We thank Ge Yang, Gaudenz Danuser, Khuloud Jaqaman, and Daniel Whisler for assistance with the adaptation and development of the software to quantitate cargo mapping analyses, and Emily Niederst for help in making the microfluidic devices. This work was supported in part by NIH-NIA grant AG032180 to L.S.B.G., and the Howard Hughes Medical Institute. L.S. was supported in part by a NIH Bioinformatics Training Grant T32 GM008806, S.E.E. was supported by a Damon Runyon Cancer Research Foundation Fellowship, NIH Neuroplasticity Training Grant AG000216, and by a grant from The Ellison Medical Foundation New Scholar in Aging Award, G.E.C was supported by an NIH/NCATS&#160;1 TL1 award TR001114 and by the Achievement Rewards for College Scientists foundation.

<ol>
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The authors have nothing to disclose.

بروتوكول المعروضة هنا تمكن ارتباط الاتجاهية حركة الجزيئات الفردية الفلورسنت البضائع تتحرك على أساس أنيبيب مع نوع النسبي وكمية من البروتينات في الخلايا العصبية الحركية المرتبطة الحية. سابقا، كان يعاير إجمالي تكوين المحرك البضائع حويصلي محور عصبي على السك...

النقل داخل الخلايا أمر حاسم في جميع أنواع الخلايا لتسليم البروتينات والأغشية، والعضيات، والجزيئات يشير الى مختلف المجالات الخلوية 1. الخلايا العصبية وخلايا متخصصة للغاية مع طويلة، توقعات الاستقطاب التي تعتمد بشكل كبير على نقل البضائع داخل الخلايا الأساسية للتسليم لمسافات طويلة لمختلف microdomains محور عصبي. وتوسط هذا النقل kinesins وdyneins - عائلتين كبيرة من البروتينات المحركات الجزيئية - التي ترتبط وتتبع الشحنات على طول الأنابيب الدقيقة الاستقطاب في تقدمي ورجعية الاتجاهات، على التوالي. في حين تتوسط أساسا حركة تراجعية من قبل داينين، مما يسهل التحرك في الاتجاه تقدمي قبل الكبيرة، عائلة متنوعة من المحركات كينيسين وظيفيا. بالتالي، نقل البضائع تقدمي محور عصبي يمكن بوساطة مختلف أفراد الأسرة من الفصيلة كينيسين 1-5. على الرغم من بعض الشحنات تتحرك باستمرار في كلا الاتجاهين، مOST الشحنات تتحرك في الاتجاهين وعكس كثير من الأحيان في طريقهم إلى وجهاتهم النهائية 1،5-13. وعلاوة على ذلك، فقد تبين أن المحركات معارضة الاتجاهية الزميلة في وقت واحد لشحنات، مما يثير السؤال عن كيفية يتم تنسيق حركة البضائع الخاضعة للتنظيم من قبل محركات عكس القطبية 5-7. معا، نقل الشحنات محور عصبي هو عملية منسقة الذي ينظمه تكوين المحركات والأنشطة البيوكيميائية الخاصة بها، والتي بدورها تعتمد على محولات مختلفة وشركاء ملزم التنظيمي 14. لوصف بأمانة آلية نقل المحاور لشحنة محددة والكشف عن التنظيم الكامن وراء هذا النقل، فإن من الأهمية بمكان لتحديد تكوين البروتينات الحركية وشركاء ملزم التنظيمي المرتبطة الشحنات الفردية خلال نقلها المباشر. أساليب أخرى، على سبيل المثال مناهج الكيمياء الحيوية، وتوفر تقديرات متوسط ​​يذكرهأو التراكيب على النقى السكان حويصلة غير متجانسة، ولكن هذه التقديرات لا تكشف عن نوع أو كمية من المحركات المرتبطة الحويصلات واحدة متحركة. أيضا، إعادة تشكيل حويصلة النقل على طول الأنابيب الدقيقة مجمعة مسبقا في المختبر مكنت قياس كمية نوع واحد من السيارات على مستوى حويصلة واحدة 15. ومع ذلك، فإن هذه التجارب لا ترتبط بشكل مباشر على كمية من المحركات مع خصائص نقل تلك الحويصلات، وقياس النقل في غياب العوامل التنظيمية الخلوية. بروتوكول يرد هنا، والذي يحدد تكوين المحرك (نوع وكمية النسبي للمحركات) من الحويصلات الانتقال من الفردية أعرب المناعي (IF) بيانات قياس التطور الطبيعي البروتينات الحركية، ويرتبط هؤلاء المعلمات إلى النقل المباشر لنفس الحويصلات بالضبط في الخلايا العصبية 16. ويستتبع هذا طريقة التعيين الدقيق لل-IF إلى العيش البيانات حركة البضائع. ويتم إنجاز ذلك من خلال growinز الخلايا العصبية قرن آمون في الماوس الأجهزة ميكروفلويديك بعد ثبوت بروتوكولات 17-19. هذه الأجهزة تسمح لتحديد والارتباط (&quot;الخرائط&quot;) من المحاور والشحنات تتحرك واحدة في طرائق ضوء المجهر الثابتة والحية (الشكل 1). و transfected الخلايا العصبية مثقف مع fluorescently البروتينات المسماة البضائع التي تم تصويرها في القرار المكانية والزمانية عالية للحصول على معلومات مفصلة الحركة أن يتم رسم في kymographs النقل. أثناء التصوير، ويتم إصلاح الخلايا العصبية مع امتصاص العرق، وبعد ذلك الملون مع الأجسام المضادة ضد البروتينات الحركية الذاتية. يتم فرضه البضائع والسيارات صور ثابتة على kymographs الحركة الحية إلى &quot;خريطة&quot; (colocalize) لهم البضائع الحية مسارات حركة 16. لربط الحركة الحية للبضائع مع جمعية البروتينات الحركية، ويتم تحليل colocalization باستخدام العرف MATLAB حزمة برامج تسمى &quot;موتور Colocalization &quot;16،20. شحنات fluorescently المسمى ومحركات تولد محدودة حيود منقط الميزات التي يمكن أن تتداخل جزئيا. لحسم الموقف من نقاط ومتداخلة، البرنامج الأول تلقائيا يناسب ظائف جاوس إلى كل وظيفة انتشار نقطة، تمثل نقاط والفلورسنت الفردية، لتحديد موقف XY الفرعية بكسل دقة بهم تنسق وشدة سعة 21-23، ومواقف للسيارات والبضائع و بعد ذلك مقارنة مع بعضها البعض لتحديد colocalization 16،20. وبالتالي، هذا الأسلوب يكلف أكثر دقة colocalization بين نقاط والفلورسنت بالمقارنة مع الطرق الأخرى 24. قوة هذه الطريقة هي القدرة على تقييم colocalization المحركات مع الشحنات الفردية في الخلايا الثابتة، والتي كانت مسارات الحركة الحية (على سبيل المثال، في الاتجاه الذي كانت تتحرك في وقت التثبيت) تفصيلأورديد. مع هذا الأسلوب، تم العثور على kinesins وdyneins لربط في الوقت نفسه إلى الحويصلات التي تحمل بروتين بريون العادي (بي ار بي -cellular C)، شحنة المخصب neuronally التي تتحرك في الاتجاهين أو تبقى ثابتة في 16 محاور. يسمح هذا التحليل في صياغة نموذج للعمل لتنظيم حركة C بي ار بي الحويصلة التي تقدمي (كينيسين) ورجعية (داينين) محركات تنسيق أنشطتها من أجل تحريك الحويصلات في كلا الاتجاهين أو أن تبقى ثابتة بينما المرتبطة البضائع . قوة أخرى هذه الطريقة هي إمكانية تطبيقه واسع محتمل للتميز colocalization / جمعية العديد من الشحنات fluorescently المسمى التي تتحرك في الواقع أي نوع من الخلايا، مع أي بروتين آخر (ق) من الفائدة. وبالتالي، يمكن أن يعيش / علاقة ثابتة يحتمل أن تسمح للكشف عن عابرة تفاعلات البروتين البضائع، العديد من الأفراد fluorescently المسمى تتحرك الجزيئات يمكن تحليلها على مدى ع المطلوبeriod من الزمن. نظرا لتطبيق واسع ونوع الأسئلة التي يمكن أن تعالج هذه الطريقة، فإن هذا البروتوكول تكون ذات فائدة لجمهور واسع من علماء الأحياء الخلايا بما في ذلك أولئك الذين يدرسون الاتجار والنقل في الخلايا العصبية أو في أنواع الخلايا الأخرى.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:579px;">Reagent and Equipment Name</td>
			<td style="width:173px;">Company</td>
			<td style="width:175px;">Catalogue Number</td>
			<td style="width:173px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">poly-L-lysine</td>
			<td>Sigma</td>
			<td>P5899-20mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-MEM (Dulbecco&#8217;s Modified Eagle Medium) High Glucose, w/ L-Glutamine, w/o Sodium Pyruvate (1X)</td>
			<td>Life Technologies</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS (Fetal Bovine Serum)</td>
			<td>Life Technologies</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal-A Medium (1X)&#160;</td>
			<td>Life Technologies</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Serum Free Supplement</td>
			<td>Life Technologies</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX I Supplement (100X)</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (1X) (Hank&#8217;s Balanced Salt Solution)</td>
			<td>Life Technologies</td>
			<td>24020117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (Dulbecco&#8217;s Phosphate Buffered Saline, no Magnesium, no Calcium (1X)</td>
			<td>Life Technologies</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin (100X)</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning cellgro Water for Cell Culture&#160;</td>
			<td>Fisher Scientific</td>
			<td>MT46000CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain</td>
			<td>USB Corporation</td>
			<td>19925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DL-cysteine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>C9768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA (bovine serum albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse I grade II</td>
			<td>Roche Applied Sciences</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>&#160;11668027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde Solution 16% EM Grade</td>
			<td>Fisher Scientific</td>
			<td>50980487</td>
			<td>Caution: Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Dispose according to official regulations.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma</td>
			<td>H-3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum</td>
			<td>Jackson Immuno Research</td>
			<td>017-000-0121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA fatty acid and IgG free</td>
			<td>Jackson Immuno Research</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td>Fisher Scientific</td>
			<td>BP2403-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
			<td>Life Technologies</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover Glass 1 1/2. 24X40mm</td>
			<td>Corning</td>
			<td>2980-244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axis microfluidic device, 450 &#181;m</td>
			<td>Millipore</td>
			<td>AX450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adobe Photoshop</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon Eclipse TE2000-U&#160;</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coolsnap HQ camera&#160;</td>
			<td>Roper Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm cell culture dish</td>
			<td>Fisher Scientific</td>
			<td>12-565-95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm cell culture dish</td>
			<td>Fisher Scientific</td>
			<td>12-565-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibodies Used:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Kinesin light chain, V-17</td>
			<td>Santa Cruz&#160;</td>
			<td>sc-13362</td>
			<td>specificity verified using KLC1-/- neurons (Ref. 16 ), recommended dilution 1:100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti Dynein Heavy Chain 1, R-325</td>
			<td>Santa Cruz&#160;</td>
			<td>sc-9115</td>
			<td>specificity verified using shRNA against DYN1HC1 (Ref. 16), recommended dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody</td>
			<td>Life Technologies</td>
			<td>A10042</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Donkey Anti-Goat IgG (H+L) Antibody IgG Antibody</td>
			<td>Life Technologies</td>
			<td>A11057</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 Donkey Anti-Goat IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>A21447</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plugins and Macros</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td></td>
			<td></td>
			<td><a href="http://imagej.nih.gov/ij/index.html">&#160;http://imagej.nih.gov/ij/index.html.&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ Kymoraph Plugin</td>
			<td></td>
			<td></td>
			<td><a href="http://www.embl.de/eamnet/html/body_kymograph.html">http://www.embl.de/eamnet/html/body_kymograph.html.</a></td>
		</tr>
	</tbody>
</table>

characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis

فهم الآليات التي المحركات الجزيئية تنسيق أنشطتهم لنقل البضائع حويصلي داخل الخلايا العصبية يتطلب التحليل الكمي للجمعيات المحرك / البضائع على مستوى حويصلة واحدة. الهدف من هذا البروتوكول هو استخدام المجهر مضان الكمي لربط (&quot;الخريطة&quot;) موقف وتوجه حركة البضائع الحية إلى كميات تكوين والنسبية المحركات المرتبطة بنفس البضائع. &quot;رسم الخرائط للشحن&quot; يتكون من التصوير الحي للبضائع fluorescently المسمى تتحرك في محاور مثقف على أجهزة ميكروفلويديك، تليها تثبيت الكيميائي أثناء تسجيل الحركة الحية، والمناعي لاحق (IF) تلطيخ من نفس المناطق بالضبط محور عصبي مع الأجسام المضادة ضد المحركات. ويتم تقييم Colocalization بين الشحنات والمحركات المرتبطة بها من خلال تخصيص موقف الفرعي بكسل تنسق لقنوات السيارات والبضائع، من خلال وظائف جاوس مناسبة للحيود ليثيومmited انتشار نقطة ظائف يمثلون الفردية مصادر نقطة الفلورسنت. يتم فرضه البضائع والسيارات صور ثابتة لاحقا إلى قطع حركة البضائع، ل&quot;الخريطة&quot; لهم لتتبع مساراتها. قوة هذا البروتوكول هو مزيج من العيش وإذا كانت البيانات لتسجيل كل من نقل الشحنات حويصلي في الخلايا الحية وتحديد المحركات المرتبطة نفس هذه الحويصلات الدقيقة. هذه التقنية تتغلب التحديات السابقة التي تستخدم أساليب الكيمياء الحيوية لتحديد متوسط ​​تكوين محرك تنقيته غير متجانسة السكان حويصلة بالجملة، لأن هذه الأساليب لا تكشف التراكيب على الشحنات المتحركة واحدة. وعلاوة على ذلك، يمكن تكييف هذا البروتوكول لتحليل مسارات النقل و / أو الاتجار أخرى في أنواع الخلايا الأخرى لربط حركة هياكل داخل الخلايا الفردية مع تكوين البروتين. قيود من هذا البروتوكول هي إنتاجية منخفضة نسبيا نظرا لانخفاض كفاءة ترنسفكأيشن مثقفالخلايا العصبية الابتدائية وحقل المحدود المتاح لعرض عالية الدقة التصوير. ويمكن أن تشمل التطبيقات المستقبلية طرق لزيادة عدد الخلايا العصبية معربا عن الشحنات fluorescently المسمى.

ويبين الشكل 1 لمحة عامة عن الجهاز ميكروفلويديك تستخدم لزراعة الخلايا العصبية قرن آمون (الشكل 1A، B). ومطلي الخلايا العصبية في خزان 1. حجم و microchannels يمنع نشر أجسام الخلايا (سوما) في حجرة محور عصبي في حين أن طول قنوات يمنع التوقعات شجيري من عبور على طول الط...

Watch this Scientific Journal Video about Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis at JoVE.com

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using &quot;Cargo Mapping&quot; Analysis

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

تميز التركيب الجزيئي للسيارات على محور عصبي نقل البضائع عن طريق &quot;شحن رسم الخرائط&quot; تحليل

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

characterizing-composition-molecular-motors-on-moving-axonal-cargo

Research

JoVE Journal

Neuroscience

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

9.3K Views.  The Scripps Research Institute. Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Video: Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

عالية الكثافة تسجيلات EEG من الفئران تتحرك بحرية باستخدام بوليميد المستندة Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

التصور والتلاعب الجيني من التشعبات والاعمده الفقريه في قشرة الدماغ والحصين ماوس باستخدام<em&gt; في الرحم</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

في الجسم الحي ثنائي الفوتون التصوير من الخبرة التي تعتمد على التغيرات الجزيئية في الخلايا العصبية القشرية

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

النظام الشمي كنموذج لدراسة أنماط نمو محور عصبي ومورفولوجيا<em&gt; في فيفو</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

التصور من محور الإسقاط نمط من الخلايا العصبية المحرك الجنينية في<em&gt; ذبابة الفاكهة</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current diagnostic and severity gradation system cannot clearly indicate its coexistence, resulting in&#160;confusion of appropriate treatment prescription. There are also constraints in conventional methods for&#160;differentiation as well. This study aims at introducing an innovative, efficient, and quick screening protocol to differentiate axonal degeneration associated with CTS, using ultrasound and nerve conduction studies (NCS). It starts by using NCS to perform orthodromic stimulation at the wrist, to obtain the sensory conduction of the median and the ulnar nerves respectively. Meanwhile, the motor conduction&#160;of the median nerve is collected by stimulating the palm, wrist, and elbow, followed by the stimulation of the ulnar nerve at the wrist,&#160;below and above the elbow. Then, an ultrasound assessment is performed, using a linear array transducer, with cross-sectional area (CSA) and perimeter (P) at the wrist and at the one-third distal forearm calipered. Ratios (R-CSA, R-P) and changes from wrist to one-third distal forearm (&#916;CSA and &#916;P) are calculated according to a standard format. Potential axonal degeneration coexisting in CTS will be screened according to the criteria of NCS and cut-off values of ultrasound measurements established in a previous study. In terms of its noninvasiveness, low cost, convenience, and efficiency, it is easy to apply ultrasound complimentarily in clinical practice to prescreen patients with potential coexisting axonal degeneration. Nevertheless, the ultrasonographic imaging cannot directly reflect axonal degeneration. It still relies on conventional but invasive methods&#160;such as electromyography (EMG) and biopsy&#160;for confirmation if needed.

Here we present a protocol using nerve conduction studies and ultrasound to screen potential axonal degeneration associated with carpal tunnel syndrome. The criteria for differentiation are established. Compared to conventional approaches, this method is noninvasive, convenient, and efficient, with an overall satisfactory accuracy, sensitivity, and specificity.

فحص تنكس محواري في متلازمة النفق الرسغ باستخدام الموجات فوق الصوتية ودراسات التوصيل العصبي

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

توليد الجيل من 3D الكولاجين القائم على Hydrogels لتحليل النمو المحوري والسلوك أثناء تطوير الجهاز العصبي

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

تحليل توطين snRNA Spliceosomal في خلايا الهيلا البشرية باستخدام الحقن المجهري

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

تحريض من منتشر إصابة الدماغ أكسونال في الفئران على أساس تسارع التناوب

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

<meta charset="utf8"></head><body>تصور البروتينات في الخلايا العصبية الحصين الأولية عن طريق تصوير الخلايا الحية

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

<meta charset="utf8"></head><body>تسليط الضوء على المؤلف: فهم تأثير البروتينات المرضية على النقل المحوري في الأمراض التنكسية العصبية

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.