Bu çalışma ve 2,10 olarak anılan, finansmanı, Yaşlılık Araştırma, Edward P. Stiles Fonu ve Kuzeybatı Louisiana Biyomedikal Araştırma Vakfı Amerikan Federasyonu MF Salvatore bir araştırma hibeler ile, kısmen sağlandı ve Ike Muslow predoctoral Kardeşliği BS Pruett için, LSU Sağlık Bilimleri Merkezi-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>

Çıkar çatışması ilan etti.

Şekil 1 &#39;de belirtildiği gibi, yukarıda detaylı yöntemler dopamin birden readouts ve sıçan veya fare ya elde SN ya VTA bir numuneden olan proteinleri düzenleyen TH, DAT ve VMAT2 vermelidir. Yine, bu Protokolün yürütülmesi yararları araştırmacı dopamin hemen her deneysel paradigma altında in vivo olarak düzenlenir ve bunu yaparken, herhangi bir gerekli hayvan sayısını azaltarak önemli deneysel kaynak tasarrufu nasıl bir operasyonel eşleştirilmiş girilmesi gerekmektedir olmalarıdır...

HPLC sisteminde: Temel sistemimizin, bir Shimadzu LC10-ADvp HPLC pompası, bir Waters WISP 717 otomatik örnek enjektör, bir 250 mm x 4.4 5 mikron Spherosorb ODS-1 C18 ters faz kolonu (Waters), bir Bioanalytical Systems (BAS) TL12 dual camsı oluşmaktadır karbon elektrot, iki BAS LC4B elektrokimyasal dedektörler ve Waters 2 veri toplama ve entegrasyon sistemi güçlendirin. Sütun 30-45 tutulur ° C (BAS LC22A kolon ısıtıcı). Mobil faz, 0.1 M sodyum fosfat (pH 3,0), 0.1 mM EDTA, 1-oktan sülfonik asit (Eastman Kodak) 0,2-0,4 mM, ve bir 0.45 mikron filtre ile filtrelenmiştir% 0.35 asetonitril (v / v) &#39;dir. Akış hızı 1.2 ml / dak taşımaktadır. Mobil faz dört litrelik gruplar pH, oktan sülfonik asit ve kolon sıcaklığı ayarlayarak ayırma için optimize edilir. Mobil faz geri kazanılır ve sürekli çözünmüş oksijen kaldırmak için helyum gazı ile temizlenir. Mobil faz, geri dönüşümümakul bir zaman süresi için iyi çözünürlük korumak için hemen hemen esastır. Mobil faz raf ömrü her dönem ilk 2-7 dakika boşa aktarmak için bir akış anahtarı (entegratör tarafından kontrol) ile korunur. Elektrotlar bir Ag/AgC1 referans elektroduna göre yaklaşık 0,78 ve 0.95V potansiyelleri az tutulur. Yüksek potansiyel olarak elektrot triptofan (NMDA ve dahili standart) tayin edilmesi için özel olarak kullanılır. 0.78 V potansiyel triptofan dışındaki monoaminler ve bileşiklerin tespiti için gürültü oranı üstün bir sinyali sağlar. Kromatogramlar güçlendirin iş istasyonunun sabit diskinde saklanan ve daha sonra işlenmiş ve veri metabolit miktarları ve grup veri derleme hesaplanması için bir Excel doğrudan aktarılır. Pompa: Shimadzu LC-10AD Hücre: BAS Çapraz Akış. Camsı karbon 0.780 ile 0.9 elektrot çalışma50 V potansiyel. Dedektör: BAS LC-4B çift kanal modunda çalıştırılır. Veri ACQ. Sistem: Waters Pro 2 güçlendirin. Enjektör: Waters WISP 717 Sütun: Waters Spherosorb ODS-1, 5 uM partikül, 4.4 mm X 250 mm. <table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Sodyum dodesil sülfat (SDS) </td><td> - JT Baker </td><td> 4095-02 </td></tr><tr><td> Trizma Base </td><td> Sigma </td><td> T1503-1KG </td></tr><tr><td> Trizma HCl </td><td> Sigma </td><td> T3253-1KG </td></tr><tr><td> Gliserin </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> Glisin </td><td> Sigma </td><td> G8898-1KG</td></tr><tr><td> Ponceau S </td><td> Fluka </td><td> 81460 </td></tr><tr><td> Bromophenol Mavi </td><td> Sigma </td><td> B8026-5G </td></tr><tr><td> Ditiyotretol </td><td> Sigma </td><td> D-9163 </td></tr><tr><td> Protein Standart 2 mg BSA </td><td> Sigma </td><td> P5619-25VL </td></tr><tr><td> Pierce BCA Protein Testi Reaktif A </td><td> Thermo Fisher Scientific- </td><td> 23223 </td></tr><tr><td> Hassas Plus, Protein Standart </td><td> Bio Rad </td><td> 161-0373 </td></tr><tr><td> [125l]-protein A, spesifik aktivite </td><td> Perkin-Elmer </td><td></td></tr></tbody></table> Tablo 2. Özel reaktifler. <table border="1"><tbody><tr><td> Reaktifler </td><td> Formüller </td></tr><tr><td> % 10 SDS </td><td> 10 g SDS, 100 mL DI H 2 0 </td></tr><tr><td> % 1 SDS (pH 8.2) </td><td><ol><li> 10 mL% 10 SDS </li><li> 60.5 mg Trizma Tabanı </li><li> 37.22 mg EDTA </li><li> 90 mL DI H 2 0 </li></ol></td></tr><tr><td> Bakır II Sülfat Çözüm </td><td><ol><li> 1 g bakır sülfat II </li><li> 25 mL DI H 2 0 </li></ol></td></tr><tr><td> 3X Örnek Tampon </td><td><ol><li> Trizma Base 2.27 g </li><li> SDS 6 g </li><li> Ditiyotretol 0.463 g </li><li> Gliserol 30 g </li><li> Bromophenol Mavi 10 mg </li><li> Dih 2 0 (ilk H 40 ml reaktiflerin üzerinde Mezür 2 0 ekleyin; hacmi 100 ml ulaşana kadar daha sonra H 2 0 ekleyin) </li><li> HCI (ekledikçe 6.85 pH ulaşmak için gerekli) </li><li> 50 2.0 ml tüpler çözüm dondurun. </li></ol> (100 ml yapar): 3X Örnek Tampon hacmi ihtiyaç = örneğinde kullanılan SDS ½ hacmi. </td></tr><tr><td> 1X Örnek Tampon </td><td> DI H 2 0 kullanarak 1X örnek tampon 3X Örnek Tampon aşağı sulandırınız </td></tr><tr><td> 10X<html> Tampon Koşu (4 L yapar) </td><td><ol><li> Trizma Bankası 121.1 g </li><li> Glisin 577 g </li><li> SDS 40 g </li></ol></td></tr><tr><td> 10X Transferi Tampon: (4 L yapar) </td><td><ol><li> Glisin 360 g </li><li> Trizma Base 96 g </li></ol></td></tr><tr><td> Ponceau </td><td><ol><li> Ponceau S. 0.5 g </li><li> Asetik asit 5 mL </li><li> DI H 2 0 95 mL </li></ol></td></tr><tr><td> .2% HCl </td><td> DI H 2 0 500ml 5.2 mL HCl </td></tr><tr><td> PVP-T20 soln engelleme. (4 L yapar) </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 + 500 ml DI H 2 0 13.9 g Tris Base) - 50 ml </li></ol></td></tr><tr><td> 10X Blot Tampon (4 L yapar) </td><td><ol><li> Tween 20 20 g </li><li> Tris Base 14 g </li><li> Tris HCI 61 g </li></ol></td></tr></tbody></table>ontent &quot;&gt; Tablo 3. Protein Testi ve Western Blot Formüller. Tirozin hidroksilaz standartları: Bu laboratuvar tarafından kullanılan kalibre TH protein ve fosforilasyon standartları sonuçta Dr John Haycock 11 laboratuar kaynaklı önceden ayarlanmış TH standartlarına karşı TH ve protein fosforilasyon stoichiometries için analiz edildi PC12 hücre ekstreleri, türetilen .

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

Dopamin MSS bir gayretle çalıştı nörotransmitterdir. Nitekim, lokomotor aktivite ve ödül ile ilgili davranışı kendi katılımı dopamin regülasyonu ile ilişkili moleküler eksikliklerin bir soruşturma beş yıl teşvik etti. Nigrostriatal ve mesoaccumbens yolların terminal alanında bölgelerde düzenlenmesi için moleküler temeli üzerine beyin odak dopamin yönetmeliğin bu soruşturmaların çoğunluğu, striatum ve nukleus akumbens. Ayrıca, bu tür çalışmaların sadece ıslak doku ağırlığı normale dopamin doku içeriğinin analizi üzerinde yoğunlaşmıştır. Böyle tirozin hidroksilaz (TH) protein, TH fosforilasyon, dopamin taşıyıcısı (DAT) ve veziküler monoamin transporter 2 (VMAT2) protein olarak, dopamin düzenleyen proteinlerin incelenmesi genellikle aynı örnek dopamin doku içeriğinin analizini içermez. Dopamin doku içeriği ve düzenleyen proteinleri (post-tra dahil olmak üzere her iki analiz yeteneğinslational değişiklikler) protein düzeyi ve TH, DAT, ya VMAT2 fonksiyonu ile dopamin arasındaki ilişkiyi yorumlama doğasında güç verir, aynı zamanda örnek ekonomisi uzatır sadece. Henüz daha az maliyet ve çevirmenize araştırmacılar &#39;seçim hemen her paradigma dopamin moleküler düzenlenmesi irdeliyoruz üretir. Biz orta beyindeki analizleri odaklanır. SN ve VTA genellikle dopamin regülasyonu en çalışmalarda ihmal rağmen, bu çekirdekler kolayca uygulama ile disseke edilir. Kapsamlı bir dopamin doku içeriği ve TH okuma, DAT, ya VMAT2 yapılabilir. Orada davranışı SN ve VTA dopamin fonksiyonunun etkisi literatürde filizlenen ve orada 1-5 eksojen maddeler veya hastalık süreçleri sıkışmaları edilir. Ayrıca, bu tür büyüme faktörleri gibi bileşikler SN veya VTA içinde nispeten büyük ölçüde, dopamin ve dopamin düzenleyen proteinler üzerinde derin bir etkiye sahiptir <sup&gt; 6-8. Bu nedenle, bu yöntem özel tedaviler davranış ve dopamin regülasyonu modüle nasıl Araştırmalarına uzatmak istiyorsanız laboratuvarlar referans sunulmuştur. Burada, çok adımlı bir yöntem dopamin doku içeriğinin analizi, TH protein düzeyleri, DAT, ya VMAT2 ve kemirgen ortabeyin gelen substantia nigra ve VTA gelen TH fosforilasyon için sunulmuştur. TH fosforilasyon analizi sadece TH aktivitesi nasıl düzenlendiği, aynı zamanda belirli bir paradigma içinde somatodendritik çekirdeklerinde etkilenen sinyal kademelerin önemli anlayışlar elde edebilirsiniz. Biz bu iki çekirdeğin ve her çekirdek (Şekil 1) için spesifik in vivo dopamin regülasyonu moleküler mekanizmaları, ortaya bir profil üreten disseke doku örnek işleme ayırmak diseksiyon tekniği örneklerle gösterecek.

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

Dopamin belirgin hücre gövdeleri ve dopamin nöron dendritler içeren Ortabeyin çekirdekler, düzenlenmiştir. Burada substantia nigra (SN) ve kemirgenlerde ventral tegmental alan (VTA) ve orta beyin çekirdeklerinde dopamin regülasyonu üzerine, böylece bir diseksiyon ve örnek işleme sonuçları maksimize etmek için yaklaşım, sonuç ve görüşlerini açıklar.

Substantia nigra ve ventral Tegmental Bölgesi Dopamin Yönetmelik Kapsamlı Profilleme

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 Görüntüleme.  Louisiana State University Health Sciences Center. Dopamin belirgin hücre gövdeleri ve dopamin nöron dendritler içeren Ortabeyin çekirdekler, düzenlenmiştir. Burada substantia nigra (SN) ve kemirgenlerde ventral tegmental alan (VTA) ve orta beyin çekirdeklerinde dopamin regülasyonu üzerine, böylece bir diseksiyon ve örnek işleme sonuçları maksimize etmek için yaklaşım, sonuç ve görüşlerini açıklar.

Video: 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.

Eşzamanlı Gerilim-kelepçe ve Amperometri ile Dopamin Film Tek Hücre Ölçümü

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

Nörobilim

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.

Ventral Chick orta beyin içinde MikroRNA ovo İfade

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.

Yetişkin Farelerde Orta beyin dopamin Nöronlar Sayısının Çevre Modülleri

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.

Lazer Yakalama Mikrodisseksiyon - Bireysel Dopamin Nöronlar İzolasyonu ve Tüm ventral tegmental Area bir Gösteri

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.

Dopaminerjik Homeostasis farelerde yüksek performanslı sıvı Kromatografi analizi ve Synaptosomal dopamin alımı kullanımı tarafından değerlendirilmesi

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.

Optik Fractionator ve standart mikroskobu ekipmanlar kullanarak fare gözlemliyorum Nigra dopaminerjik nöron sayısının stereological tahmin

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.

Dopamin D1-Alfa reseptör kemirgen Nucleus Accumbens bir yenilikçi RNA Situ içinde hafiye teknoloji tarafından içinde tanımlaması

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.

Akut omurilik dilimleri hazırlanması gözlemliyorum Gelatinosa nöronlar bütün hücreli yama-kelepçe kayıt için

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.

Multipl skleroz bireyler için kapsamlı otopsi programı

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.

Phasic Dopamin'in Ventral Tegmental Alan Reseptör Reseptör Reseptör Düzenlemesini Değerlendirmek için Hızlı Taramalı Döngüsel Voltammetri (CIS-FSCV) ile Kombine infüzyon ve stimülasyon

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