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Fecal-pellet surveys provide density estimates of pellet groups, which offer a quick and reliable index of population densities for white-tailed deer Odocoileus virginianus and other wildlife species. Comparisons of differing fecal-pellet survey methodologies have previously shown that they vary in the precision. However, researchers made these comparisons outside of the United States in regions with lower white-tailed deer densities. In this study, we compared pellet-group density estimates and precision from line transects, quadrat sampling, and strip transects. At each site, three observers surveyed simultaneously with each being responsible for one method. Like other studies, we found that each method produced similar estimates of pellet-group density, but quadrat sampling produced the most precise estimates. Furthermore, all three methods suggested that the central region of Indiana had both the highest pellet-group density and the highest precision. These results suggest a more homogenous distribution of pellet groups in areas with higher white-tailed deer densities, which may increase the precision of all methods. Our results suggest that quadrat sampling may be the most effective method for estimating pellet-group densities within woodlots of the U.S. Midwest, and that precision may increase in areas with higher white-tailed deer densities. This study not only improves deer management within the Midwest but provides guidelines for other studies to potentially advance the conservation and management of other species.

Many studies have examined the performance of stump sprouts across variables such as tree diameter, light availability, and site quality. However, most of these studies created large openings following even-aged silviculture, which is not typically employed in small woodlots typical of the Midwest United States. To address this, we evaluated sprouting performance after felling stems of six common midstory species, simulating a midstory removal treatment. We created 510 stumps across nine sites in Indiana and measured the probability of stumps sprouting, sprout density, and the height of the tallest sprout after 1 year following cutting for hackberry (Celtis occidentalis L.), red maple and sugar maple (Acer rubrum L. and A. saccharum Marsh., respectively), northern red oak (Quercus rubra L.), sassafras (Sassafras albidum Nutt.), and white ash (Fraxinus Americana L.). We observed that hackberry, northern red oak, red maple, and white ash stumps sprouted prolifically, but sugar maple and sassafras stumps did not. Furthermore, we found that white-tailed deer (Odocoileus virginianus) herbivory depressed sprout heights, whereas the effects of diameter and light availability were variable. Our results suggest that midstory removals may result in vigorous sprouting; thus, follow-up treatments such an herbicide may be needed to reduce competition.

IntroductionMicroCT of the three-dimensional fascicular organization of the human vagus nerve provides essential data to inform basic anatomy as well as the development and optimization of neuromodulation therapies. To process the images into usable formats for subsequent analysis and computational modeling, the fascicles must be segmented. Prior segmentations were completed manually due to the complex nature of the images, including variable contrast between tissue types and staining artifacts.MethodsHere, we developed a U-Net convolutional neural network (CNN) to automate segmentation of fascicles in microCT of human vagus nerve.ResultsThe U-Net segmentation of ~500 images spanning one cervical vagus nerve was completed in 24 s, versus ~40 h for manual segmentation, i.e., nearly four orders of magnitude faster. The automated segmentations had a Dice coefficient of 0.87, a measure of pixel-wise accuracy, thus suggesting a rapid and accurate segmentation. While Dice coefficients are a commonly used metric to assess segmentation performance, we also adapted a metric to assess fascicle-wise detection accuracy, which showed that our network accurately detects the majority of fascicles, but may under-detect smaller fascicles.DiscussionThis network and the associated performance metrics set a benchmark, using a standard U-Net CNN, for the application of deep-learning algorithms to segment fascicles from microCT images. The process may be further optimized by refining tissue staining methods, modifying network architecture, and expanding the ground-truth training data. The resulting three-dimensional segmentations of the human vagus nerve will provide unprecedented accuracy to define nerve morphology in computational models for the analysis and design of neuromodulation therapies.

Invasive woody perennials pose an immense threat to the diversity and function of many ecosystems, including forests in the eastern United States. While herbicide treatments have proven effective in controlling many plant invasions, there is considerable interest in the refinement of herbicide prescriptions to improve efficacy and prevent non-target damage. Adjuvants are widely utilized to improve herbicide efficacy, but research on novel adjuvants is often lacking. Furthermore, adjuvant research has generally focused on enhancement of foliar herbicide absorption, and few studies focus on adjuvant utility for other herbicide delivery techniques such as cut stump treatments. We evaluated 2XL—a cocktail of cellulase enzymes derived from fungi—as a potential herbicide adjuvant for use with glyphosate applied in a cut stump treatment due to its ability to degrade a key component of cell walls. We conducted a field experiment using the cut stump method of treatment (cut surface treated with herbicide) on a problematic invasive shrub, Amur honeysuckle [Lonicera maackii (Rupr.) Herder]. We tested combinations of three concentrations of 2XL with five concentrations of glyphosate and hypothesized that low concentrations of glyphosate combined with 2XL would be as effective in limiting the resprouting of L. maackii as higher concentrations of glyphosate without the enzymes. Our results indicated that 2XL did not improve glyphosate efficacy for reducing the number of resprouting stems or the length of the longest resprouting stem within the same or following year as treatment. Limited data indicated the combination of 2XL and glyphosate applied at 30 g L−1 slightly increased resprouting in the year following treatment. While 2XL did not improve glyphosate efficacy, our results showed effective control of L. maackii at the lowest concentration of glyphosate tested (30 g L−1), suggesting that concentrations lower than those typically applied may be effective in controlling L. maackii within parameters similar to those tested here.

Conventional clinical histopathology analysis is based on bright-field microscopy of thinly sliced tissue specimens. Although bright-field microscopy is a simple and robust method for examining microscope slides, preparing the slides needed is a lengthy and labor-intensive process. Slide-free histopathology technologies address this problem by directly imaging intact, minimally processed tissue samples using advanced optical imaging systems, bypassing the extended workflow now required for preparation of tissue sections. This manuscript explains the technical basis of slide-free microscopy, reviews common slide-free optical microscopy techniques, and discusses the opportunities and challenges involved in clinical implementation.

New tools for monitoring and manipulating neural activity have been developed with steadily improving functionality, specificity, and reliability, which are critical both for mapping neural circuits and treating neurological diseases. This review focuses on the use of an invertebrate animal, the marine mollusk

Anatomical tracing studies examining the vagal system can conflate details of sensory afferent and motor efferent neurons. Here, we used a serotype of adeno-associated virus that transports retrogradely and exhibits selective tropism for vagal afferents, to map their soma location and central termination sites within the nucleus of the solitary tract (NTS). We examined the vagal sensory afferents innervating the trachea, duodenum, stomach, or heart, and in some animals, from two organs concurrently. We observed no obvious somatotopy in the somata distribution within the nodose ganglion. The central termination patterns of afferents from different organs within the NTS overlap substantially. Convergence of vagal afferent inputs from different organs onto single NTS neurons is observed. Abdominal and thoracic afferents terminate throughout the NTS, including in the rostral NTS, where the 7th cranial nerve inputs are known to synapse. To address whether the axonal labeling produced by viral transduction is so widespread because it fills axons traveling to their targets, and not just terminal fields, we labeled pre and postsynaptic elements of vagal afferents in the NTS . Vagal afferents form multiple putative synapses as they course through the NTS, with each vagal afferent neuron distributing sensory signals to multiple second-order NTS neurons. We observe little selectivity between vagal afferents from different visceral targets and NTS neurons with common neurochemical phenotypes, with afferents from different organs making close appositions with the same NTS neuron. We conclude that specific viscerosensory information is distributed widely within the NTS and that the coding of this input is probably determined by the intrinsic properties and projections of the second-order neuron.

Objective. Vagus nerve stimulation (VNS) is Food and Drug Administration-approved for epilepsy, depression, and obesity, and stroke rehabilitation; however, the morphological anatomy of the vagus nerve targeted by stimulatation is poorly understood. Here, we used microCT to quantify the fascicular structure and neuroanatomy of human cervical vagus nerves (cVNs). Approach. We collected eight mid-cVN specimens from five fixed cadavers (three left nerves, five right nerves). Analysis focused on the ‘surgical window’: 5 cm of length, centered around the VNS implant location. Tissue was stained with osmium tetroxide, embedded in paraffin, and imaged on a microCT scanner. We visualized and quantified the merging and splitting of fascicles, and report a morphometric analysis of fascicles: count, diameter, and area. Main results. In our sample of human cVNs, a fascicle split or merge event was observed every ∼560 µm (17.8 ± 6.1 events cm−1). Mean morphological outcomes included: fascicle count (6.6 ± 2.8 fascicles; range 1–15), fascicle diameter (514 ± 142 µm; range 147–1360 µm), and total cross-sectional fascicular area (1.32 ± 0.41 mm2; range 0.58–2.27 mm). Significance. The high degree of fascicular splitting and merging, along with wide range in key fascicular morphological parameters across humans may help to explain the clinical heterogeneity in patient responses to VNS. These data will enable modeling and experimental efforts to determine the clinical effect size of such variation. These data will also enable efforts to design improved VNS electrodes.

Forests in eastern North America are experiencing high densities of white-tailed deer (Odocoileus virginianus) and encroachment by invasive plants, both of which threaten native biodiversity. We review the literature on deer and invasive plant impacts focusing on studies that simultaneously evaluate the consequences of both. Deer have more frequent and more consistently negative effects than invasive plants. Widespread deer impacts now threaten many native plant species through much of their range. In contrast, invasive plant effects currently remain more localized and/or of smaller extent within forests. Deer impacts are also cumulative, hitting preferred plant species especially hard as they decline in density. This generates difficult-to-reverse legacy effects. Invasive plant effects, in contrast, tend to be more diffuse and may be more readily reversed. High deer populations also shift physical and chemical conditions in soils promoting “invasion cascades” involving non-native earthworms and certain introduced plants. Removing invasive plants without reducing deer populations can increase deer impacts on native species. Management should be integrated to address both deer and invasive plants. To safeguard and restore native biota when resources are limited, however, it may be most effective for managers to first reduce deer populations before investing in efforts to reduce invasive populations (except when invasions are at an early stage). We should rethink and reform traditional approaches to managing deer so that we can better integrate land vegetation with wildlife management to achieve broad public objectives. Interacting effects of high ungulate populations and invasive plants deserve further study to determine whether similar recommendations apply to other regions.

Increased regurgitant blood flow has been linked to endocardial cushion defects and resultant congenital heart diseases (CHDs). Prenatal alcohol exposure (PAE) has been shown to alter early blood flow resulting in abnormal endocardial cushions and CHDs. Compounds, including folic acid (FA), mitigate PAE effects and prevent CHDs, but few studies have assessed their effects on blood flow. We modeled binge drinking in quail embryos at gastrulation. Embryos were exposed to ethanol alone, FA (3.2 μg/egg) alone, and the two simultaneously. We quantified in cardiac looping stages (equivalent to 4 wk of human gestation), the regurgitant blood flow with Doppler optical coherence tomography (OCT) and endocardial cushion volumes using OCT imaging. Incidences of abnormal body curvature and heart rates were also measured. Embryos exposed to ethanol showed significantly increased regurgitant blood flow compared with controls, whereas embryos given FA with ethanol had significantly reduced regurgitant blood flow but without returning to control levels. Ethanol exposure led to significantly smaller, abnormal endocardial cushions, and the addition of FA improved their size, but they remained smaller than controls. Abnormal body curvatures after PAE were reduced in incidence but not fully prevented by FA. FA supplementation partially alleviated PAE-induced abnormal cardiovascular function and morphology. Normal blood flow and endocardial cushions are both required to produce a healthy four-chambered heart. These findings support that FA supplementation should begin early in pregnancy to prevent heart as well as neural tube defects. Investigations into the efficacy of combinations of compounds to prevent PAE-induced defects are warranted. NEW & NOTEWORTHY State-of-the-art biophotonic tools captured blood flow and endocardial cushion volumes in tiny beating quail embryo hearts, an accessible model for studying four-chambered heart development. Both hemodynamic flow and endocardial cushion volumes were altered with ethanol exposure but normalized when folic acid was introduced with ethanol. Folic acid supplementation preserved hemodynamic function that is intimately involved in sculpting the heart from the earliest stages of heart development.

Recent advances in optical tissue clearing and three-dimensional (3D) fluorescence microscopy have enabled high resolution in situ imaging of intact tissues. Using simply prepared samples, we demonstrate here “digital labeling,” a method to segment blood vessels in 3D volumes solely based on the autofluorescence signal and a nuclei stain (DAPI). We trained a deep-learning neural network based on the U-net architecture using a regression loss instead of a commonly used segmentation loss to achieve better detection of small vessels. We achieved high vessel detection accuracy and obtained accurate vascular morphometrics such as vessel length density and orientation. In the future, such digital labeling approach could easily be transferred to other biological structures.

Optical coherence tomography (OCT) has been used to investigate heart development because of its capability to image both structure and function of beating embryonic hearts. Cardiac structure segmentation is a prerequisite for the quantification of embryonic heart motion and function using OCT. Since manual segmentation is time-consuming and labor-intensive, an automatic method is needed to facilitate high-throughput studies. The purpose of this study is to develop an image-processing pipeline to facilitate the segmentation of beating embryonic heart structures from a 4-D OCT dataset. Sequential OCT images were obtained at multiple planes of a beating quail embryonic heart and reassembled to a 4-D dataset using image-based retrospective gating. Multiple image volumes at different time points were selected as key-volumes, and their cardiac structures including myocardium, cardiac jelly, and lumen, were manually labeled. Registration-based data augmentation was used to synthesize additional labeled image volumes by learning transformations between key-volumes and other unlabeled volumes. The synthesized labeled images were then used to train a fully convolutional network (U-Net) for heart structure segmentation. The proposed deep learning-based pipeline achieved high segmentation accuracy with only two labeled image volumes and reduced the time cost of segmenting one 4-D OCT dataset from a week to two hours. Using this method, one could carry out cohort studies that quantify complex cardiac motion and function in developing hearts.

Understanding peripheral nerve micro-anatomy can assist in the development of safe and effective neuromodulation devices. However, current approaches for imaging nerve morphology at the fiber level are either cumbersome, require substantial instrumentation, have a limited volume of view, or are limited in resolution/contrast. We present alternative methods based on MUSE (Microscopy with Ultraviolet Surface Excitation) imaging to investigate peripheral nerve morphology, both in 2D and 3D. For 2D imaging, fixed samples are imaged on a conventional MUSE system either label free (via auto-fluorescence) or after staining with fluorescent dyes. This method provides a simple and rapid technique to visualize myelinated nerve fibers at specific locations along the length of the nerve and perform measurements of fiber morphology (e.g., axon diameter and g-ratio). For 3D imaging, a whole-mount staining and MUSE block-face imaging method is developed that can be used to characterize peripheral nerve micro-anatomy and improve the accuracy of computational models in neuromodulation. Images of rat sciatic and human cadaver tibial nerves are presented, illustrating the applicability of the method in different preclinical models.

S-nitrosothiols exert multiple effects on neural processes in the central and peripheral nervous system. This study shows that intravenous infusion of S-nitroso-L-cysteine (SNO-L-CYS, 1 μmol/kg/min) in anesthetized male Sprague Dawley rats elicits (a) sustained increases in minute ventilation, via increases in frequency of breathing and tidal volume, (b) a decrease in Alveolar-arterial (A-a) gradient, thus improving alveolar gas-exchange, (c) concomitant changes in arterial blood-gas chemistry, such as an increase in pO

High-elevation five-needle pine trees are a group of Pinus species in the subgenus Strobus that occur at the edges of plant growth near the alpine tree line. These species are ecologically very important and are also threatened by climate-driven insect outbreaks and an exotic pathogen. Volatile organic compounds (VOCs) play central roles in the environmental adaptation of plants and in their defense against insects and pathogens. For example, the VOCs emitted by some high-elevation five-needle pine species attract female, tree-killing mountain pine beetles (MPB, Dendroctonus ponderosae) in the pioneering phase whereas VOCs from other species strongly repel this foremost herbivore, but the mechanism is unknown. We collected and compared headspace VOCs from foliage of eight species of high-elevation five-needle pines in Europe and North America. Overall, VOCs differed quantitatively among species with few qualitative differences. Despite species emitting essentially the same compounds, Random Forest analysis correctly classified 117 of the 126 trees sampled by using VOCs and identified the most important compounds for species classification and for separating species resistant from those susceptible to MPB or white pine blister rust (Cronartium ribicola). These VOC 'fingerprints' resulted largely from species emitting distinctive ratios of compounds, rather than through presence of species-specific compounds. Importantly, these Pinus species vary greatly in resistance to the main herbivore (MPB) and pathogen (white pine blister rust) causing tree mortality. Thus, these findings provide insights and should guide research into understanding resistance and in developing tools to manage these important trees. For instance, studies into the functions of five-needle pine VOCs in defense against abiotic or biotic stressors should focus on blend ratios rather than on individual compounds.

Vagus nerve stimulation (VNS) is a method to treat drug-resistant epilepsy and depression, but therapeutic outcomes are often not ideal. Newer electrode designs such as intra-fascicular electrodes offer potential improvements in reducing off-target effects but require a detailed understanding of the fascicular anatomy of the vagus nerve. We have adapted a section-and-image technique, cryo-imaging, with UV excitation to visualize fascicles along the length of the vagus nerve. In addition to offering optical sectioning at the surface via reduced penetration depth, UV illumination also produces sufficient contrast between fascicular structures and connective tissue. Here we demonstrate the utility of this approach in pilot experiments. We imaged fixed, cadaver vagus nerve samples, segmented fascicles, and demonstrated 3D tracking of fascicles. Such data can serve as input for computer models of vagus nerve stimulation.