Financement pour le travail cela, et comme cité 2,10, a été fourni, en partie, par un octroi de subventions de recherche à MF Salvatore de la Fédération américaine de recherche sur le vieillissement, Edward P. Le Fonds d&#39;affectation spéciale et Stiles Biomedical Research Foundation du Nord-Ouest en Louisiane, et à la norme BS Pruett de la Bourse Muslow Ike prédoctoral, LSU Health Sciences Center-Shreveport.

<ol>
	<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>

Pas de conflits d&#39;intérêt déclarés.

Comme il est indiqué dans la figure 1, les méthodes décrites ci-dessus devrait produire plusieurs lectures de la dopamine et son TH régulation des protéines, DAT, et VMAT2 d&#39;un échantillon de SN ou VTA obtenu à partir de chez le rat ou la souris. Encore une fois, les avantages de la réalisation de ce protocole sont que l&#39;enquêteur peut obtenir un indicateur de vue opérationnel appariés de la façon dont la dopamine est régulée in vivo dans pratiquement n&#39;importe quel paradigme ...

Système HPLC: Le système de base se compose d&#39;un Shimadzu LC10-ADVP pompe HPLC, un échantillon des eaux WISP 717 injecteur automatique, d&#39;un 250 X 4,4 mm 5 microns Spherosorb ODS-1 C18 colonne en phase inverse (Waters), un Bioanalytical Systems (BAS) TL12 vitreux double électrode de carbone, deux BAS LC4B détecteurs électrochimiques, et quelques eaux Empower 2 collecte de données et système d&#39;intégration. La colonne est maintenue à 30-45 ° C (chauffe BAS colonne LC22A). La phase mobile est de 0,1 M de phosphate de sodium (pH 3,0), EDTA 0,1 mM, 0,2-0,4 mm 1-octane acide sulfonique (Eastman Kodak-), et de l&#39;acétonitrile 0,35% (v / v), filtré à travers un filtre de 0,45 micron. Le débit est de 1,2 ml / min. Quatre lots litre de la phase mobile sont optimisés pour des séparations en ajustant le pH, d&#39;acide sulfonique et d&#39;octane température de la colonne. La phase mobile est recyclé, et est continuellement purgé avec un gaz d&#39;hélium pour éliminer l&#39;oxygène dissous. Le recyclage de la phase mobileest presque indispensable de maintenir une bonne résolution pour une période de temps raisonnable. La phase mobile durée de vie est maintenu par l&#39;aide d&#39;un détecteur de débit (contrôlé par l&#39;intégrateur) pour détourner de perdre le premier 2-7 min de chaque course. Les électrodes sont maintenues à des potentiels d&#39;environ 0,78 et 0,95 V par rapport à une électrode de référence Ag/AgC1. L&#39;électrode à la plus élevée possible est utilisé exclusivement pour la détermination de tryptophane (et de la norme interne NMDA). Le potentiel 0,78 V fournit un signal supérieure à bruit pour la détection des monoamines et des composés, autres que le tryptophane. Les chromatogrammes sont stockés sur le disque dur du poste de travail Empower, puis traitées et les données transférées directement dans un tableur Excel pour le calcul des montants de métabolites et de compilation des données du groupe. Pompe: Shimadzu LC-10AD Cellulaire: BAS Croix débit. Carbone vitreux électrode de travail à 0,780 et 0,950 potentiel V. Détecteur: BAS LC-4B fonctionnant en mode dual channel. Données Acq. Système: Waters Empower Pro 2. Injecteur: WISP Waters 717 Colonne: Waters Spherosorb ODS-1, 5 uM de particules, 4,4 mm X 250 mm. <table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Dodécylsulfate de sodium (SDS) </td><td> - JT Baker </td><td> 4095-02 </td></tr><tr><td> Base de Trizma </td><td> Sigma </td><td> T1503-1KG </td></tr><tr><td> Trizma HCl </td><td> Sigma </td><td> T3253-1KG </td></tr><tr><td> Glycérol </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> Bleu de bromophénol </td><td> Sigma </td><td> B8026-5G </td></tr><tr><td> Dithiothreitol </td><td> Sigma </td><td> D-9163 </td></tr><tr><td> Standard Protéines 2 mg de BSA </td><td> Sigma </td><td> P5619-25VL </td></tr><tr><td> Pierce BCA Protein Assay Reagent A </td><td> Thermo Fisher Scientific- </td><td> 23223 </td></tr><tr><td> Precision Plus Protein standard </td><td> Bio Rad </td><td> 161-0373 </td></tr><tr><td> [125I]-protéine A, l&#39;activité spécifique </td><td> Perkin-Elmer </td><td></td></tr></tbody></table> Tableau 2. Réactifs spécifiques. <table border="1"><tbody><tr><td> Réactifs </td><td> Formules </td></tr><tr><td> SDS à 10% </td><td> 10 g de SDS, 100 ml DI H 2 0 </td></tr><tr><td> 1% (pH à 8,2) SDD </td><td><ol><li> 10 mlSDS à 10% </li><li> 60,5 mg de base Trizma </li><li> 37,22 mg d&#39;EDTA </li><li> 90 ml DI H 2 0 </li></ol></td></tr><tr><td> Solution de sulfate de cuivre II </td><td><ol><li> 1 Cuivre II sulfate de g </li><li> 25 ml DI H 2 0 </li></ol></td></tr><tr><td> Sample Buffer 3X </td><td><ol><li> Trizma Base de 2,27 g </li><li> SDS 6 g </li><li> Dithiothreitol 0,463 g </li><li> Glycérol 30 g </li><li> Le bleu de bromophénol 10 mg </li><li> DIH 2 0 (d&#39;abord ajouter ci-dessus réactifs à 40 mL de H 2 0 dans une éprouvette graduée, puis ajouter H 2 0 jusqu&#39;à ce que le volume atteint 100 ml) </li><li> HCl (ajouter au besoin pour atteindre un pH de 6,85) </li><li> Congeler solution dans des tubes de 50 ml 2.0. </li></ol> (Fait 100 ml): Volume de tampon d&#39;échantillon nécessaire 3X = volume ½ de la SDD utilisé dans l&#39;échantillon. </td></tr><tr><td> 1X tampon d&#39;échantillon </td><td> Diluer le tampon de l&#39;échantillon jusqu&#39;à 3X 1X tampon d&#39;échantillon à l&#39;aide DI H 2 0 </td></tr><tr><td> 10X<html> Exécution de tampon (Donne 4 L) </td><td><ol><li> Trizma Base de 121,1 g </li><li> Glycine 577 g </li><li> SDS 40 g </li></ol></td></tr><tr><td> Tampon de transfert 10X: (Donne 4 L) </td><td><ol><li> Glycine 360 ​​g </li><li> Trizma Base de 96 g </li></ol></td></tr><tr><td> Ponceau </td><td><ol><li> Ponceau S de 0,5 g </li><li> Acide acétique à 5 ml </li><li> DI H 2 0 95 ml </li></ol></td></tr><tr><td> 0,2% Solution HCl </td><td> 5,2 ml de HCl dans 500 ml de DI H 2 0 </td></tr><tr><td> PVP-T20 blocage Soln. (Donne 4 L) </td><td><ol><li> PVP-40 40 g </li><li> dPBS 38,2 g </li><li> Tween 20 2 g </li><li> Thimerisol 0,4 g </li><li> 1M Tris pH 7,6 (60,6 g de Tris HCl + 13,9 g de Tris base dans 500 ml H 2 0 DI) - 50 ml </li></ol></td></tr><tr><td> Tampon 10X Blot (Donne 4 L) </td><td><ol><li> Tween 20 20 g </li><li> Tris Base 14 g </li><li> Tris-HCl 61 g </li></ol></td></tr></tbody></table>ontenu &quot;Protein Assay&gt; Tableau 3. et de l&#39;Ouest Formules buvard. Normes de la tyrosine hydroxylase: Les calibrés TH normes de protéines et de la phosphorylation utilisés par ce laboratoire proviennent de PC12 extraits cellulaires, qui ont été analysés pour la teneur en protéines TH et stoechiométries phosphorylation contre un TH normes préalablement calibrées qui a finalement origine du laboratoire du Dr John Haycock 11 .

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

La dopamine est un neurotransmetteur vigoureusement étudiée dans le SNC. En effet, son implication dans l&#39;activité locomotrice et la récompense liée au comportement a favorisé cinq décennies d&#39;enquête sur les carences moléculaires associés à la réglementation la dopamine. La majorité de ces demandes de régulation de la dopamine dans le cerveau sur mise au point de la base moléculaire de sa réglementation dans les régions de champ terminales des voies nigrostriataux et mesoaccumbens; striatum et le noyau accumbens. En outre, ces études se sont concentrées sur l&#39;analyse du contenu des tissus de la dopamine avec la normalisation du poids des tissus seulement humide. Enquête sur les protéines qui régulent la dopamine, comme la tyrosine hydroxylase (TH) des protéines, la phosphorylation TH, transporteur de la dopamine (DAT), et vésiculaires transporteur de monoamines 2 (VMAT2) protéine souvent ne comprennent pas l&#39;analyse du contenu des tissus de la dopamine dans le même échantillon. La capacité à analyser à la fois le contenu des tissus de la dopamine et de ses protéines régulatrices (y compris post-tramodifications nslational) ne donne pas seulement le pouvoir inhérent à l&#39;interprétation de la relation de la dopamine au niveau des protéines et la fonction de TH, DAT ou VMAT2, mais s&#39;étend également l&#39;économie de l&#39;échantillon. Cela se traduit par un coût moindre, tout en produisant un aperçu de la régulation moléculaire de la dopamine dans pratiquement n&#39;importe quel paradigme de choix des enquêteurs. Nous nous concentrons les analyses dans le mésencéphale. Bien que le SN et VTA sont généralement négligé dans la plupart des études de régulation de la dopamine, ces noyaux sont facilement disséqué avec la pratique. Une lecture complète du contenu du tissu de la dopamine et TH, DAT ou VMAT2 peut être effectuée. Il est en plein essor la littérature sur l&#39;impact de la fonction de dopamine dans le SN et VTA sur le comportement, et les empiétements de substances exogènes ou des procédés qui y sont la maladie 1-5. En outre, des composés tels que les facteurs de croissance ont un effet profond sur les protéines de la dopamine et la dopamine-régulation, dans une mesure relativement plus grande dans le SN ou VTA <sup&gt; 6-8. Par conséquent, cette méthode est présentée à titre de référence aux laboratoires qui veulent étendre leurs enquêtes sur la façon dont les traitements spécifiques moduler le comportement et régulation de la dopamine. Ici, une méthode multi-étape est présentée pour les analyses de la teneur des tissus dopamine, les teneurs en protéines de TH, DAT ou VMAT2 et la phosphorylation de la TH substantia nigra et VTA de mésencéphale rongeurs. L&#39;analyse de la phosphorylation de TH peut donner des informations importantes sur la façon dont non seulement l&#39;activité TH est réglementée, mais aussi les cascades de signalisation affectées dans les noyaux somato dans un paradigme donné. Nous allons illustrer la technique de dissection de séparer ces deux noyaux et le traitement d&#39;échantillons de tissus disséqués qui produit un profil révélant les mécanismes moléculaires de régulation de la dopamine in vivo, spécifiques pour chaque noyaux (figure 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

La dopamine est nettement réglementée dans les noyaux du mésencéphale, qui contiennent les corps cellulaires et les dendrites des neurones dopaminergiques. Nous décrivons ici une approche de dissection et manipulation d&#39;échantillons de maximiser les résultats, et donc les conclusions et les points de vue, sur la réglementation de la dopamine dans les noyaux du mésencéphale de la substantia nigra (SN) et l&#39;aire tegmentale ventrale (VTA) chez les rongeurs.

Profilage global de régulation de la dopamine dans la substance noire et aire tegmentale ventrale

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

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Neuroscience

25.6K vues.  Louisiana State University Health Sciences Center. La dopamine est nettement réglementée dans les noyaux du mésencéphale, qui contiennent les corps cellulaires et les dendrites des neurones dopaminergiques. Nous décrivons ici une approche de dissection et manipulation d'échantillons de maximiser les résultats, et donc les conclusions et les points de vue, sur la réglementation de la dopamine dans les noyaux du mésencéphale de la substantia nigra (SN) et l'aire tegmentale ventrale (VTA) chez les rongeurs....

Vidéo: 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.

Mesure Single Cell de la libération de dopamine avec Voltage-clamp simultanée et Amperometry

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

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In ovo Expression of MicroRNA in Ventral Chick Midbrain

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Dans Expression in ovo de microARN dans ventrale Poussin mésencéphale

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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).

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.

Modulations environnementaux du mésencéphale Nombre de dopamine neurones chez la souris adulte

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.

Microdissection laser - une démonstration de l&#39;isolement de la dopamine neurones individuels et de l&#39;Espace ventrale tegmentale entière

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.

Évaluation de l’homéostasie des dopaminergiques chez les souris par l’utilisation de l’analyse de chromatographie liquide à haute performance et l’absorption de la Dopamine

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.

Stéréologique Estimation du nombre de neurones dopaminergiques dans la Substantia Nigra de souris à l’aide de l’optique fractionnateur et l’équipement de microscopie Standard

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.

Identification de la Dopamine D1-Alpha du récepteur au sein de rongeurs noyau Accumbens par une ARN innovantes In Situ la technologie de détection

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.

Préparation de tranches médullaires aiguës pour l’enregistrement de Patch-clamp de la cellule entière dans les neurones de Substantia 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.

Programme complet d'autopsie pour les personnes atteintes de sclérose en plaques

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.

Perfusion et stimulation combinées avec voltampérométrie cyclique à balayage rapide (CIS-FSCV) pour évaluer la régulation du récepteur de la zone tegmentale ventrale de la dopamine phasique

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