Predicting sport fish mercury contamination in heavily managed reservoirs: Implications for human and ecological health.

Mercury (Hg) is a concerning contaminant due to its widespread distribution and tendency to accumulate to harmful concentrations in biota. We used a machine learning approach called random forest (RF) to test for different predictors of Hg concentrations in three species of Colorado reservoir sport fish. The RF approach indicated that the best predictors of 864 mm northern pike (Esox lucius) Hg concentrations were covariates related to salmonid stocking in each study system, while system-specific metrics related to productivity and forage base were the best predictors of Hg concentrations of 381 mm smallmouth bass (Micropterus dolomieu), and walleye (Sander vitreus). Protecting human and ecological health from Hg contamination requires an understanding of fish Hg concentrations and variability across the landscape and through time. The RF approach could be applied to identify potential areas/systems of concern, and predict whether sport fish Hg concentrations may change as a result of a variety of factors to help prioritize, focus, and streamline monitoring efforts to effectively and efficiently inform human and ecological health.

Multi-amplicon microbiome data analysis pipelines for mixed orientation sequences using QIIME2: Assessing reference database, variable region and pre-processing bias in classification of mock bacterial community samples.

Microbiome research relies on next-generation sequencing and on downstream data analysis workflows. Several manufacturers have introduced multi-amplicon kits for microbiome characterization, improving speciation, but present unique challenges for analysis. The goal of this methodology study was to develop two analysis pipelines specific to mixed-orientation reads from multi-hypervariable (V) region amplicons. A secondary aim was to assess agreement with expected abundance, considering database and variable region. Mock community sequence data (n = 41) generated using the Ion16S™ Metagenomics Kit and Ion Torrent Sequencing Platform were analyzed using two workflows. Amplicons from V2, V3, V4, V6-7, V8 and V9 were deconvoluted using a specialized plugin based on CutPrimers. A separate workflow using Cutadapt is also presented. Three reference databases (Ribosomal Database Project, Greengenes and Silva) were used for taxonomic assignment. Bray-Curtis, Euclidean and Jensen-Shannon distance measures were used to evaluate overall annotation consistency, and specific taxon agreement was determined by calculating the ratio of observed to expected relative abundance. Reads that mapped to regions V2-V9 varied for both CutPrimers and Cutadapt-based methods. Within the CutPrimers-based pipeline, V3 amplicons had the best agreement with the expected distribution, tested using global distance measures, while V9 amplicons had the worst agreement. Accurate taxonomic annotation varied by genus-level taxon and V region analyzed. For the first time, we present a microbiome analysis pipeline that employs a specialized plugin to allow microbiome researchers to separate multi-amplicon data from the Ion16S Metagenomics Kit into V-specific reads. We also present an additional analysis workflow, modified for Ion Torrent mixed orientation reads. Overall, the global agreement of amplicons with the expected mock community abundances differed across V regions and reference databases. Benchmarking data should be referenced when planning a microbiome study to consider these biases related to sequencing and data analysis for multi-amplicon sequencing kits.

Differences in Circulating Extracellular Vesicle and Soluble Cytokines in Older Versus Younger Breast Cancer Patients With Distinct Symptom Profiles.

Because extracellular vesicle (EV)-associated cytokines, both encapsulated and surface bound, have been associated with symptom severity, and may vary over the lifespan, they may be potential biomarkers to uncover underlying mechanisms of various conditions. This study evaluated the associations of soluble and EV-associated cytokine concentrations with distinct symptom profiles reported by 290 women with breast cancer prior to surgery. Patients were classified into older (≥60 years, n = 93) and younger (< 60 years, n = 197) cohorts within two previously identified distinct symptom severity profiles, that included pain, depressive symptoms, sleep disturbance, and fatigue (i.e., High Fatigue Low Pain and All Low). EVs were extracted using ExoQuick. Cytokine concentrations were determined using Luminex multiplex assay. Mann Whitney U test evaluated the differences in EV and soluble cytokine levels between symptom classes and between and within the older and younger cohorts adjusting for Karnofsky Performance Status (KPS) score, body mass index (BMI), and stage of disease. Partial correlation analyses were run between symptom severity scores and cytokine concentrations. Results of this study suggest that levels of cytokine concentrations differ between EV and soluble fractions. Several EV and soluble pro-inflammatory cytokines had positive associations with depressive symptoms and fatigue within both age cohorts and symptom profiles. In addition, in the older cohort with High Fatigue Low Pain symptom profile, EV GM-CSF concentrations were higher compared to the All Low symptom profile (p < 0.05). Albeit limited by a small sample size, these exploratory analyses provide new information on the association between cytokines and symptom profiles of older and younger cohorts. Of note, unique EV-associated cytokines were found in older patients and in specific symptom classes. These results suggest that EVs may be potential biomarker discovery tools. Understanding the mechanisms that underlie distinct symptom class profiles categorized by age may inform intervention trials and offer precision medicine approaches.

BDNF Val66Met Polymorphism Reduces the Fatigue-Like Effects of 5-Fluorouracil on Voluntary Wheel-Running Activity in Mice.

Fatigue is a persistent and debilitating symptom following cancer treatments such as chemotherapy. Recent clinical studies have suggested a common single-nucleotide polymorphism of brain-derived neurotrophic factor (BDNF), Val66Met (rs6265), may be related to the severity of fatigue following cancer treatment. In this study, we tested transgenic mice homozygous for the human Val66Met BDNF gene and wild-type controls. We injected three doses of 5-fluorouracil (5FU) as a model of chemotherapy treatment, and we used changes in voluntary wheel running activity (VWRA) as a measure of fatigue-like behavior. Prior to 5FU injection, we found that during the baseline wheel-running period, the Val66Met mice lost more weight than WT controls. We next administered 5FU and saw a robust fatigue-like phenotype that lasted about 2 weeks. During the first week, the fatigue-like phenotype was less severe in the Val66Met mice and unrelated to the age of the mice. In contrast, during the second week after 5FU treatment, the fatigue-like phenotype was unrelated to the BDNF genotype but was more severe in middle aged mice compared to young mice. We conclude that the BDNF polymorphism may play a direct, protective role against chemotherapy-induced fatigue.

Habitat restoration for brown trout (Salmo trutta) has limited effects on macroinvertebrate communities in a historically metal-contaminated stream.

Quantifying the success of stream remediation or restoration projects that are designed to improve water quality or habitat, respectively, is often challenging because of insufficient posttreatment monitoring, poorly defined restoration goals, and failure to consider fundamental aspects of ecological theory. We measured the effects of habitat restoration on aquatic and terrestrial prey resources in a system recovering from the long-term effects of mining pollution. The study was conducted in the Upper Arkansas River, a Rocky Mountain stream located in central Colorado, USA. Remediation of California Gulch, a United States Environmental Protection Agency (USEPA) Superfund Site that discharged metals from past mining operations into the stream, was completed in 2000, resulting in significant improvements in water quality, benthic macroinvertebrate communities, and brown trout (Salmo trutta) populations. A large-scale restoration project designed to improve habitat and increase the density and biomass of brown trout was completed in 2014. To assess the effectiveness of these habitat improvements on invertebrate communities in this system, we sampled sites for 9 years before (2010-2014) and after (2015-2018) restoration was completed. In contrast to our expectations, we observed few changes in the abundance of aquatic or terrestrial invertebrates after restoration. The most common response was an overall reduction in abundance resulting from significant instream disturbances during and immediately after restoration, followed by a gradual return to pretreatment conditions. Despite reductions in prey abundance, the number of prey items in the diet of brown trout increased significantly after restoration. We discuss several explanations for these responses, including the effects of residual metals, increased predation by brown trout, and the recalcitrance of novel communities dominated by metal-tolerant species. Our results suggest that the effectiveness of remediation and restoration differed between macroinvertebrates and fish. Benthic macroinvertebrates were more dependent on water quality improvements at the watershed scale, whereas brown trout populations responded to both improvements in water quality and reach-scale improvements in habitat. Integr Environ Assess Manag 2022;18:1047-1055. © 2021 SETAC.

Inflammation plays a causal role in fatigue-like behavior induced by pelvic irradiation in mice.

Fatigue is a persistent and debilitating symptom following radiation therapy for prostate cancer. However, it is not well-understood how radiation targeted to a small region of the body can lead to broad changes in behavior. In this study, we used targeted pelvic irradiation of healthy male mice to test whether inflammatory signaling mediates changes in voluntary physical activity levels. First, we tested the relationship between radiation dose, blood cell counts, and fatigue-like behavior measured as voluntary wheel-running activity. Next, we used oral minocycline treatments to reduce inflammation and found that minocycline reduces, but does not eliminate, the fatigue-like behavioral changes induced by radiation. We also used a strain of mice lacking the MyD88 adaptor protein and found that these mice also showed less fatigue-like behavior than the wild-type controls. Finally, using serum and brain tissue samples, we determined changes in inflammatory signaling induced by irradiation in wild-type, minocycline treated, and MyD88 knockout mice. We found that irradiation increased serum levels of IL-6, a change that was partially reversed in mice treated with minocycline or lacking MyD88. Overall, our results suggest that inflammation plays a causal role in radiation-induced fatigue and that IL-6 may be an important mediator.

Metals Alter Membership but Not Diversity of a Headwater Stream Microbiome.

Metal contamination from mining or natural weathering is a common feature of surface waters in the American west. Advances in microbial analyses have created the potential for routine sampling of aquatic microbiomes as a tool to assess the quality of stream habitat. We sought to determine if microbiome diversity and membership were affected by metal contamination and identify candidate microbial taxa to be used to indicate metal stress in stream ecosystems. We evaluated microbiome membership from sediments at multiple sites within the principal drainage of an EPA superfund site near the headwaters of the Upper Arkansas River, Leadville, CO. From each sample, we extracted DNA and sequenced the 16S rRNA gene amplicon on the Illumina MiSeq platform. We used the remaining sediments to simultaneously evaluate environmental metal concentrations. We also conducted an artificial stream mesocosm experiment using sediments collected from two of the observational study sites. The mesocosm experiment had a two-by-two factorial design: (i) location (upstream or downstream of contaminating tributary), and (ii) treatment (metal exposure or control). We found no difference in diversity between upstream and downstream sites in the field. Similarly, diversity changed very little following experimental metal exposure. However, microbiome membership differed between upstream and downstream locations and experimental metal exposure changed microbiome membership in a manner that depended on origin of the sediments used in each mesocosm.IMPORTANCE Our results suggest that microbiomes can be reliable indicators of ecosystem metal stress even when surface water chemistry and other metrics used to assess ecosystem health do not indicate ecosystem stress. Results presented in this study, in combination with previously published work on this same ecosystem, are consistent with the idea that a microbial response to metals at the base of the food web may be affecting primary consumers. If effects of metals are mediated through shifts in the microbiome, then microbial metrics, as presented here, may aid in the assessment of stream ecosystem health, which currently does not include assessments of the microbiome.

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

Fatigue and cognitive deficits are often co-occurring symptoms reported by patients after radiation therapy for prostate cancer. In this study, we induced fatigue-like behavior in mice using targeted pelvic irradiation to mimic the clinical treatment regimen and assess cognitive behavioral changes. We observed that pelvic irradiation produced a robust fatigue phenotype, a reduced rate of spontaneous alternation in a Y-maze test, and no behavioral change in an open field test. We found that reversal learning for fatigued mice was slower with respect to time, but not with respect to effort put into the test, suggesting that fatigue may impact the ability or motivation to work at a cognitive task without impairing cognitive capabilities. In addition, we found that mice undergoing pelvic irradiation show lower whole-brain levels of mature BDNF, and that whole-brain proBDNF levels also correlate with spontaneous alternation in a Y-maze test. These results suggest that changes in BDNF levels could be both a cause and an effect of fatigue-related changes in behavior.

[Corrigendum] Cancer­related fatigue during combined treatment of androgen deprivation therapy and radiotherapy is associated with mitochondrial dysfunction.

Following the publication of the article, the authors realized that they have overlooked acknowledging the assistance they received from the Murine Phenotyping Core at NHLBI. Therefore, the Acknowledgements section of the Declarations should also have stated the following: 'We would like to thank the Murine Phenotyping Core at NHLBI for all their help with the mouse experiments, including Dr Danielle Springer, Audrey Noguchi, Michele Allen, Heather Potts and Morteza Peiravi.' The authors regret their oversight in failing to include this information in the Acknowledgements section of their paper. [the original article was published in International Journal of Molecular Medicine 45: 485­496, 2020; DOI: 10.3892/ijmm.2019.4435].

Cancer­related fatigue during combined treatment of androgen deprivation therapy and radiotherapy is associated with mitochondrial dysfunction.

Combined androgen deprivation therapy (ADT) and radiation therapy (RT) is the standard of care treatment for non­metastatic prostate cancer (NMPC). Despite the efficacy, treatment­related symptoms including fatigue greatly reduce the quality of life of cancer patients. The goal of the study is to examine the influence of combined ADT/RT on fatigue and understand its underlying mechanisms. A total of 64 participants with NMPC were enrolled. Fatigue was assessed using the Functional Assessment of Cancer Therapy­Fatigue. Mitochondrial function parameters were measured as oxygen consumption from peripheral blood mononuclear cells (PBMCs) extracted from participants' whole blood. An ADT/RT­induced fatigue mouse model was developed, with fatigue measured as a reduction in voluntary wheel­running activity (VWRA) in 54 mice. Mitochondrial function was assessed in the ADT/RT mouse brains using western blot analysis of glucose transporter 4 (GLUT4) and transcription factor A, mitochondrial (TFAM). The results demonstrated that fatigue in the ADT group was exacerbated during RT compared with the non­ADT group. This effect was specific to fatigue, as depressive symptoms were unaffected. PBMCs of fatigued subjects exhibited decreased ATP coupling efficiency compared to non­fatigued subjects, indicative of mitochondrial dysfunction. The ADT/RT mice demonstrated the synergistic effect of ADT and RT in decreasing VWRA. Brain tissues of ADT/RT mice exhibited decreased levels of GLUT4 and TFAM suggesting that impaired neuronal metabolic homeostasis may contribute to fatigue pathogenesis. In conclusion, these findings suggest that fatigue induced by ADT/RT may be attributable to mitochondrial dysfunction both peripherally and in the central nervous system (CNS). The synergistic effect of ADT/RT is behaviorally reproducible in a mouse model and its mechanism may be related to bioenergetics in the CNS.

Context-Dependent Responses of Aquatic Insects to Metals and Metal Mixtures: A Quantitative Analysis Summarizing 24 Yr of Stream Mesocosm Experiments.

Modernizing water quality criteria to predict how contaminants affect natural aquatic communities requires that we utilize data obtained across multiple lines of evidence, including laboratory, mesocosm, and field studies. We report the results of 29 mesocosm experiments conducted from 1994 to 2017 at the Colorado State University Stream Research Laboratory (Fort Collins, CO, USA). The primary goal of the present study was to quantify responses of aquatic insect communities collected from 8 different locations to different combinations of cadmium (Cd), copper (Cu), iron (Fe), and zinc (Zn). Treatments that included Cu or Fe, either alone or in combination with other metals, were especially toxic to aquatic insects. The results showed that effects of metals were context dependent and varied significantly among the 8 sites where communities were collected. In particular, effects on communities from smaller streams were significantly greater than those from larger streams. Our analyses also showed that several morphological (body size, shape, gills, degree of sclerotization) and life history (voltinism) traits were significantly correlated with sensitivity to metals. Across all taxa and experiments, aquatic insects broadly classified as small (maximum body length <8 mm) were significantly more sensitive to metals than medium or large individuals. These findings demonstrate the advantages of integrating results of mesocosm experiments with species traits to develop a mechanistic understanding of biotic and abiotic factors that influence community responses to contaminants. Environ Toxicol Chem 2019;38:2486-2496. © 2019 SETAC.

Comparing passive measures of fatigue-like behavior in mice.

Fatigue is a very common and costly symptom associated with various diseases and disorders. Nonetheless, understanding the pathobiology and developing of therapies for fatigue have been difficult, partly because of a lack of consensus on the measures to phenotype this behavior, both in clinical settings and in animal studies. Here, we describe a fatigue-like behavior induced in mice by abdominal irradiation and compare three different methods of measuring changes in physical activity over time: running wheels, video home cage monitoring, and telemetry. These methods collect data passively and continuously, requiring no disruption of animals' normal home cage behavior. In our experiments, all three methods reported a fatigue-like behavior, exhibited by a reduction in physical activity following abdominal irradiation. Video tracking showed the largest fatigue effect size (Cohen's D = 1.78) over four days of monitoring, and was the only method showing a significant decrease in activity during the light period. Telemetry and running wheels showed a similar effect size (D = 1.68 and 1.65, respectively), but running wheels showed different circadian patterns of physical activity. In addition, we conducted rotarod and inverted grid suspension tests, which suggested that fatigue-like behavior was not the result of gross motor abnormalities.

mGluR5 mediates post-radiotherapy fatigue development in cancer patients.

Cancer-related fatigue (CRF) is a common burden in cancer patients and little is known about its underlying mechanism. The primary aim of this study was to identify gene signatures predictive of post-radiotherapy fatigue in prostate cancer patients. We employed Fisher Linear Discriminant Analysis (LDA) to identify predictive genes using whole genome microarray data from 36 men with prostate cancer. Ingenuity Pathway Analysis was used to determine functional networks of the predictive genes. Functional validation was performed using a T lymphocyte cell line, Jurkat E6.1. Cells were pretreated with metabotropic glutamate receptor 5 (mGluR5) agonist (DHPG), antagonist (MPEP), or control (PBS) for 20 min before irradiation at 8 Gy in a Mark-1 γ-irradiator. NF-κB activation was assessed using a NF-κB/Jurkat/GFP Transcriptional Reporter Cell Line. LDA achieved 83.3% accuracy in predicting post-radiotherapy fatigue. "Glutamate receptor signaling" was the most significant (p = 0.0002) pathway among the predictive genes. Functional validation using Jurkat cells revealed clustering of mGluR5 receptors as well as increased regulated on activation, normal T cell expressed and secreted (RANTES) production post irradiation in cells pretreated with DHPG, whereas inhibition of mGluR5 activity with MPEP decreased RANTES concentration after irradiation. DHPG pretreatment amplified irradiation-induced NF-κB activation suggesting a role of mGluR5 in modulating T cell activation after irradiation. These results suggest that mGluR5 signaling in T cells may play a key role in the development of chronic inflammation resulting in fatigue and contribute to individual differences in immune responses to radiation. Moreover, modulating mGluR5 provides a novel therapeutic option to treat CRF.

Taltirelin alleviates fatigue-like behavior in mouse models of cancer-related fatigue.

Fatigue affects most cancer patients and has numerous potential causes, including cancer itself and cancer treatment. Cancer-related fatigue (CRF) is not relieved by rest, can decrease quality of life, and has no FDA-approved therapy. Thyrotropin-releasing hormone (TRH) has been proposed as a potential novel treatment for CRF, but its efficacy against CRF remains largely untested. Thus, we tested the TRH analog, taltirelin (TAL), in mouse models of CRF. To model fatigue, we used a mouse model of chemotherapy, a mouse model of radiation therapy, and mice bearing colon 26 carcinoma tumors. We used the treadmill fatigue test to assess fatigue-like behavior after treatment with TAL. Additionally, we used wild-type and TRH receptor knockout mice to determine which TRH receptor was necessary for the actions of TAL. Tumor-bearing mice displayed muscle wasting and all models caused fatigue-like behavior, with mice running a shorter distance in the treadmill fatigue test than controls. TAL reversed fatigue-like behavior in all three models and the mouse TRH1 receptor was necessary for the effects of TAL. These data suggest that TAL may be useful in alleviating fatigue in all cancer patients and provide further support for evaluating TAL as a potential therapy for CRF in humans.

A Mouse Model of Fatigue Induced by Peripheral Irradiation.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

Changes in Sport Fish Mercury Concentrations from Food Web Shifts Suggest Partial Decoupling from Atmospheric Deposition in Two Colorado Reservoirs.

Partial decoupling of mercury (Hg) loading and observed Hg concentrations ([Hg]) in biotic and abiotic samples has been documented in aquatic systems. We studied two Colorado reservoirs to test whether shifts in prey for sport fish would lead to changes in [Hg] independent of external atmospheric Hg deposition. We compared sport fish total mercury concentrations ([T-Hg]) and macroinvertebrate (chironomids and crayfish) methylmercury concentrations ([MeHg]) before and after food web shifts occurred in both reservoirs. We also monitored wet atmospheric Hg deposition and sediment [T-Hg] and [MeHg] at each reservoir. We found rapid shifts in Hg bioaccumulation in each reservoir's sport fish, and these changes could not be attributed to atmospheric Hg deposition. Our study shows that trends in atmospheric deposition, environmental samples (e.g., sediments), and samples of species at the low trophic levels (e.g., chironomids and crayfish) may not accurately reflect conditions that result in fish consumption advisories for high trophic level sport fish. We suggest that in the short-term, monitoring fish [Hg] is necessary to adequately protect human health because natural and anthropogenic perturbations to aquatic food-webs that affect [Hg] in sport fish will continue regardless of trends in atmospheric deposition.

Exploratory Investigation of Early Biomarkers for Chronic Fatigue in Prostate Cancer Patients Following Radiation Therapy.

BACKGROUND: Fatigue is one of the most debilitating adverse effects of cancer therapy. Identifying biomarkers early during cancer therapy may help us understand the biologic underpinnings of the persistence of fatigue following therapy. OBJECTIVE: We aimed to identify early biomarkers of fatigue by examining correlations of levels of cytokines during external beam radiation therapy (EBRT) with persistence of fatigue 1 year following treatment completion in men with nonmetastatic prostate cancer (NM-PC). METHODS: A sample of 34 men with nonmetastatic prostate cancer scheduled to receive EBRT were followed up at baseline (T1), midpoint of EBRT (T2), and 1 year following EBRT (T3). Demographic and clinical data were obtained by chart review. The Functional Assessment of Cancer Therapy-Fatigue was administered to measure fatigue levels. Plasma cytokine levels were determined at T1 and T2 using the Bio-Rad Bio-Plex Cytokine Assay Kits. RESULTS: Significant correlations were observed between levels of interleukin 2 (IL-3), IL-8, IL-9, IL-10, IL-16, interferon γ-induced protein 10, interferon α2, interferon γ, and stromal cell-derived factor 1α at T2 with worsening of fatigue from T1 to T3. CONCLUSIONS: Immunological changes prior to chronic fatigue development may reflect the long-term response to radiation therapy-induced damage. IMPLICATIONS FOR PRACTICE: Early biomarkers for chronic fatigue related to cancer therapy will help advance our understanding of the etiology of this distressing symptom and will help nurses identify patients at risk of developing chronic fatigue after cancer treatment. This information will also aid in patient education, as well as symptom management.

Telehealth delivery of cognitive-behavioral intervention to youth with autism spectrum disorder and anxiety: A pilot study.

Youth with autism spectrum disorders frequently experience significant symptoms of anxiety. Empirically supported psychosocial interventions exist, yet access is limited, especially for families in rural areas. Telehealth (i.e. videoconferencing) has potential to reduce barriers to access to care; however, little is known about the feasibility or efficacy of directly intervening with youth with autism spectrum disorders through this modality. This study details the pilot testing of a telehealth version of an empirically supported intervention targeting anxiety in youth with autism spectrum disorders. The primary focus of this study is on feasibility, with evaluation of outcomes as a starting point for future randomized trials. In all, 33 families of youth with autism spectrum disorders and significant anxiety symptoms participated in this study (Telehealth Facing Your Fears (FYF) Intervention: n = 17; Wait-list control: n = 16). Youth of all functioning levels were included. Acceptability was strong; however, the usability of the technology was problematic for some families and impeded some sessions significantly. Fidelity of the telehealth version to the critical elements of the original, in vivo version was excellent. More work is needed to improve delivery of exposure practices and parent coaching. Preliminary efficacy analyses are promising, with improvements observed in youth anxiety over time (relative to a comparison group waiting for live intervention) and parent sense of competence (within group). Clearly, stronger designs are necessary to evaluate efficacy sufficiently; however, this study does provide support for further investigation of clinic-to-home videoconferencing as a direct intervention tool for youth with autism spectrum disorders and their parents.

Voltage-Sensitive Dye Imaging of Population Signals in Brain Slices.

In a bright-field measurement from a vertebrate brain stained by superfusing a solution of the dye over the surface, each pixel in a camera receives light from a substantial number (thousands) of neurons and neuronal processes (population signals). Because of scattering and out-of-focus light, this will be true even if the pixel size corresponds to a small area of the brain. In this situation, the voltage-sensitive dye signal will be a population average of the change in membrane potential of all of these neurons and processes. Many investigators have published voltage-sensitive dye imaging studies of population activities in brain slices. Their methods, including choice of dyes, illumination intensity, and imaging device, vary across a large spectrum. Here we present a protocol for visualizing spatiotemporal patterns in rodent neocortex in vitro. Detecting these patterns requires high-sensitivity imaging in single trials, because averaging will obscure the complex dynamics of the spatiotemporal patterns.