The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.

وكانت جميع البروتوكولات الماوس وفقا للمبادئ التوجيهية المعاهد الوطنية للصحة وتمت الموافقة من قبل لجنة الدراسات الحيوانية من كلية الطب بجامعة واشنطن (سانت لويس، MO). 1. إعداد لوحة اختبار واسوار <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنظيف قبالة سطح الزجاج. </li><li style=";text-align:right;direction:rtl"> تأمين T-نوع خيوط التحقيق الحرارية إلى السطح في منتصف لوحة من الزجاج مع الشريط المختبر. </li><li style=";text-align:right;direction:rtl"> وضع مرفقات الحيوان على لوحة الزجاج في خط واحد على طول منتصف اللوحة. </li><li style=";text-align:right;direction:rtl"> كاتب التحقيق الحرارية من خلال مركز الضميمة الحيوانية والاندماج في مسجل البيانات. تحويل مسجل بيانات على حين تفعيل هذا ميزة لصناعة السيارات في الاغلاق، وإرفاق مسجل بيانات إلى جهاز الكمبيوتر باستخدام كابل المقدمة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إذا تسجيل درجة الحرارة لوحة أثناء التجربة، افتح البرنامج مسجل بيانات لبدء تسجيل درجات الحرارة لوحة. </li><li style=";text-align:right;direction:rtl"> إذا لزم الأمر، وضبط البرنامج لإعادةالحبل درجة الحرارة لوحة مرة واحدة كل ثانية. </li><li style=";text-align:right;direction:rtl"> بدء تسجيل درجات الحرارة باستخدام برنامج حزم مع مسجل بيانات الحراري. </li></ol></li><li style=";text-align:right;direction:rtl"> فصل العلبة السوداء مع تدرج لمنع التفاعل البصري بين الفئران. </li><li style=";text-align:right;direction:rtl"> موقف يعكس تحت الزجاج بحيث الجانب السفلي من العبوات مرئيا من وضع الجلوس المريح. </li></ol> 2. الاحتباس الحراري / تبريد لوحة زجاج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ملء صناديق الألومنيوم مع المياه تحسنت، والجليد الرطب، أو الثلج الجاف والموقف منها بشكل مناسب على لوحة زجاجية (يمكن أيضا الحزم رقائق الألومنيوم مليئة الثلج الجاف أن تستخدم لتبريد الزجاج؛ الشكل 1) (2). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> للاختبار في 30 ° C، ضع صناديق الألومنيوم حوالي 0.25 &#39;&#39; بعيدا عن حظائر الحيوانات (الشكل 2B) 2. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحديد شكل دائري الماء الساخن على جانبي لوحة زجاجية. تعيين دائري إلى 45 - 60 درجة مئوية، ولناالبريد إلى ملء صناديق الألومنيوم مع وجود تدفق مستمر من الماء الساخن (الشكل 1C) 2. </li><li style=";text-align:right;direction:rtl"> ضع المروجون مثل أن الماء الساخن من صناديق الألومنيوم يستنزف مباشرة مرة أخرى في خزان للدائري على كل جانب (الشكل 1C) 2. </li></ol></li><li style=";text-align:right;direction:rtl"> للاختبار في RT، وترك صناديق فارغة (الشكل 2) 2. </li><li style=";text-align:right;direction:rtl"> للاختبار في 17 ° C، ضع صناديق حوالي 0.25 &#39;&#39; بعيدا عن حظائر الحيوانات على جانبي وملء مع الثلج الرطب (الشكل 2) 2. </li><li style=";text-align:right;direction:rtl"> للاختبار في 12 ° C، ضع صناديق حوالي 1.25 &#39;&#39; بعيدا عن العبوات على جانبي وملء مع الثلج الجاف (الشكل 2) 2. </li><li style=";text-align:right;direction:rtl"> للاختبار في 5 ° C، ضع صناديق حوالي 0.25 &#39;&#39; بعيدا عن العبوات على جانبي وملء مع الثلج الجاف (الشكل 2) 2.<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عندما التبريد الزجاج مع الثلج الجاف، تأكد من وجود تهوية كافية لمنع CO 2 تراكم في الغرفة. </li></ol></li></ol></li><li style=";text-align:right;direction:rtl"> الانتظار للزجاج للوصول إلى مجموعة ودرجة الحرارة المطلوبة. </li><li style=";text-align:right;direction:rtl"> إضافة الفئران لالمرفقات على لوحة. ملاحظة: يمكن استخدام مولد الضوضاء البيضاء لتقليل الاضطرابات الضوضاء. </li><li style=";text-align:right;direction:rtl"> انتظر الفئران ليتأقلم. ملاحظة: في منشأتنا هذا يستغرق ما يقرب من 2.5 ساعة، ولكن هذا قد تتفاوت تفاوتا كبيرا على أساس السكن الحيوانية وشروط التعامل معها. </li><li style=";text-align:right;direction:rtl"> الحفاظ على الزجاج في نطاق درجات الحرارة المطلوبة عن طريق ضمان أن يتم الاحتفاظ صناديق كامل من الماء درجة حرارة والجليد الرطب، أو الثلج الجاف. ملاحظة: مع جهازنا في حاجة إلى صناديق ليتم تعبئتها مع الثلج تقريبا كل 90 دقيقة. ملاحظة: لC حالة 17 °، فإنه من المفيد لتفريغ معظم المياه من مربعات الألومنيوم من خلال فتحات التصريف قبل إعادة تعبئتها مع الثلج. وهذا الاستقرار في درجة الحرارة أفضل، والعلاقات العامةتجاوز الحدث ملاحظة: إن المبلغ المحدد من الثلج الجاف تختلف موسميا، ولكن بصفة عامة حفظ مربعات أكثر من ¼ الكاملة على طول من مربع سوف تبقي درجة حرارة ثابتة. </li></ol> 3. اختبار الفئران في درجات الحرارة الثابتة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خارج الغرفة السلوكية، وملء دلو الجليد حوالي نصف كامل من الثلج الجاف. </li><li style=";text-align:right;direction:rtl"> باستخدام مطرقة أو مطرقة، سحق الثلج الجاف الى مسحوق ناعم. ملاحظة: سوف الملئ دلو تجعل من الصعب سحق تماما الثلج الجاف الى مسحوق. </li><li style=";text-align:right;direction:rtl"> باستخدام شفرة حلاقة مستقيمة أو مقص، وقطع رأس قبالة حقنة 3 مل. </li><li style=";text-align:right;direction:rtl"> باستخدام إبرة 21 G، كزة 3 الثقوب على طرفي نقيض من الحقنة (ما مجموعه 6 فتحات). ملاحظة: هذه الثقوب يقلل من الضغط الناتجة عن التسامي في حين ضغط الثلج الجاف. يمكن إعادة استخدام المحاقن قطع للتجارب متعددة. </li><li style=";text-align:right;direction:rtl"> أخذ الحقنة، ومسحوق الثلج الجاف، وساعة توقيت باليد إلى غرفة السلوكية. </li><li style=";text-align:right;direction:rtl"> ملء غرفة حقنة نصف كامل من مسحوق الثلج الجاف. عقد نهاية قطع من حقنة ضد كائن مسطح، وضغط بقوة مسحوق باستخدام المكبس. احذر؛ المكبس من البلاستيك قد ينحني أو كسر من الضغط. إذا حدث هذا، استبدال المكبس من حقنة جديدة. </li><li style=";text-align:right;direction:rtl"> تمديد غيض من مضغوط بيليه الثلج الجاف الماضي حافة حقنة. </li><li style=";text-align:right;direction:rtl"> الفئران الاختبار التي هي تماما في بقية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في 30 ° C، 23 ° C و 17 ° C، والفئران التي لديها اختبار كل الكفوف 4 على الزجاج وليس تتحرك، ولكن ليس نائما تماما (19). </li><li style=";text-align:right;direction:rtl"> في 12 ° C و 5 ° C، الفئران الاختبار التي هي على الكفوف 2 أو 4 الكفوف وعدم التحرك أو القفز. </li></ol></li><li style=";text-align:right;direction:rtl"> باستخدام المرايا الاستهداف، بلطف ولكن بحزم اضغط بيليه دافق شقة ضد سطح الزجاج تحت hindpaw الماوس (الشكل 1A) 2. بدء الموقت اليد. </li><li style=";text-align:right;direction:rtl"> إيقاف الموقت وإزالة بيليه عندما يتحرك الماوس بعيدا عن الزجاج يبرد. ملاحظة: إن الحركة الانسحاب يمكن أن يكون الرأسي أو الأفقي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إذا كان الماوس يتحرك لفترة وجيزة جدا مخلب ثم يعود إلى السطح التبريد، ومواصلة توقيت وتحفيز حتى يجعل الماوس تحرك دائم بعيدا. ملاحظة: يستخدم مختبرنا وقت التحفيز الأقصى من 20 ثانية بالنسبة للفئران في معظم الحالات. </li></ol></li><li style=";text-align:right;direction:rtl"> كرر هذا الإجراء الاختبار حتى يتم جمع لا يقل عن 3 القيم على كل مخلب من كل حيوان. محاكمات منفصلة اختبار الكفوف المعاكس على نفس الماوس بنسبة 7 دقيقة على الأقل، ومحاكمات منفصلة متتالية على أي مخلب واحد من قبل لا يقل عن 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> إذا لزم الأمر، استخدم سمك مختلفة من الزجاج لتوليد معدلات مختلفة من التبريد (الشكل 3) 1. ملاحظة: ويرتبط معدل التبريد عكسيا مع سمك الزجاج. </li></ol> 4. اختبار الفئران خلال الباردة التكيف ملاحظة: هذا هو بروتوكول بديل الذي يسمح الاختبار وبلات الزجاجالبريد يبرد، بدلا من مرة واحدة قد استقر لوحة والفئران تكيفت تماما مع البيئة الباردة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اتبع الإرشادات المذكورة في القسم 1 لإعداد الجهاز. </li><li style=";text-align:right;direction:rtl"> اتبع الإرشادات الواردة في القسم 3 لأخذ القياسات الأساسية في RT (الشكل 7A) 2. </li><li style=";text-align:right;direction:rtl"> قبل صناديق تبريد الألومنيوم مع الثلج الجاف. </li><li style=";text-align:right;direction:rtl"> مرة واحدة وقد تم قياس الإختفاء انسحاب خط الأساس، وضع صناديق precooled على لوحة حوالي 1.25 &#39;&#39; بعيدا عن العبوات على جانبي (الشكل 7A، السهم المسمى &quot;وأضاف الجليد الجاف&quot;) 2. </li><li style=";text-align:right;direction:rtl"> اتبع الإرشادات الواردة في القسم 3 لأخذ القياسات كما يبرد لوحة من الزجاج، أخذ القياسات كلما كان ذلك ممكنا. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPV1+in+the+spinal+cord+by+oxidized+linoleic+acid+metabolites+contributes+to+inflammatory+hyperalgesia.">Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (44), 18820-18824 (2009).</li><li>Fujita, F., Uchida, K., Takaishi, M., Sokabe, T., Tominaga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Temperature+Affects+the+Temperature+Threshold+for+TRPM8+Activation+through+Interaction+of+Phosphatidylinositol+4,5-Bisphosphate.">Ambient Temperature Affects the Temperature Threshold for TRPM8 Activation through Interaction of Phosphatidylinositol 4,5-Bisphosphate.</a> Journal of Neuroscience. 33 (14), 6154-6159 (2013).</li><li>Rohacs, T., Lopes, C. M., Michailidis, I., Logothetis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI(4,5)P2+regulates+the+activation+and+desensitization+of+TRPM8+channels+through+the+TRP+domain.">PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain.</a> Nature neuroscience. 8 (5), 626-634 (2005).</li><li>Daniels, R. L., Takashima, Y., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+the+neuronal+cold+sensor+TRPM8+is+regulated+by+phospholipase+C+via+the+phospholipid+phosphoinositol+4,5-bisphosphate.">Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate.</a> The Journal of biological chemistry. 284 (3), 1570-1582 (2009).</li><li>Zhang, H., 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=Neurokinin-1+receptor+enhances+TRPV1+activity+in+primary+sensory+neurons+via+PKCepsilon:+a+novel+pathway+for+heat+hyperalgesia.">Neurokinin-1 receptor enhances TRPV1 activity in primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 27 (44), 12067-12077 (2007).</li><li>Wang, H., Zylka, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mrgprd-expressing+polymodal+nociceptive+neurons+innervate+most+known+classes+of+substantia+gelatinosa+neurons.">Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 29 (42), 13202-13209 (2009).</li></ol>

The authors have nothing to disclose

The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and genetic neuron ablation studies26. It can be modified to measure responses to a variety of rates of cooling using different thicknesses of glass, and the size of the cold stimulus applied can be altered using different sized syringes. 
The most critical step for performing this assay properly is cooling or warming the glass plate evenly during testing. Carefully arranging the aluminum boxes so that they are evenly spaced on either side of the animal enclosures will help generate more uniform temperatures. Marking the glass plate to standardize aluminum box placement between experiments will also improve the reproducibility of the assay, although these markings may have to be periodically recalibrated to account for seasonal variations in building temperature.
It can also be challenging to maintain a consistent glass plate temperature over the course of the experiments. Ensuring that the aluminum boxes are always evenly filled with dry ice/wet ice/warmed water will yield experiments with stable plate temperatures. Additionally, under the 17 &#176;C testing condition it may become more difficult to maintain the temperature as the wet ice melts. We recommend draining excess water from the melted ice out of the aluminum boxes before refilling with more wet ice. 
The main limitation is that the mouse paw must be in contact with the glass surface in order to ensure a consistent cold stimulus. This can be an issue in some neuropathic models, such as the chronic constriction injury, which can cause paw curling. Other challenges to obtaining reproducible results with this assay include that the mice must be fully at rest, which requires adequate acclimation periods. If the mice are too active for testing, it may help to allow longer acclimation periods. Finally, groups that are planning to utilize this assay must also ensure that there is sufficient ventilation in the room in order to prevent CO2 buildup when using large amounts of dry ice.

قياس الاستجابة الباردة في القوارض المهم لتحسين فهم الآليات المحتملة للحساسية الباردة في البشر في إطار كل من الظروف العادية والمرضية. والباردة أخمصي الفحص (CPA)، وضعت أصلا منذ عدة سنوات 1، يهدف إلى توليد استنساخه، لا لبس فيها الاستجابات السلوكية الفئران لحافز البارد ألقاها في RT. وقد أتاحت المزيد من التحسينات الأخيرة من هذا الاختبار قياس استنساخه من حساسية الباردة في مجموعة واسعة من درجات الحرارة 2. تم تصميم كلا الإصدارين أيضا أن يكون نسبيا عالية الإنتاجية، وغير مكلفة للاستخدام. أحرز قدر كبير من التقدم في فهم آليات حساسية الباردة باستخدام أساليب سلوكية أخرى. أسلوب واحد هو اختبار التبخر الأسيتون، والتي تنطوي على اللمس أو الرش الأسيتون على مخلب الماوس وقياس مقدار الوقت الذي تنفق الماوس عبها 3،4 مخلب. للأسف،خزينا الردود على الأسيتون التبخر التي كتبها الإحساس الرطب ورائحة الأسيتون. أيضا، والتحفيز الباردة التي يتم تطبيقها في اختبار الأسيتون التبخر يمكن أن تختلف على أساس كمية من الاسيتون التطبيقية، ويصعب قياسها كميا. وأخيرا، لم يصب الفئران لها ردود الحد الأدنى لالأسيتون في الأساس، مما يجعل من المستحيل قياس التسكين في غياب فرط الحساسية مع هذا الأسلوب. فحص الكلاسيكي آخر لردود الباردة هو فحص الذيل نفض الغبار، حيث يتم قياس زمن الوصول إلى الانسحاب بعد مغمورة الذيل في 5،6 الماء البارد. في حين أن الاستجابات السلوكية في هذا الاختبار هي لا لبس فيها ويقيس الفحص الردود على درجة حرارة معينة، لا بد من ضبط النفس الحيوانات أثناء الاختبار، والتي يمكن أن تغير الاستجابة الباردة من خلال موصوفة وصفا جيدا التوتر الناجم عن آليات مسكن 7. آخر أداة تستخدم عادة هي اختبار لوحة البارد، الذي يقيس السلوكيةردود الفئران بعد يتم وضعها على طبق من ذهب المبردة بلتيير 8-10. بينما توفر هذه الأداة المعلومات حول استجابات الحيوان في درجات حرارة معينة، فإنه كما استخدمت بشكل غير متسق. وتقاس مجموعات مختلفة أنواع مختلفة من الاستجابات بما في ذلك عدد من القفزات 8،11، الكمون لأول رد 8،11- 13، وعدد من مخلب يرفع 11،13،14 مع نتائج مختلفة جدا. لوحة فحص البارد هو أيضا منخفضة نسبيا الإنتاجية كما حيوان واحد فقط يمكن اختبارها في كل مرة، وأنه يتطلب وجود جهاز بلتيير مكلفة وهشة. 2-لوحة اختبار تفضيل درجة الحرارة هو مشتق تستخدم عادة للاختبار لوحة الباردة يقيس كمية النسبي من الوقت الذي تنفق الحيوانات في 2 وحات متصلة من درجات حرارة مختلفة 9،15- 17. آخر مماثل فحص شيوعا هو فحص التدرج الحراري، حيث مقدار الوقت الذي تنفق الفئران في مناطق مختلفة في درجة الحرارةتتراوح بين 5 ° C و 45 ° C على لوحة معدنية طويلة تقاس 16. في حين أن هذه المقايسات تسمح المقارنة بين درجات الحرارة، وأنه من غير الواضح ما إذا كان يمثل سلوك النفور درجة الحرارة أو لتفضيل درجة الحرارة. وأخيرا، تم استخدام لوحة الباردة الديناميكي فحص لقياس مدى الفئران تستجيب لتغير درجات الحرارة المحيطة 18. يتضمن هذا الأسلوب وضع الفئران على جهاز بلتيير RT والتعلية ذلك وصولا الى 1 درجة مئوية في حين قياس مدى تقفز الفئران أو لعق الكفوف في درجات حرارة لوحة مختلفة. في حين أن هذا اختبارات كيف الفئران على التكيف مع بيئة التبريد، وأنها لا توفر وسيلة لاختبار مدى الفئران تستجيب لحافز البارد في الإعداد لدرجة الحرارة المحيطة برودة. بالإضافة إلى ذلك، فإنه يتطلب معدات باهظة الثمن لأداء ولا توفر وسيلة لتأقلم الفئران لاختبار المعدات قبل قياس حساسيتها الباردة. واستكمالا لهذه المقايسات، وCPA يختبر acclimaردود تيد لحافز البارد واضحة المعالم في مجموعة متنوعة من تتراوح درجة الحرارة، أو أثناء عملية التكيف مع درجات الحرارة المحيطة الباردة. فإنه يمكن اختبار ما يصل الى 14 الفئران في وقت واحد مع الجهاز الحالي لدينا، مع إمكانية زيادتها غير مكلفة تصل لاختبار الإنتاجية العالية.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Name</td>
			<td style="width:320px;">Company</td>
			<td style="width:353px;">Catalog Number</td>
			<td style="width:303px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-type thermocouple probe</td>
			<td>Physitemp</td>
			<td>IT-24p</td>
			<td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plate</td>
			<td>Local glass company (in St. Louis, Stemmerich Inc)</td>
			<td>N/A</td>
			<td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#39;&#39;, but 3/16&#39;&#39; and 1/8&#39;&#39; are also useful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Data logger</td>
			<td>Extech</td>
			<td>EA15</td>
			<td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&#38;prodid=408)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mL Syringe</td>
			<td>BD</td>
			<td align="right">309657</td>
			<td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>If using Extech logger, any Pcwill work</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum boxes</td>
			<td>Washington University in St. Louis machine shop</td>
			<td>N/A</td>
			<td>boxes are 3&#39; long, 4.5&#39;&#39; wide, and 3&#39;&#39; tall with a sealed lid.&#160; There is a 1/2&#39;&#39; hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated water circulator</td>
			<td>VWR</td>
			<td></td>
			<td>Any water circulator model with a pump will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 gauge needle</td>
			<td>BD</td>
			<td align="right">305165</td>
			<td>The exact needle size is not important</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hand timer</td>
			<td>N/A</td>
			<td></td>
			<td>Any hand timer will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror</td>
			<td>N/A</td>
			<td></td>
			<td>Any flat mirror will work</td>
		</tr>
	</tbody>
</table>

a simple and inexpensive method for determining cold sensitivity and adaptation in mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

طريقة بسيطة وغير مكلفة لتحديد الباردة الحساسية والتكيف في الفئران

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

a-simple-inexpensive-method-for-determining-cold-sensitivity

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

14.7K Views.  Washington University in St. Louis. The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Video: A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

رواية جهاز وطريقة لتدعيم المخدرات

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

طريقة عامة لتقييم الحضانة لالتوق السكروز في الجرذان

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

A قراءات الجزيئية من التكيف على المدى الطويل في الشم<em&gt; C. ايليجانس</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

بحكم الرحم الصعق الكهربائي وإإكسبلنتس نصف الكرة المجموع: طريقة بسيطة للدراسات التجريبية التنمية القشرية في وقت مبكر

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

و، الفحص السلوكية قابلة غير مكلفة لقياس الإيثانول التخدير الحساسية والتسامح السريع في<em&gt; ذبابة الفاكهة</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

طريقة لاستهداف وعزل مجرى الهواء-التعصيب الخلايا العصبية الحسية في الفئران

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

وبسيطة خطوة واحدة بروتوكول تشريح لجبل لالجامع إعداد الكبار<em&gt; ذبابة الفاكهة</em&gt; أدمغة

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

رواية الفحص لالباردة حس الألم في<em&gt; ذبابة الفاكهة</em&gt; يرقات

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

استزراع في فيفو--مثل مورين Astrocytes استخدام بروتوكول أوسام سريعة وبسيطة وغير مكلفة

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

نهج بسيط للحث على العصب الذاتية التجريبية في الفئران C57BL/6 للتقييمات الفنية ولاثيل

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...