中脑多巴胺神经元的数目在成年小鼠环境的调变
Summary
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.
Abstract
在大脑或“大脑可塑性”持久的变化背后以下疾病或损伤适应行为和大脑修复。此外，我们的环境之间的相互作用可以引起大脑的可塑性。越来越多的研究，试图找出其中的环境刺激大脑的可塑性有利于治疗大脑和行为障碍。两个环境的操作中，描述了增加或减少的酪氨酸羟化酶免疫阳性的数量（TH +中多巴胺（DA）的合成中的限速酶）的神经元，在成年小鼠中脑。第一配对包括雄性和雌性小鼠持续1周，其中约12％的男性增加中脑TH +神经元一起，但大约12％的女性降低中脑TH +神经元。所述第二壳体包括小鼠连续2周在含有运行车轮，玩具，绳索，排料等，“富集环境'（EE），我通过在雄性约14％ncreases脑TH +神经元。此外，协议中描述的同时注入药物直接进入脑在这些环境的操作，以帮助识别潜在的环境引起的大脑可塑性的机制。例如，EE-诱导更多的脑TH +神经元的突触输入的同时封锁到脑神经元废止。总之，这些数据表明，有关环境的信息是通过突触输入中继到中脑的神经元，以打开或关闭的“DA”基因的表达。因此，适当的环境刺激，或药物的基本机制的靶向，可能是用于治疗与中脑多巴胺失衡有关的脑和行为障碍有用（ 例如，帕金森氏病，注意力缺陷多动障碍，精神分裂症，和药物成瘾）。
Introduction
DArgic信号通过在中脑腹侧被盖区（VTA）和黑质致密部（SNC）的神经元被认为是为奖励激励的认知，情感和运动行为的重要。但是，过多或过少的中脑多巴胺信令引起各种神经病症的许多禁用症状（ 例如 ，帕金森氏病，注意力缺陷多动障碍，精神分裂症，和药物成瘾）。药物提高或降低的DA信令缓解这些症状，但它们也产生副作用可归因于失调信令和脱靶效应。药物功效也随时间下降，由于脑的代偿反应。因此面临的挑战是在一个更有针对性和生理方式恢复正常脑多巴胺信令，以及一个偏爱的方法是通过增加或减少中脑多巴胺神经元的数目。
证据已经积累了SEVERAL几十年来，参与代谢和贩运DA等儿茶酚胺类物质在成熟的成体细胞的基因和蛋白质的表达是可以修改的（1回顾）。在中脑，酪氨酸羟化酶免疫阳性的数量（TH + DA中合成的限速酶）的神经元减小然后增大以下的神经毒素给药2,3，而TH免疫阴性（TH-）的神经元的数量示出了相反的模式（ 即增加然后下降3）。这是与损失一致，那么获得了“DA型”的一些细胞。 TH +和TH-致密的神经元的数量也已表明下列即改变这些细胞4,5的电活动的各种处理中相等，但相反的方向发生变化。例如，输注小电导的钙活化的钾（SK）通道拮抗剂蜂毒明肽进入脑2周（以相同的量）减少TH +和增加的数目的NUTH-的黑质神经元MBER 4,5。与此相反，注入对SK通道激动剂1- EBIO的增加TH +的数量和减小（以相同的量）TH-致密的神经元4,5的数量。类似的变化被视为承接各种针对黑质神经元的活动，包括一些有针对性的传入输入4个处理。致密DArgic神经元的数目的由神经元活性和传入输入此表观调节引起该环境或行为可以影响黑质致密的神经元的数量的可能性。暴露在不同的环境中确实成年小鼠具有更多或更少的中脑（SNC和VTA）TH +神经元，并且至少其中的一些环境引起的变化由突触输入的中脑6并发封锁被废除。这种沟通的目的是：（1）提供有关如何实现我们的环保操作和药物输注进一步详情; （2）提供进一步的数据支持我们的论点，即电子nvironment调节中脑多巴胺神经元的数目， 经由传入输入。
Protocol
注：对动物的所有实验程序批准了神经科学和心理健康动物伦理委员会的弗洛里学院，并符合澳大利亚国家卫生和医学研究理事会公布的做法，动物科学目的的维护和使用的代码（ 第 7版， 2004年）。 1.环境手法性别配对使用性成熟（> 8周龄），年龄匹配的雄性和雌性小鼠。 注意：我们通常使用C57BL / 6小鼠，但不得不使用Swiss小鼠在这个协议中相?…
Representative Results
经受这些环境操纵成年小鼠已经改变脑的号码（SNC和VTA），但不LC，TH +神经元，和EE加要么印防己毒素或荷包牡丹碱并发脑灌注（GABA A受体拮抗剂）废除EE-诱导更致密TH +神经元。这些数据此前公布的6人。本资料汇编中如以前的研究的一部分进行重复实验，但还没有在其他地方发表。 具体来说，已配对的成年雌性小鼠成年雄性小鼠有更多的TH +黑质致密部和腹?…
Discussion
操作环境
这些环境的操纵（性别配对和丰富环境）的设计背后的动机是为了确定是否对环境，和/或行为的环境提示时，与变化的中脑多巴胺神经元的数量有关。因此，重点是提供环境和刺激行为有可能从事脑DA信号。这些措施包括配对异性和丰富环境，包括访问运行车轮，绳索，梯子，隧道，和对象的探索，玩耍，攀爬，隐藏和鸟巢。这两种环境都应该促进奖励激励?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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’s Operational Infrastructure Support Grant.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Isofluorane | Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia | AHN3640 | |
| ALZET Osmotic pump 1002 | DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530 | 0004317 | |
| ALZET Brain infusion kit 1 | DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530 | 0004760 | |
| ALZET cannula holder 1 | DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530 | 0008860 | |
| Vertex Monomer Self-curing (dental acrylic solvent) | Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands | n/a | |
| Vertex Self Curing (dental acrylic powder) | Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands | n/a | |
| METACAM (Meloxicam) | Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia | L10100 | |
| Sodium Pentobarbitone | Lethabarb, Virbac, Milperra, NSW, Australia | 571177 | |
| Normal goat serum | chemicon-temecula, CA | S26-Litre | |
| Triton X-100 | Merck Millipore Headquarters , 290 Concord road, Billerica, MA 01821 | 1.08603.1000 | |
| Polyclonal rabbit anti-tyrosine hydroxylase | Merck Millipore Headquarters , 290 Concord road, Billerica, MA 01821 | AB152 | |
| Polyclonal biotinylated goat anti-rabbit | Dako Australia Pty. Ltd., Suite 4, Level 4, 56 Berry street, North Sydney, NSW, Australia 2060 | EO432 | |
| Avidin peroxidase | Sigma-aldrich, Castle Hill, NSW 1765 AU | A3151-1mg | |
| Diamino-benzidine | Sigma-aldrich, Castle Hill, NSW 1765 AU | D-5637 | |
| Stereo Investigator | MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495 | n/a |
Referências
- Aumann, T., Horne, M. Activity-dependent regulation of the dopamine phenotype in substantia nigra neurons. Journal of neurochemistry. 121, 497-515 (2012).
- Sauer, H., Oertel, W. H. Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neurociência. 59, 401-415 (1994).
- Stanic, D., Finkelstein, D. I., Bourke, D. W., Drago, J., Horne, M. K. Timecourse of striatal re-innervation following lesions of dopaminergic SNpc neurons of the rat. The European journal of neuroscience. 18, 1175-1188 (2003).
- Aumann, T. D., et al. Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons. Journal of neurochemistry. 116, 646-658 (2011).
- Aumann, T. D., et al. SK channel function regulates the dopamine phenotype of neurons in the substantia nigra pars compacta. Experimental neurology. 213, 419-430 (2008).
- Aumann, T. D., Tomas, D., Horne, M. K. Environmental and behavioral modulation of the number of substantia nigra dopamine neurons in adult mice. Brain and behavior. 3, 617-625 (2013).
- Paxinos, G., Franklin, K. B. J. . The mouse brain in stereotaxic coordinates. , (2001).
- Schultz, W. Behavioral dopamine signals. Trends in neurosciences. 30, 203-210 (2007).
- Sun, P., Smith, A. S., Lei, K., Liu, Y., Wang, Z. Breaking bonds in male prairie vole: long-term effects on emotional and social behavior, physiology, and neurochemistry. Behavioural brain research. 265, 22-31 (2014).
- Aponso, P. M., Faull, R. L., Connor, B. Increased progenitor cell proliferation and astrogenesis in the partial progressive 6-hydroxydopamine model of Parkinson’s disease. Neurociência. 151, 1142-1153 (2008).
- Frielingsdorf, H., Schwarz, K., Brundin, P., Mohapel, P. No evidence for new dopaminergic neurons in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A. 101, 10177-10182 (2004).
- Lie, D. C., et al. The adult substantia nigra contains progenitor cells with neurogenic potential. The Journal of neuroscience : the official journal of the Society for Neuroscience. 22, 6639-6649 (2002).
- Chen, Y., Ai, Y., Slevin, J. R., Maley, B. E., Gash, D. M. Progenitor proliferation in the adult hippocampus and substantia nigra induced by glial cell line-derived neurotrophic factor. Experimental neurology. 196, 87-95 (2005).
- Yoshimi, K., et al. Possibility for neurogenesis in substantia nigra of parkinsonian brain. Ann Neurol. 58, 31-40 (2005).
- Shan, X., et al. Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease-like mice. Stem Cells. 24, 1280-1287 (2006).
- Zhao, M., et al. Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A. 100, 7925-7930 (2003).
- Baker, H., Kawano, T., Margolis, F. L., Joh, T. H. Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat. The Journal of neuroscience : the official journal of the Society for Neuroscience. 3, 69-78 (1983).
- Black, I. B., Chikaraishi, D. M., Lewis, E. J. Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion. Brain research. 339, 151-153 (1985).
- Zigmond, R. E., Chalazonitis, A., Joh, T. Preganglionic nerve stimulation increases the amount of tyrosine hydroxylase in the rat superior cervical ganglion. Neuroscience letters. 20, 61-65 (1980).
- Biguet, N. F., Rittenhouse, A. R., Mallet, J., Zigmond, R. E. Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion. Neuroscience letters. 104, 189-194 (1989).
- Richard, F., et al. Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold. Journal of neuroscience research. 20, 32-37 (1988).
- Schalling, M., Stieg, P. E., Lindquist, C., Goldstein, M., Hokfelt, T. Rapid increase in enzyme and peptide mRNA in sympathetic ganglia after electrical stimulation in humans. Proc Natl Acad Sci U S A. 86, 4302-4305 (1989).
- Liaw, J. J., He, J. R., Barraclough, C. A. Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons. Brain research. Molecular brain research. 13, 171-174 (1992).
- Watabe-Uchida, M., Zhu, L., Ogawa, S. K., Vamanrao, A., Uchida, N. Whole-brain mapping of direct inputs to midbrain dopamine neurons. Neuron. 74, 858-873 (2012).
- Tepper, J. M., Lee, C. R. GABAergic control of substantia nigra dopaminergic neurons. Progress in brain research. 160, 189-208 (2007).
- Hannan, A. J. Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity. Neuropathology and applied neurobiology. 40, 13-25 (2014).
- Nithianantharajah, J., Hannan, A. J. Enriched environments, experience-dependent plasticity and disorders of the nervous system. Nature reviews. Neuroscience. 7, 697-709 (2006).
- Chmiel, D. J., Noonan, M. Preference of laboratory rats for potentially enriching stimulus objects. Laboratory animals. 30, 97-101 (1996).
- Hanmer, L. A., Riddell, P. M., Williams, C. M. Using a runway paradigm to assess the relative strength of rats’ motivations for enrichment objects. Behavior research methods. 42, 517-524 (2010).
- Burrows, E. L., McOmish, C. E., Hannan, A. J. Gene-environment interactions and construct validity in preclinical models of psychiatric disorders. Progress in neuro-psychopharmacology & biological psychiatry. 35, 1376-1382 (2011).
- Van de Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. M., Baumans, V. Strength of preference for nesting material as environmental enrichment for laboratory mice. Applied Animal Behaviour Science. 55, 369-382 (1998).
- Rampon, C., et al. Effects of environmental enrichment on gene expression in the brain. Proceedings of the National Academy of Sciences of the United States of America. 97, 12880-12884 (2000).
- Eckert, M. J., Abraham, W. C. Effects of environmental enrichment exposure on synaptic transmission and plasticity in the hippocampus. Current topics in behavioral neurosciences. 15, 165-187 (2013).
- Sztainberg, Y., Chen, A. An environmental enrichment model for mice. Nature protocols. 5, 1535-1539 (2010).
- Sale, A., Berardi, N., Maffei, L. Environment and brain plasticity: towards an endogenous pharmacotherapy. Physiological reviews. 94, 189-234 (2014).
Citar este artigo
Tomas, D., Prijanto, A. H., Burrows, E. L., Hannan, A. J., Horne, M. K., Aumann, T. D. Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice. J. Vis. Exp. (95), e52329, doi:10.3791/52329 (2015).