Gli autori vorrei ringraziare il Post Diploma di maturità Scholars Program Office e Novartis Institutes for BioMedical Research nel suo complesso per il finanziamento di questo studio.

1. preparazione delle celle
Nota: Questo protocollo, tra cui la generazione dei dati, utilizza cellule HEK293 trasformate con un baculovirus codifica NR1 e NR2A cellule.
<ol><li>Il numero appropriato di cellule HEK293 di semi e aggiungere il virus NR1 e/o NR2A alle concentrazioni finali appropriate (da 1.00 µ l). Per un piatto di 384 pozzetti, utilizzare 10.000 cellule/pozzetto in un volume finale di 30 µ l.</li>
	<li>In alternativa, seme cellule HEK293-NR1-NR2A presso il numero di cella appropriata e il volume (per un piatto di 384 pozzetti, uso 10.000 cellule/pozzetto in un volume finale di 30 µ l). Aggiungere tetraciclina ad una concentrazione di 2 µ g/mL per indurre l'espressione di NR2A e aggiungere la protezione composta (100 µM MDL105, 519 o 5 µM CGP07066724).</li>
	<li>Nota: Il baculovirus codifica NR1 e NR2 dovrebbe avere un titolo approssimativo tra 5 x 109 - 10 x 109 unità infettive per millilitro24.</li>
</ol>2. misurazione dell'attività del recettore NMDA
<ol><li>Preparare una piastra 384 pozzetti con cellule HEK293 trasformate con cellule NR1/NR2 o HEK293-NR1-NR2A in presenza sia composto protettivo. Utilizzare un fondo chiaro, telaio nero, piastra rivestita di poli-D-lisina. Nota: Protezione con 100 µM MDL105, 519 viene utilizzato per conferire la sensibilità a glicina, mentre 5 µM CGP070667 viene utilizzato per conferire sensibilità al glutammato.</li>
	<li>Incubare la piastra a 37 ° C e 5% CO2 per 16 ore (una notte).</li>
	<li>Rimuovere la piastra dall'incubatrice e scartare delicatamente il supporto cellulare rallentando lanciare la piastra sopra un contenitore di rifiuti organici compatibili. Picchiettare delicatamente la piastra su un tovagliolo di carta per pulire il supporto i bordi della piastra e drenare qualsiasi media rimanenti.</li>
	<li>Preparare il calcium6-colorante solubilizzanti un flaconcino di colorante calcium6 (1 dimensioni piastra) in 10 mL di tampone di incubazione (HBSS pH 7.5, 20 mM HEPES, 1 mM MgCl2, probenecid 1 mM). Incubare la piastra per 2 ore a 37 ° C e 5% CO2.</li>
	<li>Rimuovere la piastra dall'incubatore e lasciare che le celle regolare a temperatura ambiente per 10 minuti.</li>
	<li>Versare delicatamente il calcium6-colorante come descritto al punto 2.3 e aggiungere 30 µ l di tampone di dosaggio (HBSS pH 7.5, 20 mM HEPES, 1,8 mM CaCl2, probenecid 1 mM).</li>
	<li>Ripetere il passaggio 2.6 due volte per un totale di 3 lavaggi. Lasciare 30 µ l di tampone del saggio sulle cellule dopo l'ultimo lavaggio.</li>
	<li>Lasciare che le celle riposare per 5 minuti a temperatura ambiente.</li>
	<li>Preparare la piastra di ligando facendo un 4 volte stock di 400 µM glicina e 400 µM glutammato (concentrazione finale di 100 µM; concentrazione ottimale di ligandi deve essere determinato come descritto di seguito). Trasferire un minimo di 25 µ l di brodo di ligando in una piastra di fondo V 384 pozzetti.</li>
	<li>Caricare la piastra ligando e cella in Franzese (sistema funzionale di Screening di stupefacenti).</li>
	<li>Misurare la fluorescenza di base (Fo) della piastra cellulare per 30 secondi. Trasferire 10 µ l di brodo ligando nella piastra cellulare utilizzando la funzione di erogazione di scenografo e misura il flusso di calcio registrando fluorescenza calcium6-colorante per 5 minuti.</li>
	<li>Calcolare il rapporto di massima fluorescenza dividendo la massima fluorescenza ottenuta mediante la fluorescenza di base (Fo).</li>
</ol>3. ottimizzazione dei livelli di espressione di NMDAR
<ol><li>Per determinare la quantità ottimale di baculovirus utilizzare, eseguire una titolazione del virus sia NR1 e NR2A. Preparare una piastra 384 pozzetti con cellule HEK293 (raccomandazione di 10.000 cellule/pozzetto, 30 µ l di media) e includono i composti di protezione come descritto al punto 2.1.</li>
	<li>Aggiungere quantità variabili del virus NR1 in righe diverse della piastra (ad esempio: aggiungere rispettivamente 0 µ l, 2 µ l, 1 µ l, 0,5 µ l e 0,25 µ l in righe A-E). Aggiungere duplicati per accuratezza dei dati.</li>
	<li>Aggiungere quantità variabili del virus NR2A in diverse colonne del piatto (ad esempio: aggiungere rispettivamente 0 µ l, 2 µ l, 1 µ l, 0,5 µ l e 0,25 µ l in righe A-E). Aggiungere duplicati per accuratezza dei dati.</li>
	<li>Incubare la piastra a 37 ° C e 5% CO2 per 16 ore (una notte).</li>
	<li>Determinare il rapporto ottimale e la quantità dei virus NR1 e NR2A eseguendo una misurazione del flusso di calcio su Franzese (Vedi sopra).</li>
</ol>4. ligando titolazione
<ol><li>Preparare le celle come descritto al punto 2.3.</li>
	<li>Preparare diluizioni di ogni ligand in presenza di saturare le concentrazioni (100 µM) altri del ligand come una soluzione di riserva 4 volte. Ad esempio: per una concentrazione di prova finale di 10 µM di glicina, preparare una soluzione stock di 40 µM di glicina compreso 400 µM glutammato.</li>
	<li>Procedere con la misurazione di Franzese come descritto al punto 2.11.</li>
</ol>5. composti test
<ol><li>Preparare le celle come descritto nel passaggio 1.</li>
	<li>Preparare la piastra di ligando con uno stock di 5 volte della concentrazione del ligando finale nel tampone.</li>
	<li>Preparare la piastra di composti con il brodo 4 volte della concentrazione di composti nel buffer di analisi finale desiderata. Per una concentrazione finale composta di 10 µM nel tampone, preparare una soluzione stock di 40 µM.<ol>
<li>Mantenere costante la concentrazione di dimetilsolfossido (DMSO) attraverso tutti i campioni di preparare una pre-diluizione del composto in DMSO prima ulteriore diluizione nel buffer di analisi.</li>
		<li>Per una reazione al dosaggio del composto nel test finale compresa tra 3 e 30 µM, preparare una pre-diluizione del composto in DMSO che vanno da 1 a 10 mM. Diluire quelli nel tampone alla concentrazione stock 4 volte. La concentrazione finale di DMSO non deve superare 0,5%. Nota: Si consiglia di testare ogni campione in triplici copie.</li>
	</ol>
</li>
	<li>Caricare il ligando, composto e piastra in Franzese.</li>
	<li>Misurare la fluorescenza di base per 30 secondi.</li>
	<li>Aggiungere 10 µ l di brodo composto e misurare la fluorescenza per 5 minuti.</li>
	<li>Aggiungere 10 µ l di brodo di ligando e misurare la fluorescenza per 5 minuti.</li>
	<li>Determinare l'integrale del rapporto fluorescenza calcolando l'area sotto la curva dei rapporti fluorescente durante gli ultimi 5 minuti della misurazione. Nota: In alternativa, il rapporto massimo di fluorescenza dopo aggiunta di legante può essere utilizzato per l'analisi.</li>
</ol>

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Il successo di questo test dipende in gran parte la salute delle cellule HEK utilizzato. Le cellule in fase di crescita esponenziale e con numero di passaggio basso dovrebbe essere usato. Questo test comporta molti trasferimenti e aggiunte di soluzioni, così uso attenzione garantirà maggiore precisione nei suoi risultati. Concentrazioni di composti e di tutti gli altri reagenti dovrebbero essere anche controllato per minimizzare gli errori. Quando si sostituisce media delle cellule con il buffer di analisi per il dosag...

Con attuali progressi in medicina, l'aspettativa di vita è aumentata in modo significativo; Tuttavia, così ha la prevalenza delle malattie età-correlate. Malattie del sistema nervoso centrale (CNS) come schizofrenia, sclerosi laterale amiotrofica (ALS), morbo di Alzheimer e morbo di Parkinson, tra gli altri, non fanno eccezione e sono stati progettati per aumentare sopra il prossimo decennio1, 2 , 3. il malfunzionamento del noto come recettori N-metil-D-aspartato (NMDAR) dei recettori ionotropi del glutammato è stato collegato al morbo di Alzheimer, schizofrenia, trauma cranico, ictus, diabete e glaucoma tra gli altri, che garantisce la bisogno di studiare la loro biologia per lo sviluppo di terapie efficaci, modificante la malattia4,5,6,7.
NMDARs sono composti da quattro monomeri o subunità4,8,9. La composizione strutturale della NMDAR Mostra variabilità inerente allo sviluppo e regionali entro il cervello7,10. NMDARs sono coinvolti nella plasticità sinaptica, cognizione e la generazione dei ritmi per respirazione e locomozione11,12,13. Come un canale voltaggio-dipendente, è in gran parte non conduttore al potenziale di membrana a riposo (-70 mV) e viene bloccato da magnesio per prevenire ulteriori permeazione degli ioni. Il canale è attivato dall'associazione di due ligandi, glutammato e glicina/D-serina, e una depolarizzazione simultanea alla membrana sinaptica mediata da recettori AMPA, un'altra sottoclasse di recettori ionotropici del glutammato. La depolarizzazione rimuove il blocco di magnesio di NMDAR, consentendo l'afflusso dei cationi, in particolare calcio14,15,16. Anche se l'attivazione di NMDAR è essenziale per la sopravvivenza delle cellule, attivazione eccessiva può portare a cellule morte17,18,19 attraverso excitotoxicity. Questo, oltre la complessa natura del recettore, rende difficile effettuare studi necessari per lo sviluppo di terapie efficaci.
Sono stati sviluppati diversi metodi per studiare la NMDAR. Tuttavia, ciascuno di essi è corredato da avvertimenti. Ad esempio, una tecnica ampiamente utilizzata è un'analisi di fluorescenza-basata che misura i cambiamenti di NMDAR-mediati in calcio intracellulare in una linea cellulare stabile sotto il controllo di un promotore inducibile tetraciclina (Tet-On)20. Tuttavia, in questo sistema, le concentrazioni di sopramassimale di ligandi che sono necessari e l'obbligo che gli inibitori di NMDAR sono presenti durante la misurazione rende quasi impossibile rilevare l'attività dell'antagonista competitivo. In altri sistemi simili, l'espressione del recettore funzionale provoca tossicità, che richiedono di bloccanti dei canali come ketamina21,22 per preservare le colture cellulari. Questi antagonisti sedersi al centro del recettore e sono difficili da lavare fuori, soprattutto in un formato basato su piastra, in modo da interferire con gli studi funzionali del recettore. Infine, nelle misurazioni elettrofisiologiche come patch di bloccaggio, c'è la velocità effettiva limitata e studi su larga scala sono molto costosi23. Nonostante, i sistemi di cui sopra sono insensibili alla stimolazione di glicina; quindi, studiando attività di glicina-dipendente del NMDAR diventa una sfida.
Qui, descriviamo un nuovo approccio per lo studio di NMDAR che supera le limitazioni discusse. La nostra tecnica sfrutta il sistema di espressione di baculovirus per esprimere il recettore a livello funzionale con un rapporto ottimale delle subunità in meno di 16 ore. Inoltre, l'uso di baculovirus consente un approccio semplice e combinatorio, che fornisce un'ampia caratterizzazione dei sottotipi distinti ricombinante NMDAR. A differenza di altri saggi, questo protocollo non richiede bloccanti dei canali a causa dell'uso di antagonisti deboli. Vantaggio più forte del metodo è quello dopo interruzione dell'antagonista debole, il recettore è sensibile alla modulazione dei siti individuali della glicina e del glutammato-associazione oltre a doppia modulazione di glutammato e glicina/D-serina ligand-legantesi siti. Il dosaggio ricapitola noto farmacologia del recettore NMDAR e gli effetti dei suoi noti modulatori positivi e negativi. Infine, generazione di questo test in vitro cellular supera la tossicità cellulare causata da afflusso del calcio eccessivo e permette per studi funzionali del recettore in un modo ad alta velocità, che può accelerare le scoperte dei modulatori NMDAR negli Stati di malattia.

<table>
	<tbody>
		<tr>
			<td>HEK-293</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Human NMDA (NR1/NR2A) Receptor Cell Line</td>
			<td>ChanTest Corporation</td>
			<td>CT6120</td>
			<td></td>
		</tr>
		<tr>
			<td>pFastBac Dual Expression Vector</td>
			<td>ThermoFisher Scientific</td>
			<td>10712-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well Clear Flat Bottom Microplate</td>
			<td>Corning Life Sciences</td>
			<td>3844</td>
			<td></td>
		</tr>
		<tr>
			<td>FLIPR Calcium 6-QF Assay Kit</td>
			<td>Molecular Devices</td>
			<td>R8192</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>D-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4250</td>
			<td></td>
		</tr>
		<tr>
			<td>L701,324</td>
			<td>Tocris</td>
			<td>907</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Boston Bio Product</td>
			<td>BB-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>E506</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>ThermoFisher Scientific</td>
			<td>P36400</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX media</td>
			<td>ThermoFisher Scientific</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>MDL105,519</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>NVP-AAM077</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP070667</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

I recettori N-metil-D-aspartato (NMDA) (NMDAR) sono classificati come recettori ionotropici per il glutammato e hanno importanti ruoli nell'apprendimento e nella memoria. Malfunzionamento NMDAR, espresso come entrambi sopra - o sotto - activity causata da mutazioni, espressione alterata, traffico o localizzazione, può contribuire a numerose malattie, soprattutto nel sistema nervoso centrale. Di conseguenza, comprensione della biologia del ricevitore, nonché facilitare la scoperta di composti e piccole molecole è cruciale nei continui sforzi per combattere le malattie neurologiche. Attuali approcci allo studio del recettore presentano limitazioni tra cui bassa velocità effettiva, costo elevato e l'impossibilità di studiare le relative abilità funzionale dovuto la necessaria presenza di bloccanti dei canali per evitare eccitotossicità mediata NMDAR. Inoltre, gli attuali sistemi di dosaggio sono sensibili alla stimolazione da glutammato solo e la mancanza di sensibilità alla stimolazione da glicina, altri co-ligando del NMDAR. Qui, presentiamo il primo test basati su piastra con potenza di alto-rendimento per studiare un recettore NMDA con sensibilità sia co-leganti, glutammato e glicina-serina/D. Questo approccio consente lo studio delle diverse composizioni di subunità NMDAR e studi funzionali del recettore in modalità glicina - e/o sensibili al glutammato. Inoltre, il metodo non richiede la presenza di inibitori durante le misurazioni. Gli effetti di modulatori allosterici positivi e negativi possono essere rilevati con questo test e la farmacologia nota di NMDAR è stata replicata nel nostro sistema. Questa tecnica supera le limitazioni dei metodi esistenti ed è conveniente. Noi crediamo che questa tecnica novella ad accelerare la scoperta di terapie per patologie mediate da NMDAR.

Prima di testare gli effetti di piccole molecole, si devono determinare i livelli di espressione ottimale di NMDARs così come le concentrazioni di ligando ottimale. Come descritto, HEK293 cellule sono state seminate a 10.000 cellule per pozzetto di una piastra a 384 pozzetti, in presenza di 5 µM CGP060667, quindi trasdotte con quantità variabili di NR1 e NR2A virus. Dopo incubazione overnight, indotta da legante di calcio è stato misurato il flusso (Figura 1</stro...

Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

Un'analisi di flusso di calcio High-throughput per studiare i recettori NMDA con sensibilità al glutammato e glicina/D-serina

L'obiettivo del presente protocollo è quello di facilitare lo studio dei recettori NMDA (NMDAR) a scala più ampia e consentire l'esame degli effetti modulatory di piccole molecole e le loro applicazioni terapeutiche.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

a-high-throughput-calcium-flux-assay-to-study-nmda-receptors-with

Research

JoVE Journal

Neuroscience

9.1K Views.  Novartis Institutes for BioMedical Research. L'obiettivo del presente protocollo è quello di facilitare lo studio dei recettori NMDA (NMDAR) a scala più ampia e consentire l'esame degli effetti modulatory di piccole molecole e le loro applicazioni terapeutiche.

Video: A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Un modo semplice per misurare sensibilità etanolo in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Ricerca

Neuroscienze

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Luce Preferenza Assay per studiare Innata e circadiano Photobehavior Regolamentato<em&gt; Drosophila</em&gt; Larve

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

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 ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

Interrogatorio funzionale di età ipotalamico neurogenesi con focale Radiological inibizione

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

High Throughput test per esaminare Preferenze deposizione delle uova dei singoli<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

Correlativo luce e microscopia elettronica allo Studio microgliali Interazioni con placche beta-amiloide

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.