We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

Tutte le procedure sperimentali descritte in questo protocollo sono stati approvati dalla cura degli animali della Hebrew University and Use Committee. 1. Preparare le soluzioni e strumenti per affettare <ol><li> Preparare 1 L di soluzione fisiologica standard (SPS) contenente gli ioni descritte nella Tabella 1. <ol><li> Preparare una soluzione madre contenente i sali a 10 volte la concentrazione finale in anticipo in una bottiglia di vetro riempita con memorizzazione 1000 ml di acqua deionizzata (conduttanza 0,055 mS / cm). Aggiungere sali (NaCl, KCl, KH 2 PO 4, e MgSO 4), e mescolare fino a completa dissoluzione. Conservare la soluzione di riserva a 4 ° C. </li><li> Effettuare la soluzione finale SPS il giorno dell&#39;esperimento. Aggiungere 100 ml della soluzione madre a 700 ml di acqua purificata, sciogliere il glucosio (3,6 g) e NaHCO3 (2,18 g) con ancoretta magnetica e quindi aggiungere acqua purificata fino a 1 L. </li><li> Equilibrare i solutiil da dare gas con il 95% O 2/5% di CO 2 per ~ 20 min. Dopo questo, aggiungere 2 ml di 1 M CaCl 2 soluzione. </li></ol></li><li> Montare i seguenti strumenti (Figura 1). <ol><li> Preparare grandi forbici o altro strumento per la decapitazione, piccole forbici chirurgiche per l&#39;apertura del cranio, pinza punta fine per il sollevamento del cranio per esporre il cervello, bisturi con lama per sezionare la parte desiderata del cervello da affettare e una piccola spatola per la manipolazione della parti del cervello sezionato. </li><li> Inoltre, preparare piccoli pezzi di carta da filtro per rimuovere il liquido in eccesso dal cervello, due piccoli piatti di Petri per il cervello per essere sezionati in due bicchieri di vetro 500 ml al contenimento di acqua purificata e riscaldata SPS, e colla cianoacrilato per incollare il cervello al fase di taglio. </li><li> Preparare una piccola spazzola per la manipolazione delle fette durante l&#39;affettatura e una bocca larga pipetta Pasteur di vetro per spostare le fette dal bagno affettare al cha recuperomber. Disinfettare gli strumenti per prevenire la crescita di batteri nei bagni fetta caldi. Inoltre, usare i guanti di laboratorio puliti. </li></ol></li><li> Preparare una camera di recupero fetta sommersa come descritto da Gibb e Edwards 15. Continuamente gas la SPS in esso con il 95% O 2/5% di CO 2 e mantenerlo a 36 ° C. Assicurarsi che la gasazione non si traduca in piccole bolle intrappolate nel bagno che potrebbero danneggiare le fette. </li><li> Caldo ~ 300 ml di SPS a 36 ° C su una piastra riscaldante con agitatore magnetico o in un bagno d&#39;acqua. Se viene usata una piastra di riscaldamento, aver cura di evitare che la soluzione dal surriscaldamento, che può provocare la precipitazione di ioni. Nel caso in cui accade, caldi SPS freschi, invece di raffreddamento del vecchio. </li><li> Portare ~ 500 ml di acqua purificata a bollire con un bollitore elettrico e, in un bicchiere, unire 300 ml di esso con acqua fredda per ottenere acqua leggermente più calda rispetto alla temperatura di taglio. Utilizzare questa riscaldato acqua purificata durante la decapitazione e utilizzare il remaizio del acqua bollita in seguito per mantenere la temperatura fisiologica del bagno affettatura. </li><li> Anestetizzare il mouse iniettando 0,1 ml di pentobarbital (60 mg / ml) per via intraperitoneale. Dopo qualche minuto verificare che l&#39;animale non risponde a forti punta o coda pizzichi per accertare la mancanza di sensibilità. </li></ol> 2. Dissezione del cervello <ol><li> Decapitare il mouse rapidamente tagliando il collo con le grandi forbici vicino alla parte posteriore del cranio, e lasciare che la testa cadere nel bicchiere con acqua purificata riscaldata per lavare via il sangue in eccesso. </li><li> Esporre il foro occipitale nel cranio sotto i muscoli del collo e la pelle, eventualmente rimuovere più del collo con le piccole forbici. </li><li> Tirare la pelle sulla parte superiore del cranio per poter vedere chiaramente le suture cranio per guidare l&#39;apertura del cranio. </li><li> Tagliare il cranio aperto inserendo l&#39;estremità inferiore delle piccole forbici attraverso il foro occipitale e subito li voltandosi versola parte laterale. Tagliare delicatamente lungo il bordo laterale del parietale fino ad una posizione dietro gli occhi e la sutura fronto; quindi, ruotare verso il centro del cranio e tagliare attraverso la linea mediana verso l&#39;altro lato del cranio (vedi linea verde tratteggiata nella Figura 2A). </li><li> Utilizzando pinze punta fine, sollevare il cranio da un angolo fronto-parietale e tirarlo in diagonale su e lateralmente per esporre il cervello (vedi freccia gialla tratteggiata nella Figura 2A). Assicurarsi che il cranio non è collegata a un osso o pelle della testa, in modo che il cervello rimarrà in posizione quando il cranio è sollevato. Nel caso in cui il cervello si sta muovendo con il cranio, utilizzare le piccole forbici per rimuovere qualsiasi tessuto connettivo tra il cranio e il cervello. </li><li> Quando il cervello è esposto, non consentono al cervello di essere asciutta. Quindi, eseguire i passi successivi, mantenendo il cranio e il cervello in una capsula di Petri riempita con riscaldato, gasati SPS. </li><li> Staccare la parte del cervello usata per l&#39;eXperiment dal resto del cervello utilizzando un bisturi e una spatola. In caso di utilizzo del cervelletto, staccare dal prosencefalo da un unico taglio attraverso il mesencefalo e ponte (lungo le linee rosse nelle figure 2B-D) e spostarlo in una piccola scatola di Petri che contiene fresco, gasati SPS. </li><li> Rimuovere il tronco cerebrale per formare una base diritta e larga per incollare il cervello a taglio fase quando affetta cervelletto nel piano orizzontale. Brevemente posizionare il cervelletto su un pezzo di carta da filtro umida in modo che il lato che è stato tagliato dal prosencefalo sia rivolta verso il basso. <ol><li> Tagliare il tronco con un bisturi per formare la base (come indicato con la linea blu nella Figura 2B), e restituire il cervelletto al SPS. Quando sono necessari altri piani di taglio, tagliare il blocco di cervelletto di conseguenza (linee blu nelle Figure 2C e D). </li></ol></li></ol> 3. Affettare il cervello <ol><li> Preparare la fase di taglio per accertare che sia dry, prima di applicare una piccola goccia di colla al centro del palco. </li><li> Sollevare il blocco cervello dal SPS con una spatola con la corretta posizione normale e rimuovere il liquido in eccesso dal cervello utilizzando piccoli pezzi di carta da filtro. Poi, con un unico movimento, far scorrere il cervello dalla spatola sulla goccia di colla sulla scena. </li><li> Per evitare che il cervello da essiccazione e la colla di corsa sui lati del tessuto, applicare alcune gocce di SPS con una pipetta Pasteur e posizionare la fase di taglio nella camera di affettatura. Allineare il blocco cervello in modo che le regioni di interesse (ad esempio, corteccia cerebellare) si trovano ad affrontare la lama e saranno così sottoposti a minor pressione meccanica prima di essere affettato. </li><li> Tagliare ogni fetta secondo le istruzioni del produttore mentre costantemente gassazione SPS nella camera di taglio e mantenendo la temperatura a 34-37 ° C. Mantenere la temperatura della camera di taglio in questa gamma riempiendo il external camera del affettatrice con riscaldata acqua purificata e sostituendo l&#39;acqua ogni volta che la temperatura scende troppo bassa. NOTA: I parametri affettatrici devono essere trovati sperimentalmente per ogni tipo di tessuto e di neuroni che sono in fase di esame. Nel caso del Campden SMZ 700 con lame in ceramica e cellule Golgi cerebellari, utilizzare 0,75 millimetri ampiezza di vibrazione a 65 Hz e velocità di avanzamento pari a 0,05 mm / sec; per i neuroni nucleari cerebellari maggiore ampiezza e frequenza (1 mm e 90 Hz, rispettivamente) e la velocità di avanzamento lento (0,01-0,02 mm / sec) sono raccomandati. Per entrambi Golgi e neuroni cerebellari nucleari, spessore di strato deve essere di 300 micron. </li><li> Durante il taglio, non consentono le fette di ripiegare su se stessi. Per prevenire questo sostenendoli delicatamente con una spazzola morbida, preferibilmente toccando la fetta solo in regioni che non sono di interesse per l&#39;esperimento. Fare attenzione per evitare che il pennello di toccare la lama vibratome; soprattutto nel caso delle lame di ceramica, anche un leggero contattopuò danneggiare la lama. </li><li> Spostare ogni fetta dalla camera di taglio per un recupero / azienda da camera utilizzando una bocca larga, fuoco lucido vetro pipetta Pasteur. </li><li> Lasciare le fette riposare nella camera per almeno 1 ora prima di iniziare l&#39;esperimento. Questo permette di degradazione del danneggiato o morire tessuti e cellule e quindi si traduce in una superficie fetta detergente. Durante la prima ora, mantenere la temperatura del SPS nella camera nell&#39;intervallo 34-36 ° C; in modo che le fette non sono toccati da bolle d&#39;aria che possono danneggiare le fette o farli galleggiare. </li></ol> 4. Esperimento <ol><li> Dopo 1 ora, lasciare che la temperatura della camera di recupero raffreddare a RT (17-25 ° C). NOTA: Questo potrebbe prolungare il periodo di tempo durante il quale le fette possono essere usate rallentando la crescita di batteri e metabolismo cellulare. Questo periodo dipende dalla regione del cervello e del tipo di cellule. Per esempio, alcuni tipi di cellule del Golgi possono essere giudicati sani a fette8 ore dopo il taglio, mentre altri tipi di durare solo 6 ore. </li><li> Dopo 1 ora è passato dal taglio, utilizzare le fette di vari esperimenti di elettrofisiologia. </li></ol>

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The authors declare that they have no competing financial interests.

Abbiamo dimostrato un metodo per preparare fettine cerebrali acute da topi nel fisiologica anziché temperatura ghiacciata. E &#39;stato dimostrato 11 che la qualità di fette ottenuti in condizioni calde è superiore rispetto a quelli preparati con freddo, a condizione che la lama affettatrice ha vibrazione verticale minima. Affettare a temperatura fisiologica può impedire artefatti fisiologici causati dalla bassa temperatura, come quelli legati ai cambiamenti nei processi metab...

Lo scopo del metodo presentato è quello di ottenere fettine cerebrali acute alta qualità per esperimenti elettrofisiologici in vitro, specialmente quando si usano anche vecchi animali adulti o. Il metodo di taglio cerebrale acuto, come descritto da Skrede e Westgaard 1 in due frasi eleganti, è diventato uno dei fondamenti della moderna ricerca neuroscientifica ed è impiegato in innumerevoli varianti in tutto il mondo. La qualità delle fette si riflette nel numero di neuroni per fetta vivente, il periodo di tempo durante il quale le cellule mantengono le loro proprietà elettrofisiologiche e morfologiche nonché l&#39;integrità del tessuto. Inoltre, la durata massima delle registrazioni stabili dipende dalla qualità delle fette. Così, lungo i decenni, il metodo affettatura originale è stato ulteriormente sviluppato da gruppi di ricerca individuali che permettono di migliorare il recupero fetta dopo il taglio 2-10, spesso complesse modificazioni della composizione di taglio osoluzioni di recupero r (come l&#39;aggiunta di ascorbato, tiourea o H 2 O 2) e intra-cardiaca pre-perfusione dell&#39;animale con soluzioni fisiologiche raffreddate. Come è stato recentemente dimostrato 11, temperatura fisiologica durante l&#39;affettatura sembra essere più vantaggioso di raffreddamento per la salute neuronale; il miglioramento è più evidente quando si lavora con adulti (2-8 mesi) roditori. Evitare sbalzi di temperatura drammatici evita artefatti dovuti a processi dipendenti dalla temperatura nelle cellule, come la plasticità 13 e ioni canali cinetica 13,14. Tali cambiamenti potrebbero influenzare potenziale di membrana e di segnalazione intracellulare di calcio, soglia di picco e picco di forma. Il metodo &quot;a caldo&quot; fetta acuta preparazione qui presentata è una procedura generale per l&#39;ottenimento di fettine di cervello acuto di alta qualità da qualsiasi regione del cervello, tra cui il cervelletto, la corteccia e l&#39;ippocampo, nuclei del tronco cerebrale 16 &lt;/ Sup&gt; e il bulbo olfattivo, sia in ratti e topi. In particolare, la procedura di affettatura temperatura fisiologica richiede che la lama di taglio vibra quasi perfettamente orizzontale ed è senza difetti strutturali. Tale precisione potrebbe non essere raggiungibile con i modelli affettatrice anziani; in tali casi, si consiglia di eseguire la preparazione fetta in condizioni di gelo-freddo come la bassa temperatura sembra rendere il tessuto più resistente ai danni meccanici, anche a costo di aberrazioni metaboliche.

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			<td height="26" style="height:26px;width:140px;">Name</td>
			<td style="width:73px;">Company</td>
			<td style="width:69px;">Catalog #</td>
			<td style="width:292px;">Comments</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
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			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
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			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
			<td style="width:292px;"></td>
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			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
			<td style="width:292px;"></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
			<td style="width:292px;"></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For monitoring SPS temperature during dissection and slicing</td>
		</tr>
	</tbody>
</table>

slice it hot: acute adult brain slicing in physiological temperature

Qui vi presentiamo un protocollo per la preparazione di fettine cerebrali acute. Questa procedura è un elemento critico per elettrofisiologici esperimenti di patch-clamp che determina in larga misura la qualità dei risultati. E &#39;stato dimostrato che omettendo la fase di raffreddamento durante la procedura di taglio è utile nell&#39;ottenere fette e cellule sane, soprattutto quando si tratta di strutture cerebrali altamente mielinizzate da animali adulti. Anche se il meccanismo preciso con cui temperatura elevata per mantenere salute neurale può essere ipotizzato solo dopo, è ovvio che, quando possibile, la temperatura in cui viene eseguito il taglio dovrebbe essere vicino a condizioni fisiologiche per evitare artefatti relativi alla temperatura. Un altro importante vantaggio di questo metodo è la semplicità del procedimento e quindi il tempo di preparazione breve. Nel metodo illustrato topi adulti sono usati ma lo stesso procedimento può essere applicato con i topi più giovani e ratti. Inoltre, le seguenti cl di patchesperimento amplificatore viene eseguita su fettine cerebellari orizzontali, ma lo stesso procedimento può essere utilizzato anche in altri piani, nonché altre aree posteriori del cervello.

Fette preparate nel modo descritto possono essere utilizzati per vari esperimenti elettrofisiologici e optogenetic. Nella Figura 3A e 3C, vi mostriamo un esempio rappresentativo di una fetta cerebellare orizzontale e una fetta coronale cerebrale corticale, rispettivamente, visto sotto interferenza differenziale (DIC) ottiche. Nella fetta cerebellare, diversi tipi di neuroni cerebellari sono facilmente riconoscibili per la loro forma e la posizione delle cellule del corpo, permettendo di...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: acuta cervello adulto affettare in fisiologica Temperatura

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Research

JoVE Journal

Neuroscience

25.9K Views.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Video: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Stato-dipendenza Effetti sulla TMS: uno sguardo al comportamento Motive fosfene

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Ricerca

Neuroscienze

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imaging Pheromone Sensing in preparazione dei tessuti Vomeronasal acuta Slice mouse

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

La chirurgia stereotassica della lesione eccitotossico di specifiche aree cerebrali nel ratto adulto

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Rilevamento di palmitoilazione proteina in colture di neuroni ippocampali di immunoprecipitazione e di acil-Biotina Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Slice Cervello biotinilazione: An<em&gt; Ex Vivo</em&gt; Approccio alla Misura Regione-specifica proteina Tratta plasma membrana in neuroni adulti

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-riprogrammazione di cellule pericyte-derivate del cervello umano adulto in neuroni indotti

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Approfondita fisiologica Analisi di popolazioni di cellule definite nel acuta del tessuto Fette del mouse Vomeronasal Organo

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Registrazione di plasticità sinaptica in fettine ippocampali Acute mantenuti in un piccolo volume riciclaggio - aspersione- e sistema di immersione-tipo camera

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Un'analisi di gradiente di temperatura per determinare le preferenze termiche delle larve di Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

A breve termine, le colture di fette libere dal cervello umano adulto

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...