Os autores agradecem a C. Rodrigo de assistência técnica especializada. Agradecemos S. Köhler (Centro de Imagem avançada, Heinrich Heine University Duesseldorf) para obter ajuda na produção de vídeo. Pesquisa no laboratório do autor foi financiado pela Associação Alemã de Pesquisa (DFG: Ro 2327 / 8-1 para CRR), a Universidade Heinrich Heine Duesseldorf (a NH) e pelo National Institutes of Health conceder R01NS032123 (a 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.

Líquido à base de transportadora, eletrodos íon-seletivo têm sido empregados com sucesso por décadas e por muitos íons, sensores altamente específicas estão disponíveis 22-26. Quando usado no espaço extracelular (ECS) de preparações de cérebro de vertebrados, deve-se ter em mente, no entanto, que esta é uma técnica bastante invasivos: enquanto a largura da ECS é apenas cerca de 20-50 nm, o diâmetro de íon-seletivo microeletrodos é cerca de 1 um (eletrodos dois canos) ou maiores (eletrodos concêntricos). As pontas de microeletrodos íon-seletivo, assim, não só irá danificar o tecido durante o seu empalamento do tecido, mas também ampliar o ECS, favorecendo uma subestimação dos transientes de íon. Apesar dessas armadilhas, transientes de iões extracelulares em resposta à atividade neuronal são notavelmente consistentes entre os diferentes laboratórios de 7,8, que atestam a confiabilidade desse método. O desempenho e adequação dos eletrodos íon-seletivoé dependente da sua sensibilidade e selectividade, que é definida pelo sensor de cocktail (&#39;ionóforo membrana líquida&#39;) utilizado. Sensor cocktails conter uma molécula transportadora especial, por exemplo, valinomicina para K + microeléctrodos selectivos que apresenta uma elevada selectividade para a 27 potássio. Não obstante, a reactividade cruzada com outros iões podem ocorrer e deve ser testado. Valinomicina apresenta uma reactividade cruzada significativa nas pesquisas de amónio, que tem que ser considerado na interpretação dos resultados (por exemplo, 11,12). Além disso, porque a resposta de tensão dos ionóforos segue um comportamento Nernstiana (cf. equação 1), a relação sinal-ruído e limiar de detecção dependem da concentração do ião a ser medido. Assim, enquanto os pequenos [K +] o transientes evocar grandes variações de tensão contra a baixo da linha de base [K +] o, pequeno [Na +] o transientes são muito mais difíceis de detectar contra o oiGH basal [Na +] o (cf. Figura 5 e 6). O desempenho dos eléctrodos selectivos de iões também é determinada pela resolução temporal, o que é fortemente regulado por a sua constante de tempo eléctrica. O último é principalmente determinada pela resistência axial do sensor, e pela capacitância distribuída ao longo do comprimento da pipeta, entre as suas soluções internas e o fluido externo. Na configuração de cano duplo, a resistência é alta, devido à longa coluna de sensor de ião de enchimento. Para um dado dieléctrica isolante (neste caso, vidro de borosilicato), a capacitância é governada pela espessura dieléctrica. Em eléctrodos de dois canos, o dieléctrico largura eleva-se a parede de vidro da pipeta. Como o vidro thins perto da ponta, a largura dielétrico cai, e a capacitância aumenta. Esses fatores se combinam para produzir eletrodos com tempos de resposta que variam de várias centenas de milissegundos a váriossegundos, uma vez que estes factores são variados. Uma grande vantagem da concepção concêntrica é que tanto a resistência axial e a capacitância para o banho são grandemente diminuídos. As derivações de pipeta concêntricos maioria da resistência do permutador de iões utilizados no enchimento, deixando apenas um remanescente nos últimos poucos micrómetros antes da ponta. Além disso, a solução de enchimento no interior da pipeta concêntrica é fisicamente afastado do banho, separadas por a espessura de duas paredes de vidro, reduzindo grandemente a capacitância. 10 Como mostrado anteriormente, o efeito combinado da reduzida resistência e capacitância é uma melhoria na resolução temporal de duas ordens de grandeza. No caso de concêntricos de Ca2 + e do pH micro-eléctrodos, os tempos de resposta de 90% foram tão baixas como 10 10-20 ms. Uma vantagem relacionada com o desenho do concêntrico é o nível de ruído mais baixo (ver Figura 8). Devido à resistência muito reduzida, transitórios de tensão a partir de qualquer ambient de ruído são minimizados. Além disso, a recuperação a partir de tais transientes é rápida, devido à constante de tempo rápido. Tais artefatos são, portanto, pequeno e rápido, e têm um efeito menos prejudicial em gravações fisiológicas (cf. Figura 8). Existem também desvantagens da técnica concêntrica. Em primeiro lugar, a sua montagem é mais complexo e demorado. Uma segunda desvantagem é a necessidade de colocar um microeléctrodo de referência separado, com a sua ponta, o que implica a utilização de qualquer um micromanipulador separado ou uma dupla manipulador especializado. Finalmente, microeléctrodos duplos de cano pode ser estendido para um design triplo de cano, permitindo a detecção de duas espécies diferentes de iões, ao mesmo tempo 28, o que não é possível para os eléctrodos concêntricos. Armadilhas mais comuns Silanização ineficiente. O passo mais importante, e obstáculo principal a fabricação de qualquer líquido-sensou microeletrodos íon-seletivo com base é o procedimento silanização. Quando eléctrodos não conseguem responder a alterações na concentração de ião específico ou responder com uma resposta sub-Nernstiana (ou seja, bem inferior a 58 mV por diferença de concentração de dez vezes), fraca eficácia de silanização é tipicamente a causa. Em nossa experiência, isso pode ocorrer se a humidade atmosférica é muito alto ou muito baixo, típico de condições no auge do verão, ou inverno, respectivamente. Se for viável a exercer algum controlo sobre a humidade ambiente, estes problemas podem ser superados. Resistência do eléctrodo é demasiado elevada. Se necessário, a resistência do tambor sensível aos iões pode ser reduzida por biselamento. Para este fim, expor a sua dica para um forte jato de um abrasivo em suspensão na água por um par de segundos. Isto fará com que a sua ponta ao máximo para quebrar e diminuir a resistência ao valor desejado. Pontes de sal. Salpontes entre os barris de iões e os eléctrodos de referência resultar em mal ou-nada, portanto, responder e pode também confunde bastante o seu desempenho na calibração. Como mencionado acima (ver ponto 1.6.), Este é principalmente um problema quando o vidro teta de cano duplo é escolhido, mas é uma ocorrência rara quando se usa o deslocamento, a técnica de cano retorcido descrito aqui. Com facilidade de fabricação em mente, o design de cano duplo original da Lux 29 pode muitas vezes ser utilizado de forma lucrativa. Este método utiliza a pré-enchimento dos barris de iões e de referência com soluções salinas, uma rápida exposição a uma solução de silano por sua aspiração e expulsão a partir da ponta, a seguir, por incorporação de permutador de iões, também através da ponta (ver 30,31) . Estes eléctrodos podem ser fabricada em cerca de 10 minutos, mas o tamanho da ponta é, tipicamente, 4 mm ou mais e que são mais propensos a falhar durante um experimento. Em contraste, os métodos de silanização que envolvem a exposição ao vapor e calor silanoing pode produzir eletrodos com dicas menores que duram dias, e às vezes semanas. Tomados em conjunto, existem vários protocolos e aproxima-se sobre a forma de preparar micro-eléctrodos selectivos de iões. Aqui, descrevemos dois procedimentos principais para a fabricação de microeletrodos dois canos, bem como concêntricos torcidos que funcionam bem e de forma confiável em nossos laboratórios, com uma taxa de sucesso global de perto de 100%. Importante, estas técnicas podem ser transferidas para a medição de outras espécies, incluindo iões de cálcio ou de pH, e será igualmente aplicável a outras preparações do que o cérebro, incluindo cavidades cheias de líquido ou líquidos em geral. Por último, mas não menos importante, microeletrodos íon-seletivo permitir a determinação da concentração de íons no interior das células. Devido ao seu tamanho relativamente grande ponta (~ 1 mm), isto, no entanto, ser possível somente em células com um corpo de grandes células, por exemplo, tal como se encontra nas preparações de invertebrados 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> Para monitorar [K +] o e modificações das mesmas, em resposta a actividade excitatória, um microeléctrodo de cano duplo de potássio-selectivo foi inserido no estrato radiante de CA1 área. Poucos minutos depois empalação, o barril selectivo de iões do eléctrodo alcançado uma linha de base estável (Figura 7A), que corresponde a uma [K +] o de cerca de 2,8 mm, um valor perto da [K +] do ACSF utilizado ( 2,5 mM). Aplicação do banho de glutamato 0,5 mM durante 10 seg induziu um aumento reversível na [K +] o por cerca de 4 mm, seguida por uma subestimativa abaixo da linha de base no valor de ~ 0,7 mM (Figura 7A). A redução da concentração de glutamato a 0,4, 0,3, e 0,2 mM provocou uma redução correspondente na amplitude de ambos o aumento transitório e a subestimativa em [K +] o (Figura 7A). oad / 53058 / 53058fig7.jpg &quot;/&gt; Figura 7. transientes extracelular de potássio em resposta a actividade excitatória. (A) [K +] o transientes, que consiste em um aumento seguido por uma subestimativa abaixo da linha de base, induzida por aplicação de banho de diferentes concentrações de glutamato durante 10 seg, como indicado. (B) Influência de uma perfusão com bloqueadores de receptores de glutamato (100 ^ M, CNQX; APV, 100 uM) e tetrodotoxina (TTX; 0,5 uM) em [K +] o transientes induzida por aplicação de banho de glutamato 0,5 mM, durante 10 seg. (C) espontânea [K +] o transientes na presença de 0mg 2+ / BIC. Experimentos mostrados na AC foram realizadas utilizando eletrodos de dois canos. Para fins de ilustração, os traços foram suavizadas com um filtro Sawitzky-Golay (largura 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">Por favor, cl</a>ick aqui para ver uma versão maior desta figura. Experiências anteriores mostraram que tais [K +] sinais de o induzida por glutamato não são alterados de forma significativa durante a aplicação da TTX, e são, portanto, em grande parte independente sobre a abertura de canais de sódio voltagem-dependentes e ação neuronal potencial de geração de 15,16. Para estudar os mecanismos subjacentes às observadas [K +] o mudanças em resposta ao glutamato, aplicou-se bloqueadores de receptores de glutamato, ou seja, CNQX (100 uM; bloqueador de receptores AMPA) e APV (100 uM; bloqueador de receptores de NMDA) na presença de TTX (0,5 ^ M). Após a perfusão de banho com esses bloqueadores, induzida por glutamato [K +] o mudanças foram praticamente abolidas, confirmando a sua dependência em relação à abertura de canais de receptores de glutamato ionotrópicos como relatado anteriormente (por exemplo, 17; Figura 7B). Para demonstrar ainda mais o tele relevância glutamatérgica de excitação para a geração de matriz extracelular [K +] sinais, fatias foram perfundidos com solução salina nominalmente Mg2 + -livre contendo 10 uM metiodeto de bicuculina (2+ 0mg / BIC). Isto alivia a tensão dependente de Mg2 + de receptores NMDA -bloco e atenua a inibição bloqueando os receptores de GABA A, fazendo com que a actividade epileptiforme recorrente espontânea na rede (por exemplo, 18,19). Como esperado 20, espontâneo, recorrente [K +] o transientes, no valor de cerca de 1,5 mM foram detectados em 0mg 2+ / salina BIC (Figura 7C). Entre essas respostas, menor [K +] o transientes com uma amplitude média de cerca de 0,2 mM ocorreu (Figura 7C). Em um segundo conjunto de experimentos, usamos [Na +] microeletrodos sensíveis ao determinar [Na +] • mudanças evocadas pela aplicação deagonistas de glutamato. Aqui, nós também comparou as características de resposta de microeletrodos de cano duplo e concêntricas que empregam dois paradigmas de aplicação diferentes. [Na +] microeléctrodos sensíveis ao foram posicionados no stratum radiatum, a uma profundidade de cerca de 50 uM. Depois de uma linha de base estável foi alcançado, o agonista de glutamato L-aspartato foi aplicado por perfusão de banho (10 mM, 120 sec, banho de perfusão a 2,5 ml / min). Como observado anteriormente 21, a aplicação de aspartato causou uma diminuição lenta na [Na +] o por cerca de 15 mM, que durou cerca de 5 minutos e, em seguida, começou a se recuperar de volta à linha de base (Figura 8A). Nomeadamente, enquanto as amplitudes de pico e a cinética dos sinais de [Na +] o determinado por ambos os eléctrodos eram virtualmente idênticos sob estas condições, os eléctrodos concêntricos exibiu uma linha de base mais estável e um nível de ruído mais baixo (Figura 8A). Para testar o r<html> esposta características dos diferentes eléctrodos sob um paradigma aplicação mais rápida, que o glutamato-pressão aplicada por meio de uma pipeta de vidro fina posicionada no estrato radiante a uma distância de 20-40 uM a partir da ponta do microeléctrodo selectivo de iões. Como esperado 21, a aplicação de glutamato (10 mM) durante 200 ms causou uma queda no [Na +] o (Figura 8B). As amplitudes de pico de [Na +] O decréscimo estavam no mesmo intervalo para ambos os tipos de eléctrodos (cano duplo: 4,5 - 13,5 mM, n = 14; concêntrica: 2,0 - 19,1 mm, n = 15). No entanto, em contraste com os resultados obtidos com a perfusão de banho lento (ver acima), não só a relação de sinal-para-ruído, mas também do curso de tempo dos sinais de [Na +] o detectados pelos dois tipos de eléctrodos diferiram significativamente. O tempo médio de pico foi de 3,5 seg para a apenas 1,3 seg de cano duplo e para os micro-eléctrodos concêntricos. homólogos ve_content &quot;&gt; Assim, estes resultados demonstram e confirmam a cinética de resposta mais rápidos de Na + microeléctrodos selectivos duplos de cano concêntricos vs, (cf. Figura 6C e 8B), como também foi observado por Ca2 + e pH selectivos 10 . Em contraste com o estudo anterior, em que a estimulação sináptica curto-burst foi realizada para evocar transientes de iões rápido induzidas-sinapticamente, houve apenas uma tendência, mas não houve diferença significativa em amplitudes de pico médias entre eléctrodos concêntricos e dois canos com a nossa aplicação paradigma. Isto foi provavelmente devido ao facto de a distância da pipeta de aplicação a partir da ponta do microeléctrodo selectivo de iões variou por um factor de dois (20-40 uM), e, consequentemente, que a concentração máxima de glutamato na região alvo não foi a mesma em diferentes experiências, obstruindo qualquer diferença existente em amplitudes de pico. (A) Top traça: Mudança na voltaGE dos barris de referência (VREF) de um eléctrodo de cano duplo (traço pontilhado preto) e um eléctrodo concêntrico (cinzento traços) em resposta a alterações na concentração do banho de Na + ([Na +] b) como indicado. Note-se que as respostas de tensão de ambos os eléctrodos são virtualmente idênticas. Traços inferiores: Alterações na tensão da Na + -potential (V Na +) de um eléctrodo de cano duplo (traço preto) e um eléctrodo concêntrico (cinzento traços) em resposta a alterações na [Na +] b. (B) parcelas Half-logarítmicas de [Na +] b versus o V Na + de ambos os eletrodos. Parcelas lineares dos dados revelam uma inclinação de cerca de 48 mV para ambos os eléctrodos. (C) Resposta da V Na + de uma (traço preto) de cano duplo e um eletrodo concêntrico (cinza trace) para uma rápida mudança na [Na +] b de 152 mm para 70 mm. Note-se que o tempo de resposta do eléctrodo concêntrica é significativamente FASter. Para fins de ilustração, os traços foram suavizadas com um filtro Sawitzky-Golay (largura 20). <img alt="Figura 8" src="/files/ftp_upload/53058/53058fig8.jpg" /> Figura 8: transientes de sódio extracelulares como detectado por eletrodos de cano duplo e concêntricos. (A) alterações transitórias da V ref e [Na +] o induzida por perfusão banho com 10 mM aspartato, durante 120 segundos, como indicado pelo bar. Os traços superiores mostram uma gravação executada com um eléctrodo de cano duplo, os traços inferiores foram registados utilizando um eléctrodo concêntrica. (B) as alterações transitórias em V ref e [Na +] O induzidas por aplicação local de pressão com glutamato 10 mM durante 0,2 segundos, tal como indicado pela seta. Os traços superiores mostram uma gravação executada com um eléctrodo de cano duplo, os traços inferiores foram registados utilizando um eléctrodo concêntrica.Linhas vermelhas pontilhadas representam ajustes lineares de o período entre o início do sinal para o seu máximo. Note-se que o tempo de resposta do eléctrodo concêntrica é significativamente mais rápido sob esta condição. Para fins de ilustração, os traços foram suavizadas com um filtro Sawitzky-Golay (largura 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</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.

Microeletrodos de cano duplo e concêntricos para a medição de Extracelular Ion Signals no tecido cerebral

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 visualizações.  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. 

Vídeo: 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.

Preparação do Tecido Cerebral de Rato imunomicroscopia

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

Neurociência

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 centrada Fresagem e Microscopia Eletrônica de Varredura do Tecido Cerebral

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.

Fazendo, testando e usando potássio íon seletivo microeletrodos em fatias de tecido do cérebro de um adulto

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.

Dissecação de locais de Ca2 + sinais em pilhas cultivadas pelos indicadores de membrana-alvo Ca2 +

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.

Imagem latente 3D de alta resolução da infecção do vírus da raiva no tecido solvente-cancelado do cérebro

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.

Rotulagem e imagem de placas amilóides no tecido cerebral usando a curcumina polifenóis naturais

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.

Fatias hipocampais horizontais do cérebro do rato

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.

Um método de limpeza de tecido hidrofóbico para tecido cerebral de rato

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.

Um ensaio baseado em placas para a medição da liberação de monoamina endógena em fatias cerebrais agudas

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.

Medição do fluxo de informações direcionais em dados fNIRS-Hyperscanning usando o Método de Coerência de Transformação de Onda Parcial

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