This material is based on the works supported by Nanyang Assistant Professorship (NAP, M4080740), Agency for Science, Technology and Research (A*STAR) Public Sector Research Funding (PSF, M4070190), A*STAR-JST (The Japan Science and Technology Agency) joint grant (M4070198), and Singapore Ministry of Education (MOE2013-T2-2-049). The authors would like to thank Mr. Roger Tan Kay Chia, Prof. Low Kin Huat, Mr. Poon Kee Chun, Mr. Chew Hock See, Mr. Lam Kim Kheong and Dr. Mao Shixin at School of MAE for their support in setting up and maintaining the research facilities. The authors thank Prof. Michel Maharbiz (U.C. Berkeley) his advice and discussion, Prof. Kris Pister and his group (U.C. Berkeley) for their support in providing the GINA used in this study.

1. Studie Tier <ol><li> Hinten einzelnen Mecynorrhina torquata Käfer (6 cm, 8 g) in getrennten Kunststoffbehälter mit Holzpellet-Bettwäsche. </li><li> Ziehen Sie jedem Käfer eine Tasse Zucker Gelee (12 ml) alle 3 Tage. </li><li> Halten Sie die Temperatur und die Feuchtigkeit der Aufzuchtraum bei 25 ° C und 60% betragen. </li><li> Testen Sie die Flugfähigkeit eines jeden Käfer vor dünnen Drahtelektroden implantiert wird. <ol><li> Werfen Sie vorsichtig mit einem Käfer in die Luft. Wenn der Käfer für länger als 10 Sekunden für 5 aufeinander folgenden Studien fliegen können, schließen, dass die Käfer regelmäßige Flugeigenschaften und verwenden es für nachfolgende Flugexperimente hat. Zurückzuerobern den Käfer, schalten alle Lichter im Raum ab, um es dunkel machen. Dies bewirkt, dass der Käfer Flug zu beenden. Hinweis: Ein Käfer spontan, wenn sie in die Luft freigesetzt, weg zu fliegen beginnt. Es ist besser , die Flugversuche in einem großen geschlossenen Raum wie die in 1 (16 x 8 x 4 m gezeigt durchzuführen <sup&gt; 3 mit einer Motion - Capture - Raum von 12,5 x 8 x 4 m 3), eine fliegende Käfer bewegt sich sehr schnell (ca. 3-5 m / sec) und große Bögen zieht , wenn in der Luft drehen. </li></ol></li></ol> 2. Elektrodenimplantation <ol><li> Anästhesieren den Käfer , indem er in einen Kunststoffbehälter gefüllt mit CO 2 für 1 min 13,16,20-24 platzieren. </li><li> Dentalwachs erweichen, indem sie in heißes Wasser für 10 sec eingetaucht wird. Platzieren Sie den narkotisierten Käfer auf einem Holzblock und immobilisieren sie mit dem erweichten Dentalwachs. Das Dentalwachs kühlt natürlich und verfestigt sich innerhalb von wenigen Minuten. </li><li> Cut isolierte Silberdrähte (127 &amp; mgr; m blank Durchmesser, 178 &amp; mgr; m Durchmesser, wenn sie mit Perfluoralkoxy beschichtet) in Längen von 25 mm als dünne Drahtelektroden für die Implantation zu verwenden. </li><li> Expose 3 mm blanke Silber durch den Isolator an beiden Enden jedes Drahtes flammend. </li><li> Präparieren Sie die Oberfläche der Kutikula des Käfers mit einem spitzen Schere mit einem sma zu erstellenll Fenster von ungefähr 4 x 4 mm auf der metepisternum (Abbildung 2c). Hinweis: Eine weiche braun gefärbte Kutikula wird dann belichtet, wie in den 2c gezeigt - e. Der 3Ax Muskel unterhalb der weichen Nagelhaut entfernt. </li><li> Pierce zwei Löcher auf der freiliegenden braun Kutikula mit einem Insektenstift (Größe 00) mit einem Abstand von 2 mm zwischen den beiden Löchern (Figur 2d). </li><li> Legen Sie zwei Drahtelektroden (einschließlich eine aktive und eine Rückelektroden hergestellt in Schritt 2.4) vorsichtig durch die Löcher und implantieren sie in jedem 3Ax Muskel in einer Tiefe von 3 mm. </li><li> Sichern Sie sich die implantierten Elektroden und halten sie an ihrem Platz zu vermeiden, Kontakt und Kurzschlüsse durch geschmolzenes Bienenwachs fallen auf die Löcher. Bei Bedarf umfließen das Bienenwachs über die Kutikula durch das Bienenwachs mit der Spitze eines heißen Lötkolben zu berühren. Das Bienenwachs erstarrt schnell und verstärkt die Implantation. Hinweis: Um zu überprüfen, ob die Implantation richtig ist, die elytra der Rübele kann die Bewegung des 3Ax Muskel während der elektrischen Stimulation zu beobachten angehoben werden. </li></ol> 3. Wireless-Rucksack Assembly Hinweis: Der Rucksack besteht aus einem eingebauten Radio - Mikrocontroller auf einem 4 geschichteten FR-4 - Board (1,6 x 1,6 cm 2). Der Rucksack wurde von einem Lithium-Polymer-Mikro (3,7 V, 350 mg, 10 mAh) angetrieben. Die Gesamtmasse des Rucksacks einschließlich der Batterie betrug 1,2 ± 0,26 g, die weniger als die Nutzlast des Käfers (30% von 10 g Körpergewicht) ist. Der Rucksack wurde vorprogrammierte drahtlose Kommunikation zu empfangen und hatte zwei Ausgangskanäle. <ol><li> Reinigen Sie die Pronotums Oberfläche (entfernen Sie die Wachsschicht auf der Kutikula) unter Verwendung von doppelseitigen Klebeband. Dann befestigen Sie den Rucksack auf dem Halsschild des Käfers mit einem Stück doppelseitiges Klebeband. </li><li> Verbinden der Enden der implantierten Elektroden mit den Ausgängen des Rucksacks. </li><li> Wickeln Sie retroreflektierende Band um die Mikrobatterie eine Markierung zu erzeugen, for Motion-Capture-Kameras zu erfassen. </li><li> Bringen Sie den Mikrobatterie an die Spitze des Rucksacks ein Stück doppelseitiges Klebeband, so dass die retroreflektierende Band kann durch Motion-Capture-Kameras erfasst werden. </li></ol> 4. Wireless Control System Hinweis: In diesem Fall wird der Begriff drahtlose Steuersystem umfasst einen Empfänger für die Fernbedienung, ein Laptop-Computer den benutzerdefinierten Flugsteuerungssoftware, eine Basisstation, der Rucksack und die Motion-Capture-System auszuführen. <ol><li> Schließen Sie die Basisstation und Empfänger der Fernbedienung auf den Laptop-Computer über USB-Anschlüsse. </li><li> Schalten Sie das Motion-Capture-System und verbinden Sie es mit dem Laptop-Computer über einen Ethernet-Anschluss. </li><li> Führen Volumenkalibrierung durch den Kalibrierungsstab winken (vom Hersteller Unternehmen der Motion-Capture-System zur Verfügung gestellt), um voll und ganz die Motion-Capture-Raum abdecken. <ol><li> Öffnen Sie die Motion-Capture-Software auf dem Desktop des Laptops. Klicken und drag auswählen, um alle Kameras auf dem Menü &quot;System&quot; der &quot;Ressourcen&quot; Panel. </li><li> Klicken Sie auf das &quot;3D-Perspektive&quot; und wählen Sie &quot;Kamera&quot;, um die Kamera-Ansicht zu ändern. Klicken Sie auf die &quot;Kamera&quot; auf die Registerkarte &quot;Tools&quot; Panel die Kalibrierung Setup zu zeigen. Klicken Sie auf &quot;Start&quot; auf &quot;Create Kamera Masken&quot; Menü Lärm von den Kameras zu beseitigen und dann &quot;Stop&quot;, nachdem das Rauschen in Blau maskiert. </li><li> Klicken Sie auf und wählen Sie &quot;5 Marker Wand &amp; L-Frame&quot; von der &quot;Wand&quot; Menü und die &quot;L-Frame&quot; Menü auf der &quot;Kamera&quot; aus. Stellen Sie die &quot;Wand Count&quot; auf 2.500, klicken Sie auf &quot;Start&quot; auf dem &quot;Kalibrieren Kameras&quot; Menü und Welle der Kalibrierungsstab durch die gesamte Motion-Capture-Raum. Der Kalibrierungsvorgang stoppt, wenn der Stab Zahl 2500 erreicht. </li><li> Wiederholen Sie den Kalibrierungsprozess, wenn die Bildfehler (am unteren Rand der &quot;Kamera&quot; Register des Panels &quot;Tools&quot;) höher als 0,3 foder jede Kamera. Nach dem Kalibrieren legte den Stab auf dem Boden in der Mitte des Motion-Capture-Raum und klicken Sie auf &quot;Start&quot; auf &quot;Set Volume Origin&quot; Menü den Ursprung des Motion-Capture-Raum zu setzen. </li></ol></li><li> Überprüfen Sie die Abdeckung des Motion-Capture-System einen Dummy-Test mit dem Bewegungspfad eines Markers durch einen Benutzer in der Motion-Capture-Raum schwenkte aufzeichnen und bestätigen, ob der Marker erkannt und verfolgt. Wenn der Marker häufig während der Erfassung, wiederholen Volumenkalibrierung verloren geht, bis der Dummy-Test erfolgreich ist. <ol><li> Klicken Sie auf die &quot;Aufnahme&quot; auf die Registerkarte &quot;Tools&quot; Panel und dann auf &quot;Start&quot; auf der &quot;Aufnahme&quot; Menü, bevor Sie die Probe Marker durch die gesamte Motion-Capture-Raum winken seine Bahn aufzunehmen. </li><li> Nach der Aufzeichnung klicken Sie auf &quot;Führt die Reconstruct Pipeline&quot;, die Positionen der Marker zu rekonstruieren und die Qualität der Aufnahme überprüfen. </li></ol></li><li> Verbinden Sie die Anschlüsse des microbattery (an dem Rucksack in Schritt 3.4) zu den Power-Pins des Rucksacks. </li><li> Testen Sie die drahtlose Kommunikation zwischen dem Laptop und dem Rucksack die benutzerdefinierte Flugsteuerungssoftware. Klicken Sie auf den &quot;Start&quot; Befehl auf der Software und überprüfen Sie die angezeigten Verbindungsstatus. </li></ol> 5. Free Flight Experiment <ol><li> Führen Sie den freien Flug Experiment in einem Flug Arena messen 16 x 8 x 4 m 3. </li><li> Geben Sie die entsprechenden Parameter für die Flugsteuerungssoftware (Spannung, Impulsbreite, Frequenz und Stimulationsdauer). Anmerkung: Zur Demonstration, fixiert wir die Spannung auf 3 V, Pulsbreite 3 ms und Stimulationsdauer 1 sec und variiert die Frequenz von 60 bis 100 Hz. <ol><li> Auf der Software-Bildschirm, Typ 3 für 3 V in der &quot;Spannung&quot; Feld 1000 für 1.000 ms in der &quot;Stimulation Dauer&quot; Box, 3 für 3 ms im &quot;Pulse Width&quot; Feld und einer gewünschten Frequenz in Hz im &quot; Frequency &quot;Box on das Befehlsfenster. </li></ol></li><li> Lassen Sie den Rucksack montierte Käfer in die Luft durch die frei im Flug Arena zu fliegen. Sie manuell die Stimulation auslösen, wenn der Käfer den Raum Motion-Capture eintritt. Drücken Sie die entsprechende Befehlstaste (links oder rechts) auf der Fernbedienung, um den Zielmuskel auf der linken oder rechten Seite des Käfers zu stimulieren. Hinweis: Sobald die Taste gedrückt wird, erzeugt der Flugsteuerungssoftware auf dem Laptop den Befehl und sendet sie an den Rucksack. Der Rucksack gibt dann den elektrischen Reiz auf den Muskel von Interesse (links oder rechts). </li><li> Beachten Sie die Reaktion des Käfers in Echtzeit während der Stimulation und rekonstruieren die Daten mit 3D-Grafik-Software. <ol><li> Wählen Sie eine der Studien in der Datenliste des &quot;Beetle Display&quot; Fenster aufgezeichnet und klicken Sie auf &quot;Export Panda&quot;, um die Daten dieser Studie auf die Analyse-Ordner kopieren und das 3D-Grafik-Modul ausgeführt werden. </li><li> Drücken Sie auf &quot;N&quot; aufdie Tastatur, um das Reizsignal mit der aufgezeichneten Bewegungsbahn zu verbinden. Drücken Sie I mit den markierten Stimulationsperioden, die Flugbahn des Käfers zu zeigen. </li></ol></li></ol>

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The authors declare that there are no conflicts of interest.

Der Implantationsprozess ist wichtig, da es die Zuverlässigkeit des Experiments beeinflusst. Die Elektroden sollten in einer Tiefe von 3 mm oder weniger in den Muskel eingesetzt werden, abhängig von der Größe des Käfers (Vermeidung von Kontakt mit dem nahe gelegenen Muskeln). Wenn die Elektroden, die in der Nähe Muskeln, unerwünschte motorische Handlungen und Verhaltensweisen berühren auftreten können, auf die Kontraktion der Muskeln in der Nähe zurückzuführen ist. Die beiden Elektroden sollte gut ausgericht...

An insect-machine hybrid system, often referred to as a cyborg insect or biobot, is the fusion of a living insect platform with a miniature mounted electronic device. The electronic device, which is wirelessly commanded by a remote user, outputs an electrical signal to electrically stimulate neuromuscular sites in the insect via implanted wire electrodes to induce user desired motor actions and behaviors. In the early stages of this research field, researchers were limited to conducting wireless recording of the muscular action of an insect, using simple analog circuits comprised of surface-mounted components1-3. The development of system-on-a-chip technology with radio frequency functionality enabled not only the wireless recording of neuromuscular signals but also the electrical stimulation of the neuromuscular sites in living insects. At present, a built-in radio microcontroller is small enough to be mounted on living insects without causing any obstructions to their locomotion4-13.
The development of the built-in radio microcontroller allows researchers to determine electrical stimulation protocols to induce desired motor actions to control the locomotion of the insect of interest. On the ground, researchers have demonstrated walking control by stimulating the neuromuscular sites of cockroaches4,12,14, spiders15, and beetles16,17. In the air, the initiation and cessation of flight were achieved using different methods such as the stimulation of the optic lobes (the massive neural cluster of a compound eye) in beetles7,9 and brain sub-regions in bees18, whereas turning control has been demonstrated by stimulating the antennae muscles and nervous system of the abdomens in moths11,19 and the flight muscles of beetles7,9,13. In most cases, a built-in radio microcontroller was integrated on a custom-designed printed circuit board to produce a miniature wireless stimulator (backpack), which was mounted on the insect of interest. This allows wireless electrical stimulation to be applied to a freely walking or flying insect. Such a microcontroller-mounted insect is what is referred to as an insect-machine hybrid system. 
This study describes the experimental protocols for building an insect-machine hybrid system, wherein a living beetle is employed as the insect platform, and instructs on how to operate the robot and test its flight control systems. The third axillary sclerite (3Ax) muscle was chosen as the muscle of interest for electrical stimulation and demonstration of left or right turning control13. A pair of thin silver wire electrodes was implanted in both the left and right 3Ax muscles. Moreover, a backpack was mounted on the living beetle. The other ends of the wire electrode were connected to the output pins of the microcontroller. The backpack was small enough for the beetle to carry in flight. Thus, this allows an experimentalist to remotely stimulate the muscle of interest of an insect in free flight and investigate its reactions to the stimulations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mecynorrhina torquata beetle</td>
			<td>Kingdom of Beetle Taiwan</td>
			<td></td>
			<td>10 g, 8 cm, pay load capacity is 30% of the body mass 
			Aproval of importing and using by Agri-Food and Veterinary Authority of Singapore (AVA; HS code: 01069000, product code: ALV002).</td>
		</tr>
		<tr>
			<td>Wireless backpack stimulator</td>
			<td>Custom</td>
			<td></td>
			<td>TI CC2431 micocontroler 
			The board is custom made based on the GINA board from Prof. Kris Pister&#8217;s lab. The layout of GINA board can be found at&#160;&#160;&#160; https://openwsn.atlassian.net/wiki/display/OW/GINA</td>
		</tr>
		<tr>
			<td>Wii Remote control</td>
			<td>Nintendo</td>
			<td></td>
			<td>Bluetooth remote control to send the command to the operator laptop</td>
		</tr>
		<tr>
			<td>BeetleCommander v1.8</td>
			<td>Custom. Maharbiz group at UC Berkeley and Sato group at NTU</td>
			<td></td>
			<td>Establish the wireless communication of the backpack and the operator laptop. Configure the stimulus parameters and log the positional data. Visualize the flight data.</td>
		</tr>
		<tr>
			<td>GINA base station</td>
			<td>Kris Pister group at UC Berkeley</td>
			<td></td>
			<td>TI MSP430F2618 and AT86RF231</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T160</td>
			<td>8 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T40s</td>
			<td>12 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Micro battery</td>
			<td>Fullriver</td>
			<td>&#160;201013HS10C&#160;</td>
			<td>3.7V, 10 mAh</td>
		</tr>
		<tr>
			<td>Retro reflective tape</td>
			<td>Reflexite</td>
			<td>V92-1549-010150</td>
			<td>V92 reflective tape, silver color</td>
		</tr>
		<tr>
			<td>PFA-Insulated Silver Wire&#160;</td>
			<td>A-M systems</td>
			<td>786000</td>
			<td>127 &#181;m bare, 177.8 &#181;m coated, 3 mm bare silver flame exposed at tips</td>
		</tr>
		<tr>
			<td>SMT Micro Header&#160;</td>
			<td>SAMTEC</td>
			<td>FTSH-110-01-L-DV</td>
			<td>0.3 x 6 mm, bend to make a 3 mm long slider to secure the electrode into the PCB header.</td>
		</tr>
		<tr>
			<td>Beeswax</td>
			<td></td>
			<td></td>
			<td>Secure the electrodes</td>
		</tr>
		<tr>
			<td>Dental Wax</td>
			<td>Vertex</td>
			<td></td>
			<td>Immobilize the beetle</td>
		</tr>
		<tr>
			<td>Insect pin</td>
			<td>ROBOZ</td>
			<td>RS-6082-30</td>
			<td>Size&#160; 00; 0.3mm Rod diameter; 0.03 mm tip width; 38 mm Length&#160; 
			Make electrode guiding holes on cuticle</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>DUMONT</td>
			<td>RS-5015</td>
			<td>Pattern #5; .05 X .01mm Tip Size; 110mm Length 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>ROBOZ</td>
			<td>RS-5620</td>
			<td>Vannas Micro Dissecting Spring Scissors; Straight; 3mm Cutting Edge; 0.1mm Tip Width; 3&#34; Overall Length&#160; 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Potable soldering iron</td>
			<td>DAIYO</td>
			<td>DS241</td>
			<td>Reflow beeswax</td>
		</tr>
		<tr>
			<td>Hotplate&#160;</td>
			<td>CORNING</td>
			<td>PC-400D</td>
			<td>Melting beeswax and dental wax</td>
		</tr>
		<tr>
			<td>Flourescent lamp</td>
			<td>Philips</td>
			<td>TL5 14W</td>
			<td>Light the entire flight arena with 30 panels (60 x 60 cm2). Each panel has 3 lamps. 
			14 W, 549 mm x 17 mm&#160;</td>
		</tr>
	</tbody>
</table>

insect-machine hybrid system: remote radio control of a freely flying beetle (mercynorrhina torquata)

Der Aufstieg von Radio-fähige digitale elektronische Geräte hat für die Untersuchung im Flug Insekten Verhalten die Verwendung von kleinen drahtlosen neuromuskuläre Recordern und Stimulatoren aufgefordert. Diese Technologie ermöglicht die Entwicklung eines insekten Maschine Hybridsystem einen lebenden Insekts Plattform in diesem Protokoll beschrieben ist. Darüber hinaus stellt dieses Protokoll, um die Systemkonfiguration und Freiflugversuchsverfahren für die Funktion der Flugmuskeln in einem untethered Insekt zu bewerten. Zur Demonstration, gezielte wir die dritte axillären sclerite (3Ax) Muskel zu kontrollieren und nach links oder rechts Drehen eines fliegenden Käfer erreichen. Eine dünne Silberdrahtelektrode wurde auf der 3Ax Muskel auf jeder Seite des Käfers implantiert. Diese verbunden wurden an die Ausgänge eines drahtlosen Rucksack (dh einer neuromuskulären elektrischen Stimulator) , montiert auf dem Halsschild des Käfers. Der Muskel wurde in freiem Flug stimuliert durch die Stimulationsseite alternierend (links oder rechts) oder Verändern der stimulation Frequenz. Der Käfer wandte sich an der ipsilateralen Seite, wenn der Muskel wurde ein abgestuftes Reaktion auf eine Erhöhung der Frequenz angeregt und ausgestellt. Der Implantationsprozess und Volumenkalibrierung der 3-dimensionale Bewegungserfassungskamerasystem müssen mit Sorgfalt durchgeführt werden, um den Muskel nicht beschädigt wird und die Spur des Markers zu verlieren, respectively. Diese Methode ist sehr vorteilhaft Insektenflug zu studieren, da sie die Funktionen des Flugmuskel von Interesse im freien Flug zu offenbaren hilft.

Die Elektrodenimplantationsverfahren ist in Figur 2 dargestellt dünne Silberdrahtelektroden wurden in die 3Ax Muskel des Käfers durch kleine Löcher auf der weichen Kutikula auf den Muskel (Abbildungen 2d - e) durchbohrt implantiert.. Diese weiche Nagelhaut wird knapp über dem apodema des basalar Muskel gefunden , nachdem der vordere Teil des metepisternum Entfernen (Figuren 2d - c). Die Elektroden wu...

Watch this Scientific Journal Video about Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata at JoVE.com

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

Insect-Maschine Hybridsystem: Remote Radio Control eines frei fliegenden Käfer (<em&gt; Mercynorrhina torquata</em&gt;)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

insect-machine-hybrid-system-remote-radio-control-freely-flying

Research

JoVE Journal

Neuroscience

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

12.1K Aufrufe.  Nanyang Technological University. This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

Serie: Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

Lokalisieren von Protein in 3D Neural Stem Cell Culture: Ein Hybrid Visualisierung Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Forschung

Neurowissenschaften

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Langfristige Verhaltens Verfolgung von Frei Schwimmen Schwach elektrische Fische

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Ein vereinfachtes Verfahren für Ultra-Low Density, Long-Term Primäre Hippocampus Kultur

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Optische Kontrolle eines Neuronal Protein eine genetisch kodierter unnatürliche Aminosäure in Neurons Verwendung

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Neuropharmakologische Manipulation von Verhaltene und Free-Fliegen-Honig-Bienen,<em&gt; Apis mellifera</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

Aufzeichnung von EEG in frei bewegten neonatalen Ratten mit einer neuartigen Methode

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

Nicht-invasive EEG-Ableitungen von frei beweglichen Ferkel

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Langfristige sensorischen Konflikt in Mäusen frei Verhalten

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Hybrid Microdrive System mit wiederherstellbarer Opto-Silikon-Sonde und Tetrode für Dual-Site High Density Recording in frei beweglichen Mäusen

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

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 ...

Insect-Maschine Hybridsystem: Remote Radio Control eines frei fliegenden Käfer (

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