Questo lavoro è stato sostenuto dal National Institutes of Health (NIH) sovvenzione DA030396. Grazie ai membri del laboratorio Drenan di discussione utile e critica del manoscritto. Un ringraziamento speciale a Kim Mi Ran per l&#39;assistenza tecnica e Jonathan Thomas Ting per avere consigli relativi adulti fettine di cervello di topo.

1. Preparazione di soluzioni per la preparazione del cervello Slice ed Elettrofisiologia <ol><li> Soluzioni per la preparazione di fette di cervello precedentemente descritte 3, 4. Preparare N-metil-D glucammina (NMDG) basata taglio e soluzione di recupero della seguente composizione (in mM): 93 N-metil-D glucammina, KCl 2,5, 1,2 NaH 2 PO 4, 30 NaHCO 3, 20 HEPES, 25 glucosio, 5 Na + ascorbato, tiourea 2, 3 Na piruvato +, 10 MgSO 4 • 7H 2 O, 0,5 CaCl 2 • 2H 2 O. Portare a 300-310 mOsm con NMDG. Regolare il pH a 7,3-7,4 con 10 N HCl. <ol class="lalpha"><li> Sciogliere MgSO 4 • 7H 2 O e CaCl 2 • 2H 2 O in acqua Millipore per fare 2 soluzioni madre M di ciascuno. </li><li> Pesare gli altri componenti nella sequenza indicata e aggiungere in un matraccio tarato parzialmente riempito con acqua Millipore e mescolare per sciogliere. Una volta che tutto è aggiunto, riempirepallone tarato con acqua Millipore. </li><li> Misurare il pH e regolare con HCl 10 N. </li><li> Misurare l&#39;osmolarità e regolare con NMDG se necessario. </li></ol></li><li> Preparare HEPES titolari fluido cerebrospinale artificiale (aCSF) della seguente composizione (in mM): NaCl 92, KCl 2,5, 1,2 NaH 2 PO 4, 30 NaHCO 3, 20, 25 HEPES glucosio, 5 Na ascorbato + 2, tiourea, 3 Na + piruvato, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 • 2H 2 O. Portare a 300-310 mOsm con saccarosio. Regolare il pH a 7,3-7,4 con 1 N HCl o NaOH. <ol class="lalpha"><li> Pesare i componenti nella sequenza indicata e aggiungere in un matraccio tarato parzialmente riempito con acqua Millipore e mescolare per sciogliere. Una volta che tutto è aggiunto, riempire pallone tarato con acqua Millipore. </li><li> Misurare il pH e regolare con NaOH o HCl, se necessario. </li><li> Misurare l&#39;osmolarità e regolare con saccarosio, se necessario. </li></ol></li><li> Preparare la soluzione standard di registrazione aCSF della seguente composizione (in mM): 124 NaCl, KCl 2,5, 1,2 NaH 2 PO 4, 24 NaHCO 3, glucosio 12,5, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 • 2H 2 O. Portare a 300-310 mOsm con saccarosio. Regolare il pH a 7,3-7,4 con 1 N HCl o NaOH. </li></ol> 2. Preparazione di fette cerebrale acuto <ol><li> Slice taglio cervello e recupero vengono eseguiti come precedentemente descritto 3, 4. Bubble due piatti di cristallo pieno di NMDG soluzione di ripristino con il 95% di ossigeno / 5% di CO 2 gas (CarboGen), un piatto sul ghiaccio, l&#39;altra a 33 ° C. </li><li> Bubble un piatto di cristallo pieno di HEPES tengono aCSF con CarboGen a temperatura ambiente. </li><li> Anestetizzare topo adulto (dai 2 ai 12 mesi) con Na + pentobarbital (200 mg / kg, ip). Procedere con procedura quando l&#39;animale non ha alcuna risposta ad un dito del piede-pizzico. </li><li> Se necessario, tagliare un campione di tessuto per la successiva coda Genotypzione dell&#39;animale. </li><li> Aprire la cavità toracica, esporre il cuore, e bloccare l&#39;aorta discendente. Lacerano l&#39;atrio destro. </li><li> Transcardiaca perfusione animale con 5-10 ml di 0-4 ° C NMDG soluzione di ripristino. </li><li> Decapitare animale, rimuovere il cervello e posizionarlo in 4 ° C NMDG soluzione di ripristino per 1 min. </li><li> Su una pista fredda lastra di metallo, tagliare il cervello con l&#39;orientamento desiderato (coronale, parasagittale, orizzontale, ecc) per includere l&#39;area di interesse. </li><li> Fissare il blocco cervello con colla sulla superficie stadio secco di un filtro vibrante (DSK-Zero 1, Dosaka), immergere il blocco cervello in 4 ° C NMDG soluzione di ripristino, e continuamente bolla la soluzione con CarboGen. </li><li> Tagliare fette della zona cerebrale di interesse. Spessore della fetta è 200-400 micron, a seconda della zona del cervello specifico di interesse, età degli animali, e esperimento da eseguire. </li><li> Subito dopo il taglio, fette incubare desiderati in carbogenated, 33 ° C NMDG recupero soluzione moltosu per esattamente 12 minuti, poi il trasferimento a HEPES camera carbogenated, temperatura di mantenimento soluzione per un periodo iniziale di 60 min. Dopo questa incubazione 60 min, fette può essere utilizzato per la registrazione. Fette sono mantenute in HEPES tengono soluzione tutto il giorno fino al loro impiego per la registrazione. </li></ol> 3. Registrazione Patch Clamp di neuroni in fettine di cervello <ol><li> Tirare una micropipetta standard di patch con una programmabile Flaming-Brown estrattore (Sutter P-97 o equivalente estrattore verticale). Pipette ottimali per giga-ohm formazione tenuta in genere hanno una resistenza di 4-6 MW </li><li> Trasferire una fetta alla camera di registrazione (RC-27L; Instruments Warner), sul palco di un microscopio in posizione verticale (Nikon FN-1). Continua superfuse (1,5 - 2,0 ml / min) la fetta con carbogenated, registrazione aCSF serie riscaldata e mantenuta a 32 ° C. In alternativa, fette può essere mantenuto a temperatura ambiente. </li><li> Individuare e centro l&#39;area del cervello di interesse utilizzandoun obiettivo aria 10 volte (Nikon Plan Fluor 10X, NA 0.3; WD: 16 mm). </li><li> Visualizza singoli neuroni con un 40X nel vicino infrarosso (IR) Obiettivo immersione in acqua (Nikon APO 40XW; NA: 0,80; WD: 3,5 mm) collegato ad IR-differenziale contrasto interferenziale (DIC) e ottiche ad alta velocità vicino infrarosso Charged Coupled Device (CCD) telecamera (Hamamatsu C-7500). In IR-DIC osservazione, neuroni sani presentano una superficie liscia, di colore grigio chiaro membrana plasmatica. </li><li> Riempire una micropipetta con una soluzione adeguata registrazione intracellulare. Per la maggior parte delle applicazioni, si usa la seguente soluzione (in mM): 135 gluconato di potassio, EGTA 5, CaCl 2 0,5, 2 MgCl 2, 10 HEPES, 2 Mg-ATP, GTP e 0,1 (pH regolato a 7,25 con Tris base, osmolarità regolato a 290 mOsm con saccarosio). Stabilire una cellula intera registrazione da un neurone visualizzato. </li></ol> 4. Applicazione locale di farmaci ai neuroni a fette <ol><li> Tirare uno standard micropipetta patch clamp come descritto sopra. Utilizzoun estrattore pipetta come un Sutter P-97 che produce due identici &quot;sorelle&quot; consigli dai lo stesso pezzo di vetro è vantaggioso quando soluzioni farmacologiche multiple devono essere utilizzati sulla stessa cella. Suggerimento dimensioni che potrebbero avere una resistenza di ~ 5 MW se sono stati riempiti con la soluzione intracellulare sopra descritta sono ottimali. </li><li> Backfill la micropipetta con una soluzione diluita di farmaco in carbogenated, aCSF registrazione standard. Puntare la punta verso il basso, e aspirare il farmaco pieno micropipetta per eliminare eventuali bolle d&#39;aria intrappolate nella colonna di soluzione. </li><li> Montare il farmaco in una micropipetta riempita portapipette collegata con la tubazione di un adeguato sistema di espulsione a pressione, ad esempio un Picospritzer III (General Valve Co.). Il titolare pipetta deve essere montato su un micromanipolatore con la stessa risoluzione manipolatori comunemente utilizzato per la registrazione di patch clamp (per esempio, Sutter MP-285). Inoltre, il manipolatore deve consentire movimento diagonale e verso la fetta. Uomomanualmente espellere pressione 3 a 4 volte nella micropipetta per costruire un&#39;adeguata pressione dietro la colonna di fluido. </li><li> Utilizzando i controlli micromanipolatore, abbassare la pipetta farmaco nel fetta mentre visualizzi la punta sotto IR-DIC ottica. Idealmente, si dovrebbe identificare visivamente e centro un neurone da registrare da, seguito dal posizionamento della pipetta farmaco vicino alla cella prima di abbassare la micropipetta registrazione nella sezione. Posizionare la pipetta farmaco può causare il movimento del tessuto in prossimità della cella registrata che potrebbe disturbare la registrazione se il farmaco pipetta viene spostata in posizione dopo aver stabilito un sigillo gigaohm. </li><li> Utilizzando video IR, posizionare la micropipetta farmaco alla superficie superiore della fetta tessuto. Eseguire una espulsione di pressione e monitor 1) prossimità della punta per il movimento dei detriti legata alla espulsione, e 2) la punta micropipetta per qualsiasi segno che la punta sia ostruiti o bloccati. Se la punta è bloccato / intasato, ritrarre il pipettE e sostituirlo con uno nuovo. </li><li> Avvicinare la cella registrata diagonalmente dall&#39;alto della fetta tale che quando in posizione finale, la punta della pipetta farmaco-riempita è 1) nello stesso piano focale (o leggermente inferiore) come cella registrata e la punta dell&#39;elettrodo di registrazione , e 2) 10 a 40 um dalla cella registrata. Se il farmaco-riempita puntale è sopra la cella registrata, la forza di espulsione dalla pressione potrebbe compromettere la tenuta gigaohm. </li><li> Registrare la risposta cellulare desiderata tenendo la cella in modalità voltage clamp o modalità morsetto corrente. Abbiamo regolarmente registrare acetilcolina e / o nicotina suscitato correnti cellulari tenendo neuroni in modalità voltage clamp. Anche se la pressione di espulsione può essere attivato manualmente utilizzando un Picospritzer III, per risultati più riproducibili si consiglia di attivare l&#39;espulsione pressione utilizzando il sistema di acquisizione (Axon Digidata 1440 e pClamp 10.3) con un TTL impulsi digitali. </li></ol> 5. ControlloDrug-riempita Micropipetta ad un traduttore piezoelettrico <ol><li> Se possibile, montare il farmaco-riempita portapipette ad un traduttore monodimensionale piezoelettrico (es Piezojena PA-100 o Burleigh PZM-150) che è in grado di accettare un ingresso analogico di tensione (di nuovo, dal sistema di acquisizione), che è poi montato sul micromanipolatore alta precisione (Sutter MP-285). Un traduttore piezoelettrico è utile perché il movimento del farmaco-riempita pipetta dentro e fuori della fetta, inclusi i tempi e la velocità di entrata / uscita, possono essere più facilmente controllata e riprodotto da esperimento a esperimento. Quando si studia nAChR, gli effetti di desensibilizzazione perdite nicotina dal farmaco-riempita pipetta, se mai verificarsi, vengono minimizzati quando la pipetta viene spostata solo vicino alla cella con un traduttore piezoelettrico per il momento della pressione di espulsione. </li><li> Con il potenziometro a controllo manuale dell&#39;amplificatore piezoelettrico controllo della tensione, selezionare la tensioneal suo valore massimo. </li><li> Utilizzando la, posizione micromanipolatore farmaco-riempita pipetta nella fetta dove la pipetta viene espulso il contenuto nella cella registrata, e ritrarre la pipetta manualmente riducendo la tensione a zero dell&#39;amplificatore piezoelettrico controllo di tensione. </li><li> Utilizzando un segnale di uscita analogico dal sistema di acquisizione di registrazione, spostare il farmaco-riempita pipetta nella fetta su un periodo di 250 msec 300 msec prima di iniziare la pressione di espulsione impulsi TTL verifica. Ritrarre la pipetta in un periodo di 250 msec, iniziando a 50 msec dopo l&#39;impulso TTL termina. </li></ol>

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Physiol. 587, 345-361 (2009).</li><li>Endo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+acetylcholine+receptor+subtypes+involved+in+facilitation+of+GABAergic+inhibition+in+mouse+superficial+superior+colliculus.">Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.</a> J. Neurophysiol. 94, 3893-3902 (2005).</li><li>Bouzat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+structural+bases+of+activation+of+Cys-loop+receptors.">New insights into the structural bases of activation of Cys-loop receptors.</a> J. Physiol. Paris. , (2011).</li></ol>

Il metodo presentato in questo articolo è ampiamente utile per studiare ligando funzione di canale ionico nella preparazione fetta del cervello. Tuttavia, ci sono un certo numero di fattori che influenzano significativamente la qualità e la riproducibilità dei dati sperimentali che derivano da utilizzando questo metodo. Per esempio, le correnti evocate sono molto sensibili al diametro della punta della pipetta farmaco-riempita. Piccoli consigli causerà difficoltà con espulsione della soluzione di farmaco, e le punt...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nome del reagente</strong</td&gt;<td<strong&gt; Azienda</strong</td&gt;<td<strong&gt; Numero di catalogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; N-metil-D glucammina</td&gt;<td&gt; Sigma</td&gt;<td&gt; M2004</td&gt;</tr&gt;<tr&gt;<td&gt; KCl</td&gt;<td&gt; Sigma</td&gt;<td&gt; P3911</td&gt;</tr&gt;<tr&gt;<td&gt; NaH<sub&gt; 2</sub&gt; PO<sub&gt; 4</sub</td&gt;<td&gt; Sigma</td&gt;<td&gt; S9638</td&gt;</tr&gt;<tr&gt;<td&gt; NaHCO<sub&gt; 3</sub</td&gt;<td&gt; Sigma</td&gt;<td&gt; S6014</td&gt;</tr&gt;<tr&gt;<td&gt; HEPES</td&gt;<td&gt; Sigma</td&gt;<td&gt; H3375</td&gt;</tr&gt;<tr&gt;<td&gt; Glucosio</td&gt;<td&gt; Sigma</td&gt;<td&gt; G5767</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Ascorbato</td&gt;<td&gt; Sigma</td&gt;<td&gt; A4034</td&gt;</tr&gt;<tr&gt;<td&gt; Tiourea</td&gt;<td&gt; Sigma</td&gt;<td&gt; T8656</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Piruvato</td&gt;<td&gt; Sigma</td&gt;<td&gt; P2256</td&gt;</tr&gt;<tr&gt;<td&gt; MgSO<sub&gt; 4</sub&gt; • 7H<sub&gt; 2</sub&gt; O</td&gt;<td&gt; Sigma</td&gt;<td&gt; 230391</td&gt;</tr&gt;<tr&gt;<td&gt; CaCl<sub&gt; 2</sub&gt; • 2H<sub&gt; 2</sub&gt; 0</td&gt;<td&gt; Sigma</td&gt;<td&gt; 223506</td&gt;</tr&gt;<tr&gt;<td&gt; NaCl</td&gt;<td&gt; Sigma</td&gt;<td&gt; S9625</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Pentobarbital</td&gt;<td&gt; Vortech Pharmaceuticals</td&gt;<td&gt; 76351315</td&gt;</tr&gt;<tr&gt;<td&gt; Gluconato di potassio</td&gt;<td&gt; Sigma</td&gt;<td&gt; G4500</td&gt;</tr&gt;<tr&gt;<td&gt; EGTA</td&gt;<td&gt; Sigma</td&gt;<td&gt; E3889</td&gt;</tr&gt;<tr&gt;<td&gt; Mg-ATP</td&gt;<td&gt; Sigma</td&gt;<td&gt; A9187</td&gt;</tr&gt;<tr&gt;<td&gt; GTP</td&gt;<td&gt; Sigma</td&gt;<td&gt; G8877</td&gt;</tr&gt;<tr&gt;<td&gt; DSK-Zero 1 vibrante affettatrice</td&gt;<td&gt; Ted Pella, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; P-97 Flaming / Brown micropipetta estrattore</td&gt;<td&gt; Sutter</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Camera di RC-27 Registrazione</td&gt;<td&gt; Warner</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; TC-344B perfusione regolatore riscaldamento</td&gt;<td&gt; Warner</td&gt;<td&gt; 640101</td&gt;</tr&gt;<tr&gt;<td&gt; SH-27B soluzione di riscaldamento</td&gt;<td&gt; Warner</td&gt;<td&gt; 640102</td&gt;</tr&gt;<tr&gt;<td&gt; Nikon FN-1</td&gt;<td&gt; Nikon</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; C-7500 CCD Videocamera</td&gt;<td&gt; Hamamatsu</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Picospritzer III</td&gt;<td&gt; Generale Valve Co.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; MP-285 Micromanipolatore</td&gt;<td&gt; Sutter</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PA-100 traduttore piezoelettrico</td&gt;<td&gt; Piezosystem jena, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 12V40 piezo amplificatore</td&gt;<td&gt; Piezosystem jena, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Axopatch 200B</td&gt;<td&gt; Molecular Devices Corp.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Digidata 1440A</td&gt;<td&gt; Molecular Devices Corp.</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices

L&#39;uso del tabacco porta a numerosi problemi di salute, tra cui il cancro, malattie cardiache, enfisema, e ictus. Dipendenza da fumo di sigaretta è un disturbo neuropsichiatrico prevalente che deriva dalle azioni biofisici e cellulari della nicotina sui recettori nicotinici (nAChR) in tutto il sistema nervoso centrale. Comprendere i vari sottotipi nAChR esistenti in aree cerebrali importanti per la dipendenza da nicotina è una delle principali priorità. Esperimenti che impiegano tecniche elettrofisiologiche, come cellula intera o patch clamp a due elettrodi registrazioni voltage clamp sono utili per la caratterizzazione farmacologica dei nAChR di interesse. NAChRs cellule esprimenti, come cellule di mammifero o di coltura tissutale oociti di Xenopus laevis, sono fisicamente isolata e sono quindi facilmente studiato utilizzando gli strumenti della moderna farmacologia. Molti progressi sono stati fatti utilizzando queste tecniche, in particolare quando il recettore bersaglio era già noto unnd espressione ectopica è facile da realizzare. Spesso, tuttavia, è necessario studiare nAChR nel loro ambiente nativo: in neuroni in fettine cerebrali acutamente raccolti da topi o ratti di laboratorio. Ad esempio, i topi che esprime &quot;ipersensibili&quot; subunità nAChR come topi α4 L9&#39;A 1 e topi α6 L9&#39;S 2, consente l&#39;identificazione univoca dei neuroni in base alla loro espressione funzionale di una subunità nAChR specifico. Sebbene cellule intere registrazioni di patch clamp da neuroni in fettine di cervello è fatto di solito dal elettrofisiologo esperto, è difficile da applicare localmente farmaci come acetilcolina o la nicotina alla cella di registrare, in una sezione del cervello. Diluizione dei farmaci nella superfusate (applicazione bagno) non è rapidamente reversibile, e U-tube sistemi non si adattano facilmente a lavorare con fettine di cervello. In questo articolo, si descrive un metodo per la rapida applicazione di nAChR-attivazione farmaci neuroni registrati in pazienti adulti mOuse fettine di cervello. Standard di cellule intere registrazioni sono fatte da neuroni in fettine, e una micropipetta secondo riempito con un farmaco di interesse viene manovrata in posizione vicino alla cella registrata. Una iniezione di aria compressa o azoto inerte nella droga-riempita pipetta provoca una piccola quantità di soluzione di farmaco ad essere espulso dalla pipetta sulla cella registrata. Usando questo metodo, nAChR mediati correnti possono essere risolti con precisione millisecondo. Tempi di applicazione del farmaco può essere facilmente variata, e il farmaco-riempita pipetta può essere retratto e sostituita con una nuova pipetta, consentendo curve concentrazione-risposta per la creazione di un singolo neurone. Sebbene descritta nel contesto di nAChR neurobiologia, questa tecnica dovrebbe essere utile per studiare molti tipi di ligando canali ionici o recettori nei neuroni da fettine cerebrali.

Nei nostri esperimenti, abbiamo regolarmente registrare da dopamina (DA) alla produzione di neuroni dell&#39;area ventrale tegmentale (VTA) e substantia nigra pars compacta (SNC). In voltage-clamp modalità di applicazione di pressione di acetilcolina o nicotina a queste cellule in genere come risultato un rapido, corrente catione attivo che raggiunge picco entro 100-200 msec (Figura 1A-B). Decadimento della corrente in larga misura dalla diffusione del farmaco dal sito di azione, e se enzimi nella fett...

Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

In questo articolo, si descrive un metodo utile per lo studio ligando funzione di canale ionico in neuroni di fettine di cervello acuto isolato. Questo metodo comporta l&#39;uso di un farmaco-riempita micropipetta per applicazione locale di farmaci per neuroni registrati utilizzando tecniche standard di patch clamp.

Applicazione locale di farmaci allo studio delle funzioni recettore nicotinico in fettine di cervello del mouse

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Drugs Acting on Autonomic Nervous System: Cholinergic Agonists and Antagonists Agents

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19.2K Views.  Purdue University. In questo articolo, si descrive un metodo utile per lo studio ligando funzione di canale ionico in neuroni di fettine di cervello acuto isolato. Questo metodo comporta l'uso di un farmaco-riempita micropipetta per applicazione locale di farmaci per neuroni registrati utilizzando tecniche standard di patch clamp.

Video: Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Calcio Risposte imaging in neuroni GFP-tagged fette di cervello di topo ipotalamici

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Imaging Fibre serotoninergici nel midollo spinale mouse Uso del CHIAREZZA / Tecnica CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

HSV-mediata Transgene Espressione di chimerici costrutti di studio comportamentale funzione di GPCR Heteromers nei topi

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Applicazione di morsetto di patch automatico guidato per immagini per lo studio dei neuroni in fettine di cervello

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Cellule intere correnti indotte dall'applicazione di Puff di GABA nelle fette del cervello

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un approccio alternativo per studiare eventi primari nella neurodegenerazione utilizzando fettine di cervello di ratto Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Funzione del recettore nicotinico dell'acetilcolina in fettine di cervello del Mouse tramite Laser fotolisi di nicotina fuse di sondaggio

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Studiare la funzione del cervello in bambini che usando magnetoencefalografia

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Impianto cronico di tutto-corticale Electrocorticographic Array nel Marmoset comune

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Valutazione dell'induzione della depressione a lungo termine nelle fette di cerebellar adulto

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Applicazione locale di farmaci allo studio delle funzioni recettore nicotinico in fettine di cervello del mouse

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