Gostaríamos de agradecer a Leah McAleer por seu apoio na realização deste experimento e sua ajuda na apresentação de pôster na Sociedade de Neurociência Meeting 2009 e esta produção de vídeo, junto com Mohamed Abdalla e Ahrens Allison. Agradecemos também Rosie Maddox, Rachel Chavana, e Linda Ju por sua assistência na coleta de dados e análises para esta experiência. Este projecto foi apoiado pelo NIH / NIDA Grant 3R01DA014640-05S1 (CLD), O Centro de Waggoner para Álcool e Research Fellowship Jones Addiction e NIH / NIAA Training Grant AA07471 (AAF). Cocaína HCl foi generosamente fornecidos pelo Inventário de Drogas NIDA e Abastecimento e do Programa de Controle.

*= Passos mais importantes <ol><li> Bem-tratados do sexo masculino Sprague-Dawley são treinados para alavanca de imprensa com recompensa alimentar (45 pellets mg de sacarose; Bio-Serv, Frenchtown, NJ). </li><li> * O cateter intravenoso é construído a partir de aço inoxidável cânula (Plastics One, VA) e tubos de Silastic. Ratos submetidos a um procedimento de cateterismo jugular cirúrgica para permitir a auto-administração de drogas. A anestesia consistiu de uma mistura de oxigênio (0,8 l / min; Airgas Southwest, Corpus Christi, TX) e isoflurano (2,5-4%; AErrane, Baxter Healthcare, Deerfield, IL) entregues através de um sistema de fornecimento de gás (VetEquip, Inc, Pleasanton , CA). Após a cirurgia, a permeabilidade do cateter é mantido por dia flushing com 0,1 ml de uma solução salina a 0,9%, heparina e Timentin solução (antibiótico). Gentamicina, um antibiótico tópico, é aplicado na calota craniana e incisão no peito para prevenir a infecção. Todos os procedimentos cirúrgicos seguiu as orientações para as cirurgias de animais estéreis. Suprimentos cirurgia foram autoclavados e instrumentos foram esterilizados em solução química e esterilizador de talão quente. Cateteres implantados foram lavados com metanol, água e ar durante a preparação e então banhadas em solução de etanol 70% no dia da cirurgia. Ratos foram preparados para a cirurgia por raspar a cabeça e no peito seguido de aplicação de uma solução de betadine. Após a cirurgia, os ratos foram monitorados por várias horas antes do início retornou à colônia animal. </li><li> Cocaína (NIDA Inventário Drogas e Abastecimento e do Programa de Controle; RTI International, Research Triangle Park, NC) utilizadas neste experimento foi dissolvido em solução salina isotônica (0,9%) nas concentrações dose adequada de acordo com os pesos dos animais. Soluções de cocaína foram, então, filtrada através de filtros de 0,2 hum seringa estéril antes do uso diário. </li><li> * O aparelho é construído a partir de dois monocomando câmaras operante (28 x 22 x 21 cm) equipados com casa e estímulo luzes e 3 conjuntos de photobeams (Med Associates, St. Albans, VT). Câmaras estão unidas por um beco construída Plexiglas (21 x 25 x 25 cm). O beco é composto por duas paredes pretas com listras brancas e duas paredes de metal, um top Plexiglas claro, e um piso de plexiglas branco. As duas câmaras têm operante de aço grelha da haste (4,8 mm) instalado sobre pisos inferiores da gaiola removível de metal. Sinais sensoriais são colocados dentro das câmaras operante para criar único, ambientes distinguíveis. Os elementos visuais consistem em branco ou preto sentiu o material anexado para a frente e para trás paredes e sobre a parte superior da gaiola. As pistas olfativas são à base de óleo aromas (rosa ou canela) saturar uma bola de algodão localizadas sob o assoalho da haste da câmara. </li><li> O procedimento experimental ocorre ao longo de vários dias, a começar com os testes de linha de base preferência (2 dias), seguido de auto-administração de drogas sessões (8 ou 16 dias), e terminando com um lugar teste de preferência condicionada (CPP) (1 dia). </li><li> * Durante a preferência inicial e procedimentos de ensaio CPP, os animais têm acesso a ambas as câmaras operante. Uma câmera de vídeo montada sobre o aparelho registra as entradas dos ratos e saídas para cada câmara. Photobeams dentro das câmaras detectar quebras feixe que são utilizados como um índice de atividade locomotora. Durante a auto-administração de drogas sessões, os animais são restritas a uma única câmara operante enquanto a resposta de alavanca e atividade locomotora (por exemplo, quebras photobeam) os dados são gravados com um Pentium 100 MHZ Med computador equipado com software PC-Med (Med Associates, Inc., St. Albans, Vermont). </li><li> Medições de preferência de base são coletados em dois dias consecutivos. Para começar, o rato é colocado no beco center com acesso a ambas as câmaras operante para um período de 20 min. Imediatamente após a colocação na câmara, a câmara de vídeo, Med-PC programa de software e timer são ativados simultaneamente. Os dados de preferência linha de base são avaliados durante visões das gravações de vídeo. </li><li> * Drogas auto-administração sessões de iniciar o dia após o teste de referência passado. Sinais sensoriais (por exemplo, revestimentos de parede branca ou preta e aromas de rosa ou canela) e um painel para bloquear o acesso ao centro de Plexiglas beco são colocados dentro da câmara antes de o animal. Para iniciar a sessão, o animal é colocado dentro da câmara, enquanto tubo da seringa de drogas montado está ligado à entrada do animal cateter. Depois de fechar a porta da câmara operante, o programa Med-PC e temporizador é iniciado e acesso a cocaína está disponível para uma hora. Cada alavanca resultados de resposta na infusão de 0,1 ml de cocaína (0,75 mg / kg / inj) ou solução salina. No final de cada sessão de auto-administração do rato é desligada da tubulação de drogas, removido da câmara e colocado na gaiola. </li><li> O dia após a sessão de auto-administração passada, os animais submetidos a uma preferência local condicionado (CPP) de teste. O procedimento é o mesmo descrito para as medições de referência (ver 7). </li><li> * Dados de Avaliação: Um experimentador cego para trabalhos de grupo determina a quantidade detempo os animais gastam em cada compartimento, visualizando o vídeo para avaliar entradas e saídas entre os compartimentos por um período de 15 min (início 5 min após a colocação dentro do aparelho). O mesmo procedimento é utilizado para determinar a preferência de base e os resultados dos testes CPP. </li><li> * Análise de Dados: Diferenças entre as medições de referência e pontuações pós-condicionado CPP são usados ​​para determinar os efeitos de drogas condicionado. Pontuações diferença são calculados subtraindo-se a quantidade de tempo gasto no compartimento pareado salina da quantidade de tempo gasto no compartimento de drogas emparelhado durante o teste de CPP (Número de segundos em Drogas pareado compartimento) - (Número de segundos em Saline pareado compartimento). Análises estatísticas sobre as mudanças diferença de pontuação a partir da linha de base para as sessões de teste podem ser realizados para determinar os efeitos de condicionamento de drogas. </li></ol> Pontuações CPP também pode ser expresso como percentual CPP Scores Diferença (%). No presente estudo, CPP% Pontuação Diferença foi calculado primeiro determinando o percentual de tempo gasto em cada câmara operante utilizando a fórmula: (Tempo em drogas (OR Saline) Câmara / Hora total em cada Secção) * 100% = Preferência Coloque condicionado (CPP). O percentual pontuação diferença foi determinada pela fórmula: (Drug pareado%) - (Saline-pareado%) = CPP% Pontuação Diferença. <img src="/files/ftp_upload/1998/1998fig1.jpg" alt="Figura 1" /> Figura 1. Relação entre a ingestão de cocaína e de preferência Coloque condicionado análises de Correlação de Pearson determinou uma correlação positiva entre o total de respostas de cocaína Reforçado Lever e% Pontuação CPP diferença para os animais que tiveram a auto-administrada cocaína durante oito sessões (por exemplo, o grupo a longo prazo;. P &lt;0,05), mas não para os animais do grupo de curto prazo (por exemplo, 4 sessões de cocaína). Diamantes abertas representam pontos de dados individuais para a cocaína a longo prazo (n = 9) e círculos cheios representam a cocaína a curto prazo (n = 8) grupos. Melhor ajuste linhas são tracejadas para longo prazo, e sólida para os pontos dados a curto prazo.

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	<li>Sanchis-Segura, C., Spanagel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+assessment+of+drug+reinforcement+and+addictive+features+in+rodents:+an+overview.">Behavioural assessment of drug reinforcement and addictive features in rodents: an overview.</a> Addict Biol. 11 (1), 2-2 (2006).</li><li>Tzschentke, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+reward+with+the+conditioned+place+preference+(CPP)+paradigm:+update+of+the+last+decade.">Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade.</a> Addict Biol. 12 (3-4), 227-227 (2007).</li><li>Koob, G. F., Bloom, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Psychopharmacology: the fourth generation of progress. , 759-759 (1995).</li><li>Bardo, M. T., Bevins, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+place+preference:+what+does+it+add+to+our+preclinical+understanding+of+drug+reward.">Conditioned place preference: what does it add to our preclinical understanding of drug reward.</a> Psychopharmacology (Berl). 153 (1), 31-31 (2000).</li><li>Panlilio, L. V., Goldberg, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-administration+of+drugs+in+animals+and+humans+as+a+model+and+an+investigative+tool.">Self-administration of drugs in animals and humans as a model and an investigative tool.</a> Addiction. 102 (12), 1863-1863 (2007).</li><li>Stefanski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+versus+passive+cocaine+administration:+differences+in+the+neuroadaptive+changes+in+the+brain+dopaminergic+system.">Active versus passive cocaine administration: differences in the neuroadaptive changes in the brain dopaminergic system.</a> Brain Res. 1157, 1-1 (2007).</li><li>Miguens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+cocaine-induced+modulation+of+glutamate+and+dopamine+transporters+after+contingent+and+non-contingent+administration.">Differential cocaine-induced modulation of glutamate and dopamine transporters after contingent and non-contingent administration.</a> Neuropharmacology. 55 (5), 771-771 (2008).</li><li>Palamarchouk, V., Smagin, G., Goeders, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-administered+and+passive+cocaine+infusions+produce+different+effects+on+corticosterone+concentrations+in+the+medial+prefrontal+cortex+(MPC)+of+rats.">Self-administered and passive cocaine infusions produce different effects on corticosterone concentrations in the medial prefrontal cortex (MPC) of rats.</a> Pharmacol Biochem Behav. 94 (1), 163-163 (2009).</li><li>Ciano, P. D. i., Blaha, C. D., Phillips, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+changes+in+dopamine+oxidation+currents+in+the+nucleus+accumbens+of+rats+by+stimuli+paired+with+self-administration+or+yoked-administration+of+d-amphetamine.">Conditioned changes in dopamine oxidation currents in the nucleus accumbens of rats by stimuli paired with self-administration or yoked-administration of d-amphetamine.</a> Eur J Neurosci. 10 (3), 1121-1121 (1998).</li><li>Twining, R. C., Bolan, M., Grigson, P. 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A produção de vídeo deste artigo foi patrocinado pela Med Associado, que produz instrumentos utilizados neste artigo.

Operante de drogas intravenosas procedimentos de auto-administração e colocar condicionado são modelos confiáveis ​​e válidas para o estudo das bases neurobiológicas da dependência de drogas e dependência 1 2 3. Ambos os métodos são amplamente utilizados em pesquisas pré-clínicas abuso de drogas e são capazes de medir as propriedades de reforço das drogas de abuso 4. No entanto, ambos os métodos têm falhas que o novo aparelho e método aqui apresentado melhora a 5. Uma desvantagem grande de procedimentos tradicionais CPP é o modo não-contingente de administração da droga. Este modo de consumo de droga tem diferentes resultados comportamentais e neuroquímicos do que auto-administração 6 7 8 9 10 11, e não é consistente com humanos consumo de drogas experiências. Além disso, ao contrário de drogas auto-administração dos procedimentos, paradigmas CPP são incapazes de medir mudanças progressivas na motivação de drogas como refletido pelo consumo de drogas aumentou; um ponto de viragem na suposta mudança de uso de drogas ao vício de drogas incontrolável. No entanto, o comportamento auto-administração de medicamentos tem limitações de interpretação também. Por exemplo, as taxas de resposta são muitas vezes utilizados para inferir o valor da recompensa, mas pode ser diretamente influenciado por efeitos motoras da droga auto-administrado, independente dos efeitos motivacionais da droga. Além disso, o número de drogas reforçado respostas também pode ser influenciada pela presença ou ausência de drogas associadas à perfusão estímulos 12. A cocaína é conhecida por ser facilmente auto-administrado por ratos e produzir robusto preferências lugar condicionado a uma variedade de doses e vias de administração 2 13 14. Os resultados do presente estudo corroboram relatos anteriores de modelos reforço de drogas. Além disso, uma relação positiva significativa entre as respostas de cocaína reforçado alavanca e pontuações CPP foi determinada no longa, mas não o grupo cocaína a curto prazo. Estes resultados sugerem que o condicionamento lugar e auto-administração não são necessariamente isomorfos medidas de recompensa. Por exemplo, é concebível que após a CPP de curto prazo da exposição à cocaína reflete aguda propriedades reforçadoras normalmente visto com o uso de drogas recreativas inicial. Por outro lado, em ratos com mais experiência de consumo de cocaína escalada, a cocaína em correspondência com níveis crescentes de reforço condicionado pode indicar mudanças progressivas na sensibilidade de drogas ou melhorados efeitos de drogas gratificante em determinadas populações. Este método apresentado tem utilidade para além de avaliar os efeitos positivos de reforço de drogas. Por exemplo, não efeitos aversivos de drogas presentes durante o uso de drogas iniciais podem surgir com a exposição prolongada da droga (por exemplo, aversões lugar condicionado, ou CPA) e seriam detectáveis ​​usando esta técnica. Usos adicionais incluem o potencial de tela para subpopulações mais sensíveis à cocaína cue-aprendizagem associativa, para avaliar a experiência mediada por mudanças na cocaína motivados comportamentos, e para detectar drogas duradoura condicionado efeitos durante a abstinência de drogas e / ou reintegração cued. Em conclusão, este aparelho ea abordagem é o tempo e de custo eficaz, pode ser usado para examinar uma ampla gama de temas relacionados ao abuso de drogas, e oferece mais flexibilidade no design experimental que CPP ou de auto-administração métodos sozinho.

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<td>Operant Conditioning Behavior (Drug Self-administration) Test Package for Rat</td>
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<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
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<td>Infrared Source and Detector (Photobeams)</td>
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<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
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<td>Med PC Software</td>
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<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
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novel apparatus and method for drug reinforcement

Modelos animais de reforço provaram ser úteis para entender os mecanismos neurobiológicos subjacentes à toxicodependência. Operante de drogas auto-administração e preferência local condicionado (CPP) procedimentos são utilizados em pesquisas expansivamente animais para modelar vários componentes de reforço de drogas, consumo e dependência em humanos. Para este estudo, foi utilizada uma nova abordagem para estudar o reforço da droga em ratos através da combinação CPP tradicional e auto-administração metodologias. Montamos um aparelho utilizando duas câmaras Med Associado operante, estímulos sensoriais, e uma zona de Plexiglas construído neutro. Essas modificações permitiram nossos experimentos para abranger aspectos motivacionais do consumo de drogas através da auto-administração e livre de drogas avaliação de droga / cue condicionado força com o teste CPP. Em nossos experimentos, ratos cocaína auto-administrado (0,75 mg / kg / inj, iv), durante ou quatro (por exemplo, a &quot;curto prazo&quot;) ou oito (por exemplo, a &quot;longo prazo&quot;), alternando-dia as sessões em um ambiente operante distintivo contendo sinais sensoriais (por exemplo, olfativo e visual). Nos dias alternados, no outro (diferente-cued) ambiente operante, salina estava disponível para auto-infusão (0,1 ml, iv). Vinte e quatro horas após a última sessão self-administration/cue-pairing, um teste foi realizado CPP. Consistente com os achados típicos CPP, houve uma preferência significativa para a câmara de associados com cocaína auto-administração. Além disso, em animais submetidos ao experimento de longa duração, uma correlação positiva entre magnitude CPP eo número de respostas de cocaína reforçado alavanca. Em conclusão, este aparelho ea abordagem é o tempo e de custo eficaz, pode ser usado para examinar uma ampla gama de temas relacionados ao abuso de drogas, e oferece mais flexibilidade no design experimental que CPP ou de auto-administração métodos sozinho.

Watch this Scientific Journal Video about Novel Apparatus and Method for Drug Reinforcement at JoVE.com

Novel Apparatus and Method for Drug Reinforcement

Operante de drogas auto-administração e preferência local condicionado (CPP) procedimentos são utilizados em pesquisas expansiva para modelo de vários componentes de reforço de drogas, consumo e dependência em humanos. Neste relatório, nós combinamos os métodos tradicionais de CPP e auto-administração como uma nova abordagem para estudar o reforço de drogas e vício em ratos.

Aparelho novo e Método para Reforço de Drogas

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

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SCIENTISTS IN ACTION

19.1K visualizações.  University of Texas at Austin. Operante de drogas auto-administração e preferência local condicionado (CPP) procedimentos são utilizados em pesquisas expansiva para modelo de vários componentes de reforço de drogas, consumo e dependência em humanos. Neste relatório, nós combinamos os métodos tradicionais de CPP e auto-administração como uma nova abordagem para estudar o reforço de drogas e vício em ratos.

Vídeo: Novel Apparatus and Method for Drug Reinforcement

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the spinal cord (Pearse et al., 2005, Gensel et al., 2006, Anderson et al., 2009), as the majority of human SCI occur here (Young, 2010; www.sci-info-pages.com). Behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. Additionally, a key priority for human patients with cervical SCI is restoration of hand/arm function (Anderson, 2004). Thus, outcome measures that assess both proximal and distal forelimb function are needed. Although there are several behavioral assays that are sensitive to different aspects of forelimb recovery in experimental models of cervical SCI (Girgis et al., 2007, Gensel et al., 2006, Ballerman et al., 2001, Metz and Whishaw, 2000, Bertelli and Mira, 1993, Montoya et al., 1991, Whishaw and Pellis, 1990), few techniques provide detailed information on the recovery of fine motor control and digit movement.
The current measurement technique, the Irvine, Beatties and Bresnahan forelimb scale (IBB), can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation. The IBB was generated by observing recovery after a unilateral C6 SCI, and involves video recording of animals eating two differently shaped cereals (spherical and doughnut) of a consistent size. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping technique.
The IBB, like other forelimb behavioral tasks, shows a consistent pattern of recovery that is sensitive to injury severity. Furthermore, the IBB scale could be used to assess recovery following other types of injury that impact normal forelimb function.

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan IBB Forelimb Scale

Here we will describe a rodent behavioral assay that can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation.

Um novo método para avaliação da função Membro Anterior Proximal e Distal no Rato: o Irvine, Beatties e Bresnahan (IBB) Escala Membro Anterior

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the ...

Neurociência

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Um novo método de Drug Administration para Multiple peixe zebra (<em&gt; Danio rerio</em&gt;) Ea quantificação da Retirada

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Avaliação da Viabilidade Neuronal Utilizando Coloração Dupla de Iodeto de Diacetate-Propidium de Fluoresceína em Cultura de Neurônio de Granulado Cerebelar

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

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

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

Gravação de EEG em Ratos Neonatais de Movimentação Livre Usando um Novo Método

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

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Romance Ensaio para Fria nocicepção em<em&gt; Drosophila</em&gt; Larvas

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Um novo método para modelar a encefalopatia traumática crônica em<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desenvolvimento de ensaio para quantificação de conteúdo elevado de Sod1 proteína mutante formação agregada em células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

Implantação de sondas de silicone crônica e gravação de lugar Hippocampal células em um aparelho de esteira enriquecido

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

A Computerized Test Battery to Study Pharmacodynamic Effects on the Central Nervous System of Cholinergic Drugs in Early Phase Drug Development

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for further development of new compounds. A computerized and thoroughly validated battery of neuropsychological and neurophysiological tests has been shown to be sensitive to detect drug-induced effects of multiple new and existing compounds. The test battery covers the main CNS domains, which have been shown to respond to drug effects and can be repeatedly administered following drug administration to characterize the concentration-effect profile of a drug.
The standard tests in the battery are saccadic eye movement, smooth pursuit eye movement, the Bowdle visual analog scale (VAS), the Bond and Lader VAS, body sway, adaptive tracking, visual verbal learning, and quantitative electroencephalography (qEEG). However, the test battery is adaptive in nature, meaning that it can be composed and adjusted with tests fit to investigate specific drug classes, or even specific receptors.
Showing effects of new cholinergic drugs designed to have a pro-cognitive outcome has been difficult. The pharmacological challenge model is a tool for early proof-of-pharmacology. Here, a marketed drug is used to induce temporary and reversible disease-like symptoms in healthy subjects, via a pharmacological mechanism related to the disease that is targeted as indication for the new compound. The test battery was implemented to investigate the potential of the nicotinic receptor antagonist mecamylamine to be used as a challenge model for cholinergic dysfunction, as seen in neurodegenerative disorders.
A worsening of scores in a dose dependent manner on the visual verbal learning test (VVLT; a test for learning and memory abilities) and the adaptive tracking test (a measure of visuomotor control and arousal), in particular, showed that the test battery is sensitive to showing acute pharmacodynamic effect after administration of anti-cholinergic drugs.

A validated computerized battery of neuropsychological and neurophysiological tests is used to study pharmacodynamic effects on the central nervous system of newly developed drugs in early phase development. To demonstrate the test battery, the acute effects of mecamylamine and the reversal of these effects by two agonist drugs are described.

Uma bateria de testes informatizados para estudar os efeitos farmacodinâmicos no sistema nervoso Central de drogas colinérgicas em fase inicial de desenvolvimento de drogas

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for ...

CMAP Scan MUNE (MScan) - A Novel Motor Unit Number Estimation (MUNE) Method

Like other methods for motor unit number estimation (MUNE), compound muscle action potential (CMAP) scan MUNE (MScan) is a non-invasive electrophysiologic method to estimate the number of functioning motor units in a muscle. MUNE is an important tool for the assessment of neuropathies and neuronopathies. Unlike most MUNE methods in use, MScan assesses all the motor units in a muscle, by fitting a model to a detailed stimulus-response curve, or CMAP scan. It thereby avoids the bias inherent in all MUNE methods based on extrapolating from a small sample of units. Like &#39;Bayesian MUNE,&#39;&#160;MScan analysis works by fitting a model, made up of motor units with different amplitudes, thresholds, and threshold variabilities, but the fitting method is quite different, and completed within five minutes, rather than several hours. The MScan off-line analysis works in two stages: first, a preliminary model is generated based on the slope and variance of the points in the scan, and second, this model is then refined by adjusting all the parameters to improve the fit between the original scan and scans generated by the model.
This new method has been tested for reproducibility and recording time on 22 amyotrophic lateral sclerosis (ALS) patients and 20 healthy controls, with each test repeated twice by two blinded physicians. MScan showed excellent intra- and inter-rater reproducibility with ICC values of &#62;0.98 and a coefficient of variation averaging 12.3 &#177; 1.6%. There was no difference in the intra-rater reproducibility between the two observers. Average recording time was 6.27 &#177; 0.27 min.
This protocol describes how to record a CMAP scan and how to use the MScan software to derive an estimate of the number and sizes of the functioning motor units. MScan is a fast, convenient, and reproducible method, which may be helpful in diagnoses and monitoring disease progression in neuromuscular disorders.

CMAP Scan MUNE MScan - A Novel Motor Unit Number Estimation MUNE Method

This protocol describes a new method to estimate the number of functioning motor units in a muscle, by fitting a model to a detailed stimulus-response curve of the compound muscle action potential. It is quick and easy to perform and analyze and has excellent reproducibility.

CMAP Scan MUNE (MScan) - um método de estimativa (MUNE) número de romance da unidade do Motor

Like other methods for motor unit number estimation (MUNE), compound muscle action potential (CMAP) scan MUNE (MScan) is a non-invasive electrophysiologic ...