<p class="jove_content"&gt; Questo lavoro è stato sostenuto dal programma intramurale del National Institute of Neurological Disorder and Stroke dei National Institutes of Health. Vorremmo anche ringraziare il Dr. George Mentis per i suoi contributi precedenti al metodo e ai dati in Figura 3.</p

Destrani fluorescenti sono stati utilizzati come strumenti anatomici e per l&#39;imaging attività neuronale 1-4. Campi et al. (2009) 4 pubblicato un protocollo per l&#39;applicazione di ioni e della tensione di coloranti sensibili ai tratti assonali con un focus sul midollo spinale come sistema modello. Qui si descrive una procedura più dettagliata per l&#39;applicazione colorante fluorescente e / o ione-sensibile alle radici taglio ventrali, radici dorsali o qualsiasi tratto neuronale del midollo spinale per studi in vitro optophysiological e morfologiche. 1. Di tipo I e tipo II Pipettes Avviare tirando due brevi tratti di tubazione in polietilene (PE90, Clay marca Adams) sulla fiamma di una lampada ad alcool 7 per la produzione di pipette con puntali conici. Una pipetta (Type-I) deve essere breve (3-7 mm) con una piccola punta che può ben tenere la radice di destinazione o del tratto (diametro esterno: 0,2-0,4 mm, diametro interno: 0,1-0,3 mm). Quindi tirare una pipetta secondo (Type-II) con un più lungo (8-12 cm) più sottile, albero e una punta molto fine (diametro esterno: 0,2-0,3 mm, diametro interno: 0.1-0.2 mm) che può essere inserito nel tipo I pipettare fino la sua punta. La seconda pipetta sottile verrà utilizzato per aspirare ed introdurre fluido cerebrospinale artificiale (ACSF) e la soluzione colorante, rispettivamente, dal tipo I pipetta. 2. Posizionamento di tipo-I Pipettare Staccare ed isolare il midollo spinale 8,9 e collocarlo in un bagno, con superfuso refrigerate ACSF (~ 16 ° C) durante tutto il processo di caricamento. Posizionare il Type-I pipettare su un portaelettrodo (H1/12 portaelettrodo, Narishige) in modo che la sua apertura posteriore può essere facilmente collegato ad una siringa (1 ml U-100 siringa da insulina, Becton Dickinson o comparabili) tramite un tubo flessibile (PharMed BPT, Cole-Parmer, # AY242002, 10 cm) (Fig.1A-B). 3. Dye Application <ol><li> Usando la siringa, prima disegnareACSF nel tipo I pipetta (Fig.1B; 2A) seguita dal tratto assonale o radice da riempire (Fig. 2B). Il tubo flessibile deve quindi essere rimosso dalla apertura posteriore. Il Tipo II-pipetta è attaccata ad un&#39;altra siringa e inserito nel tipo I pipettare premuto il tratto assonale o radice. Usando la siringa collegata al tipo II pipetta, aspirare ACSF in modo che il residuo ACSF copre appena il tratto assonale (Fig.1C; 2C-D). Poi ritirare il tipo II pipetta dal tipo I pipetta. Monitorare il livello ACSF per qualche minuto per verificare una buona tenuta sul tratto assonale o nervi (vedi sezione 3 Risoluzione dei problemi di &quot;buona tenuta&quot;). </li><li> Sciogliere il colorante in ACSF o 0,2% di Triton X-100 in acqua bidistillata e renderlo il Type-II pipetta facendo attenzione a non includere bolle d&#39;aria. Inserire il Tipo II-pipetta contenente il colorante nel tipo I pipetta e nella soluzione residua ACSF (Fig. 2E). Rilasciare lentamenteil colorante nella ACSF mediante una leggera pressione positiva (Fig. 2F). Fare attenzione a non introdurre eventuali bolle d&#39;aria o di provocare lo spostamento del tessuto bersaglio dall&#39;interno della punta. Ritirare il tipo II pipetta dopo sufficiente quantità di colorante è stata rilasciata (3-5 ml di soluzione colorante). </li></ol> 4. Incubazione Incubare il tessuto al buio tra 6 e 20 ore a seconda del tipo di esperimento. Una volta che un tempo sufficiente è stato consentito per il riempimento, tirare delicatamente l&#39;elettrodo lontano dal tessuto lasciando il tessuto pronto per l&#39;imaging e l&#39;istologia. Risoluzione dei problemi <ol><li> Se il tessuto bersaglio non entra facilmente nella punta della pipetta di tipo I o è sciolto una volta dentro, allora il diametro della punta è la dimensione sbagliata. In questo caso, tirare e utilizzare una pipetta differente. </li><li> Prima di aggiungere il colorante alla ACSF del tipo I pipetta, assicurarsi che ACSF sia sufficiente in modo che possa essere raggiunto con laType-II puntale (Fig. 2D). Altrimenti, un traferro esisterà una volta che il colorante viene aggiunto. Se questo accade, non aggiungere il colorante e rimuovere il tratto assonale dalla pipetta e poi tirarla indietro con ACSF sufficiente per essere raggiunto dalla Type-II pipetta. </li><li> Se il livello ACSF in tipo I aumenta pipette dopo che è stato aspirato con il Tipo II-pipetta, significa che il sigillo con il tessuto non è buona. Utilizzare un elettrodo diverso, altrimenti il ​​colorante viene diluito nel corso della procedura di etichettatura. </li><li> Se una bolla d&#39;aria viene introdotta nel tipo I pipetta mentre iniettando il colorante, defluire la bolla facendo avanzare il tipo-II vicino punta della pipetta per la bolla e delicatamente applicando una pressione negativa debole usando la siringa attaccata. </li></ol> 5. Risultati rappresentativi Motoneuroni Per illustrare l&#39;applicazione del metodo, caricati contemporaneamente, le afferenze sensoriali e interneuroni spinali con tre differenti Dextcorse di coniugati coloranti fluorescenti (Fig. 3A). Come illustrato in fig. 3B, tre classi di neuroni sono etichettati; afferenze sensoriali (rosso), interneuroni sporgenti all&#39;interno del funicolo ventrale (verde) e motoneuroni (blu). <img alt="Figura 1" src="/files/ftp_upload/4008/4008fig1.jpg" /> Figura 1. Schema che mostra Pipettare di tipo I e tipo II montaggio. (A) Un tipo I pipetta (luce blu) è posto su un portaelettrodo in modo che sia indietro apertura può essere facilmente collegata al tubo elastomero flessibile (B) e in cui uno di tipo II pipetta può essere inserito (C). <img alt="Figura 2" src="/files/ftp_upload/4008/4008fig2.jpg" /> Figura 2. Schematico che mostra il processo di caricamento dei motoneuroni con coloranti fluorescenti. (A) di tipo A-I pipetta si trova vicino alla radice ventrale da riempire. (B) di aspirazione viene applicata alla pipetta per disegnare ACSF nella pipetta seguita dalla radice. (C-D) Staccare il tubo flessibile in elastomero dal backend di tipo-I pipetta e ridurre la quantità di ACSF applicando aspirazione alla Type-II pipetta. (E) Inserire la punta della pipetta Type-II contenente il colorante dissolto nel ACSF rimanente nel tipo I pipetta. (EF) Lentamente riempire la Type-I pipetta con il colorante e quindi rimuovere la Type-II pipetta. <img alt="Figura 3" src="/files/ftp_upload/4008/4008fig3.jpg" /> Figura 3. Applicazione della procedura di riempimento al midollo spinale topo isolato. (A) schematico che mostra tre tipo I pipette per riempire diverse classi neuronali nel midollo spinale. Motoneuroni (blu, blu destrano Cascade) e sacrale afferenze sensoriali (rosso, Texas-Red destrano) sono stati riempita attraverso le radici ventrali e dorsali, rispettivamente. Fluoresceina destrano è stato utilizzato per caricare interneuroni spinali (verde) i cui assoni salire attraverso il funicolo ventrale (VF). 1 mg di ciascun colorante destrano (10.000 MW; Molecular Probes)è stato sciolto in 6 microlitri di acqua distillata contenente 0,2% Triton X-100 (B). Immagine confocale di una sezione del midollo sacrale di un midollo spinale in cui i neuroni sono stati retro-etichettato come descritto in A. Si noti che le afferenti etichettate sensoriali sono principalmente omolaterale con alcune sporgenze poco controlaterali. I neuroni motori sono etichettati omolaterale alla radice ventrale riempita mentre le interneuroni sono etichettati controlaterale al lato del riempimento (testa freccia). Calibrazione bar 200 micron.

<ol>
	<li>Nance, D. M., Burns, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextrans+as+sensitive+anterograde+neuroanatomical+tracers:+applications+and+pitfalls.">Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls.</a> Brain Res. Bull. 25, 139-145 (1990).</li><li>Glover, J. C., Petursdottir, G., Jansen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextran-amines+used+as+axonal+tracers+in+the+nervous+system+of+the+chicken+embryo.">Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo.</a> J. Neurosci. Methods. 18, 243-254 (1986).</li><li>McPherson, D. R., McClellan, A. D., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+neuronal+activity+in+tissue+labeled+by+retrograde+transport+of+Calcium+Green+Dextran.">Optical imaging of neuronal activity in tissue labeled by retrograde transport of Calcium Green Dextran.</a> Brain Research Protocols. 1, 157-164 (1997).</li><li>Fields, D. R., Shneider, N., Mentis, G. Z., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+nervous+system+activity.">Imaging nervous system activity.</a> Curr. Protoc. Neurosci. Chapter 2, Unit 2.3 (2009).</li><li>O'Donovan, M. J., Ho, S., Sholomenko, G., Yee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+neurons+retrogradely+and+anterogradely+labelled+with+calcium-sensitive+dyes.">Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes.</a> J. Neurosci. Methods. 46, 91-106 (1993).</li><li>Wenner, P., Tsau, Y., Cohen, L. B., O'Donovan, M. J., Dan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dye+recording+using+retrogradely+transported+dye+in+the+chicken+spinal+cord:+staining+and+signal+characteristics.">Voltage-sensitive dye recording using retrogradely transported dye in the chicken spinal cord: staining and signal characteristics.</a> J. Neurosci. Methods. 70, 111-120 (1996).</li><li>Garudadri, S., Gallarda, B., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+Cord+Electrophysiology+II:+Extracellular+Suction+Electrode+Fabrication.">Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication.</a> J. Vis. Exp. (48), e2580 (2011).</li><li>Smith, J. C., Feldman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+brainstem-spinal+cord+preparations+for+study+of+motor+systems+for+mammalian+respiration+and+locomotion.">In vitro brainstem-spinal cord preparations for study of motor systems for mammalian respiration and locomotion.</a> J. Neurosci. Methods. 21, 321-333 (1987).</li><li>Meyer, A., Gallarda, B., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+Cord+Electrophysiology.">Spinal Cord Electrophysiology.</a> J. Vis. Exp. (35), e1660 (2010).</li><li>Shneider, N. A., Mentis, G. Z., Schustak, J., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+reduced+sensorimotor+connections+form+with+normal+specificity+despite+abnormal+muscle+spindle+development:+the+role+of+spindle-derived+neurotrophin+3.">Functionally reduced sensorimotor connections form with normal specificity despite abnormal muscle spindle development: the role of spindle-derived neurotrophin 3.</a> J. Neurosci. 29, 4719-4735 (2009).</li><li>Mentis, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+functional+impairment+of+sensory-motor+connectivity+in+a+mouse+model+of+spinal+muscular+atrophy.">Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy.</a> Neuron. 69, 453-467 (2011).</li><li>Blivis, D., Mentis, Z., O'Donovan, J. M. G., Lev-Tov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+sacral+neurons+involved+in+activation+of+the+lumbar+central+pattern+generator+for+locomotion+in+the+neonatal+rodent+spinal+cord.">Studies of sacral neurons involved in activation of the lumbar central pattern generator for locomotion in the neonatal rodent spinal cord.</a> Soc. Neurosci. Abstr. 564.8, (2009).</li><li>O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+spatiotemporal+organization+of+neural+activity+in+the+developing+spinal+cord.">Imaging the spatiotemporal organization of neural activity in the developing spinal cord.</a> Dev. Neurobiol. 68, 788-803 (2008).</li><li>Kasumacic, N., Glover, J. C., Perreault, M. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+patterns+of+vestibular-mediated+synaptic+inputs+to+axial+and+limb+motoneurons+in+the+neonatal+mouse+assessed+by+optical+recording.">Segmental patterns of vestibular-mediated synaptic inputs to axial and limb motoneurons in the neonatal mouse assessed by optical recording.</a> J. Physiol. (Lond). 588, 4905-4925 (2010).</li><li>Szokol, K., Glover, J. C., Perreault, M. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+functional+synaptic+connections+between+medullary+reticulospinal+neurons+and+lumbar+descending+commissural+interneurons+in+the+neonatal+mouse.">Organization of functional synaptic connections between medullary reticulospinal neurons and lumbar descending commissural interneurons in the neonatal mouse.</a> J. Neurosci. 31, 4731-4742 (2011).</li></ol>

<p class="jove_content"&gt; Non abbiamo nulla da rivelare.</p

<p class="jove_content"&gt; Descriviamo qui un protocollo semplice e conveniente per l&#39;introduzione di coloranti in neuroni, nervi e tratti della colonna vertebrale. Questo metodo consiste nell&#39;esporre i percorsi identificati anatomiche a una soluzione altamente concentrata colorante per la durata del processo di caricamento. Ciò si traduce in etichettatura retrogrado del sito bersaglio con sfondo poco rispetto microiniezioni, applicazione bagno e tecniche di elettroporazione. Tuttavia, il metodo è limitato ai siti nel sistema nervoso...

<table border="1"&gt;<tbody&gt;<tr&gt;<td colspan="2"<strong&gt; Nome del materiale</strong</td&gt;<td<strong&gt; Azienda</strong</td&gt;<td<strong&gt; Numero di catalogo</strong</td&gt;<td<strong&gt; Commenti</strong</td&gt;</tr&gt;<tr&gt;<td rowspan="2"&gt; Tubi</td&gt;<td&gt; PE90 (IDxOD: 0,034 &quot;x0.050&quot;; Spessore parete: 0.008 &quot;)</td&gt;<td&gt; Clay Adams Marca Intermedic</td&gt;<td&gt; 427.421</td&gt;<td&gt; Per Tipo I e Tipo II pipette</td&gt;</tr&gt;<tr&gt;<td&gt; NSF-51 (IDxOD: 1/16x1/8; Spessore parete: 1/32)</td&gt;<td&gt; PharMed BPT, Cole-Parmer</td&gt;<td&gt; AY242002</td&gt;<td&gt; Per Tubi flessibili</td&gt;</tr&gt;<tr&gt;<td&gt; Siringa</td&gt;<td&gt; Siringa da insulina da 1 ml U-100</td&gt;<td&gt; Becton Dickinson</td&gt;<td&gt; 329.650</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Needle</td&gt;<td&gt; 19G x1-1/2 &quot;Metal Hub Needle</td&gt;<td&gt; MONOJECT</td&gt;<td&gt; 200.136</td&gt;<td&gt; Collegamento di tipo II pipetta siringa</td&gt;</tr&gt;<tr&gt;<td&gt;</td&gt;<td&gt; Alcohol Lamp</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Rubinetto</td&gt;<td&gt; Valvola a tre vie con Maschio Adattatore Luer slip</td&gt;<td&gt; Baxter Healthcare Corp.</td&gt;<td&gt; 2C6241</td&gt;<td&gt; Usa con siringa</td&gt;</tr&gt;<tr&gt;<td rowspan="3"&gt; Titolare</td&gt;<td&gt; Supporto H-1 elettrodo</td&gt;<td&gt; Narishige</td&gt;<td&gt; H-1/12</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Magnet supporto</td&gt;<td&gt; Narishige</td&gt;<td&gt;-8 GJ</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Micromanipolatore</td&gt;<td&gt; Narishige</td&gt;<td&gt; M-3333</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Pinzetta</td&gt;<td&gt; S &amp; T DUMONT svizzera 00632-11</td&gt;<td&gt; DUMONT</td&gt;<td&gt; JF-5 TC</td&gt;<td&gt;</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"&gt; * Questi sono materiali suggeriti. Può essere sostituito con qualsiasi prodotti compatibili</p

retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes

Etichettatura retrograda di neuroni è uno standard 1,2 metodo anatomica che è stata anche utilizzata per caricare e calcio voltaggio-coloranti sensibili in neuroni 3-6. Generalmente, i coloranti vengono applicate come cristalli solidi o mediante iniezione a pressione locale utilizzando pipette di vetro. Tuttavia, ciò può causare diluizione della intensità etichettatura colorante e ridotta, in particolare quando sono necessarie diverse ore per diffusione colorante. Qui mostriamo una tecnica semplice e di basso costo per l&#39;introduzione coloranti fluorescenti e ioni sensibile in neuroni con una pipetta di aspirazione polietilene riempito con la soluzione colorante. Questo metodo offre un modo affidabile per mantenere un&#39;elevata concentrazione del colorante in contatto con assoni tutta la procedura di caricamento.

Watch this Scientific Journal Video about Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes at JoVE.com

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Descriviamo una tecnica semplice ea basso costo per introdurre alta concentrazione di coloranti fluorescenti e calcio-sensibili in neuroni o qualsiasi tratto neuronale mediante una pipetta di aspirazione polietilene.

Caricamento in corso retrogrado di Nervi, tratti e radici spinali con le tinture fluorescenti

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

retrograde-loading-nerves-tracts-spinal-roots-with-fluorescent

Research

JoVE Journal

Neuroscience

12.2K Views.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Descriviamo una tecnica semplice ea basso costo per introdurre alta concentrazione di coloranti fluorescenti e calcio-sensibili in neuroni o qualsiasi tratto neuronale mediante una pipetta di aspirazione polietilene.

Video: Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Studiare Piscine vescicole sinaptiche con fotoconversione di coloranti Styryl

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Ricerca

Neuroscienze

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Imaging ottico di neuroni nel ganglio Granchio Stomatogastric con tensione sensibili Coloranti

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Promozione della sopravvivenza e la differenziazione delle cellule staminali neurali con fibrina e fattore di crescita Cocktails dopo gravi lesioni del midollo spinale

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Etichettatura retrograda di cellule gangliari della retina in Zebrafish adulti con coloranti fluorescenti

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

In Misure Vivo elettrofisiologici sui nervi mouse sciatico

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Imaging potenziale di membrana con due tipi di geneticamente codificati fluorescenti di sensori di tensione

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Analisi Neuroanatomical retrogrado dei motoneuroni frenico in topi

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Corsi di formazione basati su attività su un tapis roulant con midollo spinale feriti ratti Wistar

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.