Este trabalho foi financiado pelo NIH / NINDS conceder 4R00 NS 057940, e SCBI conceder 200-12 da medula espinhal e financiar Brain Research Lesão do Estado de Indiana Department of Health.

1. Fazendo um dispositivo simples para a cirurgia undercut <ol><li> O dispositivo undercut criamos consiste em três partes (Fig. 1): (1) uma placa de suporte de aço inoxidável ou de plástico que permite a conexão de um tubo guia e uma agulha, e se senta do outro lado da janela craniana durante a cirurgia (2); um tubo de guia que contém uma agulha em posição e permite a rotação da agulha, e (3) uma agulha que é dobrado 90 graus em ~ 3 mm da ponta, e é móvel por rotação e de inserção. </li><li> Corte um pedaço de 1-1,5 mm de espessura, 7 ~ 10 x 30 mm de aço inoxidável ou retangular de plástico transparente (1) para fazer a placa de suporte. Prepare um de 1,5 polegadas calibre 22 (BD empresa, # 305156) e um de 1,5 polegadas de calibre 25 (BD empresa, # 305127) agulha da seringa. Para fazer um tubo-guia, corte a extremidade de plástico e na ponta inferior da agulha da agulha da seringa de calibre 22 para que seu comprimento total é de cerca de 31-32 mm (2). Areia ao fim de metal para torná-la plana e lisa. O comprimento final desse tubo orientador deve ser de cerca de 5-6 mm mais curta do que a agulha da seringa de calibre 25. </li><li> Use cola de cianoacrilato para fixar o tubo guiando para a placa de suporte, certificando-se que a agulha é perpendicular à borda do metal. </li><li> Inserir a agulha da seringa de calibre 25 dentro do tubo-guia, e dobrar a agulha a 90 graus em 2,5-3 mm da ponta (Fig. 1). </li><li> Ajustar o intervalo vertical em movimento da agulha colando um pequeno tubo de plástico para a extremidade da agulha para cima (Fig. 1, pare de agulha). A faixa final, movimento da agulha determina o quão profundo a agulha pode ser inserida no córtex, e deve ser 1,6-1,8 mm para P21 ratos e 1,3-1,5 mm para P21 camundongos. Este comprimento precisa ser ajustado para diferentes espécies e idades dos animais. </li><li> Anexar um fio de cobre pequeno ou um pequeno pedaço de fita adesiva sobre a extremidade superior da agulha na mesma direção como a ponta dobrada (Fig.1 indicador de agulha). O fio ou fita indica o ângulo de viragem da agulha durante a cirurgia undercut (Fig. 1). </li></ol> 2. Preparação de animais <ol><li> Todos os instrumentos cirúrgicos precisam ser autoclavado, esterilizado com um esterilizador de bolas de gude, ou desinfetados com álcool 70%. </li><li> Usamos Sprague / Dawley em idade pós-natal 20-22 dias (P20-22) ou CD1 ratos da mesma idade. Diferentes linhagens de camundongos ou ratos pode ser usado para projetos específicos. </li><li> Anestesiar o rato com uma injeção ip de cetamina 80 mg / kg e xilazina 8 mg / kg. O procedimento cirúrgico começa depois que o animal não responde a pitada cauda. Durante a cirurgia, se necessário, uma dose de reforço de um terço da dose original do cocktail anestésico pode ser dada para restaurar o estado anestésico original. </li><li> Cortar o cabelo no couro cabeludo do animal com um aparador de pêlos elétrico. Aplique uma pequena quantidade de pomada oftálmica para os olhos do animal para proteção contra o ressecamento durante a anestesia. Desinfectar o couro cabeludo usando a 10% de solução de iodo-povidona, seguido por etanol 70%. </li><li> Montar o animal em um aparelho estereotáxico para manter a cabeça em uma posição estável fixo. Durante toda a cirurgia, nós manter o animal em uma almofada de aquecimento para evitar a hipotermia. </li><li> Faça uma incisão ântero-posterior da linha média no couro cabeludo utilizando um bisturi, que se estende desde a lambda entre os olhos. Use hemostats para puxar a pele para o lado e expor o crânio deixado suficientemente. </li><li> Faça um corte retangular na caveira da esquerda, e use o bisturi para raspar periósteo. Esta etapa irá reduzir o sangramento e facilitar a perfuração no crânio. </li></ol> 3. Fazendo undercuts <ol><li> Adicionar uma pequena quantidade de salina estéril para a área do crânio exposta, e, em seguida, usar vários Q-dicas para limpar o sangue e seque a área. </li><li> Sob um microscópio cirúrgico, iniciar a perfuração de um sulco retangular (~ 5 x 7 mm em ratos, 4 x 5 mm em camundongos) no centro do crânio esquerda (acima de aproximadamente esquerda córtex sensório-motor). Aplicação de uma gota de solução salina no crânio facilita a perfuração e calor se dissipa. Após cerca de 2 / 3 da profundidade do osso é perfurado, remova o excesso salina e limpe a área de perfuração com um Q-Tip. Lenta e cuidadosamente perfurar mais fundo até que a peça central do osso é móvel ao toque suave de uma pinça. </li><li> Remova cuidadosamente a peça central do osso através da inserção de uma ponta afiada de uma pinça na borda do osso e, lentamente, levantando a pinça para expor o hemisfério esquerdo. </li><li> Sob o microscópio cirúrgico, segure o dispositivo minar, e orientar a agulha em uma direção perpendicular parasagital ea placa de suporte à linha média, inclinação do aparelho ligeiramente caudalmente de forma a manter a visualização da ponta da agulha e região-alvo cortical. Com a ponta da agulha para uma área de 1-2 milímetros lateral à sutura sagital superior, no meio da janela do crânio, e evitar a penetração direta dos grandes vasos. </li><li> Inserir a agulha em um sentido horizontal através da dura-máter e sob a pia, de modo a poupar os vasos sanguíneos. Sente-se na parte inferior do dispositivo undercutem ambas as bordas da janela do crânio de modo que a agulha está posicionada perpendicular à superfície cortical (Fig. 1). Descansando o dispositivo nas bordas tanto da janela do crânio com a mão irá reduzir significativamente ou eliminar a mão trêmula durante o procedimento a seguir. Levante a agulha debaixo da pia e desça lentamente para criar um corte transcortical até que a agulha não pode ir mais fundo. ~ Girar 135 ° de distância da linha média para criar uma questão semi-círculo branco / camada profunda VI undercut. Em seguida, levante a agulha novamente para debaixo da pia. Incline o dispositivo para trás e retire a agulha. </li><li> Opcionalmente, pode-se repetir o procedimento acima (passo 2.5), mas sem virar a agulha de forma a criar um corte adicional transcortical nas bordas laterais da janela. Isso irá criar um isolamento mais completo cortical. </li><li> Coloque um pedaço de filme plástico (6 x 6 mm) para a janela do crânio para a proteção, e sutura no couro cabeludo. Colocar o animal em uma almofada aquecida até que esteja totalmente recuperado da anestesia. </li></ol> 4. Resultados representativos: Coronal fatias corticais podem ser preparados para confirmar o sucesso da cirurgia undercut. Em fatias preparadas&gt; 2 semanas após a cirurgia, cortes transcortical e minar são discerníveis no âmbito do objectivo de baixa potência de um microscópio (Fig. 2). O córtex parcialmente isolado geralmente se torna um pouco mais fino, e atividade epileptiforme pode ser detectada na maioria dos fatias corticais com a gravação de campo potencial (Fig. 3). Na formação de contraste, de grandes buracos na substância branca ou em camadas profundas cortical, dramática afinamento do córtex lesionado, ou um corte errante estar acima ou abaixo da substância branca do cérebro fará inutilizável para novas experiências. <img src="/files/ftp_upload/2840/2840fig1.jpg" alt="Figura 1"/> Figura 1. Estrutura e aplicação de um dispositivo de minar. Um tubo guia (2) é colado em uma placa de suporte (1) que é feito de aço inoxidável ou plástico transparente. A agulha da seringa (3) é inserido através do tubo guia e dobrados em ~ 3 mm até a ponta. Uma agulha parar feita de tubo de plástico é colado sobre a extremidade superior da agulha de modo que a faixa vertical em movimento da agulha é limitada a 1,2 mm ~ e ~ 1,5 mm para uso em P21 camundongos e ratos, respectivamente. Um segmento de fio de cobre de pequeno porte é preso sob a alça agulha como indicador para a orientação da agulha torta. Note-se que o dispositivo é inclinado e em contacto com ambas as extremidade da janela do crânio de modo que um corte pode ser feita paralelamente à superfície pial. <img src="/files/ftp_upload/2840/2840fig2.jpg" alt="Figura 2"/> Figura 2. A imagem representativa da fatia undercut. A. Imagem fluorescente de uma fatia que foi preparado de duas semanas após lesão undercut em um rato P48. A ferida de corte foi rotulado por fluorescentes DII corante. As setas brancas da direita indicam corte transcortical, e as setas na parte inferior indicam minar que passaram embora a fronteira entre a camada IV e substância branca. <img src="/files/ftp_upload/2840/2840fig3.jpg" alt="Figura 3"/> Figura 3. Gravação de campo potencial de minar uma fatia. Campo registro do potencial de uma fatia do cérebro undercut exibiu atividade epileptiforme, sugerindo hiperexcitabilidade do tecido lesado cortical.

<ol>
	<li>Hoffman, S. N., Salin, P. A., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neocortical+epileptogenesis+in+vitro.">Chronic neocortical epileptogenesis in vitro.</a> J Neurophysiol. 71, 1762-1773 (1994).</li><li>Topolnik, L., Steriade, M., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperexcitability+of+intact+neurons+underlies+acute+development+of+trauma-related+electrographic+seizures+in+cats+in+vivo.">Hyperexcitability of intact neurons underlies acute development of trauma-related electrographic seizures in cats in vivo.</a> Eur J Neurosci. 18, 486-496 (2003).</li><li>Graber, K., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Models of Seizures and Epilepsy. , 477-493 (2005).</li><li>Nita, D. A., Cisse, Y., Timofeev, I., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+propensity+to+seizures+after+chronic+cortical+deafferentation+in+vivo.">Increased propensity to seizures after chronic cortical deafferentation in vivo.</a> J Neurophysiol. 95, 902-913 (2006).</li><li>Sharpless, S. K., Halpern, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrical+excitability+of+chronically+isolated+cortex+studied+by+means+of+permanently+implanted+electrodes.">The electrical excitability of chronically isolated cortex studied by means of permanently implanted electrodes.</a> Electroencephalogr Clin Neurophysiol. 14, 244-255 (1962).</li><li>Echlin, F. A., Battista, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+Seizures+from+Chronic+Isolated+Cortex.">Epileptiform Seizures from Chronic Isolated Cortex.</a> Arch Neurol. 9, 154-170 (1963).</li><li>Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptogenic+neurons+and+circuits.">Epileptogenic neurons and circuits.</a> Adv Neurol. 79, 665-684 (1999).</li><li>Marin-Padilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+neuropathology+and+impact+of+perinatal+brain+damage.+II:+white+matter+lesions+of+the+neocortex.">Developmental neuropathology and impact of perinatal brain damage. II: white matter lesions of the neocortex.</a> J Neuropathol Exp Neurol. 56, 219-235 (1997).</li><li>Jin, X., Prince, D. A., Huguenard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitatory+synaptic+connectivity+in+layer+v+pyramidal+neurons+of+chronically+injured+epileptogenic+neocortex+in+rats.">Enhanced excitatory synaptic connectivity in layer v pyramidal neurons of chronically injured epileptogenic neocortex in rats.</a> J Neurosci. 26, 4891-4900 (2006).</li><li>Li, H., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+in+chronically+injured,+epileptogenic+sensory-motor+neocortex.">Synaptic activity in chronically injured, epileptogenic sensory-motor neocortex.</a> J Neurophysiol. 88, 2-12 (2002).</li><li>Salin, P., Tseng, G. F., Hoffman, S., Parada, I., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+sprouting+in+layer+V+pyramidal+neurons+of+chronically+injured+cerebral+cortex.">Axonal sprouting in layer V pyramidal neurons of chronically injured cerebral cortex.</a> J Neurosci. 15, 8234-8245 (1995).</li><li>Avramescu, S., Nita, D. A., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortical+post-traumatic+epileptogenesis+is+associated+with+loss+of+GABAergic+neurons.">Neocortical post-traumatic epileptogenesis is associated with loss of GABAergic neurons.</a> J Neurotrauma. 26, 799-812 (2009).</li><li>Avramescu, S., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+strength+modulation+after+cortical+trauma:+a+role+in+epileptogenesis.">Synaptic strength modulation after cortical trauma: a role in epileptogenesis.</a> J Neurosci. 28, 6760-6772 (2008).</li></ol>

Não há conflitos de interesse declarados.

Minar o modelo é um sistema altamente eficiente para o estudo da epileptogênese pós-traumático. A cirurgia típica leva apenas cerca de 20-30 minutos para terminar, e evocou ou atividade epileptiforme espontânea pode ser gravado em fatias de a maioria dos animais de duas semanas após a cirurgia 1-2. Mais importante, este modelo imita os aspectos de alterações após uma lesão cerebral traumática, tais como hemorragia, inflamação, edema axotomia e morte neuronal 7. Não só tem atividade ...

<table border="1"><tbody><tr><td> Nome do material </td><td> Tipo </td><td> Companhia </td><td> Número Catálogo </td></tr><tr><td> Foredom micromotor kit </td><td> equipamento </td><td> Foredom </td><td> K.1070 </td></tr><tr><td> Agulha da seringa de 1,5 polegadas de calibre 22 </td><td> material </td><td> Empresa BD </td><td> 305156 </td></tr><tr><td> 1,5 agulha da seringa polegadas calibre 25, </td><td> material </td><td> Empresa BD </td><td> 305127 </td></tr><tr><td> Cola de cianoacrilato </td><td> material </td><td> Ted Pella </td><td> 14450 </td></tr></tbody></table>

preparing undercut model of posttraumatic epileptogenesis in rodents

Córtex parcialmente isolado (&quot;minar&quot;) é um modelo animal de epileptogênese pós-traumático. O procedimento cirúrgico envolve o corte através do córtex sensório-motor e por baixo da substância branca (minar) para que uma região específica do córtex cerebral é em grande parte isolado do córtex e regiões vizinhas subcortical 1-3. Após uma latência de duas ou mais semanas após a cirurgia, descargas epileptiformes podem ser gravados em fatias de cérebro de roedores 1; e convulsões elétricas ou comportamento pode ser observado in vivo a partir de outras espécies como gato e macaco 4-6. Este modelo animal bem estabelecido é eficiente para gerar e imita diversas características importantes da lesão cerebral traumática. No entanto, é tecnicamente desafiador tentar fazer precisas lesões corticais no cérebro de pequenos roedores com uma mão livre. Com base no procedimento estabelecido inicialmente no laboratório do Dr. David Príncipe na Universidade Stanford 1, aqui apresentamos uma técnica aprimorada para realizar uma cirurgia para a elaboração deste modelo em camundongos e ratos. Demonstramos como fazer um dispositivo cirúrgico simples e usá-lo para obter um melhor controle da profundidade de corte e ângulo de gerar resultados mais precisos e consistentes. O dispositivo é fácil de fazer, eo procedimento é rápido para aprender. A geração deste modelo animal fornece um sistema eficiente para o estudo sobre os mecanismos de epileptogênese pós-traumático.

Watch this Scientific Journal Video about Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents at JoVE.com

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Córtex parcialmente isolado (&quot;minar&quot;) é um modelo animal eficiente de epileptogênese pós-traumático. Aqui demonstramos como fazer um novo dispositivo cirúrgico e usá-lo para fazer lesões mais precisas e consistentes para gerar este modelo.

Preparando Modelo de rebaixo de epileptogênese Pós-Traumático em Roedores

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

preparing-undercut-model-of-posttraumatic-epileptogenesis-in-rodents

Research

JoVE Journal

Neuroscience

9.3K visualizações.  Indiana University School of Medicine. Córtex parcialmente isolado ("minar") é um modelo animal eficiente de epileptogênese pós-traumático. Aqui demonstramos como fazer um novo dispositivo cirúrgico e usá-lo para fazer lesões mais precisas e consistentes para gerar este modelo.

Vídeo: Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

Habituação e Prepulse Inibição de sobressalto acústico em Roedores

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

Neurociência

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

Em larga escala de gravação de Neurônios por sondas de silicone móveis em Comportando Roedores

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

A configuração de Baixo Custo para audiometria comportamental em roedores

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro e análise dos ritmos circadianos em Running rodas Atividade em Roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

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 ...

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

Cabeça de base acrílica Resina de moldagem técnica de fixação em roedores

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

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

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

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

Extrusão hidráulico da medula espinhal e Isolamento de gânglios das raízes dorsais em roedores

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

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Estimulação transcraniana de elétrica cerebral em roedores alertas

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

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 ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Farmacoterapia intracraniana e ensaios de dor em roedores

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...