Bu çalışma Müsküler Distrofi Derneği, NIH (NS062320) tarafından desteklenmiştir.

1. Muskarinik asetilkolin reseptör (mAChR) antagonistleri subkutan uygulanması <ol><li> 1.5mL reaksiyon tüp içinde steril serum fizyolojik uyuşturucu eriterek mAChR antagonisti aseptik koşullarda, uygun doz, (bkz. Tablo) altında hazırlayın. Aşağıdaki antagonistleri kullanıldı: atropin, Methoctramine, 4-DAMP AFDX-116, AFDX-384, MT 7. </li><li> A1cc insülin enjektörü çözüm 50μl çizin ve her bir fare için ayrı bir şırınga kullanın. Ayrıca, sadece kontrol grubunda her fare için serum fizyolojik içeren şırınga hazırlar. Enjekte etmek için hazır olana kadar buz üzerinde şırınga tutun. </li><li> Uygun anestezi derinliği tutam ayak yanıt eksikliği gözlemleyerek, ketamin ksilizin kokteyl (120 mg / kg ketamin, 8 mg / kg xylazine) fareler anestezi. </li><li> Uygun sedasyon sonra, boyun kaudal bölge başkanı rostral bölgeyi kapsayan saç tıraş,% 70 etanol ile uzak kalan saç silin. </li><li> Place stereomicroscope altında fare ve şırınga, iğne (Şekil 1A, B) hafifçe yukarı çekerken LAL kas iğne paralel eklemek. Bu bağ dokusu üzerinde iğne sokulur ve LAL kas kendisi doğrudan zarar vermez sağlar. Iğne ucu LAL kas kaudal yönünü kapsayacak şekilde yerleştirin. </li><li> Çok yavaş şırınga geri çekilirken doğru LAL kas üzerinde çözüm enjekte etmeye başlar; toplam enjeksiyon süresi yaklaşık 1 dakika olmalıdır. Düzgün bir şekilde enjekte edilirse, çözüm örten deride en az bir saat kalacak, yaklaşık 1 cm çapında bir &quot;dome&quot; oluşturmalıdır. Düzgün enjekte çözüm sağ LAL kas tüm rostral ve kaudal bölgeleri kapsayacaktır. </li><li> 1 ila 7 gün boyunca her 12 saatte bir aynı işlemi tekrarlayın. Diğer studies1 için, büyüme faktörleri, her 12 saatte bir 21 gün kadar enjekte etmek için aynı protokol kullanılır. 21 güne kadar, anestezi 2x bir gün ile uygulanan farelerde anormal tepkiler gözlendi. </li></ol> 2. LAL kaslarının diseksiyonu <ol><li> Pentobarbital sodyum aşırı dozda (390 mg / kg) ile fareler Euthanize. Ölüm torakotomi sonra kalp atışı eksikliği değerlendirerek güvence altına alınmıştır. </li><li> 1 pin ve burundan 1 pin her pençe, bir diseksiyon çanak dorsal tarafına fareyi yukarı aşağı pin. </li><li> Sol, sol kürek kemiği çevresinde bölgeye kulak sadece proksimal bölgeden LAL birlikte kesmek için küçük yaylı makas kullanılarak, sadece deri yoluyla ilk kesi olun. </li><li> Bu bölgede cilt dikkatlice çıkarın, ama bu doğru LAL kas ek puan olarak sağ kulak çok yakın kesme önlemek. </li><li> Başın yüzeysel yüzeyinde orta hat boyunca sağ ve sol LAL kasları karşılayan lipid merkezi bir konuma sahip yağ dokusu şeridi, bulun. Küçük yaylı makas kullanılarak, omuz (Şekil 1C) ulaşana kadar sağ sol LAL kas proksimal kenarından yağ dokusu (sol kulak doğru) 1cm kesti. </li><li> Bir kez kesi, geriye doğru LAL kas kas ventral tarafında ortaya çıkarmak için küçük forseps ile soyma yapılır. Forseps (Şekil 1C) ile sağ LAL kas dikkatle yukarı çekerken, bağ dokusu ve fasya kesin. </li><li> Kulağının dibinde kaudal sonuna doğru LAL etrafında kesin. , Kesim, sağ kulak rostral sonuna doğru hareket, sağ LAL (Şekil 1C) devredildi tutarak devam edin . LAL kendisi zarar riski daha bu aşamada daha fazla doku dahil etmek daha iyidir. Kas, pipet 1X PBS kas üzerinde kurumaya başlar. </li><li> Place 1X PBS, dorsal tarafına içeren 30mm kültür Sylgard çanak LAL disseke. Küçük böcek iğneli kas dört köşe aşağı pin. </li><li> Sağ LAL kas dorsal ve ventral yüzeyleri küçük forseps ve yaylı makas, temiz, bağ dokusu kullanma. Ventral yüzeyi temizlemek için kas ters çevirin ve yeniden-pin. 2-3 kaslar aynı 30mm çanak yerleştirilmiş olabilir. </li></ol> 3. LAL NMJs Triple immün: 1. Gün <ol><li> Başında immün önce, gerekli tüm reaktifler hazırlamak. 1X PBS,% 2 sığır serum albumin BSA) / PBS,% 2 0.1M Glycine BSA / PBS,% 2 BSA / PBS, ve% 4 1X PBS paraformaldehid (PFA)% 0.2 Triton X100 ihtiyaç duyulacaktır. -20 ° C derin dondurucuda Pre-soğuk metanol. </li><li> Bu işlem sırasında çanak dorsal tarafına tutturulmuş LAL kasları bırakın. 1X PBS çözümü atın ve kas karşılamak için yeterli% 4 (PFA) ekleyebilirsiniz. Çalkalayıcı yerleştirin çanak 20 dakika boyunca 2 veya 3 hız ayarlayın. Aksi belirtilmedikçe, aşağıdaki adımları çanak çalkalayıcı üzerinde yer olmalıdır. </li><li> % 4 PFA atın kas ve 1X PBS 3 kez, 10 &#39;, her ile yıkayın. </li><li> Son 1X PBS 2% 30 &#39;BSA / PBS çözüm 0.1M Glycine yıkayın ve eklemek atın. </li><li> 0.1M Glycine çözümü bir atınd eklemek 1X PBS 1:200 15 &#39;lik bir konsantrasyon rodamin konjuge α-bungarotoxin içeren. </li><li> Α-bungarotoxin çözüm atın ve 1X PBS, 3 kez 10 &#39;her kas yıkayın. </li><li> Ön soğutmalı metanol ekleyerek doku Permeabilize ve tam 5 dakika boyunca -20 ° C derin dondurucuda çanak, hiçbir ihtiyaç sallayarak. </li><li> Metanol çıkarın ve 3 kez 10 &#39;her 1X PBS ile yıkayın. Ile son yıkama ve blok doku atın% 0.2 triton X-100,% 2 BSA / PBS 1 saat. Yıkama ve engelleme adımları oda sıcaklığında yapılır. </li><li> 4, titreme, gecede kasları inkübe ° C primer antikorları bloke çözüm (bkz. Tablo) seyreltilmiş bir kokteyl. </li></ol> 4. LAL NMJs Triple immün: 2. Gün <ol><li> 1X PBS 3 kez, 10 &#39;oda sıcaklığında her kasları yıkayın. </li><li> Oda sıcaklığında 1 saat (bkz. Tablo) çözümü engelleme ikincil antikor kokteyli ekleyin. Foto-Alexa-Fluor 647 floresan beyazlatma önlemek için kasların bu adımı itibaren ışıktan korunmalıdır. </li><li> 1X PBS 3 kez, 10 &#39;, her kasları yıkayın. </li><li> 1X PBS içinde kalan bağ dokusu Temiz, LAL kasların lateral sınırları kesilmiş ve slaytlar dorsal tarafına monte cam kapak fişleri ve montaj medya kullanarak. Yerinde kapak kayma güvenliğini sağlamak için açık bir oje kullanın. </li><li> Analiz için hazır olana kadar -20 ° C&#39;de ışık ve mağaza slaytlar koruyun. </li></ol> 5. LAL NMJs Konfokal görüntüleme <ol><li> Yüksek çözünürlüklü konfokal görüntüler, Leica TCS 4D konfokal mikroskop Leica Planı Apo 63X yağ objektif (1.4NA) ile elde edilir. </li><li> Floresein ve Alexa 647 sinyalleri aynı anda sırasıyla, lazer çizgileri 488 nm (Argon, 488 nm, 20 mW) ve 633 nm (HeNe, 633 nm, 10 mW) ile tarandı. Sırayla, rodamin sinyal sonra lazer hattı ile 561 nm (DPSS, 561, 20 mW) taranır. Optik bölümleri 0.3 mikron aralıklarla toplanan ve z-düzleminde görüntü sayısı 60-80 arasında değişiyordu. Iğne deliği Airy 1 (107,97 um) göre ayarlanır. Görüntü formatı 1024 x 1024 (X, Y), az 1 234,32 mm x 234,32 mm ve 229,05 nm x 229,05 nm piksel boyutunda bir görüntü boyutu 400Hz hızlı bir zoom faktörü. </li><li> Leica TCS-NT kazanım yazılım sonra maksimum yoğunluğu projeksiyonları z serisi görüntüleri yeniden oluşturmak için kullanılır. </li><li> Multipanel görüntüleri Adobe Photoshop kullanarak, parlaklık ve kontrast ayarlanır. </li><li> Tipik bir LAL kas için, biri yaklaşık 75-100 tr yüz NMJs analiz etmek gerekir. Synaptic istikrar gibi pre-, peri-ve postsinaptik unsurların mekansal uyumu (yani sinir, Schwann hücreleri ve kas) gibi özellikleri ve sinaptik alan ölçümleri (yani sinaps boyutu) tarafından belirlenir. </li></ol> Temsilcisi sonuçları: Nikotinik AChRs postsinaptik kümeleri (Kırmızı Şekil 2) tam apposed tek bir kas lifi, yetişkin bir memeli NMJ, tek bir motor akson son derece sinir terminal farklılaşmış milleri (Yeşil Şekil 2) formunu ince dalları ayrıntılarına . Perisynaptically, terminal Schwann hücreleri sıkı presinaptik sinir terminalleri (Blue Şekil 2) tüm dallarını kapsamaktadır. Bu üçlü organizasyonun yapısal ve işlevsel bütünlüğü ciddi alt tipi özel mAChR inhibitörleri günlük uygulama tarafından rahatsız. Burada sunulan örnek (Şekil 3), 4-DAMP, M1, M3, M4 ve M5 mAChR alt tipleri için yüksek afinite ile mAChR antagonisti (Şekil 3B, kas yüzeyi boyunca sayısız NMJs sinir terminallerine seçici eleme çağrıştırıyor C). Buna ek olarak, terminal Schwann hücrelerinin süreç uzantısı olmadan parlak S100 etiketleme (Şekil 3B, 3C) ile kanıtlanan 8 anormal suskun. Postsynaptically, kas lifleri normal ve nAChRs, hiçbir kayıp vardır (Şekil 3B, 3C). <img src="/files/ftp_upload/3124/3124fig1.jpg" alt="Şekil 1"/> Şekil 1 LAL kas Anatomik yeri ve organizasyonu. Rostral (rLAL), kaudal (cLAL) ve LAL sinir (LALn) ve buna karşılık gelen uç plakası bantları yeri (A, içerlek) gösterilmiştir. LAL kas göre iğne konumu ve enjeksiyon işlemi (B) gösterilmiştir. Kesi noktaları diseksiyon prosedürü (C) gösterilmiştir. <img src="/files/ftp_upload/3124/3124fig2.jpg" alt="Şekil 2"/> Şekil 2 NMJ Üçlü organizasyonu. Yüksek büyütmeli bir fare NMJ konfokal kez. Tek bir akson terminal Schwann hücresi ve süreçler (terminali Schwann hücre gövdeleri A &#39;, yıldızlarla noktası) tarafından sıkıca kapalı ince terminal dalların (A), ayrıntılarına. Postsynaptically bir kas lifi, nikotinik AChRs bir küme hassas sinir terminaline dalları apposed.yalleri ve terminal Schwann hücresi. <img src="/files/ftp_upload/3124/3124fig3.jpg" alt="Şekil 3"/> Şekil 3 LAL kasları 4-DAMP, mAChR antagonisti ile tedavi. LAL kasların Düşük ve yüksek büyütmeli konfokal kez araç ya da 4-DAMP ile tedavi edildi. Araç ile tedavi edilen kas (4 DAMP tedavi kas eksikliği sinir terminalleri (A-A&#39;&#39;&#39;), sayısız NMJs aksine B-B &#39;, C-C&#39;&#39;&#39;). Şekil 2B kutulu alan Şekil 2C yakınlaştırılmış.

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Çıkar çatışması ilan etti.

Burada sunulan yöntem memeli NMJs istikrar ve bakım sinyalizasyon alt tipi özel mAChR önceden tanınmayan rolleri soruşturma izin verir. Bu yöntem ayrıca nörotrofik faktörler ve farmakolojik ajanların etkilerini test etmek için faydalı olacaktır. Örneğin, laboratuar Silier Nörotrofik Faktör (CNTF) yetişkin farelerde neredeyse tüm LAL sinir terminallerinden çimlenme yol açtığını <a>1</a> . Bu sonuç ca az çimlenme orta bildirdi CNTF ile tedavi edilen arka bacak kasları, önceki çalışmalarla ...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> ketamin </td><td> Hospira </td><td> NDC0409-2051-05 </td><td> Doz: 120mg/kg </td></tr><tr><td> ksilizin </td><td> Lloyd Laboratuvarları </td><td> LA33806 </td><td> Doz: 8mg/kg </td></tr><tr><td> atropin </td><td> Sigma-Aldrich </td><td> A0132 </td><td> (&gt;% 98 saflıkta); Doz: 0.2mg/kg - 20mg/kg </td></tr><tr><td> atropin </td><td> Voigt Global Dağıtım </td><td> AT105 </td><td> İlaç grade </td></tr><tr><td> Methoctramine </td><td> Sigma-Aldrich </td><td> M105 </td><td> Doz: 100 - 400M </td></tr><tr><td> 4-DAMP </td><td> Sigma-Aldrich </td><td> D142 </td><td> Doz: 2.5mg/kg </td></tr><tr><td> AFDX-116 </td><td> Bioscience Tocris </td><td> 1105 </td><td> 250M </td></tr><tr><td> AFDX-384 </td><td> Bioscience Tocris </td><td> 1345 </td><td> 50M - 500M </td></tr><tr><td> MT 7 </td><td> Peptitler Uluslararası </td><td> PMT-4340-s </td><td> 0.1M - 1M </td></tr><tr><td> 1X Fosfat Tuzlu, pH 7.4 Tamponlu </td><td> Invitrogen </td><td> 10010049 </td><td></td></tr><tr><td> Paraformaldehit </td><td> Balıkçı </td><td> T353-500 </td><td> 10g/100mL deiyonize distile su eriterek ilk% 10&#39;luk olun; 1X PBS ile% 4, pH 7.4 &#39;e ayarlamak </td></tr><tr><td> Pentobarbitol Sodyum </td><td> Virbac Hayvan Sağlık </td><td> NDC-051.311-050-01 </td><td> Doz: 390mg/kg </td></tr><tr><td> Sylgard </td><td> Dow Corning </td><td> Bölüm # 184 </td><td> Ihtiyaçlarına bağlı olarak birden fazla ölçekli kültür çanak kullanabilirsiniz (30mm, 60mm, 100mm), kit ile birlikte gelen yönergeleri izleyin </td></tr><tr><td> 0.1M Glycine </td><td> Sigma-Aldrich </td><td> G-7126 </td><td> % 2 BSA / PBS 0.185g için 25ml </td></tr><tr><td> % 2 Sığır serum albumin (% 2 BSA) </td><td> Sigma-Aldrich </td><td> A3059-100g </td><td> 1X PBS 100 ml içine 2g BSA çözülür </td></tr><tr><td> % 2% 0.2 Triton X 100 BSA / PBS (Engelleme Tampon) </td><td> Sigma-Aldrich </td><td> T9284-100 ml </td><td> 0.2ml/100mL% 2 BSA / PBS çözülür </td></tr><tr><td> α-bungarotoxin </td><td> Invitrogen </td><td> T1175 </td><td> 1:200, konsantrasyon kullanın </td></tr><tr><td> SMI-312 </td><td> Sternberger Monoclonals </td><td> SMI312 </td><td> 1:1000 konsantrasyon kullanın </td></tr><tr><td> SV2 </td><td> Gelişim Çalışmaları Hibridoma Bankası </td><td> SV2-Süzüntü </td><td> 1:10 konsantrasyonda kullanın </td></tr><tr><td> S100 </td><td> Dako </td><td> Z0311 </td><td> 1:400, konsantrasyon kullanın </td></tr><tr><td> FITC-keçi anti-fare IgG1 </td><td> Roche </td><td> 03117731001 </td><td> Konsantrasyonu 1:200 kullanın, ama arka planda yüksek ise, 1:400 deneyin </td></tr><tr><td> Alexa Fluor 647 konjuge keçi anti-tavşan </td><td> Invitrogen </td><td> A21244 </td><td> 1:200, konsantrasyon kullanın </td></tr><tr><td> Vectashield floresan montaj medya </td><td> Vektör laboratuvarlar </td><td> H-1000 </td><td> Bu sert ayarlanmış bir medya değil, açık bir oje ile kapak kayma güvenliğini sağlamak için gerekecektir. </td></tr><tr><td> Küçük Bahar Makas </td><td> Güzel Bilim Araçları </td><td> 15002-08 </td><td></td></tr><tr><td> Diseksiyon forseps </td><td> Güzel Bilim Araçları </td><td> 11295-51 </td><td></td></tr></tbody></table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

Gastroknemius ve tibialis anterior gibi kemirgenler, Hind bacak kasları memeli NMJs oluşumu ve korunması için gerekli sinyalleri in vivo farmakolojik çalışmalarda sık sık kullanılır . Ancak, subkutan veya intramusküler uygulamadan sonra bu kasların içine ilaç penetrasyonu genellikle eksik ya da düzensiz ve birçok NMJs etkilenmez. Mini pompaları gibi cihazlar ile sistemik yönetim Spatiotemporal etkilerini artırabilir olsa da, bu yaklaşımın invaziv doğası karıştırıcı inflamatuvar yanıtları ve / veya doğrudan kas hasarına neden olabilir. Ayrıca, zaman alıcı bir seri kesit ve kapsamlı immün gerektirdiğinden bir arka bacak kas NMJs tam analizi zordur. Fare LAL boyun dorsum yüzeysel olarak bulunan ince bir kas, düz levha. Bu, hızlı kasılan kas fonksiyonları kulak kepçesi taşımak için. Kafatası orta hat kaynaklanan ve her kepçesi kıkırdak kısmının yanal uzatmak rostral ve kaudal kısımları içerir. Kas çıkar Stylomastoid foramen olarak kaudal projeler yüz sinirinin bir şube tarafından verilir. Biz ve diğerleri, hem de kısa ve uzun vadeli NMJs ve kasları ilaçların in vivo etkilerinin araştırılması için avantaj sunan uygun bir hazırlık olması amacıyla LAL bulduk . İlk olarak, yüzeysel bir konumda, hafif anestezi altında ilaçlar birden fazla yerel uygulamaları kolaylaştırır. İkincisi, inceliği (2-3 kat kas liflerinin), kas içinde hemen hemen tüm NMJs görselleştirme ve analiz izin verir. Üçüncü olarak, onun innervasyon deseni ile birlikte sinir bozulmadan diseksiyon kolaylığı, ek elektrofizyolojik analizi, in vitro 9,5 izin verir. Belki de son ve en önemlisi, uygulanan küçük bir hacim (~ 50μl) kolayca tüm kas yüzeyi kapsayan tüm NMJs ilaç düzgün ve uzun süreli maruz kalma sağlar ve sistemik bir yaklaşım 1,8 ihtiyacını ortadan kaldırır .

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Levator auris longus (LAL) genç erişkin farelerin kas muskarinik sinyalizasyon inhibitörleri tekrarlanan yönetim ve wholemounts nöromüsküler kavşak sonraki immün (NMJs) için prosedürleri açıklar. LAL kas ifşa ettiği için benzersiz avantajları vardır.<em&gt; In vivo</em&gt; NMJs farmakolojik etkileri.

Subkutan Muskarinik Antagonistleri Yönetim ve Farelerde levator Auris longus&#39;un Triple-immün

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K Görüntüleme.  Arcadia University. Levator auris longus (LAL) genç erişkin farelerin kas muskarinik sinyalizasyon inhibitörleri tekrarlanan yönetim ve wholemounts nöromüsküler kavşak sonraki immün (NMJs) için prosedürleri açıklar. LAL kas ifşa ettiği için benzersiz avantajları vardır. In vivo NMJs farmakolojik etkileri.

Video: Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Sıçanlar İlaç Öz-yönetim sırasında Ultrasonik seslenmelere Değerlendirilmesi

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Nörobilim

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Kronik Nikotin İdaresi fareler Knock-in içinde floresan etiketli Nikotinik Reseptör Spektral Konfokal Görüntüleme

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Gaz kromatografisi ile operant etanol kendi kendine uygulama ve etanol belirleme sırasında etanol mikrodivaliz

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Yengeç Parmak Propriyoseptör ve Gerilim reseptörleri: Öğrenci Laboratuvarı Egzersizler

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

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, ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Fareler Soğuk Hassasiyeti ve Uyum belirlenmesi için Basit ve ucuz Yöntem

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Video Görüntüleme ve Spatiotemporal Haritalar Fare Gastrointestinal Motilite Analiz

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Gerçek zamanlı yönetim fMRI psikoterapi eğitimi için bir iletişim kuralı

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Levator Auris Longus Gerilim kelepçe koşullar altında memeli nöromuskuler iletim Sınavı Hazırlık

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.