This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

كان البحث على ذبابة الفاكهة السوداء البطن في الامتثال مع جامعة المبادئ التوجيهية للبحوث ألبرتا. 1. يطير مجموعة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع 20 الذباب باستخدام ثاني أكسيد الكربون 2 (ز) تخدير ووضعه في 25 ملم × 95 ملم جمع قارورة تحتوي على المواد الغذائية. </li><li style=";text-align:right;direction:rtl"> مخزن قارورة تحتوي على الذباب أفقيا لتجنب محاصرة الذباب في أي السوائل التي قد تتراكم في الجزء السفلي من القارورة. </li><li style=";text-align:right;direction:rtl"> احتضان الذباب للا يقل عن 21 ساعة على 22 ° C في 45٪ الرطوبة في الحاضنة لمدة ما يقرب من 15 ساعة. تعيين حاضنة مع ضوء 12 ساعة: دورة الظلام. </li></ol> 2. تسلق الفحص <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في صباح اليوم التالي، ونقل 20 الذباب من قنينة واحدة في كوب 250 مل تخرج اسطوانة. بمناسبة موقف اسطوانة ليبقيه ثابتا كل يوم. استخدام واحدة اسطوانة الزجاج في التركيب الوراثي لمنع التلوث المتبادل بين الأنماط الجينية. يغسل في نهاية كل تجربة وريال عمانياألمد لهم بين الأنماط الجينية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إجراء تجارب في الضوء المحيط (أو الضوء الأحمر إذا كان هناك خلل محتمل في رؤية) في درجة حرارة ورطوبة 22 ° C و 40٪ على التوالي. لتجنب إيقاع الساعة البيولوجية يخزي، نفذ دائما التجارب في نفس الوقت من اليوم. </li></ol></li><li style=";text-align:right;direction:rtl"> ختم الجزء العلوي من اسطوانة مع فيلم الحاجز (فيلم الشمع) لمنع هروب أي الذباب (الشكل 2). </li><li style=";text-align:right;direction:rtl"> إعداد كاميرا الفيديو على حامل ثلاثي الأرجل. الكاميرا التركيز على خط 190 مل من 250 مل تخرج اسطوانة (17.5 سم). </li><li style=";text-align:right;direction:rtl"> حساب عدد من الذباب الميت في الجزء السفلي من الاسطوانة وفي قارورة الغذاء. تسجيل هذا الرقم كما في وفيات. </li><li style=";text-align:right;direction:rtl"> بخفة جدا الاسطوانة ضد سادة رغوة الخلايا المغلقة مرارا وتكرارا مع ما يكفي من القوة لإزاحة الذباب على السطح السفلي الداخلي. الاستفادة من 5-10 مرات بينما تستخدم اليد الأخرى للضغط سجل على الكاميرا. </li><li style=";text-align:right;direction:rtl"> اضغط على &quot;سجل&quot; الموجود على الكاميرا. </li><li style=";text-align:right;direction:rtl"> بدء فيدEO كاميرا تسجيل والاستفادة من اسطوانة ست مرات في نمط غير إيقاعية متميزة. </li><li style=";text-align:right;direction:rtl"> إجراء كل محاكمة لمدة 2 دقيقة من الوقت الذي يتم استغلالها آخر الذباب وتسجيل عدد من الذباب عبور ارتفاع 17.5 سم (190 مل) في كل نقطة زمنية المختار (قياس كل 10 ثانية). ملاحظة: إن مل وضع علامات على اسطوانة تختلف من نموذج اسطوانة واحدة إلى أخرى اعتمادا على قطر. لتجنب الخطأ، وقياس ارتفاع على كل اسطوانة المستخدمة. </li><li style=";text-align:right;direction:rtl"> وبمجرد أن المحاكمة قد انتهت، والتخلص من الذباب في الايثانول 95٪. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 2،1-2،9 حتى يتم اختبار جميع مكررات مع الذباب جديدة في كل مرة. ملاحظة: على الرغم من 5 مكررات قد يكون كافيا مع الطفرة التي لها تأثير قوي على الحركة، 10 مكررات البيولوجية لل20 الذباب (200 الذباب) ويوصى للكشف عن الفروق الصغيرة. </li><li style=";text-align:right;direction:rtl"> عند الانتهاء من التجربة، وغسل اسطوانات في غسالة الصحون المختبر وO جاف / N لإعادة استخدامها. </li></ol> تحليل 3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Analyأشرطة الفيديو زي كل تطير المحاكمة. كل 10 ثانية، وتسجيل عدد من الذباب الذي اجتياز خط المرمى. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إذا ذبابة يصعد إلى أسفل أو السقوط، سجل أن يطير -1 والاعتماد الطاير المقبل لعبور خط الهدف في نفس العدد الطاير أن قفز إلى أسفل أو سقط. على سبيل المثال، إذا انخفض ذبابة ال 15 تحت خط الهدف، الطاير المقبل لعبور خط (16 تشرين تطير) تعتبر ذبابة ال 15 وليس ال 16. </li></ol></li><li style=";text-align:right;direction:rtl"> طرح معدل الوفيات الناجمة عن العدد الإجمالي من الذباب (20) للحصول على عدد من الذباب التي تبقى في المحاكمة. في كل نقطة زمنية، يجب الحصول على جزء من الذباب فوق خط المرمى. </li><li style=";text-align:right;direction:rtl"> رسم كل نسبة في كل نقطة زمنية (انظر الشكل 3). </li><li style=";text-align:right;direction:rtl"> تحليل أداء عند نقطة البيانات 120 ثانية وأداء الطالب اختبار t عند وجود 2 مجموعات أو ANOVA والاختبار البعدي للمقارنات متعددة (مع تعديل Bonferroni للتخطيط وتوكي للتعاون غير المخطط لهmparisons). وكولموجوروف-سمرنوف 17 تتم أيضا للتأكد من الوضع الطبيعي والتباين على قدم المساواة ولكن أيضا لمقارنة توزيع مجموعة متحولة إلى عنصر التحكم. </li><li style=";text-align:right;direction:rtl"> لتقديم البيانات عبر الشيخوخة، ورسم النسبة المئوية للذباب تسلق في 120 ثانية مع الذباب من مختلف الأعمار (2 أيام، 1 أسبوع، 2 أسابيع) لمعرفة ما إذا كان هناك عجز التدريجي (الشكل 4). </li></ol>

<ol>
	<li>Auluck, P. K., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+prevention+of+Parkinson+disease+in+Drosophila.">Pharmacological prevention of Parkinson disease in Drosophila.</a> Nature medicine. 8, 1185-1186 (2002).</li><li>Bonini, N. M., Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neurodegenerative+disease+modeling+using+Drosophila.">Human neurodegenerative disease modeling using Drosophila.</a> Annual review of neuroscience. 26, 627-656 (2003).</li><li>Bolduc, F. V., Bell, K., Cox, H., Broadie, K. S., Tully, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excess+protein+synthesis+in+Drosophila+fragile+X+mutants+impairs+long-term+memory.">Excess protein synthesis in Drosophila fragile X mutants impairs long-term memory.</a> Nature neuroscience. 11, 1143-1145 (2008).</li><li>McBride, S. M., 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=Pharmacological+rescue+of+synaptic+plasticity,+courtship+behavior,+and+mushroom+body+defects+in+a+Drosophila+model+of+fragile+X+syndrome.">Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome.</a> Neuron. 45, 753-764 (2005).</li><li>Parker, L., Padilla, M., Du, Y., Dong, K., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+epilepsy:+bss+is+a+gain-of-function+mutation+in+the+para+sodium+channel+gene+that+leads+to+seizures.">Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures.</a> Genetics. 187, 523-534 (2011).</li><li>Marley, R., Baines, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+persistent+Na++current+contributes+to+seizure+in+the+slamdance+bang-sensitive+Drosophila+mutant.">Increased persistent Na+ current contributes to seizure in the slamdance bang-sensitive Drosophila mutant.</a> Journal of neurophysiology. 106, 18-29 (2011).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+Mutants+Of+Drosophila+Isolated+By+Countercurrent+Distribution.">Behavioral Mutants Of Drosophila Isolated By Countercurrent Distribution.</a> Proceedings of the National Academy of Sciences of the United States of America. 58, 1112-1119 (1967).</li><li>Ganetzky, B., Flanagan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+relationship+between+senescence+and+age-related+changes+in+two+wild-type+strains+of+Drosophila+melanogaster.">On the relationship between senescence and age-related changes in two wild-type strains of Drosophila melanogaster.</a> Experimental gerontology. 13, 189-196 (1978).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> Journal of visualized experiments : JoVE. , (2012).</li><li>Toma, D. P., White, K. P., Hirsch, J., Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+involved+in+Drosophila+melanogaster+geotaxis,+a+complex+behavioral+trait.">Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait.</a> Nature genetics. 31, 349-353 (2002).</li><li>Inagaki, H. K., Kamikouchi, A., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+simple+gravity+sensing+in+Drosophila+melanogaster.">Methods for quantifying simple gravity sensing in Drosophila melanogaster.</a> Nature protocols. 5, 20-25 (2010).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental gerontology. 40, 386-395 (2005).</li><li>Botella, J. 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=The+Drosophila+carbonyl+reductase+sniffer+prevents+oxidative+stress-induced+neurodegeneration.">The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.</a> Current biology : CB. 14, 782-786 (2004).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+Parkinson's+disease.">A Drosophila model of Parkinson's disease.</a> Nature. 404, 394-398 (2000).</li><li>Chakraborty, 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=Characterization+of+a+Drosophila+Alzheimer's+disease+model:+pharmacological+rescue+of+cognitive+defects.">Characterization of a Drosophila Alzheimer's disease model: pharmacological rescue of cognitive defects.</a> PLoS One. 6, e20799 (2011).</li><li>Orso, G., 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=Disease-related+phenotypes+in+a+Drosophila+model+of+hereditary+spastic+paraplegia+are+ameliorated+by+treatment+with+vinblastine.">Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine.</a> J Clin Invest. 115, 3026-3034 (2005).</li><li>Lehmann, E. L., D'Abrera, H. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nonparametrics : statistical methods based on ranks. , (2006).</li><li>Sherwood, N. T., Sun, Q., Xue, M., Zhang, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+spastin+regulates+synaptic+microtubule+networks+and+is+required+for+normal+motor+function.">Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function.</a> PLoS biology. 2, e429 (2004).</li><li>Inlow, J. K., Restifo, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+comparative+genetics+of+mental+retardation.">Molecular and comparative genetics of mental retardation.</a> Genetics. 166, 835-881 (2004).</li><li>Palladino, M. J., Hadley, T. J., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+paralytic+mutants+are+enriched+for+those+causing+neurodegeneration+in+Drosophila.">Temperature-sensitive paralytic mutants are enriched for those causing neurodegeneration in Drosophila.</a> Genetics. 161, 1197-1208 (2002).</li></ol>

The authors have nothing to disclose.

وقد ثبت بالفعل أن تكون ذبابة الفاكهة نموذجا ممتازا في مرض 14 والظروف العصبية الأخرى 1،2 باركنسون. بالإضافة إلى الأدوات الجينية المتاحة في ذبابة الفاكهة، والحفظ جدا الجينوم من أجل الجينات المسؤولة عن الاضطرابات العصبية 19. ظهور أساليب الفحص ?...

يتم التعرف على الاضطرابات العصبية المدمرة مثل مرض باركنسون والتصلب الجانبي الضموري، والشلل النصفي التشنجي وراثي على نحو متزايد. لسوء الحظ، فإن معظم هذه الاضطرابات العصبية لا تزال دون معالجة. الاستخدام السريري على نطاق واسع، الاختبارات الجينية مشاركات الجينوم على نطاق مثل كله التسلسل exome أدى إلى تزايد عدد الجينات بالتورط في الاضطرابات قاطرة الإنسان. على الرغم من هذا التقدم، والتقدم المرضي من أوائل حتى مراحل متأخرة، لا يزال بعيد المنال في هذه الاضطرابات. يوفر ذبابة الفاكهة واحدة مع الأدوات الوراثية لدراسة متطلبات الجين بطريقة المكانية والزمانية التي تسيطر عليها. بالإضافة إلى ذلك، وقد ثبت ذبابة الفاكهة مفيد في فحص المخدرات لظروف عصبية مثل الشلل الرعاش 1، 2 مرض الزهايمر أو الإعاقة الفكرية 3،4 5،6 والصرع وغيرها. كان هدفنا لتطوير فعالة من حيث التكلفةوفحص موثوق بها من شأنها أن تسمح تحليل الإنتاجية العالية التي من شأنها أن تكون لا تزال حساسة بما يكفي للكشف عن تغيرات صغيرة في الأداء الحركي. هناك عدة فحوصات تستخدم لتحديد آثار الطفرات الوراثية و / أو الظروف البيئية على السلوك تسلق ذبابة الفاكهة. معظم المقايسات الاستفادة من الميل الطبيعي للذباب في الصعود، والمعروفة باسم انجذاب بالجاذبية السلبية، أو فحص التسلق. بنزر 7 اقترح في عام 1967 أن جهاز مكافحة الحالية المستخدمة لدراسة انجذاب ضوئي ويمكن أيضا أن تستخدم لدراسة gravitaxis. ومنذ ذلك الحين، Ganetsky 8 وغيرها الكثير 9 -12 قد بنيت على فحص الأولي. المبدأ هو وضع عدد معروف من الذباب في قارورة والاستفادة من قنينة بقوة ضد سطح صلب، مما تسبب في الذباب ان يسقط الى أسفل القارورة. كما هو سلوك فطري، فإن الذباب تحاول الصعود إلى أعلى القارورة، بدلا من الجاذبية. هذا الاختبار هو الكمي وmeasuوارتفعت الدقة كم عدد الذباب الماضي علامة على القارورة خلال فترة الوقت المحدد. أصبح قياس سرعة بدلا من العدد الكلي للذباب تسلق معلمة موثوقة وأظهرت عيوب في الحالات التي يكون فيها عدد من المعايير الذباب ليس كبيرا (13). وقد أثبت الفحص تسلق مفيدة في دراسة العديد من الاضطرابات العصبية بما في ذلك مرض باركنسون 14. ومع ذلك، لاحظنا أن العيوب قاطرة قد لا تكون قابلة للكشف في وقت حيث ينظر التنكس العصبي بالفعل في دراسات باثولوجية 14. وبالتالي، استخدام الفحص التقليدي قد تحد من القدرة على دراسة المراحل المبكرة من المرض والتسبب. ظهور عيوب قاطرة خلال مراحل لاحقة من أمراض قد تعكس مرض الذي تقدم هو متقدمة جدا لإنقاذ كاملة. هذا يثير مشكلة محتملة مع حساسية مقايسة تسلق التقليدي. عدم القدرة المحتملة للالتقليديينtional تسلق فحص للكشف عن العيوب قاطرة خفيفة ويمكن أن يعزى إلى الارتفاع الذي يطلب فيه من الذباب في الصعود. التقليدية التدابير فحص 15،16 عدد من الذباب لتسلق بنجاح على ارتفاع 2-5 سم في 10 إلى 20 ثانية.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td></td>
			<td></td>
			<td>Any digital camcorder will do. Make sure they can focus on close object.</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Kimble</td>
			<td>20028W</td>
			<td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td>
		</tr>
		<tr>
			<td>computer</td>
			<td></td>
			<td></td>
			<td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point</td>
		</tr>
	</tbody>
</table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

عيوب الحركي الناتجة عن الاضطرابات العصبية يمكن أن يكون أحد أعراض ظهور المرض في وقت متأخر، بعد سنوات من الانحطاط تحت الإكلينيكي، والاستراتيجيات العلاجية الحالية وبالتالي ليست شافية. من خلال استخدام كامل تسلسل exome، وقد تم تحديد عدد متزايد من الجينات تلعب دورا في الحركة البشري. على الرغم من تحديد هذه الجينات، فإنه ليس من المعروف كيف أن هذه الجينات هي الحاسمة لسير قاطرة العادي. ولذلك، فإن فحص موثوق بها، التي تستخدم الكائنات نموذج لتوضيح دور هذه الجينات من أجل تحديد أهداف جديدة للالفائدة العلاجية، هناك حاجة أكثر من أي وقت مضى. لقد قمنا بتصميم نسخة توعيتهم للمقايسة انجذاب بالجاذبية السلبية التي تسمح للكشف عن العيوب أكثر اعتدالا في وقت سابق، ولديه القدرة على تقييم هذه العيوب بمرور الوقت. ويتم تنفيذ في كوب فحص تخرج اسطوانة، التي أغلقت مع فيلم الحاجز الشمع. عن طريق زيادة المسافة عتبة للتسلق إلى 17.5 جم وزيادة مدة التجربة لمدة 2 دقيقة لاحظنا وجود حساسية أكبر في الكشف عن الخلل التنقل خفيفة. ويكلف فحص فعال و لا يتطلب تدريبا مكثفا للحصول على نتائج استنساخه للغاية. وهذا يجعل من تقنية ممتازة لفحص المخدرات مرشح في المسوخ ذبابة الفاكهة مع عيوب الحركة.

تسلق هو سلوك قوي وقابلة للتكرار. في الواقع، في يوم من الأيام القديمة الذباب من النوع البري تصل إلى أداء تسلق مسافة الهدف بسرعة (25-30 ثانية). الذباب متحولة تمثل مجموعة من الأداء بين معتدلة (أو تأخر) لإكمال عدم القدرة على الصعود إلى الهدف. نحن لتوضيح هذا هنا مع اثنين من الأ?...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

التحليل الكمي من العيوب تسلق في نموذج ذبابة الفاكهة من الاضطرابات العصبية

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

quantitative-analysis-climbing-defects-drosophila-model

Research

JoVE Journal

Neuroscience

18.3K Views.  University of Alberta. We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets. 

Video: Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

السابقين التثقيف المجراة من الجامعة، تطوير الأمخاخ ذبابة الفاكهة

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

تقييم الظواهر الاعصاب في<em&gt; ذبابة الفاكهة</em&gt; الخلايا العصبية الدوبامين عن طريق تسلق فحوصات والجامعة المناعية الدماغ

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

سريع الصغرى، الرحلان الشاردي من الغلوتامات وGABA: أداة مفيدة لبحث تكامل متشابك

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

عالية الدقة التحليل الكمي Immunogold من غشاء المستقبلات في الشبكية نقاط الاشتباك العصبي الشريط

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

تحليل متشابك التحوير من<em&gt; ذبابة الفاكهة</em&gt; خلايا مستقبلة للضوء بعد التعرض للضوء لفترات طويلة

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

استخدام الاشراط الكلاسيكي اييبلينك تتبع لتقييم هيبوكامبال الخلل في نموذج الفئران من اضطرابات طيف الكحول الجنيني

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

تقييم نفاذية حاجز الدم في الدماغ بالتسريب الوريدي الزلال المسمى فيتك في نموذج ماوس لأمراض الأعصاب

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

التحليل الكمي لتعقيد أربوريزاتيون الجذعية العصبية في المورفولوجية

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

بيولوجيا الخلية كمية من نيوروديجينيريشن في المورفولوجية من خلال تحليل غير منحازة للبروتينات فلوريسسينتلي المعلمة باستخدام إيماجيج

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

عزل نويات الحبل الشوكي الكبار لتسلسل الحمض النووي الريبي المفرد-نواة موازية على نطاق واسع

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...