The authors thank Dr. Amir Rattner for helpful comments on the manuscript. Supported by the Howard Hughes Medical Institute.

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The authors have nothing to disclose.

إتقان أساليب تشريح المذكورة أعلاه يتطلب الصبر فقط، مطرد من ناحية، وعدد قليل من الأدوات تشريح جيدة. تشريح الجلد الظهري من السهل نسبيا، ولكن الذيل والجلد القدم تشريح - وخاصة في سن مبكرة بعد الولادة - هي أكثر تحديا. في سن مبكرة قبل الولادة (على سبيل المثال، قبل E15)، وا...

الجلد هو واحد من أكبر الأجهزة في الجسم، مع وظائف هامة في somato-ضجة كبيرة، والعزل / الحراري، والدفاع المناعي 1. وقد فهم الأساس الجزيئي والخلوي للتنمية وظيفة الجلد من الاهتمام منذ فترة طويلة بسبب الأهمية الأساسية للالجلد كنظام البيولوجية وصلته الأمراض الجلدية. يحتوي الجلد الثدييات مجموعة متنوعة من هياكل متعددة الخلايا، بما في ذلك طبقات من الخلايا القرنية الطبقية، والنسيج الضام الجلدي، عدة أنواع من بصيلات الشعر والغدد الدهنية والعضلات مقف الشعرة، والأوعية الدموية، وما لا يقل عن اثني عشر فئات متميزة من وارد (الحسية) وصادر العصبية الألياف (الشكل 1). وترتبط مناطق مختلفة من الجسم مع أنواع مختلفة من الجلد بشكل مميز. في معظم الثدييات، وتغطي ما يقرب من سطح الجسم كله مع الجلد التي تعج بصيلات الشعر. [البشر والفئران الخلد عارية تشكل استثناءات لعشرهو النمط.] هو الشعر مفقودة من السطوح الراحي من اليدين والقدمين، والتي ترتبط أيضا مع أنماط المتخصصة البشرة (dermatoglyphs) والغدد خارجية الإفراز، والنهايات العصبية الحسية. الأحداث الخلوية والجزيئية التي تتحكم في النمو، والتمايز، والترتيب المكاني للخلايا داخل بصيلات الشعر هي التي تحظى باهتمام خاص حيث أن كل المعروضات المسام، في مصغرة، العديد من الميزات المركزية للتوالد 2. وتشمل هذه الميزات وجود الخلايا الجذعية والخلايا الجذعية المتخصصة، والهجرات الخلية فشلت وجه التحديد، وتجميع هياكل متعددة الخلايا من مكونات متميزة جنيني. توضح هذه المقالة الطرق للتشريح، وتحديد ووضع العلامات، والجلد الماوس التصوير باعتباره ورقة ثنائية الأبعاد سليمة، ويشار إليه على أنه &quot;جبل بأكمله&quot; أو &quot;جبل مسطح&quot; التحضير. منذ الجلد الماوس رقيقة نسبيا، فمن الممكن أن الصورة من خلال سمك كامل من التزلج بالارضن باستخدام المجهر متحد البؤر التقليدية. نهج جبل مسطح لتصوير الجلد الثدييات هو مفيد من الناحية الفنية لأنه يتجاوز الحاجة إلى باجتزاء المادية، مما يسمح الهياكل من أجل إعادة بنائها بالكامل من قبل باجتزاء البصرية. منذ ما يقرب تتم معالجة الجلد بأكمله ككائن واحد، وشقة جبل يسهل أيضا النهج التصوير من مناطق متعددة من سطح الجسم مع الحفاظ على المعلومات حول الموقف والتوجه النسبية لمحاور الجسم. أخيرا، والهياكل داخل الجلد وعادة ما تكون موجودة في الأنماط التي تتكرر على فترات منتظمة، مما يسهل جمع الصور من ممثلي متعددة من بنية معينة. هذه الخصائص هي مألوفة لبيولوجيا عصبية الذين يعملون على شبكية العين، وهي جزء ثنائي الأبعاد من الجهاز العصبي المركزي الذي تتمتع بمزايا مماثلة لدراسات مورفولوجيا الخلايا العصبية 3. في جبل النهج شقة الموصوفة هنا هو من الفاءده خاصةذ لدراسة الهياكل التي تظهر التنظيم المكاني على نطاق واسع نسبيا داخل الطائرة ثنائية الأبعاد من الجلد. مثال واحد من التنظيم المكاني على نطاق واسع هو قطبية منسقة من بصيلات الشعر والهياكل المرتبطة بصيلات الشعر - مجموعات الخلايا ميركل، مقف العضلات الشعرة والغدد الزهمية، والنهايات العصبية 4. موجهة بصيلات الشعر في زاوية مع الاحترام لطائرة من الجلد، والمكون من ناقلات المسام التي تقع داخل الطائرة 2 الأبعاد من الجلد يسلك عموما التوجه فيما يتعلق محاور الهيئة التي تحدد بدقة لكل موقف على الجسم. على سبيل المثال، بصيلات الشعر على نقطة مرة أخرى من منقاري إلى الذيلية والشعر على السطح الظهري للقدم إشارة من الأقرب إلى القاصي. يتم التحكم جريب الشعر التوجه مستو قطبية الخلية إشارة (PCP؛ تسمى أيضا قطبية الأنسجة 5). تم اكتشاف هذا النظام الإشارات في ذبابة الفاكهة حيث صغيرةتم العثور على مجموعة من الجينات PCP الأساسية للتحكم في اتجاه الشعر جليدية وشعيرات. ثلاثة orthologues الثدييات من الجينات الأساسية PCP - homolog مقلى 6 (Fzd6، كما يشار إلى FZ6)، كادهيرين صندوق تعديل العولمة الأوروبي بفارق سبع تمريرة G-نوع مستقبلات 1 (Celsr1)، ومثل فانغ 2 (Vangl2) - تلعب أدوارا مماثلة في الثدييات الجلد وتنسيق توجهات بصيلات الشعر مع محاور الجسم. دراسات FZ6 الفئران خروج المغلوب (Fzd6 tm1Nat، يشار إليها فيما FZ6 - / -) تشير إلى أن الخلل الأساسي في غياب إشارات PCP هو التوزيع العشوائي الأولي أو الفوضى من التوجه بصيلات الشعر، مع عدم وجود تأثير على هيكل لا يتجزأ من بصيلات 6-8. يعمل نظام غير PCP الثانية في وقت لاحق إلى تعزيز التوافق المحلية من بصيلات المجاورة، الأمر الذي يؤدي إلى إنتاج أنماط الشعر على نطاق واسع مثل جدلات وخصلات. والمثال الثاني على نطاق واسعويعتبر التنظيم المكاني داخل الجلد في الأشكال التضاريسية من العرش محوار الحسية. الخلايا العصبية الحسية التي يعصب الجلد لديهم أجسام الخلايا في الجذر الظهرية والعقد الثلاثي التوائم. هذه الخلايا العصبية كشف عن درجة الحرارة، والألم، وحكة، وأنواع مختلفة من التشوهات الميكانيكية التي تؤثر على الجلد والشعر 9. ويمكن تقسيمها إلى أنواع فرعية على أساس القطر محوار وسرعة التوصيل، ومحطة بنية نهاية العصبية، وأنماط التعبير من المستقبلات، وقنوات، والجزيئات الأخرى. بسبب كثافة عالية من تعصيب داخل الجلد، التحليلات التي تنطوي على تصور كل المحاور (على سبيل المثال، المناعية المضادة للخيط عصبي) أو حتى جميع محاور عصبية من فئة واحدة (كما رأينا عندما تم وضع علامة على نوع من الخلايا واحد عن طريق التعبير عن مراسل الفلورسنت) عموما يكشف تراكب كثيفة من المحاور التي تجعل من المستحيل تحديد مورفولوجية جذع الفردية. للتحايل على هذه المشكلة، ونحن قد استخدمت متفرق للغاية موجهة وراثيا لabeling لإنتاج عينات الجلد الظهرية التي هي تصور الفرد العرش محوار معزولة جيدا عن طريق التعبير عن مراسل النسيجية، الإنسان الفوسفاتيز القلوي المشيمة 10. هذا النهج يسمح التصور لا لبس فيها الفرد الأشكال التضاريسية محوار الشجرة وتعريف أنواع الخلايا العصبية الحسية الجسدية بناء على معايير مورفولوجية.

<table border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Name of Material/ Equipment</td>
			<td style="width:256px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-bromo-4-chloro-indolyl phosphate (BCIP)</td>
			<td>Roche</td>
			<td>11383221001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AM1-43</td>
			<td>Biotium</td>
			<td>70024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AM4-65</td>
			<td>Biotium</td>
			<td>70039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma</td>
			<td>402834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma&#160;</td>
			<td>B-6630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Confocal microscope</td>
			<td style="width:256px;">Zeiss</td>
			<td style="width:145px;">LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3-alpha smooth muscle actin antibody</td>
			<td>Sigma&#160;</td>
			<td>C6198</td>
			<td>1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokeratin-8&#160;</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I-c</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection tools&#160;</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>scissors and forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric razor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoromount G</td>
			<td>EM Sciences</td>
			<td>17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin</td>
			<td>Sigma</td>
			<td>HT501320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Pyrex&#160;</td>
			<td></td>
			<td>6 cm and 10 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plates</td>
			<td>Amersham Biosciences</td>
			<td>SE202P-10</td>
			<td>10 cm x 8 cm x 1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair remover&#160;</td>
			<td>Nair</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horizontal rotating platform&#160;</td>
			<td style="width:256px;">Hoefer</td>
			<td style="width:145px;">PR250 Orbital shaker</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>Fine Science Tools&#160;</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine/xylazine</td>
			<td>Sigma</td>
			<td>K113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitroblue tetrazolium (NBT)</td>
			<td>Roche&#160;</td>
			<td>11383213001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oil Red O</td>
			<td>Sigma</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:256px;">VWR</td>
			<td style="width:145px;">55411-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Alexa-dye conjugated&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard-184</td>
			<td>Fisher Scientific</td>
			<td>NC9020938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plastic dishes</td>
			<td></td>
			<td></td>
			<td>10 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td></td>
			<td></td>
			<td>6- and 12-well&#160;</td>
		</tr>
	</tbody>
</table>

flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology

الجلد هو نسيج غير متجانس للغاية. وتشمل الهياكل داخل بصيلات الشعر عن طريق الجلد والعضلات مقف الشعرة، التخصصات البشرة (مثل مجموعات الخلايا ميركل) والغدد الدهنية والأعصاب والنهايات العصبية، والشعيرات الدموية. يتم التحكم في الترتيب المكاني لهذه الهياكل بإحكام على نطاق والمجهرية - كما رأينا، على سبيل المثال، في ترتيب منظم من أنواع الخلايا داخل بصيلات الشعر واحد - وعلى نطاق والعيانية - كما يراها توجهات متطابقة تقريبا الآلاف من الشعر جريبات داخل المنطقة المحلية من الجلد. تصور هذه الهياكل دون باجتزاء جسديا الجلد ممكن لأن للهندسة 2 الأبعاد لهذا الجهاز. في هذا البروتوكول، وتبين لنا أن الجلد الماوس يمكن تشريح، ثابتة، permeabilized، الملون، وأوضحت أنها سليمة اثنين الكائن الأبعاد، وهو جبل مسطح. بروتوكول يسمح التصور من السهل الهياكل الجلد في مجملها من خلال سمك كامل من مناطق واسعة من الجلد عن طريق التقيدباجتزاء كال وإعادة الإعمار. ويمكن أيضا صورا لهذه الهياكل أن تكون متكاملة مع المعلومات حول الموقف والتوجه النسبية لمحاور الجسم.

التصوير Brightfield من flatmounts الجلد يمكن استخدامها لصورة afferents الحسية الجلدية (الشكل 3A 10) وأنماط بصيلات الشعر على أساس الميلانين تصبغ (الشكل 4). التصوير متحد البؤر من flatmounts الجلد يمكن استخدامها لتحديد هندسة (1) مجموعات الخلايا ميركل، تصور مع المضادة ل?...

Watch this Scientific Journal Video about Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology at JoVE.com

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

يحتوي الجلد الثدييات مجموعة متنوعة من هياكل - مثل بصيلات الشعر والنهايات العصبية - التي تظهر أنماط مميزة من التنظيم المكاني. تحليل الجلد مثل جبل مسطح يستفيد من الهندسة 2 الأبعاد من هذا النسيج لإنتاج كامل سمك صور عالية الدقة من الهياكل الجلد.

شقة التصوير من جبل ماوس الجلد وتطبيقه على تحليل الشعر جريب الزخرفة والحسية أكسون الصرف

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

flat-mount-imaging-mouse-skin-its-application-to-analysis-hair

Research

JoVE Journal

Neuroscience

21.2K Views.  Howard Hughes Medical Institute, Johns Hopkins University School of Medicine.  يحتوي الجلد الثدييات مجموعة متنوعة من هياكل - مثل بصيلات الشعر والنهايات العصبية - التي تظهر أنماط مميزة من التنظيم المكاني. تحليل الجلد مثل جبل مسطح يستفيد من الهندسة 2 الأبعاد من هذا النسيج لإنتاج كامل سمك صور عالية الدقة من الهياكل الجلد....

Video: Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

متعددة الماوس تشريحي عصبي التصوير بالرنين المغناطيسي

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

تحضير الأنسجة وImmunostaining من الألياف العصبية الحسية ماوس التعصيب الجلد والعظام الاطراف

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

تحليل التصوير من الخلايا العصبية إلى الخلايا الدبقية في التفاعل منهاج الثقافة ميكروفلويديك (MCP) على أساس الخلايا العصبية والخلايا الدبقية اكسون نظام المشاركة في الثقافة

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

كله من جبل Immunolabeling الشم مستقبلات الخلايا العصبية في<em&gt; ذبابة الفاكهة</em&gt; الهوائي

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

كله جبل التصوير من جنين فأر التوقعات الحسية أكسون

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

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 ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

جبل كله تشريح والمناعي من الكبار ماوس القوقعة

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

المشتركة تسجيل من ميكانيكيا بتنشيط وارد الناتج والأعصاب الطرفية وسمها في ماوس الشعر المسام سناني الشكل النهايات

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

رصد بصري الأعصاب الطرفية المعيشة وصفها في الشعر المسام سناني الشكل النهايات لل<em&gt; فيفو السابقين</em&gt; ماوس الأذن الجلد

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

التصحيح المشبك تسجيل من خلايا النجمي عديم الاستطالات في جبل شقة إعداد Deafferentated شبكية العين الفأر

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...