We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

وأجريت التجارب على الحيوانات فقط وفقا للمعاهد القومية للصحة الدليل لرعاية واستخدام الحيوانات المختبرية (NIH النشر رقم 80-23، منقحة 2012) وتمت الموافقة على الدراسات التي المؤسسي رعاية الحيوان واستخدام لجان البحث في ستوني جامعة بروك (# 1618 و # 1792). 1. vHipp-تحقق نموا مطردا متشابك المشارك الثقافات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الفئران التضحية (يوم بعد الولادة 0-3، من النوع البري (WT) أو nAChRs α7 خط الماوس المعدلة وراثيا) مع CO 2. قطع رأس كل الجرو وحفظ الذيل في أنبوب 1.5 مل الطرد المركزي لمدة رجعي التنميط الجيني. قم بتنفيذ الخطوات التالية في غطاء تعقيمها. </li><li style=";text-align:right;direction:rtl"> إزالة الجزء العلوي من الجمجمة مع مقص وملقط لفضح الدماغ كله. بدءا من المرحلة حيث قشرات الدماغ جزء من كل caudally أخرى، الحصول على قسم الاكليلية التي تحتوي على القشور الخلفي بما في ذلك vHipp 5،21 تشريح تحت المجهر. </li><li style=";text-align:right;direction:rtl"> تشريح الخامسشرائح الأنسجة هيب تحتوي المنطقة CA1-مرفد وفقا لوضع أطلس مخ الفأر 22 (الشكل 1A). نقل إلى صحن الثقافة 35 ملم تحتوي على وسائل الإعلام ثقافة الباردة (4 ° C)، مقطعة إلى شرائح رقيقة صغيرة (حوالي 100 ميكرون × 100 ميكرون microslices). </li><li style=";text-align:right;direction:rtl"> قبل احتضان 12 مم بولي-D-يسين / coverslips الزجاج المطلي laminin داخل 24 جيدا لوحات زراعة الأنسجة مع وسائل الإعلام الثقافة (~ 500 ميكرولتر) لمدة 30 دقيقة على الأقل في الحاضنة عند 37 درجة مئوية. إزالة وسائل الاعلام قبل الطلاء microslices. </li><li style=";text-align:right;direction:rtl"> لوحة مع ماصة باستير مصقول النار على مركز للساترة مع الحد الأدنى من وسائل الإعلام (~ 50 ميكرولتر، التي تحتوي على ~ 20 قطعة من microslices) لتسهيل الحجز على إإكسبلنتس. لوحة microslices vHipp القادمة من حيوان واحد (إما + / + أو - / - التركيب الوراثي للخط α7 المعدلة وراثيا) على كل ساترة. </li><li style=";text-align:right;direction:rtl"> بعد تسوية لل explants على ساترة، إضافة بلطف وسائط إضافية (~ 100 ميكرولتر) من خلال الالجدار ه بحيث أن مستوى وسائل الإعلام مرتفع بما يكفي لتغطية كامل لل explants على المحيط، ولكن ليس تلك في وسط ساترة. </li><li style=";text-align:right;direction:rtl"> بعد O / N الحضانة في 37 ° C، 5٪ CO 2 الحاضنة، تفريق الخلايا العصبية تحقق نموا مطردا من أيام الجنينية 18 إلى ما بعد الولادة يوم 1 الفئران WT وإضافة إلى إإكسبلنتس. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تشريح خارج الأنسجة تحقق نموا مطردا وفقا لوضع أطلس مخ الفأر 20 (الشكل 1B). </li><li style=";text-align:right;direction:rtl"> مقطعة إلى قطع صغيرة (حوالي 500 ميكرون × 500 ميكرون microslices) ونقل إلى أنبوب 15 مل. </li><li style=";text-align:right;direction:rtl"> علاج مع 0.25٪ التربسين (2 مل) لمدة 15 دقيقة عند 37 ° C. غسل قطع الأنسجة ثلاث مرات مع وسائل الاعلام غسل الباردة (5 مل، 4 ° C)، يليه غسل ​​واحد في وسائل الإعلام ثقافة البارد (5 مل، 4 ° C). تسمح تعليق للراحة لمدة 5 دقائق في كل خطوة غسل، ثم تخلصي من طاف بعناية. </li><li style=";text-align:right;direction:rtl"> فصل الخلايا عن طريق سحن لطيف في 2 مل من وسائل الإعلام الثقافة مع ماصة باستير طفيفة مصقول النار. </li><li style=";text-align:right;direction:rtl"> عبرتعليق خلية فر إلى أنبوب آخر 15 مل وأجهزة الطرد المركزي في 2000 دورة في الدقيقة س لمدة 5 دقائق. إزالة طاف و resuspend الخلايا في وسائل الإعلام الثقافة في 1 مل لكل 6 الجراء. تفريق الخلايا عن طريق pipetting بلطف في وسائل الإعلام عدة مرات مع باستور ماصة مصقول النار. </li><li style=";text-align:right;direction:rtl"> إضافة 0.25 مل من الخلايا تحقق نموا مطردا انتشروا في كل ساترة مع إإكسبلنتس vHipp. </li><li style=";text-align:right;direction:rtl"> الحفاظ على الثقافات في ترطيب 37 درجة مئوية، و 5٪ CO 2 الحاضنة. وضع لوحات الثقافة على المبلل منصات شاش معقم لتحقيق الاستقرار ميكانيكيا والحفاظ على الرطوبة، وتسهيل تشكيل المشبك. </li></ol></li></ol> 2. سيتولوجية مناعية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إصلاح الثقافات (5-7 أيام في المختبر) في بارافورمالدهيد 4٪ / 4٪ سكروز / PBS (~ 500 ميكرولتر في ساترة، 20 دقيقة، RT). </li><li style=";text-align:right;direction:rtl"> ثقافات Permeabilize مع 0.25٪ تريتون X-100 / PBS (~ 500 ميكرولتر في ساترة، 5 دقائق، RT). </li><li style=";text-align:right;direction:rtl"> ثقافات كتلة مع 10٪ مصل حمار عادي في برنامج تلفزيوني (~ 500 ميكرولتر في ساترة، 30 دقيقة، RT) قبل antibodذ تلطيخ. احتضان مع الأجسام المضادة الأولية O / N عند 4 درجات مئوية. استخدام الأجسام المضادة الأولية التالية: nAChRs المضادة (α 4 -ECD 1: 500، مضادة للα 5 وحدات فرعية، 1: 500)، نقل الغلوتامات مكافحة حويصلي 1 (1: 250)، ومكافحة GAD65 (1: 100). </li><li style=";text-align:right;direction:rtl"> يغسل الأجسام المضادة الأولية خارجا مع PBS (3 مرات / ~ 500 ميكرولتر في ساترة، 5 دقائق) ثم احتضان الثقافات في الأجسام المضادة الثانوية مترافق لاليكسا 488 (~ 500 ميكرولتر في ساترة، 1: 500) أو اليكسا 594 (~ 500 ميكرولتر في ساترة ، 1: 500) لمدة 1 ساعة على 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> احتضان الثقافات في αBgTx مترافق لاليكسا 594 (~ 500 ميكرولتر في ساترة، 1: 1000 في وسائل الإعلام الثقافة) لمدة 45 دقيقة عند 37 درجة مئوية قبل التثبيت لوضع العلامات α7 سطح * nAChRs. </li><li style=";text-align:right;direction:rtl"> جبل وختم coverslips. </li><li style=";text-align:right;direction:rtl"> التقاط الصور مع المجهر مضان مجهزة أهداف الخطة-امزيغ (20X مع 0.8 NA أو 63X النفط مع 1.4 NA) وكاميرا CCD. </li></ol> 3. FM1-43 القائم الحويصلي فيوجن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحفاظ على الثقافات (5-7 أيام في المختبر) في غرفة التصوير، جبل الغرفة على الغزل القرص المجهر متحد البؤر. يروي باستمرار (1 مل / دقيقة) مع HBS كوكتيل في RT. </li><li style=";text-align:right;direction:rtl"> وقف نضح وتحميل الثقافات مع 10 ميكرومتر FM1-43 في 56 ملي K + ACSF (~ 500 ميكرولتر) لمدة 90 ثانية (تلوين)، وغسل صبغ الخارجي بعيدا لمدة 15 دقيقة مع الكالسيوم 2+ HBS مجانية تحتوي على ADVASEP-7 (0.1 ملم) لكنس بغشاء FM1-43. </li><li style=";text-align:right;direction:rtl"> تحفيز الثقافات مع 56 ملي K + ACSF (~ 500 ميكرولتر) دون FM1-43 لمدة 120 ثانية (destaining)، تحميل الثقافات مع FM1-43 باستخدام نفس الظروف، ويغسل مرة أخرى مع الكالسيوم 2+ HBS مجانية تحتوي على ADVASEP-7 ل 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> الحصول FM1-43 الصور مضان مع القرص الغزل المجهر متحد البؤر مجهزة هدف خطة-امزيغ (60X المياه مع 1.4 NA، الإثارة 488 نانومتر، وانبعاث 530 نانومتر) والتقاط الصور مع كاميرا CCD. </li><li style=";text-align:right;direction:rtl"> جمع الأساس الصور FM1-43 مضان (كل 2 ثانية لمدة 1 دقيقة) كما تحكم مسبقا النيكوتين. تطبيق النيكوتين (1 ميكرومتر) من خلال نضح السريع (2 مل / دقيقة) لمدة 1 دقيقة، ثم يغسل النيكوتين خارجا مع HBS كوكتيل. إبقاء الوقت الفاصل بين الصور التقاط قبل وأثناء وبعد التطبيق النيكوتين كل 2 ثانية لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> تحديد FM1-43 كثافة مضان قبل (F تلطيخ) وبعد (F destaining) تطبيق النيكوتين في كل حبة متشابك. </li><li style=";text-align:right;direction:rtl"> حساب المبلغ الإجمالي من نشره مضان FM1-43 على طول المحاور vHipp عن طريق قياس الفرق من FM1-43 كثافة مضان قبل وبعد تطبيق النيكوتين (ΔF = F تلطيخ -F destaining). تحليل جزء من انخفاض كثافة مضان بعد النيكوتين مع المعادلة التالية: F = انخفاض٪ ΔF / F تلطيخ. </li></ol> 4. التصوير الكالسيوم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شطف الثقافات (5-7 أيام في المختبر) بسرعة مع HBS العادية، ثم تحميل الثقافات مع 5 ميكرومتر فلوو-4 كا 2+ مؤشر (AM استر) و 0.02٪ Pluronic F-127 في HBS (2 مل) لمدة 30 دقيقة عند 37 درجة مئوية و CO 2 5٪. </li><li style=";text-align:right;direction:rtl"> تغسل فلوو-4 حل مع HBS (3 مرات / 5 دقائق). عودة الثقافات إلى الحاضنة (37 ° C / 5٪ CO 2) لمدة 30 دقيقة على الأقل. </li><li style=";text-align:right;direction:rtl"> الحفاظ على الثقافات في نفس غرفة التصوير ونفس الشروط كما هو موضح في FM1-43 مقرها قسم الانصهار الحويصلي (راجع الخطوة 3.1). </li><li style=";text-align:right;direction:rtl"> جمع الصور فلوو-4 مضان من التوقعات محور عصبي من vHipp شرائح صغيرة مع هدف الخطة-امزيغ (60X المياه مع 1.4 NA، الإثارة 488 نانومتر، وانبعاث 530 نانومتر) وكاميرا CCD. </li><li style=";text-align:right;direction:rtl"> جمع الأساسية فلوو-4 صور مضان (كل 10 ثانية لمدة 2 دقيقة) عن السيطرة قبل النيكوتين، وتطبيق النيكوتين (1 ميكرومتر) من خلال نضح السريع (2 مل / دقيقة) لمدة 1 دقيقة، ثم يغسل النيكوتين خارجا مع HBS كوكتيل. إبقاء الوقت الفاصل بين الصور التقاط قبل وأثناء وبعد التطبيق النيكوتين كل 10 ثانية لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> حفظ جميع الأطر من الخام فلوو-4 صور مضان والتصدير على شكل سلسلة منصور تنسيق TIFF لمزيد من التحليل. </li><li style=";text-align:right;direction:rtl"> جمع شدة متكاملة من فلوو-4 مضان على طول المحاور vHipp قبل وبعد تطبيق النيكوتين. </li><li style=";text-align:right;direction:rtl"> تطبيع كثافة مضان متكاملة مع المعادلة التالية: ΔF / F 0 = (FF 0) / F 0، حيث F 0 هو قبل النيكوتين كثافة مضان متكامل لتصحيح الخلفية وF هو كثافة مضان متكاملة على طول المحاور vHipp في كل نقطة زمنية بعد تطبيق النيكوتين. تحليل ورسم تطبيع كثافة مضان متكاملة. </li></ol>

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+alpha4*+nicotinic+receptors+revealed+by+superecliptic+phluorin:+effects+of+a+beta4+amyotrophic+lateral+sclerosis-associated+mutation+and+chronic+exposure+to+nicotine.">Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.</a> J Biol Chem. 286 (36), 31241-31249 (2011).</li><li>Colombo, S. F., Mazzo, F., Pistillo, F., Gotti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+trafficking+and+up-regulation+of+nicotinic+ACh+receptors.">Biogenesis, trafficking and up-regulation of nicotinic ACh receptors.</a> Biochem Pharmacol. 86 (8), 1063-1073 (2013).</li><li>St John, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+trafficking+of+nicotinic+acetylcholine+receptors.">Cellular trafficking of nicotinic acetylcholine receptors.</a> Acta Pharmacol Sin. 30 (6), 656-662 (2009).</li><li>Zhong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+type+III+neuregulin+1+is+required+for+sustained+enhancement+of+hippocampal+transmission+by+nicotine+and+for+axonal+targeting+of+alpha7+nicotinic+acetylcholine+receptors.">Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors.</a> J Neurosci. 28 (37), 9111-9116 (2008).</li><li>Jacobowitz, D. M., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemoarchitectonic Atlas of the Developing Mouse Brain. , (1998).</li><li>Betz, W. J., Mao, F., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+fluorescent+staining+and+destaining+of+living+vertebrate+motor+nerve+terminals.">Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.</a> J Neurosci. 12 (2), 363-375 (1992).</li><li>Amaral, E., Guatimosim, S., Guatimosim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+fluorescent+styryl+dye+FM1-43+to+visualize+synaptic+vesicles+exocytosis+and+endocytosis+in+motor+nerve+terminals.">Using the fluorescent styryl dye FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor nerve terminals.</a> Methods Mol Biol. 689, 137-148 (2011).</li><li>Garduno, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+alpha4beta2+nicotinic+acetylcholine+receptors+increase+glutamate+release+and+serotonin+neuron+excitability+in+the+dorsal+raphe+nucleus.">Presynaptic alpha4beta2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.</a> J Neurosci. 32 (43), 15148-15157 (2012).</li><li>Guo, J. Z., Liu, Y., Sorenson, E. M., Chiappinelli, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+and+exogenous+ACh+activates+different+nicotinic+receptors+to+enhance+evoked+glutamatergic+transmission+in+the+lateral+geniculate+nucleus.">Synaptically released and exogenous ACh activates different nicotinic receptors to enhance evoked glutamatergic transmission in the lateral geniculate nucleus.</a> J Neurophysiol. 94 (4), 2549-2560 (2005).</li><li>Szabo, S. I., Zelles, T., Vizi, E. S., Lendvai, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+nicotine+on+spiking+activity+and+Ca2++dynamics+of+dendritic+spines+in+rat+CA1+pyramidal+neurons.">The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons.</a> Hippocampus. 18 (4), 376-385 (2008).</li></ol>

الكتاب ليس لديهم ما يكشف.

وصف إعداد شارك في ثقافة إعادة يستسلم بطني الدوائر قرن آمون-المتكئة في المختبر. هذا الفحص تصاريح إعداد بسيط نسبيا ويمكن الاعتماد عليها لمحات المكانية والزمانية التي تفعيل nAChRs قبل متشابك تنتزع تعزيز نقل glutamatergic 5، 21. يتم ...

تعديل الكوليني من الدائرة استثارة يساهم في الجوانب الأساسية للمعرفة، وتغيير التشكيل الكوليني هو سمة من سمات اضطرابات الاعصاب والعصبية والنفسية بما في ذلك مرض الزهايمر ومرض باركنسون والفصام والإدمان 1-4. آلية المتبعة في تسهيل الكوليني من انتقال متشابك في الجهاز العصبي المركزي هي عن طريق تفعيل مباشر من nAChRs المحلية في مواقع ما قبل متشابك. تفعيل هذه المستقبلات ما قبل متشابك يؤدي إلى زيادة الكالسيوم داخل الخلايا 2+ ([كا 2+] ط) في محطات ما قبل متشابك - سواء بصورة مباشرة، وذلك بسبب تصرف الكالسيوم عالية نسبيا لبعض الأنواع الفرعية nAChR، وبشكل غير مباشر، عبر إشارات بين الخلايا شلالات 5، وبالتالي تعزيز الإفراج العصبي. في الواقع، وقد تم ربط تفعيل nAChRs قبل متشابك مع التغيرات في الإفراج عن مجموعة واسعة من الناقلات العصبية بما في ذلك الغلوتامات، GABA، أدن تشافيز، لالثاني الدوبامين 10/06. وإن كان قد درس هذه العملية بصورة غير مباشرة باستخدام الطرق الكهربية في مختلف نقاط الاشتباك العصبي، للصحفيين البصري [كا 2+] ط وإعادة تدوير حويصلة متشابك تسمح بمزيد من القياس المباشر والدقيق زمنيا للظواهر ما قبل متشابك. وقد ثبت التعريب قبل متشابك من nAChRs مقنع مع وضع العلامات المناعية الذهب مباشرة من nAChRs في مجهرية الإلكترون (EM) مستوى 11،12. كما تم استخدام عدة تقنيات أخرى للتصدي لتوطين nAChR غير مباشرة، بما في ذلك الكشف عن مواقع nAChRs subunit- الوهم بروتين فلوري في الخلايا العصبية مثقف 13،14، تسجيل الكهربية من التيارات nAChR في محطات متشابك 15،16، رصد النيكوتين الناجم عن التغيرات في [كا 2+] أنا في النهايات العصبية متشابك بواسطة التصوير الحي الخلية 17، والرصد غير المباشر لإطلاق النواقل العصبية في محطة متشابك من قبلتقنيات التصوير الخلية الحية مع مؤشرات الفلورية، بما في ذلك إيماس من الحويصلات المشبكية ينظر إليها من قبل styryl الأصباغ متقابلة الزمر FM (FM1-43 وFM4-64) و / أو synapto-pHluorin وللصحفيين العصبي الفلورسنت معين، مثل CNiFERs لأدن تشافيز وiGluSnFr لالغلوتامات 18-20. عموما، هذه المناهج الحالية لتحديد توطين ما قبل متشابك من nAChRs معقدة، وتتطلب أنظمة وتقنيات خاصة للسماح تحديد موثوقة ورصد الفسيولوجية للنشاط قبل متشابك. نحن هنا وصف البروتوكولات ومعدات للنظام المشترك الثقافة في المختبر من قرن آمون البطني (vHipp) - النواة المتكئة (تحقق نموا مطردا) الدائرة التي توفر الوصول المباشر إلى تحديد وتحليل كل المكونات السابقة واللاحقة متشابك من انتقال متشابك. وتبين لنا أمثلة على توطين ما قبل متشابك من nAChRs والتصوير الخلية الحية من nAChR بوساطة كا 2+ الإشارات وإطلاق الناقلات العصبية منظمة العمل العربيةالمحاور نانوغرام vHipp. A امتدادا طبيعيا (واضحة) من بروتوكول المعروضة هنا هو إعداد الاتصالات قبل وبعد متشابك تتألف من الخلايا العصبية من الأنماط الجينية المختلفة. بهذه الطريقة يمكن تقييم مساهمة منتج جين معين إلى ما قبل و / أو آليات ما بعد متشابك التشكيل مباشرة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
		</tr>
		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>tetrodotoxin&#160;</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6, 56 mM Potassium ACSF pH=7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>56 mM</td>
		</tr>
		<tr>
			<td>MgSO4.7H</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td>1.3 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

إعداد العاملين يتكون من جينات خيالية شارك الثقافات الدوائر vHipp-تحقق نموا مطردا في المختبر. التوقعات الصادرة عن microslices vHipp، كمدخل المحاور قبل متشابك، يمكن أن تجعل الاتصالات متشابك مع أهداف ما بعد متشابك، والخلايا العصبية تفرق من تحقق نموا مطردا. النيكوتين التي يس...

Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

تصوير لايف من النيكوتين المستحثة اشارة الكالسيوم وناقل عصبي الإصدار جنبا إلى جنب بطني الحصين المحاوير

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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8.9K Views.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Video: Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

تعديل العمليات العصبية والنقل الميتوكوندريا : تصوير مباشر في الخلايا العصبية الحصين خلال فترات طويلة

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

يعيش بين محاور التصوير الجذر الظهرية بعد بضع الجذور

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

تحليل الفئران خلايا شوان التنمية جنبا إلى جنب المحوار النامي

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

الكالسيوم فوسفات ترنسفكأيشن من الخلايا العصبية الحصين الابتدائية

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

التصوير المباشر ل<em&gt; ذبابة الفاكهة</em&gt; اليرقات Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

عزل العقد الجذرية الظهرية والثقافة الأولية لدراسة الإفراج عن ناقل عصبي

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

السابقين فيفو تصوير الكالسيوم لتصور استجابات الدماغ للإشارات الغدد الصماء في المورفولوجية

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

مجهر ريفليكتوميتريك الطيفية على محاور عصبية ميليناتيد في الموقع

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

السابقين فيفو تصوير الكالسيوم الخاصة بالخلية إشارات في المشبك الثلاثية للحجاب الحاجز الماوس

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.