وقد تم تمويل هذا العمل عن طريق الدعم من خلال مؤسسة ماكنير ، NARSAD ، ومنح NINDS R00NS064171 - 03.

1. الدماغ في تثبيت الوضع الطبيعي * تحضير حقنة 10 مل (28 إبرة قياس) مليئة الفوسفات مخزنة المالحة (PBS). * تحضير حقنة 10 مل (28 إبرة قياس) مليئة بارافورمالدهيد 4 ٪ (PFA) في برنامج تلفزيوني. الاحتياطي إلى ترخيص إضافي 5-10 PFA / لتثبيت برنامج تلفزيوني آخر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضخ تجريبي intraperitoneally الفأر مع جرعة قاتلة من آفيرتين أو Nembutal. </li><li style=";text-align:right;direction:rtl"> بمجرد مخدرا الماوس ، والرطب في البطن مع الإيثانول وتأمين الماوس إلى أسفل الدرج الطبقات مع الفلين والشمع ، أو الاستومر سيليكون باستخدام دبابيس تشريح. البطن مع مواجهة وتأمين الكفوف أربعة إلى السطح -- نشرها على أوسع نطاق ممكن. </li><li style=";text-align:right;direction:rtl"> الاستيلاء على الجلد مع ملقط على مستوى القص ، وقطع بالعرض لفضح الكبد. قطع أفقيا ثم ارتفعت خلال الضلوع والحجاب الحاجز. إزالة الأنسجة رفرف الصدري وتستمر حتى قطع القلب للوصول. </li><li style=";text-align:right;direction:rtl"> بيرس أو قص الأذين الأيمن لاستنزاف الدم تعميمها. </li><li style=";text-align:right;direction:rtl"> تدفق الدم المتبقية من الدورة الدموية التي نضح من خلال برنامج تلفزيوني البطين الأيسر. </li><li style=";text-align:right;direction:rtl"> الوصول إلى ثقب الإبرة نفسها ، إصلاح الماوس كله عن طريق نضح اللاحقة PBS / منهاج العمل من خلال البطين الأيسر. </li></ol> 2. تشريح الدماغ واستخراج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة الجلد من الرأس والعنق والجمجمة عن طريق خفض حول قناة الأذن والعين المدارات ، يليها سحب الجلد الأمامية لفضح الجمجمة. * انظر الشكل رقم 2 لتوضيح الخطوات 2.2) إلى 2.5) </li><li style=";text-align:right;direction:rtl"> باستخدام مقص العظام ، وقطع بالعرض من العين عن طريق مأخذ عظام المحارة الأنفية. للقيام بذلك كل جانب. ينبغي أن يكون إقطع الأمامي للعين وبصيلات الشم. </li><li style=";text-align:right;direction:rtl"> قطع بالطول من كل قناة الأذن إلى تجويف العين. </li><li style=";text-align:right;direction:rtl"> باستخدام مقص تشريح الصغيرة ، وقطع لوحة العظم القذالي تغمر المخيخ من قناة الأذن واحدة إلى أخرى. </li><li style=";text-align:right;direction:rtl"> مع مقص تشريح نفسه ، خفضت الأمامية على طول خط الوسط على مستوى البصيلات الشمية. </li><li style=";text-align:right;direction:rtl"> باستخدام الملقط ، وإزالة كل لوحة العظام من الدماغ ، مع الحرص على إزالة أي نوع من الأنسجة الضامة بحيث أن الدماغ لا يزال سليما عندما رفع قبالة العظام. </li><li style=";text-align:right;direction:rtl"> قص قطع الأعصاب البصرية والجمجمة والفقرات العنقية العليا ، وإزالة تثبيتي في الدماغ. </li><li style=";text-align:right;direction:rtl"> في مرحلة ما بعد الإصلاح لمدة 1-2 ساعات في 4 درجات مئوية. اغسل 3 مرات كل 15 دقيقة في برنامج تلفزيوني. </li></ol> 3. Agarose التضمين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تذوب 2 ٪ عالية القوة ، وانخفاض نقطة agarose هلام نوع IB (سيغما رقم الكتالوج : A0576) في برنامج تلفزيوني مع المالحة فرن الميكروويف. </li><li style=";text-align:right;direction:rtl"> نقل agarose المنصهر إلى أنبوب اختبار. مكان أنبوب الاختبار في حمام ماء دافئ أو الحاضنة (40 ° -41 درجة مئوية) حتى موازنة درجة الحرارة. </li><li style=";text-align:right;direction:rtl"> الغراء أنسجة المخ ثابتة على المكبس من المحاقن مع عينة cyanoacrylate الغراء (3A الشكل). فرشاة طبقة رقيقة من محلول السكروز ، PBS المشبعة على سطح أنسجة المخ لتسهيل الافراج في وقت لاحق من الحلبة agarose من شرائح الدماغ. </li><li style=";text-align:right;direction:rtl"> سحب المكبس مع أنسجة المخ في السكن المكبس (الشكل 3B). </li><li style=";text-align:right;direction:rtl"> ماصة أو صب agarose الدافئة في السكن المكبس ، الذي يغطي أنسجة المخ. تجنب احتباس فقاعات الهواء في agarose. وفقاعات الهواء في agarose تؤثر سلبا على جودة شريحة. </li><li style=";text-align:right;direction:rtl"> المشبك المحقنة مع العينة (° 0 إلى -25 درجة مئوية) الباردة كتلة تقشعر لها الأبدان لمدة 1 دقيقة لضبط agarose (الشكل 3C). </li></ol> 4. Sectioning شريحة الدماغ مع Compresstome <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تجميع العينات حقنة تحتوي على أنسجة المخ في Compresstome (الشكل 3D). </li><li style=";text-align:right;direction:rtl"> محاذاة حافة شفرة الحلاقة بشكل وثيق مع مخرج المحقنة العينة (الشكل 3E). </li><li style=";text-align:right;direction:rtl"> تعبئة خزان العازلة مع الحل برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> تدوير شريحة سمك ميكرومتر الى الأمام لبثق كتلة أنسجة الدماغ agarose من حقنة العينة. </li><li style=";text-align:right;direction:rtl"> اضغط على زر &quot;ابدأ&quot; في وحدة التحكم من Compresstome للشروع في عملية sectioning. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 4.4) إلى 4.5) حتى يتم الحصول على المطلوب في تحديد شرائح سمك. </li><li style=";text-align:right;direction:rtl"> شرائح الدماغ الحصاد مع فرشاة أو ماصة للقوارير أو الآبار مملوءة PBS تلوين الشكل (3F). </li></ol> 5. تبادل البصرية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد المجلد 75 ٪ : المجلد حل الجلسرين : برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> صب (أو ماصة) قبالة حجم نصف PBS غسل الدماغ من الشرائح التي تم جمعها. </li><li style=";text-align:right;direction:rtl"> إضافة مرة أخرى حجم إزالة مع الجلسرين / برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> السماح للتوازن مع مزج لطيف في 4 درجات مئوية حتى تغرق شرائح. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 6.2) إلى 6.4) حتى تصل إلى تركيز الجلسرين النهائية 75 ٪. </li><li style=";text-align:right;direction:rtl"> استبدال حل مع الجلسرين 90 ٪ في برنامج تلفزيوني ، والسماح للتوازن. * ملاحظة : احرص على عدم إدخال فقاعات الهواء في حين اضاف حلول الجلسرين. صب المزيج ببطء وحلول بلطف ، منذ فقاعات الهواء سوف تتداخل مع equilibra موحدةوسيتم تنفيذ نشوئها والى شريحة متزايدة. </li></ol> 6. شريحة متزايدة للتصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع لاصقة على الوجهين الختم (SA - S - 1L ، غريس بيو مختبرات) في مستطيل مفتوح لتناسب مساحة للعرض شريحة متناهية الصغر (Superfrost زائد ، 25 X 75 X 1 ملم ، والقط. # 48311-703 ، VWR ) مع الجانبين 2-3 مم واسعة. </li><li style=";text-align:right;direction:rtl"> مكان شرائح الدماغ داخل مركز للمستطيل باستخدام شريط لاصق ماصة أو فرشاة الرسم. إزالة الزائدة الجلسرين مع التطلع لطيف. </li><li style=";text-align:right;direction:rtl"> ترتيب شرائح الدماغ على الشريحة في الترتيب المطلوب مع فرشاة الرسم. </li><li style=";text-align:right;direction:rtl"> انخفاض الغطاء برفق زجاج (24 X 60 مم ، القط. # 48383-139 ، VWR) على شريط لاصق ، مع الحرص على عدم إدخال فقاعات الهواء. بلطف تطبيق الضغط على الزجاج غطاء لضمان الاتصال مع شريط لاصق. </li><li style=";text-align:right;direction:rtl"> نضح الجلسرين الزائد من الحواف بين الشرائح وساترة. </li><li style=";text-align:right;direction:rtl"> وختم الشريحة ساترة باستخدام طلاء الأظافر واضحة. </li><li style=";text-align:right;direction:rtl"> السماح ليجف قبل التصوير ، أو تخزين طويل الأجل في صناديق الشريحة في 4 درجات مئوية. </li></ol> 7. ممثل النتائج : وقد أصبحت معالجة ، والتصوير ، وتحليل fluorescently المسمى أنسجة المخ التي لا غنى عنها لدراسة البيولوجيا العصبية. كثير من هذه التحقيقات تتطلب التلاعب الجيني متطورة للحصول على التعبير مراسل فرعية في الخلايا العصبية المستهدفة ، تليها كل من يحلل صورة منخفضة وعالية الدقة. في كثير من الأحيان قيود التجريبية والتقنية تجعل من المستحيل الحصول على هذه الأنواع من البيانات من نفس الحيوان ، أو بطريقة سريعة. إعداد مسح بصريا ، والمقاطع الدماغ شبه سميكة لمكافحة الإيدز التصوير فلوري في هذا التحدي. مثال على شريحة سميكة الدماغ سليمة المسمى مع فيروس داء الكلب المعدلة وراثيا هندسيا لEGFP التعبير ، والتقط باستخدام المجهر epifluorescent ومبائر هو مبين في الشكل (4) والشكل (5) على التوالي. التصوير epifluorescent استخدمنا M205 ايكا اتحاد كرة القدم ، والحصول على الصور مبائر استخدمنا LSM زايس 510. بسبب البساطة النسبية للبروتوكول المرفق ، وهذه الطريقة هي قادرة على إنتاج بيانات الصور مفيدة من نسيج التعبير عن صحفيين الفلورسنت مع فترة زمنية أقل من يوم واحد ، ومتوافق مع كل من المجهري الإضاءة الخافتة والعالية الدقة. إذا كان محقق تختار إدماج أساليب إضافية الوسم بعد الوفاة ، وتلطيخ المناعى ، أو جعل أرق المقاطع ، وبروتوكول يطول تبعا لذلك. ومع ذلك ، فإن الطريقة الموصوفة أعلاه يمثل طريقة بسيطة والإنتاجية العالية نسبيا من أجل فحص أنماط التعبير مراسل حيوية في الدماغ سليمة. <img src="/files/ftp_upload/2807/2807fig1.jpg" alt="الشكل 1"/> الشكل 1. مخطط تدفق diagraming التثبيت ، تشريح ، تشريح ، تبادل المعلومات ، وإجراءات متزايدة من شرائح الدماغ شبه سميكة. <img src="/files/ftp_upload/2807/2807fig2.jpg" alt="الشكل 2"/> الشكل 2. مخطط لقطع خطوات متتابعة لاستخراج مخ الفأر سليمة. <img src="/files/ftp_upload/2807/2807fig3.jpg" alt="الشكل 3"/> الشكل 3 خطوات لتحميل وشريحة أنسجة المخ باستخدام Compresstome. أ) الموضع من أنسجة المخ على خفض استخدام superglue المكبس. ب) رسم أسفل أنسجة المخ التي شنت في المكبس لتضمين agarose. C) التقوية القابس الدماغ agarose باستخدام كتلة ضغط مبردة. D) الإدراج من المكونات الدماغ والمكبس agarose في غرفة قطع Compresstome. E) محاذاة razorblade إلى الجهاز المكبس. F) ، وجمع قطع من شرائح الدماغ في غرفة عازلة Compresstome. <img src="/files/ftp_upload/2807/2807fig4.jpg" alt="الشكل 4"/> الشكل 4. الصور المجهري الضوء على شريحة سميكة من الجلسرين تطهيرها أنسجة المخ من خلال التعبير عن القشرة الخارجية الخضراء المعززة بروتين فلوري (EGFP). شريحة) الاكليل من خلال مخ الفأر (200 ميكرون سميكة) المسمى مع EGFP ناقلات فيروسية في التعبير عن وتصويرها باستخدام دقة منخفضة المجسام epifluorescent. مقياس شريط ، 2 ملم. <img src="/files/ftp_upload/2807/2807fig5.jpg" alt="الشكل 5"/> الشكل 5. صورة عالية التكبير طبقة fluorescently المسمى 06/05 العصبونات القشرية في الدماغ شريحة سميكة تطهيرها. أ) صورة عالية الدقة مبائر لإسقاط الحد الأقصى Z - المكدس (150 ميكرون سميكة) من خلال تسليط الضوء على المنطقة في (الشكل 4). مقياس بار ، و 25 ميكرون.

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جيان تشيانغ كونغ موظف في شركة Precisionary ، والصكوك ، التي تقوم بتصنيع منتج المستخدمة في هذه المقالة.

نظرا للتطبيق على نطاق واسع من استخدام البروتينات الفلورية لاستهداف الخلايا العصبية فرعية للتحقيق عبر المجهر الضوئي ، والحاجة إلى سرعة الشاشة والصورة وتحليل الشبكات العصبية داخل أنسجة الدماغ سليمة أصبحت لا تقدر بثمن. التقدم الت...

<table border="1"&gt;<tbody&gt;<tr&gt;<th&gt; اسم البند</th&gt;<th&gt; الشركة</th&gt;<th&gt; فهرس العدد</th&gt;<th&gt; تعليقات (اختياري)</th&gt;</tr&gt;<tr&gt;<td&gt; مقص العظام</td&gt;<td&gt; FST</td&gt;<td&gt; 16044-10</td&gt;<td&gt; أو ما يعادلها</td&gt;</tr&gt;<tr&gt;<tdمقص تشريح&gt;</td&gt;<td&gt; FST</td&gt;<td&gt; 14084-08</td&gt;<td&gt; أو ما يعادلها</td&gt;</tr&gt;<tr&gt;<td&gt; نوع IB agarose</td&gt;<td&gt; سيغما</td&gt;<td&gt; A0576</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Compresstome</td&gt;<td&gt; آلات Precisionary</td&gt;<td&gt; VF - 200</td&gt;<td&gt; - vibratomes أخرى متوافقة</td&gt;</tr&gt;<tr&gt;<td&gt; لاصقة مزدوجة من جانب</td&gt;<td&gt; جريس بيو مختبرات</td&gt;<td&gt; SA - S - 1L</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Superfrost بلس الشرائح</td&gt;<td&gt; VWR</td&gt;<td&gt; 48311-703</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; غطاء من الزجاج</td&gt;<td&gt; VWR</td&gt;<td&gt; 48383-139</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; الجلسرين</td&gt;<td&gt; EMD شركة الكيماويات</td&gt;<td&gt; GX0185 - 6</td&gt;<td&gt; أو ما يعادلها</td&gt;</tr&gt;</tbody&gt;</table&gt;

a rapid approach to high-resolution fluorescence imaging in semi-thick brain slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Watch this Scientific Journal Video about A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices at JoVE.com

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

نحن هنا وصفا لطريقة سريعة وبسيطة لصورة fluorescently المسمى الخلايا في الدماغ شبه شرائح سميكة. عن طريق تحديد ، تشريح ، وإزالة أنسجة الدماغ بصريا وصفنا كيف يمكن استخدام معيار التصوير epifluorescent مبائر أو لتصور الخلايا الفردية والشبكات العصبية داخل الأنسجة العصبية سليمة.

نهج السريع إلى التصوير الإسفار عالية الدقة في الدماغ شرائح شبه سميكة

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

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JoVE Journal

Neuroscience

16.2K Views.  Baylor College of Medicine (BCM). نحن هنا وصفا لطريقة سريعة وبسيطة لصورة fluorescently المسمى الخلايا في الدماغ شبه شرائح سميكة. عن طريق تحديد ، تشريح ، وإزالة أنسجة الدماغ بصريا وصفنا كيف يمكن استخدام معيار التصوير epifluorescent مبائر أو لتصور الخلايا الفردية والشبكات العصبية داخل الأنسجة العصبية سليمة.

Video: A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

والفحص عن عضوي النمط شريحة الوقت الفاصل بين التصوير عالية الدقة للهجرة الخلايا العصبية في الدماغ بعد الولادة

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

عالية الدقة التصوير بالرنين المغناطيسي الوظيفي للطرق الدماغ المتوسط ​​الإنسان

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

عالية الدقة<em&gt; في فيفو</em&gt; دليل بروتوكول تجزئة للالإنسان الحصين الحقول الفرعية عن طريق التصوير بالرنين المغناطيسي 3T

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

عالية الدقة الهيكلي التصوير بالرنين المغناطيسي من تحت القشرة الإنسان<I&gt; في فيفو</I&gt; وتشريح الجثث

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

في فيفو الدماغ أسلوب التصوير النهج القائم على الكالسيوم إضاءة الحيوية المؤشر GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

تصوير [كنفوكل] عالية الدقة لحاجز الدم في الدماغ: التصوير، وإعادة البناء ثلاثي الأبعاد، والتقدير الكمي ترانسسيتوسيس

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

تصوير الكالسيوم اثنين-فوتون في Dendrites العصبية في الدماغ شرائح

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

عالية الدقة حجم التصوير من الخلايا العصبية باستبعاد استخدام Fluorescence الأسلوب وكرست أجهزة موائع جزيئية

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

وثمة نهج بديل لدراسة الأحداث الرئيسية في نيوروديجينيريشن باستخدام شرائح المخ الفئران السابقين فيفو

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

التصوير ثلاثي الأبعاد عالي الدقة للعدوى بفيروس داء الكلب في أنسجة الدماغ التي تم تطهيرها من المذيبات

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...