culturing-rat-sympathetic-neurons-from-embryonic-superior-cervical

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

Saint Mary's College of California

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students.

The genomics education partnership: successful integration of research into laboratory classes at a diverse group of undergraduate institutions.

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The popularity and prevalence of energy beverages makes it essential to examine the interactions between the ingredients and their effects on the safety of these beverages. In this study, we used in vitro assays to examine the effects of two energy beverages on mesenchymal, epithelial and neuronal cells. Our results showed that treatment of epithelial and mesenchymal cells with either energy beverage resulted in a dose dependent delay in wound closure, in a scratch wound healing assay. In rat embryonic fibroblasts, treatment with the energy beverages led to decreased lamellipodia formation and decreased proliferation/viability; whereas in MDCK cells, energy beverage treatment resulted in actin disorganization without any effects on cell proliferation. This suggests that the mechanisms underlying delayed wound healing might be different in the two cell types. Interestingly, the delays in both cell types could not be mimicked by treatment of caffeine, taurine and glucose alone or in combinations. Furthermore, treatment of chick forebrain neuronal cultures with energy beverages resulted in a dose dependent inhibition of neurite outgrowth. The cellular assays used in this study provide a consistent, qualitative and quantitative system for examining the combinatorial effects of the various ingredients used in energy beverages.

The effects of energy beverages on cultured cells.

La popularit&eacute; et la pr&eacute;valence des boissons &eacute;nerg&eacute;tiques est donc essentiel d&#39;&eacute;tudier les interactions entre les ingr&eacute;dients et leurs effets sur la s&eacute;curit&eacute; de ces boissons. Dans cette &eacute;tude, nous avons utilis&eacute; des tests in vitro pour examiner les effets de deux boissons &eacute;nerg&eacute;tiques sur les cellules neuronales, &eacute;pith&eacute;liales et m&eacute;senchymateuses. Nos r&eacute;sultats ont montr&eacute; que le traitement des cellules &eacute;pith&eacute;liales et m&eacute;senchymateuses avec une boisson &eacute;nerg&eacute;tique entra&icirc;n&eacute; un retard d&eacute;pendant dose dans la fermeture de la plaie, dans un essai de cicatrisation gratter. Dans les fibroblastes embryonnaires de rat, traitement avec les boissons &eacute;nerg&eacute;tiques conduit &agrave; a diminu&eacute; la formation lamellipodia et diminue la prolif&eacute;ration/viabilit&eacute; ; consid&eacute;rant que dans les cellules MDCK, traitement boisson &eacute;nerg&eacute;tique a d&eacute;sorganisation actine sans aucun effet sur la prolif&eacute;ration cellulaire. Ceci sugg&egrave;re que les m&eacute;canismes qui sous-tendent la cicatrisation retard&eacute;e peuvent &ecirc;tre diff&eacute;rents dans les deux types cellulaires. Fait int&eacute;ressant, les retards dans les deux types de cellules ne pourraient pas &ecirc;tre imit&eacute;s par le traitement de la caf&eacute;ine, la taurine et le glucose, seuls ou en combinaison. En outre, traitement des cultures de neurones du cerveau ant&eacute;rieur poussin avec une boisson &eacute;nerg&eacute;tique a entra&icirc;n&eacute; une inhibition d&eacute;pendante de la dose de la croissance des neurites. Les analyses cellulaires utilis&eacute;es dans cette &eacute;tude offrent un syst&egrave;me coh&eacute;rent, qualitatif et quantitatif pour examiner les effets de combinatoires des diff&eacute;rents ingr&eacute;dients utilis&eacute;s dans les boissons &eacute;nerg&eacute;tiques.

Les effets des boissons &eacute;nerg&eacute;tiques sur des cellules cultiv&eacute;es.

Die Popularit&auml;t und Verbreitung des Energie-Getr&auml;nke macht es notwendig, die Interaktionen zwischen den Zutaten und deren Auswirkungen auf die Sicherheit dieser Getr&auml;nke zu pr&uuml;fen. In dieser Studie haben wir in-vitro-Tests untersuchen die Auswirkungen der beiden Energie-Getr&auml;nke auf Mesenchymale, epitheliale und neuronalen Zellen verwendet. Unsere Ergebnisse zeigten, dass die Behandlung der epithelialen und mesenchymalen Zellen mit entweder Energie-Getr&auml;nk einer Dosis abh&auml;ngige Verz&ouml;gerung bei Schlie&szlig;ung der Wunde, in ein kratzen Assay f&uuml;r die Wundheilung gef&uuml;hrt. In Ratte embryonalen Fibroblasten Behandlung mit der Energie-Getr&auml;nke f&uuml;hrten zu Lamellipodium Bildung sank und sank Verbreitung/Lebensf&auml;higkeit; w&auml;hrend in MDCK Zellen, Energie Getr&auml;nkebehandlung Aktin Desorganisation ohne Auswirkungen auf die Zellproliferation gef&uuml;hrt. Dies deutet darauf hin, dass die Mechanismen, die verz&ouml;gerte Wundheilung in der zwei Zelltypen unterschiedlich sein k&ouml;nnte. Interessanterweise konnte die Verz&ouml;gerungen bei der beide Zelltypen nicht durch Behandlung von Koffein, Taurin und Glucose allein oder in Kombinationen imitiert werden. Dar&uuml;ber hinaus f&uuml;hrte die Behandlung von K&uuml;ken Stirnhirn neuronale Kulturen mit Energie-Getr&auml;nke eine Dosis-abh&auml;ngige-Hemmung der Neurit Auswuchs. Die zellul&auml;re Assays, die in dieser Studie verwendeten vorsehen, dass ein System konsistent, qualitative und quantitative Pr&uuml;fung der kombinatorischen Auswirkungen der die verschiedenen Zutaten in Energie-Getr&auml;nke.

Die Auswirkungen der Energie-Getr&auml;nke auf kultivierten Zellen.

La popolarit&agrave; e la prevalenza di bevande energetiche rende indispensabile per esaminare le interazioni tra gli ingredienti e i loro effetti sulla sicurezza di queste bevande. In questo studio, abbiamo usato saggi in vitro per esaminare gli effetti delle due bevande di energia sulle cellule mesenchimali, epiteliali e neuronali. I nostri risultati hanno mostrato che il trattamento delle cellule epiteliali e mesenchimali con una bevanda di energia ha portato a un ritardo dipendente dalla dose in chiusura della ferita, in una ferita scratch test di guarigione. In fibroblasti embrionali di topo, il trattamento con le bevande di energia ha portato alla diminuita formazione lamellipodia e ridotta proliferazione/redditivit&agrave;; considerando che nelle cellule MDCK, trattamento di bevanda di energia provocato disorganizzazione dell&#39;actina senza effetti sulla proliferazione cellulare. Questo suggerisce che i meccanismi sottostanti la guarigione della ferita ritardata potrebbero essere diversi nei due tipi di cellule. Curiosamente, i ritardi in entrambi i tipi di cella non potrebbero essere imitati da trattamento di caffeina, taurina e glucosio da solo o in combinazione. Inoltre, trattamento del pulcino proencefalo neuronale culture con bevande di energia ha provocato un&#39;inibizione dipendente dalla dose di escrescenza dei neuriti. I cellulare dosaggi utilizzati in questo studio forniscono un sistema coerenza, qualitativo e quantitativo per esaminare gli effetti combinatoriali dei vari ingredienti usati nelle bevande energetiche.

Gli effetti delle bevande energetiche su cellule in coltura.

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La popularidad y la prevalencia de bebidas de energ&iacute;a hace que sea esencial para examinar las interacciones entre los ingredientes y sus efectos sobre la seguridad de estas bebidas. En este estudio, utilizamos ensayos in vitro para examinar los efectos de dos bebidas de energ&iacute;a en las c&eacute;lulas neuronales, epiteliales y mesenquimales. Nuestros resultados mostraron que el tratamiento de las c&eacute;lulas epiteliales y mesenquimales con cualquier bebida de energ&iacute;a dio lugar a un retardo dependiente de dosis en el cierre de la herida, en un ensayo de cicatrizaci&oacute;n rasgu&ntilde;o. En fibroblastos embrionarios de la rata, el tratamiento con las bebidas de energ&iacute;a llevado a disminuido lamellipodia formaci&oacute;n y disminuido la proliferaci&oacute;n/viabilidad; mientras que en las c&eacute;lulas MDCK, tratamiento de bebidas de energ&iacute;a dio lugar a la desorganizaci&oacute;n de la actinia sin ning&uacute;n efecto sobre la proliferaci&oacute;n celular. Esto sugiere que los mecanismos subyacentes de retraso de la cicatrizaci&oacute;n pueden ser diferentes en los dos tipos celulares. Curiosamente, los retrasos en ambos tipos celulares no podr&iacute;an ser m&iacute;mico por tratamiento de cafe&iacute;na, taurina y glucosa, solas o combinadas. Adem&aacute;s, el tratamiento de culturas neuronal de polluelo prosenc&eacute;falo con bebidas de energ&iacute;a dio lugar a una inhibici&oacute;n dependiente de la dosis de neurita consecuencia. Los ensayos celulares utilizados en este estudio proporcionan un sistema coherente, cualitativo y cuantitativo para examinar los efectos de la combinatorios de los varios ingredientes usados en las bebidas de energ&iacute;a.

Los efectos de bebidas de energ&iacute;a en las c&eacute;lulas cultivadas.

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.

A course-based research experience: how benefits change with increased investment in instructional time.

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.

A central support system can facilitate implementation and sustainability of a Classroom-based Undergraduate Research Experience (CURE) in Genomics.

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 higher 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% versus 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 versus 8.4-8.8 genes per block), indicating higher 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.

Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution.

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. 

Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons.

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.

Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity.

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.

Computing neurite outgrowth and arborization in superior cervical ganglion neurons.

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.

Vidya Chandrasekaran

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Saint Mary's College of California

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Department of Biology