Ringrazio Anthony Taylor per la formazione iniziale in in vivo registrazione intracellulare e A Maxwell Brown e David per aiutare lo sviluppo iniziale della tecnica di colorazione intracellulare. Ringrazio M. Argento per aiutare con la macro collocalization. Molti studiosi con i quali ho collaborato visione fornito nello sviluppo di questa tecnica tra cui R. Donga, M. Moritani, P. Luo, R. Ambalavanar. Questa tecnica è stata sviluppata con il supporto considerevole dalle sovvenzioni NIH DE10132, DE15386 e RR017971.

1. Preparazione degli animali <ol><li> Anestetizzare ratto con sodio pentobarbital (IP 50mg/kg) e posto su una piastra elettrica. Radere la pelle sovrastante il cranio posteriore con tagliaunghie animale. Controllare l&#39;animale per assicurare che un livello chirurgico di livello di anestesia è stata ottenuta con test per l&#39;assenza di un riflesso ritiro e vocalizzazione quando le dita sono schiacciati come pure l&#39;assenza di un riflesso palpebrale. Controllare il livello di anestesia ogni 15 minuti e mantenere un livello chirurgico di anesthetisa con iniezioni di 15mg/kg di sodio pentobarbital ogni 45 minuti. </li><li> Utilizzare una tecnica asettica e fare un&#39;incisione nella regione inguinale appena distalmente alla piega formata dal addome e interno coscia e inserire una cannula (1 mm di diametro, Clay Adams) nella vena femorale, e l&#39;arteria femorale. Fai una incisione sulla linea mediana nella regione sottomandibolare, riflettono i muscoli infraioidea. Fai una piccola incisione nella trachea e inserire una cannula (2mm di diametro) per consentire la ventilazione. Tye una sutura intorno alla trachael cannual per fissarlo in place.Monitor pressione arteriosa sistemica sistolica e diastolica mediante la cannula arteriosa. Oltre a monitorare il ritiro e riflesso palpebrale ora monitorare la pressione del sangue per valutare il livello di anestesia. Somministrare l&#39;anestesia aggiuntiva in caso di necessità attraverso la cannula venosa. </li><li> Posizionare il topo in un frame stereotassico ed eseguire una craniotomia per esporre il cervelletto. Fissare la cannula tracheale di un ventilatore roditori. Ventilare l&#39;animale con un volume di 2 cm 3 ad un tasso di 100/min con aria umidificata. Una pressione positiva di fine espirazione di 1 cm H 2 O dovrebbe essere mantenuto per prevenire il collasso del polmone. Ogni 15 minuti hyperinflate i polmoni per prevenire atelettasia. Poi coprire la superficie del cervello con olio minerale riscaldato (30 ° C). Fare attenzione a evitare il seno grande venoso situato direttamente sotto la giunzione tra il parietale e ossa interparietale. </li><li> Applicare la colla al cianoacrilato una canna accoppiato ad un vibratore elettromagnetico e allegare l&#39;asta nel diastema della mandibola. Spostare i segnali mandibola comando utilizzando il vibratore sia da un uscita A / D del computer o di un generatore di segnale. </li><li> Posizionare un siliver-argento elettrodo di messa a terra cloruro sotto la pelle adiacente alla craniotomia. </li></ol> 2. Elettrodo di preparazione <ol><li> Fabbricare microelettrodi in vetro di quarzo o di alluminio con un estrattore orizzontale microelettrodo. </li><li> Riempire il microelettrodo con biotinamide 5-10% sciolto in KCl 0.25M e 0.5M Tris HCl (pH 7,6) o del 2% tetramethlyrhodamine in soluzione salina (pH 6). Controllo dell&#39;impedenza dell&#39;elettrodo con un tester elettrodo. Per registrare da assoni di grande diametro fare elettrodi con impedenza di 60-80M Ω, per gli assoni piccoli e interneuroni fanno elettrodi con impedenza di 80-150MΩ. </li><li> Posizionare il microelettrodo nella fase capo della elettrometro. Visualizza l&#39;elettrodo attraverso un piccolo telescopio (20X con un reticolo allegato) che è fissato in posizione dietro la testa dell&#39;animale. Utilizzando il telescopio è importante perché permette di essere stereotaxically elettrodi posizionati con estrema precisione. </li></ol> 3. Registrazione elettrofisiologica e colorazione intracellulare <ol><li> Fai una piccola apertura nella pia madre e compensare il feedback capacità in modo che quando l&#39;elettrodo tocca il cervello un segnale di feedback viene prodotto. Questo consente un accurato posizionamento della superficie del cervello. </li><li> Produrre ripetuto spostamento della mandibola e far avanzare l&#39;elettrodo nel cervello con un motore passo-passo. </li><li> Riconoscere impalements neuronale da improvvisi cali di potenziale DC e identificare intrasomatica impalements neuronale in corso di attività sinaptica. Buzzing l&#39;elettrodo con sovracompensazione di capacità o toccando raramente producono una penetrazione di successo cellula. A causa della alta impedenza degli elettrodi, le risposte neuronali sono raramente osservati prima della penetrazione. </li><li> Dopo aver infilzare un neurone e la penetrazione è considerato stabile, caratterizzano la risposta neuronale utilizzando rampa e tenere premuto e il movimento della mascella sinusoidale. 5 Mappa del campo recettivo del neurone sondando la pelle intorno alla testa e all&#39;interno della cavità orale con un non-conduttivo sonda come un bastone di legno. Se rilevanti per lo studio, esaminare la risposta del neurone ad altri stimoli funzionalmente rilevanti quali la contrazione muscolare e stimoli nocivi. 6 afferenti primari nocicettivi recettori neuronali rispondere agli stimoli nocicettivi. Anesthesetics generale come il sodio pentobarbital Non blocco di conduzione assonale nei neuroni afferenti primari ma deprimono la trasmissione sinaptica. Così conduzione assonale nelle primarie assone afferente neuronale è conservato. </li><li> Iniettare corrente (DC, 1-4NA) per un tempo di iniezione totale di 15-70nA minuti. Monitorare la penetrazione degli elettrodi durante l&#39;iniezione di corrente e sospendere se potenziale di membrana diventa più positivo di quello-30mV.</li></ol> 4. Lavorazione dei tessuti <ol><li> Eutanasia degli animali per overdose di pentobarbital (140mg/kg IV) e profumato con un risciacquo vascolare (0,9% NaCl 38 ° C che contiene 500 unità di eparina e 1ml 2% xylocaina seguito da paraformaldeide 4% in 0.1M tampone fosfato (pH 7,4) . </li><li> Togliere il cervello e la sezione è a 50-100μm con un vibratome sia nel frontale, piano sagittale o orizzontale. Secions sono raccolti flottante libero in 25 ° C PBS. </li><li> Processo del cervello per DAB incubando in 1-2% di siero normale di capra e 1% Triton X-100 a 0.01M PBS seguita da incubazione nel complesso avidina biotina (1:50 Elite Vectastain). Reagire sezioni utilizzando nichel-DAB con H 2 O 2. Al processo per il Texas Red, sezioni incubare in complesso avidina-biotina (1:50) in PBS notte a 4 ° C e incubare nel 4% Texas Red avidina DCS in PBST a 4 ° C durante la notte. Sezioni di contrasto con una macchia fluorescente Nissl (NeuroTrace) 20 minuti a 23 ° C. </li><li> Se il neurone è stato iniettato con rodamina, visualizzare il neurone iniettato direttamente con un microscopio a fluorescenza (Es 545nm). </li></ol> 5. La combinazione di metodo con marcatura retrograda, immunocitochimica, l&#39;imaging confocale, analisi quantitativa colocalizzazione Il successo di combinare questa tecnica con altri metodi dipende in gran parte buona etichettatura intracellulare. <ol><li> Il metodo qui visualizzati possono essere facilmente combinati con retrograda etichettatura neuronale. 7-10 Per fare questo, anestetizzare l&#39;animale e con tecnica asettica, iniettare un tracciante neuronale come la perossidasi del rafano (20% perossidasi di rafano. Sigma VI, Sigma) e 1% germe di grano aggultinated perossidasi (Sigma) in regioni periferiche come bersaglio il muscolo massetere. Questo tracciante neuronale sarà assunto in assoni periferici e trasportati attraverso il trasporto assonale al somata motoneuroni. Per il muscolo massetere, 15μl di tracciante viene iniettato nel muscolo con una sterile microsiringa da 10 microlitri. Gli animali vengono poi poste su una piastra elettrica e seguito fino alla ripresa dall&#39;anestesia e poi messo nelle loro gabbie per il recupero. Dopo 24 ore, anestetizzare gli animali e fisiologicamente caratterizzano i neuroni e intracellulare loro macchia. Poi l&#39;animale profumato come descritto sopra e sezioni di tessuto di processo per la presenza di HRP utilizzando tetrametilbenzidina (TMB) come cromogeno ed tungstato di sodio come lo stabilizzatore e intensificato con il cobalto. Sezioni posto in 1% di sodio tungstato sciolto in PBS 0,1 M (pH 6,5) e 0,0007% TMB dissolto in etanolo e acetone assoluto a 15 ° C per 20 min. Poi reagire sezioni di tessuto con l&#39;aggiunta di 1,0 ml di 0,3% H 2 O 2 per 100 ml di soluzione di incubazione per 60 min. Lavare il tessuto in PBS 0,1 M (pH 6,5) e il luogo in diaminobenzidina 0,05% (pH 7,4), 0,02% di cobalto, e 0,01% di H 2 O 2 a 0,1 M PBS (pH 7,4) per 10 minuti. a 37 ° C. Processo il tessuto per la visualizzazione del neurone iniettati con biotinamide come descritto e visualizzare le relazioni tra i neuroni sensoriali etichettato e una varietà di motoneuroni. </li><li> La tecnica qui può anche facilmente essere combinato con immnocytochemistry 6. Per fare un esempio, immunocytochemically processo del cervello per sinaptofisina di localizzare con precisione all&#39;interno delle sinapsi dei neuroni etichettati (Fig. 3). Per fare questo, incubare sezioni di cervello di topo anti-sinaptofisina anticorpi (1:10.000) per 2 giorni a 4 ° C e incubare anti-mouse FITC (1:400) per 1h a 23 ° C. </li><li> Neuroni marcati con marcatori fluorescenti o trattati per l&#39;imaging fluorescenti sono l&#39;ideale per l&#39;imaging confocale. Figura 3 è un esempio di una sezione ottica ottenuta con microscopia confocale attraverso un bouton assone. (Fig. 3). Generare animazioni dei neuroni etichettati con l&#39;acquisizione di più sezioni ottiche attraverso il neurone etichettati (Fig. 4). </li><li> I neuroni etichettati con questo metodo può essere utilizzato per l&#39;analisi quantitativa colocalizzazione. Per fare questo, utilizzare una macro di software disponibili al pubblico in collaborazione con NIH immagine (che si trova a http://phy.ucsf.edu/ ° idl / colocalization.htm). Sinaptofisina localizzare all&#39;interno dei terminali singolo assone combinando etichettatura intracellulare con immunocitochimica sinaptofisina 6. </li></ol> 6. Rappresentante dei risultati: Una panoramica dei risultati rappresentativi che si possono ottenere con questo metodo sono illustrati nella Figura 1. Questo singolo neurone tronco encefalico è stato elettrofisiologico registrato durante il movimento della mandibola e, come si può chiaramente vedere, la risposta del neurone (Figura 1 in basso a sinistra, la luce blu) è stata modulata durante il movimento. Questo neurone è stato iniettato con biotinamide dopo caratterizzazione elettrofisiologica e successivamente trattati per la visualizzazione. Il neurone ricostruito (Figura 1 medio, verde) può essere realeTed ad un punto di riferimento anatomico, in questo caso il nucleo motore del trigemino designato (contorno rosso). Basati sulla risposta neuronale durante il movimento e la ricostruzione questo neurone può essere identificato come un neurone secondario fuso muscolare afferenti. La figura 2 illustra un esempio rappresentativo della risposta fisiologica di un neurone durante lo spostamento della mandibola. La risposta del neurone è rappresentato come frequenza di emissione istantanea. Si noti che la risposta neuronale imita strettamente spostamento mandibolare indicando che questo neurone particolare fornisce un feedback sensoriale legati alla posizione della mandibola. Figura 3 è un&#39;immagine forte ingrandimento di un assone intracellulare macchiato combinato con colorazione di Nissl sinaptofisina e una macchia. Si noti la colocalizzazione di sinaptofisina (giallo) all&#39;interno del bouton assone. Figura 4 è una animazione di un singolo neurone fisiologicamente caratterizzato e intracellulare etichettati. <img src="/files/ftp_upload/2650/2650fig1.jpg" alt="Figura 1"/> Figura 1. Panoramica del metodo. In alto a sinistra: lo spostamento della mandibola. Medio registrazione intracellulare (verde) da solo neurone (giallo). La morfologia di questo neurone è stato ricostruito dopo la registrazione intracellulare e iniezione. Contorno rosso indica la posizione del nucleo motore del trigemino. In basso a sinistra: risposta fisiologica di questo neurone durante il movimento della mascella. <img src="/files/ftp_upload/2650/2650fig2.jpg" alt="Figura 2"/> Figura 2. Rappresentante risposta fisiologica di un neurone singolo muscolo sensoriali registrate in vivo durante il movimento della mandibola. Si noti la somiglianza della risposta neuronale con spostamento della mandibola. <img src="/files/ftp_upload/2650/2650fig3.jpg" alt="Figura 3"/> Figura 3. Arborizzazione terminale assonale con boutons sinaptica (gonfiori rosso) di un livello intracellulare macchiato di neuroni sensoriali che hanno risposto durante muscolare sondaggio. Il conseguente trattamento immunocitochimica per sinaptofisina mostra la localizzazione del sinaptofisina all&#39;interno del bouton assonale (giallo). Green è un colorante fluorescente Nissl. Figura 4. Animazione di assone fuso muscolare neurone primario afferente la cui risposta fisiologica è stato registrato in vivo durante il movimento mandibolare, L&#39;assone è stato poi intracellulare colorati ed elaborati per la visualizzazione. <a href="/files/ftp_upload/2650/collateral4.avi">Scaricare un video ad alta risoluzione di Figura 4 qui</a> <a href="/files/ftp_upload/2650/medres.mov">Scaricare un video a media risoluzione di Figura 4 qui</a>

<ol>
	<li>Cuellar, C. A., Tapia, J. A., Juarez, V., Quevedo, J., Linares, P., Marinez, L., Manjarrez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+sinusoidal+electrical+waves+along+the+spinal+cord+during+a+fictive+motor+task.">Propagation of sinusoidal electrical waves along the spinal cord during a fictive motor task.</a> J. Neurosci. 29, 798-810 (2010).</li><li>Frigon, A., Gossard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+specialized+rhythm-generating+mechanisms+in+the+adult+mammalian+spinal+cord.">Evidence for specialized rhythm-generating mechanisms in the adult mammalian spinal cord.</a> J. Neurosci. 30, 7061-7071 (2010).</li><li>Wang, W., Chan, S. S., Heldman, D. A., Moran, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+cortical+representation+of+hand+translation+and+rotation+during+reaching.">Motor cortical representation of hand translation and rotation during reaching.</a> J. Neurosci. 30, 958-962 (2010).</li><li>Ma, C., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+investigating+cortical+control+of+stand+and+squat+in+conscious+behavioral+monkeys.">A method for investigating cortical control of stand and squat in conscious behavioral monkeys.</a> J. Neurosci. Meth. 192, 1-6 (2010).</li><li>Dessem, D., Donga, R., Luo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary-+and+secondary-like+jaw-muscle+spindle+afferents+have+characteristic+topographic+distributions.">Primary- and secondary-like jaw-muscle spindle afferents have characteristic topographic distributions.</a> J. Neurophysiol. 77, 2925-2944 (1997).</li><li>Dessem, D., Moritaini, A., Ambalavanar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+craniofacial+muscle+primary+afferent+neurons+synapse+in+both+the+rostral+and+caudal+brain+stem.">Nociceptive craniofacial muscle primary afferent neurons synapse in both the rostral and caudal brain stem.</a> J. Neurophysiol. 98, 214-223 (2007).</li><li>Luo, P., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inputs+from+identified+jaw-muscle+spindle+afferents+to+trigeminothalamic+neurons+in+the+rat:+a+double-labeling+study+using+retrograde+HRP+and+intracellular.">Inputs from identified jaw-muscle spindle afferents to trigeminothalamic neurons in the rat: a double-labeling study using retrograde HRP and intracellular.</a> J. Comp. Neurol. 353, 50-66 (1995).</li><li>Dessem, D., Luo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jaw-muscle+spindle+afferent+feedback+to+the+cervical+spinal+cord+in+the+rat.">Jaw-muscle spindle afferent feedback to the cervical spinal cord in the rat.</a> J. Comp. Neurol. 128, 451-459 (1999).</li><li>Luo, P., Wong, R., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+of+jaw-muscle+spindle+afferents+to+the+caudal+brainstem+in+rats+demonstrated+using+intracellular+biotinamide.">Projection of jaw-muscle spindle afferents to the caudal brainstem in rats demonstrated using intracellular biotinamide.</a> J. Comp. Neurol. 358, 63-78 (1995).</li><li>Luo, P., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+anatomy+of+physiologically+identified+jaw-muscle+spindle+afferent+terminations+onto+retrogradely+labeled+jaw-elevator+motoneurons+in+the+rat.">Ultrastructural anatomy of physiologically identified jaw-muscle spindle afferent terminations onto retrogradely labeled jaw-elevator motoneurons in the rat.</a> J. Comp. Neurol. 406, 384-401 (1999).</li><li>Hassani, O. K., Henny, P., Lee, M. G., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+intermingled+with+orexin+and+MCH+neurons+in+the+lateral+hypothalamus+discharge+maximally+during+sleep.">GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep.</a> Eur. J. Neurosci. 32, 448-457 (2010).</li><li>Inokawa, H., Yamada, H., Matsumoto, N., Muranishi, M., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacellular+labeling+of+tonically+active+neurons+and+phasically+active+neurons+in+the+rat+striatum.">Juxtacellular labeling of tonically active neurons and phasically active neurons in the rat striatum.</a> Neuroscience. 168, 395-404 (2010).</li></ol>

Gli esperimenti sugli animali sono stati eseguiti conformemente al guidlines e regolazione di cui la Guida per la cura e l&#39;uso di animali da laboratorio (NIH Publication No. 86-23, riveduta 1985) e l&#39;Università del Maryland e cura degli animali Usa comitato.

Il metodo qui illustrato è una tecnica potente che fornisce informazioni importanti nella funzione di singoli neuroni e di come la risposta dei singoli neuroni contribuisce a circuiti neuronali. 9 Questa conoscenza è fondamentale per comprendere la funzione senso-motoria. La più grande forza di questa tecnica è che consente sia la determinazione di un gran numero di parametri su un neurone tra cui la fisiologia, la morfologia e la morfologia sinaptica e la distribuzione. Quando combinato con altre tecnich...

<table border="1"><tbody><tr><th> Nome del reagente o attrezzature </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> vibratore elettromagnetico </td><td> Ling Dynamic Systems </td><td> V101 </td><td></td></tr><tr><td> generatore di segnale </td><td> Sistemi di feedback </td><td> PFG605 </td><td> in grado di produrre il segnale di uscita trapezoidale </td></tr><tr><td> elettrodo di vetro </td><td> Sutter Instruments </td><td> AF100-68-10 </td><td> con filamento </td></tr><tr><td> elettrodo estrattore </td><td> Sutter Instruments </td><td> Modello P-2000 e P-80 </td><td></td></tr><tr><td> biotinamide </td><td> Laboratori Vector </td><td> SP-1120 </td><td> conservato a 4 ° C </td></tr><tr><td> Texas Red avidina DCS </td><td> Laboratori Vector </td><td> A-2016 </td><td></td></tr><tr><td> tetramethlyrhodamine </td><td> Molecular Probes </td><td> D-3308 </td><td> 3000 peso molecolare, lisina fissabile </td></tr><tr><td> topo anti-sinaptofisina anticorpi </td><td> Chemicon </td><td> MAB5258 </td><td></td></tr><tr><td> fluorescente macchia Nissl </td><td> Neurotrace, Molecular Probes </td><td> N-21480 </td><td></td></tr><tr><td> elettrodo tester </td><td> Winston Elettronica </td><td> BL-1000-B </td><td> per misurare l&#39;impedenza degli elettrodi </td></tr><tr><td> elettrometro </td><td> Axon Instruments </td><td> Axoprobe 1A, 2B Axoclamp </td><td></td></tr></tbody></table>

physiological, morphological and neurochemical characterization of neurons modulated by movement

Il ruolo dei singoli neuroni e la loro funzione nei circuiti neuronali è fondamentale per capire i meccanismi neuronali delle funzioni sensoriali e motorie. Maggior parte delle indagini di meccanismi sensomotori avvalersi né esame di neuroni, mentre un animale è 1,2 statico o registrare l&#39;attività neuronale extracellulare durante un movimento. 3,4 Anche se questi studi hanno fornito lo sfondo fondamentale per la funzione sensomotoria, che o non valutare le informazioni funzionali che si verifica durante un movimento o sono limitate nella loro capacità di caratterizzare completamente il fenotipo anatomia, fisiologia e neurochimici del neurone. Una tecnica è qui che permette la caratterizzazione completa di singoli neuroni nel corso di un movimento vivo. Questa tecnica può essere utilizzata non solo per studiare i neuroni afferenti primari, ma anche per caratterizzare motoneuroni e interneuroni sensomotorie. Inizialmente la risposta di un singolo neurone viene registrata con metodi elettrofisiologici durante vari movimenti della mandibola seguita dalla determinazione del campo recettivo per il neurone. Un tracciante neuronale viene poi iniettata livello intracellulare nel neurone e il cervello viene elaborata in modo che i neuroni possono essere visualizzati con la microscopia ottica, elettronica e confocale (Fig. 1). La morfologia dettagliata del neurone è caratterizzata poi ricostruita in modo che la morfologia neuronale può essere correlato con la risposta fisiologica del neurone (Fig. 2,3). In questa comunicazione dei dettagli delle chiavi importanti e suggerimenti per il successo di questa tecnica sono forniti. Preziose informazioni aggiuntive possono essere determinate per il neurone in fase di studio, combinando questo metodo con altre tecniche. Retrograda etichettatura neuronale può essere usato per determinare i neuroni con cui le sinapsi dei neuroni etichettati; permettendo così la determinazione dettagliata dei circuiti neuronali. Immunocitochimica può essere combinato con questo metodo per esaminare neurotrasmettitori all&#39;interno del neurone etichettati e per determinare i fenotipi chimica dei neuroni con cui le sinapsi dei neuroni etichettati. Il neurone marcata può essere effettuato anche per la microscopia elettronica per determinare le caratteristiche ultrastrutturali e microcircuiti del neurone etichettati. In generale questa tecnica è un potente metodo per caratterizzare accuratamente i neuroni in movimento durante vivo permettendo quadro chiaro della funzione del neurone in funzione senso-motoria.

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Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Una tecnica è descritta per quantificare in vivo la risposta fisiologica dei neuroni dei mammiferi durante il movimento e correlare la fisiologia del neurone con morfologia neuronale, fenotipo neurochimico e microcircuiti sinaptici.

Fisiologici, caratterizzazione morfologica e neurochimica dei neuroni modulati Movimenti

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

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13.7K Views.  University of Maryland. Una tecnica è descritta per quantificare in vivo la risposta fisiologica dei neuroni dei mammiferi durante il movimento e correlare la fisiologia del neurone con morfologia neuronale, fenotipo neurochimico e microcircuiti sinaptici.

Video: Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Eye Movement Monitoraggio della Memoria

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Ricerca

Neuroscienze

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Coerenza tra funzione cerebrale corticale e prestazioni neurocognitive durante condizioni di gravità Cambiato

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Rapida determinazione della soglia termica nocicettivo in ratti diabetici

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Odorizzante indotte risposte registrate da neuroni recettori olfattivi con la tecnica della pipetta di aspirazione

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Rilevamento di palmitoilazione proteina in colture di neuroni ippocampali di immunoprecipitazione e di acil-Biotina Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

Misurazione del calcio totale in neuroni da Electron Probe microanalisi a raggi X

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-riprogrammazione di cellule pericyte-derivate del cervello umano adulto in neuroni indotti

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Elettrofisiologici e morfologica Caratterizzazione neuronali Microcircuiti in Acute Fette cervello Utilizzando accoppiati di patch-clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funzionale e morfologica Valutazione della membrana innervazione da Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Immunocolorazione delle sezioni biocitina-riempite e trasformati per neurochimici Marcatori

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...