This video article is sponsored by Cerebos Pacific Limited. This work is supported by National Medical Research Council Collaborative Research Grant (NMRC-CBRG-0041/2013) and Ministry of Education Academic Research Funding (MOE AcRF- Tier 1 - T1-2012 Oct -02).

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برعاية الوصول المفتوح لهذه المقالة الفيديو التي Cerebos المحيط الهادئ المحدودة.

شريحة الحصين الحادة هو نظام نموذجا ممتازا لدراسة LTP والعمليات اللدونة الفنية الأخرى مثل STC وعبر الالتقاط. أنه يحفظ الكثير من الشبكة الهيكلية الصفحي من الدوائر الحصين، يسمح المواقع القطب دقيقة وتقدم إلى جانب، منصة مفتوحة للتلاعب neuropharmacological السريع دون حاجز الدم في الدماغ. توضح هذه المقالة منهجية لإعداد شرائح الحصين الحادة قابلة للحياة من الفئران الشباب البالغين واستخدامها لتحقيق STC وعبر العنونة. وقد أكدت الأبحاث السابقة أن نوع الجنس والعمر للحيوانات من العوامل الهامة التي يجب مراعاتها لاستخدامها في الدراسات الكهربية. وتستخدم 27،28 الحيوانات الكبار لذلك الشباب مع وظائف مستقبلات الكبار أعرب بالكامل (فئران ويستار الذكور الذين تتراوح أعمارهم بين 5-7 أسابيع). 23 عدم التماثل في الاتصالات بين الحصين اليسار واليمين وقد لوحظ في القوارض و29تم الإبلاغ عن اختلافات كبيرة في NMDA مستقبلات التعبير وكذلك 34. وقد استخدمنا الحصين الأيمن من أجل أن تكون متسقة مع دراساتنا LTP السابقة. 23،32 ومع ذلك، أي من الحصين يمكن استخدامها طالما يتم الحفاظ على الاتساق. كما هو الحال في أي بروتوكول، فمن المهم جدا لأداء العزل وتقطيع الإجراءات بسرعة ولكن مع الحرص على أن لا تمدد الأنسجة التالفة، وأصدرت جافة أو ميتة. الاختلافات في درجة الحموضة ودرجة الحرارة والتركيب الايوني من الحلول يمكن أن يكون لها تأثير عميق على الجدوى من شرائح والنتائج. وبالتالي ينبغي تجنب مثل هذه الاختلافات. وقد لوحظ أن الغلوتامات إطلاق الكالسيوم التي تعتمد على مستقبلات التي تحدث خلال خطوات إعداد يمكن أن تؤثر بشكل لا رجعة فيه تخليق البروتين في الأنسجة العصبية 35،36، 37. ويمكن استخدام ماكينات تقطيع النسيج اليدوي تساعد على تقليل ذلك عن طريق السماح للعملية أن يتم الانتهاء بسرعة جدا بالمقارنة مع السادسbraslicers. ومع ذلك، العديد من المختبرات أيضا تستخدم على نحو فعال vibraslicers مع الاحتياطات اللازمة للحفاظ على سلامة شريحة. ثمة عامل آخر مهم هو أن تنظر إلى فترة حضانة طويلة قبل بدء التجارب. وقد لوحظ أن يكون حاسما حقا لتحقيق الاستقرار في مستويات الدولة وتفعيل كيناز التمثيل الغذائي في شرائح بعد أن تسبب الاضطرابات أثناء إعداد 23. هذا الاستقرار ضروري من أجل التناسق في التسجيلات على المدى الطويل. نعيد التأكيد على هذه الملاحظة واقتراح الساعات حضانة طويلة حوالي 3 ساعات. ومن المعروف أن العديد من المعلمات التحفيز للحث على LTP، ولكن الآليات الجزيئية أثارت في كل حالة قد لا تكون هي نفسها (للمراجعة نرى 38). هذا يمكن أن تؤثر على متانة وغيرها من خصائص LTP التي، بدورها، يمكن أن تؤثر على نتائج متشابك علامات والقبض على التجارب. وبالتالي فمن المهم للتحقق من صحة نماذج التحفيز وخصائصمن LTP المنتزعة تحت ظروف المختبر الأداء والحفاظ على الاتساق. ونحن عموما لا تعتبر التجارب مع كبير جدا وابلا من الألياف قبل المشبكي ومع fEPSPs القصوى أقل من 0.5 فولت والتجارب التي تنطوي على تغييرات كبيرة في كرة الألياف أثناء يتم رفض أيضا التسجيلات. وعلاوة على ذلك، في حين أن أداء مسار اثنين أو ثلاثة مسار-التجارب، فمن المهم لضمان استقلال الطريق. هذا يمكن القيام بها مع إقران النبض بروتوكول التيسير 28. واحد السلبي للنظم تسجيل واجهة هو تشكيل قطرات التكثيف على الأقطاب خلال ساعات تسجيل طويلة بسبب الاختلافات في درجات الحرارة والرطوبة بين الغرفة والمناطق المحيطة بها. تحتاج هذه القطرات إلى أن نشف بعناية من وقت لآخر. وإلا فإن قطرات يمكن أن يتقطر على شرائح وتؤدي إلى اضطراب أو حتى فقدان الإشارات. نعالج هذه عادة بذ النشاف بمهارة قطرات تسترشد تحت المجهر باستخدام ورق الترشيح مرهف الفتيل، دون لمس الأقطاب الكهربائية. ومع ذلك، فإن أفضل حل هو استخدام نظام التدفئة المركزية، مثل نظام ETC التي وضعها الباحثون في جامعة أدنبرة. وعلى صعيد الختامية، ومجموعة متنوعة من المنهجيات موجودة في المختبرات في جميع أنحاء العالم التي تستخدم لإعداد شرائح الحصين لأغراض تجريبية مختلفة. كل من الإجراء يقدم بعض المزايا على الآخر. واحد يحتاج إلى تحسين بعناية التفاصيل الدقيقة للبروتوكول لتتناسب مع الغرض من التجربة. نأمل أن هذه المادة تساعد في تحسين بعض جوانب المنهجية لدراسة العمليات في وقت متأخر من النقابي مثل STC وعبر الالتقاط.

The encoding and storage of information in the brain still remains the most significant and keenly pursued challenge in neuroscience. Over the years, long-term potentiation (LTP) and long-term depression (LTD) have emerged as the leading cellular correlates of memory1,2. These activity dependent changes, which exhibit input specificity and associativity, result in the stabilization of memory traces in the neuronal networks 1,3,4. The maintenance of the two forms of synaptic plasticity requires the synthesis of plasticity-related products (PRPs)5-10. Synapse specificity that involves the interaction of newly synthesized protein only with specific activated synapses expressing LTP or LTD, is critical to memory. This specificity is explained by the concept of &#8216;Synaptic Tagging and Capture&#8217; (STC), where the PRPs interact with recently active, &#8216;tagged&#8217; synapses11,12. The STC process offers a framework for associative properties of memories at the cellular level. It provides us with a conceptual basis of how short-term forms of plasticity are transformed into long-lasting forms of plasticity in an associative and time-dependent manner13.
During the process of STC, a strong tetanization in one input that leads to protein synthesis dependent late-LTP, results in the reinforcement of a protein synthesis independent early-LTP induced in another independent input on to the same population of neurons into a persistent one13. The setting of a local synaptic tag by a transient neural activity and the synthesis of the diffusible PRPs by the strong neural activity are the two key events during STC13,14. The capture of the PRPs by the recently potentiated &#8216;tagged&#8217; synapses is fundamental to the maintenance of long-term potentiation. Many studies have been done to confirm the existence of STC phenomenon15-17 and identify the candidate &#8216;tags&#8217;18 and &#8216;PRPs&#8217;19. Calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase1/2 (ERK1/2); CaMKIV, Protein Kinase M (PKM) and brain-derived neurotrophic factor (BDNF) are some of the candidate molecules for &#8216;tag&#8217; and &#8216;PRP&#8217; respectively19-21. The synaptic tagging model has further been expanded to include the positive associative interactions between LTP and LTD - the &#8220;synaptic cross-tagging&#8221;22. In synaptic cross-tagging, a late LTP/ LTD in one synaptic input transforms the opposite protein synthesis-independent early-LTD/LTP in an independent input into its long-lasting form or vice versa22. 
The hippocampal slice preparation is the most widely used model in the studies of long-term synaptic plasticity23,24. Much of the understanding about the concepts of synaptic tagging and cross- tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the labs are still unable to study these processes. Experimental conditions for the preparation of rat hippocampal slices and the recording of stable late-LTP/LTD for extended hours are extremely important to study synaptic tagging/cross-tagging23,25,26. This article describes the detailed experimental procedures for studying 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.

<table border="0" cellpadding="0" cellspacing="0">
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			<td>I. Dissection Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1. Bandage scissors</td>
			<td>KLS Martin, Germany</td>
			<td>21-195-23-07</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>B-Braun/Aesculap, Germany</td>
			<td>LX553R</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Iris scissors</td>
			<td>B-Braun/Aesculap, Germany</td>
			<td>BC140R,</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>BC100R</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Bone rongeur</td>
			<td>World Precision Instruments (WPI), Germany</td>
			<td>14089-G</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Scalpel</td>
			<td>World Precision Instruments (WPI), Germany;</td>
			<td>500236-G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>B-Braun/Aesculap, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>BB73</td>
			<td></td>
		</tr>
		<tr>
			<td>5. Scalpel blade#11</td>
			<td>B-Braun/Aesculap, Germany</td>
			<td>BB511</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>6. Sickle scaler</td>
			<td>KLS Martin, Germany</td>
			<td>38-685-00</td>
			<td></td>
		</tr>
		<tr>
			<td>7. Angled forceps</td>
			<td>B-Braun/Aesculap, Germany</td>
			<td>BD321R</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>8. Anesthetizing/Induction chamber</td>
			<td>MIP Anesthesia Technologies (Now, Patterson Scientific), Oregon</td>
			<td>AS-01-0530-LG</td>
			<td></td>
		</tr>
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			<td></td>
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			<td>II. ACSF component chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1. Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Magnesium sulphate heptahydrate (MgSO4.7H20)</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Calcium chloride dihydrate (CaCl2.2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>5. Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>6. Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>7. D-Glucose anhydrous (C6H12O6)</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>III. Electrophysiology Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1. Microscope</td>
			<td>Olympus, Japan</td>
			<td>Model SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Temperature Controller</td>
			<td>Scientific Systems Design Inc. Canada</td>
			<td>PTC03</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Differential AC Amplifier</td>
			<td>AM Systems, USA</td>
			<td>Model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Isolated Pulse Stimulator</td>
			<td>AM Systems, USA</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>5. Oscilloscope</td>
			<td>Rhode &#38; Schwarz</td>
			<td>HM0722</td>
			<td></td>
		</tr>
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			<td>6. Digital-Analog Converter</td>
			<td>Cambridge Electronic Design Ltd. Cambridge, UK</td>
			<td>CED-Power 1401-3</td>
			<td></td>
		</tr>
		<tr>
			<td>7. Interface Brain Slice Chamber</td>
			<td>Scientific Systems Design Inc. Canada</td>
			<td>BSC01</td>
			<td></td>
		</tr>
		<tr>
			<td>8. Tubing Pump</td>
			<td>Ismatec, Idex Health &#38; Science, Germany</td>
			<td>REGLO-Analog</td>
			<td></td>
		</tr>
		<tr>
			<td>9. Carbogen Flowmeter</td>
			<td>Cole-Parmer</td>
			<td>03220-44</td>
			<td></td>
		</tr>
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			<td>10. Fiber Light Illuminator</td>
			<td>Dolan-Jenner Industries</td>
			<td>Fiber Lite MI-150</td>
			<td></td>
		</tr>
		<tr>
			<td>11. Micromanipulators</td>
			<td>Marzhauser Wetzlar, Germany</td>
			<td>00-42-101-0000 (MM-33)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>00-42-102-0000 (MM-32)</td>
			<td></td>
		</tr>
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			<td>12. Electrodes</td>
			<td>AM Systems, USA</td>
			<td>571000 (Stainless steel; 0.010, 5M&#8486;, 8 degree)</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

تم استخدام منهجية وصفها لدراسة أشكال طويلة الأمد من LTP / LTD والتفاعلات النقابي في مثل وضع علامات متشابك وعبر القبض على من شرائح الحصين الحادة من الفئران الكبار. وقد ثبت 23 هذه التقنية فعالة لإجراء التجارب مع كل من الفئران (ويستار) ومجموعة متنوعة من الماوس سلالات 30،31. وقد استخدمت منهجية بنجاح للتسجيلات LTP مستقرة تصل إلى 8-12 ساعة. 32 و&#39;الوسم&#39; التي وضعتها تكزيز ضعيفة من مدخل واحد (S1) يلتقط الناجمة عن تكزيز قوية من المدخلات أخرى مستقلة ولكن متداخلة &quot;عبوات المرضى الجاهزة&quot; (S2، الشكل 3B، والدوائر شغل) وبذلك تحول شكل المتحللة خلاف ذلك من LTP (في وقت مبكر -LTP) في S1 إلى واحد طويل الأمد (الشكل 3B، والدوائر المفتوحة) (على سبيل المقارنة من المبكر LTP الناجمة عن WTET نرى 20،33). عبوات المرضى الجاهزة التي استولت عليها الضعفاءبحاجة الى مجموعة تكزيز العلامة لا تأتي بالضرورة من التي يسببها STET في وقت متأخر من LTP ولكن يمكن أيضا أن تقدم من قبل SLFS التي يسببها أواخر LTD. ويشار إلى هذا النوع من التفاعل الإيجابي بين النقابي LTP وLTD باسم &quot;عبر وضع علامات / القبض على&quot;. وWTET التي يسببها المبكر LTP في S1 يحصل المسلحة في وقت متأخر من LTP (الشكل 3C، والدوائر المفتوحة) عن طريق الاستيلاء على عبوات المرضى الجاهزة التي تقدمها SLFS التي يسببها أواخر LTD في S2 (الشكل 3C، والدوائر شغل). إحصائية تم الإبقاء التقوية كبيرة أو الاكتئاب في S1 و S2 في كلتا الحالتين بالمقارنة مع خط الأساس الخاصة بها (يلكوكسن الاختبار؛ P &lt;0.05). للتفاعل العلامة PRP أن يحدث، النظام الزمني من الحدثين (ضعيف قبل قوامها / قوي قبل ضعيفا) ليست حاسمة طالما لا يزال النافذة الزمنية بين الحدثين في نطاق 30-60 دقيقة. قد يكون من الحكمة لتشمل متشابك الثالث، مستقلا، ولكن التداخل فيوضع واستخدامه كوسيلة لمراقبة خط الأساس لمراقبة استقرار التسجيلات. يجب التحقق من صحة بروتوكولات التحفيز الكهربائية المستخدمة للحث على أشكال المبكر بها والمتأخرة من LTP / LTD في التجارب المدخلات واحد لالاتساق والموثوقية قبل استخدامها في التجارب STC. كما نود أن نؤكد على أهمية منهجية إعداد شريحة موضح في البروتوكول منذ نجاح هذه التجارب يعتمد بشكل كبير على نوعية الشرائح. <img alt="الشكل 1" src="/files/ftp_upload/53008/53008fig1.jpg" /> الشكل 1. (A) الأدوات المستخدمة في تشريح الحصين: (أ) ضمادة المقص (ب) ايريس المقص (ج) مقراض العظام (د) ملعقة رقيقة، (ه) المبضع عدد 11) و (قشارة المنجلية (ز) لينة فرشاة الطلاء -bristle (ح) من البلاستيك ماصة باستير (ط) ورقة فلتر (85mm و) (ي) ورقة فلتر (30MM) (ك) أكواب زجاج (ل) كتل الألومنيوم التبريد لتتناسب مع طبق بتري والأكواب(م) طبق بتري (B) دليل المروحية الأنسجة. (أ) منصة (ب) قطع ذراع مع شفرة صاحب (ج) رنيه ميكرومتر، القرار 10 ميكرون. <a href="https://www.jove.com/files/ftp_upload/53008/53008fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الرقم 2" src="/files/ftp_upload/53008/53008fig2.jpg" /> الشكل 2. الكهربية انشاء للتسجيلات الميدان المحتملين تتكون من (A) التحفيز والتشجيع (ب) مكبر للصوت التفاضلية (ج) التناظرية إلى الرقمية تحويل (د) راسم (ه) جهاز كمبيوتر مع برنامج الحصول على (و) Vibration- مقاومة فوق المنضدة (ز) المجهر مع&gt; 4X التكبير (ح) واجهة الدماغ شريحة غرفة (ط) نظام نضح لACSF وكربوجين العرض (ي) تحكم في درجة الحرارة (ك) مصدر إضاءة (ل) المتلاعبين مع أصحاب الكهربائي. (B) واجهة الدماغ شريحةغرفة (C) و (D) شرائح قرن آمون في غرفة واجهة. (E) غير القابل للصدأ القطب الصلب مختومة في الزجاج الشعرية. <a href="https://www.jove.com/files/ftp_upload/53008/53008fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (3)" src="/files/ftp_upload/53008/53008fig3.jpg" /> الشكل 3. (A) تمثيل تخطيطي من قرن آمون شريحة ومسرى موقع عرضية لتسجيل حقل المحتملة: في هذا التمثيل، يتم وضع قطبين تحفيز (S1 و S2) في المشععة طبقة من المنطقة CA1 لتحفيز دولتين مستقلتين ولكن تداخل مدخلات متشابك على الخلايا العصبية CA1 الهرمية. قطبين تسجيل خارج الخلية، واحدة لتسجيل الميدان الكامن الاستثاري بعد المشبكي (احتمال مثير آخر متشابك) من مقصورة شجيري قمي وآخر لتسجيل الجسدية ارتفاع عدد السكان من الخلايا الهرميةالهيئات، وتقع في المشععة الطبقة والطبقة الهرمية على التوالي. CA1- قرن آمون المنطقة 1، CA3- قرن آمون المنطقة 3، التلفيف المسنن DG-والألياف ضمانات SC- شافر، S1- تحفيز الكهربائي 1، S2 تحفيز الكهربائي 2. (B) ضعيف أمام نموذج قوي لدراسة STC: تكزيز الضعيفة (WTET) يتم تطبيقها على S1 (الدوائر المفتوحة) لإحداث المبكر LTP تليها تكزيز قوية (STET) من S2 (الدوائر شغل) في 30 دقيقة للحث في وقت متأخر من LTP. من المبكر LTP في S1 يحصل المسلحة في وقت متأخر من LTP تظهر علامات والقبض على التفاعل (ن = 6) (C) ضعيف أمام نموذج قوي لدراسة شاملة لوضع علامات: هو فعل المبكر LTP التي كتبها WTET في S1 (دوائر مفتوحة)، يليه من تحريض أواخر LTD في S2 (الدوائر شغل) باستخدام SLFS بعد 30 دقيقة. في S1، يتم تحويل المبكر LTP إلى وقت متأخر من LTP دائم 6 ساعات تظهر عبر وضع علامات والقبض على (ن = 6). سهم واحد يمثل ضعف تكزيز بطلب للحصول على حمل المبكر LTP. الثلاثي من السهام يمثلتكزيز قوية لإحداث أواخر LTP. السهم المكسور يمثل نقطة الوقت الذي تم تطبيقه SLFS للمدخلات متشابك التمثيلي للحث أواخر LTD. أشرطة الخطأ تشير SEM. <a href="https://www.jove.com/files/ftp_upload/53008/53008fig3large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a>

Watch this Scientific Journal Video about Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents at JoVE.com

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

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 ...

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13.7K Views.  National University of Singapore. 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.

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

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

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

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

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

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). ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

تسجيلات الكهربية المزدوجة للSynaptically-استجابات Astroglial والخلايا العصبية في الحصين شرائح الحاد

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 ...

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 ...

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 ...

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

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

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