We are grateful for the support of the NREF foundation.

The authors have no disclosures.

بعد عمل معلما قبل Benabid وآخرون في استخدام التحفيز العميق للمخ لعلاج المرض والهزة الضروري باركنسون، وقد تم التحقيق في تقنية جراحية DBS باهتمام كبير خلال العقد الماضي لعلاج العديد من الاضطرابات العصبية 6،10،43. يتم إجراء دراسات DBS التي تستهدف مختلف المناطق العصب...

التاريخ وراء تطوير تحفيز الدماغ العميق كأسلوب جراحة الأعصاب يعود إلى 1870s في حين استكشاف إمكانية تحفيز كهربائيا الدوائر الدماغ 2. بدأ استخدام التحفيز عالية التردد المزمن كعلاج لاضطرابات العصبية في 1960s 3. في وقت لاحق في 1990s مع ظهور زرع المزمن DBS أقطاب 4-6، واصل عدد من الاضطرابات العصبية التي كانت تعامل من قبل DBS في الزيادة. وقد استخدم تحفيز الدماغ العميق لأول مرة في الولايات المتحدة لعلاج الهزة الضروري 6. اليوم يتم استخدام الجراحة على نطاق واسع لعلاج الاضطرابات العصبية التي هي غير قابل للعلاج عن طريق التدخل الدوائي حاليا. يستخدم DBS حاليا لعلاج اضطرابات الحركة من مرض باركنسون وخلل التوتر 7-9. الزهايمر نوع الخرف، مرض هنتنغتون، والصرع، وآلام والأمراض العصبية والنفسية مثل الاكتئاب، والوسواس القهري، توريت7؛ ق متلازمة والإدمان هي بعض الشروط قابلة للعلاج بواسطة DBS 10-12. بينما عملية جراحية DBS وافقت ادارة الاغذية والعقاقير لعلاج مرض باركنسون، خلل التوتر والهزة الأساسية، واستخدام DBS لعلاج الحالات الأخرى المذكورة أعلاه هي في مراحل مختلفة من المختبر والدراسات السريرية التي تقدم الكثير من الأمل للمرضى 13،14. سريريا، يتم إجراء عملية جراحية DBS على مرحلتين. المرحلة الأولى تتضمن لتحديد المواقع جراحيا الأقطاب DBS في الموقع التشريحي المستهدف باستخدام مزيج من المواقع الإشعاعي، CT، MRI، وكذلك قراءات مسرى مكروي لتعزيز دقة. المرحلة الثانية تتضمن زرع مولد النبض في صدر المريض العلوي وتركيب تمديد يؤدي من فروة الرأس إلى مولد النبض. استنادا إلى حالة عصبية، والعديد من المخططات البرمجة للمولد النبض تم موحدة وسيتم استخدامها لتقديم الجهد المطلوب. في المجلد النهائييتم التوصل المئوية بطريقة تدريجية بحيث الحصول على أفضل استجابة سريرية مع الحد الأدنى من الجهد 15. ومع ذلك، في دراساتنا، على عكس يزرع DBS المزمنة المستخدمة سريريا، من أجل البساطة، ونحن لجأت إلى دراسة لمرة واحدة التحفيز عالية التردد (لمدة 1 ساعة) في النماذج الحيوانية لدينا. ويركز الجزء من البحث مجموعتنا على التحقيق في استخدام جراحة DBS لعلاج الصرع المعالجة الحرارية. النهج الجراحية المجسم باستخدام التحفيز الترددات العالية قد تم استكشافها من قبل العديد من الآخرين كخيار فعال لعلاج الصرع طبيا الحرارية التي تشكل حوالي 30٪ من جميع حالات الصرع 10،16،17. التحفيز الدماغي استهداف السطح القشري وكذلك نواة المخيخ العميقة التي استخدمت في الماضي كأهداف لعلاج الصرع 10،18،19. وبالإضافة إلى ذلك، كما تم حاول تحفيز الحصين ولكن مع نتائج متفاوتة 20،21. بعض الآخر التحقيقوتشمل أهداف DBS لعلاج الصرع قشرة الدماغ، المهاد، نواة تحت المهاد والعصب المبهم 8. ومع ذلك، في أعقاب نتائج العديد من الدراسات في السنوات القليلة الماضية، النواة المهادية الأمامية (ATN) برز كهدف DBS الأكثر شيوعا لعلاج الصرع 10،22. على أساس المعرفة حول الدوائر تشريحي عصبي والنتائج من النماذج الحيوانية، وقد ركزت العديد من الدراسات على تأثير علاجي من التحفيز العميق للدماغ من ATN في علاج الصرع 23-26. وATN هو جزء من الدائرة الحوفي، ويقع في منطقة من الدماغ الذي يؤثر على وتيرة مصادرة. الدراسات التي حماني وآخرون، لقد اختبرت فعالية ATN-DBS في بيلوكاربين الناجم عن نموذج الصرع ووجد أن التحفيز ATN الثنائي الإختفاء عن المضبوطات التي يسببها بيلوكاربين وصرعية وضع 24 لفترات طويلة. وعلاوة على ذلك، تم العثور على التحفيز الترددات العالية من ATN للحد من وتيرة مصادرة في نموذج بنتيلين تترازول (PTZ) من الجيش الشعبيilepsy 25،27-29. وذكرت لي وآخرون، انخفاض متوسط ​​في وتيرة مصادرة بنحو 75٪ على التحفيز العميق للمخ المزمن من ATN في علاج الصرع الجزئي صهر 30. وقد أظهرت دراسة سريرية حديثة حول الصرع المعالجة الحرارية نتائج واعدة بعد الجراحة DBS استهداف النواة المهادية الأمامية (ATN) 22. تجربة سريرية عشوائية متعددة المراكز مع 110 المرضى الذين يخضعون DBS الثنائي من ATN لعلاج الصرع المقاوم (SANTE محاكمة) تدل على وجود انخفاض في وتيرة الاستيلاء عليها ما يقرب من 40٪ 31. ألمح النتائج من هذه الدراسة أيضا على أفضل تأثير مضاد للصرع تأخر لوحظ في 2-3 أشهر بعد الجراحة. مزيد من الدراسات قبل تودا وآخرون، تؤكده مع هذه النتائج حيث أظهرت تكوين الخلايا العصبية يحدث في وقت آخر في وقت لاحق DBS (أيام 3-5) في النماذج الحيوانية 1. وبالإضافة إلى ذلك، إنسيناس وآخرون، وأفادت neurogene الحصينجهاز الأمن والمخابرات في الماوس الكبار التلفيف المسنن بعد التحفيز الترددات العالية من ATN 32. وأفادت الدراسات السابقة 33-35 انخفاض تكوين الخلايا العصبية قرن آمون في بعض الحالات الصرع مثل الصرع المزمن الفص الصدغي وجود علاقة مع عجز التعلم، وضعف الذاكرة وعفوية المضبوطات السيارات المتكررة. وعلاوة على ذلك، كان هناك انخفاض في العوامل السلف الخلايا الجذعية العصبية مثل FGF2 وIGF-1 في قرن آمون صرع مزمنة في النماذج الحيوانية 33. النظر في هذا، والاستراتيجيات التدخلية مثل DBS التي تظهر زيادة من تكوين الخلايا العصبية في التلفيف المسنن هي السبل مثيرة للأبحاث. وقد شجعت هذه النتائج لنا لاستكشاف المزيد عميقا في الآلية الكامنة وراء العلاج تكوين الخلايا العصبية بعد DBS لعلاج الصرع. لقد استهدفت ATN على حد سواء من جانب واحد (البيانات لم تبلغ)، وكذلك على المستوى الثنائي (في نتائج تمثيلية) وينظر neurotrophin مرتفعة (BDNF) التعبير في التلفيف المسنن الفئران. لدينا جurrent الفرضية هي أن عامل التغذية العصبية التعبير يبدأ تعبير سلسلة الجين الذي يبلغ ذروته في تكوين الخلايا العصبية التي تترجم إلى تأثير مضاد للصرع من عملية جراحية DBS. في هذه الورقة، نقدم طرقنا لعملية جراحية DBS استهداف ATN في الفئران تليها تحليل التعبير الجيني كنهج جذابة لدراسة الآلية الكامنة وراء فوائد DBS.

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			<td>Vendor Name</td>
			<td>Catalog No.&#160;</td>
			<td>Comments</td>
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			<td height="20" style="height:20px;">Stereotactic frame</td>
			<td>Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
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			<td>Dremmel</td>
			<td>7700, 7.2 V</td>
			<td></td>
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			<td>BD</td>
			<td>372610</td>
			<td></td>
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			<td>Patterson Veterinary</td>
			<td>07-803-6637</td>
			<td>Schedule III Controlled Substance, procurement, use and storage according to institutional rules</td>
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			<td height="20" style="height:20px;">Xylazine</td>
			<td>Patterson Veterinary</td>
			<td>07-808-1947</td>
			<td></td>
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			<td height="20" style="height:20px;">Buprenorphine</td>
			<td>Patterson Veterinary</td>
			<td>07-850-2280</td>
			<td>Schedule III Controlled Substance, procurement, use and storage according to institutional rules</td>
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			<td height="20" style="height:20px;">Surgical staples</td>
			<td>ConMed Corporation</td>
			<td>8035</td>
			<td></td>
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			<td height="20" style="height:20px;">Sutures (3-0)&#160;</td>
			<td>Harvard Apparatus</td>
			<td>72-3333</td>
			<td></td>
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			<td height="20" style="height:20px;">Syringe (1 ml, 29 1/2 G)</td>
			<td>BD</td>
			<td>329464</td>
			<td>Sterile, use for Anesthesia administration intraperitoneally</td>
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			<td height="20" style="height:20px;">Syringe (3 ml, 25 G)</td>
			<td>BD</td>
			<td>309570</td>
			<td>Sterile, use for Analgesia administration subcutaneously</td>
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			<td height="20" style="height:20px;">Needles</td>
			<td>BD</td>
			<td>305761</td>
			<td>Sterile, use for clearing broken bone pieces from the burr holes</td>
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			<td height="20" style="height:20px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>S25309B</td>
			<td>Use for general sterilization&#160;</td>
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			<td height="20" style="height:20px;">Eye Lubricant</td>
			<td>Fisher Scientific</td>
			<td>19-898-350</td>
			<td></td>
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			<td height="20" style="height:20px;">Stimulator</td>
			<td>Medtronic</td>
			<td>Model 3628</td>
			<td></td>
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			<td height="20" style="height:20px;">DBS electrodes</td>
			<td>Rhodes Medical Instruments, CA</td>
			<td>SNEX100x-100mm</td>
			<td>Electrodes are platinum, concentric and bipolar</td>
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			<td height="20" style="height:20px;">Betadine (Povidone-Iodine)&#160;</td>
			<td>PDI</td>
			<td>S23125</td>
			<td>Single use swabsticks, use for sterilizing the scalp before making incision&#160;</td>
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			<td></td>
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			<td colspan="2" height="25" style="height:25px;">&#160;Brain Dissection and Hippocampal tissue isolation</td>
			<td></td>
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			<td>Vendor Name</td>
			<td>Catalog No.&#160;</td>
			<td>Comments</td>
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			<td height="20" style="height:20px;">Acrylic Rodent Brain Matrix</td>
			<td>Electron Microscopy Sciences</td>
			<td>175-300</td>
			<td>www.emsdiasum.com</td>
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			<td height="20" style="height:20px;">Razor Blade</td>
			<td>V W R</td>
			<td>55411-050</td>
			<td></td>
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			<td height="20" style="height:20px;">Guillotine Scissors</td>
			<td>Clauss</td>
			<td>18039</td>
			<td>For decapitation, make sure these scissors are maintained in clean and working condition</td>
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			<td height="20" style="height:20px;">Scissors</td>
			<td>Codman Classic</td>
			<td>34-4098</td>
			<td>Use for removing the brain from the skull</td>
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			<td height="20" style="height:20px;">Forceps</td>
			<td>Electron Microscopy Sciences</td>
			<td>72957-06</td>
			<td>Use for removing the brain from the skull and for handling during dissection</td>
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			<td height="20" style="height:20px;">Phosphate Buffered Saline&#160;</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td></td>
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			<td></td>
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			<td colspan="2" height="25" style="height:25px;">&#160;RNA Extraction and cDNA Preparation</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Reagent/Equipment</td>
			<td>Vendor Name</td>
			<td>Catalog No.&#160;</td>
			<td>Comments</td>
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			<td height="20" style="height:20px;">Tri Reagent</td>
			<td>Sigma</td>
			<td>T9424</td>
			<td>Always use in a fume hood and wear protective goggles while handling; avoid contact with skin</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Syringe (3 ml, 25 G)</td>
			<td>BD</td>
			<td>309570</td>
			<td>Use for tissue homogenization</td>
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			<td height="20" style="height:20px;">Chloroform</td>
			<td>Fisher Scientific</td>
			<td>BP1145-1</td>
			<td>Always use in a fume hood and wear protective goggles while handling; avoid contact with skin</td>
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			<td height="20" style="height:20px;">Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A416-1</td>
			<td></td>
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			<td height="20" style="height:20px;">Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0561</td>
			<td></td>
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			<td height="20" style="height:20px;">Dnase I Kit</td>
			<td>Ambion</td>
			<td>AM1906</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Superscript First Strand Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>11904-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="25" style="height:25px;">&#160;Quantitative PCR</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagent/Equipment</td>
			<td>Vendor Name</td>
			<td>Catalog No.&#160;</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYBR Green PCR Kit</td>
			<td>Qiagen</td>
			<td>204143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Custom Oligos</td>
			<td>Invitrogen</td>
			<td>10668051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Plates (96 wells)</td>
			<td>Denville Scientific</td>
			<td>C18080-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical Adhesive Sheets</td>
			<td>Thermo Scientific</td>
			<td>AB1170</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease free Water</td>
			<td>Thermo Scientific</td>
			<td>SH30538-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Real Time PCR Machine</td>
			<td>Applied Biosystems</td>
			<td>7500</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of gene expression changes in the rat hippocampus after deep brain stimulation of the anterior thalamic nucleus

التحفيز العميق للدماغ (DBS) جراحة، واستهداف مناطق مختلفة من الدماغ مثل العقد القاعدية، المهاد، والمناطق تحت المهاد، هو علاج فعال لعدة اضطرابات الحركة التي فشلت في الاستجابة للدواء. وقد بدأ التقدم الأخير في مجال جراحة DBS إلى توسيع نطاق تطبيق هذه التقنية الجراحية لظروف أخرى متنوعة مثل السمنة المرضية، والاكتئاب والوسواس القهري. وعلى الرغم من هذه المؤشرات التوسع، لا يعرف الكثير عن الآليات الفسيولوجية الأساسية التي تسهل التأثيرات المفيدة لعملية جراحية DBS. نهج واحد على هذا السؤال هو لإجراء تحليل التعبير الجيني في الخلايا العصبية التي تستقبل التحفيز الكهربائي. وقد أظهرت الدراسات السابقة وأثار ذلك تكوين الخلايا العصبية في التلفيف المسنن الفئران في DBS استهداف نواة الأمامية من المهاد 1. وتستخدم عملية جراحية DBS استهداف ATN على نطاق واسع لعلاج الصرع المقاوم. وبالتالي، من ينتيريس الكثيرر بالنسبة لنا لاستكشاف التغيرات النسخي الناجمة عن تحفيز كهربائيا ATN. في هذا المخطوط، وصفنا منهجيات لدينا لهدايته stereotactically جراحة DBS استهداف ATN في البالغين فئران ويستار الذكور. نحن أيضا مناقشة الخطوات اللاحقة للتشريح الأنسجة والعزلة RNA، وإعداد [كدنا والكمي RT-PCR لقياس التغيرات في التعبير الجيني. هذه الطريقة يمكن تطبيقها وتعديلها لتحفيز العقد القاعدية ومناطق أخرى من الدماغ شيوعا المستهدفة سريريا. دراسة التعبير الجيني هو موضح هنا تفترض نهجا الجين المستهدف مرشح لاكتشاف اللاعبين الجزيئية التي يمكن توجيه آلية لDBS.

الأرقام 1A و 1B تظهر التعبير النسبي للعامل التغذية العصبية وGABRD نسبة إلى السيطرة الجين β-الأكتين. عامل التغذية العصبية، وكثيرا ما يرتبط في neurotrophin مع آثار اعصاب في العديد من الأمراض العصبية 38-41. ولذلك فمن المثير للاهتمام أن تحليل الشخصية التعبير ...

Watch this Scientific Journal Video about Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus at JoVE.com

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

تحليل التغييرات التعبير الجيني في الفئران الحصين بعد ديب تحفيز الدماغ الأمامي من مهادي نواة

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

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JoVE Journal

Neuroscience

10.8K Views.  Brigham & Women's Hospital, Harvard Medical School. The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Video: Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS : استخدام بروتوكول ثيتا المتفجر لاستكشاف Mechasnism من المرونة في الأشخاص الذين يعانون من متلازمة X الهشة والتوحد

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

وافق إجراء بروتوكول ادارة الاغذية والعقاقير لعلاج الاكتئاب : جهاز TMS NeuroStar

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

الحبل الشوكي الكهربية ثانيا : خارج الخلية التصنيع الكهربائي شفط

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

الأمثل تحليل الحامض النووي والتعبير الجيني من المناطق، الصغيرة تشريحيا المعرفة من الدماغ

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

تسليخ مجهري ليزر لقطة من السكان المخصب من الخلايا العصبية أو الخلايا العصبية واحدة لتحليل التعبير الجيني بعد إصابات في الدماغ

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

المجهرية من التعبير الجيني في الدماغ اسماك الزرد الكبار عن طريق حقن مكروي Cerebroventricular [أليغنوكليوتيد] Morpholino

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

تحفيز الدماغ العميق مع الرنين المغناطيسي الوظيفي في وقت واحد في القوارض

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

باستخدام الإسفار خلية المنشط الفرز لفحص الخلية من نوع محدد التعبير الجيني في الأنسجة دماغ الفئران

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

تسريب التجسيمي من بلازميدة قليلة القسيمات اميلويد بيتا في الحصين ماوس

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

مسرى مكروي تسترشد زرع الأقطاب الكهربائية في نواة تحت المهاد الجرذان على المدى الطويل العميق تحفيز الدماغ

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...