preconcentration of polybrominated diphenyl ethers (PBDEs) in liquid matrix samples. In this proposed method, a magnetic effervescent tablet, containing CO₂ sources, ionic liquids and Fe₃S₄ magnetic nanoparticles (MNPs), combines extractant dispersion and magnetic recovery into one-step. Fe₃S₄ was synthesized, characterized and applied it for the first time to the newly developed method, and its extraction recoveries (ERs) for PBDEs were 20.8-32.0% higher than those of conventional Fe₃O₄ MNPs. The increased ERs of Fe₃S₄ resulted from its larger specific surface area and pore size. Some important parameters were rigorously optimized, such as kinds of magnetic nanoparticles, effervescent agents, extraction solvents and their volumes, elution solvents, extraction temperature and salt addition. Under the optimized conditions, the META-IL-DLLME method combined with HPLC-DAD analysis gave the linear ranges of 0.1-0.5-100 µg L^-1 with correlation coefficients of > 0.9990. The ERs ranged from 80.7% to 99.3%, and the limits of detection and quantitation were 0.012-0.078 µg L^-1 and 0.04-0.26 µg L^-1, respectively. The intra- and inter-day precisions, expressed as relative standard deviations (RSD, n = 6), were 1.32-4.83% and 1.99-4.25%, respectively. To evaluate its matrix effect, the relative recoveries of PBDEs from tap and river water, skim and whole milk, pregnant women and women serum samples at three fortification levels (2.0, 5.0 and 20.0 µg L^-1) were in the range of 77.3-106.7%. Overall, the commercial Fe₃O₄ MNPs can only be used for magnetic separation in microextraction procedures, while Fe₃S₄ MNPs gave the higher adsorption and extraction efficiency for organic analytes besides the convenient magnetic separation. Therefore, the results obtained in this study provide a superior alternative for the conventional magnetic separation and adsorbent material. Also, this newly developed method has a great potential in routine monitoring of liquid matrix samples.

Growing season length (GSL) is a key unifying concept in ecology that can be estimated from eddy covariance-derived estimates of net ecosystem production (NEP). Previous studies disagree on how increasing GSLs may affect NEP in evergreen coniferous forests, potentially due to the variety of methods used to quantify GSL from NEP. We calculated GSL and GSL-NEP regressions at eleven evergreen conifer sites across a broad climatic gradient in western North America using three common approaches: (1) variable length (3-7 days) regressions of day of year versus NEP, (2) a smoothed threshold approach, and (3) the carbon uptake period, followed by a new approach of a method-averaged ensemble. The GSL and the GSL-NEP relationship differed among methods, resulting in linear relationships with variable sign, slope, and statistical significance. For all combinations of sites and methods, the GSL explained between 6% and 82% of NEP with p-values ranging from 0.45 to < 0.01. These results demonstrate the variability among GSL methods and the importance of selecting an appropriate method to accurately project the ecosystem carbon cycling response to longer growing seasons in the future. To encourage this approach in future studies, we outline a series of best practices for GSL method selection depending on research goals and the annual NEP dynamics of the study site(s). These results contribute to understanding growing season dynamics at ecosystem and continental scales and underscore the potential for methodological variability to influence forecasts of the evergreen conifer forest response to climate variability.

Immediate early and constitutively expressed products of the Homer1 gene regulate the functional assembly of post-synaptic density proteins at glutamatergic synapses to influence excitatory neurotransmission and synaptic plasticity. Earlier studies of Homer1 gene knock-out (KO) mice indicated active, but distinct, roles for IEG and constitutively expressed Homer1 gene products in regulating cognitive, emotional, motivational and sensorimotor processing, as well as behavioral and neurochemical sensitivity to cocaine. More recent characterization of transgenic mice engineered to prevent generation of the IEG form (a.k.a Homer1a KO) pose a critical role for Homer1a in cocaine-induced behavioral and neurochemical sensitization of relevance to drug addiction and related neuropsychiatric disorders. Here, we extend our characterization of the Homer1a KO mouse and report a modest pro-depressant phenotype, but no deleterious effects of the KO upon spatial learning/memory, prepulse inhibition, or cocaine-induced place-conditioning. As we reported previously, Homer1a KO mice did not develop cocaine-induced behavioral or neurochemical sensitization within the nucleus accumbens; however, virus-mediated Homer1a over-expression within the nucleus accumbens reversed the sensitization phenotype of KO mice. We also report several neurochemical abnormalities within the nucleus accumbens of Homer1a KO mice that include: elevated basal dopamine and reduced basal glutamate content, Group1 mGluR agonist-induced glutamate release and high K+-stimulated release of dopamine and glutamate within this region. Many of the neurochemical anomalies exhibited by Homer1a KO mice are recapitulated upon deletion of the entire Homer1 gene; however, Homer1 deletion did not affect NAC dopamine or alter K+-stimulated neurotransmitter release within this region. These data show that the selective deletion of Homer1a produces a behavioral and neurochemical phenotype that is distinguishable from that produced by deletion of the entire Homer1 gene. Moreover, the data indicate a specific role for Homer1a in regulating cocaine-induced behavioral and neurochemical sensitization of potential relevance to the psychotogenic properties of this drug.

The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

Borders are important as they demarcate developing tissue into distinct functional units. A key challenge is the discovery of mechanisms that can convert morphogen gradients into tissue borders. While mechanisms that produce ultrasensitive cellular responses provide a solution, how extracellular morphogens drive such mechanisms remains poorly understood. Here, we show how Bone Morphogenetic Protein (BMP) and Fibroblast Growth Factor (FGF) pathways interact to generate ultrasensitivity and borders in the dorsal telencephalon. BMP and FGF signaling manipulations in explants produced border defects suggestive of cross inhibition within single cells, which was confirmed in dissociated cultures. Using mathematical modeling, we designed experiments that ruled out alternative cross inhibition mechanisms and identified a cross-inhibitory positive feedback (CIPF) mechanism, or "toggle switch", which acts upstream of transcriptional targets in dorsal telencephalic cells. CIPF explained several cellular phenomena important for border formation such as threshold tuning, ultrasensitivity, and hysteresis. CIPF explicitly links graded morphogen signaling in the telencephalon to switch-like cellular responses and has the ability to form multiple borders and scale pattern to size. These benefits may apply to other developmental systems.

Background

Protein kinase C epsilon (PKCε) is emerging as a potential target for the development of pharmacotherapies to treat alcohol use disorders, yet little is known regarding how a history of a highly prevalent form of drinking, binge alcohol intake, influences enzyme priming or the functional relevance of kinase activity for excessive alcohol intake.

Methods

Immunoblotting was employed on tissue from subregions of the nucleus accumbens (NAc) and the amygdala to examine both idiopathic and binge drinking-induced changes in constitutive PKCε priming. The functional relevance of PKCε translocation for binge drinking and determination of potential upstream signaling pathways involved were investigated using neuropharmacologic approaches within the context of two distinct binge drinking procedures, drinking in the dark and scheduled high alcohol consumption.

Results

Binge alcohol drinking elevated p(Ser729)-PKCε levels in both the NAc and the central nucleus of the amygdala (CeA). Moreover, immunoblotting studies of selectively bred and transgenic mouse lines revealed a positive correlation between the propensity to binge drink alcohol and constitutive p(Ser729)-PKCε levels in the NAc and CeA. Finally, neuropharmacologic inhibition of PKCε translocation within both regions reduced binge alcohol consumption in a manner requiring intact group 1 metabotropic glutamate receptors, Homer2, phospholipase C, and/or phosphotidylinositide-3 kinase function.

Conclusions

Taken together, these data indicate that PKCε signaling in both the NAc and CeA is a major contributor to binge alcohol drinking and to the genetic propensity to consume excessive amounts of alcohol.