Nós estamos gratos ao Robert Graffin e Pauline Duhant pelo apoio técnico.

O protocolo experimental foi realizado em conformidade com as directivas do Conselho das Comunidades Europeias para o experimento Animal (2010/63/UE, 86/609/CEE e 87-848/CEE) e foi aprovado pelo Animal ética Comissão de Universidade de Namur (ética projeto n ° 17-284 ). A Figura 1 descreve as duas abordagens respectivas: transdiaphragmatic ou injeções transtorácicos. Usar camundongos C57bl/6J machos (n = 18), a idade de 3 a 4 meses no estudo.
1. preparação da solução do CTB
<ol><li>Para injeções de transdiaphragmatic:<ol>
<li>Dissolva o poder do CTB em água estéril na concentração de 0,2% (p/v).</li>
		<li>Carrega 7,5 µ l da solução CTB (0,2% p/v) em um estéril 10-µ l-microseringa com uma agulha de calibre 33 anexada (chanfro sem corte ou curto) para cada rato.</li>
	</ol>
</li>
	<li>Para injeção transtorácica:<ol>
<li>Dissolva o poder do CTB em água estéril na concentração de 0,1% (p/v).</li>
		<li>Carrega 20 µ l de solução CTB (0,1% w/v) em uma seringa estéril de insulina-500-µ l com uma agulha de calibre 27 chanfrada para cada rato. Nota: Certifique-se de usar água destilada e esterilizada e dissolver adequadamente o pó do CTB. A solução pode ser mantida a 4 ° C por um mês (não congelar). Um precipitado pode formar após alguns dias. Trazer a solução à temperatura ambiente e misture bem com uma pipeta antes do uso.</li>
	</ol>
</li>
</ol>2. preparação antes da Intrapleural injeções
<ol><li>Esterilize instrumentos cirúrgicos antes da cirurgia, utilizando o esterilizador autoclave ou grânulo de vidro. Prepare um casaco de banco limpo para fazer a cirurgia e para dispor os instrumentos cirúrgicos. Nota: Qualquer material utilizado durante o procedimento cirúrgico deve ser estéril. Instrumentos que não podem ser esterilizados, tais como o microseringa deve ser limpo com um desinfectante (Clorexidina) ou deve ser descartáveis apenas. O cirurgião deve lavar suas mãos com um desinfectante (esfoliante de clorexidina) antes do início da cirurgia. O cirurgião deve vestir um vestido limpo, uma máscara e luvas estéreis.</li>
	<li>Pesar o animal e administrar uma dose apropriada de anestesia: injeção intraperitoneal de anestésico cocktail (por exemplo, a cetamina 100mg/kg e xilazina 5 mg/kg).</li>
	<li>Aperte o dedo do pé e/ou verificar se há a perda do reflexo palpebral para determinar se o mouse está devidamente anestesiado. Aplica uma pomada de vet para proteger a córnea.</li>
	<li>Raspe com cuidado a pele ventral (para o procedimento de transdiaphragmatic) ou a lado direito torácica pele (para procedimento transtorácica), usando o cortador de cabelo elétrico. Raspe bem e suficientemente ampla para evitar cabelo entrando no campo da cirurgia.</li>
	<li>Assegurar condições assépticas por aplicação tópica de solução de iodo 10% sobre a área depilada. Esfregue o local cirúrgico com a solução de iodo, tendo o cuidado de esfregar no centro do local em direção a periferia.</li>
	<li>Use uma almofada homeotérmicos durante a cirurgia para manter a temperatura do corpo do animal.</li>
</ol>3. Intrapleural injeções usando a abordagem de Transdiaphragmatic
<ol><li>Coloque o mouse anestesiado em posição supina sobre uma almofada de aquecimento. Coloque uma gaze enrolada sob o pescoço do animal. Para um rato adulto, use uma gaze enrolada de 0,5 polegada de espessura. Fita esta almofada ao Conselho de cirurgia para evitar o movimento, se houver.</li>
	<li>Usando uma lâmina de bisturi, faça uma incisão na pele ventral ao longo da linha mediana: fazer uma incisão do processo xifoide à região umbilical ao esticar a pele lateralmente com a outra mão para fazer a pele esticada. Não exerça demasiada pressão sobre a lâmina para não danificar os órgãos subjacentes.</li>
	<li>Usando tesouras pequenas, cuidadosamente retire a pele dos músculos ao redor da incisão abdominais. Este procedimento ajudará na costura dos músculos e a pele separada no final da cirurgia.</li>
	<li>Realize laparotomia usando tesouras pequenas. Abra a cavidade abdominal através da realização de uma incisão de caseado ao nível do umbigo. Faça uma incisão ao longo da linha branca, os músculos abdominais ("linea alba") até o processo xifoide.</li>
	<li>A fim de visualizar a superfície abdominal, do diafragma, retrai os músculos abdominais, usando afastadores comerciais ou caseiros. Nota: Estes retractores podem ser feitos de clipes de papel de tamanho midi moldadas em L-gancho19.</li>
	<li>Estique-se ambos os lados da laparotomia e fita para baixo os afastadores para revestir o banco. Otimize a iluminação do campo de visão para que a superfície abdominal, do diafragma é não mais na penumbra. O mais poderoso dispositivo de iluminação para esta finalidade é uma lâmpada de LED com um feixe de luz orientável.</li>
	<li>Usando uma pinça macia em uma mão, levante o apêndice xyphoid e puxar para baixo os lóbulos laterais do fígado (Figura 2A). Usando tesouras pequenas na outra mão, cuidadosamente corte o ligamento suspensor, sem danificar a vesícula biliar ou do diafragma (Figura 2B).</li>
	<li>Usando uma pinça macia em uma mão, levante o apêndice xyphoid. Segure a seringa Hamilton na outra mão. Alvo de três locais de injecção no diafragma para cobrir respectivamente o esterno, a medial e as regiões crural (Figura 3A, inserir-se na Figura 3B).</li>
	<li>Como a espessura do diafragma é em torno de 0,37 mm em ratos7, insira a agulha não mais de 1 ou 2 mm além da folha de diafragma para impedir os danos de pulmão durante a respiração. Entrega 2,5 µ l de solução CTB (0,2% p/v) através do diafragma direito em cada local (Figura 4B). Estabilização do diafragma não é necessária.</li>
	<li>Repita este procedimento para os três sites ipsilaterais da injeção (figura 1A) e contralaterally para uma rotulagem bilateral da piscina PhMN.</li>
	<li>Para fechar o site de laparotomia, suture os músculos abdominais com reabsorvíveis sutura 4-0. Uso interrompidos pontos espaçados de 3 mm. pele de grampo fechado com grampos de 9,0 mm ferida. Aperte os grampos para impedir que o animal retirar agrafos. Espaço grampeia separado aproximadamente 5 mm. Não aperte os clipes de ferida, como isso pode levar a cicatrização prejudicada.</li>
	<li>Limpa micro seringa com água destilada para evitar o entupimento. Lentamente, elaborar e expulsar 2 - 3 vezes. Não extrair o ar na seringa.</li>
</ol>4. Intrapleural injeção usando abordagem transtorácica
Nota: Este método é inspirado o procedimento descrito em ratos4 e foi adaptado ao rato.
<ol><li>Coloque o mouse anestesiado em esquerda posição decúbito lateral, sobre uma almofada de aquecimento (Figura 4A).</li>
	<li>Identifica as sexta e sétima costelas sob a prega do cotovelo através da palpação manual (Figura 4B).</li>
	<li>Enquanto o assistente se estende direito dianteiro - e -membros posteriores (Figura 5A), inserir a seringa orientada cranialmente e tangencialmente sob a sexta ou sétima vértebra, 3 mm de profundidade da pele, com o bisel para baixo (figura 1B e Figura 5B).</li>
	<li>Eleve a agulha suavemente para confirmar, levantando as costelas, se ele está bem posicionado dentro da cavidade torácica (Figura 5, as inserções na figura 1B).</li>
	<li>Estabilize a respiração movimentos do tórax, aplicando leve pressão com dois dedos na parede torácica.</li>
	<li>Mantendo a seringa na outra mão, entrega 20 µ l de solução CTB (0,1% w/v) em uma única injeção.</li>
</ol>5. no pós-operatório cuidados
<ol><li>Imediatamente após o procedimento, coloque o mouse na posição de decúbito lateral direita sobre uma almofada homeotérmicos para recuperação. Isto assegura o tracer para espalhar no lado direito da cavidade pleural e permite o monitoramento de respiração do animal.</li>
	<li>1 mL de solução salina estéril i.p. Administre por via subcutânea. Fornece acompanhamento injeções se o animal for exibida, desidratados e/ou apático.</li>
	<li>Administrar por via subcutânea 0,1 mg/kg de buprenorfina, duas vezes por dia durante o primeiro dois dias depois da cirurgia, para minimizar qualquer dor potencial. Nota: Muitas instituições recomendaria analgesia multimodal de procedimentos invasivos, tais como a laparotomia retratado aqui. Isto pode incluir a utilização de um não-esteroides anti-inflamatórios (AINEs) e/ou de um agente anestésico local no local da incisão. Esses medicamentos normalmente são entregues antes da primeira incisão para minimizar a dor de dar corda.</li>
	<li>Monitorar os animais diariamente até a eutanásia.</li>
</ol>

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Os autores não têm nada para divulgar.

O protocolo descrito neste documento pode ser aplicado a qualquer tipo de ratos adultos ou qualquer paradigma experimental em que a integridade do diafragma-PhMN circuito deve ser avaliada. Por exemplo, a esclerose lateral amiotrófica (ela) e lesão medular cervical (cSCI) são condições associadas com perda de PhMN, anterógrada degeneração dos axônios frênico e subsequente compromisso respiratório. Modelos animais de ALS ou cSCI imitam histopatológicos e funcionais respiratórios déficits observados em doenç...

O objetivo do estudo é apresentar um método confiável para identificar os neurônios motores frênico (PhMN) em seções de rato da medula espinhal. Injeção de um traçador fluorescente neuroanatômica na cavidade pleural foi escolhida como o método de entrega para alcançar as projeções frênica neuromusculares sobre o diafragma e usar o transporte retrógrado ao longo dos axônios frênico para rótulo frênico corpos celulares. Duas técnicas de entrega intrapleural são descritas: transdiaphragmatic versus transtorácica.
Frênico neurônios são células de relé espinhal cujos axônios convergem em nervos frênico, que em última análise, inervam o diafragma. Estes são os neurônios motores inferiores recebendo o drive inspiratório do centro respiratório bulbar e retransmitindo-as junções de diafragma neuro-muscular (JNM). PhMN estão estruturados em duas colunas de motor, uma para cada hemicord, correndo ao longo da espinha meados-cervical. Na maioria das espécies mamíferas, incluindo os seres humanos, as colunas de motor frênica espalhar de níveis C3 a C61,2,3. Nós e os outros confirmaram que PhMN concentrada nos níveis C3-C5 no rato e o rato da medula espinhal4,5,6,7,8. A distribuição topográfica das células frênica não é ao acaso; os neurônios motores que inervam a parte esternal do diafragma são mais densamente distribuídos na parte cranial da frota de veículos frênica (C3), Considerando que os neurônios motores que inervam a parte crural são mais caudal (C5)9. Além disso, PhMN estão agrupadas vària no Corno ventral cinzenta. A nível de C3, os clusters de células frênico minto lateralmente, então eles se deslocar em uma direção ventrolateral e encontram-se ventromedially com a mais caudal níveis10,11.
Dado o seu papel vital durante a inspiração, é de extrema importância para identificar com precisão a PhMN na medula espinhal saudável mas também seguir seu destino durante condições patológicas, tais como doenças degenerativas ou lesões traumáticas da medula espinhal. Desde que PhMN não diferem morfologicamente outros neurônios motor cervicais, identificação de PhMN baseia-se na entrega alvo dos marcadores neuroanatômica, tanto ao nível dos centros respiratórios primária8, ou diafragma MNJ7 ou em o nervo frênico4. O tracer é retomado pelas fibras nervosas e transportado até os frênico corpos celulares na coluna cervical, onde ele pode ser visualizado usando sistemas de deteção direta ou indireta. Retrógrada ou anterógrada traçadores são comercialmente disponíveis com uma ampla gama de conjugados. Notável, cada marcador é dotado não, baixas ou altas habilidades para rastreamento sináptica trans.
No estudo atual, nós escolhemos a subunidade beta a toxina da cólera (CTB) acrescida com Alexa Fluor 555 (doravante referido como CTB-fluoróforo) como uma etiqueta fluorescente, permitindo uma visualização directa de PhMN em seções congeladas da medula espinhal. CTB normalmente é descrito como um tracer monosynaptic embora dados experimentais tendem a mostrar uma passagem de transneuronal12. CTB tem a capacidade de vincular o gangliosídeo GM1 na membrana plasmática do fim do nervo. CTB é internalizada através de Clatrina dependente ou - independente mecanismos e tráfegos através da rede trans-Golgi em retículo endoplasmático em uma moda retrógrada13,14. A internalização e o transporte retrógrado parecem ser dependente sobre o citoesqueleto de actina15,16 , bem como sobre a rede de microtúbulos17.
Para demonstrar a utilidade do CTB como um traçador neuroanatômica retrógrada rotulagem diafragma-PhMN circuitos, CTB-fluoróforo foi entregue intrapleurally. CTB foi administrado usando duas técnicas: a primeira que incluía uma laparotomia e múltiplas injecções de transdiaphragmatic; o segundo, menos invasiva, usado uma única injeção transtorácica. Quatro dias mais tarde, fluorescente-etiquetadas PhMNs foram quantificados na medula espinhal cervical de ambos de saudável e de feridas spinally (C4) animais.

<table>
	<tbody>
		<tr>
			<td>Glass-bead sterilizer Steri 250</td>
			<td>Keller</td>
			<td>31-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td>F.S.T.</td>
			<td>14058-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft tweezers</td>
			<td>F.S.T.</td>
			<td>11042-08</td>
			<td></td>
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retrograde neuroanatomical tracing of phrenic motor neurons in mice

Frênico neurônios motores são cervicais neurônios motores provenientes de C3 a C6 níveis em espécies de mamíferos mais. Axonal projeções convergem em nervos frênico que inervam o diafragma respiratório. Em fatias de medula espinhal, neurônios frênico não pode ser identificados de outros neurônios motores em critérios morfológicos ou bioquímicos. Nós fornecemos a descrição dos procedimentos para a visualização do neurônio motor frênico corpos celulares em camundongos, seguir intrapleural injeções de beta cólera toxina subunidade (CTB) conjugada com um fluoróforo. Este marcador fluorescente neuroanatômica tem a capacidade de ser pego na junção neuromuscular de diafragma, efectuar-se retrogradely ao longo dos axônios frênico e alcançar os frênico corpos celulares. Duas abordagens metodológicas da entrega CTB intrapleural são comparadas: transdiaphragmatic versus transtorácicos injeções. Ambas as abordagens são bem sucedidas e resultam em número semelhante de CTB-rotulado frênico neurônios motores. Em conclusão, essas técnicas podem ser aplicadas para visualizar ou quantificar os neurônios motores frênico em vários estudos experimentais tais como aqueles focados no circuito frênico-diafragma.

Camundongos C57bl/6J machos (n = 18), com idade de 3 a 4 meses foram incluídos no estudo. No dia 0 do experimento, 8 ratos sofreram uma contusão unilateral do C4, lado direito, de acordo com o protocolo publicado7,18. Como procedimento de Souza, 10 ratos foi submetido a uma laminectomia em cima C4 sem contusão. No dia 3, os ratos estavam preparados para as injeções intrapleural do CTB-fluoróforo de acordo com os dois procedi...

Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Aqui, descrevemos um protocolo para identificar frênico neurônios em ratos após entrega intrapleural de fluoróforo conjugado beta de subunidade de toxina de cólera. Duas técnicas são comparadas para injetar a cavidade pleural: transdiaphragmatic versus transtorácicos abordagens.

Rastreamento neuroanatômica retrógrado do frênico neurônios em ratos

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

retrograde-neuroanatomical-tracing-of-phrenic-motor-neurons-in-mice

Research

JoVE Journal

Neuroscience

12.6K visualizações.  Université de Namur. Aqui, descrevemos um protocolo para identificar frênico neurônios em ratos após entrega intrapleural de fluoróforo conjugado beta de subunidade de toxina de cólera. Duas técnicas são comparadas para injetar a cavidade pleural: transdiaphragmatic versus transtorácicos abordagens.

Vídeo: Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

Avaliação do Equilíbrio e Coordenação Motor em camundongos usando o feixe de equilíbrio

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

Neurociência

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Medindo Coordenação Motora em Ratos

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

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.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<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 ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracelularmente Identificar os neurônios motores de um pool do motor muscular em<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Condicional Genetic transsináptica Tracing no Embryonic do cérebro do rato

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

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

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

Funcional e morfológica de Avaliação de Diafragma Innervation por frênico Neurônios Motores

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

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Neuronal Tracing em explantes cérebro guiadas a laser

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Visualização do Padrão de Projeção Axonal de Neurônios de Motor Embrionários em<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Na Vivo Imagem de dois fotões de neurônios Cortical em camundongos Neonatais

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Rastreamento neuroanatômica retrógrado do frênico neurônios em ratos

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