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.

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			<td height="21" style="height:21px;width:379px;">ALZET Brain infusion kit 1</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0004760</td>
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			<td height="21" style="height:21px;width:379px;">ALZET cannula holder 1</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0008860</td>
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			<td height="21" style="height:21px;width:379px;">Vertex Monomer Self-curing (dental acrylic solvent)</td>
			<td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td>
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			<td height="21" style="height:21px;width:379px;">Vertex Self Curing (dental acrylic powder)</td>
			<td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td>
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			<td height="21" style="height:21px;width:379px;">METACAM (Meloxicam)</td>
			<td>Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia</td>
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			<td height="21" style="height:21px;width:379px;">Sodium Pentobarbitone</td>
			<td>Lethabarb, Virbac, Milperra, NSW, Australia</td>
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			<td>chemicon-temecula, CA</td>
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			<td height="21" style="height:21px;width:379px;">Triton X-100</td>
			<td>Merck Millipore Headquarters&#160;, 290 Concord road, Billerica, MA 01821</td>
			<td style="width:119px;">1.08603.1000</td>
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			<td height="21" style="height:21px;width:379px;">Polyclonal rabbit anti-tyrosine hydroxylase</td>
			<td>Merck Millipore Headquarters&#160;, 290 Concord road, Billerica, MA 01821</td>
			<td style="width:119px;">AB152</td>
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			<td height="21" style="height:21px;">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 style="width:119px;">EO432</td>
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			<td height="21" style="height:21px;">Avidin peroxidase</td>
			<td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td>
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			<td height="21" style="height:21px;">Diamino-benzidine</td>
			<td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td>
			<td style="width:119px;">D-5637</td>
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			<td height="21" style="height:21px;">Stereo Investigator</td>
			<td>MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495</td>
			<td style="width:119px;">n/a</td>
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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

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Aplicação de uma condutância do receptor NMDA em neurônios dopaminérgicos Rat Midbrain Usando a Técnica Clamp dinâmico

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Neurociência

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

Estudar a Integração da Adult-nascido Neurônios

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Um<em&gt; In-vitro</em&gt; Preparação de neurônios entéricos isolados e células gliais do plexo mioentérico do Rato Adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

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 ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-reprogramação de células pericyte derivados do Adulto Cérebro Humano em neurônios induzidos

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Captura Laser Microdissection - uma demonstração do Isolamento de Pessoa dopamina Neurônios e região ventral tegmental Entire

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcelulares Gravações patch-clamp do domínio somatodendrítico da substância negra dopamina Neurônios

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantificação de Endosome e Moosidades de Lisosoma em Neurônios Cultivados Usando Sondas Fluorescentes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Avaliação da homeostase Dopaminergic em camundongos pelo uso de análise de cromatografia líquida de alta performance e captação da dopamina Synaptosomal

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Uso de zinco sináptica histoquímica para revelar as diferentes regiões e lâminas no cérebro em desenvolvimento e adulto

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...