We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

ملاحظة: وقد تمت الموافقة على جميع الإجراءات بما في ذلك المواد الحيوانية جنة المؤسسي الحيوانات الأخلاقيات (هيئة الطاقة الذرية) في معهد علم الجينوم والبيولوجيا التكاملية، نيودلهي (IGIB / AEC / 10/2013). ويتم تعديل هذا البروتوكول خصيصا لاستهداف تسليم Antagomir-29 في الدماغ وضربة قاضية من مير-29. 1. استراتيجية تصميم Antagomir <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استرداد تسلسل ميرنا ناضجة من miRBase 11 <a href="http://www.mirbase.org/">(http://www.mirbase.org/).</a> </li><li style=";text-align:right;direction:rtl"> استرداد متواليات من أفراد الأسرة ميرنا المتعلقة باستخدام الرابط &quot;جين العائلة&quot; في السجل miRBase <a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">(http://www.mirbase.org/cgi-bin/mirna_summary.pl؟fam=MIPF0000009).</a> </li><li style=";text-align:right;direction:rtl"> عكس تكمل التسلسل: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a> يوفر أداة ملائمة لعكس تسلسل المتممة. </li><li style=";text-align:right;direction:rtl"> بمناسبة المواقف التي يمكن تعديلها من قبل LNA باستخدام الإرشادات التالية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اختيار بقايا ثايمين وضعت 3-4 قواعد بصرف النظر عن تعديل LNA، منذ البريميدينات LNA هي أكثر استقرارا من البيورينات 12. </li><li style=";text-align:right;direction:rtl"> اختيار بقايا السيتوزين مفصولة 3-4 بقايا إذا الخطوة السابقة تنتج أقل من خمسة تعديلات. </li><li style=";text-align:right;direction:rtl"> أولويات التعديلات الخمسة إلى 5 &#39;نهاية antagomir، لأن هذا يتجنب تسلسل البذور. </li></ol></li></ol> 2. Antagomir-نيوروبيبتيدي إعداد مجمع ملاحظة: هذه هي الخطوة الأكثر أهمية في البروتوكول ويحتاج التوحيد. لتوحيد بروتوكول أي قليل النوكليوتيد fluorescently المسمى (FLO) يمكن استخدامها في مكان antagomir 9. يجب أن يكون الببتيدات عالية النقاء (HPLC الصف، 98٪ نقاء) وترد .Sequences والمسؤول عن الببتيد وantagomir في الجدول 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قرر عدد من موليه من antagomir ليتم حقنه على أساس تهمة المولي على antagomir، سميتها والوزن من الفأرة. احسب عدد مولات الببتيد ليتم حقنه على أساس نسبة تهمة المولي من antagomir: الببتيد. ملاحظة: توحيد نسبة تهمة المولي من antagomir: الببتيد لسمية والتسليم الفعال في دماغ الفأر. استخدام نسب مختلفة تهمة المولي من قليل النوكليوتيد fluorescently المسمى: الببتيد لتوحيد. وجدنا أن Antagomir-29 و الرقابة سواء كانت سامة في 4microgram / غرام من وزن الجسم من الماوس. واستخدمت Antagomir-29 والسيطرة على 2microgram / غرام من وزن الجسم، ولكن من المرجح أن تختلف عن كل الرنا الميكروي هذا التركيز الفعال. </li><li style=";text-align:right;direction:rtl"> تمييع الببتيد وantagomir في أنابيب microfuge منفصلة عن الحلول الأسهم العقيمة مع 10٪ D-الجلوكوز إلى التركيز النهائي على النحو المرغوب فيه المحسوبة في الخطوة 1. </li><li style=";text-align:right;direction:rtl"> الحفاظ على microfuge أنبوب مع حل الببتيد في دوامة في معتدلة السرعة vortexing ل. </li><li style=";text-align:right;direction:rtl"> إضافة 1/3 الثالثة من لحل ntagomir إلى أنبوب حل الببتيد، ببطء، وانخفاض الحكمة، في حين يتم مزجه جيدا في خلاط دوامة. </li><li style=";text-align:right;direction:rtl"> مواصلة خلط الحلول على دوامة لمدة 1 دقيقة المقبل ومن ثم السماح للخليط الوقوف لمدة 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> كرر الخطوة 4 و 5 مرتين أكثر لمزج ما تبقى من antagomir الحل مع حل الببتيد في نفس microfuge أنبوب. إضافة بطيئة أمر بالغ الأهمية لتشكيل المجمعات أحادية تفريق تكون فعالة في ترنسفكأيشن. السريعة بالإضافة إلى ذلك يمكن أن يؤدي إلى تجميع المجمعات وهطول الأمطار بهم. </li><li style=";text-align:right;direction:rtl"> احتضان المجمع لمدة 30 دقيقة في RT دون vortexing ل. خلال هذا الوقت من الحضانة، والتأقلم الفئران إلى المختبر حيث سيتم إجراء الحقن. </li></ol> 3. الذيل الوريد حقن مجمع Antagomir-نيوروبيبتيدي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لكبح جماح الحيوان، ضع الماوس في رادع أو decapicone من الحجم المناسب. </li><li style=";text-align:right;direction:rtl"> تنظيف السطح من الذيل باستخدام مسحة القطن العلاقات العامةفي ماء دافئ (حوالي 40 درجة مئوية) غارقة ه. وهذا تشجيع توسع الأوعية وزيادة وضوح الوريد. </li><li style=";text-align:right;direction:rtl"> الاقتراب من الذيل مع حجم صغير (0،5-1،0 سم مكعب) حقنة الانسولين في زاوية 15-20 درجة. يجب الحرص على عدم إدخال أي الهواء في حقنة. تبدأ في الجزء الأعلى من الذيل. حقن المجمع. إزالة الإبرة وتطبيق مسحة مطهر مباشرة إلى موقع الحقن (حوالي 5-10 ثانية) لوقف أي نزيف. </li><li style=";text-align:right;direction:rtl"> استبدال الماوس في قفص التهوية بشكل فردي في مجموعة من 3-5، مع كوز الذره والمناديل الورقية كما تخصيب اليورانيوم. لا تبقي الفئران uninjected وحقن في نفس القفص. </li></ol> 4. السلوكي فحوصات ملاحظة: الحفاظ على الفاصل الزمني من 3-4 ساعة في المقايسات بين الحقن والسلوكية للسماح للانتعاش بعد الحقن. جلب الماوس إلى غرفة هادئة. لا تخل هذه الحيوانات خلال هذه الفترة من التأقلم. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التأقلم الماوس إلىغرفة جديدة لمدة 30 دقيقة ثم بدء فحوصات السلوكية التالية. الحفاظ على فجوة من 10 دقيقة بين كل فحص السلوكي. </li><li style=";text-align:right;direction:rtl"> الشبك Hindlimb: ملاحظة: هذا الاختبار يبين ضعف الحركية وإعاقة للجهاز العصبي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رفع الماوس بلطف من خلال عقد بالقرب من قاعدة الذيل في الخلفية واضحة. </li><li style=";text-align:right;direction:rtl"> التحقق من موقف hindlimb لمدة 10-15 ثانية. النوع البري splays الماوس hindlimbs بعيدا عن البطن، في حين أن الماوس رنحي يميل إلى التراجع واحد أو كلا hindlimbs نحو البطن أكثر من 50٪ من وقت علقت 13. </li><li style=";text-align:right;direction:rtl"> عودة الماوس إلى القفص </li></ol></li><li style=";text-align:right;direction:rtl"> الحافة الاختبار: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رفع الماوس برفق عن طريق الضغط على الذيل ويبقيه على الحافة من القفص. </li><li style=";text-align:right;direction:rtl"> مراقبة الماوس على وجه التحديد في ركن من أركان القفص. النوع البري الماوس يمكن أن تمر زوايا بسهولة دون خوف وفقدان التوازن. الماوس رنحي يفقد توازنه، تجميد أو يهز في حين أن المشي على طول الحافة القفص وعند الزوايا. </li></ol></li><lط&gt; فحص الفأر الطباعة القدم: ملاحظة: للحصول على هذا الاختبار حفاظ على ورقة ماصة على استعداد، على أرضية مدرج الضيق (~ 70 سم طويلة، ~ على نطاق 5 سم، وبها ~ الجدران 5 سم عالية). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عقد الماوس بلطف بواسطة يد واحدة وتطبيق حبر على أطرافه الخلفية بواسطة فرشاة. </li><li style=";text-align:right;direction:rtl"> وضع ورقة ماصة على أرضية مدرج ضيق. </li><li style=";text-align:right;direction:rtl"> السماح الماوس على المشي أو الجري على ورقة ماصة في خط مستقيم في المدرج واحدة من نهاية إلى أخرى. </li><li style=";text-align:right;direction:rtl"> تكرار هذه العملية مرتين أخريين مع كل فأر باستخدام ورقة ماصة جديدة. </li><li style=";text-align:right;direction:rtl"> قياس المسافة بين خطوتين متتاليتين في حركة إلى الأمام. لا تشمل آثار أقدام القليلة الأولى والأخيرة حيث الحيوان هو مجرد بدء والانتهاء من شوطه، على التوالي. </li></ol></li></ol>

<ol>
	<li>Roshan, R., Ghosh, T., Scaria, V., Pillai, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+novel+therapeutic+targets+in+neurodegenerative+diseases.">MicroRNAs: novel therapeutic targets in neurodegenerative diseases.</a> Drug discovery today. 14, 1123-1129 (2009).</li><li>Maes, O. C., Chertkow, H. M., Wang, E., Schipper, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA:+Implications+for+Alzheimer+Disease+and+other+Human+CNS+Disorders.">MicroRNA: Implications for Alzheimer Disease and other Human CNS Disorders.</a> Current Genomics. 10, 154-168 (2009).</li><li>Soifer, H. S., Rossi, J. J., Saetrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+Disease+and+Potential+Therapeutic+Applications.">MicroRNAs in Disease and Potential Therapeutic Applications.</a> Mol Ther. 15, 2070-2079 (2007).</li><li>Bader, A. G., Brown, D., Winkler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Promise+of+MicroRNA+Replacement+Therapy.">The Promise of MicroRNA Replacement Therapy.</a> Cancer research. 70, 7027-7030 (2010).</li><li>Stenvang, J., Petri, A., Lindow, M., Obad, S., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+microRNA+function+by+antimiR+oligonucleotides.">Inhibition of microRNA function by antimiR oligonucleotides.</a> Silence. 3, 1-17 (2012).</li><li>Trang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+Delivery+of+Tumor+Suppressor+microRNA+Mimics+Using+a+Neutral+Lipid+Emulsion+Inhibits+Lung+Tumors+in+Mice.">Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice.</a> Molecular Therapy. 19, 1116-1122 (2011).</li><li>Barbash, S., Hanin, G., Soreq, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotactic+Injection+of+MicroRNA-expressing+Lentiviruses+to+the+Mouse+Hippocampus+CA1+Region+and+Assessment+of+the+Behavioral+Outcome.">Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome.</a> J Vis Exp. (76), e50170 (2013).</li><li>Kumar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transvascular+delivery+of+small+interfering+RNA+to+the+central+nervous+system.">Transvascular delivery of small interfering RNA to the central nervous system.</a> Nature. 448, 39-43 (2007).</li><li>Roshan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-specific+knockdown+of+miR-29+results+in+neuronal+cell+death+and+ataxia+in+mice.">Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice.</a> RNA. 20, 1287-1297 (2014).</li><li>Kaur, H., Wengel, J., Maiti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+DNA&#8722;RNA+Heteroduplex+Formation:+Effects+of+Locked+Nucleic+Acid+Nucleotides+Incorporated+into+the+DNA+Strand.">Thermodynamics of DNA&#8722;RNA Heteroduplex Formation: Effects of Locked Nucleic Acid Nucleotides Incorporated into the DNA Strand.</a> Biochemistry. 47, 1218-1227 (2008).</li><li>Griffiths-Jones, S., Grocock, R. J., Van Dongen, S., Bateman, A., Enright, 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=miRBase:+microRNA+sequences,+targets+and+gene+nomenclature.">miRBase: microRNA sequences, targets and gene nomenclature.</a> Nucleic Acids Research. 34, D140-D144 (2006).</li><li>Kaur, H., Babu, B. R., Maiti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+Chemistry+and+Therapeutic+Applications+of+Locked+Nucleic+Acid+(LNA).">Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LNA).</a> Chemical Reviews. 107, 4672-4697 (2007).</li><li>Guyenet, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Composite+Phenotype+Scoring+System+for+Evaluating+Mouse+Models+of+Cerebellar+Ataxia.">A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia.</a> J. Vis. Exp. (39), (2010).</li><li>Bergen, J. M., Park, I. -. K., Horner, P. J., Pun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+Approaches+for+Neuronal+Delivery+of+Nucleic+Acids.">Nonviral Approaches for Neuronal Delivery of Nucleic Acids.</a> Pharmaceutical Research. 25, 983-998 (2008).</li><li>Zou, L. -. L., Ma, J. -. L., Wang, T., Yang, T. -. B., Liu, C. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Penetrating+Peptide-Mediated+Therapeutic+Molecule+Delivery+into+the+Central+Nervous+System.">Cell-Penetrating Peptide-Mediated Therapeutic Molecule Delivery into the Central Nervous System.</a> Current Neuropharmacology. 11, 197-208 (2013).</li><li>Hwang, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brain-targeted+rabies+virus+glycoprotein-disulfide+linked+PEI+nanocarrier+for+delivery+of+neurogenic+microRNA.">A brain-targeted rabies virus glycoprotein-disulfide linked PEI nanocarrier for delivery of neurogenic microRNA.</a> Biomaterials. 32, 4968-4975 (2011).</li></ol>

The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi...

ظهرت MicroRNAs كأهداف علاجية جديدة بسبب الدور العالمي في تنظيم التعبير الجيني وأدلة مباشرة للمشاركة في المرض. ويجري بحث MiRNAs بنشاط لإمكاناتهم كأهداف المخدرات 1،2. وعلاوة على ذلك، ترتبط التعديلات في التعبير ميرنا مع العديد من الأمراض 3 والمحاكاة من هذه التغييرات اضطراب الاصطناعي من ميرنا التعبير يمكن استخدامها لدراسة مسارات الخلوية المشاركة في مظاهر المرض. الأنسجة تسليم محددة من ميرنا المخدرات تستهدف حاليا تحديا كبيرا لتطوير الأدوية ميرنا القائمة. Antagomirs ويحاكي ميرنا وكلاء واعدة لاحداث بلبلة مستويات ميرنا 4-6. ومع ذلك، السمات الخاصة التي تعزز خصوصيتها والفعالية يجب أن تكون دمجها في تصميم antagomirs قبل أنها يمكن أن تستخدم في الجسم الحي اضطراب ميرنا التعبير. MicroRNAs هي ذات الصلة خاصة<html> كأهداف في الاعصاب غير قابل للشفاء حاليا والأمراض العصبية التطورية. حاجز الدم في الدماغ يشكل قيدا لتسليم antagomirs في الدماغ. يتم استخدام الحقن المجسم على نطاق واسع في نماذج القوارض لتسليم الجزيئات في مواقع محددة في الدماغ 7. فهو يتطلب مهارة والاستثمار المكثف في الأجهزة والوقت. حقن المجسم هي الغازية، تنطوي عملية جراحية، تسبب ما لا يقل عن قاصر الإصابة وتقتصر على التسليم المحلي. استخدام الخلايا اختراق الببتيدات مع تفضيل الخلايا العصبية التي تستهدف يمكن مواجهة هذه القيود لأنها يمكن أن يتم تسليمها عبر الطريق عبر الأوعية الدموية ولكن اختراق حاجز الدم في الدماغ. هذا الببتيد المستمدة من فيروس داء الكلب البروتين السكري (RVG)، وكان يستخدم في السابق لتسليم سيرنا ضد فيروس التهاب الدماغ الياباني في الفئران 8. لقد وجدنا أن استخدام الببتيد لantagomir التسليم، miRNAs يمكن أن تكون فعالة ترسيتها في مخ الفأر 9. ontent &quot;&gt; إن التحدي الرئيسي الثاني للميرنا تدق إلى أسفل ينشأ من صغر حجم miRNAs وجود إيسفورمس تسلسل ارتباطا وثيقا. نحن نأخذ مثال MMU-مير 29 عائلة التي تتكون من ثلاثة إيسفورمس ترتبط ارتباطا وثيقا، مير-29A و b و c. كما المعدلة Antagomirs عموما على طول العمود الفقري لزيادة استقرارها وجعلها مقاومة للهجوم من قبل nucleases. مغلق الأحماض النووية (LNAs) توفر ميزة أخرى أنها تعزز الاستقرار الحراري وتؤدي إلى استهداف تدهور أكثر وخارجها عائق الفراغية 10. تقديم تعديلات على طول العمود الفقري يمكن أن تكون فعالة ولكنها مكلفة. لقد رأينا في وقت سابق أن التعديلات يتجاوز العدد الأمثل قد لا تزيد من تعزيز فعالية. وبالتالي، فإن تصميم antagomir ينطوي على تعديل الأمثل للantagomir. لمجمع antagomir غير تساهمي مع الببتيد موجه للعصب، واتهم سباعي لتمديد NONA أرجينين يستخدم. D-ارجينينوتستخدم المخلفات لأنها تمنح أعلى الاستقرار لأنها ليست عرضة للانشقاق من قبل البروتياز. سباعي إلى NONA أرجينين مساحات العمل خلية اختراق كلاء فعالة قدر، على الرغم من أنها لا تضفي نوع من الخلايا خصوصية. من خلال ربط تساهمي الببتيد RVG إلى رابط NONA أرجينين، وهو موجه للعصب، تم إنشاء خلية اختراق الببتيد. بقايا موجبة الشحنة من الببتيد تتفاعل مع الشحنة السالبة العمود الفقري الحمض النووي، لتشكيل المجمعات. هذه المجمعات يمكن استخدامها ل transfect فعال DNA أو RNA في الخلايا المستزرعة والحية في الأنسجة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

باستخدام الإجراء المقدمة هنا والمجمعات من قليل النوكليوتيد 50microgram fluorescently المسمى (FLO) و ~ 850microgram RVG الببتيد من 1:15 المولي نسبة المسؤول: تم إعداد (FLO الببتيد) وحقنه مرة واحدة فقط خلال الوريد الذيل. وقد استخدم المجمع من غير موجه للعصب مصفوفة فيروس داء الكلب (RVM) الببتيد وFLO كع...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

بديل بسيط لحقن المجسم لالدماغ ضربة قاضية محددة من ميرنا

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

a-simple-alternative-to-stereotactic-injection-for-brain-specific

Research

JoVE Journal

Neuroscience

12.6K Views.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Video: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

والشعيرية والتعامل مع الاختبارات Capellini : تدابير كمية بسيطة من وظيفة forepaw حاذق في الجرذان والفئران

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

تسليم الجينات إلى الدماغ الجرذ بعد الولادة من غير البطين حقن البلازميد وElectroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

التجسيمي جراحة الآفات Excitotoxic من مناطق محددة في الدماغ في الجرذ الكبار

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

جنبا إلى جنب علم البصريات الوراثي وتجميد كسر طبق الاصل Immunolabeling لدراسة ترتيب المدخلات معين من الغلوتامات المستقبلات في اللوزة ماوس

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

بديل والمصادق عليها طريقة حقن للوصول إلى الفضاء تحت الشبكية<i&gt; عبر</i&gt; نهج Transcleral الخلفي

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

طريقة بسيطة لقياس التغيرات في السلوك المكافأة التماس طريق<em&gt; ذبابة الفاكهة</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

ميكروديسيكشن التقاط المناظير باستخدام الأشعة الليزر الموجهة بأطلس الدماغ المناطق المتأثرة بالإصابات الرضية

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

جيل بسيطة من ثقافة عالية الغلة المستحث الخلايا العصبية من الخلايا الليفية الجلد الكبار البشرية

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

الجمع بين أوبتوجينيتيكس ميكرورناس الاصطناعية لتوصيف "تأثيرات الجينات ضربة قاضية" في وظيفة Presynaptic داخل "الدوائر العصبية سليمة"

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

وثمة نهج بديل لدراسة الأحداث الرئيسية في نيوروديجينيريشن باستخدام شرائح المخ الفئران السابقين فيفو

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...