Les auteurs tiennent à remercier C. Roderigo pour l&#39;assistance technique d&#39;experts. Nous remercions S. Köhler (Centre d&#39;imagerie avancée, Université Heinrich Heine de Düsseldorf) de l&#39;aide dans la production vidéo. Recherche dans le laboratoire de l&#39;auteur a été financé par l&#39;Association allemande pour la recherche (DFG: Ro 2,327 / 8-1 à CRR), l&#39;Université Heinrich Heine de Düsseldorf (NH) et par les Instituts nationaux de la Santé accorde R01NS032123 (à MC).

<ol>
	<li>Somjen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+regulation+in+the+brain:+implications+for+pathophysiology.">Ion regulation in the brain: implications for pathophysiology.</a> Neuroscientist. 8, 254-267 (2002).</li><li>Dietzel, I., Heinemann, U., Hofmeier, G., Lux, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-induced+changes+in+extracellular+Na++and+Cl-+concentration+in+relation+to+changes+in+the+size+of+the+extracellular+space.">Stimulus-induced changes in extracellular Na+ and Cl- concentration in relation to changes in the size of the extracellular space.</a> Exp Brain Res. 46, 73-84 (1982).</li><li>Nicholson, C., ten Bruggencate, G., Stockle, H., Steinberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+potassium+changes+in+extracellular+microenvironment+of+cat+cerebellar+cortex.">Calcium and potassium changes in extracellular microenvironment of cat cerebellar cortex.</a> J Neurophysiol. 41, 1026-1039 (1978).</li><li>Rice, M. E., Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate-+and+aspartate-induced+extracellular+potassium+and+calcium+shifts+and+their+relation+to+those+of+kainate,+quisqualate+and+N-methyl-D-aspartate+in+the+isolated+turtle+cerebellum.">Glutamate- and aspartate-induced extracellular potassium and calcium shifts and their relation to those of kainate, quisqualate and N-methyl-D-aspartate in the isolated turtle cerebellum.</a> Neuroscience. 38, 295-310 (1990).</li><li>Prince, D. A., Lux, H. D., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+extracellular+potassium+activity+in+cat+cortex.">Measurement of extracellular potassium activity in cat cortex.</a> Brain Res. 50, 489-495 (1973).</li><li>Deitmer, J. W., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+regulation+and+proton+signalling+by+glial+cells.">pH regulation and proton signalling by glial cells.</a> Prog Neurobiol. 48, 73-103 (1996).</li><li>Chesler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+modulation+of+pH+in+the+brain.">Regulation and modulation of pH in the brain.</a> Physiol Rev. 83, 1183-1221 (2003).</li><li>Kofuji, P., Newman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Regulation of potassium by glial cells in the central nervous system. , (2009).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-selective+microelectrodes+and+diffusion+measurements+as+tools+to+explore+the+brain+cell+microenvironment.">Ion-selective microelectrodes and diffusion measurements as tools to explore the brain cell microenvironment.</a> J Neurosci Methods. 48, 199-213 (1993).</li><li>Fedirko, N., Svichar, N., Chesler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+use+of+high-speed,+concentric+h+-+and+Ca2+-selective+microelectrodes+suitable+for+in+vitro+extracellular+recording.">Fabrication and use of high-speed, concentric h+- and Ca2+-selective microelectrodes suitable for in vitro extracellular recording.</a> J Neurophysiol. 96, 919-924 (2006).</li><li>Stephan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kir4.1+channels+mediate+a+depolarization+of+hippocampal+astrocytes+under+hyperammonemic+conditions+in+situ.">Kir4.1 channels mediate a depolarization of hippocampal astrocytes under hyperammonemic conditions in situ.</a> Glia. 60, 965-978 (2012).</li><li>Haack, N., Rose, C. R., Lei, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+Calibration+and+Application+of+Potassium-Selective+Microelectrodes.">Preparation, Calibration and Application of Potassium-Selective Microelectrodes.</a> Microelectrodes. , 87-105 (2014).</li><li>Roos, A., Boron, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH.">Intracellular pH.</a> Physiol Rev. 61, 296-434 (1981).</li><li>Kleinhans, C., Kafitz, K. W., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+Intracellular+Sodium+Imaging+Combined+with+UV-mediated+Focal+Uncaging+of+Glutamate+in+CA1+Pyramidal+Neurons.">Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons.</a> Journal of visualized experiments : JoVE. , (2014).</li><li>Hertz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+astrocytic+Na+,K+-ATPase+and+glycogenolysis+for+K+homeostasis+in+mammalian+brain.">Roles of astrocytic Na ,K -ATPase and glycogenolysis for K homeostasis in mammalian brain.</a> J Neurosci Res. , (2014).</li><li>Pumain, R., Heinemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-+and+amino+acid-induced+calcium+and+potassium+changes+in+rat+neocortex.">Stimulus- and amino acid-induced calcium and potassium changes in rat neocortex.</a> J Neurophysiol. 53, 1-16 (1985).</li><li>Vargova, L., Jendelova, P., Chvatal, A., Sykova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate,+AMPA,+NMDA+Induced+Changes+in+Extracellular+Space+Volume+and+Tortuosity+in+the+Rat+Spinal+Cord.">Glutamate, AMPA, NMDA Induced Changes in Extracellular Space Volume and Tortuosity in the Rat Spinal Cord.</a> J Cereb Blood Flow Metab. 21, 1077-1089 (2001).</li><li>Fellin, T., Gomez-Gonzalo, M., Gobbo, S., Carmignoto, G., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+glutamate+is+not+necessary+for+the+generation+of+epileptiform+neuronal+activity+in+hippocampal+slices.">Astrocytic glutamate is not necessary for the generation of epileptiform neuronal activity in hippocampal slices.</a> J Neurosci. 26, 9312-9322 (2006).</li><li>Rouach, N., Koulakoff, A., Abudara, V., Willecke, K., Giaume, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglial+metabolic+networks+sustain+hippocampal+synaptic+transmission.">Astroglial metabolic networks sustain hippocampal synaptic transmission.</a> Science. 322, 1551-1555 (2008).</li><li>Lux, H. D., Heinemann, U., Dietzel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+changes+and+alterations+in+the+size+of+the+extracellular+space+during+epileptic+activity.">Ionic changes and alterations in the size of the extracellular space during epileptic activity.</a> Adv Neurol. 44, 619-639 (1986).</li><li>Zanotto, L., Heinemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartate+and+glutamate+induced+reductions+in+extracellular+free+calcium+and+sodium+concentration+in+area+CA1+of+'in+vitro'+hippocampal+slices+of+rats.">Aspartate and glutamate induced reductions in extracellular free calcium and sodium concentration in area CA1 of 'in vitro' hippocampal slices of rats.</a> Neurosci Lett. 35, 79-84 (1983).</li><li>Borrelli, M. J., Carlini, W. G., Dewey, W. C., Ransom, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+making+ion-selective+microelectrodes+suitable+for+intracellular+recording+in+vertebrate+cells.">A simple method for making ion-selective microelectrodes suitable for intracellular recording in vertebrate cells.</a> J Neurosci Methods. 15, 141-154 (1985).</li><li>Ammann, D., Anker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutral+carrier+sodium+ion-selective+microelectrode+for+extracellular+studies.">Neutral carrier sodium ion-selective microelectrode for extracellular studies.</a> Neurosci Lett. 57, 267-271 (1985).</li><li>Deitmer, J. W., Schlue, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Na++and+Ca2++in+leech+Retzius+neurones+during+inhibition+of+the+Na+-K++pump.">Intracellular Na+ and Ca2+ in leech Retzius neurones during inhibition of the Na+-K+ pump.</a> Pflugers Arch. 397, 195-201 (1983).</li><li>Schwiening, C. J., Thomas, 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=Relationship+between+intracellular+calcium+and+its+muffling+measured+by+calcium+iontophoresis+in+snail+neurones.">Relationship between intracellular calcium and its muffling measured by calcium iontophoresis in snail neurones.</a> J Physiol. 491 (Pt 3), 621-633 (1996).</li><li>Chesler, 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=Intracellular+pH+of+astrocytes+increases+rapidly+with+cortical+stimulation.">Intracellular pH of astrocytes increases rapidly with cortical stimulation.</a> Am J Physiol. 253, R666-R670 (1987).</li><li>Ammann, D., Chao, P. S., Simon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valinomycin-based+K++selective+microelectrodes+with+low+electrical+membrane+resistance.">Valinomycin-based K+ selective microelectrodes with low electrical membrane resistance.</a> Neurosci Lett. 74, 221-226 (1987).</li><li>Deitmer, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicarbonate-dependent+changes+of+intracellular+sodium+and+pH+in+identified+leech+glial+cells.">Bicarbonate-dependent changes of intracellular sodium and pH in identified leech glial cells.</a> Pflugers Arch. 420, 584-589 (1992).</li><li>Lux, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+recording+ion+specific+microelectrodes:+their+use+in+pharmacological+studies+in+the+CNS.">Fast recording ion specific microelectrodes: their use in pharmacological studies in the CNS.</a> Neuropharmacology. 13, 509-517 (1974).</li><li>Nicholson, C., Phillips, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+diffusion+modified+by+tortuosity+and+volume+fraction+in+the+extracellular+microenvironment+of+the+rat+cerebellum.">Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum.</a> J Physiol. 321, 225-257 (1981).</li><li>Chesler, M., Chan, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-induced+extracellular+pH+transients+in+the+in+vitro+turtle+cerebellum.">Stimulus-induced extracellular pH transients in the in vitro turtle cerebellum.</a> Neuroscience. 27, 941-948 (1988).</li><li>Thomas, 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=Intracellular+sodium+activity+and+the+sodium+pump+in+snail+neurones.">Intracellular sodium activity and the sodium pump in snail neurones.</a> J Physiol. 220, 55-71 (1972).</li></ol>

The authors declare no competing interests.

À base de liquide transporteur, électrodes sélectives d&#39;ions ont été utilisés avec succès depuis des décennies et pour de nombreux ions, des capteurs très spécifiques sont disponibles 22-26. Lorsqu&#39;il est utilisé dans l&#39;espace extracellulaire (ECS) des préparations de cerveau des vertébrés, il faut cependant garder à l&#39;esprit, que ce soit une technique très invasive: tandis que la largeur de l&#39;ECS est seulement autour de 20-50 nm, le diamètre de l&#39;ion-sélective microélectrodes est d&#39;environ 1 pm (électrodes à deux coups) ou plus (électrodes concentriques). Les conseils de microélectrodes sélectives d&#39;ions seront donc non seulement endommager les tissus pendant leur empalement du tissu, mais aussi agrandir l&#39;ECS, favorisant une sous-estimation des transitoires d&#39;ions. Malgré ces écueils, les transitoires d&#39;ions extracellulaires en réponse à l&#39;activité neuronale sont remarquablement cohérents entre les différents laboratoires 7,8, attestant de la fiabilité de cette méthode. La performance et la pertinence des électrodes sélectives d&#39;ionsest fonction de leur sensibilité et de sélectivité, qui est défini par le cocktail de capteur (&#39;ionophore à membrane liquide &quot;) utilisé. Cocktails de capteurs contiennent une molécule porteuse spéciale, par exemple valinomycine K + microélectrodes sélectifs qui présente une haute sélectivité pour le potassium 27. Nonobstant, la réactivité croisée avec d&#39;autres ions peut se produire et doit être testé. Valinomycine présente une réactivité croisée significative pour l&#39;ammonium, qui doit être considéré lors de l&#39;interprétation des résultats (par exemple 11,12). En outre, parce que la tension de réponse des ionophores en résulte un comportement de Nernst (cf. équation 1), le rapport signal sur bruit et seuil de détection dépend de la concentration de l&#39;ion à mesurer. Ainsi, tandis que les petites [K +] o transitoires évoquer de grandes variations de tension contre le faible niveau de référence [K +] o, petite [Na +] o transitoires sont beaucoup plus difficiles à détecter contre le salutgh référence [Na +] o (cf. Figure 5 et 6). La performance des électrodes sélectives d&#39;ions est également déterminée par la résolution temporelle, qui est largement régi par la constante de temps électrique. Celle-ci est principalement déterminée par la résistance axiale du capteur, et par la capacité répartie le long de la longueur de la pipette, entre ses solutions interne et le fluide externe. Dans la configuration à deux coups, la résistance est élevée, en raison de la longue colonne de capteur d&#39;ions remblayée. Pour un diélectrique isolante suivant (dans ce cas verre borosilicate), la capacité est régie par l&#39;épaisseur diélectrique. Dans électrodes à deux coups, la largeur diélectrique revient à la paroi de verre de la pipette. Comme le verre amincit près de la pointe, la largeur diélectrique tombe, et la capacité augmente. Ces facteurs se combinent pour produire des électrodes avec des temps de réponse qui vont de plusieurs centaines de millisecondes à plusieurssecondes, que ces facteurs sont variés. Un avantage majeur de la conception concentrique est que la résistance axiale et à la capacité du bain sont grandement diminuées. Les pipettes shunts concentriques plupart de la résistance de l&#39;échangeur d&#39;ions remblayé, laissant seulement un vestige dans les dernières micromètres avant la pointe. En outre, la solution de remplissage à l&#39;intérieur de la pipette concentrique est physiquement éloignée de la baignoire, séparés par l&#39;épaisseur de deux parois de verre, ce qui réduit considérablement la capacité. 10 Comme indiqué précédemment, l&#39;effet combiné de la résistance et de la capacité réduite est une amélioration de la résolution temporelle de deux ordres de grandeur. Dans le cas de concentriques Ca2 + et de pH microélectrodes, les temps de réponse de 90% étaient aussi bas que 10-20 ms 10. Un avantage connexe de la conception concentrique est le faible niveau de bruit (cf. Figure 8). En raison de la résistance considérablement réduit, les transitoires de tension de toute Ambient bruit sont minimisés. En outre, la récupération de ces transitoires est rapide, en raison de la constante de temps rapide. Ces artefacts sont donc petit et rapide, et ont un effet moins perturbateur sur les enregistrements physiologiques (cf. Figure 8). Il ya aussi des inconvénients de la technique concentrique. Tout d&#39;abord, leur assemblage est plus complexe et prend du temps. Un deuxième inconvénient est la nécessité de placer un micro-électrode de référence séparée avec sa pointe, entraînant l&#39;utilisation soit d&#39;un micromanipulateur séparé ou un double manipulateur spécialisé. Enfin, deux coups microélectrodes peuvent être étendus à une conception triple canon, ce qui permet la détection de deux espèces différentes d&#39;ions en même temps 28, ce qui est impossible pour des électrodes concentriques. Pièges les plus courants Silanisation inefficace. L&#39;étape la plus importante, et principal obstacle dans la fabrication de tout liquide-sensou microélectrode sélective d&#39;ions à base de la procédure de silanisation. Lorsque les électrodes ne parviennent pas à répondre aux changements dans la concentration d&#39;ions spécifiques, ou de répondre avec une réponse sous-nernstienne (bien au moins de 58 mV par différence de concentration dix fois), une mauvaise efficacité de silanisation est généralement la cause. Dans notre expérience, cela peut se produire si l&#39;humidité atmosphérique est trop élevée ou trop faible, typique des conditions à la hauteur de l&#39;été, ou en hiver, respectivement. Si il est possible d&#39;exercer un certain contrôle sur l&#39;humidité ambiante, ces problèmes peuvent être surmontés. Résistance d&#39;électrode est trop élevée. Si nécessaire, la résistance du corps sensible aux ions peut être réduite par chanfreinage. À cette fin, exposer sa pointe à un puissant jet d&#39;un abrasif en suspension dans l&#39;eau pendant quelques secondes. Cela entraînera sa pointe de upmost à casser et à réduire la résistance à la valeur souhaitée. Ponts sel. Seldes ponts entre les barils d&#39;ions et de référence entraînent électrodes mal ou aucun-réponse et peuvent donc aussi confondre grandement leur performance dans l&#39;étalonnage. Comme mentionné ci-dessus (voir point 1.6.), Cela est principalement un problème lorsque deux coups de verre thêta est choisi, mais est un événement rare lorsque vous utilisez le, la technique du canon tordu décalage décrit ici. Avec la facilité de fabrication à l&#39;esprit, la conception à double canon original de Lux 29 peut souvent être utilisé avec profit. Cette méthode utilise pré-remplissage des ions et de référence de barils avec des solutions salines, une exposition rapide à une solution de silane par son aspiration et l&#39;expulsion de la pointe, à la suite par incorporation de l&#39;échangeur d&#39;ions, également par la pointe (voir 30,31) . Ces électrodes peuvent être fabriquées dans environ 10 min, mais leur taille de la pointe est généralement 4 pm ou plus et ils sont plus enclins à l&#39;échec lors d&#39;une expérience. En revanche, les méthodes de silanisation qui impliquent l&#39;exposition aux vapeurs de silane et de la chaleurING peut produire des électrodes avec de petits conseils qui derniers jours et parfois des semaines. Pris dans leur ensemble, il existe plusieurs protocoles et des approches sur la façon de préparer des microélectrodes sélectives d&#39;ions. Ici, nous avons décrit deux procédures principales pour la fabrication de microélectrodes à deux coups ainsi que concentriques torsadés qui fonctionnent bien et de manière fiable dans nos laboratoires, avec un taux de réussite global de près de 100%. Fait important, ces techniques pourront être transférés à la mesure d&#39;autres espèces, y compris les ions calcium ou le pH, et sera également applicable à d&#39;autres préparations que le cerveau, y compris des cavités ou des liquides remplis de liquide en général. Dernier point, mais non le moindre, microélectrodes sélectives d&#39;ions permettent de déterminer les concentrations d&#39;ions à l&#39;intérieur des cellules. En raison de leur taille relativement grande de pointe (~ 1 pm), cette volonté, toutefois, être possible que dans les cellules avec un corps à grandes cellules, par exemple, comme on en trouve dans les préparations invertébrés 28,32.

An author's middle initial was omitted from the publication, <a href="http://www.jove.com/video/53058/double-barreled-concentric-microelectrodes-for-measurement">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue</a>. The author&#39;s name has been updated to:
Christine R. Rose
from:
Christine Rose

A correction was made to Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.

Erratum: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical signaling in the brain is based on the flux of ions across plasma membranes. Major ion movements into and from the extracellular space are not only mediated by passage through voltage-gated ion channels, but also by postsynaptic ionotropic receptors as well as ion transporters. Neuronal activity is thus accompanied by complex changes in the extracellular concentration of all major ions 1. For example, influx of sodium into neurons during excitatory activity has been shown to result in a decrease in the extracellular sodium concentration ([Na+]o) 2. The same holds true for the extracellular calcium concentration because calcium ions rapidly enter both pre- and postsynaptic structures 3. At the same time, potassium moves the opposite way and this mediates an increase in the extracellular potassium concentration ([K+]o) in the low mM range 4,5. Synaptic activity also causes changes in extracellular pH that are partly mitigated by concomitant glial membrane fluxes that change intraglial pH 6,7. These activity-related changes in extracellular ion concentrations have significant functional consequences. For example, even small increases in [K+]o depolarize neurons as well as glial cells thereby altering neuronal excitability, and several mechanisms exist to remove excess potassium 8. Failure of these may result in epileptiform activity of neurons or phenomena like spreading depression 1. 
Because of their critical importance, quantitative determination of extracellular ion concentrations is often necessary and required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. For decades, double-barreled ion-selective microelectrodes have proven to be excellent tools for such measurements in brain tissue 9. For many ions, highly specific sensors with low cross-reactivity for other ions are available. In addition to the classical double-barreled electrodes, so-called concentric electrodes were recently introduced. The latter provide a superior time resolution, but take a little more time and effort to construct 10. 
In the following, we will describe the preparation and calibration of these two types of ion-selective microelectrodes. We then show how these electrodes can be employed in brain slice preparations for measurement of changes in [K+]o or [Na+]o induced by excitatory activity following different stimulation paradigms including bath and pressure application of drugs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Abrasive&#160;&#160;</td>
			<td>MicroPolish&#160;</td>
			<td>Buehler GmbH</td>
			<td>Dissolved in A.dest</td>
		</tr>
		<tr>
			<td>Borosilicate-glass capillaries</td>
			<td>1405059</td>
			<td>Hilgenberg</td>
			<td>Application pipette; 75 mm x 2 mm, wall thickness 0.3 mm</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC 150 F-15&#160;</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the sensor of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC100-F-15</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the reference of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-200TF-15&#160;</td>
			<td>Science Products</td>
			<td>Concentric, outer channel. o.d. 2.0 mm&#160;&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-120TF-10</td>
			<td>Science Products</td>
			<td>Concentric, inner channel. o.d. 1.2 mm</td>
		</tr>
		<tr>
			<td>Digidata</td>
			<td>1322A</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrometer amplifier with headstage</td>
			<td></td>
			<td>Custom-made</td>
			<td>Rin = 10T&#937; and Ibias=50fA-1pA (commercially available&#160; alternatives: e. g. Dagan IX2-700, with headstage (10 Gig feedback resistor) or EPMS-07, NPI, Tamm, Germany)</td>
		</tr>
		<tr>
			<td>Experimental chamber</td>
			<td></td>
			<td>Custom-made</td>
			<td>Commercially available from e.g.&#160; Warner Instruments,USA;&#160; Scientifica, UK</td>
		</tr>
		<tr>
			<td>Furnace</td>
			<td></td>
			<td>Heraeus</td>
			<td>Must stay constant at 200&#176;C</td>
		</tr>
		<tr>
			<td>Hard sticky wax / dental wax&#160;</td>
			<td>Deiberit 502&#160;</td>
			<td>Siladent Dr. Boehme &#38; Schoeps GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td></td>
			<td>Custom-made</td>
			<td>Must stay constant at 40&#176;C</td>
		</tr>
		<tr>
			<td>Microelectrodes holder made of plexiglas</td>
			<td></td>
			<td>Custom-made</td>
			<td>Double-barreled: o.d.&#160; capillaries 1.5 mm, concentric: o.d.&#160; capillaries 2 mm</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td></td>
			<td>Leitz</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>MD4R</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>M205C</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Plan 0.8xLWD</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model PP-830</td>
			<td>Narishige</td>
			<td>Concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model P-97</td>
			<td>Sutter Instruments&#160;</td>
			<td>Sensor of concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic drug ejection system</td>
			<td>Picospritzter Type II</td>
			<td>General Valve TM Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>Travel dovetail stage</td>
			<td>DT 25/M</td>
			<td>Thorlabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-component glue</td>
			<td>&#160;Araldite</td>
			<td>Huntsman advanced materials GmbH</td>
			<td>One may also use a small stripe of aluminum foil to stick the capillaries together&#160;</td>
		</tr>
		<tr>
			<td>Silverwire</td>
			<td>99.9%&#160;</td>
			<td>Wieland Edelmetalle</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicer / Vibratome</td>
			<td>Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>AxoScope 8.1&#160;</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical puller&#160;</td>
			<td>Type PE-2</td>
			<td>Narishige Scientific Instruments</td>
			<td>With a revolvable chuck for&#160; double-barreled microelectrodes</td>
		</tr>
		<tr>
			<td>x/y translational stage</td>
			<td></td>
			<td>Custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Compound</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>1(S),9(R)-(&#8722;)-Bicuculline methiodide</td>
			<td>Sigma aldrich</td>
			<td>14343</td>
			<td>Competitive antagonist of GABAA receptors (light-sensitive); CAUTION toxic</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma aldrich</td>
			<td>C-127</td>
			<td>AMPA/kainate receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-AP5</td>
			<td>Alfa Aesar</td>
			<td>J64210</td>
			<td>NMDA receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td>CAUTION: Flammable, acute toxicity (oral, dermal, inhalation) and corrosive to metals and skin</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma aldrich</td>
			<td>A9256</td>
			<td>Activates NMDA and non-NMDA and EAATs</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma aldrich</td>
			<td>G1626</td>
			<td>Activates NMDA-R, AMPA-R, QA-R and KA-R),&#160; mGluRs and EAATs</td>
		</tr>
		<tr>
			<td>Potassium ionophore I - cocktail B</td>
			<td>Fluka&#160;</td>
			<td>60403</td>
			<td>Based on valinomycin; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Sodium ionophore II - cocktail A</td>
			<td>Fluka&#160;&#160;</td>
			<td>71178</td>
			<td>Based on ETH 157</td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Ascent Scientific</td>
			<td>Asc-055</td>
			<td>Inhibitor of voltage-dependent Na+ channels; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Water, ultra pure</td>
			<td>Sigma aldrich</td>
			<td>W3500&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

<html> Pour surveiller [K +] o et les changements qui y sont en réponse à l&#39;activité excitatrice, une microélectrode double canon potassium sélective a été inséré dans le stratum radiatum de la zone CA1. Quelques minutes après empalement, le canon à ions sélectif de l&#39;électrode atteint une ligne de base stable (figure 7A), ce qui correspond à un [K +] o d&#39;environ 2,8 mM, une valeur proche de la [K +] de l&#39;ACSF utilisé ( 2,5 mM). L&#39;application du bain de 0,5 mM de glutamate pendant 10 s induit une augmentation réversible de [K +] o d&#39;environ 4 mM, suivi d&#39;un sous-dépassement en dessous de la ligne de base d&#39;un montant de 0,7 mM ~ (figure 7A). L&#39;abaissement de la concentration de glutamate à 0,4, 0,3, et 0,2 mM a provoqué une réduction correspondante de l&#39;amplitude à la fois de l&#39;augmentation transitoire et le sous-dépassement en [K +] o (figure 7A). OAD / 53058 / 53058fig7.jpg &quot;/&gt; Figure 7. transitoires de potassium extracellulaires en réponse à l&#39;activité excitatrice. (A) [K +] o transitoires, constitué par une augmentation suivie d&#39;un sous-dépassement en dessous de la ligne de base, induite par l&#39;application du bain de différentes concentrations de glutamate pendant 10 secondes comme indiqué. (B) Influence d&#39;une perfusion avec des bloqueurs de récepteurs de glutamate (CNQX, 100 uM; APV, 100 pM) et la tétrodotoxine (TTX; 0,5 uM) de [K +] o transitoires induits par l&#39;application du bain de 0,5 mM de glutamate pendant 10 s. (C) spontanée [K +] o transitoires en présence de 0mg 2+ / BIC. Les expériences présentées dans AC ont été effectuées en utilisant des électrodes à double canon. À titre d&#39;illustration, des traces ont été lissées avec un filtre Sawitzky-Golay (largeur 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">S&#39;il vous plaît cla</a>beurk ici pour voir une version plus grande de cette figure. Des expériences antérieures ont montré que ces [K +] o signaux induite par le glutamate sont pas significativement modifiés lors de l&#39;application de TTX, et sont donc largement indépendante sur l&#39;ouverture des canaux sodiques voltage-dépendants et de l&#39;action neuronale potentiel de production de 15,16. Pour étudier les mécanismes sous-jacents les observées [K +] o changements en réponse au glutamate, nous avons appliqué les bloqueurs des récepteurs de glutamate, à savoir CNQX (100 M; bloqueur des récepteurs AMPA) et APV (100 M; bloqueur des récepteurs NMDA), en présence de TTX (0,5 uM). Après perfusion de salle de bain avec ces bloqueurs, induite par le glutamate [K +] o changements étaient pratiquement aboli, confirmant leur dépendance à l&#39;ouverture de canaux de ionotropes récepteurs du glutamate tel que rapporté avant (par exemple 17; figure 7B). Pour démontrer davantage tIl pertinence de glutamatergique excitation pour la génération de extracellulaire [K +] signaux, les tranches ont été perfusés avec nominalement Mg 2+ solution saline exempt contenant 10 uM bicuculline méthiodure (0mg 2+ / BIC). Cela soulage tension dépendant de Mg2 + -bloc des récepteurs de NMDA et atténue l&#39;inhibition par blocage des récepteurs GABA A, ce qui provoque l&#39;activité épileptiforme spontanée récurrent dans le réseau (par exemple 18,19). Comme prévu 20, spontanée, récurrente [K +] o transitoires, un montant d&#39;environ 1,5 mM ont été détectés dans 0mg 2+ / BIC solution saline (figure 7C). Entre ces réponses, plus petite [K +] o transitoires avec une amplitude moyenne d&#39;environ 0,2 mM a eu lieu (Figure 7C). Dans une deuxième série d&#39;expériences, nous avons utilisé [Na +] microélectrodes -sensibles pour déterminer [Na +] o changements évoqués par application deagonistes du glutamate. Ici, nous avons également comparé les caractéristiques des microélectrodes à double canon et concentriques employant deux paradigmes d&#39;application différents réponse. [Na +] des microélectrodes -sensibles été positionné dans le stratum radiatum à une profondeur d&#39;environ 50 uM. Après une ligne de base stable a été atteint, le glutamate agoniste de L-aspartate a été appliqué par perfusion de bain (à 10 mM, 120 secondes, le bain perfusion à raison de 2,5 ml / min). Comme observé précédemment 21, l&#39;application de l&#39;aspartate a provoqué une diminution lente dans [Na +] o d&#39;environ 15 mm, qui a duré environ 5 min, puis a commencé à recouvrer à la ligne de base (figure 8A). Notamment, tandis que les amplitudes des pics et cinétique des [Na +] o signaux déterminés par les deux électrodes étaient pratiquement identiques dans ces conditions, électrodes concentriques présentaient une base plus stable et un niveau de bruit inférieur (figure 8A). Pour tester la r<html> EPONSE caractéristiques des différentes électrodes dans un paradigme d&#39;application plus rapide, nous glutamate pression appliquée à travers une pipette en verre bien positionné dans le stratum radiatum à une distance de 20-40 um de la pointe de la microélectrode sélective d&#39;ions. 21 Comme on s&#39;y attendait, l&#39;application de glutamate (10 mM) pendant 200 ms a provoqué une baisse de la o (figure 8B) [Na +]. Les amplitudes de pointe de la [Na +] o diminution étaient dans la même gamme pour les deux types d&#39;électrodes (deux coups: de 4,5 à 13,5 mM, n = 14; concentriques: de 2,0 à 19,1 mM, n = 15). Cependant, contrairement aux résultats obtenus avec une lente perfusion de bain (voir ci-dessus), non seulement le rapport signal à bruit, mais également au cours du temps de la [Na +] o signaux détectés par les deux types d&#39;électrodes diffère de manière significative. Le délai moyen de pic était de 3,5 sec pour le double-canon et seulement 1,3 s pour les microélectrodes concentriques. homologues ve_content &quot;&gt; Ainsi, ces résultats démontrent et confirment la cinétique de réponse plus rapides de Na + microélectrodes sélectifs deux coups concentriques contre, (cf. Figure 6C et 8B), a également été noté que pour le Ca 2+ et pH-sélectifs 10 . Contrairement à la première étude, dans laquelle la stimulation synaptique à court éclater a été réalisée pour évoquer transitoires d&#39;ions rapides synaptiquement-induites, il y avait seulement une tendance, mais aucune différence significative dans amplitudes maximales moyennes entre électrodes concentriques et deux coups avec notre application paradigme. Cela est probablement dû au fait que la distance de la pipette d&#39;application de la pointe de la microélectrode sélective d&#39;ions varie d&#39;un facteur de deux (20 à 40 um) et, par conséquent, que la concentration de glutamate maximale à la région cible était pas le même dans les différentes expériences, obstruant toute différence existante dans amplitudes de pointe. (A) Haut retrace: Variation de la Voltage des barils de référence (V ref) d&#39;une électrode à double canon (trace noire pointillée) et une électrode concentrique (trace grise) en réponse à des changements dans le bain Na + concentration ([Na +] b) comme indiqué. A noter que les réponses de tension des deux électrodes sont pratiquement identiques. Traces de fond: Les changements dans la tension de (V +) de Na Na + d&#39;une électrode à double canon (trace noire) et une électrode concentrique (trace grise) en réponse à des changements dans [Na +] b. Parcelles demi-logarithmiques de la [Na +] (b) B par rapport à la V Na + des deux électrodes. Terrain linéaire des données révèlent une pente d&#39;environ 48 mV pour les deux électrodes. (C) Réponse du V Na + d&#39;un double canon (trace noire) et une électrode concentrique (trace grise) à un changement rapide dans le [Na +] b à partir de 152 à 70 mm. A noter que le temps de l&#39;électrode concentrique de réponse est considérablement faster. À titre d&#39;illustration, des traces ont été lissées avec un filtre Sawitzky-Golay (largeur 20). <img alt="Figure 8" src="/files/ftp_upload/53058/53058fig8.jpg" /> Figure 8: transitoires de sodium extracellulaires telles que détectées par des électrodes à double canon et concentriques. (A) des changements transitoires de V ref et [Na +] o induite par la perfusion de bain avec 10 aspartate mM pendant 120 secondes, comme indiqué par la barre. Les tracés supérieurs montrent un enregistrement effectué avec une électrode à double canon, les traces faibles ont été enregistrés en utilisant une électrode concentrique. (B) Les variations transitoires dans V ref et [Na +] o induite par l&#39;application de la pression locale avec 10 mM de glutamate pour 0.2 sec comme indiqué par la flèche. Les tracés supérieurs montrent un enregistrement effectué avec une électrode à double canon, les traces faibles ont été enregistrés en utilisant une électrode concentrique.Pointillés rouges représentent ajustements linéaires de la période entre le début du signal à son maximum. A noter que le temps de l&#39;électrode concentrique de réponse est nettement plus rapide dans ces conditions. À titre d&#39;illustration, des traces ont été lissées avec un filtre Sawitzky-Golay (largeur 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a>

Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Microélectrodes à double canon et concentrique pour la mesure de signaux extracellulaires Ion dans le tissu cérébral

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

double-barreled-concentric-microelectrodes-for-measurement

Research

JoVE Journal

Neuroscience

13.4K vues.  Heinrich Heine University Düsseldorf. We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations. 

Vidéo: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Préparation du tissu cérébral de souris pour microscopie électronique

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Recherche

Neurosciences

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

Ion Beam ciblée de fraisage et de microscopie électronique à balayage du tissu cérébral

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

Fabrication, des essais et à l’aide de microélectrodes sensibles aux ions Potassium dans des tranches de tissu du cerveau adulte

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Dissection des signaux locaux Ca2 + dans les cellules cultivées par axés sur la Membrane Ca2 + indicateurs

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

Imagerie 3D haute résolution de l'infection par le virus de la rage dans un tissu cérébral éclairci par solvant

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Étiquetage et imagerie des plaques amyloïdes dans les tissus cérébraux à l'aide de la curcumine polyphénol naturelle

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Tranches hippocampiques horizontales du cerveau de souris

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Une méthode de nettoyage des tissus hydrophobes pour le tissu cérébral de rat

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Un test sur plaque pour la mesure de la libération endogène de monoamine dans les tranches cérébrales aiguës

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

Measurement of the Directional Information Flow in fNIRS-Hyperscanning Data using the Partial Wavelet Transform Coherence Method

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural synchronization (INS) during social interactions, functional near-infrared spectroscopy (fNIRS) is one of the most popular techniques for hyperscanning naturalistic social interactions because of its relatively high spatial resolution, sound anatomical localization, and exceptionally high tolerance of motion artifacts. Previous fNIRS-based hyperscanning studies usually calculate a time-lagged INS using wavelet transform coherence (WTC) to describe the direction and temporal pattern of information flow between individuals. However, the results of this method might be confounded by the autocorrelation effect of the fNIRS signal of each individual. For addressing this issue, a method termed partial wavelet transform coherence (pWTC) was introduced, which aimed to remove the autocorrelation effect and maintain the high temporal-spectrum resolution of the fNIRS signal. In this study, a simulation experiment was performed first to show the effectiveness of the pWTC in removing the impact of autocorrelation on INS. Then, step-by-step guidance was offered on the operation of the pWTC based on the fNIRS dataset from a social interaction experiment. Additionally, a comparison between the pWTC method and the traditional WTC method and that between the pWTC method and the Granger causality (GC) method was drawn. The results showed that pWTC could be used to determine the INS difference between different experimental conditions and INS's directional and temporal pattern between individuals during naturalistic social interactions. Moreover, it provides better temporal and frequency resolution than the traditional WTC and better flexibility than the GC method. Thus, pWTC is a strong candidate for inferring the direction and temporal pattern of information flow between individuals during naturalistic social interactions.

This protocol describes partial wavelet transform coherence (pWTC) for calculating the time-lagged pattern of interpersonal neural synchronization (INS) to infer the direction and temporal pattern of information flow during social interaction. The effectiveness of pWTC in removing the confounds of signal autocorrelation on INS was proved by two experiments.

Mesure du flux d’informations directionnelles dans les données d’hyperscan fNIRS à l’aide de la méthode de cohérence de transformation partielle des ondelettes

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural ...