Name: optogenetic activation of afferent pathways in brain slices and modulation of responses by volatile anesthetics
Uploaded: 2020-07-23T15:00:00
Description: Watch this Scientific Journal Video about Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics at JoVE.com

Gli autori ringraziano Bryan Krause per il supporto tecnico e la guida su questo progetto.
Questo lavoro è stato supportato dall'International Anesthesia Research Society (IMRA to AR), dal National Institutes of Health (R01 GM109086 al MIB) e dal Dipartimento di Anestesiologia, Scuola di Medicina e Sanità Pubblica, Università del Wisconsin, Madison, WI, USA.

Tutte le procedure che coinvolgono gli animali descritte in questo protocollo sono state approvate dalla University of Wisconsin-Madison School of Medicine e dal Public Health Animal Care and Use Committee.
1. Topi da riproduzione per esprimere la proteina reporter fluorescente in sottopopolazioni di interneuroni
<ol><li>Accoppia il topo maschio tdTomato omoziogo e Cre-dipendente con il topo femmina omozigote SOM-Cre o il topo femmina omozigote PV-Cre. NOTA: Altre popolazioni neuronali sp...

<ol>
	<li>Baumgart, J. 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=Isoflurane+inhibits+synaptic+vesicle+exocytosis+through+reduced+Ca2++influx,+not+Ca2+-exocytosis+coupling.">Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling.</a> Proceedings of the National Academy of Sciences U.S.A. 112 (38), 11959-11964 (2015).</li><li>Herring, B. E., Xie, Z., Marks, J., Fox, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+inhibits+the+neurotransmitter+release+machinery.">Isoflurane inhibits the neurotransmitter release machinery.</a> Journal of Neurophysiology. 102 (2), 1265-1273 (2009).</li><li>Xie, 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=Interaction+of+anesthetics+with+neurotransmitter+release+machinery+proteins.">Interaction of anesthetics with neurotransmitter release machinery proteins.</a> Journal of Neurophysiology. 109 (3), 758-767 (2013).</li><li>Crick, F., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+consciousness.">A framework for consciousness.</a> Nature Neurosciences. 6 (2), 119-126 (2003).</li><li>Koch, C., Massimini, M., Boly, M., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+consciousness:+progress+and+problems.">Neural correlates of consciousness: progress and problems.</a> Nature Reviews Neurosciences. 17 (5), 307-321 (2016).</li><li>Dehaene, S., Changeux, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+approaches+to+conscious+processing.">Experimental and theoretical approaches to conscious processing.</a> Neuron. 70 (2), 200-227 (2011).</li><li>Friston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+theory+of+cortical+responses.">A theory of cortical responses.</a> Philosophical Transactions of the Royal Society of London B Biological Sciences. 360 (1456), 815-836 (2005).</li><li>Mashour, G. A., Hudetz, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bottom-Up+and+Top-Down+Mechanisms+of+General+Anesthetics+Modulate+Different+Dimensions+of+Consciousness.">Bottom-Up and Top-Down Mechanisms of General Anesthetics Modulate Different Dimensions of Consciousness.</a> Frontiers in Neural Circuits. 11, 44 (2017).</li><li>Voss, L. J., Garcia, P. S., Hentschke, H., Banks, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Effects+of+General+Anesthetics+on+Cortical+Network+Activity+Using+Ex+Vivo+Preparations.">Understanding the Effects of General Anesthetics on Cortical Network Activity Using Ex Vivo Preparations.</a> Anesthesiology. 130 (6), 1049-1063 (2019).</li><li>Redinbaugh, M. 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=Thalamus+Modulates+Consciousness+Via+Layer-Specific+Control+of+Cortex.">Thalamus Modulates Consciousness Via Layer-Specific Control of Cortex.</a> Neuron. 105 (4), 0896 (2020).</li><li>Carhart-Harris, R. L., Friston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REBUS+and+the+Anarchic+Brain:+Toward+a+Unified+Model+of+the+Brain+Action+of+Psychedelics.">REBUS and the Anarchic Brain: Toward a Unified Model of the Brain Action of Psychedelics.</a> Pharmacological Reviews. 71 (3), 316-344 (2019).</li><li>Mak-McCully, R. 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=Coordination+of+cortical+and+thalamic+activity+during+non-REM+sleep+in+humans.">Coordination of cortical and thalamic activity during non-REM sleep in humans.</a> Nature communications. 8 (1), 15499 (2017).</li><li>Alkire, M. T., Hudetz, A. G., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consciousness+and+anesthesia.">Consciousness and anesthesia.</a> Science. 322 (5903), 876-880 (2008).</li><li>Sanders, R. D., Maze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noradrenergic+trespass+in+anesthetic+and+sedative+states.">Noradrenergic trespass in anesthetic and sedative states.</a> Anesthesiology. 117 (5), 945-947 (2012).</li><li>Hemmings, H. C., 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=Towards+a+Comprehensive+Understanding+of+Anesthetic+Mechanisms+of+Action:+A+Decade+of+Discovery.">Towards a Comprehensive Understanding of Anesthetic Mechanisms of Action: A Decade of Discovery.</a> Trends in Pharmacological Sciences. 40 (7), 464-481 (2019).</li><li>Nourski, K. V., 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=Auditory+Predictive+Coding+across+Awareness+States+under+Anesthesia:+An+Intracranial+Electrophysiology+Study.">Auditory Predictive Coding across Awareness States under Anesthesia: An Intracranial Electrophysiology Study.</a> Journal of Neurosciences. 38 (39), 8441-8452 (2018).</li><li>Liu, X., 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=Propofol+disrupts+functional+interactions+between+sensory+and+high-order+processing+of+auditory+verbal+memory.">Propofol disrupts functional interactions between sensory and high-order processing of auditory verbal memory.</a> Human Brain Mapping. 33 (10), 2487-2498 (2012).</li><li>Hentschke, H., Raz, A., Krause, B. M., Murphy, C. A., Banks, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+cortical+network+activity+by+the+general+anesthetic+isoflurane.">Disruption of cortical network activity by the general anesthetic isoflurane.</a> British Journal of Anaesthesiology. 119 (4), 685-696 (2017).</li><li>Lee, U., 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=Disruption+of+frontal-parietal+communication+by+ketamine,+propofol,+and+sevoflurane.">Disruption of frontal-parietal communication by ketamine, propofol, and sevoflurane.</a> Anesthesiology. 118 (6), 1264-1275 (2013).</li><li>Ferrarelli, F., 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=Breakdown+in+cortical+effective+connectivity+during+midazolam-induced+loss+of+consciousness.">Breakdown in cortical effective connectivity during midazolam-induced loss of consciousness.</a> Proceedings of the National Academy of Science U.S.A. 107 (6), 2681-2686 (2010).</li><li>Ku, S. W., Lee, U., Noh, G. J., Jun, I. G., Mashour, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+inhibition+of+frontal-to-parietal+feedback+connectivity+is+a+neurophysiologic+correlate+of+general+anesthesia+in+surgical+patients.">Preferential inhibition of frontal-to-parietal feedback connectivity is a neurophysiologic correlate of general anesthesia in surgical patients.</a> PLoS.One. 6 (10), 25155 (2011).</li><li>Lee, 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=Network+Properties+in+Transitions+of+Consciousness+during+Propofol-induced+Sedation.">Network Properties in Transitions of Consciousness during Propofol-induced Sedation.</a> Scientific Reports. 7 (1), 16791 (2017).</li><li>Murphy, 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=Propofol+anesthesia+and+sleep:+a+high-density+EEG+study.">Propofol anesthesia and sleep: a high-density EEG study.</a> Sleep. 34 (3), 283-291 (2011).</li><li>Murphy, C., Krause, B., Banks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+effects+of+isoflurane+on+cortico-cortical+feedback+afferent+responses+in+murine+non-primary+neocortex.">Selective effects of isoflurane on cortico-cortical feedback afferent responses in murine non-primary neocortex.</a> British Journal of Anaesthesiology. 123 (4), 488-496 (2019).</li><li>Raz, 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=Preferential+effect+of+isoflurane+on+top-down+versus+bottom-up+pathways+in+sensory+cortex.">Preferential effect of isoflurane on top-down versus bottom-up pathways in sensory cortex.</a> Frontiers in System Neurosciences. 8, 191 (2014).</li><li>Schrouff, 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=Brain+functional+integration+decreases+during+propofol-induced+loss+of+consciousness.">Brain functional integration decreases during propofol-induced loss of consciousness.</a> Neuroimage. 57 (1), 198-205 (2011).</li><li>Petreanu, L., Mao, T., Sternson, S. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subcellular+organization+of+neocortical+excitatory+connections.">The subcellular organization of neocortical excitatory connections.</a> Nature. 457 (7233), 1142-1145 (2009).</li><li>Cruikshank, S. J., Urabe, H., Nurmikko, A. V., Connors, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-Specific+Feedforward+Circuits+between+Thalamus+and+Neocortex+Revealed+by+Selective+Optical+Stimulation+of+Axons.">Pathway-Specific Feedforward Circuits between Thalamus and Neocortex Revealed by Selective Optical Stimulation of Axons.</a> Neuron. 65 (2), 230-245 (2010).</li><li>Gredell, J. A., Turnquist, P. A., MacIver, M. B., Pearce, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+diffusion+and+partition+coefficients+of+propofol+in+rat+brain+tissue:+implications+for+studies+of+drug+action+in+vitro.">Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro.</a> BJA: British Journal of Anaesthesia. 93 (6), 810-817 (2004).</li><li>Benkwitz, C., 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=Determination+of+the+EC50+amnesic+concentration+of+etomidate+and+its+diffusion+profile+in+brain+tissue:+implications+for+in+vitro+studies.">Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies.</a> Anesthesiology. 106 (1), 114-123 (2007).</li><li>Franks, N. P., Lieb, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+actions+of+volatile+general+anaesthetics+at+molecular+and+cellular+levels.">Selective actions of volatile general anaesthetics at molecular and cellular levels.</a> British Journal of Anaesthesia. 71 (1), 65-76 (1993).</li><li>Honemann, C. W., Washington, J., Honemann, M. C., Nietgen, G. W., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partition+coefficients+of+volatile+anesthetics+in+aqueous+electrolyte+solutions+at+various+temperatures.">Partition coefficients of volatile anesthetics in aqueous electrolyte solutions at various temperatures.</a> Anesthesiology. 89 (4), 1032-1035 (1998).</li><li>Au - Segev, A., Au - Garcia-Oscos, F., Au - Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> Journal of Visualized Experiments. (112), e54024 (2016).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophysical Journal. 96 (5), 1803-1814 (2009).</li><li>Lin, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+channelrhodopsin+variants:+features,+limitations+and+future+developments.">A user's guide to channelrhodopsin variants: features, limitations and future developments.</a> Experimental Physiology. 96 (1), 19-25 (2011).</li><li>Banks, M. I., Pearce, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+actions+of+volatile+anesthetics+on+GABAA+IPSCs:+dissociation+of+blocking+and+prolonging+effects.">Dual actions of volatile anesthetics on GABAA IPSCs: dissociation of blocking and prolonging effects.</a> Anesthesiology. 90 (1), 120-134 (1999).</li><li>Hagan, C. E., Pearce, R. A., Trudell, J. R., MacIver, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+measures+of+volatile+anesthetics+in+the+aqueous+phase+using+calcium+sensitive+electrodes.">Concentration measures of volatile anesthetics in the aqueous phase using calcium sensitive electrodes.</a> Journal of Neuroscience Methods. 81, 177-184 (1998).</li></ol>

In questo manoscritto viene descritto un protocollo per valutare le risposte intra ed extracellulari a vie afferenti attivate selettivamente in fette di cervello ex vivo.
L'uso di strumenti optogenetici e schemi di registrazione paralleli consente ai ricercatori di sondare le risposte delle popolazioni locali agli input afferenti provenienti da regioni cerebrali distanti, registrando simultaneamente da popolazioni mirate di interneuroni. L'uso della tecnologia optogenetica consente di preserva...

Gli anestetici volatili sono stati utilizzati ovunque in una varietà di contesti clinici e accademici per più di un secolo. Classi distinte di anestetici hanno bersagli molecolari unici, spesso non sovrapposti, 1,2,3, ma quasi tutti producono incoscienza. Mentre i loro effetti comportamentali sono abbastanza prevedibili, i meccanismi con cui gli anestetici inducono la perdita di coscienza sono in gran parte sconosciuti. Gli anestetici possono infine influenzare sia il livello che il contenuto della coscienza attraverso azioni sui circuiti corticotalamici, interrompendo l'integrazione delle informazioni in tutta la gerarchia corticale 4,5,6,7,8,9. Più in generale, la modulazione dei circuiti corticotalamici può svolgere un ruolo negli stati sperimentali10 o farmacologicamente 11 alterati di coscienza, e può anche essere implicata nel sonno 12 e nei disturbi fisiopatologici della coscienza13,14.
L'elusività dei meccanismi alla base della perdita e del ritorno della coscienza durante l'anestesia può essere attribuita in parte ad azioni non lineari e sinergiche degli anestetici a livello cellulare, di rete e di sistema15. L'isoflurano, ad esempio, sopprime l'attività all'interno delle regioni cerebrali selezionate 16,17,18, altera la connettività tra regioni cerebrali distanti 19,20,21,22,23 e diminuisce le risposte sinaptiche in modo specifico del percorso 24,25 . Quali effetti degli anestetici, dal livello molecolare a quello sistemico, siano necessari o sufficienti per effettuare la perdita di coscienza rimane poco chiaro. Oltre alle indagini cliniche sostanziali della coscienza utilizzando tecniche non invasive 19,20,26, è importante che gli sperimentatori cerchino di districare le distinte interazioni cellulari e di rete che servono l'esperienza cosciente.
Semplificando le complesse interazioni presenti nel cervello intatto, le fette di cervello ex vivo consentono lo studio di componenti isolati dei sistemi dinamici del cervello9. Una preparazione ridotta delle fette combina i vantaggi delle strutture anatomiche relativamente intatte dei circuiti neurali locali con la versatilità delle manipolazioni in vitro. Tuttavia, fino a poco tempo fa, vincoli metodologici hanno precluso lo studio delle proprietà sinaptiche e circuitali degli input a lungo raggio nelle fette cerebrali27,28; Il percorso tortuoso dei tratti di fibre corticotalamici rendeva quasi impossibile l'attivazione di vie afferenti indipendenti mediante stimolazione elettrica.
Studiare gli effetti degli agenti anestetici sui preparati delle fette di cervello presenta ulteriori sfide. In assenza di un sistema respiratorio e circolatorio intatto, gli agenti anestetici devono essere applicati in bagno e le concentrazioni devono essere accuratamente abbinate alle concentrazioni stimate nel sito di effetto. Per molti anestetici per via endovenosa, il lento tasso di equilibrio nel tessuto rende laboriose le indagini farmacologiche tradizionali29,30. Studiare gli effetti degli anestetici gassosi volatili nei preparati ex vivo è più trattabile, ma presenta anche delle sfide. Questi includono la conversione di dosi a pressione parziale inalate in concentrazioni acquose e la necessità di un sistema di consegna modificato del farmaco al tessuto attraverso il liquido spinale cerebrale artificiale31.
Qui, vengono descritti metodi con cui i ricercatori possono sfruttare le proprietà fisico-chimiche ben documentate dell'isoflurano anestetico volatile per la somministrazione di farmaci a fette di cervello ex vivo, attivare input specifici del percorso e dello strato in un'area corticale di interesse con elevata risoluzione spaziotemporale e condurre registrazioni laminari simultanee e registrazioni mirate di patch clamp da popolazioni selezionate di neuroni. Combinate, queste procedure consentono ai ricercatori di misurare i cambiamenti indotti dall'anestetico volatile in diverse proprietà di risposta elettrofisiologica osservabili, dal livello sinaptico a quello locale.

<table>
	<tbody>
		<tr>
			<td>2.5x broadfield objective lens</td>
			<td>Olympus</td>
			<td>MPLFLN2.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>40x water immersion objective lens</td>
			<td>Olympus</td>
			<td>LUMPLFLN40XW</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2/5% CO2 mixture</td>
			<td>Airgas</td>
			<td>Z02OX95R2003045</td>
			<td></td>
		</tr>
		<tr>
			<td>A16 probe</td>
			<td>NeuroNexus</td>
			<td>A16x1-2mm-100-177-A16</td>
			<td>16-channel probe</td>
		</tr>
		<tr>
			<td>AAV2-hSyn-hChR2(H134R)-EYFP</td>
			<td>Karl Deisseroth Lab, UNC Vector Core</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic gas monitor (POET II)</td>
			<td>Criticare</td>
			<td>602-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, Magnesium Salt</td>
			<td>Sigma Aldrich</td>
			<td>A9187</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td>The Jackson Laboratory</td>
			<td>007914</td>
			<td>Cre-dependent tdTomato mouse</td>
		</tr>
		<tr>
			<td>B6;129P2-Pvalbtm1(cre)Arbr/J</td>
			<td>The Jackson Laboratory</td>
			<td>008069</td>
			<td>PV-Cre mouse</td>
		</tr>
		<tr>
			<td>Belly Dancer Shaker</td>
			<td>Thomas Scientific</td>
			<td>1210H86-TS</td>
			<td>for equilibration of sealed gas bags</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Generic brand</td>
			<td></td>
			<td>for silver chloriding patch clamp electrode</td>
		</tr>
		<tr>
			<td>Bupivicaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Dot Scientific</td>
			<td>DSC20010</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Capillary glass (patch clamp recordings)</td>
			<td>King Precision Glass, Inc.</td>
			<td>KG-33</td>
			<td>Borosilicate, ID: 1.1mm, OD: 1.7mm, Length: 90.0mm</td>
		</tr>
		<tr>
			<td>Capillary glass (viral injections)</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>3.5&#34;</td>
		</tr>
		<tr>
			<td>Control of junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>SM8</td>
			<td>for control of junior micromanipulator</td>
		</tr>
		<tr>
			<td>Control of manipulators and shifting table</td>
			<td>Luigs and Neumann</td>
			<td>SM7</td>
			<td>for control of multichannel electrode and shifting table</td>
		</tr>
		<tr>
			<td>Digidata 1440A + Clampex 10</td>
			<td>Molecular Devices</td>
			<td>1440A</td>
			<td>Digitizer and software</td>
		</tr>
		<tr>
			<td>E-3603 tubing</td>
			<td>Fisher Scientific</td>
			<td>14171208</td>
			<td>for delivery of 95% O2/5% CO2 gas mixture to incubation chamber + application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Dot Scientific</td>
			<td>DSE57060</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>ERP-27 EEG Reference/Patch Panel</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Filling needle</td>
			<td>World Precision Instruments</td>
			<td>50821912</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Filter cube for imaging EYFP</td>
			<td>Olympus</td>
			<td>U-MRFPHQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific</td>
			<td>09801E</td>
			<td>lay over slice template during preparation of tissue block</td>
		</tr>
		<tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>2.5x2.5 Box filament</td>
		</tr>
		<tr>
			<td>Gas dispersion tube</td>
			<td>Sigma Aldrich</td>
			<td>CLS3953312C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe (100 mL)</td>
			<td>Sigma Aldrich</td>
			<td>Z314390</td>
			<td>for filling gas-sealed bags</td>
		</tr>
		<tr>
			<td>Gluconic Acid, Potassium Salt (K-gluconate)</td>
			<td>Dot Scientific</td>
			<td>DSG37020</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Dot Scientific</td>
			<td>DSG32040</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>GTP, Sodium Salt</td>
			<td>Sigma Aldrich</td>
			<td>G8877</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Headstage-probe adaptor</td>
			<td>NeuroNexus</td>
			<td>A16-OM16</td>
			<td>adaptor to connect 16-channel probe to headstage input</td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>VWR International</td>
			<td>76192-096</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Dot Scientific</td>
			<td>DSH75030</td>
			<td>ACSF,intracellular solution</td>
		</tr>
		<tr>
			<td>HS-16 Headstage</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol (70%)</td>
			<td>VWR International</td>
			<td>101223-746</td>
			<td></td>
		</tr>
		<tr>
			<td>Junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>210-100 000 0090-R</td>
			<td>for manipulation of patch clamp electrode</td>
		</tr>
		<tr>
			<td>LED Light Source Control Module</td>
			<td>Mightex</td>
			<td>BLS-PL02_US</td>
			<td>optogenetic light source control</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Amplifier</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Power Supply</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4)</td>
			<td>Dot Scientific</td>
			<td>DSM24300</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>mCherry, Texas Red filter cube</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>for imaging tdTomato fluorescent reporter</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Fisher Scientific</td>
			<td>NC9044962</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe pump</td>
			<td>World Precision Instruments</td>
			<td>UMP3-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultiClamp 700A</td>
			<td>Molecular Devices/Axon Instruments</td>
			<td>700A</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Nitrogen (for air table)</td>
			<td>Airgas</td>
			<td>NI200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>Fisher Scientific</td>
			<td>501460083</td>
			<td>stretched over horseshoe of flattened platinum wire, slice rest on top of this during recordings</td>
		</tr>
		<tr>
			<td>Nylon, cut from pantyhose</td>
			<td>Generic brand</td>
			<td></td>
			<td>small piece to create slice platform in incubation chamber, single fibers to create platinum harp</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Fisher Scientific</td>
			<td>NC1697520</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Dot Scientific</td>
			<td>307</td>
			<td>For transferring tissue to rig</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>VWR International</td>
			<td>BT124000</td>
			<td>2 cm, flattened, to make platinum harp</td>
		</tr>
		<tr>
			<td>Polygon400</td>
			<td>Mightex</td>
			<td>DSI-E-0470-0617-000</td>
			<td>optogenetic light delivery system, comes with PolyScan2 software</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Dot Scientific</td>
			<td>DSP41000</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Potassium Phosphate (KH2PO4)</td>
			<td>Dot Scientific</td>
			<td>DSP41200</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire blade (for vibratome)</td>
			<td>VWR International</td>
			<td>100492-502</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>sc-361445</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealed gas bag</td>
			<td>Fisher Scientific</td>
			<td>109236</td>
			<td></td>
		</tr>
		<tr>
			<td>Shifting table for microscope</td>
			<td>Luigs and Neumann</td>
			<td>380FMU</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (HCO3-)</td>
			<td>Dot Scientific</td>
			<td>DSS22060</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Dot Scientific</td>
			<td>DSS23020</td>
			<td>ACSF, intracellular solution</td>
		</tr>
		<tr>
			<td>Ssttm2.1(cre)Zjh/J (SOM-IRES-Cre)</td>
			<td>The Jackson Laboratory</td>
			<td>013044</td>
			<td>SOM-Cre mouse</td>
		</tr>
		<tr>
			<td>Stereotaxic instrument</td>
			<td>Kopf</td>
			<td>Model 902</td>
			<td>Dual Small Animal</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Staples</td>
			<td>886833</td>
			<td>to fix tissue block to specimen stage during slice preparation</td>
		</tr>
		<tr>
			<td>Surgical drill</td>
			<td>RAM Products Inc.</td>
			<td>DIGITALMICROTORQUE</td>
			<td>Microtorque II</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with LuerLock tip</td>
			<td>Fisher Scientific</td>
			<td>309628</td>
			<td>for application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with slip tip</td>
			<td>WW Grainger, Inc.</td>
			<td>19G384</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>VWR International</td>
			<td>66064-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica Biosystems</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wypall towels</td>
			<td>Fisher Scientific</td>
			<td>19-042-427</td>
			<td></td>
		</tr>
	</tbody>
</table>

optogenetic activation of afferent pathways in brain slices and modulation of responses by volatile anesthetics

Gli anestetici influenzano la coscienza in parte attraverso le loro azioni sui circuiti talamocorticali. Tuttavia, la misura in cui gli anestetici volatili influenzano i componenti cellulari e di rete distinti di questi circuiti rimane poco chiara. Le fette di cervello ex vivo forniscono un mezzo attraverso il quale i ricercatori possono sondare componenti discreti di reti complesse e districare potenziali meccanismi alla base degli effetti degli anestetici volatili sulle risposte evocate. Per isolare potenziali effetti farmacologici specifici del tipo di cellula e del percorso nelle fette di cervello, i ricercatori devono essere in grado di attivare in modo indipendente le vie delle fibre afferenti, identificare popolazioni di cellule non sovrapposte e applicare anestetici volatili al tessuto in soluzione acquosa. In questo protocollo, vengono descritti metodi per misurare le risposte optogeneticamente evocate a due vie afferenti indipendenti alla neocorteccia in fette di cervello ex vivo. Le risposte extracellulari vengono registrate per l'attività della rete di test e le registrazioni mirate del patch clamp dell'intera cellula sono condotte in interneuroni positivi alla somatostatina e alla parvalbumina. Viene descritto il rilascio di concentrazioni fisiologicamente rilevanti di isoflurano attraverso il liquido cerebrospinale artificiale per modulare le risposte cellulari e di rete.

Una sequenza temporale dei passaggi descritti nel protocollo è mostrata nella Figura 1. Gli input corticali provenienti da aree corticali di ordine superiore o da nuclei talamici non primari hanno campi terminali parzialmente sovrapposti nello strato 1 della corteccia visiva non primaria24. Per isolare vie afferenti talamocorticali o corticocorticali indipendenti, è stato iniettato un vettore virale contenente ChR2 e un reporter fluorescente eYFP in Po o TCG. Le cel...

Watch this Scientific Journal Video about Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics at JoVE.com

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Le fette di cervello ex vivo possono essere utilizzate per studiare gli effetti degli anestetici volatili sulle risposte evocate agli input afferenti. L'optogenetica è impiegata per attivare indipendentemente afferenze talamocorticali e corticocorticali alla neocorteccia non primaria, e le risposte sinaptiche e di rete sono modulate con isoflurano.

Attivazione optogenetica delle vie afferenti nelle fette cerebrali e modulazione delle risposte mediante anestetici volatili

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Research

JoVE Journal

Neuroscience

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Calcio Risposte imaging in neuroni GFP-tagged fette di cervello di topo ipotalamici

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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Neuroscienze

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Attivazione Optogenetic di zebrafish neuroni somatosensoriali con Chef-tdTomato

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo optogenetic dissezione di circuiti paura nel cervello fette

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Cellule intere correnti indotte dall'applicazione di Puff di GABA nelle fette del cervello

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Registrazione e modulazione di attività Epileptiform in fettine di cervello del roditore accoppiate a matrici di microelettrodi

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

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Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

In Vivo Intracerebral Stereotaxic Iniezioni per la stimolazione optogenetica degli ingressi a lungo raggio nelle fette di cervello del topo

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Modulazione non invasiva e mappatura robotica della Corteccia motoria nel cervello in via di sviluppo

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...