التمويل: تم دعم هذا العمل من خلال منحة مؤسسة Bertarelli (DJL)، منحة MED-EL (DJL)، والمعاهد الوطنية للصحة المنح DC01089 (MCB).

ملاحظة: يتم تنفيذ كافة الإجراءات التجريبية وفقا للجنة رعاية الحيوان واستخدام ماساتشوستس العين والأذن مستوصف ومدرسة هارفارد الطبية، والتي تتبع المبادئ التوجيهية رعاية الحيوان الوطنية، بما في ذلك سياسة الصحة العامة خدمة للرعاية الإنسانية واستخدام الحيوانات المختبرية، و دليل ILAR، وقانون رعاية الحيوان. الإجراءات التجريبية المذكورة أدناه التعرض التفاصيل من DCN الأيسر. استخدام الأدوات المعقمة أثناء أداء الجراحة البقاء على قيد الحياة. 1. حج القحف الابتدائية والظهرية القوقعة نواة التعرض <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خدر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير ماوس، الذين تتراوح أعمارهم بين 8-12 أسابيع، وزنها 18-24 غ، مع زيلازين 20 ملغ / كغ والكيتامين 100 ملغ / كغ عن طريق إدارة داخل الصفاق. تأكيد التخدير المناسبة من خلال رصد معدل ضربات القلب ومعدل التنفس، فضلا عن أخمص القدمين قرصة الانسحاب لا ارادي. مكان البيطري مرهم على العينين لمنع جفاف بينما تحت التخدير. مرة واحدة على نحو كاف تحت التخدير، والشعر يحلق المغطي رانه فروة الرأس لتوفير إمكانية الوصول دون عائق إلى موقع الجراحية. </li></ol></li><li style=";text-align:right;direction:rtl"> المواقع الجراحي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضع الماوس بشكل آمن في حامل التجسيمي الحيوانات الصغيرة، والذي عقد في مكان من قبل المشبك الخطم. </li><li style=";text-align:right;direction:rtl"> تأكد من أن المشبك خطم واهية بما يكفي للسماح للتنفس كافية ولكن ضيق بما يكفي لشل تماما الرأس من الفأرة. إذا كان رئيس الماوس واهية، ومن المرجح أن تسقط من صاحب الرأس أثناء الجزء حج القحف الداخلي الحيوان. </li><li style=";text-align:right;direction:rtl"> وضع أقطاب كهربائية الدماغ السمعية زرع في الأزياء القياسية، والذي يسمح لرصد معدل ضربات القلب. وينبغي رصد معدل التنفس عن طريق التصور. لاحظ أن معدل ضربات القلب الطبيعي ومعدل التنفس وسوف تختلف تبعا للعمر من الفأرة. </li><li style=";text-align:right;direction:rtl"> يتم وضع ميزان الحرارة ويتم ضمان euthermia عن طريق وضع على بطانية التدفئة homeothermic. </li></ol></li><li style=";text-align:right;direction:rtl"> شق الجلد والتعرض للعظام الجداريين والقفوية من الجمجمة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحت microscمكتب مستشار رئيس الوزراء، وجعل شق عمودي من خلال الجلد تبدأ في خط الوسط، مباشرة بين الصيوان، وتمتد إلى الجزء الذيلية من قفا. بعد خط الوسط شق الجلد، تهجير الجلد أفقيا. تصور العضلات التي تغطي ترك الجانب الخلفي من الجمجمة. </li><li style=";text-align:right;direction:rtl"> إزالة العضلات التي تغمر الأيسر الجدارية، بين الجداريين، والعظام القفوية من الجمجمة عن طريق التفكك المرفقات العضلات بالعظام كل منها مع مشرط أو مقص القزحية. مراقبة درجة صغيرة من النزيف على طول حواف قطع العضلات. تقليل ذلك عن طريق ضغط لطيف مع قضيب من القطن ذات الرؤوس لمدة 10-15 ثانية. </li></ol></li><li style=";text-align:right;direction:rtl"> المغطي حج القحف القوقعة نواة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحديد خطوط خياطة ذات الصلة، بما في ذلك خطوط الغرز السهمي وامدا (الشكل 1، لوحة اليسار). </li><li style=";text-align:right;direction:rtl"> باستخدام رينجرز، وجعل حج القحف على العظم الجداري، غادر من خط الوسط، ~ 2 ملم الذيلية إلى خط امدا خياطة. هذه المنطقة يعتلي على DCN. </li><li style=";text-align:right;direction:rtl"> Followinز حج القحف، ومراقبة طبقة رقيقة من الجافية أن يعتلي المخيخ. باستخدام شفرة مشرط، وإزالة الجافية. (الشكل 1، لوحة الأوسط). ملاحظة: إزالة الجافية قد تتسبب درجة صغيرة من النزيف. والقصد من هذا عملية استخدام شفرة مشرط لإزالة الجافية إلى تقليل كمية فقدان الدم أثناء التطلع المخيخ، والتي سوف تجمع على سطح الدماغ وتحديد غامضة من DCN. إجمالي حجم دم الفأر هو ~ 1.5 مل لا ينبغي أن تتجاوز&gt; من 15٪. إذا تجاوز نزيف هذا المبلغ، وينبغي توفير الفأر مع مع مكملات السوائل. </li><li style=";text-align:right;direction:rtl"> إزالة الدم متخثر تتراكب المخيخ مع قضيب من القطن طرف. اختياريا، بالتنقيط بلطف المالحة باستخدام نقطة الأسنان على المخيخ لمسح الدم بعيدا الدم. </li></ol></li><li style=";text-align:right;direction:rtl"> مخيخي الطموح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام الشفط الفرنسي 5، نضح الجزء الجانبي الأكثر من المخيخ الأيسر تغمر DCN حتى DCN هو مقارنةالسياقية (الشكل 1، لوحة يمين). إزالة ما يقرب من 1/4 إلى 1/3 من المخيخ الأيسر. المعلم الرئيسي المحاذي لDCN هو أمبولة القناة الهلالية العلوية. ملاحظة: الطموح من المخيخ هي الخطوة الرئيسية للبروتوكول. يتم تنفيذ تطلع موجهة من المخيخ أكبر قدر من النجاح في حال حدوثه في محاولة واحدة وليس مع يمر متعددة من شفط وهذا سوف يسبب النزيف. للمساعدة في الطموح، تعيين البؤري للمجهر على عمق المتوقع للCN، والتي ستكون البعيدة قليلا إلى السطح من المخيخ. ووضع البؤري للCN تحسين التصور وضمان وجود صورة حادة. </li><li style=";text-align:right;direction:rtl"> ومن المتوقع التالية الطموح، المزيد من النزيف، السائل النخاعي (CSF) بناء المتابعة، وتشريد المخيخ على DCN. غرس 0.5 سم مكعب من المياه المالحة عقيمة بسرعة في حج القحف لمنع تخثر الدم. مزيج من اللمس لطيف مع نقطة الأسنان وشفط قد تكون ثمتستخدم لازالة مسار لرؤية مباشرة للDCN. عدم الاتصال مباشرة سطح DCN. </li></ol></li></ol> 2. حقن مكروي الضغط لبوساطة الفيروسات نقل الجينات والإنعاش الجراحي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد DCN خالية من الدم المغطي وCSF ومرئيا بوضوح، وجعل إبر دقيقة الضغط في DCN باستخدام 10 ميكرولتر هاميلتون حقنة على مدى فترة 2 دقيقة. </li><li style=";text-align:right;direction:rtl"> إدخال الإبرة مع مياداة مجهرية حتى غيض لم يعد مرئية تحت سطح DCN. </li><li style=";text-align:right;direction:rtl"> لتحقيق الأداء الأمثل، واستخدام 33 أو 34 إبرة قياس (استخدام حقنة gastight مع إبرة قياس 34) مع شطبة الضحلة، مثل 45 درجة، للحد من صدمة حادة وتوطين حجم الحقن داخل DCN، وهي رقيقة و بنية الدماغ الضحلة (&lt;300 ميكرون سميكة). ملاحظة: أدوات حقن مكروي أخرى يمكن استخدامها مقرا لها. من الناحية المثالية، ينبغي أن يكون أداة حقن مرونة للسماح لبعض طفيف بند، إذا لزم الأمر لاستهداف مباشرة DCN. وعلاوة على ذلك، كما بتحريك الماوس قليلا في جميع أنحاء الداخلي بسبب التنفس، يجب أن تكون أداة قوية بما يكفي لتحمل كميات ضئيلة من الحركة. </li></ol> 3. استعادة الجراحي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> على الفور حقن التالية، وإعادة تقارب الجلد والسماح الماوس لاستعادة الانتعاش في الإجراء القياسي. سوف تبقى المخيخ وندبا في ملء تجويف الناجمة عن تطلع المخيخ. باستمرار مراقبة الحيوانات حتى يتم استعاد وعيه كافية للحفاظ على الاستلقاء القصية. مراقبة معدل ضربات القلب ومعدل التنفس، وكذلك القدرة على إطعام. يتم إرجاع أية الحيوان الذي خضع لعملية جراحية لقفص مع الحيوانات الأخرى حتى تعافى تماما: ملاحظة. </li><li style=";text-align:right;direction:rtl"> إذا يبدأ الماوس على المشي في دوائر بعد العملية (&lt;5٪ من الحالات)، وعادة 2-4 ساعة بعد إغلاق شق الجلد، وتضحي فورا الماوس، لأنه على الأرجح تغذية الوقت صعبة. تيومن المرجح الآثار الجانبية له نظرا لتطلع المخيخ. يجب مراقبة الحيوانات لآلام بعد العمل الجراحي، ويجب أن تعطى المخدرات المناسبة لضمان راحة الحيوان على أساس المعايير المؤسسية. قد تكون المضادات الحيوية اللازمة، إذا أدلة على العدوى. </li></ol> 4. الثانوي حج القحف والقوقعة نواة التعرض <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد 2-4 أسابيع للشفاء وبوساطة الفيروسات الجينات نقل الحضانة، وإعادة تخدير واحد حقن سابقا الماوس وكرر الخطوة 1،1-1،5 فترة حضانة الأمثل يختلف مع نوع الجين المنقول. </li><li style=";text-align:right;direction:rtl"> إزالة ندبة الأنسجة اكثر من موقع حج القحف من قبل مجموعة من ملاقط، مشرط، ورينجرز. </li><li style=";text-align:right;direction:rtl"> بعد تصور حج القحف، وتحديد مجموعة من المخيخ وندبا التي تغمر DCN المتبقية. استخدام شفط الفرنسي 5 إلى نضح المخيخ المغطي / ندبا (على غرار تطلع المخيخ السابق). </li><li style=";text-align:right;direction:rtl"> التالية الطموح، ونتوقع المزيد من النزيف، CSF بuild المتابعة، والبتات المتبقية من المخيخ. إدخال بسرعة المالحة في حج القحف لمنع تخثر الدم. استخدام مزيج من لطيف اللمس نقطة الأسنان والشفط لتمهيد الطريق لرؤية مباشرة للDCN. </li><li style=";text-align:right;direction:rtl"> مراقبة سطح DCN. إجراء تجارب علم وظائف الأعضاء على أساس علم البصريات الوراثي كما DCN الآن يمكن الوصول إليها. إدخال التحفيز خفيفة، على سبيل المثال مع الألياف البصرية (الشكل 2)، لدفع التجربة على أساس علم البصريات الوراثي. </li></ol> 5. علم الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وبعد الانتهاء من التجارب، والموت ببطء الفأر مع جرعة زائدة من الكيتامين. يروي الماوس مع محلول ملحي تليها بارافورمالدهيد 4٪. </li><li style=";text-align:right;direction:rtl"> استخراج الدماغ من الجمجمة وبعد الإصلاح، لمدة 2 ساعة. Cryoprotect الدماغ في 30٪ سكروز لمدة 24-48 ساعة. قسم الدماغ باستخدام ناظم البرد قياسي باستخدام 60 ميكرون أقسام. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+single+auditory+nerve+fibers+in+the+mouse.">Response properties of single auditory nerve fibers in the mouse.</a> J Neurophysiol. 93 (1), 557-569 (2005).</li><li>Rolls, A., 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=Optogenetic+disruption+of+sleep+continuity+impairs+memory+consolidation.">Optogenetic disruption of sleep continuity impairs memory consolidation.</a> Proc Natl Acad Sci. 108 (32), 13305-13310 (2011).</li><li>Huff, M., Miller, R., Deisseroth, K., Moorman, D., LaLumiere, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraining+optogenetic+manipulations+of+basolateral+amygdala+activity+modulate+consolidation+of+inhibitory+avoidance+memory+in+rats.">Posttraining optogenetic manipulations of basolateral amygdala activity modulate consolidation of inhibitory avoidance memory in rats.</a> Proc Natl Acad Sci. 110 (9), 3597-3602 (2013).</li><li>Stortkuhl, K., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Smell+of+Blue+Light:+A+New+Approach+toward+Understanding+an+Olfactory+Neuronal+Network.">The Smell of Blue Light: A New Approach toward Understanding an Olfactory Neuronal Network.</a> Front Neurosci. 5 (72), (2011).</li><li>Hira, R., 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=Transcranial+optogenetic+stimulation+for+functional+mapping+of+the+motor+cortex.">Transcranial optogenetic stimulation for functional mapping of the motor cortex.</a> J Neurosci Methods. 179 (2), 258-263 (2009).</li><li>Ayling, O., Harrison, T., Boyd, J., Foroshkov, A., Murphy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+light-based+mapping+of+motor+cortex+by+photoactivation+of+channelrhodopsin-2+transgenic+mice.">Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.</a> Nat Methods. 6 (3), 219-224 (2009).</li><li>Boyden, E., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat. Neurosci. 8, 1263-1268 (2005).</li></ol>

الكتاب ليس لديهم ما يكشف.

وتصف هذه الورقة تقنية التصور المباشر للDCN في نموذج الفئران للتلاعب من النظام السمعي المركزي. النهج المبين التصور المباشر يوفر مزايا هامة على البديل الرئيسي، والتي هي نهج التجسيمي. في المقام الأول، والتصور المباشر للDCN يسمح للتأكيد فوري للموقع من جذع الدماغ، في حين أ?...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

<table><tbody><tr><td>Stereotaxic holder</td><td>Stoelting</td><td>51500</td><td></td></tr><tr><td>Homeothermic blanket</td><td>Harvard</td><td>507214</td><td></td></tr><tr><td>Scalpel blade #11</td><td>Fine Surgical Tools</td><td>10011-00</td><td></td></tr><tr><td>Iris scissor</td><td>Fine Surgical Tools</td><td>14084-08</td><td></td></tr><tr><td>5 French suction</td><td>Symmetry Surgical</td><td>2777914</td><td></td></tr><tr><td>Dental Points</td><td>Henry Schein</td><td>100-8170</td><td></td></tr><tr><td>Bone rongeur</td><td>Fine Surgical Tools</td><td>16020-14</td><td></td></tr><tr><td>10 &amp;micro;l Hamilton syringe</td><td>Hamilton&amp;nbsp;</td><td>7633-01</td><td></td></tr><tr><td>34 gauge, needle</td><td>Hamilton&amp;nbsp;</td><td>207434</td><td></td></tr><tr><td>Rongeurs</td><td>Fine Surgical Tools</td><td>16021-14</td><td></td></tr></tbody></table>

direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

وقد تم إلى حد كبير هبط التحقيق في استخدام بوساطة فيروس نقل الجينات لاعتقال أو عكس فقدان السمع إلى النظام السمعي المحيطي. وقد درست قليل من الدراسات نقل الجينات إلى النظام السمعي المركزي. النواة الظهرية قوقعة (DCN) من الدماغ، الذي يحتوي على الخلايا العصبية من الدرجة الثانية من المسار السمعي، هو موقع محتمل لنقل الجينات. في هذا البروتوكول، ويتجلى تقنية لالتعرض المباشر والحد الأقصى من DCN الفئران عن طريق نهج الحفرة الخلفية. هذا النهج يسمح لأي عملية جراحية حادة أو البقاء على قيد الحياة. وبعد رؤية مباشرة للDCN، مجموعة من التجارب ممكنة، بما في ذلك حقن opsins في نواة القوقعة والتحفيز لاحق من الألياف البصرية بالإضافة إلى ضوء الليزر الأزرق. التجارب الفسيولوجيا العصبية الأخرى، مثل التحفيز الكهربائي واقتفاء أثر حاقن العصبية هي أيضا مجدية. إن مستوى visualizaنشوئها ومدة التحفيز جعل تحقيقه هذا النهج ينطبق على مجموعة واسعة من التجارب.

الجزئي مخيخي الطموح يوضح الوصول إلى القوقعة نواة بعد إزالة الجلد والعضلات التي تغمر الجمجمة، ومعالم سطح الجمجمة، مثل خطوط الدرز الإكليلي وLAMDA، ​​وإظهار توطين التقريبي للحج القحف. وبعد حج القحف مع رينجرز، هو تصور المخيخ. طم...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

التصور المباشر من الفئران الظهرية القوقعة نواة لOptogenetic تحفيز من المسار السمعي

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

Research

JoVE Journal

Neuroscience

9.2K Views.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

Video: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

كانالوستومي كنهج العمليات جراحية لإيصال الأدوية المحلية إلى آذان الداخلية من الفئران الكبار والأطفال حديثي الولادة

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetics

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

الليزر مسح ضوئي للدوائر الدماغ الأمامي المستهدفة Optogenetically

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

الابتدائية الثقافة وElectroporation البلازميد الجهاز الفئران كورتي.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biology

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

في الجسم الحي Optogenetic تحفيز الجهاز العصبي المركزي القوارض

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

دليل ل<em&gt; في الجسم الحي</em&gt; تسجيل وحدة واحدة من التي تم تحديدها Optogenetically القشرية التثبيطي Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

تحفيز Optogenetic من العصب السمعي

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

حديثي الولادة الفئران كوكلار تقنية إكسبلانت باعتبارها<em&gt; في المختبر</em&gt; أداة الفحص في أبحاث السمع

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Medicine

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

في في المختبر إسفين شريحة التحضير لتقليد في اتصال الدائرة العصبية فيفو

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

مصفوفة Optrode للتعديل البصري الوراثي المتزامن والتسجيل العصبي الكهربائي

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://app.jove.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

تحضير شرائح الدماغ الإسفيني لتصور الخلايا العصبية السمعية

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain ...

التصور المباشر من الفئران الظهرية القوقعة نواة لOptogenetic تحفيز من المسار السمعي

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