The RNA-Binding Protein HuR Is Integral to the Function of Nociceptors in Mice and Humans.

HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators.

Global analyses of mRNA expression in human sensory neurons reveal eIF5A as a conserved target for inflammatory pain.

Nociceptors are a type of sensory neuron that are integral to most forms of pain. Targeted disruption of nociceptor sensitization affords unique opportunities to prevent pain. An emerging model for nociceptors are sensory neurons derived from human stem cells. Here, we subjected five groups to high-throughput sequencing: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. Co-culture of nociceptors and astrocytes promotes expression of transcripts enriched in DRG tissues. Comparisons of the hiPSC models to tissue samples reveal that many key transcripts linked to pain are present. Markers indicative of a range of neuronal subtypes present in the DRG were detected in mature hiPSCs. Intriguingly, translation factors were maintained at consistently high expression levels across species and culture systems. As a proof of concept for the utility of this resource, we validated expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissues and hiPSC samples. eIF5A is subject to a unique posttranslational hypusine modification required for its activity. Inhibition of hypusine biosynthesis prevented hyperalgesic priming by inflammatory mediators in vivo and diminished hiPSC activity in vitro. Collectively, our results illuminate the transcriptomes of hiPSC sensory neuron models. We provide a demonstration for this resource through our investigation of eIF5A. Our findings reveal hypusine as a potential target for inflammation associated pain in males.

Continental-scale tree-ring-based projection of Douglas-fir growth: Testing the limits of space-for-time substitution.

A central challenge in global change research is the projection of the future behavior of a system based upon past observations. Tree-ring data have been used increasingly over the last decade to project tree growth and forest ecosystem vulnerability under future climate conditions. But how can the response of tree growth to past climate variation predict the future, when the future does not look like the past? Space-for-time substitution (SFTS) is one way to overcome the problem of extrapolation: the response at a given location in a warmer future is assumed to follow the response at a warmer location today. Here we evaluated an SFTS approach to projecting future growth of Douglas-fir (Pseudotsuga menziesii), a species that occupies an exceptionally large environmental space in North America. We fit a hierarchical mixed-effects model to capture ring-width variability in response to spatial and temporal variation in climate. We found opposing gradients for productivity and climate sensitivity with highest growth rates and weakest response to interannual climate variation in the mesic coastal part of Douglas-fir's range; narrower rings and stronger climate sensitivity occurred across the semi-arid interior. Ring-width response to spatial versus temporal temperature variation was opposite in sign, suggesting that spatial variation in productivity, caused by local adaptation and other slow processes, cannot be used to anticipate changes in productivity caused by rapid climate change. We thus substituted only climate sensitivities when projecting future tree growth. Growth declines were projected across much of Douglas-fir's distribution, with largest relative decreases in the semiarid U.S. Interior West and smallest in the mesic Pacific Northwest. We further highlight the strengths of mixed-effects modeling for reviving a conceptual cornerstone of dendroecology, Cook's 1987 aggregate growth model, and the great potential to use tree-ring networks and results as a calibration target for next-generation vegetation models.

The revolution of crossdating in marine palaeoecology and palaeoclimatology.

Over the past century, the dendrochronology technique of crossdating has been widely used to generate a global network of tree-ring chronologies that serves as a leading indicator of environmental variability and change. Only recently, however, has this same approach been applied to growth increments in calcified structures of bivalves, fish and corals in the world's oceans. As in trees, these crossdated marine chronologies are well replicated, annually resolved and absolutely dated, providing uninterrupted multi-decadal to millennial histories of ocean palaeoclimatic and palaeoecological processes. Moreover, they span an extensive geographical range, multiple trophic levels, habitats and functional types, and can be readily integrated with observational physical or biological records. Increment width is the most commonly measured parameter and reflects growth or productivity, though isotopic and elemental composition capture complementary aspects of environmental variability. As such, crossdated marine chronologies constitute powerful observational templates to establish climate-biology relationships, test hypotheses of ecosystem functioning, conduct multi-proxy reconstructions, provide constraints for numerical climate models, and evaluate the precise timing and nature of ocean-atmosphere interactions. These 'present-past-future' perspectives provide new insights into the mechanisms and feedbacks between the atmosphere and marine systems while providing indicators relevant to ecosystem-based approaches of fisheries management.

Gold nanostructure microelectrode arrays for in vitro recording and stimulation from neuronal networks.

An ideal microelectrode array (MEA) design should include materials and structures which exhibit biocompatibility, low electrode polarization, low impedance/noise, and structural durability. Here, the fabrication of MEAs with indium tin oxide (ITO) electrodes deposited with self-similar gold nanostructures (GNS) is described. We show that fern leaf fractal-like GNS deposited on ITO electrodes are conducive for neural cell attachment and viability while reducing the interfacial impedance more than two orders of magnitude at low frequencies (100-1000 Hz) versus bare ITO. GNS MEAs, with low interfacial impedance, allowed the detection of extracellular action potentials with excellent signal-to-noise ratios (SNR, 20.26 ± 2.14). Additionally, the modified electrodes demonstrated electrochemical and mechanical stability over 29 d in vitro.

The MNK-eIF4E Signaling Axis Contributes to Injury-Induced Nociceptive Plasticity and the Development of Chronic Pain.

Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4ES209A ). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2-/- mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2-/- mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in the excitability of peripheral nociceptive neurons, but the precise mechanisms controlling these changes are not elucidated. Emerging evidence demonstrates that mRNA translation regulation pathways are key factors in changes in nociceptor excitability. Our work demonstrates that a single phosphorylation site on the 5' cap-binding protein eIF4E is a critical mechanism for changes in nociceptor excitability that drive the development of chronic pain. We reveal a new mechanistic target for the development of a chronic pain state and propose that targeting the upstream kinase, MAPK interacting kinase 1/2, could be used as a therapeutic approach for chronic pain.

Chronic recording and electrochemical performance of Utah microelectrode arrays implanted in rat motor cortex.

Multisite implantable electrode arrays serve as a tool to understand cortical network connectivity and plasticity. Furthermore, they enable electrical stimulation to drive plasticity, study motor/sensory mapping, or provide network input for controlling brain-computer interfaces. Neurobehavioral rodent models are prevalent in studies of motor cortex injury and recovery as well as restoration of auditory/visual cues due to their relatively low cost and ease of training. Therefore, it is important to understand the chronic performance of relevant electrode arrays in rodent models. In this report, we evaluate the chronic recording and electrochemical performance of 16-channel Utah electrode arrays, the current state-of-the-art in pre-/clinical cortical recording and stimulation, in rat motor cortex over a period of 6 mo. The single-unit active electrode yield decreased from 52.8 ± 10.0 ( week 1) to 13.4 ± 5.1% ( week 24). Similarly, the total number of single units recorded on all electrodes across all arrays decreased from 106 to 15 over the same time period. Parallel measurements of electrochemical impedance spectra and cathodic charge storage capacity exhibited significant changes in electrochemical characteristics consistent with development of electrolyte leakage pathways over time. Additionally, measurements of maximum cathodal potential excursion indicated that only a relatively small fraction of electrodes (10-35% at 1 and 24 wk postimplantation) were capable of delivering relevant currents (20 µA at 4 nC/ph) without exceeding negative or positive electrochemical potential limits. In total, our findings suggest mainly abiotic failure modes, including mechanical wire breakage as well as degradation of conducting and insulating substrates. NEW & NOTEWORTHY Multisite implantable electrode arrays serve as a tool to record cortical network activity and enable electrical stimulation to drive plasticity or provide network feedback. The use of rodent models in these fields is prevalent. We evaluated chronic recording and electrochemical performance of 16-channel Utah electrode arrays in rat motor cortex over a period of 6 mo. We primarily observed abiotic failure modes suggestive of mechanical wire breakage and/or degradation of insulation.

Adult mouse sensory neurons on microelectrode arrays exhibit increased spontaneous and stimulus-evoked activity in the presence of interleukin-6.

Following inflammation or injury, sensory neurons located in the dorsal root ganglia (DRG) may exhibit increased spontaneous and/or stimulus-evoked activity, contributing to chronic pain. Current treatment options for peripherally mediated chronic pain are highly limited, driving the development of cell- or tissue-based phenotypic (function-based) screening assays for peripheral analgesic and mechanistic lead discovery. Extant assays are often limited by throughput, content, use of tumorigenic cell lines, or tissue sources from immature developmental stages (i.e., embryonic or postnatal). Here, we describe a protocol for culturing adult mouse DRG neurons on substrate-integrated multiwell microelectrode arrays (MEAs). This approach enables multiplexed measurements of spontaneous as well as stimulus-evoked extracellular action potentials from large populations of cells. The DRG cultures exhibit stable spontaneous activity from 9 to 21 days in vitro. Activity is readily evoked by known chemical and physical agonists of sensory neuron activity such as capsaicin, bradykinin, PGE2, heat, and electrical field stimulation. Most importantly, we demonstrate that both spontaneous and stimulus-evoked activity may be potentiated by incubation with the inflammatory cytokine interleukin-6 (IL-6). Acute responsiveness to IL-6 is inhibited by treatment with a MAPK-interacting kinase 1/2 inhibitor, cercosporamide. In total, these findings suggest that adult mouse DRG neurons on multiwell MEAs are applicable to ongoing efforts to discover peripheral analgesic and their mechanisms of action. NEW & NOTEWORTHY This work describes methodologies for culturing spontaneously active adult mouse dorsal root ganglia (DRG) sensory neurons on microelectrode arrays. We characterize spontaneous and stimulus-evoked adult DRG activity over durations consistent with pharmacological interventions. Furthermore, persistent hyperexcitability could be induced by incubation with inflammatory cytokine IL-6 and attenuated with cercosporamide, an inhibitor of the IL-6 sensitization pathway. This constitutes a more physiologically relevant, moderate-throughput in vitro model for peripheral analgesic screening as well as mechanistic lead discovery.

A boundary current drives synchronous growth of marine fishes across tropical and temperate latitudes.

Entrainment of growth patterns of multiple species to single climatic drivers can lower ecosystem resilience and increase the risk of species extinction during stressful climatic events. However, predictions of the effects of climate change on the productivity and dynamics of marine fishes are hampered by a lack of historical data on growth patterns. We use otolith biochronologies to show that the strength of a boundary current, modulated by the El Niño-Southern Oscillation, accounted for almost half of the shared variance in annual growth patterns of five of six species of tropical and temperate marine fishes across 23° of latitude (3000 km) in Western Australia. Stronger flow during La Niña years drove increased growth of five species, whereas weaker flow during El Niño years reduced growth. Our work is the first to link the growth patterns of multiple fishes with a single oceanographic/climate phenomenon at large spatial scales and across multiple climate zones, habitat types, trophic levels and depth ranges. Extreme La Niña and El Niño events are predicted to occur more frequently in the future and these are likely to have implications for these vulnerable ecosystems, such as a limited capacity of the marine taxa to recover from stressful climatic events.

Rising synchrony controls western North American ecosystems.

Along the western margin of North America, the winter expression of the North Pacific High (NPH) strongly influences interannual variability in coastal upwelling, storm track position, precipitation, and river discharge. Coherence among these factors induces covariance among physical and biological processes across adjacent marine and terrestrial ecosystems. Here, we show that over the past century the degree and spatial extent of this covariance (synchrony) has substantially increased, and is coincident with rising variance in the winter NPH. Furthermore, centuries-long blue oak (Quercus douglasii) growth chronologies sensitive to the winter NPH provide robust evidence that modern levels of synchrony are among the highest observed in the context of the last 250 years. These trends may ultimately be linked to changing impacts of the El Niño Southern Oscillation on midlatitude ecosystems of North America. Such a rise in synchrony may destabilize ecosystems, expose populations to higher risks of extinction, and is thus a concern given the broad biological relevance of winter climate to biological systems.

A patterned polystyrene-based microelectrode array for in vitro neuronal recordings.

Substrate-integrated microelectrode arrays (MEAs) are non-invasive platforms for recording supra-threshold signals, i.e. action potentials or spikes, from a variety of cultured electrically active cells, and are useful for pharmacological and toxicological studies. However, the MEA substrate, which is often fabricated using semiconductor processing technology, presents some challenges to the user. Specifically, the electrode encapsulation, which may consist of a variety of inorganic and organic materials, requires a specific substrate preparation protocol to optimize cell adhesion to the surface. Often, these protocols differ from and are more complex than traditional protocols for in vitro cell culture in polystyrene petri dishes. Here, we describe the fabrication of an MEA with indium tin oxide microelectrodes and a patterned polystyrene electrode encapsulation. We demonstrate the electrochemical stability of the electrodes and encapsulation, and show viable cell culture and in vitro recordings.

The value of crossdating to retain high-frequency variability, climate signals, and extreme events in environmental proxies.

High-resolution biogenic and geologic proxies in which one increment or layer is formed per year are crucial to describing natural ranges of environmental variability in Earth's physical and biological systems. However, dating controls are necessary to ensure temporal precision and accuracy; simple counts cannot ensure that all layers are placed correctly in time. Originally developed for tree-ring data, crossdating is the only such procedure that ensures all increments have been assigned the correct calendar year of formation. Here, we use growth-increment data from two tree species, two marine bivalve species, and a marine fish species to illustrate sensitivity of environmental signals to modest dating error rates. When falsely added or missed increments are induced at one and five percent rates, errors propagate back through time and eliminate high-frequency variability, climate signals, and evidence of extreme events while incorrectly dating and distorting major disturbances or other low-frequency processes. Our consecutive Monte Carlo experiments show that inaccuracies begin to accumulate in as little as two decades and can remove all but decadal-scale processes after as little as two centuries. Real-world scenarios may have even greater consequence in the absence of crossdating. Given this sensitivity to signal loss, the fundamental tenets of crossdating must be applied to fully resolve environmental signals, a point we underscore as the frontiers of growth-increment analysis continue to expand into tropical, freshwater, and marine environments.

Long-term growth-increment chronologies reveal diverse influences of climate forcing on freshwater and forest biota in the Pacific Northwest.

Analyses of how organisms are likely to respond to a changing climate have focused largely on the direct effects of warming temperatures, though changes in other variables may also be important, particularly the amount and timing of precipitation. Here, we develop a network of eight growth-increment width chronologies for freshwater mussel species in the Pacific Northwest, United States and integrate them with tree-ring data to evaluate how terrestrial and aquatic indicators respond to hydroclimatic variability, including river discharge and precipitation. Annual discharge averaged across water years (October 1-September 30) was highly synchronous among river systems and imparted a coherent pattern among mussel chronologies. The leading principal component of the five longest mussel chronologies (1982-2003; PC1(mussel)) accounted for 47% of the dataset variability and negatively correlated with the leading principal component of river discharge (PC1(discharge); r = -0.88; P < 0.0001). PC1(mussel) and PC1(discharge) were closely linked to regional wintertime precipitation patterns across the Pacific Northwest, the season in which the vast majority of annual precipitation arrives. Mussel growth was also indirectly related to tree radial growth, though the nature of the relationships varied across the landscape. Negative correlations occurred in forests where tree growth tends to be limited by drought while positive correlations occurred in forests where tree growth tends to be limited by deep or lingering snowpack. Overall, this diverse assemblage of chronologies illustrates the importance of winter precipitation to terrestrial and freshwater ecosystems and suggests that a complexity of climate responses must be considered when estimating the biological impacts of climate variability and change.

Evidence of climate-driven ecosystem reorganization in the Gulf of Mexico.

The Gulf of Mexico is one of the most ecologically and economically valuable marine ecosystems in the world and is affected by a variety of natural and anthropogenic phenomena including climate, hurricanes, coastal development, agricultural runoff, oil spills, and fishing. These complex and interacting stressors, together with the highly dynamic nature of this ecosystem, present challenges for the effective management of its resources. We analyze a compilation of over 100 indicators representing physical, biological, and economic aspects of the Gulf of Mexico and find that an ecosystem-wide reorganization occurred in the mid-1990s. Further analysis of fishery landings composition data indicates a major shift in the late 1970s coincident with the advent of US national fisheries management policy, as well as significant shifts in the mid-1960s and the mid-1990s. These latter shifts are aligned temporally with changes in a major climate mode in the Atlantic Ocean: the Atlantic Multidecadal Oscillation (AMO). We provide an explanation for how the AMO may drive physical changes in the Gulf of Mexico, thus altering higher-level ecosystem dynamics. The hypotheses presented here should provide focus for further targeted studies, particularly in regard to whether and how management should adjust to different climate regimes or states of nature. Our study highlights the challenges in understanding the effects of climatic drivers against a background of multiple anthropogenic pressures, particularly in a system where these forces interact in complex and nonlinear ways.

A 3D In Vitro Cortical Tissue Model Based on Dense Collagen to Study the Effects of Gamma Radiation on Neuronal Function.

Studies on gamma radiation-induced injury have long been focused on hematopoietic, gastrointestinal, and cardiovascular systems, yet little is known about the effects of gamma radiation on the function of human cortical tissue. The challenge in studying radiation-induced cortical injury is, in part, due to a lack of human tissue models and physiologically relevant readouts. Here, a physiologically relevant 3D collagen-based cortical tissue model (CTM) is developed for studying the functional response of human iPSC-derived neurons and astrocytes to a sub-lethal radiation exposure (5 Gy). Cytotoxicity, DNA damage, morphology, and extracellular electrophysiology are quantified. It is reported that 5 Gy exposure significantly increases cytotoxicity, DNA damage, and astrocyte reactivity while significantly decreasing neurite length and neuronal network activity. Additionally, it is found that clinically deployed radioprotectant amifostine ameliorates the DNA damage, cytotoxicity, and astrocyte reactivity. The CTM provides a critical experimental platform to understand cell-level mechanisms by which gamma radiation (GR) affects human cortical tissue and to screen prospective radioprotectant compounds.

Phenotypic Screening of Prospective Analgesics Among FDA-Approved Compounds using an iPSC-Based Model of Acute and Chronic Inflammatory Nociception.

Classical target-based drug screening is low-throughput, largely subjective, and costly. Phenotypic screening based on in vitro models is increasingly being used to identify candidate compounds that modulate complex cell/tissue functions. Chronic inflammatory nociception, and subsequent chronic pain conditions, affect peripheral sensory neuron activity (e.g., firing of action potentials) through myriad pathways, and remain unaddressed in regard to effective, non-addictive management/treatment options. Here, a chronic inflammatory nociception model is demonstrated based on induced pluripotent stem cell (iPSC) sensory neurons and glia, co-cultured on microelectrode arrays (MEAs). iPSC sensory co-cultures exhibit coordinated spontaneous extracellular action potential (EAP) firing, reaching a stable baseline after ≈27 days in vitro (DIV). Spontaneous and evoked EAP metrics are significantly modulated by 24-h incubation with tumor necrosis factor-alpha (TNF-α), representing an inflammatory phenotype. Compared with positive controls (lidocaine), this model is identified as an "excellent" stand-alone assay based on a modified Z' assay quality metric. This model is then used to screen 15 cherry-picked, off-label, Food and Drug Administration (FDA)-approved compounds; 10 of 15 are identified as "hits". Both hits and "misses" are discussed in turn. In total, this data suggests that iPSC sensory co-cultures on MEAs may represent a moderate-to-high-throughput assay for drug discovery targeting inflammatory nociception.

Biology and pathophysiology of symptomatic neuromas.

ABSTRACT: Neuromas are a substantial cause of morbidity and reduction in quality of life. This is not only caused by a disruption in motor and sensory function from the underlying nerve injury but also by the debilitating effects of neuropathic pain resulting from symptomatic neuromas. A wide range of surgical and therapeutic modalities have been introduced to mitigate this pain. Nevertheless, no single treatment option has been successful in completely resolving the associated constellation of symptoms. While certain novel surgical techniques have shown promising results in reducing neuroma-derived and phantom limb pain, their effectiveness and the exact mechanism behind their pain-relieving capacities have not yet been defined. Furthermore, surgery has inherent risks, may not be suitable for many patients, and may yet still fail to relieve pain. Therefore, there remains a great clinical need for additional therapeutic modalities to further improve treatment for patients with devastating injuries that lead to symptomatic neuromas. However, the molecular mechanisms and genetic contributions behind the regulatory programs that drive neuroma formation-as well as the resulting neuropathic pain-remain incompletely understood. Here, we review the histopathological features of symptomatic neuromas, our current understanding of the mechanisms that favor neuroma formation, and the putative contributory signals and regulatory programs that facilitate somatic pain, including neurotrophic factors, neuroinflammatory peptides, cytokines, along with transient receptor potential, and ionotropic channels that suggest possible approaches and innovations to identify novel clinical therapeutics.

Otolith oxygen isotopes measured by high-precision secondary ion mass spectrometry reflect life history of a yellowfin sole (Limanda aspera).

RATIONALE: The oxygen isotope ratio (δ(18)O value) of aragonite fish otoliths is dependent on the temperature and the δ(18)O value of the ambient water and can thus reflect the environmental history of a fish. Secondary ion mass spectrometry (SIMS) offers a spatial-resolution advantage over conventional acid-digestion techniques for stable isotope analysis of otoliths, especially given their compact nature. METHODS: High-precision otolith δ(18)O analysis was conducted with an IMS-1280 ion microprobe to investigate the life history of a yellowfin sole (Limanda aspera), a Bering Sea species known to migrate ontogenetically. The otolith was cut transversely through its core and one half was roasted to eliminate organic contaminants. Values of δ(18)O were measured in 10-µm spots along three transects (two in the roasted half, one in the unroasted half) from the core toward the edge. Otolith annual growth zones were dated using the dendrochronology technique of crossdating. RESULTS: Measured values of δ(18)O ranged from 29.0 to 34.1‰ (relative to Vienna Standard Mean Ocean Water). Ontogenetic migration from shallow to deeper waters was reflected in generally increasing δ(18)O values from age-0 to approximately age-7 and subsequent stabilization after the expected onset of maturity at age-7. Cyclical variations of δ(18)O values within juvenile otolith growth zones, up to 3.9‰ in magnitude, were caused by a combination of seasonal changes in the temperature and the δ(18)O value of the ambient water. CONCLUSIONS: The ion microprobe produced a high-precision and high-resolution record of the relative environmental conditions experienced by a yellowfin sole that was consistent with population-level studies of ontogeny. Furthermore, this study represents the first time that crossdating has been used to ensure the dating accuracy of δ(18)O measurements in otoliths.

Electrically-evoked oscillating calcium transients in mono- and co-cultures of iPSC glia and sensory neurons.

Activated glia are known to exhibit either neuroprotective or neurodegenerative effects, depending on their phenotype, while participating in chronic pain regulation. Until recently, it has been believed that satellite glial cells and astrocytes are electrically slight and process stimuli only through intracellular calcium flux that triggers downstream signaling mechanisms. Though glia do not exhibit action potentials, they do express both voltage- and ligand-gated ion channels that facilitate measurable calcium transients, a measure of their own phenotypic excitability, and support and modulate sensory neuron excitability through ion buffering and secretion of excitatory or inhibitory neuropeptides (i.e., paracrine signaling). We recently developed a model of acute and chronic nociception using co-cultures of iPSC sensory neurons (SN) and spinal astrocytes on microelectrode arrays (MEAs). Until recently, only neuronal extracellular activity has been recorded using MEAs with a high signal-to-noise ratio and in a non-invasive manner. Unfortunately, this method has limited compatibility with simultaneous calcium transient imaging techniques, which is the most common method for monitoring the phenotypic activity of astrocytes. Moreover, both dye-based and genetically encoded calcium indicator imaging rely on calcium chelation, affecting the culture's long-term physiology. Therefore, it would be ideal to allow continuous and simultaneous direct phenotypic monitoring of both SNs and astrocytes in a high-to-moderate throughput non-invasive manner and would significantly advance the field of electrophysiology. Here, we characterize astrocytic oscillating calcium transients (OCa2+Ts) in mono- and co-cultures of iPSC astrocytes as well as iPSC SN-astrocyte co-cultures on 48 well plate MEAs. We demonstrate that astrocytes exhibit OCa2+Ts in an electrical stimulus amplitude- and duration-dependent manner. We show that OCa2+Ts can be pharmacologically inhibited with the gap junction antagonist, carbenoxolone (100 µM). Most importantly, we demonstrate that both neurons and glia can be phenotypically characterized in real time, repeatedly, over the duration of the culture. In total, our findings suggest that calcium transients in glial populations may serve as a stand-alone or supplemental screening technique for identifying potential analgesics or compounds targeting other glia-mediated pathologies.

Long-term tropical cyclones activity shapes forest structure and reduces tree species diversity of U.S. temperate forests.

Increasing tropical cyclone (TC) pressure on temperate forests is inevitable under the recent global increase of the intensity and poleward migration of TCs. However, the long-term effects of TCs on large-scale structure and diversity of temperate forests remain unclear. Here, we aim to ascertain the legacy of TCs on forest structure and tree species richness by using structural equation models that consider several environmental gradients and use an extensive dataset containing >140,000 plots with >3 million trees from natural temperate forests across eastern United States impacted by TCs. We found that high TC activity (a combination of TC frequency and intensity) leads to a decrease in maximum tree sizes (height and diameter), an increase in tree density and basal area, and a decline in the number of tree species and recruits. We identified TC activity as the strongest predictor of forest structure and species richness in xeric (dry) forests, while it had a weaker impact on hydric (wet) forests. We highlight the sensitivity of forest structure and tree species richness to impacts of likely further increase of TC activity in interaction with climate extremes, especially drought. Our results show that increased TC activity leads to the homogenization of forest structure and reduced tree species richness in U.S. temperate forests. These findings suggest that further declines in tree species richness may be expected because of the projected increase of future levels of TC activity.