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In JoVE (1)
- Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling
Other Publications (4)
Articles by Norman Atkins in JoVE
Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling
Norman Atkins, Charlie M. Miller, Joseph R. Owens, Fred W. Turek
Center for Sleep and Circadian Biology, Northwestern University
RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.
Other articles by Norman Atkins on PubMed
Proceedings of the National Academy of Sciences of the United States of America. Aug, 2008 | Pubmed ID: 18719122
A significant challenge to understanding dynamic and heterogeneous brain systems lies in the chemical complexity of secreted intercellular messengers that change rapidly with space and time. Two solid-phase extraction collection strategies are presented that relate time and location of peptide release with mass spectrometric characterization. Here, complex suites of peptide-based cell-to-cell signaling molecules are characterized from the mammalian suprachiasmatic nucleus (SCN), site of the master circadian clock. Observed SCN releasates are peptide rich and demonstrate the co-release of established circadian neuropeptides and peptides with unknown roles in circadian rhythms. Additionally, the content of SCN releasate is stimulation specific. Stimulation paradigms reported to alter clock timing, including electrical stimulation of the retinohypothalamic tract, produce releasate mass spectra that are notably different from the spectra of compounds secreted endogenously over the course of the 24-h cycle. In addition to established SCN peptides, we report the presence of proSAAS peptides in releasates. One of these peptides, little SAAS, exhibits robust retinohypothalamic tract-stimulated release from the SCN, and exogenous application of little SAAS induces a phase delay consistent with light-mediated cues regulating circadian timing. These mass spectrometry-based analyses provide a new perspective on peptidergic signaling within the SCN and demonstrate that the integration of secreted compounds with information relating time and location of release generates new insights into intercellular signaling in the brain.
Endogenous Peptide Discovery of the Rat Circadian Clock: a Focused Study of the Suprachiasmatic Nucleus by Ultrahigh Performance Tandem Mass Spectrometry
Molecular & Cellular Proteomics : MCP. Feb, 2010 | Pubmed ID: 19955084
Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body's circadian rhythms is an ongoing research area. This important time-keeping system requires a complex suite of peptide hormones and transmitters that remain incompletely characterized. Here, capillary liquid chromatography and FTMS have been coupled with tailored software for the analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, peptide extraction, identification, and characterization from tandem FTMS data with <5-ppm mass accuracy produced a hyperconfident list of 102 endogenous peptides, including 33 previously unidentified peptides, and 12 peptides that were post-translationally modified with amidation, phosphorylation, pyroglutamylation, or acetylation. This characterization of endogenous peptides from the SCN will aid in understanding the molecular mechanisms that mediate rhythmic behaviors in mammals.
PloS One. 2010 | Pubmed ID: 20830308
Neuropeptides are critical integrative elements within the central circadian clock in the suprachiasmatic nucleus (SCN), where they mediate both cell-to-cell synchronization and phase adjustments that cause light entrainment. Forward peptidomics identified little SAAS, derived from the proSAAS prohormone, among novel SCN peptides, but its role in the SCN is poorly understood.
Frontiers in Neuroendocrinology. Oct, 2011 | Pubmed ID: 21334363
The chemical complexity of cell-to-cell communication has emerged as a fundamental challenge to understanding brain systems. This is certainly true for the hypothalamus, where neuropeptide signals are heterogeneous, localized and dynamic. Thus far, most hypothalamic peptidomic studies have centered on the entire structure; however, recent advances in collection strategies and analytical technologies have enabled direct, high-resolution peptidomic profiles focused on two regions of interest, the suprachiasmatic and supraoptic nuclei, including their sub-regions and individual cells. Suites of peptides now can be identified and probed for function. High spatial and analytical sensitivities reveal that discrete hypothalamic nuclei have distinct peptidomic signatures. Peptidomic discovery not only reveals unanticipated complexity, but also peptides previously unknown that act as key circuit components. Analysis of tissue releasates identifies peptides secreted into the extracellular environment and available for transmitting intercellular signals. Direct sampling techniques define peptide-releasate profiles in spatial, temporal and event-dependent patterns. These approaches are providing remarkable new insights into the complexity of neuropeptidergic cell-to-cell signaling central to neuroendocrine physiology.