The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

Questo metodo richiede l&#39;utilizzo di soggetti animali, consentite dalla Laval University comitato etico degli animali (protocollo # 2012-170). 1. Impostazione e preparazione <ol><li> Soluzioni <ol><li> Preparare soluzioni aCSF secondo le seguenti ricette 7,17. Altre ricette con variazioni di concentrazione sono disponibili in letteratura. Soluzioni memoria Stock a 4 ° C per un massimo di un mese. <ol><li> Soluzione salina: aggiungere 75,39 g di NaCl (129 mm finale); 2,5 g di KCl (3,35 mM finale); 0,81 g di NaH2PO4 (0,58 mM finale); 2,33 g di MgCl2 (1,15 mM finale); 1,85 g di CaCl2 (1,26 mM). Sciogliere in 800 ml di acqua distillata poi riempire a 1 L con acqua distillata. </li><li> Soluzione di bicarbonato: aggiungere 17,65 g di NaHCO 3 (21 mm finale). Sciogliere in 900 ml di acqua distillata poi riempire a 1 L con acqua distillata. Variazioni della concentrazione di bicarbonato provocheranno variazioni di pH. </li><li> Soluzione di glucosio: aggiungi 54.06g di glucosio (30 mM finale). Sciogliere in 400 ml di acqua distillata poi riempire a 500 ml con acqua distillata. </li></ol></li><li> Preparare aCSF diluendo 100 ml di soluzione salina, 100 ml di soluzione di bicarbonato, e 50 ml di soluzione di glucosio in 1 L di acqua distillata. Conservare a 4 ° C fino al momento dell&#39;uso. </li><li> Preparare un elettrodo di vetro riscaldando e si estende un tubo di vetro fino a rompersi. Sand giù la punta prima dell&#39;uso. L&#39;elettrodo può essere riutilizzato molte volte finché rimane pulito. </li></ol></li><li> Setup sperimentale Nota: I dati di set-up sono presentati nella figura 2. <ol><li> Accendere l&#39;amplificatore, averager, sistema di acquisizione dati, controllo della temperatura e della pompa in movimento. Controllare l&#39;amplificazione del segnale (guadagno = 10.000), la filtrazione (soglia bassa, 10 Hz; soglia alta, 5 kHz), e la frequenza di campionamento della conversione da analogico a digitale del segnale grezzo (2,5 kHz). </li><li> Riempire una bottiglia con aCSF e bolla con CarboGen (95% O 2 </sub&gt;, 5% di CO 2). Inducono una bubbled- e riscaldato aCSF flusso nella camera di registrazione (volume 5 ml) a 4 ml / min. Mantenere la temperatura nella camera di registrazione a 26 (± 1) ° C. pH deve essere 7,4 in queste condizioni standard. </li><li> Conservare 50 ml di RT e CarboGen-bolle aCSF in una siringa da 50 ml in prossimità della camera di dissezione. </li><li> Accendere il computer e avviare il software di registrazione. </li></ol></li></ol> 2. Dissection <ol><li> Pesare e determinare visivamente il sesso dell&#39;animale (i maschi hanno un ano-genitale distanza più lunga, mentre le femmine hanno un breve tratto ano-genitale, i maschi genitali sono spesso oscuro mentre genitali femminili sono di colore rosa). </li><li> Anestetizzare i roditori neonati da una delle seguenti opzioni: cryoanesthesia (immergere completamente l&#39;animale in ghiaccio per 4 - 5 min 18), iniezione (equitensine a 4 ml / kg) 19 o inalazione di anestetici volatili (isoflurano 20 o etere 21). Confirm un piano adeguata di anestetici dall&#39;assenza di un ritiro riflesso zampa. </li><li> Posto l&#39;animale in panchina, faccia ventrale giù. Sezione della parte rostrale della testa coronale con un bisturi a livello di bregma (visibile attraverso la pelle e il cranio). Eseguire questo passaggio subito dopo che l&#39;animale è anestetizzato. </li><li> Sezione coronale il corpo con un bisturi sotto i membri anteriori. </li><li> Togliere la pelle, i muscoli, tessuti grassi e viscere con le forbici chirurgiche e sottili e pinze. Dal momento che le ossa sono morbidi a questa età, essere prudenti per non danneggiare il sistema nervoso. Eseguire questo passaggio in panchina. </li><li> Posizionare la preparazione nella camera dissezione. Utilizzare il aCSF memorizzato nella siringa per ossigenare preparazione. Da questo punto alla fine della registrazione, utilizzare un microscopio. </li><li> Sulla faccia dorsale del preparato, tagliare il cranio e vertebre dal rostrale alla parte caudale lungo l&#39;asse medio con forbici chirurgiche e sottili e pinze. Aprire il cranio taglioe vertebre, al fine di esporre il tessuto nervoso. Utilizzare il aCSF strored nella siringa per ossigenare la preparazione. </li><li> Con le pinze, rimuovere la membrana aracnoidea, un sottile tessuto che ricopre la superficie del tessuto nervoso. Conservare la pia madre e vasi sanguigni contro il tessuto nervoso. Utilizzare il aCSF memorizzato nella siringa per ossigenare preparazione. </li><li> Posizionare la preparazione con il dorso rivolto verso il basso, e con attenzione abbassare il tronco encefalico e del midollo spinale tagliando i nervi e tessuto connettivo con le forbici mentre si tiene la preparazione sul posto. Un approccio dorsale è anche possibile. Mantenere le radici e nervi più a lungo possibile. Togliere le ossa per isolare il tronco encefalico e del midollo spinale. Utilizzare il aCSF memorizzato nella siringa per ossigenare preparazione. </li><li> Rimuovere il cervelletto e rimanendo strutture rostrali da loro sezionamento con il bisturi. Utilizzare il aCSF memorizzato nel ago per ossigenare preparazione. </li><li> Opzionalmente seconda sperimenprogettazione tal, rimuovere il ponte con il bisturi da una sezione anteriore coronale alla inferiore arteria cerebellare 22. Notare che mantenendo tutta ponte rallenterà il ritmo in ratti e completamente inibirla in topi. Preparazioni che includono solo la parte caudale del ponte mostrano una attività respiratoria simile con una frequenza stabile alla radice C4. </li></ol> 3. Registrazione <ol><li> Mettere il preparato in camera di registrazione, ventrale a faccia in su. Fissare la preparazione con perni nella parte più bassa del midollo spinale e rostrale-maggior parte del tronco cerebrale. </li><li> Utilizzando una siringa collegata all&#39;elettrodo dal suo ago, indurre una depressione nell&#39;elettrodo (diametro punta di vetro: 150 - 225 micron) tirando il pistone della siringa, per riempire parzialmente l&#39;elettrodo con aCSF. </li><li> Utilizzando un micromanipolatore, posizionare accuratamente l&#39;elettrodo vicino ad una delle quarte radici ventrali. Altre radici ventrali potrebbero anche presentare una delle vie respiratorie come l&#39;attività (e.g., XII nervo cranico, C1 radice ventrale). </li><li> Inducono una depressione nel serbatoio dell&#39;elettrodo tramite la siringa estraendo lo stantuffo per aspirare delicatamente il rootlet nervo. Quindi spostare accuratamente l&#39;elettrodo di applicare contro il midollo spinale. Nota: Idealmente la dimensione del rootlet corrisponde alla dimensione dell&#39;apertura elettrodo e crea una tenuta tra i compartimenti interni ed esterni dell&#39;elettrodo così. Poiché gli amplificatori differenziali sono comunemente utilizzati per tali registrazioni, ogni apertura tra l&#39;interno dell&#39;elettrodo e la camera di registrazione riduce la qualità del segnale e rende difficile per eliminare il rumore di fondo. </li><li> Avviare la registrazione. </li><li> Registra il ritmo prodotto dalla preparazione in condizioni di normossia (cioè, aCSF gorgogliare con CarboGen: 95% O 2 e 5% CO 2) per almeno 20 minuti per determinare i parametri di base della preparazione. </li><li> Passare la perfusione da CarboGen-bolle aCSF astimolo aCSF (cioè, con il 95% gorgogliare N 2 e 5% di CO 2 per ipossia stimolo) per 15 min. Alter durata dell&#39;esposizione a seconda del protocollo sperimentale. Registrare la durata dell&#39;esposizione sulla registrazione degli animali e scheda tecnica. Utilizzare tubi in acciaio inossidabile tra il flacone e la camera aCSF quando possibile per evitare la diffusione di gas verso l&#39;esterno del tubo. </li><li> Accendere la perfusione torna a aCSF CarboGen-bolle standard per almeno 15 minuti per una registrazione di recupero. Registrare questo sulla registrazione degli animali e scheda tecnica. </li><li> Terminare la registrazione. </li></ol> 4. Analisi statistica <ol><li> Dal segnale integrato, calcolare la frequenza del numero di burst per minuto (espressa in burst / min), l&#39;ampiezza come differenza tra la linea di base e il picco del burst (espressa in mV), la durata di scoppio della durata da l&#39;inizio e la fine del burst (espresso in s), l&#39;area di scoppio come l&#39;area sotto la curve del burst sul segnale integrato (espressa in mV · s) (Figura 3). </li><li> Calcolare la frequenza, ampiezza, durata scoppiare e zona scoppiare sotto normossia (baseline), ipossia (stimolo) e post-ipossia condizioni (post-stimolo) di recupero come la media degli ultimi 5 minuti delle registrazioni per ogni condizione. Non analizzare la prima porzione di ciascuna condizione perché c&#39;è un ritardo tra l&#39;accensione e perfusione aCSF omogeneizzazione nella camera di registrazione. </li><li> Express poi stimolo (cioè, ipossia) e post-stimolo (cioè, dopo ipossia) valori di recupero come percentuale dei valori di base per la registrazione corrispondente. Esprimere i risultati come media ± SD </li></ol>

<ol>
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The authors have no competing financial interests or conflicts of interests to disclose.

Quantificazione accurata dell&#39;attività respiratoria può essere impegnativo. Infatti, la respirazione è una funzione che può essere sia automatica e volontaria, e che è modulata in base all&#39;ambiente, le esigenze del corpo, lo stato emotivo ed il comportamento. Il vantaggio di questa tecnica è l&#39;isolamento degli elementi neurali responsabili della produzione del comando respiratoria. Così, registrazioni elettrofisiologiche di preparati midollo spinale e tronco-pletismografia sono tecniche complementari ...

La respirazione è un complesso e vitale attività controllata dal cervello, permettendo diossigeno (O 2) e l&#39;assorbimento di anidride carbonica (CO 2) eliminazione. Il drive respiratorio centrale è generato da una complessa rete situata nel tronco cerebrale in entrambi i mammiferi 1, 2 anfibi, rettili, uccelli 3 4 5 e pesci. Anche se lo studio di respirazione può essere elaborata in vivo, precise indagini meccanicistiche richiedono l&#39;accesso diretto alla rete di controllo respiratorio. A tal fine, Adrian e Buytendijk sviluppato una ridotta preparazione pesci rossi, in cui elettrodi posti sulla superficie del tronco cerebrale record ritmo generato associato ventilazione gill 5. Questo approccio è stato in seguito adattato da Suzue nel 1984 6 per l&#39;impiego nei roditori appena nati. L&#39;avvento di questa preparazione ha portato a significativi progressi nella neurobiologia respiratoria. Poiché è relativamente semplice, la tecnica presentato here è suscettibile di una vasta gamma di ricerche di base di comportamenti motori ritmici e le loro origini nei roditori appena nati. L&#39;obiettivo generale di questo metodo è quello di registrare il correlato neurale di attività inspiratoria, un ritmo respiratorio simile chiamato respirazione fittizia, prodotta dalla rete respiratoria. Questo metodo può essere impiegato in una vasta gamma di obiettivi di ricerca, puntando risposte inspiratori alle variazioni respiratorie o farmacologia in entrambi i selvatici tipo 7 e 8 animali transgenici. Dato che gli esperimenti vengono eseguiti a bassa temperatura, senza afferenti sensoriali, e in condizioni in cui le concentrazioni di glucosio e O 2 ai aCSF sono alti, questioni sono state sollevate per quanto riguarda la rilevanza fisiologica del segnale registrato. Mentre vi sono chiare differenze tra in vivo e in vitro condizioni (ad es., La frequenza di raffiche inspiratori) resta il fatto che la presenza digli elementi fondamentali della rete respiratoria 6 permettono di studiare un ritmo robusto associato ad una funzione omeostatica vitale 9,10. La logica dietro lo sviluppo e l&#39;utilizzo di questa tecnica è quello di facilitare l&#39;accesso diretto agli elementi del tronco della rete respiratoria, che sono difficilmente accessibili in vivo, soprattutto nei neonati. Il tronco encefalico è posto in condizioni rigorosamente controllate: il ritmo registrato non è modulato da input afferenti periferici dai polmoni o gli organismi carotidei, permettendo lo studio di concentrarsi sul drive respiratorio centrale stessa 11. Così, questo accesso è utilizzato per applicare stimoli e registrare il segnale di uscita. In contrasto con pletismografia registrazioni, il ritmo respiratorio è modulata da tutti i suoi componenti in tutto il corpo (ad es., Distensione polmonare, chemosensori periferiche), rendendo difficile applicare stimoli precisi. In unratto ewborn, il protocollo consiste nel registrare il quarto segnale ventrale root su un tronco isolato e midollo spinale troncato, mantenuto in fluido cerebrospinale artificiale (aCSF). Il ritmo generato dai preparativi del midollo spinale tronco-è composto da singole raffiche lenti che sono collegati al segnale inspiratorio 9. Isolato preparativi del midollo spinale tronco-sono facilmente registrabili in ratti da post-natale giorno 0-4 (P0 - P4) 7. Questo approccio è comunemente utilizzato per valutare la risposta ipossica della rete respiratoria, e anche la risposta ai ipercapnia, acidosi o droghe. Un protocollo acuta all&#39;ipossia è presentato qui. Questa stimolazione è ottenuto dal prelievo di O 2 nel aCSF; questo approccio è comunemente utilizzato per valutare la tolleranza e la risposta agli insulti ipossici. Il protocollo induce una depressione ritmo dal primo minuto fino alla fine dell&#39;esposizione ipossia (Figura 1) 12. Questa depressione è invertitadurante la post-ipossica 12 recupero. Per quanto riguarda il disegno sperimentale, è importante notare che il ponte, si trovano nella parte rostrale del tronco cerebrale, ha un&#39;azione inibitoria sul generatore ritmo 8. Così, preparazioni di piena del tronco encefalico e del midollo spinale rostrale mostrano un ritmo inferiore. L&#39;inclusione del ponte del campione isolata per la registrazione è determinato secondo il fine dell&#39;esperimento 13; studio dell&#39;influenza pontino sulla rete midollo allungato richiederebbe registrazioni con e senza il ponte di confrontare i risultati 14. Inoltre, uno dei vantaggi di questa tecnica è la possibilità di estendere la parte rostrale della preparazione per includere mesencefalico e / o regioni diencefaliche 15,16, che consenta di valutare l&#39;effetto di queste regioni della rete respiratoria ponto-midollare.

<table><tbody><tr><td>Sylgard</td><td>Sigma Aldrich</td><td>761036-5EA</td><td>Use under hood</td></tr><tr><td>NaCl</td><td>Bioshop</td><td>SOD002</td><td></td></tr><tr><td>KCl</td><td>Bioshop</td><td>POC888</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>CCL444</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>MAG510</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Bioshop</td><td>SPM306</td><td></td></tr><tr><td>D-glucose</td><td>Bioshop</td><td>GLU501</td><td></td></tr><tr><td>Carbogen</td><td>Linde</td><td>343-02-0006&amp;nbsp;</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments, Hamden, CT, USA</td><td>TC-324B</td><td></td></tr><tr><td>Suction electrode</td><td>A-M Systems, Everett, WA, USA</td><td>model 573000</td><td></td></tr><tr><td>Differential AC amplifier</td><td>A-M Systems, Everett, WA, USA</td><td>model 1700</td><td></td></tr><tr><td>Moving averager</td><td>CWE, Ardmore, PA, USA</td><td>model MA-821</td><td></td></tr><tr><td>Data acquisition system</td><td>Dataq Instruments, Akron, OH, USA</td><td>model DI-720</td><td></td></tr><tr><td>LabChart software</td><td>ADInstruments, Colorado Springs, CO, USA</td><td></td><td></td></tr><tr><td>Prism sofware</td><td>Graphpad, La Jolla, CA, USA</td><td></td><td></td></tr><tr><td>Dissection chamber</td><td>Plastic box (e.g. petri box) will do</td><td></td><td></td></tr><tr><td>Recording chamber</td><td>Home made</td><td></td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Micromanipulator</td><td>World Precision Instrument Inc, Sarasota, FL, USA</td><td>KITE-R</td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Peristaltic pump</td><td>Gilson, Middleton, WI, USA</td><td>MINIPULS 3</td><td></td></tr><tr><td>Faraday Cage</td><td>Home made</td><td></td><td></td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr></tbody></table>

electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

Come accennato nell&#39;introduzione, uno dei vantaggi più importanti di questa tecnica è l&#39;accesso diretto al tronco cerebrale applicare vari stimoli. Come esempio, ipossia stato applicato qui. Figura 1. AB mostra una registrazione completa protocollo, con entrambe le condizioni normossia e ipossia. Figura 1.CE visualizza il ritmo registrata in condizioni di normossia (cioè, aCSF gorgogliare con 95% O 2 e 5% CO 2 a 26 ° C). Come già illustrato in...

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Elettrofisiologia su isolati tronco cerebrale, midollo spinale Ammassi di Newborn Roditore Consente Neural Network respiratoria registrazione Uscita

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

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Research

JoVE Journal

Neuroscience

8.5K Views.  Laval University. The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Video: Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Elettrofisiologia II: extracellulare fabbricazione di elettrodi di aspirazione

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Ricerca

Neuroscienze

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord Elettrofisiologia

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Biologia

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

L&#39;induzione di uno Stato Isoelectric cervello per studiare l&#39;impatto di endogeno Synaptic attività su NEURONALE<em&gt; In Vivo</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

Registrazione di EEG nei ratti neonatali liberamente in movimento utilizzando un metodo novello

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

acuto<em&gt; In Vivo</em&gt; Registrazioni elettrofisiologiche dei potenziali del campo locale e dell&#39;attività multi-unità dal percorso iperdirezionale nei ratti anestetizzati

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Registrazioni simultanee di potenziali di campo locale corticale, elettrocardiogramma, elettromiogramma e respirazione ritmo da un ratto liberamente commovente

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Preparazione di ritmicamente-attivo In Vitro neonatale del roditore del tronco cerebrale-spinale e fetta sottile

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Acquisizione e analisi dei dati nel cervello evocato Audiometria di risposta nei topi

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

In Vivo Registrazione intracellulare di Motoneuroni Spinali Ratti Identificati dal Tipo durante la stimolazione a corrente diretta trans-spinale

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

<meta charset="utf8"></head><body>Registrazioni multielettrodo in silicio angolato per strutture profonde del tronco encefalico nei topi

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

<meta charset="utf8"></head><body>Autore in primo piano: Studio delle vie del sonno indotte dall'anestesia e dell'eccitabilità neuronale nei topi

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Registrazioni acute multi-elettrodo a unità singola dal tronco encefalico di topi fissati alla testa

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

Elettrofisiologia su isolati tronco cerebrale, midollo spinale Ammassi di Newborn Roditore Consente Neural Network respiratoria registrazione Uscita

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