This work has been supported by NINDS, RO1NS52699 and MH84874 to SA. 
We would like to thank Dr. Dave Featherstone (Department of Biological Sciences, University for Illinois at Chicago) for providing us with the suture glue used in the immunohistochemistry work. We thank Michael Alpert for his comments and proofreading of the manuscript.

<ol>
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The authors do not have any competing financial interests or other conflicts of interest to disclose.

بروتوكول التفكك الكبير الذي لدينا هو العائد محاور الشبكي معزولة خالية من التوقعات بعد المشبكي الشكل 2F، ولكن مع ذلك الذي يحتفظ ظيفية محطات قبل المشبكي 4C الشكل والشكل 4D. غياب العمليات بعد المشبكي معارضة المحطة قبل المشبكي يسمح تسجيل الوصول المباشر إ...

انتقال متشابك هو عملية سريعة ودقيقة للغاية. العمل غزو محتمل من المحطة قبل المشبكي يؤدي إلى فتح VGCCs تقع في الغشاء الإفراج جهه، والزيادة الناتجة في كاليفورنيا قبل المشبكي 2+ بوصفها الزناد عن الانصهار الحويصلة والعصبي الإفراج 1. كل هذه الخطوات تحدث داخل مئات ميكروثانية 2، وبالتالي تتطلب اقتران مكاني ضيق من VGCCs إلى حويصلة الانصهار الآلات 3. وقد تميزت الكالسيوم قبل المشبكي 2+ تدفقات المقام الأول من خلال النهج التصوير باستخدام الأصباغ الكالسيوم 2+ الحساسة 4. دمج الكالسيوم 2+ المخازن المؤقتة التي تعدل الكالسيوم في الخلايا العصبية قبل المشبكي 2+ وقد استخدم لتوصيف غير مباشر العلاقة بين الكالسيوم والنقل العصبي قبل المشبكي 3. بالإضافة إلى ذلك، تحوير قبل المشبكي مجانا كا 2+ تركيز الكالسيوم عن طريق uncaging 2+ 5 أو تسجيل macroscopiج كا استخدمت 2+ التيارات جنبا إلى جنب مع التدابير الانصهار الحويصلة و / أو الإطلاق. مثل قياسات السعة 2 6 أو بعد المشبكي الردود على معالجة نفس السؤال. ومع ذلك، تميز كا 2+ التيارات مباشرة في وجه الإفراج عنهم، وقسم متخصص للغشاء قبل المشبكي حيث تتم ترجمة الاستقطاب في غشاء الكالسيوم 2+ التيارات اثار الانصهار حويصلة متشابك وإطلاق ناقل عصبي، هو جزء لا يتجزأ من الحصول على قياس دقيق لكا 2+ شرط حويصلة متشابك الانصهار. وبالإضافة إلى ذلك، فإن القدرة على تميز مباشرة الكالسيوم 2+ التيارات في محطات قبل المشبكي فردية، إلى جانب قياسات دقيقة في وقت واحد الانصهار حويصلة والافراج يسمح التوضيح الدقيق للعلاقة بين توقيت مدار الساعة من إمكانات العمل، قبل المشبكي الكالسيوم 2+ الحالية، الانصهار حويصلة والافراج عنهم. الوصول إلى غشاء الإفراج الوجهغير متوفرة في معظم محطات قبل المشبكي المقرر ان تغلق بدل من التشعبات بعد المشبكي. وكانت هذه صعوبة الوصول عقبة رئيسية في توصيف VGCCs لأنه يمنع القياسات المباشرة الحالية في محطات قبل المشبكي فردية. وحتى الآن لم توصيف المباشر من قبل المشبكي الكالسيوم 2+ التيارات في محطات قبل المشبكي فردية ممكن في نقاط الاشتباك العصبي المركزي، وقد تتحقق إلا في اثنين من نوع كأسي محطات قبل المشبكي. الكأس من نوع المشبك من فرخ العقدة الهدبية 7-10 والفئران الكؤوس 11،12. في جميع محطات قبل المشبكي الأخرى بما في ذلك المشبك الشبكي العملاق في الجلكى الحبل الشوكي 13، وعدم الوصول إلى غشاء قبل المشبكي الإفراج الوجه وقد استلزم استخدام الأساليب غير المباشرة مثل التصوير الكالسيوم 2+ لدراسة الكالسيوم قبل المشبكي 2+ التدفقات. <img alt="الشكل 1" fس: محتوى العرض = &quot;5in&quot; SRC = &quot;/ الملفات / ftp_upload / 51925 / 51925fig1highres.jpg&quot; العرض = &quot;500&quot; /&gt; الرقم 1. الجلكى المشبك الشبكي العملاق. (أ) عبر قسم من الجلكى الحبل الشوكي مما يدل توجيه ظهراني البطني. وتتميز محاور الشبكي مع أستريكس الأخضر (ب) إعادة الإعمار 3-D من المشبك الشبكي في النخاع الشوكي الجلكى تظهر المحوار قبل المشبكي الشبكي جعل العديد أون الاتصالات بسنت (تميزت السهام الخضراء) على عصبون بعد المشبكي 13. وقد وصفت محطات قبل المشبكي مع اليكسا فلور 488 هيدرازيد phalloidin مترافق (الأخضر)، بينما يتم شغل عصبون بعد المشبكي مع اليكسا فلور 568 هيدرازيد (الحمراء). محاور الشبكي الجلكى العملاقة، وتقع في المنطقة البطنية من النخاع الشوكي مواز للمحور منقاري الذيلية-1A الشكل، شكل متعددة أون الاتصالات متشابك بسنت على الخلايا العصبية للالشوكي القرن البطني الشكل 1B 14 13. وقد سجلت العيانية خلية كاملة الكالسيوم 2+ التيارات من محاور الشبكي في النخاع الشوكي سليمة 13،15. ومع ذلك، محاولات عمياء السابقة في القياس المباشر من الكالسيوم أثبتت 2+ التيارات في محاور الشبكي في الجلكى الحبل الشوكي سليمة باستخدام الخلية المرفقة تقنية المشبك التصحيح الفاشل 13 بسبب عدم وجود الوصول إلى غشاء قبل المشبكي الإفراج الوجه بسبب العمليات بعد المشبكي معارضة الشكل 1B. غشاء الإفراج وجه أحرز في السابق الوصول إليها عن طريق إزالة الخلايا العصبية بعد المشبكي 11، اضطراب الميكانيكية المشبك قبل تسجيل 12 أو المعالجة الأنزيمية إلى جانب التفكك الميكانيكية 16. نظرا لمنظمة معقدة من الحبل الشوكي، فإنه يكون من الصعب للغاية تحديد الخلايا العصبية بعد المشبكي وسحب ذلك ميكانيكيا أو التشويش عشرالبريد المشبك. ومن هنا، قررنا استخدام المعالجة الأنزيمية 17 تليها التفكك الميكانيكية. باستخدام هذا النهج، قمنا بتطوير وإعداد فصلها تماما من الحبل الشوكي الجلكى أن ينتج محاور الشبكي منعزلة قابلة للحياة مع محطات قبل المشبكي وظيفية خالية من أي عمليات بعد المشبكي، وبالتالي توفير وصول غير مقيد إلى محطات قبل المشبكي الفردية. بالتزامن مع معيار مقلوب المجهر والتصوير مضان، وتمكننا من تحديد واستهداف محطات قبل المشبكي fluorescently التي تم تحديدها الفردية، مع ماصة التصحيح يحتوي على محلول التسجيل الذي يعزل الكالسيوم 2+ التيارات والشكل الشكل 4C 4D، لتسجيل باستخدام الخلوي تعلق تقنية الجهد المشبك. وقد سجلت كاليفورنيا 2+ التيارات مباشرة في الغشاء قبل المشبكي الإفراج مواجهة فردية محطات قبل المشبكي الشكل 4F. هذا هو الهامة فياختراق الإقليم الشمالي في مجال انتقال متشابك لأنه هو الأول من نوعه التسجيل التي يتعين الاضطلاع بها في نقاط الاشتباك العصبي المركزي.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">0.2 &#181;m syringe filter</td>
			<td style="width:211px;">EMD Millipore</td>
			<td style="width:119px;">SLGV004SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">22x60 mm coverslips</td>
			<td style="width:211px;">Fisherbrand</td>
			<td style="width:119px;">12545J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Advasep-7</td>
			<td style="width:211px;">Cydex Pharmaceuticals</td>
			<td style="width:119px;">ADV7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Alexa-633 conjugated goat anti-rabbit secondary antibody</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A21070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Antifreeze</td>
			<td style="width:211px;">Prestone</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Boric acid</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">B7660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bright field light source</td>
			<td style="width:211px;">Dolan-Jenner</td>
			<td style="width:119px;">Fiberlite 180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Calcium chloride</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Collagenase Type IA from Cloristridium histolyticum</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cover slip</td>
			<td style="width:211px;">Fisher-Scientific</td>
			<td style="width:119px;">12-545-J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dextrose</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9559</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Digital CCD Camera</td>
			<td style="width:211px;">Hamamatsu</td>
			<td style="width:119px;">C8484-03G01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection fine forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection microscope</td>
			<td style="width:211px;">Leica Biosystems</td>
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			<td>To prepare, mix elastomer A and elastomer B 10:1 by weight</td>
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</table>

acute dissociation of lamprey reticulospinal axons to enable recording from the release face membrane of individual functional presynaptic terminals

انتقال متشابك هو عملية سريعة للغاية. إمكانات العمل مدفوعة تدفق الكالسيوم 2+ في محطة قبل المشبكي، من خلال قنوات الكالسيوم الجهد بوابات (VGCCs) الموجود في غشاء الوجه الإفراج عنهم، هو على الزناد لانصهار حويصلة والافراج عن العصبي. حاسمة بالنسبة لسرعة انتقال متشابك هو التزامن المكاني والزمني بين وصول إمكانات العمل، VGCCs وآلية إطلاق ناقل عصبي. القدرة على تسجيل مباشرة الكالسيوم 2+ التيارات من الغشاء الإفراج وجه محطات قبل المشبكي الفردية أمر حتمي لفهم دقيق للعلاقة بين الكالسيوم قبل المشبكي 2+ وإطلاق ناقل عصبي. الوصول إلى غشاء قبل المشبكي الإفراج الوجه لتسجيل الكهربية غير متوفر في معظم الاستعدادات والكالسيوم قبل المشبكي 2+ تميزت الدخول باستخدام تقنيات التصوير وmeasureme الحالي العيانيةاليلة - التقنيات التي ليس لديها قرار زمني يكفي لتصور دخول الكالسيوم 2+. لم يكن توصيف VGCCs مباشرة في محطات قبل المشبكي واحدة ممكن في نقاط الاشتباك العصبي المركزي وتمت حتى الآن تحققت بنجاح إلا في الكأس من نوع المشبك من العقدة الهدبية وفرخ في كؤوس زهرية الفئران. عالجنا هذه المشكلة بنجاح في المشبك الشبكي العملاق من الحبل الشوكي الجلكى من خلال تطوير إعداد فصلها تماما من الحبل الشوكي يمكن أن ينتج محاور الشبكي المعزولة مع محطات قبل المشبكي وظيفية خالية من الهياكل بعد المشبكي. يمكننا تسمية fluorescently وتحديد محطات قبل المشبكي الفردية واستهدافهم للتسجيل. باستخدام هذا الإعداد، واتسمت VGCCs مباشرة في وجه الافراج عن محطات قبل المشبكي الفردية باستخدام المناعية والكهربية النهج. وقد سجلت كاليفورنيا 2+ التيارات مباشرة في إطلاق الوجه غشاء سمحطات قبل المشبكي فردية جمعة، أول مثل هذا التسجيل التي يتعين الاضطلاع بها في نقاط الاشتباك العصبي المركزي.

<html> هذه العوائد بروتوكول التفكك صحية وظيفية محاور الشبكي معزولة خالية من التوقعات بعد المشبكي الشكل 2F، ولكنها مع ذلك تحتفظ محطات قبل المشبكي وظيفية قادرة على أثار حويصلة متشابك إكسو والإلتقام الشكل 4C و 4D الشكل. أجزاء من المناطق المعزولة م?...

Watch this Scientific Journal Video about Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals at JoVE.c

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

التفكك الحاد من الجلكى الشبكي المحاوير لتمكين تسجيل من الإصدار الوجه غشاء من محطات قبل المشبكي الوظيفية الفردية

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

acute-dissociation-lamprey-reticulospinal-axons-to-enable-recording

Research

JoVE Journal

Neuroscience

8.7K Views.  University of Illinois at Chicago. Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals. 

Video: Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

المنطقة Subventricular اون الوجه : يلطخ Wholemount والتدفق البطانة العصبية

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation من الخلايا العصبية من الدماغ القوارض شرائح لدراسة نقل صورة متشابك ومحطات قبل المشبكي

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

الجهد حساسة من تسجيل صبغ محاور عصبية، التشعبات والعمود الفقري شجيري الخلايا العصبية الفردية في شرائح الدماغ

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

الليزر Nanosurgery من مخيخي محاور عصبية<em&gt; في فيفو</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

قياس العمود الفقري قبل المشبكي في الفئران عن طريق تثبيط الظهرية جذر تسجيل المحتملة<em&gt; في فيفو</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

التصوير كاليفورنيا<sup&gt; 2+</sup&gt; حيوية في محطات المخروط مبصرة أكسون من شبكية العين الفأر

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

تصوير لايف من النيكوتين المستحثة اشارة الكالسيوم وناقل عصبي الإصدار جنبا إلى جنب بطني الحصين المحاوير

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

تسجيل اللدونة متشابك في شرائح هيبوكامبال الحاد تحتفظ في صغيرة الحجم-إعادة التدوير، ونضح-، وغمر من نوع نظام الدائرة

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

في فيفو تتبع في محطة Presynaptic الأعصاب الحركية المورفولوجية جزيء واحد

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.