Ringraziamo Manfred Heckmann per utili discussioni durante stabilisce il metodo. Inoltre, ringraziamo Claudia Sommer per l&#39;assistenza tecnica e Frank Schubert per il supporto la produzione del video. Il lavoro è stato supportato dal Ministero federale dell&#39;Istruzione e della Ricerca (BMBF), Germania, FKZ: 01EO1002 e il Centro Interdisciplinare per la Ricerca Clinica (IZKF) di Jena University Hospital.

Tutte le procedure sperimentali di cui al seguente protocollo sono stati approvati dalle autorità statali della Turingia (Thüringer Landesamt für Verbraucherschutz, Reg.-Nr. 02-044/12). 1. Preparativi per Experiment <ol><li> Fabbricazione di elettrodi di aspirazione <ol><li> Tirare una micropipetta con un capillare di vetro borosilicato di serie con un estrattore micropipetta, ad esempio, un elettrodo di patch standard. </li><li> Freno l&#39;elettrodo a punta diametro di 0,5-1 mm (leggermente più grande del diametro delle radici dorsali) utilizzando un file di diamante. </li><li> Polish calore la punta in modo da non danneggiare la radice dorsale quando viene risucchiato dentro una torcia laboratorio standard sarà adatto. </li><li> Montare il filo di vetro su un supporto di elettrodo collegato ad una siringa attraverso il quale la pressione negativa può essere applicata. </li></ol></li><li> Preparazione di soluzioni <ol><li> Anestesia iniettabile per topi: Miscelare 0,75 ml di ketamina 10%, 0,24 ml xylazina 2%nd 5 ml di soluzione fisiologica 0,9%. 10 microlitri / g di peso corporeo (BW) della soluzione viene iniettata per via intraperitoneale (ip). </li><li> Liquido cerebrospinale artificiale (aCSF): diluire in acqua bidistillata i seguenti sali (in mm): 134 NaCl, KCl 3; KH 2 PO 4 1,25; MgSO 4 • H 2 O 2; NaHCO 3 25; CaCl 2, 2, D- glucosio 10. Aggiungere H 2 O 2 ad una concentrazione finale di 0,003%. Utilizzare una soluzione sempre preparata al momento. </li></ol></li><li> Preparazione della configurazione di registrazione (Figura 2) <ol><li> Collegare l&#39;amplificatore ad una interfaccia PC per la digitalizzazione dei dati. </li><li> Utilizzare un programma basato su PC o un timer analogico per innescare uno stimolatore impulso quadrato e l&#39;acquisizione dei dati sul disco rigido del PC. </li><li> Utilizzare silverwires chlorided collegati alla porta elettrodi vetro standard, per la stimolazione e la registrazione. </li><li> Montare tre manipolatori per la stimolazione, la registrazione e l&#39;elettrodo di riferimento, rispettivamente, intorno a un sterecornice otactic per topi, in modo che tutti gli elettrodi hanno accesso al midollo spinale preparato in seguito. </li><li> Collegare tubi e siringhe ai titolari elettrodi essere mela per applicare una pressione negativa. </li></ol></li></ol> 2. Commenti generali per la sperimentazione animale e preparazione degli animali Procedura di registrazione <ol><li> Eseguire tutti gli esperimenti con i topi secondo le direttive del rispettivo comitato cura degli animali istituzionale e l&#39;uso. Eseguire tutti gli interventi chirurgici e le registrazioni in anestesia profonde assicurando che la sofferenza degli animali è ridotta al minimo. </li><li> Anestetizzare l&#39;animale mediante iniezione ip di ketamina / xilazina (125 mg e 8 mg per g BW di ketamina e xilazina, rispettivamente 10 ml / g di peso corporeo della soluzione preparata come descritto sopra). Se necessario, durante le registrazioni a lungo termine, ulteriori iniezioni possono essere effettuate ip o im Nota: ripetitivi im iniezioni di 0,05-0,1 ml di soluzione di ketamina / xylazina nelle cosce hanno dimostrato di essere adattoper mantenere l&#39;animale in anestesia profonda fino a 3 ore. </li><li> Utilizzare veterinario unguento sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Fissare la testa dell&#39;animale in un telaio stereotassico e utilizzare una piastra elettrica con sonda rettale e anello riflesso per controllare la temperatura corporea dell&#39;animale durante l&#39;esperimento. Fissazione del midollo spinale in un telaio stereotassico non è necessario. </li><li> Prima di iniziare la preparazione, verificare la profondità della narcosi da reflex eyeblink e contrazioni tra le dita dei topi. Riflessi dovrebbero essere aboliti. </li><li> Aprire la pelle lungo la linea mediana sopra il midollo spinale dal livello toracico superiore ad abbassare le zone lombari per ottenere un campo operativo chiaro con un bisturi. Non usare le forbici per fare incisioni cutanee. Perdere con cura la pelle dal tessuto sottostante. Durante le fasi successive mantengono la ferita inumidito dello 0,9% soluzione salina. </li><li> Tagliare tendini e tessuto connettivo su entrambi i lati delle vertebre lombari da livelli toracici usando bisturi e forbici. </li&gt; <li> Rimuovere i processi spinosi e resto del tessuto connettivo intorno alle vertebre con una piccola pinza. </li><li> Rompere con cautela vertebre con la pinza a partire dai livelli lombari (L4/L5) sotto un microscopio da dissezione. Spingere la punta della pinza nello spazio tra le vertebre e il midollo spinale e sollevare pezzi di ossa a parte. Non danneggiare la dura ed evitare una pressione al midollo spinale. Entrambi sono fondamentali per il successo. Mantenere il midollo spinale inumidito durante l&#39;intera procedura. Procedere a livelli medio-toracici. </li></ol> 3. Separazione delle radici dorsali e DRP registrazione (Figura 2) <ol><li> Aprire la dura madre con cura utilizzando un ago sottile (30 G) con una punta piegata. Utilizzare aCSF per inumidire da ora in poi. </li><li> Separare le radici dorsali, per quanto possibile con il calibro e tagliare due radici adiacenti come distale possibile. Vigorosa tirando le radici durante separazione colpisce il successo dell&#39;esperimento. </li><li> Abbassare l&#39;elettrodo di aspirazione verso il basso per il faiRSAL radici. Aggiungere tanto aCSF al midollo spinale possibile perché liquido aiuta a succhiare nelle radici dorsali. </li><li> Succhiare la fine taglio di una radice dorsale in una pipette di vetro applicando una pressione negativa attraverso una siringa. Se necessario, spostare la radice dorsale di fronte all&#39;apertura elettrodo utilizzando un ago sottile. </li><li> Se la radice dorsale si trova a secco entro l&#39;elettrodo di aspirazione, aggiungere un po &#39;aCSF alla sua punta, mentre con cura succhia fino a quando la pipetta è sufficientemente pieno di aCSF. </li><li> Dopo la radice dorsale viene aspirata, sollevare la punta dell&#39;elettrodo dal midollo spinale. Fate attenzione che nessun &quot;ponte acqua&quot; tra il puntale e il midollo spinale corto circuito l&#39;elettrodo di registrazione / stimolazione. </li><li> Ripetere i passaggi 3,3-3,6 per una radice adiacente omolaterale. </li><li> Posizionare l&#39;elettrodo di riferimento più vicino possibile alle radici dorsali e tenerlo inumidito applicando aCSF. </li><li> Stabilendo la configurazione della registrazione, una radice è già scelto per la stimolazione, ilaltra per la registrazione. Aumentare la tensione di graduale durante la registrazione della seconda radice dorsale è fatto in modo pinza corrente. Si può notare una breve deviazione verso il basso che è seguita da una lenta, lunga durata deflessione verso l&#39;alto rappresenta il DRP. </li><li> Regolare la tensione stimolo per sopramassimale livelli e registrare diverse scansioni (almeno 20 - 30 spazza / radice dorsale a 0.1 Hz, filtra tra 0,3 Hz-3 kHz). </li><li> Registrare treni corti di tre stimoli (100 Hz) per ottenere informazioni circa la sommatoria tempo-dipendente della DRP. </li><li> Passare la registrazione e il sito di stimolazione per ulteriori registrazioni controlaterale. </li><li> Animali non sono destinati a sopravvivere alla procedura. Sacrifica animali dopo l&#39;ultima registrazione per decapitazione, mentre ancora sotto anestesia profonda (ketamina / xylazina, vedi sopra, passo 2.2). </li></ol> 4. Analisi dei dati <ol><li> Trasferire i dati al programma di analisi (ad esempio Sigma Plot, Igor Pro, o MATLAB). </li><li> Calcola tracce medi from 20-30 spazza. </li><li> Prendere la massima ampiezza della deflessione tensione (rispetto al basale; DRP ampiezza di picco) per ulteriore analisi (Figura 3). </li><li> Calcolare il rapporto tra l&#39;ampiezza picco DRP dopo tre impulsi e del singolo impulso per ottenere una misura della sommatoria dipendente dal tempo di potenziali successive. </li></ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+inhibition+of+the+spinal+monosynaptic+reflex+pathway.">Presynaptic inhibition of the spinal monosynaptic reflex pathway.</a> J. Physiol. 161, 282-297 (1962).</li><li>Manjarrez, E., Rojas-Piloni, J. G., Jimenez, I., Rudomin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+synaptic+transmission+from+segmental+afferents+by+spontaneous+activity+of+dorsal+horn+spinal+neurones+in+the+cat.">Modulation of synaptic transmission from segmental afferents by spontaneous activity of dorsal horn spinal neurones in the cat.</a> J. 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Gli autori dichiarano interessi finanziari concorrenti.

Registrazioni-extra e intracellulari elettrofisiologici di attività neuronale e potenziali sinaptici in vivo sono allo stato dell&#39;arte delle tecniche di indagine delle funzioni neuronali del sistema nervoso centrale e fisiopatologia. Integrazione spinale è fondamentale per la funzione motoria, ad esempio, il movimento degli arti e per la percezione sensoriale multimodale. Inibizione presinaptica è un meccanismo fondamentale in questo processo computazionale garantire risposte adeguate a input se...

Inibizione presinaptica è uno dei più potenti meccanismi inibitori nel midollo spinale. Inibisce potenziali postsinaptici eccitatori (EPSPS) in motoneuroni monosinaptica eccitati senza modificare il potenziale di membrana post-sinaptica e l&#39;eccitabilità dei motoneuroni 1-3. Depolarizzazione afferenti primari (PAD) indotta da GABAergici sinapsi axo-assonale su fibre presinaptiche sensoriali è il meccanismo sottostante 4-7 (vedi anche Figure1a). Queste sinapsi contengono GABA A e GABA B recettori GABA A (R e GABA B R). Attività GABA A R porta ad un aumento della conduttanza cloruro che suscita PAD dovuto alla distribuzione ione locale. Questo blocca depolarizzazione la propagazione dei potenziali d&#39;azione nei terminali dell&#39;assone e riduce la loro forza con conseguente riduzione del Ca 2 +-afflusso e una riduzione del rilascio trasmettitore. Attivazione dei recettori GABA B non fat contribuiscono al PAD ma porta ad una riduzione di Ca 2 +-afflusso migliorando così inibizione presinaptica. Mentre l&#39;attivazione di GABA A R sembra essere coinvolto nella inibizione breve termine, GABA B R sono coinvolti nella modulazione a lungo termine 8-10. Oltre al GABA, che rappresenta la maggior parte del PAD e l&#39;inibizione presinaptica, altri sistemi di trasmettitori possono anche modulare e contribuire a questo meccanismo 11,12. Alterazioni patologiche inibizione presinaptica sembrano determinanti in diversi stati patologici esempio infiammazione periferica e dolore neuropatico 13,14, così come anormale trasformazione dolore centrale 15, lesioni del midollo spinale 16, e malattie del SNC con ipereccitabilità motore mediata da difettoso trasmissione GABAergica 17, 18. Pertanto, la stima inibizione presinaptica la pena di indagare sulle condizioni patologiche sperimentali a livello del midollo spinale in vivo </em&gt;. PAD dà luogo a potenziali volumi condotti forniscono una misura diretta della inibizione presinaptica nel midollo spinale. Tali potenzialità sono chiamati potenziali radici dorsali (DRP) e possono essere misurati dal spinale radice spinale dorsale dopo stimolazione delle radici dorsali adiacenti 7. Le prime misure di DRP sono stati riportati in gatti e rane 19 e sono state intensamente studiate nei gatti da Eccles, Schmidt e altri nei primi anni 1970 3,4,20,21. Mentre le registrazioni in vivo di DRP nei gatti 22 e 23 ratti sono stati ampiamente utilizzati, misurazioni nei topi sono stati condotti quasi esclusivamente in ex vivo isolato preparazioni midollo spinale 15,24. Qui, descriviamo un metodo per registrare DRP in topi anestetizzati in vivo permette una misura diretta di inibizione presinaptica nell&#39;organismo intatto.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

Inibizione presinaptica è uno dei più potenti meccanismi inibitori nel midollo spinale. Il meccanismo fisiologico alla base è una depolarizzazione delle fibre afferenti primarie mediate da GABAergici sinapsi axo-assonale (primaria depolarizzazione afferente). La forza di primaria depolarizzazione afferente può essere misurata mediante registrazione dei potenziali volumi condotto alla radice dorsale (potenziali radice dorsale, DRP). Le alterazioni patologiche di inibizione presinaptica sono cruciali nella elaborazione centrale anormale di alcune condizioni di dolore e in alcuni disturbi di ipereccitabilità del motore. Qui, si descrive un metodo di registrazione DRP in vivo nei topi. La preparazione del midollo spinale radici dorsali nella procedura di registrazione utilizzando elettrodi di aspirazione animale anestetizzato e sono spiegato. Questo metodo permette di misurare GABAergica DRP e quindi stimare inibizione presinaptica spinale nel topo vivente. In combinazione con modelli di topi transgenici, la registrazione DRP può serve come un potente strumento per studiare associata a malattia del midollo fisiopatologia. In vivo la registrazione ha diversi vantaggi rispetto alle ex vivo isolato preparazioni midollo spinale, ad esempio, la possibilità di registrazione simultanea o manipolazione di reti sopraspinali e induzione di DRP dalla stimolazione dei nervi periferici.

Tracce tipiche DRP sono mostrati in Figura 3. La stimolazione manufatto di rilievo è di solito seguita da una breve deviazione verso il basso. Successivamente una lenta, lunga durata deflessione verso l&#39;alto, che rappresenta il DRP è chiaramente distinguibile. In un sottogruppo di registrazioni, i riflessi spinali sono visibili come piccole punte sulla parte superiore del DRP. In normali topi wild-type, i riflessi spinali appaiono più spesso quando la tensione è eccessiva stimolazione. Come i ri...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergica inibizione presinaptica è un potente meccanismo inibitorio nel midollo spinale importante per il motore e l&#39;integrazione del segnale sensoriale in reti midollo spinale. Sottostante afferenti primari depolarizzazione può essere misurata mediante registrazione dei potenziali radice dorsale (DRP). Qui mostriamo un metodo di registrazione in vivo di DRP nei topi.

Misurare spinale Presinaptiche inibizione nei topi radice dorsale Recording potenziale<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

measuring-spinal-presynaptic-inhibition-mice-dorsal-root-potential

Research

JoVE Journal

Neuroscience

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

13.9K Views.  Jena University Hospital, Jena, Germany.  GABAergica inibizione presinaptica è un potente meccanismo inibitorio nel midollo spinale importante per il motore e l'integrazione del segnale sensoriale in reti midollo spinale. Sottostante afferenti primari depolarizzazione può essere misurata mediante registrazione dei potenziali radice dorsale (DRP). Qui mostriamo un metodo di registrazione in vivo di DRP nei topi. 

Video: Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Ricerca

Neuroscienze

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultaneo<em&gt; Ex vivo</em&gt; Test funzionale di due retine per<em&gt; In vivo</em&gt; Sistema Elettroretinogramma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Procedura di registrazione poligrafica per la misurazione del sonno nei topi

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Estrusione idraulica del midollo spinale e isolamento dei gangli dorsali nei roditori

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Radice dorsale Ganglion iniezione e dorsale radice Lesioni Crush come un modello per sensoriale Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomia per accesso del ganglio di radice dorsale lombare e l'iniezione nei suini

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Inquirenti rigenerazione dell'assone dei mammiferi: In Vivo elettroporazione del ganglio di radice dorsale del topo adulto

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Uso della registrazione in fibra singola in vivo e della radice dorsale intatta Ganglion con il nervo Sciatico attaccato per esaminare il meccanismo del fallimento della conduzione

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Isolamento rapido dei macrofagi di Ganglion dorsale

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...