Name: whole-cell patch-clamp recordings from morphologically- and neurochemically-identified hippocampal interneurons
Uploaded: 2014-09-30T13:00:00
Description: Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons at JoVE.com

Forfatterne ønsker å takke Ina Wolter for henne utmerket teknisk assistanse. VGAT-Venus transgene rotter ble generert av Drs. Y. Yanagawa, M. Hirabayashi og Y. Kawaguchi i Statens institutt for Fysiologiske Sciences, Okazaki, Japan, ved hjelp pCS2-Venus levert av Dr. A. Miyawaki.

Etikk Uttalelse: Alle prosedyrer og dyr vedlikehold ble utført i henhold til institusjonelle retningslinjer, den tyske dyrevernloven, Europarådsdirektiv 86/609 / EØF om beskyttelse av dyr, og retningslinjer fra lokale myndigheter (Berlin, T-0215/11 ) 1. Utarbeidelse av Akutt-hippocampus Slices <ol><li> Ta en transgen rotte (17 til 24 dager gammel), uttrykker den fluorescerende Venus / YFP protein under vGAT arrangøren, hvilke etiketter flertallet av kortikale hemmende interneurons <sup...

<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. 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=Differential+GABAB-Receptor-Mediated+Effects+in+Perisomatic-+and+Dendrite-Targeting+Parvalbumin+Interneurons.">Differential GABAB-Receptor-Mediated Effects in Perisomatic- and Dendrite-Targeting Parvalbumin Interneurons.</a> Journal of Neuroscience. 33 (18), 7961-7974 (2013).</li><li>Uematsu, 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=Quantitative+chemical+composition+of+cortical+GABAergic+neurons+revealed+in+transgenic+Venus-expressing+rats.">Quantitative chemical composition of cortical GABAergic neurons revealed in transgenic Venus-expressing rats.</a> Cerebral Cortex. 18, 315-330 (2008).</li><li>Bischofberger, J., Engel, D., Li, L., Geiger, J. R. 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>

Vi beskriver en metode som kombinerer elektrofysiologiske og nevroanatomi teknikker for å funksjonelt karakter morphologically- og neurochemically-identifiserte nevroner in vitro; spesielt de ulike typer av kortikale hemmende ins. Viktige aspekter av prosedyren er: (1) pre-utvalg av potensielle INs; (2) intracellulært opptak og neuron visualisering; og til slutt (3) morfologisk og immuncytokjemisk analyse av registrerte ins. Selv om denne studien har adressert PV-Ins spesielt, kan den beskrevne protokoll benyttes for ...

Hippocampus nevrale kretser har lenge vært gjenstand for intens gransking, både med hensyn til anatomi og fysiologi, på grunn av sin viktige rolle i læring og hukommelse samt romlig navigasjon i både mennesker og gnagere. Likeledes gjør fremtredende, men enkel laminær organisering av hippocampus denne regionen et yndet tema for undersøkelser rettet mot strukturelle og funksjonelle egenskaper kortikale nettverk. Hippocampal kretser består av eksitatoriske hovedceller (&gt; 80%) og et mindre (10-20%), men svært mangfoldig kohort av inhibitoriske interneuroner fra 1-3. Interneuroner slipper γ-aminosmørsyre (GABA) fra sine axon terminaler som fungerer på rask ionotrofiske GABA A-reseptorer (GABA A Rs) og langsomme metabotrope GABA B-reseptorer (GABA B RS) 4. Disse hemmende mekanismer motvekt eksitasjon og regulere oppstemthet av hovedceller, og dermedir timing og mønster av utslippet. Imidlertid GABA frigjøres fra interneuroner virker ikke bare på hovedceller, men også på interneuroner seg 5,6. Pre og postsynaptiske reseptorer megle feedback regulering og hemmende gjensidig samspill mellom de ulike typer interneuron. Disse hemmende mekanismer i Interneuron nettverk antas å være sentral i den generasjonen og utforming av aktivitets befolkningen mønstre, spesielt svingninger ved ulike frekvenser 7. Hel-cell patch-clamp opptak er en veletablert metode for undersøkelse av iboende egenskaper og synaptiske interaksjoner av nerveceller. Imidlertid, på grunn av den store variasjonen av Interneuron typer, undersøkelse av inhiberende interneuroner krever streng identifisering av de registrerte celler. Som hippocampus Interneuron typer er preget av forskjellige morfologiske funksjoner og neurochemical markør uttrykk, kombinert anatomiske og immuncytokjemisk examination kan være et middel for å fastslå nøyaktig interneuron identitet 6,8,9. I den foreliggende beskrivelsen beskriver vi en eksperimentell tilnærming der hel-celle patch-clamp opptak fra én nerveceller eller postsynaptisk-koplede par er kombinert med intracellulær merking, etterfulgt av post-hoc morfologisk og immuncytokjemisk analyse, slik at for karakterisering av langsomt GABA B reseptormediert inhiberende virkninger i identifiserte interneuroner. Som et eksempel, fokuserer vi på den ene store typen interneuron, en undergruppe av de såkalte &quot;kurv celler&quot; (BC), som innervates soma og proksimale dendritter av sine postsynaptiske mål og er preget av en &quot;fast spiking&quot; (FS) slippes mønster, et axon tett dekker cellekroppen lag, og ekspresjon av kalsium-bindende protein parvalbumin (PV) 10,11. Disse interneurons vise store postsynaptiske hemmende strømninger, samt fremtredende presynaptisk modulering av deres synaptisk utgang, som reaksjon på GABA-B R-aktivering 12. Kombinasjonen av teknikkene som er beskrevet her kan anvendes like godt for å undersøke iboende eller synaptiske mekanismer i en rekke andre identifiserte neuron typer.

<table border="0" cellpadding="0" cellspacing="0" width="725">
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			<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 hemmende interneurons spille en sentral rolle innen nevrale kretser i hjernen. Interneuroner omfatter en liten undergruppe av neuronal populasjon (10-20%), men viser en høy grad av fysiologisk, morfologiske og neurochemical heterogenitet, som gjenspeiler deres ulike funksjoner. Derfor etterforskning av interneurons gir viktig innsikt i organisasjonen prinsipper og funksjon av nevrale kretser. Dette krever imidlertid en integrert fysiologiske og nevroanatomi metode for utvelgelse og identifikasjon av individuelle Interneuron typer. Hel-cell patch-clamp opptak fra akutte hjerne skiver av transgene dyr, uttrykker fluorescerende proteiner under arrangører av Interneuron spesifikke markører, gir en effektiv metode for å målrette og elektrofysiologisk karakter iboende og synaptiske egenskapene til spesifikke Interneuron typer. Kombinert med intracellulære fargestoff merking, kan denne tilnærmingen bli utvidet med post-hoc morphological og immuncytokjemisk analyse, slik systematisk identifisering av innspilte nevroner. Disse metodene kan skreddersys for å passe et bredt spekter av vitenskapelige spørsmål angående funksjonelle egenskaper ulike typer kortikale nevroner.

Forutsatt at skive kvaliteten er merkbart bra, opptak fra både CA1 PCer og FS-ins kan oppnås med minimal vanskeligheter. Den transgene rotte linje uttrykker Venus / YFP under vGAT promoter 13 ikke utvetydig identifisere FS-ins, eller faktisk BCs. Men opptak fra INs i og rundt str. Pyramide, der tettheten av FS-ins er typisk høy 1, resulterer i en høy sannsynlighet for å velge FS-Ins (figur 2B). FS-ins kan være preget av sine karakteristiske fysiologiske egenskaper so...

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

Hel-celle Patch-clamp Opptak fra Morphologically- og Neurochemically-identifiserte hippocampus interneuroner

Kortikale nettverk styres av en liten, men mangfoldig sett av hemmende interneurons. Funksjonell undersøkelse av interneurons krever derfor målrettet opptak og streng identifikasjon. Beskrevet her er en kombinert tilnærming med hel-celle opptak fra én eller postsynaptisk-koblede par av nevroner med intracellulære merking, post-hoc morfologisk og immuncytokjemisk analyse.

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

Research

JoVE Journal

Neuroscience

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 ...

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.  ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch Clamp Recordings i Indre ørehår celler isolert fra sebrafisk

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

Hele Cell Patch Clamp for Gransker Mekanismer for Infrared Neural Stimulering

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

Patch Clamp Recordings fra embryonale Sebrafisk 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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Sammenkoblede hele cellen Recordings i organotypic hippocampus Slices

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

Hel-celle Patch-clamp Recordings i hjernen skiver

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

Subcellulære Patch-klemme Opptak fra Somatodendritic Domain of Nigral dopaminneuroner

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

Hele-celle Patch-klemmeopptak for elektrofysiologisk bestemmelse av ion selektivitet i Channelrhodopsins

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

Auditory hjernestammen respons og ytre hår cellen hele celle Patch klemme opptak i Postnatal rotter

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 ...