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

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

NOTE: All experimental procedures on animals were approved by the Florey Institute of Neuroscience &#38; Mental Health Animal Ethics Committee and conform to Australia&#8217;s National Health and Medical Research Council published code of practice for the care and use of animals for scientific purposes (7th edition, 2004).
1. Environmental Manipulations
<ol>
	<li>Gender Pairing
	<ol>
		<li>Use sexually mature (&#62;8 week old), age-matched male and female mice. 
		NOTE: We ty...

<ol>
	<li>Aumann, T., Horne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+regulation+of+the+dopamine+phenotype+in+substantia+nigra+neurons.">Activity-dependent regulation of the dopamine phenotype in substantia nigra neurons.</a> Journal of neurochemistry. 121, 497-515 (2012).</li><li>Sauer, H., Oertel, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+degeneration+of+nigrostriatal+dopamine+neurons+following+intrastriatal+terminal+lesions+with+6-hydroxydopamine:+a+combined+retrograde+tracing+and+immunocytochemical+study+in+the+rat.">Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat.</a> Neuroscience. 59, 401-415 (1994).</li><li>Stanic, D., Finkelstein, D. I., Bourke, D. W., Drago, J., Horne, M. 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D., 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=SK+channel+function+regulates+the+dopamine+phenotype+of+neurons+in+the+substantia+nigra+pars+compacta.">SK channel function regulates the dopamine phenotype of neurons in the substantia nigra pars compacta.</a> Experimental neurology. 213, 419-430 (2008).</li><li>Aumann, T. D., Tomas, D., Horne, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+behavioral+modulation+of+the+number+of+substantia+nigra+dopamine+neurons+in+adult+mice.">Environmental and behavioral modulation of the number of substantia nigra dopamine neurons in adult mice.</a> Brain and behavior. 3, 617-625 (2013).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2001).</li><li>Schultz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+dopamine+signals.">Behavioral dopamine signals.</a> Trends in neurosciences. 30, 203-210 (2007).</li><li>Sun, P., Smith, A. S., Lei, K., Liu, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+bonds+in+male+prairie+vole:+long-term+effects+on+emotional+and+social+behavior,+physiology,+and+neurochemistry.">Breaking bonds in male prairie vole: long-term effects on emotional and social behavior, physiology, and neurochemistry.</a> Behavioural brain research. 265, 22-31 (2014).</li><li>Aponso, P. M., Faull, R. L., Connor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+progenitor+cell+proliferation+and+astrogenesis+in+the+partial+progressive+6-hydroxydopamine+model+of+Parkinson's+disease.">Increased progenitor cell proliferation and astrogenesis in the partial progressive 6-hydroxydopamine model of Parkinson's disease.</a> Neuroscience. 151, 1142-1153 (2008).</li><li>Frielingsdorf, H., Schwarz, K., Brundin, P., Mohapel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+new+dopaminergic+neurons+in+the+adult+mammalian+substantia+nigra.">No evidence for new dopaminergic neurons in the adult mammalian substantia nigra.</a> Proc Natl Acad Sci U S A. 101, 10177-10182 (2004).</li><li>Lie, D. 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=The+adult+substantia+nigra+contains+progenitor+cells+with+neurogenic+potential.">The adult substantia nigra contains progenitor cells with neurogenic potential.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 22, 6639-6649 (2002).</li><li>Chen, Y., Ai, Y., Slevin, J. R., Maley, B. E., Gash, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+proliferation+in+the+adult+hippocampus+and+substantia+nigra+induced+by+glial+cell+line-derived+neurotrophic+factor.">Progenitor proliferation in the adult hippocampus and substantia nigra induced by glial cell line-derived neurotrophic factor.</a> Experimental neurology. 196, 87-95 (2005).</li><li>Yoshimi, K., 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=Possibility+for+neurogenesis+in+substantia+nigra+of+parkinsonian+brain.">Possibility for neurogenesis in substantia nigra of parkinsonian brain.</a> Ann Neurol. 58, 31-40 (2005).</li><li>Shan, X., 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=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.">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.</a> Stem Cells. 24, 1280-1287 (2006).</li><li>Zhao, M., 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=Evidence+for+neurogenesis+in+the+adult+mammalian+substantia+nigra.">Evidence for neurogenesis in the adult mammalian substantia nigra.</a> Proc Natl Acad Sci U S A. 100, 7925-7930 (2003).</li><li>Baker, H., Kawano, T., Margolis, F. L., Joh, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transneuronal+regulation+of+tyrosine+hydroxylase+expression+in+olfactory+bulb+of+mouse+and+rat.">Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 3, 69-78 (1983).</li><li>Black, I. B., Chikaraishi, D. M., Lewis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-synaptic+increase+in+RNA+coding+for+tyrosine+hydroxylase+in+a+rat+sympathetic+ganglion.">Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion.</a> Brain research. 339, 151-153 (1985).</li><li>Zigmond, R. E., Chalazonitis, A., Joh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+the+amount+of+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases the amount of tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 20, 61-65 (1980).</li><li>Biguet, N. F., Rittenhouse, A. R., Mallet, J., Zigmond, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+mRNA+levels+for+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 104, 189-194 (1989).</li><li>Richard, F., 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=Modulation+of+tyrosine+hydroxylase+gene+expression+in+rat+brain+and+adrenals+by+exposure+to+cold.">Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold.</a> Journal of neuroscience research. 20, 32-37 (1988).</li><li>Schalling, M., Stieg, P. E., Lindquist, C., Goldstein, M., Hokfelt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+increase+in+enzyme+and+peptide+mRNA+in+sympathetic+ganglia+after+electrical+stimulation+in+humans.">Rapid increase in enzyme and peptide mRNA in sympathetic ganglia after electrical stimulation in humans.</a> Proc Natl Acad Sci U S A. 86, 4302-4305 (1989).</li><li>Liaw, J. J., He, J. R., Barraclough, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+tyrosine+hydroxylase+mRNA+levels+in+A1,+A2+and+locus+ceruleus+neurons+following+electrical+stimulation+of+A1+noradrenergic+neurons.">Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.</a> Brain research. 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. 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Environmental manipulations
The motivation behind the design of these environmental manipulations (gender pairing and environmental enrichment) was to determine whether the environment, and/or behavior prompted by the environment, is associated with changes in the number of midbrain DA neurons. The focus was therefore on providing environments and stimulating behaviors that are likely to engage midbrain DA signaling. These included pairing with the opposite gender, and environmental e...

DArgic signaling by neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the midbrain is thought to be important for reward-motivated cognitive, emotive and motor behaviors. However, too much or too little midbrain DA signaling causes many disabling symptoms in a variety of neurological disorders (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction). Drugs that increase or decrease DA signaling alleviate these symptoms, however they also produce side-effects attributable to dysregulated signaling and off-target effects. Drug efficacy also declines over time due to compensatory responses of the brain. The challenge therefore is to restore normal midbrain DA signaling in a more targeted and physiological way, and a favored approach is by increasing or decreasing the number of midbrain DA neurons.
Evidence has been accumulating for several decades that the expression of genes and proteins involved in metabolizing and trafficking DA and other catecholamines in mature adult cells is modifiable (reviewed in1). In midbrain, the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in DA synthesis) neurons decreases then increases following neurotoxin administration2,3, while the number of TH immunonegative (TH-) neurons shows the opposite pattern (i.e. increases then decreases3). This is consistent with loss then gain of the &#8216;DA phenotype&#8217; by some cells. The number of TH+ and TH- SNc neurons has also been shown to change in equal but opposite directions following various treatments that alter the electrical activity of these cells4,5. For example, infusion of the small-conductance, calcium-activated potassium (SK) channel antagonist apamin into midbrain for 2 weeks decreases the number of TH+ and increases (by the same amount) the number of TH- SNc neurons4,5. In contrast, infusion of the SK channel agonist 1-EBIO increases the number of TH+ and decreases (by the same amount) the number of TH- SNc neurons4,5. Similar changes were seen following a variety of treatments targeting SNc neuronal activity, including some which targeted afferent inputs4. This apparent regulation of the number of SNc DArgic neurons by neuronal activity and afferent input raises the possibility that the environment or behavior can influence the number of SNc neurons. Indeed adult mice exposed to different environments have more or less midbrain (SNc and VTA) TH+ neurons, and at least some of these environment-induced changes are abolished by concurrent blockade of synaptic input in midbrain6. The aims of this communication are to: (1) provide further details about how to implement our environmental manipulations and drug infusions; and (2) provide further data supporting our contention that the environment regulates the number of midbrain DA neurons, via afferent input.

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			<td height="21" style="height:21px;width:379px;">Name of Material/ Equipment</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:379px;">Isofluorane</td>
			<td style="width:636px;">Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia</td>
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			<td height="21" style="height:21px;width:379px;">ALZET Osmotic pump 1002</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0004317</td>
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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>
			<td style="width:119px;">n/a</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>
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			<td>chemicon-temecula, CA</td>
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			<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>
			<td style="width:119px;">A3151-1mg</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

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).

Adult mice subjected to these environmental manipulations have altered numbers of midbrain (SNc and VTA), but not LC, TH+ neurons, and EE plus concurrent midbrain infusion of either picrotoxin or bicuculline (GABAA receptor antagonists) abolishes EE-induction of more SNc TH+ neurons. These data were previously published in6. The present data were compiled in replicate experiments performed as part of that previous study, but have not been published elsewhere.
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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.

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

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Neuroscience

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