עבודה זו מומנה על ידי תרומות של המכון הלאומי להפרעות נוירולוגיות ושבץ (NS-19108), קרן מחקר מיגרנה, וקרן הלבן ריצ&#39;רד פ &#39;Kraig. גב &#39;פ&#39; מרשה Kraig סייעו בהכנת התקשורת בתרבות ותחזוקה של תרבויות פרוסה. אנו מודים ילנה גרינברג הערות ותיקונים אותה על גרסה סופית של מאמר זה.

<ol>
	<li>Aptowicz, C. O., Kunkler, P. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+plasticity+in+hippocampal+slice+cultures+involves+changes+in+voltage-gated+Na++channel+expression.">Homeostatic plasticity in hippocampal slice cultures involves changes in voltage-gated Na+ channel expression.</a> Brain Res. 998, 155-163 (2004).</li><li>Beattie, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synaptic+strength+by+glial+TNFalpha.">Control of synaptic strength by glial TNFalpha.</a> Science. 295, 2282-2285 (2002).</li><li>Breder, C. D., Dewitt, D., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+inducible+cyclooxygenase+in+rat+brain.">Characterization of inducible cyclooxygenase in rat brain.</a> J Comp Neurol. 355, 296-315 (1995).</li><li>Caggiano, A. O., Breder, C. D., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+elevation+of+cyclooxygenase-2,+but+not+lipoxygenase,+in+regions+synaptically+distant+from+spreading+depression.">Long-term elevation of cyclooxygenase-2, but not lipoxygenase, in regions synaptically distant from spreading depression.</a> J Comp Neurol. 376, 447-462 (1996).</li><li>Caggiano, A. O., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eicosanoids+and+nitric+oxide+influence+induction+of+reactive+gliosis+from+spreading+depression+in+microglia+but+not+astrocytes.">Eicosanoids and nitric oxide influence induction of reactive gliosis from spreading depression in microglia but not astrocytes.</a> J Comp Neurol. 369, 93-108 (1996).</li><li>Caggiano, A. O., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+and+4-aminopyridine+prevent+the+lipopolysaccharide-induced+outwardly+rectifying+potassium+current+and+interleukin-1beta+production+in+cultured+rat+microglia.">Prostaglandin E2 and 4-aminopyridine prevent the lipopolysaccharide-induced outwardly rectifying potassium current and interleukin-1beta production in cultured rat microglia.</a> J Neurochem. 70, 2357-2368 (1998).</li><li>Caggiano, A. O., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E+receptor+subtypes+in+cultured+rat+microglia+and+their+role+in+reducing+lipopolysaccharide-induced+interleukin-1beta+production.">Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production.</a> J Neurochem. 72, 565-575 (1999).</li><li>Calabrese, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+therapies+for+stroke+and+traumatic+brain+injury+often+display+U-shaped+dose+responses:+occurrence,+mechanisms,+and+clinical+implications.">Drug therapies for stroke and traumatic brain injury often display U-shaped dose responses: occurrence, mechanisms, and clinical implications.</a> Crit Rev Toxicol. 38, 557-577 (2008).</li><li>Calabrese, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+stress+response+terminology:+Integrating+the+concepts+of+adaptive+response+and+preconditioning+stress+within+a+hormetic+dose-response+framework.">Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework.</a> Toxicol Appl Pharmacol. 222, 122-128 (2007).</li><li>Calabrese, E. J., Baldwin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormesis:+the+dose-response+revolution.">Hormesis: the dose-response revolution.</a> Annu Rev Pharmacol Toxicol. 43, 175-197 (2003).</li><li>Hansel, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CD4++T-cell+gene+expression+in+allergic+subjects+using+two+different+microarray+platforms.">Analysis of CD4+ T-cell gene expression in allergic subjects using two different microarray platforms.</a> Allergy. 63, 366-369 (2008).</li><li>Hulse, R. E., Kunkler, P. E., Fedynyshyn, J. P., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+multiplexed+bead-based+cytokine+immunoassays+for+rat+serum+and+brain+tissue.">Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue.</a> J Neurosci Methods. 136, 87-98 (2004).</li><li>Hulse, R. E., Swenson, W. G., Kunkler, P. E., White, D. M., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+IgG+is+neuroprotective+via+enhancing+microglial+recycling+endocytosis+and+TNF-alpha.">Monomeric IgG is neuroprotective via enhancing microglial recycling endocytosis and TNF-alpha.</a> J Neurosci. 28, 12199-12211 (2008).</li><li>Hulse, R. E., Winterfield, J., Kunkler, P. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+clasmatodendrosis+in+hippocampal+organ+culture.">Astrocytic clasmatodendrosis in hippocampal organ culture.</a> Glia. 33, 169-179 (2001).</li><li>Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+and+Microglial+Hormetic+Involvement+in+Neurological+Health+and+Migraine.">TNF-alpha and Microglial Hormetic Involvement in Neurological Health and Migraine.</a> International Dose-Respose Society. , (2010).</li><li>Kunkler, P. E., Hulse, R. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+cytokine+protein+expression+profiles+from+spreading+depression+in+hippocampal+organotypic+cultures.">Multiplexed cytokine protein expression profiles from spreading depression in hippocampal organotypic cultures.</a> J Cereb Blood Flow Metab. 24, 829-839 (2004).</li><li>Kunkler, P. E., Hulse, R. E., Schmitt, M. W., Nicholson, C., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+current+source+density+analysis+in+hippocampal+organotypic+culture+shows+that+spreading+depression+occurs+with+uniquely+reversing+currents.">Optical current source density analysis in hippocampal organotypic culture shows that spreading depression occurs with uniquely reversing currents.</a> J Neurosci. 25, 3952-3961 (2005).</li><li>Kunkler, P. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+astrocytosis+from+excitotoxic+injury+in+hippocampal+organ+culture+parallels+that+seen+in+vivo.">Reactive astrocytosis from excitotoxic injury in hippocampal organ culture parallels that seen in vivo.</a> J Cereb Blood Flow Metab. 17, 26-43 (1997).</li><li>Kunkler, P. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+waves+precede+electrophysiological+changes+of+spreading+depression+in+hippocampal+organ+cultures.">Calcium waves precede electrophysiological changes of spreading depression in hippocampal organ cultures.</a> J Neurosci. 18, 3416-3425 (1998).</li><li>Kunkler, P. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P/Q+Ca2++channel+blockade+stops+spreading+depression+and+related+pyramidal+neuronal+Ca2++rise+in+hippocampal+organ+culture.">P/Q Ca2+ channel blockade stops spreading depression and related pyramidal neuronal Ca2+ rise in hippocampal organ culture.</a> Hippocampus. 14, 356-367 (2004).</li><li>Lambertsen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+protect+neurons+against+ischemia+by+synthesis+of+tumor+necrosis+factor.">Microglia protect neurons against ischemia by synthesis of tumor necrosis factor.</a> J Neurosci. 29, 1319-1330 (2009).</li><li>Lee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-protein+signaling+10+promotes+dopaminergic+neuron+survival+via+regulation+of+the+microglial+inflammatory+response.">Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.</a> J Neurosci. 28, 8517-8528 (2008).</li><li>Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormesis+and+disease+resistance:+activation+of+cellular+stress+response+pathways.">Hormesis and disease resistance: activation of cellular stress response pathways.</a> Hum Exp Toxicol. 27, 155-162 (2008).</li><li>Ning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+simultaneous+gene+profiling+of+84+drug-metabolizing+genes+in+primary+human+hepatocytes.">Systematic and simultaneous gene profiling of 84 drug-metabolizing genes in primary human hepatocytes.</a> J Biomol Screen. 13, 194-201 (2008).</li><li>Oppenheim, J., Feldman, M., Oppenheim, J. J., Feldman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in Cytokine Reference. 1, (2001).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29, e45-e45 (2001).</li><li>Stellwagen, D., Beattie, E. C., Seo, J. Y., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+AMPA+receptor+and+GABA+receptor+trafficking+by+tumor+necrosis+factor-alpha.">Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.</a> J Neurosci. 25, 3219-3228 (2005).</li><li>Stellwagen, D., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+scaling+mediated+by+glial+TNF-alpha.">Synaptic scaling mediated by glial TNF-alpha.</a> Nature. 440, 1054-1059 (2006).</li><li>Streit, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+as+neuroprotective,+immunocompetent+cells+of+the+CNS.">Microglia as neuroprotective, immunocompetent cells of the CNS.</a> Glia. 40, 133-139 (2002).</li><li>Wilde, G. J., Pringle, A. K., Sundstrom, L. E., Mann, D. A., Iannotti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+and+augmentation+of+ischaemia-related+neuronal+death+by+tumour+necrosis+factor-alpha+in+vitro.">Attenuation and augmentation of ischaemia-related neuronal death by tumour necrosis factor-alpha in vitro.</a> Eur J Neurosci. 12, 3863-3870 (2000).</li></ol>

אין ניגודי אינטרסים הכריז.

שני מושגים חשובים ביסודו חשוב התיחום של מערכת איתות ציטוקינים המעורבים neuroprotection קר נפשית מראש מומחשים איורים 6 ו -7. ראשית, ציטוקינים הם ריכוז נמוך מאוד מולקולות איתות במוח הנורמלי. עם זאת, שינויים פיזיולוגיים ריכוז ציטוקינים יש פוטנציאל עצום לשנות את מבנה המוח ותפקודו (כלומר, פנוטיפ) בגלל היכולת שלהם לשנות את ביטוי הגנים (איור 6). יתר על כן, הציטוקינים הם מיותרים מאוד pleiotropic בכך ציטוקינים מרובים יכולה להיות השפעה דומה ציטוקין בודד יכולה להיות השפעה משתנה (איור 7). לכן, כדי לקבוע במדויק את המולדת ציטוקינים-בסיסים neuroprotection מהקור-נפשית מראש (או גירויים אחרים נפשית מראש פיזיולוגיים), ניתוח מורכב של משתנים הקשורים איתות חייבת להיות נחושה. מטרה זו מושגת באמצעות אסטרטגיות assay multiplexed. זה יהיה להקים את ציטוקינים &quot;חתימה&quot; של neuroprotection קר נפשית מראש. <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="איור 6" /> איור 6. כוחה של ציטוקינים איתות במוח. האיורים להעביר את הכוח איתות עצום של ריכוזים פיזיולוגיים של ציטוקינים במוח לעומת ריכוזי מוכרת היטב עמיתיהם אחרים. ריכוז מיוצג ההופכי של המרחק, החל זיף חתול בודד כנקודת התייחסות. נתרן (מ -1 10 מאויר על ידי גרגר פלפל) ואשלגן (10 מ -3 מאויר על ידי עגבניה) נמצאים במרחב ביניים המוח ברמות של סביב 150 ו - 3 מ&quot;מ, בהתאמה, יש מוכרים היטב תפקידים תאים עצביים לתפקד אלקטרו. כמו כן, ה-pH (כלומר, ~ 10 -7 M רמות של יוני מימן ואייר כמו 780 מטרים לאורך שפת האגם של שיקגו) וסידן (כלומר, ~ 10 -8 M רמות כפי שמוצג 7.8 ק&quot;מ לראות תמונת לווין גוגל שפת האגם של שיקגו מ מקורמיק המקום לנקודה חרטום בהייד פארק ליד אוניברסיטת שיקגו). בנוסף, העצביים המשוחררים לחלל ביניים על ריכוזים אלה משפיעים על הפעילות המקומית אזור במוח. לעומת זאת, ציטוקינים (כפי שמוצג המרחק מכדור הארץ למאדים) יכול לשנות את תפקוד המוח בריכוזים יותר מעשר מיליון פעמים פחות. <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="איור 7" /> איור 7. איתות אינטראקטיבית של מסלולים ציטוקינים מולדים. ציטוקינים הם מיותרים מאוד pleiotropic בכך ציטוקינים מרובים יכולה להיות השפעה דומה ציטוקין בודד יכולה להיות השפעה משתנה. גיוון כזה נובעת איתות אינטראקטיביים מורכבים המתרחשת ברמה של ligands, קולטנים, ו phosphoproteins. מורכבות נוספת נובעת מהעובדה כי המוח מורכב של סוגי תאים שונים, שכל אחד מהם מסוגל ציטוקינים תא קולטן ספציפי מולדת, ושינויים phosphoprotein בנושא. לשם המחשה כאן, רק ציטוקינים מולדת איתות המסלולים (הנגזרות מחקרים תא החיסון) מוצגים. לשם הפשטות, המוח הוא נמשך כמו תא בודד (קו לבן) מראה את יחסי הגומלין פוטנציאל מולד ציטוקינים (IL-1α ו-IL-1β (המכונה כאן כמו IL-1α / β), TNF-α, IL-6, IFN-γ ו IL-10), קולטנים (IL-1R1, TNFR1, IL-6/gp130, IFNγR, ו-IL-10R), ו phosphoproteins (כלומר, קינאזות ERK1 / 2, p38 (p38-MAPK) ו JNK) ואת גורמי תעתוק (ATF-2, NFκB ו STAT3). לדוגמה, TNF-α אותות דרך TNFR1 כדי JNK, p38 ו-MAPK ERK1 / 2, אשר מפעילה ביטוי גנים באמצעות ATF-2. TNF-α גם משנה את ביטוי הגנים ישירות באמצעות הפעלת NFκB. יחד, הפעלה של גורמי שעתוק אלה מעוררות מוגברת (חץ) וירידה (סוף בוטה) ביטוי של ציטוקינים הקולטנים שלהם כפי שצוין. חשוב לציין, מסלולים אלה מראים כי שינויים ציטוקינים אחד (למשל, TNF-α) ההשפעה של ייצור (למשל, IL-1β) ציטוקינים אחרים. לכן, כדי להקים במדויק את ציטוקינים-בסיסים neuroprotection מהקור-נפשית מראש, ניתוח מורכב של משתנים הקשורים איתות חייבת להיות נחושה. זה יהיה להקים את ציטוקינים מולדת &quot;חתימה&quot; של קר נפשית מראש. (תמונה נערך מנתוני התייחסות # 25).

הכנת תרבויות Slice <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> Millicell-CM בתרבית רקמה מוסיף (30 מ&quot;מ) </td><td> תרמו פישר </td><td> PICM0-3050 </td><td></td></tr><tr><td> בינוני בסל הנשר </td><td> Invitrogen </td><td> 21010 </td><td></td></tr><tr><td> ארל s פתרון מאוזן מלח </td><td> סיגמא </td><td> E2888 </td><td></td></tr><tr><td> סוס בסרום </td><td> Invitrogen </td><td> 26050-088 </td><td></td></tr><tr><td> Glutamax </td><td> Invitrogen </td><td> 35050 </td><td></td></tr><tr><td> גנטמיצין </td><td> Invitrogen </td><td> 15710-064 </td><td></td></tr><tr><td> Fungizone </td><td> Invitrogen </td><td> 15295 </td><td></td></tr><tr><td> D-גלוקוז </td><td> סיגמא </td><td> G8769 </td><td></td></tr><tr><td> BSL-1 קטר ברדס </td><td> אופה </td><td></td><td></td></tr><tr><td> McIlwain המסוק רקמה </td><td> Brinkmann </td><td></td><td></td></tr><tr><td> טפלון דיסקים </td><td> Brinkmann </td><td> 023-49-310-1 </td><td></td></tr><tr><td> מרית </td><td> תרמו פישר </td><td> 14-373-25A </td><td></td></tr><tr><td> Curved איריס מספריים </td><td> המדע כלים פיין </td><td> 14091-09 </td><td></td></tr><tr><td> מספריים ישר ארוך </td><td> המדע כלים פיין </td><td> 14002-14 </td><td></td></tr><tr><td> לונג מלקחיים </td><td> תרמו פישר </td><td> 13-812-40 </td><td></td></tr><tr><td> בזווית מלקחיים </td><td> המדע כלים פיין </td><td> 11808-02 </td><td></td></tr><tr><td> קצר מלקחיים </td><td> תרמו פישר </td><td> 13-812-38 </td><td></td></tr><tr><td> # 5 Inox מלקחיים </td><td> המדע כלים פיין </td><td> 11254-20 </td><td></td></tr><tr><td> 2 איריס spatulae </td><td> המדע כלים פיין </td><td> 10094-13 </td><td></td></tr><tr><td> 150 x 15 מ&quot;מ צלחות פטרי </td><td> תרמו פישר </td><td> 08-757-148 </td><td></td></tr><tr><td> 60 x 15 מ&quot;מ צלחות פטרי </td><td> תרמו פישר </td><td> 08-757-100B </td><td></td></tr><tr><td> Stereomicroscope Wild M8 </td><td> Wild Heerbrugg </td><td></td><td></td></tr><tr><td> מלח איזון גיי של פתרון </td><td> סיגמא </td><td> G9779 </td><td></td></tr><tr><td> Blak-Ray למדידת גלים קצרים סגול מטר </td><td> UVP </td><td> J-225 </td><td></td></tr><tr><td> 6-Falcon היטב מנות תרבות </td><td> תרמו פישר </td><td> 08-772-1B </td><td></td></tr><tr><td> Neurobasal </td><td> Invitrogen </td><td> 21103</td><td></td></tr><tr><td> תוסף B27 </td><td> Invitrogen </td><td> 17504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td> ג&#39;מיני Bioproducts </td><td> 400-160-010 </td><td></td></tr><tr><td> חומצה אסקורבית </td><td> סיגמא </td><td> A4544 </td><td></td></tr><tr><td> CO 2 Analyzer </td><td> בכרך </td><td> # 2820 </td><td></td></tr></tbody></table> לפני ההקרנה של Vitality תרבויות Slice <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> Sytox גרין </td><td> Invitrogen </td><td> S7020 </td><td></td></tr><tr><td> DMIBRE הפוכה מיקרוסקופ </td><td> לייקה </td><td></td><td></td></tr></tbody></table> הקרה נפשית מראש <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> BSL-2 פומה הוד </td><td> NuAire </td><td></td><td></td></tr></tbody></table> Excitotoxic פגיעה <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> CoolSnap fx מצלמת CCD </td><td> Photometrics </td><td></td><td></td></tr><tr><td> Fluoroscein </td><td> Invitrogen </td><td> F36915 </td><td></td></tr><tr><td> MetaMorph תוכנה </td><td> מולקולרית התקנים </td><td> נ 7.5.04 </td><td></td></tr><tr><td> 100 hemacytometer עמוק מיקרומטר </td><td> האוסר מדעי </td><td></td><td></td></tr><tr><td> N-methyl-D-aspartate </td><td> Calbiochem </td><td> 454575 </td><td></td></tr><tr><td> SigmaStat תוכנה </td><td> Systat תוכנה בע&quot;מ </td><td> נ 3.5 </td><td></td></tr></tbody></table> עמית טיפולים להחיל עם הקרה נפשית מראש <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> sTNFR1 </td><td> R &amp; D מערכות </td><td> 425-R1-050 </td><td></td></tr><tr><td> שור סרום אלבומין </td><td> סיגמא </td><td> A-4503 </td><td></td></tr></tbody></table> בידוד RNA <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> RNAl ater </td><td> Ambion </td><td> AM7021 </td><td></td></tr><tr><td> מיקרו RNEasy Kit </td><td> Qiagen </td><td> 74004 </td><td></td></tr><tr><td> β-mercaptoethanol </td><td> דיג </td><td> 03446-100 </td><td></td></tr><tr><td> Diposable 1.5 RNAse ללא העלי </td><td> דרום ג&#39;רזי כלי Precision ו, עובש בע&quot;מ </td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td> Promega </td><td> N261A </td><td></td></tr><tr><td> טריס [hydroxymethyl] aminomethane </td><td> סיגמא </td><td> T3069 </td><td></td></tr><tr><td> EDTA (ethylenediaminetetraacetic חומצה disodium מימה מלח </td><td> סיגמא </td><td> # E7889 </td><td></td></tr></tbody></table> RNA כימות <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> RiboGreen Assay </td><td> Invitrogen </td><td> R11491 </td><td></td></tr><tr><td> שמרים tRNA </td><td> Invitrogen </td><td> 15401-011 </td><td></td></tr><tr><td> 1.5 צנטריפוגות צינורות מ&quot;ל </td><td> ארה&quot;ב מדעי </td><td> 16155500 </td><td></td></tr><tr><td> גם 96-assay פלורסנט צלחת </td><td> Nunc </td><td> DK-4000 </td><td></td></tr><tr><td> 96 גם צלחת פלורסנט הקורא </td><td> טקאן </td><td> ספייר מה&quot;ניו II </td><td></td></tr></tbody></table> PCR כמותי <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> שעתוק iScript הפוכה ערכת </td><td> Bio-Rad </td><td> 170-8891 </td><td></td></tr><tr><td> SYBR גרין </td><td> Invitrogen </td><td> S7563 </td><td></td></tr><tr><td> תקי פלטינום פולימראז </td><td> Invitrogen </td><td> RNA Ultrasense 11732-927 </td><td></td></tr><tr><td> iCycler thermocycler </td><td> Bio-Rad </td><td> iCycler אופטי מודול </td><td></td></tr><tr><td> iCycler תוכנת המערכת </td><td> Bio-Rad </td><td> נ 3.1 </td><td></td></tr></tbody></table> כמותי PCR עבור microarrays <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> RNase חוץ </td><td> מולקולרית Bioproducts </td><td> # 7000 </td><td></td></tr><tr><td> RT 2 סטרנד ראשון Kit </td><td> SABiosciences </td><td> C-03 </td><td></td></tr><tr><td> SYBR גרין מבוססי PCR לערבב </td><td> SABiosciences </td><td> PA-011 </td><td> ספציפיות iCycler </td></tr><tr><td> RT 2 Profiler מערך ה-PCR </td><td> SABiosciences </td><td> פרן-011A </td><td></td></tr></tbody></table> Proteomic Assay <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג/ דגם מספר </td><td> תגובות </td></tr><tr><td> תמוגה Cell ערכת </td><td> Bio-Rad </td><td> 171-304011 </td><td></td></tr><tr><td> Microcentrifuge </td><td> פישר סיינטיפיק </td><td> מיקרו 7 </td><td></td></tr><tr><td> BSA מניות חלבון </td><td> Bio-Rad </td><td> 500-0007 </td><td></td></tr><tr><td> Assay BCA חלבון ערכת </td><td> Thermo Scientific </td><td> 23225 </td><td></td></tr><tr><td> חיסונית 96 microwell צלחות </td><td> Nunc </td><td> 449824 </td><td></td></tr><tr><td> איטום קלטת </td><td> Bio-Rad </td><td> 2239444 </td><td></td></tr><tr><td> 96 גם צלחת הקורא </td><td> פרקין אלמר </td><td> 1420-mulilabel לדלפק </td><td></td></tr><tr><td> Bioplex עכברוש TNF-α ציטוקינים assay </td><td> Bio-Rad </td><td> X0000001T </td><td></td></tr></tbody></table> Immunostaining <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג / דגם מספר </td><td> תגובות </td></tr><tr><td> 16% Paraformaldehyde </td><td> אלפא Aesar </td><td> 43368 </td><td></td></tr><tr><td> ליזין </td><td> סיגמא </td><td> L-6001 </td><td></td></tr><tr><td> סודיום פוספט </td><td> פישר סיינטיפיק </td><td> S374-1 </td><td></td></tr><tr><td> נתרן periodate </td><td> סיגמא </td><td> S-1878 </td><td></td></tr><tr><td> נתרן אזיד </td><td> סיגמא </td><td> S-2002 </td><td></td></tr><tr><td> מי חמצן (30%) </td><td> סיגמא </td><td> H1009 </td><td></td></tr><tr><td> עיזים בסרום </td><td> קולורדו החברה סרום </td><td> CS 0920 </td><td></td></tr><tr><td> טריטון X-100 </td><td> סיגמא </td><td> T-8787 </td><td></td></tr><tr><td> מכתים מנות </td><td> טד פלה, Inc </td><td> 14511 </td><td></td></tr><tr><td> פלייט שאכר </td><td> Lab-Line מכשירים, Inc </td><td> 3520 </td><td></td></tr><tr><td> # 1 סנן מאמרים (185 מ&quot;מ) </td><td> Whatman </td><td> 1001-185 </td><td></td></tr><tr><td> 3,3-diaminobenzidine </td><td> סיגמא </td><td> D8001 </td><td></td></tr><tr><td> דימתיל sulfoxide </td><td> סיגמא </td><td> D8418 </td><td></td></tr><tr><td> אתנול </td><td> Pharmco-AAPER </td><td> 111000200 </td><td></td></tr><tr><td> קסילן </td><td> פישר סיינטיפיק </td><td> X3S-4 </td><td></td></tr><tr><td> התקשורת הרכבה Permount </td><td> פישר סיינטיפיק </td><td> SP15-100 </td><td></td></tr><tr><td> Cryostat </td><td> לייקה </td><td> 3 ס&quot;מ 050 S </td><td></td></tr><tr><td> רקמות-Tek </td><td> טד פלה, Inc </td><td> 27050 </td><td></td></tr><tr><td> עדין קצה מברשת צבע </td><td> טד פלה </td><td> 11806 </td><td></td></tr><tr><td> PAP חסימת עט </td><td> טד פלה, Inc </td><td> 22309 </td><td></td></tr><tr><td> SFX אות משפר </td><td> Invitrogen </td><td> A31631 </td><td></td></tr><tr><td> להאריך Antifade </td><td> Invitrogen </td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

פציעה נוירולוגיות הוא הגורם השכיח של תחלואה ותמותה מן ההרדמה הכללית ניתוחים הקשורים שניתן להקל על ידי פיתוח יעיל, קל לניהול וטיפולים נפשית מראש בטוח. אנו מבקשים להגדיר את האותות העצביים האחראים החיסונית קר נפשית מראש כאמצעי לזיהוי מטרות הרומן לפיתוח הרפוי כדי להגן על המוח לפני תחילת פציעה. ברמה נמוכה הפרו דלקתי מתווך איתות שינויים לאורך זמן חיוניים neuroprotection קר נפשית מראש. איתות זה עולה בקנה אחד עם העקרונות הבסיסיים של hormesis מיזוג פיזיולוגיים, אשר דורשים גירויים irritative להגיע גודל סף עם מספיק זמן הסתגלות לגירויים להגנה להיות ברור. לפיכך, התוויית איתות החיסון המעורבים neuroprotection קר נפשית מראש דורש כי מערכות ביולוגיות מניפולציות ניסיוני בתוספת היכולות הטכניות ולייצר מאוד רגיש. הגישה שלנו היא להשתמש תרבויות פרוסה בהיפוקמפוס כמו במודל חוץ גופית כי מקרוב משקף שלהם עם עמיתיהם vivo רב סינפטיים רשתות עצביות מושפע macroglia בוגרת שקט / microglia. מצב זה גליה חשוב במיוחד עבור microglia שכן הם המקור העיקרי של ציטוקינים, אשר פעיל בטווח femtomolar. כמו כן, תרבויות פרוסה יכול להישמר במבחנה במשך מספר שבועות, וזה מספיק זמן כדי לעורר גירויים הפעלת ולהעריך התגובות אדפטיבית. לבסוף, תנאים סביבתיים ניתן לשלוט בצורה מדויקת באמצעות תרביות פרוסה כך ציטוקינים כי האיתות של קר נפשית מראש ניתן למדוד, חיקה, ומתנגן לנתח את ההיבטים צומת קריטי. איתות ציטוקין מנתחת המערכת דורשת שימוש בטכניקות multiplexed רגיש לשחזור. אנו משתמשים כמותי PCR עבור TNF-α למסך להפעלת microglial ואחריו כמותי ההקרנה בזמן אמת qPCR מערך להעריך רקמות רחב שינויים ציטוקינים. האחרון הוא אמצעי רגיש ביותר לשחזור למדוד מערכת ציטוקינים מרובים איתות שינויים בו זמנית. שינויים משמעותיים הם אישרו עם qPCR ממוקד ולאחר מכן גילוי חלבון. אנחנו בדיקה עבור מבוססי רקמות ציטוקינים שינויים חלבון באמצעות זרם multiplexed microsphere מבחני cytometric באמצעות טכנולוגיית Luminex. תא ספציפי ייצור ציטוקינים נקבעת עם אימונוהיסטוכימיה פעמיים התווית. יחדיו, זו רקמת המוח הכנה וסגנון השימוש, מצמידים את האסטרטגיות החקירה הציע, יכול להיות גישה אופטימלית לזיהוי מטרות פוטנציאליות לפיתוח הרפוי הרומן הזה יכול לחקות את היתרונות של קר נפשית מראש.

Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

Strategies for Study of Neuroprotection from Cold-preconditioning

אנו מבקשים להגדיר את האותות העצביים האחראים החיסונית קר נפשית מראש כאמצעי לזיהוי מטרות הרומן לפיתוח הרפוי כדי להגן על המוח לפני תחילת פציעה. אנו מציגים אסטרטגיות עבודה כזו הדורשים מערכות ביולוגיות, מניפולציות ניסיוני בתוספת היכולות הטכניות, כי הם לשעתקו מאוד רגיש.

אסטרטגיות לחקר neuroprotection מן הקרה נפשית מראש

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

strategies-for-study-of-neuroprotection-from-cold-preconditioning

Research

JoVE Journal

Neuroscience

14.8K Views.  The University of Chicago Medical Center. אנו מבקשים להגדיר את האותות העצביים האחראים החיסונית קר נפשית מראש כאמצעי לזיהוי מטרות הרומן לפיתוח הרפוי כדי להגן על המוח לפני תחילת פציעה. אנו מציגים אסטרטגיות עבודה כזו הדורשים מערכות ביולוגיות, מניפולציות ניסיוני בתוספת היכולות הטכניות, כי הם לשעתקו מאוד ר...

Video: Strategies for Study of Neuroprotection from Cold-preconditioning

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

הדם במוח חמצון מדידה על בסיס מרווה חמצן תלויי של זרחני

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

לייזר Microdissection לכידה של תסיסנית נוירונים היקפיים

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

הכנת פרוסות בהיפוקמפוס של חולדות חריפה העכברים הטרנסגניים לחקר שינויים Synaptic במהלך ההזדקנות עמילואיד פתולוגיה

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation של נוירונים בין פרוסות המוח מכרסמים לחקר הילוכים Synaptic ותמונה מסופי Presynaptic

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

תמונה רציפה-הלבנה להתוות תאי שווא יחידים בצומת Neuromuscular

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

ממברנה דיי הדמיה אפשרית של ventromedial היפותלמוס נוירונים מעכברים בוגרים ללמוד גלוקוז חישה

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

בידוד של CA1 גרעיני שברים מועשרים מSlices בהיפוקמפוס לחקר פעילות תלויה גרעיני יבוא של חלבונים Messenger synapto-גרעיניים

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Analyzer הנייד מבוססת עכבה בזמן אמת ככלי להתוות מסלולים מולקולריים מעורבים ברעילות העצבית וneuroprotection בקו תאים עצבי

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic תרבויות פרוסה בהיפוקמפוס כמודל מחקר Neuroprotection, Invasiveness של תאים סרטניים

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

זמן תלויי תגובת הרשת בתגובה לגירוי של תרבויות תאים עצביים על מערכי מיקרו-אלקטרודה

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...