Financial support from the Flanders Research Foundation FWO (contract n. 12C9112N to DL), the 7th Framework Programme of the European Commission (Marie Curie Network &#8220;C7&#8221;, contract n. 238214; ICT Future Emerging Technology &#8220;ENLIGHTENMENT&#8221; project, contract n. 306502), the Interuniversity Attraction Poles Program initiated by the Belgian Science Policy Office (contract n. IUAP-VII/20), and the University of Antwerp is kindly acknowledged.

<ol>
	<li>Saleem, A. B., Ayaz, A., Jeffery, K. J., Harris, K. D., Carandini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+visual+motion+and+locomotion+in+mouse+visual+cortex.">Integration of visual motion and locomotion in mouse visual cortex.</a> Nature neuroscience. 16, 1864-1869 (2013).</li><li>Ahrens, M. B., Li, J. 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=Brain-wide+neuronal+dynamics+during+motor+adaptation+in+zebrafish.">Brain-wide neuronal dynamics during motor adaptation in zebrafish.</a> Nature. 485 (7399), 471-477 (2012).</li><li>Paz, J. T., Davidson, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closed-loop+optogenetic+control+of+thalamus+as+a+tool+for+interrupting+seizures+after+cortical+injury.">Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury.</a> Nature neuroscience. 16 (1), 64-70 (2013).</li><li>Wallach, A., Eytan, D., Gal, A., Zrenner, C., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+response+clamp.">Neuronal response clamp.</a> Frontiers in neuroengineering. 3 (April), 3 (2011).</li><li>Linaro, D., Couto, J., Giugliano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Command-line+cellular+electrophysiology+for+conventional+and+real-time+closed-loop+experiments.">Command-line cellular electrophysiology for conventional and real-time closed-loop experiments.</a> Journal of neuroscience. 230, 5-19 (2014).</li><li>Sharp, A., O&#8217;Neil, M., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> Journal of neurophysiology. 69 (3), 992-995 (1993).</li><li>Robinson, H. P., Kawai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+digitally+synthesized+synaptic+conductance+transients+to+measure+the+integrative+properties+of+neurons.">Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.</a> Journal of neuroscience methods. 49 (3), 157-165 (1993).</li><li>Vervaeke, K., Hu, H., Graham, L. J., Storm, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+effects+of+the+persistent+Na++current+on+neuronal+excitability+and+spike+timing.">Contrasting effects of the persistent Na+ current on neuronal excitability and spike timing.</a> Neuron. 49 (2), 257-270 (2006).</li><li>White, J. A., Klink, R., Alonso, A., Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+from+voltage-gated+ion+channels+may+influence+neuronal+dynamics+in+the+entorhinal+cortex.">Noise from voltage-gated ion channels may influence neuronal dynamics in the entorhinal cortex.</a> Journal of neurophysiology. 80 (1), 262-269 (1998).</li><li>Destexhe, a., Rudolph, M., Fellous, J. M., Sejnowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuating+synaptic+conductances+recreate+in+vivo-like+activity+in+neocortical+neurons.">Fluctuating synaptic conductances recreate in vivo-like activity in neocortical neurons.</a> Neuroscience. 107 (1), 13-24 (2001).</li><li>Fellous, 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=Regulation+of+Persistent+Activity+by+Background+Inhibition+in+an+In+Vitro+Model+of+a+Cortical+Microcircuit.">Regulation of Persistent Activity by Background Inhibition in an In Vitro Model of a Cortical Microcircuit.</a> Cerebral Cortex. 13 (11), 1232-1241 (2003).</li><li>Gal, A., Eytan, D., Wallach, A., Sandler, M., Schiller, J., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+excitability+over+extended+timescales+in+cultured+cortical+neurons.+The+Journal+of+neuroscience.">Dynamics of excitability over extended timescales in cultured cortical neurons. The Journal of neuroscience.</a> the official journal of the Society for Neuroscience. 30 (48), 16332-16342 (2010).</li><li>Wang, Y., Toledo-Rodriguez, 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=Anatomical,+physiological+and+molecular+properties+of+Martinotti+cells+in+the+somatosensory+cortex+of+the+juvenile+rat.">Anatomical, physiological and molecular properties of Martinotti cells in the somatosensory cortex of the juvenile rat.</a> The Journal of physiology. 561 (Pt 1), 65-90 (2004).</li><li>Wang, Y., Gupta, A., Toledo-Rodriguez, M., Wu, C. Z., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical,+physiological,+molecular+and+circuit+properties+of+nest+basket+cells+in+the+developing+somatosensory+cortex).">Anatomical, physiological, molecular and circuit properties of nest basket cells in the developing somatosensory cortex).</a> Cerebral cortex (New York, N.Y). 12 (4), 395-410 (1991).</li><li>Brette, R., Piwkowska, Z., 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=High-resolution+intracellular+recordings+using+a+real-time+computational+model+of+the+electrode.">High-resolution intracellular recordings using a real-time computational model of the electrode.</a> Neuron. 59 (3), 379-391 (2008).</li><li>Rutishauser, U., Kotowicz, A., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+closed-loop+presentation+of+sensory+stimuli+conditional+on+the+internal+brain-state+of+awake+animals.">A method for closed-loop presentation of sensory stimuli conditional on the internal brain-state of awake animals.</a> Journal of neuroscience. 215 (1), 139-155 (2013).</li><li>Margrie, T., Brecht, M., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo,+low-resistance,+whole-cell+recordings+from+neurons+in+the+anaesthetized+and+awake+mammalian+brain.">In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain.</a> Pflugers Archiv European Journal of Physiology. 444 (4), 491-498 (2002).</li><li>Graham, L., Schramm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Dynamic-Clamp+Manipulation+of+Extrinsic+and+Intrinsic+Conductances:+Functional+Roles+of+Shunting+Inhibition+and+I+BK+in+Rat+and+Cat+Cortex.">In Vivo Dynamic-Clamp Manipulation of Extrinsic and Intrinsic Conductances: Functional Roles of Shunting Inhibition and I BK in Rat and Cat Cortex.</a> Dynamic Clamp: From Principles to Applications. , (2008).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-channel recording. , (1995).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamping. , (2002).</li><li>Davie, J. T., Kole, M. H. P., 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=Dendritic+patch-clamp+recording.">Dendritic patch-clamp recording.</a> Nature Protocols. 1 (3), 1235-1247 (2006).</li><li>Gold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Axon Guide for Electrophysiolog., &amp; Biophysics Laboratory Techniques... , (2007).</li><li>Mainen, Z. F., Sejnowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+spike+timing+in+neocortical+neurons.">Reliability of spike timing in neocortical neurons.</a> Science. 268 (5216), 1503-1506 (1995).</li><li>Buzs&#225;ki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+potential+threshold+of+hippocampal+pyramidal+cells+in+vivo+is+increased+by+recent+spiking+activity.">Action potential threshold of hippocampal pyramidal cells in vivo is increased by recent spiking activity.</a> Neuroscience. 105 (1), 121-130 (2001).</li><li>Koch, C., Segev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Neuronal Modeling: From Synapses to Networks. , (1988).</li><li>Silberberg, G., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disynaptic+inhibition+between+neocortical+pyramidal+cells+mediated+by+Martinotti+cells.">Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells.</a> Neuron. 53 (5), 735-746 (2007).</li><li>Berger, T. K., Silberberg, G., Perin, R., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+bursts+self-inhibit+and+correlate+the+pyramidal+network.">Brief bursts self-inhibit and correlate the pyramidal network.</a> PLoS biology. 8 (9), (2010).</li><li>Tsodyks, M., Pawelzik, K., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+networks+with+dynamic+synapses.">Neural networks with dynamic synapses.</a> Neural computation. 10 (4), 821-835 (1998).</li><li>Kapfer, C., Glickfeld, L. L., Atallah, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supralinear+increase+of+recurrent+inhibition+during+sparse+activity+in+the+somatosensory+cortex.">Supralinear increase of recurrent inhibition during sparse activity in the somatosensory cortex.</a> Nature. 10 (6), 743-753 (2007).</li></ol>

The authors have nothing to disclose.

في هذا النص بروتوكول الكامل للتنفيذ في الوقت الحقيقي، وصفت حلقة مغلقة خلية واحدة تجارب الكهربية، وذلك باستخدام تقنية المشبك التصحيح ومجموعة أدوات برنامج تم تطويره مؤخرا دعا LCG. لتحسين جودة التسجيلات فمن الأهمية بمكان أن الإعداد تسجيل أن ترتكز بشكل صحيح، محمية والاه...

<html> في السنوات الأخيرة، تطورت الكهربية الخلوية من التقليدي نموذج حلقة مفتوحة العاملين في التيار الكهربائي والمشبك الحالي تجارب لحديثة بروتوكولات حلقة مغلقة. تقنية حلقة مغلقة أشهرها وربما كان المشبك دينامية 6،7، الأمر الذي مكن حقن الاصطناعية من القنوات ايون الجهد بوابات الاصطناعية لتحديد غشاء الخلايا العصبية الجهد 8، ودراسة متعمقة للآثار غير القطعية الخفقان على القنوات الأيونية على ديناميكيات استجابة الخلايا العصبية 9، فضلا عن الترفيه في المختبر من واقعية في vivo- مثل النشاط خلفية متشابك 10. وتشمل غيرها من النماذج حلقة مغلقة التي تم اقتراحها المشبك رد الفعل 11، للدراسة في المختبر توليد النشاط المستمر مكتفية ذاتيا، والاستجابة المشبك 4،12، للتحقيق في الآليات الخلوية الكامنة استثارة الخلايا العصبية. ontent &quot;&gt; هنا نحن تصف الإطار القوي الذي يسمح بتطبيق مجموعة متنوعة من البروتوكولات الكهربية المغلقة حلقة في سياق كامل الخلية التسجيلات المشبك التصحيح التي أجريت في شرائح الدماغ الحادة. نظهر كيفية تسجيل الجسدية غشاء الجهد عن طريق التسجيلات المشبك التصحيح في الخلايا العصبية الهرمية من القشرة الحسية الجسدية من الفئران الأحداث وتطبيق ثلاثة بروتوكولات مختلفة حلقة مغلقة باستخدام LCG، وهو برنامج الأدوات القائمة على سطر الأوامر نموا في مختبر البيولوجيا العصبية النظرية وNeuroengineering. لفترة وجيزة، والبروتوكولات المذكورة هي، أولا الحقن الآلي من سلسلة من الطول الموجي المشبك التحفيز الحالية، ذات الصلة لتوصيف مجموعة كبيرة من خصائص الغشاء الإيجابي والسلبي. وقد اقترحت هذه للقبض على النمط الظاهري الكهربية من خلية من حيث الخصائص ردها على سلسلة نمطية من الطول الموجي التحفيز. تعرف علي اكواد الإلكتروني للخلية (على سبيل المثال، انظر &amp; #160؛ 13،14)، يتم استخدام هذه المجموعة من الاستجابات الكهربائية من قبل العديد من المختبرات لتصنيف موضوعي الخلايا العصبية على أساس خصائصها الكهربائية. ويشمل ذلك تحليل للعلاقة ثابتة نقل المدخلات والمخرجات (منحنى فاي)، بواسطة تقنية مبتكرة ينطوي على حلقة مغلقة، ومراقبة الوقت الحقيقي من معدل اطلاق النار عن طريق (PID) وحدة تحكم يتناسب-يتجزأ المشتقة والثانية في الترفيه واقعية في الجسم الحي تشبه خلفية النشاط متشابك في المختبر في الاستعدادات 10، والثالث على اتصال الاصطناعي في الوقت الحقيقي من اثنين من الخلايا العصبية الهرمية سجلت في وقت واحد عن طريق GABAergic عصبون الظاهري، الذي هو محاكاة بواسطة الكمبيوتر. بالإضافة إلى ذلك، LCG تطبق تقنية تعرف باسم نشط الكهربائي التعويض (AEC) 15، والذي يسمح بتنفيذ البروتوكولات المشبك الديناميكية باستخدام قطب واحد. وهذا يسمح تعويض آثار غير مرغوب فيها (أrtifacts) من تسجيل الكهربائي التي تنشأ عندما يتم استخدامها لتقديم المحفزات داخل الخلايا. وتستند هذه الطريقة على تقدير غير حدودي من الخواص الكهربائية ما يعادل الدائرة تسجيل. التقنيات والبروتوكولات التجريبية وصفها في هذه الورقة يمكن تطبيقها بسهولة في التقليدي المفتوح حلقة الجهد والتجارب المشبك الحالية ويمكن أن تمتد إلى الاستعدادات الأخرى، مثل الخلية 4،16 أو تسجيلات داخل الخلايا في الجسم الحي 17،18. الجمعية دقيق لالإعداد لالمشبك التصحيح خلية كاملة الكهربية هو خطوة مهمة للغاية بالنسبة مستقرة، تسجيلات عالية الجودة. في ما يلي افترضنا أن مثل هذا الإعداد التجريبية متاحة بالفعل للاختبارات، ونركز اهتمامنا على وصف استخدام LCG. وأشار القارئ إلى 19-22 للحصول على نصائح إضافية حول التحسين والتصحيح.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tissue slicer</td>
			<td>Leica</td>
			<td>VT-1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td>1.5/0.84 mm OD/ID, with filament</td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>TMC</td>
			<td>20 Series</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DMLFS</td>
			<td>40X Immersion Objective</td>
		</tr>
		<tr>
			<td>Manipulators</td>
			<td>Scientifica</td>
			<td>PatchStar</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifiers</td>
			<td>Axon Instruments</td>
			<td>MultiClamp 700B</td>
			<td>Computer controlled</td>
		</tr>
		<tr>
			<td>Data acquisition card</td>
			<td>National Instruments</td>
			<td>PCI-6229</td>
			<td>Supported by Comedi Linux Drivers</td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Dell</td>
			<td>Optiplex 7010 Tower</td>
			<td>OS: real-time Linux</td>
		</tr>
		<tr>
			<td>Oscilloscopes</td>
			<td>Tektronix</td>
			<td>TDS-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Pump</td>
			<td>Gibson</td>
			<td>MINIPULS3</td>
			<td>Used with R4 Pump head (F117606)</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Multichannel Systems</td>
			<td>TC02</td>
			<td>PH01 Perfusion Cannula</td>
		</tr>
		<tr>
			<td>Manometer</td>
			<td>Testo</td>
			<td>510</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert</td>
			<td>WB14</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>ACSF, ICS</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S3139</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>K-Gluconate</td>
			<td>Sigma</td>
			<td>G4500</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>Na2-GTP</td>
			<td>Sigma</td>
			<td>51120</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td>ICS</td>
		</tr>
	</tbody>
</table>

real-time electrophysiology: using closed-loop protocols to probe neuronal dynamics and beyond

علم الأعصاب التجريبي يشهد اهتماما متزايدا في تطوير وتطبيق الرواية وغالبا ما تكون معقدة والبروتوكولات حلقة مغلقة، حيث تطبق التحفيز يعتمد في الوقت الحقيقي على الاستجابة للنظام. وتتراوح التطبيقات التي صدرت مؤخرا عن تنفيذ نظم الواقع الافتراضي لدراسة الاستجابات الحركية في كل من الفئران 1 و 2 في الزرد، للسيطرة على النوبات التالية السكتة الدماغية القشرية باستخدام optogenetics 3. والميزة الرئيسية لتقنيات حلقة مغلقة تكمن في القدرة على سبر خصائص أعلى من الأبعاد التي لا يمكن الوصول إليها مباشرة أو التي تعتمد على متغيرات متعددة مثل استثارة الخلايا العصبية 4 والموثوقية، وفي الوقت نفسه تحقيق أقصى قدر من الإنتاجية التجريبية. في هذه المساهمة وفي سياق الكهربية الخلوية، ونحن تصف كيفية تطبيق مجموعة متنوعة من البروتوكولات حلقة مغلقة لدراسة خصائص استجابة الخلايا العصبية القشرية هرمي، التوصيةأورديد داخل الخلية مع تقنية المشبك التصحيح في شرائح الدماغ الحادة من القشرة الحسية الجسدية من الفئران الأحداث. كما لا يقدم أي برمجيات المصدر المتاحة تجاريا أو فتح جميع الميزات المطلوبة لأداء بكفاءة التجارب وصفها هنا، تم تطوير برنامج مربع أدوات جديدة تسمى LCG 5، الذي يعظم إعادة استخدام التعليمات البرمجية للكمبيوتر ويسهل تنفيذ النماذج التجريبية رواية هيكل وحدات. يتم تحديد الطول الموجي التحفيز باستخدام المدمجة الفوقية الوصف ويتم وصف البروتوكولات التجريبية الكاملة في ملفات التكوين يستند إلى نص. بالإضافة إلى ذلك، LCG لديه واجهة سطر الأوامر التي هي مناسبة لتكرار التجارب وأتمتة البروتوكولات التجريبية.

في الأجزاء السابقة، التي وصفناها كيفية استخدام برنامج LCG الأدوات لتوصيف الخصائص الكهربية للخلايا هرمية L5 وإعادة في الجسم الحي تشبه النشاط متشابك في إعداد شريحة. استخدام واجهة سطر الأوامر وبروتوكول شبه الآلي يفضل استنساخ وكفاءة التجربة، التي يمكن أن يكون لها تأ...

Watch this Scientific Journal Video about Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond at JoVE.com

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

في الوقت الحقيقي الكهربية: استخدام البروتوكولات ذات الحلقات المغلقة للتحقيق العصبية حيوية وما بعدها

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

real-time-electrophysiology-using-closed-loop-protocols-to-probe

Research

JoVE Journal

Neuroscience

11.4K Views.  University of Antwerp. Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board. 

Video: Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

على منهاج تجريبي لدراسة الأداء حلقة مغلقة من الدماغ والآلة واجهات

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

تسجيلات صفيف متعدد القطب من الانهيارات الجليدية الخلايا العصبية في الثقافات عضوي النمط

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

في وقت واحد رسم المخ، في الوقت الحقيقي قياس تركيز اللاكتات والتلاعب Optogenetic من نشاط الخلايا العصبية في القشرة الدماغية القوارض

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

محلل خلية مقرها الممانعة الحقيقي في الوقت كأداة لتحديد المسارات الجزيئية تشارك في العصبية والحمايه العصبيه في خط الخلية العصبية

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

حلقة مغلقة العصبية الروبوتية تجارب لاختبار خصائص الحاسوبية من الشبكات العصبية

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

الحضانة لفترات طويلة الحادة العصبية الأنسجة للالكهربية والكالسيوم التصوير

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems are already found in clinical applications, and are important tools for basic brain research. A particularly interesting recent development is the integration of closed-loop approaches with optogenetics, such that specific patterns of neuronal activity can trigger optical stimulation of selected neuronal groups. However, setting up an electrophysiological system for closed-loop experiments can be difficult. Here, a ready-to-apply Matlab code is provided for triggering stimuli based on the activity of single or multiple neurons. This sample code can be easily modified based on individual needs. For instance, it shows how to trigger sound stimuli and how to change it to trigger an external device connected to a PC serial port. The presented protocol is designed to work with a popular neuronal recording system for animal studies (Neuralynx). The implementation of closed-loop stimulation is demonstrated in an awake rat.

This protocol demonstrates how to use an electrophysiological system for closed-loop stimulation triggered by neuronal activity patterns. Sample Matlab code that can be easily modified for different stimulation devices is also provided.

استخدام العصبية النشاط Spiking لتحريك المحفزات حلقه مغلقه في التجارب الفسيولوجية العصبية

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

تتبع العتبة الكهربائية ذات الحلقة المغلقة في الوقت الفعلي مفتوحة المصدر لأبحاث الألم الانتقالية

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...

التحفيز العميق للدماغ لعلاج الاكتئاب الشديد

Closed-Loop Neurostimulation for Biomarker-Driven, Personalized Treatment of Major Depressive Disorder

Deep brain stimulation involves the administration of electrical stimulation to targeted brain regions for therapeutic benefit. In the context of major depressive disorder (MDD), most studies to date have administered continuous or open-loop stimulation with promising but mixed results. One factor contributing to these mixed results may stem from when the stimulation is applied. Stimulation administration specific to high-symptom states in a personalized and responsive manner may be more effective at reducing symptoms compared to continuous stimulation and may avoid diminished therapeutic effects related to habituation. Additionally, a lower total duration of stimulation per day is advantageous for reducing device energy consumption. This protocol describes an experimental workflow using a chronically implanted neurostimulation device to achieve closed-loop stimulation for individuals with treatment-refractory MDD. This paradigm hinges on determining a patient-specific neural biomarker that is related to states of high symptoms and programming the device detectors, such that stimulation is triggered by this read-out of symptom state. The described procedures include how to obtain neural recordings concurrent with patient symptom reports, how to use these data in a state-space model approach to differentiate low- and high-symptom states and corresponding neural features, and how to subsequently program and tune the device to deliver closed-loop stimulation therapy.

تسليط الضوء على المؤلف: الفائدة العلاجية لتحفيز الدماغ العميق ذو الحلقة المغلقة في علاج الاكتئاب 

Deep brain stimulation triggered by a patient-specific neural biomarker of a high-symptom state may better control symptoms of major depressive disorder compared to continuous, open-loop stimulation. This protocol provides a workflow for identifying a patient-specific neural biomarker and controlling the delivery of therapeutic stimulation based on the identified biomarker.

التحفيز العصبي ذو الحلقة المغلقة للعلاج الشخصي المدفوع بالعلامات الحيوية للاضطراب الاكتئابي الشديد

Deep brain stimulation involves the administration of electrical stimulation to targeted brain regions for therapeutic benefit. In the context of major ...