وأيد هذا العمل من قبل المعاهد الوطنية للصحة والمعهد الوطني للاضطرابات العصبية والسكتة الدماغية (NINDS) (NS049442).&gt;

إعادة التعليق β اميلويد ببتيد لقد بدأت قبل الحصول على استعداد : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Aβ 1-42 (مسحوق مجفف بالتجميد) </li><li style=";text-align:right;direction:rtl"> 1،1،1،3،3،3 - Hexafluoro - 2 - بروبانول (HFIP) </li><li style=";text-align:right;direction:rtl"> منخفضة ملزم أنابيب البولي بروبلين microcentrifuge (1.5 مل) </li><li style=";text-align:right;direction:rtl"> GasTight هاميلتون المحاقن مع وقف الهبوط تفلون (250 ميكرولتر) </li><li style=";text-align:right;direction:rtl"> Dimethylsulfoxide (DMSO) </li></ul><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تسمح Aβ مجفد 1-42 لكي تتوازن في درجة حرارة الغرفة لمدة 30 دقيقة لتجنب التكثيف عند فتح القارورة الببتيد. </li><li style=";text-align:right;direction:rtl"> تحت غطاء الدخان ، وإعادة تعليق Aβ 1-42 الببتيد في HFIP الجليد الباردة للحصول على حل من 1 ملم ودوامة الحل لبضع ثوان. </li><li style=";text-align:right;direction:rtl"> باستخدام محقنة زجاجية هاميلتون GasTight مع المكونات تفلون ، تقسيم بسرعة Aβ 1-42 / HFIP حل بالتساوي في قوارير البولي بروبلين الثلاث وختم القوارير. </li><li style=";text-align:right;direction:rtl"> ثم يتم تحضين قارورة لمدة 2 ساعة للسماح للmonomerization Aβ. المقبل ، فتح قنينة والتركيز على حل Aβ 1-42 / HFIP ظل فراغ باستخدام جهاز للطرد المركزي SpeedVac (800 غرام ، درجة حرارة الغرفة) وحتى لوحظ الفيلم الببتيد واضحة في الجزء السفلي من قنينة. الاختيار بعناية درجة الحرارة داخل أجهزة الطرد المركزي لتجنب تدهور SpeedVac الببتيد (حد أقصى 25 درجة مئوية). </li><li style=";text-align:right;direction:rtl"> ختم القنينات وتخزين aliquots في -80 درجة مئوية. ويمكن تخزين فيلم Aβ لمدة 6 أشهر. </li><li style=";text-align:right;direction:rtl"> اعادة تعليق فيلم واحد عن طريق إضافة DMSO للحصول على تركيز تصل إلى 5 ملي Aβ 1-42. يصوتن في حمام مائي لمدة 10 دقيقة كاملة لضمان اعادة التعليق. </li><li style=";text-align:right;direction:rtl"> قسامة هذا Aβ 5mM 1-42 حل في قوارير البولي بروبلين. ختم كل قارورة وتخزينها في -20 درجة مئوية. يجب إذابة Aliquots مرة واحدة فقط. حذار من تكثيف الماء داخل الثلاجة. الببتيدات في حل تتحلل بسهولة بالغة. </li></ol> Oligomerization لقد بدأت قبل الحصول على استعداد : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Aβ 1-42 / DMSO (5mM) </li><li style=";text-align:right;direction:rtl"> الفوسفات العقيمة العازلة </li><li style=";text-align:right;direction:rtl"> منخفضة ملزم أنابيب البولي بروبلين microcentrifuge (1.5 مل) </li><li style=";text-align:right;direction:rtl"> منخفضة ملزم أنابيب البولي بروبلين الطرد المركزي (50 مل) </li></ul><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمييع Aβ الحصول 5mM 1-42 / DMSO قسامة مع العازلة الفوسفات العقيمة (تصل إلى 100 ​​ميكرولتر). </li><li style=";text-align:right;direction:rtl"> دوامة لمدة 30 ثانية ثم احتضان لمدة 12 ساعة في 4 درجات مئوية </li></ol> الحصين شريحة العلاج عند معالجة شرائح الحصين ، فمن المهم لتجنب التصاق غير محددة الببتيد على الأكواب ، والأسطح أنابيب التروية ، وغرفة تسجيل. استخدام تفضيلي منخفض ملزمة أنابيب البولي بروبيلين والحاويات. تجنب استخدام الأواني الزجاجية أو البلاستيكية عامة وير. وينبغي أن تكون وسائل الاعلام نضح المصل أو الزلال الحرة. Aβ نضح لقد بدأت قبل الحصول على استعداد : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Oligomerized Aβ 1-42 </li><li style=";text-align:right;direction:rtl"> تسجيل العازلة (مم : 124 كلوريد الصوديوم ، 4.4 بوكل ، 1 نا 2 هبو 4 ، 25 NaHCO 3 ، 2 CaCl 2 ، 2 MgCl 2 ، والجلوكوز 10) </li><li style=";text-align:right;direction:rtl"> الكهربية تسجيل انشاء غرفة مع واجهة </li><li style=";text-align:right;direction:rtl"> الحصين شرائح عرضية المحتضنة في المخزن المؤقت لتسجيل الاوكسيجين 90 دقيقة على الأقل في مرحلة ما بعد تشريح (T = 29 درجة مئوية ، معدل ثابت نضح = 2 مل / دقيقة) </li></ul><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> على الفور قبل التجربة ، وتمييع Aβ oligomerized 1-42 قسامة لتركيز العمل المناسبة (200 نيوتن متر أو أعلى) في أنبوب البولي بروبلين العازلة مع تسجيل قبل الاوكسيجين. </li><li style=";text-align:right;direction:rtl"> ويروي Aβ 1-42 حل لمدة 20 دقيقة لشرائح قبل تحريض اللدونة متشابك. </li></ol> تحريض اللدونة والمتابعة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عادة ، النموذج الأكثر استخداما على نطاق واسع من المرونة متشابك هو الكمونية. يمكن أن يتسبب ذلك من خلال تحفيز كزازي من المشبك معينة في قرن آمون. نسجل الردود الحقل من نقاط الاشتباك العصبي بين التوقعات والضمانات شافير والخلايا العصبية CA1 الهرمية في الطبقة المشععة. التحفيز لدينا كزازي يتكون من تحفيز انفجار ثيتا التي تضم 3 قطارات مفصولة فترات 15 ثانية. كل قطار يتكون من 10 طلقات نارية عند 5 هرتز حيث انفجر يستتبع كل 5 نبضات عند 100 هرتز. تطبيق التحفيز كزازي مباشرة بعد انتهائه ، نضح من Aβ oligomerized. </li><li style=";text-align:right;direction:rtl"> مباشرة بعد التحفيز كزازي التبديل إلى نضح مع تسجيل عازلة طبيعية. الحفاظ على معدل ثابت التروية ، وتسجيل درجة حرارة الغرفة والأوكسجين المناسبة طوال فترة التجربة. </li></ol> ممثل النتائج والمشاكل المحتملة وAβ oligomerized وفقا لبروتوكول الكلاسيكية ستاين وآخرون كول 1 ، يولد مونومرات Aβ ومجموعة متنوعة من oligomers من أحجام مختلفةق (ديمر ، ترايمر ، الخ) ونضح من oligomerized Aβ 1-42 يؤدي إلى تناقص في LTP Aβ شرائح المعالجة مقارنة مع شرائح السيطرة. تحت ظروف تجريبية لدينا ، حيث يتم استخدام 3-5 الشهر القديمة C57BL/6J الفئران الذكور ، الكمونية في 200 نانومتر Aβ شرائح المعالجة على 150 ٪ من متوسط ​​القيم الأساسية في ساعتين بعد التحفيز كزازي ، في حين أن شرائح تحكم القيم والكمونية على ٪ متوسط ​​baseline2 200-250. في بعض الحالات ، قد تفشل المعاملة مع Aβ للحد من الكمونية الحصين. أخطاء فادحة عدة التي قد تحدث تشمل الفيلم المفرطة أثناء التجفيف وتركيز Aβ oligomerization غير مكتملة. قد نضح في Aβ شرائح الحصين تكون مصدرا آخر للقلق بسبب الخصائص الفيزيائية للالببتيد Aβ في الحل ، الذي هو عرضة للالتصاق غير محددة للمواد البلاستيكية. وتناقش هذه القضايا من المشاكل أدناه.

<ol>
	<li>Stine, W. B., Dahlgren, K. N., Krafft, G. A., LaDu, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+conditions+for+amyloid-beta+peptide+oligomerization+and+fibrillogenesis.">In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.</a> J Biol Chem. 278, 11612-11622 (2003).</li><li>Vitolo, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+-peptide+inhibition+of+the+PKA/CREB+pathway+and+long-term+potentiation:+reversibility+by+drugs+that+enhance+cAMP+signaling.">Amyloid beta -peptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling.</a> Proc Natl Acad Sci U S A. 99, 13217-13221 (2002).</li><li>Puzzo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picomolar+amyloid-beta+positively+modulates+synaptic+plasticity+and+memory+in+hippocampus.">Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus.</a> J Neurosci. 28, 14537-14545 (2008).</li><li>Lewczuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sample+collection+tubes+on+cerebrospinal+fluid+concentrations+of+tau+proteins+and+amyloid+beta+peptides.">Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid beta peptides.</a> Clin Chem. 52, 332-334 (2006).</li><li>Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+synaptic+dysfunction+in+Alzheimer's+disease.">Mechanisms of synaptic dysfunction in Alzheimer's disease.</a> Histol Histopathol. 10, 509-519 (1995).</li><li>Cullen, W. K., Suh, Y. H., Anwyl, R., Rowan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+of+LTP+in+rat+hippocampus+in+vivo+by+beta-amyloid+precursor+protein+fragments.">Block of LTP in rat hippocampus in vivo by beta-amyloid precursor protein fragments.</a> Neuroreport. 8, 3213-3217 (1997).</li><li>Itoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+of+long-term+potentiation+in+hippocampal+slices+of+beta-amyloid-infused+rats.">Impairments of long-term potentiation in hippocampal slices of beta-amyloid-infused rats.</a> Eur J Pharmacol. 382, 167-175 (1999).</li><li>Walsh, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+secreted+oligomers+of+amyloid+beta+protein+potently+inhibit+hippocampal+long-term+potentiation+in+vivo.">Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.</a> Nature. 416, 535-539 (2002).</li><li>Lambert, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nonfibrillar+ligands+derived+from+Abeta1-42+are+potent+central+nervous+system+neurotoxins.">nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.</a> Proc Natl Acad Sci U S A. 95, 6448-6453 (1998).</li><li>Gong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+hydrolase+Uch-L1+rescues+beta-amyloid-induced+decreases+in+synaptic+function+and+contextual+memory.">Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory.</a> Cell. 126, 775-788 (2006).</li><li>Trinchese, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+calpains+improves+memory+and+synaptic+transmission+in+a+mouse+model+of+Alzheimer+disease.">Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.</a> J Clin Invest. 118, 2796-2807 (2008).</li></ol>

وقد أجريت تجارب على الحيوانات وفقا للمبادئ التوجيهية واللوائح التي وضعتها IACUC جامعة كولومبيا.

على النحو الموصوف أعلاه ، قد تحدث مشاكل عدة في إعداد Aβ oligomeric النتيجة في LTP ايتأثر. طريقة واحدة لتقييم ما إذا كان تدهور Aβ أو عدم وجود oligomerization Aβ قد حدث هو تقييم إعداد Aβ باستخدام TRIS - Tricine PAGE / تحليل الغربية التنشيف والتحليلية تنبيذ فائق (وليس وصفها في هذا البروتوكول). عند?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>A&beta; 1-42 (lyophilized) </td>
<td>&nbsp;</td>
<td>American Peptide Co.</td>
<td>62-0-80</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>52517</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Biotium</td>
<td>90082</td>
<td>&nbsp;</td>
<tr>
<td>50mL Polypropylene Centrifuge Tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>05-538-67</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml MaxyClear microtubes Maximum recovery</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT-150-L-C</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-Buffered Saline pH 7.4</td>
<td>&nbsp;</td>
<td>Invitrogen-Gibco</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>GASTIGHT Syringes 250 &mu;L</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>1725</td>
<td>&nbsp;</td>
<tr>
<td>Bath sonicator</td>
<td>&nbsp;</td>
<td>Branson</td>
<td>3510</td>
<td>&nbsp;</td>
<tr>
<td>Savant SpeedVac Concentrator System</td>
<td>&nbsp;</td>
<td>Various distributors</td>
<td>SC 110A</td>
<td>&nbsp;</td>		</tbody>
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preparation of oligomeric &beta;-amyloid1-42 and induction of synaptic plasticity impairment on hippocampal slices

ضعف الاتصالات متشابك من المحتمل أن تكمن وراء التغييرات الطفيفة التي تحدث في نساوي المراحل المبكرة من مرض الزهايمر ق (AD). ومن المعروف β اميلويد (Aβ) ، الببتيد التي تنتج في كميات عالية في ميلادي ، للحد بعيدة المدى (الكمونية) ، وهي الخلوية ربط التعلم والذاكرة. في الواقع ، بسبب ضعف LTP Aβ هو نموذج تجريبي لدراسة مفيدة متشابك اختلالات في النماذج ميلادي والفرز على المخدرات قادرة على التخفيف من العاهات أو العودة متشابك من هذا القبيل. وقد أظهرت الدراسات التي تنتج Aβ تعطل LTP تفضيلي عبر شكله oligomeric. هنا نقدم بروتوكول مفصلة للعرقلة من قبل LTP نضح الاصطناعية oligomerized Aβ1 - 42 على شرائح الببتيد الحصين الحادة. في هذا الفيديو ، ونحن المخطط إجراء خطوة بخطوة لإعداد Aβ oligomeric<sub&gt; 1-42</sub&gt;. ثم ، ونحن نتابع تجربة الفردية التي يتم تخفيض شرائح LTP في الحصين يتعرض لAβ oligomerized<sub&gt; 1-42</sub&gt; مقارنة مع شرائح في تجربة الرقابة حيث لا Aβ<sub&gt; 1-42</subوكان&gt; التعرض حدث.

Watch this Scientific Journal Video about Preparation of Oligomeric Î²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices at JoVE.com

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

ميزة واحدة لمرض الزهايمر هو ارتفاع Aβ<sub&gt; 1-42</sub&gt; الببتيد. هنا نقدم مشروع بروتوكول لإعداد Aβ الاصطناعية<sub&gt; 1-42</sub&gt; oligomers ، الذي ينال الحصين بعيدة المدى ، وهي ربط الخلوي للذاكرة. هذا الإجراء مفيدا للتحقيق في آليات فحص الأمراض والمخدرات التي يسببها Aβ.

إعداد Oligomeric β اميلويد 1-42 والتعريفي للعاهة اللدونة متشابك على شرائح الحصين

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

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Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

23.0K Views.  Columbia University. ميزة واحدة لمرض الزهايمر هو ارتفاع Aβ 1-42 الببتيد. هنا نقدم مشروع بروتوكول لإعداد Aβ الاصطناعية 1-42 oligomers ، الذي ينال الحصين بعيدة المدى ، وهي ربط الخلوي للذاكرة. هذا الإجراء مفيدا للتحقيق في آليات فحص الأمراض والمخدرات التي يسببها Aβ.

Video: Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

إعداد شرائح الحصين الحادة من الجرذان والفئران المعدلة وراثيا لدراسة التعديلات متشابك وامراض الشيخوخة خلال اميلويد

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

المقررة الإنتشار الأفقي والإيماس من غشاء بروتينات في الخلايا العصبية مثقف باستخدام الاسترداد الإسفار وPhotobleaching الإسفار الخسارة

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

بعد تضمين التعريف Immunogold من البروتينات متشابك في الثقافات شريحة الحصين

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

تحسين إعداد والحفاظ على شرائح الحصين ماوس لتسجيل مستقرة جدا وقابلة للتكرار من التقوية بعيدة المدى

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

تسريب التجسيمي من بلازميدة قليلة القسيمات اميلويد بيتا في الحصين ماوس

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

التحقيق في متشابك وسم / القبض على والصليب التقاط باستخدام الحاد شرائح الحصين من القوارض

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

قياس الالتقام حويصلة متشابك في الخلايا العصبية هيبوكامبال مثقف

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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 ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

تقييم كثافة المشبك في الدماغ القوارض هيبوكامبال شرائح

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...