الكتاب أود أن أشكر إينا فولتر لها المساعدة التقنية الممتازة. تم إنشاؤها VGAT-فينوس الفئران المعدلة وراثيا من قبل الدكاترة. Y. ياناغاوا، M. Hirabayashi وY. كاواجوتشي في المعهد الوطني للعلوم الفسيولوجية، أوكازاكي، اليابان، وذلك باستخدام PCS2-فينوس التي يقدمها الدكتور A. Miyawaki.

<ol>
	<li>Freund, T. F., 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=Interneurons+of+the+hippocampus.">Interneurons of the hippocampus.</a> Hippocampus. 6 (4), 347-470 (1996).</li><li>Meyer, A. H., Katona, I., Blatow, M., Rozov, A., Monyer, H. <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+labeling+of+parvalbumin-positive+interneurons+and+analysis+of+electrical+coupling+in+identified+neurons.">In vivo labeling of parvalbumin-positive interneurons and analysis of electrical coupling in identified neurons.</a> Journal of Neuroscience. 22, 7055-7064 (2002).</li><li>Vida, I., Halasy, K., Szinyei, C., Somogyi, P., Buhl, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unitary+IPSPs+evoked+by+interneurons+at+the+stratum+radiatum-stratum+lacunosum-moleculare+border+in+the+CA1+area+of+the+rat+hippocampus+in+vitro.">Unitary IPSPs evoked by interneurons at the stratum radiatum-stratum lacunosum-moleculare border in the CA1 area of the rat hippocampus in vitro.</a> Journal of Physiolology. 506, 755-773 (1998).</li><li>Mody, I., De Koninck, Y., Otis, T. S., Soltesz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+the+cleft+at+GABA+synapses+in+the+brain.">Bridging the cleft at GABA synapses in the brain.</a> Trends Neurosci. 17 (12), 517-525 (1994).</li><li>Bartos, 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=Fast+synaptic+inhibition+promotes+synchronized+gamma+oscillations+in+hippocampal+interneuron+networks.">Fast synaptic inhibition promotes synchronized gamma oscillations in hippocampal interneuron networks.</a> PNAS. 99, 13222-13227 (2002).</li><li>Cobb, S. 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=Synaptic+effects+of+identified+interneurons+innervating+both+interneurons+and+pyramidal+cells+in+the+rat+hippocampus.">Synaptic effects of identified interneurons innervating both interneurons and pyramidal cells in the rat hippocampus.</a> Neuroscience. 79 (3), 629-648 (1997).</li><li>Bartos, M., Vida, I., Jonas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+mechanisms+of+synchronized+gamma+oscillations+in+inhibitory+interneuron+networks.">Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks.</a> Nature Review Neurosci. 8, 45-56 (2007).</li><li>Ascoli, G. 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=Petilla+terminology+nomenclature+of+features+of+GABAergic+interneurons+of+the+cerebral+cortex.">Petilla terminology nomenclature of features of GABAergic interneurons of the cerebral cortex.</a> Nature Review Neurosci. 9, 557-568 (2008).</li><li>Klausberger, T., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+diversity+and+temporal+dynamics+the+unity+of+hippocampal+circuit+operations.">Neuronal diversity and temporal dynamics the unity of hippocampal circuit operations.</a> Science. 321, 53-57 (2008).</li><li>Buhl, E. H., Szil&#225;gyi, T., Halasy, K., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+properties+of+anatomically+identified+basket+and+bistratified+cells+in+the+CA1+areas+of+the+rat+hippocampus+in+vitro.">Physiological properties of anatomically identified basket and bistratified cells in the CA1 areas of the rat hippocampus in vitro.</a> Hippocampus. 6, 294-305 (1996).</li><li>Kawaguchi, Y., Katsumara, H., Kosaka, T., Heizmann, C. W., Hama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+spiking+cells+in+rat+hippocampus+(CA1+region)+contain+the+calcium-+binding+protein+parvalbumin.">Fast spiking cells in rat hippocampus (CA1 region) contain the calcium- binding protein parvalbumin.</a> Brain Res. 416 (2), 369-374 (1987).</li><li>Booker, S. 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P., Jonas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+recording+from+mossy+fiber+terminals+in+hippocampal+slices.">Patch-clamp recording from mossy fiber terminals in hippocampal slices.</a> Nature Protocols. 1, 2075-2081 (2006).</li><li>Houston, C. M., Bright, D. P., Sivilotti, L. G., Beato, M., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Chloride+Ions+Regulate+the+Time+Course+of+GABA-Mediated+Inhibitory+Synaptic+Transmission.">Intracellular Chloride Ions Regulate the Time Course of GABA-Mediated Inhibitory Synaptic Transmission.</a> Journal of Neuroscience. 29 (33), 10416-10423 (2009).</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=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annual Review Physiology. 46, 455-472 (1984).</li><li>Schindelin, 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=Fiji+an+open-source+platform+for+biological-image+analysis.">Fiji an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Geiger, J. 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=Patch-clamp+recording+in+brain+slices+with+improved+slicer+technology.">Patch-clamp recording in brain slices with improved slicer technology.</a> Pflugers Archives. 443, 491-501 (2002).</li><li>Malinow, R., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+enhancement+shown+by+whole-cell+recordings+of+long-term+potentiation+in+hippocampal+slices.">Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices.</a> Nature. 346, 177-180 (1990).</li><li>Vida, I., Bartos, M., Jonas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shunting+inhibition+improves+robustness+of+gamma+oscillations+in+hippocampal+interneuron+networks+by+homogenizing+firing+rates.">Shunting inhibition improves robustness of gamma oscillations in hippocampal interneuron networks by homogenizing firing rates.</a> Neuron. 49 (1), 107-117 (2006).</li><li>Neu, A., F&#246;ldy, C., Soltesz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+origin+of+CB1-dependent+tonic+inhibition+of+GABA+release+at+cholecystokinin-positive+basket+cell+to+pyramidal+cell+synapses+in+the+CA1+region+of+the+rat+hippocampus.">Postsynaptic origin of CB1-dependent tonic inhibition of GABA release at cholecystokinin-positive basket cell to pyramidal cell synapses in the CA1 region of the rat hippocampus.</a> Journal of Physiology. 578 (1), 233-247 (2007).</li><li>Baude, A., Bleasdale, C., Dalezios, Y., Somogyi, P., Klausberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoreactivity+for+the+GABAA+receptor+alpha1+subunit,+somatostatin+and+Connexin36+distinguishes+axoaxonic,+basket,+and+bistratified+interneurons+of+the+rat+hippocampus.">Immunoreactivity for the GABAA receptor alpha1 subunit, somatostatin and Connexin36 distinguishes axoaxonic, basket, and bistratified interneurons of the rat hippocampus.</a> Cerebral Cortex. 17, 2094-2107 (2007).</li></ol>

تعلن الكتاب أنه ليس لديهم المصالح المالية المتنافسة.

نحن تصف الطريقة التي تجمع بين تقنيات الكهربية وتشريحي عصبي لتوصيف الخلايا العصبية وظيفيا morphologically- وحددت neurochemically في المختبر؛ ولا سيما أنواع متنوعة من الوظائف المثبطة القشرية. الجوانب الرئيسية لهذا الإجراء هي: (1) الاختيار من الوظائف المحتملة؛ (2) تسجيل داخل الخلايا و?...

وكانت الدوائر العصبية قرن آمون منذ فترة طويلة موضوع تدقيق مكثف، فيما يتعلق بكل من علم التشريح وعلم وظائف الأعضاء، وذلك بسبب الدور الأساسي في التعلم والذاكرة، فضلا عن الملاحة المكانية في كل من البشر والقوارض. وبالمثل، فإن منظمة الصفحي بارزة، ولكن بسيطة من الحصين يجعل هذه المنطقة وهو موضوع مفضل من الدراسات التي تتناول الخصائص التركيبية والوظيفية للشبكات القشرية. وتتألف الدوائر من خلايا قرن آمون مثير الرئيسية (&gt; 80٪) وأصغر (10-20٪)، ولكن فوج متنوعة للغاية من interneurons المثبطة 1-3. Interneurons الافراج حامض γ-امينوبوتيريك (GABA) من المحطات محوار بهم الذي يعمل في ionotropic سريع GABA A مستقبلات (GABA A روبية) وبطيئة metabotropic مستقبلات GABA B (GABA B روبية) 4. هذه الآليات المثبطة موازنة الإثارة وتنظيم استثارة الخلايا الرئيسية، وبالتاليتوقيت الأشعة تحت الحمراء ونمط التصريف. ومع ذلك، GABA سراحه من interneurons تعمل ليس فقط على الخلايا الرئيسية، ولكن أيضا على interneurons أنفسهم 5،6. قبل وبعد المشبكي مستقبلات توسط تنظيم ردود الفعل والتفاعلات المتبادلة المثبطة بين أنواع مختلفة من عصبون. ويعتقد أن هذه الآليات المثبطة في شبكات عصبون أن تكون مركزية في جيل وتشكيل أنماط النشاط السكاني، في التذبذبات خاصة على ترددات مختلفة 7. خلية كاملة تسجيل التصحيح، المشبك هي طريقة راسخة لفحص الخصائص الذاتية والتفاعلات متشابك من الخلايا العصبية. ولكن نظرا لتنوع كبير من أنواع عصبون، والتحقيق في interneurons المثبطة ويتطلب التشخيص الدقيق للخلايا المسجلة. كما تتميز أنواع عصبون قرن آمون بواسطة سمات مميزة المورفولوجية والكيميائية العصبية علامة التعبير، التشريحية مجتمعة والبريد immunocytochemicalيمكن xamination توفر وسيلة لتحديد الهوية الدقيق 6،8،9 عصبون. في هذه الورقة وصفنا هذا النهج التجريبي الذي يتم الجمع بين خلية كاملة تسجيلات التصحيح المشبك من الخلايا العصبية واحدة أو أزواج جانب synaptically مع وضع العلامات داخل الخلايا، تليها بعد خاصة التحليل الصرفي وimmunocytochemical، مما يسمح لتوصيف بطيئة GABA B مستقبلات بوساطة التأثيرات المثبطة interneurons في تحديدها. وكمثال على ذلك، ونحن نركز على نوع واحد رئيسي للعصبون، مجموعة فرعية من ما يسمى &quot;خلايا سلة&quot; (BC)، والتي يعصب سوما والتشعبات القريبة من أهدافها بعد المشبكي ويتميز &quot;ارتفاعه السريع&quot; (FS) تصريف نمط، محور عصبي تغطي طبقة كثيفة جسم الخلية، والتعبير من البروتين parvalbumin ملزم الكالسيوم (PV) 10،11. تعرض هذه التيارات interneurons المثبطة بعد المشبكي كبيرة، وكذلك قبل بارزتعديل متشابك من إنتاجها متشابك، ردا على GABA B R تفعيل 12. مزيج من الأساليب المذكورة هنا يمكن تطبيق جيد على قدم المساواة للتحقيق آليات فعلية أو متشابك في مجموعة متنوعة من غيرها من أنواع الخلايا العصبية التي تم تحديدها.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:175px;">Name</td>
			<td style="width:148px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Transgenic vGAT-venus rats</td>
			<td style="width:148px;">-</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">see Uematsu et al., 2008</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Venus (515&#160;nm) goggles</td>
			<td style="width:148px;">BLS Ltd., Hungary</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">-</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Dissection tools</td>
			<td style="width:148px;">i.e. FST</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">For brain removal</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">Vibratome</td>
			<td style="width:148px;">Leica</td>
			<td style="width:152px;">VT1200S</td>
			<td style="width:251px;">Or other high end vibratome with minimal vertical oscillation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Slice holding chambers</td>
			<td style="width:148px;">-</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">Custom-made in lab</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">Upright IR-DIC microscope</td>
			<td style="width:148px;">Olympus, Japan</td>
			<td style="width:152px;">BX50WI</td>
			<td style="width:251px;">With micromanipulator system; i.e. Luigs and Neumann, Kleindiek etc.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">CCD camera</td>
			<td style="width:148px;">Till Photonics</td>
			<td style="width:152px;">VX55</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">505 nm LED system</td>
			<td style="width:148px;">Cairn Research</td>
			<td style="width:152px;">OptiLED system</td>
			<td style="width:251px;">Or mercury lamp or other LED system i.e. CooLED.&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">Multiclamp 700B&#160;</td>
			<td style="width:148px;">Axon Instruments</td>
			<td style="width:152px;"></td>
			<td style="width:251px;">Alternatively 2x Axopatch 200B amplifiers&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">WinWCP acquisition software</td>
			<td style="width:148px;">John Dempster, Strathclyde University</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">Any quality acquisition software could be used, i.e. EPHUS, pClamp, Igor etc.&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Electrode Puller</td>
			<td style="width:148px;">Sutter</td>
			<td style="width:152px;">P-97</td>
			<td style="width:251px;">Used with box-filament</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">Borosilicate pipette glass</td>
			<td style="width:148px;">Hilgenberg, Germany</td>
			<td style="width:152px;">1405020</td>
			<td style="width:251px;">2&#160;mm outer, 1&#160;mm inner diameter, no filament</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">Peristaltic pump</td>
			<td style="width:148px;">Gilson</td>
			<td style="width:152px;">Minipuls</td>
			<td style="width:251px;">Other pumps or gravity feed could be used instead</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Digital Thermometer</td>
			<td style="width:148px;">-</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">Custom made</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Digital Manometer</td>
			<td style="width:148px;">Supertech, Hungary</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="17">
			<td height="34" rowspan="2" style="height:34px;width:175px;">Isolated constant voltage stimulator</td>
			<td rowspan="2" style="width:148px;">Digitimer, Cambridge</td>
			<td rowspan="2" style="width:152px;">DS-2A</td>
			<td rowspan="2" style="width:251px;">-</td>
		</tr>
		<tr height="17">
		</tr>
		<tr height="17">
			<td height="34" rowspan="2" style="height:34px;width:175px;"><a name="RANGE!A19:D34">Biocytin</a></td>
			<td rowspan="2" style="width:148px;">Invitrogen</td>
			<td rowspan="2" style="width:152px;">B1592</td>
			<td rowspan="2">Otherwise known as &#949;-Biotinoyl-L-Lysine&#160;</td>
		</tr>
		<tr height="17">
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">DL-AP5(V) disodium salt</td>
			<td style="width:148px;">Abcam Biochemicals</td>
			<td style="width:152px;">ab120271</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">DNQX disodium salt</td>
			<td style="width:148px;">Abcam Biochemicals</td>
			<td style="width:152px;">ab120169</td>
			<td style="width:251px;">Alternatively NBQX or CNQX</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Gabazine (SR95531)</td>
			<td style="width:148px;">Abcam Biochemicals</td>
			<td style="width:152px;">ab120042</td>
			<td style="width:251px;">Alternatively bicuculline methiodide</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">R-Baclofen</td>
			<td style="width:148px;">Abcam Biochemicals</td>
			<td style="width:152px;">ab120325</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">CGP-55,845 hydrochloride</td>
			<td style="width:148px;">Tocris</td>
			<td style="width:152px;">1248</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Streptavidin 647</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:152px;">S32357</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">anti-PV mouse monoclonal antibody</td>
			<td style="width:148px;">Swant, Switzerland</td>
			<td style="width:152px;">235</td>
			<td style="width:251px;">Working concentration 1:5000-1:10,000</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">anti-mouse secondary antibody&#160;</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:152px;">A11030</td>
			<td style="width:251px;">If using Venus or GFP rodent using a red-channel (i.e. 546 nm) is advisable.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Normal Goat Serum</td>
			<td style="width:148px;">Vector Labs</td>
			<td style="width:152px;">S-1000</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Microscopy slides</td>
			<td style="width:148px;">-</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">Any high quality brand&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Glass coverslips</td>
			<td style="width:148px;">-</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">Usually 22 x 22 mm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Agar spacers</td>
			<td style="width:148px;">-</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">Agar block, cut on vibratome to 300 &#956;m</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:175px;">Laser scanning confocal microscope</td>
			<td style="width:148px;">Olympus, Japan</td>
			<td style="width:152px;">Fluoview FV1000</td>
			<td style="width:251px;">Or other comparable system</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:175px;">Fiji (Fiji is just ImageJ)</td>
			<td>http://fiji.sc/Fiji</td>
			<td style="width:152px;">-</td>
			<td style="width:251px;">See Schindelin et al., 2012</td>
		</tr>
	</tbody>
</table>

whole-cell patch-clamp recordings from morphologically- and neurochemically-identified hippocampal interneurons

GABAergic interneurons المثبطة تلعب دورا مركزيا في الدوائر العصبية في الدماغ. تشمل Interneurons مجموعة فرعية صغيرة من السكان العصبية (10-20٪)، ولكن تظهر مستوى عال من الفسيولوجية، الصرفي، وعدم التجانس الكيميائية العصبية، والتي تعكس وظائفها المتنوعة. لذلك، والتحقيق في interneurons يوفر نظرة ثاقبة على مبادئ تنظيم وظيفة الدوائر العصبية. هذا، ومع ذلك، يتطلب نهجا الفسيولوجية وتشريحي عصبي متكامل لاختيار وتحديد أنواع عصبون الفردية. خلية كاملة تسجيل من شرائح الدماغ الحادة من الحيوانات المعدلة وراثيا، معربا عن بروتينات الفلورسنت تحت المروجين للعلامات عصبون محددة التصحيح، المشبك، ويوفر وسيلة فعالة لاستهداف وelectrophysiologically تميز الخصائص الذاتية ومتشابك من أنواع عصبون محددة. جنبا إلى جنب مع وضع العلامات الصبغة داخل الخلايا، ويمكن تمديد هذا النهج مع ما بعد مو خاصةتحليل rphological وimmunocytochemical، مما يتيح تحديد منهجي من الخلايا العصبية المسجلة. هذه الأساليب يمكن أن تكون مصممة لتناسب مجموعة واسعة من المسائل العلمية المتعلقة بالخصائص الوظيفية لأنواع مختلفة من الخلايا العصبية القشرية.

شريطة أن جودة شريحة جيدة بشكل ملحوظ، وتسجيل من كلا CA1 أجهزة الكمبيوتر وFS الإضافية يمكن أن يتحقق بأقل قدر من الصعوبة. خط الفئران المعدلة وراثيا معربا عن فينوس / YFP تحت المروج vGAT 13 لا تحدد بشكل قاطع FS الإضافية، أو في الواقع BCS. لكن التسجيلات من دائرة الهجرة والتجنيس ?...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons at JoVE.com

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

يتم التحكم في الشبكات القشرية من قبل مجموعة صغيرة، ولكنها متنوعة من interneurons المثبطة. ولتحقيق الوظيفي للinterneurons يتطلب تسجيل المستهدفة وتحديد دقيق. الموصوفة هنا هو نهج مجتمعة تشمل تسجيلات خلية كاملة من أزواج واحدة أو جانب synaptically من الخلايا العصبية بين الخلايا مع وضع العلامات، خاصة بعد المورفولوجية والتحليل immunocytochemical.

خلية كاملة تسجيلات التصحيح، المشبك من Morphologically- وحددت Neurochemically-الحصين Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

whole-cell-patch-clamp-recordings-from-morphologically

Research

JoVE Journal

Neuroscience

24.3K Views.  Charité Universitätmedizin.  يتم التحكم في الشبكات القشرية من قبل مجموعة صغيرة، ولكنها متنوعة من interneurons المثبطة. ولتحقيق الوظيفي للinterneurons يتطلب تسجيل المستهدفة وتحديد دقيق. الموصوفة هنا هو نهج مجتمعة تشمل تسجيلات خلية كاملة من أزواج واحدة أو جانب synaptically من الخلايا ال...

Video: Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

تسجيلات المشبك التصحيح من الخلايا العصبية في الشبكية ماوس إعداد غامق تكييفها شريحة

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

المقترنة تسجيلات المشبك من تصحيح للسيارات ، والخلايا العصبية في العضلات الهيكلية الهدف اسماك الزرد

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

تسجيلات المشبك التصحيح في الأذن الداخلية خلايا الشعر المعزولة من اسماك الزرد

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

كله المشبك التصحيح الخلايا لدراسة آليات التحفيز العصبية الأشعة تحت الحمراء

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

تسجيلات المشبك التصحيح من الخلايا الجنينية الزرد ماوتنر

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

تقرن تسجيلات خلية كاملة في عضوي شرائح الحصين

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

تسجيلات خلية كاملة التصحيح، المشبك في الدماغ شرائح

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

التحت خلوية تسجيلات التصحيح، المشبك من المجال Somatodendritic من سودائي الدوبامين العصبية

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

كامل خلية التصحيح المشبك تسجيلات لتحديد الكهربية من أيون الانتقائية في تشانلرودوبسينز

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

استجابة جذع الدماغ السمعية والخارجي الشعر خلية تسجيل المشبك تصحيح كامل الخلية في الفئران بعد الولادة

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...