Wir danken Herrn Dr. Meng-Chin Lin und Frau Yuan Dong für die technische Unterstützung. Diese Arbeit wurde durch ein Stipendium der National Institutes of Health HD053767 (Unterauftrag NLW) und durch Mittel aus dem Institut für Physiologie und Büro des Vizekanzlers für Forschung, University of California-Los Angeles (NLW) unterstützt.

1. Präparation der Gehirne von Adult Medaka <ol><li> Anesthetize erwachsenen männlichen oder weiblichen (Abbildung 1A) durch Eintauchen in 5 ml MS-222 (150 mg / L, pH 7,4); warten Sie ein paar Minuten nach der Kiemen Bewegungen vor enthaupten aufgehört. Alle Verfahren wurden von der Institutional Animal Care und Verwenden Ausschuss der University of California-Los Angeles genehmigt. </li><li> Enthaupte den Fisch in Fisch Kochsalzlösung am kaudalen Ende des Verschlusses mit einer Schere in einer 60-mm-Petrischale. </li><li> Übertragen Fischkopf zu einer 35-mm-Durchmesser Petrischale, halb Fisch mit Kochsalzlösung gefüllt und innen mit Sylgard, die eine Vertiefung in sie zu positionieren und sichern den Kopf für Gehirnsezierung hat. Position und sichern Sie den Kopf dorsal-Seite mit Insekten Pins. </li><li> Schneiden Sie vorsichtig die dorsale Teil des Schädels um ihren Umfang mit einer feinen Schere und entfernen Sie sie vorsichtig mit einer Pinzette, Aussetzen der dorsale Teil des Gehirns vollständig einschließlich der vordere Teil des Rückenmarks. </li> &lt;li&gt; Heben Sie das Rückenmark vorsichtig mit feinen Spitze Zange durchtrennen und die bilateralen Binde Nerven mit spitzen Schere: Hirnnerven, Spinalnerven und Sehnerven an der Bauchseite des Gehirns, und die olfaktorischen Nerven am vorderen Teil des das Gehirn. </li><li> Setzen Sie den vollständig seziert Gehirn in einer frischen Petrischale mit Fisch Kochsalzlösung gefüllt, und überprüfen Sie sorgfältig unter die Lupe, um sicherzustellen, das ganze Gehirn intakt ist (einschließlich der Hypophyse) ohne Schnitte oder Einstiche. Beschädigte Gehirne sind nicht für Experimente verwendet. </li></ol> 2. Montage des Gehirn in einem Recording Kammer <ol><li> Setzen Sie einen kleinen Tropfen Sekundenkleber oder andere schnell wirkende Kleber mit einer Nadel in der Mitte der Aufnahme Kammer und verteilt den Kleber etwa 1 cm 2. </li><li> Schnell das Gehirn übertragen und kleben Sie es in einem ventral-Seite nach oben. </li><li> 1 ml Kochsalzlösung Fisch so dass sie die Aufnahme Kammer füllt und deckt das Gehirn. Kontinuierlich perfundierendas Gehirn mit belüfteten Fisch Kochsalzlösung mit einer Geschwindigkeit von mindestens 200 ml / min. </li><li> Mit der feinen Spitze-Pinzette vorsichtig die Hirnhäute über die Oberfläche der Aufnahme Bereich des Gehirns. </li></ol> 3. Elektrophysiologie <ol><li> Lose Patch Aufnahme von Aktionspotential Brennen <ol><li> Glas-Mikroelektroden (6-10 MQ) werden aus Borosilikatglas Pipetten (1B150F-4, World Precision Instruments) unter Verwendung einer Elektrode Abzieher (z. B. Sutter P-87) gebaut, und zurück mit dem losen-patch Recording-Lösung auf halber Höhe der gefüllte Elektrode. </li><li> Verwendung eines aufrecht Fluoreszenzmikroskop mit einem gekühlten charge-coupled device (CCD)-Kamera, um den GFP-exprimierenden TN-GnRH3 Neuronen durch 10x (1B), dann 40x Wasserimmersionsobjektiven (Abbildung 1C). Sie werden an oder nahe der ventralen Oberfläche der Riechkolben, knapp vor dem telencephalon entfernt. </li><li> Wechseln Sie zu einem Video-Monitor, bietetEchtzeit-Bilder aus dem Mikroskop, um den TN-GnRH3 Neuron Cluster (Abbildung 1C) zu lokalisieren. </li><li> Positionieren der Mikroelektrode in das Bad, so daß die Spitze der Elektrode über dem Neuron Ziel ist. Bewerben konstanten leichten Überdruck der Mikroelektroden, so dass sie nicht verstopft werden, und sanft nähern sich dem Ziel Neuron mit der Spitze der Elektrode. </li><li> Überprüfen und überwachen die Elektrode Widerstand in Voltage-Clamp-Modus mit AxoGraph Software und Verstärker Axoclamp 200B mit Axograph Software (Axon Instruments). Wenn man von dem Videomonitor, dass die Spitze der Elektrode auf der Oberfläche des Neurons und der AxoGraph Anzeige, dass der Widerstand leicht ändert, lassen Sie die Überdruck und mit leichtem Unterdruck, wenn notwendig, einen niedrigen Widerstand Siegel machen das Neuron (&lt;100 M). </li><li> Wechseln Sie zu Current-Clamp-Modus, und notieren Sie die Membran Spannung kontinuierlich ohne Strom-Eingang, Einstellung der Waageder Spannung und der Geschwindigkeit der Aufnahme, wenn nötig. Die Daten werden gesammelt und durch Powerlab Software (ADInstruments Inc.) analysiert. </li><li> Nimm Baseline elektrische Aktivität in normalen Fisch Kochsalzlösung für ca. 5 min vor jeder Behandlung (Abbildung 2A). Bath Perfusion von Arzneimitteln, Hormonen, Peptiden, etc. zugegeben, um Fisch Kochsalzlösung weiterhin mit einer Rate von 200 ml / min, durch eine Auswaschung in normale Fische Kochsalzlösung 5, 6, 11 gefolgt. </li></ol></li><li> Whole cell recording des Membranpotentials Die gesamte Patch-Clamp-Elektrophysiologie Verfahren ist das gleiche wie das der extrazellulären lose Patch Aufnahme oben beschriebenen Verfahren aus den Schritten 3.1.1 - 3.1.4. Dann werden die Verfahren entsprechend abweichen: <ol><li> Überprüfen und überwachen die Elektrode Widerstand in Voltage-Clamp-Modus. Wenn man von dem Videomonitor, dass die Spitze der Elektrode auf der Oberfläche des Neurons und der auf dem Oszilloskop ist, dass sich der Widerstand leicht freigeben positiveDruck und gelten ein wenig Unterdruck auf einen hohen Widerstand Siegel auf dem Neuron (~ 3 Giga Ω) zu machen. Bewerben sanfte Saugwirkung durch den Mund zu zerreißen die gepatchte Membran, du wirst einen plötzlichen Abfall des Widerstandes auf etwa 120-250 MOhm sehen. </li><li> Wechseln Sie zu Current-Clamp-Modus, und notieren Sie die Membran Spannung kontinuierlich ohne Stromaufnahme, die Anpassung der Skala von Spannung und Geschwindigkeit der Aufnahme, wenn nötig. </li><li> Nimm Baseline elektrische Aktivität in normalen Fisch Kochsalzlösung für ca. 5 min vor jeder Behandlung, wie oben in 3.1.7 (2B) beschrieben. </li></ol></li></ol>

<ol>
	<li>Kah, O., Lethimonier, C., Lareyre, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotrophin-releasing+hormone+(GnRH)+in+the+animal+kingdom.">Gonadotrophin-releasing hormone (GnRH) in the animal kingdom.</a> J. Soc. Biol. 198 (1), 53-60 (2004).</li><li>Ramakrishnan, S., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+from+conspecifics+alter+electrical+activity+of+gonadotropin-releasing+hormone+neurons+in+the+terminal+nerve+via+visual+signals.">Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (1), R135-R141 (2009).</li><li>Abe, H., Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neuromodulation+by+a+nonhypophysiotropic+GnRH+system+controlling+motivation+of+reproductive+behavior+in+the+teleost.">Mechanisms of neuromodulation by a nonhypophysiotropic GnRH system controlling motivation of reproductive behavior in the teleost.</a> 57 (6), 665-674 (2011).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodotoxin-resistant+persistent+Na++current+underlying+pacemaker+potentials+of+fish+gonadotrophin-releasing+hormone+neurones.">Tetrodotoxin-resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin-releasing hormone neurones.</a> J. Physiol. 482 (Pt. 1), 1-6 (1995).</li><li>Zhao, Y., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Kisspeptin1+on+Electrical+Activity+of+an+Extrahypothalamic+Population+of+Gonadotropin-Releasing+Hormone+Neurons+in+Medaka.">Effects of Kisspeptin1 on Electrical Activity of an Extrahypothalamic Population of Gonadotropin-Releasing Hormone Neurons in Medaka.</a> PLoS One. 7 (5), e37909 (2012).</li><li>Wayne, N. L., 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=Whole-cell+electrophysiology+of+gonadotropin-releasing+hormone+neurons+that+express+green+fluorescent+protein+in+the+terminal+nerve+of+transgenic+medaka+(Oryzias+latipes).">Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).</a> Biol. Reprod. 73 (6), 1228-1234 (2005).</li><li>Okubo, K., 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=Forebrain+gonadotropin-releasing+hormone+neuronal+development:+insights+from+transgenic+medaka+and+the+relevance+to+X-linked+Kallmann+syndrome.">Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.</a> Endocrinology. 147 (3), 1076-1084 (2006).</li><li>Okubo, K., 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=A+novel+form+of+gonadotropin-releasing+hormone+in+the+medaka,+Oryzias+latipes.">A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes.</a> Biochem. Biophys. Res. Commun. 276 (1), 298-303 (2000).</li><li>Ramakrishnan, 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=Acquisition+of+spontaneous+electrical+activity+during+embryonic+development+of+gonadotropin-releasing+hormone-3+neurons+located+in+the+terminal+nerve+of+transgenic+zebrafish+(Danio+rerio).">Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio).</a> Gen. Comp. Endocrinol. 168 (3), 401-407 (2010).</li><li>Abraham, E., 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=Targeted+gonadotropin-releasing+hormone-3+neuron+ablation+in+zebrafish:+effects+on+neurogenesis,+neuronal+migration,+and+reproduction.">Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction.</a> Endocrinology. 151 (1), 332-340 (2010).</li><li>Wayne, N. L., Kuwahara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-endorphin+alters+electrical+activity+of+gonadotropin+releasing+hormone+neurons+located+in+the+terminal+nerve+of+the+teleost+medaka+(Oryzias+latipes.">Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes.</a> Gen. Comp. Endocrinol. 150 (1), 41-47 (2007).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+types+of+gonadotrophin-releasing+hormone+neurones+and+steroid-sensitive+sexually+dimorphic+kisspeptin+neurones+in+teleosts.">Three types of gonadotrophin-releasing hormone neurones and steroid-sensitive sexually dimorphic kisspeptin neurones in teleosts.</a> J. Neuroendocrinol. 21 (4), 334-338 (2009).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology. , (2003).</li></ol>

Die Autoren haben nichts zu offenbaren.

GnRH 3: GFP transgenen Fische bieten einzigartige Modelle, die neurophysiologischen Mechanismen neuronaler Integration und Regulierung in zentralen Steuerung von Verhaltensweisen, die sowohl direkt als auch indirekt in der Reproduktion 3, 8-10 beteiligt sind studieren. Einer der wesentlichen Vorteile dieses Modells besteht darin, dass viele GnRH3 Neuronen, die GFP in der Nähe der ventralen Oberfläche des Gehirns sind, so dass für relativ einfachen Zugang zu den Neuronen für elektrophysiologische ...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>BX50W (Upright)</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axoclamp 200B </td>
 </tr>
 <tr>
 <td>A-D converter</td>
 <td>Computer Interference Corp.</td>
 <td></td>
 <td>Digidata ITC-18 </td>
 </tr>
 <tr>
 <td>Cooled CCD camera</td>
 <td>PCO Computer Optics </td>
 <td></td>
 <td>Sensicam</td>
 </tr>
 <tr>
 <td>Xenon lamp</td>
 <td>Sutter Instruments Co.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>GFP filter set</td>
 <td>Chroma Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging Software</td>
 <td>Intelligent Imaging Innovations</td>
 <td></td>
 <td>Slidebook software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axograph software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>AD Instruments Inc.</td>
 <td></td>
 <td>PowerLab </td>
 </tr>
 <tr>
 <td>Headstage for electrophysiology</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>CV 203BU </td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Sutter Instrument Co </td>
 <td>MP-285 </td>
 <td></td>
 </tr>
 <tr>
 <td>Recording Chamber Platform</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>P1 </td>
 </tr>
 <tr>
 <td>Recording Chamber</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>RC-26G </td>
 </tr>
 <tr>
 <td>Electrode Puller</td>
 <td>Sutter instruments</td>
 <td></td>
 <td>P87 </td>
 </tr>
 <tr>
 <td>Filament for electrode puller</td>
 <td>Sutter Instruments</td>
 <td>FB330B</td>
 <td>3.0 mm wide trough filament</td>
 </tr>
 <tr>
 <td>1.5 mm glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B150-4</td>
 <td>Microelectrode for recording</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309586</td>
 <td>3 ml</td>
 </tr>
 <tr>
 <td>MS-222 </td>
 <td>Sigma</td>
 <td>E10521-10G</td>
 <td>Ethyl 3-aminobenzoate methanesulfonate salt</td>
 </tr>
 <tr>
 <td>Fish saline</td>
 <td></td>
 <td></td>
 <td>mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES </td>
 </tr>
 <tr>
 <td>Electrode solution (loose-patch) </td>
 <td></td>
 <td></td>
 <td>mM: 150 NaCl; 3.5 KCl; 2.5 CaCl2; 1.3 MgCl2; 10 HEPES; 10 glucose </td>
 </tr>
 <tr>
 <td>Electrode solution (whole-cell patch) </td>
 <td></td>
 <td></td>
 <td>mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl2; 1 MgCl2; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine </td>
 </tr>
 </tbody>
</table>

recording electrical activity from identified neurons in the intact brain of transgenic fish

Das Verständnis der Zellphysiologie von neuronalen Schaltkreise, die komplexe Verhalten regulieren stark verbessert, indem Modellsystemen in denen diese Arbeiten in einer intakten Zubereitung bei der neuronalen Schaltkreise des ZNS intakt durchgeführt werden kann. Wir verwenden transgene Fische in dem Gonadotropin-Releasing-Hormon (GnRH) Neuronen genetisch mit dem grün fluoreszierenden Protein für die Identifizierung im intakten Gehirn sind verschlagwortet. Fische haben mehrere Populationen von GnRH Neuronen und ihre Funktionen sind abhängig von ihrer Lage im Gehirn und dem GnRH-Gen, dass sie 1 auszudrücken. Wir haben unsere Demonstration am GnRH3 Neuronen in den Terminal Nerven (TN) mit den Riechkolben mit dem intakten Gehirn von transgenen Medaka Fisch (Abbildung 1B und C) verbunden befindet konzentriert. Studien zeigen, dass Medaka TN-GnRH3 Neuronen neuromodulatory sind, als ein Sender von der externen Umgebung des zentralen Nervensystems; they spielen keine direkte Rolle bei der Regulierung der Hypophysen-Gonaden-Funktionen, wie auch die bekannte Hypothalamus GNRH1 Neuronen 2, 3. Das Tonikum Muster der spontanen Aktionspotentiale Abfeuern von TN-GnRH3 Neuronen eine intrinsische Eigenschaft 4-6, die Frequenz von denen durch visuelle Hinweise von Artgenossen 2 und dem Neuropeptid kisspeptin 1 5 moduliert. In diesem Video, verwenden wir eine stabile Linie von transgenen Medaka in dem TN-GnRH3 Neuronen exprimieren ein Transgen enthält die Promotor-Region des Gnrh3 verknüpft enhanced green fluorescent protein 7, Ihnen zu zeigen, wie man Neuronen identifizieren und überwachen ihre elektrische Aktivität im gesamten Gehirns Zubereitung 6.

Ein Beispiel für bilaterale Cluster von GFP-markierten TN-GnRH3 Neuronen aus dem Gehirn herausgeschnitten Medakafisches sind in den 1B und 1C gezeigt. Jeder Cluster enthält etwa 8-10 GnRH Neuronen. Die spontanen neuronalen Aktivitäten des Ziel-TN-GnRH3 wurden in Current-Clamp-Modus (I = 0) mit typischen Feuerraten von 0,5-6 Hz aufgezeichnet. Das Muster der Aktionspotential Brennen ist typischerweise ein Tonic oder gegen Muster, mit einer ziemlich regelmäßigen Interspike Intervall. ...

Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

In diesem Video zeigen wir Ihnen, wie die elektrische Aktivität von einzelnen Neuronen identifiziert in einer ganzen Gehirn Vorbereitung, die komplexe neuronale Schaltkreise bewahrt aufzeichnen. Wir verwenden transgene Fische in der Gonadotropin-Releasing-Hormon (GnRH) Neuronen genetisch mit einem fluoreszierenden Protein zur Identifikation der intakten Zubereitung markiert sind.

Aufzeichnung der elektrischen Aktivität von identifizierten Neuronen im intakten Gehirn von transgenen Fischen

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

recording-electrical-activity-from-identified-neurons-intact-brain

Research

JoVE Journal

Neuroscience

13.4K Aufrufe.  University of California, Los Angeles. In diesem Video zeigen wir Ihnen, wie die elektrische Aktivität von einzelnen Neuronen identifiziert in einer ganzen Gehirn Vorbereitung, die komplexe neuronale Schaltkreise bewahrt aufzeichnen. Wir verwenden transgene Fische in der Gonadotropin-Releasing-Hormon (GnRH) Neuronen genetisch mit einem fluoreszierenden Protein zur Identifikation der intakten Zubereitung markiert sind.

Serie: Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

Gleichzeitige Aufnahme von Calcium-Signale von identifizierten Neuronen und Fressverhalten von Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Forschung

Neurowissenschaften

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograde Fluoreszenzmarkierung Ermöglicht Gezielte Extracellular Einzel-Einheit Aufnahme von identifizierten Neuronen<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Voltage-sensitive Dye Aufnahme von Axonen, Dendriten und dendritischen Spines einzelner Neuronen in Hirnschnitten

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Die intrazelluläre Aufnahme, Sinnes Field Mapping und Kultivierung identifizierten Neuronen in der Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Elektrophysiologische Aufnahme im Gehirn von erwachsenen Zebrafisch Intact

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Ein Leitfaden zur<em&gt; In vivo</em&gt; Single-Unit-Aufnahme von Optogenetically Identifizierte kortikalen inhibitorischen Inter

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforated-Patch-Clamp-Aufzeichnung der Maus Riechzellen in Intact Neuroepithel: Funktionelle Analyse von Neuronen, die eine bestimmte Geruchsrezeptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Aufzeichnung von Temperatur-induzierte neuronale Aktivität durch Calcium Überwachen von Änderungen in der Riechkolben von<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Neuronalen Aktivität im primären somatosensorischen Cortex mit Thy1Imaging-GCaMP6s transgene Mäuse

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Drahtlose elektrophysiologische Aufzeichnung von Neuronen durch bewegliche Tetrodes in frei schwimmenden Fischen

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...