Un soutien a été fourni par startup-fonds du Département de biologie et pharmacologie chimique à l&#39;Université de Pittsburgh School of Medicine. Accusé de membres du laboratoire Jacob qui ont contribué à la présentation de la vidéo: Nicolas Graff.

Tous les protocoles décrits ci-dessous sont conformes à la IACUC et NI comités d&#39;éthique de l&#39;Université de Pittsburgh School of Medicine. Une. Préparation des cultures de neurones de l&#39;hippocampe en culture de tissus capot Note: Utiliser une technique stérile et réactifs tout au long de protocole 1. <ol><li> Préparer le poly-D-lysine (0,1 mg / ml dans H 2 O) des lamelles de verre revêtu en tant que substrat pour la culture des neurones. <ol><li> Placez 4-5 lamelles rondes de verre à l&#39;intérieur de chaque boîte de culture de tissu de 3,5 cm. </li><li> Coin 70 ul de poly-D-lysine sur chaque lamelle couvre-objet de 12 mm. Remarque: pour l&#39;imagerie en temps réel, un fond de verre de 3,5 cm boîte de culture de tissu peut être utilisé (repérer 200 pi poly-D-lysine sur incorporé 14 mm lamelle de verre). </li><li> Laissez les plats préparés dans la culture de tissus capot O / N. Pour minimiser la poly-D-lysine évaporation et maintenir les surfaces stériles dans la hotte de culture de tissus, fermer la fenêtre à guillotine et éteignez ee soufflante O / N. Remarque: les lampes UV ne sont pas utilisés pendant l&#39;incubation O / N. </li><li> Le lendemain matin, laver la vaisselle 3x avec 2 ml de H 2 O. </li><li> Après avoir retiré le dernier H 2 O lavage, ajouter 2 ml de milieu à chaque 3,5 cm plat et laissez plats dans l&#39;incubateur jusqu&#39;à ce que prêt à plaquer les neurones à l&#39;étape 1.4. </li></ol></li><li> Neurones de l&#39;hippocampe sont préparés à partir de jour embryonnaire 18-19 (E18-19) (rats modifiés de Goslin 22). </li><li> Transfecter des neurones dissociés fraîchement le jour de mise en culture avec de 1 à 4 pg d&#39;ADN de construction maxiprepped. Note: en général 3 pg d&#39;ADN est transfecté dans 1-2000000 neurones, avec une viabilité de 50% et une efficacité de transfection de 60% ​​(par exemple, en commençant par les neurones: 2000000 ((2 x 10 6 x neurones 0,5) 0,6) prévoit environ 1 million de neurones;. 600000. qui sont transfectées 1 quantités différentes de construction d&#39;ADN peuvent être transfectées lors de la résolution des problèmes potentiels desur-expression, suivi par une caractérisation appropriée de construction localisation et leur fonction. </li><li> Plate neurones transfectées sur des poly-D-lysine lamelles enrobées à une densité finale d&#39;approximativement 200 000 neurones par 3,5 cm plat. Remarque: pour un fond de verre plat, assiette 40,000-50,000 neurones sur la surface vitrée de 14 mm. </li><li> Remplacez le support 4-24 heures après la préparation des cultures de neurones, puis permettent aux neurones de se développer dans l&#39;incubateur jusqu&#39;à 14-17 jours in vitro (DIV) ou stade désiré. </li></ol> 2. Endocytose Assay ATTENTION: Note sur α-bungarotoxine et déchets de tubocurarine et la manipulation: <ol><li> Manipulation de tous les neurotoxines peptidiques est recommandé dans les lignes directrices de biosécurité de niveau 2 (BL-2) protocoles de recherche. Tout l&#39;équipement de protection individuelle approprié doit être porté. Poudres de toxine lyophilisés doivent être reconstitués selon les instructions du fabricant. Toxine déchets should être éliminés en conformité avec les directives de santé et sécurité de l&#39;environnement de l&#39;institution gouvernementale. </li><li> Pour éliminer les agrégats peptidiques potentiels qui se sont formées au cours du stockage, des aliquotes α-bungarotoxine doivent être centrifugés brièvement avant utilisation, et le surnageant doivent être utilisées pour l&#39;expérience. </li><li> α-bungarotoxine (Bgt) les stocks sont remises en suspension à une concentration de 1 mg / ml dans H 2 O stérile et stocké dans des aliquotes de 10 ul à -20 ° C. Les stocks BGT fluorescence couplés sont utilisés à 3 pg / ml. Tubocurarine est utilisé à une concentration finale de 150 uM. </li></ol><ol><li> Réglez paillasse bloc chauffant à 16 ° C dans une chambre froide avec de l&#39;aluminium mince ou plaque d&#39;acier inoxydable sur le dessus. Alternativement, si disponible, un haut dispositif de refroidissement / chauffage de banc peut être utilisé pour chaque étape nécessitant 16 ° C. </li><li> Frais extracellulaire salines Hepes tamponné (HBS contenant en mm: 135 NaCl, KCl 4,7, 10 HEPES, 11 glucose, 1,2 MgCl 2 et 2,5CaCl2, pH 7,4) à 16 ° C dans un bain d&#39;eau. </li><li> Transfert des plats de neurones à la plaque d&#39;aluminium à 16 ° C et laisser refroidir pendant 5 minutes. </li><li> Retirez le support (garder milieux conditionnés et réserve dans l&#39;incubateur pour l&#39;étape 2.8, points de temps de dosage d&#39;endocytose) et le remplacer par 1 ml de 16 ° C HBS + tubocurarine (150 M) pendant 2 min. </li><li> Retirer HBS + tubocurarine à conteneur à déchets dûment et remplacer par 1 ml de 16 ° C HBS + tubocurarine (150 M) + α-bungarotoxine Alexa 594 [3 pg / ml], l&#39;incubation à 16 ° C pendant 15 min. </li><li> Retirer HBS + + tubocurarine α-bungarotoxine marquée à l&#39;Alexa 594 conteneur de déchets et laver la vaisselle 3x avec 2 ml de 16 ° C HBS (les lavages peuvent être collectées par l&#39;intermédiaire d&#39;une aspiration dans une fiole à vide contenant 50 ml d&#39;eau de Javel à 50%). </li><li> Pour les expériences d&#39;imagerie en direct de séries chronologiques suivantes endocytose récepteur à l&#39;aide d&#39;un fond de verre de 3,5 cm boîte de culture, effectuer les étapes 2.1 au 2.6, puis transférer plat à platine chauffante(Dispositif à effet Peltier) sur microscope, et commencer l&#39;imagerie confocale avec un ou configuration FRBR de microscope. </li><li> Transfert T = 0 point de temps lamelles dans un plat de RT paraformaldéhyde à 4% / 4% de saccharose pendant 20 min, en continuant avec d&#39;autres lamelles à l&#39;étape 2.9. </li><li> Pour d&#39;autres points dans le temps, remplacer HBS avec conditionnée équilibrée 37 ° C médias (réservé à l&#39;étape 2.4) et revenir à l&#39;incubateur pour l&#39;endocytose à 37 ° C. Note: suggéré points supplémentaires de temps de T = 15, 30, et 60. </li><li> Aux points de temps nécessaire, prendre des plats de l&#39;incubateur, lavez rapidement deux fois avec 2 ml de RT DPBS (dPBS pas de calcium, magnésium), puis fixer à 4% de paraformaldéhyde / 4% de saccharose pendant 20 min. </li><li> Après 20 min de fixation, retirez du paraformaldéhyde 4% / 4% de saccharose à perdre récipient et laver la vaisselle deux fois avec 2 ml RT DPBS. </li><li> Incuber les lamelles à température ambiante pendant 10 min dans une solution de bloc d&#39;immunofluorescence (solution de bloc = sérum de cheval à 5%, BSA à 0,5% dans du DPBS) contenant 0,2% de Triton X-100 à perméabilitéiser les neurones et permettre immunomarquage anti-GFP de la piscine et / ou à l&#39;étiquetage d&#39;autres protéines d&#39;intérêt récepteur intracellulaire. </li><li> Retirer bloc + 0,2% de Triton X-100 et incuber dans une solution de lamelles couvre-bloc d&#39;immunofluorescence sans Triton X-100 pendant 20-30 min. </li><li> Effectuer des incubations d&#39;anticorps primaires de lamelles couvre-objet en bloc pendant plusieurs heures à la température ambiante ou O / N à 4 ° C. Remarque: l&#39;incubation des lamelles avec les primaires à 4 ° CO / N produit régulièrement des résultats améliorés d&#39;immunofluorescence. </li><li> Laver 3 fois avec du DPBS lamelles couvre-objet (5 min à chaque étape de lavage). </li><li> Incuber les lamelles couvre-objet avec des anticorps secondaires dans la solution de bloc pour une heure à température ambiante. </li><li> Laver 3 fois avec du DPBS lamelles couvre-objet (5 min à chaque étape de lavage). </li><li> Des lamelles de montage, la manutention chaque lamelle individuelle: supprimer l&#39;excès de liquide de l&#39;arrière de la lamelle avec un tissu de laboratoire, puis inversez lamelle sur 4 pi de milieu de montage sur une lame de verre. </li><li> Permettre diapositives à sécher à la température ambiante couverts pendant 30 min puis stminerai à 4 ° C jusqu&#39;au moment de réaliser la microscopie. </li><li> Acquisition et l&#39;analyse de l&#39;expérience avec la microscopie confocale (aveugle à condition expérimentale) l&#39;image. Huile 60X NA 1.49 objectif d&#39;immersion avec les lasers suivants: gaz Argon 488 nm, 561 diode, diode 640. <ol><li> En utilisant les mêmes paramètres d&#39;acquisition d&#39;images, d&#39;acquérir des images de la section de Z simples avec le corps des cellules neuronales et plusieurs processus dendritiques dans la discussion. </li><li> Quantifier α-bungarotoxine Alexa signal de fluorescence GFP et immunocoloration 20 um de long 04.03 dendrites proximales par neurone, en utilisant le même seuil pour l&#39;analyse de toutes les données d&#39;endocytose. </li><li> Analyser les données de 10 à 12 neurones pour chaque point de temps, répétant l&#39;expérience avec plusieurs cultures de neurones indépendants. </li></ol></li></ol>

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Les auteurs n&#39;ont rien à révéler.

Les techniques fixes et vivants basés BBS décrites ici peuvent être utilisés pour suivre récepteur ou autre membrane plasmique protéine traite des lignées de cellules, les neurones et d&#39;autres cellules primaires. Cette méthode a été utilisée avec succès pour étudier l&#39;insertion de membrane et la suppression des canaux ioniques et des GPCR ligand-dépendants et évaluer les changements dans le trafic en raison de la présence d&#39;agonistes des récepteurs et des modulateurs. Aspects clés comprenne...

L&#39;utilisation de la fluorescence couplé α-bungarotoxine à étudier la localisation du récepteur et la dynamique a été lancée dans des études sur le récepteur nicotinique de l&#39;acétylcholine 13-15, cible endogène de la toxine. Par la suite, l&#39;incorporation du peptide minimal Bgt liaison (BBS) a été utilisée pour étudier la traite des canaux ioniques excitateurs et inhibiteurs ligand-dépendants et G récepteurs couplés aux protéines 2,10,16-21. Cette technique basée sur la BBS offre des avantages à d&#39;autres approches utilisées pour les études de trafic tels que les méthodes de biotinylation de surface, marquage de l&#39;anticorps de cellules vivantes avec des anticorps à des épitopes extracellulaires, et la récupération de fluorescence après photoblanchiment (FRAP). Au cours de la surface cellulaire biotinylation amines libres sont modifiés de manière covalente, avec le potentiel d&#39;affecter l&#39;activité cellulaire. Études basées anticorps ont souvent été entravés par le regroupement ou le plafonnement de l&#39;antigène de surface, ce qui peut modifier les événements de la traite. En raison de l&#39;étape de blanchiment pour FRAP ses études, une préoccupation importante est endommager la structure cellulaire sous-jacent. Un avantage supplémentaire est que l&#39;étiquette BBS constructions peuvent aussi être utilisées pour des méthodes biochimiques avec le trafic de récepteur couplé à la biotine bungarotoxine à ânes. Cette technique est facilement applicable à des lignées cellulaires et des cellules primaires. Pour une utilisation dans des cellules qui expriment l&#39;acétylcholine nicotinique (nAChR) des récepteurs, l&#39;antagoniste tubocurarine nAChR doit être utilisé dans le protocole comme indiqué. Exécution d&#39;une simple surface étiquetage Bgt (équivalent au point de temps T = 0 pour le protocole d&#39;endocytose) sur les cellules non transfectées en l&#39;absence de tubocurarine fournira la preuve de nAChR endogène. Une considération importante pour l&#39;utilisation de cette technique est l&#39;insertion appropriée du BBS de sorte qu&#39;il est présent à un emplacement extracellulaire lorsque la protéine d&#39;intérêt est délivré à la membrane plasmique. Par exemple, les domaines N-terminaux des sous-unités GABAAR résident dans la lumière au cours de la traite vésiculairesficking et devenir extracellulaire après l&#39;insertion du récepteur dans la membrane plasmatique, ce qui permet le marquage spécifique de récepteurs de surface cellulaire et l&#39;évaluation de leur retrait de la surface de la cellule par endocytose événements. Nous avons précédemment montré que l&#39;addition de GFP, myc, ou BBS épitopes de ce domaine de sous-unités GABAAR est fonctionnellement silencieuse. Les contrôles standard doivent être effectuées afin de s&#39;assurer que la protéine marquée est exprimée à des niveaux similaires à un produit d&#39;assemblage non marqué, qu&#39;il localisé de manière appropriée, et que cela n&#39;affecte pas la fonction du récepteur. Cette caractérisation de constructions transfectées aidera également à dépannage surexpression préoccupations.

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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

Il est de plus en plus évident que les récepteurs de neurotransmetteurs, dont les récepteurs ionotropiques GABAA (GABAAR), exposition traite très dynamique et la mobilité de la surface de la cellule 1-7. Pour étudier la localisation des récepteurs de surface cellulaire et l&#39;endocytose, la technique décrite ici combine l&#39;utilisation d&#39;α-bungarotoxine fluorescent avec des cellules exprimant des constructions contenant une α-bungarotoxine (Bgt) du site de liaison (BBS). Le BBS (WRYYESSLEPYPD) est basé sur la sous-unité α du récepteur nicotinique de l&#39;acétylcholine de muscle, qui se lie avec une grande affinité Bgt 8,9. Incorporation du site BBS permet la localisation de surface et des mesures de l&#39;insertion ou du retrait du récepteur avec application de la fluorescence exogène Bgt, comme décrit précédemment dans le cheminement de GABAA et GABAB récepteurs métabotropiques 2,10. En plus du site de BBS, on a inséré une GFP sensible au pH (pHGFP 11) entre les acides aminés 4 et 5 de la sous-unité mature par GABAAR m-typestratégies de biologie et de clonage PCR olecular (voir la figure 1) 12. Le BBS est en 3 &#39;de la GFP reporter sensible au pH, séparés par une alanine / acide proline lieur 13-amino. Pour le trafic études décrites dans cette publication qui sont basées sur des échantillons fixés, le pHGFP sert un journaliste du total marqué GABAAR niveaux de sous-unité protéique, permettant la normalisation de la Bgt marqué population de récepteur à récepteur population totale. Cela minimise cellule à Bgt coloration variabilité du signal résultant de supérieur ou inférieur expression de base des sous-unités GABAAR marqués. En outre, l&#39;étiquette pHGFP permet une identification facile des cellules de construction exprimant des expériences en direct ou fixes imagerie.

Caractérisation d&#39;une construction BBS marqué comprend des contrôles importants, tels que la détermination de si la protéine exprimée assemble correctement (en particulier avec des récepteurs constitués de plusieurs sous-unités), des trafics à la surface de la cellule et se localise de façon appropriée. Synapses inhibitrices sont composées de GABA A grappes de surface des récepteurs qui sont colocalisées avec l&#39;inhibiteur géphyrine de protéine d&#39;échafaudage et sont apposés à des bornes in...

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

Ici, nous démontrons l&#39;utilisation de colorant fluorescent Alexa couplé à α-bungarotoxine à mesurer récepteur GABA A de localisation à la surface et l&#39;endocytose dans les neurones de l&#39;hippocampe. Grâce à l&#39;utilisation de constructions portant une étiquette extracellulaire qui se lie à court α-bungarotoxine, l&#39;analyse de la membrane plasmique protéine endocytose trafic peut être atteint.

L&#39;utilisation d&#39;un α-bungarotoxine Reliure Tag du site à étudier récepteur GABA A membrane localisation et le trafic

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 vues.  University of Pittsburgh School of Medicine.  Ici, nous démontrons l'utilisation de colorant fluorescent Alexa couplé à α-bungarotoxine à mesurer récepteur GABA A de localisation à la surface et l'endocytose dans les neurones de l'hippocampe. Grâce à l'utilisation de constructions portant une étiquette extracellulaire qui se lie à court α-bungarotoxine, l'analyse de la membrane plasmique protéine endocytose trafic peut être atteint. 

Vidéo: 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.

Analyse quantitative des vésicules reconstitution Piscine synaptique dans des cultures de neurones granulaires du cervelet utilisant des colorants FM

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

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Neurosciences

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.

Utiliser MazeSuite et fonctionnelle proche infrarouge pour étudier l&#39;apprentissage de la navigation spatiale

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.

Imagerie pHluorin-étiqueté insertion du récepteur de la membrane plasmique dans les neurones primaires de souris en culture

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.

Application locale de médicaments pour l&#39;étude des fonctions récepteur de l&#39;acétylcholine nicotinique dans des tranches de cerveau de souris

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.

Rapide Micro-iontophorèse de glutamate et le GABA: un outil utile d&#39;étudier l&#39;intégration synaptique

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.

Potentiel de membrane Dye Imaging de ventromédian hypothalamus neurones de souris adulte à étudier 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 ...

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.

Tranche de cerveau Biotinylation: Une<em&gt; Ex Vivo</em&gt; Approche de Mesure région spécifique membrane de protéines plasmatiques traite des adultes neurones

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.

Localisation des gènes des récepteurs odorants dans les antennes acridiennes par ARN<em&gt; In Situ</em&gt; Hybridation

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.

Images en direct de la protéine GLUT4 traite des neurones hypothalamiques primaire de souris au microscope de déconvolution

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.

Une autre approche pour étudier les événements primaires dans la neurodégénérescence utilisant des coupes de cerveau de Rat Ex Vivo

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