Questo lavoro è stato sostenuto da SFB 604, DFG MO 1421/2-1 e Krebshilfe 107.089 (HM). AS è destinatario di un premio Young Investigator dalle Tumore dei Bambini Foundation (New York, USA).

Il presente studio è stato eseguito secondo la Protezione degli Animali legge della Repubblica Federale di Germania (Tierschutzgesetz der Bundesrepublik Deutschland) ed è stato approvato dall&#39;Ufficio di Stato della Turingia per la sicurezza alimentare e la tutela dei consumatori (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz). 1. Impostazione delle Misure <ol><li> Anestetizzare i topi da isoflurano / O 2 inalazione - per apertura di anestesia 3%, per manutenzione 2% isoflurano in ossigeno al 100% (Figura 1). Confermare anestesia sufficienti testando riflessi semplici come i riflessi e test di sensibilità per il dolore di basso grado del movimento. L&#39;uso della pomata sugli occhi per prevenire la secchezza durante l&#39;anestesia è consigliato ma non indispensabile in quanto la procedura richiede in genere solo 30 min / animale in totale. In caso di esperimenti di sopravvivenza, amministrare tempestiva più analgesici che agiscono per la gestione del dolore postoperatorio. </li><li> Accorciare il pelo che copre gli arti posteriori con un rasoio elettrico ed eseguire depilazione con una crema depilazione disponibili in commercio mentre gli animali sono già in analgesia. Al fine di mantenere lo status di libero-patogeno durante tutta la procedura, indossare guanti asettici e utilizzare sempre strumenti accuratamente puliti con il 70% di etanolo. </li><li> Corpo Controllo stabilità della temperatura da una piastra riscaldante controllato in retroazione e sonda termo rettale (Figura 2). Se necessario, si prega di utilizzare un telino sterile per coprire la piastra di riscaldamento tra gli animali al fine di mantenere un ambiente sperimentale sterile. Inoltre, si raccomanda di utilizzare una piastra di riscaldamento, che è controllata elettronicamente mediante un sensore integrato per limitare la temperatura di riscaldamento a ≤ 40 ° C per evitare danni ai tessuti. <img alt="Figura 1" fo:content-width="4in" fo:src="/files/ftp_upload/51181/51181fig1highres.jpg" src="/files/ftp_upload/51181/51181fig1.jpg" /> Figura 1. Expinstallazione erimental mostrando un mouse anestetizzato con gli arti posteriori rasati. </li><li> Prendere elettrocardiografia (ECG) registrazioni per monitorare la frequenza cardiaca come parametro vitale. Installare tre elettrodi per registrazioni ECG come segue: un elettrodo sotto la pelle di ogni zampa anteriore e un elettrodo sotto la pelle nella zona del collo. </li><li> Elettrodi luogo ad anello con gel di contatto per ottimizzare la resistenza conducibilità / trasferimento. L&#39;elettrodo di rivelazione (marcato in nero) è posto nella posizione in cui il muscolo gastrocnemio ha il suo diametro massimo. L&#39;elettrodo di riferimento (indicata in rosso) è posto appena sotto l&#39;elettrodo di rilevamento. Nota: Si prega di vedere la &#39;tavola Materiale&#39; per i dettagli apparecchiature. </li><li> Contrassegnare le posizioni corrette di misura con distanze predeterminate ma coerenti tra prossimale e distale stimolazione del nervo sciatico e l&#39;elettrodo di piombo. Proposta sperimentale: Ad una distanza di 4 mm dal elettrodo di rilevamento, la stimolazione distale avrà luogo.In una distanza di 16 mm e l&#39;elettrodo di rilevamento, la stimolazione prossimale sarà effettuata (Figura 2). </li></ol><img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig2highres.jpg" src="/files/ftp_upload/51181/51181fig2.jpg" /> Figura 2. Immagine rappresentativa che mostra la situazione sperimentale appena prima dell&#39;inizio delle misurazioni. La freccia bianca indica la posizione del sensore (nero) e di riferimento (rosso) elettrodo al muscolo gastrocnemio di arto posteriore sinistra. La stimolazione con elettrodi ad ago verrà effettuata in posizioni definite in relazione allo elettrodo di rilevamento nero. Il punto di stimolazione distale (segno nero con &quot;ds&quot; al posteriore arto sinistro) ha una distanza di 4 mm dal elettrodo di rilevamento; il luogo della stimolazione prossimale (segno nero con &quot;ps&quot;) dista 16 mm. La linea rossa sulla destra dell&#39;arto posteriore mostra la ca<html> iMate corso anatomica del nervo sciatico. Inoltre, le posizioni di massima dei relativi muscoli degli arti posteriori sono indicati come punti di riferimento. L&#39;asterisco indica la sonda termica rettale. 2. Misura <ol><li> Principio: Eseguire una serie di stimolazioni nervose con impulsi ad onda quadra singoli ripetutamente generati da 0,1 durata msec da monouso elettrodi ad ago 28 G monopolare (tasso di ripetizione di 200 msec; vedi Figura 3A). Per l&#39;analisi dei dati off-line, si consiglia di acquisire simultaneamente segnali di stimolazione insieme con la curva di risposta funzione neuromuscolare (Figura 3B) a causa di stimolazione del nervo. Media, una serie di risposte massime (&quot;rappresentante neuromuscolare risposta funzione&quot;) per la successiva analisi dei dati. Al fine di produrre dati affidabili, registrare e successivamente la media di almeno 3 indipendenti, curve di risposta ottimali per sito di stimolazione e animale. p_upload/51181/51181fig3highres.jpg &quot;src =&quot; / files/ftp_upload/51181/51181fig3.jpg &quot;/&gt; Figura 3. Procedura di acquisizione e analisi dei dati (presentazione schematica). Ripetutamente impulsi generati vengono applicati al nervo sciatico tramite elettrodi ad ago (riga superiore nella Figura 3A). Contemporaneamente, diversi corrispondenti curve di risposta neuromuscolare sono iscritte (riga inferiore in Figura 3A). Quando mediati e ingrandita, curve di risposta neuromuscolari dovute a impulsi di stimolazione (riga superiore nella Figura 3B) mostrano le seguenti proprietà caratteristiche (riga inferiore nella Figura 3B): latenza della risposta del segnale così come la durata e l&#39;ampiezza del segnale sono indicati e possono essere ottenuto per i calcoli successivi e statistiche. Conduzione del segnale irregolare e / o registrazioni non ottimali solito risultato in varie deformazioni segnale con più di uno positivo e uno negativo deflessione o deformazione del segnale(Forma bimodale) con ampiezza ridotta (Figura 3C). </li><li> Eseguire stimolazione prossimale con un elettrodo ad ago in posizione determinata (&quot;ps&quot; in Figura 2). <ol><li> Per conseguire migliori condizioni di registrazione con ampiezze massime, visualizzare le curve di risposta reali simultanee al processo di stimolazione. In questo modo, lo sperimentatore è in grado di valutare immediatamente la forma delle curve di risposta e la dimensione dell&#39;ampiezza. </li><li> Se necessario, leggermente e con attenzione manipolare la posizione e / o l&#39;angolo dell&#39;ago stimolazione rispetto al nervo sciatico. Questo dolce ottimizzazione delle condizioni di stimolazione consente di raggiungere ampiezze costanti con il valore massimo possibile e una curva di risposta di tipica forma bifasico (Figura 4). </li></ol></li><li> Eseguire stimolazione distale con un elettrodo ad ago in posizione determinata (&quot;ds&quot; in Figura 2). </li><li> Dopo completion di misura, di trasferire il mouse testato per una gabbia separata fino a quando non ha ripreso conoscenza sufficiente per mantenere decubito sternale. Non lasciare un animale incustodito e in compagnia di altri animali fino a quando non si è completamente ripreso dall&#39;anestesia. Somministrare tempestivamente più analgesici che agiscono per la gestione del dolore postoperatorio. La somministrazione sistemica di farmaci anti-infiammatori non steroidei (FANS) e gli oppioidi sono raccomandati per 1-3 giorni. </li><li> In alternativa, sacrificare il mouse ancora anestetizzato in modo rapido e umano senza alcun ulteriore dolore per l&#39;animale, ad esempio dislocazione del collo. </li></ol><img alt="Figura 4" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig4highres.jpg" src="/files/ftp_upload/51181/51181fig4.jpg" /> Figura 4. Illustrativa per determinare le registrazioni CMAP con ampiezze massime. Una serie completa di registrazione è presentata. (A </strong&gt;) Punto di inserimento con la minima risposta CMAP. (B) i risultati di circolazione stimolazione Lieve aghi nelle registrazioni CMAP con ampiezze massime. (C) Altre modifiche di posizionamento dell&#39;ago producono registrazioni CMAP con diverse ampiezze comprese vicino-massimo ampiezze. (D) La stimolazione sostituzione dell&#39;ago con le registrazioni di serie CMAP di ampiezze quasi massimo. Nota: decremento Tipica in ampiezze CMAP può verificarsi durante la stimolazione ripetitiva ottimale del sito di stimolazione 12, 13. Gli asterischi indicano le registrazioni CMAP con ampiezze massime rappresentati per la media. 3. Analisi <ol><li> Estrarre i parametri di conduzione nervosa basate sul rappresentante set di dati di risposta funzione neuromuscolare utilizzando un pacchetto software appropriato (ad es AtisaPro). Nota: Si prega di gestire la determinazione dei dati in funzione del tempo con particolare attenzione perché la determinazione del punto di flesso del composto pentola azione motoriaziale (CMAP) insorgenza e la cessazione può essere difficile. Una procedura con la riproducibilità verificato è dato in figura 3B, in cui vengono utilizzati tangenti su deviazioni di segnale dopo l&#39;esordio e prima di terminazione. </li><li> Calcola &#39;latenza del segnale&#39; e di valori &#39;CMAP&#39;. <ol><li> Latency &#39;rappresenta il ritardo di tempo tra stimolo e CMAP esordio, mentre l&#39;intervallo di tempo tra la comparsa dei CAMP iniziale deflessione negativa per siglare il ritorno ai valori di base si chiama&#39; durata CMAP &#39;. Utilizzare la differenza tra latenze distali e prossimali di calcolare la velocità di conduzione insieme alla distanza tra i siti di stimolazione distale e prossimale. velocità di conduzione = latenza / distanza tra i siti di stimolazione </li><li> CMAP &#39;(composto potenziale d&#39;azione del motore) ampiezza rappresenta la grandezza tra i punti positivi e negativi massimo turnaround del segnale CMAP (dato in mV). CMAP = valore &#39;punto di svolta positivo&#39; - valore &#39;negativo po turnaroundint &#39; </li></ol></li></ol>

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Il protocollo descritto fornisce un metodo semplice e affidabile per determinare nervo sciatico proprietà di conduzione su topi anestetizzati, senza la necessità di esporre il nervo di interesse. Tuttavia, questa procedura sperimentale provoca lesioni dei tessuti da puntura. E &#39;quindi una scelta ragionevole sacrificare gli animali dopo aver terminato le registrazioni. Tuttavia, rispetto ad altre procedure più invasive, che richiedono l&#39;esposizione del nervo prima registrazioni, danno tissutale è relativament...

Misure elettrofisiologiche sono uno strumento indispensabile per indagare l&#39;integrità funzionale dei nervi periferici in ambienti clinici e di laboratorio. Negli esseri umani, un gran numero di disturbi neuromuscolari e neuropatie diagnostico si basa su misure elettrofisiologiche. Misurando proprietà nervose come velocità di conduzione o potenziali ampiezze del segnale, è possibile caratterizzare l&#39;origine approssimativa delle malattie nervose periferiche. La velocità di conduzione nervosa è altamente dipendente rapida propagazione del segnale attivata da mielinizzazione. Pertanto, i processi demielinizzanti in genere mostrano una diminuita velocità di conduzione 4. Il potenziale d&#39;azione composto di motore (CMAP) - la correlazione con il numero di assoni funzionali - è un indicatore di danno assonale, quando ha ridotto significativamente 5. Così, mediante metodi elettrofisiologici eziologia di danni ai nervi perifericipossono essere discriminati, come ad neuropatie ereditarie 6,7, 8,9, neuropatia diabetica cronica polineuropatia infiammatoria demielinizzante (CIDP) 10, o neuropatie metaboliche 11. Normalmente, nella domanda umana sono preferiti registrazioni non invasive sul nervo surale o ulnare. Nei topi, è semplice da analizzare le proprietà nervosi del nervo sciatico, il nervo più grande del sistema nervoso periferico (PNS) contenente sia grande - e assoni di piccolo calibro del sistema motorio e sensoriale. La procedura come dimostrato qui è un metodo rapido, semplice e affidabile per misurare tutti i valori forfettari rilevanti per elettrofisiologia sui nervi periferici nel topo intatto. Prendendo registrazioni da un organismo conservato, condizioni fisiologiche dell&#39;ambiente nervo sono garantite.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Concentric Needle Electrodes (Stimulation)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td style="width:119px;">9013S0901</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital Ring Electrodes (Recording)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td>9013S0302</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ToM - Tower of Measurement (A/D converter)</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AtisaPro, Data acquisition &#38; analysis software</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HSE-Stimulator T</td>
			<td colspan="2">Hugo Sachs Elektronik, Hugstetten, Germany</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurements on mouse sciatic nerves

Studi elettrofisiologici consentono una classificazione razionale delle varie malattie neuromuscolari e sono di aiuto, insieme alle tecniche neuropatologiche, nella comprensione della fisiopatologia 1. Qui si descrive un metodo per eseguire studi elettrofisiologici sul mouse del nervo sciatico in vivo. Gli animali vengono anestetizzati con isoflurano per garantire analgesia per i topi testati e indisturbato ambiente di lavoro durante le misurazioni che prendono circa 30 min / animale. Una temperatura costante di 37 ° C corpo è mantenuto da una piastra riscaldante e continuamente misurata da una sonda rettale termo 2. Inoltre, un elettrocardiogramma (ECG) viene normalmente registrato durante le misurazioni al fine di monitorare costantemente lo stato fisiologico degli animali studiati. Registrazioni elettrofisiologiche vengono eseguite sul nervo sciatico, il nervo più grande del secoloe del sistema nervoso periferico (PNS), fornendo il mouse arto posteriore con tratti di fibre sia motorie e sensoriali. Nel nostro protocollo, nervo sciatico rimanere in situ e, pertanto, non devono essere estratto o esposto, consentendo misurazioni senza alcuna irritazioni nervose avverse con registrazioni effettive. Utilizzando opportuni elettrodi ad ago 3 svolgiamo sia prossimale e stimolazioni nervose distali, registrando i potenziali trasmessi con elettrodi di rilevamento a muscoli gastrocnemio. Dopo l&#39;elaborazione dei dati, i valori molto consistenti per la velocità di conduzione nervosa (NCV) e il composto potenziale d&#39;azione del motore (CMAP) affidabile e, i parametri chiave per la quantificazione del funzionamento dei nervi periferici lordo, possono essere raggiunti.

<html> Abbiamo condotto una serie di misurazioni elettrofisiologiche in vivo sul nervo sciatico di 12 topi in totale per questo studio: 6 animali di ogni genere. Le misurazioni sono state effettuate con il protocollo presentato e consegnato i seguenti risultati: Entrambi i topi maschi e femmine mostrano una velocità di conduzione del nervo sciatico medio di circa 20 m / sec (Figura 5). Questo è coerente con altre misurazioni in letteratura. Inoltre, dimostra che no...

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves at JoVE.com

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Le misurazioni del nervo proprietà di conduzione in vivo esemplificano un potente strumento per caratterizzare i vari modelli animali di malattie neuromuscolari. Qui, presentiamo un protocollo semplice e affidabile con cui può essere eseguita l&#39;analisi elettrofisiologica sul nervo sciatico di topi anestetizzati.

In Misure Vivo elettrofisiologici sui nervi mouse sciatico

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

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In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

33.1K Views.  Fritz Lipmann Institute.  Le misurazioni del nervo proprietà di conduzione in vivo esemplificano un potente strumento per caratterizzare i vari modelli animali di malattie neuromuscolari. Qui, presentiamo un protocollo semplice e affidabile con cui può essere eseguita l'analisi elettrofisiologica sul nervo sciatico di topi anestetizzati. 

Video: In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Le misurazioni elettrofisiologiche da un sistema olfattivo Moth

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

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Neuroscienze

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 ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Le misurazioni elettrofisiologiche e Analisi di nocicezione nei neonati umani

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimaging funzionale con l&#39;impiego dell&#39;ecografia emato-encefalica e la barriera di Turbativa del Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo a due fotoni di imaging dei cambi molecolari esperienza-dipendenti nei neuroni corticali

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Gene Transfer di cellule di Schwann nel nervo sciatico roditori da elettroporazione

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Analisi delle cellule immuni in singolo nervo sciatico e del ganglio della radice dorsale da un singolo Mouse mediante citometria a flusso

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

In Vivo Elettrofisiologici misurazione del potenziale di azione muscolare composto da arti anteriori in modelli murini di degenerazione del motoneurone

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Nel calcio in Vivo Imaging delle cellule ciliate linea laterale in Zebrafish larvale

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...