O financiamento para a esse trabalho, e como citado 2,10, foi fornecido, em parte, por uma premiação de pesquisa para concessão MF Salvatore da Federação Americana para Pesquisas sobre o Envelhecimento, Edward P. A Stiles Trust Fund e da Fundação de Pesquisa Biomédica da Northwest Louisiana, e BS Pruett da Sociedade Muslow Ike predoctoral, LSU Health Sciences Center-Shreveport.

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	<li>Trevitt, J. T., Carlson, B. B., Nowend, K., Salamone, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substantia+nigra+pars+reticulate+is+a+highly+potent+site+of+action+for+the+behavioral+effects+of+the+D1+antagonist+SCH23390+in+rat.">Substantia nigra pars reticulate is a highly potent site of action for the behavioral effects of the D1 antagonist SCH23390 in rat.</a> Psychopharmacology. 156, 32-41 (2001).</li><li>Salvatore, M. F., Pruett, B. S., Spann, S. L., Dempsey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+reveals+a+role+for+nigral+tyrosine+hydroxylase+ser31+phosphorylation+in+locomotor+activity+generation.">Aging reveals a role for nigral tyrosine hydroxylase ser31 phosphorylation in locomotor activity generation.</a> PLoS ONE. 4, e8466 (2009).</li><li>Rossato, J. 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M., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+infusion+of+brain-derived+neurotrophic+factor+into+the+ventral+tegmental+area+induces+long-lasting+potentiation+of+cocaine+seeking+after+withdrawal.">A single infusion of brain-derived neurotrophic factor into the ventral tegmental area induces long-lasting potentiation of cocaine seeking after withdrawal.</a> J. Neurosci. 24, 1604-1611 (2004).</li><li>Hoffer, B. J., Hoffman, A., Bowenkamp, K., Huettl, P., Hudson, J., Martin, D., Lin, L. F., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+line-derived+neurotrophic+factor+reverses+toxin-induced+injury+to+midbrain+dopaminergic+neurons+in+vivo.">Glial cell line-derived neurotrophic factor reverses toxin-induced injury to midbrain dopaminergic neurons in vivo.</a> Neurosci. Lett. 182, 107-111 (1994).</li><li>Salvatore, M. F., Zhang, J. L., Large, D. M., Wilson, P. E., Gash, C. R., Thomas, T. C., Haycock, J. W., Bing, G., Stanford, J. A., Gash, D. M., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striatal+GDNF+administration+increases+tyrosine+hydroxylase+phosphorylation+in+the+rat+striatum+and+substantia+nigra.">Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra.</a> J. Neurochem. 90, 245-254 (2004).</li><li>Lu, L., Wang, X., Wu, P., Xu, C., Zhao, M., Morales, M., Harvey, B. K., Hoffer, B. J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ventral+tegmental+area+glial+cell-line+derived+neurotrophic+factor+in+incubation+of+cocaine+craving.">Role of ventral tegmental area glial cell-line derived neurotrophic factor in incubation of cocaine craving.</a> Biol. Psychiatry. 66, 137-145 (2009).</li><li>Lavicky, J., Dunn, 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=Corticotropin-releasing+factor+stimulates+catecholamine+release+in+hypothalamus+and+prefrontal+cortex+in+freely+moving+rats+as+assessed+by+microdialysis.">Corticotropin-releasing factor stimulates catecholamine release in hypothalamus and prefrontal cortex in freely moving rats as assessed by microdialysis.</a> J. Neurochem. 60, 602-612 (1993).</li><li>Salvatore, M. F., Pruett, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dichotomy+of+tyrosine+hydroxylase+and+dopamine+regulation+between+somatodendritic+and+terminal+field+areas+of+nigrostriatal+and+mesoaccumbens+pathways.">Dichotomy of tyrosine hydroxylase and dopamine regulation between somatodendritic and terminal field areas of nigrostriatal and mesoaccumbens pathways.</a> PLoS ONE. 7, e29867 (2012).</li><li>Salvatore, M. F., Garcia-Espana, A., Goldstein, M., Deutch, A. Y., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+tyrosine+hydroxylase+phosphorylation+in+the+nigrostriatal+and+mesolimbic+systems+in+vivo:+Effects+of+acute+haloperidol+and+related+compounds.">Stoichiometry of tyrosine hydroxylase phosphorylation in the nigrostriatal and mesolimbic systems in vivo: Effects of acute haloperidol and related compounds.</a> J. Neurochem. 75, 225-232 (2000).</li><li>Salvatore, M. F., Waymire, J. C., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization-stimulated+catecholamine+biosynthesis:+involvement+of+protein+kinases+and+tyrosine+hydroxylase+phosphorylation+sites+in+situ.">Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ.</a> J. Neurochem. 79, 349-360 (2001).</li><li>Haycock, J. W., Lew, J. Y., Garcia-Espana, A., Lee, K. Y., Harada, K., Meller, E., Goldstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+serine-19+phosphorylation+in+regulating+tyrosine+hydroxylase+studied+with+site-+and+phosphospecific+antibodies+and+site-directed+mutagenesis.">Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis.</a> J. Neurochem. 71, 1670-1675 (1998).</li><li>Lindgren, N., Xu, Z. Q., Linskog, M., Herrera-Marschitz, M., Goiny, M., Haycock, J. W., Goldstein, M., Hokfelt, T., Fisone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tyrosine+hydroxylase+activity+and+phosphorylation+at+ser19+and+ser40+via+activation+of+glutamate+NMDA+receptors+in+rat+striatum.">Regulation of tyrosine hydroxylase activity and phosphorylation at ser19 and ser40 via activation of glutamate NMDA receptors in rat striatum.</a> J. Neurochem. 74, 2470-2477 (2000).</li></ol>

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Conforme descrito na Figura 1, os métodos descritos acima devem produzir leituras múltiplas de dopamina e da sua regulação TH proteínas, DAT e VMAT2 a partir de uma amostra de SN ou VTA obtido a partir de qualquer um rato ou ratinho. Novamente, os benefícios de levar a cabo este protocolo são que o investigador pode obter leituras operacionalmente-combinados de como a dopamina é regulada in vivo sob virtualmente qualquer paradigma experimental e, ao fazê-lo, salvo significativas meios experimentais, re...

HPLC sistema: O sistema de base é constituído por uma bomba Shimadzu HPLC LC10-ADvp, um Waters WISP 717 automático amostra injector, um 250 x 4,4 mm 5 micron Spherosorb ODS-1 coluna de fase inversa C18 (Waters), uma Bioanalytical Systems (BAS) TL12 vítreo dupla eletrodo de carbono, dois detectores de BAS LC4B eletroquímicos, e um Empower Waters 2 de coleta de dados e integração de sistemas. A coluna é mantida a 30-45 ° C (BAS aquecedor de coluna LC22A). A fase móvel é fosfato de sódio 0,1 M (pH 3,0), EDTA 0,1 mM, 0,2-0,4 mM 1-octano sulfónico (Eastman-Kodak), e acetonitrilo 0,35% (v / v), filtrada através de um filtro de 0,45 micron. Taxa de fluxo é de 1,2 ml / min. Quatro lotes litro de fase móvel são optimizados para separações por ajuste do pH, ácido sulfónico octano e temperatura da coluna. A fase móvel é reciclado, e é continuamente purgado com gás hélio para remover o oxigénio dissolvido. A reciclagem da fase móvelé quase essencial para manter uma boa resolução por um período de tempo razoável. A fase móvel vida de prateleira é mantida por meio de um interruptor de fluxo (controlado pelo integrador) para desviar a perder a 2-7 min antes de cada ensaio. Os eléctrodos são mantidas em potenciais de cerca de 0,78 e 0.95V com respeito a um eléctrodo de referência Ag/AgC1. O eléctrodo na maior potencial é utilizada exclusivamente para a determinação do triptofano (eo NMDA padrão interno). O potencial 0,78 V fornece um sinal superior à relação de ruído para a detecção das monoaminas e compostos, com excepção do triptofano. Os cromatogramas são armazenados no disco rígido da estação de trabalho Empower, e, posteriormente, processado e os dados transferidos diretamente para uma planilha Excel para o cálculo das quantidades de metabolitos e compilação de dados do grupo. Bomba: Shimadzu LC-10AD Cell: BAS Cross Flow. Carbono vítreo eletrodo de trabalho em 0,780 e 0,950 potencial V. Detector: BAS LC-4B operado em modo dual channel. Dados Acq. Sistema: Águas Empower Pro 2. Injector: Waters WISP 717 Coluna: Waters Spherosorb ODS-1, 5 mM de partícula, 4,4 mm x 250 mm. <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Dodecil sulfato de sódio (SDS) </td><td> - JT Baker </td><td> 4095-02 </td></tr><tr><td> Trizma Base de Dados </td><td> Sigma </td><td> T1503-1KG </td></tr><tr><td> Trizma HCl </td><td> Sigma </td><td> T3253-1KG </td></tr><tr><td> Glicerina </td><td> Sigma </td><td> G8773-500 mL </td></tr><tr><td> PVP-40 </td><td> Sigma </td><td> PVP40-1KG </td></tr><tr><td> DPBS </td><td> Gibco </td><td> 21600-069 </td></tr><tr><td> Tween20 </td><td> Sigma </td><td> P1379-500 mL </td></tr><tr><td> Glycine </td><td> Sigma </td><td> G8898-1KG</td></tr><tr><td> Ponceau S </td><td> Fluka </td><td> 81460 </td></tr><tr><td> Azul de Bromofenol </td><td> Sigma </td><td> B8026-5G </td></tr><tr><td> Ditiotreitol </td><td> Sigma </td><td> D-9163 </td></tr><tr><td> Proteína padrão 2 mg de BSA </td><td> Sigma </td><td> P5619-25VL </td></tr><tr><td> Pierce BCA Protein Assay Reagent Um </td><td> Thermo-Fisher Scientific </td><td> 23223 </td></tr><tr><td> Precision Plus padrão de proteína </td><td> Bio Rad </td><td> 161-0373 </td></tr><tr><td> [125I] Uma proteína, atividade específica </td><td> Perkin-Elmer </td><td></td></tr></tbody></table> Tabela 2. Reagentes específicos. <table border="1"><tbody><tr><td> Reagentes </td><td> Fórmulas </td></tr><tr><td> 10% de SDS </td><td> 10 g de SDS, 100 mL DI H 2 0 </td></tr><tr><td> SDS a 1% (pH para 8,2) </td><td><ol><li> 10 mL10% de SDS </li><li> 60,5 mg de Trizma base </li><li> 37,22 mg de EDTA </li><li> 90 mL DI H 2 0 </li></ol></td></tr><tr><td> Cobre solução de sulfato II </td><td><ol><li> 1 g de cobre sulfato II </li><li> 25 mL DI H 2 0 </li></ol></td></tr><tr><td> Tampão de amostra 3X </td><td><ol><li> Trizma base de dados de 2,27 g </li><li> SDS 6 g </li><li> Ditiotreitol 0,463 g </li><li> Glicerol 30 g </li><li> Azul de Bromofenol 10 mg </li><li> DIH 2 0 (inicialmente adicionar acima reagentes a 40 mL de H 2 0 em um cilindro graduado, em seguida, adicionar H 2 0 até que o volume atinge 100 mL) </li><li> HCl (adicionar, conforme necessário para alcançar o pH de 6,85) </li><li> Congelar solução em 50 tubos de 2,0 mL. </li></ol> (Faz 100 mL): Volume de tampão de amostra necessário 3X = ½ volume de SDS utilizados na amostra. </td></tr><tr><td> 1X tampão de amostra </td><td> Diluir Tampão de amostra 3X para baixo para 1X tampão de amostra usando DI H 2 0 </td></tr><tr><td> 10X<html> Tampão de corrida (faz 4 L) </td><td><ol><li> Trizma Base de Dados de 121,1 g </li><li> Glycine 577 g </li><li> SDS 40 g </li></ol></td></tr><tr><td> Tampão de Transferência 10X: (Faz 4 L) </td><td><ol><li> Glicina 360 g </li><li> Trizma base de dados de 96 g </li></ol></td></tr><tr><td> Encarnado vivo </td><td><ol><li> Ponceau S. 0,5 g </li><li> Ácido Acético 5 mL </li><li> DI H 2 0 95 mL </li></ol></td></tr><tr><td> 0,2% solução de HCl </td><td> 5,2 ml de HCl em 500mL de DI H 2 0 </td></tr><tr><td> PVP-T20 de bloqueio Soln. (Faz 4 L) </td><td><ol><li> PVP-40 40 g </li><li> DPBS 38,2 g </li><li> Tween20 2 g </li><li> Thimerisol 0,4 g </li><li> 1M Tris pH 7,6 (60,6 g Tris HCl + 13,9 g Base de Dados de Tris em 500 mL DI H 2 0) - 50 mL </li></ol></td></tr><tr><td> Tampão 10X Blot (faz 4 L) </td><td><ol><li> Tween 20 20 g </li><li> Tris Base de Dados de 14 g </li><li> Tris-HCl 61 g </li></ol></td></tr></tbody></table>ONTEÚDO &quot;Protein Assay&gt; Tabela 3. e ocidentais Fórmulas Blotting. Padrões tirosina hidroxilase: Os padrões de proteína TH calibrados e fosforilação utilizados por este laboratório são derivados de extratos de células PC12, que foram analisadas para teor de proteína TH e estequiometrias fosforilação contra um TH padrões previamente calibrados que em última análise, oriundos do laboratório do Dr. John Haycock 11 .

comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area

A dopamina é um neurotransmissor vigorosamente estudada no SNC. Na verdade, o seu envolvimento na atividade locomotora e recompensa comportamentos relacionados promoveu cinco décadas de investigação sobre as deficiências moleculares associados com a regulação da dopamina. A maioria destas investigações de regulação de dopamina no cérebro o foco sobre a base molecular para a sua regulação em regiões terminais de campo das vias nigrostiatais e mesoaccumbens; estriado e nucleus accumbens. Além disso, esses estudos têm-se concentrado na análise do teor de tecido da dopamina, com normalização ao peso do tecido apenas molhado. Investigação das proteínas que regulam a dopamina, tais como tirosina hidroxilase (TH), proteína de fosforilação TH, do transportador de dopamina (DAT) e transportador monoamina vesicular 2 (VMAT2) proteína muitas vezes não incluem a análise do teor de dopamina no tecido da mesma amostra. A capacidade de analisar tanto o conteúdo do tecido da dopamina e suas proteínas de regulação (incluindo pós-tramodificações nslational) não só dá o poder inerente ao interpretar a relação da dopamina com o nível da proteína ea função de TH, DAT, ou VMAT2, mas também se estende economia amostra. Isso se traduz em menor custo, e ainda produz insights sobre a regulação molecular da dopamina em praticamente qualquer paradigma de escolha dos investigadores. Nós nos concentramos nas análises no mesencéfalo. Embora o SN e VTA são normalmente negligenciados na maioria dos estudos de regulação da dopamina, esses núcleos são facilmente dissecados com a prática. Uma leitura abrangente do conteúdo tecido dopamina e TH, DAT, ou VMAT2 pode ser realizado. Não é florescente literatura sobre o impacto da função dopaminérgica no SN e VTA sobre o comportamento, e as imposições de substâncias exógenas ou processos doença nisso 1-5. Além disso, os compostos, tais como factores de crescimento têm um efeito profundo sobre as proteínas da dopamina e da dopamina-regulador, a uma extensão comparativamente maior no SN ou VTA <sup&gt; 6-8. Portanto, esta metodologia é apresentada como referência para laboratórios que desejam estender suas investigações sobre como tratamentos específicos modular o comportamento e regulação da dopamina. Aqui, um método multi-passo é apresentado para as análises de teor de tecido de dopamina, os níveis proteicos de TH, DAT ou VMAT2 e fosforilação TH da substantia nigra e VTA do mesencéfalo de roedores. A análise da fosforilação TH pode produzir insights significativos em não só como actividade TH é regulada, mas também as cascatas de sinalização afectadas nos núcleos somatodendritic num dado paradigma. Iremos ilustrar a técnica de dissecação para segregar estes núcleos duas eo processamento da amostra de tecido dissecado que produz um perfil revelando mecanismos moleculares de regulação da dopamina in vivo, específicas para cada núcleos (Figura 1).

Watch this Scientific Journal Video about Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area at JoVE.com

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

A dopamina é distintamente regulada nos núcleos mesencéfalo, que contêm a célula corpos e dendrites de os neurónios de dopamina. Descrevemos aqui uma abordagem dissecção e manipulação da amostra para maximizar os resultados, e, assim, conclusões e compreensão, na regulação da dopamina nos núcleos mesencéfalo da substantia nigra (SN) e área tegmental ventral (VTA) em roedores.

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 ...

comprehensive-profiling-dopamine-regulation-substantia-nigra-ventral

Research

JoVE Journal

Neuroscience

25.6K visualizações.  Louisiana State University Health Sciences Center. A dopamina é distintamente regulada nos núcleos mesencéfalo, que contêm a célula corpos e dendrites de os neurónios de dopamina. Descrevemos aqui uma abordagem dissecção e manipulação da amostra para maximizar os resultados, e, assim, conclusões e compreensão, na regulação da dopamina nos núcleos mesencéfalo da substantia nigra (SN) e área tegmental ventral (VTA) em roedores.

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

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

Medição única célula de liberação de dopamina com simultânea de tensão-clamp e amperometria

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Neurociência

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Em Expression in ovo de microRNA em Ventral Pintinho Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

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

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

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, ...

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 ...

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) ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

Stereological estimativa do número de neurônios Dopaminergic na substância negra do Mouse usando o fracionador óptico e equipamento padrão de microscopia

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Identificação de Receptor de dopamina D1-alfa dentro roedores núcleo Accumbens por uma inovador RNA In Situ tecnologia da deteção

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Preparação de fatias aguda da medula espinhal para a gravação de células inteiras remendo-braçadeira nos neurônios da substância Gelatinosa

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

Programa de autópsia abrangente para indivíduos com esclerose múltipla

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

Infusão e Estimulação Combinada com Voltammetry Cíclica de Varredura Rápida (CIS-FSCV) para avaliar a regulação do receptor da área tegmental ventral da dopamina fástica

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...