Dendrites are the primary site of synaptic activity in neurons and changes in synapses are often the first pathological stage in neurodegenerative diseases. Molecular studies of these changes rely on morphological analysis of the imaging of somas and dendritic arbors of cultured or primary neurons. As research on preventing or reversing synaptic degeneration develops, demands increase for user-friendly 2D neurite analyzers without undermining accuracy and reproducibility. The most common method of 2D neurite analysis is manual by using ImageJ. This method relies completely on the user's ability to distinguish the shape and size of dendrites and trace morphology with a series of straight connected lines. Semi-automatic methods have also been developed, such as the NeuronJ plugin for ImageJ. These methods still rely on the user to identify the start and end of the dendrites, but automatically determine the shape, reducing the likelihood of user bias and speeding the process. Some automatic methods have been developed through image processing software, like ImagePro. These programs tend to be expensive, but have been shown to be fast and effective, limiting user interaction. In this study, we compare three methods of neurite analysis-ImageJ, NeuronJ, and ImagePro-in measuring the soma size, number of dendrites, and length of dendrites per cell of embryonic sympathetic rat neurons with BMP-7-induced dendritic growth. Our results indicate that ImageJ and NeuronJ measurements were of similar effectiveness and consistent throughout various images and multiple trials. NeuronJ required less user interaction in measuring the length of dendrites than the manual method and therefore, was faster and less labor intensive. Conversely, ImagePro tended to be inconsistent across images, overestimating both soma size and the number of dendrites per cell while underestimating the length of dendrites. Overall, NeuronJ, in conjunction with ImageJ, is the most reliable and efficient method of 2D neurite analysis tested in the present study.

Dendrites are the primary site of synaptic activity in neurons and changes in synapses are often the first pathological stage in neurodegenerative diseases. Molecular studies of these changes rely on morphological analysis of the imaging of somas and dendritic arbors of cultured or primary neurons. As research on preventing or reversing synaptic degeneration develops, demands increase for user-friendly 2D neurite analyzers without undermining accuracy and reproducibility. The most common method of 2D neurite analysis is manual by using ImageJ. This method relies completely on the user's ability to distinguish the shape and size of dendrites and trace morphology with a series of straight connected lines. Semi-automatic methods have also been developed, such as the NeuronJ plugin for ImageJ. These methods still rely on the user to identify the start and end of the dendrites, but automatically determine the shape, reducing the likelihood of user bias and speeding the process. Some automatic methods have been developed through image processing software, like ImagePro. These programs tend to be expensive, but have been shown to be fast and effective, limiting user interaction. In this study, we compare three methods of neurite analysis-ImageJ, NeuronJ, and ImagePro-in measuring the soma size, number of dendrites, and length of dendrites per cell of embryonic sympathetic rat neurons with BMP-7-induced dendritic growth. Our results indicate that ImageJ and NeuronJ measurements were of similar effectiveness and consistent throughout various images and multiple trials. NeuronJ required less user interaction in measuring the length of dendrites than the manual method and therefore, was faster and less labor intensive. Conversely, ImagePro tended to be inconsistent across images, overestimating both soma size and the number of dendrites per cell while underestimating the length of dendrites. Overall, NeuronJ, in conjunction with ImageJ, is the most reliable and efficient method of 2D neurite analysis tested in the present study.

Chemical exposures have been implicated as environmental risk factors that interact with genetic susceptibilities to influence individual risk for complex neurodevelopmental disorders, including autism spectrum disorder, schizophrenia, attention deficit hyperactivity disorder and intellectual disabilities. Altered patterns of neuronal connectivity represent a convergent mechanism of pathogenesis for these and other neurodevelopmental disorders, and growing evidence suggests that chemicals can interfere with specific signaling pathways that regulate the development of neuronal connections. There is, therefore, a growing interest in developing screening platforms to identify chemicals that alter neuronal connectivity. Cell-cell, cell-matrix interactions and systemic influences are known to be important in defining neuronal connectivity in the developing brain, thus, a systems-based model offers significant advantages over cell-based models for screening chemicals for effects on neuronal connectivity. The embryonic zebrafish represents a vertebrate model amenable to higher throughput chemical screening that has proven useful in characterizing conserved mechanisms of neurodevelopment. Moreover, the zebrafish is readily amenable to gene editing to integrate genetic susceptibilities. Although use of the zebrafish model in toxicity testing has increased in recent years, the diverse tools available for imaging structural differences in the developing zebrafish brain have not been widely applied to studies of the influence of gene by environment interactions on neuronal connectivity in the developing zebrafish brain. Here, we discuss tools available for imaging of neuronal connectivity in the developing zebrafish, review what has been published in this regard, and suggest a path forward for applying this information to developmental neurotoxicity testing.

Neuronal connectivity is dependent on size and shape of the dendritic arbor. However, mechanisms controlling dendritic arborization, especially in the peripheral nervous system, are not completely understood. Previous studies have shown that bone morphogenetic proteins (BMPs) are important initiators of dendritic growth in peripheral neurons. In this study, we examined the hypothesis that post-transcriptional regulation mediated by microRNAs (miRNAs) is necessary for BMP-7-induced dendritic growth in these neurons. To examine the role of miRNAs in BMP-7-induced dendritic growth, microarray analyses was used to profile miRNA expression in cultured sympathetic neurons from the superior cervical ganglia of embryonic day 21 rat pups at 6 and 24 h after treatment with BMP-7 (50 ng/mL). Our data showed that BMP-7 significantly regulated the expression of 43 of the 762 miRNAs. Of the 43 miRNAs, 22 showed robust gene expression; 14 were upregulated by BMP-7 and 8 were downregulated by BMP-7. The expression profile for miR-335, miR-664-1*, miR-21, and miR-23b was confirmed using qPCR analyses. Functional studies using morphometric analyses of dendritic growth in cultured sympathetic neurons transfected with miRNA mimics and inhibitors indicated that miR-664-1*, miR-23b, and miR-21 regulated early stages of BMP-7-induced dendritic growth. In summary, our data provide evidence for miRNA-mediated post-transcriptional regulation as important downstream component of BMP-7 signaling during early stages of dendritic growth in sympathetic neurons.

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons.

Previous studies have shown that bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, the downstream signaling molecules that mediate the dendrite promoting activity of BMPs are not well characterized. Here we test the hypothesis that reactive oxygen species (ROS)-mediated signaling links BMP receptor activation to dendritic growth. In cultured rat sympathetic neurons, exposure to any of the three mechanistically distinct antioxidants, diphenylene iodinium (DPI), nordihydroguaiaretic acid (NGA) or desferroxamine (DFO), blocked de novo BMP-induced dendritic growth. Addition of DPI to cultures previously induced with BMP to extend dendrites caused dendritic retraction while DFO and NGA prevented further growth of dendrites. The inhibition of the dendrite promoting activity of BMPs by antioxidants was concentration-dependent and occurred without altering axonal growth or neuronal cell survival. Antioxidant treatment did not block BMP activation of SMAD 1,5 as determined by nuclear localization of these SMADs. While BMP treatment did not cause a detectable increase in intracellular ROS in cultured sympathetic neurons as assessed using fluorescent indicator dyes, BMP treatment increased the oxygen consumption rate in cultured sympathetic neurons as determined using the Seahorse XF24 Analyzer, suggesting increased mitochondrial activity. In addition, BMPs upregulated expression of NADPH oxidase 2 (NOX2) and either pharmacological inhibition or siRNA knockdown of NOX2 significantly decreased BMP-7 induced dendritic growth. Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that ROS-mediated signaling positively modulates dendritic complexity in peripheral neurons.

In their 2012 report, the President's Council of Advisors on Science and Technology advocated "replacing standard science laboratory courses with discovery-based research courses"-a challenging proposition that presents practical and pedagogical difficulties. In this paper, we describe our collective experiences working with the Genomics Education Partnership, a nationwide faculty consortium that aims to provide undergraduates with a research experience in genomics through a scheduled course (a classroom-based undergraduate research experience, or CURE). We examine the common barriers encountered in implementing a CURE, program elements of most value to faculty, ways in which a shared core support system can help, and the incentives for and rewards of establishing a CURE on our diverse campuses. While some of the barriers and rewards are specific to a research project utilizing a genomics approach, other lessons learned should be broadly applicable. We find that a central system that supports a shared investigation can mitigate some shortfalls in campus infrastructure (such as time for new curriculum development, availability of IT services) and provides collegial support for change. Our findings should be useful for designing similar supportive programs to facilitate change in the way we teach science for undergraduates.

There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.