Il sostegno è stato fornito da avvio-fondi del Dipartimento di Biologia Farmacologia e Chimica presso l&#39;Università di Pittsburgh School of Medicine. Riconoscimento dei membri del laboratorio Giacobbe che hanno contribuito alla presentazione del video: Nicholas Graff.

Tutti i protocolli descritti di seguito sono in conformità con la IACUC e IRB commissioni di revisione istituzionale dell&#39;Università di Pittsburgh School of Medicine. 1. Preparazione delle Culture neuronale ippocampale in Tissue Culture Hood Nota: utilizzare una tecnica sterile e reagenti per tutta protocollo 1. <ol><li> Preparare poli-D-lisina (0,1 mg / ml in H 2 O) vetrini rivestiti come substrato per la coltura neuronale. <ol><li> Mettere 4-5 vetrini tondi all&#39;interno di ogni 3,5 centimetri di tessuto piatto di coltura. </li><li> Spot 70 microlitri poli-D-lisina su ogni vetrino 12 millimetri. Nota: per l&#39;imaging dal vivo, un fondo di vetro da 3,5 cm di tessuto piatto di coltura può essere utilizzato (posto 200 microlitri poli-D-lisina sul incorporato 14 millimetri coprioggetto di vetro). </li><li> Lasciare i piatti preparati nel tessuto della cultura cofano O / N. Per ridurre al minimo poli-D-lisina evaporazione e mantenere le superfici sterili nella cappa coltura tissutale, chiudere il telaio della finestra e spegnere °e soffiante O / N. Nota: luci UV non vengono utilizzati durante l&#39;incubazione O / N. </li><li> La mattina seguente, lavare i piatti 3x con 2 ml di H 2 O. </li><li> Dopo aver rimosso l&#39;ultimo H 2 O lavaggio, aggiungere 2 ml di media per ogni piatto 3,5 centimetri e lasciare i piatti in incubatrice fino al momento piastra neuroni nella fase 1.4. </li></ol></li><li> Neuroni dell&#39;ippocampo sono preparati dal giorno embrionale 18-19 (E18-19) ratti (modificati dalla Goslin 22). </li><li> Transfettare neuroni appena dissociati il ​​giorno della coltura con 1-4 mg di maxiprepped DNA costrutto. Nota: tipicamente 3 pg di DNA viene transfettato in 1-2 milioni di neuroni, con una vitalità del 50% ed una efficienza di trasfezione del 60% (ad esempio partendo con 2 milioni di neuroni: ((2 x 10 6 neuroni x 0.5) 0.6) fornisce circa 1 milione di neuroni,.. 600.000 dei quali sono transfettate 1 diverse quantità di costrutto di DNA possono essere trasfettate durante la risoluzione dei potenziali problemi disovra-espressione, seguita da un&#39;opportuna caratterizzazione della localizzazione e della funzione costrutto. </li><li> Piatto i neuroni trasfettati sui vetrini rivestiti di poli-D-lisina con una densità finale di circa 200.000 neuroni per 3,5 centimetri piatto. Nota: per un fondo di vetro piatto, piatto 40,000-50,000 neuroni sulla superficie vetrata 14 millimetri. </li><li> Sostituire i media 4-24 ore dopo la preparazione di colture neuronali, poi permettono ai neuroni di svilupparsi in incubatrice fino a 14-17 giorni in vitro (DIV) o stadio desiderato. </li></ol> 2. Endocitosi Assay ATTENZIONE: Nota sulla α-bungarotossina e dei rifiuti e la gestione tubocurarine: <ol><li> Manipolazione di tutte le neurotossine peptide è raccomandato entro le linee guida di protocolli di ricerca Biosafety Level-2 (BL-2). Tutti corretta attrezzatura di protezione personale devono essere indossati. Polveri tossina liofilizzato deve essere ricostituito secondo le istruzioni del produttore. Tossina shou rifiutild essere smaltita in conformità con le linee guida dell&#39;ente in materia di salute ambientale e sicurezza sul lavoro. </li><li> Per rimuovere eventuali aggregati peptidici che possono formarsi durante la conservazione, aliquote α-bungarotossina devono essere centrifugati brevemente prima dell&#39;uso, e il supernatante dovrebbero essere utilizzati per l&#39;esperimento. </li><li> α-bungarotossina (Bgt) stock sono risospese ad una concentrazione di 1 mg / ml in H 2 O sterile e memorizzati in 10 microlitri aliquote a -20 ° C. Scorte Bgt fluorescente accoppiati vengono utilizzati a 3 mg / ml. Tubocurarina viene utilizzato ad una concentrazione finale di 150 mM. </li></ol><ol><li> Set banco di blocco riscaldatore superiore a 16 ° C in camera fredda con sottile in alluminio o lamiera di acciaio inox sulla parte superiore. In alternativa, se disponibile, un dispositivo top di raffreddamento / riscaldamento banco può essere utilizzato per ogni passo richiede 16 ° C. </li><li> Raffreddare extracellulare saline Hepes tamponata (HBS contenenti in mm: 135 NaCl, 4.7 KCl, 10 HEPES, 11 glucosio, 1.2 MgCl 2, e 2,5CaCl 2, pH 7,4) a 16 ° C in un bagno d&#39;acqua. </li><li> Trasferimento piatti neurone a lastra di alluminio a 16 ° C e raffreddare per 5 min. </li><li> Rimuovere i supporti (mantenere mezzi condizionati e riserva in incubatrice per il passaggio 2.8, endocitosi punti di tempo saggio) e sostituirlo con 1 ml di 16 ° C HBS + tubocurarina (150 micron) per 2 minuti. </li><li> Rimuovere HBS + tubocurarine di contenitore per rifiuti etichettati e sostituirlo con 1 ml di 16 ° C HBS + tubocurarina (150 micron) + α-bungarotossina Alexa 594 [3 mg / ml], incubando a 16 ° C per 15 min. </li><li> Rimuovere HBS + tubocurarina + α-bungarotossina Alexa 594 per etichettato contenitore di rifiuti e lavare piatti 3x con 2 ml di 16 ° C HBS (i lavaggi possono essere raccolti tramite aspirazione in una beuta da vuoto contenente 50 ml di 50% candeggina). </li><li> Per gli esperimenti di serie temporali di immagini dal vivo seguenti endocitosi con un fondo di vetro 3,5 centimetri piatto di coltura, eseguire i passaggi 2,1-2,6, quindi trasferire il piatto al palco riscaldato(Dispositivo di Peltier) sul microscopio, e iniziare imaging con una confocale o setup microscopio TIRF. </li><li> Trasferimento T coprioggetto = 0 punto temporale in un piatto di RT paraformaldeide 4% / 4% di saccarosio per 20 minuti, continuando con altri coprioggetto al punto 2.9. </li><li> Per gli altri punti temporali, sostituire HBS con condizionata equilibravano 37 ° C supporti (riservato nel passo 2.4) e tornare a incubatore per endocitosi a 37 ° C. Nota: suggerito punti aggiuntivi per il tempo di T = 15, 30, e 60. </li><li> A intervalli di tempo necessario, prendere piatti da incubatrice, lavare rapidamente due volte con 2 ml di RT DPBS (DPBS senza calcio, magnesio), quindi fissare in paraformaldeide al 4% / 4% di saccarosio per 20 min. </li><li> Dopo 20 minuti la fissazione, rimuovere paraformaldeide al 4% / 4% di saccarosio sprecare contenitore e lavare i piatti due volte con 2 ml di RT DPBS. </li><li> Incubare coprioggetti a temperatura ambiente per 10 min in soluzione di blocco immunofluorescenza (soluzione blocco = siero di cavallo 5%, 0,5% BSA in DPBS) contenente 0,2% Triton X-100 di permeabilitàizzare i neuroni e abilitare immunostaining anti-GFP della piscina recettore intracellulare e / o l&#39;etichettatura di altre proteine ​​di interesse. </li><li> Rimuovere blocco + 0,2% Triton X-100 e incubare coprioggetto in soluzione blocco immunofluorescenza senza Triton X-100 per 20-30 min. </li><li> Eseguire incubazioni anticorpi primari di vetrini in blocco per diverse ore a temperatura ambiente o O / N a 4 ° C. Nota: l&#39;incubazione dei vetrini con primarie a 4 ° CO / N produce abitualmente risultati migliori immunofluorescenza. </li><li> Lavare coprioggetto 3x con DPBS (5 min per ogni fase di lavaggio). </li><li> Incubare coprioggetto con anticorpi secondari in soluzione blocco per 1 ora a RT. </li><li> Lavare coprioggetto 3x con DPBS (5 min per ogni fase di lavaggio). </li><li> Coprioggetti Mount, la manipolazione ogni vetrino singolarmente: rimuovere il liquido in eccesso dal retro del vetrino con il tessuto di laboratorio, quindi capovolgere coprioggetto su 4 ml di mezzo di montaggio su un vetrino. </li><li> Consenti vetrini ad asciugare a temperatura ambiente coperto per 30 minuti poi vminerale a 4 ° C fino al momento dell&#39;esecuzione microscopia. </li><li> Acquisizione delle immagini e l&#39;analisi di sperimentare con la microscopia confocale (ciechi a condizione sperimentale). 60X olio NA 1.49 obiettivo ad immersione con i seguenti laser: gas Argon 488 nm, 561 diodo, diodo 640. <ol><li> Utilizzando le stesse impostazioni di acquisizione dell&#39;immagine, acquisire immagini singole sezioni Z con il corpo cellulare neuronale e diversi processi dendritiche a fuoco. </li><li> Quantificare α-bungarotossina segnale fluorescente Alexa e GFP immunostaining lungo 20 micron di 3-4 dendriti prossimali per neurone, utilizzando la stessa soglia per l&#39;analisi di tutti i dati endocitosi. </li><li> Analizzare i dati 10-12 neuroni per ogni punto temporale, ripetendo l&#39;esperimento con diverse colture neuronali indipendenti. </li></ol></li></ol>

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Le tecniche fisse e live basati BBS qui descritti possono essere utilizzati per monitorare recettore o altra membrana plasmatica traffico di proteine ​​in linee cellulari, neuroni e altre cellule primarie. Questo metodo è stato usato con successo per studiare l&#39;inserimento e la rimozione della membrana dei canali ionici ligando-dipendenti e GPCR e valutare variazioni di traffico per la presenza di agonisti dei recettori e modulatori. Gli aspetti chiave includono la localizzazione appropriata del tag in una posi...

L&#39;uso di fluorescenza accoppiato α-bungarotossina studiare localizzazione recettore e dinamiche è stato sperimentato in studi del acetilcolina recettore nicotinico 13-15, Obiettivo endogeno di una tossina. Successivamente, l&#39;incorporazione del minimo peptide vincolante Bgt (BBS) è stato utilizzato per studiare il traffico di entrambi i canali ionici ligando-dipendenti eccitatori e inibitori e proteine ​​G recettori accoppiati 2,10,16-21. Questa tecnica basata su BBS offre vantaggi ad altri approcci utilizzati per lo studio di traffico come i metodi biotinilazione superficie, l&#39;etichettatura degli anticorpi di cellule vive con anticorpi verso epitopi extracellulari, e il recupero di fluorescenza dopo photobleaching (FRAP). Durante superficie cellulare biotinilazione ammine libere sono covalentemente modificati, con il potenziale per influenzare l&#39;attività cellulare. Studi basati anticorpo sono stati spesso ostacolati da antigene di superficie di clustering o tappatura, che possono alterare gli eventi di traffico. A causa del passaggio sbiancante per FRAP sTUDI, una preoccupazione importante sta danneggiando la struttura cellulare sottostante. Un ulteriore vantaggio è che BBS etichettato costrutti possono essere utilizzati anche per metodologie biochimiche con traffico biotina-recettore accoppiato bungarotoxin per valutare. Questa tecnica è facilmente applicabile a linee cellulari e cellule primarie. Per l&#39;uso in cellule che esprimono nicotinico per l&#39;acetilcolina (nAChR) recettori, l&#39;antagonista tubocurarina nAChR deve essere utilizzato in tutto il protocollo, come indicato. Esecuzione di etichettatura Bgt semplice superficie (equivalente al punto di tempo T = 0 per il protocollo endocitosi) sulle cellule non trasfettate in assenza di tubocurarina fornirà la prova di endogeno nAChR. Una considerazione importante per l&#39;utilizzo di questa tecnica è opportuno inserimento del BBS in modo che sia presente in un extracellulari quando la proteina di interesse è trasportato alla membrana plasmatica. Ad esempio, i domini N-terminali delle subunità GABAAR risiedono nel lume vescicolari durante trafla tratta e diventare extracellulare dopo l&#39;inserimento del recettore nella membrana plasmatica, consentendo un&#39;etichettatura specifica di recettori di superficie delle cellule e la valutazione della loro rimozione dalla superficie cellulare da eventi endocitiche. Abbiamo precedentemente dimostrato che l&#39;aggiunta di GFP, myc, o BBS epitopi a questo dominio di subunità GABAAR è funzionalmente silenzioso. Controlli standard devono essere effettuate per garantire che la proteina marcata è espressa a livelli simili a un costrutto non etichettato, che opportunamente localizzato, e che non influenza la funzione del recettore. Questa caratterizzazione di costrutti trasfettate sarà anche un aiuto per la risoluzione di problemi iperespressione.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:196px;">Catalog Number</td>
			<td style="width:187px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B35450</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
			<td style="width:196px;">cS703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:196px;">P6407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">640-0702</td>
			<td></td>
		</tr>
	</tbody>
</table>

using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

E &#39;sempre più evidente che recettori per i neurotrasmettitori, tra cui i recettori ionotropici GABA A (GABAAR), mostre traffico altamente dinamico e la mobilità di superficie delle cellule 1-7. Per studiare recettore cellulare localizzazione superficie e endocitosi, la tecnica qui descritta combina l&#39;uso di fluorescente α-bungarotossina con cellule che esprimono costrutti contenenti un α-bungarotossina (Bgt) sito di legame (BBS). Le BBS (WRYYESSLEPYPD) è basato sulla subunità α del muscolo recettore nicotinico, che si lega con alta affinità Bgt 8,9. Incorporazione del sito BBS consente localizzazione superficie e misure di inserimento recettore o la rimozione con applicazione di fluorescente esogeno Bgt, come precedentemente descritto nel monitoraggio di GABAA e GABAB recettori metabotropici 2,10. Oltre al sito BBS, abbiamo inserito un GFP sensibile al pH (pHGFP 11) tra aminoacidi 4 e 5 del maturo GABAAR subunità da m normastrategie di biologia e clonazione PCR olecular (vedi figura 1) 12. Il BBS è 3 &#39;del reporter GFP sensibile al pH, separati da un 13-amminoacido alanina / prolina linker. Per il traffico di studi descritti in questa pubblicazione che si basano su campioni fissati, il pHGFP serve come reporter del totale tagged GABAAR livelli della proteina subunità, che consente la normalizzazione della Bgt etichettato popolazione di recettori per la popolazione recettore totale. Questo minimizza cella a cella Bgt variabilità segnale colorazione risultante dalla maggiore o minore espressione basale delle subunità GABAAR contrassegnate. Inoltre, il tag pHGFP consente una facile identificazione di cellule che esprimono costrutto per esperimenti di imaging dal vivo o fissi.

Caratterizzazione di un costrutto BBS targhetta include controlli importanti come determinare se la proteina espressa assembla correttamente (in particolare con i recettori composti da più subunità), traffici alla superficie cellulare e localizza appropriato. Sinapsi inibitorie sono composti da GABA A cluster di superficie recettore che colocalizza con l&#39;inibitoria della proteina scaffold gephyrin e sono apposto ai terminali presinaptici inibitori, individuati dal vescicolare inibitorio trasportatore di aminoacidi...

Watch this Scientific Journal Video about Using an Î±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Qui mostriamo l&#39;uso di colorante fluorescente Alexa accoppiato ad α-bungarotossina per misurare GABA A localizzazione superficiale recettore e endocitosi nei neuroni dell&#39;ippocampo. Attraverso l&#39;uso di costrutti recanti un breve tag extracellulare che lega α-bungarotossina, analisi di proteine ​​di membrana plasmatica traffico endocitico può essere raggiunto.

L&#39;utilizzo di un α-bungarotossina Binding Tag sito per studiare GABA un recettore di membrana localizzazione e il traffico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

Research

JoVE Journal

Neuroscience

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

15.7K Views.  University of Pittsburgh School of Medicine.  Qui mostriamo l'uso di colorante fluorescente Alexa accoppiato ad α-bungarotossina per misurare GABA A localizzazione superficiale recettore e endocitosi nei neuroni dell'ippocampo. Attraverso l'uso di costrutti recanti un breve tag extracellulare che lega α-bungarotossina, analisi di proteine ​​di membrana plasmatica traffico endocitico può essere raggiunto. 

Video: Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Ricerca

Neuroscienze

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

Utilizzando MazeSuite e funzionale Near Infrared Spectroscopy per studiare l&#39;apprendimento in navigazione spaziale

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imaging pHluorin-tagged inserimento recettore alla membrana plasmatica nella Primaria neuroni mouse coltivate

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

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

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Potenziale di membrana Dye Imaging di ventromediale dell&#39;ipotalamo neuroni da topi adulti per studiare glucosio Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

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 ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localizzazione dei geni del recettore degli odoranti nell&#39;antenna di Locust da RNA<em&gt; In Situ</em&gt; Ibridazione

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Immagini in diretta della proteina GLUT4 tratta di neuroni ipotalamici primario del Mouse usando la microscopia di Deconvolution

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

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, ...