Name: simultaneous electroencephalography, real-time measurement of lactate concentration and optogenetic manipulation of neuronal activity in the rodent cerebral cortex
Uploaded: 2012-12-19T12:00:00
Description: Watch this Scientific Journal Video about Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral C

Forskning finansieret af Department of Defense (Defense Advanced Research Projects Agency, Young Faculty Award, Grant Number N66001-09-1-2117) og NINDS (R15NS070734).

1. Kirurgisk Fremstillinq af dyr 1. Eksperimentelle fag Anvender mus af B6.Cg-Tg (Thy1-COP4/eYFP) 18Gfng / J transgene linje 9, JAX stamme # 7612) eller andre mus, der udtrykker det blå lys-sensitive kation-kanal, Channelrhodopsin-2, i cerebrale corticale neuroner. Anvendelsen af blåt lys til hjernebarken af B6.Cg-Tg (Thy1-COP4/eYFP) 18Gfng / J transgene linje forårsager de pyramidale neuroner udtrykker Channelrhodopsin-2 til at depolarisere og undergå ...

<ol>
	<li>Magistretti, P., Zigmond, M. J., Bloom, F. E., Landis, S. C., Roberts, J. L., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Energy+Metabolism.">Brain Energy Metabolism.</a> Fundamental Neuroscience. , 389-413 (1999).</li><li>Maquet, 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=Cerebral+glucose+utilization+during+sleep-wake+cycle+in+man+determined+by+positron+emission+tomography+and+[18F]2-fluoro-2-deoxy-D-glucose+method.">Cerebral glucose utilization during sleep-wake cycle in man determined by positron emission tomography and [18F]2-fluoro-2-deoxy-D-glucose method.</a> Brain Res. 513 (1), 136-143 (1990).</li><li>Buchsbaum, M. S., 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=Regional+cerebral+glucose+metabolic+rate+in+human+sleep+assessed+by+positron+emission+tomography.">Regional cerebral glucose metabolic rate in human sleep assessed by positron emission tomography.</a> Life Sci. 45 (15), 1349-1356 (1989).</li><li>Kennedy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+cerebral+glucose+utilization+in+non-rapid+eye+movement+sleep.">Local cerebral glucose utilization in non-rapid eye movement sleep.</a> Nature. 297 (5864), 325-327 (1982).</li><li>Pappenheimer, J. R., Koski, G., Fencl, V., Karnovsky, M. L., Krueger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+sleep-promoting+factor+S+from+cerebrospinal+fluid+and+from+brains+of+sleep-deprived+animals.">Extraction of sleep-promoting factor S from cerebrospinal fluid and from brains of sleep-deprived animals.</a> J. Neurophysiol. 38 (6), 1299-1311 (1975).</li><li>Borbely, A. A., Achermann, P., Kryger, M. H., Roth, T., Dement, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+homeostasis+and+models+of+sleep+regulation.">Sleep homeostasis and models of sleep regulation.</a> Principles and Practice of Sleep Medicine. , 377-390 (2004).</li><li>Destexhe, A., Contreras, D., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+analysis+of+local+field+potentials+and+unit+discharges+in+cat+cerebral+cortex+during+natural+wake+and+sleep+states.">Spatiotemporal analysis of local field potentials and unit discharges in cat cerebral cortex during natural wake and sleep states.</a> J. Neurosci. 19 (11), 4595-4608 (1999).</li><li>Astrup, J., Sorensen, P. M., Sorensen, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+glucose+consumption+related+to+Na+-K++transport+in+canine+brain.">Oxygen and glucose consumption related to Na+-K+ transport in canine brain.</a> Stroke. 12 (6), 726-730 (1981).</li><li>Arenkiel, B. 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=In+vivo+light-induced+activation+of+neural+circuitry+in+transgenic+mice+expressing+channelrhodopsin-2.">In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.</a> Neuron. 54 (2), 205-218 (2007).</li><li>Mateo, C. <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+Optogenetic+Stimulation+of+Neocortical+Excitatory+Neurons+Drives+Brain-State-Dependent.">In Vivo Optogenetic Stimulation of Neocortical Excitatory Neurons Drives Brain-State-Dependent.</a> Curr. Biol. , (2011).</li><li>Wisor, J. P., Clegern, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+short-term+slow+wave+sleep+homeostasis+and+its+disruption+by+minocycline+in+the+laboratory+mouse.">Quantification of short-term slow wave sleep homeostasis and its disruption by minocycline in the laboratory mouse.</a> Neurosci. Lett. 490 (3), 165-169 (2011).</li><li>El Yacoubi, 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=Behavioral,+neurochemical,+and+electrophysiological+characterization+of+a+genetic+mouse+model+of+depression.">Behavioral, neurochemical, and electrophysiological characterization of a genetic mouse model of depression.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6227-6232 (2003).</li><li>Tsunematsu, T., 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=Acute+optogenetic+silencing+of+orexin/hypocretin+neurons+induces+slow-wave+sleep+in+mice.">Acute optogenetic silencing of orexin/hypocretin neurons induces slow-wave sleep in mice.</a> J. Neurosci. 31 (29), 10529-10539 (2011).</li><li>Le, S., Gruner, J. A., Mathiasen, J. R., Marino, M. J., Schaffhauser, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+ex+vivo+receptor+occupancy+and+wake-promoting+activity+of+selective+H3+receptor+antagonists.">Correlation between ex vivo receptor occupancy and wake-promoting activity of selective H3 receptor antagonists.</a> J. Pharmacol. Exp. Ther. 325 (3), 902-909 (2008).</li><li>Burmeister, J. J., Palmer, M., Gerhardt, 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=L-lactate+measures+in+brain+tissue+with+ceramic-based+multisite+microelectrodes.">L-lactate measures in brain tissue with ceramic-based multisite microelectrodes.</a> Biosens. Bioelectron. 20 (9), 1772-1779 (2005).</li><li>Cardin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+optogenetic+stimulation+and+recording+of+neurons+in+vivo+using+cell-type-specific+expression+of+Channelrhodopsin-2.">Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2.</a> Nat. Protoc. 5 (2), 247-254 (2010).</li><li>Destexhe, A., Contreras, D., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortically-induced+coherence+of+a+thalamic-generated+oscillation.">Cortically-induced coherence of a thalamic-generated oscillation.</a> Neuroscience. 92 (2), 427-443 (1999).</li><li>Liu, Z. W., Faraguna, U., Cirelli, C., Tononi, G., Gao, X. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+wake-related+increases+and+sleep-related+decreases+in+synaptic+strength+in+rodent+cortex.">Direct evidence for wake-related increases and sleep-related decreases in synaptic strength in rodent cortex.</a> J. Neurosci. 30 (25), 8671-8675 (2010).</li><li>Iwai, Y., Honda, S., Ozeki, H., Hashimoto, M., Hirase, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+head-mountable+LED+device+for+chronic+stimulation+of+optogenetic+molecules+in+freely+moving+mice.">A simple head-mountable LED device for chronic stimulation of optogenetic molecules in freely moving mice.</a> Neurosci. Res. 70 (1), 124-127 (2011).</li></ol>

Fremgangsmåderne præsenteres her, at man kan måle forholdet mellem søvn og ændringer i hjernen koncentrationen i den glycolytiske mellemprodukt lactat på en tidsskala ikke tidligere var muligt. Dyr gennemgå spontane overgange mellem kølvand, NREMS og REMS. Desuden er vi i stand til at anvende optogenetic stimuli, mens dyrene gennemgå disse overgange. Data indsamlet indtil nu viser, at både spontane og inducerede bølger indvirkning på udlæsningen af ​​en lactatoxidase-baseret biosensor. <p class="jov...

<table border="1"><tbody><tr><td> Component </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> BASI Mouse Guide Kanyle </td><td> Pinnacle Teknologi / BASI Inc </td><td> 7032 </td><td></td></tr><tr><td> Laktat Biosensoren </td><td> Pinnacle Technology </td><td> 7004 </td><td></td></tr><tr><td> Hovedmonteret </td><td> Pinnacle Technology </td><td> 8402 </td><td></td></tr><tr><td> Sleep / biosensor Recording system </td><td> Pinnacle Technology </td><td> 8400-K1-SL </td><td> 2 EEG kanaler, 1 EMG kanal, og 1 biosensor </td></tr><tr><td> Forbunden Mouse in-vitro Calibration kit </td><td> Pinnacle Technology </td><td> 7000-K1-T </td><td></td></tr><tr><td> Fiber Optic Guide Kanyle </td><td> Plast One </td><td> C312G </td><td> 21 Gauge Guide Kanyle </td></tr><tr><td> Dummy Kanyle </td><td> Plast One </td><td> C312DC </td><td> 21 Gauge Dummy </td></tr><tr><td> Diamond Fiber Scribe </td><td> Thorlabs </td><td> S90W </td><td></td></tr><tr><td> Fiber Connector Crimp Tool </td><td> Thorlabs </td><td> CT042 </td><td></td></tr><tr><td> Furkaturerne Tubing </td><td> Thorlabs </td><td> FT030 </td><td> 03.0 mm </td></tr><tr><td></td><td> Thorlabs </td><td> T10S13 </td><td> Max Dia. 0,012 </td></tr><tr><td> Furkaturerne Tube Stripper </td><td> Thorlabs </td><td> FTS3 </td><td></td></tr><tr><td> Bare Hard Metalbeklædning Multimode Fiber </td><td> Thorlabs </td><td> BFL37-200 </td><td> 200 um Core, 0,37 NA </td></tr><tr><td> Wire Snips / Kevlar Shears </td><td> Thorlabs </td><td> T865 </td><td></td></tr><tr><td> Fiber Optic Epoxy </td><td> Thorlabs </td><td> F112 </td><td></td></tr&gt; <tr><td> Fiber Stripper Tool </td><td> Thorlabs </td><td></td><td></td></tr><tr><td> Glas Polering Plate </td><td> Thorlabs </td><td> CTG913 </td><td></td></tr><tr><td> Rubber Polishing Pad </td><td> Thorlabs </td><td> NRS913 </td><td></td></tr><tr><td> Eye Loupe </td><td> Thorlabs </td><td> JEL10 </td><td></td></tr><tr><td> Kim Wipes </td><td> Thorlabs </td><td> KW32 </td><td></td></tr><tr><td> Trykluft </td><td> Thorlabs </td><td> CA3 </td><td></td></tr><tr><td> Polering Puck </td><td> Thorlabs </td><td> D50-xx </td><td></td></tr><tr><td> Fiber Inspektion omfang </td><td> Thorlabs </td><td> CL-200 </td><td></td></tr><tr><td> Polering Films </td><td> Thorlabs </td><td> LFG5P, LFG3P, LFG1P, LFG03P </td><td></td></tr><tr><td> FC / PC stik ende </td><td> Thorlabs </td&gt; <td> 30126G2-240 </td><td> 240 um Boring, SS Endetyller </td></tr><tr><td> MC Stimulus Unit </td><td> Multi-Channel Systems </td><td> STG-4002 </td><td></td></tr><tr><td> MC Stimulus Software </td><td> Multi-Channel Systems </td><td> MC-Stimulus V 2.1.5 </td><td></td></tr><tr><td> Blå Laser </td><td> CrystaLaser </td><td> CL473-050-0 </td><td></td></tr><tr><td> Laser Strømforsyning </td><td> CrystaLaser </td><td> CL2005 </td><td></td></tr><tr><td> Fiber Optic drejeleddet </td><td> Doriske Lenses </td><td> FRJ-v4 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabel 2. Forsyninger og udstyr. </td></tr></tbody></table>

simultaneous electroencephalography, real-time measurement of lactate concentration and optogenetic manipulation of neuronal activity in the rodent cerebral cortex

Selvom hjernen udgør mindre end 5% af kroppen efter vægt, den udnytter omkring en fjerdedel af glucose som kroppen i hvile 1. Funktionen af ​​non rapid eye movement søvn (NREMS), den største del af søvn ved tiden, er usikker. Men et iøjnefaldende træk ved NREMS er en væsentlig reduktion i forekomsten af cerebral glucoseudnyttelse i forhold til vågenhed 2-4. Dette og andre fund har ført til den udbredte opfattelse, at søvnen har en funktion relateret til cerebral metabolisme. Men de underliggende mekanismer reduktion i cerebral glucosemetabolisme i NREMS stadig at blive belyst. Et fænomen forbundet med NREMS, der kan påvirke cerebral stofskifte er forekomsten af langsomme bølger, svingninger ved frekvenser på mindre end 4 Hz, i elektroencefalogram 5,6. Disse langsomme bølger detekteret på niveauet af kraniet eller cerebrale kortikale overflade afspejlersvingninger af underliggende neuroner mellem en depolariseret / op stat og en hyperpolariseret / ned tilstand 7. Under ned tilstand, behøver celler ikke undergå aktionspotentialer i intervaller på op til adskillige hundrede millisekunder. Restaurering af ioniske koncentrationsgradienter efterfølgende til virkningspotentialer udgør en væsentlig metabolisk belastning af cellen 8 fravær af virkningspotentialer under ned associeret med NREMS kan bidrage til nedsat metabolisme i forhold til at vågne. To tekniske udfordringer skulle løses for at denne hypotetiske forhold, der skal testes. For det første var det nødvendigt at måle cerebral glycolytisk metabolisme med en tidsmæssig opløsning reflekterende af dynamikken i den cerebrale EEG (dvs. i løbet sekunder i stedet for minutter). For at gøre dette, målte vi koncentrationen af ​​lactat, produktet af aerobe glycolyse, og derfor en udlæsning af den mængde glucose metabolisme i hjernerne hos mus. Lactat blevmåles under anvendelse af en lactatoxidase baseret realtid sensor indlejret i den frontale cortex. Den følende mekanisme består af en platin-iridium elektrode omgivet af et lag af lactatoxidase molekyler. Metabolisme af lactat af lactatoxidase producerer hydrogenperoxid, som frembringer en strøm i platin-iridium elektrode. Så en rampe op af cerebral glycolyse tilvejebringer en stigning i koncentrationen af ​​substrat for lactatoxidase, som derefter afspejles i øget strøm ved måleelektroden. Det var desuden nødvendigt at måle disse parametre, samtidig med at manipulere ophidselse af den cerebrale cortex, for at isolere denne variabel fra andre aspekter af NREMS. Vi udtænkte et eksperimentelt system til samtidig måling af neuronal aktivitet via elecetroencephalogram, måling af glycolytisk flux via en lactat biosensor, og manipulation af cerebral cortical neuronal aktivitet via optogenetic aktivering af PyraMIDAL neuroner. Vi har udnyttet dette system til at dokumentere forholdet mellem søvn-relateret elektroencephalografisk bølgeformer og de øjeblik til øjeblik dynamik laktat koncentration i hjernebarken. Protokollen kan være nyttige for enhver person interesseret i at studere i frit opfører gnavere, forholdet mellem neuronal aktivitet målt på elektroencephalografisk niveau og cellulære energetik i hjernen.

<html> Som vist i figur 2, en mus udstyret til optogenetic stimulation og laktat / EEG / EMG dataindsamling undergik spontane søvn / vågner tilstandsovergange mens EEG, EMG og cerebral laktat koncentrationen blev overvåget kontinuerligt. Strøm ved den laktat sensor steg i perioder med lav amplitude EEG og faldt i perioder med høj amplitude EEG. Som vist i figur 3, begge kanaler for EEG reagerer på optogenetic stimuli leveret i den frontale cortex. <p class="j...

Watch this Scientific Journal Video about Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral C

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

En procedure er beskrevet for at manipulere med aktiviteten af ​​cerebrale kortikale pyramidale neuroner optogenetically mens elektroencefalogram, electromyogram, og cerebral laktat koncentration overvåges. Eksperimentelle optagelser udføres på kabel-forankrede mus, mens de gennemgår spontane søvn / vågner cyklusser. Optogenetic udstyr samles i vores laboratorium, kontrolapparatet er kommercielt tilgængeligt.

Samtidig Elektroencefalografi, Real-time måling af laktat Koncentration og Optogenetic Manipulation af neuronale aktivitet i Rodent Cerebral Cortex

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

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Metoder til Undersøgelse af Neuronal morfogenese:<em&gt; Ex vivo</em&gt; RNAi Elektroporation i embryoniske murin Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualisering og genetisk manipulering af dendritter og udløberne i mus cerebrale cortex og hippocampus under anvendelse<em&gt; I utero</em&gt; Elektroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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

Dobbelte Elektrofysiologiske Optagelser af synaptisk-evoked astrogliale og Neuronal respons i Akut hippocampusskiver

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

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetisk manipulation af Cerebellær granuleneuroner<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; At studere Neuronal morfologi og Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Inducerende plasticitet astrocytisk receptorer ved manipulation af neuronfyring priser

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo optogenetic Stimulering af Rodent centralnervesystemet

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

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Optogenetic medrivning af hippocampus Theta svingninger i opfører sig mus

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Kombinerede transkranial magnetisk Stimulation og electroencefalografi i den Dorsolateral præfrontale Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Samtidige Cryosectioning af flere gnaver hjerner

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...