فإن الكتاب أود أن أنوه جامعة ديلي ستودي دي ميلانو لدعم إليانا دي Cairano (زمالة ما بعد الدكتوراه) وستيفانيا موريتي (زمالة الدكتوراه). وأيد هذا العمل من قبل برنامج أبحاث جامعة PUR لCP ونود أن نشكر البروفيسور جيريمي م هينلي، كلية الكيمياء الحيوية، جامعة بريستول، المملكة المتحدة، لpHluorin بناء والدكتور Dotti فرانشيسكو للمساعدة في تحليل البيانات، وسيلفيا Marsicano للحصول على المساعدة الفنية.

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The authors declare that they have no competing financial interests.

<html> وتعرض هذه الورقة على بروتوكول لصورة وتحليل ديناميات الحويصلات في الخلايا المفرزة، وذلك باستخدام ناقلات ترميز [كدنا الفلورسنت وTIRFM. العناصر الرئيسية في التصوير الناجحة التي قام بها TIRFM هي اختيار نموذج الخلوية وترنسفكأيشن الخلية مع المؤشرات البصرية المشفرة وراثي?...

انتقال متشابك الكيميائية بين الخلايا العصبية هو آلية رئيسية للاتصال في الجهاز العصبي. وهي تعتمد على إطلاق النواقل العصبية من خلال دورة ديناميكية من الانصهار الحويصلة واسترجاعها في الموقع قبل المشبكي. وقد تم تحديد العديد من البروتينات المشاركة في ديناميات الحويصلة. ومع ذلك، لا تزال المساهمة المحددة لهذه الظاهرة إلى توضيح 1. فهمنا يقتصر جزئيا بحقيقة أن المقايسات الأكثر استخداما لإكسو / الإلتقام ليست دائما الأنسب. العديد من الدراسات المتعلقة الانصهار الحويصلة وديناميكية تعتمد على تقنيات الكهربية. توفر هذه التقنية على قرار الزمني الأمثل وممتازة للتحقيق الانصهار الأولي من الحويصلات إلى غشاء البلازما ولكن غير قادرة على الكشف عن العديد من الأحداث الجزيئية الكامنة التي تدعم وظيفة قبل المشبكي. الإلكترون المجهري، على الجانب الآخر، يوفر أرقى morphologicaل وصف لكل خطوة واحدة، ولكن الجانب الديناميكي لهذا الحدث لا يمكن القبض، لأن العينات يجب أن تكون ثابتة لكي يتم تحليلها. ظهور تقنيات التسجيل البصري الجديدة 2،3، في تركيبة مع التقدم في الفلورسنت تطوير تحقيقات الجزيئية 4-6، وتمكن التصور من العمليات exocytic في الخلايا الحية، وبالتالي توفير مستويات جديدة من المعلومات حول بنية متشابك وظيفة. الدراسات الأولية استغلال الأصباغ التي تعتمد على النشاط styryl (FM1-43 والأصباغ العضوية ذات الصلة) 7،8. دولة من بين الفن وتقنيات التصوير توظف المتغيرات الحساسة الرقم الهيدروجيني للالبروتين الأخضر نيون (GFP) (pHluorin) المربوطة إلى الحويصلات اللمعية البروتينات 9. يتم تبديل هذه التحقيقات عادة قبالة عندما تكون موجودة في الحويصلات بسبب انخفاض درجة الحموضة اللمعية. بعد الاندماج مع غشاء البلازما، يتعرض الداخلية الحويصلة إلى الفضاء خارج الخلية محايد، ودرجة الحموضة<html> يزيد فجأة، ويخفف من وتبريد تعتمد على بروتون من pHluorin ويظهر إشارة الفلورسنت بسرعة. كما تغير في pHluorin أسرع من الحدث الانصهار، من خلال رصد زيادات مضان، الانصهار الحويصلة مع غشاء يمكن قياسها وتحليلها. لأن endocytosed سطح جزيئات الموسومة pHluorin، إشارة مضان يعود بعد ذلك إلى المستوى القاعدي، وبالتالي فإن نفس التركيبة يمكن أن تستخدم أيضا لمراقبة الحويصلة إعادة تدوير 9. في حين أن الموسومة حويصلة درجة الحموضة استشعار يضمن التصور فقط من تلك الحويصلات التي تلتحم حقا مع غشاء البلازما، مطلوب التصوير في القرار المكانية والزمانية عالية لوصف بالتفصيل الخطوات المتبعة في إكسو / العمليات التقامي. هذه التقنية البصرية التي توفر الدقة المكانية والزمانية الضروري هو مجموع المجهري التأمل الداخلي مضان (TIRFM)، وهو تطبيق مضان المجهري 10. &quot;&gt; يحدث الانعكاس الكلي الداخلي في واجهة بين الزجاج غطاء الانزلاق والعينة. عندما يصل مسار الضوء الزجاج غطاء للانزلاق مع زاوية الحادث أكبر من الزاوية الحرجة، لا ينتقل ضوء الإثارة في العينة ولكن تنعكس تماما الظهر. وفي ظل هذه الظروف، وهو زائل أشكال موجة الضوء في واجهة ويكاثر على المدى المتوسط ​​مع أقل كثافة ضوئية (العينة). وبما أن شدة المجال زائل يضمحل بشكل كبير، مع البعد عن واجهة (مع عمق تغلغل حوالي 100 نانومتر) فقط fluorophores في أقرب القرب من غطاء الانزلاق يمكن أن يكون سعيدا في حين أن أولئك بعيدا عن الحدود ليست كذلك. في الخلايا transfected مع GFP-يبني، هذا العمق يتوافق مع البروتينات وأعرب على غشاء البلازما أو في هياكل حويصلي الاقتراب منه. كما fluorophores في داخل الخلية لا يمكن متحمس، والتقليل من مضان الخلفية، وصورة مع إشارة / الفئران خلفية عالية جداويتكون الإعلام والتوعية 11. العديد من الخصائص تجعل TIRFM أسلوب الاختيار لرصد الحويصلات ديناميات. على النقيض من ذلك الكمال وإشارة إلى الضوضاء نسبة عالية تسمح للكشف عن الإشارات منخفضة جدا مستمدة من الحويصلات واحدة. الحصول على الصور يعتمد على الشرائح الالكترونية في كل إطار يوفر القرار الزماني اللازم لاكتشاف عمليات ديناميكية للغاية. وأخيرا، فإن التعرض الحد الأدنى من الخلايا لضوء في أي طائرة أخرى في العينة يقلل بشدة الضيائية وتمكن تسجيل طويل دائم الوقت الفاصل 12. يبقى تحليل البيانات الجانب الأكثر تحديا وحاسم من هذه التقنية. إن أبسط طريقة لمراقبة الانصهار الحويصلة هي لقياس تراكم البروتينات مراسل فلوري على سطح الخلية، على مر الزمن 13. كما يزيد من الانصهار، صافي الزيادة إشارة مضان كذلك. ومع ذلك، وهذه الطريقة قد يقلل من شأن هذه العملية، لا سيما في الخلايا الكبيرة وفي ظروف الراحة،لأن العمليات الإلتقام وphotobleaching من تعويض الزيادة في كثافة مضان بسبب حويصلة إيماس. طريقة بديلة هو اتباع كل الانصهار حدث واحد 14. هذا الأسلوب الأخير هو حساس جدا ويمكن أن تكشف عن تفاصيل مهمة حول آليات الاندماج. ومع ذلك، فإنه يتطلب اختيار اليدوية الأحداث واحدة، لأن إجراءات مؤتمتة بالكامل لمتابعة الحويصلات وللتسجيل تذبذب إشاراتها الفلورسنت لا تتوفر دائما. مراقبة ديناميات الحويصلة تتطلب خلايا أخذ العينات في وتيرة عالية. وهذا يولد كمية كبيرة من البيانات التي بالكاد يمكن تحليلها يدويا. على اقتراح من هذه الورقة هو تحسين تقنية التصوير TIRFM لرصد القاعدية وحفز الإفراج العصبي في خط الخلية العصبية SH-5YSY، ووصف، خطوة بخطوة، وهو إجراء المتقدمة في المختبر لتحليل البيانات، على حد سواء عند مستويات كلها خلية واحدة حويصلة.

<table border="0" cellpadding="0" cellspacing="0" width="1414">
	<tbody>
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			<td height="21" style="height:21px;width:259px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td style="width:371px;"></td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Axio Observer Z1</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">491912-9850-000</td>
			<td>inverted microscope 
			<a href="http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction">http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Multiline Argon Laser Lasos 77</td>
			<td style="width:199px;">Lasos</td>
			<td style="width:119px;">00000-1312-752</td>
			<td>multi-line (458/488/514 nm), 100mW argon-ion laser 
			<a href="http://www.lasos.com/products/argon-ion-laser">http://www.lasos.com/products/argon-ion-laser</a></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:259px;">Laser TIRF slider</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">423681-9901-000</td>
			<td><a href="http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html">http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">100x Objective</td>
			<td style="width:199px;">Zeiss</td>
			<td>421190-9900-000</td>
			<td>Oil, NA 1.45 Alpha-Plan 
			<a href="https://www.micro-shop.zeiss.com/?l=en&amp;p=us&amp;f=o&amp;a=v&amp;m=a&amp;id=421190-9900-000&amp;ss=1">https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=o&#38;a=v&#38;m=a&#38;id=421 
			190-9900-000&#38;ss=1</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">CCD Camera RetigaSRV Fast 1394&#160;</td>
			<td style="width:199px;">QImaging</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf">http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">LAMBDA 10-3 optical filter changer with SmartShutter</td>
			<td style="width:199px;">Sutter Instrument Company</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.sutter.com/IMAGING/lambda103.html">http://www.sutter.com/IMAGING/lambda103.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Software</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image ProPlus 6.3 Software</td>
			<td style="width:199px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>spot selection, ROI selection, fluorescence intensity determination 
			<a href="http://www.mediacy.com/index.aspx?page=IPP">http://www.mediacy.com/index.aspx?page=IPP</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Excel</td>
			<td style="width:199px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>photobleaching correction, whole-cell and single-vesicle analyses 
			<a href="http://office.microsoft.com/it-it/excel/">http://office.microsoft.com/it-it/excel/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 4.00&#160;</td>
			<td>GraphPad Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>statistical analysis 
			<a href="http://www.graphpad.com/scientific-software/prism/">http://www.graphpad.com/scientific-software/prism/</a></td>
		</tr>
	</tbody>
</table>

tirfm and ph-sensitive gfp-probes to evaluate neurotransmitter vesicle dynamics in sh-sy5y neuroblastoma cells: cell imaging and data analysis

الحويصلات متشابك الافراج عن الناقلات العصبية في المشابك الكيميائية من خلال دورة ديناميكية من الانصهار واسترجاعها. رصد نشاط متشابك في الوقت الحقيقي، وتشريح الخطوات مختلفة من إكسو-الإلتقام على مستوى واحد حويصلة حاسمة لفهم وظائف متشابك في الصحة والمرض. وراثيا ترميز تحقيقات حساسة للدرجة الحموضة المستهدفة مباشرة إلى الحويصلات متشابك وانعكاس إجمالي الداخلية المجهر مضان (TIRFM) تقديم القرار المكانية والزمانية اللازمة لمتابعة ديناميات الحويصلة. مجال زائل الناتجة عن الانعكاس الكلي الداخلي يمكن أن تثير فقط fluorophores وضعها في طبقة رقيقة (&lt;150 نانومتر) فوق غطاء زجاجي على الخلايا التي تلتزم، بالضبط حيث عمليات إكسو-الإلتقام تحدث. هي مناسبة الناتجة صور عالية التباين مثالي لالحويصلات التتبع والتحليل الكمي من الأحداث الانصهار. في هذا البروتوكول، SH-SY5Y ن البشريويقترح خلايا euroblastoma باعتباره نموذجا قيما لدراسة إطلاق سراح العصبي في مستوى واحد-الحويصلة التي كتبها TIRFM، بسبب سطحها مستو وجود حويصلات تفرقوا. يتم توفير الطرق لزراعة SH-SY5Y كما الخلايا الملتصقة وtransfecting لهم synapto-pHluorin، فضلا عن تقنية لأداء TIRFM والتصوير. وأخيرا، قدم استراتيجية تهدف إلى تحديد، عد، وتحليل الأحداث الانصهار عند مستويات كلها خلية واحدة حويصلة. للتحقق من صحة نهج التحليل إجراء التصوير والبيانات، ويتم تحليل ديناميات حويصلات pHluorin الموسومة تحت يستريح وحفز (إزالة إستقطاب تركيزات البوتاسيوم) الظروف. الاستقطاب غشاء يزيد من وتيرة الأحداث الانصهار ويؤدي إلى زيادة موازية من صافي إشارة مضان سجلت في خلية كاملة. تحليل احدة حويصلة يكشف تعديلات للسلوك الانصهار الحدث (الارتفاع المتزايد الذروة والعرض). وتشير هذه البيانات عشرفي الاستقطاب البوتاسيوم ليس فقط يؤدي الى الافراج عن ناقل عصبي هائل ولكن يغير أيضا آلية الانصهار الحويصلة وإعادة التدوير. مع التحقيق الفلورسنت المناسب، وهذا الأسلوب يمكن أن تستخدم في الأنظمة الخلوية المختلفة لتشريح آليات إفراز التأسيسي وحفز.

وقد صممت هذه الإجراءات التصوير TIRF وتحليل البيانات ووصف لدراسة الحويصلات ديناميكية في الأنظمة الخلوية. يمكن استخدام هذه التقنية لتحديد آثار يشير الجزيئات والمخدرات على الأحداث الانصهار وديناميكية العصبي حويصلة 17. باستخدام بروتينات غشاء البلازما الموسومة GFP، ...

Watch this Scientific Journal Video about TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis at JoVE.com

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM والحساسة للدرجة الحموضة GFP-تحقيقات في تقييم ناقل عصبي الحويصلة حيوية في خلايا SH-SY5Y العصبية: التصوير خلية وتحليل البيانات

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

tirfm-ph-sensitive-gfp-probes-to-evaluate-neurotransmitter-vesicle

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Neuroscience

10.7K Views.  Università degli Studi di Milano. This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Video: TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

تعيش خلية التصوير الحسي للخلايا السليمة في الجهاز السلائف ذبابة الفاكهة شرانق

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

زرع الخلايا الشمية مغمد لتقييم الانتعاش وظيفية بعد إصابة العصب المحيطي

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

تصور جسيم داخلي حيوية في محطات عصب المعيشة مع رباعية الابعاد التصوير الإسفار

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

الثقافات شريحة عضوي النمط لدراسة ديناميات دبقية قليلة التغصن وتكون الميالين

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

فرز خلية العصبية الجذعية وخلايا السلف من الفأر Subventricular المنطقة الكبار وحية على التصوير من حيوية دورة الخلية الخاصة

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

تحليل متشابك التحوير من<em&gt; ذبابة الفاكهة</em&gt; خلايا مستقبلة للضوء بعد التعرض للضوء لفترات طويلة

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

عزل العقد الجذرية الظهرية والثقافة الأولية لدراسة الإفراج عن ناقل عصبي

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

في المختبر التصوير الكمي المقايسة لPhaagocytosis من خلايا الورم الأرومي العصبي الميت من قبل iPSC-الضامة

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from &#946; cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis.
This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.

This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis cycle.

قياس معدل دوران الدهون الغشائية باستخدام التناظرية الفلورية الحساسة للأس الهيدروجيني ND6

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

تحليلات أكتين دايناميكز، اقتران مخلب وقوة الجر للنمو مخروط مقدما

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...