وقد تم تمويل هذا العمل من قبل NINDS -- NIH R01NS060756

1. قبل البدء في إعداد شرائح الحصين. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد تقطيع الأنسجة عن طريق وضع قطعة من ورقة تفلون وتصاعد شفرة جديدة. </li><li style=";text-align:right;direction:rtl"> مسح نسيج الثقافة (TC) هود مع الايثانول 70 ٪ ومجموعة الداخل مجهر تشريح. تعقيم هود ، المجهر ، وتقطيع اللحم والأنسجة وتشريح جميع الصكوك لمدة 15 دقيقة للأشعة فوق البنفسجية. </li><li style=";text-align:right;direction:rtl"> إعداد ست لوحات TC جيدا. إضافة 750 ميكرولتر ثقافة شريحة وسائل الإعلام (SCM) في مكان جيد وإدراج ثقافة خلية في كل بئر. تأكد من الأغشية الرطبة تماما مع عدم وجود فقاعات تحتها. وضع لوحات في حاضنة على 35 درجة مئوية بالغاز مع 5 ٪ CO 2 لحين الحاجة إليها. </li><li style=";text-align:right;direction:rtl"> صب 50 مل + ACSF منخفضة نا (تشريح الحل) في كوب 100 مل ووضعه على الجليد الملح المزيج. فقاعة المنخفض الصوديوم + ACSF مع 5 ٪ CO 2 / 95 ٪ O 2 حتى يتغير لونها وACSF أشكال مزيج من الطين حل المجمدة والسائلة (10-20 دقيقة). </li><li style=";text-align:right;direction:rtl"> الحصول على الفئران P5 - P7 الجرو. ويمكن استخدام ما يصل الى ثلاثة الجراء. </li></ol> 2. الحصين شرائح التحضير. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع رأس الحيوان مع مقص أداة حادة. قطع الجلد وفضح الجمجمة. فتح الجمجمة عن طريق قطع من جانب إلى آخر على طول خط interaural ومن ثم على طول الدرز السهمي مع مقص صغير. ويمكن إجراء خفض اختياري من جانب إلى آخر في الجبهة لتسهيل إزالة العظام والتعرض للدماغ. يستخرج الدماغ بسرعة مع ملعقة ملعقة صغيرة مدورة ووضعه في الطين من تشريح حل البرد لمدة دقيقة 1 ~. ~ صب 10 مل من محلول الباردة الجليد تشريح على طبق 60 ملم ونقل الدماغ من الدورق إلى الطبق. ينبغي تغطية تشريح الدماغ مع الحل. </li><li style=";text-align:right;direction:rtl"> تحت المجهر تشريح : ضع الدماغ والاحتفاظ بها في خط الوسط مع ملقط تشريح الضغط إلى أسفل الطبق 60 ملم. استخدام أداة تشريح الحصين للفصل بين نصفي الكرة الأرضية تاركة الدماغ المتوسط. ويتعرض بعد ذلك الحصين على كل الكرة الأرضية. ثم بلطف الحصين مغرفة بها مع تشريح الحصين الأداة. استخدام إبرة تشريح لعزل تماما الحصين وتنظيفه قدر المستطاع. </li><li style=";text-align:right;direction:rtl"> استخدام غيض مقصوص من طرف P1000 ماصة التصفية ، نضح بلطف الحصين وتحويلها إلى ورقة تفلون على تقطيع اللحم والأنسجة. موقف الحصين على جانبها مقعرة. </li><li style=";text-align:right;direction:rtl"> محاذاة عمودية الحصين لنصل للحصول على مقاطع الاكليلية واستنزاف فائض السائل. </li><li style=";text-align:right;direction:rtl"> الحصين كل شريحة ميكرون 400. </li><li style=";text-align:right;direction:rtl"> ~ صب 10 مل الباردة المجلس الاعلى للقضاة في صحن 60 ملم ونقل شرائح الحصين من تقطيع اللحم وباستخدام آخر مقصوص P1000 طرف مرشح المجلس الاعلى للقضاة والباردة. تجنب جعل فقاعات. </li><li style=";text-align:right;direction:rtl"> مع مساعدة من ملعقة ملعقة والقزحية مباشرة منفصلة بلطف شرائح من بعضها البعض. </li><li style=";text-align:right;direction:rtl"> شرائح منفصلة واضحة المعالم وغير التالفة من الشرائح المتضررة. </li></ol> 3. الحصين شرائح الثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تقديم لوحات ستة جيدا مع المجلس الاعلى للقضاة وإدراج ثقافة خلية من الحاضنة. مع مساعدة من طرف آخر P1000 مقصوص تصفية ونقل الشرائح الفردية على الغشاء. مكان شرائح 4-5 في الغشاء. يجب الحرص على عدم وضع الشرائح وثيقة إما إلى الجدار إدراج أو قريبة من بعضها البعض. عند الضرورة ، واستخدام ملعقة لقزحية شرائح منفصلة. إزالة الزائدة المتوسطة. شرائح اتصال أقل قدر ممكن مرة واحدة كانت على الغشاء. </li><li style=";text-align:right;direction:rtl"> تحرك الصفيحة العودة إلى الحاضنة والثقافة في 35 درجة مئوية و 5 ٪ CO 2. </li><li style=";text-align:right;direction:rtl"> SCM تغيير كل 48 ساعة داخل غطاء TC بواسطة الشفط مع المجلس الأعلى للقضاء ماصة باستور. إضافة 750 ميكرولتر من قبل المجلس الاعلى للقضاة تحسنت جديدة لكل بئر. تأكد من أن يتم تشكيل أي فقاعات تحت الغشاء. </li></ol> 4. الحلول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> انخفاض الصوديوم + ACSF -- حل تشريح للثقافات شريحة منزوع الأيونات ومعقمة لإضافة H 2 O : <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> ل 500 مل </td><td> ل 1000 مل </td><td> التركيز النهائي </td></tr><tr><td> CaCl 2 (1 م) </td><td> 0.5 مل </td><td> 1 مل </td><td> 1 ملم </td></tr><tr><td> D - غلوكوز </td><td> 0،901 ز </td><td> 1،802 ز </td><td> 10 ملي </td></tr><tr><td> بوكل </td><td> 0،149 ز </td><td> 0،298 ز </td><td> 4 ملم </td></tr><tr><td> MgCl 2 (1 م) </td><td> 2.5 مل </td><td> 5 مل </td><td> 5 ملم </td></tr><tr><td> NaHCO 3 </td><td> 1،092 ز </td><td> 2،184 ز </td><td> 26 مم </td></tr><tr><td> السكروز </td><td> 40 غ </td><td> 80 غ </td><td> 234 ملي </td></tr><tr><td> محلول الفينول الأحمر 0.5 ٪ في DPBS </td><td> 0.5 مل </td><td> 1 مل </td><td> 0.1 ٪ V / V </td></tr></tbody></table> مزيج ~ 30 دقيقة Sterilإيزي قبل المرور عبر فلتر 0.22μm جعل aliquots 50 مل وتخزينها في 4 درجات مئوية لا تزيد على 2 أشهر. </li><li style=";text-align:right;direction:rtl"> شريحة متوسطة الثقافة (SCM) <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> ل 500 مل </td><td> ل 1000 مل </td><td> التركيز النهائي </td></tr><tr><td> MEM النسر المتوسطة </td><td> 4.2 غرام </td><td> 8.4 غرام </td><td> 8.4 غرام / لتر </td></tr><tr><td> حصان المصل الحرارة المعطل </td><td> 100 مل </td><td> 200 مل </td><td> 20 ٪ </td></tr><tr><td> L - الجلوتامين (200 ملم) </td><td> 2.5 مل </td><td> 5 مل </td><td> 1 ملم </td></tr><tr><td> CaCl 2 (1 م) </td><td> 0.5 مل </td><td> 1 مل </td><td> 1 ملم </td></tr><tr><td> MgSO 4 (1 م) </td><td> 1 مل </td><td> 2 مل </td><td> 2 ملم </td></tr><tr><td> الانسولين (1 ملغ / مل) ، الذائب في حمض الهيدروكلوريك 0.01 N </td><td> 0.5 مل </td><td> 1 مل </td><td> 1 ملغم / لتر </td></tr><tr><td> حمض الاسكوربيك ، والحل (25 ٪ W / V) </td><td> 0.024 مل </td><td> 0.048 مل </td><td> 0.00125 ٪ </td></tr><tr><td> D - غلوكوز </td><td> 1.16g </td><td> 2.32g </td><td> 13 ملم </td></tr><tr><td> NaHCO 3 </td><td> 0.22g </td><td> 0.44g </td><td> 5.2 ملي </td></tr><tr><td> Hepes </td><td> 3.58g </td><td> 7.16g </td><td> 30 ملم </td></tr></tbody></table> مزيج حتى حل شامل وتقديمهم إلى درجة حرارة الغرفة. ضبط الرقم الهيدروجيني ل7،27-7،28 مع هيدروكسيد الصوديوم N 1 قياس الأسمولية. ضبط إلى 320 ملمول / كغ مع منزوع الأيونات ومعقمة O. 2 H ويتوقع أن تضيف ما يقرب من 25-40 مل. الأسمولية الاختيار مرة أخرى. *** الحموضة قد تتغير قليلا حين تعديل الأسمولية ، وهذا على ما يرام ، فإنه من المهم أن الأسمولية في النطاق الصحيح (317-323). تعقيم قبل المرور عبر فلتر 0.22 ميكرون. جعل aliquots 20 مل وتخزينها لمدة تصل إلى 2-3 أسابيع في 4 درجات مئوية. Gluatamine المخزون : إعداد عند تركيز 200 ملم ومخزن في -20 درجة مئوية في aliquots من 2.5 مل. حمض الاسكوربيك الأسهم : إعداد بتركيز 25 ٪ (W / V) وتخزينها في -20 درجة مئوية في aliquots من 100 ميكرولتر. </li></ol> 5. ممثل النتائج : وينبغي أن ننظر شرائح بيضاء تحت نطاق تشريح دون بقع سوداء واضحة المعالم وغير التالفة CA1 ، CA3 ، والمناطق التلفيف المسنن. ويعتبر التلوث الجرثومي وبسهولة نقل بقع سوداء في المتوسط ​​أو تعكر المجلس الأعلى للقضاء. عند وضعها تحت المجهر ، يجب على سطح شريحة تبدو نظيفة بعد 4 أيام في الثقافة مع الهيئات واضحة وdiscernibly الخلية. إذا لم يكن هناك أجسام الخلايا واضحة وينظر الكثير من الحطام ويغطي السطح بعد 4 ايام ، ومن ثم لا شريحة صحية.

<ol>
	<li>Martin, S. J., Grimwood, P. D., Morris, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+and+memory:+an+evaluation+of+the+hypothesis.">Synaptic plasticity and memory: an evaluation of the hypothesis.</a> Annu Rev Neurosci. 23, 649-711 (2000).</li><li>Bliss, T. V., Collingridge, G. L., Morris, R. G. I. n. t. r. o. d. u. c. t. i. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+potentiation+and+structure+of+the+issue.">Long-term potentiation and structure of the issue.</a> Philos Trans R Soc Lond B Biol Sci. 358, 607-611 (2003).</li><li>Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The synaptic organization of the brain. , (2004).</li><li>Malinow, R., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+enhancement+shown+by+whole-cell+recordings+of+long-term+potentiation+in+hippocampal+slices.">Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices.</a> Nature. 346, 177-180 (1990).</li><li>Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+green+fluorescent+protein+(GFP)+into+hippocampal+neurons+through+viral+infection.">Introduction of green fluorescent protein (GFP) into hippocampal neurons through viral infection.</a> Cold Spring Harb Protoc. , (2010).</li><li>Shi, S., Hayashi, Y., Esteban, J. A., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-specific+rules+governing+AMPA+receptor+trafficking+to+synapses+in+hippocampal+pyramidal+neurons.">Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons.</a> Cell. 105, 331-343 (2001).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li><li>Esteban, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKA+phosphorylation+of+AMPA+receptor+subunits+controls+synaptic+trafficking+underlying+plasticity.">PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity.</a> Nat Neurosci. 6, 136-143 (2003).</li><li>Mainen, Z. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+in+living+brain+slices.">Two-photon imaging in living brain slices.</a> Methods. 18, 231-239 (1999).</li><li>Barria, A., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-specific+NMDA+receptor+trafficking+to+synapses.">Subunit-specific NMDA receptor trafficking to synapses.</a> Neuron. 35, 345-353 (2002).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Simoni, A. D. e., Griesinger, C. B., Edwards, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+rat+CA1+neurones+in+acute+versus+organotypic+slices:+role+of+experience+in+synaptic+morphology+and+activity.">Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity.</a> J Physiol. 550, 135-147 (2003).</li></ol>

الإعلان عن أي تضارب في المصالح.

ويستند هذا الأسلوب على طريقة وصف لأول مرة من قبل Stoppini آخرون (11) وتقدم على نحو سريع لشرائح ثقافة الحصين. الجانب الأكثر أهمية من هذا البروتوكول هو الحفاظ على شرائح معقمة ، وبالتالي من الأهمية بمكان استخدام تقنيات مناسبة ومعقمة بشكل صحيح لتطهير وتعقيم جميع ال?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell culture inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Slicer</td>
<td>&nbsp;</td>
<td>Stoelting</td>
<td>51425/51415</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZX7-ILLD2-100</td>
<td>&nbsp;</td>
<tr>
<td> Hippocampus dissecting tool</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>10099-15</td>
<td>&nbsp;</td>
<tr>
<td>Large utility scissors. Perfection</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>37500-00/37000-00 Right/ Left handed</td>
<td>&nbsp;</td>
<tr>
<td>Iris Spatula</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>10093-13</td>
<td>&nbsp;</td>
<tr>
<td>Straight spatula</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>10094-13</td>
<td>&nbsp;</td>
<tr>
<td>Rounded spoon micro spatula</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57949-039</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting single cutting edge needle</td>
<td>&nbsp;</td>
<td>Electron Microscopy Science</td>
<td>72946</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting tweezers</td>
<td>&nbsp;</td>
<td>Dummont</td>
<td>#2</td>
<td>&nbsp;</td>
<tr>
<td>Small dissecting scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>14060-10</td>
<td>&nbsp;</td>
<tr>
<td>MEM Eagle medium</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>50-019 PB</td>
<td>&nbsp;</td>
<tr>
<td>Horse serum heat inactivated</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26050-88</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3881</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4 (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2773</td>
<td>&nbsp;</td>
<tr>
<td>Insulin (1 mg/ml), dissolved in HCl 0.01 N</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Ascorbic Acid, solution (25%)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4544</td>
<td>&nbsp;</td>
<tr>
<td>D-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6014</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7523</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5016</td>
<td>&nbsp;</td>
<tr>
<td>Phenol Red Solution 0.5% in DPBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P0290</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9272</td>
<td>&nbsp;</td>		</tbody>
		</table>
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organotypic hippocampal slice cultures

قرن آمون ، وهو مكون من الجهاز الحوفي ، ويلعب دورا هاما في الذاكرة طويلة المدى والملاحة الفضائية 1. يمكن أن الخلايا العصبية الحصين تعديل قوام صلاتهم بعد فترات وجيزة من تفعيل قوي. ويمكن لهذه الظاهرة ، والمعروفة باسم بعيدة المدى (الكمونية) تستمر لساعات أو أيام ، وأصبحت آلية أفضل مرشح للتعلم والذاكرة 2. بالإضافة إلى ذلك ، بذلت التشريح محددة جيدا ، والتواصل بين الحصين 3 أنه نظام نموذجي لدراسة نقل الكلاسيكية متشابك ومتشابك 4 اللدونة. كما فهمنا للعلم وظائف الأعضاء من نقاط الاشتباك العصبي الحصين نما وأصبح اللاعبون الجزيئية التي تم تحديدها ، وأصبح من الضروري التعامل مع البروتينات متشابك حتمية. ثقافات الحصين عضوي النمط توفر إمكانية للتلاعب الجيني سهلة ودقيقة ولكن التدخل الدوائي الحفاظ على منظمة متشابك التي تعتبر ضرورية لفهم وظيفة المشبك في سياق طبيعي أكثر من الأساليب الروتينية ثقافة فصل الخلايا العصبية. هنا نقدم طريقة لتحضير شرائح الحصين والثقافة التي يمكن تكييفها بسهولة إلى مناطق أخرى من الدماغ. هذا الأسلوب يتيح سهولة الوصول إلى شرائح للتلاعب الجيني باستخدام أساليب مختلفة مثل العدوى الفيروسية أو 5،6 biolistics 7. بالإضافة ، يمكن استردادها بسهولة لشرائح فحوصات البيوكيميائية 8 ، أو نقلها إلى المجاهر التصوير 9 أو 10 تجارب الكهربية.

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures at JoVE.com

Organotypic Hippocampal Slice Cultures

نحن تصف طريقة لتحضير شرائح الحصين عضوي النمط التي يمكن تكييفها بسهولة إلى مناطق أخرى من الدماغ. وضعت شرائح الدماغ على الأغشية التي يسهل اختراقها وسائل الاعلام الثقافة يسمح له شكل واجهة. هذا الأسلوب يحافظ على بنية الإجمالي للقرن آمون لمدة تصل الى 2 أسابيع في الثقافة.

عضوي النمط الثقافات شريحة الحصين

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Research

JoVE Journal

Neuroscience

36.9K Views.  University of Washington School of Medicine. نحن تصف طريقة لتحضير شرائح الحصين عضوي النمط التي يمكن تكييفها بسهولة إلى مناطق أخرى من الدماغ. وضعت شرائح الدماغ على الأغشية التي يسهل اختراقها وسائل الاعلام الثقافة يسمح له شكل واجهة. هذا الأسلوب يحافظ على بنية الإجمالي للقرن آمون لمدة تصل الى 2 أسابيع في الثق?...

Video: Organotypic Hippocampal Slice Cultures

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

وعضوي النمط الثقافة شريحة الحصين النموذجي لفحص الإصابات متعلق بالخلايا العصبية

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

مخيخي الثقافات عضوي النمط : التحديات والكشف عن أفكارك

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

الثقافات شريحة عضوي النمط من المخ الأوسط بطني الجنينية : نظام لدراسة التنمية متعلق بالخلايا العصبية الدوبامينية في المختبر</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

على تحليل من الخليوي بركينيي علم الصرف شجيرى في الثقافات شريحة عضوي النمط

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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

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

الثقافات شريحة عضوي النمط لدراسة ديناميات دبقية قليلة التغصن وتكون الميالين

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

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

تقرن تسجيلات خلية كاملة في عضوي شرائح الحصين

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

وتحليل وعائية عصبية إعادة عرض في Entorhino-الحصين الثقافات شريحة عضوي النمط

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

أورجانوتيبيك شريحة هيبوكامبال الثقافات كنموذج للدراسة نيوروبروتيكتيون واختزاع الخلايا السرطانية

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

الرحم السابقين انهانسر والثقافات أورجانوتيبيك شريحة من أدمغة الفأر الجنينية للعيش-التصوير لترحيل إينتيرنيورونس جابايرجيك

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...