Ce travail a été financé par des subventions de l&#39;Institut national des troubles neurologiques et des maladies (NS-19108), la Fondation pour la recherche Migraine, et la Fondation blanche à Richard P. Kraig. Mme Marcia P. Kraig aidé à la préparation des milieux de culture et d&#39;entretien des cultures tranche. Nous tenons à remercier Yelena Grinberg pour ses commentaires et révisions sur une version finale de cet article.

<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>

Deux concepts fondamentalement important important à la délimitation du système de signalisation des cytokines impliquées dans la neuroprotection froid préconditionnement sont illustrés dans les figures 6 et 7. Tout d&#39;abord, les cytokines sont extrêmement faibles molécules de signalisation de concentration dans le cerveau normal. Néanmoins, physiologiques des changements de concentration de cytokines ont un énorme potentiel de modifier la structure du cerveau et la fonction (c.-à-phénotype) en raison de leur capacité à modifier l&#39;expression des gènes (Figure 6). De plus, les cytokines sont très redondantes et pléiotrope en ce que plusieurs cytokines peuvent avoir des effets similaires et une seule cytokine peut avoir des effets variables (figure 7). Ainsi, pour établir avec précision les cytokines innées des bases pour la neuroprotection du froid préconditionnement (ou d&#39;autres stimuli physiologiques de préconditionnement), analyse composite des variables de signalisation connexes doit être déterminée. Ceci est accompli grâce à des stratégies d&#39;analyse multiplexés. Ceci permettra d&#39;établir la cytokine «signature» de froidpréconditionnement neuroprotection. <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="Figure 6" /> Figure 6. Puissance de signalisation des cytokines cerveau. Les illustrations transmettre le pouvoir immense de signalisation de concentrations physiologiques de cytokines cérébrales par rapport aux concentrations d&#39;autres bien connus homologues. Concentration est représentée comme étant l&#39;inverse de la distance, en commençant par une tige souple à ressort unique comme point de référence. De sodium (10 -1 M illustrée par un grain de poivre) et de potassium (10 -3 M illustrée par la tomate) sont présents dans l&#39;espace interstitiel du cerveau à des niveaux de l&#39;ordre de 150 et 3 mm, respectivement, et ont bien reconnu rôles dans fonction électrophysiologique des cellules neuronales. De même, le pH (par exemple, ~ 10 -7 M niveaux d&#39;ions hydrogène et illustrés comme 780 mètres le long au bord du lac de Chicago) et de calcium (par exemple, ~ 10 -8 M niveaux montré que 7,8 kilomètres vu partir d&#39;une image satellite de Google ainsi que Chicago du lac de McCormick Place à Promontory Point, à Hyde Park, près de l&#39;Université de Chicago). En outre, des neurotransmetteurs libérés de l&#39;espace interstitiel au cours de ces concentrations affecter l&#39;activité locale région du cerveau. En revanche, les cytokines (comme le montre la distance entre la Terre et Mars) peuvent altérer le fonctionnement du cerveau à des concentrations de plus de dix millions de fois moins. <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="Figure 7" /> Figure 7. Signalisation interactifs de voies de cytokines innées. Cytokines sont très redondants et pléiotrope en ce que plusieurs cytokines peuvent avoir des effets similaires et une seule cytokine peut avoir des effets variables. Cette diversité provient de signalisation interactif complexe qui se produit au niveau des ligands, des récepteurs et des phosphoprotéines. Complexité supplémentaire provient du fait que le cerveau se compose de différents types de cellules, chaque cellule capable de cytokine spécifique innée, récepteur, et liée phosphoprotéine changes. À titre d&#39;exemple ici, seules les voies de signalisation des cytokines innées (dérivée des études de cellules immunitaires) sont affichés. Par souci de simplicité, le cerveau est dessinée comme une seule cellule (ligne blanche) montrant les interactions potentielles de cytokines innées (IL-1α et l&#39;IL-1β (appelée ici l&#39;IL-1α / β), le TNF-α, IL-6, IFN-γ et l&#39;IL-10), les récepteurs (IL-1R1, TNFR1, IL-6/gp130, IFNyR, et IL-10R), et phosphoprotéines (c.-à-kinases ERK1 / 2, P38 (P38-MAPK) et JNK) facteurs de transcription et (ATF-2, NFkB, et STAT3). Par exemple, le TNF-α signaux via TNFR1 à JNK, p38-MAPK et ERK1 / 2, ce qui déclenche l&#39;expression des gènes par l&#39;intermédiaire ATF-2. TNF-α modifie également l&#39;expression des gènes directement par l&#39;activation NFkB. Ensemble, l&#39;activation de ces facteurs de transcription evoke augmenté (flèche) et une diminution (bout franc) l&#39;expression de cytokines et de leurs récepteurs comme indiqué. Fait important, ces voies de montrer que les changements dans une cytokine (par exemple, le TNF-α) influence prouction d&#39;autres (par exemple, l&#39;IL-1β) des cytokines. Ainsi, pour déterminer de manière précise les cytokines bases pour la neuroprotection du froid préconditionnement, analyse composite des variables de signalisation connexes doit être déterminée. Ceci permettra d&#39;établir la cytokine innée &quot;signature&quot; du froid préconditionnement. (L&#39;image a été compilé à partir des données de référence n ° 25.)

Préparation des cultures Slice <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> Millicell-CM inserts de culture de tissus (30 mm) </td><td> Thermo Fisher </td><td> PICM0-3050 </td><td></td></tr><tr><td> Moyen de base de Eagle </td><td> Invitrogen </td><td> 21010 </td><td></td></tr><tr><td> Earle s Balanced Salt Solution </td><td> Sigma </td><td> E2888 </td><td></td></tr><tr><td> Sérum de cheval </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> Gentamicine </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-glucose </td><td> Sigma </td><td> G8769 </td><td></td></tr><tr><td> BSL-1 hotte </td><td> Boulanger </td><td></td><td></td></tr><tr><td> Tissue chopper McIlwain </td><td> Brinkmann </td><td></td><td></td></tr><tr><td> Disques de téflon </td><td> Brinkmann </td><td> 023-49-310-1 </td><td></td></tr><tr><td> Spatule </td><td> Thermo Fisher </td><td> 14 à 373-25A </td><td></td></tr><tr><td> Courbe ciseaux à iris </td><td> Outils Fine Science </td><td> 14091-09 </td><td></td></tr><tr><td> Longs ciseaux droits </td><td> Outils Fine Science </td><td> 14002-14 </td><td></td></tr><tr><td> Pince à bec long </td><td> Thermo Fisher </td><td> 13-812-40 </td><td></td&gt; </tr><tr><td> Pinces angulaires </td><td> Outils Fine Science </td><td> 11808-02 </td><td></td></tr><tr><td> Pince courte </td><td> Thermo Fisher </td><td> 13-812-38 </td><td></td></tr><tr><td> N ° 5 Inox forceps </td><td> Outils Fine Science </td><td> 11254-20 </td><td></td></tr><tr><td> 2 Iris spatules </td><td> Outils Fine Science </td><td> 10094-13 </td><td></td></tr><tr><td> 150 x 15 mm boîte de Pétri </td><td> Thermo Fisher </td><td> 08-757-148 </td><td></td></tr><tr><td> 60 x 15 mm boîte de Pétri </td&gt; <td> Thermo Fisher </td><td> 08 à 757-100B </td><td></td></tr><tr><td> Sauvage M8 stéréomicroscope </td><td> Wild Heerbrugg </td><td></td><td></td></tr><tr><td> La solution Gey équilibre salin </td><td> Sigma </td><td> G9779 </td><td></td></tr><tr><td> Blak-Ray mètre de mesure ultraviolet à ondes courtes </td><td> UVP </td><td> J-225 </td><td></td></tr><tr><td> 6-et boîtes de culture Falcon </td><td> Thermo Fisher </td><td> 08-772-1B </td><td></td></tr><tr><td> Neurobasal </td><td> Invitrogen </td><td> 21103</td><td></td></tr><tr><td> B27 supplément </td><td> Invitrogen </td><td> 17504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td> Bioproduits Gémeaux </td><td> 400-160-010 </td><td></td></tr><tr><td> L&#39;acide ascorbique </td><td> Sigma </td><td> A4544 </td><td></td></tr><tr><td> Analyseur de CO 2 </td><td> Bacharach </td><td> # 2820 </td><td></td></tr></tbody></table> Pré-sélection pour la vitalité des cultures Slice <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td><strong&gt; Société </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> Sytox vert </td><td> Invitrogen </td><td> S7020 </td><td></td></tr><tr><td> DMIBRE microscope inversé </td><td> Leica </td><td></td><td></td></tr></tbody></table> Froid Préconditionnement <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> BSL-2 Hotte </td><td> NuAire </td><td></td><td></td></tr></tbody></table> Blessure excitotoxique <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> CooISNAP fx caméra CCD </td><td> Photométrie </td><td></td><td></td></tr><tr><td> Fluorescéine </td><td> Invitrogen </td><td> F36915 </td><td></td></tr><tr><td> MetaMorph logiciel </td><td> Molecular Devices </td><td> v 7.5.04 </td><td></td></tr><tr><td> 100 hématimètre um de profondeur </td><td> Hausser scientifique </td><td></td><td></td></tr><tr><td> N-méthyl-D-aspartate </td><td> Calbiochem </td><td> 454575 </td><td></td></tr><tr><td> SigmaStat logiciel </td><td> Systat Software Inc </td><td> version 3.5 </td><td></td></tr></tbody></table> Co-traitements appliqués à froid Préconditionnement <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> sTNFR1 </td><td&gt; R &amp; D Systems </td><td> 425-R1-050 </td><td></td></tr><tr><td> Sérum Albumine Bovine </td><td> Sigma </td><td> A-4503 </td><td></td></tr></tbody></table> RNA Isolation <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> Ater rne </td><td> Ambion </td><td> AM7021 </td><td></td></tr><tr><td> Micro RNeasy Kit </td><td> Qiagen </td><td> 74004 </td><td></td></tr><tr><td> β-mercaptoéthanol </td><td> Pêcheur </td><td> 03446-100 </td><td></td></tr><tr><td> Diposable 1,5 RNAse free Pilon </td><td> Outil de précision du Sud Jersey et à la moisissure, Inc </td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td> Promega </td><td> N261A </td><td></td></tr><tr><td> Tris [hydroxyméthyl] aminométhane </td><td> Sigma </td><td> T3069 </td><td></td></tr><tr><td> EDTA (acide éthylènediamine-hydrate de sel disodique </td><td> Sigma </td><td> # E7889 </td><td></td></tr></tbody></table> Quantification ARN <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> RiboGreen Assay </td><td> Invitrogen </td><td> R11491 </td><td></td></tr><tr><td> ARNt de levure </td><td> Invitrogen </td><td> 15401-011 </td><td></td></tr><tr><td> Tubes de 1,5 ml à centrifuger </td><td> USA Scientific </td><td> 16155500 </td><td></td></tr><tr><td> Plaque de 96 puits dosage par fluorescence </td><td> Nunc </td><td> DK-4000 </td><td></td></tr><tr><td> 96-et lecteur de plaque fluorescente </td><td> Tecan </td><td> Safire II </td><td></td></tr></tbody></table> PCR quantitative <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> kit iScript transcription inverse </td><td> Bio-Rad </td><td> 170-8891 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7563 </td><td></td></tr><tr><td> Platinum Taq polymérase </td><td> Invitrogen </td><td> ARN Ultrasense 11732-927 </td><td></td></tr><tr><td> iCycler thermocycleur </td><td> Bio-Rad </td><td> iCycler Module Optique </td><td></td></tr><tr><td> Système logiciel iCycler </td><td> Bio-Rad </td><td> version 3.1 </td><td></td></tr></tbody></table> PCR quantitative pour puces à ADN <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> RNase Away </td><td> Molecular BioProducts </td><td> # 7000 </td><td></td></tr><tr><td> RT 2 Kit First Strand </td><td> SABiosciences </td><td> C-03 </td><td></td></tr><tr><td> SYBR Green PCR basée mélange </td><td> SABiosciences </td><td> PA-011 </td><td> Spécifique pour iCycler </td></tr><tr><td> RT 2 Profiler PCR array </td><td> SABiosciences </td><td> PARN-011A </td><td></td></tr></tbody></table> Essai protéomique <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue/ Numéro de modèle </td><td> Commentaires </td></tr><tr><td> Kit de lyse cellulaire </td><td> Bio-Rad </td><td> 171-304011 </td><td></td></tr><tr><td> Microcentrifugeuse </td><td> Fisher Scientific </td><td> Micro 7 </td><td></td></tr><tr><td> Stocks protéine BSA </td><td> Bio-Rad </td><td> 500-0007 </td><td></td></tr><tr><td> Kit BCA protein assay </td><td> Thermo Scientific </td><td> 23225 </td><td></td></tr><tr><td> Immuno des plaques de 96 puits MicroWell </td><td> Nunc </td><td> 449824 </td><td></td></tr><tr><td> Bande d&#39;étanchéité </td><td> Bio-Rad </td><td> 2239444 </td><td></td></tr><tr><td> Lecteur de plaque à 96 puits </td><td> Perkin Elmer </td><td> 1420-mulilabel contre </td><td></td></tr><tr><td> Bioplex Rat test cytokine TNF-α </td><td> Bio-Rad </td><td> X0000001T </td><td></td></tr></tbody></table> Immunocoloration <table border="1"><tbody><tr><td> Nom de réactif / Equipement </td><td> Entreprise </td><td> Catalogue / Numéro de modèle </td><td> Commentaires </td></tr><tr><td> Paraformaldéhyde 16% </td> &lt;td&gt; Alfa Aesar </td><td> 43368 </td><td></td></tr><tr><td> Lysine </td><td> Sigma </td><td> L-6001 </td><td></td></tr><tr><td> Phosphate de sodium </td><td> Fisher Scientific </td><td> S374-1 </td><td></td></tr><tr><td> Periodate de sodium </td><td> Sigma </td><td> S-1878 </td><td></td></tr><tr><td> L&#39;azoture de sodium </td><td> Sigma </td><td> S-2002 </td><td></td></tr><tr><td> Peroxyde d&#39;hydrogène (30%) </td><td> Sigma </td><td> H1009 </td><td></td></tr><tr><td> Sérum de chèvre</td><td> Colorado Serum Company </td><td> CS 0920 </td><td></td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T-8787 </td><td></td></tr><tr><td> Bacs de coloration </td><td> Ted Pella, Inc </td><td> 14511 </td><td></td></tr><tr><td> Plate Shaker </td><td> Lab-Line Instruments, Inc </td><td> 3520 </td><td></td></tr><tr><td> # 1 Papiers filtres (185 mm) </td><td> Whatman </td><td> 1001-185 </td><td></td></tr><tr><td> 3,3-diaminobenzidine </td><td> Sigma </td><td> D8001 </td><td> &lt;/ Td&gt; </tr><tr><td> Le diméthylsulfoxyde </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> L&#39;éthanol </td><td> Pharmco-AAPER </td><td> 111000200 </td><td></td></tr><tr><td> Xylène </td><td> Fisher Scientific </td><td> X3-4 </td><td></td></tr><tr><td> Permount milieu de montage </td><td> Fisher Scientific </td><td> SP15-100 </td><td></td></tr><tr><td> Cryostat </td><td> Leica </td><td> cm 3 050 S </td><td></td></tr><tr><td> Tissue-Tek </td><td> Ted Pella, Inc </td><td> 27050 </td><td></td></tr><tr><td> À pointe fine pinceau </td><td> Ted Pella </td><td> 11806 </td><td></td></tr><tr><td> Stylo PAP blocage </td><td> Ted Pella, Inc </td><td> 22309 </td><td></td></tr><tr><td> SFX signal de renforceur </td><td> Invitrogen </td><td> A31631 </td><td></td></tr><tr><td> ProLong Antifade </td><td> Invitrogen </td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

Lésion neurologique est une cause fréquente de morbidité et de mortalité liés à l&#39;anesthésie générale et les procédures chirurgicales qui pourraient être atténués par le développement d&#39;efficace, facile à administrer et traitements sécuritaires préconditionnement. Nous cherchons à définir les neurones de signalisation immunitaires responsables de froid préconditionnement comme moyen d&#39;identifier de nouvelles cibles pour le développement de thérapies pour protéger le cerveau avant l&#39;apparition des blessures. Faible niveau de médiateur pro-inflammatoire de signalisation des changements au fil du temps sont essentielles à froid préconditionnement neuroprotection. Cette signalisation est compatible avec les principes fondamentaux de l&#39;hormèse conditionnement physiologique, qui exigent que les stimuli irritatifs atteindre une amplitude seuil suffisamment de temps pour s&#39;adapter aux stimuli de protection à devenir évidents. En conséquence, la délimitation de la signalisation immunitaire impliquée dans le froid-préconditionnement neuroprotection exige que les systèmes biologiques et les manipulations expérimentales plNous capacités techniques sont hautement reproductibles et sensibles. Notre approche consiste à utiliser des cultures de tranches d&#39;hippocampe comme un modèle in vitro qui reflète le mieux leur homologues en vivo avec multi-synaptiques des réseaux neuronaux matures et influencés par de repos macroglia / microglie. Cet état gliale est particulièrement important pour les microglies, car ils sont la principale source de cytokines, qui sont actifs dans le domaine femtomolaire. En outre, les cultures tranche peuvent être maintenues in vitro pendant plusieurs semaines, ce qui est un délai suffisant pour évoquer des stimuli d&#39;activation et d&#39;évaluer les réponses adaptatives. Enfin, les conditions d&#39;environnement peut être contrôlée avec précision en utilisant des cultures de tranches de sorte que la signalisation des cytokines de préconditionnement à froid peut être mesurée, imité, et modulé à disséquer les aspects noeud critique. Analyses des cytokines du système de signalisation nécessitent l&#39;utilisation de techniques sensibles et reproductibles multiplexés. Nous utilisons PCR quantitative pour le TNF-α à l&#39;écran pour acte microglialeIvation suivie en temps réel quantitative PCR quantitative dépistage tableau pour évaluer les changements de cytokines tissu entier. Ce dernier est un moyen très sensibles et reproductibles pour mesurer de multiples changements de cytokines système de signalisation simultanément. Des changements importants sont confirmés par PCR quantitative ciblée et la détection de protéines. Nous sonder pour les tissus à base de modifications des protéines cytokine en utilisant des dosages multiplexés flux de microsphères de cytométrie en utilisant la technologie Luminex. La production de cytokine spécifique des cellules est déterminée à double étiquette immunohistochimie. Dans l&#39;ensemble, cette préparation du tissu cérébral et le style de l&#39;utilisation, couplée aux stratégies proposées enquête, peut être une approche optimale pour identifier des cibles potentielles pour le développement de nouveaux traitements qui pourraient imiter les avantages du froid préconditionnement.

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

Nous cherchons à définir les neurones de signalisation immunitaires responsables de froid préconditionnement comme moyen d&#39;identifier de nouvelles cibles pour le développement de thérapies pour protéger le cerveau avant l&#39;apparition des blessures. Nous présentons des stratégies pour de tels travaux qui exigent des systèmes biologiques, des manipulations expérimentales ainsi que des capacités techniques qui sont hautement reproductible et sensible.

Stratégies pour l&#39;étude de neuroprotection du froid préconditionnement

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 vues.  The University of Chicago Medical Center. Nous cherchons à définir les neurones de signalisation immunitaires responsables de froid préconditionnement comme moyen d'identifier de nouvelles cibles pour le développement de thérapies pour protéger le cerveau avant l'apparition des blessures. Nous présentons des stratégies pour de tels travaux qui exigent des systèmes biologiques, des manipulations expérimentales ainsi que des capacités techniques qui sont hautement reproductible et sensible.

Vidéo: 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.

Mesure cérébrale oxygénation sanguine à base d&#39;oxygène-dépendant trempe de phosphorescence

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

Recherche

Neurosciences

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.

Microdissection laser des Drosophile Neurones périphériques

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.

Préparation des coupes d&#39;hippocampe aiguë chez des rats et des souris transgéniques pour l&#39;étude des altérations synaptiques au cours du vieillissement et de l&#39;amylose pathologie

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 des neurones à partir de tranches de cerveau de rongeurs pour étudier la transmission synaptique et de l&#39;image terminaisons présynaptiques

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.

Séquentielle photo-blanchiment sur le tracé de cellules de Schwann simple à la jonction neuromusculaire

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.

Potentiel de membrane Dye Imaging de ventromédian hypothalamus neurones de souris adulte à étudier 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 ...

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.

Isolement de CA1 nucléaires enrichies fractions de coupes d&#39;hippocampe pour étudier l&#39;activité dépendante nucléaire importation de Synapto nucléaires protéines Messenger

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.

Analyseur portable à base d&#39;impédance en temps réel comme un outil pour délimiter voies moléculaires impliquées dans la neurotoxicité et la neuroprotection dans une lignée cellulaire neuronale

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.

Hippocampe organotypiques comme modèle d’étude Neuroprotection et son caractère invasif des cellules tumorales

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.

Augmentation de la réponse du réseau à la stimulation des cultures cellulaires neuronales sur les tableaux de microélectrodes

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