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.

Tutti i protocolli topo erano in conformità con le linee guida del National Institutes of Salute e sono state approvate dal comitato Animal Studies di Washington University School of Medicine (St. Louis, MO). 1. Preparare la piastra di prova e Contenitori <ol><li> Pulire la superficie del vetro. </li><li> Fissare la T-tipo di sonda termocoppia filamento alla superficie al centro della lastra di vetro con il nastro di laboratorio. </li><li> Posizionare dello stabulario sulla lastra di vetro in una singola linea lungo il centro del piatto. </li><li> Infilare la sonda della termocoppia attraverso il recinto degli animali centro e collegarlo alla data logger. Girare il data logger sulla mentre disattivare la funzione di spegnimento automatico, e collegare il logger al computer con il cavo fornito. <ol><li> Se si registra la temperatura della piastra durante l&#39;esperimento, aprire il software datalogger per iniziare a registrare le temperature della piastra. </li><li> Se necessario, modificare il software di ricavo la temperatura della piastra ogni secondo. </li><li> Iniziare il rilevamento della temperatura utilizzando il software fornito con il data logger termico. </li></ol></li><li> Separare il recinto con inserti neri per impedire l&#39;interazione visiva tra i topi. </li><li> Posizione rispecchia sotto il vetro in modo tale che la parte inferiore delle recinzioni è visibile da una posizione seduta comoda. </li></ol> 2. riscaldamento / raffreddamento della lastra di vetro <ol><li> Riempire scatole di alluminio con acqua riscaldata, ghiaccio umido, o ghiaccio secco e posizionare in modo appropriato sulla lastra di vetro (pacchetti di stagnola alluminio riempiti con ghiaccio secco può essere utilizzato anche per raffreddare il vetro; Figura 1) 2. <ol><li> Per la prova a 30 ° C, posizionare le scatole di alluminio circa 0,25 &#39;&#39; lontano dai recinti degli animali (Figura 2b) 2. <ol><li> Impostare un circolatore acqua riscaldata su entrambi i lati della piastra di vetro. Impostare il circolatore a 45 - 60 ° C, e civia e per riempire le scatole di alluminio, con un flusso costante di acqua calda (Figura 1C) 2. </li><li> Posizionare i circolatori tale che l&#39;acqua calda dalle scatole di alluminio drena direttamente nel serbatoio del circolatore su ciascun lato (Figura 1C) 2. </li></ol></li><li> Per il test a temperatura ambiente, lasciare le caselle vuote (Figura 2) 2. </li><li> Per la prova a 17 ° C, posizionare le caselle circa 0,25 &#39;&#39; lontano dai recinti di animali su entrambi i lati e riempire con ghiaccio umido (Figura 2) 2. </li><li> Per la prova a 12 ° C, posizionare le caselle circa 1,25 &#39;&#39; di distanza dalle recinzioni su entrambi i lati e riempire con ghiaccio secco (Figura 2) 2. </li><li> Per le prove a 5 ° C, posizionare le caselle circa 0,25 &#39;&#39; distanti custodie su entrambi i lati e riempire con ghiaccio secco (Figura 2) 2.<ol><li> Quando il raffreddamento del bicchiere con ghiaccio secco, assicurarsi che vi sia sufficiente ventilazione per evitare l&#39;accumulo di CO 2 nella stanza. </li></ol></li></ol></li><li> Attendere che il vetro per raggiungere il campo di temperatura desiderata. </li><li> Aggiungere i topi ai contenitori sulla piastra. NOTA: Un generatore di rumore bianco può essere utilizzato per ridurre i disturbi acustici. </li><li> Attendere che i topi si adatti. NOTA: Nella nostra struttura questo richiede circa 2,5 ore, ma puo &#39;variare notevolmente in base stabulazione degli animali e le condizioni di manipolazione. </li><li> Mantenere vetro nel range di temperatura desiderato garantendo che le scatole siano pieni di acqua riscaldata, ghiaccio umido o ghiaccio secco. NOTA: Con il nostro apparato le caselle devono essere riempite con ghiaccio circa ogni 90 min. NOTA: Per la condizione di 17 ° C, è utile svuotare la maggior parte dell&#39;acqua dalle scatole di alluminio attraverso i fori di scarico prima di riempire con ghiaccio. Ciò stabilizzare la temperatura migliore e prtroppopieno evento NOTA: La quantità esatta del ghiaccio secco varia stagionalmente, ma in generale mantenendo caselle più di ¼ completi lungo tutta la lunghezza della scatola manterrà costante la temperatura. </li></ol> 3. Test del Mice a temperature fisse <ol><li> Al di fuori della sala del comportamento, riempire un secchio di ghiaccio pieno per metà di ghiaccio secco. </li><li> Con un martello o mazzuolo, schiacciare il ghiaccio secco in una polvere finissima. NOTA: eccessivo del secchio renderà difficile schiacciare completamente il ghiaccio secco in polvere. </li><li> Utilizzando una lama di rasoio o forbici, tagliare la parte superiore di una siringa da 3 ml. </li><li> Utilizzando un ago 21 G, colpire 3 fori ai lati opposti della siringa (totale di 6 buche). NOTA: Questi fori diminuirà la pressione generata dalla sublimazione comprimendo il ghiaccio secco. La siringa cut-off può essere riutilizzato per diversi esperimenti. </li><li> Prendere la siringa, polvere ghiaccio secco, e un cronometro portatile nella stanza comportamentale. </li><li> Riempire la camera di siringa di mezzo pieno di polvere di ghiaccio secco. Tenere l&#39;estremità tagliata del siringa contro un oggetto piatto, e saldamente comprimere la polvere utilizzando lo stantuffo. Stai attento; lo stantuffo di plastica può piegarsi o rompersi dalla pressione. Se questo accade, sostituire lo stantuffo di una nuova siringa. </li><li> Estendere la punta del pellet ghiaccio secco compressa oltre il bordo della siringa. </li><li> Topi di prova che sono completamente a riposo. <ol><li> A 30 ° C, 23 ° C e 17 ° C, i topi che hanno tutte 4 zampe sul vetro e non si muove, ma non completamente addormentato 19. </li><li> Al 12 ° C e 5 ° C, i topi di prova che sono su 2 zampe o 4 zampe e non in movimento o saltare. </li></ol></li><li> Utilizzando gli specchi per il targeting, delicatamente ma con fermezza premere il filo pellet piatto contro la superficie di vetro sotto il mouse hindpaw (Figura 1A) 2. Avviare la mano-timer. </li><li> Arrestare il timer e rimuovere il pellet quando il mouse si allontana dal vetro raffreddato. NOTA: Lo spostamento di retrocessione può essere verticale o orizzontale. <ol><li> Se il mouse si muove molto brevemente la zampa e poi ritorna alla superficie di raffreddamento, continuano i tempi e stimolando il mouse fino a quando fa una mossa permanente di distanza. NOTA: Il nostro laboratorio utilizza un tempo massimo di 20 sec stimolo per topi nella maggior parte dei casi. </li></ol></li><li> Ripetere questa procedura di test fino a quando vengono raccolti almeno 3 valori per ogni zampa di ciascun animale. Prove separate test zampe opposte sulla stessa mouse almeno 7 min, e prove consecutive separati per ciascuna zampa di almeno 15 min. </li><li> Se necessario, utilizzare diversi spessori di vetro per generare differenti tassi di raffreddamento (Figura 3) 1. NOTA: Il tasso di raffreddamento è inversamente correlato con lo spessore del vetro. </li></ol> 4. Verifica del Mice Durante fredda Adattamento NOTA: Questo è un protocollo alternativo che consente di testare la piattaforma di vetroe si raffredda, piuttosto che una volta che il piatto si è stabilizzata ei topi hanno completamente adattato per l&#39;ambiente freddo. <ol><li> Seguire le istruzioni di cui alla sezione 1 per impostare l&#39;apparecchio. </li><li> Seguire le istruzioni riportate nella sezione 3 di prendere misurazioni di base a RT (Figura 7A) 2. </li><li> Pre-raffreddare le scatole in alluminio con ghiaccio secco. </li><li> Una volta latenze ritiro di base sono stati misurati, posizionare le caselle preraffreddato sul piatto circa 1,25 &#39;&#39; di distanza dalle recinzioni su entrambi i lati (Figura 7A, freccia etichettato come &quot;ghiaccio secco ha aggiunto&quot;) 2. </li><li> Seguire le istruzioni riportate nella sezione 3 di prendere misure come la lastra di vetro si raffredda, le misurazioni il più spesso possibile. </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...

Misurare reattività freddo nei roditori è importante per migliorare la comprensione dei potenziali meccanismi di sensibilità freddo negli esseri umani sia in condizioni normali e patologiche. The Cold plantare Assay (CPA), originariamente sviluppato diversi anni fa 1, è stato progettato per generare risposte riproducibili, inequivocabili murine comportamentali a uno stimolo freddo consegnato a RT. Miglioramenti più recenti di questo test hanno permesso la misurazione riproducibile di sensibilità fredda in un ampio intervallo di temperature 2. Entrambe le versioni sono inoltre progettati per essere relativamente alto rendimento, e poco costoso da utilizzare. Una grande quantità di progressi sono stati compiuti nella comprensione dei meccanismi di sensibilità a freddo utilizzando altri metodi comportamentali. Un metodo è il test di evaporazione acetone, che comporta tamponando o spruzzando acetone sulla zampa mouse e misurando la quantità di tempo che il mouse spende portando l&#39;3,4 zampa. Purtroppo,le risposte alla acetone evaporazione sono confusi dalla sensazione umida e l&#39;odore del acetone. Inoltre, lo stimolo fredda che viene applicato durante la prova di evaporazione acetone può variare in base alla quantità di acetone applicata, ed è difficile da quantificare. Infine, i topi hanno risposte indenni minime quantità di acetone al basale, rendendo impossibile misurare l&#39;analgesia in assenza di ipersensibilità con questo metodo. Un altro test classico per le risposte a freddo è il test coda flick, dove la latenza di recesso viene misurata dopo la coda è immerso in 5,6 acqua fredda. Mentre le risposte comportamentali in questo saggio sono inequivocabili e il test misurano risposte ad una temperatura specifica, gli animali devono essere trattenuti durante la prova, che possono alterare la risposta fredda attraverso ben definite meccanismi analgesici da stress 7. Un altro strumento comunemente usato è il test della piastra fredda, che misura il comportamentorisposte di topi dopo che sono posti su una piastra di Peltier raffreddato 8-10. Mentre questo strumento fornisce informazioni sulle risposte animale a temperature specifiche, è stato anche utilizzato incoerente; diversi gruppi hanno misurato diversi tipi di risposte incluso il numero di salti 8,11, la latenza alla prima risposta 8,11- 13, e il numero di ascensori paw 11,13,14 con risultati molto diversi. Il saggio piastra fredda è relativamente bassa capacità, come solo un animale può essere testato per volta, e richiede un dispositivo Peltier costosi e fragili. Il test preferenza temperatura 2-piastra è un derivato comunemente usato per il test della piastra fredda che misura la quantità relativa di tempo che gli animali passano su 2 piastre collegate delle diverse temperature 9,15- 17. Un altro simile test comunemente usato è il saggio gradiente termico, in cui la quantità di tempo che i topi passano in differenti zone di temperaturacompresa tra 5 ° C e 45 ° C su una piastra metallica lunga 16 viene misurata. Mentre questi test permettono il confronto delle temperature, non è chiaro se il comportamento rappresenti avversione temperatura o di preferenza temperatura. Infine, la dinamica dosaggio piastra fredda è stato usato per misurare quanto topi rispondono alle mutevoli temperatura ambiente 18. Questo metodo prevede il posizionamento mouse su un dispositivo Peltier RT e rampa verso il basso per 1 ° C durante la misurazione di quanto i topi saltano o leccarsi le zampe a temperature diverse placche. Mentre questo test come topi adattarsi ad un ambiente di raffreddamento, non fornisce un modo per testare come topi rispondono ad uno stimolo freddo nella cornice di una temperatura ambiente più fresco. Inoltre, richiede attrezzature costose effettuare e non fornisce un modo per acclimatare topi all&#39;apparecchiatura prova prima di misurare la loro sensibilità fredda. Per completare questi test, il CPA testa il ACCLIMAted risposte ad uno stimolo fredda ben definito in una varietà di campi di temperatura, o durante il processo di adattamento a temperature ambiente freddo. Si può testare fino a 14 topi alla volta con il nostro apparato attuale, con la possibilità di essere a buon mercato in scala per i test high-throughput.

<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.

Le risposte comportamentali indotte da topi a partire da 30 ° C, 23 ° C, 17 ° C, e 12 ° C sono altamente riproducibili (Figura 4A) 20. Per misurare lo stimolo freddo generato sotto il hindpaw, i topi sono stati anestetizzati con ketamina / xilazina / acepromazina cocktail e le zampe sono state fissate sul vetro sulla cima di una termocoppia di tipo T filamento (Figura 4B) 20. Il vetro è stato raffreddato o riscaldato alla gamma di test desiderato. Sebbene la p...

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.

Un metodo semplice e poco costoso per la determinazione fredda sensibilità e adattamento nei topi

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

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Neuroscience

14.6K Views.  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.

Video: 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.

I vermicelli e test Manipolazione Capellini: Semplici misure quantitative della funzione zampe anteriori abile nel ratto e nel topo

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

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Neuroscienze

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.

Un modo semplice per misurare sensibilità etanolo 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 ...

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.

Un metodo generale per la valutazione di incubazione della brama di saccarosio in ratti

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.

Una lettura molecolare di lungo termine adattamento olfattivo nel<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 elettroporazione e Espianti intero emisfero: un semplice metodo sperimentale per gli studi del primo sviluppo corticale

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.

Un poco costoso, test comportamentale scalabile per la misurazione etanolo sedazione sensibilità e tolleranza Rapid 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.

Un metodo per identificare e isolare Airway-innervano neuroni sensoriali nei topi

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.

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti<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.

Nuovo metodo di analisi per Cold nocicezione in<em&gt; Drosophila</em&gt; Le larve

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.

Coltura In Vivo-piace murini astrociti utilizzando il protocollo AWESAM veloce, semplice e poco costoso

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 ...