Name: environmental modulations of the number of midbrain dopamine neurons in adult mice
Uploaded: 2015-01-20T14:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice at JoVE.com

This study was supported by the National Health and Medical Research Council of Australia (NHMRC) Project grant 1022839. AJH is an Australian Research Council (ARC) FT3 Future Fellow (FT100100835). The Florey Institute of Neuroscience and Mental Health acknowledges support from the Victorian Government&#8217;s Operational Infrastructure Support Grant.

NOTA: Todos os procedimentos experimentais em animais foram aprovados pelo Instituto Florey do Comitê de Ética Animal Health Neuroscience &amp; Mental e em conformidade com da Austrália Nacional de Saúde e Pesquisa Médica do Conselho publicou código de boas práticas para o cuidado e uso de animais para fins científicos (7 ª edição, 2004). 1. As manipulações ambientais <ol><li> O emparelhamento de Gênero <ol><li> Use sexualmente maduros (&gt; 8 semanas de idade), os ratos machos e fêmeas pareados por idade. NOTA: Normalmente usamos camundongos C57BL / 6, mas tiveram os mesmos resultados usando camundongos Swiss neste protocolo. Normalmente usamos n = 8 machos e fêmeas n = 8 para cada experimento, dividido em n = 2 pares macho-macho (n = 4 machos), n = 2 pares fêmea-fêmea (n = 4 mulheres), e n = 4 pares macho-fêmea (n = 4 machos e 4 fêmeas = n). </li><li> Após a chegada no animal instalação de detenção, casa-grupo dos camundongos por sexo para&gt; 3 dias para se aclimatar. Aqui e througHout o emparelhamento sexo, manter estímulos ambientais externos (por exemplo, temperatura ambiente, a luz: ciclo escuro, comida, água, manuseio e limpeza) constante ou distribuir igualmente a todos os ratos. </li><li> Aleatoriamente atribuir a cada rato a um par macho-macho, um par fêmea-fêmea, ou um par macho-fêmea. Colocar cada par em uma gaiola limpa em isolamento (2 ratos / gaiola) com acesso ad libitum à comida e água, e simplesmente os alojar deste modo continuamente durante 7 dias. NOTA: pecuária rotina é OK, durante este período, mas deve ser distribuída igualmente para todos os ratos. Tome cuidado para evitar o emparelhamento da ninhada masculinos e femininos. </li></ol></li><li> Enriquecimento Ambiental <ol><li> Use sexualmente maduros (&gt; 8 semanas de idade), do sexo masculino ou feminino ratos da mesma idade. NOTA: Normalmente usamos n = 18 ratos para cada experimento, divididas em grupo n = 6 / tratamento. </li><li> Após a chegada na zona de exploração de animais mantê-los alojados em padrão idêntico alojados em grupo (SH) (por exemplo, </em&gt; Veja abaixo) condições, nesta fase, por&gt; 3 dias para se aclimatar. Aqui e em todo o experimento, manter estímulos ambientais externos (por exemplo, temperatura ambiente, a luz: ciclo escuro, comida, água, manuseio e limpeza) constante ou distribuir igualmente a todos os ratos. </li><li> Para começar o enriquecimento ambiental, atribuir aleatoriamente cada mouse para um dos 3 grupos: SH; roda de corrida (RW), ou ambiente enriquecido (EE) e colocar cada grupo de ratinhos em conjunto numa gaiola limpa idêntico com acesso ad libitum a comida e água. NOTA: Aqui, maiores gaiolas de retenção de ratos de todos os grupos, medindo 27 cm de largura, 42 cm de comprimento e 16 cm de profundidade foram usados. <ol><li> Além disso, fornecer as seguintes condições ambientais para cada grupo: SH compreendendo apenas maca (pelotas de papel ou serragem) no chão; RW SH compreendendo mais 2 rodas de correr; EE compreendendo RW além de brinquedos (cordas, escadas, túneis, e objetos como vazios rolos de papel toalha e pedaços de papel de seda) com os quais a explore, brincar, escalar, esconder, e ninho. </li><li> Abrigá-los desta forma continuamente por 14 dias. NOTA: pecuária rotina é OK, durante este período, mas deve ser distribuída igualmente para todos os ratos. </li></ol></li><li> Camundongos EE sujeita a adicional &#39;super-enriquecimento&#39; (SE), colocando-os juntos em uma grande gaiola contendo novas brinquedos para uma hora / dia (mesma hora todos os dias), 5 dias / semana. NOTA: Aqui, a 46 centímetros de largura, 69 centímetros de comprimento e 40 cm de profundidade banheira de plástico foi utilizado. <ol><li> Manter a novidade por apresentar um conjunto diferente de brinquedos cada sessão. Brinquedos que são limpos para ser re-apresentado à ratinhos com água e sabão e etanol a 80% para remover odores. </li><li> Após cada sessão de SE, retornar os ratos para sua gaiola EE. SH Handle e RW camundongos o mesmo que os ratos SE (exceto para si SE) ao longo deste período (por exemplo, remover e voltar cada SH e RW rato de e para a sua gaiola). </li></ol></li></ol></li></ol><ol><li> Preparação <ol><li> Use bombas osmóticas estéreis e de infusão de cérebro kits, que estão disponíveis comercialmente. O dia antes da implantação, utilizando uma técnica asséptica, primos dos implantes, preenchendo cada bomba, tubo de ligação e de uma cânula com uma solução de droga ou veículo estéril (tal como descrito nas instruções fornecidas com as bombas). Ligá-los juntos e incubar em solução salina estéril durante a noite a 37 ° C. Incubar implantes drogas e cheio de veículos separadamente para evitar a contaminação cruzada. </li></ol></li><li> Cirurgia. NOTA: Dependendo do país do experimentador, licenças especiais de experimentação animal ou dos subsídios são necessários para perseguir esses tipos de cirurgia e experiências pós-operatório. <ol><li> Esterilizar todos os equipamentos cirúrgicos e empregar uma técnica asséptica por toda parte. Anestesiar um mouse (por exemplo, usando 1-2% isoflurano no ar) e colocá-lo em um stereheadframe otaxic. Verifique a profundidade da anestesia durante o processo e administrar mais anestésico como necessário (por exemplo, ausência de retirada do membro de aperto nocivo pata é indicativo de anestesia adequada). Garantir a olhos são protegidos de dessecação por pomada lubrificante ocular, e que a temperatura corporal do rato permanece normal durante todo o procedimento. </li><li> Fazer uma incisão na linha média através da pele a partir de entre os olhos e terminada 2cm posterior para a extremidade traseira do crânio. Blunt dissecar a pele longe da fáscia para baixo a parte de trás do mouse para criar uma &#39;bolsa&#39; subcutânea grande o suficiente para caber confortavelmente a bomba e tubo de ligação uma vez que a cânula é implantado (o tubo que liga não deve ser dobrado após a conclusão). Raspe limpar a fáscia de sobre a face dorsal do crânio. </li><li> Usando um diâmetro burr dental aproximadamente 1,5 mm, drill down no crânio no estereotáxica apropriado coordenadas uté uma camada fina, flexível de osso continua. Peel esta camada de distância usando uma pinça fina (isso evita danos a dura-máter subjacente e do cérebro com a rebarba dental). Verifique se o crânio é limpo de quaisquer fragmentos de sangue e osso, e não que haja sangramento ainda mais. </li><li> Com a cânula realizada por um detentor de uma cânula, colocar a bomba e tubo de ligação para o &#39;bolso&#39; subcutâneo que recobre as costas do mouse. Em seguida, posicionar a ponta da cânula nas coordenadas estereotáxicas adequadas e sobre a superfície do cérebro. Baixe cuidadosamente a cânula para o cérebro, mas parar de aproximadamente 1 mm de profundidade necessária. NOTA: A profundidade restante será negociado após a aplicação da primeira camada de acrílico dental. </li><li> Certifique-se de novo a superfície do crânio está limpa e seca, em seguida, preparar um pequeno volume de acrílico dental e use um palito para alisá-lo em toda a área exposta do crânio, inclusive no burr-buraco no crânio em torno the cânula. Antes do acrílico endurece, abaixe a cânula ponta a profundidade restante no alvo. </li><li> Prepare um pequeno volume de acrílico dental e camada esta em relação ao primeiro, bem como sobre o suporte de plástico branco para a cânula, para fixar a cânula no lugar. Repita com camadas adicionais de acrílico como necessário. </li><li> Uma vez que o acrílico tem endurecido, retire cuidadosamente o suporte da cânula e cortar o plástico guia cânula removível branco usando uma lâmina de bisturi aquecida com uma chama de gás butano. Por último, suturar a pele fechada durante todo o implante. </li></ol></li><li> O tratamento pós-cirúrgico de Animais <ol><li> Aplicar pomada anti-séptico para as margens de pele, e administrar um agente anti-inflamatório para o rato (por exemplo meloxicam, 3 mg / kg sc). Remover o mouse do quadro, coloque-o sob uma lâmpada de calor, e observar até que ele recupere a consciência. Não devolva um rato que foi submetido a cirurgia para a companhia de outros animais até que esteja totalmente recuperado. NOTA: Expmanipulações erimental como emparelhamento de gênero e meio ambiente enriquecido pode ser iniciada ou retomada no dia seguinte a cirurgia, no mínimo. </li><li> Monitore seu peso corporal, comportamento geral e aparência diária. Trate sinais de infecção (por exemplo, inchaço, vermelhidão, pus) ao redor de incisões cirúrgicas com pomada anti-séptico tópico. Trate quaisquer sinais de doença, dor, stress ou desconforto (por exemplo, perda de&gt; 10% do peso corporal, retraimento social, a falta de preparação, &quot;amaciamento&quot;, disfunção do movimento, convulsões) com analgésicos e / ou antibióticos sistêmicos como necessário. </li><li> Eutanásia ratos se&gt; 15% de perda de peso ou sintomas da doença, dor, stress ou desconforto são não-reembolsáveis ​​no prazo de 1 semana de tratamento curativo. </li></ol></li></ol> 3. tecido cerebral preparação, transformação imunohistoquímica, e estereologia <ol><li> Perfusão <ol><li> Imediatamente após manipulações / drogas ambientais, preparar o cérebro para o estudo. <br/&gt; NOTA: Nas experiências relatadas aqui o número de neurónios TH + SNc foram medidos e comparados em cada um dos diferentes grupos de tratamento. </li><li> Primeiro administrar uma sobredose de anestésico para matar os ratos (por exemplo, 100 mg / kg ip de sódio pentobarbitona). Uma vez anestesiados, mas antes o coração pára de bater, coloque o mouse sobre as suas costas e gravata ou de definir ambos os membros. </li><li> Usando um bisturi de cortar a pele que recobre o tórax, em seguida, uma incisão através do músculo logo abaixo da caixa torácica para dentro da cavidade abdominal. Coloque um grampo no apêndice xifóide da caixa torácica e levante as costelas para cima e longe do fígado expondo o diafragma. </li><li> Corte através do diafragma e através das costelas lateralmente em ambos os lados utilizando tesouras grandes até a caixa torácica pode ser dobrada para trás sobre a cabeça para expor os pulmões e coração. Usando uma tesoura fina fazer uma pequena incisão no átrio direito como um ponto de saída de sangue e soluções perfundidos, em seguida, cortar a base do Ventri esquerdacle e colocar uma cânula-se através do ventrículo esquerdo, o átrio esquerdo e na aorta. </li><li> Fixar a cânula no lugar depois a bomba quente (37 ° C) heparinizados e 0,1 M de fosfato de solução salina tamponada fisiológica (PBS) através da vasculatura até que a solução que sai do átrio direito é totalmente livre de sangue. Em seguida, a bomba de frio (4 ° C) a solução fixadora (tal como 4% de paraformaldeído em PBS) através da vasculatura, até que todo o rato está bem fixado. Remover o cérebro e o lugar em PBS com 30% de sacarose durante 2-3 dias até os dissipadores cerebrais. NOTA: Outros tipos de preparação do tecido pode ser aplicada dependendo da experiência no laboratório correspondente. </li></ol></li><li> Preparação de Cortes congelados de livre flutuação <ol><li> Corte cortes seriados através das regiões do cérebro de interesse e recolher estes em PBS. NOTA: Nós cortamos 40 mm de espessura, utilizando um criostato. Outros tipos de corte de tecido pode ser aplicada dependendo da especialização no correslaboratório ng. </li></ol></li><li> A imuno-histoquímica <ol><li> Execute imunohistoquímica padrão para proteínas de interesse. Incubar secções em 5% de soro normal de cabra e 0,3% de triton X-100 em PBS à temperatura ambiente durante 30 min, em seguida imunorreagem com anticorpo policlonal de coelho anti-TH (1: 400) a 4 ° C durante 48 hr, em seguida, policlonal biotinilado de cabra anti -rabbit (1: 1000) à temperatura ambiente durante 2 h. </li><li> Em seguida, incuba-se avidina-peroxidase (1: 500) à temperatura ambiente durante 1 hora, em seguida, em cobalto-níquel e intensificou-diamino- benzidina (0,5 mg / ml) à temperatura ambiente durante 18-20 min; para a última 3-5 min de incubação a diamino-benzidina adicionar peróxido de hidrogénio (0,01%) para catalisar a precipitação cromagénio. Lave os cortes três vezes para cada 10 min em PBS antes, depois, e entre cada uma das etapas anteriores. </li><li> Monte as secções em lâminas de microscópio gelatinizadas, ar secá-las, então Nissl stain (vermelho neutro), desidratar em álcool, claro (X-3B), e lamela. </li></ol></li><li&gt; Estereologia <ol><li> Estimar o número total de neurônios TH + e TH SNc (LC e VTA) e utilizando métodos estereológicos imparciais. Certifique-se de que o stereologist é cego para o tratamento recebido. Excluir células gliais com base na soma diâmetro &lt;5 um e contam apenas as células com um núcleo visível. </li><li> Identificar o SNc (e VTA e LC) pelas localizações espaciais de células TH + e as estruturas anatómicas / limites de acordo com o atlas do cérebro de Paxinos e Watson 7. Contagem de células TH + no interior de um quadro de contagem (55 x 55 mm = 3,025 uM 2) em intervalos pré-determinados regularmente (x = 140 mm, y = 140 uM para SNc; x = 100 um, y = 100 mm para VTA e LC) ao longo de cada núcleo em cada quarta secção. </li></ol></li></ol>

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Molecular brain research. 13, 171-174 (1992).</li><li>Watabe-Uchida, M., Zhu, L., Ogawa, S. K., Vamanrao, A., Uchida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+mapping+of+direct+inputs+to+midbrain+dopamine+neurons.">Whole-brain mapping of direct inputs to midbrain dopamine neurons.</a> Neuron. 74, 858-873 (2012).</li><li>Tepper, J. M., Lee, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+control+of+substantia+nigra+dopaminergic+neurons.">GABAergic control of substantia nigra dopaminergic neurons.</a> Progress in brain research. 160, 189-208 (2007).</li><li>Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+and+brain+repair:+harnessing+the+therapeutic+effects+of+cognitive+stimulation+and+physical+activity+to+enhance+experience-dependent+plasticity.">Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity.</a> Neuropathology and applied neurobiology. 40, 13-25 (2014).</li><li>Nithianantharajah, J., Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriched+environments,+experience-dependent+plasticity+and+disorders+of+the+nervous+system.">Enriched environments, experience-dependent plasticity and disorders of the nervous system.</a> Nature reviews. Neuroscience. 7, 697-709 (2006).</li><li>Chmiel, D. J., Noonan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+of+laboratory+rats+for+potentially+enriching+stimulus+objects.">Preference of laboratory rats for potentially enriching stimulus objects.</a> Laboratory animals. 30, 97-101 (1996).</li><li>Hanmer, L. A., Riddell, P. M., Williams, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+runway+paradigm+to+assess+the+relative+strength+of+rats'+motivations+for+enrichment+objects.">Using a runway paradigm to assess the relative strength of rats' motivations for enrichment objects.</a> Behavior research methods. 42, 517-524 (2010).</li><li>Burrows, E. L., McOmish, C. E., Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interactions+and+construct+validity+in+preclinical+models+of+psychiatric+disorders.">Gene-environment interactions and construct validity in preclinical models of psychiatric disorders.</a> Progress in neuro-psychopharmacology &amp; biological psychiatry. 35, 1376-1382 (2011).</li><li>Van de Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. M., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strength+of+preference+for+nesting+material+as+environmental+enrichment+for+laboratory+mice.">Strength of preference for nesting material as environmental enrichment for laboratory mice.</a> Applied Animal Behaviour Science. 55, 369-382 (1998).</li><li>Rampon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+on+gene+expression+in+the+brain.">Effects of environmental enrichment on gene expression in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12880-12884 (2000).</li><li>Eckert, M. J., Abraham, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+exposure+on+synaptic+transmission+and+plasticity+in+the+hippocampus.">Effects of environmental enrichment exposure on synaptic transmission and plasticity in the hippocampus.</a> Current topics in behavioral neurosciences. 15, 165-187 (2013).</li><li>Sztainberg, Y., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+environmental+enrichment+model+for+mice.">An environmental enrichment model for mice.</a> Nature protocols. 5, 1535-1539 (2010).</li><li>Sale, A., Berardi, N., Maffei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+brain+plasticity:+towards+an+endogenous+pharmacotherapy.">Environment and brain plasticity: towards an endogenous pharmacotherapy.</a> Physiological reviews. 94, 189-234 (2014).</li></ol>

The authors have nothing to disclose.

Manipulações ambientais A motivação por trás do design dessas manipulações ambientais (emparelhamento de gênero e de enriquecimento ambiental) foi determinar se o meio ambiente, e / ou comportamento solicitado pelo ambiente, está associado a alterações no número de neurônios DA mesencéfalo. O foco foi, portanto, no fornecimento de ambientes e estimular comportamentos que são propensos a se envolver sinalização DA mesencéfalo. Estes emparelhamento incluído com o se...

Sinalização DArgic pelos neurônios na área tegmental ventral (VTA) e substantia nigra pars compacta (SNC) do mesencéfalo é pensado para ser importante para os comportamentos cognitivos, emotivos e motoras motivou-recompensa. No entanto, muita ou pouca sinalização DA mesencéfalo causa muitos sintomas incapacitantes em uma variedade de distúrbios neurológicos (por exemplo, doença de Parkinson, déficit de atenção e hiperatividade, esquizofrenia e dependência de drogas). Drogas que aumentam ou diminuem a sinalização DA aliviar estes sintomas, no entanto, eles também produzem efeitos colaterais atribuíveis à sinalização desregulada e efeitos fora do alvo. A eficácia do fármaco também diminui ao longo do tempo, devido a respostas compensatórias do cérebro. Por isso, o desafio é restaurar a sinalização DA mesencéfalo normal em uma forma mais direccionada e fisiológico, e uma abordagem favorecido é aumentando ou diminuindo o número de neurônios DA mesencéfalo. A evidência foi acumulando para several décadas que a expressão de genes e proteínas envolvidas no metabolismo e DA tráfico e outras catecolaminas em células de adulto é modificável (revisto em 1). No mesencéfalo, o número de tirosina-hidroxilase imunopositivas (TH +, a enzima limitante da velocidade na síntese de DA), em seguida, diminui, aumenta neurónios após a administração da neurotoxina 2,3, enquanto que o número de TH immunonegative (TH) neurónios mostra o padrão oposto (isto é, aumenta em seguida, diminui 3). Isto é consistente com a perda, em seguida, ganhar do &quot;DA fenótipo &#39;por algumas células. O número de neurónios TH + TH e SNc também foi mostrado para mudar em direcções iguais mas opostos na sequência de vários tratamentos que alteram a actividade eléctrica destas células 4,5. Por exemplo, a infusão de pequeno-condutância, potássio activados por cálcio (SK) canal antagonista apamina em mesencéfalo durante 2 semanas, diminui o número de TH + e aumentos (pela mesma quantidade) o number de TH SNc neurônios 4,5. Em contraste, a infusão do agonista do canal SK-1 EBIO aumenta o número de TH + e diminui (pela mesma quantidade) o número de neurónios TH SNc 4,5. Alterações semelhantes foram vistos na sequência de uma variedade de tratamentos que visam a atividade neuronal do SNC, incluindo alguns que visava inputs aferentes 4. Esta aparente de regulação do número de neurónios SNc DArgic por actividade neuronal aferente e entrada levanta a possibilidade de que o ambiente ou comportamento pode influenciar o número de neurónios SNc. Na verdade ratos adultos expostos a diferentes ambientes têm mais ou menos mesencéfalo (SNc e VTA) TH + neurônios, e pelo menos algumas dessas mudanças induzidas pelo meio ambiente são abolidas pelo bloqueio simultâneo de entrada sináptica no mesencéfalo 6. Os objectivos desta comunicação são os seguintes: (1) fornecer mais detalhes sobre como implementar nossas manipulações ambientais e infusões de drogas; e (2) fornecer mais dados de apoio a nossa afirmação de que o environment regula o número de neurônios DA mesencéfalo, através da entrada de aferentes.

<table><tbody><tr><td>Isofluorane</td><td>Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia</td><td>AHN3640</td><td></td></tr><tr><td>ALZET Osmotic pump 1002</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004317</td><td></td></tr><tr><td>ALZET Brain infusion kit 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004760</td><td></td></tr><tr><td>ALZET cannula holder 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0008860</td><td></td></tr><tr><td>Vertex Monomer Self-curing (dental acrylic solvent)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>Vertex Self Curing (dental acrylic powder)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>METACAM (Meloxicam)</td><td>Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia</td><td>L10100</td><td></td></tr><tr><td>Sodium Pentobarbitone</td><td>Lethabarb, Virbac, Milperra, NSW, Australia</td><td>571177</td><td></td></tr><tr><td>Normal goat serum</td><td>chemicon-temecula, CA</td><td>S26-Litre</td><td></td></tr><tr><td>Triton X-100</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>1.08603.1000</td><td></td></tr><tr><td>Polyclonal rabbit anti-tyrosine hydroxylase</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>AB152</td><td></td></tr><tr><td>Polyclonal biotinylated goat anti-rabbit</td><td>Dako Australia Pty. Ltd., Suite 4, Level 4, 56 Berry street, North Sydney, NSW, Australia 2060</td><td>EO432</td><td></td></tr><tr><td>Avidin peroxidase</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>A3151-1mg</td><td></td></tr><tr><td>Diamino-benzidine</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>D-5637</td><td></td></tr><tr><td>Stereo Investigator</td><td>MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495</td><td>n/a</td><td></td></tr></tbody></table>

environmental modulations of the number of midbrain dopamine neurons in adult mice

Mudanças duradouras no cérebro ou &quot;plasticidade cerebral&quot; subjazem o comportamento adaptativo e reparação cerebral após doença ou lesão. Além disso, as interações com o meio ambiente pode induzir plasticidade cerebral. Cada vez mais, a pesquisa está tentando identificar quais ambientes estimular a plasticidade cerebral benéfica para o tratamento de desordens cerebrais e comportamentais. Duas modificações ambientais que são descritos aumentar ou diminuir o número de tirosina-hidroxilase imunopositivas (TH +, a enzima limitante da velocidade de dopamina (DA síntese)) neurónios do mesencéfalo do rato adulto. O primeiro inclui o emparelhamento masculino e camundongos fêmeas juntos continuamente durante uma semana, o que aumenta TH mesencéfalo + neurônios em aproximadamente 12% no sexo masculino, mas diminui TH mesencéfalo + neurônios em aproximadamente 12% no sexo feminino. A segunda compreende ratos habitação continuamente por 2 semanas em &quot;ambientes enriquecidos&quot; (EE) contendo rodas de corrida, brinquedos, cordas, material de nidificação, etc., que increases TH mesencéfalo + neurônios em cerca de 14% no sexo masculino. Além disso, um protocolo é descrito por concomitantemente infundindo drogas diretamente no mesencéfalo durante estas manipulações ambientais para ajudar a identificar os mecanismos subjacentes a plasticidade do cérebro por motivos ambientais. Por exemplo, EE-indução de mais TH mesencéfalo + neurônios é abolida pelo bloqueio simultâneo de entrada sináptica em neurônios do mesencéfalo. Juntos, estes dados indicam que a informação sobre o meio ambiente é retransmitida via de entrada sináptica nos neurônios do mesencéfalo para ligar ou desligar a expressão de genes &#39;da&#39;. Assim, a estimulação ambiental adequada, ou droga segmentação dos mecanismos subjacentes, pode ser útil para o tratamento de distúrbios cerebrais e comportamentais associados com desequilíbrios no mesencéfalo DA (por exemplo, doença de Parkinson, déficit de atenção e hiperatividade, esquizofrenia e dependência de drogas).

Ratos adultos submetidos a essas manipulações ambientais alteraram número de mesencéfalo (SNC e VTA), mas não LC, TH + neurônios, e EE mais infusão mesencéfalo simultânea de picrotoxin ou bicuculina (GABA os antagonistas do receptor) abole EE-indução de mais SNc TH + neurônios. Estes dados foram previamente publicados em 6. Os presentes dados foram compilados em experiências duplicadas realizados como parte desse estudo anterior, mas não foram publicados em outros lugares. <p clas...

Watch this Scientific Journal Video about Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice at JoVE.com

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Modulações Ambientais do número de Midbrain dopamina Neurônios no Adulto Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

environmental-modulations-number-midbrain-dopamine-neurons-adult

Research

JoVE Journal

Neuroscience

8.7K visualizações.  The University of Melbourne. This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Vídeo: Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Avaliação da função sensório-motora em rato modelos de doença de Parkinson

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Comportamento

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Biologia

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Profiling abrangente do Regulamento dopamina em substância negra e área tegmental ventral

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Neurociência

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Um modelo de rato induzida por laser de crônica Ocular Hypertension para Caracterizar defeitos visuais

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicina

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Modelo murino para a doença de Parkinson: a partir de 6-OH lesão dopamina para teste comportamental

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Modelos de interação gene-ambiente para desmasca mecanismos de suscetibilidade na doença de Parkinson

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Membrana Potencial Dye imagem de Ventromedial Hipotálamo Neurônios de camundongos adultos para estudar Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Os métodos para a modulação e Análise de NF-kB-dependente Adulto Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primária de neurônios dopaminérgicos mouse

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolamento, cultura e Long-Term Manutenção da Primária mesencefálico Dopaminergic neurônios dos cérebros de roedores Embryonic

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...