Gli autori desiderano ringraziare Anni Lee e Maureen Byrne per il loro aiuto nello stabilire il luogo protocollo esteso preferenza condizionata.

Tutte le procedure sono condotte in conformità con il Weill Cornell Medical College Institutional Animal Care e Usa regole del Comitato. 1. Preparazione e configurazione di consegna stereotassico di vettori virali <ol><li> Se uno strumento, tampone sterile, o della mano indossando guanti sterili è contaminata da entrare in contatto con una superficie non sterile, eliminare o ri-sterilizzare lo strumento con un caldo tallone sterilizzatore, scartare il tampone o passare a nuovi guanti sterili. </li><li> Posizionare una gabbia del mouse con biancheria da letto su un blocco di calore per riscaldare per il recupero post-operatorio. </li><li> Impostare rasoio elettrico per radersi la testa e l&#39;etanolo e iodio per sterilizzare il cuoio capelluto. </li><li> Pulire le orecchie bar e bocca stretta della stereotax con il 70% di etanolo. </li><li> Utilizzando un 5 microlitri siringa Hamilton, redigere 5 ml di vettore virale (1 x 10 6 particelle virali / mL) e istituito nel supporto siringa sul stereotax. </li><li> Riscaldamento a microonde pad o accendere pad elettrica su una° posto su stereotax. Coprire con un tampone assorbente pulita per ridurre il rischio di ustioni. Controllo della temperatura corporea del mouse (tra 97-99,5 ° F) frequentemente durante procedura. </li><li> Iniettare topo (peso compreso tra 22-35 g) con una miscela di ketamina (100 mg / kg) e xilazina (20 mg / kg). Una volta che un topo è anestetizzato, punta della coda pizzico di verificare i riflessi del mouse e osserva qualsiasi movimento o risposta per garantire che il mouse è adeguatamente anestetizzato. </li><li> Radersi la testa tra le orecchie e gli occhi. </li><li> Pulire la testa rasata con alternanza di etanolo e iodio sterili applicatori di cotone. </li><li> Passa il mouse a pad riscaldato stereotax. </li></ol> 2. Microiniezione stereotassica di vettori virali <ol><li> Indossare guanti sterili durante l&#39;intervento chirurgico. Se i guanti sono contaminati in qualsiasi momento toccando superfici non sterili, cambiare i guanti. </li><li> Zero bar e con i denti bar scale orecchio. </li><li> Set denti in bar dente. </li><li> Vite in museruola. Se dimenando o baffo mossazione è notato, in qualsiasi momento, somministrare una dose di richiamo ip (~ 0,05 cc) di una miscela di ketamina (100 mg / kg) e xilazina (20 mg / kg). </li><li> Vite in barre orecchio. </li><li> Lubrificare gli occhi con PuraLube. </li><li> Tagliare cuoio capelluto aperta a appena dietro le orecchie con un bisturi. </li><li> Luogo bulldog clip su angoli del cuoio capelluto per sbucciare la pelle dal cranio. Bregma e lambda dovrebbe essere facilmente visibile. </li><li> Assicurati di sutura dal bregma al lambda sia allineata. Se la sutura è storto riaggiustare posizionamento della testa. </li><li> Verificare che tutte le impostazioni sulla stereotax sono azzerati. </li><li> Mettere la punta dell&#39;ago in atto a bregma. </li><li> Misurare tutte le coordinate per entrambi bregma e lambda. Questo include coordinate dorsale / ventrale, mediale / laterale e anteriore / posteriore. </li><li> Regolare la testa fino a quando le coordinate ventrali per bregma e lambda sono a 0,02 millimetri l&#39;uno dall&#39;altro. </li><li> Una volta allineata, dal bregma medialmente andare fuori alle coordinate mediale / laterale (M / L). Assicurarei lati sinistro e destro sono allineate all&#39;interno di 0,02 mm di vicenda. </li><li> Punta dell&#39;ago ri-center su bregma e da lì muoversi nella direzione anteriore / posteriore (A / P) per la coordinata anteriore / posteriore desiderato. </li><li> Se necessario, ago angolo in una direzione e assicurarsi che il stereotax è bloccato. Uscire alla M / L coordinata sinistra (+ / - 1,52) </li><li> Misurare la posizione ventrale della parte superiore del cranio da utilizzare come riferimento dopo il pozzo viene perforato. </li><li> Praticare un foro con una punta sterile alla coordinata desiderata. Controllare che l&#39;ago può entrare senza interruzioni attraverso il cranio. </li><li> Abbassare l&#39;ago alla coordinata ventrale desiderato. </li><li> Una volta che la coordinata è raggiunto, dip 0,015 millimetri sotto per ~ 10 sec per creare una piccola tasca per il vettore virale. </li><li> Iniettare il volume desiderato di vettore virale a una velocità di ~ 0,1 ml / min. Attendere 3 minuti per il vettore virale per diffondere completamente. Sollevare la punta dell&#39;ago 0,015 millimetri e attendere un ulteriore 2 min. </li><li>Immergere l&#39;ago (se facendo iniezioni angolati) nella direzione opposta. Ancora, registrare la posizione ventrale come riferimento per la parte superiore del cranio. </li><li> Ripetete la stessa procedura sul primo lato di iniettare vettore virale sul lato opposto (se facendo iniezioni bilaterali). </li><li> Applicare la cera ossea per entrambi i fori per sigillarli con il fine di legno di una sterile cotton-fioc. </li><li> Suturare il cuoio capelluto. </li><li> Applicare il blocco del nervo alla zona ferita. </li><li> Rimuovere il mouse da stereotax e mettere in gabbia riscaldata sul blocco di calore per recuperare per 45 min. </li><li> Monitorare i segni di dolore, tra cui: riduzione dell&#39;attività complessiva o irrequietezza, diminuzione del consumo di cibo e acqua o maggiore vocalizzazione. Buprenorfina (0,05 - 0,2 mg / kg) può essere somministrato due volte al giorno per una settimana, se necessario, per il dolore. Alternative accettabili includono: meperidina (20-60 mg / kg), pentazocina (10 mg / kg), nalbufina (4-8 mg / kg). </li></ol> 3. Condizionata Luogo Preferenza: Acquisition, Extinction e Ripristino <ol><li> Topi posto nella camera centrale di un luogo apparato preferenza a tre camere (Med Associates Inc., St. Albans, VT, USA) per un secondo periodo di 60 assuefazione con porte a ghigliottina chiusi. </li><li> Dopo assuefazione, porte a ghigliottina aperti e consentire topi libera esplorazione di tutti e 3 camere per 1.200 secondi, con il tempo trascorso in ciascuna camera di essere registrati utilizzando il software MedPC IV (Med Associates, Inc.). </li><li> Assegnare topi per camere di condizionamento in base al loro rendimento durante il pre-test. Usando un disegno di parte, coppia somministrazione del farmaco durante i successivi 3 giorni con il vano che era meno preferito durante la linea di base pre-test. </li><li> Nel corso dei prossimi 3 giorni di condizionamento, iniettare topi con la cocaina (10 mg / kg, ip), durante la sessione del mattino e li limita nella loro camera assegnata per 1.200 sec immediatamente dopo l&#39;iniezione. Topi tornare alle loro gabbie a casa dopo la sessione. Dopo 4 ore, iniettare topi con soluzione salina (0,01 ml / gdi peso corporeo) e relegarli alla camera opposto per il secondo turno pomeridiano 1.200. </li><li> Per eseguire il test per l&#39;acquisizione, posto topi in camera centrale con porte a ghigliottina chiuse per un secondo periodo di adattamento 60. Porte a ghigliottina aperte e consentire l&#39;esplorazione libera per 1.200 secondi, con il tempo trascorso in tutte le camere registrati dal software MedPC IV (Med Associates, Inc.). Calcola preferenza sottraendo la quantità di tempo trascorso nella camera salina-accoppiati dalla quantità di tempo trascorso nella camera di cocaina ea coppia. </li><li> Per spegnere la preferenza luogo, esporre topi per due sessioni di 20 min di libera esplorazione quotidiana dello stesso apparato, separati da almeno 2 ore, con inizio 24 ore dopo la prova di acquisto. Tempo trascorso in tutte le camere occorre nuovamente registrata e la grandezza del CPP iniziale deve essere confrontata con quella di ogni sessione estinzione. </li><li> Determinare se i topi hanno spento la preferenza indotta da farmaci da valutare se la preferenza per un parcamera di particolare attraverso due giorni di estinzione consecutivi è significativamente inferiore rispetto al punteggio di preferenza acquisizione. </li><li> Reintegrare topi con una dose primaria di cocaina (5 o 10 mg / kg, ip), e ri-esporre l&#39;apparecchio che consente di esplorazione libera e registrare il tempo trascorso in ogni vano. </li><li> Somministrare soluzione eutanasia Euthasol (150 mg / kg). Transcardially perfusione topi con 4% paraformaldeide (PFA) ed effettuare un esame istologico per confermare il posizionamento corretto microiniezione. </li></ol>

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Regionalmente e temporalmente specifici ablazione genica tramite microiniezione stereotassico di vettori virali combinati con CPP che include estinzione e le fasi di rimessa permette di indagine dei contributi specifici di geni di tre distinte fasi di comportamenti di dipendenza simile. Mentre knockout condizionale che viene realizzato utilizzando il sistema Cre-loxP tradizionale prevede spazio-temporalmente limitato ablazione genica, stereotaxically microinjecting Cre-ricombinasi in aree distinte del cervello di topi f...

La cocaina è uno psicostimolante altamente rinforzo. A seguito di esposizione ripetuta, diversi adattamenti molecolari e cellulari che si verificano in ricompensa rilevanti circuiti cerebrali che si ritiene di provocare compulsivo di droga in cerca di comportamento, provocando alti tassi di recidiva che presentano un grave problema clinico 1. La cocaina esercita questi effetti comportamentali di lunga durata di regolare l&#39;espressione genica. Per studiare gli adattamenti derivanti da uso cronico di cocaina, roditori modelli preclinici sono stati ampiamente utilizzati. Uno di questi modelli è il luogo preferenza paradigma condizionata (CPP). Tale modello prevede lo sviluppo di una associazione tra un ambiente appreso precedentemente neutro e le proprietà gratificanti della cocaina. Dopo diversi abbinamenti di cocaina con una particolare camera, sono ammessi animali di esplorare liberamente gli ambienti di cocaina-accoppiati e non-cocaina-appaiati e se preferiscono il vano droga appaiati, si dice che essi hanno acquisito un cocaina-indotta place preferenza. Inoltre, a seguito di un periodo di formazione estinzione, questo paradigma può essere usato per studiare al contesto specifico ripristino di cocaina comportamento di ricerca. Rispetto ad altri modelli comportamentali di dipendenza comportamento simile, come sensibilizzazione psicomotorio, che viene utilizzato per studiare lungo termine indotta dalla cocaina comportamentale e plasticità molecolare 2,3 e di auto-somministrazione (SA), che si ritiene più esattamente imitare coinvolgente comportamento simile trovato negli esseri umani, il paradigma CPP è una procedura semplice per studiare la cocaina apprendimento contestuale 4. Protocolli CPP possono essere convenientemente estesi per includere l&#39;estinzione e le fasi di reintegrazione, in modo simile a SA, che consentono di studiare i meccanismi alla base di craving e la ricaduta 5-7 e che si ritiene di ricapitolare gli aspetti di ciò che avviene in umana droga in cerca di comportamento e droga - e cue-indotto ricaduta 8-10. Un meccanismo che sta alla base delcocaina-indotta plasticità comportamentale che è caratteristica di ciascuna delle distinte fasi di CPP, compresa l&#39;acquisizione, estinzione e reintegrazione, è l&#39;attivazione di firme uniche di espressione genica in differenti regioni cerebrali. Per testare direttamente quali geni all&#39;interno di regioni cerebrali premio rilevanti mediare cocaina-indotta cambiamenti comportamentali, è utile essere in grado di manipolare selettivamente in maniera regionalmente specifico. Un modo per raggiungere questo obiettivo è quello microinject vettori ricombinanti adenoassociated virali (rAAV) che esprimono la ricombinasi Cre che utilizzano la chirurgia stereotassica, in aree distinte del cervello dei topi che hanno geni bersaglio affiancati da siti LoxP (topi floxed). Questo metodo consente il controllo temporale e regionale molto preciso quando e dove i geni vengono asportate, in entrambi i neuroni divisione e non la divisione, senza indurre risposte immunitarie 11-13. Questo livello di controllo rappresenta un importante vantaggio rispetto tecnologia tradizionale Cre-loxP di allevamento topi floxed with topi esprimenti ricombinasi Cre sotto il controllo di un promotore del gene endogeno, in quanto i tempi e la distribuzione di delezione del gene possono essere più strettamente regolata. Inoltre, virali knockout genico mediata da vettori aggira potenziali effetti compensativi inerenti allo sviluppo che possono verificarsi utilizzando strategie di eliminazione diretta tradizionali. Oltre alla cocaina CPP, che è descritto qui, la microiniezione di rAAV-Cre nel cervello di topi floxed per valutare il ruolo di specifici geni può essere applicato ubiquitariamente a paradigmi comportamentali che coinvolgono fasi distinte, tra cui l&#39;auto-amministrazione e psicomotorio sensibilizzazione. Ad esempio, il nostro laboratorio ha utilizzato la tecnologia rAAV-Cre per studiare il ruolo del Ca v 1.2 di tipo L Ca 2 + canali in cocaina psicomotorio sensibilizzazione 14. In particolare, rAAV-Cre stato microiniettato nel nucleo accumbens (NAc) di topi con il gene che codifica Ca v 1.2 floxed, per dimostrare che Ca v </sub&gt; 1.2 che agisce in questa regione media la fase di espressione di sensibilizzazione psicomotoria 14,15. Tuttavia, la strategia rAAV-Cre non può essere utilizzato se non esistono topi con un gene floxed di interesse, come era la nostra esperienza nel valutare il ruolo del Ca v 1.3 di tipo L Ca 2 + canali di sensibilizzazione psicomotoria. Quindi, una limitazione di utilizzo rAAV-Cre si presenta se non esistono topi condizionali per un particolare gene di interesse. Tuttavia, rAAVs che esprimono siRNA può essere utilizzato per geni bersaglio atterramento, come abbiamo fatto per esaminare il ruolo del Ca V 1.3 canali 14,15. Microinjecting rAAV-Cre in regioni cerebrali discrete di topi floxed e poi testarle nel paradigma CPP permette di indagine i geni specifici che mediano le varie fasi del comportamento di dipendenza, come e dove operano. L&#39;utilizzo di questo paradigma ha aiutato nella nostra comprensione di come la somministrazione ripetuta di cocaina in sostanza dirotta il brains circuito ricompensa causando cambiamenti adattativi nel molecolari di trasduzione del segnale e l&#39;espressione genica che portano allo stato di tossicodipendente 1,16,17.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Conditioned place preference activity chambers</td>
			<td>Med Associates, Inc., St. Albans, VT, USA</td>
			<td>MED-CPP-MS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment system for mouse</td>
			<td>David Kopf Instruments, Tujunga, CA, USA</td>
			<td>model 900</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton syringes</td>
			<td>Hamilton Company, Reno, Nevada, USA</td>
			<td>7634-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-Cre-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7004</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

stereotaxic microinjection of viral vectors expressing cre recombinase to study the role of target genes in cocaine conditioned place preference

Microinjecting virali (rAAV) vettori ricombinanti adenoassociated esprimono Cre ricombinasi in distinte regioni del cervello del mouse in modo selettivo i geni knockout di interesse permette maggiore controllo temporalmente e specifici per ogni regione di delezione genica, rispetto ai metodi esistenti. Mentre delezione condizionale può essere ottenuto anche con accoppiamento topi che esprimono ricombinasi Cre sotto il controllo di promotori genici specifici con i topi che portano un gene floxed, microiniezione stereotassico consente per il targeting delle aree cerebrali discrete a sperimentatore-determinati punti di tempo di interesse. Nel contesto di cocaina luogo preferenza condizionata, e altri paradigmi comportamentali della cocaina come l&#39;auto-somministrazione o psicomotorio sensibilizzazione che può coinvolgere recesso, estinzione e / o fasi di rimessa, questa tecnica è particolarmente utile a esplorare il contributo unico di geni bersaglio di questi distinti fasi di modelli comportamentali di cocaina indotta plasticità. Specificamente,Questa tecnica permette di ablazione selettiva di geni bersaglio durante fasi distinte di un comportamento di testare il loro contributo al comportamento nel tempo. In definitiva, questa comprensione permette terapeutica più mirati che sono meglio in grado di affrontare i fattori di rischio più potenti che si presentano nel corso di ogni fase del comportamento di dipendenza.

CPP Dopo aver eseguito CPP sui topi microiniettati, si dovrebbe verificare che il gruppo di controllo a iniezione (rAAV-GFP) topi hanno normalmente acquisito preferenza per la camera di farmaco-accoppiato (Figura 1A, 1B). I topi sono considerati di aver acquisito la preferenza per una particolare camera di preferenza quando la cocaina (tempo trascorso nella camera di cocaina-accoppiato meno tempo trascorso in camera salina-accoppiato) è significativamente più alto rispetto al...

Watch this Scientific Journal Video about Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference at JoVE.com

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

Questo articolo viene descritto come microinject vettori virali nel cervello di topo e poi prova in un luogo climatizzato preferenza paradigma che comprende una fase di acquisizione, l&#39;estinzione e la reintegrazione.

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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Behavior

18.5K Views.  Weill Cornell Graduate School of Biomedical Sciences. Questo articolo viene descritto come microinject vettori virali nel cervello di topo e poi prova in un luogo climatizzato preferenza paradigma che comprende una fase di acquisizione, l'estinzione e la reintegrazione.

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

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

Iniezione stereotassica di microRNA-esprimono lentivirus al mouse Hippocampus CA1 regione e valutazione dei risultati Behavioral

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

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A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

Una procedura per studiare l&#39;effetto della prolungata restrizione alimentare sulla Cerco eroina nei ratti in astinenza

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Preferenza Grasso: un modello Novel of Eating Comportamento in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

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 ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Roditore cervello microiniezione di studio molecolare substrati di Motivato Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

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.

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 ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

Posizionare e risposta apprendimento nel campo aperto Torre Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

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 ...

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 ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

Assessing the Coherence of Parents&#039; Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valutazione della coerenza delle brevi narrazioni dei genitori per quanto riguarda il loro bambino utilizzando la procedura di campionamento del discorso di cinque minuti

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...