We wish to thank Mr Wabbel for preparing the wires and Mr Tietsch for constructing the plugs and cages according to our plans. This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 688). Felix Fluri holds a fellowship of the Interdisziplin&#228;re Zentrum f&#252;r Klinische Forschung (IZKF), University Clinics W&#252;rzburg, Germany.

As experiências com animais foram aprovados pela Universidade de Würzburg e as autoridades estaduais legais (Lower Franconia, número de aprovação: 54-2531.01-102 / 13) e realizada de acordo com as recomendações para a investigação em curso experimental estuda 17 ea corrente de Pesquisa Animal: Relatório de Em Diretrizes experimentos in vivo (http://www.nc3rs.org.uk/arrive-guidelines). 1. Anestesia <ol><li> Verificar o sistema de anestésico para assegurar quantidades adequadas de abastecimento de gás (oxigénio) e isoflurano para a duração do processo. Conecte o nosecone com a barra incisivo do instrumento estereotáxico e colocar o bar incisivo em -3.3 mm. </li><li> Ligar o fornecimento de gás (2 L / min). Coloque o rato em uma caixa e selar o topo. Ligue o vaporizador isoflurano a 3,5%. </li><li> Quando o rato é reclinada, comutar o sistema de modo que o gás flui para o anestésico cone de protecção, que está fixado à barra de incisivo. </li><li> Remover o rat da câmara de caixa e raspar a área entre as orelhas e os olhos; usando um cotonete embebido com Jodosept PVP, limpe a área raspada para remover qualquer cabelo solto. </li><li> Colocar o rato no nariz cónico (Figura 1) e continuar a anestesia com isoflurano 2,5% em O 2 (1 L / min). Verifique o nível de anestesia por beliscar a área interdigital. Se o rato é anestesiado adequadamente, os reflexos defensivos são suprimidos (isto é, retirada do pé). </li><li> Monitorar a respiração e a resposta à estimulação durante o procedimento e ajustar o vaporizador conforme necessário. </li><li> Aplicar veterinário pomada nos olhos para evitar a secura sob anestesia. Monitorizar e manter a temperatura corporal a 37 ± 0,5 ° C por um sistema de aquecimento controlado por realimentação. </li></ol> 2. Cirurgia <ol><li> Mantenha o campo cirúrgico esterilizado durante toda a cirurgia. Uma vez que as mãos do cirurgião são estéreis eo campo operacional é estéril, mover apenas carefully e lembre-se para não quebrar a esterilidade. Isso inclui ter também um campo estéril (ou seja, cortinas impermeáveis ​​estéreis), no qual se pode definir para baixo instrumentos. </li><li> Injectar 0,2 mL de mepivacaína subcutaneamente no centro da área rapada. Mepivacaína é um anestésico local, que tem uma duração de acção de até 3 horas. Ele vai anestesiar ainda mais a área cirúrgica. </li><li> Utilizando um bisturi, fazer uma incisão na linha média a partir entre as orelhas e estendendo-se para 2 cm. Assegurar que o periósteo (membrana brilhante sob a pele) também é incisado. Expor o crânio com quatro grampos (Figura 2). </li><li> Usando um cotonete, remova cuidadosamente o periósteo, até as suturas coronal e sagital são expostos; depois disso, estancar o sangue com lã de algodão. </li><li> Determinar as coordenadas da bregma, utilizando uma agulha fixada num suporte da sonda, e em seguida, marcar a ponta da agulha com uma caneta de feltro preto. Utilizando o anterior / posterior (AP), da linha média / lateral,(ML) e dorsoventrais parafusos (DV) da unidade, posicione a ponta da agulha diretamente sobre o bregma. </li><li> Aqui o AP e ML leituras escala vernier: subtrair 3.6 mm a partir da leitura do AP e 2,5 mm a partir da leitura mL para implantação eléctrodo na STN direito, ou adicionar 2.5 mm para implantação eléctrodo na STN esquerda. Esta posição será marcada pelo corante da caneta com ponta de feltro após a diminuição da ponta da agulha para a superfície do crânio. </li><li> Prenda a broca de dentista para o titular grande sonda do instrumento estereotáxico. Mova a broca de dentista para a área calculada - ou seja, o ponto marcado no crânio. Olhando através do microscópio, um furo (diâmetro cerca de 1 mm) através do crânio até que a dura-máter é visível (o crânio é de cerca de 1 mm de espessura). Retrair a dura usando uma pinça micro-dissecação ou uma agulha estéril. A dura-máter é resistente o suficiente para destruir a ponta do eletrodo. </li><li> Perfurar um furo com a broca de dentista em cada escama frontal, e no interparietal oposto squama para o buraco eletrodo. Desligue o suporte da sonda do instrumento estereotáxico. Não faça uma sutura no crânio como vasos venosos siga as suturas sob o crânio. </li><li> Parafuso de uma parafuso de osso em cada um dos cinco furos. Evite enfiar os parafusos muito profundo. Para parafusos de aço inoxidável (M1.6), 2-3 voltas do parafuso vai realizar adequadamente o parafuso sem colocar pressão sobre o cérebro. O número de espiras vai depender do passo do parafuso. Prender o suporte da sonda com o eléctrodo na micromanipulador (Figura 3). </li><li> Utilizando os parafusos de accionamento AP, ML e DV, mover o suporte de sonda com o eléctrodo até que a ponta está quase a tocar a bregma. Observe as leituras de escala vernier AP, ML e DV no bregma. Quando as leituras são feitas, elevar o eléctrodo de alguns milímetros para impedir o eléctrodo de raspagem do crânio durante o movimento. Para determinar as coordenadas da posição em que o eléctrodo tem de ser inserido empara o buraco, adicionar 3,6 mm para a leitura AP e adicionar (ou subtrair) 2,5 mm para a leitura ML. </li><li> Usando os parafusos AP e ML unidade, mova o eletrodo para a posição calculada. Neste ponto, a ponta do eléctrodo deve estar situado directamente sobre o orifício perfurado eléctrodo. Em seguida, olhando através do microscópio, diminuir o eléctrodo ao nível da dura-máter (Figura 4). Este nível serve como nível zero na direção DV. Em seguida, inserir suavemente a ponta do eléctrodo no cérebro, olhando através do microscópio. </li><li> Ligue o pino de eléctrodo para a peça de ligação do sistema de gravação. Coloque uma gaiola de Faraday (ou substitui-la com uma folha de alumínio) sobre o rato do instrumento estereotáxico na (Figura 5). Aterrar o instrumento estereotáxico com o contrapeso da sala que está sendo trabalhado em. </li><li> Inicie o sistema de gravação. Se disponível, use também um alto-falante para obter um sinal acústico de descargas / salves de unidades individuais durante advancing do eletrodo. </li><li> Insira lentamente o eletrodo dentro do cérebro, registrando a atividade elétrica durante o avanço do eletrodo. A uma profundidade de entre 7,5 e 8,1 mm a partir da dura-máter, a actividade eléctrica específica do STN é geralmente detectável (Figura 6). A atividade típica de neurônios no STN é caracterizado por um padrão de disparo irregular e uma taxa de disparo de alta (média de frequência: 40,9 ± 12,9 Hz) 18. </li><li> Durante a gravação, a anestesia reduzir, tanto quanto possível (por exemplo, de 0,8-1,0%); animais de baixa anestesiados mostram uma atividade cerebral elétrica mais clara. </li><li> Pincelar fora qualquer sangue ou fluido cerebrospinal que foi deslocado para a superfície do crânio durante a descida do eléctrodo. </li><li> Misturar-se uma pequena quantidade de cimento dentário e aplicá-lo em torno do eléctrodo e cerca de quatro dos cinco parafusos usando uma pequena espátula (Figura 7). O quinto parafuso será usado para fixar o fio de aterramento do plugue. </li><li> Desligue o eléctrodo a partir do pino de suporte do eléctrodo e do conector do sistema de gravação, quando o cimento dental é fixo. </li><li> Solte o parafuso que não foi fixado pelo cimento dental. Coloque a ficha sobre o pino do eléctrodo. Fixe o fio de aterramento do plugue com o quinto parafuso (Figura 8). </li><li> Misture cimento dental e aplicá-lo ao redor da vela. Como o cimento engrossa, moldá-lo ao redor da vela para formar um tampão. Evitar arestas vivas do cimento dental que pode prejudicar o animal e removê-los durante o endurecimento (Figura 9A e B). </li><li> Debride bordas da ferida e fechá-los com um fio de sutura na frente e por trás da tampa. Desinfectar as bordas da ferida. </li><li> Ligue a ficha de cabeça para o fio que é fixo em um giro. Remover o rato do instrumento estereotáxico. </li><li> Aplicar tramadol (12,5 mg / kg, por via intraperitoneal) no final da intervenção e, em seguida, uma vez por dia durante 2-3 dias. Coloque o rato em uma gaiola limpa com thermal apoio, corrigir o giro sobre esta gaiola (Figura 10) e inspecioná-lo cuidadosamente durante 1 h. </li><li> Não deixe um animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. Não devolva um animal que tenha sido submetido a cirurgia para a companhia de outros animais até que totalmente recuperado. </li></ol> 3. Estimulação <ol><li> Determinar a resistência no animal antes da estimulação usando um medidor de impedância. </li><li> Ligue a ficha da peça giratória com um arame e as fichas na outra extremidade do fio com a corrente de saída e a saída para o fio de terra do estimulador. Ligue o estimulador com um computador a fim de programar o estimulador. </li><li> Escolha os parâmetros de estimulação no programa; por exemplo, os parâmetros usados ​​na doença de Parkinson são o comprimento do pulso: 60 ms; freqüência: 130 Hz. Estimular o rato com um aumento da amplitude da corrente até discinesia são reconhecidos. Reduzir the intensidade elétrica em 10-20% abaixo da intensidade que provocou discinesia ou até sinais neurológicos desaparecem eo animal está confortável. Pulsos rectangulares monofásicas foram utilizados neste estudo. </li><li> Depois de concluir o experimento, a eutanásia do animal com isoflurano: Ajustar o fluxo isofurane ou concentração de 5% ou mais. Continue exposição isoflurano até 1 min após a respiração pára. </li></ol>

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The authors declare that they have no competing financial interests.

<html> Este estudo apresenta um conjunto passo-a-passo de instruções para a implantação de um eletrodo monopolar crônico na STN de ratos. Apesar de eléctrodos de tungsténio com baixa impedância são frequentemente utilizados para DBS 18,19, um eletrodo monopolar de platina / irídio (Pt / Ir) foi empregada que tinha uma impedância de cerca de 1 MQ. Eléctrodos de Pt / Ir são também utilizados em doentes com doença de Parkinson devido às suas propriedades favoráveis: eles demonstram erosão mínim...

A estimulação cerebral profunda (DBS) é um tratamento baseado na entrega de impulsos elétricos através de eletrodos implantados nas estruturas cerebrais específicas, tais como o globo pálido interno Globus 1, o núcleo subtalâmico (STN) 2-4 ou o tálamo ventral intermediário 5. Nas últimas duas décadas, este tratamento foi estabelecida como uma poderosa ferramenta terapêutica para a doença de Parkinson 1 - 4, distonia 6 e tremor 7, e também é usado para modular a dor crónica 7, distúrbios psiquiátricos (isto é, o transtorno obsessivo-compulsivo 8, depressão maior 9) ou epilepsia refratária 10,11. Além disso, DBS pode, no futuro, tornar-se uma opção de tratamento para a hipertensão arterial refratária 12 ou hipotensão ortostática 13. Os mecanismos fisiológicos subjacentes aos efeitosde DBS permanecem pouco compreendidos. Estudos em roedores anestesiados forneceram uma visão sobre respostas neurais a estimulação de alta frequência que imitam clinicamente aplicado DBS 14. No entanto, estes estudos não só falta comprovação do efeito comportamental DBS, mas também resultar em variabilidade considerável dependendo dos parâmetros de estimulação aplicado 14. Para investigar de forma mais concisa os efeitos comportamentais e os mecanismos subjacentes da DBS em roedores conscientes, uma estimulação set-up é necessário que preencha os requisitos específicos. DBS é usado principalmente como uma terapia a longo prazo (por exemplo, doença de Parkinson, a dor crónica). Assim, a estimulação set-up em roedores deve ser concebido de modo a que a unidade é constituída por um eléctrodo com um tampão, bem como um fio a partir da ficha para um estimulador externo; e esta unidade deve ser leve, mas inquebrável quando fixada no crânio. Além disso, a liberdade de circulação é indispensável para ratos durante stimulação durante um período prolongado. As estruturas alvo de DBS são pequenos; por exemplo, a STN em ratos tem um comprimento de 1,2 mm e um volume de 0,8 mm 3,15. Portanto, os eléctrodos devem ser concebidos de tal modo que o núcleo está não lesionado durante a inserção e necessidades direccionamento para ser mais preciso. Como a maioria dos estudos realizados em roedores DBS ter usado marco baseado estereotáxica de inserção do eléctrodo para a estrutura de alvo, a taxa de erro pode ser relativamente alta, mesmo quando se utiliza as coordenadas de acordo com Paxinos e Watson 16. Isso resulta em um maior número de animais necessários para chegar a um resultado estatisticamente significativo. No presente estudo, de uma técnica de implantação de eléctrodo é introduzido, o que tem como alvo STN com alta precisão utilizando um sistema de microrecording durante o avanço do eléctrodo. Além disso, um sistema de estimulação que é apresentada não só permitir um elevado grau de mobilidade para o animal, mas também garante estimulada stimulati contínuana via fixação segura do fio de estimulação (que é protegida por uma mola de aço inoxidável) no cabeçote do rato.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pt/Ir electrode</td>
			<td>FHC Inc.</td>
			<td>UE</td>
			<td>Custom-made: Specification: UEPSEGSECN1M</td>
		</tr>
		<tr>
			<td>Plugs</td>
			<td>GT Labortechnik (Arnstein/Germany)</td>
			<td></td>
			<td>Custom-made</td>
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			<td>Pin header</td>
			<td>DISTRELEC</td>
			<td>143-95-324</td>
			<td>single-row, 90&#176; 1x3 datamate, Type M80-8420342</td>
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			<td>Socket</td>
			<td>DISTRELEC</td>
			<td>143-95-621</td>
			<td>single-row,straight 2 mm pole no.1x3 datamate, Type M80-8400342</td>
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			<td>Stainless steel spring</td>
			<td>Plastics ONE</td>
			<td>SS0102</td>
			<td>Part-#: .120 X .156 Spring ID (mm): 3.0&#160; Spring OD (mm): 4.0</td>
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			<td>Dental cement/Paladur</td>
			<td>Heraeus Kulzer</td>
			<td>64707938</td>
			<td>Liquid, 500 ml</td>
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			<td>Dental cement/Paladur</td>
			<td>Heraeus Kulzer</td>
			<td>64707954</td>
			<td>Powder, rose, 500g</td>
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			<td>Head screw</td>
			<td>Hummer &#38; Reiss</td>
			<td>V2ADIN84 M1.6x3</td>
			<td></td>
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			<td>Jodosept PVP</td>
			<td>Vetoquinol</td>
			<td></td>
			<td>435678/E04</td>
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			<td>Mepivacain 1%</td>
			<td>AstraZeneca</td>
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			<td>PZN03338515</td>
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			<td>Epinephrine</td>
			<td>Sanofi-Aventis</td>
			<td></td>
			<td>PZN00176118</td>
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			<td>Tramadolhydrochloride</td>
			<td>Rotexmedica</td>
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			<td>38449.00.00</td>
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microelectrode guided implantation of electrodes into the subthalamic nucleus of rats for long-term deep brain stimulation

A estimulação cerebral profunda (DBS) é uma terapia eficaz e amplamente utilizado para várias perturbações neurológicas, tais como doença, distonia ou tremor de Parkinson idiopática. DBS é baseada na entrega de estímulos eléctricos a estruturas anatómicas específicas profundos do sistema nervoso central. No entanto, os mecanismos subjacentes ao efeito de DBS permanecem enigmáticos. Isto levou a um interesse na investigação do impacto de DBS em modelos animais, especialmente em ratos. Como DBS é uma terapia de longo prazo, a investigação deve ser centrada em mudanças genético-moleculares dos circuitos neurais que ocorrem várias semanas após o DBS. A longo prazo em ratos DBS é um desafio porque os ratos mover nas suas gaiolas, o que causa problemas em manter em lugar de fios que conduz a partir da cabeça do animal para o estimulador. Além disso, as estruturas alvo para a estimulação do cérebro do rato são pequenas e, portanto, os eléctrodos não podem ser facilmente colocados na posição desejada. Assim, um set-up para a estimulação de longa duraçãoção de ratos por meio de eletrodos de platina / irídio com uma impedância de cerca de 1 MQ foi desenvolvido para este estudo. Um eletrodo com estas especificações permite não só a estimulação adequada, mas também a gravação de estruturas cerebrais profundas para identificar a área alvo para DBS. No nosso set-up, um eletrodo com um conector para o cabo foi incorporado no cimento dental com quatro parafusos de fixação garantidos para o crânio. O fio da tomada ao estimulador foi protegido por uma mola de aço inoxidável. Um giratória foi ligado ao circuito para impedir que o fio fique emaranhada. No geral, esta estimulação set-up oferece um elevado grau de mobilidade livre para o rato e permite que o bujão de cabeça, bem como a ligação do fio entre o bujão e o estimulador, para reter a força de longa duração.

Implantação de um eletrodo na STN de um rato usando um sistema de gravação - como aqui apresentados - é um procedimento eficaz e preciso para DBS que demora cerca de 1 hora por animal. Este modelo é um procedimento bastante menor: de 10 ratos submetidos à cirurgia, todos sobreviveram a intervenção. Vinte e quatro horas após a intervenção, o estado de cada rato foi monitorizada e nenhum animal conseguiu mais do que 1 de 3 pontos de acordo com o código de gravidade. Durante o período de estimulação contín...

Watch this Scientific Journal Video about Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation at JoVE.com

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Microeletrodos guiada implantação de eletrodos no subtalâmica Núcleo de Ratos por longo prazo Estimulação Profunda do Cérebro

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

microelectrode-guided-implantation-electrodes-into-subthalamic

Research

JoVE Journal

Neuroscience

19.4K visualizações.  University Hospital Wuerzburg. A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Vídeo: Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Usando o protocolo Theta-Burst para Explorar Mechasnism de Plasticidade em Indivíduos com Síndrome do X Frágil e Autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Neurociência

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

O dispositivo NeuroStar TMS: Conduzindo a FDA Aprovado protocolo para o tratamento da depressão

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

A polido e reforçado Janela afinado crânio para longo prazo de imagens do cérebro de camundongo

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Implantação de fibra óptica para Estimulação Crônica optogenética do Tecido Cerebral

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

Estimulação Cerebral Profunda com fMRI simultâneo em Roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Análise das Alterações Gene expressão no Rat Hippocampus Após Estimulação Cerebral Profunda do Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Cânula de implantação para a Cisterna Magna de roedores

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Crônica implantação de matriz Electrocorticographic todo-cortical no sagui comum

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Avaliação da indução de depressão em longo prazo em fatias Cerebelares adultas

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Preparação de Eletrodos de Estimulação Nervosa Periférica para Implantação Crônica em Ratos

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...