The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.

Alle Mausprotokolle wurden in Übereinstimmung mit National Institutes of Health Leitlinien und wurden von der Tierstudien Ausschuss der Washington University School of Medicine (St. Louis, MO) zugelassen. 1. Vorbereitung der Testplatte und Gehäuse <ol><li> Reinigen Sie die Glasoberfläche. </li><li> Fixierung des T-Typ Faser Thermoelementsonde an der Oberfläche in der Mitte der Glasplatte mit Labor Band. </li><li> Legen Sie die Tiergehege auf der Glasplatte in einer Linie entlang der Mitte der Platte. </li><li> Schrauben Sie die Thermoelementsonde durch das Zentrum Tiergehege und stecken Sie in den Datenlogger. Drehen Sie den Datenlogger an, während Deaktivierung des Auto-Shutdown-Funktion, und befestigen Sie den Datenlogger an den Computer mit dem mitgelieferten Kabel. <ol><li> Wenn die Aufzeichnung der Plattentemperatur während des Experiments, öffnen Sie die Datenlogger-Software Aufnahme Plattentemperaturen zu beginnen. </li><li> Falls erforderlich, stellen Sie die Software neuKabel die Plattentemperatur einmal pro Sekunde. </li><li> Starten Sie die Aufnahme Temperaturen mit Hilfe der Software mit dem Thermodatenlogger verpackt. </li></ol></li><li> Trennen Sie das Gehäuse mit schwarzen Einsätzen, um visuelle Interaktion zwischen Mäusen verhindern. </li><li> Position unterhalb des Glasspiegel, so dass die Unterseite der Gehäuse aus einer bequemen Sitzposition sichtbar ist. </li></ol> 2. Erwärmung / Abkühlung der Glasplatte <ol><li> Füllen Aluboxen mit erwärmtem Wasser, nassem Eis oder Trockeneis und positionieren Sie sie angemessen auf die Glasplatte (Aluminiumfolie Pakete mit Trockeneis gefüllt können auch, um das Glas zu kühlen verwendet werden; Abbildung 1) 2. <ol><li> Für die Prüfung bei 30 ° C, positionieren Sie die Aluboxen etwa 0,25 &#39;&#39; von den Tiergehegen (2B) 2. <ol><li> Legen Sie einen beheizten Wasser Zirkulator auf beiden Seiten der Glasplatte. Stellen Sie den Thermostat auf 45 - 60 ° C, und unsE ist die Alu-Boxen mit einem stetigen Strom von heißem Wasser (1C) 2 zu füllen. </li><li> Positionieren Sie die Umwälzpumpen, dass das heiße Wasser aus den Aluboxen entwässert direkt zurück in das Reservoir des Thermostaten auf jeder Seite (Abbildung 1 C) 2. </li></ol></li><li> Für die Prüfung bei RT, lassen Sie die Felder leer (Abbildung 2) 2. </li><li> Für die Prüfung bei 17 ° C, stellen Sie die Boxen etwa 0,25 &#39;&#39; von den Tiergehegen auf beiden Seiten füllen und mit nassem Eis (Abbildung 2) 2. </li><li> Für die Prüfung bei 12 ° C, stellen Sie die Boxen etwa 1,25 &#39;&#39; von den Gehäusen zu beiden Seiten und füllen mit Trockeneis (Abbildung 2) 2. </li><li> Für die Prüfung bei 5 ° C, stellen Sie die Boxen etwa 0,25 &#39;&#39; von den Gehäusen zu beiden Seiten und füllen mit Trockeneis (Abbildung 2) 2.<ol><li> Beim Abkühlen des Glases mit Trockeneis, stellen Sie sicher, für eine ausreichende Belüftung, um CO 2 Aufbau im Raum zu verhindern. </li></ol></li></ol></li><li> Warten Sie, bis das Glas, um den gewünschten Temperaturbereich zu erreichen. </li><li> Fügen Sie die Maus, um die Gehäuse auf der Platte. ANMERKUNG: Eine Rauschgenerator verwendet werden, um Lärmbelästigungen zu verringern. </li><li> Warten Sie, bis die Mäuse zu akklimatisieren. HINWEIS: In unserer Einrichtung das dauert etwa 2,5 Stunden, aber das kann deutlich auf Basis von Tierhaltung und Umgangsbedingungen variieren. </li><li> Halten Sie das Glas auf dem gewünschten Temperaturbereich, indem sichergestellt wird, dass die Boxen voll von erwärmtem Wasser, nassem Eis oder Trockeneis gehalten. HINWEIS: Mit unserem Gerät müssen die Kisten mit Eis etwa jede 90 Minuten wieder aufgefüllt werden. HINWEIS: Für die 17 ° C Bedingung, ist es hilfreich, das meiste Wasser aus den Aluminium-Boxen durch die Ablauflöcher vor dem Nachfüllen mit Eis zu leeren. Dadurch wird die Temperatur besser, und pr stabilisierenVeranstaltungsüberlauf HINWEIS: Die genaue Höhe der Trockeneis wird saisonal variieren, aber in der Regel halten die Boxen mehr als ¼ voll über die gesamte Länge der Box wird die Temperatur konstant zu halten. </li></ol> 3. Prüfung der Mäuse bei Fest Temperaturen <ol><li> Außerhalb des Verhaltensraum, füllen einen Eiskübel etwa zur Hälfte mit Trockeneis. </li><li> Mit einem Hammer oder Gummihammer, quetschen Sie das Trockeneis zu einem feinen Pulver. HINWEIS: Durch Überladen des Eimer wird es schwierig, vollständig das Trockeneis zu Pulver zermalmen. </li><li> Mit einem geraden Rasierklinge oder einer Schere, schneiden Sie die Spitze einer 3-ml-Spritze. </li><li> Unter Verwendung einer 21 G Nadel, stoßen 3 Löcher auf gegenüberliegenden Seiten der Spritze (insgesamt 6 Löcher). HINWEIS: Diese Löcher werden den Druck durch Sublimation erzeugt verringern, während Zusammendrücken des Trockeneis. Der Cut-off-Spritze für mehrere Versuche wieder verwendet werden. </li><li> Nehmen Sie die Spritze, Trockeneis-Pulver und ein Hand Stoppuhr in die Verhaltensraum. </li><li> Füllen Sie die Spritze Kammer zur Hälfte mit Trockeneispulver. Halten Sie das Schnittende der Spritze vor einem flachen Gegenstand, und fest komprimieren das Pulver mit dem Kolben. Achtung; die Kunststoff-Kolben kann sich verbiegen oder brechen aus dem Druck. Wenn dies geschieht, ersetzen Sie den Kolben aus einer neuen Spritze. </li><li> Verlängern Sie die Spitze des Druck Trockeneis-Pellets über den Rand der Spritze. </li><li> Versuchsmäuse, die vollständig in Ruhe. <ol><li> Bei 30 ° C, 23 ° C und 17 ° C, Testmäuse, die alle 4 Pfoten auf dem Glas und nicht bewegen, aber nicht vollständig eingeschlafen 19. </li><li> Bei 12 ° C und 5 ° C, Testmäuse, die auf 2 oder 4 Pfoten Pfoten und nicht bewegen oder springen werden. </li></ol></li><li> Mit den Spiegel für Targeting, vorsichtig, aber fest drücken Sie die Flach Pellet bündig an der Glasoberfläche unter dem Maushinterpfote (1A) 2. Starten Sie den Handauslöser. </li><li> Stoppen Sie den Timer und nehmen Sie die Tablette, wenn die Maus bewegt sich weg von der gekühlten Glas. HINWEIS: Diese Rücklaufbewegung kann vertikal oder horizontal sein. <ol><li> Wenn die Maus bewegt sich nur kurz die Pfote und dann gibt es an der Kühlfläche, weiterhin Timing und anregend, bis die Maus macht eine dauerhafte Abkehr. Hinweis: Unser Labor verwendet eine maximale Anregung Zeit von 20 sec für Mäuse in der Mehrzahl der Fälle. </li></ol></li><li> Wiederholen Sie diesen Testverfahren bis mindestens 3 Werte auf jeder Pfote jedes Tieres gesammelt werden. Separate Studien testen gegen Pfoten auf der gleichen Maus um mindestens 7 min, und einen separaten aufeinanderfolgenden Studien auf eine einzelne Pfote um mindestens 15 min. </li><li> Falls erforderlich, mit verschiedenen Dicken von Glas an verschiedene Abkühlungsgeschwindigkeiten (Figur 3) 1 zu erzeugen. HINWEIS: Die Kühlrate ist umgekehrt proportional mit der Dicke des Glases korreliert. </li></ol> 4. Prüfung der Mäuse während der kalten Anpassung Hinweis: Dies ist ein alternatives Protokoll, das Testen als Glas plat erlaubtE abkühlt, anstatt einmal die Platte stabilisiert hat und die Mäuse wurden vollständig an die kalte Umgebung angepasst. <ol><li> Folgen Sie den in Abschnitt 1 Einrichten des Geräts aufgeführten Hinweise. </li><li> Folgen Sie den in Kapitel 3 werden auf Baseline-Messungen bei Raumtemperatur (7A) 2 nehmen Anweisungen. </li><li> Pre-Kühlung der Aluminiumboxen mit Trockeneis. </li><li> Sobald Ausgangsrückzugslatenzen gemessen wurden, positionieren Sie die vorgekühlte Boxen auf der Platte etwa 1,25 &#39;&#39; von den Gehäusen zu beiden Seiten (7A, Pfeil mit der Aufschrift &quot;Trockeneis hinzugefügt&quot;) 2. </li><li> Befolgen Sie die in Abschnitt 3, Maß zu nehmen, wie die Glasplatte abkühlt, die Messungen so oft wie möglich Anweisungen. </li></ol>

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The authors have nothing to disclose

The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge...

Mess kalten Reaktionsfähigkeit bei Nagern ist wichtig für ein besseres Verständnis der möglichen Mechanismen der kalten Empfindlichkeit beim Menschen unter normalen und pathologischen Bedingungen. Der Kalte Plantar Assay (CPA), ursprünglich entwickelt, einige Jahre vor 1, wurde entwickelt, um reproduzierbare und eindeutig murine Verhaltensreaktionen auf einen Kältereiz bei RT ausgeliefert zu generieren. Neuere Verbesserungen dieses Tests wurden auf einem großen Temperaturbereich 2 die reproduzierbare Messung der kalten Empfindlichkeit erlaubt. Beide Varianten sind auch entworfen, um mit relativ hohem Durchsatz und kostengünstig zu handhaben sein. Ein Großteil der Fortschritte im Verständnis der Mechanismen der kalten Empfindlichkeit mit anderen Verhaltensmethoden vorgenommen. Eine Methode ist die Acetonverdampfungstest, der Tupfen beinhaltet oder Sprühen Aceton auf der Maus Pfote und Messung der Menge an Zeit, die der Maus verbringt schnippte die Pfote 3,4. Leiderdie Antworten auf die Acetonverdampfung werden durch den nassen Gefühl und den Geruch des Aceton verwechselt. Auch kann die Kältereiz, die in der Acetonverdampfungstest angewendet wird auf die Menge an Aceton aufgetragen variieren, und ist schwierig zu quantifizieren. Schließlich müssen unverletzten Mäusen minimal antwortet Aceton als Ausgangswert, so dass es unmöglich ist, Analgesie in Abwesenheit von Überempfindlichkeit bei dieser Methode zu messen. Eine weitere klassische Test für Kälte Reaktionen ist die Tail-Flick-Test, in dem die Latenz zum Rücktritt wird gemessen, nachdem der Schwanz ist in kaltem Wasser 5,6 eingetaucht. Während die Verhaltensreaktionen in diesem Test eindeutig sind, und der Assay misst Antworten auf eine bestimmte Temperatur, müssen die Tiere während der Prüfung zurückgehalten werden, die kalte Reaktionsfähigkeit durch gut beschriebene spannungsinduzierte analgetische Mechanismen 7 verändern kann. Ein weiteres häufig verwendetes Werkzeug ist die kalte Platte Test, der die VerhaltensmaßeReaktionen von Mäusen, nachdem sie auf einem Peltier-Kühlplatte 8-10 platziert werden. Während dieses Tool liefert Informationen über Tier Reaktionen bei bestimmten Temperaturen, wurde auch uneinheitlich verwendet; verschiedenen Gruppen haben verschiedene Arten von Reaktionen einschließlich Anzahl der Sprünge 8,11, die Latenz bis zum ersten Ansprechen 8,11- 13 gemessen wird, und die Anzahl der Pfote hebt 11,13,14 mit sehr unterschiedlichen Ergebnissen. Die Kaltplattentest ist auch relativ geringen Durchsatz, da nur ein Tier zu einem Zeitpunkt getestet werden, und es ist eine teure und zerbrechliche Peltier-Vorrichtung erfordert. Das 2-Plattentemperatur Präferenztest ist eine häufig verwendete Derivat der Kaltplattentest, das die relative Zeitdauer, die Tiere ausgeben 2 verbundenen Platten mit unterschiedlichen Temperaturen 9,15- 17 misst. Ein weiteres ähnliches häufig verwendete Test ist der Wärmegradient Assay, in dem die Zeitdauer, die Mäuse zu verbringen in unterschiedlichen Temperaturzonenim Bereich zwischen 5 ° C und 45 ° C auf einer langen Metallplatte gemessen 16. Obwohl diese Assays erlauben den Vergleich von Temperaturen, ist es unklar, ob das Verhalten darstellt Temperatur Abneigung oder Temperatur bevorzugt. Schließlich ist die dynamische Kaltplattenassay wurde verwendet, um zu messen, wie Mäusen, auf sich ändernde Umgebungstemperatur 18 reagieren. Diese Methode geht darum, Mäuse auf einem RT Peltier-Vorrichtung und Rampen es bis zu 1 ° C während der Messung, wie viel die Mäuse springen oder lecken ihre Pfoten auf verschiedenen Plattentemperaturen. Während dieses Tests, wie Mäusen Anpassung an eine Kühlumgebung, es keine Möglichkeit zu testen, wie Mäusen, die Kältereiz zu reagieren in der Einstellung eines kühleren Umgebungstemperatur. Zusätzlich erfordert es eine teure Ausrüstung durchzuführen und bietet keine Möglichkeit, Mäuse zu Prüfeinrichtungen vor der Messung ihrer kalten Empfindlichkeit akklimatisieren. Um diese Tests zu ergänzen, überprüft der CPA die ACCLIMAted Antworten auf eine gut definierte Kältereiz bei einer Vielzahl von Temperaturbereichen, oder während des Prozesses der Anpassung an kalten Umgebungstemperaturen. Es kann bis zu 14 Mäuse in einer Zeit mit unseren aktuellen Gerät zu testen, mit dem Potenzial, für High-Throughput-Tests kostengünstig skaliert werden.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Name</td>
			<td style="width:320px;">Company</td>
			<td style="width:353px;">Catalog Number</td>
			<td style="width:303px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-type thermocouple probe</td>
			<td>Physitemp</td>
			<td>IT-24p</td>
			<td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plate</td>
			<td>Local glass company (in St. Louis, Stemmerich Inc)</td>
			<td>N/A</td>
			<td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#39;&#39;, but 3/16&#39;&#39; and 1/8&#39;&#39; are also useful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Data logger</td>
			<td>Extech</td>
			<td>EA15</td>
			<td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&#38;prodid=408)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mL Syringe</td>
			<td>BD</td>
			<td align="right">309657</td>
			<td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>If using Extech logger, any Pcwill work</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum boxes</td>
			<td>Washington University in St. Louis machine shop</td>
			<td>N/A</td>
			<td>boxes are 3&#39; long, 4.5&#39;&#39; wide, and 3&#39;&#39; tall with a sealed lid.&#160; There is a 1/2&#39;&#39; hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated water circulator</td>
			<td>VWR</td>
			<td></td>
			<td>Any water circulator model with a pump will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 gauge needle</td>
			<td>BD</td>
			<td align="right">305165</td>
			<td>The exact needle size is not important</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hand timer</td>
			<td>N/A</td>
			<td></td>
			<td>Any hand timer will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror</td>
			<td>N/A</td>
			<td></td>
			<td>Any flat mirror will work</td>
		</tr>
	</tbody>
</table>

a simple and inexpensive method for determining cold sensitivity and adaptation in mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Die Verhaltens-Antworten von Mäusen ausgelöst beginnend bei 30 ° C, 23 ° C, 17 ° C und 12 ° C sind in hohem Maße reproduzierbar (4A) 20. Um die Kältereiz unter der Hinterpfote erzeugt messen, wurden Mäuse mit einer Ketamin / Xylazin / Acepromazin anästhesiert Cocktail und die Pfoten wurden auf dem Glas auf einem T-Typ-Thermoelement Filament (4B) 20 befestigt. Das Glas gekühlt, erwärmt, um die gewünschte Testbereich wurde. Obwohl die Platte gleichmäßi...

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Eine einfache und kostengünstige Methode zur Bestimmung der Überempfindlichkeit auf Kälte und Anpassung an der Maus

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

a-simple-inexpensive-method-for-determining-cold-sensitivity

Research

JoVE Journal

Neuroscience

14.7K Aufrufe.  Washington University in St. Louis. The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Serie: A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Die Vermicelli und Capellini Handhabung Tests: Einfache quantitative Maßnahmen der geschickten Vorderpfote Funktion bei Ratten und Mäusen

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Forschung

Neurowissenschaften

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Ein einfacher Weg zu Ethanol Empfindlichkeit in Flies Measure

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Eine allgemeine Methode zur Bewertung der Inkubation von Saccharose Craving in Ratten

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

A Molecular Auslesen Langfristige Olfactory Anpassung in<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero Elektroporation und Whole Hemisphäre Explantate: A Simple Experimentelle Methode für Studien der frühen kortikalen Entwicklung

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Eine preiswerte, skalierbare Behavioral Assay zur Messung von Ethanol Sedierung Empfindlichkeit und Rapid Toleranz in<em&gt; Drosophila</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Eine Methode zum Ziel und Isolieren Airway-innervating sensorischen Neuronen bei Mäusen

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Eine einfache Ein-Schritt-Dissection-Protokoll für Whole-mount Vorbereitung von Adult<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Neuartige Assay für kalte Nozizeption in<em&gt; Drosophila</em&gt; Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

In VivoKultivierung-wie Murine Astrozyten über das schnelle, einfache und kostengünstige AWESAM-Protokoll

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...