Gli autori desiderano ringraziare C. Roderigo per l&#39;assistenza tecnica di esperti. Ringraziamo S. Köhler (Center of Advanced Imaging, Heinrich Heine università Duesseldorf) per un aiuto nella produzione video. La ricerca in laboratorio dell&#39;autore è stato finanziato dal Research Association tedesco (DFG: Ro 2327 / 8-1 a CRR), l&#39;Università Heinrich Heine Duesseldorf (NH) e dal National Institutes of Health di Grant 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. 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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. 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The authors declare no competing interests.

Liquido a base porta-elettrodi ionoselettivi sono stati impiegati con successo per decenni e per molti ioni, sensori altamente specifici sono disponibili 22-26. Quando utilizzato nello spazio extracellulare (ECS) di preparazioni cerebrali vertebrati, si deve tenere presente, tuttavia, che questa è una tecnica molto invasiva: mentre la larghezza della ECS è solo circa 20-50 nm, il diametro di ione-selettivo microelettrodi è di circa 1 micron (elettrodi a doppia canna) o più grandi (elettrodi concentrici). Le punte dei microelettrodi ionoselettive saranno quindi non solo danneggiare il tessuto durante la loro impalamento del tessuto, ma anche ingrandire la ECS, favorendo una sottovalutazione dei transitori di ioni. Nonostante queste insidie, transitori ioni extracellulari in risposta all&#39;attività neuronale sono notevolmente coerenti tra i diversi laboratori 7,8, attestanti l&#39;affidabilità di questo metodo. Le prestazioni e l&#39;idoneità di elettrodi ionoselettividipende dalla loro sensibilità e selettività, che è definita dal cocktail sensore (&#39;ionoforo membrana liquida&#39;) utilizzato. Cocktail sensori contengono una speciale molecola carrier, per es valinomicina K + microelettrodi -selettivo che presenta un&#39;elevata selettività per il potassio 27. Nonostante, cross-reattività con altri ioni può avvenire e deve essere testato. Valinomicina presenta una significativa reattività crociata per ammonio, che deve essere considerato quando si interpretano i risultati (ad esempio, 11,12). Inoltre, poiché la tensione di risposta dei ionofori segue un comportamento Nernst (cfr equazione 1), il rapporto segnale-rumore e soglia di rilevamento dipendono dalla concentrazione dello ione da misurare. Così, mentre piccolo [K +] o transienti evocare grandi variazioni di tensione contro la bassa linea di base [K +] o, piccolo [Na +] o transitori sono molto più difficili da individuare contro hibasale gh [Na +] o (vedere la figura 5 e 6). Le prestazioni di elettrodi ionoselettivi è determinato anche dalla risoluzione temporale, che è largamente governata dalla sua costante di tempo elettrica. Quest&#39;ultima è determinata principalmente dalla resistenza assiale del sensore, e dalla capacità distribuita lungo la lunghezza della pipetta, tra le soluzioni interne e il fluido esterno. Nella configurazione a doppia canna, la resistenza è alta, a causa della lunga colonna di sensore ione ripiena. Per un dato dielettrico isolante (in questo caso vetro borosilicato), la capacità è governato dallo spessore dielettrico. In elettrodi a doppia canna, la larghezza dielettrico equivale alla parete di vetro della pipetta. Quando il vetro si assottiglia in prossimità della punta, la larghezza dielettrico cade, e la capacità aumenta. Questi fattori si combinano per produrre elettrodi con tempi di risposta che vanno da alcune centinaia di millisecondi a diversesecondi, come questi fattori sono molteplici. Un importante vantaggio del disegno concentrico è che sia la resistenza assiale e la capacità al bagno sono notevolmente ridotte. I derivatori pipetta concentrici maggior parte della resistenza dello scambiatore di ioni ripiena, lasciando solo un residuo negli ultimi micrometri prima della punta. Inoltre, la soluzione di riempimento all&#39;interno della pipetta concentrica è fisicamente distanziato dal bagno, separato dallo spessore di due pareti di vetro, riducendo notevolmente la capacità. Come illustrato in precedenza 10, l&#39;effetto combinato della ridotta resistenza e capacità è un miglioramento della risoluzione temporale di due ordini di grandezza. In caso di Ca 2+ concentrici e pH microelettrodi, 90% i tempi di risposta erano partire da 10-20 msec 10. Un vantaggio relativo del disegno concentrica è il livello di rumore inferiore (vedi figura 8). Grazie alla resistenza notevolmente ridotto, transitori di tensione da qualsiasi ambienrumore t sono ridotti al minimo. Inoltre, il recupero da tali transitori è rapido, a causa della costante di tempo veloce. Tali manufatti sono quindi piccolo e veloce, e hanno un effetto meno dirompente sulle registrazioni fisiologiche (vedi Figura 8). Ci sono anche degli svantaggi della tecnica concentrica. Innanzitutto, il loro assemblaggio è più complesso e richiede tempo. Un secondo inconveniente è la necessità di posizionare un microelettrodo riferimento separato con la punta, che comporta l&#39;utilizzo della propria micromanipolatore separato o un doppio manipolatore specializzata. Infine, microelettrodi doppia canna possono essere estesi ad un disegno tripla canna, consentendo rilevamento di due specie ioniche differenti allo stesso tempo 28, che non è possibile per elettrodi concentrici. Insidie ​​più comuni Silanizzazione inefficiente. Il passo più importante, e ostacolo principale nella fabbricazione di qualsiasi liquido-senso basate microelettrodo ionoselettivo è la procedura silanizzazione. Quando gli elettrodi non riescono a rispondere ai cambiamenti nella concentrazione di ioni specifici, o di rispondere con una risposta sub-Nernst (vale a dire, ben meno di 58 mV per dieci volte differenza di concentrazione), scarsa efficacia di silanizzazione è in genere la causa. Nella nostra esperienza, questo può verificarsi se l&#39;umidità atmosferica è troppo alto, o troppo basso, tipico delle condizioni piena estate o inverno, rispettivamente. Se è fattibile per esercitare un certo controllo dell&#39;umidità ambientale, questi problemi possono essere superati. Resistenza dell&#39;elettrodo è troppo alta. Se necessario, la resistenza della canna sensibile agli ioni può essere ridotta di bisellatura. A tal fine, esporre la sua punta a un forte getto di abrasivo sospeso in acqua per un paio di secondi. Questo farà sì che la punta upmost per rompere e abbassare la resistenza al valore desiderato. Ponti salini. Saleponti tra le botti di ioni e di riferimento determinano elettrodi scarsamente o niente di rispondere e possono quindi anche confondere notevolmente le loro prestazioni nella calibrazione. Come accennato in precedenza (vedi punto 1.6.), Questo è soprattutto un problema quando si sceglie a doppia canna di vetro theta, ma è un evento raro quando si utilizza l&#39;offset, tecnica botte contorto qui descritto. Con la facilità di fabbricazione in mente, il design a doppia canna originale di Lux 29 può essere spesso utilizzato proficuamente. Questo metodo utilizza pre-riempimento delle botti di ioni e di riferimento con soluzioni saline, un&#39;esposizione veloce per una soluzione di silano con la sua aspirazione e l&#39;espulsione dalla punta, seguendo per incorporazione di scambiatore ionico, anche tramite la punta (vedi 30,31) . Questi elettrodi possono essere realizzati in circa 10 minuti, ma il loro formato di punta è in genere 4 micron o più e sono più inclini a fallire nel corso di un esperimento. Al contrario, i metodi silanizzazione che comportano l&#39;esposizione al vapore silano e caloreing può produrre elettrodi con punte più piccoli che durano giorni, e, a volte settimane. Nel loro insieme, ci sono diversi protocolli e approcci su come prepararsi microelettrodi ionoselettive. Qui, abbiamo descritto due principali procedure per la fabbricazione di microelettrodi due canne e concentrici intrecciati che funzionano bene e affidabile nei nostri laboratori, con un tasso di successo complessivo prossimo al 100%. È importante sottolineare che queste tecniche saranno trasferibili alla misurazione di altri ioni specie, tra cui il pH o di calcio, e sarà anche essere applicabile ad altri preparati che il cervello, comprese le cavità piene di liquido o di fluidi in genere. Ultimo, ma non meno importante, microelettrodi ionoselettive consentono la determinazione delle concentrazioni di ioni all&#39;interno delle cellule. A causa della loro relativamente grandi dimensioni punta (~ 1 micron), tale volontà, però, sarà possibile solo in cellule con un corpo a grandi cellule, quali ad esempio, ha trovato in preparati invertebrati 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> Per monitorare [K +] o e sue variazioni in risposta all&#39;attività eccitatoria, un microelettrodo doppia canna potassio-selettivo è stato inserito nel radiatum strato dell&#39;area CA1. Pochi minuti dopo impalamento, la canna ionoselettivo dell&#39;elettrodo raggiunto una linea di base stabile (Figura 7A), corrispondente ad un [K +] o di circa 2,8 mm, un valore prossimo al [K +] del ACSF utilizzato ( 2,5 mm). Bagno applicazione di glutammato 0,5 mM per 10 sec indotto un aumento reversibile [K +] o di circa 4 mm, seguito da un undershoot al basale pari a ~ 0,7 mM (Figura 7A). L&#39;abbassamento della concentrazione di glutammato a 0,4, 0,3, e 0,2 mM causato una corrispondente riduzione dell&#39;ampiezza sia l&#39;aumento transitorio e sottomodulazioni [K +] o (Figura 7A). OAD / 53058 / 53058fig7.jpg &quot;/&gt; Figura 7. transitori potassio extracellulari in risposta all&#39;attività eccitatoria. (A) [K +] o transitori, consistente in un aumento seguito da un undershoot inferiore al valore basale, indotta da bagno applicazione di diverse concentrazioni di glutammato per 10 secondi come indicato. (B) L&#39;influenza di una perfusione con bloccanti del recettore del glutammato (CNQX, 100 micron, APV, 100 micron) e tetrodotossina (TTX; 0,5 micron) su [K +] o transienti indotta dalla applicazione bagno di glutammato 0.5 mm per 10 sec. (C) spontaneo [K +] o transienti in presenza di 0 mg 2+ / BIC. Gli esperimenti riportati in AC sono stati eseguiti utilizzando elettrodi a doppia canna. A scopo illustrativo, le tracce sono state levigate con un filtro Sawitzky-Golay (larghezza 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">Si prega di cl</a>ick qui per vedere una versione più grande di questa figura. Esperimenti precedenti hanno dimostrato che tali [K +] o segnali glutammato indotto non risultano significativamente modificati durante l&#39;applicazione di TTX, e sono quindi in gran parte indipendente sull&#39;apertura dei canali del sodio voltaggio-dipendenti e l&#39;azione neuronale potenziale di generazione di 15,16. Per studiare i meccanismi alla base delle osservati [K +] o cambiamenti in risposta al glutammato, abbiamo applicato bloccanti del recettore del glutammato, cioè CNQX (100 micron; bloccante dei recettori AMPA) e APV (100 micron; bloccante dei recettori NMDA) in presenza di TTX (0,5 mM). Al Bagno perfusione con questi bloccanti, glutammato indotta [K +] o cambiamenti erano praticamente abolito, confermando la loro dipendenza l&#39;apertura di canali recettori del glutammato ionotropi come riportato in precedenza (ad esempio, 17; Figura 7B). Per dimostrare ulteriormente tegli rilevanza di glutamatergico di eccitazione per la generazione di extracellulare [K +] i segnali, le fette sono stati perfusi con nominalmente Mg 2 + -free salina contenente 10 mM bicucullina methiodide (0mg 2+ / BIC). Questo allevia voltaggio-dipendenti Mg 2+ -block dei recettori NMDA e smorza l&#39;inibizione bloccando i recettori GABA A, provocando spontanea attività epilettiforme ricorrente nella rete (ad esempio, 18,19). Come previsto 20, spontanea, ricorrente [K +] o transienti, pari a circa 1,5 mm sono stati rilevati in 0mg 2+ / salina BIC (Figura 7C). Tra queste risposte, più piccolo [K +] o transienti con una ampiezza media di circa 0,2 mM verificato (figura 7C). In una seconda serie di esperimenti, abbiamo utilizzato [Na +] microelettrodi sensitive per determinare [Na +] o cambiamenti evocati mediante l&#39;applicazione diagonisti glutammato. Qui, abbiamo anche confrontato le caratteristiche di risposta di microelettrodi a due canne e concentriche che impiegano due diversi paradigmi di applicazione. [Na +] microelettrodi -sensitive erano posizionati nella radiatum strato a una profondità di circa 50 micron. Dopo una linea di base stabile è stato raggiunto, il glutammato agonista L-aspartato è stato applicato per bagno perfusione (10 mM, 120 sec, bagno perfusione a 2,5 ml / min). Come osservato in precedenza 21, applicazione di aspartato ha causato una lenta diminuzione del [Na +] o di circa 15 mm, che è durato circa 5 minuti e poi ha iniziato a recuperare di nuovo al basale (Figura 8A). In particolare, mentre le ampiezze di picco e cinetica delle [Na +] o segnali determinati da entrambi gli elettrodi erano virtualmente identiche in queste condizioni, elettrodi concentrici mostravano una linea di base stabile e una maggiore rumorosità inferiore (Figura 8A). Per verificare il r<html> esponse caratteristiche dei diversi elettrodi sotto un paradigma un&#39;applicazione più rapida, abbiamo glutammato pressione applicata attraverso una pipetta di vetro fine posizionata nella radiatum strato a una distanza di 20-40 micron dalla punta del microelettrodo ione-selettivo. Come previsto 21, applicazione di glutammato (10 mM) per 200 ms causato un calo nella o [Na +] (Figura 8B). Le ampiezze di picco del o diminuzione [Na +] erano nello stesso intervallo per entrambi i tipi di elettrodi (doppia canna: 4,5 - 13.5 mM, n = 14; concentrici: 2.0 - 19.1 mM, n = 15). Tuttavia, in contrasto con i risultati ottenuti con lenta perfusione vasca (vedi sopra), non solo il rapporto segnale-rumore, ma anche l&#39;andamento temporale dei [Na +] o segnali rilevati dai due tipi di elettrodi differivano significativamente. Il tempo medio di picco è stato 3,5 secondi per la doppia canna e solo 1,3 secondi per i microelettrodi concentrici. controparti ve_content &quot;&gt; Quindi, questi risultati dimostrano e confermano la cinetica di risposta più veloci del concentriche rispetto microelettrodi a doppia canna Na + -selettivo, (vedi Figura 6C e 8B), come è stato anche notato per Ca 2+ e pH-selettivi 10 . A differenza del primo studio, in cui la stimolazione sinaptica breve scoppio è stata effettuata per evocare transitori ioni veloce sinapticamente-indotti, c&#39;era solo una tendenza, ma nessuna differenza significativa nelle ampiezze di picco medie tra elettrodi concentrici e doppia canna con la nostra applicazione paradigma. Questo è probabilmente dovuto al fatto che la distanza della pipetta applicazione dalla punta del microelettrodo ionoselettivo variava di un fattore di due (20-40 micron), e di conseguenza, che la concentrazione glutammato massimo in corrispondenza della zona di destinazione non era la stessa in diversi esperimenti, ostacolando qualsiasi differenza esistente in ampiezze di picco. (A) In alto tracce: Variazione della Voltage delle canne di riferimento (VREF) di una doppia canna elettrodo (nero traccia tratteggiata) ed un elettrodo concentrica (grigio traccia) in risposta ai cambiamenti nella vasca di concentrazione di Na + ([Na +] b) come indicato. Si noti che le risposte tensione di entrambi gli elettrodi sono praticamente identici. Tracce di fondo: variazioni della tensione della Na + -potential (V Na +) di una doppia canna elettrodo (traccia nera) ed un elettrodo concentrica (grigio traccia) in risposta alle variazioni di [Na +] b. (B) trame mezza logaritmiche del [Na +] b rispetto al V Na + di entrambi gli elettrodi. Trame lineare dei dati rivelano una pendenza di circa 48 mV per entrambi gli elettrodi. (C) Reazione del V Na + di una doppia canna (trace nero) e un elettrodo concentrica (grigio trace) ad una variazione rapida della [Na +] b da 152 mM a 70 mM. Si noti che il tempo di risposta dell&#39;elettrodo concentrica è significativamente faster. A scopo illustrativo, le tracce sono state levigate con un filtro Sawitzky-Golay (larghezza 20). <img alt="Figura 8" src="/files/ftp_upload/53058/53058fig8.jpg" /> Figura 8: transitori di sodio extracellulari rilevate tramite elettrodi a doppia canna e concentriche. (A) i cambiamenti transitori V ref e [Na +] o indotta da bagno perfusione con aspartato 10 mm per 120 sec, come indicato dalla barra. Le tracce superiori mostrano una registrazione eseguita con un elettrodo a due canne, le tracce inferiori sono stati registrati utilizzando un elettrodo concentrica. (B) cambiamenti transitori V ref e [Na +] o indotta applicando pressione locale con glutammato 10 mM per 0,2 sec come indicato dalla freccia. Le tracce superiori mostrano una registrazione eseguita con un elettrodo a due canne, le tracce inferiori sono stati registrati utilizzando un elettrodo concentrica.Linee rosse tratteggiate rappresentano attacchi lineari del periodo tra l&#39;inizio del segnale al massimo. Si noti che il tempo di risposta dell&#39;elettrodo concentrica è significativamente più veloce in questa condizione. A scopo illustrativo, le tracce sono state levigate con un filtro Sawitzky-Golay (larghezza 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa 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.

Microelettrodi doppia canna e concentrico per la misurazione di extracellulare Ion Segnali in tessuto cerebrale

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 Views.  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. 

Video: 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.

Preparazione del tessuto cerebrale del mouse per la microscopia Immunoelectron

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

Ricerca

Neuroscienze

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 concentrato di fresatura e Microscopia Elettronica a Scansione di tessuto cerebrale

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

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Progettazione e assemblaggio di un ultraleggero motorizzato per Microdrive croniche Recordings neurali in animali di piccola taglia

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

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.

Facendo, test e l'utilizzo di microelettrodi selettivo di ioni potassio in fettine di tessuto del cervello 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.

Dissezione del locale Ca2 + segnali in cellule coltivate di targeting per membrana Ca2 + indicatori

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.

Imaging 3D ad alta risoluzione dell'infezione da virus della rabbia nel tessuto cerebrale con la rimozione del solvente

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.

Etichettatura e imaging di placche amiloidi nel tessuto cerebrale utilizzando la curcumina polifenolo naturale

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.

Fette ippocampali orizzontali del cervello del topo

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.

Un metodo di compensazione del tessuto idrofobico per il tessuto cerebrale del ratto

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 basato su piastre per la misurazione del rilascio di monoamina endogena in fette cerebrali acute

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