Name: assessment of cocaine-induced behavioral sensitization and conditioned place preference in mice
Uploaded: 2016-02-18T13:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice at JoVE.com

Gli autori ringraziano Karen Dietz e Shari Birnbaum per la precedente input su considerazioni di progettazione comportamentali e Lauren Peca per un aiuto con test comportamentali. Gli autori riconoscono anche il generoso sostegno della Simons Foundation (Simons Fondazione Initiative Autism Research Grant a CWC), NIDA (DA008277, DA027664, e DA030590 a CWC, F32DA027265 a LNS e F32DA036319 a RDP), la Fondazione FRAXA ricerca e Eleanor e Miles Shore Fellowship Program (supporto borsa di studio a LNS), e il John Kaneb Fellowship Program (supporto borsa di studio per MT).

Tutte le procedure sperimentali sono state approvate dal Comitato Istituzionale cura degli animali e Usa McLean Hospital. NOTA: La seguente protocollo descrive un approccio unico al CPP e locomotore sensibilizzazione, molti dettagli di cui differiscono da altri protocolli di successo (ad esempio, luce- vs test scuro fase, consecutiva contro somministrazione intermittente, etc.). Novizi possono desiderare iniziare con questi protocolli, o semplicemente usarli come guide, adattando alterazioni dalla letteratura sulla base del presupposto dell&#39;esperimento (s) a portata di mano. sono descritti metodi di misurazione automatizzati; tuttavia, è possibile utilizzare non automatizzato per ogni saggio (cioè, registrazione video, mano scoring). 1. place-preference <ol><li> Attrezzature e locale di installazione: <ol><li> Maniglia topi sperimentale per 1-3 minuti ogni giorno, per almeno 3-5 giorni prima del test. NOTA: Non manico topi possono trovare rimozione dalla camera di stressful, che possono interferire con o alterare condizionata. </li><li> Ottenere quattro o più apparati CPP tre camerata, preferibilmente dotati di photobeams per la raccolta automatica dei dati 18. Assicurarsi che ogni camera ha due camere grandi, visually- e tattilmente-distinte che collegano ad una camera più piccola, neutro tramite porte che può essere sollevato / abbassato per controllare l&#39;accesso. Coperchi in ciascuna camera dovrebbero aprire per l&#39;inserimento / rimozione di topi e di essere montato con le luci di piccole dimensioni, controllabili singolarmente (dimmerabili) (uno per camera). NOTA: CPP disegni da camera variano e possono essere acquistati in commercio o costruiti dai ricercatori. Per il motivo &quot;bilanciata&quot;, un regime raccomandato è una camera grande con pareti bianche e pavimenti griglia metallica e l&#39;altra con pareti nere e pavimenti bar. La camera centrale dovrebbe avere pareti grigie e solido pavimento in Plexiglass grigio. Per facilitare la descrizione, queste camere saranno indicati come &quot;bianco&quot;, &quot;nero&quot; e &quot;middle&quot;.I coperchi devono essere chiare Plexiglass. configurazioni alternative vano (uno o due da camera) sono anche possibili e discusso altrove (vedi la discussione). </li><li> Imposta la stanza come sarà durante i test: spegnere o impostare plafoniere per l&#39;impostazione minima e chiudere la porta. Utilizzare un misuratore di luce all&#39;interno di ogni camera e impostare le luci coperchio in modo che le camere medie sono leggermente più brillante (15-20 lux) di camere in bianco e nero (6-10 lux), al fine di scoraggiare i topi da passare il tempo lì. NOTA: Se il tempo medio trascorso in mezzo è tanto o più che nelle camere bianche o nere, aumentare ulteriormente il contrasto di illuminazione descritto al punto 1.1.3. In alternativa, utilizzare pavimenti meno attraente per la metà. (Ad esempio, ~ 220 fori di diametro 0,5 cm, distribuiti uniformemente in 6 x 3.5 x ¼ &quot;plexiglass), ma evitare di fare questa camera di avversione. Pilota alterazioni per garantire che diminuisce nel tempo di mezzo non sono causa di diminuzioni di esplorazioni totali (incroci) . </li><li> Programma raccolta automatizzata dei dati ( &quot;procedura&quot; Figura 1A) o raccogliere manualmente i dati in base ai seguenti parametri. Set prove per iniziare alla prima pausa trave in entrambi i camera di condizionamento. Impostare le sessioni di &quot;test&quot; per essere 20 minuti di lunghezza e per monitorare il tempo trascorso e del fascio si rompe in ciascuna camera. Impostare le sessioni di &quot;condizionamento&quot; di essere 30 minuti lungo e (opzionalmente) per misurare le interruzioni del fascio in ciascuna camera. Impostare tutte le luci della camera per illuminare durante il processo. </li><li> Preparare pieno volume di soluzione di cocaina necessaria per l&#39;esperimento a portata di mano. Sciogliere cocaina HCl in 0,9% NaCl (soluzione fisiologica), basando la concentrazione finale in un volume di iniezione di 0,1 ml / 10 g di peso corporeo (ad esempio, per una dose di 5 mg / kg, la concentrazione della soluzione è di 0,5 mg / ml). miscela vortex per 30-45 secondi, filtro sterile (0,2 micron filtri per siringa) e conservare a temperatura ambiente. </li></ol></li><li> test: NOTA: La timeline generale per CPP è pre-test, condizionata epoi post-test. Il pre-test può essere separato dalla condizionamento da parte di 1-3 giorni; tuttavia, condizionata e il giorno post-test dovrebbe aver luogo nei giorni consecutivi (Figura 2A). Questa temporizzazione deve essere mantenuto lo stesso per tutte le coorti nello stesso esperimento. <ol><li> Test durante fase leggera degli animali con la stessa apparecchiatura per ogni animale di tutti giorni. </li><li> Ogni giorno di prova (ad esempio, i test e giorni di condizionamento), spostare topi per l&#39;anticamera del comportamento e consentire loro di sedersi indisturbati nelle loro gabbie casa per 1-1,5 ore prima del processo. Scegliere una posizione dove i topi possono essere spostati rapidamente da loro gabbia all&#39;apparato, con il minimo disturbo, quando inizia il processo. Accendere tutte le apparecchiature in modo che eventuali rumori connessi con il test (ad esempio, ventilatori Tecnica) sono presenti. </li><li> Pulire accuratamente ogni apparato (pareti interne, pavimenti e vassoi) con lieve, alcool, glicole-Ethernet o a base di ammoniaca disinfezione salviette prima e dopo ogni topo (utilizzare il same il tipo di detergente per tutto l&#39;esperimento). Non trascurare pavimentazione inferiori. </li><li> Controllare che le luci della stanza siano impostati in modo appropriato (spento o in grigio) all&#39;inizio di ogni giornata. </li><li> Procedere con la seguente seconda che si tratta di un &quot;test&quot; o giorno &quot;condizionamento&quot;: <ol><li> On Trial giorni 1 e 6 (vale a dire, il pre e post-test), posizionare le porte inter-camera in posizione aperta (Figura 2B). </li><li> Caricare il programma informatico &quot;test&quot; e inserire gli ID degli animali (Figura 1A), quindi emettere un comando di avvio (Figura 1B), se applicabile. </li><li> Delicatamente inferiore ogni topo nella camera di metà del suo apparato assegnato, di fronte alla parete di fondo, e dolcemente chiudere il coperchio. Una volta che tutte le camere sono stati caricati, lasciare la sala prove e ridurre al minimo il rumore. </li><li> Lascia i topi all&#39;interno di ogni apparato fino a quando le prove per tutti i mouse hanno chiuso / finito (Figura 1C). dati degli studi di esportazione. </li></ol></li> &lt;li&gt; Appena prima o dopo il pre-test, pesare gli animali. Utilizzare questi pesi per il calcolo della dose durante il condizionamento. </li><li> Al fine di prepararsi per le prove di condizionamento, calcolare pre-test &quot;preferenza&quot; di ogni mouse per le camere in bianco e nero dal tempo trascorso in ciascuno di questi dall&#39;altro (cioè, &quot;nero meno nero&quot; e &quot;bianco minus bianco&quot; sottraendo; vedere Figura 3, Colonne 9 e 10). </li><li> Dal momento che i topi con forti preferenze iniziali rendono difficile il bilanciamento, stabilire un limite accettabile per i punteggi di preferenza (ad esempio, &lt;33% del tempo di prova totale) ed escludere i topi che superano da calcoli. Utilizzare un limite liberale (&lt;66% del tempo di prova totale) per massimizzare l&#39;inclusione quando logoramento è probabile che dopo il test è completo (ad esempio, a causa di off-bersaglio manipolazioni chirurgiche). NOTA: I topi che superano il limite può ancora essere testati, con un tentativo di mantenere le loro preferenze pre-test equilibrata. Più tardi, ttopi hese possono essere esclusi dalla analisi, se necessario, per bilanciare i punteggi pre-test. Se estremi pregiudizi pre-test (ad esempio,&gt; 800 sec) colpiscono in modo sproporzionato un gruppo, considerare alterando gli ambienti di condizionamento e / o utilizzando altri saggi. </li><li> Scegli la camera di bianco o nero come la camera di droga abbinamento per ogni mouse, nel frattempo sommando i corrispondenti punteggi di preferenza pre-test all&#39;interno di ogni gruppo con le seguenti priorità in mente: NOTA: Fare attenzione a bilanciare i punteggi tra i gruppi all&#39;interno di ciascuna coorte, nonché attraverso qualsiasi coorti precedenti. <ol><li> Rendere le somme per tutti i gruppi come equivalenti possibile. </li><li> Rendere le somme il più vicino possibile allo zero (cioè, scegliere il lato preferito per alcuni topi e il lato non preferito per gli altri). Se un quasi-somma zero è impossibile per qualsiasi dato gruppo, ri-regolare tutti gli altri a corrispondere al meglio alla miglior punteggio ottenibile per il gruppo di limitazione, favorendo gruppo leggermente negativo riassume soprapositivo. </li><li> Per quanto possibile, mantenere le assegnazioni di nero contro bianco e preferita rispetto a camere non preferite per abbinamenti di droga anche all&#39;interno di ogni gruppo. </li><li> Poiché non è sempre possibile per raggiungere gli obiettivi di cui sopra, correggere eventuali deviazioni rendendo opposte considerazioni di bilanciamento in coorti successive; tuttavia, cercare di evitare la produzione di coorti con selvaggiamente diverse preferenze media pre-test. </li></ol></li><li> Su condizionata giorni 2-5, posizionare porte inter-camera in posizione chiusa (Figura 2C). </li><li> Preparare singole siringhe con la cocaina (giorni 2 e 4) o soluzione salina (giorni 3 e 5) soluzione, come descritto al punto 1.1.5 e sulla base del peso corporeo valutate al pre-test. NOTA: dose deve essere scelto in relazione alle aspettative sperimentali, considerazioni di cui in premessa, e potenziali effetti pavimento e soffitto. Spesso è meglio per condurre esperimenti indipendenti utilizzando almeno due dosi differenti. </li><li> Load &quot;cID animale onditioning programma per elaboratore &quot;e inserire (Figura 1A), quindi emettere un comando di avvio (Figura 1B), se applicabile. </li><li> Scruff e iniettare ogni topo (ip), subito li abbassamento nella camera di bianco o nero appropriata del loro apparato assegnato di fronte alla parete di fondo, poi dolcemente chiudere il coperchio. Una volta che tutte le camere sono stati caricati, lasciare la sala prove e ridurre al minimo il rumore. </li><li> Rimuovere i topi da loro camera come vicino esattamente 30 min fattibile (cioè, il primo topi vengono rimossi dalla loro camere mentre altri topi sono ancora condizionata). Rimuovere gli animali il più silenziosamente possibile, senza introdurre rumore. </li></ol></li><li> Analisi statistica: <ol><li> Scegliere un metodo di analisi. In entrambi i casi il tempo sottrarre trascorso sul lato salina-accoppiato durante la post-test da tempo trascorso sul lato cocaina accoppiato durante la post-test (la cocaina - salina, sec) o utilizzare il tempo trascorso nella camera di droga abbinato post-testmeno il tempo di pre-test speso per la camera di droga-accoppiato. NOTA: se si usa il primo metodo, anche grafici lineari trama del tempo medio trascorso nel mezzo, camere saline- e droga-accoppiato durante i pre e post-test per ogni gruppo. Rispetto al pre-test, il post-test dovrebbe mostrare un aumento del tempo nel lato droga abbinato ed è diminuito il tempo trascorso nel lato salina-accoppiato (vedi figura 6, in basso, e discussione per la spiegazione). </li><li> A seconda della natura e del numero di gruppi a confronto, utilizzare un t-test, uno o due Way ANOVA, a seconda dei casi, possibilmente con l&#39;analisi post-hoc, per analizzare uno dei punteggi sottrazione sopra presentato. NOTA: cocaina punteggi di preferenza tendono ad essere variabile, e, inoltre, possono essere negativi (ad esempio, indicare l&#39;avversione al lato della droga-accoppiato). I topi che mostrano l&#39;avversione non devono essere rimossi (a meno che siano valori erratici statistici), dal momento che questo risultato è normale e probabilmente importanti per determinare le differenze tra grOups. Prevedono la necessità dimensioni del campione di 12 a 30 animali per gruppo, a seconda delle dimensioni effetto del trattamento. </li></ol></li></ol> 2. locomotore Sensibilizzazione <ol><li> Attrezzature e Camera Set-Up <ol><li> Ottenere un 4 x 8 (X x Y) Fotocellule serie (dimensioni esterne 11.5 &quot;x 20&quot;). Costruire una camera decappottabile in plexiglas nero (dimensioni interne 22 1/6 &quot;x 13 ¾&quot; x 9 3/8 &quot;) per ospitare l&#39;array (Figura 4A). </li><li> Preparare la sala prove in modo che una luce rossa (a soffitto ea parete) può essere utilizzato durante il test. </li><li> Preparare sessioni programma separato per assuefazione e iniezione prove quotidiane. <ol><li> Impostare prove di assuefazione ad essere tra i 30-60 min e iniezione prove per essere compreso tra 60-120 min (Figura 5B). La lunghezza raccomandata per ciascuna è di 60 min. Mantenere la lunghezza di prova consistente in più coorti all&#39;interno dello stesso esperimento. </li><li> Set prove per iniziare recording sulla prima pausa trave che si verifica dopo il segnale di avvio è stata avviata (Figura 5B). Per il processo di iniezione, impostare il segnale di avvio in modo che scatole possono essere avviati singolarmente (non all&#39;unisono), se possibile. </li><li> Impostare le interruzioni del fascio da registrare in definiti dall&#39;utente &quot;bidoni&quot;, preferibilmente di 5 minuti ciascuno (Figura 5B). </li></ol></li><li> Preparare pieno volume di soluzione di cocaina necessaria per l&#39;esperimento a portata di mano. Sciogliere cocaina HCl in 0,9% NaCl (soluzione fisiologica), basando la concentrazione finale in un volume di iniezione di 0,1 ml / 10 g di peso corporeo (ad esempio, per una dose di 5 mg / kg, la concentrazione della soluzione è di 0,5 mg / ml). miscela vortex per 30-45 secondi, filtro sterile (0,2 micron filtri per siringa) e conservare a temperatura ambiente. </li></ol></li><li> analisi NOTA: La fase iniziale di test viene eseguito per 10-11 giorni consecutivi. I topi ricevono due prove quotidiane: assuefazione (senza iniezione) e l&#39;iniezione. Somministrare soluzione salina per i primi tre di fi nostri giorni (vedi la discussione per importanza di assuefazione fisiologica), e la cocaina per i prossimi sette utilizzando la stessa dose (ad es., 15 mg / kg / die). <ol><li> Ogni giorno, ambientarsi topi nelle loro gabbie a casa per l&#39;anticamera comportamentale per 30 minuti a 1 ora. </li><li> Preparare pulito radura gabbie abitative acrilico del mouse di dimensioni standard, con un sottilissimo strato di lettiera fresca (ad esempio, i chip di pino), in modo da non oscurare photobeams, se del caso (Figura 4C &amp; D). </li><li> Inserire gabbie contro l&#39;asse Y della matrice photobeam vicino ad una estremità, in modo che cinque travi sono distanziati in modo uniforme lungo la sua lunghezza. Travi asse X non vengono utilizzati in questo test (Figura 4C &amp; D). </li><li> Prendere i topi dalla loro gabbia casa, in ordine casuale, collottola e pesare. Rimuovere ogni topo dalla barca pesare dalla base della loro coda (con il supporto) e posto direttamente nella loro gabbia motorio assegnato. Coprire ogni gabbia con un coperchio del filtro-top standard. </li><li> Preparare singole siringhe wesimo salina o soluzione di cocaina, come descritto al punto 2.1.7, sulla base del peso corporeo del giorno corrente. Iniziare con una dose di 15 o 20 mg / kg, e prendere in considerazione un secondo esperimento usando una dose maggiore o minore (vedi la discussione di fattori rilevanti). </li><li> Una volta che gli studi di assuefazione hanno finito per tutte le gabbie, caricare il programma di iniezione. </li><li> Uno alla volta, togliere i topi dalla loro gabbia di prova, collottola e dare il loro iniezione (ip). Prima di tornare il mouse per la sua gabbia di prova, avviare rapidamente il segnale di partenza per quella gabbia (Figura 5C, in basso). </li><li> Dopo tutte le prove di iniezione sono finiti, tornare topi per loro gabbie casa e la loro camera abitazioni. </li><li> Se lo si desidera, consentire topi sottoposti ad una serie di periodi di sospensione e sfide droga, come il seguente: stessa dose originale, metà dose, doppia dose, poi salina, permettendo sette giorni prima della sfida stessa dose e 06:57 giorni prima di ogni ulteriore sfida. Qualunque sia la scelta di sfide, eseguire manutenzione sun periodi di sospensione simili in tutta coorti all&#39;interno di un esperimento. NOTA: Se la dose iniziale è elevato (ad esempio, 30 mg / kg o superiore), la doppia dose deve essere saltato o sostituita con una dose inferiore. La sfida salina rivela alcuna attivazione locomotoria condizionata dall&#39;ambiente cocaina-abbinato sola, e nel processo, la quantità di locomozione sensibilizzata che la cocaina-dipendente (vedi Discussione). </li></ol></li><li> Analisi statistica <ol><li> Scegliere la porzione (s) di ogni prova che verrà utilizzato per l&#39;analisi. La maggior parte locomozione indotta dalla cocaina nei roditori si verifica entro il primo ~ 15-30 minuti dopo l&#39;iniezione di droga. A seconda variabili coinvolte, considera di analisi locomozione cumulativo per più finestre di tempo (ad esempio, la prima 15, 30, 60 e / o 120 min) o concentrarsi su spezzoni indipendenti del processo. </li><li> Utilizzando il tempo cornice scelta, sommare le pause del fascio per ciascun animale al giorno per le prove di adattamento e di iniezione separatamente e poi unverage le somme per ogni gruppo. Trama significa e gli errori standard della media (SEM) in un grafico a linee su tutti i giorni. Come trattamenti possono alterare come topi rispondono al farmaco precoce e / o in ritardo in ogni prova quotidiana, anche tracciare pause medi fascio gruppo da 5 min bin nel corso di ogni prova quotidiana. NOTA: Tracciare le medie attività giornaliere per prove di adattamento e di iniezione (ad esempio, prima 15 min) fa apparire artificialmente che locomozione viene smorzata mediante iniezione di soluzione salina, ma ricordate che l&#39;attività ha (molto probabilmente) rifiutato a questo livello entro la fine di assuefazione ogni giorno . </li><li> Analizzare giorni saline e trattamento farmacologico consecutivi separatamente utilizzando misure ripetute (RM) ANOVA avere il fattore entro soggetti del giorno / ora e compresi eventuali fattori tra soggetti nella progettazione, a seconda dei casi. Utilizzare un t-test o One-Way ANOVA per indagare le differenze di gruppo il giorno 1 di cocaina (esposizione acuta), e una ANOVA multivariata per le sfide. Se del caso, seguire significance con i test post-hoc o Univariata ANOVA. </li></ol></li></ol>

Gli autori dichiarano di non avere interessi finanziari concorrenti.

Questo protocollo illustra metodi place preference e locomotore sensibilizzazione, ciascuno dei quali può essere utilizzato dal laboratorio media di valutare aspetti della plasticità comportamentale indotta da farmaci. Come con la maggior parte dei test comportamentali, ci sono altre considerazioni degne di là del protocollo di base. Innanzitutto, ciascuna di queste tecniche può essere concepito come avente due fasi, l&#39;induzione ed espressione. &quot;Asincrono&quot; comprende lo sviluppo del comportamento per CP...

Research aimed at understanding drug addiction using animal models must take a variety of approaches to address each of the assorted components that obstruct treatment success, including reward/reinforcement/motivation and withdrawal and relapse, as well as the general persistence that further complicates these issues in addiction. Since rewarding experiences associated with taking a drug of abuse are thought to motivate subsequent use, studies focusing on drug-context associations may be particularly useful for understanding brain mechanisms that contribute to drug taking and seeking. One such assay, conditioned place preference (CPP) is a high-throughput method for comparing group differences in reward sensitivity. The traditional interpretation of the task involves classical, or Pavlovian, conditioning, where a conditioned stimulus (CS) is paired with an unconditioned stimulus (UCS), and after multiple pairings, the CS elicits the same behavior as the UCS (however, see39,40). Theoretically, animals learn to associate an interoceptive state (reward or aversion) with contextual cues. The relative aversive or appetitive intensity of the interoceptive state is then assessed by then determining the animal's preference for the contextual cues. The use of place conditioning to measure drug-reward associations dates back to at least 1957, to a study using morphine on rats in a Y-maze3,4. Over the past several decades, variations on this method have been widely used to study place preference and aversion in rodents to various stimuli, and it remains particularly useful in the study of associations induced by drugs of abuse. In drug-addiction research, the assay has been used to assess the rewarding properties of a number of drugs and the contribution of different brain systems and proteins to drug reward (for reviews, see5-7,44). While there are superior methods of assessing factors that contribute to drug addiction, namely drug self-administration, CPP is a simple and much more accessible approach to measuring reward function. 
Most current protocols for conditioned place preference and aversion (CPA) use an apparatus that allows rodents to have access to two distinct chambers, either via a doorway or smaller connecting chamber. Distinctions between the two chambers are often based, at a minimum, on visual and tactile cues, including wall color and floor texture, but sometimes include other elements, such as olfactory cues. "Biased" designs typically attempt to reverse a pre-existing, innate preference for one chamber over the other, such as the one that rodents generally show for a black chamber over white. "Unbiased" designs aim to create a preference to one of two chambers that were initially equally appealing by randomly counterbalancing assignment to either chamber within a group. A "balanced" design is used when animals show small preferences, but do not, as a group, favor the same chamber. Goals of this latter design are to produce 1) pre-test preference scores for the (eventual) cocaine-paired chamber that are not significantly different between experimental groups and 2) negligible preference for the cocaine-paired chamber at pre-test, either positive or negative8. The balanced design is ideal for use with the described chambers, which utilize contradicting biases for wall color (black over white) and flooring (wire over bar), resulting in a roughly equal distribution of small preferences for both the black and white sides in different animals. Balancing calculations are described in further detail below.
During conditioning, animals are exposed to a drug and quickly placed into one of these two environments for a limited time period. Exposure is typically via intraperitoneal (i.p.) or sometimes subcutaneous (s.c.) injection, although paradigms for intravenous (i.v.) self-administration9, and intracranial infusions38 in a place preference apparatus have also been developed. These pairings are complemented by non-drug (vehicle) pairings of the same length conducted in the opposite chamber, which can take place on the same day as drug pairings or on separate days. In general, when allowed to explore the apparatus after conditioning, animals will spend more time where they received a rewarding drug (i.e., one that humans and animals will voluntarily self-administer), while they will avoid a place where they were given a drug that induced illness (e.g., lithium chloride). Several studies have been dedicated to optimizing the conditions for place preference to different drugs of abuse (for review, see7). Cocaine doses (i.p.) for mice generally range from 1 to 20 mg/kg, with doses less than 5 mg/kg often used to parse high sensitivity in one group. Two or more drug pairings are typically required for adult mice10, and the length of these pairings is an important consideration. Very low doses of cocaine require an immediate and brief conditioning, likely because this method captures the most rewarding period of the exposure. Delayed or very long conditioning periods can result in no preference, or may even induce aversion11,12. Here is presented a basic method for obtaining conditioned place preference to cocaine in adult mice.
While the CPP assay is an ideal method for assessing reward-related learning and memory of drug-context associations, behavioral sensitization is arguably easier to perform and allows the assessment of changes that develop over repeated treatment. Also known as reverse-tolerance, behaviors undergoing sensitization are incrementally enhanced over repeated exposures to a particular drug of abuse, especially psychostimulants, and cross-sensitization is known to occur between some, but not all, of these drugs. One of the first assessments of cocaine-induced locomotor sensitization, in particular, in rodents was published in 197613. A number of labs have shown that sensitized locomotion is detectable long after drug cessation, depending on the original length, location and dose of exposure14-17, and the current protocol has been used to detect sensitization as long as 10 months following seven days (30 mg/kg) of cocaine treatment in mice18. The test can be performed using either photobeam or video-tracking technologies, in apparatuses of differing sizes and shapes, making it simple for many labs to perform. The robust nature, simplicity and persistence of locomotor sensitization makes its assessment an ideal part of examining basic mechanisms of long-lasting changes in drug-induced behavior. 
As is expanded upon in the discussion, an important consideration when performing the locomotor sensitization assay is whether drug is given in the home- or test-cage environment. To take advantage of the robust sensitization that occurs when drug administration occurs outside of the home cage, this protocol employs this method. However, it has been observed that when animals are not adequately habituated to a new environment before drug exposure, a novelty-induced ceiling effect occurs on Day 1, which can partially or fully mask the progressive nature of sensitization. It is likely that this represents synergistic locomotor-activating effects of the drug together with novelty, and while the mechanisms underlying such effects may be interesting, the method described is designed to reduce the role of novelty and allow the effects of the drug to be measured more independently. While it is expected this method will be useful in the assessment of other locomotor-sensitizing drugs, it has primarily evaluated its effectiveness with cocaine in C57BL/6 mice.

<table><tbody><tr><td>Cocaine Hydrochloride USP</td><td>Mallinckrodt Pharmaceuticals</td><td>0406-1520</td><td>Purchase and use (Schedule II controlled substance) for research purposes requires compliance and licensure according to state and federal law.&amp;nbsp;</td></tr><tr><td>Conditioned Place Preference,&amp;nbsp; Three Compartment Apparatus with Manual Doors and Lights for Mouse</td><td>Med-Associates Inc.</td><td>MED-CPP-MS &amp;amp; MED-CPP-3013</td><td>Our laboratory has used these boxes; however, many alternative boxes are available &amp;amp; acceptable.</td></tr><tr><td>PAS-Home Cage Activity Monitoring Photobeam Arrays</td><td>San Diego Instruments</td><td>2325-0223 &amp;amp; 7500-0221</td><td>Our lab houses these arrays inside of custom built chambers, as described in the text.&amp;nbsp; There are alternatives available.</td></tr><tr><td>Disposable Sani-Cloth disenfecting wipes</td><td>PDI</td><td>13872</td><td></td></tr></tbody></table>

assessment of cocaine-induced behavioral sensitization and conditioned place preference in mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

Risultati rappresentativi del dosaggio CPP sono mostrate in Figura 6 con wild-type topi C57BL / 6N a circa nove settimane di età. Il disegno dello studio era un fattoriale misto 2 x 3, con una variabile entro soggetti di prova (pre e post) e tra soggetti variabile del trattamento (soluzione salina e cocaina 5 e 10 mg / kg). Una RM ANOVA ha mostrato una significativa interazione tra test e trattamento (F 2,20 = 3.68, p &lt;0,05), che è stato interpretato in luogo de...

Watch this Scientific Journal Video about Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice at JoVE.com

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

Valutazione della cocaina indotta sensibilizzazione comportamentale e conditioned place preference nei topi

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

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16.9K Views.  Harvard Medical School, McLean Hospital. This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

Video: Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microiniezione stereotassica di vettori virali che esprimono Cre ricombinasi per studiare il ruolo di geni bersaglio in cocaina condizionata place preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

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Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Nuovo Apparato e metodo per il rinforzo della droga

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

Neuroscienze

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Behavioral Valutazione del sistema vestibolare invecchiamento mouse

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

Analisi completa di Trascrizione Dynamics da campioni di cervello seguito Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Una Procedura di osservare Context-indotta Rinnovo pavloviano condizionata alcol-seeking comportamento in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Sensation operante in Cerco il mouse

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

Reintegrazione di droga in Cerca nei topi utilizzando il paradigma di Conditioned Place Preference

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Metodi per l&#39;auto-somministrazione per via endovenosa in un modello murino

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

Un protocollo di Conditioned Place Preference per l'incubazione di Craving nei ratti di misura

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Un protocollo per la misurazione Cue reattività in un modello del ratto di disturbo da uso di cocaina

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...