Ce travail a été soutenu par la compagnie d&#39;assurance vie &quot;AG2R-La Mondiale-&quot;, l&#39;Ecole des Neurosciences de Paris (ENP), l&#39;Agence Nationale de la Recherche &quot;ANR-09-NEUR-004&quot; dans le cadre de «ERA-NET NEURON »du FP7 programme par la Commission européenne et la Fondation Pasteur. Sébastien Wagner a été soutenu par la Fondation Letten.

1. Calibrage optique: Mesure de LED <ol><li> Fixez une barrette de DEL à un dissipateur thermique activement refroidi par un ventilateur et apposer cette aparatus LED / radiateur à une lentille de collimation. </li><li> Remplacer la lampe utilisée en fond clair l&#39;éclairage à LED / radiateur / ventilateur / appareil de l&#39;objectif. Cet appareil doit être soigneusement placé de sorte que le faisceau LED collimaté se déplace le long d&#39;une trajectoire rectiligne optiques vers la lentille de condenseur. Assurez-vous que le dissipateur / ventilateur est correctement mise à la terre commune du système. </li><li> Conduire le tableau LED avec une alimentation qui peut donner des impulsions rapides et carrés de courant. Cette alimentation peut être contrôlée par une impulsion de 5V TTL provenant d&#39;un générateur d&#39;impulsions. </li><li> Centrer le faisceau collimaté le long du trajet de la lumière défini entre le diaphragme de champ et de la lentille condenseur. Idéalement, le faisceau LED peu trop le diaphragme de champ complètement ouvert. Typiquement, un faisceau plus étroitement collimaté LED va remplir cette ouverture moins, et va produire MorPower E au détriment de l&#39;uniformité. Dans notre configuration, nous avons augmenté l&#39;uniformité de lumière en choisissant une lentille de collimation qui a projeté une image légèrement élargie de la matrice de LED à son plan conjugué au niveau du diaphragme consenser. </li><li> Atteindre éclairage de Köhler en concentrant le condenseur afin que l&#39;image du diaphragme de champ (le diaphragme le plus proche de la source de lumière) est axé sur la chambre tranche (fig. 1). Un tissu mince de papier pour objectif peuvent agir comme un écran de projection pour visualiser l&#39;image focalisée sur le diaphragme de champ à d&#39;autres profondeurs. </li><li> Percer une série de trous de diamètre connu dans un matériau opaque. Placez un de ces trous petits sur le capteur d&#39;une puissance-mètre optique. Placer le mesureur de puissance sur la scène spécimen et le centre du compteur de pouvoir sur l&#39;image focalisée sur le diaphragme de champ en déplaçant simplement le compteur de puissance jusqu&#39;à ce qu&#39;il donne une valeur maximale. Apposez le wattmètre dans cette position. </li><li> Totalement ouvert toutes les ouvertures (diaphragme et aperaturediaphragme de champ). Systématiquement passer la tranche attachée de chambre / wattmètre par rapport à la lightpath optiques et de calculer l&#39;uniformité de la puissance optique dans la zone éclairée. Construire l&#39;intrigue d&#39;uniformité pour votre système. Si le microscope est correctement configuré avec illumination Kohler centrée au foyer de l&#39;objectif, la puissance maximale doit être directement en dessous de l&#39;objectif, et les régions en dehors de cette focalisation doit maintenant recevoir une quantité connue de puissance en fonction de l&#39;intrigue uniformité. </li><li> Pour chaque taille de trou d&#39;épingle, de construire une courbe standard de puissance optique vs surface sténopé. En ajustant le courant d&#39;entrée du tableau de LED, de produire cette courbe plusieurs niveaux de puissance et de chaque courbe de calculer la puissance par mm2. Si le tableau de LED est utilisé pour l&#39;optique patch, assurez-vous d&#39;introduire des éléments optiques nécessaires pour patcher (condenseurs, les piqûres et les filtres) pour savoir combien la lumière est transmise sous un éclairage patcher. </li><li> Retournement du wattmètre pourface à l&#39;objectif, calculer l&#39;intensité de l&#39;éclairage à 470nm lorsque la lampe au mercure est allumé. </li><li> Ajouter une tranche en direct à la chambre, et de reconstruire les courbes standard pour déterminer la puissance lumineuse transmise à travers les tissus du cerveau de diffusion. </li></ol> 2. Procédure de tranchage et d&#39;électrophysiologie Partie A: Préparation Trancher <ol><li> Anesthetize (60 mg / kg de kétamine et de xylazine 2mg/kg) et décapiter la souris. Disséquer le cerveau dans le liquide céphalorachidien artificiel (ACSF, en mm: 124 NaCl, KCl 3, 1,3 MgSO 4, 26 NaHCO 3, 1,25 NaHPO 4, 20 de glucose, 2 CaCl 2; ~ 310mOsm, pH 7,4 lors barbotage avec un mélange de O2 95% et 5% de CO 2 5,1), en prenant soin de ne pas endommager les bulbes olfactifs. Séparez les deux hémisphères et le lieu sur une gélose à la surface ventrale, même avec un bord (pour les sections horizontales). </li><li> Collez l&#39;agar-agar et la surface dorsale de chaque hémisphère cortical à l&#39;vibrAtome Chuck, et lentement, remplissez la baignoire avec glacée ACSF. Trancher de la surface ventrale dans 300 sections um, le transfert de chaque section pour réchauffé (34-36 ° C) et oxygénées ACSF, leur permettant de récupérer pendant 30-45 minutes. </li></ol> Partie B: Mesure Patch en vrac du Seuil à Spike <ol start="3"><li> Après avoir porté les tranches à la température ambiante pendant 30 minutes, placez doucement une tranche dans la chambre d&#39;enregistrement de la chambre de microscope sous perfusion constante de l&#39;ACSF oxygénée. Au risque de trop stimuler CHR2 neurones infectés, la présence de EYFP-CHR2 peut être confirmée en vertu épifluorescence (fig. 2a) </li><li> Tirez électrodes de verre sur un extracteur de pipette (Sutter P-97). Remplissez cette électrode avec l&#39;ACSF. Lorsqu&#39;il est placé dans le bain ACSF la résistance de pointe devrait être entre 7-10 MOhms. </li><li> Sous un éclairage fluorescent, de localiser dans la tranche d&#39;une saine CHR2-EYFP neurone avec une morphologie mature. Également localiser le soma ce neurone dans le cadre de l&#39;optique patcher. </li><li> Avec une pression positive lumière passait à travers la pointe de l&#39;électrode, l&#39;électrode inférieure correctif vers le neurone identifié fluorescentes. Quand le contact est fait de membrane, de libérer rapidement la pression positive et appliquez une petite quantité et brève de l&#39;aspiration à travers la pointe. Un sceau de giga-ohms doit être faite entre la membrane plasmique et les murs de l&#39;électrode de patch. </li><li> Même si un giga-joint n&#39;est pas formé, si l&#39;électrode est suffisamment proche d&#39;une activité des neurones fluorescents dopage devrait produire un potentiel de champ mesurable locales. Activer CHR2 dans ce neurone par le clignotement des doses différentes de la lumière. Parce que la lumière dose est fonction de deux LED d&#39;alimentation et de la durée, calculer la quantité de lumière est nécessaire pour évoquer un potentiel d&#39;action à des puissances et des durées multiples (fig. 2b). Aussi observer combien de dopage se produit sous une lampe à mercure d&#39;éclairage. </li></ol> 3. Les résultats représentatifs: Sur notre microscope (Olympus BX51WI), notre LED est in ligne avec deux ouvertures et un condenseur, maintenant ainsi le chemin optique d&#39;origine de l&#39;usine installée lampe à arc. En fermant la fois le diaphragme de champ et le diaphragme aperature, nous pouvons obtenir un contraste suffisant pour fond clair enregistrements de patch-clamp (Fig. 1b). Avec tous les diaphragmes totalement ouverte, nous exposent la tranche à puissance lumineuse maximale pour channelrhodopsin activation (figure 1A). Sur notre microscope, cette configuration patcher produit à faible densité qui est d&#39;environ trois ordres de grandeur inférieure à la densité maximale plein champ (4,1 uW / mm 2 versus 6,88 mW / mm 2). Nous voyons l&#39;étiquetage robuste de l&#39;adulte née granules bulbe olfactif et des neurones periglomerular semaines après l&#39;infection de la migration des neuroblastes lentiviral dans le flux migratoire rostrale (fig. 2a) Un enregistrement lâche-correctif à partir d&#39;un seul adulte-né CHR2-EYFP exprimer cellules granulaires indique que une stimulation de 5 ms chez Maximeuh la puissance (6,88 mW / mm 2) est suffisante pour évoquer dopage (fig. 2b). Depuis le niveau d&#39;expression varie entre les cellules, la quantité de lumière qui passe le seuil à pic sera variable et devrait être décrit statistiquement pour chaque type de cellule d&#39;intérêt. <img src="/files/ftp_upload/3379/3379fig1.jpg" alt="Figure 1" /> Figure 1. DEL de configuration réseau pour le plein champ photostimulation et l&#39;électrophysiologie tranche de patch-clamp. Pour activer channelrhodopsin (CHR2) nous projettent un faisceau collimaté à travers les ouvertures dos ouvert et l&#39;optique condenseur (a). Cette configuration peut être modifiée en optique à contraste élevé patcher par la fermeture complète du diaphragme de champ et la modulation de la largeur du diaphragme de champ (b). Abréviations: a. ventilateur, radiateur b., c. LED array, d. lentille de collimation, e. miroir, diaphragme de champ f., g. ouverture de diaphragme, condenseur h., i. SAstade de mple, J. objectif. <img src="/files/ftp_upload/3379/3379fig2.jpg" alt="Figure 2" /> Figure 2. Image d&#39;une tranche horizontale de 300 um bulbe olfactif pour le patch-clamp et de l&#39;ensemble du champ de photostimulation (a). Lentivirally cellules infectées par des granules pour adultes-né exprimer CHR2-EYFP peut être vu rayonnant à partir du noyau du bulbe olfactif. La lumière-dose requise pour évoquer dopage peuvent être trouvés en augmentant la durée du flash à LED (b). Le seuil de cette cellule a été granules 5ms à intensité LED complet (2.43mW/mm 2). Échelle en (a) = 500 um, de l&#39;échelle en (b) = 50 ms.

<ol>
	<li>Nissant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+promotes+synaptic+plasticity+in+the+olfactory+bulb.">Adult neurogenesis promotes synaptic plasticity in the olfactory bulb.</a> Nature Neuroscience. 12, 728-730 (2009).</li><li>Apicella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+Cells+in+Piriform+Cortex+Receive+Convergent+Input+from+Distinct+Olfactory+Bulb+Glomeruli.">Pyramidal Cells in Piriform Cortex Receive Convergent Input from Distinct Olfactory Bulb Glomeruli.</a> Journal of Neuroscience. 30, 14255-14260 (2010).</li><li>Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=genetically+targeted+optical+control+of+neural+activity.">genetically targeted optical control of neural activity.</a> Nature. 8, 1263-1263 (2005).</li><li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb.">where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> The Journal of Neuroscience. 30, 17023-17034 (2010).</li><li>Grubb, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+the+first+synapse+in+olfaction:+development+and+adult+neurogenesis.">Functional maturation of the first synapse in olfaction: development and adult neurogenesis.</a> The Journal of neuroscience. 28, 2919-2932 (2008).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit-breakers:+optical+technologies+for+probing+neural+signals+and+systems.">Circuit-breakers: optical technologies for probing neural signals and systems.</a> Nature reviews. Neuroscience. 8, 577-581 (2007).</li><li>Grossman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-site+optical+excitation+using+ChR2+and+micro-LED+array.">Multi-site optical excitation using ChR2 and micro-LED array.</a> Journal of neural engineering. 7, 16004-16004 (2010).</li><li>Dhawale, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-redundant+odor+coding+by+sister+mitral+cells+revealed+by+light+addressable+glomeruli+in+the+mouse.">Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse.</a> Nature neuroscience. 13, 1404-1412 (2010).</li><li>Weick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+control+of+transplantable+human+ESC-derived+neurons+via+optogenetic+targeting.">Functional control of transplantable human ESC-derived neurons via optogenetic targeting.</a> Stem cells. 28, 2008-2016 (2010).</li><li>Toni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+born+in+the+adult+dentate+gyrus+form+functional+synapses+with+target+cells.">Neurons born in the adult dentate gyrus form functional synapses with target cells.</a> Nature Neuroscience. 11, 901-907 (2008).</li><li>Grubb, M. S., Burrone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2+Localised+to+the+Axon+Initial+Segment.">Channelrhodopsin-2 Localised to the Axon Initial Segment.</a> PLoS ONE. 5, e13761-e13761 .</li><li>Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdala+circuitry+mediating+reversible+and+bidirectional+control+of+anxiety.">Amygdala circuitry mediating reversible and bidirectional control of anxiety.</a> Nature. 471, 358-362 (2011).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Ces dernières années ont vu une explosion de la popularité des outils de recherche en neurosciences pour optogénétique 6. En conséquence, il est plus important d&#39;abaisser la barrière d&#39;entrée pour les laboratoires qui désirent commencer à utiliser ces nouveaux outils. Nous décrivons ici comment procéder à une rénovation simple et peu coûteuse et l&#39;étalonnage d&#39;un classique de patch-clamp banc afin qu&#39;il puisse faire plein champ stimulation optique de channelrhodopsin exprim...

<table border="1">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue No.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>100 mg/ml</td>
			<td>Imalg&egrave;ne 1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>2%</td>
			<td>Rompun</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbps;</td>
			<td>Sigma </td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>P5405</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>M1880</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5011</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>C7902</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>A9539</td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>P-97</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>1.5 mm O.D./1.17 mm I.D.</td>
			<td>Harvard Apparatus</td>
			<td>GC150T-10</td>
		</tr>
		<tr>
			<td>LED array</td>
			<td>&nbsp;</td>
			<td>Bridgelux</td>
			<td>BXRA-C2000</td>
		</tr>
		<tr>
			<td>Collimating lens</td>
			<td>40 mm beam diameter</td>
			<td>Thor Labs</td>
			<td>LEDC1</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>2.8 amp</td>
			<td>A1W Electronik</td>
			<td>HKO2800</td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>&nbsp;</td>
			<td>Thor Labs</td>
			<td>PM 100</td>
		</tr>
		<tr>
			<td>Heatsink </td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Fan</td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>&nbsp;</td>
			<td>Leica</td>
			<td>VT1200S</td>
		</tr>
	</tbody>
</table>

using affordable led arrays for photo-stimulation of neurons

Électrophysiologie tranche standard a permis aux chercheurs de sonder les composantes individuelles des circuits neuronaux par l&#39;enregistrement des réponses électriques des cellules individuelles en réponse à des manipulations électriques 1,2 ou pharmacologiques. Avec l&#39;invention de méthodes pour le contrôle des neurones génétiquement optiquement ciblées (optogénétique), les chercheurs ont désormais un niveau sans précédent de contrôle sur des groupes spécifiques de neurones dans la préparation tranche standard. En particulier, photosensibles channelrhodopsin-2 (CHR2) permet aux chercheurs d&#39;activer des neurones avec 3,4 lumière. En combinant l&#39;étalonnage minutieux des LED à base de photostimulation CHR2 à l&#39;électrophysiologie tranche de la norme, nous sommes en mesure de sonder avec plus de détails sur le rôle de l&#39;adulte-né interneurones dans le bulbe olfactif, premier relais central du système olfactif. En utilisant l&#39;expression virale des CHR2-YFP spécifiquement chez l&#39;adulte-né neurones, nous pouvons contrôler sélectivement les jeunes adultes-né des neurones dans un milieu d&#39;une ancienneneurones d matures. Notre contrôle optique utilise un système simple et peu coûteux à LED, et nous montrons comment ce système peut être calibré pour bien comprendre comment la lumière est nécessaire pour évoquer l&#39;activité de dopage dans les neurones unique. Ainsi, de brefs éclairs de lumière bleue peut contrôler à distance le modèle tirs de CHR2-cellules transduites nouveau-né.

Watch this Scientific Journal Video about Using Affordable LED Arrays for Photo-Stimulation of Neurons at JoVE.com

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Adulte-né neurones exprimant CHR2 peuvent être manipulés dans des préparations tranche électrophysiologiques afin d&#39;examiner leur contribution à la fonction de olfactive circuits neuronaux.

L&#39;utilisation abordable tableaux LED pour photo-stimulation des neurones

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

using-affordable-led-arrays-for-photo-stimulation-of-neurons

Research

JoVE Journal

Neuroscience

18.5K vues.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). Adulte-né neurones exprimant CHR2 peuvent être manipulés dans des préparations tranche électrophysiologiques afin d'examiner leur contribution à la fonction de olfactive circuits neuronaux.

Vidéo: Using Affordable LED Arrays for Photo-Stimulation of Neurons

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Évolutif fluidique Arrays Injecteur pour virale ciblage d&#39;Intact circuits du cerveau en 3-D

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Recherche

Neurosciences

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Diffusion latérale et l&#39;exocytose des protéines membranaires dans des neurones en culture évaluée à l&#39;aide de récupération de fluorescence et de fluorescence de perte de photoblanchiment

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagerie épines dendritiques des neurones de l&#39;hippocampe de rat primaire utilisant structuré Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

La production et l&#39;isolement des axones de neurones sensoriels de l&#39;analyse biochimique Utilisation des filtres poreux

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

Externe Excitation Neurones Utilisation de champs électriques et magnétiques et des cultures dans One- à deux dimensions

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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 In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Un modèle in vitro pour étudier l’agrégation de Tau à l’aide de la transduction à médiation Lentivirale des neurones humains

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Implantation chronique de multiples réseaux d'électrodes polymères flexibles

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).